Drug-resistant Ovarian Cancer Cell Lines Research Articles

The TGFBI gene and protein expression in topotecan resistant ovarian cancer cell lines

PurposeThe primary limiting factor in achieving cures for patients with cancer, particularly ovarian cancer, is drug resistance. The mechanisms of drug resistance of cancer cells during chemotherapy may include compounds of the extracellular matrix, such as the transforming growth factor-beta-induced protein (TGFBI). In this study, we aimed to analyze the TGFBI gene and protein expression in different sensitive and drug-resistant ovarian cancer cell lines, as well as test if TGFBI can be involved in the response to topotecan (TOP) at the very early stages of treatment. Materials and methodsIn this study, we conducted a detailed analysis of TGFBI expression in different ovarian cancer cell lines (A2780, A2780TR1, A2780TR2, W1, W1TR, SKOV-3, PEA1, PEA2 and PEO23). The level of TGFBI mRNA (QPCR), intracellular and extracellular protein (Western blot analysis) were assessed in this study. ResultsWe observed upregulation of TGFBI mRNA in drug-resistant cell lines and estrogen-receptor positive cell lines, which was supported by overexpression of both intracellular and extracellular TGFBI protein. We also showed the TGFBI expression after a short period of treatment of sensitive ovarian cancer cell lines with TOP. ConclusionThe expression of TGFBI in ovarian cancer cell lines suggests its role in the development of drug resistance.

MiRNA-mRNA integrative analysis reveals epigenetically regulated and prognostic miR-103a with a role in migration and invasion of carboplatin-resistant ovarian cancer cells that acquired mesenchymal-like phenotype

BackgroundDNA methylation, histone modifications, and miRNAs affect ovarian cancer (OC) progression and therapy response. PurposeIdentification of epigenetically downregulated miRNAs in drug-resistant OC cell lines with a possible role in drug resistance and/or drug-induced mesenchymal-like phenotype. MethodsMiRNA profiling was performed on parental and carboplatin-resistant OC cells, MES-OV and MES-OV CBP. RT-qPCR validation, epigenetic modulation and other CBP-resistant OC cell lines were used to select miRNAs of interest. The integration of miRNA-predicted target genes and differentially expressed genes (DEGs), pathway and functional analysis were used for forecasting their biological role. Data mining was performed to determine their possible prognostic and predictive values. ResultsMiRNA profiling revealed 48 downregulated miRNAs in OC cells whose drug sensitivity and metastatic potential were impacted by epigenetic modulators. Of the fourteen selected, nine were validated as changed, and seven of these restored their expression upon treatment with epigenetic inhibitors. Only three had similar expression patterns in other OC cell lines. MiRNA-mRNA integrative analysis resulted in 56 target DEGs. Pathway analysis revealed that these genes are involved in cell adhesion, migration, and invasion. The functional analysis confirmed the role of miR-103a–3p, miR-17–5p and miR-107 in cell invasion, while data mining showed their prognostic and predictive values. Only miR-103a–3p was epigenetically regulated at the constitutive level. ConclusionHigh throughput miRNA and cDNA profiling coupled with pathway analysis and data mining delivered evidence for miRNAs which can be epigenetically regulated in drug-resistant, mesenchymal-like OC cells as possible markers to combat therapy-induced short overall survival and tumor metastatic potential.

Bioactive Hexapeptide Reduced the Resistance of Ovarian Cancer Cells to DDP by Affecting HSF1/HSP70 Signaling Pathway.

