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. Author manuscript; available in PMC: 2014 Jun 19.
Published in final edited form as: Cell Cycle. 2009 Oct 7;8(19):3191–3198. doi: 10.4161/cc.8.19.9751

Involvement of MKP-1 and Bcl-2 in acquired cisplatin resistance in ovarian cancer cells

Juan Wang 1, Jun-Ying Zhou 1, Lianfeng Zhang 2, Gen Sheng Wu 1,*
PMCID: PMC4063298  NIHMSID: NIHMS521436  PMID: 19755862

Abstract

Although cisplatin is a very effective anticancer agent against several types of cancer including ovarian cancer, the mechanisms of acquired resistance are not fully understood. By chronically exposing cisplatin to ovarian cancer cell lines, we established two cisplatin-resistant cell lines OV433 and tOV112D. Our results indicate that the mechanisms underlying their cisplatin resistance are distinct. In OV433 cells, cisplatin resistance is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). By knocking down MKP-1 expression by siRNA or inhibiting MKP-1 expression by its pharmacological inhibitor triptolide, cisplatin-resistant OV433 cells became cisplatin-sensitive and subsequently increased cisplatin-induced apoptosis. In tOV112D cells, on the other hand, acquired cisplatin resistance is associated with increased levels of Bcl-2 protein. By inhibiting the activity of Bcl-2 protein with its pharmacological inhibitor gossypol or knocking down Bcl-2 expression by siRNA, cisplatin-resistant tOV112D cells became cisplatin-sensitive and subsequently increased cisplatin-induced apoptosis. therefore, our data suggest that the mechanisms of acquired cisplatin resistance vary among ovarian cancer cells, which involve upregulation of molecules associated with the cell survival pathways.

Keywords: cisplatin resistance, MKP-1, Bcl-2, apoptosis

Introduction

Cisplatin is a very effective anticancer agent against several types of cancer including epithelial ovarian cancer. The mechanism of its action is generally accepted that cisplatin is a DNA-damaging agent that forms cisplatin-DNA adducts, which causes DNA damage, leading to the induction of cell cycle arrest and apoptosis.1 In patients with ovarian cancer, the major obstacle in cisplatin-based chemotherapy is the development of resistant cell populations following the relapse of an initially responsive malignancy. It has been shown that ~80% of patients initially respond to cisplatin-based chemotherapy, but 75% of patients relapse initial treatment within 2 years due to the development of cisplatin resistance.2,3 Therefore, there is an urgent need to understand the mechanisms of cisplatin resistance.

The mechanisms of acquired cisplatin resistance are not fully understood, but they are believed to be multifactorial in nature. They include insufficient DNA binding, increased DNA repair, and altered expression and activation of genes involved in the cell death pathways, such as p53, Bcl-2 and JNK.4 Because a common feature of cancer cells to acquire chemoresistance is their ability to blunt apoptosis induced by chemotherapy, understanding how cancer cells evade anticancer drug induced-apoptosis may provide insights into the mechanisms of chemoresistance, including cisplatin resistance.5 Since members of the Bcl-2 family are key regulators of the mitochondrial apoptosis pathway, they have been implicated in chemoresistance.6 The Bcl-2 family contains both anti-apoptotic members, such as Bcl-2 and Bcl-XL, and proapoptotic members, such as Bax and Bid. In general, increased expression of the anti-apoptotic Bcl-2 family members protects cancer cells from death induced by a variety of agents, such as UV irradiation, cytotoxic drugs, growth factor withdrawal and p53 overexpression. However, the role of Bcl-2 family members in cisplatin resistance in ovarian cancer cells is controversial.7,8

