Combination of curcumin and bicalutamide enhanced the growth inhibition of androgen-independent prostate cancer cells through SAPK/JNK and MEK/ERK1/2-mediated targeting NF-κB/p65 and MUC1-C

Jing Li
Songtao Xiang
†
Qiouhong Zhang
Jingjing Wu
Qing Tang
Jianfu Zhou
Lijun Yang
Zhiqiang Chen
Swei Sunny Hann
Combination of curcumin and bicalutamide enhanced the growth inhibition of androgen-independent prostate cancer cells through SAPK/JNK and MEK/ERK1/2-mediated targeting NF-κB/p65 and MUC1-C
10.1186/s13046-015-0168-zR E S E A R C H A R T I C L E Open Access
Background: Prostate cancer is one of the most common malignancies in men. The mucin 1 (MUC1) heterodimeric oncoprotein is overexpressed in human prostate cancers with aggressive pathologic and clinical features, resulting in a poor outcome. However, the functional role for MUC1 C-terminal domain (MUC1-C) in androgen-independent prostate cancer occurrence and development has remained unclear.Methods: Cell viability was measured by MTT assays. Western blot analysis was performed to measure the phosphorylation and protein expression of SAPK/JNK and ERK1/2, and MUC1-C, NF-κB subunit p65 and p50. Exogenous expression of MUC1-C, NF-κB subunit p65 was carried out by transient and electroporated transfection assays.Results: We showed that curcumin inhibited the growth of androgen-independent prostate cancer cells and a synergy was observed in the presence of curcumin and bicalutamide, the androgen receptor antagonist. To further explore the potential mechanism underlining this, we found that curcumin increased the phosphorylation of ERK1/2 and SAPK/JNK, which was enhanced by bicalutamide. In addition, curcumin reduced the protein expression of MUC1-C and NF-κB subunit p65, which were abrogated in the presence of the inhibitors of MEK/ERK1/2 (PD98059) and SAPK/JNK (SP60015). A further reduction was observed in the combination of curcumin with bicalutamide. Moreover, while exogenous expression of MUC1-C had little effect on curcumin-reduced p65, the overexpression of p65 reversed the effect of curcumin on MUC1-C protein expression suggesting that p65 is upstream of MUC1-C. Intriguingly, we showed that exogenous expression of MUC1-C feedback diminished the effect of curcumin on phosphorylation of ERK1/2 and SAPK/JNK, and antagonized the effect of curcumin on cell growth.Conclusion:Our results show that curcumin inhibits the growth of androgen-independent prostate cancer cells through ERK1/2-and SAPK/JNK-mediated inhibition of p65, followed by reducing expression of MUC1-C protein.More importantly, there are synergistic effects of curcumin and bicalutamide. The negative feedback regulatory loop of MUC1-C to ERK1/2 and SAPK/JNK further demonstrates the role of MUC1-C that contributes to the overall responses of curcumin. This study unveils the potential molecular mechanism by which combination of curcumin with bicalutamide enhances the growth inhibition of androgen-independent prostate cancer cells.

Introduction

Prostate cancer is one of the most common cancer types and the second leading cause of cancer related death in men in the world [1,2]. The androgen deprivation therapy (ADT), which suppresses or reduces androgens binding to the androgen receptor (AR), is a well-known treatment strategy for advanced, recurrent and even metastatic prostate cancer; however, the long term therapeutic outcomes of ADT on prostate cancer remain uncertain, and are associated with considerable known adverse effects that affect the quality of life in prostate cancer patients [3]. Also, development of resistance to ADT is a major obstacle for the management of advanced prostate cancer [4]. Therapies with AR antagonists such as bicalutamide (Casodex) and androgen withdrawal initially regress tumors but development of various compensatory mechanisms including AR bypass signaling leading to tumor growth, and eventually develop more aggressive castration resistant prostate cancer (CRPC) [5]. Therefore, search for the novel therapeutic approaches based on various combinations of anticancer drugs and procedures to minimize the resistance in order to enhance the therapeutic efficacy are strongly required. Curcumin, an active natural polyphenol derived from the root of Curcuma longa, has been shown great potential as a novel therapeutic agent due to its pharmacological safety and efficacy in treating a wide variety of cancers [6,7]. These facts, tested and confirmed in many different cancer types [8], have paved the way for research aimed at elucidating the potential beneficial effects of combining curcumin and various anti-cancer drugs in order to establish more efficient and less toxic cancer treatment modalities.

Cell surface-associated mucin 1 (MUC1) (previously known as PUM, MCKD1), a highly glycosylated transmembrane heterodimeric protein and a transmembrane member of the mucin family, is highly expressed in various human malignant tumors including prostate cancers and is correlated with a poor prognosis [9,10]. MUC1 cytoplasmic tail can interact with many signaling pathways, and act as a co-transcription factor to activate various genes involved in tumor progression and metastasis. In many tumor types, expression of MUC1 correlates with aggressive, metastatic phenotype, limited response to therapy and poor survival [10,11]. The MUC-1 C-terminal subunit (MUC1-C) is a single-pass transmembrane protein that interacts with receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and others, at the cell membrane and contributes to activation of other kinase signaling pathways that induce proliferation and tumor growth [12][13][14]. MUC1-C also binds directly to nuclear factor NF-kappaB (NF-κB) p65 and promotes NF-κB-mediated gene transcription [9]. Thus, mucins including MUC1-C are considered important markers for early diagnosis and targeted therapy due to their unique expression pattern during cancer progression. Studies have provided substantial evidence for the involvement of transmembrane MUC1-C in altered cell signaling, tumor growth, and metastasis [15,16].

In this study, we explored the potential mechanism by which combination of curcumin with bicalutamide in the inhibition growth of androgen-independent prostate cancer cells.

Materials and methods

Cell culture and chemicals

The prostate cancer cell lines (PC3, DU145, LNCaP) were obtained from the Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China. All cell lines have been tested and authenticated for absence of Mycoplasma, genotypes, drug response, and morphology in the Laboratory. Cells were grown in F-12K or DMEM (1:1) medium (obtained from GIBCO, Life Technologies, Grand Island, NY, USA) with supplemented 10 % fetal bovine serum. The polyclonal antibody against both MUC1 and MUC1-C (

Western blot analysis

The detailed procedure was reported previously [17,18]. Briefly, protein concentrations were determined by the Bio-Rad protein assay. Equal amounts of protein from whole cell lysates were solubilized in 5 × SDS-sample buffers and separated on 12 % SDS polyacrylamide gels. Membranes were incubated with antibodies against MUC1-C, p65, the phosphor and total ERK1/2, and SAPK/ JNK. The membranes were washed and incubated with a secondary antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling Technology, Inc., Beverly, MA, USA). The membranes were washed again and transferred to freshly made ECL solution (Immobilon Western; Millpore, Billerica, MA, USA), followed by observing the signals under the Molecular Imager ChemiDoc XRS Gel Imagine System (Bio-Rad, Hercules, CA, USA) and documenting the results.

Cell viability assay

Cell viability was measured using the 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay [18]. Briefly, prostate cancer cells were harvested, counted and seeded in a 96-well microtitre plate. The cells were treated with increasing concentrations of curcumin for up to 72 hrs. After incubation, 20 μl MTT solution (5 g/L) was added to each well and prostate cancer cells were incubated at 37°C for additional 4 h. Supernatant was removed, then 200 μL solvent dimethyl sulfoxide was added to each well and oscillated for 10 min. Absorbance at 490 nm was determined through the use of ELISA reader (Perkin Elmer, Victor × 5, Waltham, MA, USA). Cell viability (% of control) was calculated as (absorbance of test sample/absorbance of control) × 100 %.

