Expression of mTOR/70S6K signaling pathway in melanoma cancer cell and the effects of dacarbazine and metformin

Background: Melanoma, also known as malignant melanoma, is a type of skin cancer that develops from the pigment-producing cells known as melanocytes. Melanomas typically occur in the skin but may rarely occur in the mouth, intestines or, eye. The study aimed to examine the expression of the mammalian target of rapamycin (mTOR)/70S6K signaling pathway in melanoma cancer. Methods: The B16F10 cell line treated with dacarbazine IC50 and different concentrations of metformin (0.5, 2, and 8 mM) for 24 hr and mTOR and 70S6k proteins expression was examined by western blotting. Cell viability was measured by MTT assay. Results: Western blot analysis showed that after different concentra¬tions of metformin and dacarbazine treatments, the mTOR and 70S6K protein expression significantly (P<0.05) decreased. Conclusion: Metformin-induced repression of mTOR/70S6k axis activity disrupts B16F10 growth. Thus, we believe that combination therapy may be a suitable potential therapeutic target for melanoma cancer.

TGF-β Increases MFGE8 Production in Myeloid-Derived Suppressor Cells to Promote B16F10 Melanoma Metastasis

There is growing evidence that myeloid-derived suppressor cells (MDSCs) are directly involved in all stages leading to metastasis. Many mechanisms for this effect have been proposed, but mechanisms of coregulation between tumor cells and MDSCs remain poorly understood. In this study, we demonstrate that MDSCs are a source of milk fat globule-epidermal growth factor (EGF) factor 8 (MFGE8), which is known to be involved in tumor metastasis. Interestingly, TGF-β, an abundant cytokine in the tumor microenvironment (TME), increased MFGE8 production by MDSCs. In addition, co-culturing MDSCs with B16F10 melanoma cells increased B16F10 cell migration, while MFGE8 neutralization decreased their migration. Taken together, these findings suggest that MFGE8 is an important effector molecule through which MDSCs promote tumor metastasis, and the TME positively regulates MFGE8 production by MDSCs through TGF-β.

The Influence of Some Axial Ligands on Ruthenium–Phthalocyanine Complexes: Chemical, Photochemical, and Photobiological Properties

This work presents a new procedure to synthesize ruthenium–phthalocyanine complexes and uses diverse spectroscopic techniques to characterize trans-[RuCl(Pc)DMSO] (I) (Pc = phthalocyanine) and trans-[Ru(Pc)(4-ampy)2] (II) (4-ampy = 4-aminopyridine). The triplet excited-state lifetimes of (I) measured by nanosecond transient absorption showed that two processes occurred, one around 15 ns and the other around 3.8 μs. Axial ligands seemed to affect the singlet oxygen quantum yield. Yields of 0.62 and 0.14 were achieved for (I) and (II), respectively. The lower value obtained for (II) probably resulted from secondary reactions of singlet oxygen in the presence of the ruthenium complex. We also investigate how axial ligands in the ruthenium–phthalocyanine complexes affect their photo-bioactivity in B16F10 murine melanoma cells. In the case of (I) at 1 μmol/L, photosensitization with 5.95 J/cm2 provided B16F10 cell viability of 6%, showing that (I) was more active than (II) at the same concentration. Furthermore, (II) was detected intracellularly in B16F10 cell extracts. The behavior of the evaluated ruthenium–phthalocyanine complexes point to the potential use of (I) as a metal-based drug in clinical therapy. Changes in axial ligands can modulate the photosensitizer activity of the ruthenium phthalocyanine complexes.

Isorhamnetin Induces Melanoma Cell Apoptosis via the PI3K/Akt and NF-κB Pathways

Malignant melanoma is characterized by its bad prognosis for aggressiveness, drug resistance, and early metastasis. Isorhamnetin (3′-methoxy-3,4′,5,7-tetrahydroxyflavone; IH) is a natural flavonoid that has been investigated for its antitumor effects in breast cancer, colon cancer, and gastric cancer through inducing cell apoptosis. Given its role in tumor inhibition, no research has been conducted concerning its effect against melanoma. In the present study, we found that IH could significantly inhibit B16F10 cell proliferation and migration and induce B16F10 cell apoptosis. The examination on molecular mechanism revealed that IH could suppress the phosphorylation of Akt and the translocation of NF-κB, which are key factors in apoptosis-related pathways. We also detected that this process was related to the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases 4 (PFKFB4) by PFKFB4 knockdown experiment. In line with in vitro study, we further provided that IH effectively inhibited tumor growth in vivo. Taken together, IH was proven to induce melanoma cell apoptosis in vitro and in vivo, which may serve as a potential agent in malignant melanoma treatment in the future.

