Resistant Colorectal Cancer Cell Lines Research Articles

Molecular Mimics of Classic P-Glycoprotein Inhibitors as Multidrug Resistance Suppressors and Their Synergistic Effect on Paclitaxel.

P-glycoprotein (Pgp) is a membrane bound efflux pump spread in a variety of tumor cells and considered as a main component of multidrug resistance (MDR) to chemotherapies. In this work, three groups of compounds (imidazolone, oxazolone and vinyl dipeptide derivatives) were synthesized aiming to develop a molecular framework that effectively suppresses MDR. When tested for their influence on Pgp activity, four compounds coded Cur1-01, Cur1-12V, Curox-1 and Curox-3 significantly decreased remaining ATP concentration indicating Pgp substrate site blocking. On the other hand, Cur-3 and Cur-10 significantly increased remaining ATP concentration, which is indicative of Pgp ATPase inhibition. The cytotoxicity of synthesized compounds was examined against Pgp expressing/highly resistant colorectal cancer cell lines (LS-174T). Compounds Cur-1 and Cur-3 showed considerable cytotoxicity with IC50 values of 7.6 and 8.9 μM, respectively. Equitoxic combination (at IC50 concentrations) of PTX and Cur-3 greatly diminished resistant cell clone from 45.7% to 2.5%, albeit with some drop in potency from IC50 of 7.9 nM to IC50 of 23.8 nM. On the other hand, combination of PTX and the non-cytotoxic Cur1-12V (10 μM) significantly decreased the IC50 of PTX to 3.8 nM as well as the resistant fraction to 16.2%. The combination test was confirmed using the same protocol but on another resistant CRC cell line (HCT-116) as we obtained similar results. Both Cur-3 and Cur1-12V (10 μM) significantly increased the cellular entrapment of Pgp probe (doxorubicin) elevating its intracellular concentration from 1.9 pmole/cell to 3.0 and 2.9 pmole/cell, respectively.

Salvianolic acid B reverses multidrug resistance in HCT‑8/VCR human colorectal cancer cells by increasing ROS levels.

Salvianolic acid B (SalB) a water-soluble phenolic compound, extracted from Salvia miltiorrhiza, has previously been demonstrated to reverse tumor multidrug resistance (MDR), including in colorectal cancer. Reactive oxygen species (ROS) are oxygen radicals generated during aerobic metabolism (superoxide and hydroxyl radicals) and superoxide easily generating free radicals (H2O2). The concept that increased ROS levels can lead to augmented tumor cell-sensitivity to chemotherapy drugs has become notable. The aim of the present study was to elucidate the role of ROS in mediating the effect of SalB on drug resistance and the correlation with drug resistance-associated protein, P-glycoprotein (P-gp), and apoptosis-associated proteins, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax). In the current study, through utilizing the multidrug resistant colorectal cancer cell line, HCT-8/VCR, it was demonstrate that SalB reversed MDR in HCT-8/VCR. In addition, SalB significantly increased ROS levels, which may have accelerated the apoptosis of HCT-8/VCR cells by downregulating Bcl-2 and increasing Bax protein expression. Furthermore the increased intracellular ROS levels may have inhibited P-gp expression at the gene and protein levels. In conclusion, the data of the current study demonstrate that SalB reversed MDR in HCT-8/VCR cells, and the effect is associated with increased ROS levels, which may downregulate P-gp expression and promote tumor cell apoptosis, which in turn increases the sensitivity of drug-resistant cells to chemotherapy drugs.

Downregulation of RLIP76 is associated with vincristine resistance in human colorectal cancer HCT-8/VCR cells.

