Avaliação de novas terapias in vitro contra o melanoma e câncer de bexiga

Abstract

Cancer is considered a multifactorial disease caused by abnormal cell proliferation and has an economic impact on the health system worldwide. The types of cancer studied in this study were melanoma and bladder cancer. For melanoma, auxotrophic strains of Mycobacterium bovis (BCG ΔLeuD and recombinant BCG ΔLeuD/Ag85B) were evaluated, and in combination with imiquimod on lineage WM1366 to study the effect on cell inhibition and on macrophage lineage J774A.1 to determine the cellular immune response. In WM1366, MTT, Annexin and qRT-PCR tests were performed, and in J774A.1 qRT-PCR was performed. For bladder cancer, organocalcogen compounds (β-arylcalcogen azide) containing tellurium (5c and 5j) on line 5637 were evaluated to evaluate cytotoxic and antitumor activity. MTT, live/dead, DAPI, migration test, qRT-PCR, and molecular docking tests were performed. The results obtained showed that for melanoma (WM1366), the IC50 of cellular inhibition of imiquimod was 42.63 ± 4.28 µM in 48 hours. The combination of ΔleuD/Ag85B+imiquimod in 40 µM showed 59.41% growth inhibition, the combination in 50 µM showed 61.30% and with 60 µM it was 80.08% growth inhibition when compared to imiquimod isolated (48.22%, 51.77%, and 57.97). Apoptosis induction showed different percentages of early and late apoptosis at a concentration of 60 µM in BCG ΔleuD+imiquimod (15.77%), BCG ΔleuD/Ag85B+imiquimod (13.96%), imiquimod (19.91%) when compared to the untreated control (3.78%) showing statistically significant differences between treatments. In gene expression in WM1366, Bax and Caspase- 3 showed an increase in BCG ΔleuD/Ag85B and ΔleuD/Ag85B+imiquimod treatments, and in J774A.1 in 24 hours, the BCG ΔleuD/Ag85B + imiquimod combination increased the mRNA levels of proinflammatory cytokines (IL-6, IL-12, TNF-α, and IFN-γ) when compared to imiquimod and untreated cells (P <0.05). On the other hand, in bladder cancer (5637) compounds 5c and 5j showed IC50 cell inhibition values of 1.57 ± 0.70 µM and 0.48 ± 0.13 µM (48h) respectively. In the analysis of apoptosis, we observed condensation of chromatin, showing 13% of apoptotic cells in 5c and 20.3% in 5j, compared to untreated cells (2.34%). In gene expression, compound 5c increased the relative expression of genes p53, caspase-3 and caspase-9, SOD and CAT, and compound 5j increased genes p53, p21, caspase-3 and caspase-9, Bax, SOD, CAT, GPx, GR and iNOS. In molecular docking, 5c showed binding affinity to Survivin, Bcl-XL, RSK2, SGK1, and 5j showed binding affinity only to the SGK1 gene. In conclusion, for melanoma, the combination of BCG ΔleuD / Ag85B + imiquimod treatment showed inhibition of cell growth in WM1366, and increased cellular immune response in J774A.1. Likewise, for bladder cancer compounds 5c and 5j showed cytotoxic activity, and an increase in the expression of pro-apoptotic proteins and antioxidant enzymes, which could suggest in general that the treatments evaluated in this study could have promising antitumor effects for these types of cancers.