Treatment with paclitaxel alone rather than combination with paclitaxel and cisplatin may be selective for cisplatin-resistant ovarian carcinoma

BACKGROUND: We have previously reported that paclitaxel (taxol) results in cisplatin sensitization to human ovarian cancer cells with cisplatin resistance in vitro. This study was designed to determine effects of taxol and its combination with cisplatin on growth of cisplatin-sensitive cell line (KF28) and the cisplatin-resistant counterpart (KFr13) in nude mice. METHODS: From 14 days after tumor inoculation treatment was initiated. Taxol (3 mg/kg) and cisplatin (2 mg/kg) were administered i.p. once a week for 5 weeks. RESULTS: In nude mice bearing cisplatin-sensitive cells (KF28), taxol followed by cisplatin and cisplatin plus taxol inhibited significantly (P < 0.05) the tumor growth rate compared with that in nude mice treated with cisplatin alone or taxol alone and cisplatin followed by taxol. On the other hand, in nude mice bearing cisplatin-resistant KFr13 cells, treatment with taxol alone inhibited completely the tumor growth rate, whereas no schedule-dependent interaction of taxol with cisplatin was observed. CONCLUSION: These results suggest that treatment with taxol alone may be superior to combination of taxol with cisplatin in patients with cisplatin-resistant ovarian carcinoma.     

Ovarian serous carcinomas acquire cisplatin resistance and increased invasion through downregulation of the high-temperature-required protein A2 (HtrA2), following repeated treatment with cisplatin

High-temperature-required protein A2 (HtrA2) is one of the serine proteases related to apoptosis. HtrA2 protein expression has been associated with cisplatin resistance and poor prognosis in ovarian serous adenocarcinoma (SAC). The aim of this study was to understand the influence of HtrA2 on repeated treatment with cisplatin. The change in HtrA2 expression in 31 ovarian cancers was investigated by immunohistochemical analysis, before and after cisplatin-based chemotherapy, and the association between HtrA2 expression after chemotherapy and prognosis was analyzed. The association between the change in HtrA2 and proteins associated with LATS1 in ovarian serous cancer cell lines after repeated treatment with cisplatin was evaluated in vitro. In immunohistochemical analysis, repeated cisplatin treatment induced downregulation of HtrA2 protein expression, before and after cisplatin-based chemotherapy in SAC. Progression-free survival and overall survival of SAC with low expression of HtrA2 were worse than those with high expression. In vitro analysis using cisplatin-sensitive ovarian cancer cell lines, KF28, and cisplatin-resistant cancer cell lines, KFr13, obtained from KF28 by repeated cisplatin treatment, showed that HtrA2 protein expression was lower in KFr13 than in KF28. Furthermore, KFr13 had a higher invasive capacity than KF28. Next, downregulation of HtrA2 transfected with an HtrA2-specific siRNA in KF28 had not only cisplatin resistance, but also more invasive capacity than those with non-specific siRNA. Repeated treatment with cisplatin downregulated the HtrA2 protein, which led to cisplatin resistance and increased invasive capacity. Thus, HtrA2 might be a biomarker of response to cisplatin treatment and prognosis, after cisplatin-based chemotherapy.     

Characteristic expression of globotriaosyl ceramide in human ovarian carcinoma-derived cells with anticancer drug resistance

The transporter protein genes and lipids in human ovarian carcinoma-derived KF28 cells with anticancer-drug-sensitive properties were compared with those in resistant cells, taxol-resistant KF28TX, cisplatin-resistant KFr13, and taxol- and cisplatin-resistant KFr13TX, to identify the molecules required for anticancer-drug resistance. In accordance with previous reports, taxol and cisplatin resistance was closely correlated with expression of the multidrug resistance 1 and bile acid export pump, and multidrug resistance-associated protein 2 genes, respectively. In addition, we found a distinct difference in glycosphingolipids between the sensitive and resistant cells. Although GlcCer was the major glycolipid (83.0%) in sensitive cells, GalCer, LacCer and, particularly, Gb(3)Cer were characteristically increased in all resistant cells, irrespective of whether the resistance was to taxol or cisplatin, and comprised 65-84% of total glycosphingolipids. GM3, which was present at 0.04 microg/mg dry weight in the sensitive cells, showed a twofold increase in the taxol-resistant cells, but was absent in the cisplatin-resistant cells. The altered glycolipid composition was proven to be due to enhanced or suppressed expression of the respective sugar transferase genes. In addition, the ceramide moiety of ceramide monohexoside in the sensitive cells constituted 83% of non-hydroxy fatty acids, but that in the resistant cells comprised 67-74% of alpha-hydroxy fatty acids. Thus, cells containing Gb(3)Cer with alpha-hydroxy fatty acids were found to survive selectively in the presence of taxol and cisplatin, and modification of the glycolipid structure was revealed to occur in association with anticancer-drug resistance.     