Transient transfection assay

The cells were seeded in 6-well dishes and grown to 50-60 % confluence. The control or MUC1-C and p65 overexpression vectors (pCMV6-AC-MUC1, pCMV6-AC-p65) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA). Briefly, cells were seeded in 6-well dishes and grown to 50-60 % confluence. For each well, 2 μg of control (pCMV6-AC) and MUC1-C and p65 plasmid DNA constructs were transfected into the cells using Lipofectamine 3000 reagent (Life Technologies, Grand Island, NY, USA) for up to 30 h based on the instruction from the provider, followed by treating with curcumin for an additional 24 or 48 h for other experiments.

Statistical analysis

Data are presented as mean ± SD from three independent experiments with triplicates. Statistical significance was determined with Student's t test (two-tailed) comparison between two groups of data set. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control condition (P < 0.05, see figure legends).

Results

The effect of curcumin and bicalutamide on growth of prostate cancer cells

We first examined the effect of curcumin on prostate cancer cell growth. We showed that curcumin inhibited the growth of multiple prostate cancer cell lines in the timeand dose-dependent manner with significant effect most observed at 40 μM at 48 h (Fig. 1a). Note that low dose showing significant response were also seen in one androgen-dependent prostate cancer cell (LNCaP) compared to other two androgen-independent prostate cancer cell lines (PC3 and DU145). We also demonstrated that bicalutamide, an androgen receptor antagonist, inhibited cell growth in dose-and time-dependent fashion in PC3 and LNCaP cells. Note that the inhibitory effect of bicalutamid was different in LNCaP and PC3 cells; lower dose and shorter time were observed in LNCaP cells (10 μM at 24 h), whereas high doses and longer time were found in PC3 (30 μM, 72 h) (Fig. 1b) and DU145 cells (not shown). Interestingly, while bicalutamide alone at lower doses (e.g., 10-20 μM) had no effect on androgen-independent prostate cancer cell growth inhibition, combination of curcumin with bicalutamide showed a significant inhibition of cell growth in not only androgen-dependent but also androgen-independent prostate cancer cells (Fig. 1c). This suggested a potential new mechanism by which the combination of curcumin and bicalutamide enhanced the growth inhibition in androgen-independent prostate cancer cells.

Curcumin and bicalutamide increased phosphorylation of ERK1/2 and SAPK/JNK

We then explored the potential signaling that may be involved in the inhibitory response by curcumin and bicalutamide in androgen-independent prostate cancer cells. We showed that curcumin increased the phosphorylation of ERK1/2 and SAPK/JNK in a time-dependent fashion with significant induction observed at 4-8 h in PC3 and DU145 cells (Fig. 2a, b). Furthermore, combination of curcumin with bicalutamide further enhanced the phosphorylation of ERK1/2 and SAPK/JNK in PC3 and DU145 cells (Fig. 2c, d).

The effect of curcumin and bicalutamide on protein expression of p65 and MUC1-C through activation of MEK/ERK/12 and SAPK/JNK Next, we further examined the potential molecular mechanism affected by curcumin in the presence or absence of bicalutamide. We showed that curcumin reduced the protein expression of p65, NF-κB subunit, and MUC1-C, a membrane-anchored mucin, in the dose-dependent manner in PC3 and DU145 cells (Fig. 3a). Note that curcumin had no effect on p50 protein expression (Fig. 3a). Interestingly, the inhibitors of MEK/ERK1/2 (PD98059) and SAPK/JNK (SP60015) blocked the effect of curcumin on both p65 and MUC1-C protein expression (Fig. 3b, c). More importantly, we found that combination of curcumin with bicalutamide further significantly reduced the protein expression of p65 and MUC1-C suggesting a synergy in this process (Fig. 3d).

Exogenous expression of p65 abrogated the effect of curcumin on MUC1-C expression and cell growth inhibition

Moreover, we tested the role of p65 in this process; we showed that exogenous expression of p65 blocked the effect of curcumin on MUC1-C expression in PC3 and DU145 cells (Fig. 4a, b); this suggested that p65 is an upstream of MUC1-C. In addition, we found that overexpression of p65 reversed curcumin-inhibited growth in PC3 and DU145 cells (Fig. 4c, d). While overexpression of MUC1-C had no effect on p65, it attenuated the effect of curcumin on cell growth inhibition and phosphorylation of ERK1/2 and SAPK/JNK

To further determine the role of MUC1-C in this process, we transfected the exogenous expression vector of MUC1-C into the cells and found that, while overexpression of MUC1-C had no effect on curcumin-reduced p65 protein expression in PC3 and DU145 cells (Fig. 5a), it significantly antagonized the effect of curcumin on cell growth inhibition (Fig. 5b). This result indicated the critical role of MUC1-C in this process. Interestingly, exogenous expression of MUC1-C significantly attenuated the curcumin-induced phosphorylation of SAPK/JNK and ERK1/2 in PC3 and DU145 cells (Fig. 5c).

Discussion

Majority of advanced prostate cancers are sensitive to androgen deprivation therapy in the first beginning, then subsequently progress to the CRPC. The reasons remain unknown. Because of the limitations of current therapeutic approaches, many patients die of recurrent and metastatic diseases. The combination with anti-inflammatory and other adjuvant therapies present a very promising treatment approach for this malignancy. Curcumin is a promising anticancer agent for various cancer types including prostate cancer cells, and involves in multiple signaling and potential targets [6,7,19]. However, the detailed molecular mechanisms underlining suppression of androgen-independent prostate cancer cell growth still remain to be elucidated. In this study, we demonstrated not only a significant inhibition of growth of prostate cancer cells by curcumin, but more importantly, a synergistic effect observed in combination of curcumin with bicalutamide, an androgen receptor antagonist, in androgen-independent prostate cancer cells. These findings implied a potential new mechanism for this synergy that was AR-unrelated. The molecular mechanisms by which curcumin inhibited growth of androgen-dependent and -independent prostate cancer cells were reported in other studies demonstrating that pathways and transcription factors other than AR-mediated and -regulated downstream genes were involved in this process [20][21][22]. However, more experiments are needed to further determine this.  Fig. 3 The effect of curcumin and bicalutamide on protein expression of p65 and MUC1-C through activation of ERK/12 and SAPK/JNK. a PC3 and DU145 cells ells were exposed to increased concentration of curcumin for 24 h. Afterwards, the expression of p65 and MUC1-C proteins was detected by Western blot. b-c PC3 and DU145 cells were treated with PD98059 (10 μM) and SP600125 (20 μM) for 2 h before exposure of the cells to curcumin (40 μM) for an additional 24 h. Afterwards, the expression of p65 and MUC1-C protein were detected by Western blot using antibodies against p65 and MUC1-C. The bar graphs represent the mean ± SD of p65 or MUC1-C /GAPDH of three independent experiments. d, PC3 and DU145 cells were treated with curcumin (40 μM) and bicalutamide (30 μM) for 24 h. Afterwards, the expression of p65 and MUC1-C protein were detected by Western blot using antibodies against p65 and MUC1-C. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from curcumin treated alone (P < 0.05)

