Pyruvate dehydrogenase kinase 1 contributes to cisplatin resistance of ovarian cancer through EGFR activation

Patients with ovarian cancer frequently develop acquired drug resistance after the long-term chemotherapy, leading to disease progression. Enhanced epithelial–mesenchymal transition (EMT) has been implicated in chemoresistance of ovarian cancer cells; however, the molecular mechanisms involved are largely undefined. Pyruvate dehydrogenase kinase 1 (PDK1), a key regulatory enzyme in glucose metabolism, has been recognized as a gatekeeper of the Warburg effect, a hallmark of cancer. In this study, the function of PDK1 in cisplatin resistance of ovarian cancer in terms of growth and EMT was investigated. PDK1 was upregulated in cisplatin-resistant ovarian cancer cells. PDK1 knockdown in resistant cells led to increased sensitivity to cisplatin-induced cell death and apoptosis. PDK1 downregulation also reversed the EMT and cell motility in cisplatin-resistant cells. In a mouse xenograft model, tumors derived from PDK1-silenced ovarian cancer cells exhibited decreased tumor growth and EMT compared with control after the cisplatin treatment. Mechanistically, PDK1 overexpression led to increased phosphorylation of EGFR, and blocking EGFR kinase activity by erlotinib reversed cisplatin resistance induced by PDK1 overexpression. Furthermore, in patients with ovarian cancer, higher PDK1 and p-EGFR levels were associated with chemoresistance. These results supported that PDK1 contributes to chemoresistance of ovarian cancer by activating EGFR. Therefore, PDK1 may serve as a promising target to combat chemoresistance of ovarian cancer.     

MiR-130a and MiR-374a Function as Novel Regulators of Cisplatin Resistance in Human Ovarian Cancer A2780 Cells

Chemoresistance remains a major obstacle to effective treatment in patients with ovarian cancer, and recently increasing evidences suggest that miRNAs are involved in drug-resistance. In this study, we investigated the role of miRNAs in regulating cisplatin resistance in ovarian cancer cell line and analyzed their possible mechanisms. We profiled miRNAs differentially expressed in cisplatin-resistant human ovarian cancer cell line A2780/DDP compared with parental A2780 cells using microarray. Four abnormally expressed miRNAs were selected (miR-146a,-130a, -374a and miR-182) for further studies. Their expression were verified by qRT-PCR. MiRNA mimics or inhibitor were transfected into A2780 and A2780/DDP cells and then drug sensitivity was analyzed by MTS array. RT-PCR and Western blot were carried out to examine the alteration of MDR1, PTEN gene expression. A total of 32 miRNAs were found to be differentially expressed in A2780/DDP cells. Among them, miR-146a was down-regulated and miR-130a,-374a,-182 were upregulated in A2780/DDP cells, which was verified by RT-PCR. MiR-130a and miR-374a mimics decreased the sensitivity of A2780 cells to cisplatin, reversely, their inhibitors could resensitize A2780/DDP cells. Furthermore, overexpression of miR-130a could increase the MDR1 mRNA and P-gp levels in A2780 and A2780/DDP cells, whereas knockdown of miR-130a could inhibit MDR1 gene expression and upregulate the PTEN protein expression .In a conclusion, the deregulation of miR-374a and miR-130a may be involved in the development and regulation of cisplatin resistance in ovarian cancer cells. This role of miR-130a may be achieved by regulating the MDR1 and PTEN gene expression.     

The negative feedback between miR-143 and DNMT3A regulates cisplatin resistance in ovarian cancer