Ovarian cancer is the leading cause of death in gynecologic malignancies. Ovarian cancer as a metastatic malignant tumor is highly recurrent and prone to drug resistance. Bioactive peptides are an emerging area of biomedical research in reducing resistance of tumor cell to drugs. In this paper, we investigated the effects and mechanisms of bioactive hexapeptide (PGPIPN) derived in milk protein on the sensitivity of ovarian cancer cells to cis-dichlorodiammine platinum (DDP). Human ovarian cancer cell lines (SKOV3 and COC1), their DDP-resistant sublines (SKOV3/DDP and COC1/DDP) and human primary ovarian cancer cells were cultured in vitro under the combined treatment of DDP (close to IC50) and different concentrations of PGPIPN. The viabilities, apoptosis and cell cycle changes were respectively measured by WST-8 and flow cytometry. The mRNA and protein expression levels of HSF1, HSP70, MDR1, ERCC1 and β-actin gene were respectively assayed by RT-qPCR and western blotting. The results showed that PGPIPN significantly increased the sensitivity of human ovarian cancer cells to DDP in inhibiting viability and inducing apoptosis in vitro. But the effects in sensitive cells were lower than DDP-resistant cells. PGPIPN significantly changed the cell cycles in all human ovarian cancer cells, which leaded to a significant increase in the percentage of cells blocked at G2/M phase and decrease the percentage of cells at G1 phases in a dose-dependent manner. PGPIPN affected the expression levels of HSF1, HSP70, MDR1 and ERCC1 genes. Compared with cells in DDP treatment alone, the expression levels of HSF1 and HSP70 in human ovarian cancer cells treated with DDP and PGPIPN together significantly decreased in dose-dependent manner. PGPIPN significantly decreased MDR1 and ERCC1 of drug-resistant ovarian cancer cell lines and human primary ovarian cancer cell in a dose-dependent manner. Pifithrin-μ (PFTμ, HSP70 inhibitor) decreased or removed the effects of peptide in increasing the sensitivity of ovarian cancer cells to DDP. This suggests that PGPIPN enhanced the sensitivity of ovarian cancer cells to DDP partially via reducing the activity of HSF1/HSP70 signaling pathway, thus inducing cell apoptosis and decreasing repairment of DNA damage.

Abstract 3627: Development of CLENs for future treatment & diagnostic detection using cell models of drug-resistant ovarian cancer

Abstract Current guidelines suggest the use of a platinum agent and a taxane as first line therapy for ovarian cancer where total surgical resection is limited. Liposomal preparations of these drugs have demonstrated therapeutic value by increasing drug uptake and reducing toxicity. The following research aims to improve target specificity by employing a multi-strategic approach to drug targeting and treatment in vitro. Cellular membrane lipid-extracted nanoliposomes (CLENs) were constructed in part from lipid extracts (LE) derived from the membrane of multidrug resistant ovarian cancer (SKOV-3-MDR) cell lines, and more conventional components of liposomes. Formulations of greatest interest were selected after comparing cellular uptake studies of conventional liposomes to various types of CLENs composed of mixed ratios of DOPC, cholesterol, and LE. Cellular uptake was measured by rhodamine fluorescence (RF) following 24 hours incubation in 48 well plates, where RF was used as an indicator of cellular uptake. To determine effectiveness of LE, formulations with and without synthetic cationic lipid DOTAP were compared. Flow cytometry was used to evaluate liposome binding. Additional studies include drug loading efficiency and SRB cytotoxicity studies involving cisplatin, and the effect of additional inclusion of MAG-C (magnetic material) in the SKOV-3 lipid extract-containing liposomes. CLENs containing 15% LE demonstrated greater uptake compared to CLENs prepared with 0% and 5% LE content. Minimal uptake was observed in non-target cancer cell lines (prostate cancer-CRL-1740 and gliobastoma-U87MG), as well as with normal pancreas, non-malignant (hTERT) cell line. The fluorescence intensity values reported for SKOV-3-MDR, CRL-1740, U87-MG and hTERT, were 437 ± 153, 295 ± 67, 57 ± 6, and 29 ± 6, respectively. The additional inclusion of DOTAP showed significant cellular uptake with and without LE. Flow cytometry showed greater initial binding of CLENs to target cells when LE was included in preparations. Incorporation efficiency of cisplatin into SKOV-3 CLENS was 67%. Chemotherapy drug-loaded CLENs is an appealing vehicle as it addresses many parameters. Early findings suggest that CLENS is a suitable vehicle for targeting and treatment using a drug-resistant ovarian cancer cell line. CLENS allows for faster initial nanoparticle binding, cell-type specific uptake, and enhances cytotoxicity. Our encouraging results corroborate this theorem, showing greater targeting with reduced uptake by non-target cells. Additional studies involving MAG-C are currently underway. Citation Format: Negar Amini, Joshua Emerson, Jenel Clement. Development of CLENs for future treatment & diagnostic detection using cell models of drug-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3627.