In this study, we demonstrated the role of SAPK/JNK and MEK/ERK1/2 signaling pathways in mediating the effect of curcumin and bicalutamide on controlling growth of androgen-independent prostate cancer cells. The activation of these kinases by curcumin or/and bicalutamide has been shown in other studies implicating in the regulation of other gene expression and subsequent cellular responses [23][24][25][26][27][28]. Consistent with ours, one report showed that activation of SAPK/JNK was involved in the curcumin-triggered intrinsic apoptotic pathway in cardiac myoblasts [23]. Also, activation of MEK/ERK1/2 pathway mediated in curcumin-induced cell cycle arrest and apoptosis in human gastric cancer cells [25]. We noticed that opposite results were also reported in other cell system [29]. It was possible that the different environment contexts, cell lines used, and other unknown factors were account for this discrepancy. Thus, more studies are required to further confirm this. Findings from other studies including gene deletion data indicated the conflicting results in essence that turn MEK/ERK1/2 and SAPK/JNK functions from oncogene to tumor suppressor, which could suggest the possible dual functions of these kinases (pro-oncogene or tumor suppressor) [30][31][32]. Therefore, the insight true role of SAPK/JNK and MEK/ ERK1/2 signaling pathways in triggering cancer cell differentiation, senescence, apoptosis, and survival appeared to be context dependent and more complicated, which needs to be clarified in the future.  Fig. 4 Exogenous expression of p65 abrogated the effect of curcumin on MUC1-C expression and cell growth inhibition. a-b PC3 and DU145 cells were transfected with the control (pCMV6) or expression constructs of p65 for 24 h before exposing the cells to curcumin for an additional 24 h. Afterwards, p65 and MUC1-C protein expression were determined by Western blot. c-d PC3 and DU145 cells were transfected with the control (pCMV6) or expression construct of p65 for 24 h before exposing the cells to curcumin for an additional 48 h. Afterwards, the cell viability was determined using the MTT assay as described in the Materials and Methods section and was expressed as percentage of control in the mean ± SD of three separate experiments. Insert on the upper panel represented the protein levels of p65 as determined using Western blot. β-actin was used as internal control. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from curcumin treated alone (P < 0.01)

To further explore the potential mechanism underlining the aforementioned, we tested the involvement of MUC1-C. Our results indicated the critical role of MUC1-C in mediating the effect of curcumin on inhibition of growth of androgen-independent prostate cancer cells. Consistent with this, other studies were observed the similar findings and suggested that MUC1 including its subunits (e.g., MUC1-C) could be a potential target of curcumin in the treatment of prostate and breast cancer cells [33,34]. Furthermore, we demonstrated an important role of NF-κB/p65 that may involve in the effects of curcumin on MUC1-C expression and prostate cancer cell growth. Curcumin was shown to be a strong inhibitor of NF-κB activity and its inhibitory effect on NF-κB related pathways led to enhance the cytotoxicity of chemotherapeutic agents in prostate cancer cells [35]. Overexpression of MUC1-C attenuated the effect of curcumin on cell growth inhibition and curcumin-induced phosphorylation of ERK1/2 and SAPK/JNK. a PC3 and DU145 cells were transfected with the control (pCMV6) or expression constructs of MUC1-C for 24 h before exposing the cells to curcumin for an additional 24 h. Afterwards, p65 and MUC1-C protein expression were determined by Western blot. b PC3 and DU145 cells were transfected with the control (pCMV6) or expression constructs of MUC1-C for 24 h before exposing the cells to curcumin for an additional 48 h. Afterwards The cell viability was determined using the MTT assay as described in the Materials and Methods section. Insert on the upper panel represented the protein levels of MUC1-C as determined by Western blot. β-actin was used as internal control. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from curcumin treated alone (P < 0.01). c PC3 and DU145 cells were transfected with the control (pCMV6) or expression constructs of MUC1-C for 24 h before exposing the cells to curcumin for an additional 8 h. Afterwards, p-SAPK/JNK and p-ERK1/2 were determined by Western blot. d The diagram the shows that curcumin inhibits the growth of androgen-independent prostate cancer cells through ERK1/2 and SAPK/JNK-mediated inhibition of p65, followed by reducing expression of MUC1-C. There is a synergy of curcumin and bicalutamide. The negative feedback regulatory loop of MUC1-C to ERK1/2 and SAPK/JNK signaling pathways also adds the important role of MUC1-C in mediating the overall responses of curcumin Moreover, reported data have demonstrated the link of MUC1-C and NF-κB signaling in other studies [36,37]. Cancer cells are dependent on intact MUC1-C function for constitutive activation of the canonical NF-κB pathway and cancer cell growth, and survival [36,37]. MUC1-C is a direct activator of NF-κB/p65 and that an inhibitor of MUC1 function is effective in blocking activation of the NF-κB pathway in nonmalignant epithelial cells [36,37]. Our results implied that MUC1-C was downstream of NF-κB. Consistent with this, early study showed that MUC1 promoter region contained NF-κB/ p65 binding site that mediated the proinflammatory cytokines-induced MUC1 promoter activity and gene expression in normal breast epithelia and breast cancer cells [38]. Thus, the function and regulation of NF-κB/ p65-MUC1-C complexes are more complicated than we could think. Nevertheless, we reasoned that regulation of p65 played a crucial role in mediating curcumininhibited MUC1-C expression in androgen-independent prostate cancer cells.

We also observed the involvement of activation of MEK/ ERK1/2 and SAPK/JNK signaling pathways in the regulation of p65 and MUC1-C affected by curcumin. The links of these kinases to the regulation of NF-κB and MUC1-C have been shown in other studies, suggesting that blockade of these kinases could reduce expression of MUC1 in several cell systems [12,[39][40][41]. More interestingly, we also showed the negative feedback regulation of ERK1/2 and SAPK/JNK by MUC1-C. These signaling axes formed a bidirectional feedback loop to mediate curcumin-inhibited prostate cancer proliferation. The feedback regulation circuit of kinases with other genes/proteins was reported in other studies demonstrating relative common autocrine or paracrine physiopathological phenomena [42,43]. One study found that sustained activation of SAPK/JNK inhibited NF-κB signaling via a feedback loop mechanism that led to an alteration in the transcription of the NF-κBinduced apoptotic gene in immortalized renal proximal tubular epithelial cells [44]. More experiments are warranted to better elucidate the in-depth mechanism for these unexplored complicated regulatory circuit.

More importantly, we demonstrated a synergy of combination of curcumin and bicalutamide, the androgen receptor antagonist, in the inhibition of p65, MUC1-C and androgen-independent prostate cancer cell growth, implying that signaling and mechanisms other than through the AR-mediated regulatory pathways and genes contributed to the overall enhanced effects. On the other hand, this also suggested the potential new mechanism of bicalutamide in controlling androgen-independent prostate cancer cell growth. Bicalutamide was found to inhibit androgenindependent prostate cancer cell growth appeared through AR-independent pathways [45,46]. One study showed that combination of bicalutamide with other therapeutic agents enhanced the CRPC growth inhibition via upregulation of insulin-like growth factor-binding protein 3 (IGFBP3) [47]. Also, previous study indicated that MUC1-C subunit suppressed AR expression through a posttranscriptional mechanism and resisted to bicalutamide treatment in androgen-dependent prostate cancer cells, this implied that inhibition of AR expression by MUC1-C led to develop more aggressive androgen-independent phenotype in prostate cancer cells that was sensitive to MUC1-C inhibition [10]. Consequently, this may facilitate the therapeutic effects of bicalutamide. We reasoned that MUC1-C could be a potential target of curcumin in suppression of androgen-independent prostate cancer cell growth, and inhibition of MUC1-C by curcumin sensitized the therapeutic effect of bicalutamide in prostate cancer cell growth. A cell based morphology experiment showed that curcumin analogs or curcumin-anti-androgen conjugates demonstrated more potent than bicalutamide alone in the cytotoxic effects on LNCaP and PC-3 cells through suppression of pseudopodia formation, which was highly related to cell migration and tumor metastasis, other than targeting AR [48]. Also, note that studies implicated the dissected mechanisms by which using different antiandrogen receptor compounds on affecting prostate cancer invasion and metastasis, resulting in opposite effects [49]; the potential risks of using these agents, such as bicalutamide, among others, on prostate cancer metastasis require to be carefully evaluated. Thus, additional studies are warranted to further explore the combination (curcumin and bicalutamide) effects on invasion and metastasis of androgen-independent prostate cancer cells.

Conclusion

This study shows that curcumin inhibits the growth of androgen-independent prostate cancer cells through MEK/ERK1/2 and SAPK/JNK-mediated inhibition of p65, followed by reducing expression of MUC1-C protein. More importantly, there is a synergy of curcumin and bicalutamide. The negative feedback regulatory loop of MUC1-C to ERK1/2 and SAPK/JNK signaling pathways further added the important role of MUC1-C in mediating to the overall responses of curcumin (Fig. 5d). This study unveils the potential molecular mechanism by which combination of curcumin and bicalutamide enhances the growth inhibition of androgen-independent prostate cancer cells.