Emerging evidence suggests that miR-143 plays an important role in the regulation of tumor sensitivity to chemotherapeutic agents. The study explores the underlying mechanism of miR-143 in reversing cisplatin resistance in ovarian cancer. The cisplatin-resistant ovarian cancer cell line A2780/CDDP was induced and established via treating A2780 cells by gradually increasing cisplatin concentrations. The IC₅₀ values of A2780/CDDP and A2780 to cisplatin were 218.10 ± 1.12 and 21.99 ± 1.12 μM, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that miR-143 was significantly decreased in A2780/CDDP cells compared with A2780 cells. miR-143 overexpression decreased cisplatin resistance in A2780/CDDP, and miR-143 inhibition decreased A2780 sensitivity to cisplatin. Results of qRT-PCR, Western blot analysis, and luciferase reporter assay indicated that the direct target of miR-143 was DNMT3A, which, in turn, was upregulated in A2780/CDDP. DNMT3A overexpression antagonized the sensitizing effect of miR-143 on A2780/CDDP to cisplatin. Knocking down of DNMT3A reduced cisplatin resistance in A2780/CDDP, while overexpression of DNMT3A increased cisplatin resistance in A2780. Methylation-specific polymerase chain reaction results showed that the methylation level in the promoter region of the miR-143 precursor gene was higher in A2780/CDDP cells than in A2780 cells. DNMT3A mediated the hypermethylation of the miR-143 precursor gene, resulting in miR-143 downregulation in A2780/CDDP. miR-143 inhibited cell growth of A2780/CDDP cell in nude mice. Our findings indicated the negative feedback between miR-143 and DNMT3A as a crucial epigenetic modifier of cisplatin resistance in ovarian cancer.     

Interference with SMO increases chemotherapy drug sensitivity of A2780/DDP cells by inhibiting the Hh/Gli signaling pathway

Aberrant activation of the Hedgehog (Hh)/Gli pathway contributes to the tumorigenesis of several human cancers, including ovarian cancers. We investigated the function of SMO on cell growth, drug resistance, and invasive ability in A2780/DDP cells. Moreover, we also tested the levels of the downstream target genes of the Hh/Gli pathway in SMO short hairpin RNA (shRNA) lentivirus-infected A2780/DDP cells. Western blot analysis results revealed that the Hh/Gli pathway was activated in cisplatin-resistant A2780/DDP cells. After infection by SMO shRNA lentivirus, the colony formation rate and invasive rate of cisplatin-resistant A2780/DDP cells were decreased. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that upon transfection with SMO shRNA, cell growth was decreased and drug sensitivity to cisplatin was upregulated. Moreover, interference with SMO decreased the expression of MMP-2, MMP-9, VEGF, and Snail in cisplatin-resistant cells. Thus, the Hh/Gli signaling pathway was aberrantly activated in A2780/DDP cells. The colony formation rate and invasive rate were decreased in SMO shRNA lentivirus–infected A2780/DDP cells. All results showed that inhibiting Hh/Gli signaling may negatively regulate the proliferation, invasion, and metastasis of cisplatin-resistant A2780/DDP cells, as well as increase the sensitivity of A2780/DDP to the chemotherapeutic drug of cisplatin.     

Biochemical and molecular properties of cisplatin-resistant A2780 cells grown in folinic acid

In this study, A2780 human ovarian carcinoma cells were grown in folinic acid in contrast to folic acid, and the molecular and biochemical properties of cisplatin-resistant A2780 cells were analyzed for changes in the dTMP synthase cycle. At concentrations of folinic acid that were optimal for cell growth (10(-8) M), the ED50 for cisplatin was 2.5 and 43 microM in the A2780S and A2780DDP cells, respectively. Resistance to cisplatin was associated with a 2-fold cross-resistance to 5-fluorodeoxyuridine and 5-fluorouracil as well as a 3-fold increase in both dTMP synthase activity and mRNA. The ED50 for methotrexate was similar in both A2780S and A2780DDP cells (1.2 microM). When both the A2780S and A2780DDP cells were grown in folinic acid, there was no significant difference in the level of dihydrofolate reductase activity. This data would suggest that cisplatin resistance is associated with changes in folate metabolism.     

