Cisplatin-resistant Human Ovarian Cancer Cells Research Articles

Abstract 4135: Cisplatin resistance promotes cell motility and migration through EGF receptor-mediated Erk and Stat3 pathways in human ovarian cancer cells

Abstract Ovarian cancer causes the highest gynecologic neoplasm-related deaths in the United States. A common feature of ovarian cancer is the wide dissemination of the disease in the peritoneum at the time of presentation by the patient, which poses an added challenge to therapy in terms of the ability to completely eliminate the disease tissue. Moreover, disease recurrence is common and frequently associated with drug resistance. Cisplatin has been the most active drug for the treatment of ovarian cancer for the last decades. Although the majority of ovarian cancer patients respond to Cisplatin therapy, most develop recurrent and resistant disease, raising the need to understand the molecular mechanisms that support the resistant phenotype and to identify novel effective therapeutic and chemo-sensitizing modalities. Here, we developed Cisplatin resistance in vitro to address the molecular and biological mechanisms that may be presented by the recurrent disease. The cell characteristic changes and the molecular basis of resistance to Cisplatin were investigated using the Cisplatin-resistant human ovarian cancer cell lines, A2780S/CP1, A2780S/CP3 and A2780S/CP5, which were derived from the parental, Cisplatin-sensitive line, A2780S and are resistant to 1, 3 and 5 μM Cisplatin, respectively. Cell morphology under bright-field microscope showed marked changes in the Cisplatin-resistant cells compared to that of the parental cells. Notably, wound healing and migration assays showed significantly enhanced cell motility and migration properties in Cisplatin-resistant clones. Furthermore, resistance to Cisplatin enhanced the ability of cells to form colonies. Molecular mechanistic analysis indicated that Cisplatin resistance in A2780S/CP3 and A2780S/CP5 cells dramatically activated the EGF receptor (EGFR)-Erk1/2MAPK and EGFR-Stat3 pathways, and upregulated the expression of cellular survival genes such as Bcl-xL and Survivin. Accordingly, treatment of the Cisplatin-resistant A2780S/CP3 and A2780S/CP5 lines with the EGFR inhibitor, Iressa or the Stat3 inhibitor, S3I-201 significantly attenuated colony survival and cell migration. Moreover, resistance to Cisplatin led to the upregulation of the expression of F-actin and cortactin, and promoted the assembly of F-actin at the front of lamellipodia in A2780S/CP3 and A2780S/CP5 lines. These results suggest that Cisplatin resistance in ovarian cancer is mediated in part through the activation of the EGF receptor-mediated Erk and Stat3 pathways that promote cell survival, motility and migration. These findings raise the possibility that EGFR or Stat3 inhibitors combined with Cisplatin may be an effective therapeutic approach for Cisplatin-resistant ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4135.

Abstract 1256: Protein kinase G type Iα activity in human ovarian cancer cells contributes to enhanced Src activation and DNA synthesis/cell proliferation

Abstract We have previously shown that basal activity of the nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG) signaling pathway protects human ovarian cancer cells against spontaneous apoptosis and confers resistance to cisplatin-induced apoptosis. The present study determines whether basal PKG activity up-regulates Src and induces cell proliferation in human ovarian cancer cells. PKG-Iα was identified as the predominant isoform of PKG expressed in both cisplatin-sensitive OV2008 and cisplatin-resistant A2780cp human ovarian cancer cells. In both cell lines, both ODQ, an inhibitor of endogenous-NO-induced cGMP biosynthesis, and DT-2, highly-specific inhibitor of PKG-Iα activity, as well as PKG siRNA caused concentration-dependent inhibition of DNA synthesis (assessed by BrdU incorporation) under normal (plus-serum) growing conditions, indicating an important role of basal cGMP/PKG-Iα activity in promoting cell proliferation. DNA synthesis in OV2008 cells was dependent on Src activation, determined using selective Src inhibitor, 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline (SKI-1). In vitro co-incubation of human recombinant Src and PKG-Iα resulted in Src-mediated tyrosine-phosphorylation of PKG-Iα and enhanced Src autophosphorylation at tyrosine-416, suggesting that Src directly tyrosine-phosphorylates PKG-Iα and the interaction between PKG-Iα and Src also leads to enhanced Src activation. The result was further confirmed by using immunoprecipitation of PKG, showing a concentration-dependent decrease in PKG tyrosine-phosphorylation by SKI in both ovarian cancer cell lines. In intact OV2008 cells, inhibition of basal PKG-Iα activity with DT-2 dramatically reduced endogenous Src activation and completely blocked the elevation of DNA synthesis stimulated by EGF. The data suggest that Src activation and DNA synthesis/cell proliferation in human ovarian cancer cells are dependent on endogenous basal PKG-Iα activity. The NO/cGMP/PKG-Iα signaling pathway may provide a novel therapeutic target for disrupting the proliferation of ovarian cancer cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1256.

