Chromomycin A5 induces bona fide immunogenic cell death in melanoma.

Purpose: Some first-line cytotoxic chemotherapics, e.g. doxorubicin, paclitaxel and oxaliplatin, induce activation of the immune system through immunogenic cell death (ICD). Tumor cells undergoing ICD function as a vaccine, releasing damage-associated molecular patterns (DAMPs), which act as adjuvants, and neoantigens of the tumor are recognized as antigens. ICD induction is rare, however it yields better and long-lasting antitumor responses to chemotherapy. Advanced metastatic melanoma (AMM) is incurable for more than half of patients. The discovery of ICD inducers against AMM is an interesting drug discovery strategy with high translational potential. Here we evaluated ICD induction of four highly cytotoxic chromomycins A (CA5-8). Methods: ICD features and DAMPs were evaluated using several in vitro techniques with metastatic melanoma cell line (B16-F10) exposed to chromomcins A5-8 such as flow cytometry, western blot, RT-PCR and luminescence. Additionally in vivo vaccination assays with CA5-treated cells in a syngeneic murine model (C57Bl/6) were performed to confirm ICD evaluating the immune cells activation and their antitumor activity. Results: B16-F10 treated with CA5-8 and doxorubicin exhibited ICD features such as autophagy and apoptosis, externalization of calreticulin, and releasing of HMGB1. However, CA5-treated cells had the best profile, also inducing ATP release, ERp57 externalization, phosphorylation of eIF2α and altering expression of transcription of genes related to autophagy, endoplasmic reticulum stress, and apoptosis. Bona fide ICD induction by CA5 was confirmed by vaccination of C57BL/6 mice with CA5-treated cells which activated antigen-presenting cells and T lymphocytes and stimulated antitumor activity. Conclusion: CA5 induces bona fide immunogenic cell death on melanoma.

Targeting glioma cells by antineoplastic activity of reversine.

Gliomas are the most common type of primary central nervous system tumors and despite great advances in understanding the molecular basis of the disease very few new therapies have been developed. Reversine, a synthetic purine analog, is a multikinase inhibitor that targets aurora kinase A (AURKA) and aurora kinase B (AURKB). In gliomas, a high expression of AURKA or AURKB is associated with a malignant phenotype and a poor prognosis. The present study investigated reversine-related cellular and molecular antiglioma effects in HOG, T98G and U251MG cell lines. Gene and protein expression were assessed by reverse transcription-quantitative PCR and western blotting, respectively. For functional assays, human glioma cell lines (HOG, T98G and U251MG) were exposed to increasing concentrations of reversine (0.4-50 µM) and subjected to various cellular and molecular assays. Reversine reduced the viability and clonogenicity in a dose- and/or time-dependent manner in all glioma cells, with HOG (high AURKB-expression) and T98G (high AURKA-expression) cells being more sensitive compared with U251MG cells (low AURKA- and AURKB-expression). Notably, HOG cells presented higher levels of polyploidy, while T98G presented multiple mitotic spindles, which is consistent with the main regulatory functions of AURKB and AURKA, respectively. In molecular assays, reversine reduced AURKA and/or AURKB expression/activity and increased DNA damage and apoptosis markers, but autophagy-related proteins were not modulated. In conclusion, reversine potently induced mitotic catastrophe and apoptosis in glioma cells and higher basal levels of aurora kinases and genes responsive to DNA damage and may predict improved antiglioma responses to the drug. Reversine may be a potential novel drug in the antineoplastic arsenal against gliomas.