AKT2 inhibition of cisplatin-induced JNK/p38 and Bax activation by phosphorylation of ASK1: implication of AKT2 in chemoresistance

Cisplatin and its analogues have been widely used for treatment of human cancer. However, most patients eventually develop resistance to treatment through a mechanism that remains obscure. Previously, we found that AKT2 is frequently overexpressed and/or activated in human ovarian and breast cancers. Here we demonstrate that constitutively active AKT2 renders cisplatin-sensitive A2780S ovarian cancer cells resistant to cisplatin, whereas phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 sensitizes A2780S and cisplatin-resistant A2780CP cells to cisplatin-induced apoptosis through regulation of the ASK1/JNK/p38 pathway. AKT2 interacts with and phosphorylates ASK1 at Ser-83 resulting in inhibition of its kinase activity. Accordingly, activated AKT2 blocked signaling down-stream of ASK1, including activation of JNK and p38 and the conversion of Bax to its active conformation. Expression of nonphosphorylatable ASK1-S83A overrode the AKT2-inhibited JNK/p38 activity and Bax conformational changes, whereas phosphomimic ASK1-S83D inhibited the effects of cisplatin on JNK/p38 and Bax. Cisplatin-induced Bax conformation change was inhibited by inhibitors or dominant negative forms of JNK and p38. In conclusion, our data indicate that AKT2 inhibits cisplatin-induced JNK/p38 and Bax activation through phosphorylation of ASK1 and thus, plays an important role in chemoresistance. Further, regulation of the ASK1/JNK/p38/Bax pathway by AKT2 provides a new mechanism contributing to its antiapoptotic effects.     

The anti-tumor effect of cross-reacting material 197, an inhibitor of heparin-binding EGF-like growth factor, in human resistant ovarian cancer

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. Cross-reacting material 197 (CRM197), a specific HB-EGF inhibitor, has been proven to represent possible chemotherapeutic agent for ovarian cancer. However, the effect of CRM197 on the resistant ovarian carcinoma cells has not been sufficiently elucidated. Here, we found that HB-EGF was over-expressed in a paclitaxel-resistant human ovarian carcinoma cell line (A2780/Taxol) and a cisplatin-resistant cell line (A2780/CDDP), as well as the xenograft mouse tissue samples with these cells. To investigate the possible significance of the HB-EGF over-expression in A2780/Taxol and A2780/CDDP cells, we inhibited HB-EGF expression by CRM197 to investigate the effect of CRM197 treatment on these cells. We observed that CRM197 significantly induced anti-proliferative activity in a dose-dependent manner with the cell-cycle arrest at the G0/G1 phase and enhanced apoptosis in A2780/Taxol and A2780/CDDP cells. The sensitive ovarian carcinoma parental cell line (A2780), A2780/Taxol and A2780/CDDP cells formed tumors in nude mice, and enhanced tumorigenicity was observed in drug-resistant tumors. Furthermore, we observed that CRM197 significantly suppressed the growth of drug-resistant ovarian cancer xenografts in vivo (p<0.001). These results suggest that CRM197 as an HB-EGF-targeted agent has potent anti-tumor activity in paclitaxel- and cisplatin-resistant ovarian cancer which over-express HB-EGF.     

Proteasome inhibitors sensitize ovarian cancer cells to TRAIL induced apoptosis

In the present study we have explored the sensitivity of ovarian cancer cells to TRAIL and proteasome inhibitors. Particularly, we have explored the capacity of proteasome inhibitors to bypass TRAIL resistance of ovarian cancer cells. For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/DDP and A2780/ADR, providing evidence that: (i) the three cell lines are either scarcely sensitive (A2780 and A2780/ADR) or moderately sensitive (A2780/DDP) to the cytotoxic effects of TRAIL; (ii) the elevated c-FLIP expression observed in ovarian cancer cells is a major determinant of TRAIL resistance of these cells; (iii) proteasome inhibitors (PS-341 or MG132) are able to exert a significant pro-apoptotic effect and to greatly enhance the sensitivity of both chemosensitive and chemoresistant A2780 cells to TRAIL; (iv) proteasome inhibitors damage mitochondria through stabilization of BH3-only proteins, Bax and caspase activation and significantly enhance TRAIL-R2 expression; (v) TRAIL-R2, but not TRAIL-R1, mediates the apoptotic effects of TRAIL on ovarian cancer cells. Importantly, studies on primary ovarian cancer cells have shown that these cells are completely resistant to TRAIL and proteasome inhibitors markedly enhance the sensitivity of these cells to TRAIL. Given the high susceptibility of ovarian cancer cells to proteasome inhibitors, our results further support the experimental use of these compounds in the treatment of ovarian cancer.     

