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Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin.

ABSTRACT: Therapeutic protocols including EGFR antibodies in the context of oxaliplatin-based regimens have variable clinical effect in colorectal cancer. Here, we tested the effect of the EGFR antibody cetuximab in different sequential combinations with oxaliplatin on the growth of colorectal cancer cells in vitro and in vivo. Cetuximab reduced the efficacy of oxaliplatin when administered before oxaliplatin but provided additive effect when administered after oxaliplatin regardless of the KRAS or BRAF mutation status of the cells. Systemic gene expression and protein phosphorylation screens revealed alternatively activated pathways regulating apoptosis, cell cycle and DNA damage response. Functional assays indicated that cetuximab-induced arrest of the cells into the G1 phase of the cell cycle was associated with reduced responsiveness of the cells to subsequent treatment with oxaliplatin. In contrast, oxaliplatin-enhanced responsiveness to subsequent treatment with cetuximab was associated with increased apoptosis, inhibition of STAT3 activity and increased EGFR down-regulation. This preclinical study indicates that optimizing the sequence of administration may enhance the antitumor effect of combination therapy with EGFR antibodies and oxaliplatin.

SUBMITTER: Narvi E

PROVIDER: S-EPMC6224565 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin.

Narvi Elli E Vaparanta Katri K Karrila Anna A Chakroborty Deepankar D Knuutila Sakari S Pulliainen Arto A Sundvall Maria M Elenius Klaus K

Scientific reports 20181108 1

Therapeutic protocols including EGFR antibodies in the context of oxaliplatin-based regimens have variable clinical effect in colorectal cancer. Here, we tested the effect of the EGFR antibody cetuximab in different sequential combinations with oxaliplatin on the growth of colorectal cancer cells in vitro and in vivo. Cetuximab reduced the efficacy of oxaliplatin when administered before oxaliplatin but provided additive effect when administered after oxaliplatin regardless of the KRAS or BRAF m ...[more]

PMID: 30410004

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Project description:The antibody cetuximab, targeting epidermal growth factor receptor (EGFR), is used to treat metastatic colorectal cancer (mCRC). Clinical trials suggest reduced benefit from the combination of cetuximab with oxaliplatin. The aim of this study was to investigate potential negative interactions between cetuximab and oxaliplatin.Thiazolyl blue tetrazolium bromide (MTT) assay and Calcusyn software were used to characterize drug interactions. Reactive oxygen species (ROS) were measured by flow cytometry and real-time polymerase chain reaction oxidative stress arrays identified genes regulating ROS production. Chromatin immunoprecipitation (ChIP) measured signal transducer and activator of transcription 1 (STAT-1) binding to dual oxidase 2 (DUOX2) promoter. SW48, DLD-1 KRAS wild-type cell lines and DLD-1 xenograft models exposed to cetuximab, oxaliplatin, or oxaliplatin + cetuximab (control [saline]; n = 3 mice per treatment group) were used. Statistical tests were two-sided.Cetuximab and oxaliplatin exhibited antagonistic effects on cellular proliferation and apoptosis (caspase 3/7 activity reduced by 1.4-fold, 95% confidence interval [CI] = 0.78 to 2.11, P = .003) as opposed to synergistic effects observed with the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 produced ROS, only oxaliplatin-mediated apoptosis was ROS dependent. Production of ROS by oxaliplatin was secondary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, P < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab reduced oxaliplatin cytotoxicity.Inhibition of STAT1 and DUOX2-mediated ROS generation by cetuximab impairs p38-dependent apoptosis by oxaliplatin in preclinical models and may contribute to reduced efficacy in clinical settings. Understanding the rationale for unexpected trial results will inform improved rationales for combining EGFR inhibitors with chemotherapeutic agents in future therapeutic use.

