Oxaliplatin-resistant Cell Lines Research Articles

Establishment and characterization of an oxaliplatin-resistant hepatic cancer cell line*

Abstract Objective The aim of the current study was to establish an oxaliplatin-resistant hepatoma cell line (HepG2/OXA) and investigate the potential mechanisms of its drug resistance. Methods The hepatoma cell subline, HepG2/OXA, resistant to oxaliplatin (OXA), was established from a parent cell line HepG2, by stepwise exposure to gradually increasing concentrations of OXA over a half-year period. Chemosenstivity of the cytotoxic drugs, OXA, cisplatin (CDDP), adriamycin (ADM), and 5-fuorouracil (5-FU), was determined in HepG2 and HepG2/OXA cells, by the Cell counting kit-8 (CCK8) assay. Cell cycle distribution of HepG2 and HepG2/OXA cells was analyzed by Flow cytometry (FCM). The expression levels of several drug resistance-related proteins, such as P-glycoprotein (P-gp), multidrug resistant protein 1 (MRP1), and excision repair-cross complementing 1 (ERCC1) protein in the two cell lines were tested by the western blot assay. Results The IC50 of OXA in HepG2/OXA and HepG2 were 136.84 µmol/L and 23.86 µmol/L, respectively. The resistance index (RI) was 5.34. HepG2 was also demonstrated to be cross-resistant to other anti-tumor agents, such as 5-FU, ADM, and CDDP. The percentage of HepG2/OXA cells in the S phase was significantly decreased compared to HepG2 cells (25.58% ± 2.36% vs 14.37% ± 2.54%, P < 0.05), while the percentage of cells in the G0/G1 and G2/M phases showed no statistical difference (respectively 55.29% ± 4.98% vs 56.73% ± 4.56%, P > 0.05, and 24.63% ± 4.81% vs 28.26% ± 3.82%, P > 0.05). The ERCC1 was found to be over expressed in HepG2/OXA cells, while there was no difference in the expressions of P-gp and MRP1 between the multiple drug resistance (MDR) phenotype cell line and its parental cell line. Conclusion HepG2/OXA showed an MDR ability; the over expression of ERCC1 might be associated with the platinum resistance of the cells, but P-gp and MRP1 are not.

Polyene phosphatidylcholine overcomes oxaliplatin resistance in human gastric cancer BGC823 cells

Intrinsic or acquired resistance to oxaliplatin (L-OHP) is a major reason of treatment failure in gastric cancer and limits therapeutic success. Here we generated an oxaliplatin resistant gastric cancer cell line, BGC823/L-OHP, to investigate the effect of a hepatoprotective compound, polyene phosphatidylcholine (PPC), on conquest of oxaliplatin resistance. BGC823/L-OHP cells showed less sensitive to L-OHP directed growth inhibition than the parental BGC823 cells. PPC treatment significantly increased anti-proliferative activity of L-OHP on resistant cells and promoted L-OHP triggered apoptosis, indicating that drug resistance was overcome. Mechanistically, L-OHP incubation stimulated upregulation of an ABC family protein, ABCF2, and the expression was inhibited by PPC. Moreover, expression levels of the stemness factor Nanog and its regulator TLR4 were notably enhanced in BGC823/L-OHP cells and reduced by PPC treatment. To conclude, PPC can overcome oxaliplatin resistance in gastric cancer cells via promoting apoptosis, inhibiting ABCF2, as well via reducing cancer stem cell-like features. The combination therapeutic strategy could serve to increase oxaliplatin effectiveness in the clinic.

Abstract A143: Repurposing disulfiram as a potential novel treatment of drug-resistant metastatic colorectal cancer

Abstract Introduction: Drug repurposing has emerged as an attractive alternative to the classical pipeline of drug development for novel anticancer drugs. We have selected disulfiram, which was FDA approved for treatment of alcoholism already in 1951. Disulfiram has been examined in various in vitro cancer model systems and has demonstrated several different mechanistic types of anticancer effects. The consensus is that coadministration of divalent metal ions, especially copper, is required for the maximal anticancer effect of disulfiram. Disulfiram has also been investigated in a few clinical trials but with a difference in efficacy. However, there are no data on the clinical effects of disulfiram in patients with colorectal cancer, and no studies on disulfiram in colorectal cancer can be found at www.clinicaltrials.org. Methods: We have established a research platform consisting of isogenic pairs of parental (sensitive) and drug-resistant human colorectal cancer cell lines (HT-29, HCT-116, LoVo) with resistance to either oxaliplatin or SN-38 (the active metabolite of irinotecan) (Jensen et al., Mol Oncol 2015 9(6):1169-85). We have tested disulfiram ± copper in this panel. MTT assay was used to estimate cell number. Results: In all 9 cell lines it was observed that addition of copper to disulfiram resulted in increased cell cell viability inhibition. Even more importantly, when combining disulfiram and copper with oxaliplatin or SN38, an additive or synergistic viability inhibitory effect was observed with oxaliplatin in SN-38 resistant cell lines and with SN-38 in oxaliplatin resistant cell lines. Conclusions: Based on these in vitro results, we are now planning a clinical phase 2 study using Simon’s two-stage min/max design enrolling metastatic colorectal cancer patients having failed first-line treatment with either oxaliplatin- or irinotecan-containing treatment. The patients will as second-line treatment receive irinotecan- or oxaliplatin-containing treatment (depending on their first-line treatment) plus disulfiram and copper. Endpoint will be objective response rate according to RECIST version 1.1. Citation Format: Jan Stenvang, Helle Keinicke, Signe L. Nielsen, Haatisha Jandu, Jiri Bartek, Nils Brünner. Repurposing disulfiram as a potential novel treatment of drug-resistant metastatic colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A143.