Cucurbit[7]uril encapsulated cisplatin overcomes cisplatin resistance via a pharmacokinetic effect

The cucurbit[n]uril (CB[n]) family of macrocycles has been shown to have potential in drug delivery where they are able to provide physical and chemical stability to drugs, improve drug solubility, control drug release and mask the taste of drugs. Cisplatin is a small molecule platinum-based anticancer drug that has severe dose-limiting side-effects. Cisplatin forms a host-guest complex with cucurbit[7]uril (cisplatin@CB[7]) with the platinum atom and both chlorido ligands located inside the macrocycle, with binding stabilised by four hydrogen bonds (2.15-2.44 ?). Whilst CB[7] has no effect on the in vitro cytotoxicity of cisplatin in the human ovarian carcinoma cell line A2780 and its cisplatin-resistant sub-lines A2780/cp70 and MCP1, there is a significant effect on in vivo cytotoxicity using human tumour xenografts. Cisplatin@CB[7] is just as effective on A2780 tumours compared with free cisplatin, and in the cisplatin-resistant A2780/cp70 tumours cisplatin@CB[7] markedly slows tumour growth. The ability of cisplatin@CB[7] to overcome resistance in vivo appears to be a pharmacokinetic effect. Whilst the peak plasma level and tissue distribution are the same for cisplatin@CB[7] and free cisplatin, the total concentration of circulating cisplatin@CB[7] over a period of 24 hours is significantly higher than for free cisplatin when administered at the equivalent dose. The results provide the first example of overcoming drug resistance via a purely pharmacokinetic effect rather than drug design or better tumour targeting, and demonstrate that in vitro assays are no longer as important in screening advanced systems of drug delivery.     

Adenosine enhances cisplatin sensitivity in human ovarian cancer cells

Ovarian cancer is the deadliest gynecologic cancer due to lack of early effective diagnosis and development of resistance to platinum-based chemotherapy. Several studies reported that adenosine concentrations are higher in tumor microenvironment than in non-tumor tissue. This finding inspired us to study the role of adenosine in ovarian cancer cells and to investigate if adenosine pathways offer new treatment options urgently needed to prevent or overcome chemoresistance. The ovarian cancer cell lines HEY, A2780, and its cisplatin-resistant subline A2780CisR were used in this study. Expression and functional activity of adenosine receptors were investigated by RT-PCR, Western blotting, and cAMP assay. A1 and A2B adenosine receptors were expressed and functionally active in all three cell lines. Adenosine showed moderate cytotoxicity (MTT-IC₅₀ values were between 700 and 900 μM) and induced apoptosis in a concentration-dependent manner by increasing levels of sub-G1 and cleaved PARP. Apoptosis was diminished by QVD-OPh, confirming caspase-dependent induction of apoptosis. Forty-eight hours pre-incubation of adenosine prior to cisplatin significantly enhanced cisplatin-induced cytotoxicity in a synergistic manner and increased apoptosis. SLV320 or PSB603, selective A1 and A2B antagonists, was not able to inhibit adenosine-induced increase in cisplatin cytotoxicity or apoptosis whereas dipyridamole, a nucleoside transporter inhibitor, completely abrogated both effects. Mechanistically, adenosine increased pAMPK and reduced pS6K which was prevented by dipyridamole. In conclusion, application of adenosine prior to cisplatin could be a new therapeutic option to increase the potency of cisplatin in a synergistic manner and thus overcome platinum resistance in ovarian cancer.     

