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 flow cytometry.

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.

METHODS: A cross-sectional study involving patients with coronavirus disease 2019 self-perceived as asymptomatic was conducted. Assessments included the subjective Malaysian Smell and Taste Questionnaire and the culturally adapted Malaysian version of the objective Sniffin' Sticks Identification smell test.

RESULTS: In 81 participants (mean age of 31.59 ± 12.04 years), with mean time from diagnosis to smell test of 7.47 ± 3.79 days, subjective assessment showed that 80.2 per cent were asymptomatic (questionnaire score of 6) and 19 per cent had mild symptoms (questionnaire score of 7 and 8). The mean objective smell test score was 10.89 ± 2.11. The prevalence of olfactory impairment was 76.6 per cent among patients with coronavirus disease 2019 self-perceived as asymptomatic. There was no association between the questionnaire and the smell test scores (p = 0.25). There was a correlation between the smell test score and the duration from diagnosis to smell test (p = 0.04).