Tartrolon D/E induz alterações típicas de morte celular imunogênica em células de melanoma metastático

Abstract

Metastatic melanoma is the most serious type of skin cancer due to its high mortality rate and the lack of effective treatment in most cases. Immunogenic cell death (ICD) is an interesting strategy when developing chemotherapeutic treatments for metastatic melanoma because of its capability in potentializing the necessary cytotoxicity for a cancer treatment by activating the adaptive immune system against neoplastic cells. This effect is caused by damage-associated molecular patterns molecules (DAMPs), released during ICD, such as ATP, HMGB1, CRT, among others. Tartrolons are promising bioactive compounds with diverse biological properties, such as antiproliferative, antimicrobial and antiprotozoan. The current study aimed to evaluate the markers ICD induction by tartrolon D/E (TRL) on murine metastatic melanoma (B16-F10 cell lineage). In order to evaluate the antiproliferative effect and the survival cell rate of the B16-F10 cells exposed to TRL, sulforhodamine B (SRB) test and clonogenic assays were performed. Moreover, the cell death mechanisms and DAMPs detection were observed by flow cytometry and luminescence detection. The antiproliferative effect of TRL was tested in concentrations ranging from 0.46 to 60 µM after 24, 48 and 72 h by SRB assay. A cytostatic effect demonstrated by the average inhibitory concentration (IC50) of 7.7 μM, 1.3 μM and 0.4 μM at 24, 48 and 72 h respectively. The cytotoxic effect, determined by the average lethal concentration LC50, was of 24.5 μM after 48 h and 20.4 μM after 72 h incubation. The survival evaluation was performed by clonogenic assay at two concentrations of TRL (12 μM and 25 μM) after exposure of 24 and 48 h. We also noticed proliferation inhibition in a time and concentration dependent fashion with complete absence of colonies in the group at 25 μM exposed by 48 h. The flow cytometry assays were carried out after 24 hours. TRL at 12 μM induced morphological changes, increasing of high granularity and cell shrinkage as well as plasmatic membrane rupture and DNA fragmentation. Additionally Hsp70 and CRT externalization were detected along with and HMGB1 releasing. Preliminary data depicted an increase in the extracellular ATP levels measured in the supernatant. In summary TRL induced cell death and immunogenic signals related to ICD. Additional studies are necessary to confirm if TRL is a bonafide ICD inducer. Nevertheless, the results obtained already suggest a promising profile as an antitumor agent against metastatic melanoma.