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Tanshinone IIA enhances the effects of TRAIL by downregulating survivin in human ovarian carcinoma cells.

ABSTRACT

Background: Tanshinone IIA (TIIA), a diterpene quinone from the medicinal plant Salvia miltiorrhiza Bunge (Lamiaceae) was shown to possess apoptotic and TRAIL-sensitizing effects. Still, the molecular mechanisms whereby TIIA induces apoptosis remain largely unknown.

Purpose: The role of survivin, an inhibitor of apoptosis protein, in TIIA-induced apoptosis has never been addressed before and hence was the primary goal of this study.

Methods: In this study, we explored the anticancer effect of TIIA in TOV-21G, SKOV3, and OVCAR3 ovarian carcinoma cells. Cytotoxicity was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of related signaling proteins.

Results: Our results illustrated that TIIA's cytotoxic effect was caused by apoptosis with the involvement of caspases activity. Moreover, TIIA downregulated survivin in a concentration-dependent manner without affecting the expression of Bcl-2, Bcl-xL, and Bax. TIIA-induced survivin downregulation is regulated by both transcriptional processes and proteasomal degradation. Using TOV-21G cells as our cellular model, we demonstrated that TIIA-induced survivin downregulation requires p38 MAPK activation. Importantly, genetic overexpression of survivin rendered cells more resistant to TIIA, indicating an essential role of survivin downregulation in TIIA-induced apoptosis. This TRAIL sensitization effect of TIIA is ascribed to survivin downregulation because the effect was abrogated in cells that overexpressed survivin.

Conclusion: Our findings provide new insights into the action modes of TIIA-mediated anticancer effects and further implicate a rational design for cancer therapeutic regimens by combining TIIA-sensitized TRAIL via downregulating survivin to elicit ovarian cancer cell death.

Keywords:

Tanshinone IIA (TILA)

Ovarian cancer

Apoptosis

Survivin

p38 mitogen-activated protein kinase

(p38 MAPK)

Tumor necrosis factor-related

apoptosis-inducing ligand (TRAIL)

Introduction

Epithelial ovarian carcinoma (EOC) is one of the most deadly cancers in women. Based on histopathological, immunohistochemical, and molecular genetic analysis, the heterogeneity of EOCs can be further categorized into serous, clear cell, endometrioid, and mucinous types (Lengyel 2010). The standard care for ovarian cancer is a combination of cytoreductive surgery and platinum-based chemotherapy (Kim et al. 2012). Despite medical advances, there remains a significant risk of recurrence and resistance to therapy, and at present, recurrent and resistant ovarian cancer is currently incurable. Resistance to chemotherapy presents a major obstacle in improving the prognosis of patients with ovarian cancer. Hence, there is an urgent need to develop smarter treatment options. Recently, there are several landmark reports in ovarian cancer proving the development of molecular-driven, patient-selective clinical trials and changes in clinical practice with lower toxicity than conventional chemotherapy (Banerjee and Kaye 2013).

Survivin is a unique member of the inhibitor of apoptosis (IAP) protein family that potently inhibits caspase activities and hence blocks the execution of apoptosis. In addition to inhibiting apoptosis, survivin is fundamental to the mitotic progression, invasion, and metastasis of EOCs (Yoshida et al. 2001). Studies from other groups demonstrated that survivin expression was correlated with poor prognosis and shorter patient survival (Cohen et al. 2003); thus, survivin is a broad-spectrum molecular target for cancer gene therapy (Athanasoula et al. 2014).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents a promising anticancer agent because of its selective proapoptotic activity against malignant cells without affecting normal cells (Lemke et al. 2014). Although TRAIL may be targeted as an antitumor strategy, most of the literature reports insufficient effects of TRAIL-induced apoptosis in ovarian cancer cells, often due to resistance to TRAIL, raising the necessity for a combinatorial strategy that employs therapeutics to circumvent the resistance mechanisms for TRAIL sensitization (Dimberg et al. 2013).

