Paclitaxel-resistant Ovarian Cancer Cell Lines Research Articles

Resensitizing Paclitaxel-Resistant Ovarian Cancer via Targeting Lipid Metabolism Key Enzymes CPT1A, SCD and FASN.

Epithelial ovarian cancer (EOC) is a lethal gynecological cancer, of which paclitaxel resistance is the major factor limiting treatment outcomes, and identification of paclitaxel resistance-related genes is arduous. We obtained transcriptomic data from seven paclitaxel-resistant ovarian cancer cell lines and corresponding sensitive cell lines. Define genes significantly up-regulated in at least three resistant cell lines, meanwhile they did not down-regulate in the other resistant cell lines as candidate genes. Candidate genes were then ranked according to the frequencies of significant up-regulation in resistant cell lines, defining genes with the highest rankings as paclitaxel resistance-related genes (PRGs). Patients were grouped based on the median expression of PRGs. The lipid metabolism-related gene set and the oncological gene set were established and took intersections with genes co-upregulated with PRGs, obtaining 229 co-upregulated genes associated with lipid metabolism and tumorigenesis. The PPI network obtained 19 highly confidential synergistic targets (interaction score > 0.7) that directly associated with CPT1A. Finally, FASN and SCD were up-stream substrate provider and competitor of CPT1A, respectively. Western blot and qRT-PCR results confirmed the over-expression of CPT1A, SCD and FASN in the A2780/PTX cell line. The inhibition of CPT1A, SCD and FASN down-regulated cell viability and migration, pharmacological blockade of CPT1A and SCD increased apoptosis rate and paclitaxel sensitivity of A2780/PTX. In summary, our novel bioinformatic methods can overcome difficulties in drug resistance evaluation, providing promising therapeutical strategies for paclitaxel-resistant EOC via taregting lipid metabolism-related enzymes.

PGAM1 Promotes Glycolytic Metabolism and Paclitaxel Resistance via Pyruvic Acid Production in Ovarian Cancer Cells.

Enhanced glycolysis occurs in most human cancer cells and is related to chemoresistance. However, detailed mechanisms remain vague. Using proteinomics analysis, we found that the glycolytic enzyme Phosphoglycerate mutase 1 (PGAM1) was highly expressed in the paclitaxel-resistant ovarian cancer cell line SKOV3-TR30, as compared to its parental cell line SKOV3. Cell Counting Kit-8 proliferation experiment, plasmids and siRNA transfection, pyruvic acid and lactic acid production detection, immunofluorescence staining of functional mitochondria and oxygen consumption rate and extracellular acidification rate measurement were uesd to assess the glycolytic metabolism and paclitaxel resistance in ovarian cancer cells. The expression and prognostic effect of PGAM1 in 180 ovarian cancer patients were analyzed. SKOV3-TR30 cells display higher glycolytic flux and lower mitochondrial function than SKOV3 cells. Down-regulation of PGAM1 in SKOV3-TR30 cells resulted in decreased paclitaxel resistance. Up-regulation of PGAM1 in SKOV3 cells led to enhanced paclitaxel resistance. Analysis of the glycolytic flux revealed that PGAM1-mediated pyruvic acid or lactic acid production could modulate the capabilities of ovarian cancer cell resistance to paclitaxel. Our data also show high expression of PGAM1 as significantly correlated with reduced overall survival and reduced progression free survival in ovarian cancer patients. PGAM1 acts to promote paclitaxel resistance via pyruvic acid and/or lactate production in ovarian cancer cells. Inhibiting PGAM1 may provide a new approach to favorably alter paclitaxel resistance in ovarian cancer.

The Profile of MicroRNA Expression and Potential Role in the Regulation of Drug-Resistant Genes in Cisplatin- and Paclitaxel-Resistant Ovarian Cancer Cell Lines.

Ovarian cancer is the most lethal gynecological malignancy. The high mortality results from late diagnosis and the development of drug resistance. Drug resistance results from changes in the expression of different drug-resistance genes that may be regulated miRNA. The main aim of our study was to detect changes in miRNA expression levels in two cisplatin (CIS) and two paclitaxel (PAC)—resistant variants of the A2780 drug-sensitive ovarian cancer cell line—by miRNA microarray. The next goal was to identify miRNAs responsible for the regulation of drug-resistance genes. We observed changes in the expression of 46 miRNA that may be related to drug resistance. The overexpression of miR-125b-5p, miR-99a-5p, miR-296-3p, and miR-887-3p and downregulation of miR-218-5p, miR-221-3p, and miR-222-3p was observed in both CIS-resistant cell lines. In both PAC-resistant cell lines, we observed the upregulation of miR-221-3p, miR-222-3p, and miR-4485, and decreased expression of miR-551b-3p, miR-551b-5p, and miR-218-5p. Analysis of targets suggest that expression of important drug-resistant genes like protein Tyrosine Phosphatase Receptor Type K (PTPRK), receptor tyrosine kinase—EPHA7, Semaphorin 3A (SEMA3A), or the ATP-binding cassette subfamily B member 1 gene (ABCB1) can be regulated by miRNA.