A systematic review of platinum and taxane resistance from bench to clinic: an inverse relationship

We undertook a systematic review of the pre-clinical and clinical literature for studies investigating the relationship between platinum and taxane resistance. Medline was searched for (1) cell models of acquired drug resistance reporting platinum and taxane sensitivities and (2) clinical trials of platinum or taxane salvage therapy in ovarian cancer. One hundred and thirty-seven models of acquired drug resistance were identified. 68.1% of cisplatin-resistant cells were sensitive to paclitaxel and 66.7% of paclitaxel-resistant cells were sensitive to cisplatin. A similar inverse pattern was observed for cisplatin vs. docetaxel, carboplatin vs. paclitaxel and carboplatin vs. docetaxel. These associations were independent of cancer type, agents used to develop resistance and reported mechanisms of resistance. Sixty-five eligible clinical trials of paclitaxel-based salvage after platinum therapy were identified. Studies of single agent paclitaxel in platinum-resistant ovarian cancer where patients had previously recieved paclitaxel had a pooled response rate of 35.3%, n=232, compared to 22% in paclitaxel na?ve patients n=1918 (p<0.01, Chi-squared). Suggesting that pre-treatment with paclitaxel may improve the response of salvage paclitaxel therapy. The response rate to paclitaxel/platinum combination regimens in platinum-sensitive ovarian cancer was 79.5%, n=88 compared to 49.4%, n=85 for paclitaxel combined with other agents (p<0.001, Chi-squared), suggesting a positive interaction between taxanes and platinum. Therefore, the inverse relationship between platinum and taxanes resistance seen in cell models is mirrored in the clinical response to these agents in ovarian cancer. An understanding of the cellular and molecular mechanisms responsible would be valuable in predicting response to salvage chemotherapy and may identify new therapeutic targets.     

Cellular efflux pump and interaction between cisplatin and paclitaxel in ovarian cancer cells

OBJECTIVE: The aim of this study was to evaluate the combination effect of paclitaxel (PTX) and cisplatin (CDDP) and to determine the mechanisms of interaction between these agents. METHODS AND RESULTS: We used human ovarian adenocarcinoma cell lines, namely a parent cell line (KF), a CDDP-resistant cell line (KFr) and a PTX-resistant cell line (KFTx).The combination effect of PTX and CDDP was synergistic on KF and KFTx and additive on KFr. The incidence of anaphase or telophase, evaluated by immunofluorescence microscopy, decreased with PTX and significantly decreased with PTX and CDDP in KF and KFTx. The concentration of PTX, which was measured by high-performance liquid chromatography, was higher in KF and KFTx cells treated with a combination of PTX and CDDP than those treated with PTX alone. Multidrug resistance gene mRNA appeared in KFTx and its expression decreased after exposure to PTX and CDDP. After exposure to CDDP, the expression of multidrug resistance-associated protein (MRP) and the concentration of glutathione increased in KF, but not in KFr or KFTx. MRP expression slightly increased in KF and KFTx after exposure to PTX. In contrast, its expression decreased in KFr. CONCLUSION: The present study suggests that CDDP enhances PTX accumulation and that the interaction of these agents is synergistic in CDDP-sensitive cells.     

Cross-talk between signalling pathways and the multidrug resistant protein MDR-1.

