Abstract
Lung cancer is heterogeneous and challenging to cope with once it has progressed. Chemotherapy is the first step once no active driver mutation has been discovered. Non-antitumor drugs have been found to be beneficial when used as adjuvants to chemotherapy. In this study, the additive effect and mechanism of metformin combined with pemetrexed in non-small-cell lung cancer (NSCLC) cells were elucidated. Three NSCLC cell lines, A549, H1975, and HCC827, were used to analyze tumor cell proliferation, colony formation and the cell cycle in vitro when exposed to metformin alone, pemetrexed alone or their combination. We found that combination treatment in three cell lines exerted antiproliferative effects through cell cycle arrest in the S phase. An ex vivo chicken chorioallantoic membrane (CAM) assay was used to examine the antiangiogenic effect of metformin combined with pemetrexed on vascular structure formation. We further created an A549 orthotopic xenograft model with an in vivo imaging system (IVIS) and explored the associated indicators involved in the tumorigenic process. The in vitro results showed that the combination of metformin and pemetrexed exhibited an antiproliferative effect in reducing cell viability and colony formation, the downregulation of cyclin D1 and A2 and the upregulation of CDKN1B, which are involved in the G1/S phase. For antiangiogenic effects, the combination therapy inhibited the vascular structure, as proven by the CAM assay. We elucidated that combination therapy could target VEGFA and Endoglin by RT-qPCR, ELISA and histopathological findings in an A549 orthotopic NSCLC xenograft model. Our research demonstrated the additive antiproliferative and antiangiogenic effects of the combination of metformin with pemetrexed in NSCLC and could be applied to clinical lung cancer therapy.
Highlights
Lung cancer remains a challenging obstacle and ranks as the leading cause of mortality worldwide, including in Taiwan (Hsu et al, 2015; Siegel et al, 2017; Tseng et al, 2019)
In three non-small-cell lung cancer (NSCLC) cell lines and normal lung cells (WI38-2RA), the flow cytometric analysis of propidium iodide (PI)-labeled cells showed that the cells accumulated in the S phase after single pemetrexed treatment (Figures 2A–D); in the combination of metformin and pemetrexed-treated NSCLC cells showed more significant cells arrested in the S phase than single pemetrexed treatment groups (Figures 2A–C)
In three NSCLC cell lines model, when metformin was added to pemetrexed, an antiproliferative effect was observed through the inhibition of colony formation (Figure 1), as well as an induced cell cycle arrest (G1/S phase) (Figure 2) through regulating the AMPK/AKT signaling pathway (Figure 3)
Summary
Lung cancer remains a challenging obstacle and ranks as the leading cause of mortality worldwide, including in Taiwan (Hsu et al, 2015; Siegel et al, 2017; Tseng et al, 2019). Lung adenocarcinoma is predominant among NSCLC, and most patients are diagnosed with an advanced or metastatic status (Levy and Doyen, 2018). Once driver mutations, such as those in epidermal growth factor receptor (EGFR) or echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion mutations, are detected, targeted therapy can be applied (Hsu et al, 2015; Mok et al, 2017; Soria et al, 2017). When patients do not harbor active driver mutations or immune checkpoint expression, chemotherapy is the mainstream treatment
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