RLIP76 is an anti-apoptotic transporter, participating in the multi-specific drug transport and resistance. In the absence of chemotherapy drugs, the knockout or inhibition of RLIP76 leads to pronounced tumor regression. RLIP76 transports anthracycline and vinca alkaloid drugs and mediates the resistance to these drugs. However, functions of RLIP76 in drug resistance colorectal cancer remain unclear. HCT-8 and the vincristine (VCR)-resistant colorectal cancer cell line HCT-8/VCR (HCT-8/V) were used in the present study. The effects of RLIP76 knockdown by the lentivirus were examined in cultured cells, including growth, apoptosis, invasion, and signaling pathways by qRT-PCR, western blot analysis and transwell assay. The relative level of RLIP76 in HCT-8 and HCT-8/V was assessed by western blot analysis, finding RLIP76 was overexpressed in HCT-8/V. Then, HCT-8/V cancer cells were transfected with lentivirus encoding RLIP76-specific shRNA (KD) and the control (NC), and no significant difference of RLIP76 level between the NC cells and cells without transfection was found, but the relative mRNA level decreased to 0.277±0.016 and protein level also reduced in KD cells. Cell functions changed after RLIP76 knockdown in HCT-8/V. The IC50 of VCR decreased from 164.4±1.734 to 13.95±2.008 (µg/ml) (p<0.05) in cell culture. The cell number reduced from 329.67±20.23 to 176.33±2.52 (p<0.05) in migration assay and from 294.67±30.07 to 153±22.11 (p<0.05) in invasion assay. Moreover, apoptotic proteins, including cleaved-caspase-8, cleaved-caspase-9, cleaved-Parp and Bax increased. The phosphorylation level of Erk also reduced significantly. The present study showed that RLIP76 is a key effector of cancer cell survival, invasion, and migration and possibly an important target to improve drug resistance and tumor treatment.

Combined Pan-RAF and MEK Inhibition Overcomes Multiple Resistance Mechanisms to Selective RAF Inhibitors.

RAF and MEK inhibitors are effective in BRAF-mutant melanoma but not in BRAF-mutant colorectal cancer. To gain additional insights into this difference, we performed a genome-scale pooled shRNA enhancer screen in a BRAF-mutant, RAF inhibitor-resistant colorectal cancer cell line exposed to the selective RAF inhibitor PLX4720. We identified multiple genes along the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) signaling axis that, when suppressed, either genetically or pharmacologically, sensitized cells to the selective RAF inhibitor through sustained inhibition of MAPK signaling. Strikingly, CRAF was a key mediator of resistance that could be overcome by the use of pan-RAF inhibitors in combination with a MEK inhibitor. Furthermore, the combination of pan-RAF and MEK inhibitors displayed strong synergy in melanoma and colorectal cancer cell lines with RAS-activating events such as RTK activation, KRAS mutation, or NF1 loss-of-function mutations. Combinations of selective RAF inhibitors, such as PLX4720 or dabrafenib, with MEK inhibitors did not incur such profound synergy, suggesting that inhibition of CRAF by pan-RAF inhibitors plays a key role in determining cellular response. Importantly, in contrast to the modest activity seen with single-agent treatment, dual pan-RAF and MEK inhibition results in the induction of apoptosis, greatly enhancing efficacy. Notably, combined pan-RAF and MEK inhibition can overcome intrinsic and acquired resistance to single-agent RAF/MEK inhibition, supporting dual pan-RAF and MEK inhibition as a novel therapeutic strategy for BRAF- and KRAS-mutant cancers.

Abstract 5465: Systematic interrogation of druggable pathways in pancreatic adenocarcinomas using pooled gene-knockdown lentiviral libraries

Abstract Despite significant advances in the development of cancer treatment in the last 20 years the best available therapies have marginally improved the survival rate of treated pancreatic cancer patients (from 3% to 6% five-year survival), making it still one the deadliest malignancies. Single standard of care cytotoxic and targeted therapies demonstrate little to no effect on patient survival. Even though we now understand some of the main genetic factors that are mutatated in pancreatic tumors, their role in drug response remains poorly understood. To identify drug-sensitizing genetic targets in cancer cells we have developed a high-throughput screening platform. In this methodology, pooled lentiviral libraries containing hundreds of knockout constructs specific for cancer-related genes are used to infect cancer cells. By treating the infected populations with therapeutic agents we are able to identify genetic targets that when inhibited synergize with existing drugs to cause increased cell death. We piloted this type of screening platform in HCT-116 cells, a KRAS mutant, MEK inhibitor resistant colorectal cancer cell line, using the MEK-inhibitor AZD-2644 and a pooled shRNA library. This study identified 4-7 target genes that when knocked down sensitized the previously resistant HCT-116 cells to AZD-2644. To conduct a more robust and representative sensitization screen we developed a lentiviral CRISPR-Cas9 library. By performing screens in a panel of pancreatic cancer cell lines, we have identified both cell line dependencies and sensitizers to targeted and cytotoxic chemotherapies. The findings from these screens provide potential mechanistic insights into the key survival signaling programs in pancreatic cancer as well as new therapeutic strategies. Citation Format: Edgar Ferrer-Lorenzo, Daniel P. Nussbaum, Peter Winter, Kris Wood. Systematic interrogation of druggable pathways in pancreatic adenocarcinomas using pooled gene-knockdown lentiviral libraries. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5465. doi:10.1158/1538-7445.AM2015-5465

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.