Lapatinib and poziotinib overcome ABCB1-mediated paclitaxel resistance in ovarian cancer.

Conventional frontline treatment for ovarian cancer consists of successive chemotherapy cycles of paclitaxel and platinum. Despite the initial favorable responses for most patients, chemotherapy resistance frequently leads to recurrent or refractory disease. New treatment strategies that circumvent or prevent mechanisms of resistance are needed to improve ovarian cancer therapy. We established in vitro paclitaxel-resistant ovarian cancer cell line and organoid models. Gene expression differences in resistant and sensitive lines were analyzed by RNA sequencing. We manipulated candidate genes associated with paclitaxel resistance using siRNA or small molecule inhibitors, and then screened the cells for paclitaxel sensitivity using cell viability assays. We used the Bliss independence model to evaluate the anti-proliferative synergy for drug combinations. ABCB1 expression was upregulated in paclitaxel-resistant TOV-21G (q < 1x10-300), OVCAR3 (q = 7.4x10-156) and novel ovarian tumor organoid (p = 2.4x10-4) models. Previous reports have shown some tyrosine kinase inhibitors can inhibit ABCB1 function. We tested a panel of tyrosine kinase inhibitors for the ability to sensitize resistant ABCB1-overexpressing ovarian cancer cell lines to paclitaxel. We observed synergy when we combined poziotinib or lapatinib with paclitaxel in resistant TOV-21G and OVCAR3 cells. Silencing ABCB1 expression in paclitaxel-resistant TOV-21G and OVCAR3 cells reduced paclitaxel IC50 by 20.7 and 6.2-fold, respectively. Furthermore, we demonstrated direct inhibition of paclitaxel-induced ABCB1 transporter activity by both lapatinib and poziotinib. In conclusion, lapatinib and poziotinib combined with paclitaxel synergizes to inhibit the proliferation of ABCB1-overexpressing ovarian cancer cells in vitro. The addition of FDA-approved lapatinib to second-line paclitaxel therapy is a promising strategy for patients with recurrent ovarian cancer.

Bifunctional chiral selenium-containing 1,4-diarylazetidin-2-ones with potent antitumor activities by disrupting tubulin polymerization and inducing reactive oxygen species production.

Organoselenium compounds have attracted growing interests as promising antitumor agents over recent years. Herein, four series of novel selenium-containing chiral 1,4-diarylazetidin-2-ones were asymmetrically synthesized and biologically evaluated for antitumor activities. Among them, compound 7 was found to be about 10-fold more potent than its prototype compound 1a, and compound 9a exhibited the most potent cytotoxicity against five human cancer cell lines, including a paclitaxel-resistant human ovarian cancer cell line A2780T, with IC50 values ranging from 1 to 3nM. Mechanistic studies revealed that compound 9a worked by disrupting tubulin polymerization, inducing reactive oxygen species (ROS) production, decreasing mitochondrial membrane potential, blocking the cell cycle in the G2/M phase, inducing cellular apoptosis and suppressing angiogenesis. Additionally, compound 9a exhibited appropriate human-microsomal metabolic stability and physicochemical properties. Importantly, compound 9a was found to inhibit tumor growth effectively in a xenograft mice model with low toxicity profile, which rendered 9a a highly promising candidate for further pre-clinical development.

CPEB4-Promoted Paclitaxel Resistance in Ovarian Cancer In Vitro Relies on Translational Regulation of CSAG2.