Targeted quantitative analysis of superoxide dismutase 1 in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells

Protein quantification in a complex protein mixture presents a daunting task in biochemical analysis. Antibody-based immunoassays are traditional methods for protein quantification. However, there are issues associated with accuracy and specificity in these assays, especially when the changes are small (e.g., <2-fold). With recent developments in mass spectrometry, monitoring a selected peptide, thus protein, in a complex biological sample has become possible. In this study, we demonstrate a simple mass spectrometry-based method for selective measurement of a moderately low abundant protein, superoxide dismutase 1 (SOD1), in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells. Selected-reaction-monitoring (SRM) technology was employed to specifically analyze the target peptides in a pair of human ovarian cancer cell lines: 2008/2008-C13*5.25 (cisplatin-sensitive/cisplatin-resistant, respectively). The observed 1.47-fold higher expression in the resistant cell line is consistent with findings by other approaches. This robust liquid chromatography/mass spectrometry (LC/MS) method provides a powerful tool for targeted proteomic verification and/or validation studies.

Tangeretin Sensitizes Cisplatin-Resistant Human Ovarian Cancer Cells through Downregulation of Phosphoinositide 3-Kinase/Akt Signaling Pathway

Combination of innocuous dietary components with anticancer drugs is an emerging new strategy for cancer chemotherapy to increase antitumor responses. Tangeretin is a citrus flavonoid known to inhibit cancer cell proliferation. Here, we show an enhanced response of A2780/CP70 and 2008/C13 cisplatin-resistant human ovarian cancer cells to various combination treatments of cisplatin and tangeretin. Pretreatment of cells with tangeretin before cisplatin treatment synergistically inhibited cancer cell proliferation. This combination was effective in activating apoptosis via caspase cascade as well as arresting cell cycle at G(2)-M phase. Moreover, phospho-Akt and its downstream substrates, e.g., NF-kappaB, phospho-GSK-3beta, and phospho-BAD, were downregulated upon tangeretin-cisplatin treatment. The tangeretin-cisplatin-induced apoptosis in A2780/CP70 cells was increased by phosphoinositide-3 kinase (PI3K) inhibition and siRNA-mediated Akt silencing, but reduced by overexpression of constitutively activated Akt and GSK-3beta inhibition. The overall results indicated that tangeretin exposure preconditions cisplatin-resistant human ovarian cancer cells for a conventional response to low-dose cisplatin-induced cell death occurring through downregulation of PI3K/Akt signaling pathway. Thus, effectiveness of tangeretin combinations, as a promising modality in the treatment of resistant cancers, warrants systematic clinical studies.

Collateral sensitivity to novel thymidylate synthase inhibitors correlates with folate cycle enzymes impairment in cisplatin-resistant human ovarian cancer cells

The cytotoxicity of two novel folate cycle inhibitors with quinoxalinic structure, 3-methyl-7-trifluoromethyl-2(R)-[3,4,5-trimethoxyanilino]-quinoxaline (453R) and 3-piperazinilmethyl-2[4(oxymethyl)-phenoxy]quinoxaline (311S), was tested against a panel of both cisplatin(cDDP)-sensitive and -resistant carcinoma cell lines. Interestingly, the cisplatin-resistant human ovarian line, C13⁎ cells, exhibited collateral sensitivity towards the two compounds when compared to its sensitive parental 2008 cells. In this resistant line, which showed elevated expression of the folate cycle enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR), due to cisplatin-resistance phenotype, collateral sensitivity correlated with the greater reduction of enzyme expression. In addition, TS and DHFR expression of the other resistant lines, the human ovarian carcinoma A2780/CP cells and the human breast cancer MDA/CH cells, were decreased in accordance with the similar sensitivity or the low level of cross-resistance to these compounds in comparison to their respective parental lines. Noteworthy, unlike 5-fluorouracil, both drugs reduced the level of TS without inducing ternary complex formation with the co-substrate and the nucleotide analogue. Median effect analysis of the interactive effects of cisplatin with the two quinoxalines mainly showed additive or synergistic cell killing, depending on schedules of drug combinations. In particular, synergistic effects were more often obtained, even on the resistant cells, when cisplatin was added at the beginning of the treatment. These results indicate that, despite the possibility of other mechanisms being involved, inhibition of TS cycle enzymes plays an important role in the pharmacology of these compounds, which might also represent a useful component in drug treatment protocols against cDDP-resistant cells.