Abstract B24: De novo and acquired resistance to first-line standard therapy in colorectal cancer: from cell lines to metastatic tumors

Abstract Introduction: Personalized medicine (PM) is a concept that has raised high expectations amongst scientists, clinicians, and patients. An emerging approach is to examine tumor biopsy material for genomic changes that are known targets of currently available therapeutic agents, with the assumption that a clinical benefit will be observed if the target is inhibited. While striking anecdotal reports are predictable from this approach, the clinical impact of these agents is limited by the inevitable development of therapeutic resistance. Our focus is on the design of parallel research programs using both in vitro and in vivo strategies, in an effort to delay or inhibit resistance. We present here preliminary data for a signature of resistance to standard first-line treatment - fluorouracil, folinic acid, oxaliplatin and bevacizumab (FOLFOX/B) using cell line models of resistance to this regimen. In parallel, we are conducting a prospective study to identify biomarkers of clinical resistance to first-line therapy in patients with metastatic colorectal cancer (CRC) (NCT00984048). Methods: Ten established CRC cell lines were treated with FOLFOX/B and categorized as resistant or sensitive based on IC50 values. In parallel, patients who consented to an initial biopsy and one at disease progression following an initial response were identified as intrinsically resistant or as having acquired resistance during treatment. CRC cell lines that were initially sensitive were rendered resistant to mimic the acquired resistance in patients, by serial passages with gradual increases in concentration of the combination regimen. We compared microarray data from three sensitive and three resistant cell lines. Results: We found a different expression pattern from microarray data comparing sensitive and resistant cell lines, thereby indicating a potential signature of resistance to FOLFOX/B. Interestingly, we found that the Src family kinase Lyn was overexpressed in resistant cells lines. Treating cells with non-cytotoxic concentration of dasatinib, a dual Src family kinase and Abl inhibitor, sensitized both the parental sensitive cells and the cells with acquired resistance to FOLFOX/B, thereby suggesting that combination treatment with dasatinib may be effective in delaying or inhibiting resistance. We have thus far collected needle core biopsies from liver metastases from forty patients who agreed to partake in this multi-center trial. Eligible patients have confirmed metastatic CRC, measurable disease, and consent to three needle-core biopsies (NCBs) of a non-resectable liver metastasis before treatment and at resistance, as well as serial blood collection throughout the study. Using standard operating procedures developed for this trial, we were able to both preserve morphology and obtain high-quality genomic material from biopsy tissue. We will determine if the resistance signature and overexpression of Lyn observed in the resistant CRC cell lines are similarly demonstrated in patients that were intrinsically resistant to FOLFOX/B. Conclusions: We have designed parallel in vitro and in vivo experiments to study resistance to standard first-line treatment for mCRC. These studies provide insight on metastatic signatures of resistance and suggest combination therapies to delay or inhibit therapeutic resistance in patients.

Abstract B245: Synergistic antiproliferative effects of the P21-activated kinase (PAK) inhibitor, PF-03758309, and standard-of-care agents in colorectal cancer models.