In addition to the apoptotic pathways regulated by Bcl-2 family members, there are other pathways that are also involved in cisplatin-induced cell death, including the mitogen-activated protein (MAP) kinase (MAPK) pathway.9 Mammalian MAPK pathways mainly consist of three subfamilies: the p38 MAPK, the extracellular signal-related kinase (ERK), and the c-Jun N-terminal protein kinase (JNK).10,11 Upon stimulation, MAPKs are activated through phosphorylation by upstream dual-specificity kinases.12 Because the activation of JNK is believed to be important for cisplatin-induced cell death, modulation of JNK activity has an important impact on cisplatin sensitivity. Consistent with this notion, members of the MAPK phosphatase (MKP) family, endogenous inhibitors of MAPKs including JNK, play an important role in negatively regulating JNK activity and cisplatin resistance.13,14

The MAP kinase phosphatases (MKPs) are a family of dual-specificity protein phosphatases,15 which include MKP-1, MKP-2, MKP-3, MKP-4, MKP-5, MKP-7, MKP-X, PAC1, hVH3, M3/6 and MK-STYX.16 These MKPs can dephosphorylate both phospho-threonine and phospho-tyrosine residues on MAPKs and thereby inactivate their activities.15 MKP-1 is the founding member of the MKP family, which can be induced by growth factor and stressful stimuli.17-20 MKP-1 uses ERK, JNK and p38 as substrates to exert its biological functions.18,21-23 Recent studies showed that MKP-1 is induced by cisplatin in human lung and ovarian cancer cells13,14 and that MKP-1 plays an important role in cisplatin resistance.13,24

In this study, we established two cisplatin-resistant ovarian cancer cell lines OV433 and TOV112D. Our results showed that these two cell lines use distinct mechanisms to acquire cisplatin-resistant phenotypes. In OV433 cells, acquired cisplatin resistance was correlated with increased MKP-1 expression. We showed that knockdown of MKP-1 by siRNA or its pharmacological inhibitor triptolide renders cisplatin-resistant OV433 cells sensitive to cisplatin. In TOV112D cells, acquired cisplatin resistance was correlated with increased Bcl-2 level. By inhibiting Bcl-2 activity with its pharmacological inhibitor gossypol or knockdown of Bcl-2 expression by siRNA, cisplatin-resistant TOV112D cells became cisplatin sensitive. Taken together, our results suggest that the mechanisms of acquired cisplatin resistance vary in the different ovarian cancer cells involving deregulation of the survival pathways.

Results

Establishment of cisplatin-resistant ovarian cancer cells

Although most ovarian cancer patients initially responded to cisplatin chemotherapy, the majority of patients developed cisplatin resistance and relapsed initial treatment within 2 years. To understand the underlying mechanisms of acquired cisplatin resistance, we initially attempted to generate acquired cisplatin-resistant ovarian cancer cell lines by chronically exposing TOV112D and OV433 cells to gradually increased concentrations of cisplatin starting with 0.1 μg/ml to 1.2 μg/ml for over 6 months. As shown in Figure 1A, cisplatin-resistant OV433 (OV433-CR) cells generated were much more resistant than their parental OV433 (OV433-P) cells to cisplatin. Likewise, cisplatin-resistant TOV112D (TOV112D-CR) cells generated were also much more resistant than their parental TOV112D (TOV112D-P) cells to cisplatin (Fig. 1B). To maintain the acquired resistance to cisplatin, we routinely cultured OV433-CR and TOV112D-CR cells in medium containing 0.8 μg/ml and 1.2 μg/ml cisplatin, respectively.

Figure 1.

Figure 1

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Establishments of two cisplatin-resistant ovarian cancer cell lines. OV433 (A) and TOV112D (B) cells were chronically exposed to gradually increased concentrations of cisplatin starting from 0.1 μg/ml to 0.8 μg/ml (OV433 cells) or 1.2 μg/ml (TOV112D cells) for over 6 months. Both parental (P) and cisplatin-resistant (CR) cells were treated with cisplatin at 10 and 25 μM for 24 hr (TOV112D cells) or 48 hr (OV433 cells) and cisplatin sensitivity was determined by the MTT assay. Data are representative of three independent experiments. **p < 0.001, statistically significant.