Contribution of intracellular ATP to cisplatin resistance of tumor cells

Decreased cellular accumulation of cisplatin is a frequently observed mechanism of resistance to the drug. Beside passive diffusion, several cellular proteins using ATP hydrolysis as an energy source are assumed to be involved in cisplatin transport in and out of the cell. This investigation aimed at clarifying the contribution of intracellular ATP as an indicator of energy-dependent transport to cisplatin resistance using the A2780 human ovarian adenocarcinoma cell line and its cisplatin-resistant variant A2780cis. Depletion of intracellular ATP with oligomycin significantly decreased cellular platinum accumulation (measured by flameless atomic absorption spectrometry) in sensitive but not in resistant cells, and did not affect cisplatin efflux in both cell lines. Inhibition of Na⁺,K⁺-ATPase with ouabain reduced platinum accumulation in A2780 cells but to a lesser extent compared with oligomycin. Western blot analysis revealed lower expression of Na⁺,K⁺-ATPase α₁ subunit in resistant cells compared with sensitive counterparts. The basal intracellular ATP level (determined using a bioluminescence-based assay) was significantly higher in A2780cis cells than in A2780 cells. Our results highlight the importance of ATP-dependent transport, among other processes mediated by Na⁺,K⁺-ATPase, for cisplatin influx in sensitive cells. Cellular platinum accumulation in resistant cells is reduced and less dependent on energy sources, which may partly result from Na⁺,K⁺-ATPase downregulation. Our data suggest the involvement of other ATP-dependent processes beside those regulated by Na⁺,K⁺-ATPase. Higher basal ATP level in cisplatin-resistant cells, which appears to be a consequence of enhanced mitochondrial ATP production, may represent a survival mechanism established during development of resistance.     

Proteomic analysis of cisplatin resistance in human ovarian cancer using 2-DE method

Platinum-based chemotherapy, such as cisplatin, is the primary treatment for human ovarian cancer. However, overcoming drug resistance has become an important issue in cancer chemotherapy. In this study, we performed 2-DE and ESI-Q-TOF MS/MS analysis to identify differential proteins expression between cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780-CP) ovarian cancer cell lines. Of the 14 spots identified as differentially expressed (±over twofold, P < 0.05) between the two cell lines, ten spots (corresponding to ten unique proteins) were positively identified by ESI-Q-TOF MS/MS analysis. These proteins include capsid glycoprotein, fructose-bisphosphate aldolase C, heterogeneous nuclear ribonucleoproteins A2/B1, putative RNA-binding protein 3, Ran-specific GTPase-activating protein, ubiquitin carboxyl-terminal hydrolase isozyme L1, stathmin, ATPSH protein, chromobox protein homolog3 and phosphoglycerate kinase 1. The proteins identified in this study would be useful in revealing the mechanisms underlying cisplatin resistance and also provide some clues for further research.     

Prolonged wild-type p53 protein accumulation and cisplatin resistance

The major limitation for the chemotherapeutic use of DNA-damaging agent cisplatin is the development of resistance in initially responsive tumors. One of the main pathways regulating cell survival following DNA damage is the p53 pathway. In this study we compared the cisplatin-induced response of p53 protein and its downstream targets p21WAF-1 and Mdm2 in the cisplatin-sensitive ovarian carcinoma cell line A2780 and its cisplatin-resistant derivative CP70. A higher dose of cisplatin and a longer exposure time was required to achieve the same level of p53, p21WAF-1, and Mdm2 protein accumulation in the cisplatin-resistant CP70 cells versus cisplatin-sensitive A2780 cells. A significant difference between the two cell lines was observed in cisplatin-induced stabilization of p53 protein. The p53 half-life increased 31-fold in CP70 cells compared to only 6-fold in A2780 cells. In contrast, there was no difference in p21WAF-1 half-life between the two cell lines. These results demonstrate that in A2780 and CP70 cells resistance to cisplatin correlates with prolonged p53 protein stabilization and accumulation.     

