Ruchika Kaul-Ghanekar

Alpha-linolenic acid-mediated epigenetic reprogramming of cervical cancer cell lines

Cervical cancer, the fourth most common cancer globally and the second most prevalent cancer among women in India, is primarily caused by Human Papilloma Virus (HPV). The association of diet with cancer etiology and prevention has been well established and nutrition has been shown to regulate cancer through modulation of epigenetic markers. Dietary fatty acids, especially omega-3, reduce the risk of cancer by preventing or reversing the progression through a variety of cellular targets, including epigenetic regulation. In this work, we have evaluated the potential of ALA (α linolenic acid), an ω-3 fatty acid, to regulate cervical cancer through epigenetic mechanisms. The effect of ALA was evaluated on the regulation of histone deacetylases1, DNA methyltransferases 1, and 3b, and global DNA methylation by ELISA. RT-PCR was utilized to assess the expression of tumor regulatory genes (hTERT, DAPK, RARβ, and CDH1) and their promoter methylation in HeLa (HPV18-positive), SiHa (HPV16-positive) and C33a (HPV-negative) cervical cancer cell lines. ALA increased DNA demethylase, HMTs, and HATs while decreasing global DNA methylation, DNMT, HDMs, and HDACs mRNA expression/activity in all cervical cancer cell lines. ALA downregulated hTERT oncogene while upregulating the mRNA expression of TSGs (Tumor Suppressor Genes) CDH1, RARβ, and DAPK in all the cell lines. ALA reduced methylation in the 5' CpG island of CDH1, RARβ, and DAPK1 promoters and reduced global DNA methylation in cervical cancer cell lines. These results suggest that ALA regulates the growth of cervical cancer cells by targeting epigenetic markers, shedding light on its potential therapeutic role in cervical cancer management.

Exploring the diversity of vaginal microbiota between healthy women and cervical cancer patients in India

Introduction. Cervicovaginal diversity has been reported as a predictive biomarker for cervical cancer risk. We recently reported the bio-therapeutic potential of vaginal probiotics from healthy Indian women against vaginal pathogens, isolated from the invasive cervical cancer (ICC) patients. Gap Statement. The cervicovaginal microflora from cervical cancer patients has not yet been reported from Indian population. Aim. The present study aimed at comparing the cervicovaginal microbiome between healthy controls (HC) and ICC patients from the Indian population. Methodology. In total, 30 vaginal swabs (15 from HC and 15 from ICC) were subjected to 16S rRNA gene sequencing. Alpha diversity was evaluated by Shannon and Chao1 index; and beta diversity by principle coordinate analysis (PCoA) of weighted and unweighted UniFrac distances. The relative abundance of the microbial taxa was done according to linear discriminant analysis effect size (LEfSe). Results. Predominance of Staphylococcus spp. in ICC and Lactobacillus gasseri in HC groups was observed. Alpha-diversity was found to be higher in ICC as compared to HC but was statistically non-significant. LEfSe analysis revealed Bacteroides fragilis and Escherichia coli as the marker genera in ICC with a marked decrease in Lactobacillus sp. Contrarily, in HC, L. gasseri, L. iners and L. fermentum were found to be abundant. Conclusion. Differences in the vaginal microbiome between healthy and ICC women could help in the early prediction of cervical cancer risk and thus in designing prevention strategies.

Panchvalkala, a traditional Ayurvedic formulation, exhibits antineoplastic and immunomodulatory activity in cervical cancer cells and C57BL/6 mouse papilloma model

