Paclitaxel-resistant Ovarian Cancer Cells Research Articles

Effects of alisol B 23-acetate on ovarian cancer cells: G1 phase cell cycle arrest, apoptosis, migration and invasion inhibition

BackgroundOvarian cancer is the first leading cause of death among gynecologic malignancies worldwide. Discovery of new chemotherapeutic drugs is still imperative for the improvement of the survival rate. PurposeThis study aims to investigate the anti-cancer potential of alisol B 23-acetate (AB23), a protostane-type triterpene isolated from the Alismatis Rhizoma, in the parental and paclitaxel-resistant ovarian cancer cells. MethodsMTT assay was performed to evaluate cell viability after treatment with AB23, along with flow cytometry for apoptosis and cell cycle analysis. Western blotting was conducted to determine the relative protein level. Wound healing and transwell assays were performed to investigate the effect of AB23 on cell migration and invasion. ResultsAB23 obviously inhibited proliferation of the three ovarian cancer cell lines, down-regulated the protein levels of CDK4, CDK6, and cyclin D1, and blocked the cell cycle progressions in G1 phase. Meanwhile, AB23 induced accumulation of the sub-G1 phase in the three cell lines in a concentration dependent manner. The protein levels of cleaved poly ADP-ribose polymerase (PARP) and the ratio of Bax/Bcl-2 were up-regulated after treatment with AB23. Further study showed that AB23 induced endoplasmic reticulum stress through IRE1 signaling pathway and silencing of IRE1α partially enhanced AB23-induced apoptosis. Wound healing and transwell assays showed that AB23 could also suppress the migration and invasion of HEY cells. Moreover, it down-regulated the protein levels of matrix metalloproteinases MMP-2 and MMP-9. ConclusionAB23 possessed anti-proliferation, anti-migration and anti-invasion activities as a single agent on 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.

Abstract 4631: AKAP12 is elevated in paclitaxel-resistant ovarian cancer cells and correlates with poor ovarian cancer patient survival

Abstract Objective: Although most ovarian cancer patients respond initially to the standard of care involving carboplatinum/paclitaxel-based chemotherapy, the majority will recur with chemotherapeutically resistant disease. Unfortunately, no clinical biomarker(s) for predicting or monitoring chemotherapeutic resistance are available. We sought to identify shed and/or secreted proteins from paclitaxel-resistant human ovarian cancer cells and identify whether any of these correlate with ovarian cancer patient outcome. Methods: A proteomic analysis of secretomes from high grade serous ovarian cancer (HGSOC) cells (OV90) was compared to that from a syngeneic paclitaxel-resistant variant (OV90-TR) and a cancer cell line developed from a chemorefractory HGSOC patient. Associations between the identified candidates and patient outcome were assessed from publicly available transcript expression data (n = 545 ovarian cancer patients) and validated independently (n = 795 ovarian cancer patients). Results: Among the 81 differentially abundant proteins identified (q<0.05) from paclitaxel-sensitive vs -resistant HGSOC cell secretomes, AKAP12 was verified to be elevated at the protein and transcript level in all models of paclitaxel-resistant HGSOC. Further, elevated AKAP12 transcript expression was found to significantly correlate with poor progression-free (Hazard Ratio = 1.487 (± 0.148), p = 0.0085 (± 0.002) and overall (Hazard Ratio = 1.22 (± 0.08), p = 0.009 (± 0.013) survival. Conclusions: These findings suggest that elevation and efflux of AKAP12 is characteristic of paclitaxel-resistant HGSOC cells and that elevated AKAP12 expression correlates with poor ovarian cancer patient survival. Citation Format: Nicholas Bateman, Elizabeth Jaworski, Guisong Wang, Elizabeth Dubil, Charlotte Marcus, Kelly Conrads, Pang-ning Teng, Brian Hood, Chad Hamilton, Larry Maxwell, Kathleen Darcy, Thomas Conrads. AKAP12 is elevated in paclitaxel-resistant ovarian cancer cells and correlates with poor ovarian cancer patient survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4631. doi:10.1158/1538-7445.AM2015-4631

Quantitative proteomic analysis of mitochondria from human ovarian cancer cells and their paclitaxel-resistant sublines.

