A Review of Osteosarcoma Therapeutics

Michael W Beaury
Megan L Kelly-Beaury
Gilbert Sharp
DrJessica A Cottrell email@example.com
Article Info Article Notes
Seton Hall University
South OrangeNJUSA

400 South Orange Ave South Orange07079NJUSA

A Review of Osteosarcoma Therapeutics
6FBFA8EB0097983BA2F96B910981C5D9Received: March 07, 2018 Accepted: April 24, 2018OsteosarcomaChemoresistanceImmunotherapyMicroRNAsPathogenesis
Osteosarcoma is a rare but deadly cancer, predominantly affecting both adolescent and young adult populations.Osteosarcoma occurs when an aggressive malignant neoplasm arises from transformed cells of mesenchymal origin, which eventually produce a malignancy in the osteoid.Diagnosis of osteosarcoma typically results from symptoms of pain or swelling in the bone, which can be confirmed through laboratory testing of alkaline phosphatase and lactate dehydrogenase levels as well the detection of microscopic and macroscopic lesions.Pathogenesis of osteosarcoma is caused by a diverse set of factors including physical agents, radiation, chromosomal aberrations and viral infection which dysregulate cellular functions.Current research focuses on understanding how microRNAs play a role in osteosarcoma and other aggressive cancers.In this review, we discuss current treatments options including chemoresistant strategies and immunotherapies that show promise at combating osteosarcoma and other cancers.

Introduction

Osteosarcoma is a low incidence or uncommon cancer, which originates in the bones and is predominantly found in adolescents and young adults.Osteosarcoma usually occurs in individuals in the range of 10 to 30 years of age, although teens are the most commonly affected.Each year, 1,000 cases of osteosarcoma are diagnosed in the United States, and 45% of those cases occur in children and adolescents.Approximately seventy percent of patients with nonmetastatic osteosarcoma can survive long-term with multidrug chemotherapy 1 .However, patients with metastatic osteosarcoma rapidly development lesions and become resistant to chemotherapy.The development of secondary tumors in these patients is a common cause of morbidity 1 .New therapies for metastatic osteosarcoma are needed to help prevent morbidity in these patients.Below we review current mechanisms of pathogenesis, diagnosis, and treatment strategies including chemoresistant therapies, immunotherapies, and microRNA derived techniques.

Mechanisms of Pathogenesis

Numerous factors have been associated with osteosarcoma pathogenesis including age, gender, environmental agents, genetic background, and viral infection.Rapidly growing bones, especially during puberty, are an easy site for osteosarcoma tumorigenesis 2 .Physical agents, ultraviolet light, and ionizing radiation are agents known to cause osteosarcoma in 2% of all cases but have not been demonstrated to have a large effect in pediatric cases 3 .Methylcholanthrene, chromium salts, beryllium oxide, zinc beryllium silicate, asbestos, and aniline dyes are chemical agents known to cause osteosarcoma.Chromium salts, with or without methylcholanthrene treatment, can transform non-tumorigenic osteoblast-like human osteosarcoma cells.Although the chromium salts alone were highly toxic to the cells, the cells that survived had a marked increase in anchorage independent growth when compared to controls.Cells treated with chromium salts and methylcholanthrene together had an even larger rate of anchorage independent growth.The cells themselves were not tumorigenic when tested in nude mice, but had altered phenotypes that demonstrated hallmarks of a stage in the carcinogenesis cascade 4 .Beryllium oxide and calcined phosphor were shown to induce osteosarcoma as well as other neoplastic growths in a rabbit model.Rabbits were injected three times a week for six to eight weeks.Osteosarcomas developed in 6 of the 9 animals that lived over 1 year and the first tumor appeared 11.5 months after the start of the experiment.The data demonstrate that beryllium compounds not only induced osteosarcoma, but also metastatic tumors of the lung, liver, spleen, kidney, heart, and urinary tract.Earlier studies support this data demonstrating that injection of beryllium compounds induced osteosarcoma in the epiphysis, tibial, scapula, and femoral in mammals such as guinea pigs and rabbits 5,6 .

Genetic changes such as chromosomal abnormalities or mutations in tumor suppressor genes and protooncogenes can also contribute to the onset of osteosarcoma.Chromosomal abnormalities in patients with Bloom syndrome, Rothmund-Thompson syndrome, Werner syndrome, Li-Fraumeni syndrome, and hereditary retinoblastoma often have a higher risk of developing multiple malignancies such as osteosarcoma 7 .

Osteosarcoma has also been linked to amplification of chromosomes 6p21, 8q24, an 12q14, loss of heterozygous chromosome a 10q21.1, and changes in chromosomes 9, 10, 13, and 17 8 .

The mutated forms of tumor suppressor genes p53 and retinoblastoma (Rb) lose their functions and are associated with various cancers.p53 and Rb genes are known to repair DNA damage or induce cell apoptosis 7,9 .If they become mutated, these protective functions are compromised and can allow the cell to become neoplastic.Both of these genes have been indicated in the pathogenesis of osteosarcoma 7,9 .Fifty percent of all cancers have a mutated p53 gene; this gene is also mutated in 22% of osteosarcomas, showing the importance of this gene mutation in cancer's progression 7,9 .Rb is important in cell cycle regulation by binding transcription factors of the E2F family until CDK4/cyclin D complex phosphorylation occurs.Mutation in Rb allows for E2Fs to allow the cell cycle to continue without regulation 10 An individual with Li-Fraumeni syndrome has a 70% chance of developing primary invasive cancer, including osteosarcoma but excluding skin cancer 11 .

Proto-oncogenes such as c-fos, c-jun, myelocytomatosis proto-oncogene protein (c-myc) have been associated with osteosarcoma.Activator protein 1 complex (AP-1) is a heterodimeric complex composed of c-fos and c-jun.AP-1 controls bone metabolism, cell proliferation, and differentiation, whereas c-myc stimulates growth and division in the nucleus.Analysis of primary skeleton neoplasms via immunohistochemistry found c-fos and c-jun expression in bone-forming lesions.Further analysis demonstrated that high-grade osteosarcomas had elevated levels of c-fos and c-jun 12 associating their expression with aggressive human osteosarcoma.Wu et al.'s data demonstrate that c-fos expression is elevated 150% in human osteosarcoma sections when compared to benign or normal tissues.Further supporting the conclusion, that c-fos is involved in the growth and spread of osteosarcoma tumor formation 13 .In another study using osteosarcoma and lung metastases, c-myc and c-fos gene and protein expression was significantly elevated in relapsed tumors and was correlated with metastasis frequency and intensity 14 .Myc overexpression has also been correlated with osteosarcoma pathogenesis and chemotherapeutic resistance.Shimizu et al. demonstrated that overexpression of c-myc in bone marrow stromal cells derived from Ink4a/ Arf null mice could generate lethal osteosarcoma cells 15 .In a different murine osteosarcoma study, TAM67 was used to conditionally inhibit AP-1 activity in highly metastatic K7M2 cells.AP-1 inhibition blocked the migration and invasion potential of these cells and increased mouse survival suggesting that AP-1 inhibition could be used a therapeutic tool to prevent invasion, metastasis, and migration of osteosarcoma tumors 16 .

Diagnosis

Patients diagnosed with osteosarcoma will normally present with pain and swelling in the metaphyseal bone of the distal femur, proximal tibia, and proximal humerus; blunt force trauma to those regions have also been noted before diagnosis, although a scientific link to trauma and osteosarcoma is unknown.Pain is typically associated with activity and overtime the pain occurs during restful periods and is also attributed to growing pains in children.In a study involving osteosarcoma symptoms, pain in the knee joint was always the first reported and was more intense when bearing weight and at night.Two-thirds of patients had a limp, and only seven percent of patients had a pathological fracture.Among these patients, the study identified that the mean total delay for diagnosis was 17 weeks 17 .

Noninvasive diagnostics methods have improved detection over the past decades and includes the use of radiography, computed tomography (CT), magnetic resonance imagining (MRI), positron emission tomography (PET) or a combination of these methods.Radiographs typically detect lesions with defects including osteolytic areas, periosteal reactions, or the development of soft tissue masses 7 .CT scans are utilized at defining fracture sites or irregularities in mineralization, the cortices, or neurovascular development 7 .Often MRIs are employed to assess soft tissue invasion, neurovascular damage, bordering joint damage, or to determine bone marrow replacement needs 7 .Ongoing research is being conducted to determine ways PET can be used to determine metabolic rates of osteosarcoma, the response rate of neoadjuvant therapy and other post treatment changes which is thoroughly reviewed by Brennan et al 18 .Still, biopsy and microscopic examination are required to confirm the diagnosis.These examinations carry additional prognostic implications such as subtype classification and histological response to neoadjuvant chemotherapy 19 .Osteosarcoma subtypes can be divided into many groups including conventional osteosarcoma (subdivided into osteoblastic, chondroblastic or fibroblastic groups), telangiectatic, small-cell, low-grade, parosteal, periosteal, and high-grade surface osteosarcomas.These subtypes are classified based upon their histological appearances 19 .

No official laboratory test exists as a diagnosis for OS.However, basic lab tests such as complete blood count, metabolic panels, and other functional tests can be useful pretreatment to assess a patients help before the onset of chemotherapy.Laboratory testing has shown that alkaline phosphatase levels can be elevated in osteosarcoma patients by approximately 40%.When patients had elevated levels of the alkaline phosphatase enzyme in the preoperative stage, their recurrence rate is found to be much higher, and they have a poorer prognosis 20 .Lactate dehydrogenase levels can also be elevated in osteosarcoma patients.In a multi-institutional osteosarcoma study, elevated lactate dehydrogenase (LDH) levels were found to be the most predictive factor for a poorer prognosis 21 .In another study that correlated LDH levels and prognostic value, researchers found that metastatic patients had a significantly higher level of LDH than patients who only had localized osteosarcoma 22 .It is still unclear whether lactate dehydrogenase and alkaline phosphatase should be used as indicators for osteosarcoma, however approximately 80% of patients present with microscopic metastatic disease or have undetectable patterns.Therefore, utilizing LDH and alkaline phosphatase levels as indicators of osteosarcoma maybe useful in early stages or in conjugation with other diagnostic measures.

Osteosarcoma Treatment Options

Following clinical presentation and diagnosis, the next step is the treatment to remove and potentially eradicate the tumors.Surgery is commonly the first step in treatment and can include removal of just the tumor or potentially the limb itself.Surgical treatment requires the complete removal of the affected tissue including areas where biopsies occurred, drainage, and other potentially contaminated tissue.Chemotherapy following surgery is the next step to ensure eradication, although chemotherapy can be given before and after in some cases.Early chemotherapeutic agents bleomycin, cyclophosphamide, and actinomycin D were frequently used, but now doxorubicin and methotrexate are now more commonly used.Clinical trials relating to many osteosarcoma treatments have been completed or actively ongoing [23][24][25] .A host of chemoresistant strategies, immunotherapies, and microRNA-derived techniques have been developed to help improve patient outcomes and is reviewed below.