Fig. 1
1The effect of curcumin and bicalutamide on growth of prostate cancer cells. a Prostate cancer cells (PC3, DU145 and LNCaP) were treated with increased concentrations of curcumin for up to 72 h to examine the cell viability. b Prostate cancer cells (PC3 and LNCaP) were treated with increased concentrations of bicalutamide for up to 72 h to examine the cell viability c PC3, DU145 and LNCaP cells were treated with curcumin (40 μM) and bicalutamide (30 μM) for 48 h to examine the cell viability. The cell viability was determined using the MTT assay as described in the Materials and Methods section and was expressed as percentage of control in the mean ± SD of three separate experiments. *indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from curcumin treated alone (P < 0.05)

Fig. 2
2Curcumin and bicalutamide increased phosphorylation of ERK1/2 and SAPK/JNK. a-b PC3 and DU145 cells were treated with curcumin (40 μM) in the indicated times, and cell lysate was harvested and the expression of the phosphorylated or total protein of ERK1/2 and SAPK/JNK were measured by Western blot analysis using corresponding antibodies. GAPDH was used as loading control. c-d PC3 and DU145 cells were treated with curcumin (40 μM) and bicalutamide (30 μM) for 8 h. Afterwards, the phosphorylation and expression of ERK1/2 and SAPK/JNK were detected by Western blot. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from curcumin treated alone (P < 0.05)

Fig. 5 Overexpression of MUC1-C attenuated the effect of curcumin on cell growth inhibition and curcumin-induced phosphorylation of ERK1/2 and SAPK/JNK. a PC3 and DU145 cells were transfected with the control (pCMV6) or expression constructs of MUC1-C for 24 h before exposing the cells to curcumin for an additional 24 h. Afterwards, p65 and MUC1-C protein expression were determined by Western blot. b PC3 and DU145 cells were transfected with the control (pCMV6) or expression constructs of MUC1-C for 24 h before exposing the cells to curcumin for an additional 48 h. Afterwards The cell viability was determined using the MTT assay as described in the Materials and Methods section. Insert on the upper panel represented the protein levels of MUC1-C as determined by Western blot. β-actin was used as internal control. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from curcumin treated alone (P < 0.01). c PC3 and DU145 cells were transfected with the control (pCMV6) or expression constructs of MUC1-C for 24 h before exposing the cells to curcumin for an additional 8 h. Afterwards, p-SAPK/JNK and p-ERK1/2 were determined by Western blot. d The diagram the shows that curcumin inhibits the growth of androgen-independent prostate cancer cells through ERK1/2 and SAPK/JNK-mediated inhibition of p65, followed by reducing expression of MUC1-C. There is a synergy of curcumin and bicalutamide. The negative feedback regulatory loop of MUC1-C to ERK1/2 and SAPK/JNK signaling pathways also adds the important role of MUC1-C in mediating the overall responses of curcumin

Cat No. ab109185) was obtained from Abcam (Cambridge, MA, USA). The antibodies against p65 (Cat No. 8242), total extracellular signal-regulated kinase1/2 (ERK1/2) (Cat No. 4695), stress-activated protein kinase/c-Jun Nterminal kinase (SAPK/JNK) (Cat No. 9258) and the phosphor-forms (Cat No. 4370 and 4668) were purchased from Cell Signaling Technology Inc (Beverly, MA, USA). Curcumin, PD98059 [(MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK)/ERK inhibitor] and SP600125 (SAPK/JNK inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Competing interestsThe authors declare that they have no competing interests.Authors' contributions SSH is fully responsible for the study designing, experiment adjustment, drafting and finalizing the manuscript. JL, STX performed most of the experiments involved. QHZ, LJY carried out transfection assays and some protein measurement by Western blot and statistical analysis. JJW, QT, JFZ conducted the densitometry, statistical analysis and participated in coordination manuscript. ZQC coordinated and provided important suggestions including some reagents, and critical read the manuscript. All authors read and approved the final manuscript.Submit your next manuscript to BioMed
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Efficacy and safety of edoxaban versus enoxaparin for the prevention of venous thromboembolism following total hip arthroplasty: STARS J-V

Takeshi Fuji
Satoru Fujita
Yohko Kawai
Mashio Nakamura
Tetsuya Kimura
Masayuki Fukuzawa
Kenji Abe
Shintaro Tachibana
Efficacy and safety of edoxaban versus enoxaparin for the prevention of venous thromboembolism following total hip arthroplasty: STARS J-V
10.1186/s12959-015-0057-xO R I G I N A L C L I N I C A L I N V E S T I G A T I O N
Background: In the absence of thromboprophylaxis, patients undergoing total hip arthroplasty (THA) are at increased risk for venous thromboembolism (VTE). The objective of this study was to compare the efficacy and safety of edoxaban with enoxaparin for the prevention of VTE after THA in Japan.Methods: This was a phase 3, double-blind, double-dummy, noninferiority study. Patients undergoing elective, unilateral primary THA were randomized to receive edoxaban 30 mg once daily (n = 307) or enoxaparin 2000 IU (equivalent to 20 mg) twice daily (n = 303) for 11 to 14 days. The primary efficacy endpoint was the incidence of VTE. Safety endpoints included the incidence of major or clinically relevant nonmajor (CRNM) bleeding. Results: The incidence of VTE, based on venography and clinical surveillance, was 2.4 % in the edoxaban group and 6.9 % in the enoxaparin group (P <0.001). The absolute difference in the incidence of VTE was −4.5 % (95 % confidence interval [CI]: −8.6, −0.9), which was within the noninferiority margin set at 8 % for the difference and established the noninferiority of edoxaban to enoxaparin. Since the upper limit of the 95 % CI of the absolute difference was less than 0 %, the superiority of edoxaban over enoxaparin was demonstrated. The incidence of major or CRNM bleeding was 2.6 % in the edoxaban group and 3.7 % in the enoxaparin group (P = 0.475). Conclusions: Oral edoxaban 30 mg once daily was superior to subcutaneous enoxaparin 2000 IU twice daily in the prevention of VTE following THA without increasing the risk for major or CRNM bleeding.

Background

Venous thromboembolism (VTE), comprising deep vein thrombosis (DVT) and pulmonary embolism (PE), is a leading cause of morbidity and mortality [1]. In the absence of thromboprophylaxis, 40 % to 60 % of patients undergoing total hip arthroplasty (THA) or total knee arthroplasty (TKA) will develop venographic evidence of DVT, and approximately 1 in 300 THA patients will experience a fatal PE [2]. The morbidity and mortality associated with VTE strongly support primary thromboprophylaxis in THA patients [3,4].

In Japan, the standard of care for thromboprophylaxis following THA is the low-molecular weight heparin (LMWH) enoxaparin 2000 IU twice daily, initiated 24 to 36 h after surgery and continued for 14 days [4][5][6]. Fondaparinux, a selective factor Xa inhibitor administered by subcutaneous injection, is another potent anticoagulant approved for the prevention of VTE in Japanese patients undergoing THA or TKA [7].

Edoxaban, a direct, once-daily factor Xa inhibitor [8], is a new oral anticoagulant that has been studied for the prevention of VTE [9,10]. In phase 2 studies of patients undergoing elective THA [10] or TKA [9], edoxaban demonstrated significant dose-related reductions in VTE compared with dalteparin and placebo, respectively. Another phase 2 study in patients following THA demonstrated no significant difference in the incidence of VTE for patients who received edoxaban compared with enoxaparin [11]. There was a low incidence of major or clinically relevant nonmajor (CRNM) bleeding observed with edoxaban in these studies. Results from the phase 2 studies in patients following THA led to the initiation of the phase 3 Studying Thrombosis After Replacement Surgery J-V (STARS J-V) trial. The objective of the STARS J-V trial was to compare the efficacy and safety of edoxaban 30 mg once daily with enoxaparin 2000 IU (equivalent to 20 mg) twice daily in Japanese patients for the prevention of VTE following elective, unilateral, primary THA.

