Dados do Resumo

Título

In Vitro Study of the Effects of Chromomycin A2 on Liver Cancer Cells

Introdução

Liver cancer is diagnosed every year in more than half a million people, with a high mortality and recurrence rate. The main chemotherapeutic agents have side effects and are associated with resistance. Since there is no effective treatment for this disease, a deeper understanding of the mechanisms associated with death and survival after conventional and other untested chemotherapy treatments in liver cancer is crucial for new perspectives and effective therapy. Chromomycins have been highlighted for presenting potent cytotoxic effects against different tumor cell lines, displaying a promising activity profile for application in new therapies.

Objetivo

The objective was to use human liver tumor cell lines to analyze cellular and molecular mechanisms associated with modulation of cell survival and death, through treatment with two DNA damage-inducing agents, one traditional - doxorubicin, and another new one considered promising - chromomycin A2.

Métodos

We evaluated the cellular vitality and cytotoxic potential of DOX and CA2 in two human liver cancer cell lines, HepG2 and Hep3B, and determined the mean inhibitory concentrations (IC50) after 48h of treatment using the MTT method. We also evaluated the reproductive viability of individual cells to proliferate and form colonies after 24 and 48h of treatment using the clonogenic assay. The observation and analysis of cell morphology after 48h of treatment with the compounds, by phase contrast microscopy and fluorescence microscopy, after DNA labeling with DAPI and microtubule labeling with anti-alpha and anti-beta tubulin antibodies. Analysis of cell cycle progression by flow cytometry by DNA quantification was performed after 24, 48, and 72h of treatment with DOX and CA2. Western blotting experiments after treatment with DOX and CA2 for 48h were performed to identify activation of DNA repair and apoptotic signaling, using anti-histone H2AX and anti-cleaved caspase 3 antibodies.

Resultados

The data revealed that both compounds have cytotoxic activity against the cell lines, highlighting that CA2 acted effectively at concentrations in the nanomolar range, while DOX acted at micromolar concentrations. Both compounds altered the characteristic morphological pattern, with a population of cells less adherent to the substrate, and another of rounded cells in suspension, with condensed nuclei indicating possible cell death by apoptosis. The adhered cells presented morphological alterations with cytoplasm extensions and constriction of microtubules. Cell cycle data showed a significant increase in the sub-G1 population in the treated cultures. Sub-G1 is composed of hypodiploid cells, and dead cells that present fragmented DNA, indicating that DOX and CA2 exert cytotoxic activity on these cells. Western blotting experiments showed phosphorylation of histone H2AX (γH2AX), indicating possible activation of DNA repair, and cleavage of Caspase-3, indicating apoptosis signaling after DOX and CA2 treatment.

Conclusões

These initial data focus on the acquisition of new knowledge in the effects of CA2 on liver tumor cells, and open perspectives for new approaches to be taken to identify potential targets to improve the sensitivity of liver cancer to chemotherapeutic agents.

Financiador do resumo

FAPESP, CAPES, CNPq

Palavras Chave

Liver cancer; doxorubicin; chromomycin A2