GnRH analog resensitizes cisplatin-resistant human ovarian cancer cells

Objective:To study the effect of GnRH analog triptorelin in resensitizing cisplatin-resistant human ovarian canc- er cells and to discuss the related mechanism.Methods:Cisplatin-resistant human ovarian cancer cell line OVCAR-3/CDDP was established in vitro.MTT assay was used to assess the inhibitory effects of triptorelin,cisplatin alone or a combination of both on OVCAR-3/CDDP cells.Flow cytometry was employed to observe the expression changes in epidermal growth factor receptor (EGFR)in different groups.Results:The drug restant index of OVCAR-3/CDDP cells was 13.42.The resensitizing fold of cisplatin combined with triptorelin was 3.80.The expression of EGFR had the most prominent decrease in OVCAR-3/CDDP cells in the combination group.Conclusion:Triptorelin can partially resensitize cisplatin-resistant OVCAR-3/CDDP cells,which might be related to the down-regulation of EGFR.

Targeting the Folate Receptor (FR): Imaging and Cytotoxicity of ReI Conjugates in FR‐Overexpressing Cancer Cells

The synthesis, characterization, in vitro imaging, and cytotoxic properties of a new folate conjugate of rhenium(I) are reported. The conjugate [FA-PEG-BQAV-Re(CO)3]+ (gamma-4) was screened against an adriamycin- and cisplatin-resistant human ovarian cancer cell line (A2780/AD) that overexpresses the folate receptor (FR). Compound gamma-4 was internalized by a folate-receptor-mediated endocytotic pathway, which results in internal accumulation of gamma-4. This was contrasted with a FR-negative Chinese hamster ovary cell line in which no internalization of gamma-4 was observed. gamma-4 was found to be cytotoxic with IC(50) values of 189 and 78 microM at 6 and 24 h, respectively, toward the FR-positive cell line. This is in contrast to the IC(50) value of 502 microM at 6 h and 84 microM at 24 h for cisplatin in the same cell line, with a significantly greater toxicity at the earlier time point. The cytotoxicity of gamma-4 as explained by interactions that occur between the rhenium(I) complex moiety and DNA is described.

Cisplatin alters nitric oxide synthase levels in human ovarian cancer cells: involvement in p53 regulation and cisplatin resistance

The present study determines if (1) basal protein levels of nitric oxide (NO) synthases (eNOS, iNOS, and nNOS) are different in cisplatin-sensitive (OV2008) and counterpart cisplatin-resistant (C13*) human ovarian cancer cells, (2) cisplatin alters NOS levels, (3) NO donor causes apoptosis and p53 upregulation, (4) NO donor sensitises C13* cells to cisplatin via p53 upregulation (determined by p53 siRNA gene-knockdown), and (5) inhibition of endogenous NOS alters cisplatin-induced apoptosis. Basal iNOS levels were higher in OV2008 cells than in C13* cells. Cisplatin upregulated iNOS, but dramatically reduced eNOS and nNOS, in OV2008 cells only. Failure of cisplatin to upregulate iNOS and downregulate eNOS/nNOS in cisplatin-resistant C13* cells may be an aetiological factor in the development of resistance. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased p53 protein levels and induced apoptosis in both cell types, and enhanced cisplatin-induced apoptosis in C13* cells in a p53-dependent manner (i.e., enhancement blocked by p53 siRNA). Specific iNOS inhibitor 1400W partially blocked cisplatin-induced apoptosis in OV2008 cells. In cisplatin-resistant C13* cells, blocking all NOSs with NG-amino-L-arginine dramatically changed these cells from cisplatin-resistant to cisplatin-sensitive, greatly potentiating cisplatin-induced apoptosis. The data suggest important roles for the three NOSs in regulating chemoresistance to cisplatin in ovarian cancer cells.