Inhibitory Effect of Cuphea aequipetala Extracts on Murine B16F10 Melanoma In Vitro and In Vivo

Cuphea aequipetala (C. aequipetala) has been used in Mexican traditional medicine since prehispanic times to treat tumors. In this paper, we evaluated the antiproliferative and apoptotic effect of the methanolic and aqueous extracts of C. aequipetala on several cancer cell lines including the B16F10 cell line of murine melanoma and carried a murine model assay. In vitro assay analyzed the effect in the cellular cycle and several indicators of apoptosis, such as the caspase-3 activity, DNA fragmentation, phosphatidylserine exposure (Annexin-V), and induction of cell membrane permeabilization (propidium iodide) in the B16F10 cells. In vivo, groups of C57BL/6 female mice were subcutaneously injected with 5x105 B16F10 cells and treated with 25 mg/mL of C. aequipetala extracts via oral. Aqueous and methanolic extracts showed a cytotoxic effect in MCF-7, HepG2, and B16F10 cell lines. The methanolic extract showed more antiproliferative effect with less concentration, and for this reason, the in vitro experiments were only continued with it. This extract was able to induce accumulation of cells on G1 phase of the cell cycle; moreover, it was able to induce DNA fragmentation and increase the activity of caspase-3 in B16F10 cells. On the other hand, in the murine model of melanoma, the aqueous extract showed a greater reduction of tumor size in comparison with the methanolic extract, showing an 80% reduction versus one of around 31%, both compared with the untreated control, indicating a better antitumor effect of C. aequipetala aqueous extract via oral administration. In conclusion, the in vitro data showed that both C. aequipetala extracts were able to induce cytotoxicity through the apoptosis pathway in B16F10 cells, and in vivo, the oral administration of aqueous extract reduces the melanoma tumoral mass, suggesting an important antitumoral effect and the perspective to search for effector molecules involved in it.

Apoptosis Caused by Triterpenes and Phytosterols and Antioxidant Activity of an Enriched Flavonoid Extract from Passiflora mucronata

Background: P. mucronata (Pm) comes from South America, Brazil and is characterized as “Maracujá de Restinga”. It is used in folk medicine for its soothing properties and in treating insomnia. Objective: The present study for the first time analyzed the antioxidant and cytotoxicity of the hydroalcoholic leaves extract and fractions from Pm. Method: The cytotoxicity test will be evaluated by different assays (MTT and CV) against human prostate cancer (PC3) and mouse malignant melanoma (B16F10) cell lines, and the antioxidant test by DPPH method. Results: β-Amyrin, oleanolic acid, β-sitosterol and stigmasterol were isolated of the most active, hexane fraction. These substances were tested against the tumor cell lines: β-sitosterol and stigmasterol showed the most relevant activity to PC3 in CV assay and, oleanolic acid to B16F10 by the MTT assay. In addition, it was possible to indicate that the mode of cell death for stigmasterol, presumably is apoptosis. In terms of antioxidant activity, the hydroalcoholic leaves extract presented higher activity (EC50 133.3 µg/mL) compared to the flower (EC50 152.3 µg/mL) and fruit (EC50 207.9 µg/mL) extracts. By the HPLC-MS, it was possible to identify the presence of flavones in the leaf extract (isoschaftoside, schaftoside, isovitexin, vitexin, isoorientin, orientin). Conclusions: P. mucronata hexane fraction showed promising cytotoxic effect against cancer cell lines, and stigmasterol contributes to this activity, inducing apoptosis of these cells. Furthermore, as other Passiflora species, Pm extract showed antioxidant activity and flavones are its major phenolic compounds.

Carnosic acid impedes cell growth and enhances anticancer effects of carmustine and lomustine in melanoma

Carnosic acid (CA), a major polyphenolic diterpene present in Rosmarinus officinalis, has been reported to have multiple functions, including antitumor activity. The MTT assay, BrdU incorporation, wound healing, and colony formation were used to detect melanoma B16F10 cell growth and proliferation. Flow cytometry was used for cell cycle detection. p21 and p27 expression was detected by Western blotting. B16F10 cell xenograft model was established, and treated with CA, carmustine (BCNU), or lomustine (CCNU). The present study found that CA exhibits significant growth inhibition and cell cycle arrest in melanoma B16F10 cells. We also found that CA triggers cell cycle arrest at G0/G1 phase, and enhances p21 expression. Additionally, CA can enhance BCNU- and CCNU-mediated cytotoxicity and cell cycle arrest in B16F10 cells. Finally, we found that CA inhibits tumor growth, and reduces the values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in vivo. The present study study concluded that CA may be safe and useful as a novel chemotherapeutic agent.