Background: Drug resistance is a major obstacle in chemotherapy for ovarian cancer, wherein the up regulation of drug-resistant genes plays an important role. The cytoplasmic polyadenylation element binding protein 4 (CPEB4) is an RNA binding protein that controls mRNA cytoplasmic polyadenylation and translation. Methods: The expression of CPEB4 in paclitaxel-resistant ovarian cancer cell lines and recurrent ovarian tumors relative to counterparts was determined by qRT-PCR, Western blotting and immunohistochemistry. The response to paclitaxel treatment was evaluated by cellular viability test and colony formation assay. RNA immunoprecipitation and poly(A) tail test were applied to examine the levels of RNA binding and cytoplasmic polyadenylation. Results: CPEB4 is elevated in paclitaxel-resistant ovarian cancer cells and recurrent ovarian tumors treated with paclitaxel-based chemotherapy. In addition, CPEB4 overexpression promotes paclitaxel resistance in ovarian cancer cells in vitro, and vice versa, CPEB4 knockdown restores paclitaxel sensitivity, indicating that CPEB4 confers paclitaxel resistance in ovarian cancer cells. Mechanistically, CPEB4 binds with the taxol (paclitaxel)-resistance-associated gene-3 (TRAG-3/CSAG2) mRNAs and induces its expression at a translational level. Moreover, CSAG2 expression is upregulated in paclitaxel-resistant ovarian carcinoma and cancer cell lines, and more importantly, siRNA-mediated CSAG2 knockdown overtly attenuates CPEB4-mediated paclitaxel resistance. Conclusion: This study suggests that the drug-resistant protein CSAG2 is translationally induced by CPEB4, which underlies CPEB4-promoted paclitaxel resistance in ovarian cancer in vitro. Thus, interfering CPEB4/CSAG2 axis might be of benefit to overcome paclitaxel-resistant ovarian cancer.

Abstract A50: Lapatinib potentiates the antitumor effects of paclitaxel treatment in resistant ovarian cancer cells

Abstract Introduction: Frontline treatment regimens for ovarian cancer routinely consist of repeated cycles of paclitaxel/platinum doublets; however, chemotherapy resistance frequently leads to recurrent or refractory disease. Novel therapeutic strategies aimed at circumventing or preventing resistance are needed for improved disease control. Experimental Procedures: Multiple paclitaxel-resistant ovarian cancer cell line models were established via repeated cycles of paclitaxel treatment in vitro. Resistance to paclitaxel was assessed using dose response cell viability assays with CellTiter-Glo 2.0 and calculation of IC50 values using a 4-parameter logistic regression model. RNAseq and whole-exome sequencing were used to examine genomic variation distinguishing resistant lines from their sensitive counterparts. Recurrent expression differences associated with paclitaxel resistance were identified, a subset of which were selected for further validation. Candidate genes and pathways associated with paclitaxel resistance were manipulated using siRNA and/or small-molecule inhibitors and assessed for the ability to alter the resistant phenotype. Synergistic interactions for drug combinations were evaluated using the Loewe Additivity model. Results: Paclitaxel-resistant ovarian cancer cell lines were derived from TOV-21G, OVCAR-3, and UWB1.289 cells. Resistant cell lines exhibited increases in paclitaxel IC50 ranging from 6.5 to 94-fold over their parental control cells. RNAseq analysis identified the transcript encoding P-glycoprotein (P-gp), ABCB1, among the most highly upregulated genes in the resistant TOV-21G and OVCAR-3 cells while no such induction of expression was seen in resistant UWB1.289 cells. Previous reports have shown several different tyrosine kinase inhibitors (e.g., lapatinib, neratinib, dasatinib, etc.) have the ability to inhibit P-gp function. We tested a panel of tyrosine kinase inhibitors for the ability to sensitize resistant P-gp-overexpressing ovarian cancer cells to paclitaxel. The most potent synergistic antiproliferative activity was observed when lapatinib was combined with paclitaxel treatment of resistant TOV-21G and OVCAR-3 cells; however, a similar interaction was not observed in resistant UWB1.289 cells that do not overexpress P-gp. The dependence of this synergistic effect on P-gp expression was confirmed by using siRNA to inhibit P-gp expression in paclitaxel-resistant TOV-21G and OVCAR-3 cells, significantly reducing paclitaxel IC50 by 20.7 and 6.2-fold, respectively, and eliminating cytotoxic synergy. Conclusions: Paclitaxel and lapatinib have synergistic anticancer properties when used to treat P-gp-overexpressing ovarian cancer cells. The addition of lapatinib to second-line dose-dense paclitaxel therapy could be an effective strategy for a subset of ovarian cancer patients with recurrent disease and warrants further clinical exploration. Citation Format: Rob McCorkle, Justin W. Gorski, Abigail Anderson, Jill M. Kolesar. Lapatinib potentiates the antitumor effects of paclitaxel treatment in resistant ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A50.