NCX-4016, a nitro-derivative of aspirin, inhibits EGFR and STAT3 signaling and mdulates Bcl-2 proteins in cisplatin-resistant human ovarian cancer cells and xenografts

We have previously reported the inhibitory effect of NCX-4016, a nitro derivative of aspirin, on the proliferation of cisplatin-resistant human ovarian cancer cells, in vitro (Bratasz et al., Proc Natl Acad Sci USA 2006; 103:3914-9). In this report we present the results of our study on the mechanistic aspects of drug action including the molecular and signaling pathways involved in an in vitro cell line, as well as in a murine tumor xenograft. We report, for the first time, that NCX-4016 significantly inhibited the growth of cisplatin-resistant human ovarian cancer xenografts in mice. We observed that the inhibitory effect of NCX-4016 on cell proliferation was associated with G1 phase cell-cycle arrest with increased activity of p53, p21 and p27 proteins. NCX-4016 modulated the Bcl-2 family of proteins, and induced apoptosis by activating Bax and cytochrome c release in a time-dependent manner. In addition, NCX-4016 selectively down-regulated the phosphorylated forms of EGFR (Tyr845, Tyr992), pAkt (Ser473, Thr305), and STAT3 (Tyr705, Ser727), in vitro and in vivo. Taken together, the results clearly suggested that NCX-4016 causes significant induction of cell-cycle arrest and apoptosis in cisplatin-resistant human ovarian cancer cells via down-regulation of EGFR/PI3K/STAT3 signaling and modulation of Bcl-2 family proteins. Thus, NCX-4016 appears to be a potential therapeutic agent for treating recurrent human ovarian carcinoma.

A potent cytotoxic photoactivated platinum complex.

We show by x-ray crystallography that the complex trans, trans, trans-[Pt(N(3))(2)(OH)(2)(NH(3))(py)] (1) contains an octahedral Pt(IV) center with almost linear azido ligands. Complex 1 is remarkably stable in the dark, even in the presence of cellular reducing agents such as glutathione, but readily undergoes photoinduced ligand substitution and photoreduction reactions. When 1 is photoactivated in cells, it is highly toxic: 13-80 x more cytotoxic than the Pt(II) anticancer drug cisplatin, and ca. 15 x more cytotoxic toward cisplatin-resistant human ovarian cancer cells. Cisplatin targets DNA, and DNA platination levels induced in HaCaT skin cells by 1 were similar to those of cisplatin. However, cisplatin forms mainly intrastrand cis diguanine cross-links on DNA between neighboring nucleotides, whereas photoactivated complex 1 rapidly forms unusual trans azido/guanine, and then trans diguanine Pt(II) adducts, which are probably mainly intrastrand cross-links between two guanines separated by a third base. DNA interstrand and DNA-protein cross-links were also detected. Importantly, DNA repair synthesis on plasmid DNA platinated by photoactivated 1 was markedly lower than for cisplatin or its isomer transplatin (an inactive complex). Single-cell electrophoresis experiments also demonstrated that the DNA damage is different from that induced by cisplatin or transplatin. Cell death is not solely dependent on activation of the caspase 3 pathway, and, in contrast to cisplatin, p53 protein did not accumulate in cells after photosensitization of 1. The trans diazido Pt(IV) complex 1 therefore has remarkable properties and is a candidate for use in photoactivated cancer chemotherapy.

Nitroaspirin (NCX-4016), an NO Donor, is Antiangiogenic Through Induction of Loss of Redox-Dependent Viability and Cytoskeletal Reorganization in Endothelial Cells

We recently reported that NCX-4016, a derivative of aspirin containing a nitro moiety that releases nitric oxide (NO) in a sustained fashion in biologic systems, is a potent cytotoxic agent inhibiting the proliferation of cisplatin-resistant human ovarian cancer cells. Therefore, we hypothesize that NCX-4016 possesses antiangiogenic properties. Our study with the bovine lung microvascular endothelial cells (BLMVECs) revealed that NCX-4016 significantly induced the loss of redox-dependent cell viability in a dose- and time-dependent manner, as assayed by the redox-sensitive Alamar blue cell viability assay. Fluorescence microscopy of cells labeled with NO-specific fluorophore (DAF-FM) confirmed that NCX-4016 generated significant levels of intracellular NO. NO donors, including S-nitroso-N-acetylpenicillamine, spermine NONOate, and isosorbide dinitrite, were less effective in causing loss of cell viability. Thiol-protectant, N-acetylcysteine, significantly attenuated the NCX-4016-induced loss of cell viability, suggesting the role of alteration of thiol-redox status therein. NCX-4016 also suppressed oxygen consumption, decreased transendothelial electrical resistance (EC barrier dysfunction), and induced actin cytoskeletal reorganization in BLMVECs. The in vitro assay with human umbilical vein ECs and BLMVECs revealed that NCX-4016, in a dose-dependent manner, significantly inhibited angiogenesis with almost complete inhibition at a 100-microM concentration, suggesting that NCX-4016 can act as an antiangiogenic drug.