Paclitaxel resistance is a major obstacle for the treatment of ovarian cancer. The chemoresistance mechanisms are partly related to the mitochondria. Identification of the relevant proteins in mitochondria will help in clarifying the possible mechanisms and in selecting effective chemotherapy for patients with paclitaxel resistance. In the present study, mitochondria from two paclitaxel-sensitive human ovarian cancer cell lines (SKOV3 and A2780) and their corresponding resistant cell lines (SKOV3-TR and A2780-TR) were isolated. Guanidine-modified acetyl-stable isotope labeling and liquid chromatography-hybrid linear ion trap Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR MS) were performed to find the expressed differential proteins. Comparative proteomic analysis revealed eight differentially expressed proteins in the ovarian cancer cells and their paclitaxel-resistant sublines. Among them, mimitin and 14-3-3 ζ/δ were selected for further research. The effects of mimitin and 14-3-3 ζ/δ were explored using specific siRNA interference in ovarian cancer cell lines and immunohistochemistry in human tissue specimens. The downregulation of mimitin and 14-3-3 ζ/δ using specific siRNA in paclitaxel-resistant ovarian cancer cells led to an increase in the resistance index to paclitaxel. Multivariate analyses demonstrated that lower expression levels of the mimitin and 14-3-3 ζ/δ proteins were positively associated with shorter progression-free survival (PFS) and overall survival (OS) in patients with primary ovarian cancer (mimitin: PFS: P = 0.041, OS: P = 0.003; 14-3-3 ζ/δ: PFS: P = 0.031, OS: P = 0.011). Mimitin and 14-3-3 protein ζ/δ are potential markers of paclitaxel resistance and prognostic factors in ovarian cancer.

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.

Elevated AKAP12 in paclitaxel-resistant serous ovarian cancer cells is prognostic and predictive of poor survival in patients.

A majority of high-grade (HG) serous ovarian cancer (SOC) patients develop resistant disease despite high initial response rates to platinum/paclitaxel-based chemotherapy. We identified shed/secreted proteins in preclinical models of paclitaxel-resistant human HGSOC models and correlated these candidate proteins with patient outcomes using public data from HGSOC patients. Proteomic analyses of a HGSOC cell line secretome was compared to those from a syngeneic paclitaxel-resistant variant and from a line established from an intrinsically chemorefractory HGSOC patient. Associations between the identified candidate proteins and patient outcome were assessed in a discovery cohort of 545 patients and two validation cohorts totaling 795 independent SOC patients. Among the 81 differentially abundant proteins identified (q < 0.05) from paclitaxel-sensitive vs -resistant HGSOC cell secretomes, AKAP12 was verified to be elevated in all models of paclitaxel-resistant HGSOC. Furthermore, elevated AKAP12 transcript expression was associated with worse progression-free and overall survival. Associations with outcome were observed in three independent cohorts and remained significant after adjusted multivariate modeling. We further provide evidence to support that differential gene methylation status is associated with elevated expression of AKAP12 in taxol-resistant ovarian cancer cells and ovarian cancer patient subsets. Elevated expression and shedding/secretion of AKAP12 is characteristic of paclitaxel-resistant HGSOC cells, and elevated AKAP12 transcript expression is a poor prognostic and predictive marker for progression-free and overall survival in SOC patients.

Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

Development of chemoresistance is a major impediment to successful treatment of patients suffering from epithelial ovarian carcinoma (EOC). Among various molecular factors, presence of MyD88, a component of TLR-4/MyD88 mediated NF-κB signaling in EOC tumors is reported to cause intrinsic paclitaxel resistance and poor survival. However, 50–60% of EOC patients do not express MyD88 and one-third of these patients finally relapses and dies due to disease burden. The status and role of NF-κB signaling in this chemoresistant MyD88negative population has not been investigated so far. Using isogenic cellular matrices of cisplatin, paclitaxel and platinum-taxol resistant MyD88negative A2780 ovarian cancer cells expressing a NF-κB reporter sensor, we showed that enhanced NF-κB activity was required for cisplatin but not for paclitaxel resistance. Immunofluorescence and gel mobility shift assay demonstrated enhanced nuclear localization of NF-κB and subsequent binding to NF-κB response element in cisplatin resistant cells. The enhanced NF-κB activity was measurable from in vivo tumor xenografts by dual bioluminescence imaging. In contrast, paclitaxel and the platinum-taxol resistant cells showed down regulation in NF-κB activity. Intriguingly, silencing of MyD88 in cisplatin resistant and MyD88positive TOV21G and SKOV3 cells showed enhanced NF-κB activity after cisplatin but not after paclitaxel or platinum-taxol treatments. Our data thus suggest that NF-κB signaling is important for maintenance of cisplatin resistance but not for taxol or platinum-taxol resistance in absence of an active TLR-4/MyD88 receptor mediated cell survival pathway in epithelial ovarian carcinoma.