ID1 promotes hepatocellular carcinoma proliferation and confers chemoresistance to oxaliplatin by activating pentose phosphate pathway

BackgroundDrug resistance is one of the major concerns in the treatment of hepatocellular carcinoma (HCC). The aim of the present study was to determine whether aberrant high expression of the inhibitor of differentiation 1(ID1) confers oxaliplatin-resistance to HCC by activating the pentose phosphate pathway (PPP).MethodsAberrant high expression of ID1 was detected in two oxaliplatin-resistant cell lines MHCC97H–OXA(97H–OXA) and Hep3B–OXA(3B–OXA). The lentiviral shRNA or control shRNA was introduced into the two oxaliplatin-resistant cell lines. The effects of ID1 on cell proliferation, apoptosis and chemoresistance were evaluated in vitro and vivo. The molecular signaling mechanism underlying the induction of HCC proliferation and oxaliplatin resistance by ID1 was explored. The prognostic value of ID1/G6PD signaling in HCC patients was assessed using the Cancer Genome Atlas (TCGA) database.ResultsID1 was upregulated in oxaliplaitin-resistant HCC cells and promoted HCC cell proliferation and oxaliplatin resistance. Silencing ID1 expression in oxaliplaitin-resistant HCC cell lines inhibited cell proliferation and sensitized oxaliplaitin-resistant cells to death. ID1 knockdown significantly decreased the expression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme of the PPP. Silencing ID1 expression blocked the activation of G6PD, decreased the production of PPP NADPH, and augmented reactive oxygen and species (ROS), thus inducing cell apoptosis. Study of the molecular mechanism showed that ID1 induced G6PD promoter transcription and activated PPP through Wnt/β-catenin/c-MYC signaling. In addition, ID1/G6PD signaling predicted unfavorable prognosis of HCC patients on the basis of TCGA.ConclusionsOur study provided the first evidence that ID1 conferred oxaliplatin resistance in HCC by activating the PPP. This newly defined pathway may have important implications in the research and development of new more effective anti-cancer drugs.

Reversion of resistance to oxaliplatin by inhibition of p38 MAPK in colorectal cancer cell lines: involvement of the calpain / Nox1 pathway.

Oxaliplatin is a major treatment for metastatic colorectal cancer, however its effectiveness is greatly diminished by the development of resistances. Our previous work has shown that oxaliplatin efficacy depends on the reactive oxygen species (ROS) produced by Nox1. In this report, we investigated Nox1 involvement in the survival mechanisms of oxaliplatin resistant cell lines that we have selected. Our results show that basal ROS production by Nox1 is increased in resistant cells. Whereas the transitory Nox1-dependent production of superoxide contributes to the cytotoxicity of oxaliplatin in sensitive cells, oxaliplatin treatment of resistant cells leads to a decrease in the production of superoxide associated with an increase of H2O2 and a decreased cytotoxicity of oxaliplatin. We have shown that calpains regulate differently Nox1 according to the sensitivity of the cells to oxaliplatin. In sensitive cells, calpains inhibit Nox1 by cleaving NoxA1 leading to a transient ROS production necessary for oxaliplatin cytotoxic effects. In contrast, in resistant cells calpain activation is associated with an increase of Nox1 activity through Src kinases, inducing a strong and maintained ROS production responsible for cell survival. Using a kinomic study we have shown that this overactivation of Nox1 results in an increase of p38 MAPK activity allowing the resistant cells to escape apoptosis. Our results show that the modulation of Nox1 activity in the context of anticancer treatment remains complex. However, a strategy to maximize Nox1 activation while inhibiting the p38 MAPK-dependent escape routes appears to be an option of choice to optimize oxaliplatin efficiency.

