CDDP-resistant Ovarian Cancer Cells Research Articles

Inhibition of ALDH1A1 Activity in Cisplatin-Resistant Ovarian Cancer Cells Alters Their Cancer Stemness, Cell Cycle Profile and Mitochondrial Respiration Rate

Introduction: Ovarian cancer is one of the leading cause of morbidity and death among women, with a five-year relative survival rate of only 30% in patients diagnosed with distant metastasis. The ovarian cancer cells initially respond to first-line platinum drug cisplatin [cis-diamminedichloroplatinum(II) (CDDP)] treatment. But, they subsequently develop resistance to CDDP and eventually exhibit chemoresistance. Aldehyde dehydrogenase 1 family member A1 (ALDH1A1) is one of the key functional markers of ovarian cancer stem cells (CSCs) that confers cancer stemness and therapeutic resistance, and is associated with poor prognosis and patient survival. In this study, we have assessed the anticancer effects of the ALDH1A1 inhibitor, A37, in CDDP-resistant ovarian cancer cells in vitro. Experimental Methods: SK-OV-3-CDDP, cisplatin-resistant ovarian cancer cells were treated with different concentrations of the small molecule inhibitor of ALDH1A1, A37. We determined the cell proliferation using water-soluble tetrazolium salt (WST-1) assay at 24 and 48 h. The distribution of cell division phases by cell cycle analysis and oxygen consumption rate (OCR) via seahorse extracellular flux analysis were assessed by flow cytometry and seahorse XFe24 analyzer, respectively. Furthermore, we examined the protein expression of key signaling molecules by western blot analysis and cancer stemness by tumorsphere formation assay. Results: Treatment of SK-OV-3-CDDP cells with A37 significantly reduced the ovarian cancer cell proliferation. Interestingly, A37 induced cell cycle arrest as observed by an increase in G1 phase of the cell cycle. Additionally, A37 reduced the mitochondrial respiration of ovarian cancer cells as observed by the decrease in basal OCR. Moreover, A37 treatment markedly decreased the expression of WW domain containing transcription regulator 1 (WWTR1) protein [also called as transcriptional co-activator with PDZ-binding motif (TAZ)], which is a key downstream effector of mammalian Hippo signaling pathway that promotes cancer stemness, metastasis and chemoresistance. Importantly, A37 reduced the number and size of the tumorspheres. Conclusions: Our study suggests that inhibiting the ALDH1A1 activity using A37 reduced the cell proliferation and induced cell cycle arrest in CDPP-resistant ovarian cancer cells. The mechanism by which A37 elicits its anticancer effects on ovarian cancer cells include impairment in mitochondrial respiration that could alter cancer cell metabolism, and a decrease in WWTR1/TAZ expression and tumorsphere formation that could suppress cancer stemness. Our findings demonstrate that inhibition of ALDH1A1 could effectively eliminate the chemoresistant ovarian cancer cells, and therefore, new strategies targeting ALDH1A1 could lead to the development of novel therapeutics for aggressive chemoresistant ovarian cancer.

Association of ALDH1A1-NEK-2 axis in cisplatin resistance in ovarian cancer cells

