Paclitaxel-resistant Ovarian Carcinoma Cell Research Articles

Anticancer regimens containing third generation taxanes SB-T-121605 and SB-T-121606 are highly effective in resistant ovarian carcinoma model.

Taxanes are widely used in the treatment of ovarian carcinomas. One of the main problems with conventional taxanes is the risk of development of multidrug resistance. New-generation synthetic experimental taxoids (Stony Brook Taxanes; SB-T) have shown promising effects against various resistant tumor models. The aim of our study was to compare the in vitro efficacy, intracellular content, and in vivo antitumor effect of clinically used paclitaxel (PTX) and SB-Ts from the previously tested second (SB-T-1214, SB-T-1216) and the newly synthesized third (SB-T-121402, SB-T-121605, and SB-T-121606) generation in PTX resistant ovarian carcinoma cells NCI/ADR-RES. The efficacy of the new SB-Ts was up to 50-times higher compared to PTX in NCI/ADR-RES cells in vitro. SB-T-121605 and SB-T-121606 induced cell cycle arrest in the G2/M phase much more effectively and their intracellular content was 10-15-times higher, when compared to PTX. Incorporation of SB-T-121605 and SB-T-121606 into therapeutic regimens containing PTX were effective in suppressing tumor growth in vivo in NCI/ADR-RES based mice xenografts at small doses (≤3mg/kg), where their adverse effects were eliminated. In conclusion, new SB-T-121605 and SB-T-121606 analogs are promising candidates for the next phase of preclinical testing of their combination therapy with conventional taxanes in resistant ovarian carcinomas.

Reverse of the resistance to paclitaxel of the heparin binding-epidermal growth factor-like growth factor inhibitor in ovarian cancer

Objective: To investigate the effect and mechanism of CRM197, the heparin binding-epidermal growth factor-like growth factor (HB-EGF) inhibitor, on the reverse of the resistance of ovarian cancer to paclitaxel. Methods: (1)The effect of CRM197 on the 50% inhibitory concentrations (IC(50)) of human ovarian carcinoma cell line A2780 and paclitaxel-resistant ovarian carcinoma cell line A2780/Taxol was tested by methyl thiazolyl tetrazolium (MTT) assay. Western blot was used to detect the effect of CRM197 on the expression of HB-EGF, epidermal growth factor receptor (EGFR) and plasma membrane glycoprotein (P-gp) protein in A2780 and A2780/Taxol cells. Real-time PCR was used to examine the MDR1 mRNA expression in these cells. (2) A2780/Taxol cells were divided into 4 groups, including the cells transfected with empty vector and saline treatment (empty vector group), MDR1 small interference RNA (siRNA) vector and saline treatment (MDR1 siRNA group), empty vector and CRM197 treatment (empty vector+CRM197 group) and MDR1 siRNA vector and CRM197 treatment (MDR1 siRNA+CRM197 group), respectively. Flow cytometry was used to detecte the effect of intracellular rhodomine 123 (Rh123) accumulation, and caspase-3 activity assay was used to test the effect of apoptosis in four groups of A2780/Taxol cells. (3) In experiments in vivo, A2780/Taxol cells were inoculated to nude mouse subcutaneously to determine the EGFR and P-gp protein expression following CRM197 treatment by immunohistochemistry. Results: (1) In vitro, MTT examination showed that the IC(50) of A2780/Taxol cells to paclitaxel in A2780/Taxol+CRM197 group [(6.4±0.3) μmol/L] was significantly lower than the IC(50) in A2780/Taxol group [ (34.1±0.5) μmol/L, P<0.01], and the reveral fold of CRM197 was 5.3. The expression level of HB-EGF protein in A2780/Taxol+CRM197 group (1.44±0.29) was significantly lower than HB-EGF protein in A2780/Taxol group (2.72±0.32), respectively (P<0.05). The expression level of EGFR protein (0.71±0.25) and P-gp protein (0.82±0.19) in A2780/Taxol+CRM197 group was significantly lower than EGFR protein (1.87±0.31) and P-gp protein (1.84±0.27) of A2780/Taxol group (P<0.05). Compared with A2780/Taxol group (1.78±0.27) , MDR1 mRNA was significantly down-regulated in A2780/Taxol+CRM197 group (0.79±0.13, P<0.05). (2) The fluorescence intensity of Rh123 of the A2780/Taxol cells in empty vector group, MDR1 siRNA group,empty vector+CRM197 group, MDR1 siRNA+CRM197 group was 33.4±1.6, 56.3±3.3, 43.5±3.1,100.4±7.4, and the pNA of the A2780/Taxol cells was (11.4±1.2) , (52.8±0.9) , (71.2±3.6) , (82.7±3.8) μmol/L. The expression levels in MDR1 siRNA+CRM197 group were both higher than the expression levels in empty vector+CRM197 group, and the expression levels in empty vector+CRM197 group, MDR1 siRNA group were both higher than the expression levels in empty vector group (P<0.05). (3) In vivo, the expression scores of EGFR protein in A2780/Taxol+CRM197 tumors (4.4±1.4) were lower than that in A2780/Taxol tumors (10.2±3.1, P<0.05). The expression scores of P-gp protein in A2780/Taxol+CRM197 tumors (3.8±1.1) were lower than that in A2780/Taxol tumors (8.8±2.7, P<0.05). Conclusion: CRM197 reverses the resistance of ovarian cancer to paclitaxel by increasing caspase-3 activity to advance apoptosis via EGFR/MDR1/P-gp pathway.