Chemoresistant Strategies

Gene expression is often altered in osteosarcomas so that the tumor cells can continue to proliferate despite chemotherapeutic treatments.Downregulation of reduced folate carriers (RFCs) are often observed in chemoresistant osteosarcomas.RFCs are located at the cell membrane and are utilized by chemotherapeutics like methotrexate to enter the cytoplasm of the cell 26 .Mutations seen in the RFC protein sequence include Leu291Pro, Ser46Asn, Ser4Pro, and Gly259Trp and prevent chemoresistant treatments from entering tumor cells [27][28][29] .These mutations alter the structure of the enzyme so that drugs like methotrexate cannot associate with the protein and enter the cell membrane 27 .An alternative to methotrexate is trimetrexate, a drug that has a similar chemotherapeutic function, but does not utilize RFC to enter the cell 30 .Limited clinical studies exist using trimetrexate 31 but transport defective tumor cells are sometimes more sensitive to this drug 32 .Therefore, trimetrexate is a potential candidate to overcome the methotrexate transport resistance 33 .Another cellular enzyme that prevents drugs from remaining in tumor cells is the P-glycoprotein pump (P-GP).The transcription factor, MDR1 (multidrug-resistant gene) can upregulate P-GP in chemoresistant tumor cells and promote the removal of chemotherapeutic drugs like doxorubicin from the cytoplasm of the cell 34,35 .To overcome P-GP expression in tumor cells, doxorubicin is delivered to cancerous tissue along with a silencing RNA (siRNA) sequence via a nanoparticle vector.The specific siRNA is modified within the cell to miRNA, which blocks the expression of P-GP allowing doxorubicin to complete its function 36 .

Other genes are often directly upregulated in osteosarcoma tissues to directly combat the presence of chemotherapeutic drugs.Glutathione S-transferase P1 (GTSP1), an enzyme that has detoxifying characteristics, inactivates various types of treatments and is often overexpressed in osteosarcoma 37,38 .Osteosarcoma patients with mutant variants of the GTSP1 protein have shown increased resistance to several therapeutic drugs including methotrexate, adriamycin and cisplatin 39 .To combat GSTP1-based resistance, an inhibitor of GTSP1, NBDHEX was developed.In vitro studies have observed that NBDHEX does not increase apoptosis in tumor cells but does prevent metastatic signaling 40 .

Signaling pathways for cell proliferation and antiapoptotic factors become overactive in several tumor cells including osteosarcoma.Chemotherapeutic drugs like rapamycin, cisplatin and doxorubicin that have inhibited these pathways in the past are now obsolete [41][42][43] .New treatment strategies and pharmaceuticals are being developed to improve these key treatment types 44,45 .For instance, rapamycin and its analog molecule, cell cycle inhibitor-779, are used in tandem in one treatment that has led to significant inhibition of the mammalian target of rapamycin (mTOR) pathway 1,46 .Doxorubicin treatment together with the insulin-like growth factor receptor 1 (IGF-R1) inhibitor, tyrphostin, increases cellular apoptosis much better than doxorubicin treatment on its own 45 .New treatments cediranib and trastuzumab were generated to specifically target vascular endothelial growth factor (VEGF) and human epidermal growth factor receptor 2 (HER2) respectively, resulting in inhibition of tumor growth 47,48 .Advances in genetic manipulation within the genome of a lentivirus can generate an abundance of short-hairpin RNA (shRNA), miRNA and cDNA sequences to knockdown specific gene expression, such as IGF-R1, BCL-2 and BCL-xL, that allows for increased sensitivity to doxorubicin and cisplatin 43,49,50 .Autophagy is a process that occurs in cells under harsh conditions where the cells' organelles and proteins are degraded but prevents the cell from undergoing apoptosis.This process is observed frequently in tumor cells and promotes chemo-resistant characteristics, as observed in osteosarcoma cell lines treated with doxorubicin and roscovitine 51,52 .Several treatments have been developed to push cancerous cells into apoptotic processes from the autophagy state.In several in vitro studies, the use of autophagy inhibitors chloroquine and 3-methyoadenine (3-MA) treated simultaneously with 5-fluorouracil, paclitaxel (PCX) and cisplatin increased apoptotic events in known autophagy osteosarcoma cells [53][54][55] .

Immunotherapies

Chemotherapeutic resistance has led to the development of new strategies in immunotherapuetics.Immunomodulation, adoptive T-cell immunotherapy, vaccines, immunologic checkpoint blockade, oncolytic virotherapy and targeted therapies have been developed.Immunomodulation adjusts the immune system in a way to target the cancer itself.For example, a synthetic lipophilic analogue of muramyl dipeptide, muramyl tripeptide phophatidylethanolamine (MTP-PE), has been encapsulated into liposomes (L-MTP-PE) as a way for targeted therapy to allow monocytes and macrophages to induce tumoricidal qualities 1,46 .Tumoricidal qualities help immune cells release factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β).The release of these proinflammatory factors promotes the removal of residual micrometatases not eliminated with surgery and chemotherapy.Induction of tumoricidal activity by macrophages induced with L-MTE-PE may be dependent on interferon-γ (IFN-γ), which enhances the liposome intake of the macrophage.

Interferons are pleiotropic cytokines that are involved in antitumor, antiangiogenic, apoptotic, antiviral, and cellular immune responses.Three subtypes of IFN, IFN-α, IFN-β, and IFN-γ, which have direct and indirect activation of T-cells and B-cells 56 .IFN-α has been shown to inhibit osteosarcoma growth and arrest growth of tumors, therefore it is commonly used as the IFN treatment of choice 46 .A small study of three patients with osteosarcoma related pulmonary metastases underwent treatment with human leukocyte interferon.Treatment reduced the tumor size temporarily 6-8 months after administration 57 .In a phase two clinical trial with 20 patients suffering from high-grade osteosarcomas, IFN-α2a treatment only caused partial tumor regression in three patients 58 .Granulocyte macrophage-colony stimulating factor (GM-CSF) has also been shown to induce differentiation and apoptosis of osteosarcoma cells.In this study, treatment of SaOS-2 human osteosarcoma cell line with GM-CSF daily promoted the differentiation and function of these cells including extracellular matrix mineralization and collagen production.However, fourteen days post GM-CSF treatment, the SaOS-2 cell line was found to have high levels of apoptotic cell death when compared with the controls via flow cytometry 59 .Interleukin-2 (IL-2) has been added to standard treatments to help increase the prognosis of patients with osteosarcoma.IL-2 can stimulate and upregulate T-cells and natural killer (NK) cells to activate lymphocytes to become lymphocyte-activated killer cells (LAK).LAK cells have the ability to target and kill tumor cells.Studies show that IL-2 treatment with complete surgical remission and can prevent recurrence and increase survival rate.This therapy has been shown to significantly increase white blood cells, decrease alkaline phosphatase, but can cause influenza-like symptoms and high fever.Despite these reversible severe side effects, heavily treated pediatric patients have a 50% better prognosis 60 .

Adoptive T-cell therapy (ACT) uses T-lymphocytes exhibiting antitumor activity to mediate responses. Genetic engineering of T-cells, T-cell receptor (TCR)-modified T-cells, chimeric antigen receptor (CAR)-modified T-cells, and NK cells, have shown promise to target tumor cells and

have been extensively reviewed and therefore are briefly summarized below 61,62 .T-lymphocytes that have been removed from a patient, expanded in vitro, genetically engineered and reintroduced into the patient have been found to be very effective at tumor regression.Genes for TCRs can be cloned into lentiviruses or retroviruses and use for infection of autologous T-cells.Synthetic receptors with extracellular single-chain variable fragments (scFv) derived from monoclonal antibodies, transmembrane domain, and intracellular domain with differentiation clusters known as CARs can also be used as effective tumor treatment 63 .CAR T-cells have been developed against human epidermal growth factor receptor 2 (HER-2) and IL-11 receptor alpha (Rα) 64,65 .For example, a majority of osteosarcoma patients, express low levels of HER2 in their osteosarcoma cells.As a result, HER2 monoclonal antibodies used to treat tumor cells are ineffective.Ahmed et al. demonstrated that genetic modification of T cells for a specific antigen such as HER-2 can cause regression of established osteosarcoma lesions in a metastaic mouse model 64 .Though CAR therapies have had serious side effects including severe respiratory distress and death 66 .NK cells have an important role in tumor surveillance and recognition and can be important to the elimination of tumor cells 67 .Recently, data demonstrated that the reduction of Killer Immunoglobulin-Like Receptor (KIR) receptorligand expression in osteosarcoma cells could increase the susceptibility of tumor cells to NK cell mediated 66,68 .Other studies support this data by demonstrating that disrupting interactions of KIR with their ligands on tumor cells in vivo can elicit antitumor response 69 .Potential treatments of the future might include inducing solidity of tumors or activating NK cells to recognize specific oncogenic features in a similar manner to T-cells, leading to NK destruction of tumors.

Vaccines using tumor-associated factors to induce an antitumor immune response have been in development.

Tumor-associated factors include gangliosides, heat shock proteins, autologous dendritic cells, tumor peptides or proteins, and autologous or allogeneic tumor cells 70 .Adjuvants, IL-2 and GM-CSF, or other immunostimulants can be used in the vaccines to enhance the response 70,71 .Immunologic checkpoint blockades, CTL antigen-4 (CTLA-4) and PD-1, are currently being researched in osteosarcoma immunotherapy.CTLA-4 is an immune regulatory molecule for attenuating antitumor responses downstream of T-cell activation.Ipilimumab, a monoclonal antibody, blocks CTLA-4 to enhance antitumor responses by inhibiting regulatory T-cells immunosuppressive capabilities 72,73 .PD-1 is part of the CD28 family and is expressed on activated T-cells.When PD-1 and PD-ligand are activated T-cell are stimulated to undergo apoptosis, which contributes to a poor cancer prognosis.Nivolumab is an antibody that blocks PD-1 and can inhibit metastasis, enhance effector T cell function, and increase cytokine production in patients with melanoma, renal-cell cancer, and non-small-cell lung cancer, although its use has been limited in osteosarcoma [74][75][76] .