TXNDC17 promotes paclitaxel resistance via inducing autophagy in ovarian cancer

Paclitaxel is recommended as a first-line chemotherapeutic agent against ovarian cancer, but drug resistance becomes a major limitation of its success clinically. The key molecule or mechanism associated with paclitaxel resistance in ovarian cancer still remains unclear. Here, we showed that TXNDC17 screened from 356 differentially expressed proteins by LC-MS/MS label-free quantitative proteomics was more highly expressed in paclitaxel-resistant ovarian cancer cells and tissues, and the high expression of TXNDC17 was associated with poorer prognostic factors and exhibited shortened survival in 157 ovarian cancer patients. Moreover, paclitaxel exposure induced upregulation of TXNDC17 and BECN1 expression, increase of autophagosome formation, and autophagic flux that conferred cytoprotection for ovarian cancer cells from paclitaxel. TXNDC17 inhibition by siRNA or enforced overexpression by a pcDNA3.1(+)-TXNDC17 plasmid correspondingly decreased or increased the autophagy response and paclitaxel resistance. Additionally, the downregulation of BECN1 by siRNA attenuated the activation of autophagy and cytoprotection from paclitaxel induced by TXNDC17 overexpression in ovarian cancer cells. Thus, our findings suggest that TXNDC17, through participation of BECN1, induces autophagy and consequently results in paclitaxel resistance in ovarian cancer. TXNDC17 may be a potential predictor or target in ovarian cancer therapeutics.

Silencing dishevelled-1 sensitizes paclitaxel-resistant human ovarian cancer cells via AKT/GSK-3β/β-catenin signalling.

Expression of dishevelled-1 (DVL1) has recently been linked to cancer progression, however, its role in resistance to cancer therapy is unclear. In this study, we aimed to explore the function of DVL1 in paclitaxel-resistant human ovarian cancer cells. The MTT assay was used to assess effects of DVL1 silencing on sensitivity of cells that were otherwise resistant to paclitaxel (Taxol). Western blotting and immunofluorescence staining were used to examine effects of DVL1 on AKT/GSK-3β/β-catenin signalling. Dishevelled-1 was found to be over-expressed in a paclitaxel-resistant cell line derived from human ovarian cancer cell line A2780 (A2780/Taxol line) as well as parental A2780 cells. Down-regulation of DVL1 (using the inhibitor 3289-8625 or siRNA (siDVL1) against DVL1) sensitized A2780/Taxol cells to paclitaxel. Over-expression of DVL1 in A2780 cells increased protein levels of P-gp, BCRP and Bcl-2, which are known targets of β-catenin. Silencing DVL1 in A2780/Taxol cells also reduced levels of these proteins, and led to accumulation of β-catenin. In addition, DVL1 aberrantly activated AKT/GSK-3β/β-catenin signalling. Inactivation of AKT signalling attenuated DVL1-mediated inhibition of GSK-3β and accumulation of β-catenin, in both A2780 and A2780/Taxol cells. Taken together, these results suggest that silencing DVL1 sensitized A2780/Taxol cells to paclitaxel, by down-regulating AKT/GSK-3β/β-catenin signalling, providing a novel strategy for chemosensitization of ovarian cancer to paclitaxel-induced cytotoxicity.

Effects of suberoylanilide hydroxamic acid (SAHA) combined with paclitaxel (PTX) on paclitaxel-resistant ovarian cancer cells and insights into the underlying mechanisms.