Danshen, the dried root of Salvia miltiorrhiza Bunge (Lamiaceae) is a well-known traditional Chinese herbal medicine that has been widely used for the prevention and treatment of cardiovascular disorders. Tanshinone 1IA (TIIA, Fig. 1 A) is one of its lipophilic constituents that has shown cytotoxic effects in several cancer cells in vitro and in some tumor xenograft models in vivo (Li et al. 2015; Munagala et al. 2015). Our previous study reveals that TIIA has both cytotoxic and TRAIL-sensitizing effects (Chang et al. 2013). The mechanisms of the effects of TIIA on human ovarian carcinoma are largely unknown. The underlying mechanisms of the effects of TIIA could provide the rationale for the development of clinical therapeutic strategies for the treatment of EOCs. Therefore, the purpose of this study was to examine the molecular target in TlIA-induced cytotoxicity. Interestingly, we found that survivin is transcriptionally downregulated and proteasome-degraded by TIIA in an array of TRAIL-resistant ovarian cancer cell lines. In addition, we found that TIIA-induced downregulation of survivin is essential for TIIA to trigger apoptosis and induce TRAIL sensitization. Our discovery provides new insights into the molecular basis of apoptosis induction by TIIA but also suggests the potential use of TIIA in the treatment of TRAIL-resistant ovarian cancer.

Materials and methods

Reagents and antibodies

Tanshinone IIA (Lot number 00020043-010, purity > 96.1%) was purchased from ChromaDex, Inc., (Irvine, USA). Purified TIIA was dissolved in DMSO at a concentration of 10 mM and stored in the dark at -20[degrees]C until use. The final concentration of DMSO was 0.1% (v/v) throughout the experiments. Recombinant human TRAIL (Gibco[R], Invitrogen, Carlsbad, CA) was prepared as a 100 [micro]g/ml stock solution and stored in aliquots at -20[degrees]C before use. The broad-spectrum caspase inhibitor z-VAD.fmk was purchased from Calbiochem (San Diego, CA, USA). MG132 (Tocris Bioscience, UK) and the p38 MAPK-specific inhibitor SB203580 (Tocris Bioscience) were prepared as 20 mM stock solutions in DMSO and stored in aliquots at -20[degrees]C before use. Geneticin disulfate salt (G418) was purchased from Sigma-Aldrich. The primary antibodies against caspase-3, caspase7, caspase-9, PARP, cleaved PARP, Bax, Bcl-2, Bcl-xL, p38 MAPK and phospho-p38 MAPK were obtained from Cell Signaling Technology (Beverly, MA, USA). The anti-survivin antibody was purchased from Abeam (Cambridge, UK).

Cell culture

The human clear cell ovarian carcinoma cell line TOV-21G (ATCC CRL-11730) and the serous cell lines SKOV3 (ATCC HTB-77), OVCAR3 (BCRC 60551), and CaOV3 (ATCC HTB-75) were used in this study. TOV-21G cells were grown in a 1:1 mixture of MCDB 105 and medium 199 with 15% fetal bovine serum (FBS). SKOV3 cells were grown in McCoy's 5[alpha] medium with 10% FBS. OVCAR3 cells were grown in RPMI-1640 medium with 20% FBS supplemented with 0.01 mg/ml insulin, and CaOV3 cells were grown in DMEM containing 10% FBS supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. The media and supplements were purchased from Invitrogen. All cell lines were cultured at 37[degrees]C and 5% C02.

Measurement of cell cytotoxicity

Cell cytotoxicity was evaluated by the CellTiter 96' [AQ.sub.ueous] One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), using 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) as previously described (Ho et al. 2007). The MTS tetrazolium compound is bioreduced by the mitochondrial activity of viable cells into a soluble colored formazan product, and the amount of colored formazan product is proportional to the number of viable cells. In brief, cells were plated at 5 x [10.sup.3] - 15 x [10.sup.3] cells/well in 96-well plates in 100 [micro]l of complete medium and allowed to attach overnight. The medium was then changed, and cells were treated with various concentrations of TIIA or TRAIL in a final volume of 100 [micro]l in 2% FBS culture medium. After 48 h of incubation, the medium in each well of the 96-well assay plates was replaced with 80 [micro]l of fresh phenol red-free culture medium, followed by the addition of 20 [micro]1 of MTS solution. The plate was then incubated for 2 h at 37[degrees]C in the humidified 5% C02 atmosphere before measuring the absorbance at 490 nm. Cell cytotoxicity was calculated as [1- ([A.sub.sample] - [A.sub.blank])/([A.sub.control] - [A.sub.blank)]] x 100%. At least three independent assays were performed to calculate the cytotoxicity for statistical analysis.