Cross-reacting material 197 reverses the resistance to paclitaxel in paclitaxel-resistant human ovarian cancer.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been proven to be a promising chemotherapeutic target for ovarian cancer. Our previous studies have demonstrated that inhibition of HB-EGF by the special inhibitor, cross-reacting material 197 (CRM197), potently inhibits the anti-tumor activity in paclitaxel-resistant ovarian cancer. Here, we found that inhibition of HB-EGF by CRM197 significantly reverses the resistance to paclitaxel in paclitaxel-resistant ovarian carcinoma cell line (A2780/Taxol). A2780/Taxol cells over-expressed HB-EGF and epidermal growth factor receptor (EGFR) and CRM197 notably suppressed the expression of HB-EGF and EGFR. Experiments performed in vitro and in vivo further suggested that CRM197 markedly down-regulated the ATP-binding cassette sub-family B member 1 (ABCB1/MDR1) messenger RNA (mRNA) expression (P = 0.01), plasma membrane glycoprotein (P-gp) protein (P = 0.009), and P-gp-mediated efflux (P = 0.007) through inhibition of nuclear factor-κB (NF-κB) expression, which were classical chemoresistance-related targets with respect to paclitaxel therapy. Meanwhile, inhibition of HB-EGF enhanced caspase-3 activity to induce apoptosis via MDR1 inhibition in A2780/Taxol cells (P = 0.038). Collectively, HB-EGF is a molecular target for the resistance of ovarian cancer to paclitaxel and CRM197 as a HB-EGF-targeted agent might be a chemosensitizing agent for paclitaxel-resistant ovarian carcinoma. Our findings provide novel possible mechanisms for HB-EGF to be a target to restore the chemosensitivity to paclitaxel.

HMSH2 expression is associated with paclitaxel resistance in ovarian carcinoma, and inhibition of hMSH2 expression invitro restores paclitaxel sensitivity.