BackgroundSuberoylanilide hydroxamic acid (SAHA) is a member of the hydroxamic acid class of the newly developed histone deacetylase inhibitors. Recently, Suberoylanilide hydroxamic acid has attracted increasing attention because of its antitumor activity and synergistic effects in combination with a variety of traditional chemotherapeutic drugs. Paclitaxel (PTX), is a natural anticancer drugs; however, resistance to paclitaxel has become a major challenge to the efficacy of this agent. The purpose of this study was to investigate the effects of the combined application of these two drugs on the paclitaxel-resistant ovarian cancer OC3/P cell line.MethodsIn the present study, the effects of Suberoylanilide hydroxamic acid or/and paclitaxel on OC3/P cells cultured in vitro were analyzed in terms of cell viability, migration, cell-cycle progression and apoptosis by CCK-8, wound healing and flow cytometry assays. Changes in cell ultrastructure were observed by transmission electron microscopy. The expression of genes and proteins related to proliferation, apoptosis and drug resistance were analyzed by quantitative real-time polymerase chain reaction and Western blot analyses.ResultsThere was no cross-resistance of the paclitaxel-resistant ovarian cancer OC3/P cells to Suberoylanilide hydroxamic acid. Suberoylanilide hydroxamic acid combined with paclitaxel significantly inhibited cell growth and reduced the migration of OC3/P cells compared with the effects of Suberoylanilide hydroxamic acid or paclitaxel alone. Q-PCR showed the combination of Suberoylanilide hydroxamic acid and paclitaxel reduced intracellular bcl-2 and c-myc gene expression and increased bax gene expression more distinctly than the application of SAHA or paclitaxel alone. Moreover, the level of mdr1 gene expression in cells treated with Suberoylanilide hydroxamic acid was lower than that of the control group (P <0.05). Western blot analysis showed that Suberoylanilide hydroxamic acid alone or in combination with paclitaxel enhanced caspase-3 protein expression and degraded ID1 protein expression in OC3/P cells.ConclusionSuberoylanilide hydroxamic acid inhibited the growth of paclitaxel-resistant ovarian cancer OC3/P cells and reduced migration by the induction of cell-cycle arrest, apoptosis and autophagy. These observations indicate the possible synergistic antitumor effects of sequential Suberoylanilide hydroxamic acid and paclitaxel treatment.

Microvesicles mediate transfer of P-glycoprotein to paclitaxel-sensitive A2780 human ovarian cancer cells, conferring paclitaxel-resistance

The overexpression of P-glycoprotein (P-gp) causes resistance to chemotherapy in human ovarian cancer. However, the underlying mechanism remains unclear. In the present study, we showed that, at membrane-bound protein level, P-gp was ‘shared’ between human ovarian cancer cells by the intercellular transfer of microvesicles (MVs). Paclitaxel-resistant human ovarian cancer cells (A2780/PTX) readily formed and released P-gp-containing MVs into the extracellular space compared with the wild-type parental line (A2780/WT). Shedding MVs bound to the chemosensitive A2780/WT cells in a time- and dose-dependent manner, transferring P-gp via the microenvironment. MV-mediated transfer of P-gp led to redistribution of the chemotherapeutic drug adriamycin in recipient cells (A2780/WT), which displayed 5- and 5-fold higher resistance to adriamycin and paclitaxel, respectively. Thus, these findings demonstrate a new mechanism of drug-resistance acquisition via MVs.

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.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Folic acid-coupled nano-paclitaxel liposome reverses drug resistance in SKOV3/TAX ovarian cancer cells