Western blotting

Cells were plated onto 60-mm dishes at a density of 5 x [10.sub.5] - 20 x [10.sup.5]/dish. After drug treatment, adherent cells were washed twice with ice-cold PBS. Cells were then disrupted in 100 [micro]l of RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 50 mM NaCl, 0.1% SDS, 1% Nadeoxycholic acid, 1 mM sodium orthovanadate, 2 mM PMSF) for 30 min on ice with gentle rocking. The cells/lysis buffer mixture was then scraped into solution, followed by centrifugation at 10,500g for 15 min at 4[degrees]C. The resulting soluble fraction was then transferred to a new tube and stored at -80[degrees]C until use. Protein concentrations were determined by the bicinchoninic acid protein assay (Pierce, Oud-Beijerland, Netherlands) with bovine serum albumin as the standard. Immunoblotting was performed as previously described. Briefly, whole-cell lysates were boiled in the sample buffer (100 mM Tris-HCl, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 10% dithiothreitol). Equal amounts of proteins (20-50 [micro]g) were separated by SDS-PAGE using a 10-15% polyacrylamide gel. After electrophoresis, the proteins were transferred onto a FluoroTrans W membrane (Pall Life Sciences, USA), blocked with 5% skim milk, and then probed with antibodies. The level of human [beta]-tubulin antibody (Sigma-Aldrich) was used as a loading control for each blot. The signals were detected with enhanced super Signal West Pico chemiluminescence (Pierce, USA).

Quantitative real-time RT-PCR

The levels of survivin mRNA were determined by quantitative RT-PCR. Total RNA extracted from mock- or drug-treated cells was isolated using the guanidinium thiocyanate method (Trizol; Invitrogen) following the manufacturer's protocol. Total RNA (10 [micro]g) was reverse-transcribed into first strand cDNA by ImProm-II[TM] Reverse Transcription reagents (Promega) using random hexamers as the primer. The survivin mRNA levels were then revealed by PCR, which was performed in a 20 [micro]l reaction volume using SYBR Green PCR Master Mix (Applied Biosystems. USA) with the following primer pairs: forward, 5'-TCCACTGCCCCACTGAGAAC3'; reverse, 5,-TGGCTCCCAGCCTTCCA-3/. Real-time RT-PCR was performed on an ABI PRISM 7300 sequence detector. The gene expression levels of survivin were normalized to that of TATA-binding protein (TBP). The final results were expressed as the ratio of the survivin mRNA copy numbers to the TBP mRNA copy num bers and presented as the mean [+ or -] SEM of three independent experiments.

Establishment of stable cell lines overexpressing survivin

For gene overexpression of survivin, our previously constructed plasmid of pcDNA3-survivin (Ho et al. 2009) was stable transfected into ovarian cancer cells. The stable transfection method is used to introduce a gene of interest into eukaryotic cells, and long-term expression for studying protein function (Wurm 2004). TOV-21G cells (0.5 x [10.sup.5]-1 x [10.sub.5]) were seeded in each well in 24-well plates to yield a 50-60% confluent monolayer. After 24 h, cells were transfected using jetPEI[TM] transfection reagent (Polyplus, USA) with 2 [micro]g/well of the survivin expression plasmid (pc[DNA.sub.3]-survivin) or empty pcDNA3 vector (control). After 48 h of transfection, cells from each well were moved to 96-well plates and selected in medium supplemented with G418 (1 mg/ml). G418 resistant stable cell lines can be generated in 1-2 months. Overexpression of survivin in these stable transfectants was verified by immunoblotting.