The multidrug resistant protein MDR-1 has been associated with the resistance to a wide range of anti-cancer drugs. Taxol is a substrate for this transporter system and is used in the treatment of a wide range of human malignancies including lung, breast and ovarian cancer. We have generated a series of ovarian cell lines resistant to this compound, all of which overexpress MDR-1 through gene amplification. We present novel evidence that a constitutive activation of the ERK1/2 MAP kinase pathway was also observed although the level of active JNK and p38 remained unchanged. Inhibition of the ERK1/2 MAP kinase pathway using UO126 or PD098059 re-sensitised the Taxol resistant cells at least 20-fold. Importantly, when Mdr-1 cDNA was stably expressed in the wild-type cell line to generate a highly Taxol-resistant sub-line, 1847/MDR5, ERK1/2 MAP kinases again became activated. This result demonstrated that the increased activity of the signalling pathway in the Taxol-resistant lines was directly attributable to MDR-1 overexpression and was not due to the effects of Taxol itself. Additionally, we demonstrated that inhibition of the P13K pathway with LY294002 sensitised the MDR-1-expressing 1847/TX0.5 cells and 1847/MDR5 cells at least 10-fold but had no effect in the wild-type cells. This finding suggests a possible role for this pathway, also, in the generation of resistance to Taxol.     

The mechanism of acquired resistance to cisplatin by a human ovarian cancer cell line

The present study was designed to elucidate the mechanism of resistance to cisplatin. A cisplatin-resistant cell line (KFr) was established from KF cells derived from human serous cystadenocarcinoma of the ovary. The DNA histogram revealed an increase of S-phase cells and a decrease of G1-phase cells in cultured KFr cells, compared to that in cultured KF cells. Although the cisplatin content in the KF cells incubated with cisplatin at 10 micrograms/ml increased in a time-dependent manner, that in the KFr cells remained unchanged during the experimental period. When 0.5 mg of cisplatin was administered ip to nude mice with KF or KFr tumor, the cisplatin content in the KFr tumor was significantly lower than that in the KF tumor. The KFr cells showed a cross-resistance to L-phenylalanine mustard, while no cross-resistance to vincristine or 5-fluorouracil was observed. These findings suggest that the mechanism of cisplatin resistance in the KFr cells involves a decrease of cisplatin accumulation in the tumor cells.     

The Mechanism of Acquired Resistance to Cisplatin by a Human Ovarian Cancer Cell Line

The present study was designed to elucidate the mechanism of resistance to cisplatin. A cisplatin-resistant cell line (KFr) was established from KF cells derived from human serous cystadenocarcinoma of the ovary. The DNA histogram revealed an increase of S-phase cells and a decrease of G₁-phase cells in cultured KFr cells, compared to that in cultured KF cells. Although the cisplatin content in the KF cells incubated with cisplatin at 10 μg/ml increased in a time-dependent manner, that in the KFr cells remained unchanged during the experimental period. When 0.5 mg of cisplatin was administered ip to nude mice with KF or KFr tumor, the cisplatin content in the KFr tumor was significantly lower than that in the KF tumor. The KFr cells showed a cross-resistance to L-phenylalanine mustard, while no cross-resistance to vincristine or 5-fluorouracil was observed. These findings suggest that the mechanism of cisplatin resistance in the KFr cells involves a decrease of cisplatin accumulation in the tumor cells.     

人紫杉醇耐药细胞株LoVo/TAX的建立及其耐药机制研究

目的:以体外建立的人结肠癌紫杉醇（paclitaxel,TAX）耐药细胞株LoVo/TAX为模型,探讨结肠癌紫杉醇获得性耐药潜在的分子机制。方法:对LoVo亲本细胞及LoVo/TAX耐药细胞进行对比分析,采用光镜及透射电镜观察细胞形态,MTT法检测细胞药物敏感性,流式细胞术检测细胞周期分布及细胞凋亡水平,实时荧光定量PCR方法和Western blot方法分别检测多药耐药相关基因、微管蛋白相关基因以及凋亡和细胞周期调控相关基因的转录和翻译水平。结果:与亲本细胞相比,LoVo/TAX对紫杉醇高度耐药,细胞形态也有明显变化,其G2/M期阻滞降低(P<0.05),凋亡率显著下降(P<0.01),MRP、MAP-Tau和CDK1基因表达水平提高(P<0.05)。结论:多药耐药相关蛋白MRP1、微管相关蛋白MAP-Tau以及细胞周期蛋白依赖性激酶1（CDK1）过表达可能是引起结肠癌细胞对紫杉醇耐药的主要原因。     

