In vitro Characterization of Novel Compounds with Anti-cancer Activity

Edgar Alonso Borrego Puerta, University of Texas at El Paso


Two libraries of chemical compounds were analyzed for their cytotoxic activity against cancer and non-cancerous cell lines. In chapter 1, fourteen novel piperidones were tested on a panel of different cell lines to identify their cytotoxic activity. These compounds were found to have cytotoxic activity toward cancer cells with a cytotoxic concentration 50 (CC50) in the micromolar range. Furthermore, these fourteen compounds have selectivity preferentially towards cancer cells. Based on selectivity, and cytotoxicity, four compounds were selected for further characterization in the HL-60 cell line. The four compounds elicit apoptosis as measured by the externalization of the phospholipid phosphatidylserine (PS), the activation of caspase 3, and DNA fragmentation. In addition, the chemical compounds induce the overproduction of Reactive Oxygen Species and the depolarization of the mitochondrial membrane. Cell cycle analyses show that the 4 compounds mainly arrest HL-60 cells in the G0/G1 phase. Finally, Western Blots indicate that the treatment with the four compounds produces the accumulation of polyubiquitinated proteins suggesting that the protein degradation pathway was affected. Additionally, the four compounds overexpressed the HMOX-1 gene that encodes a protein that is overexpressed by proteasome inhibitors treatment. In Chapter 2, more than 5000 compounds from a commercial library were analyzed to find a potent chemical compound against cancer cells. A new pyrazole, referred to as B6, was discovered for having the highest cytotoxic activity at the low micromolar range. B6 was found to be cytotoxic in a panel of twenty-three different cell lines including leukemias and cell lines derived from different solid tumors. The MDA-MB-231 cell line was chosen due to its sensitivity to various anti-cancer compounds. B6 was found to induce apoptosis as measured by PS externalization, the activation of caspase 3, and DNA fragmentation. However, B6 did not overproduce reactive oxygen species (ROS) nor the depolarization of the mitochondria membrane, suggesting another apoptotic pathway. To get an insight into the mechanism of action we performed transcriptomic analyses. After 6 h of treatment, we observed the up-regulation of 116 genes and the down-regulation of 87 genes; while after 24 hours of treatment, we observed 198 up-regulated and 532 down-regulated. A comparison to Connectivity Map (CMap) revealed that cells treated with the B6 compound displayed a gene expression pattern similar to the gene signatures expressed by tubulin inhibitors. Moreover, molecular docking analyses revealed that B6 interacts with tubulin in an in silico model. Thus, we hypothesized that B6 could be a tubulin inhibitor. Tubulin inhibitors disturb the organization of microtubules; therefore, we captured images by using fluorescent microscopy. The images showed that B6 disrupts microtubule organization. In addition, the B6 compound arrests the cell cycle in the G2/M phase. Finally, a microtubule polymerization assay revealed that B6 inhibits tubulin polymerization. The findings presented in this dissertation show promising results for these compounds as they can potentially be used as they can feasibly be used in cancer therapy.

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Recommended Citation

Borrego Puerta, Edgar Alonso, "In vitro Characterization of Novel Compounds with Anti-cancer Activity" (2022). ETD Collection for University of Texas, El Paso. AAI30000486.