MEG3 is a prognostic factor for CRC and promotes chemosensitivity by enhancing oxaliplatin-induced cell apoptosis.

A major reason for the failure of advanced colorectal cancer (CRC) treatment is the occurrence of chemoresistance to oxaliplatin-based chemotherapy. Recently, studies have shown that long non-coding RNAs (lncRNAs) play an important role in drug resistance. Using HiSeq sequencing methods, we identified that lncRNAs show differential expression levels in oxaliplatin-resistant (OxR) and non-resistant CRC patients. RT-qPCR was then performed in tissues and serum samples, and lncRNA MEG3 was verified to be downregulated in non-responding patients and to have considerable discriminating potential to identify responding patients from non-responding patients. Moreover, decreased serum MEG3 expression was associated with poor chemoresponse and low survival rate in CRC patients receiving oxaliplatin treatment. Subsequently, OxR cell lines were established, and MEG3 was significantly downregulated in HT29 OxR and SW480 OxR cells. In addition, overexpression of MEG3 with pMEG3 reversed oxaliplatin resistance in both CRC cell lines. Flow cytometric apoptosis analysis indicated that MEG3 promoted CRC cell apoptosis. More importantly, MEG3 enhanced oxaliplatin-induced cell cytotoxicity in CRC. In conclusion, our integrated approach demonstrated that decreased expression of lncRNA MEG3 in CRC confers potent poor therapeutic efficacy, and that MEG3 promotes chemosensitivity by enhancing oxaliplatin-induced cell apoptosis. Thus, overexpression of MEG3 may be a future direction by which to develop a novel therapeutic strategy to overcome oxaliplatin resistance of CRC patients.

Screening of 129 FDA approved anti-cancer drugs in colorectal cancer cell lines resistant to oxaliplatin or irinotecan (SN38).

642 Background: In metastatic colorectal cancer (mCRC) only 3 cytotoxic drugs (oxaliplatin, irinotecan and fluorouracil (5-FU)) are approved and the first and second line response rates are about 50% and 10-15%, respectively. Thus, new treatment options are needed. Novel anti-cancer drug candidates are primarily tested in an environment of drug resistance and the majority of novel drug candidates fail during clinical development. Therefore, “repurposing” of drugs has emerged as a promising strategy to apply established drugs in novel indications. The aim of this project was to screen established anti-cancer drugs to identify candidates for testing in mCRC patients relapsing on standard therapy. Methods: We applied 3 parental (drug sensitive) CRC cell lines (HCT116, HT29 and LoVo) and for each cell line also an oxaliplatin and irinotecan (SN38) resistant cell line. We obtained 129 FDA approved anti-cancer drugs from the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI) ( https://dtp.cancer.gov/ ). The parental HT29 cell line and the drug resistant sublines HT29-SN38 and HT29-OXPT were exposed to 3 concentrations of each of the anti-cancer drugs. The effect on cell viability was analyzed by MTT assays. Nine of the drugs were analyzed for effect in the LoVo and HCT116 and the SN38- and oxaliplatin-resistant derived cell lines. Results: None of the drugs caused evident differential response between the resistant and sensitive cells or between the SN38 and oxaliplatin resistant cells. The screening confirmed the resistance as the cells displayed resistance to drugs in the same class as the one they were made resistant to. Of the drugs, 45 decreased cell viability in the HT29 parental and oxaliplatin- or SN-38 resistant cell lines. Nine drugs were tested in all nine CRC cell lines and eight decrease cell viability in the nine cell lines. These included drugs in different classes such as epigenetic drugs, antibiotics, mitotic inhibitors and targeted therapies. Conclusions: This study revealed several possible new “repurposing” drugs for CRC therapy, by showing that 45 FDA-approved anti-cancer drugs decrease cell viability in CRC cell lines with acquired drug resistance.

A novel dual-functioning ruthenium(II)–arene complex of an anti-microbial ciprofloxacin derivative — Anti-proliferative and anti-microbial activity

7-(4-(Decanoyl)piperazin-1-yl)-ciprofloxacin, CipA, (1) which is an analogue of the antibiotic ciprofloxacin, and its ruthenium(II) complex [Ru(η6-p-cymene)(CipA-H)Cl], (2) have been synthesised and the x-ray crystal structures of 1·1.3H2O·0.6CH3OH and 2·CH3OH·0.5H2O determined. The complex adopts a typical pseudo-octahedral ‘piano-stool’ geometry, with Ru(II) π-bonded to the p-cymene ring and σ-bonded to a chloride and two oxygen atoms of the chelated fluoroquinolone ligand. The complex is highly cytotoxic in the low μM range and is as potent as the clinical drug cisplatin against the human cancer cell lines A2780, A549, HCT116, and PC3. It is also highly cytotoxic against cisplatin- and oxaliplatin-resistant cell lines suggesting a different mechanism of action. The complex also retained low μM cytotoxicity against the human colon cancer cell line HCT116p53 in which the tumour suppressor p53 had been knocked out, suggesting that the potent anti-proliferative properties associated with this complex are independent of the status of p53 (in contrast to cisplatin). The complex also retained moderate anti-bacterial activity in two Escherichia coli, a laboratory strain and a clinical isolate resistant to first, second and third generation β-lactam antibiotics.