Inhibition of ALDH1A1 activity decreases expression of drug transporters and reduces chemotherapy resistance in ovarian cancer cell lines

The high mortality of ovarian cancer patients results from the failure of treatment caused by the inherent or acquired chemotherapy drug resistance. It was reported that overexpression of aldehyde dehydrogenase A1 (ALDH1A1) in cancer cells can be responsible for the development of drug resistance. To add the high expression of the drug transporter proteins the ALDHA1 is considered as a molecular target in cancer therapy. Therefore, we analysed drug-resistant ovarian cancer cell lines according to ALDHA1 expression and the association with drug resistance.The expression of ALDH1A1, P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) was determined using a microarray and confirmed by Q-PCR, western blot and fluorescence analysis. ALDH1A1 activity was determined using an Aldefluor assay. The impact of all-trans retinoic acid (ATRA) and diethylaminobenzaldehyde (DEAB) on chemotherapy resistance was assessed by the MTT chemosensitivity assay.The most abundant expression of ALDH1A1 was noted in paclitaxel- and topotecan-resistant cell lines where two populations of ALDH-positive and ALDH-negative cells could be observed. Those cell lines also revealed the overexpression of P-gp and BCRP respectively, and were able to form spheres in non-adherent conditions. Pre-treatment with ATRA and DEAB reduced chemotherapy resistance in both cell lines. ATRA treatment led to downregulation of the ALDH1A1, P-gp and BCRP proteins. DEAB treatment led to downregulation of the P-gp protein and BCRP transcript and protein.Our results indicate that ALDH1A1-positive cancer cells can be responsible for drug resistance development in ovarian cancer. Developing more specific ALDH1A1 inhibitors can increase chemotherapy effectiveness in ovarian cancer.

Downregulation of vimentin expression increased drug resistance in ovarian cancer cells.

Cisplatin and other platinum-based drugs have been widely used in the treatment of ovarian cancer, but most patients acquire the drug resistance that greatly compromises the efficacy of drugs. Understanding the mechanism of drug resistance is important for finding new therapeutic approaches. In the present study, we found that the expression of vimentin was downregulated in drug-resistant ovarian cancer cell lines A2780-DR and HO-8910 as compared to their respective control cells. Overexpression of vimentin in A2780-DR cells markedly increased their sensitivity to cisplatin, whereas knockdown of vimentin in A2780, HO-8910-PM and HO-8910 cells increased the resistance to cisplatin, demonstrating that vimentin silencing enhanced cisplatin resistance in ovarian cancer cells. Quantitative proteomic analysis identified 95 differentially expressed proteins between the vimentin silenced A2780 cells (A2780-VIM-KN) and the control cells, in which downregulation of endocytic proteins and the upregulation of exocytotic proteins CHMP2B and PDZK1 were proposed to contribute the decreased cisplatin accumulation in vimentin knockdown cells. Silencing of vimentin induced upregulation of cancer stem cell markers and both A2780-DR and A2780-VIM-KN cells were more facile to form spheroids than control cells under serum-free culture condition. Our results also revealed that vimentin knockdown increased the 14-3-3 mediated retention of Cdc25C in the cytoplasm, leading to inactivation of Cdk1 and the prolonged G2 phase arrest that allowed the longer period of time for cells to repair cisplatin-damaged DNA. Taken together, we demonstrated that vimentin silencing enhanced cells' resistance to cisplatin via prolonged G2 arrest and increased exocytosis, suggesting that vimentin is a potential target for treatment of drug resistant ovarian cancer.