Oncolytic virotherapy is a new treatment approach utilizing replication-competent viruses to selectively infect and damage cancer tissue without the harm to normal tissue 77 including osteosarcoma 78 .Adenoviruses are double stranded DNA (dsDNA) viruses associated with mild respiratory infections, alimentary and conjunctiva infections.Adenoviruses infect cells using receptormediated endocytoses, releasing early genes to begin transcription, with these genes binding to Rb and p53 proteins 79 .Attenuated adenovirus mutants have been shown to be capable of lysing p53-deficient tumor cells but not cells expressing functional tumor suppressor protein p53.Injection of this adenovirus into human cervical carcinomas in a nude mouse model was capable of reducing tumor size 80 .A phase 1 study using Onyx-15, an adenovirus that targeted p53-deficient cells was well tolerated by patients with head and neck cancer.Five of the 22 patients were found to have some response to therapy and as a result further investigations were necessary 81,82 .Another possible oncolytic virus utilized herpes simplex viruses (HSV).HSVs are neurotropic dsDNA viruses with two serotypes, HSV-1 and HSV-2, which infects the mucosa of the mouth, eyes, and anogenital tract 83,84 .HSV natural infection causes the host to halt protein synthesis, therefore stopping HSV protein synthesis.The primary neuropathogenicity gene in HSV is γ-34.5 with its protein causing dephosphorylation of eIF-2 thereby removing the inhibition of protein synthesis 83,85 .Ras signaling pathway is commonly mutated in cancer, which suppresses the protein kinase (PKR) that inhibits γ-34.5 thereby, allowing HSV to replicate in cancer cells 86 .ICP-6 is a subunit of ribonucleotide reductase essential in DNA viral replication and is highly expressed in cancer cells.HSV ICP-6 is mutated causing it to only be able to divide in cancer cells and not normal cells.Therefore, a vaccine with HSV having ICP-6/γ-34.5 deletion may allow it to specifically target cancer cells 87,88 .

MicroRNAs

Small non-coding RNA sequences are transcribed from the genomes of animal and plant cells as well as the genetic material of some viral families.Although they do not generate proteins of their own, some of these noncoding sequences are able to regulate protein expression.These sequences are known as microRNA (miRNA).Once transcribed, the pre-miRNA is modified by RNase III enzyme Drosha that forms secondary structures with the RNA sequence before entering the cytoplasm.The double stranded miRNA is then recognized by the RNase III endonuclease known as Dicer, which breaks the secondary structures creating miRNA strands that have the capability of silencing gene expression 89 .The mature miRNA can either bind directly to a complementary messenger RNA (mRNA) or enter the active site of a ribosome, bind to and prevent movement of the complementary mRNA within.Both mechanisms inhibit protein synthesis of the mRNA and limit gene expression 89 .This process is a normal occurrence in healthy cells, miRNAs regulate the expression of necessary enzymes and transcription factors that if overexpressed may influence cellular issues.In fact, the study of miRNA over the past two decades has created hypotheses that the development of cancer may be due to irregular expression of miRNA in equal proportion to sporadic mutations of the particular genes 90 .

Several miRNAs have been observed specifically as chemo-resistant and radiation resistant factors in treating osteosarcoma by targeting tumor suppressor genes that allow apoptosis of the cell, inhibit cell proliferation and migration from the tissue 91 .Human apurinic/apyrimidinic (AP) endonuclease APE1 is an endonuclease responsible for repairing DNA damage after exposure to radiation treatment.miRNA-513a-5p is generated to maintain levels of APE1 expression.Osteosarcomas and other aggressive cancers have overexpression of APE1 producing unregulated repair of oncolytic genes as well as downregulation of the miRNA-513a-5p 92,93 .Reintroducing miRNA-513a-5p into osteosarcoma cell lines that have reduced expression of APE1 caused cells to undergo apoptosis after DNA damage to radiotherapy 92 .Natural upregulation of miRNA-224 inhibits Ras-related C3 botulinum toxin substrate 1 (Rac1), a GTPase that when overexpressed in tumor cells is involved in cell proliferation and metastasis.However, overexpression of miRNA-224 creates a negative feedback that allows Rac1 to inhibit the sequences' production and allow Rac1 to perform its function unhindered by cisplatin treatment 94 .Osteosarcoma cell lines can undergo successful apoptosis if treated with exogenous miRNA-224 and cisplatin simultaneously 94 .Expression of miRNA-138 as well increases tumor sensitivity to cisplatin treatment.EZH2, an inhibitor of the caspase-3 enzyme, is inhibited by miRNA-138, which allows the cell to enter an apoptotic state after chemotherapy treatment 95 .Some miRNA sequences are known to increase chemoresistance of osteosarcoma cell lines.Upregulation of miRNA-21 impedes sprouty homolog 1 (Spry1) and sprouty homolog 2 (Spry2) gene production, important inhibitors of tyrosine kinase receptor signaling from certain growth factors 96 .Spry1 and Spry2 function are a major part of cisplatin therapy to reduce tumor characteristics, therefore upregulation of miRNA-21 increases cisplatin resistance of bone tumor and promotes uncontrolled cell proliferation 97 .Upon treatment with cisplatin and doxorubicin, upregulation of miRNA-140-5p is observed to induce autophagy of the osteosarcoma cell lines and results in cell death 98 .However, another sequence is upregulated, miRNA-184, after treatment with doxorubicin that increases cell survival by blocking apoptosis inhibitor BCL2L1, contrary to the expression and function of miRNA-140-5p 99 .miRNA-367 can also prevent apoptosis of cancer cells.miRNA-367 specifically conquers apoptosis through the downregulation of BNIP3L/Nix and the upregulation of BCL-xL 100 .Other miRNAs that correlate with poor chemotherapeutic remediation in pediatric osteosarcoma include miRNA-221 and miRNA 210 101,102 .miRNA-221 prevents expression of NF-κB inhibitors which maintains cell proliferation despite treatments 103 .miRNA-210 promotes and maintains the initiation of the cell cycle by acting upon E2F3 cell cycle regulator, MNT the myc antagonist and homeobox proteins, despite signals from chemo-therapeutic conditions 55,104,105 .Repressing expression of certain oncolytic miRNA sequences is equally as important as maintaining expression of tumor suppressor genes in other miRNA sequences.This strategy will allow an unhindered chemotherapy treatment to successfully fight against osteosarcoma and other aggressive cancers.

Conclusions

Osteosarcoma is a rare but deadly cancer affecting pediatric patients and in some cases adults.With its rarity, treatment options are limited and require innovative solutions to find effective options, which eradicate tumor cells and tissue without the need for amputation.Emerging treatment options utilize or combine tools harnessed from the host tissue response, chemoresistant therapeutics, immunotherapies, and microRNA therapy to effectively combat and eliminate cancer cells with the ultimate goal of improving patient prognosis and survival rate.

Strategies and developments of immunotherapies in osteosarcoma. J Wan, X Zhang, T Liu, Oncol Lett. 11

Stature of young people with malignant bone tumors. S J Cotterill, C M Wright, M S Pearce, Pediatr Blood Cancer. 422004

Osteosarcoma (osteogenic sarcoma). P Picci, Orphanet J Rare Dis. 262007

Transformation of non-tumorigenic osteoblastlike human osteosarcoma cells by hexavalent chromates: alteration of morphology, induction of anchorage-independence and proteolytic function. A S Rani, S Kumar, Carcinogenesis. 131992

Osteosarcoma induced by beryllium oxide. F R Dutra, E J Largent, Am J Pathol. 261950

Experimental production of bone sarcomas in the rabbit by a single local injection of beryllium. A Mazabraud, Bull Cancer. 621975

Osteosarcoma: a review of diagnosis, management, and treatment strategies. D S Geller, R Gorlick, Clin Adv Hematol Oncol. 8

Biology and therapeutic advances for pediatric osteosarcoma. N Marina, M Gebhardt, L Teot, Oncologist. 92004

Osteosarcoma treatment: state of the art. H T Ta, C R Dass, P F Choong, Cancer Metastasis Rev. 282009

Making gene therapy for osteosarcoma a reality. M L Broadhead, J C Clark, P F Choong, Expert Rev Anticancer Ther. 10

Multiple primary malignancies in osteosarcoma patients. Incidence and predictive value of osteosarcoma subtype for cancer syndromes related with osteosarcoma. E I Hauben, J Arends, J P Vandenbroucke, Eur J Hum Genet. 112003

Immunohistochemical detection of c-fos and c-jun expression in osseous and cartilaginous tumours of the skeleton. A Franchi, A Calzolari, G Zampi, Virchows Arch. 4321998

The proto-oncogene c-fos is over-expressed in the majority of human osteosarcomas. J X Wu, P M Carpenter, C Gresens, Oncogene. 51990

C-myc and c-fos in human osteosarcoma: prognostic value of mRNA and protein expression. G Gamberi, M S Benassi, T Bohling, Oncology. 551998

c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis. T Shimizu, T Ishikawa, E Sugihara, Oncogene. 29

Inhibition of AP-1 transcriptional activity blocks the migration, invasion, and experimental metastasis of murine osteosarcoma. V D Leaner, J F Chick, H Donninger, Am J Pathol. 1742009

Initial symptoms and delayed diagnosis of osteosarcoma around the knee joint. K L Pan, W H Chan, Y Y Chia, J Orthop Surg. 18

PET imaging of osteosarcoma. W Brenner, K H Bohuslavizki, J F Eary, J Nucl Med. 442003

Osteosarcoma: a comprehensive review. A Misaghi, A Goldin, M Awad, SICOT J.. 412

Prognostic significance of alkaline phosphatase measurements in patients with osteogenic sarcoma receiving chemotherapy. W P Thorpe, J J Reilly, S A Rosenberg, Cancer. 431979

Adjuvant chemotherapy of high-grade osteosarcoma of the extremity. Updated results of the Multi-Institutional Osteosarcoma Study. M P Link, A M Goorin, M Horowitz, Clin Orthop Relat Res. 1991

Prognostic significance of serum lactate dehydrogenase in osteosarcoma of the extremity: experience at Rizzoli on 1421 patients treated over the last 30 years. G Bacci, A Longhi, S Ferrari, Tumori. 902004

Outcomes of patients with sarcoma enrolled in clinical trials of pazopanib combined with histone deacetylase, mTOR, Her2, or MEK inhibitors. V Dembla, R Groisberg, K Hess, 15963

Characteristics and outcomes of patients with advanced sarcoma enrolled in early phase immunotherapy trials. R Groisberg, D S Hong, A Behrang, J Immunother Cancer. 5100

Osteosarcoma: Accelerating Progress Makes for a Hopeful Future. A J Saraf, J M Fenger, R D Roberts, Front Oncol. 84

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques. C M Hattinger, G Reverter-Branchat, D Remondini, Eur J Cell Biol. 822003

Functional analysis of altered reduced folate carrier sequence changes identified in osteosarcomas. W F Flintoff, H Sadlish, R Gorlick, Biochim Biophys Acta. 16902004

Mechanisms of methotrexate resistance in osteosarcoma. W Guo, J H Healey, P A Meyers, Clin Cancer Res. 51999

Tumor necrosis factor-alpha and ceramide induce cell death through different mechanisms in rat mesangial cells. Y L Guo, B Kang, L J Yang, Am J Physiol. 2761999

High dose trimetrexate with leucovorin protection in recurrent childhood malignancies: a phase II trial. T Meyers P Trippett, R Gorlick, ASCO Annual Meeting. 1999889

Pharmacogenomics of genes involved in antifolate drug response and toxicity in osteosarcoma. C M Hattinger, E Tavanti, M Fanelli, Expert Opin Drug Metab Toxicol. 13

