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Plumbagin promotes human hepatoma SMMC-7721 cell apoptosis via caspase-3/vimentin signal-mediated EMT

Background/aims Plumbagin (PL) has been shown to effectively inhibit tumor growth and migration of hepatocellular carcinoma cells in previous studies, but the specific mechanism for this remains unclear. The purpose of this study was to investigate the effects of PL-induced apoptosis in epithelial–mesenchymal transition (EMT) of human hepatocellular carcinoma (HCC) in vivo and in vitro. Methods SMMC-7721 cells were cultured, an EMT model was induced in vitro by TGF-β1, cell proliferation was detected by the MTT assay, cell invasion was analyzed by the Transwell invasion assay, and the apoptosis rate was measured by flow cytometry. RT-PCR was used to detect vimentin, E-cadherin, N-cadherin and snail mRNA, and Western blotting was used to detect the vimentin, caspase-3, PARP-1, E-cadherin, N-cadherin and snail protein expression levels. HE staining and TUNEL staining, immunohistochemistry and immunofluorescence were used to detect the expression levels of bax and bcl-2 in hepatocarcinoma xenografts and to evaluate their apoptosis in vivo. Results The in vitro results showed that PL inhibited the proliferation of EMT model cells, increased the apoptosis rate of the EMT model, and significantly decreased the vimentin, PARP-1, N-cadherin and snail protein levels, but significantly increased E-cadherin and caspase-3 protein expression. In addition, the in vivo results indicated that PL can affect the expression of bax/bcl-2 apoptotic marker proteins. Conclusion PL may induce apoptosis of human hepatocellular carcinoma cells undergoing epithelial–mesenchymal transition by increasing the caspase-3 protein level and cleaving vimentin.


Introduction
Hepatocellular carcinoma (HCC) is one of the most commonly occurring cancers and is the second leading cause of cancer-related deaths from malignancy worldwide. 1 Despite advances in surgery and chemotherapy, HCC is associated with a poor 5-year survival rate due to its uncontrolled invasion and metastasis under the presently available treatments, including surgery and chemotherapy. Recurrence and metastasis are the major obstacles to improve survival for HCC patients. 2 Thus, there is a critical need for improving HCC therapies, which can be developed from the identification of new and effective chemotherapeutic agents and a better understanding of the molecular mechanisms involved.
Emerging evidence suggests that the epithelial-tomesenchymal transition (EMT) is activated during HCC development, growth, progression, and metastasis. 3 It has been proposed that EMT facilitates metastatic dissemination of HCC cells from a primary organ to secondary sites; thus, intervention of this process may represent a novel strategy to prevent HCC metastasis. The expression of vimentin, along with the expression of mesenchymal markers, such as N-cadherin and the loss of E-cadherin, are key molecular markers of EMT. 4 A recent study found that vimentin, a core molecule in the EMT, is overexpressed in many epithelialoriginating malignant tumor tissues or cancer cell lines (such as liver cancer and lung cancer). As an important signaling molecule, vimentin is involved in tumor cell growth, invasion and metastasis, 5,6 and when vimentin is cleaved by caspase, the cleaved fragments can be involved in tumor cellular apoptosis. 7 Hence, agents that can downregulate the activation of vimentin would have the potential to inhibit the development of cancer. Cells undergoing EMT become migratory, invasive and gain resistance to apoptosis. The failure of tumor cells to undergo apoptosis translates into malignancy potential and chemotherapeutic resistance.
A large number of phytochemicals are considered to regulate several molecular and metabolic processes, such as cell cycle regulation, apoptosis activation, angiogenesis and metastatic suppression, that can hinder cancer progression. 8,9 Plumbagin (5-hydroxy-2-methyl-1,4-napthoquinone, PL), an active naphthoquinone isolated from the roots of the medicinal plant Plumbago zeylanica L. (Figure 1A), is the main active ingredient of Plumbago. 10 PL has been demonstrated to have a wide spectrum of pharmacological effects, including anti-inflammatory, anti-angiogenesis, and anticancer, antibacterial, antifungal, hepatoprotective and neuroprotective effects in preclinical models. In recent years, the anticancer effect of PL has drawn a great deal of interest. PL has been shown to exert antitumor properties by inhibiting proliferation and inducing apoptosis in vitro in a broad range of cancer types, including breast cancer, non-small cell lung cancer, and HCC. [11][12][13][14] It also exhibits pro-apoptotic and radio-sensitizing activities in different tumor cells and animal models, both in vitro and in vivo. Currently, the anti-hepatoma effect of PL also causes widespread concern. In a previous study, PL was found to be cytotoxic against the HEPA-3B hepatoma cell line; the effect of PL on liver cancer inhibited the migration and invasion of the liver cancer cells through downregulation of MMP-2 and uPA. In addition, PL was found to induce significant regression of the tumors in a 3-methyl-4-dimethyl aminoazobenzene (3MeDAB)-induced hepatoma model in male Wistar rats. However, no studies exist regarding the effects of PL on apoptosis of epithelialmesenchymal transition cells in HCC.
In this study, a human HCC xenograft model was established in nude mice, and an EMT model of liver cancer was generated from TGF-β stimulation. The effect of PL on the apoptosis of human HCC epithelial-mesenchymal transition cells was investigated to explore its potential mechanism of  Figure 1 Chemical structure of plumbagin (PL) (A). SMMC-7221 cells were treated with10 ng/ml TGF-β1 for 48 h, EMT model preparation (B). The expression levels of E-cadherin and N-cadherin were detected by immunofluorescence to verify whether the EMT model was successfully established, error bars and mean ± SD, **P<0.01, ***P<0.001 vs control group (C). The morphology of TGF-β-induced cells changed from a rounded shape to a fusiform shape. Cell nuclei were defined by DAPI (blue). Scale bar, 25 μm (D, E). Mice SMMC-7721 cells were grown to approximately 80% confluency, and concentrated cells were regulated at a density of 2.5×10 7 cell/ml (the number of living cells in Trypan blue exclusion assay was over 95%). Nude mice were subcutaneously injected with 0.2 ml of cell suspension containing SMMC-7721; after approximately one week, round, small colliculi were observed (tumors of 0.5 cm 2 in diameter). The mice were randomly divided into 5 groups, which included the control group, positive thalidomide control group, and PL (Sigma, SLBG3436V, US) low-, middle-, and high-dose groups; each group had 6 nude mice. After tumor formation, the mice were peritoneally injected with normal saline (0.