Arsenic trioxide and tetraarsenic oxide induce cytotoxicity and have a synergistic effect with cisplatin in paclitaxel-resistant ovarian cancer cells

Background: Arsenic compounds (As2O3 and As4O6) have demonstrated anticancer effects in various malignancies. In this study, the cytotoxicity of arsenic compounds on ovarian cancer cell lines and the anticancer activity of the combination of arsenic compounds and cisplatin IN chemoresistant ovarian cancer cells were investigated.Methods: We investigated the cytotoxicity of As2O3 and As4O6 and their combinations with cisplatin in the paclitaxel-sensitive ovarian cancer cell lines SKOV3ip1 and HeyA8 and paclitaxel-resistant ovarian cancer cell lines SKOV3TRip2 and HeyA8-MDR. Growth and apoptosis were evaluated by MTT assay and annexin V assay using flow cytometry, respectively. For detection of apoptotic cells, immunofluorescence was performed using a cleaved caspase-3 antibody. Cell-cycle distribution was determined by propidium iodide staining and flow cytometry.Results: Treatment of each cell line with As2O3 or As4O6 led to a marked dose-dependent inhibition of cell growth. As2O3 and As4O6 treatment induced caspase-3-dependent apoptosis in all cell lines compared to the respective control groups (p < .05). As2O3 and As4O6 induced apoptosis of paclitaxel-sensitive and -resistant cancer cell lines following G2/M cell cycle arrest (p < .05). A synergistic effect was achieved by combining cisplatin with As2O3 or As4O6 in the paclitaxel-resistant ovarian cancer cell lines.Conclusions: As2O3 and As4O6 can inhibit cell growth and induce apoptosis in paclitaxel-sensitive and -resistant ovarian cancer cell lines. Their combination with cisplatin resulted in a synergistic effect in paclitaxel-resistant cancer cell lines. These results suggest that arsenic compounds may be given in monotherapy or combination therapy with cisplatin for treating paclitaxel-resistant ovarian cancer.

In vitro selection of DNA aptamers recognizing drug-resistant ovarian cancer by cell-SELEX

Ovarian cancer is regarded as the most lethal gynecologic malignancy with poor prognosis and high mortality rate. Drug-resistance was thought to be the main obstacle to improving overall survival rate of ovarian cancer. New ligands for drug-resistant ovarian cancer cells have potential for the development of diagnosis and therapy of ovarian cancer. In present work, we reported two aptamers, HF3–58 and HA5–68 generated by cell-SELEX, against a paclitaxel-resistant ovarian cancer cell line (A2780T). Both two aptamers exhibited high selectivity and strong affinity to target cells with low nanomolar dissociation constants. The binding of aptamers to target cells was independent of divalent ions, and was tolerant of incubation temperature and nucleases in serum. Molecular targets of the two aptamers were preliminarily demonstrated to be two different glycoproteins on cell surface of A2780T cells. Owing to the structure stability and high resistance to nuclease, these two aptamers had good performance in the detection of drug-resistant ovarian cancer cells in human serum.

Abstract 3083A: C6-ceramide nanoliposomes sensitize paclitaxel resistant ovarian cancer cells to chemotherapy treatment

Abstract Ovarian cancer is the fifth leading cause of cancer deaths among women, and the majority of women are diagnosed at advanced stages resulting in poorer outcomes and survival rates. One of the driving forces behind these outcomes is the development of chemoresistance, and subsequent proliferation and invasion of tumor cells. In a patient-derived xenograft model of ovarian cancer, we identified the sphingosine pathway as significantly enriched in tumors after treatment with conventional chemotherapy. In order to target the sphingosine pathway, we investigated the potential effects of combining paclitaxel with C6-ceramide nanoliposomes in paclitaxel-resistant ovarian cancer cell lines, HeyA8-MDR and SKOV3-TR. Ceramide has been shown to be a key player in several processes important for tumor suppression including apoptosis, autophagy, and cell cycle arrest. We administered paclitaxel and C6-ceramide to HeyA8-MDR and SKOV3-TR cell lines in vitro and measured cell viability and expression of apoptotic and autophagic proteins. Results showed that in both cell lines, C6-ceramide significantly decreases the IC50 dose of paclitaxel, compared to control ghost nanoliposome treated cells. The IC50 of paclitaxel decreased in HeyA8-MDR cells from 400nM to 150nM, and in SKOV3-TR cells from 1000nM to 450nM. Western blot analyses indicate that combination treatment increases expression of apoptotic (cleaved caspase-3) factors in HeyA8-MDR cells, and autophagic (LC3) factors in SKOV3-TR cells, over single agent treatment or control. Paclitaxel is known to increase expression of apoptotic factors while decreasing expression of autophagic factors. Ceramide may increase both apoptotic and autophagic factors. In an orthotopic mouse model with the HeyA8-MDR line, combination treatment of paclitaxel and C6-ceramide resulted in significantly decreased tumor burden (1.38g +/- 0.74), compared to control ghost (p = 0.022, 2.81g +/- 1.57), C6-ceramide alone (p = 0.078, 2.50g +/- 1.58), and paclitaxel alone (p = 0.005, 2.48g +/- 0.75) groups. The combination of C6-ceramide and paclitaxel may prove useful in elucidating and targeting underlying chemoresistance mechanisms in ovarian cancer. Citation Format: Danielle C. Llaneza, Tye G. Deering, Samantha G. Sherwood, John R. Cornelison, Mark Kester, Charles N. Landen. C6-ceramide nanoliposomes sensitize paclitaxel resistant ovarian cancer cells to chemotherapy treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3083A. doi:10.1158/1538-7445.AM2017-3083A