Biochemical pharmacology of the lipophilic antifolate, trimetrexate. R C Jackson, D W Fry, T J Boritzki, Adv Enzyme Regul. 221984

Intrinsic and acquired resistance to methotrexate in acute leukemia. R Gorlick, E Goker, T Trippett, N Engl J Med. 3351996

The co-expression of p53 protein and P-glycoprotein is correlated to a poor prognosis in osteosarcoma. Y B Park, H S Kim, J H Oh, Int Orthop. 242001

Molecular basis of preferential resistance to colchicine in multidrug-resistant human cells conferred by Gly-185----Val-185 substitution in P-glycoprotein. A R Safa, R K Stern, K Choi, Proc Natl Acad Sci U S A. 871990

Doxorubicin loaded Polymeric Nanoparticulate Delivery System to overcome drug resistance in osteosarcoma. M Susa, A K Iyer, K Ryu, BMC Cancer. 93992009

The role of glutathione-S-transferase in anticancer drug resistance. D M Townsend, K D Tew, Oncogene. 222003

Overexpression of resistancerelated proteins (metallothioneins, glutathione-S-transferase pi, heat shock protein 27, and lung resistance-related protein) in osteosarcoma. Relationship with poor prognosis. H Uozaki, H Horiuchi, T Ishida, Cancer. 791997

Germline genetic polymorphisms may influence chemotherapy response and disease outcome in osteosarcoma: a pilot study. R E Windsor, S J Strauss, C Kallis, Cancer. 118

Targeting glutathione-S transferase enzymes in musculoskeletal sarcomas: a promising therapeutic strategy. M Pasello, M C Manara, F Michelacci, Anal Cell Pathol (Amst). 34

The insulin-like growth factor system and cancer. D Leroith, C T RobertsJr, Cancer Lett. 1952003

Targeting the mTOR signaling network for cancer therapy. F Meric-Bernstam, A M Gonzalez-Angulo, J Clin Oncol. 272009

VEGF and EMMPRIN expression correlates with survival of patients with osteosarcoma. Q Zhou, Y Zhu, Z Deng, Surg Oncol. 20

Evaluation of the mammalian target of rapamycin pathway and the effect of rapamycin on target expression and cellular proliferation in osteosarcoma cells from dogs. I K Gordon, F Ye, M S Kent, Am J Vet Res. 692008

IGF1R-targeted therapy and its enhancement of doxorubicin chemosensitivity in human osteosarcoma cell lines. F Luk, Y Yu, W R Walsh, Cancer Invest. 29

Rapamycin inhibits ezrinmediated metastatic behavior in a murine model of osteosarcoma. X Wan, A Mendoza, C Khanna, Cancer Res. 652005

Phase II trial of trastuzumab in combination with cytotoxic chemotherapy for treatment of metastatic osteosarcoma with human epidermal growth factor receptor 2 overexpression: a report from the children's oncology group. D Ebb, P Meyers, H Grier, J Clin Oncol. 30

Initial testing of the VEGFR inhibitor AZD2171 by the pediatric preclinical testing program. J M Maris, J Courtright, P J Houghton, Pediatr Blood Cancer. 502008

Lentivirus-mediated shRNA targeting insulin-like growth factor-1 receptor (IGF-1R) enhances chemosensitivity of osteosarcoma cells in vitro and in vivo. Y H Wang, J Xiong, S F Wang, Mol Cell Biochem. 341

Upregulated expression of microRNA-214 is linked to tumor progression and adverse prognosis in pediatric osteosarcoma. Z Wang, H Cai, L Lin, Pediatr Blood Cancer. 61

Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. K Degenhardt, R Mathew, B Beaudoin, Cancer Cell. 102006

Autophagy: a novel mechanism of synergistic cytotoxicity between doxorubicin and roscovitine in a sarcoma model. L A Lambert, N Qiao, K K Hunt, Cancer Res. 682008

Cytoprotective role of autophagy during paclitaxel-induced apoptosis in Saos-2 osteosarcoma cells. H J Kim, S G Lee, Y J Kim, Int J Oncol. 42

Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model. J Li, N Hou, A Faried, Eur J Cancer. 46

Expression of Beclin1 in osteosarcoma and the effects of down-regulation of autophagy on the chemotherapeutic sensitivity. Z Zhang, Z Shao, L Xiong, J Huazhong Univ Sci Technolog Med Sci. 292009

S P D' Angelo, W D Tap, G K Schwartz, R D , Sarcoma immunotherapy: past approaches and future directions. 2014391967

Effect of human leukocyte interferon on the metastatic lung tumor of osteosarcoma: case reports. H Ito, K Murakami, T Yanagawa, Cancer. 461980

Phase II study of recombinant alfa-2a interferon in patients with advanced bone sarcomas. J H Edmonson, H J Long, S Frytak, Cancer Treat Rep. 711987

Effect of human granulocyte macrophage-colony stimulating factor on differentiation and apoptosis of the human osteosarcoma cell line SaOS-2. L Postiglione, Di Domenico, G Giordano-Lanza, G , Eur J Histochem. 472003

Feasibility of high-dose interleukin-2 in heavily pretreated pediatric cancer patients. W Schwinger, V Klass, M Benesch, Ann Oncol. 162005

Adoptive immunotherapy for cancer: harnessing the T cell response. N P Restifo, M E Dudley, S A Rosenberg, Nat Rev Immunol. 12

Adoptive immunotherapy for cancer. M Ruella, M Kalos, Immunol Rev. 257

HLA class I antigen down-regulation in primary ovary carcinoma lesions: association with disease stage. M Vitale, G Pelusi, B Taroni, Clin Cancer Res. 112005

Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression. N Ahmed, V S Salsman, E Yvon, Mol Ther. 172009

Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma. N Rainusso, V S Brawley, A Ghazi, Cancer Gene Ther. 19

Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. R A Morgan, J C Yang, M Kitano, Mol Ther. 18

Oncogenic stress sensed by the immune system: role of natural killer cell receptors. D H Raulet, N Guerra, Nat Rev Immunol. 92009

KIR receptor-ligand incompatibility predicts killing of osteosarcoma cell lines by allogeneic NK cells. D Delgado, D E Webster, K B Desantes, Pediatr Blood Cancer. 55

Killer cell inhibitory receptors for MHC class I molecules regulate lysis of melanoma cells mediated by NK cells, gamma delta T cells, and antigen-specific CTL. A B Bakker, J H Phillips, C G Figdor, J Immunol. 1601998

Phase I/II trial of autologous tumor cell line-derived vaccines for recurrent or metastatic sarcomas. R Dillman, N Barth, S Selvan, Cancer Biother Radiopharm. 192004

A pilot study of consolidative immunotherapy in patients with high-risk pediatric sarcomas. C L Mackall, E H Rhee, E J Read, Clin Cancer Res. 142008

Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, doubleblind, multicentre, phase 2, dose-ranging study. J D Wolchok, B Neyns, G Linette, Lancet Oncol. 11

Ipilimumab augments antitumor activity of bispecific antibody-armed T cells. H Yano, Thakur, E N Tomaszewski, J Transl Med. 12191

PD-1 blockade inhibits hematogenous spread of poorly immunogenic tumor cells by enhanced recruitment of effector T cells. Y Iwai, S Terawaki, T Honjo, Int Immunol. 172005

Blockade of B7-H1 or B7-DC induces an anti-tumor effect in a mouse pancreatic cancer model. K Okudaira, R Hokari, Y Tsuzuki, Int J Oncol. 352009

Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer. M Sznol, L Chen, Clin Cancer Res. 19

Oncolytic virotherapy. S J Russell, K W Peng, J C Bell, Nat Biotechnol. 30

Oncolytic viruses for potential osteosarcoma therapy. P Hingorani, V Sampson, C Lettieri, Adv Exp Med Biol. 804

HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. R P Tomko, R Xu, L Philipson, Proc Natl Acad Sci U S A. 941997

An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. J R Bischoff, D H Kirn, A Williams, Science. 2741996

Use of replicating oncolytic adenoviruses in combination therapy for cancer. R L Chu, D E Post, F R Khuri, Clin Cancer Res. 102004

A phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer. I Ganly, D Kirn, G Eckhardt, Clin Cancer Res. 62000

Advance in herpes simplex viruses for cancer therapy. S Liu, M Dai, L You, Sci China Life Sci. 56

Genital infection with herpes simplex virus types 1 and 2 in women from natal, Brazil. C A Miranda, E G Lima, D B De Lima, ISRN Obstet Gynecol. 3236572014

Viruses with deletions in antiapoptotic genes as potential oncolytic agents. T C Liu, D Kirn, Oncogene. 242005

Activated MEK suppresses activation of PKR and enables efficient replication and in vivo oncolysis by Deltagamma(1)34.5 mutants of herpes simplex virus 1. K D Smith, J J Mezhir, K Bickenbach, J Virol. 802006

Differential susceptibility of pediatric sarcoma cells to oncolysis by conditionally replicationcompetent herpes simplex viruses. N S Bharatan, M A Currier, T P Cripe, J Pediatr Hematol Oncol. 242002

Effective oncolytic vaccinia therapy for human sarcomas. S He, P Li, C H Chen, J Surg Res. 175

RNA interference. G J Hanno, Nature. 4182002

J Hayes, P P Peruzzi, S Lawler, MicroRNAs in cancer: biomarkers, functions and therapy. 20

Altered expression of miR-21, miR-31, miR-143 and miR-145 is related to clinicopathologic features of colorectal cancer. O Slaby, M Svoboda, P Fabian, Oncology. 722007

miR-513a-5p regulates radiosensitivity of osteosarcoma by targeting human apurinic/apyrimidinic endonuclease. Y Qing, N Dai, Y Cun, Onco Targets Ther. 2016

Apurinic/apyrimidinic endonuclease 1 regulates angiogenesis in a transforming growth factor beta-dependent manner in human osteosarcoma. X Jiang, J Shan, N Dai, Cancer Sci. 106

MicroRNA-224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1. S Geng, L Gu, F Ju, J Cell Mol Med. 20

MiR-138 Acts as a Tumor Suppressor by Targeting EZH2 and Enhances Cisplatin-Induced Apoptosis in Osteosarcoma Cells. Z Zhu, J Tang, J Wang, PLoS One. 11e0150026

Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis. C C Lee, A J Putnam, C K Miranti, Oncogene. 232004

MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells. V Vanas, B Haigl, V Stockhammer, PLoS One. 11e0161023

miR-140-5p attenuates chemotherapeutic drug-induced cell death by regulating autophagy through inositol 1,4,5-trisphosphate kinase 2 (IP3k2) in human osteosarcoma cells. R Wei, G Cao, Z Deng, 36

MicroRNA-184 Modulates Doxorubicin Resistance in Osteosarcoma Cells by Targeting BCL2L1. B C Lin, D Huang, C Q Yu, Med Sci Monit. 22