Abstract B1-02: Correlation between oncogenic mutations, signaling pathways, and efficacy of platinum-based drugs against colorectal cancers

Abstract The purpose of this study is to correlate genetic mutations, amino-acid variants, signaling pathways with platiunum based drug activity to shed light on personalized treatment of cancer. Platinum anticancer drugs such as cisplatin, carboplatin, and oxalipaltin are widely used to treat a variety of cancers. Although DNA is the molecular target for these platinum therapies, their efficacies, toxicities, and resistance mechanisms vary widely among various categories and sub-categories of cancers. To comprehend this great variability, an integrated analysis was performed to determine the impact of somatic mutations on protein functions, signaling pathways, and drug activity (sensitivity or resistance) among the US National Cancer Institute (NCI) 60 human tumor cell lines based on Z-scores to predict a priori treatment outcome using CellMiner (http://discover.nci.nih.gov/cellminer). Specifically, somatic mutations of significantly mutated genes from the cancer genome atlas (TCGA) were analyzed along with the driver genes (oncogenes and tumor suppressor genes) from catalogue of somatic mutations in cancer (COSMIC) for total 188 genes that belong to more than 20 different signaling pathways. The functional impact of individual amino-acid variant for each gene and its correlation with the activity of carboplatin, cisplatin and oxaliplatin for each cancer cell line were explored. Particular attention was given to colon cancer for which nearly 40% of tumors are known to have mutated KRAS (Kirsten rat sarcoma viral oncogene homolog) gene. Resulting analysis revealed that colon cancer cell lines with KRAS mutations for codon 12 (G12V-mutant from SW620 cell line) and codon 13 (G13D-mutant from HCT116 and HCT15 cell lines) correlated with the sensitivity to oxaliplatin. Conversely, oxaliplatin resistant HCC2998 colon cancer cell line did not show any correlation with mutated KRAS for codon 146 (A146T-mutant). Notably, all the colon cancer cell lines were resistant to both carboplatin and cisplatin with no correlation to the KRAS mutants. Based on our integrated analysis we further predicted gene networks related to oxaliplatin activity for colon cancer. The network includes the epidermal growth factor (EGFR) signaling pathway that involves PIK3CA, PIK3CG and MTOR from the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) (PIK3/mTOR) pathway, and JAK3 from Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in addition to KRAS from Mitogen-activated protein kinases (MAPK) pathway. We conclude that instead of considering all mutations of a gene in the same way to assess their clinical significance, it may be beneficial to categorize them into different classes based on their functional impact and efficacies towards the anti-cancer drugs for personalized treatment. Similar analytical approach is being extended to non-small cell lung and ovarian cancers where platinum therapies are widely used. S. Tripathi gratefully acknowledges the support through a training fellowship from a grant by the Cancer Prevention Research Institute of Texas (Grant No. RP140113 to R.N.Bose). Citation Format: Swarnendu Tripathi, Louiza Belkacemi, Margaret S. Cheung, Rathindra N. Bose. Correlation between oncogenic mutations, signaling pathways, and efficacy of platinum-based drugs against colorectal cancers. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-02.

Abstract A2-48: Correlation between oncogenic mutations, signaling pathways and efficacy of platinum based drugs against colorectal cancers