Cellular apoptosis assay
Each group of cells was treated in strict accordance with the operating instructions of the apoptosis detection kit. The cells were digested with EDTA-free trypsin, centrifuged at 300 g a 4°C for 5 min and collected. The cells were washed twice with precooled PBS, centrifuged at 300 g and 4°C for 5 min, and 10-50×10 4 cells were collected. The cells were resuspended in 100 µl of 1×binding buffer, and then 5 µl of annexin V-FITC and 5 µl of PI staining solution were added, mixed gently, and incubated in the dark at room temperature for 10 min. Next, 400 µl of 1×binding buffer was added and mixed, and the sample was filtered with a nylon mesh (200 mesh) and added to a round-bottom flow cytometry tube. The cells were detected by flow cytometry within 1 h. The apoptosis rate = (number of apoptotic cells/total number of cells observed) ×100%.

RT-PCR
SMMC-7721 cells were harvested in medium containing 10% fetal bovine serum at a final concentration of 2×10 6 cells/mL and incubated for 24 hrs in 24-well plates. Next, plumbagin was added and the sample was incubated for 48 hrs. Total RNA was extracted from the SMMC-7721 cells by TRIzol reagent (Takara, Japan), and the total RNA content was measured by a Nano-100 differential spectrophotometer. The extracted 4.284 μg of RNA binds to reverse transcriptase to generate cDNA. The search for gene sequences and the primer design was conducted in-house, according to Gene Bank's guidelines. Real-time PCR for mRNA detection was performed using SYBR Green PCR Master Mix (Ambion, Carlsbad, CA, USA). The mRNA levels of E-cadherin, Ncadherin, vimentin and Snail were evaluated. The primer sets used for PCR amplification are as follows in Table 1.