Elevated MKP-1 protein and enzymatic activity in OV433 cells and elevated Bcl-2 protein in TOV112D cells are associated with acquired cisplatin resistance

Our previous study indicated that MKP-1 plays an important role in cisplatin resistance in ovarian cancer cells.14 To determine whether MKP-1 plays a role in acquired cisplatin resistance in these two cell lines, we treated OV433-P and -CR cells with or without cisplatin and then examined total and phosphorylated MKP-1 protein expression. As shown in Figure 2A, the basal levels of total and phosphorylated MKP-1 protein were elevated in OV433-CR cells as compared to OV433-P cells. More importantly, the levels of both total and phosphorylated MKP-1 protein were significantly increased in OV433-CR cells than their counterpart OV433-P cells in response to cisplatin treatment. These results suggest that an increase in MKP-1 expression in the absence or presence of cisplatin treatment may be responsible for acquired cisplatin resistance in OV433-CR cells. Since MKP-1 phosphatase activity is critical for its function, we asked whether OV433-CR cells have a higher level of MKP-1 enzymatic activity than OV433-P cells. Figure 2B shows that MKP-1 phosphatase activity is higher in OV433-CR cells than OV433-P cells in the absence of cisplatin treatment. Further, MKP-1 enzymatic activity was much higher in OV433-CR cells than OV433-P cells in response to cisplatin treatment. Thus, these data establish a correlation between increased MKP-1 activity and acquired cisplatin resistance in OV433 cells.

Figure 2.

Figure 2

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The levels of MKP-1 and Bcl-2 protein and MKP-1 enzymatic activity in ovarian cancer cells. (A) increased MKP-1 protein level in cisplatin-resistant OV433 cells. OV433-P and -CR cells were treated with cisplatin (50 μg/ml) for 8 hr and total protein was then extracted. the levels of Bcl-2, MKP-1, phosphorylated MKP-1 (p-MKP-1), PARP were determined by western blot analysis. β-actin was used as a loading control. (B) increased enzymatic activity in cisplatin-resistant OV433 cells. OV433-P and -CR cells were treated with cisplatin as described in A and MKP-1 phosphatase activity was assayed using pNPP protein phosphatase assay kit. Data are representative of three independent experiments. **p < 0.001, statistically significant. (C) increased Bcl-2 expression in cisplatin-resistant TOV112D cells. TOV112D-P and -CR cells were treated with cisplatin (50 μg/ml) for 5 hr. Total protein was then extracted and the levels of proteins (as indicated) were determined by western blot analysis.

To determine whether increased MKP-1 expression and its enzymatic activity also apply to TOV112D cells, we examined the levels of total and phosphorylated MKP-1 protein. As shown in Figure 2C, the levels of MKP-1 protein remained unchanged between TOV112D-P and TOV112D-CR cells in the absence of cisplatin treatment. In fact, upon cisplatin treatment, there was a decrease in total and phosphorylated MKP-1 protein in TOV112D-CR cells as compared to TOV112D-P cells, which suggests that MKP-1 may not be involved in acquired cisplatin resistance in this cell line. However, we found that the levels of Bcl-2 were significantly elevated in TOV112D-CR cells than TOV112D-P cells (Fig. 2C). Since Bcl-2 is an antiapoptotic molecule of the Bcl-2 family, our result suggests that Bcl-2 might play an important role in acquired cisplatin resistance in TOV112D cells.

Inhibition or knockdown of MKP-1 expression sensitizes cisplatin-resistant OV433 cells to cisplatin

