Study of pradimicin-IRD molecular interaction with DNA and its tumor cell mechanism of action.

Author(s)

Larissa Costa de Almeida

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2022-07-19

Examining board members

Leticia Veras Costa Lotufo; Flavio da Silva Emery; Luciana Biagini Lopes; Clarissa Ribeiro Reily Rocha

• Advisor: Leticia Veras Costa Lotufo; João Agostinho Machado Neto

Abstract

Pradimicins are cytotoxic substances produced by microorganisms. In a previous work by our group, we described a new pradimicin called pradimicin-IRD, with activity in tumor cells, and certain selectivity for colon cancer cells. Thus, the aim of this work was to investigate the mechanism of this natural substance in a panel of colon cells. The initial results have shown pradimicin-IRD as a DNA-damaging agent against colon cancer cells. Cellular and molecular analyzes indicate that pradimicin-IRD induces DNA damage (phosphorylation of H2AX protein and induction of p21 expression) independent of p53 expression, cell cycle arrest, and apoptosis (increased cleaved caspase-3 and PARP1 expression) in colorectal carcinoma cells. Despite a similar molecular structure to anthracyclines, including doxorubicin, anticancer activity of pradimicin-IRD differs from that of doxorubicin in terms of potency, p21 induction, cell cycle arrest, and TP53 independent response. Based on these results, we further investigated the molecular interaction of pradimicin-IRD with DNA and the DNA repair pathways related to the DNA damage response promoted by this molecule. Cellular and molecular assays, such as molecular docking, transcriptomic of DNA repair genes (PCR array), PCR, Western blot, MTT, and clonogenic assay were carried out. Our results show pradimicin-IRD as a DNA intercalating agent and a potential inhibitor of DNA-binding proteins. Transcriptomic analysis revealed DNA repair functions related to pradimicin-IRD modulated genes, such as nucleotide excision repair, telomere maintenance and double-strand break repair. Validation of genes related to these functions highlighted the PCNA gene for presenting DNA-pradimicin-PCNA (molecular docking) interaction and reduction of their protein levels in pradimicin-IRD treatments. In addition, hTERT and POLH showed reduced mRNA levels after 6 hours of pradimicin-IRD treatment. Moreover, POLH-deficient cells displayed higher resistance to pradimicin-IRD than POLH-proficient cells and the compound prevented formation of the POLH/DNA complex (molecular docking). Since the modulation of DNA repair genes by pradimicin-IRD is TP53- independent, unlike doxorubicin, dissimilarities between mechanism of action and DNA damage response of doxorubicin and pradimicin-IRD open new insights for further studies of pradimicin-IRD as a new antineoplastic agent. (AU)

• FAPESP's process: 18/17595-0 - Study of pradimicina-IRD molecular interaction with DNA and its tumor cell mechanism of action
• Grantee: Larissa Costa de Almeida
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)