Western blotting
Each group of cells was collected in the logarithmic growth phase (5-10×10 6 cell/ml). The cells were washed twice with cold PBS, and then 500 µl of cold lysate was added. After mixing, the cell sample was shaken at 4°C for 15-20 min, and then centrifuged at 16,000 g and 4°C for 15 min. The protein concentration of the supernatant was measured by the BCA method. Subsequently, 4×SDS sample treatment solution was added, and the samples were subjected to 100°C heating for 5 min for denaturation. After SDS-PAGE electrophoresis, the proteins were transferred from SDS polyacrylamide gels to nitrocellulose (PVDF) membranes. After the transfer was completed, the PVDF membranes were blocked for1 h in 5% nonfat dry milk in TBST buffer at room temperature. The appropriate dilutions of primary antibodies were added as follows: mouse anti-caspase-3 (1:300, Santa Cruz, CA, USA, sc-7272), mouse anti-vimentin (1:300, Santa Cruz, CA, USA, sc-6260), mouse anti-PARP-1 (1:300, Santa Cruz, CA, USA,sc-8007), rabbit anti-β-actin Signaling Technology, USA, 3879) and then incubated overnight at 4°C. The suitable secondary antibodies were then added, respectively: HRP-labeled goat anti-mouse IgG (1:12000) and goat anti-rabbit IgG (1:10000). The samples were then incubated at room temperature for 1 h. The CL chECL chemiluminescence method was utilized for visualization. X-ray film exposure, chemiluminescence development, film scanning, and quantitative analysis were conducted the US NIH ImageJ software. The anti-β-actin antibody was used as an internal loading control. The gray value ratio of the target band and internal loading control represent the results.

Histological assessment
The tumors were embedded in paraffin and stained with a Hematoxylin & Eosin Staining Kit to observe the morphological changes of liver cancer cells, the basement membrane integrity of the tumor angiogenesis, and the phenome apoptosis and necrosis in the tumor tissues. Cell apoptosis in tumor tissues was detected by the TUNEL assay using the conventional labeling index (LI), and 5 high-power fields were selected at 400X (Leica, DMI3000, Germany); all cells per high-power field were counted. The labeling index = the number of positive cells/ horizons of all cells; the apoptosis index of each case (AI) equals the average horizon labeling index value.

Immunohistochemistry
To further detect the tumors tissue apoptosis levels, we next examined the apoptosis-related proteins Bax and Bcl-2 in hepatocellular carcinoma xenografts by immunohistochemistry. The tumor tissue sections were deparaffinized, rehydrated and boiled in 0.01 M sodium citrate for antigen retrieval. The endogenous peroxidase activity was quenched. The sections were incubated with rabbit anti-Bax (1:50, Sanying, Wuhan, China) and Bcl-2 (1:50, Abcam, Cambridge, MA) overnight at 4°C. The sections were incubated with biotinylated secondary antibodies and a streptavidin-HRP complex (Zsbio, Wuhan, China). The staining was visualized using DAB (Boster, Wuhan, China). Representative images were taken using an Olympus camera (Nikon Instruments, Melvil). Optical density analysis was performed on the immunohistochemical images using IPP6.0 software. Four 400-fold images were taken for each slice to perform optical density analysis. The density was defined as the average optical density.

Immunofluorescence staining
Tissue from treated tumors was fixed with 4% paraformaldehyde for 25 min and permeabilized with 0.1% Triton X-100 for 10 min. The samples were then blocked with 2% bovine serum albumin (BSA) for 30 min at 37°C, followed by incubation with primary antibody against Bax (1:50, Sanying, Wuhan, China) and Bcl-2 (1:50, Abcam, Cambridge, MA) at 4°C overnight. The sections were subsequently incubated with the corresponding secondary antibody for 1 h at room temperature. The nuclei were stained with DAPI for 3 min, washed twice with PBS, and then images were captured using a DMI-4000B inverted fluorescence microscope (Olympus BX53). Fluorescence intensity analysis was performed on immunofluorescence images using IPP6.0 software. Three 400-fold images were selected for each slice for fluorescence intensity analysis, and the density (mean) was the average fluorescence intensity.

Statistical analysis
Statistical analysis was performed using SPSS 17.0. Comparisons between two groups were made using ANOVA, and each mean pairwise comparison was analyzed by using the q test. P-values are represented by * or #.