Our previous work indicated that MKP-1 plays an important role in intrinsic cisplatin resistance in ovarian cancer.14 To determine the role of MKP-1 in acquired cisplatin resistance, we attempted to inhibit MKP-1 expression to overcome resistance in acquired cisplatin resistance. It has been shown that triptolide, a diterpenoid triep-oxide, is a potent inhibitor of MKP-1.25 We treated OV433-P and -CR cells with cisplatin (10 μM) in the presence or absence of 1 μM triptolide for 48 hr and cell proliferation was determined by the MTT assay. Figure 3A shows that cisplatin alone and in combination with triptolide caused ~60% and ~70% growth inhibition of OV433-P cells, respectively while triptolide alone had no effect. By contrast, in OV433-CR cells, cisplatin alone caused 17% growth inhibition and triptolide alone had a minimal effect on growth inhibition (7%). However, the combination of cisplatin with triptolide decreased OV433-CR cell proliferation by 40%, suggesting that triptolide can sensitize cisplatin-resistant OV433-CR cells to cisplatin. To determine whether triptolide affects MKP-1 expression, we examined the level of MKP-1 in cells with or without cisplatin treatment in the absence or presence of triptolide. As shown in Figure 3B, cisplatin induced MKP-1 expression in OV433-P cells and such induction was even higher in OV433-CR cells. Triptolide inhibited both the basal and cisplatin-induced MKP-1 protein levels, confirming the role of triptolide in inhibiting MKP-1 expression.

Figure 3.

Figure 3

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Effect of triptolide on MKP-1 expression and cisplatin sensitivity in OV433 cells. (A) the effect of cisplatin, triptolide or their combination on growth inhibition. OV433-P and -CR were treated with 10 μM cisplatin (Cis), 1 μM triptolide (Tript), or their combination (Tript + Cis) for 48 hr, and the growth inhibition was determined by the MTT assay. Data are representative of three independent experiments. **p < 0.001, statistically significant. (B) increased cisplatin-induced apoptosis by triptolide. OV433-P and -CR cells were left untreated or treated with cisplatin, triptolide or their combination as described in (A). Total protein was extracted and the levels of MKP-1 and PARP cleavage were determined by western blot analysis. β-actin was used as a loading control.

Since the combination of cisplatin with triptolide decreased cell proliferation in OV433-CR (Fig. 3A), we asked whether such effect is due to enhanced cisplatin-induced apoptotic cell death by examining the cleavage of PARP, a hallmark of apoptosis. As shown in Figure 3B, in OV433-P cells, cisplatin caused PARP cleavage and the combination of cisplatin with triptolide did not significantly increase PARP cleavage while triptolide alone did not have any effect on PARP cleavage. By contrast, in OV433-CR cells, there was minimal PARP cleavage caused by cisplatin treatment while triptolide alone did not affect PARP cleavage. However, the combination of cisplatin with triptolide increased PARP cleavage. Triptolide-caused sensitization of cisplatin-resistant OV433-CR cells to cisplatin suggests that triptolide may be useful for reversing cisplatin resistance in ovarian cancer cells.

Although triptolide is a potent inhibitor of MKP-1,25 since triptolide can target other proteins,26-28 the results obtained with triptolide treatment may not reflect the role of MKP-1 in cisplatin resistance in these cells. To this end, we alternatively used small interfering RNA (siRNA) silencing technology to knock down MKP-1 and then examined the effect of MKP-1 knockdown on cisplatin sensitivity. As shown in Figure 4A, MKP-1 siRNA could efficiently knock down MKP-1 in both OV433-P and OV433-CR in the absence and presence of cisplatin, as compared to cells transfected with control siRNA. To determine the effect of knockdown of MKP-1 on cisplatin sensitivity, OV433-P and -CR cells transfected with MKP-1 siRNA or control siRNA were treated with cisplatin (10 μM) for 48 hr, and cell growth inhibition was determined by the MTT assay. As shown in Figure 4B, 10 μM cisplatin treatment caused ~53% growth inhibition in cells transfected with control siRNA and ~67% growth inhibition in cells transfected with MKP-1 siRNA, which is consistent with a role of MKP-1 in cisplatin resistance.13 In contrast, knockdown of MKP-1 in OV433-CR cells significantly increased cisplatin sensitivity as compared to control siRNA transfected cells (16% vs. 40%) (Fig. 4B), which is consistent with the results obtained with triptolide treatment. Importantly, knockdown of MKP-1 increased PARP cleavage (Fig. 4A). Thus, these data suggest that MKP-1 plays an important role in acquired cisplatin resistance in OV433-CR cells.

Figure 4.

