Background: Oxadiazoles, triazoles, and their respective precursors have been shown to exhibit various
pharmacological properties, namely antitumour activities. Cytotoxic activity was reported for these compounds in
various cancer cell lines.
Aim and Objectives: In this study, we aim at investigating the mechanism of apoptosis by N-(4-chlorophenyl)-2-(4-
(3,4,5-trimethoxybenzyloxy)benzoyl)-hydrazinecarbothioamide, a triazole precursor, henceforth termed compound
P7a, in breast cancer cell line, MCF-7. We first screen a series of analogues containing (3,4,5-trimethoxybenzyloxy)
phenyl moiety in breast cancer cell lines (MCF-7 and MDA-MB-231) to select the most cytotoxic compound and
demonstrate a dose- and time-dependent cytotoxicity. Then, we unravel the mechanism of apoptosis of P7a in MCF-7 as
well as its ability to cause cell cycle arrest.
Methods: Synthesis was performed as previously described by Kareem and co-workers. Cytotoxicity of analogues
containing (3,4,5-trimethoxybenzyloxy)phenyl moiety against MCF-7 and MDA-MB-231 cell lines was evaluated
using the MTS assay. Flow cytometric analyses was done using Annexin V/PI staining, JC-1 staining and ROS
assay. The activity of caspases using a chemoluminescence assay and western blot analysis was conducted to study
the apoptotic pathway induced by the compound in MCF-7 cells. Lastly, cell cycle analysis was conducted using
Results: Upon 48 hours of treatment, compound P7a inhibited the proliferation of human breast cancer cells with
IC50 values of 178.92 ± 12.51μM and 33.75 ± 1.20μM for MDA-MB-231 and MCF-7, respectively. Additionally,
compound P7a showed selectivity towards the cancer cell line, MCF-7 compared to the normal breast cell line,
hTERT-HME1, an advantage against current anticancer drugs (tamoxifen and vinblastine). Flow cytometric analyses
using different assays indicated that compound P7a significantly increased the proportion of apoptotic cells,
increased mitochondria membrane permeabilisation and caused generation of ROS in MCF-7. In addition, cell cycle
analysis showed that cell proliferation was arrested at the G1 phase in the MCF-7 cell line. Furthermore, upon
treatment, the MCF-7 cell line showed increased activity of caspase-3/7, and caspase-9. Lastly, the western blot
analysis showed the up-regulation of pro-apoptotic proteins along with up-regulation of caspase-7 and caspase-9,
indicating that an intrinsic pathway of apoptosis was induced.
Conclusion: The results suggest that compound P7a could be a potential chemotherapeutic agent for breast cancer.