Establishment and verification of the SMMC-7721 EMT model
To observe the morphological changes of the SMMC-7721 cells during EMT, HE staining was performed. The results showed that the control adherent cells grew well, and displayed an epithelioid form by fluorescence microscopy, and ovoid, polygonal or irregular shapes, such as stars. SMMC-7721 cells treated with 10 ng/ml TGF-β1 showed a slender morphology. The number of cells in the model group was significantly increased, and the cells exhibited a long spindle shape ( Figure 1B). The expression levels of E-cadherin and N-cadherin were detected by immunofluorescence to verify whether the EMT model was successfully established. Epithelial-mesenchymal transition (EMT) mainly involved E-cadherin to N-cadherin translocation. The expression levels of E-cadherin and N-cadherin in SMMC-7721 cells treated with TGF-β1 for 48 hrs were then examined, as shown in Figure 1C-E. Compared with the control group, the E-cadherin expression levels were decreased and N-cadherin expression levels were increased in SMMC-7721 cells treated with TGF-β1.

PL inhibits the proliferation of EMT induced by TGF-β in SMMC-7721 cells
To investigate the effects of PL in human HCC cells, SMMC-7721 cells were exposed to various concentrations of PL for 24 h and 48 h and then subjected to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PL was found to inhibit SMMC-7721 cell viability in a concentrationand time-dependent manner (Figure 2A). The inhibitory effect of the 24 h group was the most effective, so a 24 h intervention time was used in the follow-up experiments.

PL suppresses cell invasion of EMT induced by TGF-β in SMMC-7721 cells
Previous studies have shown the crucial role of PL in tumor cell proliferation, apoptosis, and migration. 15,16 The effect of PL on the invasion of SMMC-7721 cells induced by TGF-β in the EMT model was further determined in this study. In this study, cell invasion was measured by the Transwell invasion assay, and it was found that cell invasion was significantly reduced after PL treatment ( Figure 2B

Regulation of PL on vimentin and apoptosis-related proteins
The mechanism of apoptosis induced by PL in epithelialmesenchymal cells of human HCC was evaluated by measuring the level of apoptosis-related proteins. As shown in Figure 3C, compared with the EMT model group, PL treatment significantly decreased vimentin and PARP-1 protein expression and increased caspase-3 expression, especially in the 8 µM group, and the difference was significant (P<0.01). Some vimentin cleavage occurred in the 8 µM group and in the Vim group, and there was a positive correlation between the degree of cleavage and the rate of early apoptosis ( Figure 3D-F).

Regulation of PL on vimentin and EMTrelated proteins
The effect of PL on TGF-β-induced epithelial-mesenchymal transition in human HCC cells was assessed by measuring the expression levels of EMT-related genes and their proteins. As shown in Figure 4A-D, compared with the EMT model group, the expression of vimentin, N-cadherin and snail mRNA was significantly decreased after PL treatment, while the expression of E-cadherin was increased, especially in the 8 μM group, and the difference was significant (P<0.01). In addition, the results of Western blot analysis ( Figure 4E-I) showed that the expression levels of vimentin, N-cadherin and snail proteins were significantly decreased after PL treatment compared with the EMT model group, while E-cadherin protein expression was increased, especially in the 8 μM high-dose group. These findings demonstrate that PL upregulates the expression of the epithelial biomarker E-cadherin, and downregulates the expression of the interstitial cell marker protein vimentin, N-cadherin and the transcriptional regulator Snail, thereby inhibiting the epithelial-mesenchymal transition of HCC in vitro.

Evaluation of apoptosis in vivo
To evaluate whether PL can induce apoptosis in vivo, tumor sections from mice were examined. In our previously    12 To determine whether PL induces apoptosis in human HCC cells. In the current study results, we measured apoptosis in tumor xenograft tissues using TUNEL staining. As shown in Figure 5A PL treatment groups were greater than that in the model group. The results of HE showed that the cells in the model group had large nuclei, deep staining, obvious nuclear aberration, disordered arrangement without polarity, more striped arrangement, and obvious vascular hyperplasia; the cells in the model group were treated with a low dose of PL. Changes of the cells in the tumor as a whole were not obvious; however, the proliferation of blood vessels was obvious. When the PL dosage was raised, the proliferation of blood vessels decreased significantly and cell apoptosis became apparent, especially in the highdose group. The expression of TUNEL was more obvious, where the positive expression of TUNEL was brownish. A light blue color was visible as nuclear staining. Compared with the model group, and there was no significant change in the low-dose PL group compared with the model group, indicating that the effect of the drug was positively correlated with the dose.