Figure 4

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Effect of MKP-1 knockdown on cisplatin sensitivity in OV433 cells. (A) increased cisplatin-induced apoptosis by MKP-1 knockdown. OV433-P and -CR cells were transfected with non-target or MKP-1 siRNAs. After 3 days, cells were left untreated or treated with cisplatin (10 μM) for 48 hr. Total protein was extracted and the levels of MKP-1 and PARP cleavage were determined by western blot analysis. β-actin was used as a loading control. (B) the role of MKP-1 knockdown in growth inhibition. OV433-P and -CR were transfected with non-target or MKP-1 siRNAs and then treated with cisplatin as described in (A). Growth inhibition was determined by the MTT assay. Data are representative of three independent experiments. **p < 0.001, statistically significant.

Inhibition of Bcl-2 activity overcomes acquired cisplatin resistance in TOV112D cells

It has been known that Bcl-2 plays a role in protecting cells from apoptosis.29 We have shown that Bcl-2 expression is significantly increased in cisplatin-resistant TOV112D-CR cells as compared to their parental TOV112D-P cells (Fig. 2C). To determine whether such increase in fact is directly associated with acquired cisplatin resistance, we used the Bcl-2 inhibitor gossypol to inhibit Bcl-2 activity and then tested the effect of such inhibition on cisplatin sensitivity. As shown in Figure 5A, gossypol treatment did not affect Bcl-2 expression in both TOV112D-P and TOV112D-CR cells as compared with untreated cells. In TOV112D-P cells, cisplatin alone caused PARP cleavage and combination of cisplatin with gossypol did not further increase PARP cleavage significantly. As expected, TOV112D-CR cells were resistant to cisplatin-induced apoptosis as exemplified by lacking PARP cleavage (Fig. 5A). Importantly, combination of gossypol with cisplatin significantly increased PARP cleavage as compared to cells treated with either agent alone (Fig. 5A). Furthermore, we treated TOV112D-P or TOV112D-CR cells with cisplatin (10 μM) in the presence or absence of 5 μM gossypol for 24 hr and cell proliferation was determined by the MTT assay. Figure 5B shows that in TOV112D-P cells, cisplatin alone or in combination with gossypol caused ~56% and 60% growth inhibition, respectively, while gossypol alone had no effect. By contrast, in TOV112D-CR cells, cisplatin alone decreased cell proliferation by 17% and gossypol alone had no effect on growth inhibition. However, combination of cisplatin with gossypol decreased TOV112D-CR growth proliferation by 55%. Thus, our results suggest that inhibition of Bcl-2 activity could overcome acquired cisplatin resistance in some ovarian cancer cells.

Figure 5.

Figure 5

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Effect of Bcl-2 inhibition on cisplatin sensitivity and apoptosis in TOV112D cells. (A) increased cisplatin sensitivity by inhibiting Bcl-2 activity. TOV112D-P and -CR cells were treated with cisplatin (10 μM) in the presence or absence of the Bcl-2 inhibitor gossypol (5 μM) for 24 hr. Total protein was extracted and Bcl-2 expression and PARP cleavage were determined by western blot analysis. β-actin was used as a loading control. (B) gossypol increased cisplatin-induced growth inhibition. Both TOV112D-P and -CR cells were treated as described in A and growth inhibition was determined by the MTT assay. Data are representative of three independent experiments. **p < 0.001, statistically significant. Con, control; Cis, cisplatin; Gos, gossypol.