Development of acquired resistance to cisplatin (CDDP) is a major obstacle in the treatment of ovarian cancer patients. According to the cancer stem cell (CSC) hypothesis, the recurrence and chemoresistance are presumed to be linked to cancer stem/progenitor cells. Here, we investigated the CSC-like phenotypes and mechanism of chemoresistance in CDDP resistant ovarian cancer cells. A well-established CDDP sensitive ovarian cancer cell line A2780 and its resistant population A2780-Cp were used. We also developed a supra resistant population (SKOV3-Cp) from a naturally CDDP resistant cell line SKOV3. Both resistant/supra resistant cell lines showed significantly higher self-renewal capability than their parental counterparts. They also showed significant resistance to apoptosis and sub-G1 arrest by CDDP treatment. Stem cell marker ALDH1 positivity rates were higher both in A2780-Cp and SKOV3-Cp cell lines than in their counterparts, quantified by Aldefluor assay kit. Hoechst 33342 dye effluxing side populations were increased up to about five folds in A2780-Cp cells and two folds in SKOV3-Cp cells compared to A2780 and SKOV3 cells, respectively. Among major stemness related genes (POU5F1/OCT4, SOX2, NANOG, NES, BMI1, KLF4 and ALDH1A1), ALDH1A1 and KLF4 were significantly overexpressed in both resistant/supra resistant cells. Silencing ALDH1A1 in A2780 and A2780-Cp cells using siRNA greatly reduced the stem cell population and sensitized cells to CDDP. Moreover, silencing of ALDH1A1 reduced the transcript and protein level of its downstream target NEK-2. We also observed the downregulation of ABC transporters (ABCB1/MDR1, ABCG2 and ABCC1/MRP1) either by ALDH1A1 or NEK-2 silencing and upreguation of ABCB1/MDR1 due to the overexpression of NEK-2. Taken together, the present study suggests that stemness gene ALDH1A1 can be involved in CDDP resistance through the upregulation of NEK-2 in ovarian cancer.

Ailanthone increases oxidative stress in CDDP-resistant ovarian and bladder cancer cells by inhibiting of Nrf2 and YAP expression through a post-translational mechanism

Chemoresistance represents one of the main obstacles in treating several types of cancer, including bladder and ovarian cancers, and it is characterized by an increase of cellular antioxidant potential. Nrf2 and YAP proteins play an important role in increasing chemoresistance and in inducing antioxidant enzymes. It has been reported that Ailanthone (Aila), a compound extracted from the Ailanthus Altissima, has an anticancer activity toward several cancer cell lines, including chemoresistant cell lines. We have examined the effect of Aila on proliferation, migration and expression of Nrf2 and YAP proteins in A2780 (CDDP-sensitive) and A2780/CP70 (CDDP-resistant) ovarian cancer cells. Furthermore, to clarify the mechanism of Aila action we extended our studies to sensitive and CDDP-resistant 253J-BV bladder cancer cells, which have been used in a previous study on the effect of Aila. Results demonstrated that Aila exerted an inhibitory effect on growth and colony formation of sensitive and CDDP-resistant ovarian cancer cells and reduced oriented cell migration with higher effectiveness in CDDP resistant cells. Moreover, Aila strongly reduced Nrf2 and YAP protein expression and reduced the expression of the Nrf2 target GSTA4, and the YAP/TEAD target survivin. In CDDP-resistant ovarian and bladder cancer cells the intracellular oxidative stress level was lower with respect to the sensitive cells. Moreover, Aila treatment further reduced the superoxide anion content of CDDP-resistant cells in correlation with the reduction of Nrf2 and YAP proteins. However, Aila treatment increased Nrf2 and YAP mRNA expression in all cancer cell lines. The inhibition of proteolysis by MG132, a proteasoma inhibitor, restored Nrf2 and YAP protein expressions, suggesting that the Aila effect was at post-translational level. In accordance with this observation, we found an increase of the Nrf2 inhibitor Keap1, a reduction of p62/SQSTM1, a Nrf2 target which leads Keap1 protein to the autophagic degradation, and a reduction of P-YAP. Moreover, UCHL1 deubiquitinase expression, which was increased in bladder and ovarian resistant cells, was down-regulated by Aila treatment.In conclusion we demonstrated that Aila can reduce proliferation and migration of cancer cells through a mechanism involving a post translational reduction of Nrf2 and YAP proteins which, in turn, entailed an increase of oxidative stress particularly in the chemoresistant lines.