Short hairpin RNA-mediated MDR1 gene silencing increases apoptosis of human ovarian cancer cell line A2780/taxol

Objective: Recurrent ovarian cancer is often resistant to drugs such as paclitaxel. Short hairpin RNA (shRNA) targeting MDR1, a gene involved in the process of drug resistance, may be a promising strategy to overcome drug resistance. Methods: Construction and identification of eukaryotic expression plasmid of shRNA targeting on MDR1 gene. The plasmid was transiently transfected into human ovarian cancer cell line A2780/Taxol. Apoptosis was determined by flow cytometry using annexin V-FITC/PI double labeling. Expression of MDR1 mRNA was detected by quantitative polymerase chain reaction (qPCR) and P-glycoprotein expression was detected using Western blot. Results: The IC50 of paclitaxel in MDR1 shRNA-transfected group was significantly reduced (1.986±0.153) μmol/ml as compared with that in negative control (5.246±0.107) μmol/ml and empty vector-transfected group (5.212±0.075) μmol/ml (P<0.05). The percent of the relative reverse sensitivity to paclitaxel on A2780/Taxol cells was 67.1%, and the apoptotic rate was significantly increased [(6.977±0.333)%] compared with control [(1.637±0.111)%] and empty vector-transfected group [(1.663±0.114)%] (P<0.05). Expressions of MDR1 mRNA and P-glycoprotein were significantly reduced compared with control (P<0.05). Conclusion: The present study demonstrated that the eukaryotic expression plasmid of shRNA targeting on MDR1 inhibited the expression of MDR1 effectively, thus enhance the sensitivity of A2780/Taxol cells to paclitaxel.     

Survivin deregulation in beta-tubulin mutant ovarian cancer cells underlies their compromised mitotic response to taxol

Taxol is one of the most successful drugs for the treatment of cancer because of its ability to target tubulin, block cell cycle progression at mitosis, and induce apoptosis. Despite the success of Taxol, the development of drug resistance hampers its clinical applicability. Herein we report that beta-tubulin mutant, Taxol-resistant ovarian cancer cells exhibit defective mitotic response to Taxol, even at high concentrations that are sufficient to trigger apoptosis. This mitotic response-defective phenotype is independent of p53 status. We have found that survivin, the mitosis regulator and inhibitor of apoptosis protein, is deregulated in these Taxol-resistant cancer cells; Taxol fails to induce survivin levels and survivin phosphorylation in these cells, in contrast to their parental drug-sensitive counterparts. Exogenous expression of wild-type survivin is able to restore the mitotic response of the resistant cells to Taxol treatment. On the other hand, exogenous expression of dominant-negative survivin abrogates the Taxol-induced mitotic response in drug-sensitive cancer cells. We have also found that overexpression of the mitotic kinase Cdk1, which phosphorylates survivin, is unable to restore the Taxol-induced mitotic response in the resistant cells. Our results show the importance of survivin for the mitotic response in the context of Taxol resistance and provide novel insights into the mechanisms of mitotic arrest and apoptosis induced by microtubule-targeting agents.     