Reduced accumulation of platinum drugs is not observed in drug-resistant ovarian cancer cell lines derived from cisplatin-treated patients

The resistance of ovarian cancer towards front-line chemotherapy, usually cisplatin or carboplatin in combination with paclitaxel or docetaxel, remains a major clinical challenge. Resistance to these agents has been largely studied using cell lines selected for resistance to agents in vitro. We examined a series of paired cell lines derived from patients with ovarian cancer prior to chemotherapy (PEO1, PEO4, PEO14 and PEA1), and following the acquisition of resistance to a platinum-based chemotherapy regimen (PEO6, PEO23 and PEA2, respectively). All resistant patient lines showed resistance to cisplatin (2-5-fold), but this did not correspond with lowered accumulation. No general cross-resistance was observed for oxaliplatin, paclitaxel or docetaxel, and paclitaxel accumulation was not affected. PEO1 cells carrying BRCA2 mutations were hypersensitive to the PARP inhibitors olaparib and velaparib, but all other cell lines expressing functional forms of BRCA2 were less sensitive. While reduced drug accumulation was not observed, we believe these pairs of lines are of use to researchers studying Pt drug resistance and experimental therapeutics against drug-resistant ovarian cancer.

Circulating peptidome to indicate the tumor-resident proteolysis.

Tumor-resident proteases (TRPs) are regarded as informative biomarkers for staging cancer progression and evaluating therapeutic efficacy. Currently in the clinic, measurement of TRP is dependent on invasive biopsies, limiting their usefulness as monitoring tools. Here we identified circulating peptides naturally produced by TRPs, and evaluated their potential to monitor the efficacy of anti-tumor treatments. We established a mouse model for ovarian cancer development and treatment by orthotopic implantation of the human drug-resistant ovarian cancer cell line HeyA8-MDR, followed by porous silicon particle- or multistage vector (MSV) - enabled EphA2 siRNA therapy. Immunohistochemistry staining of tumor tissue revealed decreased expression of matrix metallopeptidase 9 (MMP-9) in mice exhibiting positive responses to MSV-EphA2 siRNA treatment. We demonstrated, via an ex vivo proteolysis assay, that C3f peptides can act as substrates of MMP-9, which cleaves C3f at L1311-L1312 into two peptides (SSATTFRL and LWENGNLLR). Importantly, we showed that these two C3f-derived fragments detected in serum were primarily generated by tumor-resident, but not blood-circulating, MMP-9. Our results suggested that the presence of the circulating fragments specially derived from the localized cleavage in tumor microenvironment can be used to evaluate therapeutic efficacy of anti-cancer treatment, assessed through a relatively noninvasive and user-friendly proteomics approach.

Noscapine Increases the Sensitivity of Drug-Resistant Ovarian Cancer Cell Line SKOV3/DDP to Cisplatin by Regulating Cell Cycle and Activating Apoptotic Pathways.

Cisplatin is a first-line chemotherapy drug against ovarian cancer. However, its strong toxic side effects and the development of cisplatin resistance in human cancer cells seriously influence the effects of chemotherapy and quality of life in patients. Noscapine (Nos), a non-toxic benzylisoquinoline alkaloid extracted from opium, has been recently reported to have anti-cancer activity, but the mechanism of that effect has not been clearly established. In the present study, we investigated cytotoxicity of Nos in combination with cisplatin (DDP) in drug-resistant human ovarian cancer cell line SKOV3/DDP in vitro and in vivo null mice xenograft model. Cell proliferation was measured by MTT assay, flow cytometry was used to analyze cell cycle and apoptosis, protein expression of several apoptotic factors was investigated by flow cytometry and immunohistochemical method, and their mRNA expression levels were determined by real-time PCR. In vitro experiments showed that Nos significantly inhibited proliferation of SKOV3/DDP cells. DDP/Nos-combined treatment notably enhanced DDP-induced inhibition of cell proliferation and increased the pro-apoptotic effect of DDP in SKOV3/DDP cells. DDP/Nos administration increased the proportion of G2/M cells, reduced both protein and mRNA expression of anti-apoptotic factors XIAP, surviving and NF-kB, and augmented protein and mRNA levels of pro-apoptotic caspase-3. In vivo experiments revealed that Nos/DDP treatment increased the apoptotic rate of xenograft tumors in null mice. Tumor volume decreased from 1.733 ± 0.155 g in mice treated with DDP alone to 1.191 ± 0.106 g in animals treated with Nos/DDP. These observations suggest that Nos increases the anti-cancer activity of DDP against the drug-resistant ovarian cancer cell line SKOV3/DDP by modulating the cell cycle and activating apoptotic pathways. The study provides a new chemotherapy strategy for the treatment of DDP-resistant human ovarian cancer.