MicroRNA-302/367 cluster governs hESC self-renewal by dually regulating cell cycle and apoptosis pathways. Z Zhang, Y Hong, D Xiang, Stem Cell Reports. 4

Prognostic evaluation of microRNA-210 expression in pediatric osteosarcoma. H Cai, L Lin, M Tang, Med Oncol. 30499

Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and colon cancer cells. B Song, Y Wang, Y Xi, Oncogene. 282009

Multifunction protein staphylococcal nuclease domain containing 1 (SND1) promotes tumor angiogenesis in human hepatocellular carcinoma through novel pathway that involves nuclear factor kappaB and miR-221. P K Santhekadur, S K Das, R Gredler, J Biol Chem. 287

Hypoxia-inducible mir-210 regulates normoxic gene expression involved in tumor initiation. X Huang, L Ding, K L Bennewith, Mol Cell. 352009

MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT. Z Zhang, H Sun, H Dai, Cell Cycle. 82009


Residual hepatocellular carcinoma after oxaliplatin treatment has increased metastatic potential in a nude mouse model and is attenuated by Songyou Yin

Wei Xiong
Zheng-Gang Ren
Shuang-Jian Qiu
Hui-Chuan Sun
Lu Wang
Bin-Bin Liu
Qi-Song Li
Wei Zhang
Xiao-Dong Zhu
Liang Liu
Wen-Quan Wang
Zhao-You Tang
Residual hepatocellular carcinoma after oxaliplatin treatment has increased metastatic potential in a nude mouse model and is attenuated by Songyou Yin
Xiong et al. BMC Cancer 2010, 10:219 RESEARCH ARTICLE BioMed Central Research article
Background: The opposite effects of chemotherapy, which enhance the malignancy of treated cancers such as hepatocellular carcinoma (HCC), are not well understood. We investigated this phenomenon and corresponding mechanisms to develop a novel approach for improving chemotherapy efficacy in HCC.Methods:Human hepatocellular carcinoma cell lines HepG2 (with low metastatic potential) and MHCC97L (with moderate metastatic potential) were used for the in vitro study. An orthotopic nude mouse model of human HCC was developed using MHCC97L cells. We then assessed the metastatic potential of surviving tumor cells after in vitro and in vivo oxaliplatin treatment. The molecular changes in surviving tumor cells were evaluated by western blot, immunofluorescence, and immunohistochemistry. The Chinese herbal extract Songyou Yin (composed of five herbs) was investigated in vivo to explore its effect on the metastatic potential of oxaliplatin-treated cancer cells.Results: MHCC97L and HepG2 cells surviving oxaliplatin treatment showed enhanced migration and invasion in vitro.Residual HCC after in vivo oxaliplatin treatment demonstrated significantly increased metastasis to the lung (10/12 vs. 3/12) when re-inoculated into the livers of new recipient nude mice. Molecular changes consistent with epithelialmesenchymal transition (EMT) were observed in oxaliplatin-treated tumor tissues and verified by in vitro experiments. The Chinese herbal extract Songyou Yin (4.2 and 8.4 g/kg) attenuated EMT and inhibited the enhanced metastatic potential of residual HCC in nude mice (6/15 vs. 13/15 and 3/15 vs. 13/15, respectively).Conclusions:The surviving HCC after oxaliplatin treatment underwent EMT and demonstrated increased metastatic potential. Attenuation of EMT by Songyou Yin may improve the efficacy of chemotherapy in HCC.

Background

Hepatocellular carcinoma (HCC) is one of the most common malignancies in many countries, including China. Radical treatment, such as surgical resection and orthotopic liver transplantation, is feasible only for patients with small tumors [1]. For the majority of patients with unresectable HCC, the response rate of systemic chemotherapy with Adriamycin, 5-fluorouracil and platinum-based combinations was reported to be approximately 20%, and no survival benefit was observed [2].

Biological deterioration after chemotherapy has recently been reported. An "opposite effect" of chemotherapy has been demonstrated in which cyclophosphamide pretreatment induced metastasis of fibrosarcoma cells in a nude mouse model [3]. Other studies also reported that in vitro exposure to chemotherapeutic agents enhanced metastatic potential in colorectal, pancreatic, breast, and ovarian carcinoma cells [4][5][6][7]. Two explanations have been proposed for this opposite effect. Chemotherapy may damage the vascular endothelium and inhibit the host antitumor system, enabling increased tumor cell extravasation and survival [3,8]. Alternatively, chemotherapy may induce epithelial-mesenchymal transition (EMT) and enhance the metastatic potential of tumor cells [4][5][6][7]. EMT is characterized by the loss of epithelial marker E-cadherin and expression of mesenchymal markers such as N-cadherin and vimentin; it is regarded as a critical step in tumor invasion and metastasis [9]. Although these studies established a relationship among in vitro chemotherapy, EMT, and enhanced tumor metastasis, the altered metastatic potential of residual cancer after in vivo chemotherapy is not completely understood.

To exclude the influence of tumor burden, vascular injury, and inhibition of host antitumor system by chemotherapy, we designed a re-inoculation experiment in a nude mouse model of human HCC to determine whether chemotherapy promotes a more malignant phenotype of residual HCC. Because platinum-based combined chemotherapy targets HCC [10,11], oxaliplatin was selected as the chemotherapeutic agent in this study. We investigated potential mechanisms, as well as the Chinese herbal extract Songyou Yin as a potential intervention.

Methods

Cell lines

In the present study, we used two human HCC cell lines: MHCC97L cells, which originated from MHCC97 [12,13] (established at the Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China), and HepG2 cells (American Type Culture Collection). MHCC97L cells show a moderate metastatic potential, and HepG2 cells show a low metastatic potential. All cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and incubated in a humidified incubator at 37°C in 5% CO 2 .

Compounds and antibodies

Treatment of tumor cells with oxaliplatin and analysis of cell morphology

MHCC97L or HepG2 cells were plated in six-well tissue culture plates (1 × 10 5 cells/well). After 24 h, the medium was replaced with DMEM containing 10% FBS and 2 μmol/L oxaliplatin. After 48 h, the medium was changed, and drug treatment was terminated. Cells were allowed to recover, and when the surviving populations reached 80% confluence, cells were passaged and exposed to oxaliplatin again for 48 h. During 6 weeks of treatment this process was repeated for a total of four 48-h exposures to oxaliplatin. Cells surviving treatment (Oxa cells) were designated MHCC97L-Oxa and HepG2-Oxa, respectively. The morphological characteristics of parental and Oxa cells were compared by microscopy (Olympus, Tokyo, Japan).

Cell migration and invasion assays

Cell migration was assessed by transwell assay (Boyden chambers) (Corning, Cambridge, MA, UK). Briefly, 6 × 10 4 cells in serum-free DMEM were seeded on a membrane (8.0-μm pore size) inserted in a well of a 24-well plate. DMEM containing 10% FBS was added to the lower chamber of each well. After 48 h, cells that had reached the underside of the membrane were stained with Giemsa (Sigma Chemical Co.) and counted at × 200 magnification. The cell invasion assay was carried out similarly, except that 10 μL of matrigel (BD Biosciences) was added to each well 6 h before cells were seeded on the membrane.

Cell proliferation assay

MHCC97L and HepG2 parental and Oxa cells were incubated in 96-well plates (5 × 10 3 cells/well) for 24, 48, or 72 h. Cell proliferation was then determined with an MTT kit (Beyotime, Shanghai, China). Results were expressed as the absorbance of each well at 570 nm (OD570).

Western blot analysis and immunofluorescence

The concentration of protein extracted from Oxa and parental cells was determined with the BCA Protein Assay Kit (Beyotime, Shanghai, China). The expression of E-cadherin, N-cadherin, vimentin, Snail, Slug, and Twist was determined by immunoblotting as previously described [14]. E-cadherin, N-cadherin, and vimentin expression in parental and Oxa cells were also demonstrated by immunofluorescence. Cells were grown on glass cover slips to 40%-50% confluence, and then fixed, permeabilized, and blocked. Cells were then incubated with primary monoclonal antibodies against E-cadherin, N-cadherin, and vimentin overnight at 4°C. The next day, slides were washed and incubated with anti-mouse and/or anti-rabbit fluorescein isothiocyanate (FITC)-and/or tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibody (Invitrogen). Cells were counterstained with 4'-6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei and visualized by fluorescence microscopy (Olympus, Tokyo, Japan).

Animals

Male BALB ⁄ c nu ⁄ nu mice (age, 4-6 weeks old; weight, approximately 20 g) were obtained from the Shanghai Institute of Materia Medica (Chinese Academy of Science) and maintained under standard pathogen-free conditions. The experimental protocol was approved by the Shanghai Medical Experimental Animal Care Commission.

Pilot study for animal model

To evaluate changes in metastatic potential of residual HCC cells after in vivo oxaliplatin treatment, residual cancer from the livers of oxaliplatin-treated and untreated nude mice were re-inoculated orthotopically into new recipient nude mice. Because the treated and untreated tumors needed to be histologically similar, a pilot study compared the histology of treated and untreated tumors at different time points after chemotherapy to determine the appropriate time points for reinoculation.

A metastatic model of human HCC in nude mice using MHCC97L cells was employed for this pilot study [12]. Briefly, MHCC97L cells (5 × 10 6 ) were injected subcutaneously into the upper left flank region of nude mice. When the subcutaneous tumor reached approximately 1 cm in length (approximately 4 weeks after injection), it was removed, minced into small pieces of equal volume (2 × 2 × 2 mm 3 ), and transplanted into the livers of 60 different nude mice. Based on the literature [15] and the results of our previous studies, a dosage for oxaliplatin of 10 mg/kg, once a week was adopted in the present study. Oxaliplatin was administered intraperitoneally (i.p.) to randomly selected mice (oxaliplatin treatment group; n = 30) on days 12, 19, and 26 after inoculation; the control group (n = 30) received 0.2 mL of 0.9% sodium chloride (i.p.) on those days. On days 1, 2, 5, 7 and 14 after the final treatment, six mice from each group were sacrificed by cervical dislocation. The necrosis and apoptosis of tumors in each group were compared.

Analysis of tumor necrosis and apoptosis

Paraffin-embedded sections were prepared for hematoxylin and eosin staining. Necrosis of tumor tissue was determined by comparing the surface of necrotic areas to that of the whole tumor [16]. Apoptosis was determined using a terminal transferase dUTP nick end labeling (TUNEL) assay kit (KeyGen, Nanjing, China) according to the manufacturer's protocol. The apoptosis rate was expressed as a ratio of apoptotic cells to total tumor cells.

Re-inoculation experiment

Analysis of tumor histology in the pilot study showed that tumor necrosis and apoptosis on days 5, 7 and 14 after the final oxaliplatin treatment were not significantly different between the control group and the treated group. We therefore used day 7 tumors for re-inoculation. Orthotopic models of human HCC were established in 36 nude mice and treated with oxaliplatin (n = 18) or sodium chloride (n = 18) using the same protocol described for the pilot study. On day 7 after the final treatment, all mice were sacrificed by cervical dislocation. Tumor fragments of equal volume (2 × 2 × 2 mm 3 ) from each mouse of the oxaliplatin-treated and untreated control groups were reinoculated into the livers of each new recipient mice correspondently. The remaining tumor tissues of both groups were fixed in 10% buffered formalin and embedded in paraffin wax for histological study.

