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Evaluation of new drug candidate for the treatment of non-small cell lung cancer

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Author(s):
Bárbara Kawamura
Total Authors: 1
Document type: Master's Dissertation
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
Defense date:
Examining board members:
Adilson Kleber Ferreira; Wallax Augusto Silva Ferreira
Advisor: Adilson Kleber Ferreira
Abstract

Lung cancer is one of the most incident and lethal malignant tumors in the world and has, in most cases, an unfavorable prognosis. Due to the relative failure of current therapeutic protocols, thus, new treatments are urgently required. The enzyme CTP: phosphoethanolamine cytidylstransferase (Pcyt-2), which uses phosphoethanolamine as substrate, is the key regulator of the Kennedy pathway for phosphatidylethanolamine (PE) production. This phospholipid is one of the most abundant in eukaryotic cells and, therefore, the reduction of its production could directly affect cell division, autophagy and apoptosis. In a previous study, it was confirmed that Pcyt-2 is a therapeutic target in lung cancer cells and two new compounds were identified, prototypes for the rational development of Pcyt-2 enzyme inhibitors: CHY-1 and SF2. The aim of this study was to evaluate the cytotoxic effects and inhibitory mechanisms on autophagy of new compounds in non-small cell lung cancer (NSCLC). The results indicated that CHY-1 has superior in vitro effect than paclitaxel and cisplatin, in addition, the sub-toxic dose combination of CHY-1 with cisplatin and CHY-1 with paclitaxel had superior cytotoxic effects than the isolated drugs. Besides, CHY-1 was able to induce endoplasmic reticulum (ER) stress and reduce autophagic flux. Possibly SF2, an intermediate of CHY-1, is structurally related to its pharmacophoric group. In the three-dimensional model with the A549 cells, it was observed that the combination of cisplatin with SF2 is more efficient when compared to individual treatments, destabilizing the spheroid due to loss of extracellular matrix. However the same results were also observed for MRC-5, a non-tumor fibroblast. SF2 has been shown to inhibit the enzyme Pcyt-2 and transport of ethanolamine to the ER, thereby reducing PE production. Using the Annexin / PI assay indicated that the combination of SF2 with cisplatin preferentially induces apoptosis, however, did not show synergistic effects. SF2 was able to decrease mitochondrial membrane potential of A549 and NCI-H460 cells, indicating mitochondrial participation in the cell death process. In addition, SF2 was able to decrease autophagic flux, observed by the reduction of proteins: ATG5, ATG7, Beclin-1 and LC3 I and II, by Western blotting, results similar to those previous shown by CHY-1 in A549 cells. By CRISPR / Cas9 technique (Clustered Regularly Interspaced Short Palindromic Repeats), A549 knockout cells were obtained for the genes involved in the autophagic process: ATG5, ATG7 and Beclin-1. The results showed that, although all these proteins are related to autophagy, the cells behaved differently for each silencing. The images obtained by MET corroborate the hypothesis that SF2 inhibits the autophagic pathway differently from chloroquine, besides inducing morphological alterations of mitochondria and RE. SF2 has the potential to become an antitumor drug by inhibiting the Pcyt-2 enzyme, leading to apoptosis and reduction of autophagic flux in NSCLC models. However, further studies and a possible modification of the molecule structure are still needed to enhance its effect and improve selectivity to tumor cells (AU)

FAPESP's process: 17/00497-3 - Development of new drug candidate for the treatment of non-small cell lung cancer: CHY-1 as a novel inhibitor of autophagy and prototype of a novel class of inhibitors of the enzyme CTP: phosphoethanolamine citidililtransferase
Grantee:Bárbara Kawamura
Support Opportunities: Scholarships in Brazil - Master