Previouse experiment showed that metformin, at doses of 2mM and higher, inhibited the proliferation of B16F10 melanoma cells. A significant increase in cytotoxicity was observed in cells treated with a fixed dose of dacarbazine (IC50) and increasing doses of metformin. All doses of metformin, especially at 2 mM, in combination with dacarbazine (134µg/ml) inhibited the growth of B16F10 melanoma cells after 24 h. Although metformin alone has increased cell viability, Combination therapy has reduced it so that changes compared to dacarbazine alone are not significant (Fig. 1B) and in following melanoma progression and pathways described. Our finding in accordance with previous confirmed combination therapy is more effective than dacarbazine therapy.
Combined treatment with metformin,as an antidiabetic drug, and dacarbazine, a well-known chemotherapeutic drug, has shednew light on the molecular pathways involved in cancer progression andthe expression levels of CSCmarkers, such as CD34 and CD44. It has been shown that CSCs, unlike bulk tumor cells, are capable of inducing tumor growth and forming colonies in culture [16, 21]. The expression levels of multiple CSC surface markers have been found to associate with invasion potential . In recent years, some studies have focused on the high expression of these CSC marker genes in cancer cell lines and solid tumors [22, 23].These results indicate that when B16F10 cancer cells were treated with different concentrations of metformin for 24 h, the expression of CSC markers was reduced and a cellular response was triggered, which, in turn, inhibited cell proliferation and metastasis. Therefore, it can be concluded that metformin, alone or in combination with dacarbazine, can reduce melanoma cancer cells. Also, it can be stated that the changes in the expression of CSC markers are a good indicator of the changes in intracellular pathways.
The reduction in CD44, despite being significant, is not so great (p < 0.05).Based on these data, it can be stated that combination therapy with metformin and dacarbazine cannot reduce the expression of this marker after 24 h and that the changes in its expression level must be evaluated over a longer time period. It is suggested to consider a longer incubation time in future researches. Some studies have shownthat the CD44 ligand-receptor complexes on dacarbazine-treated melanoma cells are less stable than on untreated cells. However, the CD44 receptor could still be activated, which, in turn, could lead to the activation of intracellular signaling and, consequently, a cellular response .
Flow cytometricanalysis of B16F10 cells revealed that combination therapy with metformin significantlydecreasedthe expression of CD34 which is in accordance with out previous reports [5, 24]. In this study, as expected, cells treated with dacarbazine and metformin at a physiological dose showed a reduction inthe expression levels of CD34 CSC marker. Thus, the combination therapy used in this study can be considered as an effective treatment for melanoma cancer. These findingsareconsistentwith those of other studies that have shown theeffects of dacarbazine on reducing the expression of CD34 marker. Our data revealed a new role for CD34 in the growth of cancerous tumors and provides new insights into a treatment that could inhibit tumor progression. Inour previous study, the cells treated withmetformin and dacarbazine generated fewer colonies than thosetreatedwithonly metformin. Colonization reflectsthe proliferation of CSCs, and it hasbeen shown that combination therapy with metformin and dacarbazinecan reduce colony formation and stemness.
Typically, melanoma metastasis is associated withthe activation of signaling pathways that are essential for embryogenesis . In recent years, some molecular pathways have been reported tobe involved in many cancers. Identifying the pathways that are responsible for embryogenesis and melanoma progression and metastasis is a key step toward a more effective treatment for melanoma cancer. Some data suggest that intracellular pathways are strongly associated with malignant melanocytic lesions, whichare caused by a combination of genetic and epigenetic changesinvolvedinneoplastic transformation[11, 26].Growing evidence indicates that metabolic reprogramming is a downstream consequence of tumor progression and that it is induced by oncogene activation. Italso has been reported that metabolicreprogramming can change metabolism and enhance oncogenic signaling to promote tumor malignancy .
The results of this study showed that metformin inhibited the proliferation of melanoma cells by suppressing the phosphorylation of AKT pathway proteins (p < 0.05), facilitated thephosphorylation of β-catenin, and increased the expression of cleaved PARP (p < 0.05).