Abstract Background: The p21-activated kinase (PAK) family of serine/threonine kinases is overexpressed in multiple cancer types including breast, ovarian, colorectal, thyroid, and pancreatic. The two groups of PAK proteins, Group I (PAK 1–3) and Group II (PAK 4–6), are critical mediators of cell survival, motility, mitosis, transcription, and translation. The goal of this study was to assess the efficacy of a PAK inhibitor, PF-03758309, with irinotecan in colorectal cancer (CRC). Methods: A panel of 27 colorectal cancer (CRC) cell lines were exposed to increasing doses of PF-03758309 (0.015–1 μM) for 72 hours and analyzed for inhibition of proliferation using the sulforhodamine B (SRB) assay. Cell lines were designated sensitive (S), intermediate (I), or resistant (R) based on IC50's &amp;lt; 0.125 μM, ≥ 0.125 and ≤ 1 μM, or &amp;gt; 1 μM, respectively. Two sensitive and two resistant cell lines were exposed to three different concentrations of either the PAKi, irinotecan or all possible combinations. Proliferation was assessed by the SRB assay and synergy was calculated using the Calcusyn software. Apoptosis was also assessed using the luminescent Caspase 3/7 assay. Athymic nude mice were injected with primary patient derived explants. Once the tumors reached 200–300mm3 the mice were dosed with PF-03758309, irinotecan, or cetuximab or the combination. Tumors were measured twice weekly and growth curves were analyzed. Results: Synergy was observed in both PF-03758309 sensitive and resistant cell lines, though the greatest synergy was seen in one of the more sensitive lines. Apoptosis was also increased in the combination when compared to the single agents. Of the explants treated, one of them demonstrated a synergistic/super-additive effect when dosed with the combination of irinotecan. One of the explants also demonstrated an added benefit with the combination of PF-03758309 and cetuximab. Further investigation into the differential sensitivities seen with PF-03758309 revealed that the more resistant CRC cell lines had higher expression of MDR1. When this was modulated either by shRNA knockdown or by addition of verapamil, we saw a shift to a more sensitive phenotype. Conclusions: The combination of the PAKi, PF-03758309 with irinotecan resulted in synergistic anti-proliferative effectsand apoptosis in CRC cell lines regardless of sensitivity. Similar effects were observed in patient-derived CRC explants with PF-03758309 and standard of care agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B245.

Diosgenin induces death receptor-5 through activation of p38 pathway and promotes TRAIL-induced apoptosis in colon cancer cells

Previously, we demonstrated that diosgenin induced apoptosis in colorectal cancer cell lines HCT-116 and HT-29. HT-29 cells have been reported to be one of the most resistant colorectal cancer cell lines to TRAIL-induced apoptosis. In this study, we investigated the effect of diosgenin on TRAIL-induced apoptosis in HT-29 cells. We showed that diosgenin sensitizes HT-29 cells to TRAIL-induced apoptosis. Mechanisms underlying this sensitization mainly involved diosgenin-induced p38 MAPK pathway activation and subsequent DR5 overexpression. Furthermore, we showed that diosgenin alone, TRAIL alone or combination treatment increased COX-2 expression and that the use of a COX-2 inhibitor further increased apoptosis induction.

Abstract C22: Identification of candidate microRNAs that correlate with sensitivity to the IGF-1R/IR inhibitor, OSI-906, in colorectal cancer (CRC) cell lines