Altered Expression of γ-Glutamylcysteine Synthetase, Metallothionein and Topoisomerase I or II during Acquisition of Drug Resistance to Cisplatin in Human Ovarian Cancer Cells

This study was designed to elucidate the mechanisms of cisplatin (CDDP) resistance using two human ovarian cancer cell lines, KF and TYK, and two CDDP-resistant lines, KFr and TYK/R, derived from the former lines. KFr and TYK/R showed about 3-fold higher resistance to the cytotoxic effects of CDDP than their parental lines. They also showed a significant increase in sensitivity to not only etoposide, but also (+)-(4S)-4, ll-diethyl-4-hydroxy-9-[(4-piperidino-piperidino)carbonyloxy]-l H -pyrano[3',4':6,7]inodolizino[l,2- b ]quinoline-3,14(4 H , 12 H )-dione hydrochloride trihydrate (CPT-11). Cellular CDDP accumulation levels in KFr and TYK/R were decreased from those of the parental cells. By contrast, the cellular glutathione (GSH) content in KFr cells was 1.7-fold higher than that in KF, whereas TYK/R cells had a 40% lower content than TYK cells. Cellular mRNA levels of drug-resistance-related genes, such as DNA topoisomerase (topo) I and topo II, glutathione S-transferase-π (GST-π;), γ-glutamylcysteine synthetase (.γ -GCS ), and metallothionein ( hMT ) genes, were compared between drug-sensitive KF or TYK and KFr or TYK/R. KFr cells had 8.5- and 24.7-fold higher mRNA levels of γ-GCS and topo II genes than KF cells while KFr had only a slight increase in GST-π mRNA level as compared with KF. By contrast, TYK/R cells had 2.9- and 1.7-fold higher hMT and topo I mRNA levels than TYK cells. Acquisition of CDDP resistance in human ovarian cancer cells thus appeared to be related mainly to expression of γ -GCS, topo II and hMT genes, and partly to that of topo I and GST-π genes, in addition to a decrease in CDDP accumulation     

Gene therapy for ovarian cancer using adenovirus-mediated transfer of cytosine deaminase gene and uracil phosphoribosyltransferase gene directed by MDR1 promoter

OBJECTIVE: To evaluate the specific killing effects of the adenoviral vector in which the CD::UPP genes were directed by the MDR1 promoter on Taxol-resistance ovarian cancer cells in vitro and in vivo. METHODS: Taxol-resistance (A2780/Taxol, SKOV3/Taxol) ovarian cancer cells and Taxol-sensitive (A2780, SKOV3) ovarian cancer cells were infected with adenovirus vector carrying the CD::UPP gene driven by the MDR1 promoter, followed with 5-fluorocytosine administration. Sensitivity to 5-fluorocytosine (5-FC) was analyzed. The AdMDR1-CD::UPP was subcutaneously injected into the xenografts of the nude mice and 5-FC intraperitoneally. The overall survival and anti-tumor effects were observed. RESULTS: In vitro, AdMDR1-CD::UPP showed a stronger cytotoxicity in A2780/Taxol cells and SKOV3/Taxol cells than that in A2780 cells and SKOV3 cells. Subcutaneous injection of AdMDR1-CD::UPP into the xenografts of mice bearing tumors of A2780/Taxol cells could significantly suppress the tumor growth and prolong survival as compared with the group of A2780 cells. CONCLUSION: AdMDR1-CD::UPP in combination with 5-FC is an effective approach to suppress the growth of Taxol-resistant ovarian cancer.     

Lonidamine as a modulator of taxol activity in human ovarian cancer cells: effects on cell cycle and induction of apoptosis

The ability of lonidamine (LND), an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxicity of Taxol (TX) was investigated in the A2780 human ovarian cancer cell line. Different cytotoxicity results were obtained as a function of treatment schedule. Specifically, TX followed by LND produced synergistic effects. Conversely, antagonistic effects were recorded when drugs were given simultaneously or according to the opposite sequence. TX induced an oligonucleosomal DNA fragmentation typical of the apoptotic process. The extent and the kinetics of DNA cleavage in samples treated with the taxane alone were similar to those of samples treated with the TX-LND sequence. Activation of Yama protease and degradation of poly (ADP-ribose) polymerase were not observed after individual or combined treatment. LND did not appreciably modify the effect exerted by TX on proteins involved in cell cycle progression (i.e., inhibition of p34^cdc2 expression) and apoptosis (i.e., upregulation of wt p53 and transactivation of p21^waf1), and only caused a slight induction of the Bax protein. LND alone did not affect tubulin polymerization in A2780 cells and, when administered after a 24 hr TX exposure, did not appreciably alter the extent of tubulin polymerization induced by the taxane. Although additional studies are needed to define the molecular basis of the TX-LND interaction, our results suggest that LND can positively modulate the antitumor activity of TX in ovarian cancer cells and indicate that the energolytic is potentially useful in combination therapy including the taxane in ovarian cancer patients. Int. J. Cancer 78:377–384, 1998.© 1998 Wiley-Liss, Inc.     