These mice, segregated into control (bearing untreated tumors, n = 18) and trial ( bearing oxaliplatin pre-treated tumors, n = 18) groups, were then kept under standard conditions. On day 42 after re-inoculation, 12 randomly selected mice from each group were sacrificed, and tumor growth and pulmonary metastasis were assessed. Tumor tissues from re-inoculated mice were also prepared for histological study. The remaining six mice from each group were kept for survival analysis.

Characterization of the Chinese herbal extract Songyou Yin

The water-soluble Chinese herbal medicine Songyou Yin (SYY), authorized by the Chinese State Food and Drug Administration (Grant No. G20070160), includes five Chinese medicinal herbal extracts in the following proportions (w/w): Salvia miltiorrhiza Bge., 14.3%; Astragalus membranaceus Bge., 14.3%; Lycium barbarum L., 23.8%; Crataegus pinnatifida Bge., 23.8% and Trionyx sinensis Wiegmann, 23.8% (all from China). High-performance liquid chromatography (HPLC) fingerprinting of Songyou Yin and its five characteristic components (see additional file 1) was carried out by the Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences (CAS), China [17]. The SYY used in this study was produced by the Caitong Detang Chinese Traditional Medicine Pharmaceutical Factory (batch number: 090401; Shanghai, China).

Songyou Yin treatment in the re-inoculation model beaing oxaliplatin pre-treated tumors

The orthotopic nude mice model bearing oxaliplatin pretreated MHCC97L xenografts was established in 84 mice using the previously described protocol. Three days after re-inoculation, mice were randomized into control (n = 21) and SYY-treated groups (n = 63). The 63 nude mice in the SYY-treated group received SYY at three different doses (2.1, 4.2, and 8.4 g/kg; each dose, n = 21) by oral gavage once per day. Mice in the control group received 0.2 mL of 0.9% sodium chloride via oral gavage at the same time. Treatment continued for 6 consecutive weeks. On day 42 after initiation of treatment, mice from the control group and each SYY dose group (each group, n = 15) were sacrificed to determine tumor volume. The tumor tissue and lungs were collected for histological analysis and a pulmonary metastasis assay. The remaining mice were maintained and used for survival analysis.

Tumor volume, lung metastasis and survival

Tumor volume was calculated as: (a × b 2 )/2, where "a" is the widest diameter and "b" is the smallest [18]. Lung metastasis was determined by examining serial sections of every lung tissue block by microscopy. Survival time was defined as the interval between the day of inoculation and the day of death.

Immunohistochemistry

Tumor tissue was fixed, embedded, and sliced into 5-μm thick sections. Immunostaining of E-cadherin, N-cadherin, vimentin, Snail, Slug, and Twist was carried out using a standard protocol [19].

Statistical analysis

In vitro cell migration, invasion, and proliferation assays were analyzed by Student's t-test. Necrosis and apoptosis of tumors from the animal model were also compared by Student's t-test. Tumor volume was compared by analysis of variance (ANOVA), the lung metastasis assay was analyzed using Fisher's exact test, and survival was compared with Kaplan-Meier method with a log-rank test. Statistical analysis was performed with SPSS 15.0 for Windows (SPSS Inc. Chicago, IL, USA). P < 0.05 was considered statistically significant.

Results

Tumor cells that survived oxaliplatin treatment showed higher motility and invasiveness and slower proliferation in vitro

In the migration assay (Figure 1), cells that survived oxaliplatin treatment migrated through the membrane in greater numbers compared with corresponding parental cells (MHCC97L-Oxa, 29.50 ± 5.28 vs. MHCC97L, 19.17 ± 2.64; P=0.002; and HepG2-Oxa, 21.17 ± 3.13 vs. HepG2, 12.33 ± 2.73; P < 0.001). In the invasion assay (Figure 2), MHCC97L-Oxa and HepG2-Oxa cells showed a 0.89-fold(P < 0.001) and 1.58-fold (P =0.001) increase, respectively, in the number of cells invading the matrigelcoated membrane, compared with untreated controls. At 48 and 72 h after plating, both MHCC97L-Oxa and HepG2-Oxa cells exhibited reduced proliferation compared with corresponding parental cells ( Figure 3B).

Tumor cells surviving oxaliplatin treatment exhibited EMT in vitro

MHCC97L-Oxa and HepG2-Oxa cells were morphologically distinct from the typical epithelial appearance of parental MHCC97L and HepG2 cells, demonstrating a spindle shape with less cell-cell adhesion and increased formation of pseudopodia ( Figure 3A). Western blot analysis ( Figure 3C) showed a significant reduction in E-cadherin expression with an upregulation of N-cadherin and vimentin in MHCC97L-Oxa and HepG2-Oxa cells. The transition from a dominant E-cadherin distribution in the cell membrane before chemotherapy to predominant Ncadherin expression after chemotherapy, known as the "cadherin switch" [20], was also demonstrated by immunofluorescence in both cell lines ( Figure 3D). Furthermore, an upregulation of the transcription factor Snail was also

Oxaliplatin treatment resulted in transient histological changes in tumor tissues primarily via apoptosis induction

To explore the feasibility of re-inoculation, the histology of tumor tissues between the control and oxaliplatin treatment group was compared at various time points. As shown in Figure 4A, no significant difference in tumor necrosis was observed between the control group and oxaliplatin treatment group. Tumors in the control group maintained a basal apoptosis rate of approximately 3% throughout the 2-week experiment; however, oxaliplatintreated tumors demonstrated significantly increased apoptosis on day 2 after the final oxaliplatin injection ( Figure 4B, C). No significant difference in apoptosis was observed between the two groups at other time points.

Residual tumors after oxaliplatin treatment exhibited enhanced metastatic potential in re-inoculated nude mice

To evaluate biological features of HCC tumors after chemotherapy, MHCC97L xenografts from the oxaliplatintreated and untreated control groups were re-inoculated into the livers of new recipient mice. Six weeks after reinoculation, tumor volumes in the trial (bearing oxaliplatin pre-treated tumors) and control (bearing untreated tumors) groups were 2624.59 ± 491.60 and 3849.72 ± 827.09 mm 3 (P < 0.001) respectively. In addition, Ki-67 positive cells were significantly reduced in oxaliplatin pre-treated tumors (see additional file 2).

Analysis of serial lung sections revealed that oxaliplatin pre-treated tumors gave rise to spontaneous pulmonary metastasis in 83% (10/12) of the mice in the trial group (see additional file 3). In contrast, pulmonary metastasis was found in only 25% (3/12) of the mice in the control group (P = 0.012).

However, no significant difference was found between the trial group and control group in mean survival time (55.17 ± 5.53 vs. 51.33 ± 4.63 days; P = 0.128).

Residual tumors after chemotherapy exhibited changes consistent with EMT in nude mice

On day 7 after chemotherapy, oxaliplatin-treated and untreated tumors were subjected to immunohistochemistry ( Figure 5). Untreated tumors showed typical membranous E-cadherin expression in the cell-cell contacts. In contrast, oxaliplatin-treated tumors showed an overall reduction of E-cadherin expression, especially at the invasive front where the expression of E-cadherin was faint or even undetectable. The mesenchymal marker Ncadherin was significantly upregulated in oxaliplatintreated tumors. N-cadherin-positive tumor cells were concentrated primarily in areas of tumor-stroma interaction and were scattered in the stroma. Increased cytoplasmic expression of vimentin, another mesenchymal marker, was also detected in oxaliplatin-treated tumors. We also analyzed the expression of transcription factors Snail, Slug, and Twist in tumor tissues. However, only Snail was detected; it was significantly upregulated in oxaliplatin-treated tumors.

A comparison was also carried out between the control group (re-inoculated with untreated tumor) and trial group (re-inoculated with oxaliplatin pre-treated tumor), producing similar results. A much more frequent and active EMT was observed in tumors of the trial group than the control group (see additional file 4).  To explore the relationship between SYY treatment and EMT, tumor tissues in SYY-treated nude mice and control mice were analyzed by immunohistochemistry. Com-pared with the control, EMT was less common in tumors from mice treated with 8.4 g/kg or 4.2 g/kg SYY. A restoration of E-cadherin expression ( Figure 6D) and a concomitant reduction in N-cadherin expression ( Figure 6E) were detected in these tumors. The expression of vimentin was low or even undetectable in most tumors in these two SYY treatment groups.

Songyou Yin inhibited metastasis of oxaliplatin pre-treated tumors and increased survival

Discussion

Chemoresistance and chemotherapy side effects in HCC treatment have been studied; however, little is known about the opposite effect of chemotherapy. Consistent with the studies on other malignant tumors [4][5][6][7], we showed that in vitro exposure of MHCC97L and HepG2 HCC cells to pulse treatment with oxaliplatin led to increased motility and invasiveness of the surviving tumor cells. However, in vivo study is more complicated; chemotherapy affects not only tumor cells but also damages blood vessels and inhibits the antitumor system of the host, which may contribute to cancer metastasis [3,8]. Furthermore, chemotherapy directly influences tumor burden, and tumor burden strongly correlates with the incidence of pulmonary metastasis (i.e., metastasis occurs more frequently with larger tumors). To avoid these confounding factors, we employed a re-inoculation model. The similarity in histology between the untreated tumors and oxaliplatin-treated tumors on day 7 after chemother-apy support the validity of this model. Finally, oxaliplatin pre-treated tumors showed a 2.3-fold increase in the incidence of spontaneous metastasis to the lungs of the new recipient mice. These results indicated that HCC cells surviving in vitro and in vivo oxaliplatin treatment have increased metastatic potential, a possible opposite effect of oxaliplatin treatment.

Accumulating evidence indicates that EMT plays an important role in chemotherapy-induced invasiveness in colorectal, pancreatic, breast and ovarian carcinoma cells in vitro. We showed here that pulse exposure to oxaliplatin stimulated transformation of HepG2 and MHCC97L HCC cells from a typical epithelial phenotype to a spindle-shaped mesenchymal phenotype, accompanied by the loss of E-cadherin and upregulation of N-cadherin and vimentin. Further evidence was provided by xenografts in nude mice; molecular alterations consistent with EMT were detected in tumors on day 7 after chemotherapy. These observations were consistent with a recent clinical investigation of breast cancer [21], which indicated that residual breast cancers after chemotherapy and endocrine therapy displayed mesenchymal features. We also detected upregulated transcription factor Snail in oxaliplatin-treated tumor tissues and cell lines. This finding, combined with results of a previous study using oxaliplatin [4], suggests that the Snail signal transduction pathway may play a central role in oxaliplatin-induced tumor progression. Finally, the decreased proliferation of oxaliplatin-treated tumor cells suggests that these tumor cells may have switched from a proliferative to a more invasive and migratory phenotype, thus providing further evidence of EMT following chemotherapy [22].