Colony formation assay

For colony formation assays, cells were plated onto 60-mm dishes at a density of 5x[10.sup.5] cells/dish overnight and then treated with the indicated doses of drugs for 24 h. Control cells were alternatively exposed to 0.1% (v/v) solvent. At the end of drug treatment, cells were washed twice with PBS and then trypsinized to determine cell numbers. The drug-treated cells were then seeded at a density of 200 cells per 60-mm dish in triplicate for each treatment. Cells were then allowed to form colonies by incubation in drug-free medium for 14 days. The cell monolayer was rinsed twice with PBS, followed by staining with 1% crystal violet solution in 30% ethanol to count the number of colonies.

Statistical analysis

All data were expressed as the mean [+ or -] SEM of at least three independent experiments. The differences between groups were examined for statistical significance using Student's t-test. A p value lower than 0.05 was used as the minimum criteria for statistical significance. Statistical significance is expressed as *p < 0.05, **p < 0.01, ***p < 0.005.

Results

Apoptosis is involved in TlIA-induced cytotoxicity in human ovarian cancer cells

A panel of human ovarian cancer cell lines, including TOV-21G, SKOV3, and OVCAR3 cells, was used to evaluate the cytotoxic effects of TIIA. Cells were treated for 48 h with various doses of TIIA (0, 10, 20 [micro]M for TOV-21G and SKOV3; 0,1, 3 [micro]M for OVCAR3, respectively). We found that TIIA exerted a concentration-dependent cytotoxic effect in the three cell lines tested (Fig. IB, upper panel). Apoptosis was evaluated by Western blot analysis of caspase-9 and PARP cleavage for 24 h. Cleavage of caspase-9 and PARP was evident in TIIA-treated TOV-21G cells, and the level of cleavage paralleled the increase of the TIIA concentration (Fig. IB, lower panel). To further confirm the caspase-dependent cell death, the broad-spectrum caspase inhibitor z-VAD.fmk was employed. Pretreatment with z-VAD.fmk suppressed the cleavage of PARP (Fig. 1C). In the presence of z-VAD.fmk, the TIIA-suppressed cell numbers were enhanced (Fig. ID, lower panel). The number of viable cells was significantly increased from 12.0 [+ or -] 1.4 without z-VAD.fmk to 37.8 [+ or -] 3.1 upon stimulation with z-VAD.fmk (Fig. IE). Collectively, these results demonstrated that TIIA-induced apoptosis was caspase-dependent in ovarian carcinoma cells.

TIIA down-regulates survivin expression at the transcriptional and proteasomal degradation levels

As shown in Fig. 2A, treatment with TIIA for 24 h reduced survivin protein expression without altering the expression of Bd-2 protein family, including Bd-2 itself, Bcl-xL anti-apoptotic protein and Bax pro-apoptotic protein (Fig. 2A). With survivin down-regulation by TIIA firmly established, we were interested to explore the mechanisms underlying this effect. We first tested whether TIIA reduces the levels of survivin mRNA. To address this, ovarian carcinoma cells were treated for 24 h with various doses of TIIA (0,10,20 [micro]M for TOV-21G and SKOV3; 0, 1, 3 [micro]M for OVCAR3, respectively). Using quantitative real-time RT-PCR, TIIA reduced the levels of survivin mRNA in all three cell lines in a concentration-dependent manner (Fig. 2B). We next probed the possibility of post-transcriptional regulation of survivin by TIIA. Because survivin is known to be regulated by proteasome-mediated degradation (Zhao et al. 2000), we then determined whether the observed downregulation of survivin by TIIA was mediated via this process. To this end, we treated cells with TIIA in the absence or presence of the proteasome inhibitor MG132 and then compared survivin modulation under these conditions. As shown in Fig. 2C, the inhibition of survivin from TIIA treatment was reversed by MG132 in SKOV3 and OVCAR3 cells, and it was a partial reversion in TOV-21G cells, suggesting that TIIA induces survivin degradation via a proteasome-dependent pathway. Together with previous results, we concluded that survivin is down-regulated by TIIA both at the levels of transcription and proteasomal degradation.

