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Adilson Kleber Ferreira



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    EP1.14 - Targeted Therapy (ID 204)

    • Event: WCLC 2019
    • Type: E-Poster Viewing in the Exhibit Hall
    • Track: Targeted Therapy
    • Presentations: 1
    • Now Available
    • Moderators:
    • Coordinates: 9/08/2019, 08:00 - 18:00, Exhibit Hall
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      EP1.14-04 - Validation of New Targets Interfering at Phosphatidylethanolamine Production Against NSCLC (Now Available) (ID 31)

      08:00 - 18:00  |  Author(s): Adilson Kleber Ferreira

      • Abstract
      • Slides

      Background

      Besides all medicine advancements, lung cancer is still the leading cancer-related cause of death worldwide. In this scenario, our group validate new targets to drug development focusing in interfering at phosphatidylethanolamine (PE) production as a breakthrough strategy, once PE has been previously described as increased in Non-Small Cell Lung Cancer (NSCLC) tumors. Two critical points in PE production should be highlighted: ethanolamine cellular uptake and the enzyme responsible for the limiting step of the main route of PE synthesis, CTP:phosphoethanolamine cytidylyltransferase (Pcyt2). Therefore, this study aimed to evaluate in vitro modulation of Pcyt2 enzyme and ethanolamine cellular uptake in A549 cells to validate the therapeutic potential of these targets for NSCLC treatment.

      Method

      Pcyt2 was silenced in A549 cell line by CRISPR-Cas9-Mediated technology using 4D-Nucleofector™ System, followed by sorting using FACSAria II and confirmation of Pcyt2 protein depletion by Western blotting. Cellular sensitivity to meclizine (a Pcyt2 inhibitor) and isopropanolamine (an ethanolamine uptake inhibitor) was assessed by MTT assay. The proliferation capacity was evaluated by plotting cell growth curves and Colony Forming Units (CFU) assay. Cells were fixed in ethanol overnight in freezer, stained with 7-Aminoactinomycin D and analyzed by flow cytometry using BD LSRFortessa. All statistical data are expressed as mean ± standard deviation (SD) and were analyzed by one-way ANOVA followed by the Tukey's test using GraphPad Prism software, version 7.

      Result

      Initially, A549 cells Pcyt2 knockout (KO) cells were established and confirmation in three different passages by Western Blotting. KO cells were resistant to meclizine (p<0.001) and more sensitive to isopropanolamine (p<0.05) than wild-type (WT) cells. Only WT cells proliferate more when cultured in media with ethanolamine supplementation (p<0.01), which is related to the S-phase arrest (p<0.05). KO cells showed proliferation changes which were not affected by meclizine treatment both in clonogenic and cell cycle assays. This data is closely related to the reduction of pRb expression and the increase of p21 expression in KO cells. On the other hand, WT cells were arrested in G0/G1 phases and their proliferation capacity was reduced (34% less colonies, p<0.01) after meclizine treatment.

      Conclusion

      Pcyt2 modulation in A549 cells both genetic and pharmacological showed the decrease of cancer cells proliferation and viability. On the other hand, supplementation of ethanolamine increase cellular proliferation. Altogether, these preliminary results confirm the importance of PE for cell metabolism and division which is an exciting starting point to deeply evaluate Pcyt2 potential as a target for NSCLC treatment.

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