Abstract 5420: PGAM1-REDD1 pathway promotes paclitaxel resistance through enhancing aerobic glycolysis in ovarian cancer cells

Abstract Aerobic glycolysis is a dominant pathway for energy production in cancer cells, but the association of this phenomenon with chemoresistance remains still uncertain. Here, we found that the expression of phosphoglycerate mutase 1 (PGAM1), a key catalytic enzyme in aerobic glycolysis, was significantly higher in paclitaxel-resistant ovarian cancer cell line SKOV3. Up-regulated PGAM1 expression by gene transfection and down-regulated PGAM1 expression by siRNA knockdown altered the process of aerobic glycolysis, paclitaxel sensitivity and DNA damage response 1 (REDD1) expression in ovarian cancer cells (SKOV3 and A2780). Further, the change of the concentration of pyruvic acid or lactic acid in culture medium and REDD1 expression by gene transfection or siRNA knockdown also altered the sensitivity to paclitaxel. REDD1 knockdown of by siRNA reversed partially paclitaxel resistance induced by PGAM1 over-expression in cancer cells, vice versa. Structural interaction between PGAM1 and REDD1 was predicted by molecular docking. Our results suggested that PGAM1-REDD1 pathway promotes paclitaxel resistance through enhancing aerobic glycolysis in ovarian cancer cells. Citation Format: Yan Feng, Weiguo Lv, Xing Xie, Yan Yu. PGAM1-REDD1 pathway promotes paclitaxel resistance through enhancing aerobic glycolysis in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5420. doi:10.1158/1538-7445.AM2017-5420

Synthetic paclitaxel-octreotide conjugate reverses the resistance of paclitaxel in A2780/Taxol ovarian cancer cell line.

The high mortality of ovarian cancer is partly due to the frequent resistance of ovarian cancer to current chemotherapy agents such as paclitaxel and platinum. Somatostatin analogue (SSTA) has been shown to inhibit the proliferation of some tumors through binding to somatostatin receptor (SSTR) and activation of Ras-, Rapl- and B-Raf-dependent extracellular signal-regulated kinase 2 (Erk2). It was reported that paclitaxel-octreotide conjugate (POC) exhibited enhanced tumor growth inhibition with reduced toxicity. In the present study, we prepared the POC and investigated its effects and mechanism for the reversal of drug resistance in paclitaxel-resistant ovarian cancer cell line. We demonstrated that treatment with POC led to more cell apoptosis than either paclitaxel or octreotide (OCT) alone. Moreover, the expression of multidrug resistance 1 (MDR1) and vascular endothelial growth factor (VEGF) mRNA, and protein decreased in a dose-dependent manner while the expression of SSTR remained stable following treatment with POC. Although the exact action, invivo effects and pharmacologic kinetics of POC remain to be investigated, we have demonstrated the feasibility for the synthesis of POC, and more significantly, provided a potential approach to overcome the resistance of ovarian cancer against taxol. The findings also shed some new light on the mechanisms underlying the development of resistance to taxol by ovarian cancer cells.

Inhibition of JAK2 Reverses Paclitaxel Resistance in Human Ovarian Cancer Cells.