Interestingly, similar changes were also detected in reinoculated tumors 6 weeks after re-inoculation. Compared with controls, oxaliplatin pre-treated tumors showed decreased growth and significantly increased EMT. This observation indicates that tumor cells surviving in vivo chemotherapy acquired intrinsic characteristics that facilitated EMT. A possible explanation for this might be derived from the survival mechanisms of tumor cells. Molecules such as interleukin (IL)-8 and integrins not only help tumor cells survive chemotherapy but stimulate EMT [6,23]. These factors may be pre-existing in minor subpopulations of tumor cells and then selected by chemotherapy, or they could be induced by chemotherapy, or both mechanisms may occur. These results, together with enhanced pulmonary metastasis rates in reinoculated mice, highlight the close relationship among oxaliplatin treatment, EMT, and metastasis. Further investigations are needed to characterize the underlying mechanisms in more detail.

It is not clear whether the increased metastatic potential of residual HCC after oxaliplatin treatment overweighs or is less important to the host than the reduced tumor burden by chemotherapy; however, the opposite effect of chemotherapy attenuates its anti-tumor effect as far as host life span is concerned. As mentioned above, previous clinical trials failed to show a correlation between the HCC patients' response to chemotherapy and prolonged survival. The findings of the present study suggest that the increased metastatic potential within residual HCC cells after chemotherapy may be partly responsible for these results. Indeed, most cancer deaths are due to metastasis in clinical trials [24]. While much attention has been paid to reducing tumor burden, the complex mechanisms involved in chemotherapy-related tumor progression require clarification, which may eventually lead to mechanism-based combination treatments to improve the efficacy of chemotherapy in HCC.

SYY is a Chinese herbal extract consisting of Salvia miltiorrhiza Bge. and four other herbs. Some components of SYY have demonstrated value in treating malignancies [25,26]. In our previous study [17], SYY effectively inhibited tumor growth and metastasis, and increased survival in a similar HCC nude mouse model bearing MHCC97H (a cell line with high metastatic potential originating from MHCC97 cells [13]) xenograft. The induction of apoptosis and downregulation of MMP-2 and VEGF were suggested to be responsible for the anticancer effect of SYY. In the present study, we further evaluated the effects of SYY on oxaliplatin pre-treated tumors to determine its effects against chemotherapyrelated cancer metastasis. We demonstrated that SYY treatment at doses of 4.2 g/kg and 8.4 g/kg significantly inhibited the metastatic potential of residual HCC after chemotherapy and prolonged mouse survival. Thus SYY Figure 6 The effect of Songyou Yin on oxaliplatin pre-treated tumors in nude mice. Tumor volume was significantly decreased in mice treated with 8.4 g/kg Songyou Yin compared with the control group (A). Songyou Yin treatment (4.2 and 8.4 g/kg) significantly decreased pulmonary metastasis (B) and prolonged survival (C) of nude mice bearing oxaliplatin pre-treated MHCC97L xenografts. Songyou Yin treatment (4.2 g/kg) restored Ecadherin expression (D) and arrested the cadherin switch (E) in tumor tissues. treatment may attenuate the opposite effect of oxaliplatin treatment, thereby improving treatment efficacy. Furthermore, the restoration of E-cadherin and arrest of cadherin switch in SYY-treated tumors suggest that EMT inhibition may play a role in SYY effects. In addition to recent studies that demonstrated EMT attenuation by a herbal prescription Wen-pi-tang-Hab-Wu-ling-san and a medicinal herb Panax notoginseng in kidney cells against fibrosis [27,28], we showed here that the arrest of EMT was also linked to the anticancer effects of traditional Chinese medicine. Expression of MMP2 and VEGF was found to be important in the EMT program, with relevance to the induction of transcription factor Snail [21,23,29]. Hence, the attenuation of EMT by SYY demonstrated in the present study supports our previous findings by providing underlying mechanisms. One limitation of the present study is the lack of detailed understanding of the relationship between EMT arrest and SYY treatment due to its complexity. Therefore, additional basic research is needed.

Conclusions

Our results suggest that residual HCC cells after oxaliplatin treatment undergo EMT and exhibit increased metastatic potential. Accordingly, attenuation of EMT by SYY may improve the efficacy of chemotherapy in HCC.

Additional material

Figure 1
1Tumor cells that survived oxaliplatin treatment showed enhanced migration in vitro.The transwell assay demonstrated that MHCC97L-Oxa and HepG2-Oxa cells migrated through the collagen membrane in greater numbers compared with parental MHCC97L and HepG2 cells.

detected in MHCC97L-Oxa and HepG2-Oxa cells (Figure 3C). However, expression of the transcription factors Twist and Slug was very low and was not affected by oxaliplatin treatment in either cell line (data not shown).

Figure 2
2Tumor cells that survived oxaliplatin treatment exhibited enhanced invasive properties in vitro. MHCC97L-Oxa and HepG2-Oxa cells showed significant increase in the numbers of cells invading the Matrigel-coated membrane compared with parental MHCC97L and HepG2 cells in the modified transwell assay. P < 0.001). With this SYY dose, maximum inhibition of pulmonary metastasis (3/15 vs. 13/15; P = 0.001) and prolonged survival was also observed (81.17 ± 7.36 days vs. 53.83 ± 4.71 days; P = 0.001) (Figure 6A-C). In contrast, 2.1 g/kg SYY treatment did not significantly reduce tumor volume, incidence of pulmonary metastasis, or mean survival time. Although 4.2 g/kg SYY did not significantly inhibit tumor growth (2470.41 ± 583.96 mm 3 vs. 2,841.87 ± 417.59 mm 3 ; P = 0.065), this dose decreased the incidence of pulmonary metastasis (6/15 vs. 13/15; P = 0.021) and increased life span (66.67 ± 5.61 days vs. 53.83 ± 4.71 days; P = 0.002).

Figure 3
3Exposure to oxaliplatin in vitro induced epithelial-mesenchymal transition (EMT) in MHCC97L and HepG2 cells. A) Tumor cells sur-viving oxaliplatin treatment were morphologically distinct from the typical epithelial appearance of parental MHCC97L and HepG2 cells, demonstrating a spindle shape with less cell-cell adhesion and increased formation of pseudopodia. B) MTT assay revealed a reduced proliferation in MHCC97L-Oxa and HepG2-Oxa cells compared with the respective parental cells. C) Western blot analysis showed molecular changes consistent with EMT in MHCC97L-Oxa and HepG2-Oxa cells (e.g., downregulation of E-cadherin and upregulation of N-cadherin, vimentin, and transcription factor Snail). GAPDH was used as an internal control. D) Reduced E-cadherin expression and increased expression of N-cadherin, and vimentin in MHCC97L-Oxa and HepG2-Oxa cells were also demonstrated by immunofluorescence.

Figure 4
4Analysis of tumor histology after oxaliplatin treatment. A) No significant difference in tumor necrosis was observed between the control group and oxaliplatin treatment group after treatment. B) Terminal transferase dUTP nick end labeling (TUNEL) analysis showed a basal level of apoptosis rate of about 3% in tumors of the control group throughout the 2-week experiment. In the oxaliplatin treatment group, significant apoptosis was observed on day 2 after the final injection (B, C). No significant difference in apoptosis was found between the two groups at other time points.

Figure 5
5Oxaliplatin-treated tumors exhibited changes consistent with epithelial-mesenchymal transition (EMT). Immunohistochemistry revealed typical membranous E-cadherin expression in the cell-cell contacts of untreated tumors. In contrast, oxaliplatin-treated tumors showed an overall reduction of E-cadherin expression, especially at the invasive front where the expression of E-cadherin was faint or even undetectable. Instead, N-cadherin was significantly upregulated in oxaliplatin-treated tumors. N-cadherin-positive tumor cells were concentrated in areas of tumor-stroma interaction and were scattered in the stroma. Increased expression of vimentin and Snail were also detected in the invasive front of oxaliplatin-treated tumors.

After 6 weeks, 8.4 g/kg SYY treatment resulted in decreased tumor volume compared with the control group (1969.95 ± 454.32 mm 3 vs. 2841.87 ± 417.59 mm 3 ;
This article is available from: http://www.biomedcentral.com/1471-2407/10/219 © 2010 Xiong et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Cancer 2010, 10:219
AcknowledgementsWe thank professor Zhao-Chong Zeng for his smart suggestions for the revi-Competing interestsThe authors declare that they have no competing interests.Authors' contributions WX, ZYT, ZGR, HCS, SJQ, LW, BBL and QSL contributed to the study design, analysis and interpretation of data. ZYT conceived the study. WX performed the experiment. WZ, WQW and LL participated in the establishment of nude mice model. XDZ participated in statistical analysis. WX and ZGR drafted the manuscript. ZYT carried out the revision and provided important suggestions. All authors approved the final manuscript.
Strategies for the management of hepatocellular carcinoma. M Schwartz, S Roayaie, M Konstadoulakis, Nat Clin Pract Oncol. 4Schwartz M, Roayaie S, Konstadoulakis M: Strategies for the management of hepatocellular carcinoma. Nat Clin Pract Oncol 2007, 4:424-432.

Hepatocellular carcinoma: current management and future trends. B I Carr, Gastroenterology. 1271SupplCarr BI: Hepatocellular carcinoma: current management and future trends. Gastroenterology 2004, 127(Suppl 1):218-224.

Induction of cancer metastasis by cyclophosphamide pretreatment of host mice: an opposite effect of chemotherapy. K Yamauchi, M Yang, K Hayashi, P Jiang, N Yamamoto, H Tsuchiya, K Tomita, A R Moossa, M Bouvet, R M Hoffman, Cancer Res. 68Yamauchi K, Yang M, Hayashi K, Jiang P, Yamamoto N, Tsuchiya H, Tomita K, Moossa AR, Bouvet M, Hoffman RM: Induction of cancer metastasis by cyclophosphamide pretreatment of host mice: an opposite effect of chemotherapy. Cancer Res 2008, 68:516-520.

Chronic oxaliplatin resistance induces epithelial-tomesenchymal transition in colorectal cancer cell lines. A D Yang, F Fan, E R Camp, G Van Buren, W Liu, R Somcio, M J Gray, H Cheng, P M Hoff, L M Ellis, Clin Cancer Res. 12Yang AD, Fan F, Camp ER, Van Buren G, Liu W, Somcio R, Gray MJ, Cheng H, Hoff PM, Ellis LM: Chronic oxaliplatin resistance induces epithelial-to- mesenchymal transition in colorectal cancer cell lines. Clin Cancer Res 2006, 12:4147-4153.