Materials and methods

Study design

This was a randomized, double-blind, double-dummy, parallel-group, enoxaparin-controlled, multicenter phase 3 trial (ClinicalTrials.gov Identifier: NCT01181167) of edoxaban in patients undergoing unilateral THA (excluding revision THA). The study protocol, information for patients, and informed consent forms were reviewed and approved by an institutional review board, and the study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines.

Patients were randomized 1:1 to receive oral edoxaban 30 mg once daily or subcutaneous enoxaparin 2000 IU twice daily for 11 to 14 days. Treatment with edoxaban (or edoxaban placebo) was administered within 6 to 24 h after surgery and once daily each morning thereafter; enoxaparin injection (or enoxaparin placebo injection) was administered 24 to 36 h after surgery and twice daily from the following day onward (Fig. 1). The enoxaparin dosing regimen was set according to the approved regimen for enoxaparin sodium in Japan. Patients were permitted to use mechanical physiotherapy, which included elastic stockings or intermittent pneumatic compression of the sole of the foot or of the lower legs and thigh. Venography of the lower limbs was performed within 24 h after either the end of treatment or after the last dose of study drug when treatment was discontinued, or within 96 h for reasons such as difficulty in establishing an intravenous line. Investigations, observations, examinations, and urine and blood sample collection (for urinalysis, edoxaban plasma concentration measurements, assessment of pharmacodynamic [PD] indices, and hematology tests) were performed at presurgical evaluation, pretreatment (postsurgery), on day seven, and on the completion day of treatment. Follow-up examination was performed at 25 to 35 days after the last dose of study drug. The occurrence of thromboembolic events, bleeding, and all other adverse events (AEs) were recorded throughout the study. If applicable, site, duration, and total time of physiotherapy use were recorded.

Selection of study population

Patients 20 to 85 years of age undergoing unilateral THA were included. Presurgical exclusion criteria included risk for bleeding (eg, history of intracranial bleeding, comorbid gastrointestinal bleeding or peptic ulcer within the previous 3 months, or prothrombin time [PT] prolongation above the upper limit of the normal [ULN]); risk for thromboembolism (eg, history of symptomatic DVT or PE, coagulation disease at high risk for thromboembolism, or history of lower limb fracture within the previous 6 months); previous TKA; expected joint replacement of the other lower limb at the same time of anesthesia; weight <40 kg; severe renal impairment (creatinine clearance [CL CR ] of <30 mL/min) [12]; evidence of hepatic dysfunction (serum aspartate aminotransferase [AST] or serum alanine aminotransferase [ALT] levels ≥2 times the ULN or total bilirubin ≥1.5 times the ULN); previous treatment with edoxaban; and current antithrombotic therapy for another complication. Postsurgical exclusion criteria included abnormal bleeding from the puncture site during spinal anesthesia, need for repeat surgery before the start of study treatment, abnormal or excessive bleeding experienced during surgery, and inability to take oral medication. All patients provided written informed consent prior to initiation of any study-related procedures. Patients discontinued treatment if they developed symptomatic DVT, PE, or major bleeding; had evidence of hepatic dysfunction; had persistent systolic blood pressure of >180 mm Hg or diastolic blood pressure of >110 mm Hg; required anticoagulant drugs, antiplatelet drugs, thrombolytic drugs, or drugs that might affect thrombus formation; needed postsurgical epidural anesthesia; experienced an AE that the investigator or subinvestigator judged was sufficient for study discontinuation; voluntarily requested to withdraw; were found to be ineligible for continued participation in the study for any reason; or if the investigator judged that the study should be discontinued for the patient.

Study endpoints

The primary efficacy endpoint was the incidence of VTE from the start of treatment to venography at the end of treatment, including asymptomatic DVT and symptomatic PE or DVT. Secondary efficacy endpoints were the incidence of symptomatic DVT, proximal DVT, symptomatic PE, or VTE-related death; the incidence of asymptomatic or symptomatic DVT; the incidence of symptomatic or proximal DVT; the incidence of symptomatic PE; the incidence of VTE-related deaths; and the incidence of allcause deaths.

Safety endpoints included the incidences of major bleeding; CRNM bleeding; major or CRNM bleeding; any bleeding (major, CRNM, and minor); and minor bleeding events from the start of treatment to the day after the end of treatment. Major bleeding was defined as fatal bleeding; clinically overt bleeding accompanied by a decrease in hemoglobin of >2 g/dL; clinically overt bleeding requiring hemotransfusion with more than four units of blood (1 unit = approximately 200 mL); retroperitoneal, intracranial, intraocular, or intrathecal bleeding; or bleeding requiring repeat surgery. CRNM bleeding was bleeding that was not characterized as major, but corresponded to any of the following: hematoma ≥5 cm in long diameter, epistaxis or gingival bleeding in the absence of external factors and lasting ≥5 min, gastrointestinal bleeding, gross hematuria persistent after 24 h of onset, or any other bleeding deemed clinically significant by the investigator. Minor bleeding was any bleeding event (including a positive occult blood reaction) that was not considered a major or CRNM event. Additional safety endpoints were the incidences of AEs and adverse drug reactions (ADRs).

Thromboembolic events were assessed by the Thromboembolic Event Assessment Committee, which made the final assessment of thromboembolic events based on blinded copies of the test results (radiographic films, etc.) collected from each study site. Bleeding events were assessed by the Bleeding Event Assessment Committee, which was responsible for the final assessment of bleeding events. Results of the assessments by these committees were defined as the final evaluation.

Edoxaban plasma concentrations were measured by liquid chromatography-tandem mass spectrometry; the lower limit of quantification was 0.764 ng/mL.

Statistical analysis

The primary analyses for the efficacy endpoints were performed for the full analysis set (FAS) and per-protocol set (PPS). The FAS was defined as all enrolled patients who received ≥1 dose of study drug. Patients who did not develop symptomatic DVT or PE, and in whom venography was not appropriately performed, were excluded from the FAS. The PPS included patients in the FAS but excluded those with violations to the inclusion/exclusion criteria, those who received prohibited concomitant medications or therapies, or whose study treatment compliance rate was <80 %. Safety analyses were performed for the safety analysis set (SAS), defined as all patients who received ≥1 dose of study drug. The pharmacokinetic (PK) analysis set included patients in the PPS with valid plasma drug concentration measured at ≥1 time points.

For all comparisons of the primary, secondary, and safety endpoints, a paired comparison was performed between treatment groups using the χ 2 test, with a two-sided significance level of 0.05 and a two-sided confidence coefficient of 0.95. In the primary efficacy analysis, the incidence of the primary endpoint and 95 % confidence intervals (CIs) were calculated for each treatment group. The difference in the incidence of the primary endpoint between edoxaban and enoxaparin and its corresponding 95 % CI were then calculated using the Farrington-Manning method to test for noninferiority [13]. The noninferiority margin was set at 8 % for the difference in the primary endpoint between the two groups, with a 1-sided significance level of 0.025. This noninferiority margin was chosen as it was less than half of the reduction in VTE incidence, estimated to be at least approximately 18 % based on the incidence of VTE in Japanese patients undergoing THA (27.3 %; 95 % CI 22.2, 32.9) given in the "Japanese Guideline for the Prevention of Venous Thromboembolism" [14] and the incidence of VTE with twice-daily enoxaparin 20 mg used in the phase 2b study of patients undergoing THA (4.1 %) [15]. If noninferiority was established, edoxaban would be considered superior to enoxaparin if the upper limit of the 95 % CI calculated for the difference in the primary endpoint between the two groups was below 0 %. Similar analyses were performed for the secondary efficacy endpoints.