EF24 Induces G2/M Arrest and Apoptosis in Cisplatin-resistant Human Ovarian Cancer Cells by Increasing PTEN Expression

We report that EF24, a synthetic compound 3,5-bis(2-flurobenzylidene)piperidin-4-one, greatly inhibits cisplatin-resistant (CR) human ovarian cancer cell proliferation. The inhibitory effect of EF24 on cell proliferation is associated with G(2)/M phase cell cycle arrest and increased G(2)/M checkpoint protein (pp53, p53, and p21) levels. Within 24 h following treatment, EF24 induced apoptosis in CR cells. The apoptosis was partially blocked by the general caspase inhibitor z-VAD. Within 12 h, EF24 induced a membranous FasL expression, consistent with a substantial decrease in the Ser(473) and Thr(308) phosphorylation of Akt, a known negative regulator of FasL transcription. Also, EF24 activated the phosphorylated PTEN and marginally up-regulated total PTEN expression through the inhibition of ubiquitin-mediated PTEN degradation. Suppression of PTEN expression with siRNA significantly reduced the p53 and p21 levels and activated Akt phosphorylation at Ser(473) and Thr(308), resulting in decreased apoptosis and increased cell survival. On the other hand, overexpression of PTEN markedly induced apoptosis. Our results clearly suggested that EF24 induced significant increase in PTEN expression. The up-regulation of PTEN inhibited Akt and MDM2, which enhanced the level of p53, thereby inducing G(2)/M arrest and apoptosis. Therefore, EF24 appears to have a potential therapeutic role in human ovarian cancer through the activation of PTEN.

Abrogation of MAPK and Akt Signaling by AEE788 Synergistically Potentiates Histone Deacetylase Inhibitor-Induced Apoptosis through Reactive Oxygen Species Generation

To evaluate the effects of combining the multiple receptor tyrosine kinase inhibitor AEE788 and histone deacetylase (HDAC) inhibitors on cytotoxicity in a broad spectrum of cancer cell lines, including cisplatin-resistant ovarian adenocarcinoma cells. Multiple cancer cell lines were treated in vitro using AEE788 and HDAC inhibitors (LBH589, LAQ824, and trichostatin A), either alone or in combination. Effects on cytotoxicity were determined by growth and morphologic assays. Effects of the combination on cell signaling pathways were determined by Western blotting, and the results were confirmed using pathway-specific inhibitors and transfection of constitutively active proteins. Cell treatment with AEE788 and HDAC inhibitors (LBH589, LAQ824, and trichostatin A) in combination resulted in synergistic induction of apoptosis in non-small cell lung cancer (MV522, A549), ovarian cancer (SKOV-3), and leukemia (K562, Jurkat, and ML-1) cells and in OV202hp cisplatin-resistant human ovarian cancer cells. AEE788 alone or in combination with LBH589 inactivated mitogen-activated protein kinase (MAPK) and Akt cascades. Inhibition of either MAPK and/or Akt enhanced LBH589-induced apoptosis. In contrast, constitutively active MAPK or Akt attenuated LBH589 or LBH589 + AEE788-induced apoptosis. Increased apoptosis was correlated with enhanced reactive oxygen species (ROS) generation. The free radical scavenger N-acetyl-l-cysteine not only substantially suppressed the ROS accumulation but also blocked the induction of apoptosis mediated by cotreatment with AEE788 and LBH589. Collectively, these results show that MAPK and Akt inactivation along with ROS generation contribute to the synergistic cytotoxicity of the combination of AEE788 and HDAC inhibitors in a variety of human cancer cell types. This combination regimen warrants further preclinical and possible clinical study for a broad spectrum of cancers.