It is known that gossypol can target other Bcl-2 family members (e.g., Bcl-XL).30 To determine the direct role of Bcl-2 in acquired cisplatin resistance, we knocked down Bcl-2 by siRNA and then examined the effect of Bcl-2 knockdown on cisplatin sensitivity. As shown in Figure 6A, Bcl-2 siRNA could efficiently knock down Bcl-2 expression in both TOV112D-P and TOV112D-CR cells, as compared to corresponding cells transfected with control siRNA. Although cisplatin-induced PARP cleavage was not significantly increased in TOV112D-P cells transfected with Bcl-2 siRNA relative to the same cells transfected with control siRNA, there was an increase in PARP cleavage in TOV112D-CR cells transfected with Bcl-2 siRNA as compared to TOV112D-CR cells transfected with control siRNA (Fig. 6A). To determine the effect of Bcl-2 knockdown on cisplatin sensitivity, TOV112D-P and -CR cells transfected with Bcl-2 siRNA or control siRNA were treated with cisplatin (10 μM) for 24 hr, and growth inhibition was determined by the MTT assay. As shown in Figure 6B, in TOV112D-P cells, 10 μM cisplatin treatment caused ~56% growth inhibition in cells transfected with control siRNA and such treatment caused ~60% growth inhibition in cells transfected with Bcl-2 siRNA. In contrast, Bcl-2 knockdown in TOV112D-CR cells significantly increased cisplatin sensitivity as compared to control siRNA transfected cells (19% vs. 49%) (Fig. 6B), which is consistent with the results obtained with gossypol treatment. Thus, these data strongly suggest that Bcl-2 plays an important role in acquired cisplatin resistance in TOV112D-CR cells.

Figure 6.

Figure 6

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Effect of Bcl-2 knockdown on cisplatin sensitivity in TOV112D cells. (A) increased cisplatin-induced apoptosis by Bcl-2 knockdown. Both TOV112D-P and -CR cells were transfected with non-target or Bcl-2 siRNAs. After 3 days, cells were left untreated or treated with cisplatin (10 μM) for 24 hr. Total protein was extracted and the levels of Bcl-2 and PARP cleavage were determined by western blot analysis. β-actin was used as a loading control. (B) the role of Bcl-2 knockdown in growth inhibition. Both TOV112D-P and -CR were transfected with non-target or Bcl-2 siRNAs and then treated with cisplatin as described in (A). Growth inhibition was determined by the MTT assay. Data are representative of three independent experiments. **p < 0.001, statistically significant.

Discussion

It is well accepted that failure to inducing apoptosis can result in chemoresistance, including cisplatin resistance. Although there are many pathways that can negatively or positively regulate apoptosis, it has been shown that the activation of survival pathways and increased expression of antiapoptotic members of the Bcl-2 family are strongly implicated in chemoresistance.

It has been known that cisplatin-induced apoptosis involves MAPK-mediated apoptosis, including JNK-mediated apoptosis. Since MAPK activity can be negatively regulated by members of the MKP family, overexpression of MKPs has been shown to protect cells from death induced by several stressful stimuli including cisplastin treatment.13,24 Our previous studies showed that cisplatin induces MKP-1 expression and that induction of MKP-1 protects cells from cisplatin-induced apoptosis.14 However, it is not known whether MKP-1 plays a role in acquired cisplatin resistance. In this study, we showed that in OV433 cells, an increase in MKP-1 protein and phosphatase activity is associated with acquired cisplatin resistance (Fig. 2A and B). Because chronic exposure of cancer cells to cisplatin may result in many changes that are associated with cell survival, it is possible that increased levels of MKP-1 protein and enzymatic activity may not account for cisplatin resistance. However, inhibition of MKP-1 expression by its pharmacological inhibitor triptolide or knockdown of its expression by siRNA sensitized cisplatin-resistant OV433 cells to cisplatin, suggesting that MKP-1 indeed plays a role in acquired cisplatin resistance in OV433 cells.