Resistance to chemotherapy is a major factor that limits the postsurgical survival of ovarian cancer patients. Janus-activated kinase 2 (JAK2) has been implicated in cancer cell survival and the development of drug resistance in ovarian cancers. In the present study, we sought to determine whether inhibition of JAK2 reverses drug resistance in OC3/TAX300 cells, a paclitaxel-resistant human ovarian cancer cell line previously established in our laboratory. OC3/TAX300 cells were transduced with lentivirus expressing small interference RNA (siRNA) against JAK2 and treated with JAK2 kinase inhibitor AG490. Treatment with JAK2-siRNA markedly decreased the messenger RNA and protein of JAK2 as determined by real-time polymerase chain reaction and Western blot analysis. OC3/TAX300 cells treated with JAK2-siRNA exhibited stalled growth, increased cell cycle arrest in G2/M phase, and enhanced apoptosis in response to paclitaxel. In keeping with this, JAK2-siRNA also inhibited the expression of multidrug resistance protein 1. To determine whether JAK2 promotes paclitaxel resistance via phosphorylation of signal transducer and activator of transcription 3 (STAT3), a transcription factor known to be involved in resistance to chemotherapy, we treated OC3/TAX300 cells with JAK2 kinase inhibitor AG490. Of note, AG490 reduced the level of p-STAT3 and inhibited the expression of multidrug resistance protein 1 in a dose-dependent manner. Collectively, we conclude that the JAK2-STAT3 pathway promotes the development of paclitaxel resistance via upregulating the expression of prosurvival and antiapoptotic genes. Targeting this pathway may be effective in reversing resistance to chemotherapy in ovarian cancers.

Nsc23925 prevents the development of paclitaxel resistance by inhibiting the introduction of P-glycoprotein and enhancing apoptosis.

Strategies to prevent the emergence of drug resistance will increase the effectiveness of chemotherapy treatment and prolong survival of women with ovarian cancer. The aim of our study is to determine the effects of NSC23925 on preventing the development of paclitaxel resistance in ovarian cancer both in cultured cells in vitro and in mouse xenograft models in vivo, and to further elucidate these underlying mechanisms. We first developed a paclitaxel-resistant ovarian cancer cell line, and demonstrated that NSC23925 could prevent the introduction of paclitaxel resistance by specifically inhibiting the overexpression of P-glycoprotein (Pgp) in vitro. The paclitaxel-resistant ovarian cancer cells were then established in a mouse model by continuous paclitaxel treatment in combination with or without NSC23925 administration in the mice. The majority of mice continuously treated with paclitaxel alone eventually developed paclitaxel resistance with overexpression of Pgp and antiapoptotic proteins, whereas mice remained sensitivity to paclitaxel and displayed lower expression levels of Pgp and antiapoptotic proteins after administered continuously with combination of paclitaxel-NSC23925. Paclitaxel-NSC23925-treated mice experienced significantly longer overall survival time than paclitaxel-treated mice. Furthermore, the combination of paclitaxel and NSC23925 therapy did not induce obvious toxicity as measured by mice body weight changes, blood cell counts and histology of internal organs. Collectively, our observations provide evidence that NSC23925 in combination with paclitaxel may prevent the onset of Pgp or antiapoptotic-mediated paclitaxel resistance, and improve the long-term clinical outcome in patients with ovarian cancer.

MiR-145 sensitizes ovarian cancer cells to paclitaxel by targeting Sp1 and Cdk6

Multidrug resistance (MDR) remains a major obstacle to effective chemotherapy treatment in ovarian cancer. In our study, paclitaxel-resistant ovarian cancer patients and cell lines had decreased miR-145 levels and expressed high levels of Sp1 and Cdk6. Introducing miR-145 into SKOV3/PTX and A2780/PTX cells led to a reduction in Cdk6 and Sp1 along with downregulation of P-gp and pRb. These changes resulted in increased accumulation of antineoplastic drugs and G1 cell cycle arrest, which rendered the cells more sensitive to paclitaxel in vitro and in vivo. These effects could be reversed by reintroducing Sp1 or Cdk6 into cells expressing high levels of miR-145, resulting in restoration of P-gp and pRb levels. Furthermore, we confirmed that both Cdk6 and Sp1 are targets of miR-145. Intriguingly, demethylation with 5-aza-dC led to reactivation of miR-145 expression in drug-resistant ovarian cancer cell lines, which also resulted in increased sensitivity to paclitaxel. Collectively, these findings begin to elucidate the role of miR-145 as an important regulator of chemoresistance in ovarian cancer by controlling both Cdk6 and Sp1.

RSF1 Is a Positive Regulator of NF-κB–Induced Gene Expression Required for Ovarian Cancer Chemoresistance