PL regulates apoptosis-related proteins in tumor tissues
Bcl-2 family proteins serve as importance regulators involved in apoptosis, acting to either inhibit or promote cell death. By analyzing the average optical density values of immunohistochemistry, as shown in Figure 5B and C, PL treatment significantly increased the expression of the pro-apoptotic protein Bax. It reduced the expression of the anti-apoptotic protein Bcl-2 in the membrane and cytoplasm of tumor cells compared to the control group. Among them, PL high dose group was the most significant (P<0.001). In addition, immunofluorescence assay showed that PL treatment reduced the nuclear immunofluorescence staining of Bcl-2 and increased the immunofluorescence staining of Bax ( Figure 6A and B). By analyzing the average optical density of immunofluorescence, the expression of proapoptotic protein Bax was increased compared with the control group, and the PL high dose group was the most significant (P<0.001). In contrast, the expression of the anti-apoptotic protein Bcl-2 was significantly reduced ( Figure 6C).

Discussion
The epithelial-mesenchymal transition (EMT) is a basic physiological and pathological phenomenon of organisms. 17 It refers to processes in which the epithelial cells acquire biological characteristics of mesenchymal cells, characterized by the loss of cell polarity, decreased expression of epithelial cell markers (such as E-cadherin) and increased expression of mesenchymal markers (eg, vimentin). 18 EMT plays a very important role in tumor invasion and metastasis and has become a hot topic of cancer research. 19,20 As a marker of EMT, vimentin is an important cytoskeletal protein expressed in mesenchymal cells and some ectodermal cells. Vimentin, microtubules, actin and other proteins form a complete cellular support network system whose structure is characterized by radial distribution, connected to the nuclear membrane on the inside, and connected to the cell membrane on the outside, with microtubules, actin and many organelles in the middle. Therefore, vimentin has several intermediate fiber functions, such as cellular morphogenesis and maintenance, cellular displacement and spreading, nuclear localization and others. 21 In addition, the main function of vimentin also includes the effects of cell adhesion and migration and is also involved in signal transduction. 22 Recent studies have shown that vimentin is closely associated with the apoptosis caused by a variety of factors, especially those related to cancer research. Vimentin is cleaved into fragments of different sizes during apoptosis. Some studies 23 have found human leukemia cells (Jurkat cells) with anti-Fas antibody. After Fas stimulation, it was found that specific cleavage of vimentin can be used as an apoptosis marker for caspase activation. In the studies of human epithelial tumor cell apoptosis caused by ultraviolet radiation, some studies 24 have found that vimentin was cleaved into three fragments of different sizes. The cleavage of vimentin is an inevitable result of the process of apoptosis, and cleavage occurs in a cell type-specific manner. 25 Vimentin mediates apoptosis primarily by cleaving caspase. Cleavage of vimentin is an early apoptotic event, with poly ADP-ribose polymerase (poly ADP-ribose polymerase, PARP) cleavage occurring almost simultaneously. After vimentin undergoes structural damage, the N-terminal peptide produced from cleavage can further induce caspase-induced vimentin cleavage, promoting apoptosis signal amplification. 26 In the expression of the apoptosis pathway, PARP is produced by the activation of caspase-3.
The results of the current study showed that among the hepatoma cells, the SMMC-7721 EMT model in the PL group and Vim cleavage group had increased caspase-3 protein levels, while vimentin and PARP-1 protein levels were reduced, showing a dose-dependency (P<0.01). Regarding cancer therapeutics, many studies have been dedicated to developing anticancer drugs that target vimentin. The tumor angiogenesis inhibitor, drunk eggplant hormone A (withaferin-A, WFA), targets vimentin, and WFA combined with vimentin causes covalent modification, inducing tumor cell apoptosis and thus exerting antitumor effects. 27 Silibinin and Clerodendrum bungei have strong anticancer activity and can inhibit prostate cancer invasion and metastasis by decreasing the expression of vimentin and reducing the activity of matrix metalloproteinase (MMP-2), thereby inhibiting prostate cancer invasion and metastasis. 