Abstract The purpose of this study is to correlate genetic mutations, amino-acid variants, signaling pathways with platiunum based drug activity to shed light on personalized treatment of cancer. Platinum anticancer drugs such as cisplatin, carboplatin, and oxalipaltin are widely used to treat a variety of cancers. Although DNA is the molecular target for these platinum therapies, their efficacies, toxicities, and resistance mechanisms vary widely among various categories and sub-categories of cancers. To comprehend this great variability, an integrated analysis was performed to determine the impact of somatic mutations on protein functions, signaling pathways, and drug activity (sensitivity or resistance) among the US National Cancer Institute (NCI) 60 human tumor cell lines based on Z-scores to predict a priori treatment outcome using CellMiner (http://discover.nci.nih.gov/cellminer). Specifically, somatic mutations of significantly mutated genes from the cancer genome atlas (TCGA) were analyzed along with the driver genes (oncogenes and tumor suppressor genes) from catalogue of somatic mutations in cancer (COSMIC) for total 188 genes that belong to more than 20 different signaling pathways. The functional impact of individual amino-acid variant for each gene and its correlation with the activity of carboplatin, cisplatin and oxaliplatin for each cancer cell line were explored. Particular attention was given to colon cancer for which nearly 40% of tumors are known to have mutated KRAS (Kirsten rat sarcoma viral oncogene homolog) gene. Resulting analysis revealed that colon cancer cell lines with KRAS mutations for codon 12 (G12V-mutant from SW620 cell line) and codon 13 (G13D-mutant from HCT116 and HCT15 cell lines) correlated with the sensitivity to oxaliplatin. Conversely, oxaliplatin resistant HCC2998 colon cancer cell line did not show any correlation with mutated KRAS for codon 146 (A146T-mutant). Notably, all the colon cancer cell lines were resistant to both carboplatin and cisplatin with no correlation to the KRAS mutants. Based on our integrated analysis we further predicted gene networks related to oxaliplatin activity for colon cancer. The network includes the epidermal growth factor (EGFR) signaling pathway that involves PIK3CA, PIK3CG and MTOR from the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) (PIK3/mTOR) pathway, and JAK3 from Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in addition to KRAS from Mitogen-activated protein kinases (MAPK) pathway. We conclude that instead of considering all mutations of a gene in the same way to assess their clinical significance, it may be beneficial to categorize them into different classes based on their functional impact and efficacies towards the anti-cancer drugs for personalized treatment. Similar analytical approach is being extended to non-small cell lung and ovarian cancers where platinum therapies are widely used. S. Tripathi gratefully acknowledges the support through a training fellowship from a grant by the Cancer Prevention Research Institute of Texas (Grant No. RP140113 to R.N.Bose). Citation Format: Swarnendu Tripathi, Louiza Belkacemi, Margaret S. Cheung, Rathindra N. Bose. Correlation between oncogenic mutations, signaling pathways and efficacy of platinum based drugs against colorectal cancers. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-48.

The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer.

BackgroundIrinotecan (SN38) and oxaliplatin are chemotherapeutic agents used in the treatment of colorectal cancer. However, the frequent development of resistance to these drugs represents a considerable challenge in the clinic. Alus as retrotransposons comprise 11% of the human genome. Genomic toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation. Whether or not reactivation of Alus occurs in SN38 and oxaliplatin resistance remains unknown.ResultsWe applied reduced representation bisulfite sequencing (RRBS) to investigate the DNA methylome in SN38 or oxaliplatin resistant colorectal cancer cell line models. Moreover, we extended the RRBS analysis to tumor tissue from 14 patients with colorectal cancer who either did or did not benefit from capecitabine + oxaliplatin treatment. For the clinical samples, we applied a concept of ‘DNA methylation entropy’ to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences- especially the Alu Y subfamily. Furthermore, we identified an enrichment of Alu Y sequences that likely results from increased integration of new copies of Alu Y sequence in the drug-resistant cell lines. In the clinical samples, SOX1 and other SOX gene family members were shown to display variable DNA methylation states in their gene regions. The Alu Y sequences showed remarkable variation in DNA methylation states across the clinical samples.ConclusionOur findings imply a crucial role of Alu Y in colorectal cancer drug resistance. Our study underscores the complexity of colorectal cancer aggravated by mobility of Alu elements and stresses the importance of personalized strategies, using a systematic and dynamic view, for effective cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1552-y) contains supplementary material, which is available to authorized users.

Luteolin sensitizes two oxaliplatin-resistant colorectal cancer cell lines to chemotherapeutic drugs via inhibition of the Nrf2 pathway.

Oxaliplatin is a first-line therapy for colorectal cancer, but cancer cell resistance to the drug compromises its efficacy. To explore mechanisms of drug resistance, we treated colorectal cancer cells (HCT116 and SW620) long-term with oxaliplatin and established stable oxaliplatin-resistant lines (HCT116-OX and SW620-OX). Compared with parental cell lines, IC50s for various chemotherapeutic agents (oxaliplatin, cisplatin and doxorubicin) were increased in oxaliplatin-resistant cell lines and this was accompanied by activation of nuclear factor erythroid-2 p45-related factor 2 (Nrf2) and NADPH quinone oxidoreductase 1 (NQO1). Furthermore, luteolin inhibited the Nrf2 pathway in oxaliplatin-resistant cell lines in a dose-dependent manner. Luteolin also inhibited Nrf2 target gene [NQO1, heme oxygenase-1 (HO-1) and GSTα1/2] expression and decreased reduced glutathione in wild type mouse small intestinal cells. There was no apparent effect in Nrf2-/- mice. Luteolin combined with other chemotherapeutics had greater anti-cancer activity in resistant cell lines (combined index values below 1), indicating a synergistic effect. Therefore, adaptive activation of Nrf2 may contribute to the development of acquired drug-resistance and luteolin could restore sensitivity of oxaliplatin-resistant cell lines to chemotherapeutic drugs. Inhibition of the Nrf2 pathway may be the mechanism for this restored therapeutic response.