The incidence of major bleeding events and corresponding 95 % CIs during the treatment period were calculated for each treatment group. The difference in the incidences of major bleeding between the edoxaban and enoxaparin groups and its 95 % CI were also calculated. Similar analyses were performed for CRNM bleeding, major or CRNM bleeding, minor bleeding, and any bleeding events.

The overall incidences of AEs and ADRs and their 95 % CIs were calculated by treatment group. Hepatic dysfunction was classified by the extent of the increase of liver function parameters above the ULN, and the frequency of abnormal changes was summarized.

The number of patients necessary to verify noninferiority was calculated to be 235 patients per treatment group, providing ≥90 % power assuming a VTE incidence of 6 %. Assuming that the proportion of patients excluded from analyses would be approximately 20 %, the planned number of patients was set at 600 (300/group).

Results

Patient disposition

Between May 2009 and January 2010, 610 patients were randomized: 307 patients to edoxaban and 303 to enoxaparin (Fig. 2). Of the randomized patients, 604 received ≥1 dose of medication and were included in the SAS. Venography was unassessable in 101 patients (48 and 53 in the edoxaban and enoxaparin groups, respectively), resulting in 503 patients included in the FAS. During the study period, 23 patients in the edoxaban group and 32 patients in the enoxaparin group discontinued treatment. In both groups, the most common reasons for discontinuation were AEs and voluntary patient withdrawal. In the edoxaban group, nine patients withdrew due to AEs, six patients voluntarily withdrew, three patients were found to be ineligible, and the investigator judged withdrawal was appropriate for two patients. In the enoxaparin group, ten patients withdrew due to AEs, eight patients voluntarily withdrew, and the investigator judged withdrawal was appropriate for three patients. There were no clinically relevant differences in baseline characteristics between the treatment groups (Table 1). Mean age was 62.8 years and mean body weight was 57.4 kg. Overall, most patients were female (86 %), <75 years of age (87 %), weighed <60 kg (63 %), and had a CL CR of ≥80 mL/min (59 %). Mean treatment duration for both groups was 12.5 days. Physiotherapy was used in at least 80 % of patients and was balanced between groups.

Outcome measures

The incidence of VTE is shown in Table 2. All thromboembolic events were asymptomatic DVT, as neither symptomatic DVT nor PE developed in either treatment group. In the FAS, the primary efficacy outcome occurred in 2.4 % (6/255) of patients receiving edoxaban and in 6.9 % (17/ 248) receiving enoxaparin, resulting in a relative risk reduction of 65.7 % (P <0.001). The absolute difference for the primary efficacy endpoint was −4.5 % (95 % CI: −8.6, −0.9) and was within the noninferiority margin, thereby establishing that edoxaban was noninferior to enoxaparin for the The incidence of symptomatic DVT, proximal DVT, symptomatic PE, or VTE-related death was 0.4 % (1/255) in the edoxaban group and 0.8 % (2/248) in the enoxaparin group ( Table 2). All events were asymptomatic, proximal DVT. The absolute difference in the incidence was −0.4 % (95 % CI: −2.5, 1.5), with no clear differences between the two groups ( Table 2). No VTE-related deaths occurred during the study. Primary and secondary efficacy outcomes for the PPS population paralleled those of the FAS.

Safety

The incidence of major bleeding was 0.7 % and 2.0 % in the edoxaban and enoxaparin groups, respectively, for an absolute difference of −1.3 % (P = 0.176) ( Table 3). Clinically relevant nonmajor bleeding was reported by 2.0 % and 1.7 % of patients in the edoxaban and enoxaparin groups, respectively (P = 0.769). There was no significant difference in the incidence of major or CRNM bleeding during the treatment period in the edoxaban group (2.6 %) compared with the enoxaparin group (3.7 %; P = 0.475). Breakdowns of major and CRNM bleeding events are provided in Table 3. The incidence of any bleeding events (major, CRNM, and minor bleeding) was 20.5 % vs 15.9 % in the edoxaban and enoxaparin groups, respectively (P = 0.151). The incidence of minor bleeding events was significantly greater in the edoxaban group compared with the enoxaparin group (P = 0.049). The presence of blood in the urine was characterized as minor bleeding, and occurred in 38 patients in the edoxaban group (12.5 %) and 34 patients in the enoxaparin group (11.3 %). No cases of fatal bleeding occurred in either group.

The overall incidence of AEs from the start of treatment to the day of the follow-up examination was 65.0 % (197/ 303) in the edoxaban group and 77.1 % (232/301) in the enoxaparin group (Table 4). The most common AEs that occurred in ≥5 % of either treatment groups were increased γ-glutamyltransferase, blood present in the urine, increased ALT, increased AST, increased blood alkaline phosphatase, and subcutaneous hemorrhage. Elevated ALT or AST levels ≥3 times the ULN was higher in the enoxaparin group (10.0 %) than in the edoxaban group (2.6 %; Table 4). The pattern of the incidence of ADRs was similar to that of AEs. All AEs and ADRs were mild or moderate in intensity. The incidences of AEs and ADRs excluding bleeding events and hepatic dysfunction were 41.6 % and 11.2 %, respectively, in the edoxaban group, Data presented as n (%) unless otherwise stated. BID, twice daily; BMI, body mass index; CL CR , creatinine clearance; IU, international unit; NSAIDs, non-steroidal anti-inflammatory drugs; QD, once daily; SD, standard deviation a Patients could be using more than one mode of physiotherapy and 44.9 % and 14.0 %, respectively, in the enoxaparin group. There was no difference in the incidence of serious AEs (SAEs) between the edoxaban (3.0 %) and enoxaparin (3.0 %) treatment groups. Nine SAEs occurred in the edoxaban group; for all of these, a causal relationship was ruled out by the investigator. In the enoxaparin group, 10 SAEs occurred; for nine of them, a causal relationship was ruled out, excluding one case of postoperative wound infection.

Plasma concentrations of edoxaban

The PK analysis set included a cohort of 250 patients. However, laboratory anomalies detected after remediation of the bioanalytic results identified 36 samples as inevaluable, leaving 214 patients in the updated PK analysis set. The mean (standard deviation) edoxaban plasma concentration in these 214 patients at predose on day seven of treatment was 17.8 (12.1) ng/mL. The plasma concentration was similar to the results obtained in the original PK analysis set of 250 patients before remediation.

Discussion

The STARS J-V trial demonstrated the noninferiority and superiority of once-daily oral edoxaban 30 mg to twicedaily subcutaneous enoxaparin 2000 IU for the prevention of VTE in Japanese patients undergoing elective unilateral THA based on venography. Although no symptomatic PE occurred, this was not unexpected, as the rate of symptomatic PE following THA or TKA has a very low event rate of 0.55 % in Japan [16]. The incidence of VTE for patients receiving edoxaban 30 mg has been previously explored in phase 2b studies of patients undergoing TKA or THA [9,11]. In a study evaluating edoxaban vs enoxaparin in patients undergoing THA in Japan and Taiwan [11], the incidence of thromboembolic events in the edoxaban 30-mg group (2.8 %) was consistent with the current study (2.4 %). In a placebo-controlled study in Japanese patients undergoing TKA [9], VTE occurred in 12.5 % of patients in the edoxaban 30-mg once-daily treatment group, which is higher than the incidence of VTE reported in the edoxaban group in this study. The higher rate of VTE may be explained by an older population in the phase 2b TKA study compared with STARS J-V (mean age 71.4 vs 62.8 years). A recent analysis by Rohatagi, et al. [17] explored the exposure vs response relationship between edoxaban and VTE rates using pharmacometric analyses. In addition to a direct relationship between edoxaban exposure and the incidence of VTE, they showed that increasing age is a risk factor for VTE [17]. In this study, the incidences of any bleeding and major or CRNM bleeding events were similar for edoxaban and enoxaparin. In addition, the rates of major or CRNM bleeding events in patients receiving edoxaban 30 mg were low in our study (2.6 %) and similar to the phase 2b studies in TKA and THA (3.9 % and 1.2 %, respectively) [9,11]. The incidence of any bleeding was higher in our study (20.5 %) compared with that observed in the edoxaban 30-mg treatment group in the phase 2 TKA trial (10.7 %) [9]. Although the rates of major bleeding and CRNM bleeding were low and similar between these studies, the incidence of minor bleeding was high for edoxaban in our study (18.8 %). Reasons for the higher rate of minor bleeding in the edoxaban group are not clear. Higher rates of bleeding may be attributed, at least in part, to the very high rate of NSAID use-98 % of patients in the edoxaban group concomitantly used NSAIDs compared to 81 % in the enoxaparin group. In a retrospective analysis of pooled data from four phase 3 studies evaluating the safety and efficacy of rivaroxaban compared with enoxaparin for the prevention of VTE after THA or TKA, the risk of bleeding increased with concomitant use of NSAIDs in either treatment group [18]. Higher rates of major bleeding following administration of low molecular weight heparin have been reported at 2-4 h postoperatively compared with 12-48 h postoperatively, suggesting that higher levels of anticoagulation earlier may be associated with more bleeding events [19]. As edoxaban was initiated with an earlier start than enoxaparin, this may contribute to the higher observed rates of minor bleeding. Lastly, no fatal bleeding or bleeding of critical sites (eg, intracranial bleeding) occurred in this study.