Chemotherapy could be used as an effective treatment for ovarian cancer and subsequent peritoneal metastasis. Administration of chemoagents in a targeted manner may bring the advantage of higher efficiency and lower drug resistance. In the present study, folate receptor (FR)-targeted nano-paclitaxel formulations were generated and tested for cytotoxicity in a peritoneal xenograft model of paclitaxel-resistant ovarian cancer and SKOV3/TAX cell lines. Immunocytochemical staining confirmed the expression of FR in both SKOV3 and SKOV3/TAX cells. The enrichment of the folic acid-coupled PEGylated nano-paclitaxel liposome (FA-NP) in FR-positive cells was visualized with fluorescence. The uptake of the FA-NP peaked at 4 h and was more robust than nontargeted PEGylated nano-paclitaxel liposome (NP). FA-NP but not NP markedly inhibited the growth of ovarian cancer cells and induced a two-fold increase in the doubling time. The cytotoxic effects of FA-NP were more potent than NP in both SKOV3 cells [50% of inhibition concentration (IC50), 5.67 vs. 50.2 μg/ml, FA-NP vs. NP] and SKOV3/TAX cells (IC50, 0.38 vs. >200 μg/ml, FA-NP vs. NP). FA-NP caused more G2-M cell cycle arrest and apoptotic changes in ovarian cancer cells than NP or regular paclitaxel. However, these effects were blunted in the presence of free FA, which competitively inhibited the receptor-mediated uptake of FA-NP particles. Intraperitoneal (i.p.) administration of FA-NP but not regular paclitaxel, NP, or vehicle significantly prolonged the survival and reduced tumor nodule number (2.9±0.3) in BALB/c nude mice. FA-NP also markedly enhanced the percentage of apoptotic cells in peritoneal xenografts of paclitaxel-resistant ovarian cancer cells (44.6±8.5 vs. 3.2±1.1% for vehicle, 22.4±5.9% for regular paclitaxel, and 35.2±7.7% for NP; P<0.05). However, intravenous administration of FA-NP at the same dose failed to induce apoptosis (20.1±6.2%; P<0.05) and inhibit tumor nodule number to the same extent as intraperitoneal administration. FA-NP reversed the drug resistance in paclitaxel-resistant SKOV3/TAX ovarian cancer cells both in vitro and in vivo. Localized and targeted administration of the FR-targeted chemoagents might prolong the survival time in patients with drug-resistant ovarian cancer.

A novel series of di-fluorinated propanedione derivatives synergistically augment paclitaxel mediated caspase 3 activation in ovarian cancer cells.

Both chronic and acute inflammatory circuits are known to be associated with malignancy and drug resistance indicating that many antiinflammatory agents can potentially act as chemotherapeutic drugs. A series of new class of propanediones with good antiinflammatory activity were shown to possess moderate cytotoxic activities. The aim of the study was to evaluate this new series of 1-(2',4'-difluorophenyl)-3-(substituted phenyl)-1,3 propanediones (PR 1-7) for their caspase dependent apoptotic activity by using a reporter gene mediated caspase-3 sensor in chemo sensitive and paclitaxel resistant ovarian cancer cells. A cellular model of paclitaxel resistance was developed in OAW42 cells stably expressing the caspase 3 sensor. The activity of caspase 3 after single and combinatorial drug treatments was determined using western blot and luciferase activity. Cell viability and cell cycle analysis were determined by MTT 3 (4,5- dimethyl thiazol-2 yl-2,5- diphenyl tetrazolium bromide (MTT) and Flow cytometric analysis (FACS) analysis. High Performance Liquid Chromatography (HPLC) analysis was performed to assess cellular uptake of the propanediones. Both nitro/methoxy (Group I) and halogen substituted propandiones at ortho, meta and para positions (Group II) showed a moderate increase in caspase-3 activity by 1.5- to 3.3-fold as compared with controls. However, no noticeable change in apoptotic cells percentage was observed. Increased intracellular uptake of Paclitaxel was observed during combinatorial treatment with one of the propanediones (PR2). Intriguingly, PR2 alone or in combination with Paclitaxel could induce a 2.5- to 2.9-fold increase in caspase-3 activity in Paclitaxel resistant cells. Our study reports a new class of propanediones that can augment the cytotoxic effect of Paclitaxel, and potentially can be used for treating Paclitaxel-resistant cancers.

Grape Seed Procyanidin Reversal of P-glycoprotein Associated Multi-Drug Resistance via Down-regulation of NF-κB and MAPK/ERK Mediated YB-1 Activity in A2780/T Cells

