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Anticancer activity and mechanisms of action of a selected diarylpentanoid treated on human papillomavirus (HPV) infected human cervical cancer cells.
thesisposted on 26.02.2017, 23:55 by Paulraj, Felicia Simone
Cervical cancer is the second most common cancer among Malaysian women and demonstrates high incidence and mortality rates. Current chemotherapy strategies are cytotoxic but are accompanied with severe side effects, thus necessitating research into alternative strategies for cervical cancer treatment. Diarylpentanoids (DAP) are curcumin analogues which demonstrate significant anti-tumorigenic activity in vitro and in vivo but its effects on cervical cancer have not been well studied. The present study aimed to investigate the potential anticancer activity and mechanisms of action of a selected DAP treated on HPV-infected HeLa and CaSki cervical cancer cells. Preliminary screening of the cytotoxic potential of 29 synthetic diarylpentanoids on cervical cancer cells by MTT assay identified MS17 (1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one) as a potential candidate for comprehensive downstream investigation as it demonstrated EC50 values of 2.6±0.9μM and 1.03±0.5μM on HeLa and CaSki cells respectively and displayed anti-proliferative activity by significantly inhibiting cell growth at 48 and 72 hours of treatment, with higher potency compared to curcumin and other analogues. Fluorescence microscopy of MS17-treated cells exhibited morphological changes consistent with apoptosis, corroborated by an increase in nucleosomal enrichment due to DNA fragmentation in HeLa and CaSki cells and activation of caspase-3 activity in CaSki cells. Quantitative real-time PCR detected significant down-regulation of HPV18- and HPV16-associated E6 and E7 oncogene expression following treatment. In order to understand the potential molecular mechanisms underlying the observed cytotoxic, anti-proliferative and apoptotic activity of MS17, microarray-based gene profiling was performed on HeLa and CaSki cells that had been treated with 2×EC50 and 3×EC50 doses of MS17 for 24 hours. Gene expression profiling revealed a dose-dependent regulation by MS17 on the differentially expressed genes (DEGs) and demonstrated unique gene profiles for mutually expressed and exclusively expressed DEGs. Furthermore the 1.5-fold increase in treatment dose correlated with a higher number of regulated DEGs and correspondingly higher fold change (FC). Grouping of top ranked genes for both cell lines according to gene ontology (GO) proposed that MS17 regulated similar biological functions to affect its cytotoxic, anti-proliferative and apoptotic activity, and exhibited dose-dependent differences in the proportion of genes belonging to each GO. Top ranking genes were associated with immune response, transcription regulator activity, metabolic process, signal transduction, unfolded protein response, cell cycle, chromatin organisation, cytoskeleton organisation, proteolysis, programmed cell death and transporter activity. Pathway analysis demonstrated that mutually regulated genes from HeLa and CaSki were associated with the regulation of immune response pathways while exclusively regulated DEGs from HeLa and CaSki were primarily associated with the regulation of metabolic and cell cycle-related pathways respectively. MicroRNA profiling by sequencing showed similar dose-dependent effects on the miRNA profile of the treated cells. Integration of miRNA and mRNA expression data suggested that MS17 modulated miRNA regulatory mechanisms over many target mRNAs from the dataset. These mRNA targets were associated with signalling pathways that are deregulated in cancers. Overall it may be suggested that MS17 has potential anticancer activity and merits further investigation for its use as a potential candidate in drug discovery research for the treatment of cervical cancer.