Telomerase activity in response to RBBP6- Cannabidiol co-treatment in cervical cancer
Abstract
Cervical cancer ranks as the 4th most cited reason for mortalities amongst women across the globe. Accordingly, in 2018, 570 000 new individuals were diagnosed of cervical cancer. Early screening, detection and treatment can reduce cervical cancer incidences by 50%, however, developing countries lack resources, trained personnel and education in the general population limits the implementation of these routine testing programs in developing countries of sub-Saharan Africa. Cheaper and alternative therapeutic options to the otherwise expensive current interventions of chemotherapy, radiation and surgical resections are urgently required.
Retinoblastoma binding protein 6, which interacts with tumour suppressor p53, is highly abundant and expressed in many cancers such as cervical cancer. Taken together, the enhanced expression of RBBP6 and association with p53, has opened up interesting research possibilities exploring the RBBP6 biomolecule as both as therapeutic agent and biomarker for cancer. Gene therapy is associated with toxic effects. Hence, the present study proposes combination therapy which relies on the use of natural anticancer agents such as cannabidiol (CBD) found in a medicinal plant, along with gene-targeted therapeutics such as RBBP6 as a novel and cost-effective strategy for eradication of advanced stages of cancer. The exact molecular mechanism by which CBD mediates anticancer effects remains to be fully elucidated. There is a speculation that CBD induces apoptosis in cervical cancer, hence, current study was commenced to elucidate whether the possible apoptotic mechanisms involved in the chemotherapeutic properties of CBD in combination with targeted gene therapy maybe resulting in decreased telomerase activity.
Co-treatment of cervical cancer cells with a silenced of RBBP6 and CBD was used to assess telomerase activity. Once telomeres lose their function, they become rearranged and unstable. This results in genetic instability and leads to an increase in telomerase activity. It is then possible that telomerase activity can be explored as a molecular biomarker for early screening prevention of cervical cancer. To explore this possibility, ME-180 cervical cancer cells were maintained and subjected to treatment with the anticancer agent, CBD. The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) viability assay was conducted to evaluate CBD effects on the viability of ME-180 cells. Furthermore, apoptotic response of cervical cancer cells to siRBBP6 and co-
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treatment with CBD was evaluated by DNA fragmentation and microscopy. Gene expression levels of RBBP6, MDM2 and p53 relative to GAPDH were evaluated by quantitative real-time PCR (qPCR). The effect of siRBBP6 and co-treatment was assessed using telomerase repeated amplification protocol (TRAP) assay.
It was found that CBD (0.5μg/ml) significantly reduces the viability of ME-180 cells, possibly by eliciting the apoptosis pathway. This was supported by observation of DNA fragmentation indicating both smearing that indicates necrosis in the combinational treatment. Microscopy revealed morphological features of apoptosis in cells treated with CBD alone and in-combination with siRBBP6. RBBP6 was successfully silenced and the combination therapy resulted in a down-regulation of RBBP6 and MDM2 expression levels and in contrast, p53 expression was found to be up-regulated. This study showed that RBBP6 promotes p53 degradation and that the p53 most likely central in telomerase activity. The likelihood that p53-dependent apoptosis is not related to telomerase activity in cervical cancer is being suggested based on the results obtained in the study. Overall, future endeavours should explore the cost effective and promising combination therapy as a powerful tool for halting cervical cancer