Overexpression or amplification of the RSF1 gene has been associated with poor prognosis in various human cancers, including ovarian cancer. In previous work, RSF1 was identified as an amplified gene that facilitated the development of paclitaxel-resistant ovarian cancer. In the present study, we further demonstrated that RSF1 expression inversely correlated with paclitaxel response in patients with ovarian cancer and the mouse xenograft model. In addition, RSF1-overexpressing paclitaxel-resistant ovarian cancer cell lines were found to express elevated levels of genes regulated by NF-κB, including some involved with the evasion of apoptosis (CFLAR, XIAP, BCL2, and BCL2L1) and inflammation (PTGS2). In addition, ectopic expression of RSF1 using Tet-off inducible SKOV3 cells significantly enhanced NF-κB-dependent gene expression and transcriptional activation of NF-κB. An RSF1 knockdown using short hairpin RNAs suppressed these same pathways. Moreover, pretreatment with NF-κB inhibitors or downregulation of NF-κB-regulated gene expression considerably enhanced paclitaxel sensitivity in RSF1-overexpressing OVCAR3 and/or RSF1-induced SKOV3 cells. A coimmunoprecipitation assay revealed that RSF1 interacts with NF-κB and CREB-binding protein, a ubiquitous coactivator for NF-κB. Recruitment of RSF1 to the NF-κB binding element in the PTGS2 and XIAP promoters was demonstrated by the chromatin immunoprecipitation assay. Furthermore, hSNF2H, a well-known binding partner of RSF1, was partially involved in the interaction between RSF1 and NF-κB. Taken together, these data suggest that RSF1 may function as a coactivator for NF-κB, consequently augmenting expression of genes necessary for the development of chemoresistance in ovarian cancer cells.

Epigallocatechin gallate and sulforaphane combination treatment induce apoptosis in paclitaxel-resistant ovarian cancer cells through hTERT and Bcl-2 down-regulation

The cellular development of resistance to chemotherapy contributes to the high mortality noted in patients affected by ovarian cancer. Novel compounds that specifically target cellular drug resistance in ovarian cancer are therefore highly desired. Previous epidemiological studies indicate that consumption of green tea and cruciferous vegetables is inversely associated with occurrence of ovarian cancer. Therefore revealing the effects and mechanisms of major components of green tea (epigallocatechin gallate, EGCG) and cruciferous vegetables (sulforaphane, SFN) on ovarian cancer cells will provide necessary knowledge for developing potential novel treatments for the disease. In this study, EGCG or SFN was used to treat both paclitaxel-sensitive (SKOV3-ip1) and -resistant (SKOV3TR-ip2) ovarian cancer cell lines alone or in combination. We found that SFN inhibits cell viability of both ovarian cancer cell lines time- and dose-dependently and that EGCG potentiates the inhibiting effect of SFN on ovarian cancer cells. Cell cycle analysis indicates SFN can arrest ovarian cancer cells in G2/M phase, while EGCG and SFN co-treatment can arrest cells in both G2/M and S phase. Combined EGCG and SFN treatment increases apoptosis significantly in paclitaxel-resistant SKOV3TR-ip2 cells after 6 days of treatment, while reducing the expression of hTERT, the main regulatory subunit of telomerase. Western blotting also indicates that SFN can down-regulate Bcl-2 (a gene involved in anti-apoptosis) protein levels in both cell types. Cleaved poly(ADP-ribose) polymerase (PARP) becomes up-regulated by 6 days of treatment with SFN and this is more pronounced for combination treatment indicating induction of apoptosis. Furthermore, phosphorylated H2AX is up-regulated after 6 days of treatment with SFN alone, and EGCG can potentiate this effect, suggesting that DNA damage is a potential cellular mechanism contributing to the inhibiting effect of EGCG and SFN combination treatment. Taken together, these results indicate that EGCG and SFN combination treatment can induce apoptosis by down-regulating of hTERT and Bcl-2 and promote DNA damage response specifically in paclitaxel-resistant ovarian cancer cell lines and suggest the use of these compounds for overcoming paclitaxel resistance in ovarian cancer treatment.

Preliminary study of the prohibitin protein and paclitaxel resistance in ovarian cancer