We have also shown that in TOV112D cells, acquired cisplatin resistance is associated with increased expression of Bcl-2 protein. In general, increased expression of the anti-apoptotic Bcl-2 family members protects cancer cells from death induced by a variety of agents, such as UV irradiation, cytotoxic drugs, growth factor withdrawal and p53 overexpression while overexpression of pro-apoptotic Bcl-2 family members can sensitize cancer cells to these treatments including chemotherapeutics. However, in ovarian cancer cells, the role of Bcl-2 family members in cisplatin sensitivity remains contradictory. On the one hand, overexpression of Bcl-2 and Bcl-XL has been shown to inhibit cisplatin-induced apoptosis, leading to cisplatin resistance.8,31 On the other hand, there are studies showing that overexpression of anti-apoptotic Bcl-2 and Bcl-XL has no effect on cisplatin resistance or even correlated with cisplatin sensitivity.7 Thus, the role of anti-apoptosis members of the Bcl-2 family in cisplatin resistance in ovarian cancer, particularly in acquired cisplatin-resistant ovarian cancer cells, is not clear. We showed that inhibition of Bcl-2 activity by gossypol restores cisplatin sensitivity in acquired cisplatin-resistant TOV112D cells, suggesting that Bcl-2 plays a role in cisplatin resistance in TOV112D-CR cells. Since gossypol also inhibits Bcl-XL activity,30 it is possible that the results obtained with gossypol may be due to the inhibition of Bcl-XL activity, leading to cisplatin sensitization. However, we showed that knockdown of Bcl-2 by siRNA in TOV112D-CR cells leads to sensitivity to cisplatin (Fig. 6), suggesting a direct role of Bcl-2 in cisplatin resistance. In addition, we have established additional two ovarian cancer cell lines with acquired cisplatin resistance and found that Bcl-2 expression is elevated in those acquired cisplatin-resistant cells (data not shown). Therefore, our results strongly suggest that increased Bcl-2 expression is the likely mechanism by which TOV112D cells acquire cisplatin resistance.

It is becoming evident that acquired chemoresistance including acquired cisplatin resistance is associated with many distinct alternations involving a variety of the signaling pathways. Among these pathways, a particular signaling pathway may play a predominant role in chemoresistance in a given tumor cell line. Consistent with this, we showed that in OV433 cells, increased MKP-1 expression plays a predominant role in acquired cisplatin resistance because (1) MKP-1 expression was increased in cisplatin-resistant OV433-CR cells, (2) inhibition of MKP-1 expression by its pharmacological inhibitor triptolide sensitized OV433-CR cells to cisplatin and (3) more importantly, knockdown of MKP-1 increased cisplatin-induced apoptosis (Figs. 3 and 4). However, it is possible that cisplatin resistance in OV433-CR cells may also be associated with many other changes that collectively confer cisplatin resistance and this need further investigation. Nevertheless, our data clearly indicated that knockdown of MKP-1 sensitizes OV433-CR cells to cisplatin, which suggests a direct role of MKP-1 in acquired cisplatin resistance. Consistent with the distinct alternations in the cell signaling pathways, we showed that TOV112D-CR cells acquire cisplatin-resistant phenotype involving increased expression of Bcl-2 protein because inhibition of Bcl-2 by gossypol and knockdown of Bcl-2 by siRNA restores cisplatin sensitivity in TOV112D-CR cells. We believe that an increase in Bcl-2 but not MKP-1 protein is a predominant event that is closely associated with acquired cisplatin resistance in TOV112D-CR.

In conclusion, we demonstrated that different ovarian cell lines use distinct mechanisms to acquire cisplatin resistance. We showed that in OV433 cells, increased MKP-1 protein may be responsible for acquired cisplatin resistance. We also showed that overexpression of Bcl-2 accounts for acquired cisplatin resistance in TOV112D cells. Since individual ovarian cancer cells acquire cisplatin resistance through distinct mechanisms, this study suggests that targeting the predominant signaling pathway associated with cisplatin resistance in individual ovarian cancer cells may overcome cisplatin resistance.

Materials and Methods

Reagents

Cisplatin, triptolide and anti-actin antibody (AC-74) were purchased from Sigma (St. Louis, MO). Mouse anti-Bcl-2 monoclonal antibody was purchased from Calbiochem (San Diego, CA). Gossypol was obtained from AXXORA (San Diego, CA). Rabbit polyclonal anti-human MKP-1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies against PARP and phosphorylated MKP-1 at serine 359 were purchased from Cell Signaling Technology (Beverly, MA).