TIIA-induced down-regulation of survivin requires p38 MAPK activation

The upstream signaling initiated by TIIA to induce survivin down-regulation was further examined. The p38 MAPK signaling was then investigated for its contributions in TIIA's actions. TOV-21 G cells were treated with 0, 10 or 20 [micro]M of TIIA for 24 h. Fig. 3A shows that TIIA increased p38 MAPK phosphorylation in a concentration-dependent manner. To examine whether p38 MAPK plays any role in TIIA-induced down-regulation of survivin, we assessed the involvement of p38 MAPK in TIIA-induced down-regulation of survivin using pharmacological inhibitor of p38 MAPK (SB203580). Cells were pretreated with or without 20 [micro]M SB203580 for 30 min to TIIA treatment for 24 h. Pretreatment of cells with a p38 MAPK inhibitor, suppressed the TIIA-induced down-regulation survivin (Fig. 3B). To further examine the role of p38 MAP kinase on the regulation of survivin, we incubated cells with 10 [micro]M of TIIA in the presence of SB203580 to investigate whether p38 MAPK was involved in the TIIA-induced downregulation of survivin expression. As shown in Fig. 3C, SB203580 not only blocked TIIA-induced downregulation of survivin expression but indeed causes a significant upregulation of the expression of these mRNAs. These results suggest that TIIA inhibits survivin expression possibly via the p38 MAPK dependent pathway.

Survivin downregulation is pivotal for TIIA-induced apoptosis

If a decrease in survivin expression is crucial for TIIA to trigger apoptosis, survivin overexpression should reverse this effect. Thus, we generated TOV-21G clones stably expressing survivin (TOV-21G/S1, S2) as well as cells stably carrying the empty vector (TOV-21 G/Vec) as a negative control. The TOV-21 G/S2 cells expressed a high level of survivin (Fig. 4A), and it was selected for further study. These transfectants were subjected to treatment with various concentrations of TIIA (0,1, 5 or 10 [micro]M) for 24 h, and PARP cleavage was then determined by immunoblotting. Compared to TOV-21 G/Vec cells, in which PARP cleavage was induced by TIIA treatment, TOV-21 21G/S1 cells

exhibited significant resistance to TIIA (Fig. 4B). We further examined the effect of TIIA on the long-term clonogenic survival of stable transfectants (TOV-21 G/Sl or control TOV-21 G/Vec). Cells were treated without or with 1, 5, or 10 [micro]M of TIIA for 24 h, followed by colony formation assays to assess the long-term survival/progression of tumor cells (TOV-21 G/Vec and TOV-21G/S1). Compared to TOV-21 G/Vec cells, we found that TOV-21 G/S1 cells exhibited significant resistance to TIIA (Fig. 4C). Treatment with 10 [micro]M TIIA resulted in an increase in colony formation (the numbers of cell colonies increased from 30.0 [+ or -] 1.1 to 57.8 [+ or -] 4.1, p < 0.01). Taken together, these results evidently underscored an essential role of survivin downregulation in TIIA-mediated cell death.

Survivin inhibition sensitizes ovarian cancer cells to TRAIL-induced apoptosis

We next assessed the ability of T1IA to restore TRAIL-induced cell death in these resistant ovarian cancer cells. Ovarian cancer cells were first treated with various doses of TRAIL (0,50 or 100 ng/ml), followed by immunoblotting to monitor survivin expression. As shown in Fig. 5A, no TRAIL-resistant ovarian cancer cell lines displayed suppressed survivin protein expression. By contrast, exposure of TRAIL-sensitive CaOV3 ovarian cancer cells to TRAIL decreased survivin expression. We then tested whether the TIIA induced sensitization to TRAIL in these cells. These cells were treated for 48 h with either TRAIL (100 ng/ml) alone, or various doses of TIIA (2, 5, 0.5 [micro]M for TOV-21G, SKOV3 and OVCAR3 cells, respectively) alone, or a combination of both drugs. As shown in Fig. 5B, treatment of TOV-21G cells with 100 ng/ml of TRAIL resulted in a 21.1 [+ or -] 3.6% induction in cell cytotoxicity, whereas 2 [micro]M of TIIA decreased the survival of TOV-21G cells by 49.6 [+ or -] 6.0%. Remarkably, when TRAIL was combined with TIIA, cytotoxicity increased to 88.5 [+ or -] 5.6%, indicating that TIIA greatly enhanced the toxicity of TRAIL (p < 0.005). Importantly, similar patterns of increased TRAIL toxicity following TIIA exposure were also observed in SKOV3 and OVCAR3 cells, excluding the possibility of the cell type-specific effect (Fig. 5B, upper panei). We observed a marked increase in apoptosis induction upon treatment with the TIIA-TRAIL combination, as evidenced by the increased levels of cleaved caspase9, -7, -3, and PARP (Fig. 5B, lower panel). It is also noteworthy that the TIIA-TRAIL combination induced induction of apoptosis accompanied by a marked downregulation of survivin, suggesting that survivin downregulation likely contributes to the TIIA-mediated sensitization to TRAIL-induced apoptosis.