Maintenance of Stemness in Oxaliplatin-Resistant Hepatocellular Carcinoma Is Associated with Increased Autocrine of IGF1

BackgroundEvidence suggests that many types of cancers are composed of different cell types, including cancer stem cells (CSCs). We have previously shown that the chemotherapeutic agent oxaliplatin induced epithelial-mesenchymal transition, which is thought to be an important mechanism for generating CSCs. In the present study, we investigate whether oxaliplatin-treated cancer tissues possess characteristics of CSCs, and explore oxaliplatin resistance in these tissues.MethodsHepatocellular carcinoma cells (MHCC97H cells) were subcutaneously injected into mice to form tumors, and the mice were intravenously treated with either oxaliplatin or glucose. Five weeks later, the tumors were orthotopically xenografted into livers of other mice, and these mice were treated with either oxaliplatin or glucose. Metastatic potential, sensitivity to oxaliplatin, and expression of CSC-related markers in the xenografted tumor tissues were evaluated. DNA microarrays were used to measure changes in gene expression as a result of oxaliplatin treatment. Additionally, an oxaliplatin-resistant cell line (MHCC97H-OXA) was established to assess insulin-like growth factor 1 secretion, cell invasion, cell colony formation, oxaliplatin sensitivity, and expression of CSC-related markers. The effects of an insulin-like growth factor 1 receptor inhibitor were also assessed.ResultsOxaliplatin treatment inhibited subcutaneous tumor growth. Tumors from oxaliplatin-treated mice that were subsequently xenografted into livers of other mice exhibited that decreasing sensitivity to oxaliplatin and increasing pulmonary metastatic potential. Among the expression of CSC-related proteins, the gene for insulin-like growth factor 1, was up-regulated expecially in these tumor tissues. Additionally, MHCC97H-OXA cells demonstrated that increasing cell invasion, colony formation, and expression of insulin-like growth factor 1 and CSC-related markers, whereas treatment with an inhibitor of the insulin-like growth factor 1 receptor suppressed these effects.ConclusionMaintenance of stemness in oxaliplatin-resistant hepatocellular carcinoma cells is associated with increased autocrine of IGF1.

Txr1: an important factor in oxaliplatin resistance in gastric cancer

Oxaliplatin-based chemotherapy is the main treatment regimen for gastric cancer (GC), but can fail because of drug resistance. We investigated the role of a recently identified drug-resistance gene, taxol-resistant gene 1 (Txr1), in oxaliplatin resistance. A retrospective study based on banked tissue was carried out. We collected clinical data from 95 patients with stage II-III GC who were treated with radical D2 surgery and standardized first-line chemotherapy with oxaliplatin; paraffin blocks of their tumor specimens were prepared for a tissue microarray in which Txr1 expression was analyzed immunohistochemically and compared with their clinical data and their 3-year disease-free survival (DFS) rate. The human GC cell line, SGC7901, was developed into the oxaliplatin-resistant cell line, SGC7901/L-OHP, using slowly increased oxaliplatin concentrations over 6months. The relationship between Txr1 expression and drug-resistance of oxaliplatin in GC was studied with drug intervention, gene silencing technology, real-time PCR and Western blot analysis. Of the 95 patients with GC, those with TXR1(-) GC had longer postoperative 3-year DFS (77.8%) than those with TXR1(+) GC (52.9%). In oxaliplatin-resistant SGC7901/L-OHP cells, the main expression location of Txr1 shifted from the nucleus to cytoplasm, and both the mRNA and protein expression of Txr1 were higher than that of the parental cells, whereas expression of thrombospondin-1 (TSP1) decreased. When the Txr1 gene was silenced, TSP1 expression increased and the oxaliplatin resistance was significantly reduced in SGC7901/L-OHP cells. Changed Txr1 expression in GC affects the efficacy of oxaliplatin-based chemotherapy. Increased Txr1 expression decreases TSP1 expression and inhibits apoptosis. Txr1 could be a target in reversing oxaliplatin resistance in GC.