To determine the effect of RNA interference with transferred pshRNA/PHB on the biological characteristics of paclitaxel-resistant ovarian cancer cell lines. Western blot and real time-PCR were used to assay the expression of PHB protein and mRNA in SKOV3/Taxol-25 and SKOV3 cell lines. The SKOV3/Taxol-25 cell lines were transiently transfected by 3 target-specific small hairpin RNA (shRNA) interference fragments with fluorescent protein named the pshRNA427/PHB1, pshRNA248/PHB2, and pshRNA136/PHB3. The empty plasmid transfection via vehicle Lipofectamine2000 served as a negative control. The expression levels of PHB protein and mRNA were detected by Western blot and real time-PCR after the transfection for 48 h. The silence effect of PHB1 and PHB3 groups was obvious. PHB1, PHB3, and the negative control groups were used for the following experiments. MTT and flow cytometry assay were used to test the cell proliferation, IC50 of paclitaxel, and cell apoptosis in the 3 groups. The expression levels of PHB protein and mRNA (2(-ΔΔCt)) were significantly higher in SKOV3/Taxol-25 cell line than those in SKOV3 cell line (P<0.05). The expression levels of PHB protein and mRNA were significantly lower in the PHB1 and PHB3 groups than those in the negative control group (P<0.05). The cell proliferations in the PHB1 and PHB3 groups were obviously slower than those in the negative control group after transfection for 48 h and 72 h (P<0.05). The IC50 of paclitaxel in the PHB1 and PHB3 groups significantly decreased after transfection for 72 h compared with the negative control group(P<0.05). The cell apoptotic rate in the PHB1 and PHB3 groups significantly increased after transfection for 48 h compared with the negative control group (P<0.05). The shRNA/PHB can effectively suppress the expression of PHB gene in paclitaxel-resistant ovarian cancer cell lines. The cell proliferation in paclitaxel-resistant cell lines with removed PHB gene is significantly reduced. The apoptotic rate and the paclitaxel sensitivity of resistant cell lines with removed PHB gene are significantly increased. PHB gene is related to paclitaxel-resistance and interfering PHB gene expression may reduce paclitaxel resistance in ovarian cancer.

Clinical implications of REST and TUBB3 in ovarian cancer and its relationship to paclitaxel resistance

The objective of this study was to examine the expression levels of RE1-silencing transcription factor (REST) and class III β-tubulin (TUBB3) in ovarian cancer and to determine if there is a correlation between their expression and resistance to chemotherapy in ovarian cancer. The protein expression of REST and TUBB3 in ovarian cancer was detected by Western blot analysis. REST expression was inhibited by small interfering ribonucleic acid (siRNA) in human ovarian cancer cell lines. The levels of REST and TUBB3 protein expression were detected by immunohistochemistry. The relationship between REST and TUBB3 expression and chemotherapy resistance, clinicopathological parameters, and prognosis of ovarian cancer was then determined. The present study found that REST was more highly expressed in the ovarian SKOV3 carcinoma cell line compared to the paclitaxel-resistant ovarian cancer cell line, SKOV3/TAX (P = 0.01). In contrast, TUBB3 was more highly expressed in SKOV3/TAX cells compared to SKOV3 cells (P = 0.01). After REST siRNA interference, TUBB3 expression increased in SKOV3 cells. REST expression was significantly higher in the paclitaxel-sensitive group compared to the paclitaxel-resistant group (70.7 % vs. 37.0 %, P < 0.05). However, TUBB3 expression was significantly lower in the paclitaxel-sensitive group compared to the paclitaxel-resistant group (47.6 % vs. 77.8 %, P < 0.05). Notably, REST was more highly expressed in TUBB3-negative cases than TUBB3-positive cases (P < 0.05). Univariate analyses indicated that both REST and TUBB3 expression were unrelated to tumor differentiation, histological type, and clinical stage (all P > 0.05). According to the Cox regression model, negative REST and positive TUBB3 protein expression was detected as independent prognostic factors (P = 0.003 and P = 0.005, respectively). REST and TUBB3 protein may be potential biomarkers for chemoresistance and prognosis in ovarian cancer.

Side population is increased in paclitaxel-resistant ovarian cancer cell lines regardless of resistance to cisplatin

ObjectivesIn recent years, cancer stem cells (CSCs) have been reported to be correlated with chemoresistance and may also be enriched in side populations (SPs). In this study, the relationship between resistance to paclitaxel (PTX) and cisplatin (CDDP) and side populations was examined in three parental PTX- and CDDP-sensitive ovarian cancer cell lines (2008, KF28, and TU-OM-1) and several other cell lines derived from these as well as the additional effects of interferon-alpha (INF-α). MethodsSP of three different parental cell lines and PTX- and/or CDDP-resistant cell lines derived from these was analyzed with flow cytometry. The expression of ABCB1 and ABCG2 in KF28 and its derived cell lines was examined. Additional cell-death effect of INF-α with PTX was also examined. ResultsIn the three parental cell lines and the PTX-sensitive cell lines derived from these lines, SP was very low. Conversely, in PTX-resistant cell lines, regardless of CDDP resistance, SP increased. ABCB1 was strongly expressed in the PTX-resistant cells, but not in their parental lines, which are sensitive to PTX. While INF-α showed only slight enhancement of the cell-death effect of PTX in PTX-sensitive cells, INF-α itself strongly induced apoptosis in PTX-resistant cells regardless of PTX concentration. ConclusionsThe SP could be correlated with resistance to PTX. SP could be a target of INF-α, and resistance to PTX might be overcome by INF-α.