According the Fig. 6 the activatedp-AKT has the potential to phosphorylate other proteins, which can lead to increased cancer cell proliferation. Glycogen synthase kinase 3β (GSK3β) is another kinase that is inhibited when phosphorylated by p-AKT . As a result of GSK3β inhibition, free β-catenin can accumulate and translocate to the nucleus and up regulate the expression of oncogenic genes, such as cyclin D1 . In addition, activated p-AKT can increase the expression of c-MYC by activating mTOR. The activation of mTOR, a kinase implicated in the regulation of apoptosis, can lead to the phosphorylation and activation of S6rpand initiation factor 4E-BP1, thus promoting protein translation and cell growth. Through these regulatory mechanisms, p-AKT exerts an anti-apoptotic effect and promotes cancer progression. mTOR activation functions asa growth factor in melanoma cells. Our results, together with the above data, show the inactivation of mTOR and a decrease in its activity after therapy,suggestingthat the mTOR pathway can beused as a target for the treatment of melanoma. Also according to the study performed by Baradaran et al,it has been reported that when metformin is transported into cells, it inhibits mitochondrial complex I (NADH) . Thus, it can be said that metformin decreases ATP synthesis and activates theAMPK pathway. Metformin has been shown to directly inhibit insulin-induced malignant cell growth in a PI3K/AKT/mTOR- and Wnt-dependent manner via the AMPK pathway. Metformin has also been found to cause a significant increase in AMPK activity by the phosphorylation of Thr and inhibition of DNA synthesis, cell mitosis, and proliferation. Furthermore, metformin has been shown to activate AMPK,which, in turn, canblock protein synthesis and mTOR signaling in many cancer cells . Our studies have demonstrated that loss of mTOR and S6rpis associated with marked dependence on AKT. The evaluation ofthe effects of treatment with metformin anddacarbazine on melanoma cells shows that these agents havestronginhibitory effects on AKT, mTOR, 4E-BP1, and S6rp pathways. Treatment with metformin, alone or in combination withdacarbazine, reduced AKT, mTOR, and rpS6phosphorylation and negatively regulatedthe activities of thesepathways. Our studies show that combination therapy with metformin and dacarbazine, compared to treatment with dacarbazine alone, can lead toa more significant decrease in the expression of phosphorylatedAKT, S6rp, and 4E-BP1, indicating that combined treatment with metformin is likely to enhance the therapeutic effects of dacarbazine.
Wnt signaling has a key role in cell survival and differentiation. Another study reported that cytoplasmic β-catenin destruction complex with APC, Axin, and GSK can cause the ubiquitination and degradation of β-catenin. Metformin activates theAMPK pathway, and this pathway can inhibit βcatenin nuclear translocation by binding to β-catenin in the cytosol[31, 33]. In the present study similar to others findings, the elevated levels of phosphorylated β-catenin following combination therapy and the activation of small molecules inhibitedtheproliferation of cancer cells. However, the changes in cyclin D1 levels were inconsistent, and metformin treatment, especially at normal doses, increased the level of this protein. The phosphorylation of β-catenin may block thetranslocation of β-catenin into the nucleus. Anincreased ratio of phosphorylated to non-phosphorylated β-catenin indicates the effectiveness of combination therapy in inhibiting the Wnt signaling pathway (p < 0.05).
Some previousstudies haveshown that chemotherapy drugs are critical mediators of alternative metabolic pathways because their use leads to some coordinate changes in phosphorylated AKT, phosphorylated β-catenin, phosphorylated S6rp, and phosphorylated 4E-BP1 . This is consistent with the results of the present study. Furthermore, ithas been noted that the inactivation of Wnt/β-catenin and PI3K pathways and the activation of AMPK following combined treatment with metformin and dacarbazine can lead to a decrease in the expression of stemness genes and cell proliferation. Stemness markers are often upregulated in melanoma tumors and promote tumor progression and metastasis. These cell-surface markers are associated with CSCs and are thought to drive the growth of many cancers which is in accordance with out previous results . Tumor growth and metastasis are determined by the complex interplay between several factors, includingCD34 and CD44 markers and intracellular pathways. Our results revealeda novel role for CD34 in both cell proliferation and stemness. The results of this study also indicated that the Wnt and PI3K pathways were involved in theupregulation of stemnessmarkers such as CD34 at the transcriptional level, conferring cancer stem cell-like properties to melanoma cells. In this respect, combination therapy with metformin and dacarbazine represents a potential therapeutic option for the treatment of melanoma cancer because this combination therapy can suppress the above-mentioned pathways.
PARP is a nuclear enzyme involved in DNA repair. PARP hyper activation induced NAD+ depletion, which led to mitochondrial dysfunction and cell death . According to the results of this study, metformin increased the expression of PARP after 24 h, which can reflect an attempt by the cells to prevent apoptosis during treatment (p < 0.05). In some other studies, the expression of PARP increased after 48 or 72 h following treatment with metformin. The characteristic ability of PARP to be activated by DNA strand breaks makes poly(ADP-ribosy)lation an immediate and drastic cellular response to DNA damage as induced by ionizing radiation, alkylating agents, and oxidants . In this study, although metformin increased the expression of PARP expression, given that it inhibited colonization and proliferation pathways and increased the expression of cleaved PARP, metformin treatment can be considered an effective treatment for several cancers .