Applications of CRISPR Cas-9 in Ovarian Cancer Research.

Ovarian cancer is a highly prevalent malignancy among women and affects a significant population worldwide. Different forms of hormonal treatments or chemotherapies are used to treat ovarian cancer, but the possible side effects, including menopausal symptoms, can be severe, forcing some patients to prematurely stop the treatment. The emerging genome editing technology, known as clustered regularly interspaced short palindromic repeats (CRISPR)-caspase 9 (Cas9), has the potential to treat ovarian cancer via gene editing strategies. Studies have reported CRISPR knockouts of several oncogenes that are involved in the pathogenesis of ovarian cancer, such as BMI1, CXCR2, MTF1, miR-21, and BIRC5, and demonstrate the potential of the CRISPR-Cas9 genome editing technique to effectively treat ovarian cancer. However, there are limitations that restrict the biomedical applications of CRISPR-Cas9 and limit the implementation of Gene therapy for ovarian cancer. These include offtarget DNA cleavage and the effects of CRISPR-Cas9 in non-target, normal cells. This article aims to review the current state of ovarian cancer research, highlight the significance of CRISPR-Cas9 in ovarian cancer treatment, and establish the groundwork for potential clinical research.

Microbiota in cancer development and treatment.

PURPOSE: Human microbiota comprises of a variety of organisms ranging from bacterial species to viruses, fungi, and protozoa which are present on the epidermal and mucosal barriers of the body. It plays a key role in health and survival of the host by regulation of the systemic functions. Its apparent functions in modulation of the host immune system, inducing carcinogenesis and regulation of the response to the cancer therapy through a variety of mechanisms such as bacterial dysbiosis, production of genotoxins, pathobionts, and disruption of the host metabolism are increasingly becoming evident. METHODS: Different electronic databases such as PubMed, Google Scholar, and Web of Science were searched for relevant literature which has been reviewed in this article. RESULTS: Characterization of the microbiome particularly gut microbiota, understanding of the host-microbiota interactions, and its potential for therapeutic exploitation are necessary for the development of novel anticancer therapeutic strategies with better efficacy and lowered off-target side effects. CONCLUSION: In this review, the role of microbiota is explained in carcinogenesis, mechanisms of microbiota-mediated carcinogenesis, and role of gut microbiota in modulation of cancer therapy.

ROS-modulated therapeutic approaches in cancer treatment.

PURPOSE: Reactive oxygen species (ROS) are produced in cancer cells as a result of increased metabolic rate, dysfunction of mitochondria, elevated cell signaling, expression of oncogenes and increased peroxisome activities. Certain level of ROS is required by cancer cells, above or below which lead to cytotoxicity in cancer cells. This biochemical aspect can be exploited to develop novel therapeutic agents to preferentially and selectively target cancer cells. METHODS: We searched various electronic databases including PubMed, Web of Science, and Google Scholar for peer-reviewed english-language articles. Selected articles ranging from research papers, clinical studies, and review articles on the ROS production in living systems, its role in cancer development and cancer treatment, and the role of microbiota in ROS-dependent cancer therapy were analyzed. RESULTS: This review highlights oxidative stress in tumors, underlying mechanisms of different relationships of ROS and cancer cells, different ROS-mediated therapeutic strategies and the emerging role of microbiota in cancer therapy. CONCLUSION: Cancer cells exhibit increased ROS stress and disturbed redox homeostasis which lead to ROS adaptations. ROS-dependent anticancer therapies including ROS scavenging anticancer therapy and ROS boosting anticancer therapy have shown promising results in vitro as well as in vivo. In addition, response to cancer therapy is modulated by the human microbiota which plays a critical role in systemic body functions.

TRPC1 protects dopaminergic SH-SY5Y cells from MPP+, salsolinol, and N-methyl-(R)-salsolinol-induced cytotoxicity.

Neurotoxins and alterations in Ca2+ homeostasis have been associated with Parkinson's disease (PD), but the role of store-operated Ca2+ entry channels is not well understood. Previous studies have shown the neurotoxicity of salsolinol and 1-methyl-4-phenylpyridinium ion on SH-SY5Y cells and cytoprotection induced by transient receptor potential protein 1 (TRPC1). In the present study, N-methyl-(R)-salsolinol was tested for its cellular toxicity and effects on TRPC1 expression. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, DAPI (4',6-diamidino-2-phenylindole), fluorescein isothiocyanate-Annexin-V/propidium iodide, western blot analysis, and JC-1 labeling revealed that the three indicated drugs could induce caspase-dependent, mitochondrial-mediated apoptosis. Exposure of SH-SY5Y cells to the indicated drugs resulted in a significant decrease in thapsigargin-mediated Ca2+ influx and TRPC1 expression. Immunocytochemistry experiments revealed that neurotoxins treatment induced TRPC1 translocation to the cytoplasm. Taken together, our results indicate that treatment with neurotoxins may alter Ca2+ homeostasis and induce mitochondrial-mediated caspase-dependent cytotoxicity, an important characteristic of PD.

RNF13 protein regulates endoplasmic reticulum stress induced apoptosis in dopaminergic SH-SY5Y cells by enhancing IRE1α stability.

Any interruption in the folding capacity of endoplasmic reticulum (ER) may result in inducing ER stress which would initiate an adaptive signaling mechanism called Unfolded Protein Response (UPR) in order to restore homeostasis, failing to which would initiate signaling pathway leading to death of the cell. Finding new mediators could help better understand the molecular mechanisms of ER stress-induced apoptosis. Our lab initiated a genetic screen method using retroviral insertion mutation system to look for genes whose inactivation would confer resistance to apoptosis. In our previous findings, Ring finger protein 13 (RNF13) was identified whose down-regulation conferred survival against ER stress-induced apoptosis. Our previous results also showed important role of RNF13 in apoptotic signaling in 293T cells as a result of strong RNF13-IRE1α interaction. In current study, using SH-SY5Y cells, overexpression of RNF13 in apoptosis assays and RT-PCR analysis has shown to induce apoptosis as well as splicing of X-box binding Protein 1 (XBP1) confirming its role in ER stress mediated cell death in this cell line as well. Western blot analysis has revealed that overexpression of both N-terminal as well as C-terminal tagged RNF13 resulted in activation of c-Jun N-terminal kinase (JNK) in SH-SY5Y cells. Our co-immunoprecipitation assays in SH-SY5Y cells also showed a strong interaction of RNF13 with IRE1α. Finally, Cycloheximide chase experiment exhibited that RNF13-IRE1α interaction increased the stability of IRE1α. Altogether, our data suggest that RNF13 may act as an important regulator of IRE1α, mediating ER stress-mediated apoptosis in neuroblastoma SH-SY5Y cells.

α-Synuclein increases U251 cells vulnerability to hydrogen peroxide by disrupting calcium homeostasis.

Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by glial cytoplasmic inclusions containing insoluble α-synuclein. Since Ca(2+) plays an important role in cell degeneration, [Ca(2+)]( i ) in α-synuclein-overexpressed human glioma cells was analyzed by Fura-2 fluorometry. Overexpression of α-synuclein increased the basal level of [Ca(2+)]( i ), and a higher Ca(2+) response to hydrogen peroxide was further observed. The effect that α-synuclein overexpression caused U251 cells to be more vulnerable to hydrogen peroxide was eliminated by Ca(2+) chelator BAPTA-AM or transient receptor potential channels blocker SKF 96365 but not by L-type Ca(2+) channel blocker nimodipine. These findings suggest that the dysregulation of cellular Ca(2+) homeostasis caused by α-synuclein under oxidative stress may contribute to the glial cell death in MSA.