Microarray-based detection and expression analysis of extracellular matrix proteins in drug‑resistant ovarian cancer cell lines.

Ovarian cancer is the most lethal gynecological malignancy. Multiple drug resistance (MDR) development leads to resistance of cancer cells to chemotherapy. Microarray methods can provide information regarding new candidate genes that can play a role in resistance to cytostatic drugs. Extracellular matrix (ECM) can influence drug resistance by inhibiting the penetration of the drug into cancer tissue as well as increased apoptosis resistance. In the present study, we report changes in the ECM and related gene expression pattern in methotrexate-, cisplatin-, doxorubicin-, vincristine-, topotecan- and paclitaxel-resistant variants of the W1 ovarian cancer cell line. The resistant variants of the W1 cell line were generated by stepwise selection of cells with an increasing concentration of the indicated drugs. Affymetrix GeneChip® Human Genome U219 Array Strips were used for hybridizations. Independent t-tests were used to determinate the statistical significance of results. Genes whose expression levels were higher than the assumed threshold (upregulated, >5-fold and downregulated, <5-fold) were visualized using the scatter plot method, selected and listed in the tables. Among the investigated genes, expression of 24 genes increased, expression of 14 genes decreased and expression of three genes increased or decreased depending on the cell line. Among the increased genes, expression of 10 increased very significantly, >20-fold. These genes were: ITGB1BP3, COL3A1, COL5A2, COL15A1, TGFBI, DCN, LUM, MATN2, POSTN and EGFL6. The expression of seven genes decreased very significantly: ITGA1, COL1A2, LAMA2, GPC3, KRT23, VIT and HMCN1. The expression pattern of ECM and related genes provided the preliminary view into the role of ECM components in cytostatic drug resistance of cancer cells. The exact role of the investigated genes in drug resistance requires further investigation.

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.

Abstract 4077: Chemosensitizing effects of the novel, small molecule DNA methylation inhibitor SGI-110 in ovarian cancer

Abstract Deoxycytosine methylation of CpG islands in promoter regions of tumor suppressor genes (TSGs) plays a prominent role in the development and progression of drug-resistant epithelial ovarian cancer (OC). We previously demonstrated in a phase I/II trial that the DNA methylation inhibitor decitabine alters DNA methylation and restores platinum sensitivity in platinum-resistant OC patients, resulting in significant clinical activity. SGI-110 (Astex Pharmaceuticals) is a DNA hypomethylating agent with demonstrated activity in restoring silenced TSG expression in cancer cells by reversal of DNA methylation. As a decitabine-deoxyguanosine dinucleotide, SGI-110 has been shown to be less prone to deamination by cytidine deaminase and could have advantages over decitabine, such as better stability, less toxicity and a more convenient and less frequent SQ administration. We examined SGI-110 for its ability to inhibit OC cell proliferation and to demethylate and induce TSGs in vitro and in vivo. A 48 hour pretreatment with SGI-110 resensitized drug-resistant ovarian cancer cell lines to cisplatin (3-fold reduction in IC50 of cisplatin). In A2780 platinum sensitive OC cells, SGI-110 pre-treatment for 5 days reduced cisplatin IC50 by &amp;gt;2-fold. Prolonged (7 days) SGI-110 treatment reduced by 2-fold the number of ALDH1+ cells in SKOV3 and A2780 cells, suggesting an effect on the stem cell population, potentially involved in drug resistance. SGI-110 induced significant demethylation of TSGs ras-associated domain family 1A (RASSF1A) and human MutL homologue-1 (MLH1) and the differentiation-associated gene HOXA10; furthermore, RASSF1A, MLH1 and HOXA10 gene reexpression was also observed after 48 hour SGI-110 treatment. SGI-110 prevented TGF-β induced epithelial to mesenchymal transition of OC cells, as measured morphologically and by quantification of E-cadherin, Zeb1, Slug and miR200c expression levels. SGI-110 suppressed E-cadherin promoter methylation induced by TGF-β. We then examined the in vivo tolerability and efficacy of SGI-110, singly and in combination with cisplatin, in nude athymic mice. Mice were injected qd or bi-weekly with SGI-110 (sc) and/or cisplatin (ip) and body weights were measured twice weekly. In the qd regimen, SGI-110 2 mg/kg plus 4 mg/kg cisplatin was tolerated. The 10 mg/kg bi-weekly dose of SGI-110 was tolerated, either alone or in combination with 2 mg/kg cisplatin (5mg/kg SGI-110 plus 4 mg/kg cisplatin was similarly tolerated). SGI-110 induced significant global hypomethylation, as determined by pyrosequencing analysis of LINE1 demethylation in peripheral blood mononuclear cells. We are currently assessing the activity of SGI-110 in combination with cisplatin to retard the growth of platinum resistant human ovarian cancer xenografts. In summary, SGI-110 combined with platinum is a promising clinical combination for the therapy of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4077. doi:1538-7445.AM2012-4077