The objective of the present study was to investigate the association between paclitaxel resistance, gene copy number, and gene expression in ovarian carcinoma, and to restore paclitaxel sensitivity in a paclitaxel-resistant ovarian carcinoma cell line by using hMSH2-targeting siRNA. Paclitaxel-resistant ovarian carcinoma cell lines OC3/TAX300 and OC3/TAX50 and their parental cell lines were analyzed by comparative genomic hybridization, and the expression levels of hMSH2 in ovarian carcinoma cell lines and tissues were determined. An siRNA targeted to hMSH2 mRNA was used to transfect a paclitaxel-resistant cell line. We assessed the morphological features, proliferation, and susceptibility to apoptosis of the transfected cells after paclitaxel treatment. Chromosome 2p21(gene locus of hMSH2) was amplified in OC3/TAX300 cells. hMSH2 was overexpressed in 93.9 and 47.6% of paclitaxel-treated and untreated ovarian carcinoma tissue samples(P=0.0001), respectively. hMSH2 was overexpressed in 93.3 and 54.2% of low-differentiated and moderate-to-highly differentiated ovarian carcinoma tissue samples(P=0.0008), respectively. hMSH2 expression was inhibited in the OC3/TAX300 cells transfected with hMSH2 siRNA. hMSH2 siRNA increased paclitaxel sensitivity, inhibited OC3/TAX300 cell proliferation(G2/M arrest), and increased susceptibility to apoptosis. hMSH2 expression was upregulated in ovarian carcinoma cell lines and tissues after paclitaxel treatment. hMSH2 overexpression is related to paclitaxel resistance and poor prognosis. Inhibition of hMSH2 expression invitro restores paclitaxel sensitivity in paclitaxel‑resistant ovarian carcinoma cell lines and indicates a new direction in adjuvant therapy for ovarian carcinoma.

Overexpression of Glycogen Synthase Kinase-3 in Ovarian Carcinoma Cells With Acquired Paclitaxel Resistance

Acquired resistance to paclitaxel, including regimens, is one of the most significant reasons for treatment failure and death in patients with ovarian cancer, but the causes of this resistance remain unclear. However, cell cycle regulation is a key mechanism by which most chemotherapeutic agents exert their cytotoxic effects. We created a paclitaxel-resistant ovarian carcinoma cell line from SKOV3 cell line, and the difference of cell cycle distribution was analyzed using flow cytometry. Analysis of human cell cycle pathway complementary DNA array was performed to identify candidate genes associated with paclitaxel resistance. Gene expression changes were validated at the messenger RNA and protein levels by real-time reverse transcriptase polymerase chain reaction and Western analysis, respectively. The ratio of Gap0/Gap1 phase in SKOV3-TR30 was significantly lower than that in SKOV3 (54.8% ± 6.3% vs 72.7% ± 7.6%, P = 0.035), and the ratio of G2/M phase in SKOV3-TR30 was significantly higher than that in SKOV3 (24.9% ± 6.0% vs 10.2% ± 3.5%, P = 0.021). Complementary DNA microarray analysis demonstrated enhanced glycogen synthase kinase-3α (GSK-3α) expression in paclitaxel-resistant ovarian carcinoma cells. Real-time reverse transcriptase polymerase chain reaction analysis revealed that the paclitaxel-resistant subline exhibited a 7.0 ± 1.8-fold increase in GSK-3α messenger RNA expression. There was a 3.34 ± 0.47-fold increase of total GSK-3 protein (GSK-3α/β) in SKOV3-TR30 cells validated by Western analysis. This study demonstrates that enhanced expression of GSK-3 is associated with acquired resistance to paclitaxel in ovarian carcinoma cells. Glycogen synthase kinase-3 overexpression may probably be a significant contributor to chemoresistance.