28 The degree of vimentin reduction and proteolytic cleavage were consistent with the trend of the proliferation inhibition rate, invasion inhibition rate and early apoptosis rate of hepatoma cells. The proliferation inhibition rate, invasion inhibition rate and early apoptosis rate of cells in the Vim cleavage group were highest in the experimental group, while the proliferation inhibition rate, invasion inhibition rate and early apoptosis rate of cells in the Caspase inhibition group were lowest in the experimental group. Our experimental findings indicated that PL (especially the 8 µM group) could significantly reduce proliferation and invasion and induce hepatoma cell apoptosis. These effects of PL were related to vimentin reduction and caspase upregulation. Tumor cell epithelial-mesenchymal transition is accompanied by changes in cell surface markers, including downregulation of the epithelial biomarker E-cadherin expression and upregulation of stromal cell marker proteins, such as vimentin and N-cadherin, and by transcriptional regulatory factors through Snail activation. 29 Ecadherin is a member of the calcium-dependent adhesion family. Decreased expression of E-cadherin leads to changes in cell morphology, decreased intercellular adhesion, and increased cellular activity, which occur during EMT, and is an important sign of, and plays an important role in, cancer infiltration. 30 Vimentin is an intermediate filament protein present in mesenchymal cells, which is positive in mesenchymal-derived tumor cells and negative in epithelial-derived tumor cells, so increased vimentin expression suggests tumor epithelial-mesenchymal transition. The expression of E-cadherin and Cytokeratin were seen to decrease. When the expression levels of N-cadherin and vimentin increased, the arrangement of the tumor cytoskeleton system changed, the biological characteristics of the cells changed, the adhesion function decreased, and it was becoming easier for the cells to detach from the primary tumor and invade or metastasize to surrounding tissues. 31 Snail is the first member of the Snail zinc finger protein family. Studies have shown that Snail is essentially a transcriptional repressor that directly inhibits E-cadherin expression by binding to the E-box junction motif of the E-cadherin promoter. In turn, EMT is induced to promote tumor invasion and distant metastasis. 32 The RT-PCR and Western blot results in this study showed that compared with the control group, the mRNA and protein expression levels of vimentin, Ncadherin and snail were induced and upregulated by TGFβ, while E-cadherin expression was downregulated. After PL treatment, compared with the EMT model group, the mRNA and protein expression levels of vimentin, N-cadherin and snail factors were downregulated, and the expression of E-cadherin was upregulated. The high dose of PL was the most effective. This indicates that PL can inhibit EMT. By inhibiting the expression levels of EMTrelated proteins, PL can inhibit the occurrence and metastasis of HCC.
Anti-apoptosis genes (B-cell lymphoma-2, Bcl-2) and pro-apoptosis genes (Bax) have been shown to be important regulators of apoptosis in cancer cells. 33,34 The ratio between the pro-apoptotic protein bax and the anti-apoptotic protein Bcl-2 determines whether the cells survive or die. 35 In a preliminary experimental study, to evaluate the antitumor effect of PL in vivo, the SMMC-7221 tumor xenograft mouse model was used and found that PL inhibits the growth of SMMC-7221 cells in xenograft tumors in vivo and inhibits tumors. PL treatment inhibited tumor growth in a dose-dependent manner according to body weight. The Bcl-2 family proteins serve as importance regulators involved in apoptosis, acting to either inhibit or promote cell death. In this study, PL treatment significantly increased the pro-apoptosis Bax protein expression, whereas it decreased the anti-apoptosis Bcl-2 protein expression in both the membranes and cytoplasm of tumor cells compared to the control group. In addition, PL treatment reduced the nuclear immunofluorescence staining for Bcl-2, while increasing the nuclear immunofluorescence staining for Bax.
Thus, this study clearly indicated that PL increases caspase-3 protein levels and cleaves vimentin via the caspase/vimentin signaling pathway. PL induces the apoptosis of human HCC epithelial-mesenchymal transition cells, In our previous studies, we also found that PL has other mechanisms that inhibit HCC tumor angiogenesis, invasion and metastasis, and induce interaction between autophagy and apoptosis, 12,14 suggesting that PL is a potential anti-therapeutic drugs for HCC, and provide more evidence for the clinical use of PL in the treatment of HCC.