Combination effect of adenovirus-mediated pro-apoptotic bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines

To develop a novel therapeutic strategy for ovarian cancer, we constructed a recombinant adenovirus which highly expresses pro-apoptotic Bax protein and examined its therapeutic effect on a series of ovarian cancer cell lines: A2780, A2780/cDDP, OVCAR-3 and SK-OV-3. A recombinant adenovirus carrying the Bax-alpha gene (AxCALNKYbax) induced high expression of the Bax-alpha protein in all the cell lines. The cytotoxic effect of Bax was observed in three ovarian cancer cell lines: the per cent reduction in the number of cells was 40.0% for cisplatin-sensitive A2780, 50.0% for cisplatin-resistant A2780/cDDP, and 64.8% for marginally cisplatin-resistant OVCAR-3. In contrast, it was only 12.3% for cisplatin-resistant SK-OV-3. Cisplatin-resistant A2780/cDDP had a p53 mutation and exhibited attenuated Bax induction after cisplatin treatment, which may explain why supplementation of Bax was effective in this chemoresistant ovarian cancer. Combination with cisplatin or paclitaxel enhanced the cytotoxic effect of Bax induction in all but one cell line including cisplatin-resistant A2780/cDDP. It appears that adenovirus-mediated Bax induction, with or without combination with conventional chemotherapy, useful strategy for the treatment of ovarian cancer.     

Docetaxel in the management of ovarian cancer

Standard first-line treatment for Stage IC-IV ovarian cancer is currently a platinum agent or a combination of a platinum agent with a taxane. The use of a taxane compound in addition to single-agent platinum is increasingly preferred to platinum alone. In countries such as the UK, the taxane paclitaxel has been approved by the government for first-line use. However, it has yet to receive US Food and Drug Administration approval in the USA for use in this context. Typically, in countries such as the UK, patients with advanced ovarian cancer receive a combination of paclitaxel and carboplatin first line, both drugs given 3-weekly by intravenous infusion. Subsequent trials have demonstrated that the second-generation taxane docetaxel can be used as a substitute for paclitaxel; sharing many of its actions but with a different toxicity profile. However, docetaxel has not yet received approval for standard use. Here, the clinical development of docetaxel and its present and future place in the management of ovarian cancer is discussed.     

The prognostic significance of specific HOX gene expression patterns in ovarian cancer

HOX genes are vital for all aspects of mammalian growth and differentiation, and their dysregulated expression is related to ovarian carcinogenesis. The aim of the current study was to establish the prognostic value of HOX dysregulation as well as its role in platinum resistance. The potential to target HOX proteins through the HOX/PBX interaction was also explored in the context of platinum resistance. HOX gene expression was determined in ovarian cancer cell lines and primary EOCs by QPCR, and compared to expression in normal ovarian epithelium and fallopian tube tissue samples. Statistical analysis included one‐way ANOVA and t‐tests, using statistical software R and GraphPad. The analysis identified 36 of the 39 HOX genes as being overexpressed in high grade serous EOC compared to normal tissue. We detected a molecular HOX gene‐signature that predicted poor outcome. Overexpression of HOXB4 and HOXB9 was identified in high grade serous cell lines after platinum resistance developed. Targeting the HOX/PBX dimer with the HXR9 peptide enhanced the cytotoxicity of cisplatin in platinum‐resistant ovarian cancer. In conclusion, this study has shown the HOX genes are highly dysregulated in ovarian cancer with high expression of HOXA13, B6, C13, D1 and D13 being predictive of poor clinical outcome. Targeting the HOX/PBX dimer in platinum–resistant cancer represents a potentially new therapeutic option that should be further developed and tested in clinical trials.