Satoru Kitazono

Antiproliferative action of metformin in human lung cancer cell lines

The oral antidiabetic agent metformin has anticancer properties, probably via adenosine monophosphate-activated protein kinase activation. In the present study, growth inhibition was assessed by a clonogenic and by a cell survival assay, apoptosis induction was assessed by Hoechst staining and caspase activities and cell cycle alteration after exposure to metformin, and the interaction of metformin with cisplatin in vitro were elucidated in four human lung cancer cell lines representing squamous, adeno-, large cell and small cell carcinoma. Clonogenicity and cell proliferation were inhibited by metformin in all the cell lines examined. This inhibitory effect was not specific to cancer cells because it was also observed in a non-transformed human mesothelial cell line and in mouse fibroblast cell lines. Inhibition of clonogenicity was observed only when the cells were exposed to metformin for a long period, (10 days) and the surviving fraction, obtained after inhibiting proliferation by increasing the dose, reached a plateau at approximately 0.1-0.3, indicating the cytostatic characteristics of metformin. Metformin induced significant apoptosis only in the small cell carcinoma cell line. A tendency of cell cycle accumulation at the G0/G1 phase was observed in all four cell lines. Cisplatin, in a dose-dependent manner, severely antagonized the growth inhibitory effect of metformin, and even reversed the effect in three cell lines but not in the adenocarcinoma cell line. The present data obtained using various histological types of human lung cancer cell lines in vitro illustrate the cytostatic nature of metformin and its cytoprotective properties against cisplatin.

Effect of metformin on residual cells after chemotherapy in a human lung adenocarcinoma cell line

Cancer chemotherapy, including molecular targeted therapy, has major limitations because it does not kill all the cancer cells; the residual cells survive until they acquire chemoresistance. In the present study, the combined effects of metformin and gefitinib were examined in vivo in a mouse xenograft model, inoculated with a human lung adenocarcinoma cell line that possesses an activating epidermal growth factor receptor mutation. The mechanism of the interaction was further elucidated in vitro. Metformin did not suppress the growth of already established tumors, nor did metformin augment tumor shrinkage by gefitinib. However, metformin significantly suppressed the regrowth of the tumor after effective treatment with gefitinib, suggesting the specific effect of metformin on the residual cells. Cytotoxicity of metformin was characterized by the absence of apoptosis induction and unremarkable cell cycle shift in vitro. The residual cell population after treatment with gefitinib was characterized by enriched cells with high expression of CD133 and CD24. Metformin was still effective on this specific cell population. Targeting residual cells after chemotherapy may represent an effective novel strategy for the treatment of cancer. Elucidating the mechanism of metformin cytotoxicity provides insights into future development of anticancer therapeutics.

Interaction and cross-resistance of cisplatin and pemetrexed in malignant pleural mesothelioma cell lines

Although cisplatin and pemetrexed are key drugs in the treatment of malignant pleural mesothelioma, their drug-drug interactions, cross-resistance and resistance mechanisms in malignant pleural mesothelioma are not well understood. In the present study, the interaction of these 2 agents was determined by clonogenic assays followed by isobologram analysis of 4 human malignant pleural mesothelioma cell lines. The cell lines were exposed to the agents using a stepwise dose-escalation method to establish drug-resistant sublines. Thymidylate synthase mRNA expression was evaluated in the drug-resistant sublines. As a consequence, cisplatin and pemetrexed had synergistic effects in 3 cell lines and an additive effect in the fourth cell line. The former 3 cell lines showed similar pemetrexed sensitivity in the parental cells and their cisplatin-resistant sublines, whereas the fourth cell line exhibited cross-resistance. In contrast, cisplatin had diverse effects on pemetrexed-resistant sublines. High thymidylate synthase expression did not correlate with natural pemetrexed resistance. Elevated thymidylate synthase expression correlated with acquired pemetrexed resistance in 2 sublines. In conclusion, cisplatin and pemetrexed showed synergistic activity and no cross-resistance in 3 of the 4 malignant pleural mesothelioma cell lines, suggesting the clinical relevance of their combination in chemotherapy. Thymidylate synthase expression did not necessarily correlate with pemetrexed resistance. The information together with the experimental model presented here would be useful for further investigating therapeutic targets of malignant mesothelioma.

Abstract 2737: Anti-proliferative action of metformin on various types of human lung cancer cell lines

[Background] Metformin has a potential to inhibit tumor growth in various cancer cell lines in vitro and in vivo. [Purpose] To elucidate anti-proliferative actions of the agent on various types of human lung cancer cell lines, and to evaluate combined effects with cisplatin. [Methods] Cell lines used consisted of four human lung cancer cell lines, RERF-LC-AI (squamous cell carcinoma), A549 (adenocarcinoma), IA-5 (large cell carcinoma) and WA-hT (small cell carcinoma). Colonogenic assay was employed to evaluate colonogenicity with exposure to metformin for 1, 24 hours and 10 days. In cell surviving assay, 100,000 cells per plate were cultured for 4 days with metformin and/or cisplatin, and survived cells were counted. [Results] Metformin inhibited colonogenecity in every cell line in a dose-dependent manner when cells were exposed to the agent for 10 days, whereas 1- and 24-hour exposure yielded not significant or only mild effects, with the degree varied depending on cell lines. With cell surviving assay at the metformin concentrations where 70% (IC70) and 30% (IC30) of the cells survived for each cell line, apoptosis was not increased over control groups in every cell line except for WA-hT that showed significant increase in apoptosis with metformin. Significant alteration in cell cycle distribution with the agent was not observed in all of the 4 cell lines. For evaluating combination treatment consisting of metformin and cisplatin, cisplatin at doses of IC50 and IC10 was combined with metformin. Inhibitory effects of metformin on cell proliferation were slightly suppressed with cisplatin at its dose of IC50 in all except for in A549. High dose (IC10) of cisplatin almost completely countervailed or even reversed the metformin effects in all cell lines, again except for A549 where a modest but significant sub-additive effect was observed. [Conclusions] Metformin has anti-proliferative effects on various types of human lung cancer cell lines. Although apoptosis was observed only in a small-cell lung cancer cell line, its effects may not depend on apoptosis in other 3 cell lines. Alteration of cell cycle distribution was not observed in all cell lines. Metformin and cisplatin were antagonistic especially with higher dose cisplatin. 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 2737. doi:1538-7445.AM2012-2737