Knockdown of MALAT1 enhances chemosensitivity of ovarian cancer cells to cisplatin through inhibiting the Notch1 signaling pathway

Long non-coding RNAs (lncRNAs) are critical regulators in chemoresistance of various tumors including ovarian cancer. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been reported to be upregulated and contributed to ovarian cancer tumorigenesis. The aim of this study was to explore the roles of MALAT1 and the underlying molecular regulatory mechanism in the chemoresistance of ovarian cancer cells. Our data demonstrated that MALAT1 and Notch1 mRNA were upregulated in ovarian cancer tissues, as well as cisplatin (CDDP)-resistant ovarian cancer cells. A positive correlation between MALAT1 and Notch1 mRNA expression was observed. MALAT1 knockdown significantly attenuated CDDP resistance, and enhanced CDDP-induced apoptosis in CDDP-resistant ovarian cancer cells. MALAT1 knockdown enhanced CDDP-induced apoptosis in vivo, as indicated by upregulation of Bax protein expression and downregulation of Bcl-2 protein expression. Additionally, MALAT1 knockdown inhibited the Notch1 pathway and ABCC1 expression in CDDP-resistant ovarian cancer cells. MALAT1 was demonstrated to interact with Notch1. Notch1 knockdown attenuated CDDP resistance, and downregulated the protein expression of ABCC1 in ovarian cancer cells. Taken together, our findings suggested that knockdown of MALAT-1 enhanced chemosensitivity of ovarian cancer cells to CDDP through inhibiting Notch1 signaling pathway.

Abstract 2511: Association of ALDH1A1 with cisplatin resistance via modulation of NEK-2 in ovarian cancer stem-like cells

Abstract Cisplatin (CDDP) resistance is the foremost obstacle in the treatment of ovarian cancer patients. According to the cancer stem cell hypothesis, the recurrence and chemoresistance are presumed to be linked to cancer stem/progenitor cells. Here, we investigated the cancer stem cell like phenotypes and mechanism of chemoresistance in CDDP resistant ovarian cancer cells. A well-established CDDP sensitive ovarian cancer cell line A2780 and its resistant clone A2780 Cp were used and we also developed a supra resistant clone (SKOV3 Cp) from a naturally CDDP resistant cell line SKOV3 by six shocks treatment. Sensitiveness to CDDP was evaluated by MTT assay and self renewal efficiency by sphere formation, noble agar and clonogenic assays. A few cancer hallmarks and stem-cell markers were characterized using flow cytometry. Molecular analysis was performed using quantitative PCR and western blotting. For the silencing of selected genes small interfering RNA (siRNA) based approaches was utilized. Both CDDP resistant A2780 Cp and supra resistant SKOV3 Cp cell lines showed significantly (P < 0.05) higher self-renewal ability in sphere formation, noble agar and clonogenic assays than their parental counterparts. They also showed significant resistance to apoptosis and sub-G1 arrest upon CDDP treatment. Stem cell marker ALDH1 positivity rates were higher both in A2780 Cp and SKOV3 Cp cell lines, quantified by ALDEFLUORTM assay kit using flow cytometry. Hoechst 33342 dye effluxing side populations were increased about five folds in A2780 Cp cells and two folds in SKOV3 Cp cells compared to A2780 and SKOV3 cells respectively. Among major stemness related genes (OCT4, SOX2, NANOG, NESTIN, BMI1, KLF4 and ALDH1A1) quantitative PCR analysis showed significantly higher expression of ALDH1A1 and KLF4 genes in both resistant and supra resistant cells than their parental clones. Of two genes, silencing ALDH1A1 in A2780 and A2780 Cp cells using siRNA greatly reduced the stem cell population as detected by ALDEFLUORTM assay and sensitized cells to CDDP. Moreover, silencing of ALDH1A1 reduce the level of its downstream target NEK-2. We also observed the downregulation of ABC transporters (ABCB1, ABCG2 and ABCC1) by silencing either ALDH1A1 or NEK-2 gene. Taken together, present study showed for the first time that stemness gene ALDH1A1 can be involved in CDDP resistance via the upregulation of NEK-2 in ovarian or solid cancer. Citation Format: Md. Hafiz Uddin, Yong Sang Song. Association of ALDH1A1 with cisplatin resistance via modulation of NEK-2 in ovarian cancer stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2511.