Curcumin induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer cells by modulating akt and p38 mAPK

Curcumin, a major active component of turmeric, is known to induce apoptosis in several types of cancer cells, but little is known about its activity in chemoresistant cells. Hence, the aim of the present study was to investigate the anticancer properties of curcumin in cisplatin-resistant human ovarian cancer cells in vitro. The results indicated that curcumin inhibited the proliferation of both cisplatin-resistant (CR) and sensitive (CS) human ovarian cancer cells almost equally. Enhanced superoxide generation was observed in both CR and CS cells treated with curcumin. Curcumin induced G2/M phase cell-cycle arrest in CR cells by enhancing the p53 phosphorylation and apoptosis through the activation of caspase-3 followed by PARP degradation. Curcumin also inhibited the phosphorylation of Akt while the phosphorylation of p38 MAPK was enhanced. In summary, our results showed that curcumin inhibits the proliferation of cisplatin-resistant ovarian cancer cells through the induction of superoxide generation, G2/M arrest, and apoptosis.

Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells

Cisplatin is an effective antitumor agent for the treatment of several carcinomas. However, the development of resistance to cisplatin represents a serious clinical problem. The effects of auranofin, a gold(I) compound clinically used as an antirheumatic agent, on cisplatin-sensitive (2008) and-resistant (C13*) cancer cells were studied. Auranofin is more effective than cisplatin in decreasing cell viability and its action is particularly marked in C13* cells, indicating that no cross-resistance occurs. Furthermore, auranofin is able to permeate C13* cells more efficiently than 2008 cells. Treatment with auranofin determines a consistent release of cytochrome c in both cell lines, while cisplatin is effective only in sensitive cells. Both auranofin and cisplatin induce apoptosis in 2008 cells, while in C13* cells only auranofin is effective. Apoptosis is accompanied by an increased production of hydrogen peroxide that, however, is inhibited by N-acetyl-L-cysteine. In resistant cells, H 2O 2 production is counteracted by a large overexpression of thioredoxin reductase that constitutes the preferred target of the inhibitory action of auranofin. This specific effect of auranofin might rationalize its ability in overcoming cisplatin resistance in human ovarian cancer cells.

Reversing chemoresistance in cisplatin-resistant human ovarian cancer cells: A role of c-Jun NH 2-terminal kinase 1

To investigate the role of activation of c-Jun NH 2-terminal kinase 1 (JNK1) in mediating cisplatin-induced apoptosis and the possibility of induction of JNK activity in triggering relation to DNA damage and drug resistance. We investigated the difference of cisplatin-induced activation of JNK pathway and H 2O 2 alteration between cisplatin-sensitive human ovarian carcinoma cell line A2780 and its resistant variant A2780/DDP. JNK, p-JNK protein, and extracellular H 2O 2 levels were determined in both A2780 and A2780/DDP cells which were transfected with dominant negative allele of JNK and recombinant JNK1 separately. Both A2780 and A2780/DDP were treated with CDDP, the JNK pathway was activated and a prolonged JNK activation was maintained for at least 12 h in A2780, and only a transient activation (3 h) was detected in A2780/DDP in response to cisplatin treatment. Inhibition of JNK activity by transfection with a dominant negative allele of JNK blocked CDDP-induced apoptosis significantly in A2780 cells. Selective stimulation of the JNK pathway by lipofectamine-mediated delivery of recombinant JNK1 led to activation of c-Jun and decrease of extracellular H 2O 2, as well as apoptosis sensitization to CDDP in A2780/DDP cells. We concluded that JNK pathway might play an important role in mediating cisplatin-induced apoptosis in A2780 cells, and the duration of JNK activation might be critical in determining whether cells survive or undergo apoptosis. The resistance to CDDP can be reversed through activating c-Jun and decreasing extracellular generation of H 2O 2 by pcDNA3(FLAG)-JNK1-wt transfection in A2780/DDP cells.

Ultrastructure alterations in adriamycin-resistant and cisplatin-resistant human ovarian cancer cell lines exposed to nonlethal ultrasound

Human ovarian cancer cell lines, SKOV3 and its adriamycin-resistant substrain SKOV3/ADR and COC1 and its cisplatin-resistant substrain COC1/DDP, were exposed to nonlethal ultrasound. Ultrastructures in sham-insonated and insonated cells were inspected by transmission electron microscopy, and cytochrome C in cytosol was determined by enzyme-linked immunosorbent assay. Ultrasound exposure led to no significant changes in SKOV3/ADR cells, but tumid mitochondria occurred in SKOV3 cells. Mitochondria changes were also detected in some exposed COC1 and COC1/DDP cells. Apoptotic bodies could be detected in either control or insonated COC1/DDP cells. A few exposed COC1/DDP cells became reticular. Cytochrome C in cytosol in exposed SKOV3/ADR cells was increased but that in exposed COC1/DDP cells was decreased. These findings revealed that the bioeffect of ultrasound on chemosensitive cells was not identical to that of chemoresistant ones, and ultrasound was a potential approach for treatment of drug-resistant ovarian cancers.