Project description:BackgroundKRAS mutations are fraught with the progression of colorectal cancer and resistance to chemotherapy. There are pathways such as extracellular regulated protein kinase 1/2 (ERK1/2) and Akt downstream and farnesylation and geranylgeranylation upstream that are activated upon mutated KRAS. Previous studies have shown that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, are effective to treat KRAS mutated colorectal cancer cells. Increased doses of oxaliplatin (L-OHP), a well-known alkylating chemotherapeutic drug, causes side effects such as peripheral neuropathy due to ERK1/2 activation in spinal cords. Hence, we examined the combinatorial therapeutic efficacy of statins and L-OHP to reduce colorectal cancer cell growth and abrogate neuropathy in mice.MethodsCell survival and confirmed apoptosis was assessed using WST-8 assay and Annexin V detection kit. Detection of phosphorylated and total proteins was analyzed the western blotting. Combined effect of simvastatin and L-OHP was examined the allograft mouse model and L-OHP-induced neuropathy was assessed using cold plate and von Frey filament test.ResultsIn this study, we examined the effect of combining statins with L-OHP on induction of cell death in colorectal cancer cell lines and improvement of L-OHP-induced neuropathy in vivo. We demonstrated that combined administration with statins and L-OHP significantly induced apoptosis and elevated the sensitivity of KRAS-mutated colorectal cancer cells to L-OHP. In addition, simvastatin suppressed KRAS prenylation, thereby enhancing antitumor effect of L-OHP through downregulation of survivin, XIAP, Bcl-xL, and Bcl-2, and upregulation of p53 and PUMA via inhibition of nuclear factor of κB (NF-κB) and Akt activation, and induction of c-Jun N-terminal kinase (JNK) activation in KRAS-mutated colorectal cancer cells. Moreover, simvastatin enhanced the antitumor effects of L-OHP and suppressed L-OHP-induced neuropathy via ERK1/2 activation in vivo.ConclusionTherefore, statins may be therapeutically useful as adjuvants to L-OHP in KRAS-mutated colorectal cancer and may also be useful in the treatment of L-OHP-induced neuropathy.

Project description:BACKGROUND: Cancer cells frequently adopt cellular and molecular alterations and acquire resistance to cytostatic drugs. Chemotherapy with oxaliplatin is among the leading treatments for colorectal cancer with a response rate of 50%, inducing intrastrand cross-links on the DNA. Despite of this drug's efficiency, resistance develops in nearly all metastatic patients. Chemoresistance being of crucial importance for the drug's clinical efficiency this study aimed to contribute to the identification and description of some cellular and molecular alterations induced by prolonged oxaliplatin therapy. Resistance to oxaliplatin was induced in Colo320 (Colo320R) and HT-29 (HT-29R) colorectal adenocarcinoma cell lines by exposing the cells to increasing concentrations of the drug. Alterations in morphology, cytotoxicity, DNA cross-links formation and gene expression profiles were assessed in the parental and resistant variants with microscopy, MTT, alkaline comet and pangenomic microarray assays, respectively. RESULTS: Morphology analysis revealed epithelial-to-mesenchymal transition in the resistant vs parental cells suggesting alterations of the cells' adhesion complexes, through which they acquire increased invasiveness and adherence. Cytotoxicity measurements demonstrated resistance to oxaliplatin in both cell lines; Colo320 being more sensitive than HT-29 to this drug (P?<?0.001). The treatment with oxaliplatin caused major DNA cross-links in both parental cell lines; in Colo320R small amounts of DNA cross-links were still detectable, while in HT-29R not. We identified 441 differentially expressed genes in Colo320R and 613 in HT-29R as compared to their parental counterparts (at least 1.5 -fold up- or down- regulation, p?<?0.05). More disrupted functions and pathways were detected in HT-29R cell line than in Colo320R, involving genes responsible for apoptosis inhibition, cellular proliferation and epithelial-to-mesenchymal transition. Several upstream regulators were detected as activated in HT-29R cell line, but not in Colo320R. CONCLUSIONS: Our findings revealed a more resistant phenotype in HT-29R as compared to Colo320R and different cellular and molecular chemoresistance patterns induced by prolonged treatment with oxaliplatin in cell lines with identical origins (colorectal adenocarcinomas).

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Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin.

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Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin.

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