MiR-203 induces oxaliplatin resistance in colorectal cancer cells by negatively regulating ATM kinase

Chemotherapy for patients with metastatic colorectal cancer (CRC) is the standard of care, but ultimately nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may help identify novel targets and drugs that will, in turn, improve therapy. Oxaliplatin is a common component combination therapeutic regimen for use in patients with metastatic CRC, but is also used as a component of adjuvant therapy for patients at risk for recurrent disease. In this study, unbiased microRNA array screening revealed that the miR-203 microRNA is up-regulated in three of three oxaliplatin-resistant CRC cell lines, and therefore we investigated the role of miR-203 in chemoresistance. Exogenous expression of miR-203 in chemo-naïve CRC cells induced oxaliplatin resistance. Knockdown of miR-203 sensitized chemoresistant CRC cells to oxaliplatin. In silico analysis identified ataxia telangiectasia mutated (ATM), a primary mediator of the DNA damage response, as a potential target of miR-203. ATM mRNA and protein levels were significantly down-regulated in CRC cells with acquired resistance to oxaliplatin. Using TCGA database, we identified a significant reverse correlation of miR-203 and ATM expression in CRC tissues. We validated ATM as a bona fide target of miR-203 in CRC cells. Mutation of the putative miR-203 binding site in the 3′ untranslated region (3′UTR) of the ATM mRNA abolished the inhibitory effect of miR-203 on ATM. Furthermore, stable knockdown of ATM induced resistance to oxaliplatin in chemo-naïve CRC cells. This is the first report of oxaliplatin resistance in CRC cells induced by miR-203-mediated suppression of ATM.

Abstract 976: CUDC-101, a hybrid molecular targeted agent, reverses multiple mechanisms of drug resistance.

Abstract CUDC-101, a hybrid molecular targeted agent, reverses multiple mechanisms of drug resistance CUDC-101 is a potent molecular targeted anticancer agent, rationally designed to simultaneously inhibit HDAC, EGFR and HER2. It exhibits potent antiproliferative and proapoptotic activities in both in vitro and in vivo assays and it is currently in Phase 1b clinical trial. Multidrug resistance (MDR) is a major unresolved obstacle to successful cancer chemotherapy. It is often associated with increased ABC transporters-mediated efflux of substrate anticancer drugs out of the cell. Platinum (Pt)-based anticancer drugs, exemplified by cisplatin and oxaliplatin, are the mainstay of treatment for most solid cancers. However, resistance to Pt anticancer drugs develops rapidly upon their administration, which can be caused by overexpression of MDR transporters and activated DNA repair mechanisms. We investigated the potentiation effect of CUDC-101 on the anticancer activity of conventional cytotoxic drugs in MDR cells with overexpression of various ABC transporters and in Pt-resistant cancer cells. CUDC-101 was found to increase the cytotoxicity and accumulation of substrate anticancer drugs in MPR-1 (doxorubicin) and MRP-2 (methotrexate) overexpressing cells, while no effect was observed in the parental sensitive cells. Mechanistically, CUDC-101 was found to inhibit MRP-1 and MRP-2 efflux function, probably by acting as an uncompetitive inhibitor and interfering with their ATPase activity. In Pt-resistant cancer cells, CUDC-101 appears to circumvent the resistance through inhibition of both MRP-2 and other DNA repair-mediated mechanisms. The combinations of CUDC-101 with cisplatin or oxaliplatin were found to display synergistic cytotoxic effect in cisplatin- or oxaliplatin-resistant cancer cell lines, respectively. Upon the concomitant administration of CUDC-101, cellular accumulation of Pt drugs and formation of DNA-Pt adducts were found to be increased whereas expression levels of DNA repair genes (e.g. ERCC1) was inhibited in Pt-resistant cells. The data advocates further development of CUDC-101 as a novel MDR reversal agent for use in combination cancer chemotherapeutic regimens. Citation Format: Kenneth K.W. To, Daniel C. Poon, XG Chen, Ge Lin, Li-wu Fu. CUDC-101, a hybrid molecular targeted agent, reverses multiple mechanisms of drug resistance. [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 976. doi:10.1158/1538-7445.AM2013-976

NMR Investigation of the Spontaneous Thermal- and/or Photoinduced Reduction of trans Dihydroxido Pt(IV) Derivatives