The incidence of all AEs in this study was lower in the edoxaban group than in the enoxaparin group. In addition, elevations in ALT or AST were seen in fewer patients in the edoxaban group vs the enoxaparin group. However, it should be noted that elevated serum transaminase levels are recognized as a class effect of heparins. These elevations are transient and reversible and considered benign [20]. Overall bleeding events were more common with edoxaban. The AEs other than bleeding events and AEs related to abnormal hepatic function test values were similar between both Data are presented as n (%) unless otherwise indicated. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BID, twice daily; CI, confidence interval; IU, international unit; QD, once daily; ULN, upper limit of normal a Classified by MedDRA/J V.12.0 Preferred Term treatment groups. In addition, the incidence of SAEs was similar in both the edoxaban and enoxaparin groups. Other oral direct factor inhibitors have been compared with subcutaneous enoxaparin 40 mg for thromboprophylaxis following elective hip replacement and have also been shown to effectively reduce the risk for VTE postsurgery without increasing the risk for clinically relevant bleeding [21][22][23]. However, in these studies, only 0 %-6.8 % of randomized patients were Asian. It should be noted that in the current study, the Japanese patient population was smaller than the population in the other THA studies, which had enrolled patients from Western countries. Additionally, the dose of enoxaparin used (2000 IU, twice daily) is a recommendation specific to Japan for the prevention of VTE [4]. Japanese patients typically have a lower body weight than patients from the United States or the European Union; notably the mean body weight in this study was 57 kg compared with a mean body weight of 78 kg for patients in the phase 3 trial for rivaroxaban in THA, which included primarily white patients (92 %) [21]. A body weight for males >57 kg is associated with increased enoxaparin exposure and increased bleeding risk for prophylactic, subcutaneous enoxaparin doses of 40 mg once daily or 30 mg twice daily [24]. Thus, these results are primarily relevant to Japanese patients.

In conclusion, the STARS J-V study demonstrated the superiority of oral once-daily edoxaban 30 mg compared with twice-daily subcutaneous injection of enoxaparin 2000 IU for the prevention of VTE in Japanese patients after THA, without an increased risk of bleeding or AEs. The favorable efficacy-to-safety balance of edoxaban suggests an attractive option for thromboprophylaxis in patients following THA.

Fig. 1
1Study design. BID, twice a day; IU, international unit; QD, once daily

Fig. 2
2Patient disposition. IU, international unit prevention of VTE. As the upper limit of the 95 % CI of the absolute difference was less than 0 %, the superiority of edoxaban over enoxaparin was demonstrated.Among patients who utilized physiotherapy, there was a significant reduction in the incidence of VTE with use of elastic stockings in the edoxaban group compared with

twice daily; CI, confidence interval; DVT, deep vein thrombosis; IU, international unit; PE, pulmonary embolism; QD, once daily; VTE, venous thromboembolism a p <0.001 as calculated by the noninferiority Farrington-Manning test; b p = 0.0157 as calculated by the superiority Farrington-Manning test; c one patient had both a proximal and a distal DVT

Table 1
1Baseline characteristicsCharacteristic
Edoxaban 30 mg
QD (n = 255)

Enoxaparin 2000 IU
BID (n = 248)

Sex

Female
220 (86.3)
212 (85.5)

Age (y)

Mean ± SD
62.8 ± 9.61
62.8 ± 9.72

<75
220 (86.3)
217 (87.5)

≥75
35 (13.7)
31 (12.5)

Weight (kg)

Mean ± SD
57.7 ± 9.72
57.0 ± 9.60

<60
160 (62.7)
157 (63.3)

≥60
95 (37.3)
91 (36.7)

BMI, mean ± SD
24.5 ± 3.52
24.2 ± 3.60

CL CR (mL/min)

Mean ± SD
89.6 ± 29.58
88.9 ± 26.49

≥80
148 (58.0)
150 (6)

≥50 to <80
93 (36.5)
91 (36.7)

<50
14 (5.5)
7 (2.8)

History of artificial joint
replacement of the
lower limbs

39 (15.3)
35 (14.1)

Duration from the end
of surgery to the start
of study treatment (h)

≥12 to <18
16 (6.3)
-

≥18 to ≤24
239 (93.7)
-

≥24 to <30
-
170 (68.5)

≥30 to <36
-
78 (31.5)

Hour:minute, Mean ± SD
21:40 (1:54)
28:37 (3:00)

Physiotherapy a

Intermittent pneumatic
compression therapy
(foot sole)

137 (53.7)
135 (54.4)

Intermittent pneumatic
compression therapy
(lower legs and thigh)

120 (47.1)
114 (46.0)

Elastic stockings
209 (82.0)
201 (81.0)

Concomitant NSAIDs
250 (98.0)
243 (98.0)

Treatment duration (days)

Mean ± SD
12.5 (1.4)
12.5 (1.5)

Table 2
2Incidence of thromboembolic eventsOutcome
Edoxaban 30 mg QD (n = 255)
Enoxaparin 2000 IU BID (n = 248)
Absolute difference %
(95 % CI)
n (%)
95 % CI
n (%)
95 % CI

Primary efficacy (any VTE)
6 (2.4)
1.1, 5.0
17 (6.9)
4.3, 10.7

Table 3
3Incidence of bleeding events during the treatment periodEvent
Edoxaban 30 mg QD (n = 303)
Enoxaparin 2000 IU BID (n = 301)
Absolute difference %
(95 % CI)

P

n (%)
95 % CI
n (%)
95 % CI

Major bleeding
2 (0.7)
0.2, 2.4
6 (2.0)
0.9, 4.3
−1.3 (−3.7, 0.7)
0.176

Subcutaneous hemorrhage
1 (0.3)
NC
2 (0.7)
NC
NC
NC

Wound hemorrhage
1 (0.