Necdin, a negative growth regulator, is a novel STAT3 target gene down-regulated in human cancer.

Cytokine and growth factor signaling pathways involving STAT3 are frequently constitutively activated in many human primary tumors, and are known for the transcriptional role they play in controlling cell growth and cell cycle progression. However, the extent of STAT3's reach on transcriptional control of the genome as a whole remains an important question. We predicted that this persistent STAT3 signaling affects a wide variety of cellular functions, many of which still remain to be characterized. We took a broad approach to identify novel STAT3 regulated genes by examining changes in the genome-wide gene expression profile by microarray, using cells expressing constitutively-activated STAT3. Using computational analysis, we were able to define the gene expression profiles of cells containing activated STAT3 and identify candidate target genes with a wide range of biological functions. Among these genes we identified Necdin, a negative growth regulator, as a novel STAT3 target gene, whose expression is down-regulated at the mRNA and protein levels when STAT3 is constitutively active. This repression is STAT3 dependent, since inhibition of STAT3 using siRNA restores Necdin expression. A STAT3 DNA-binding site was identified in the Necdin promoter and both EMSA and chromatin immunoprecipitation confirm binding of STAT3 to this region. Necdin expression has previously been shown to be down-regulated in a melanoma and a drug-resistant ovarian cancer cell line. Further analysis of Necdin expression demonstrated repression in a STAT3-dependent manner in human melanoma, prostate and breast cancer cell lines. These results suggest that STAT3 coordinates expression of genes involved in multiple metabolic and biosynthetic pathways, integrating signals that lead to global transcriptional changes and oncogenesis. STAT3 may exert its oncogenic effect by up-regulating transcription of genes involved in promoting growth and proliferation, but also by down-regulating expression of negative regulators of the same cellular processes, such as Necdin.

Antiproliferative activity of a phenolic extract from a native Chilean Amaranthaceae plant in drug-resistant ovarian cancer cell lines

Antiproliferative activity of a phenolic extract from a native Chilean Amaranthaceae plant in drug-resistant ovarian cancer cell lines

Abstract A33: in ovarian cancer cell lines resistant to microtubule-stabilizing agents, expression of IGF2 and ABCB1 is increased and IGF1R inhibition restores drug sensitivity