Development and characterization of gemcitabine-resistant pancreatic tumor cells. A N Shah, J M Summy, J Zhang, S I Park, N U Parikh, G E Gallick, Ann Surg Oncol. 14Shah AN, Summy JM, Zhang J, Park SI, Parikh NU, Gallick GE: Development and characterization of gemcitabine-resistant pancreatic tumor cells. Ann Surg Oncol 2007, 14:3629-3637.

Progression and enhancement of metastatic potential after exposure of tumor cells to chemotherapeutic agents. J E De Larco, B R Wuertz, J C Manivel, L T Furcht, Cancer Res. 61De Larco JE, Wuertz BR, Manivel JC, Furcht LT: Progression and enhancement of metastatic potential after exposure of tumor cells to chemotherapeutic agents. Cancer Res 2001, 61:2857-2861.

Chemoresistance to paclitaxel induces epithelial-mesenchymal transition and enhances metastatic potential for epithelial ovarian carcinoma cells. H Kajiyama, K Shibata, M Terauchi, M Yamashita, K Ino, A Nawa, F Kikkawa, Int J Oncol. 31Kajiyama H, Shibata K, Terauchi M, Yamashita M, Ino K, Nawa A, Kikkawa F: Chemoresistance to paclitaxel induces epithelial-mesenchymal transition and enhances metastatic potential for epithelial ovarian carcinoma cells. Int J Oncol 2007, 31:277-283.

Promotion of pulmonary metastasis in mice by bleomycin-induced endothelial injury. F W Orr, I Y Adamson, L Young, Cancer Res. 46Orr FW, Adamson IY, Young L: Promotion of pulmonary metastasis in mice by bleomycin-induced endothelial injury. Cancer Res 1986, 46:891-897.

Epithelial-mesenchymal transitions in tumour progression. J P Thiery, Nat Rev Cancer. 2Thiery JP: Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2002, 2:442-454.

Phase II study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma. A X Zhu, L S Blaszkowsky, D P Ryan, J W Clark, A Muzikansky, K Horgan, S Sheehan, K E Hale, P C Enzinger, P Bhargava, K Stuart, J Clin Oncol. 24Zhu AX, Blaszkowsky LS, Ryan DP, Clark JW, Muzikansky A, Horgan K, Sheehan S, Hale KE, Enzinger PC, Bhargava P, Stuart K: Phase II study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma. J Clin Oncol 2006, 24:1898-1903.

Gemcitabine plus oxaliplatin (GEMOX) in patients with advanced hepatocellular carcinaoma (HCC): results of a phase II study. S Louafi, V Boige, M Ducreux, L Bonyhay, T Mansourbakht, T De Baere, A Asnacios, L Hannoun, T Poynard, J Taïeb, Cancer. 109Louafi S, Boige V, Ducreux M, Bonyhay L, Mansourbakht T, de Baere T, Asnacios A, Hannoun L, Poynard T, Taïeb J: Gemcitabine plus oxaliplatin (GEMOX) in patients with advanced hepatocellular carcinaoma (HCC): results of a phase II study. Cancer 2007, 109:1384-1390.

New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis. J Tian, Z Y Tang, S L Ye, Y K Liu, Z Y Lin, J Chen, Q Xue, Br J Cancer. 81Tian J, Tang ZY, Ye SL, Liu YK, Lin ZY, Chen J, Xue Q: New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis. Br J Cancer 1999, 81:814-821.

Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. Y Li, Z Y Tang, S L Ye, Y K Liu, J Chen, Q Xue, J Chen, D M Gao, W H Bao, World J Gastroenterol. 7Li Y, Tang ZY, Ye SL, Liu YK, Chen J, Xue Q, Chen J, Gao DM, Bao WH: Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol 2001, 7:630-636.

JS-K, a novel non-ionic diazeniumdiolate derivative, inhibits Hep 3B hepatoma cell growth and induces c-Jun phosphorylation via multiple MAP kinase pathways. Z Ren, S Kar, Z Wang, M Wang, J E Saavedra, B I Carr, J Cell Physiol. 197Ren Z, Kar S, Wang Z, Wang M, Saavedra JE, Carr BI: JS-K, a novel non-ionic diazeniumdiolate derivative, inhibits Hep 3B hepatoma cell growth and induces c-Jun phosphorylation via multiple MAP kinase pathways. J Cell Physiol 2003, 197:426-434.

Radiosensitization by oxaliplatin in a mouse adenocarcinoma: influence of treatment schedule. A Cividalli, F Ceciarelli, E Livdi, P Altavista, G Cruciani, P Marchetti, D T Danesi, Int J Radiat Oncol Biol Phys. 52Cividalli A, Ceciarelli F, Livdi E, Altavista P, Cruciani G, Marchetti P, Danesi DT: Radiosensitization by oxaliplatin in a mouse adenocarcinoma: influence of treatment schedule. Int J Radiat Oncol Biol Phys 2002, 52:1092-1098.

Effects of cryotherapy or chemotherapy on apoptosis in a non-small-cell lung cancer xenografted into SCID mice. V Forest, M Peoc'h, L Campos, D Guyotat, J M Vergnon, Cryobiology. 50Forest V, Peoc'h M, Campos L, Guyotat D, Vergnon JM: Effects of cryotherapy or chemotherapy on apoptosis in a non-small-cell lung cancer xenografted into SCID mice. Cryobiology 2005, 50:29-37.

Songyou Yin" inhibits tumor growth and prolongs survival in nude mice bearing human hepatocellular carcinoma xenograft with high metastatic potential. X Y Huang, L Wang, Z L Huang, Q Zheng, Q S Li, Z Y Tang, J Cancer Res Clin Oncol. 135Herbal extractHuang XY, Wang L, Huang ZL, Zheng Q, Li QS, Tang ZY: Herbal extract "Songyou Yin" inhibits tumor growth and prolongs survival in nude mice bearing human hepatocellular carcinoma xenograft with high metastatic potential. J Cancer Res Clin Oncol 2009, 135:1245-1255.

High-dose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential. L Wang, Z Y Tang, L X Qin, X F Wu, H C Sun, Q Xue, S L Ye, Hepatology. 32Wang L, Tang ZY, Qin LX, Wu XF, Sun HC, Xue Q, Ye SL: High-dose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential. Hepatology 2000, 32:43-48.

Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. Q Gao, S J Qiu, J Fan, J Zhou, X Y Wang, Y S Xiao, Y Xu, Y W Li, Z Y Tang, J Clin Oncol. 25Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, Xu Y, Li YW, Tang ZY: Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. J Clin Oncol 2007, 25:2586-2593.

Cadherin switching. M J Wheelock, Y Shintani, M Maeda, Y Fukumoto, K R Johnson, J Cell Sci. 121Wheelock MJ, Shintani Y, Maeda M, Fukumoto Y, Johnson KR: Cadherin switching. J Cell Sci 2008, 121:727-735.

Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. C J Creighton, X Li, M Landis, J M Dixon, V M Neumeister, A Sjolund, D L Rimm, H Wong, A Rodriguez, J I Herschkowitz, C Fan, X Zhang, X He, A Pavlick, M C Gutierrez, L Renshaw, A A Larionov, D Faratian, S G Hilsenbeck, C M Perou, M T Lewis, J M Rosen, J C Chang, Proc Natl Acad Sci. 106Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A, Rimm DL, Wong H, Rodriguez A, Herschkowitz JI, Fan C, Zhang X, He X, Pavlick A, Gutierrez MC, Renshaw L, Larionov AA, Faratian D, Hilsenbeck SG, Perou CM, Lewis MT, Rosen JM, Chang JC: Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci USA 2009, 106:13820-13825.

Reduced proliferation and enhanced migration: two sides of the same coin? Molecular mechanisms of metastatic progression by YB-1. V Evdokimova, C Tognon, T Ng, P H Sorensen, Cell Cycle. 8Evdokimova V, Tognon C, Ng T, Sorensen PH: Reduced proliferation and enhanced migration: two sides of the same coin? Molecular mechanisms of metastatic progression by YB-1. Cell Cycle 2009, 8:2901-2906.

Molecular signature and therapeutic perspective of the epithelial-to-mesenchymal transitions in epithelial cancers. M Sabbah, S Emami, G Redeuilh, S Julien, G Prévost, A Zimber, R Ouelaa, M Bracke, De Wever, O Gespach, C , Drug Resist Updat. 11Sabbah M, Emami S, Redeuilh G, Julien S, Prévost G, Zimber A, Ouelaa R, Bracke M, De Wever O, Gespach C: Molecular signature and therapeutic perspective of the epithelial-to-mesenchymal transitions in epithelial cancers. Drug Resist Updat 2008, 11:123-151.

Cancer metastasis: building a framework. G P Gupta, J Massagué, Cell. 127Gupta GP, Massagué J: Cancer metastasis: building a framework. Cell 2006, 127:679-695.

Reversing effect of Tanshinone on malignant phenotypes of human hepatocarcinoma cell line. S L Yuan, R M Huang, X J Wang, Y Song, G Q Huang, World J Gastroenterol. 4Yuan SL, Huang RM, Wang XJ, Song Y, Huang GQ: Reversing effect of Tanshinone on malignant phenotypes of human hepatocarcinoma cell line. World J Gastroenterol 1998, 4:317-319.

Astragalus saponins induce growth inhibition and apoptosis in human colon cancer cells and tumor xenograft. M M Tin, C H Cho, K Chan, A E James, J K Ko, Carcinogenesis. 28Tin MM, Cho CH, Chan K, James AE, Ko JK: Astragalus saponins induce growth inhibition and apoptosis in human colon cancer cells and tumor xenograft. Carcinogenesis 2007, 28:1347-1355.

Wen-pi-tang-Hab-Wu-ling-san, an oriental herbal prescription, attenuates epithelialmesenchymal transdifferentiation stimulated by TGF-beta1 in kidney cells. S I Lee, H J Kim, M C Baek, K M Park, Y Park, C H Yoon, Y C Boo, Phytother Res. 21Lee SI, Kim HJ, Baek MC, Park KM, Park Y, Yoon CH, Boo YC: Wen-pi-tang- Hab-Wu-ling-san, an oriental herbal prescription, attenuates epithelial- mesenchymal transdifferentiation stimulated by TGF-beta1 in kidney cells. Phytother Res 2007, 21:548-553.

Ginsenoside Rg1, a major active component isolated from Panax notoginseng, restrains tubular epithelial to myofibroblast transition in vitro. X S Xie, M Yang, H C Liu, C Zuo, H J Li, J M Fan, J Ethnopharmacol. 122Xie XS, Yang M, Liu HC, Zuo C, Li HJ, Fan JM: Ginsenoside Rg1, a major active component isolated from Panax notoginseng, restrains tubular epithelial to myofibroblast transition in vitro. J Ethnopharmacol 2009, 122:35-41.

Epithelialmesenchymal transitions: the importance of changing cell state in development and disease. H Acloque, M S Adams, K Fishwick, M Bronner-Fraser, M A Nieto, J Clin Invest. 119Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA: Epithelial- mesenchymal transitions: the importance of changing cell state in development and disease. J Clin Invest 2009, 119:1438-1449.