The relationship between p38MAPK and apoptosis during paclitaxel resistance of ovarian cancer cells

To investigate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and cell apoptosis during the paclitaxel resistance of ovarian carcinoma cell lines, flow cytometry (FCM) and PI staining were employed to determine the effect of p38MAPK inhibitor SB203580 on the apoptosis of A2780/Taxol cells, a drug-resistant human ovarian carcinoma cell line. p38MAPK protein expression in SB203580-treated cells was immunochemically measured. The 50% inhibition concentration (IC(50)) of paclitaxel on A2780/Taxol cells was determined by MTT assay. MDR-1 mRNA, and expression of p38MAPK and phospho-p53 protein were detected by RT-PCR and Western blotting, respectively. The apoptosis rate of A2780/Taxol cells was (19.7+/-1.04)% 24 h after SB203580 treatment. A significant difference in apoptosis rate was found among experiment group, control group and untreated group (P<0.05). The relative reversal rate of A2780/Taxol cells to paclitaxel was (57.18+/-2.01)%. As compared with the control group and the untreated group, p38MAPK protein and MDR-1 mRNA in SB203580-treated cells was substantially decreased. The expression of p53 protein was significantly increased. It is concluded that p38MAPK pathway is related to paclitaxel resistance of ovarian carcinoma, and blockade of this pathway can promote the apoptosis of the drug-resistant cells and reverse the drug-resistance. Moreover, p38MAPK-mediated apoptosis in paclitaxel-resistant ovarian carcinoma cells depends on the activation of p53.

Weakened spindle checkpoint with reduced BubR1 expression in paclitaxel-resistant ovarian carcinoma cell line SKOV3-TR30

Paclitaxel sensitivity has recently been associated with the spindle checkpoint. The aim of our study is to investigate the status of spindle checkpoint and the alteration of its major components in phenotype with acquired paclitaxel resistance in ovarian carcinoma. A paclitaxel-resistant ovarian carcinoma cell line, SKOV3-TR30, with the resistant ability of 27-fold greater than its parental cell line, was derived from SKOV3 cell line. The competence of spindle checkpoint and the expression of the major spindle checkpoint proteins (BubR1 and Mad2) in SKOV3-TR30 cells were investigated. Mitotic index and flow cytometric analysis revealed that SKOV3-TR30 cells were not arrested in M phase in contrast to SKOV3, which showed a clear mitotic arrest in the presence of paclitaxel or nocodazole. The expressions of securin and cyclin B in SKOV3-TR30 cells were significantly lower than those in SKOV3 cells, indicating the premature degradation of APC substrates in SKOV3-TR30 cells in response to spindle damagers. Chromosome spread analysis showed increased rate of premature sister chromatid segregation in SKOV3-TR30 cells. The expression of spindle checkpoint protein BubR1 was evidently lower in SKOV3-TR30 cells than that in SKOV3 cells. However, there was no significant difference in Mad2 expression between SKOV3-TR30 and SKOV3 cells. This study demonstrates that weakened spindle checkpoint with reduced expression of BubR1, but not Mad2, is associated with acquired paclitaxel resistance in ovarian carcinoma cells.

Taxol-resistance-associated gene-3 (TRAG-3/CSAG2) expression is predictive for clinical outcome in ovarian carcinoma patients

An obstacle in chemotherapy of ovarian cancer is the development of drug resistance. Taxol (paclitaxel)-resistance-associated gene-3 (TRAG-3/CSAG2) was found to be overexpressed in a paclitaxel-resistant ovarian carcinoma cell line. However, clinical impact of TRAG-3 in ovarian carcinoma has not been demonstrated previously. For demonstration of potential clinical impact of TRAG-3, immunohistochemistry was applied to determine TRAG-3 protein expression in specimens obtained from ovarian carcinoma patients (n=37) who received a paclitaxel-based chemotherapy at two different time points, initial laparotomy before chemotherapy, and secondary cytoreduction after chemotherapy. The TRAG-3-specific immunohistochemical staining was correlated with clinical outcome. In ovarian carcinoma specimens obtained at the initial laparotomy, an advantage in overall (P < 0.001) and progression-free (P = 0.003) survival for patients with weak TRAG-3 expression could be demonstrated. Tumor specimens excised at secondary cytoreduction procedure were not predictive for clinical outcome. In summary, TRAG-3 was found to be a prognostic factor for the prediction of clinical outcome after the application of paclitaxel-based chemotherapy.