The expression and function of P-glycoprotein (P-gp) is associated with the phenotype of multi-drug resistance (MDR), leading chemotherapy failure of patients suffered with cancer. Grape seed procyanidin(GSP) is a natural polyphenol supplement with anti-inflammatory effect. Present study assessed a new use of GSP on the MDR reversal activity and its possible molecular mechanisms in MDR1-overpressing paclitaxel resistant ovarian cancer cells. Our results showed GSP significantly enhanced the cytotoxicity of paclitaxel and adriamycin in paclitaxel resistant A2780/T cells but its parental A2780 cells. Furthermore, GSP strongly inhibited P-gp expression by blocking MDR1 gene transcription, as well as, increased the intracellular accumulation of the P-gp substrate rhodamine-123 in A2780/T cells. Nuclear factor-κB(NF-κB) activity, IκB degradation level and NF-κB/p65 nuclear translocation induced by lipopolysaccharide (LPS) and receptor activator for nuclear factor-κB ligand (RANKL) were markedly inhibited by pre-treatment with GSP. Meanwhile, GSP inhibited MAPK/ERK pathway by decreasing the phosphorylation of ERK1/2, resulting in reduced the Y-box binding protein 1 (YB-1) activation with blocking its nuclear translocation. Moreover, the up-regulation of P-gp expression, the activation of AKT/NF-κB and MAPK/ERK pathway induced by LPS was attenuated by GSP administration. Compared with PDTC and U1026, inhibitor of NF-κB and MAPK/ERK respectively, GSP showed the same tendency of down-regulating NF-κB and MAPK/ERK mediated YB-1 activities. Thus, GSP reverses P-gp associated MDR by inhibiting the function and expression of P-gp through down-regulation of NF-κB activity and MAPK/ERK pathway mediated YB-1 nuclear translocation, offering insight into the mechanism of reversing MDR by natural polyphenol supplement compounds. GSP could be a new potential MDR reversal agent used for combination therapy with chemotherapeutics in clinic.

An Antimitotic and Antivascular Agent BPR0L075 Overcomes Multidrug Resistance and Induces Mitotic Catastrophe in Paclitaxel-Resistant Ovarian Cancer Cells

Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC50 = 2–7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.

Abstract 4500: Tubulin binding agent BPR0L075 induces iitotic catastrophe in paclitaxel-resistant ovarian cancer cells that is independent of caspase-3 activation.

Abstract Paclitaxel resistance is a significant clinical problem. In the current study, we evaluated antiproliferative effects of a tubulin binding agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC50 = 2-7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage and DNA fragmentation. Immunoblot analysis shows that survivin protein expression was depleted in resistant cells. BPR0L075 induced cell death in the paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms, supported by the absence of caspase-3 cleavage and ineffectiveness of caspase-3 inhibitor in protecting the resistant cells from BPR0L075 induced cell death. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a promising antimitotic agent in controlling taxane-resistant ovarian cancer cells. Citation Format: Xinli Liu, Weimin Sun, Xiaolei Wang, Erxi Wu. Tubulin binding agent BPR0L075 induces iitotic catastrophe in paclitaxel-resistant ovarian cancer cells that is independent of caspase-3 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4500. doi:10.1158/1538-7445.AM2013-4500

Abstract 973: Salinomycin sensitized paclitaxel-resistant human ovarian cancer cells and induced apoptosis.

Abstract Ovarian cancer has the highest mortality rate among gynecologic malignancies in the world. Drug resistance is the main cause of treatment failure and mortality in cancer patients. The objective of this study was to establish a paclitaxel (PTX)-resistant human ovarian carcinoma cell line (OVCAR3) and to sensitize chemoresistant cells. The drug-resistant tumor cell lines were established in vitro by stepwise sequential exposure to increased concentration of PTX. For sensitization, OVCAR3/PTX cells were treated with different concentration of salinomycin alone or combined with PTX. The cell viability and cell cycle distribution was measured by MTT and flow cytometric analysis. The transcriptional change of mRNA was examined by RT-PCR. The induction of apoptosis was measured by caspase-3 activity test, DNA fragmentation assay and western blot analysis. The cell viability was significantly reduced by combination treatment in a dose dependent manner. The flow cytometry result showed an increase in sub-G1 phase. Combination treatment enhanced the sensitivity of OVCAR3/PTX cells to PTX with down-regulation of P-glycoprotein, XIAP and survivin. Taken together, we demonstrated potential effect of salinomycin in sensitization of PTX resistant ovarian tumor growth. Our results suggest salinomycin in combination with PTX may be a beneficial chemotherapeutic strategy, especially in patients with tumors refractory to PTX alone. Citation Format: Chi-Heum Cho, Hyun-Gyo Lee, Bidur Parajuli, So-Jin Shin, Sang-Hoon Kwon, Soon-Do Cha. Salinomycin sensitized paclitaxel-resistant human ovarian cancer cells and induced apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 973. doi:10.1158/1538-7445.AM2013-973