Antiproliferative effect of β-elemene in chemoresistant ovarian carcinoma cells is mediated through arrest of the cell cycle at the G2-M phase

Elemene is a natural antitumor plant drug. However, the effect of elemene on cell growth in ovarian cancer is unknown. In this study, we show that beta-elemene inhibited the proliferation of cisplatin-resistant human ovarian cancer cells and their parental cells, but had only a marginal effect in human ovary cells, indicating differential inhibitory effects on cell growth between ovarian cancer cells and normal ovary cells. We also demonstrated for the first time that beta-elemene markedly enhanced cisplatin-induced growth inhibition in resistant cells compared to sensitive cells. In addition, cell cycle analysis revealed a synergistic effect of beta-elemene and cisplatin on the induction of cell cycle G2-M arrest in our resistant ovarian carcinoma cells. Furthermore, we showed that treatment of these cells with both drugs downregulated cyclin B1 and Cdc2 expression, but elevated the levels of p53, p21waf1/cip1, p27kip1 and Gadd45. Finally, the combination of beta-elemene and cisplatin was found to increase the phosphorylation of Cdc2 and Cdc25C, which leads to a reduction in Cdc2-cyclin B1 activity. These novel findings suggest that beta-elemene sensitizes chemoresistant ovarian carcinoma cells to cisplatin-induced growth suppression partly through modulating the cell cycle G2 checkpoint and inducing cell cycle G2-M arrest, which lead to blockade of cell cycle progression.

Ultrastructure alterations in adriamycin-resistant and cisplatin-resistant human ovarian cancer cell lines exposed to nonlethal ultrasound

Human ovarian cancer cell lines, SKOV3and its adriamycin-resistant substrain SKOV3/ADR and COC1 and its cisplatin-resistant substrain COC1/DDP, were exposed to nonlethal ultrasound. Ultrastructures in sham-insonated and insonated cells were inspected by transmission electron microscopy, and cytochrome C in cytosol was determined by enzyme-linked immunosorbent assay. Ultrasound exposure led to no significant changes in SKOV3/ADR cells, but tumid mitochondria occurred in SKOV3cells. Mitochondria changes were also detected in some exposed COC1 and COC1/DDP cells. Apoptotic bodies could be detected in either control or insonated COC1/DDP cells. A few exposed COC1/DDP cells became reticular. Cytochrome C in cytosol in exposed SKOV3/ADR cells was increased but that in exposed COC1/DDP cells was decreased. These findings revealed that the bioeffect of ultrasound on chemosensitive cells was not identical to that of chemoresistant ones, and ultrasound was a potential approach for treatment of drug-resistant ovarian cancers.

Reversal of multidrug resistance by small interfering double-stranded RNAs in ovarian cancer cells

Objective Cisplatin (DDP) resistance is a major barrier to overcome before chemotherapy can become curative for most patients presenting with ovarian cancer. In this study, we investigated the effect of siRNAs on expression of p-gp, GST-pi mRNA and protein in cisplatin-resistant human ovarian cancer cells in order to restore sensitivity to DDP. Methods Small interfering double-stranded RNAs (siRNA) were designed to target p-glycoprotein (p-gp) and glutathione S-transferases (GST) mRNA as a strategy to inhibit both resistant gene expression at the mRNA level. Using Real-Time PCR and western blotting assay the changes of the RNA and protein levels of both drug resistant genes were studied. Results Transfection of MDR-1 and GST siRNAs into human multi-drug resistance (MDR) ovarian cancer cell lines, COC1/DDP and SKOV3/DDP, resulted in a time-dependent inhibition of both gene expressions with the decline of the IC 50 values but had no effect on the expression of a-Tubulin. Inhibition of P-gp and GST expression by siRNA enhanced the intracellular accumulation of and restored sensitivity to DDP. Conclusions These studies suggest that p-gp and GST siRNAs are effective inhibitors of MDR gene expression and reverse the resistance of ovarian carcinomas. Our studies may provide a new insight to develop siRNAs as a novel therapeutic tool for the treatment of ovarian carcinomas.