Table 4
4Adverse events, adverse drug reactions, and abnormal changes in hepatic functionEdoxaban 30 mg
QD (n = 303)

Enoxaparin 2000 IU
BID (n = 301)

Adverse events

Patients with events
197 (65.0)
232 (77.1)

95 % CI
59.5, 70.2
72.0, 81.5

Number of events
448
676

Adverse events reported
by ≥5 % of patients in
either treatment group a

ALT increased
36 (11.9)
126 (41.9)

AST increased
17 (5.6)
97 (32.2)

γ-glutamyltransferase
increased

44 (14.5)
79 (26.2)

Blood urine present
38 (12.5)
34 (11.3)

Blood alkaline phosphatase
increased

14 (4.6)
40 (13.3)

Hemorrhage subcutaneous
12 (4.0)
21 (7.0)

Adverse drug reactions

Patients with events
121 (39.9)
177 (58.8)

95 % CI
34.6, 45.5
53.2, 64.2

Number of events
198
439

Hepatic function test
parameter

ALT or AST

≥1.5 x ULN
43 (14.2)
135 (44.9)

≥3 x ULN
8 (2.6)
30 (10.0)

≥5 x ULN
2 (0.7)
11 (3.7)

AcknowledgementsThis study (ClinicalTrials.gov Identifier: NCT01181167) was sponsored by Daiichi Sankyo Co., Ltd (Tokyo, Japan). The study sponsors were involved in the design of the study and the collection and analysis of the data. Medical writing and editorial support was provided by Courtney Zeni, PhD, and Meryl Gersh, PhD, of AlphaBioCom, LLC (King of Prussia, PA). Additional writing support was provided by Kei Ibusuki, MS, of Daiichi Sankyo Co., Ltd.AppendixAuthors' contributions TF, SF, YK, MN, TK, and ST were involved in the concept and design of the study, interpretation of the data, critical revising of the manuscript, and provided final approval to submit the manuscript for publication. MF was involved in the concept and design of the study, interpretation of the data, critical review of the manuscript, and provided final approval to submit the manuscript for publication. KA was involved in analysis of the data, critical review of the mansucript, and provided final approval to submit the manuscript for publication.
The event adjudication committee and investigators are as follows: Data Monitoring Committee. − S Sponsor's Medical Experts, M Tachibana, Y Nakamura, T Koretsune, M Yamashita ; − H. Origasa, S Murata, Yanagimoto, Sponsor's Medical Experts − S. Tachibana, M. Nakamura, Y. Koretsune, T. Yamashita. The event adjudication com- mittee and investigators are as follows: Data Monitoring Committee − H. Origasa, M. Murata, S. Yanagimoto;

Thromboembolic Event Assessment Committee − H. Thromboembolic Event Assessment Committee − H.

. K Nakamura, S Nakamura, S Fujita, − S Nakata; Bleeding Event Assessment Committee, Y Fujita, − M Kawai; Study Investigators, K Tsuji, T Kitamura, J Masuda, T Ito, K Sano, K Sato, T Kawamae, M Kikuchi, T Saito, T Kodama, T Matsuda, S Sasaki, K Hasegawa, N Maeda, T Mitsugi, K Mori, A Uchiyama, S Hyodo, T Kondo, K Matsumoto, T Suzuki, S Tani, O Iwasada, S Obayashi, T Kitamura, T Sato, K Yamakawa, H Ogawa, J Iwaki, S Tamura, H Osawa, K Ohashi, M Fujiwara, K Kakiuchi, H Nakata, S Kagiyama, S Kushitani, H Saito, H Tokunaga, N Iida, T Shibanuma, T Nakai, N Matsubara, H Nakagawa, N Kishimoto, Y Ikeda, T Nanba, M Sato, J Tsubouchi, T Nakashiro, Y Tani, M Ito, Y Naito, F Esaki, Y Higuchi, K Doiguchi, M Yonemori, Nagayama, Nakamura, K. Nakamura, S. Fujita, S. Nakata; Bleeding Event Assessment Committee − S. Fujita, Y. Kawai; Study Investigators − M. Tsuji, K. Kitamura, T. Masuda, J. Ito, T. Sano, K. Sato, K. Kawamae, T. Kikuchi, M. Saito, T. Kodama, T. Matsuda, T. Sasaki, S. Hasegawa, K. Maeda, N. Mitsugi, T. Mori, K. Uchiyama, A. Hyodo, S. Kondo, T. Matsumoto, K. Suzuki, T. Tani, S. Iwasada, O. Obayashi, S. Kitamura, T. Sato, T. Yamakawa, K. Ogawa, H. Iwaki, J. Tamura, S. Osawa, H. Ohashi, K. Fujiwara, M. Kakiuchi, K. Nakata, H. Kagiyama, S. Kushitani, S. Saito, H. Tokunaga, H. Iida, N. Shibanuma, T. Nakai, T. Matsubara, N. Nakagawa, H. Kishimoto, N. Ikeda, Y. Nanba, T. Sato, M. Tsubouchi, J. Nakashiro, T. Tani, Y. Ito, M. Naito, Y. Esaki, F. Higuchi, Y. Doiguchi, K. Yonemori, M. Nagayama;

Sponsor Representatives − T. Kimura (Project Leader), M. Fukuzawa (Study Leader), K. Abe (Statistician). Sponsor Representatives − T. Kimura (Project Leader), M. Fukuzawa (Study Leader), K. Abe (Statistician).

Adverse drug reaction; AE: Adverse event; ALT: Alanine aminotransferase; aPTT: Activated partial thromboplastin time; AST: Aspartate aminotransferase; CI: Confidence interval; CRNM: Clinically relevant nonmajor. Adr Abbreviations, Abbreviations ADR: Adverse drug reaction; AE: Adverse event; ALT: Alanine aminotransferase; aPTT: Activated partial thromboplastin time; AST: Aspartate aminotransferase; CI: Confidence interval; CRNM: Clinically relevant nonmajor;

DVT: Deep vein thrombosis; FAS: Full analysis set; INR: International normalized ratio; LMWH: Low-molecular weight heparin. DVT: Deep vein thrombosis; FAS: Full analysis set; INR: International normalized ratio; LMWH: Low-molecular weight heparin;

PD: Pharmacodynamics; PE: Pulmonary embolism; PK: Pharmacokinetics. PD: Pharmacodynamics; PE: Pulmonary embolism; PK: Pharmacokinetics;

PPS: Per-protocol set; PT: Prothrombin time; SAE: Serious adverse event. PPS: Per-protocol set; PT: Prothrombin time; SAE: Serious adverse event;

Safety analysis set; STARS J-V: Studying Thrombosis After Replacement Surgery J-V; THA: Total hip arthroplasty; TKA: Total knee arthroplasty. SAS. SAS: Safety analysis set; STARS J-V: Studying Thrombosis After Replacement Surgery J-V; THA: Total hip arthroplasty; TKA: Total knee arthroplasty;

Pharmaceutical Company; served on the speakers' bureau for Daiichi Sankyo; and received royalties from Century Medical and Showa Ikakogyo. S. Fujita has been a consultant for Daiichi Sankyo, Astellas, and GlaxoSmithKline. Y. Kawai has been a consultant for Daiichi Sankyo and Toyama Chemical. M. Nakamura has been a consultant for Daiichi Sankyo. Daiichi Sankyo, Bayer, Astellas, Kaken Glaxosmithkline, Ono , VTE: Venous thromboembolism. Competing interests T. Fuji has been a consultant for. Abe are employees of Daiichi Sankyo Co., LtdVTE: Venous thromboembolism. Competing interests T. Fuji has been a consultant for Daiichi Sankyo, Bayer, Astellas, GlaxoSmithKline, Kaken, and Ono Pharmaceutical Company; served on the speakers' bureau for Daiichi Sankyo; and received royalties from Century Medical and Showa Ikakogyo. S. Fujita has been a consultant for Daiichi Sankyo, Astellas, and GlaxoSmithKline. Y. Kawai has been a consultant for Daiichi Sankyo and Toyama Chemical. M. Nakamura has been a consultant for Daiichi Sankyo. S. Tachibana has been a consultant for Daiichi Sankyo and GlaxoSmithKline. T. Kimura, M. Fukuzawa, and K. Abe are employees of Daiichi Sankyo Co., Ltd.

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Lovenox® (enoxaparin sodium injection) for subcutaneous and intravenous use. Package insertLovenox® (enoxaparin sodium injection) for subcutaneous and intravenous use. [Package insert].

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