Abstract Overexpression of insulin-like growth factor 2 (IGF2) is associated with worse outcome in ovarian cancer patients; previously, our group has shown that IGF2 is upregulated after Taxol treatment of ovarian cancer cells and leads to activation of AKT. Thus, we hypothesized that IGF2 may be involved in drug resistance. The Taxol-resistant cell line HEY-T30 was developed in our laboratory from chemo-naive, sensitive HEY ovarian cancer cells by repeated Taxol treatment in tissue culture. HEY-T30 is 40-fold resistant to Taxol (IC50: 2.6 nM versus 105.9 nM), as determined by sulforhodamine B cytotoxicity assays. In these cells, IGF2 mRNA levels, measured by quantitative PCR, are increased 8.8-fold compared with its expression in the parental cell line. In addition, HEY-T30 have a genomic amplification of ABCB1 (mdr1; encoding the drug efflux pump p-glycoprotein) detected by array CGH, associated with increased ABCB1 mRNA expression. Alterations in IGF2 and ABCB1 were assessed in an additional drug-resistant ovarian cancer cell line, OVCAR8-D30, developed in our laboratory. OVCAR8-D30 is 2-fold-resistant to discodermolide (IC50: 56 nM versus 115 nM), another microtubule-stabilizing drug with a distinct chemical structure. This resistant cell line also showed an increase (4-fold) in IGF2 expression compared to its parental cell line, as well as a 19-fold increase in ABCB1 expression. To determine the effect of IGF pathway inhibition in Taxol-resistant cells, NVP-AEW541 (a small molecule inhibitor of the IGF1R, the major receptor for IGF2) was used. By concurrent treatment with NVP-AEW541, HEY-T30 cells were significantly re-sensitized to Taxol (IC50 from 105.9 nM to 17.0 nM). The effect of combined Taxol and NVP-AEW541 treatment on cell cycle distribution was evaluated by flow cytometry. Eighteen hours after Taxol (75 nM, 125 nM and 250 nM) and NVP-AEW541 (1 μM) treatment alone or in combination, more cells were arrested in G2/M with combination treatment compared to Taxol treatment alone at the higher Taxol concentrations (75 nM: 23.3% to 26.4% with NVP, 125 nM: 23.8% to 44.0% with NVP, and 250 nM: 34.6% to 74.1% with NVP) as determined by flow cytometry. At this time point, caspase activity analysis by flow cytometry detection of FAM-VAD-FMK FLICA reagent, showed no significant differences between the two treatments (75 nM: 6.7% and 9.5% with NVP, 125 nM: 10.0% and 11.5% with NVP, and 250 nM: 12.4% and 14.9% with NVP). These data suggest that treatment with the IGF1R inhibitor NVP-AEW541 can restore Taxol sensitivity in cells with multiple aberrations that confer drug-resistance (IGF2 and ABCB1 over-expression). We conclude that IGF2 has a prominent role in resistance to microtubule-stabilizing drugs in ovarian cancer cell lines and we therefore propose IGF2 to be a valuable target in future molecular therapies for chemo-resistant ovarian cancer. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A33

Cell type-specific methylation of an intronic CpG island controls expression of the MCJ gene.

Over 50% of human genes are associated with CpG islands and DNA methylation within such CpG islands has been clearly correlated with inhibition of expression. Whereas changes in DNA methylation play a key role in a number of human diseases, in particular cancer, in normal DNA CpG islands are nearly always methylation free, regardless of the expression status of the associated gene. Only limited evidence supports a role for DNA methylation in controlling tissue-specific expression in adult somatic tissue. Loss of expression of the MCJ gene has previously been linked to increased chemotherapeutic drug resistance in ovarian cancer. We report that loss of expression of MCJ in drug-resistant ovarian cancer cell lines depends on methylation of a CpG island within its first exon, but is independent of methylation within the promoter region. Furthermore, cell type-specific expression of the MCJ gene in normal cells also depends on the methylation status of the CpG island within its first exon. The MCJ CpG island is methylated and the gene is not expressed in cells of epithelial origin, but unmethylated and expressed in cells of lymphocyte or fibroblast origin. Chromatin immunoprecipitation assays determined that MCJ CpG island methylation was associated with loss of histone acetylation in ovarian epithelial cells compared with unmethylated fibroblast cells. Reduced acetylation was observed not only within the CpG island, but also within the promoter region, suggesting that CpG island methylation may direct alterations in chromatin structure within the promoter region, leading to gene inactivation.