Forkhead Box Protein C2 Promotes Epithelial-Mesenchymal Transition, Migration and Invasion in Cisplatin-Resistant Human Ovarian Cancer Cell Line (SKOV3/CDDP)

Background/Aims: Forkhead Box Protein C2 (FOXC2) has been reported to be overexpressed in a variety of human cancers. However, it is unclear whether FOXC2 regulates epithelial-mesenchymal transition (EMT) in CDDP-resistant ovarian cancer cells. The aim of this study is to investigate the effects of FOXC2 on EMT and invasive characteristics of CDDP-resistant ovarian cancer cells and the underlying molecular mechanism. Methods: MTT, Western blot, scratch wound healing, matrigel transwell invasion, attachment and detachment assays were performed to detect half maximal inhibitory concentration (IC₅₀) of CDDP, expression of EMT-related proteins and invasive characteristics in CDDP-resistant ovarian cancer cell line (SKOV3/CDDP) and its parental cell line (SKOV3). Small hairpin RNA (shRNA) was used to knockdown FOXC2 and analyze the effect of FOXC2 knockdown on EMT and invasive characteristics of SKOV3/CDDP cells. Also, the effect of FOXC2 upregulation on EMT and invasive characteristics of SKOV3 cells was analyzed. Furthermore, the molecular mechanism underlying FOXC2-regulating EMT in ovarian cancer cells was determined. Results: Compared with parental SKOV3 cell line, SKOV3/CDDP showed higher IC₅₀ of CDDP (43.26μM) (P<0.01) and acquired EMT phenotype and invasive characteristics. Gain- and loss-of-function assays indicated that shRNA-mediated FOXC2 knockdown could reverse EMT and reduce the capacity of migration, invasion, attachment and detachment in SKOV3/CDDP cell line and upregulation of FOXC2 could induce the reverse effects in parental SKOV3 cell line. Furthermore, it was found that activation of ERK or AKT/GSK-3β signaling pathways was involved in FOXC2-promoting EMT in CDDP-resistant ovarian cancer cells. Conclusions: Taken together, these data demonstrate that FOXC2 may be a promoter of EMT phenotype in CDDP-resistant ovarian cancer cells and a potential therapeutic target for the treatment of advanced ovarian cancer.

Forkhead Box Protein C2 (FOXC2) Promotes the Resistance of Human Ovarian Cancer Cells to Cisplatin In Vitro and In Vivo

Background/Aims: FOXC2 has been reported to play a role in tumor progression, but the correlations of FOXC2 with the cisplatin (CDDP) resistance of ovarian cancer cells are still unclear. The purpose of the present study is to investigate the roles of FOXC2 in the CDDP resistance of ovarian cancer cells and its possible mechanisms. Methods: Quantitative real-time PCR (qRT-PCR) was performed to detect the expression of FOXC2 mRNA in CDDP-resistant or sensitive ovarian cancer tissues and cell lines (SKOV3/CDDP and SKOV3). Gain- and loss-of-function assays were performed to analyze the effects of FOXC2 knockdown or overexpression on the in vitro and in vivo sensitivity of ovarian cancer cells to CDDP and its possible molecular mechanisms. Results: The relative expression level of FOXC2 mRNA in CDDP-resistant ovarian cancer tissues was higher than that in CDDP-sensitive tissues. Also, the expression of FOXC2 mRNA and protein in CDDP-resistant ovarian cancer cell line (SKOV3/CDDP) cell line was higher than that in its parental cell line (SOKV3). Small hairpin RNA (shRNA)-mediated FOXC2 knockdown significantly increased the in vitro and in vive sensitivity of SKOV3/CDDP cells to CDDP by enhancing apoptosis, while upregulation of FOXC2 significantly decreased the in vitro and in vivo sensitivity of SKOV3 cells to CDDP by reducing apoptosis. Furthermore, FOXC2 activates the Akt and MAPK signaling pathways, and then induced the decreased expression of Bcl-2 protein and the increased expression of Bax and cleaved caspase-3 proteins. Conclusions: FOXC2 mediates the CDDP resistance of ovarian cancer cells by activation of the Akt and MAPK signaling pathways, and may be a potential novel therapeutic target for overcoming CDDP resistance in human ovarian cancer.

