In the urinary system, the most common malignancy is bladder cancer, ranking as the 4th most common malignancy in men and the 11th most common malignancy in women in the United States. The incidence and mortality of bladder cancer in males is approximately four times that of females [1]. In China in 2019, there were 549,393 cases of bladder cancer, with an initial incidence of 7.2 per 100,000, ranking as 10th among all tumors. There were almost the same number of new cases in China (82,270) as there were in the in the USA (82,501) [2]. Clinically, about 70% of bladder cancers are NMIBC and 30% are MIBC. NMIBC has a low mortality rate and a high recurrence rate, whereas about 50% of cases of MIBC are potentially fatal [3]. For patients with NMIBC, the preferred treatment is TURBT with conventional postoperative infusion chemotherapy using the Bacillus Calmette-Guérin (BCG) vaccine (BCG), epirubicin (EPI), pirubicin (THP), and other cytotoxic drugs to prevent postoperative tumor recurrence and progression [4]. Despite active postoperative adjuvant treatment, the 5-year recurrence rate remains high (50-70%) among patients [5, 6].
Metformin safely reduces insulin resistance and lowers blood glucose [7, 8]. It is commonly used in the clinic to treat type II diabetes mellitus and has become popular in tumor treatment research because of its extensive anti-tumor effects. Metformin has anti-tumor effects in a variety of tumors, such as breast cancer, colon cancer, stomach cancer, pancreatic cancer, prostate cancer, lung cancer, and liver cancer [9,10,11,12,13,14]. Our current understanding of the mechanism by which metformin inhibits tumor cell invasion, migration, and proliferation can be roughly divided into the following pathways [9,10,11,12,13,14,15]: (1) Metformin inhibits the downstream mTOR pathway by activating the AMP-activated protein kinase (AMPK) signaling pathway to inhibit tumors; (2) Metformin inhibits tumors by activating reactive oxygen species (ROS) and the C-Jun N-Terminal Kinase 1 (JNK) signaling cascade; (3) Metformin can enhance the cytotoxicity of chemotherapy drugs. However, there has been only one study on the molecular mechanism of metformin when used in bladder cancer [16]. Our results indicated that under 5, 10, 15, and 20 mmol/L metformin treatment for 48 h, T24 and 5637 cell survival was lower compared with that of the control group. The migration ability of T24 and T5637 cells treated with metformin was significantly weakened in a concentration-dependent manner, as assessed using wound healing and Transwell chamber assays. Thus, metformin can inhibit the migration and proliferation abilities of T24 and T5637 cells, which suggested that it would inhibit bladder cancer progression.
Programmed cell death (PCD) comprises mainly apoptosis (type I) and autophagy (type II) (17). The most common type of PCD is apoptosis, which is under the control of both intracellular and extracellular signals. Apoptosis is characterized by changes in cell morphology, e.g., mitosis and DNA condensation, production of membranous vesicles, cell contraction, and the development of apoptotic bodies. In last 30 years, apoptotic signaling pathways have been intensively studied in many types of tumor cells. Tumor apoptosis-related protein include the caspase family and the B-cell CLL/lymphoma 2 (Bcl-2) family, that latter of which has vital functions mitochondria-mediated apoptosis. The pro-apoptotic BCL2 associated X protein (Bax) promotes mitochondrial cytochrome C through regulating the permeability of the mitochondrial membrane and activating the caspase cascade reaction, which ultimately leads to cell apoptosis. In our apoptosis experiment, the level of the apoptosis promoter cleaved-caspase 3 was increased by the different concentrations of metformin. In addition, flow cytometry showed that a higher proportion of T24 and T5637 bladder cancer cells underwent apoptosis after treatment with different concentrations of metformin.
Xem thêm : Essiac
Tumorigenesis and development are complex processes and extensive research on the molecular mechanism of tumors has revealed that tumorigenesis and development are related to multiple signaling pathways, among which PI3K/Akt /mTOR is the canonical pathway. Domestic and foreign scholars have discovered that this signaling pathway plays a crucial role in the occurrence and development of breast cancer, cervical cancer, pancreatic cancer, and other tumors [17]. In ovarian cancer, Parashar established MCW-OV-SL-3, an endometrioid subtype of ovarian cancer cell line, which is tumorigenic and highly metastatic, similar to the A-2780 ovarian cancer cell line. They observed that aberrant PI3K/Akt/ERK signaling promotes cancer stemness characteristics, chemoresistance, and EMT in this cell line. Meanwhile, inhibition of PI3K/Akt/ERK signaling using a PI3K/Akt dual kinase inhibitor abolished oncogenic features such as cancer stemness, chemoresistance, and EMT [18]. In breast cancer, the PI3K pathway presents mutations of genes that encode the catalytic and the regulatory subunits. The most frequent mutations are in PIK3CA, expecially in exon 9 and 20, identified from tumor tissue and/or circulating DNA, in all breast cancer subtypes. Breast cancer mutations also appear in receptor tyrosine kinase receptors, such as HER2, and phosphorylation of this receptor leads to PI3K/Akt/mTOR activation. PI3K mutations, PTEN methylation, and Akt activation will result in hormonal therapy resistance [19]. In colon cancer, metformin monotherapy was more effective than active vitamin D3 (VD3) against cancer, resulting in higher levels of p21, p27, phosphatase and tensin homolog (PTEN), BCL2-associated X protein (BAX), cytochrome C (Cyto-C),and caspase-3(Casp-3). Metformin also inhibited colon oncogenesis involving abnormal upregulation of the cyclin D1 (CCND1), CCND3, B-cell lymphoma 2 (BCL2), and PI3K/Akt/mTOR networks; and induced higher rates of apoptosis, both in vivo and in vitro. Although all dual regimens showed enhanced regulation of the PI3K/PTEN/Akt/mTOR pathway, together with higher expression of cell cycle inhibitors and pro-apoptotic molecules compared with monotherapy, the Metformin/5-fluorouracil combination was better than the other dual therapies. In contrast, triple therapy regimens exhibited the best anticancer effects associated with cell cycle arrest and apoptosis compared with single and dual regimens, possibly through enhanced attenuation of the PI3K/Akt/mTOR oncogenic pathway [20]. In summary, PI3K/Akt signaling is associated with cell autophagy, apoptosis, proliferation, and inflammation. In-depth study of the PI3K signaling pathway has important clinical significance for tumor therapy. In addition, in this pathway, mTOR acts a downstream regulator, exerting a vital function in autophagy and protein synthesis [21,22,23,24]. Although there have been few studies about metformin in bladder cancer, analysis of reviews and published studies revealed that more than 40% of patients with urothelial carcinoma had a dysregulated PI3K/AKT/mTOR pathway [25,26,27,28,29,30]. These observations inspired us to consider whether metformin also has an inhibitory effect in bladder cancer and if this effect is related to the PI3K pathway. In our in vitro study, we found that metformin indeed inhibited the PI3K/Akt/mTOR signaling pathway, and different concentrations of metformin reduced the levels of phosphorylated PI3K, Akt, and mTOR.
In conclusion, metformin inhibits bladder cancer T24 and 5637 cell migration and proliferation, activates the caspase cascade signaling pathway, and induces cell apoptosis. The mechanism might be associated with inhibition of the activation of the PI3K/Akt/mTOR signaling pathway.
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