Additive Antiproliferative and Antiangiogenic Effects of Metformin and Pemetrexed in a Non-Small-Cell Lung Cancer Xenograft Model

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.

Transmembrane p24 trafficking protein 2 regulates inflammation through the TLR4/NF-κB signaling pathway in lung adenocarcinoma

“…Human alveolar epithelium (HAE, normal), as well as A549, HCC827, HCL-H1299, and HCL-H524 (NSCLC) cell lines, was used in this study. Cells were maintained in Ham’s F12K medium (Life Technologies, MA, USA) and RPMI-1640 medium (Thermo Fisher Scientific, Inc., MA, USA), as described previously [ 18 ].…”

Advances in metformin‑based metabolic therapy for non‑small cell lung cancer (Review)

“…This study demonstrated that metformin prevents the tobacco carcinogen-induced development of lung tumors via inhibition of Akt, upstream of mTOR, and indirect inhibition of mTOR. In three other xenogeneic models of A549 cell origin, treatment with metformin significantly reduced tumor growth and metastatic capacity in vivo , and reduced the expression of proteins such as Ki-67, proliferating cell nuclear antigen (PCNA), Akt and Myc ( 72 , 78 , 81 ). In a study by Moro et al ( 73 ) on patient-derived xenografts (PDXs), metformin (100 mg/kg/day) partially inhibited the tumor growth of PDXs with wild-type LKB1 (maximum inhibition rate, 50.5±14.8%), but had no significant inhibitory effect on LKB1-mutant PDXs, and with increasing doses of metformin, p-AMPK expression was increased and Ki67 expression was decreased, indicating that LKB1-deficient tumors have an impaired ability to adapt to metabolic stress induced by metformin treatment.…”

“…In a study by Ko et al ( 77 ), treatment of H2087 cells with 0.25-4 mM metformin revealed a decrease in cell colonization and invasion, and upregulated expression of phosphorylated (p)-ERK. Wang et al ( 78 ) treated A549, H1975 and HCC827 cells with 0.2 mM metformin and observed that not only did they inhibit cell proliferation, but they also induced cell cycle arrest in the S phase and increased apoptosis.…”

Therapeutic Effects of Kefir Peptides on Hemophilia-Induced Osteoporosis in Mice With Deficient Coagulation Factor VIII

“…To prepare femur tissue sections for hematoxylin and eosin (H&E) staining, the femur bones were decalcified in Decalcifier I ® solution (Leica Microsystems Inc., Buffalo Grove, IL, USA), dehydrated in a series of 50, 60, 70, 80, 90 and 100% ethanol, embedded in paraffin and longitudinally sectioned at 2-3 μm (Chen et al, 2021;Wang et al, 2021). The femur tissue sections were submitted to H&E staining using a Sakura model DRS-60A automatic slide stainer (Tissue-Tex DRS, Sakura, and Japan).…”