Chitosan coated polylactic acid nanoparticle-mediated combinatorial delivery of cisplatin and siRNA/Plasmid DNA chemosensitizes cisplatin-resistant human ovarian cancer cells.

Development of resistance toward anticancer drugs results in ineffective therapy leading to increased mortality. Therefore, overriding resistance and restoring sensitivity to anticancer drugs will improve treatment efficacy and reduce mortality. While numerous mechanisms for drug resistance in cancer have previously been demonstrated, recent studies implicate a role for proteasome and the autophagy regulatory protein P62/SQSTM1 (P62) in contributing to drug resistance. Specifically, reduction in the expression of the β5 subunit of the proteasome and/or enhanced P62 protein expression is known to contribute to cancer drug resistance such as cisplatin (CDDP) in ovarian cancer cells. Therefore, we hypothesized that restoration of β5 expression and/or suppression of P62 protein expression in CDDP-resistant ovarian cancer cells will lead to restoration of sensitivity to CDDP and enhanced cell killing. To test our hypothesis we developed a biodegradable multifunctional nanoparticle (MNP) system that codelivered P62siRNA, β5 plasmid DNA, and CDDP and tested its efficacy in CDDP resistant 2008/C13 ovarian cancer cells. MNP consisted of CDDP loaded polylactic acid nanoparticle as inner core and cationic chitosan (CS) consisting of ionically linked P62siRNA (siP62) and/or β5 expressing plasmid DNA (pβ5) as the outer layer. The MNPs were spherical in shape with a hydrodynamic diameter in the range of 280-350 nm, and demonstrated encapsulation efficiencies of 82% and 78.5% for CDDP and siRNA respectively. MNPs efficiently protected the siRNA and showed superior serum stability compared to naked siRNA as measured by gel retardation and spectrophotometry assays. The MNPs successfully delivered siP62 and pβ5 to cause P62 knockdown and restoration of β5 expression in 2008/C13 cells. Combined delivery of siP62, pβ5, and CDDP using the MNPs resulted in a marked reduction in the IC50 value of CDDP in 2008/C13 cells from 125 ± 1.3 μM to 98 ± 0.6 μM (P < 0.05; 21.6% reduction) when compared to the reduction in the IC50 of CDDP observed in cells that had only siP62 delivered (IC50 = 106 ± 1.1 μM; P < 0.05; 15.2% reduction) or pβ5 delivered (IC50 = 115 ± 2.8 μM; 8% reduction) via MNPs. Finally, our studies showed that the CDDP resistance index in 2008/C13 cells was reduced from 4.62 for free CDDP to 3.62 for MNP treatment. In conclusion our study results demonstrated the efficacy of our MNP in overcoming CDDP resistance in ovarian cancer cells.

Combination of FTY720 with cisplatin exhibits antagonistic effects in ovarian cancer cells: Role of autophagy

Combination therapy with different anticancer drugs has been proven to be an effective strategy for the treatment of various types of cancers, including ovarian cancer. We have previously reported that FTY720 exhibited potent cytotoxic effects in ovarian cancer cells through the necrotic pathway, which differs from the killing effect of cisplatin (CDDP). In the present study, we report that the combination of FTY720 with CDDP yields an unexpected antagonistic effect towards the cytotoxicity of CDDP in a variety of ovarian cancer cell lines, including both CDDP-sensitive and -resistant cells. The antagonistic activity of FTY720 appears ascribable to its effect in autophagy induction. A significant increase in baseline autophagy was observed in CDDP-resistant ovarian cancer cells, compared with the sensitive cells. Blockade of autophagy by either a pharmacological inhibitor (3-MA) or siRNA-mediated knockdown of autophagic gene expression enhances CDDP-induced apoptotic cell death. Notably, by inhibiting autophagy, 3-MA can convert the combination of FTY720 with CDDP from an antagonistic into an additive effect towards killing ovarian cancer cells. Collectively, the findings suggest that a combination of an autophagy regulator with the CDDP-based regime could effectively modulate its efficacy for the treatment of ovarian cancer.