Abstract Background: MicroRNAs (miRNAs) are small ∼20–22 nucleotide noncoding RNAs, thatwhich function as antisense regulators of gene expression by targeting specific mRNAs, and thereby modulate a variety of cellular signaling pathways. Deregulation of miRNA expression contributes to specific oncogenic events in human cancers and therefore may contribute to have a role in resistance to targeted cancer therapies. This study was based upon the hypothesis Here, we hypothesized that miRNA expression profiles wouldcan predict sensitivity to the novel IGF-1R tyrosine kinase inhibitor, OSI-906, in colorectal cancer (CRC) cell lines. Materials and Methods: To evaluate the sensitivity of CRC cells to OSI-906, a panel of 27 lines was exposed to increasing concentrations of the drug and assessed for proliferation using the sulforhodamine B assay. Six cell lines were sensitive (S, IC50 &amp;lt; 1.5 mol/L) and 21 were resistant (R, IC50 &amp;gt; 1.5 mol/L) to OSI-906. Baseline total RNA was isolated from 4 S and 4 R cell lines for miRNA and mRNA profiling on Dharmacon miRNA microarray and Affymetrix HG-U133 Plus 2.0 microarray platforms, respectively. For miRNA microarray data pre-processing, 342 miRNA probes with relative intensity of p ≤ 0.01 in at least 2 of the 8 experiments were kept for further data analysis. For mRNA microarray analysis, probe sets representing the same gene were collapsed based on maximum values and the expression levels were converted to a rank-based matrix. Anti-sense and mimic-miRNAs were used to study the effects of loss- and gain-of-function for the identified candidate miRNAs. Results: Significance Analysis of Microarray was used to determine the differentially expressed miRNAs in cell lines S or R to OSI-906 S vs. R cell lines profiles. Thirteen13 miRNAs were identified which showed a positive correlation with OSI-906 sensitivity. Three and 10 miRNAs were under- orand over-expressed in OSI-906 S cell lines, while respectively. gGene expression analysis revealed 78 and 42 genes under- orand over-expressed in OSI-906 S cell lines (p &amp;lt; 0.005), respectively. By comparing the predicted miRNA targets and differentially expressed genes, we identified two candidate miRNAs (hsa-miR-181a and hsa-miR-224) where the expression of their potential target genes, metallothioneins 1E and 2A (MT1E and MT2A) showed an inverse correlation with OSI-906 sensitivity. This observation was confirmed by qRT-PCR on both miRNAs and their target genes. To assess the loss- and gain-of-function effects of the candidate miRNAs, we delivered mimic- and anti-sense miRNAs to the OSI-906 R and S cell lines, respectively. Interestingly, over-expression of the miR-181a and miR-224 mimics in the OSI-906 R cell lines resulted in increased sensitivity to the drug, whereas. However, silencing of these miRNAs in the S cell lines did not demonstrateshow the reverse effect. Conclusion: These results suggest that loss of the candidate miRNA expression can confer OSI-906 resistance. Restoring the expression of these candidate miRNAs may induce sensitivity to OSI-906 in resistant CRC cell lines and may have clinical significant application in the targeted therapy of patients with advanced CRC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C22.

Identification of 5-fluorouracil response proteins in colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

Colorectal cancer (CRC) is the second most prevalent cause of cancer-related deaths in the Western world. 5-Fluorouracil (5-FU) is a standard chemotherapeutic drug to treat CRC. However, the response rate is less than 20% and patients who have responded to 5-FU may become resistant. Therefore there is an urgent need to examine the 5-FU response proteins so that patients with no response to 5-FU can change to other treatment strategies promptly. In this study, the proteomic expression profile in a CRC cell line SW480 before and after 5-FU treatment was examined using 2-dimensional electrophoresis technology. Fourteen proteins with differential expression were identified using mass spectrometry and 7 of them were validated using immunocytochemical (ICC) staining. Protein identification indicated that cyclophilin A, cytokeratin 19 (CK19), cytokeratin 8 (CK8), ras-related nuclear protein, heat shock protein 27 (hsp27) and peroxiredoxin 6 (Prx 6) were upregulated whereas heat shock protein 60 (hsp60), cytokeratin 18 (CK18), cytokeratin 9 (CK9), carbamoylphosphate synthetase I, alpha-enolase, heat shock protein 70 (hsp70), nm23 and beta-actin were down-regulated. Seven of the 14 proteins detected were validated by ICC staining, which showed that the expression of hsp27, Prx 6 and hsp70 correlated with that from proteomics profiling. Our results suggest that hsp27, Prx 6 and hsp70 are potential 5-FU response proteins and they may represent potential targets for further evaluation in other 5-FU-sensitive and -resistant CRC cell lines.

Cerivastatin enhances the cytotoxicity of 5-fluorouracil on chemosensitive and resistant colorectal cancer cell lines

Cerivastatin is one of the synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used for the treatment and prevention of hypercholesterolaemia. The observation that patients receiving this drug had a lower incidence at cancer led to our interest in using it as a putative anticancer agent. In this study, we tested the cytotoxicity of cerivastatin on a panel of 5-fluorouracil (5FU) sensitive and resistant cell lines in vitro. Cerivastatin was cytotoxic to both 5FU sensitive and resistant cells. Cerivastatin significantly augmented the cytotoxic effect of 5FU on drug sensitive (6–22-fold) and resistant (229–310-fold) cell lines. Cerivastatin and 5FU acted synergistically. Cerivastatin inhibited nuclear factor κB DNA binding activity. The enhancing effect of cerivastatin on 5FU was partially mevalonate pathway independent. Cerivastatin may allow successful 5FU therapy in chemoresistant patients.