Survivin downregulation is responsible for TIIA-mediated TRAIL sensitization

As TIIA enhances the effects of TRAIL, we next probed whether survivin downregulation by TIIA accounts for this effect. To address this, we first tested whether ectopic survivin expression could rescue the TIlA-sensitizing effect on TRAIL-induced cell death. TOV-21G/S1 cells overexpressing survivin as well as TOV-21G/Vec cells carrying the empty vector were treated for 24 h with TRAIL (100 ng/ml) alone, TIIA (2 [micro]M) alone or a combination of both drugs, followed by immunoblotting for survivin expression. We further analyzed another biochemical hallmark of apoptosis, PARP cleavage, in the TOV-21G/S1 cell line. Contrary to inducing PARP cleavage in control clone cells, the combination of both drugs failed to show an increase in cleaved PARP levels in cells overexpressing survivin (Fig. 6A). These results suggest that TIIA can promote TRAIL-induced apoptosis through a caspase cascade involving survivin. We next evaluated the effects of TIIA and TRAIL on long-term survival in both TOV-21G/Vec and TOV-21G/S1 cells treated with either drug alone or in combination (Fig. 6B). As shown in Fig. 6B, co-treatment of TOV-21G/Vec cells with TIIA significantly augmented the inhibitory effect of TRAIL on the capacity of TOV-21G cells to form colonies. On the contrary, this sensitization was compromised in TOV-21G/S1 cells (lower panel), suggesting that the overexpression of survivin in these cells prevents TIIA-induced decreases in survivin levels and consequently blocks TRAIL sensitization by TIIA. Our results firmly supported the notion that TIIA induces TRAIL sensitization by downregulating survivin.

Discussion

TIIA, the major active compound extracted from medicinal plant S. miltiorrhiza has been shown to induce apoptosis and inhibit cell growth in several cancer cell lines. The mechanisms underlying TIIA-induced antitumor effect in EOCs are unknown. However, this is the first study showing that TIIA is a survivin inhibitor and that a decrease in survivin expression is essential for TIIA's effects on apoptosis induction and TRAIL sensitization (Fig. 7).

Our study shows that TIIA's cytotoxicity probably occurred through apoptosis, as revealed by the cleavage of caspase-9 and PARP and further validated by the cleaved PARP after treatment with the pan-caspase inhibitor z-VAD.fmk. In addition, TIIA can inhibit survivin expression in a dose-dependent manner at various levels, including transcription and proteasomal degradation. In fact, survivin has generally been considered to exert a cytoprotective effect via caspase inhibition, encompassing both initiator caspase-9 and effector caspase-3 (Cheung et al. 2013). Thus, tumor cells are sensitive to the induction of apoptosis when survivin is downregulated. Survivin expression has been reported as a novel prognostic factor in several human malignancies (Alaggio et al. 2013; Krieg et ai. 2013; Lv et al. 2014), and its expression level is correlated with poor prognosis and shorter patient survival in EOCs (Cohen et al. 2003). Recently, survivin has been considered an attractive target for anticancer therapy because it exhibits low expression in most normal cells and high expression in cancer cells (Kelly et al. 2011). Numerous studies illustrated that targeting survivin could become a potential approach for anticancer therapeutics (Groner and Weiss 2014). Therefore, the downregulation of survivin by TIIA provides a useful clue for new therapeutic strategies for EOCs.