Alpha-TEA plus cisplatin reduces human cisplatin-resistant ovarian cancer cell tumor burden and metastasis

A novel nonhydrolyzable ether-linked acetic acid analog of vitamin E, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (alpha-TEA) in combination with cisplatin, reduces tumor burden of A2780/cp70 (cp70) cisplatin-resistant human ovarian cancer cells xenografted into immune compromised nude mice. Two xenograft studies were conducted using cp70 cells stably expressing green fluorescent protein (cp70-GFP) subcutaneously transplanted into NU/NU mice. For studies 1 and 2, alpha-TEA was formulated in liposomes and delivered by aerosol such that approximately 36 microg and 72 microg of alpha-TEA were deposited in the respiratory tract of each mouse each day, respectively. Cisplatin at 5 mg/kg was administered by intraperitoneal injections once weekly for the first 3 weeks in Study 1 and on the third and 10th days following treatment initiation in Study 2. The combination alpha-TEA + cisplatin treatment reduced tumor burden and metastasis of cp70-GFP cells in comparison to control mice or mice treated with alpha-TEA or cisplatin singly. A significant reduction (P < 0.001) in growth of subcutaneous transplanted tumors was obtained with alpha-TEA + cisplatin for both studies. Visible metastases were observed in the lungs of animals from control and cisplatin-treated groups but not in animals from the alpha-TEA- or alpha-TEA + cisplatin-treated groups. The alpha-TEA + cisplatin significantly reduced the total number of lung and axillary lymph node micrometastasis (P < 0.03 and P < 0.0001, respectively). Analyses of tumor sections showed the alpha-TEA + cisplatin treatment group, in comparison to control, to have a significantly lower level of cell proliferation (Ki-67 staining; P < 0.0001) and a significantly higher level of apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling [TUNEL]; P < 0.0001). In summary, combinations of alpha-TEA + cisplatin significantly reduced tumor burden and metastases in a xenograft model of cisplatin-resistant human ovarian cancer cells. These data show promise for combination alpha-TEA + cisplatin chemotherapy for ovarian cancer.     

Polyphenols bearing cinnamaldehyde scaffold showing cell growth inhibitory effects on the cisplatin-resistant A2780/Cis ovarian cancer cells

Ovarian carcinoma remains the most lethal among gynecological cancers. Chemoresistance is a clinical problem that severely limits treatment success. To identify potent anticancer agents against the cisplatin-resistant human ovarian cancer cell line A2780/Cis, 26 polyphenols bearing a cinnamaldehyde scaffold were synthesized. Structural differences in their inhibitory effect on clonogenicity of A2780/Cis cells were elucidated using comparative molecular field analysis and comparative molecular similarity indices analysis. Structural conditions required for increased inhibitory activity can be derived based on the analysis of their contour maps. The two most active compounds (16 and 19) were selected and further characterized their biological activities. We found that compounds 16 and 19 trigger cell cycle arrest at the G2/M phase and apoptotic cell death in cisplatin-resistant A2780/Cis human ovarian cancer cells. The molecular mechanism of compound 16 was elucidated using in vitro aurora A kinase assay, and the binding mode between the compound 16 and aurora A kinase was interpreted using in silico docking experiments. The findings obtained here may help us develop novel plant-derived polyphenols used for potent chemotherapeutic agents. In conclusion, compounds 16 and 19 could be used as promising lead compounds for the development of novel anticancer therapies in the treatment of cisplatin-resistant ovarian cancers.     

Mitochondrial proteomic analysis of Cisplatin resistance in ovarian cancer

Epithelial ovarian cancer (EOC) is the leading cause of death among women with gynecologic malignancies and accounts for approximately 6% of cancer deaths among women. Cisplatin and its analogues form the backbone of the most active chemotherapy regimens in advanced EOC; however, development of platinum resistance is common and typically marks a transition in which curing the patient is no longer possible. An emerging theme in many cancers is that mitochondrial dysfunction contributes to an aggressive carcinogenic phenotype. We hypothesized that changes in the mitochondrial proteome are required to support development of cisplatin resistance in human EOC. To investigate this hypothesis, an organellar proteomics approach was utilized to quantify alterations in protein abundance in mitochondria enriched from isogenic cisplatin-sensitive (A2780) and -resistant (A2780-CP20) human EOC cells. Protein isolates from mitochondria-enriched fractions were analyzed by high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), and relative abundance of identified proteins was quantified by spectral counting. Pathway analyses revealed significant increases in notch signaling pathways, cell survival, and alternate apoptotic pathways in the A2780-CP20 subtype. Among the alterations identified in the mitochondrial proteomic composition in cisplatin-resistant EOC cells, activated leukocyte cell adhesion molecule (AKAP12) and A kinase anchoring protein 12 (AKAP12) were elevated, while nestin was diminished in the mitochondrial fraction of A2780-CP20 relative to A2780. This was verified by immunoblot analysis. These results confirm that important changes in the mitochondrial proteome, many of which promote evasion of apoptosis and tumor invasiveness and metastasis, are present in cisplatin-resistant EOC.     