Cell lines, culture conditions and treatment

The human ovarian cancer cell lines TOV112D and OV433 were kindly provided by Dr. Samuel Mok (Brigham and Women's Hospital, Boston, MA), as described previously.32 These cells were maintained in MCDB105/M199 supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air.

siRNA transfection for knockdown of MKP-1 and Bcl-2

On-TARGETplus SMARTpool siRNAs for MKP-1, Bcl-2 and corresponding control siRNA were purchased from Dharmacon Research (Lafayette, CO). The transfection was performed as suggested by Dharmacon with slight modifications, as described previously.13 Briefly, OV433 and TOV112D cells were plated at 6 × 105 cells per well in 6-well plates. The next day, cells were transfected with MKP-1 siRNA (OV433 cells), Bcl-2 siRNA (TOV112D cells) or non-target control siRNA using Lipofectamine 2000 (Invitrogen, CA). After 3 days, transfected cells were left untreated or treated with cisplatin (10 μM) for 24 hr (TOV112D cells) or 48 hr (OV433 cells) and then harvested for examining the expression of MKP-1, Bcl-2 and PARP by western blot analysis. To determine cisplatin sensitivity, transfected cells were placed at 8,000 cells per well in 96-well plates and then treated with or without cisplatin (10 μM) for 24 or 48 hr, and cell proliferation was determined by the MTT assay.

MTT assay

MTT assay was previously described.33 Briefly, cells were left untreated or treated with cisplatin in the presence or absence of either triptolide (1 μM) or gossypol (5 μM) for various periods of time. After incubation with MTT solution, isopropanol was added to dissolve the formazan crystals. OD was measured using a Vmax Microplate Reader (Molecular Devices, Sunnyvale, CA) at 590 nm. Cell proliferation was calculated from the mean of pooled data from three separate experiments with five wells.33

Western blot analysis

Cell lysates were prepared as described previously,34 and protein concentration was determined using the Protein Assay Kit (Bio-Rad, Hercules, CA). Cell lysates (100 μg) were electrophoresed through 12 or 15% denaturing polyacryl-amide gels and transferred to a PVDF membrane (Millipore, Bedford, MA). The blots were probed or reprobed with the antibodies, and bound antibody was detected using Enhanced Chemiluminescence (ECL) or ECLplus Reagent (Amersham Pharmacia, Biotech, Piscataway, NJ) according to the manufacturer's protocol.

Assay of MKP-1 phosphatase activity

MKP-1 phosphatase activity was measured using immunoprecipitated MKP-1. Immunoprecipitation (IP) was performed as described previously35 with slight modification. Briefly, cells were lysed in lysis buffer, and 500 μg of supernatant protein was incubated with 2 μg of anti-phosphorylated MKP-1 antibody or IgG at 4°C for 2 hr, followed by incubation with protein A/G PLUS for 1 hr. Immunoprecipitates were washed 4 times with cell lysis buffer. The precipitates were used to measure MKP-1 phosphatase activity. Phosphatase activities of immunoprecipitated MKP-1 were analyzed as described previously36 using pNPP protein phosphatase assay kit from AnaSpec (San Jose, CA) according to manufacturer's instructions. Specifically, 50 μL of pNPP reaction mixture was added to 50 μL of MKP-1 immunoprecipitates and the reaction was performed at 30°C for 1 h. Optical absorbance measured at 405 was obtained in a microplate reader. Nonspecific hydrolysis of pNPP was assessed in IgG immunoprecipitates, which served as a control.

Statistical analysis

Statistical analyses were performed using student's t test. The data were presented as the mean ± SD, and p value <0.05 was considered significant.

Acknowledgements

We thank Dr. Samuel Mok for providing ovarian cancer cell lines. Grant support: NIH grant R01 CA100073 and Gail Purtan Ovarian Cancer Research Fund (G.S.W.).

Abbreviations

MAPK

mitogen-activated protein (MAP) kinase

MKP-1

MAPK phosphatase-1

ERK

extracellular signal-regulated kinase

JNK

c-Jun NH2-terminal kinase

siRNA

small interfering RNA

FBS

fetal bovine serum

IP

immunoprecipitation

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