Metformin anticancer: Reverses tumor hypoxia induced by bevacizumab and reduces the expression of cancer stem cell markers CD44/CD117 in human ovarian cancer SKOV3 cells

Abstract

Background: The occurrence and development of solid tumors depend on the blood supply in the tumor microenvironment (TME). Blocking angiogenesis is a new therapeutic strategy to inhibit tumor growth. The anti-angiogenic drug bevacizumab has been approved for gynecological malignancies, especially for advanced recurring cervical cancers and recurring ovarian cancers (OC). Studies in OC have shown a limited effect of bevacizumab in the general population, with a slight improvement in progression-free survival (PFS) and no effect on overall survival (OS). This might be related to the bevacizumab’s role in aggravating the hypoxia in the TME, which helps maintain the stemness of ovarian cancer stem cells (CSCs) and promotes the invasion and metastasis of cancer cells. Drugs that target CSCs, such as metformin, may enhance the efficacy of anti-vascular therapies. Therefore, this study aimed to evaluate the effect of metformin combined with bevacizumab on the proliferation of OC cells both in vitro and in vivo, as well as on tumor hypoxia and tumor stem cell markers of human ovarian cancer SKOV3 cells.Methods: The OC cell model SKOV3 was treated with metformin, bevacizumab, and cisplatin alone or in combinations. Cell Counting Kit-8 (CCK-8) was used to measure the rate of cell proliferation. Metformin and bevacizumab were studied in vivo in nude mice. SKOV3 cells were transplanted subcutaneously in nude mice, and different drug interventions were performed after tumor formation, including blank control, bevacizumab alone, metformin alone, cisplatin alone, bevacizumab + metformin, bevacizumab + cisplatin, metformin + cisplatin, and bevacizumab + metformin + cisplatin treatments. The growth of transplanted tumors was routinely monitored and visualized by the tumor growth curve. We used flow cytometry to examine the proportion of CD44+/CD117+ CSCs in each group. The immunohistochemistry (IHC) method was applied to detect expressions of vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α), and microvascular density-associated factor CD34 in tumor cells. The limit dilution method was used to re-inject tumor cells in nude mice to examine the tumor recurrence rate.Results: Combination therapy of metformin and bevacizumab significantly reduced the proliferation rate of SKOV3 cells and the growth rate of transplanted tumors in nude mice compared with the monotherapy effects. In vivo results showed that metformin significantly reduced the proportion of CD44+/CD117+ CSCs (p < 0.01). Although bevacizumab increased the proportion of CD44+/CD117+ CSCs, the addition of metformin did offset this fluctuating trend. The combination of bevacizumab, metformin, and cisplatin efficiently decreased the proportion of CSCs in the OC animal model. IHC results exhibited that expressions of VEGF, CD34, and HIF-1α in transplanted tumors were decreased by metformin alone compared with the control (p < 0.05). In the bevacizumab treatment, VEGF, and CD34 expressions were decreased, while that of HIF-1α was increased, suggesting that the degree of hypoxia was differentially aggravated after the bevacizumab treatment. The VEGF, CD34, and HIF-1α expressions in the bevacizumab + metformin + cisplatin group were the lowest among all other treatment groups (p < 0.05). Subcutaneous statistics of nude mice reseeded by the limit dilution method showed that the tumor recurrence rate in the bevacizumab + metformin + cisplatin group was relatively lower.Conclusion: Metformin, bevacizumab combined with platinum-based chemotherapy can significantly inhibit the growth of ovarian cancer cells and transplanted tumors, which is due to the reduction of the proportion of CD44+/CD117+ CSCs and the alleviation of hypoxia in the tumor microenvironment. Therefore, this may be a reasonable and promising treatment regimen.

Gamma/delta T cells as cellular vehicles for anti-tumor immunity

“…Even if γδ T cells could infiltrate solid tumors, their cytotoxicity can be suppressed by hypoxic conditions in the TME due to apoptosis via PD-1 and reduced expression of NKG2D. In brain tumors, the use of metformin, a repurposed drug that has been shown to elicit an anti-tumor effect ( 75 , 76 ), reduced hypoxia and rescued the anti-tumor effect of γδ T cells ( 72 ). In oral cancers, blockade of PD-1 or targeting hypoxia-inducible factor-1α could also help to overcome tumor hypoxia ( 73 ).…”

CD44 and its implication in neoplastic diseases

“…It has been shown that suppressing the expression of CD44 250 or blocking its interaction with other ligands 251 , 252 is a promising strategy in cancer therapy. Antibody against CD44 has been reported to be used in this way in CD44‐positive cancer.…”

Direct antitumor activity of bevacizumab: an overlooked mechanism?

“…As our understanding of bevacizumab’s pharmacological characteristics deepens, numerous findings challenge longstanding beliefs. This is evident in bevacizumab’s capacity to induce direct cytotoxic effects in various tumor cells expressing high VEGFA levels ( Miranda-Goncalves et al, 2017 ; Zhao et al, 2018 ; Suo et al, 2020 ; Alonso-Diez et al, 2021 ; Fan et al, 2022 ; Wei et al, 2023 ). At the molecular level, studies proposed mechanisms through which bevacizumab induces tumor cell death.…”

“…However, the tide of evidence is shifting. Increasingly, studies suggest that bevacizumab does exert direct cytotoxic effects on encountered tumor cells ( Miranda-Goncalves et al, 2017 ; Zhao et al, 2018 ; Suo et al, 2020 ; Alonso-Diez et al, 2021 ; Fan et al, 2022 ; Wei et al, 2023 ), with even bevacizumab-induced cell death observed in certain transplant tumor models ( Ramezani et al, 2017 ; Zhao et al, 2018 ; Ramezani et al, 2019 ; Xiang et al, 2022 ). These emerging findings challenge the notion that bevacizumab lacks direct anti-tumor activity, prompting a need for rigorous scrutiny and scientific exploration.…”