ΔNp63α Confers Tumor Cell Resistance to Cisplatin through the AKT1 Transcriptional Regulation

Strategies to address resistance to platin drugs are greatly needed in human epithelial cancers (e.g., ovarian, head/neck, and lung) where platins are used widely and resistance occurs commonly. We found that upon ΔNp63α overexpression, AKT1 and phospho-AKT1 levels are upregulated in cancer cells. Investigations using gel-shift, chromatin immunoprecipitation and functional reporter assays implicated ΔNp63α in positive regulation of AKT1 transcription. Importantly, we found that ΔNp63α, AKT1, and phospho-AKT levels are greater in 2008CI3 CDDP-resistant ovarian cancer cells than in 2008 CDDP-sensitive cells. siRNA-mediated knockdown of ΔNp63α expression dramatically decreased AKT1 expression, whereas knockdown of either ΔNp63α or AKT1 decreased cell proliferation and increased death of ovarian and head/neck cancer cells. Conversely, enforced expression of ΔNp63α increased cancer cell proliferation and reduced apoptosis. Together, our findings define a novel ΔNp63α-dependent regulatory mechanism for AKT1 expression and its role in chemotherapeutic resistance of ovarian and head/neck cancer cells.

Intraperitoneal administration of telomerase-specific oncolytic adenovirus sensitizes ovarian cancer cells to cisplatin and affects survival in a xenograft model with peritoneal dissemination

Despite tremendous development in chemotherapy for ovarian cancer over the past few decades, the prognosis of advanced cases with massive peritoneal dissemination is still unsatisfactory, and novel treatment modalities that can combine with chemotherapy are urgently needed. We recently developed virotherapy for solid tumors using telomerase-specific replication-selective adenoviruses (Telomelysin: OBP-301), in which the human telomerase reverse transcriptase (hTERT) gene promoter has been inserted to direct tumor-specific E1 gene expression. In this study, we investigated the anti-tumor effects of OBP-301, combined with cisplatin (CDDP), on ovarian cancer cells. In vitro treatment of SKOV3 cells with OBP-301 at a multiplicity of infection (MOI) of 0.01–100 induced significant cell death in a dose-dependent manner, with moderate cytotoxicity at an MOI of 1–10 and maximal cytotoxicity at an MOI of 100. In contrast, OBP-301 treatment of normal human cells showed no significant cell death at an MOI of 1–10 and exhibited modest cytotoxicity at an MOI of 100. The effects of low-dose CDDP at 0.5–1 μM, which induced only 20% cell death, were significantly augmented by combination with OBP-301 at an MOI of 1–10, finally achieving 40% cell death. Such enhancement of CDDP sensitivity was also observed in CDDP-resistant ovarian cancer cells. The combinatorial effects were further tested using a xenograft mouse model of SKOV3 with peritoneal dissemination. After intraperitoneal administration of OBP-301, we confirmed that injected OBP-301 fused with the green fluorescent protein (GFP) gene (OBP-401) was preferentially localized to peritoneal disseminations, as determined by fluorescence imaging. Treatment of mice with CDDP at low dose (0.5 mg kg–1) had modest effects, showing a 10% decrease in disseminations, whereas combination with intraperitoneal administration of OBP-301 at an MOI of 10 led to enhanced effects, achieving an approximately 80% decrease in disseminations. Kaplan–Meier analysis showed improved overall survival of mice treated with CDDP plus OBP-301 compared with CDDP alone. These findings support the therapeutic potential of intraperitoneal administration of OBP-301 to sensitize ovarian cancer cells to CDDP.