The initial aim of the present work was the synthesis of the axial disuccinato Pt(IV) derivative of [PtCl2(cis-1,4-DACH)] (Kiteplatin, 1 in Figure 1 ) (DACH = diaminocyclohexane), which contains an isomeric form of the diamine ligand present in oxaliplatin (i.e., 1R,2R-DACH). The interest in this compound stems from its activity on several cisplatin and oxaliplatin-resistant cell lines. Oxidation of 1 with hydrogen peroxide affords cis,trans,cis-[PtCl2(OH)2(cis-1,4-DACH)] (2) which was treated with succinic anhydride in suitable solvents. To our surprise, in dimethylformamide (DMF) (50-70 °C or under light irradiation) or in dimethylsulfoxide (DMSO) (under light irradiation) the formation of the succinato complex cis,trans,cis-[PtCl2{OC(O)CH2CH2C(O)OH}2(cis-1,4-DACH)] (3) was accompanied by reduction to 1. It was found that solvolysis of 2 and formation of a μ-oxo dinuclear species (5) is the key step. The dinuclear species can then undergo reduction to a 1:1 mixture of 1 and 2 with concomitant elimination of oxygen (1/2 O2 in the form of H2O2). The whole process is fostered by heat and/or light, which could favor solvolysis of 2 as well as decomposition of hydrogen peroxide to water and oxygen so preventing the reoxidation of 1 to 2. Because of its peculiar behavior, compound 5 could be exploited also for the development of a technology for water splitting.

247 Alterations of Cellular and Molecular Patterns in Oxaliplatin Resistant Colorectal Adenocarcinoma Cell Lines

247 Alterations of Cellular and Molecular Patterns in Oxaliplatin Resistant Colorectal Adenocarcinoma Cell Lines

Trabectedin and Its C Subunit Modified Analogue PM01183 Attenuate Nucleotide Excision Repair and Show Activity toward Platinum-Resistant Cells

PM01183 is a novel marine-derived covalent DNA binder in clinical development. PM01183 is structurally similar to trabectedin (yondelis, ecteinascidin-743) except for the C subunit, and this modification is accompanied by different pharmacokinetics in cancer patients. We here characterize the interaction of PM01183 with the nucleotide excision repair (NER) pathway in comparison with trabectedin. Our results show for the first time that although neither PM01183 nor trabectedin is repaired by NER, both compounds are able to interfere with the NER machinery thereby attenuating the repair of specific NER substrates. We further show that the NER activity is increased in 3 of 4 cellular models with acquired resistance to cisplatin or oxaliplatin, confirming the involvement of NER in the resistance to platinum derivatives. Importantly, both PM01183 and trabectedin show unchanged or even enhanced activity toward all 4 cisplatin- and oxaliplatin-resistant cell lines. We finally show that combinations of PM01183 and cisplatin were mostly synergistic toward both parental and cisplatin-resistant ovarian carcinoma cells as indicated by Chou and Talalay analysis. These data show that the C subunit of trabectedin can be subjected to at least some structural modifications without loss of activity or NER interaction. While PM01183 and trabectedin appear functionally similar in cellular models, it is likely that the differences in pharmacokinetics may allow different dosing and scheduling of PM01183 in the clinic that could lead to novel and/or increased antitumor activity. Taken together, our results provide a mechanistic basis to support clinical trials of PM01183 alone or in combination with cisplatin.

Abstract 1704: A role for glucosylceramide synthase in resistance to oxaliplatin in colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the U.S. Nearly 25 percent of patients who present with CRC have already developed metastatic disease. While combination therapies for metastatic CRC have improved median survival times, most patients inevitably become resistant to chemotherapy. Chemoresistance is thus the major cause of treatment failure in CRC patients, and accordingly, the development of novel therapies aimed at overcoming chemoresistance is of paramount importance. Many commonly used chemotherapeutic agents act by inducing cell death by increasing cellular levels of the bioactive sphingolipid, ceramide. In addition to serving well-known structural functions in the cell membrane, sphingolipids are increasingly recognized as important cellular signaling molecules. Studies have demonstrated that human colorectal tumors contain decreased concentrations of ceramide in comparison to normal colonic mucosa. Furthermore, multi-drug resistant cancer cells often display an altered sphingolipid content, with the presence of increased levels of the pro-survival ceramide metabolites. In this study we have examined the role of the ceramide-metabolizing enzyme, glucosylceramide synthase (GCS), in the mechanism of resistance to oxaliplatin. These studies utilize a series of oxaliplatin-resistant isogenic colorectal cancer cell lines. While much evidence has been presented establishing the role of GCS in resistance to a variety of chemotherapeutic agents, a role for GCS in oxaliplatin resistance has yet to be examined. Our studies demonstrate that oxaliplatin-resistant CRC cell lines have ∼ 2-fold more GCS protein with increased levels of the pro-survival ceramide metabolite, glucosylceramide, in comparison to the parental cell lines. We further demonstrate that inhibition of GCS by either pharmacological inhibitors or an RNAi-mediated gene knockdown results in reduced cellular glucosylceramide levels, with a concomitant increased sensitivity towards oxaliplatin. Furthermore, inhibition of GCS causes a reversion of an observed epithelial-mesenchymal transition phenotype observed in oxaliplatin-resistant CRC cell lines. Our results may offer a possible strategy with which to augment chemotherapeutic drug effectiveness in CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1704. doi:10.1158/1538-7445.AM2011-1704