Abstract 2735: Effects of metformin on a non-small cell lung cancer cell line with an EGFR mutation

Background:Metformin, one of the most widely prescribed oral agents for treating diabetes mellitus, has been speculated to have antineoplastic effects. The effects, however, have not been fully understood. Experimental Design:A human non-small-cell lung cancer cell line, PC9 (with an EGFR mutation of the exon 19 E746-A750 deletion), was used to evaluate antineoplastic effects of metformin in vitro and in vivo. For in vitro experiments, the cells were exposed to metformin, gefitinib or cisplatin to observe inhibitory effects on cell proliferation. At 24, 48 and 72 hours after the start of the culture with the agents, cell cycle analysis, measurements of caspase 3, 8 and 9 activities, and evaluation with Hoechst staining and APOPercentage TM assay, respectively, were performed. For in vivo experiments in SCID mouse with a xenograft subcutaneous tumor, gefitinib (150 mg/kg, oral gavage) or metformin (250 mg/kg, i.p.) was daily administered to the mice. Results:Gefitinib at a concentration of 0.03 μM, cisplatin of 1.5 μg/ml and metformin of 10 mM exerted equivalent inhibitory effects on cell proliferation in vitro. At these particular concentrations, metformin did not induce significant apoptosis over control as assessed by Hoechst staining, APOPercent assay and caspase 3 activity, whereas gefitinib and cisplatin did induce apoptosis dramatically. Metformin failed to alter cell cycle distribution, whereas gefitinib and cisplatin caused G1/G0 an S phase accumulation, respectively. Metformin did not inhibit growth of already grown subcutaneous tumors, whereas the tumors shrank dramatically with gefitinib treatment. After almost complete disappearance of the tumors by gefitinib, however, they regrew by gefitinib withdrawal. Metformin did significantly suppress the regrowth of the tumors after gefitinib withdrawal. Conclusions: Metformin has inhibitory effects on growth of a human non-small cell lung cancer cell line with a sensitive EGFR mutation in vitro and in vivo, and its mechanism may not depend on apoptosis or cell cycle alteration. Although the agent does not suppress growth of already grown tumors, it significantly suppresses tumor regrowth after gefitinib withdrawal. 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 2735. doi:1538-7445.AM2012-2735

Abstract 3821: Interaction and cross-resistance of cisplatin and pemetrexed in malignant pleural mesothelioma cell lines

Background: Malignant pleural mesothelioma (MPM) is one of the most lethal cancers with a rapidly increasing incidence worldwide. Although cisplatin and pemetrexed are recognized as key drugs, and their combination chemotherapy is a standard therapy for MPM, their drug-to-drug interactions, cross-resistance, and resistance mechanisms are not well understood. Methods: The interaction between cisplatin and pemetrexed was evaluated by colonogenic assays followed by isobologram analysis in 4 human MPM cell lines. The cell lines were exposed to the agents using a stepwise dose-escalation method to establish drug-resistant sublines. Thymidylate synthase (TS) mRNA expression was evaluated by RT-PCR, and gene expression profiles were assessed by cDNA microarray analysis and gene ontology analysis using Ingenuity Pathway Analisis (IPA) in the drug-resistant sublines. Results: Cisplatin and pemetrexed had synergistic effects in 3 cell lines and an additive effect in the fourth cell line. The former 3 cell lines showed similar sensitivity to pemetrexed in the parental cells and their cisplatin-resistant sublines, whereas the fourth cell line exhibited cross-resistance. In contrast, cisplatin had diverse effects on pemetrexed-resistant sublines. High TS expression did not correlate with natural pemetrexed resistance and its elevatedexpression correlated with acquired pemetrexed resistance in 2 sublines. Microarray analysis disclosed that the genes which related to “cellular development”,” small molecule biochemistry,” “cellular movement,” “cell death, and “cellular growth and proliferation” affected drug-resistant mechanisms deeply, and that this tendency was common to cisplatin-resistant sublines and pemetrexed-resistant sublines. Conclusion: Cisplatin and pemetrexed showed synergistic activity and no cross-resistance in 3 of the 4 MPM cell lines, suggesting the clinical relevance of their combination chemotherapy. TS expression did not necessarily correlate with pemetrexed resistance. Multiple mechanisms of molecular and cellular functions may underlie acquired resistance to cisplatin and pemetrexed in MPM. 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 3821. doi:1538-7445.AM2012-3821