Bim protein degradation contributes to cisplatin resistance

Cisplatin is the first-line chemotherapy for the treatment of several cancers. However, the development of cisplatin resistance represents a major clinical problem, and the mechanisms of acquired resistance are not fully understood. Here we show that degradation of the Bcl-2 homology 3-only proapoptotic protein Bim plays an important role in cisplatin resistance in ovarian cancer. Specifically, we show that treatment of ovarian cancer cells with cisplatin caused Bim phosphorylation and subsequent degradation and that its degradation is associated with cisplatin resistance. We also show that cisplatin treatment caused the activation of ERK, which correlated with Bim phosphorylation and degradation. By inhibiting ERK phosphorylation with the MEK inhibitor and knocking down ERK expression with siRNA, we show that Bim phosphorylation and degradation were blocked, which suggests that Bim is phosphorylated by ERK and that such phosphorylation is responsible for cisplatin-induced Bim degradation. We show that ERK was activated in cisplatin-resistant OV433 cells as compared with their counterpart parental OV433 cells. We also show that Bim was phosphorylated and degraded in cisplatin-resistant OV433 cells but not in the parental OV433 cells. Importantly, we show that inhibition of Bim degradation by the proteasome inhibitor MG132 sensitized resistant OV433 cells to cisplatin-induced death. Taken together, our data indicate that degradation of Bim via ERK-mediated phosphorylation can lead to cisplatin resistance. Therefore, these findings suggest that cisplatin resistance can be overcome by the combination of cisplatin and the proteasome inhibitors in ovarian cancer cells.     

RNA interference-triggered reversal of ABCC2-dependent cisplatin resistance in human cancer cells

The adenosine triphosphate binding cassette (ABC)-transporter ABCC2 (MRP2/cMOAT) can mediate resistance against the commonly used anticancer drugs cisplatin and paclitaxel. To overcome the ABCC2-depending drug resistance, two specific anti-ABCC2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS. Since both siRNAs showed biological activity, for stable inhibition of ABCC2 a corresponding short hairpin RNA (shRNA)-encoding expression vector was designed. By treatment of A2780RCIS cells with this construct, the expressions of the targeted ABCC2 encoding mRNA and transport protein were inhibited. These effects were accompanied by reversal of resistance against cisplatin and paclitaxel. Thus, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that siRNA- and shRNA-mediated RNAi-based gene therapeutic approaches may be applicable in preventing and reversing ABCC2-depending drug resistance.     

Role of Autophagy in Cisplatin Resistance in Ovarian Cancer Cells

Cisplatin-based treatment is the first line chemotherapy for several cancers including ovarian cancer. The development of cisplatin resistance results in treatment failure, but the underlying mechanisms are not fully understood. Here we show that the induction of autophagy plays an important role in cisplatin resistance in ovarian cancer cells. Specifically, we show that cisplatin resistance is correlated with autophagy induction in a panel of ovarian cancer cells but not in immortalized human ovarian surface epithelial cells. Mechanistically, cisplatin treatment activates ERK and subsequently promotes autophagy. The inhibition of ERK activation with MEK inhibitors or knockdown of ERK expression with siRNA decreases cisplatin-induced autophagy and subsequently sensitizes ovarian cancer cells to cisplatin-induced apoptosis. In ovarian cancer cells that have developed acquired cisplatin resistance, both ERK activation and autophagy induction are increased. Importantly, knockdown of ERK or inhibition of autophagy promotes cisplatin-induced apoptosis in acquired cisplatin-resistant cells. Collectively, our data indicate that ERK-mediated autophagy can lead to cisplatin resistance and suggest that cisplatin resistance can be overcome by inhibition of autophagy in ovarian cancer cells.