Panchvalkala, an Ayurvedic traditional formulation has references in Charak Samhita and Bhavaprakasha Nighantu for the treatment of women with endometriosis-related problems, leucorrhea and vaginal ailments. The formulation comprises of equal ratios of the barks from Ficus glomerata, Ficus virens, Ficus religiosa, Ficus benghalensis, and Thespesia populnea. The present study aimed to evaluate the anticancer and immunomodulatory activity of aqueous extract of Panchvalkala (PVaq) against cervical cancer in vitro and in vivo. The effect of PVaq on disruption of mitochondrial membrane potential in cervical cancer cell lines, SiHa and HeLa, was studied by using JC1 dye. The expression of generic caspases in the cells after treatment with PVaq was evaluated by ELISA kit. The expression of pRb, p53, E6 and E7 proteins were evaluated by western blotting. Acute oral toxicity and DRF studies were performed in Swiss albino mice by following OECD guidelines 423 and 407, respectively. Tumor retardation study was done in C57BL/6 mouse papilloma model. The mice were divided into six groups: No tumor control (NTC), Tumor control (TC), Cisplatin (Cis) (4 mg/kg b.w.), PVaq 100, 200 mg/kg b.w and combination of PVaq (200 mg/kg b.w.) and Cisplatin (4 mg/kg b.w.). The mice were orally gavaged with PVaq daily for 14 days and cisplatin was given intravenously on every 1st, 5th and 9th day. Hematological and biochemical parameters were studied by using hematology analyzer and kits, respectively. E6 and E7 gene expression in the tumor samples was determined by qPCR. Th1 and Th2 cytokine levels were determined by ELISA. PVaq induced mitochondrial depolarization in SiHa and HeLa, and increased the expression of generic caspases, resulting into apoptosis. PVaq upregulated the expression of tumor suppressor proteins (p53 and pRb) and reduced the expression of viral oncoproteins (E6 and E7). Acute toxicity study displayed non-toxicity of PVaq while DRF study ensured its safe dose for further efficacy studies. PVaq reduced tumor volume and weight in mouse papilloma model and induced immunomodulation in the animals. It increased serum levels of IL-2 (Th1) with a concomitant decrease in IL-10 (Th2) cytokines. The drug did not affect body weight, food consumption and organ histopathology of the animals. PVaq exhibited anticancer and immunomodulatory activities against cervical cancer cells and female mouse papilloma model.

Cervicovaginal microbiota isolated from healthy women exhibit probiotic properties and antimicrobial activity against pathogens isolated from cervical cancer patients

Abnormal cervicovaginal microbiota play an important role in HPV persistence and progression to cervical cancer. The present study aimed at isolating and identifying potential probiotics from vaginal swabs of healthy women and evaluating their activity against vaginal pathogens isolated from cervical cancer patients. Based on probiotic, acid-bile tolerance and antimicrobial properties, 13 lactic acid bacteria (LAB) from the healthy group were identified by MALDI TOF MS (Matrix Assisted Laser Desorption and Ionisation, Time Of Flight Mass Spectrometry). Among these, four strains, Lactobacillus gasseri P36Mops, Limosilactobacillus fermentum P37Mws, Lactobacillus delbrueckii P31Mcs and Enterococcus faecium P26Mcm, exhibited significant antimicrobial activity against 8 vaginal pathogens (Staphylococcus haemolyticus P41Tcs, Escherichia coli P30Tcs, E. coli P79Bcm, Enterococus faecalis P29Mops, E. faecalis P50Tws, E. faecalis P68Tcb, S. haemolyticus P48Bcb and S. haemolyticus P58Bcb) isolated from precancerous and cervical cancer patients. 16S rRNA sequencing of four potential probiotics revealed congruency with the MALDI-TOF MS identification and phylogenetic analysis showed genetic relationship with previously reported LAB strains. The selected LAB showed strain specific hydrophobicity (35.88-56.70%), auto-aggregation (35.26-61.39%) and antibiotic susceptibility. Interestingly, L. gasseri P36Mops was resistant to five standard antibiotics routinely used against urogenital or vaginal infections. LCMS (Liquid Chromatography Mass Spectrometry) analyses of the CFS (cell-free supernatant) of the four potential probiotics revealed the presence of metabolites such as N-(1-deoxy-1-fructosyl)valine, hygroline, acetoxy-2-hydroxy-16-heptadecen-4-one, avocadyne 4-acetate, avocadyne 2-acetate, taraxinic acid glucosyl ester, 6-hydroxypentadecanedioic acid, with reported antimicrobial activity. The overall data suggest the bio-therapeutic potential of the identified vaginal probiotics against cervical cancer-associated pathogens.