The p38 MAPK pathway has been implicated in the induction of apoptosis by varying extracellular stimuli (Dolado et al. 2007). It was previously reported that p38 MAPK was involved in the downregulation of survivin by several chemosensitizers for cancer therapy (Cao et al. 2011; Hsiao et al. 2007; Liu et al. 2010, 2014). Our results revealed the activation of p38 MAPK in TIIA-treated TOV-21G cells, and the p38 MAPK inhibitor SB203580 reversed the downregulation of survivin after TIIA treatment. To our knowledge, this is the first report demonstrating that p38 MAPK is involved in the downregulation of survivin following treatment with TIIA in EOC cells.

TRAIL represents a promising anticancer agent due to its selective killing of tumor cells but not normal cells. However, resistance to TRAIL remains a challenge facing the development of anticancer therapy. High levels of survivin expression have also been observed in cancer cell lines resistant to TRAIL (Ryu et al. 2012; Van Geelen et al. 2004). A number of anticancer agents are reported to enhance the sensitivity of cancer cells to TRAIL via downregulation of survivin. We recently found that TRAIL alone did not cause significant cell death, whereas TRAIL treatment in combination with TIIA led to a marked increase in TRAIL-elicited cytotoxicity in EOC cell lines (Chang et al. 2013). These findings led us to investigate whether TIIA could counteract survivin up-regulation and consequently increase the sensitivity of cancer cells to TRAIL. In this study, TRAIL did not suppress survivin levels in TRAIL-resistant ovarian cancer cell lines (Fig. 5A). However, three TRAIL-resistant EOC cell lines could be made sensitive to TRAIL by co-treatment with TIIA, which was identified as a survivin inhibitor. Importantly, the sensitization was abrogated when survivin is overexpressed in TOV-21G/S1 cells (Fig. 6B). Based on these observations, we suggest a critical role for survivin downregulation in mediating TIIA-induced apoptosis and the augmentation of TRAILinduced apoptosis.

Conclusions

Taken together, our results demonstrate that survivin downregulation is pivotal to TIIA-induced apoptosis and further suggest a potential for TIIA to increase sensitivity to anticancer modalities, including TRAIL for treatment of ovarian cancer. Accordingly, we recommend that this combination effect should be further evaluated in animal models for clinical practice.

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

ARTICLE INFO

Article history:

Received 4 December 2014

Revised 20June 2015

Accepted 23 June 2015

Abbreviations

TIIA        tanshinone IIA
TRAIL       tumor necrosis factor-related apoptosis-inducing ligand
EOC         epithelial ovarian carcinoma
MTS         [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy
            phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]
IAP         inhibitor of apoptosis
z-VAD.fmk   N-benzyloxycarbonyl Val-Ala-Asp (O-methyl)-fluorome
            thylketone
PARP        poly (ADP-ribose) polymerase
p38 MAPK    p38 mitogen-activated protein kinase
RT-PCR      reverse transcriptase polymerase chain reaction


Acknowledgments

The authors thank Dr. Chia-Che Chang (National Chung Hsing University) for his guidance and helpful discussions and Dr. Shyh-Chang Chen (Taichung Veterans General Hospital) for technical assistance. This work was supported by grants from the Taichung Veterans General Hospital and Central Taiwan University of Science and Technology, Taichung, Taiwan (TCVGH-CTUST1027704, TCVGH-CTUST1037704) and from Agricultural Research Institute and Central Taiwan University of Science and Technology, Taichung, Taiwan (CTU101-TARI-004).

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Jyun-Yi Lin (a), Yu-Min Ke (b,c), Jui-Sheng Lai (d), Tsing-Fen Ho (a), *

(a) Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan

(b) Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan

(c) Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan

(d) Division of Biotechnology, Taiwan Agricultural Research Institute, Taichung, Taiwan

* Corresponding author. Tel.: +886 4 22391647; fax: +886 4 22396761.

E-mail address: tfho@ctust.edu.tw, tf693588@gmail.com (T.-F. Ho).

http://dx.doi.Org/10.1016/j.phymed.2015.06.012
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Author:Lin, Jyun-Yi; Ke, Yu-Min; Lai, Jui-Sheng; Ho, Tsing-Fen
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Sep 15, 2015
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