ßhCG mediates immune suppression through upregulation of CD11b+ Gr1+ myeloid derived suppressor cells, CD206+ M2 macrophages, and CD4+ FOXP3+ regulatory T-cells in BRCA1 deficient breast cancers.

BRCA1 mutation is reported in about 70% of all triple negative breast cancers (TNBC), while BRCA1 defect due to promoter hypermethylation is seen in about 30%-60% of sporadic breast cancers. Although PARP inhibitors and platinum-based chemotherapy are used to treat these cancers, more efficient therapeutic approaches are required to overcome the resistance to treatment. Our previous findings have reported elevated ßhCG expression but not αhCG in BRCA1 deficient breast cancers. As ßhCG causes immune suppression in pregnancy, this study explored the immunomodulatory effect of ßhCG in BRCA1mutated/deficient TNBC. We observed that Th1, Th2, and Th17 cytokines are upregulated in the presence of ßhCG in BRCA1 defective cancers. In NOD-SCID and syngeneic mouse models, ßhCG increases the frequency of Myeloid-derived suppressor cells in tumour tissues and contributes to macrophage reprogramming from antitumor M1 to pro-tumour M2 phenotype. ßhCG reduces the CD4+ T-cell infiltration while increasing the density of CD4+ CD25+ FOXP3+ regulatory T-cell in BRCA1 deficient tumour tissues. In contrast, xenograft tumours with ßhCG knocked down TNBC cells did not show these immune suppressive effects. We have also shown that ßhCG upregulates pro-tumorigenic markers arginase1(Arg1), inducible nitric oxide synthase, PD-L1/PD-1, and NFκB in BRCA1 defective tumours. Thus, for the first time, this study proves that ßhCG suppresses the host antitumor immune response and contributes to tumour progression in BRCA1 deficient tumours. This study will help develop new immunotherapeutic approaches for treating BRCA1 defective TNBC by regulating ßhCG.

BRCA1 promoter hypermethylation in human placenta: a hidden link with ß-hCG expression.

Gestational trophoblastic diseases (GTD) are group of pregnancy-related tumors characterized by abnormal levels of 'ß-hCG' with higher incidence in South-East Asia, especially India. Our laboratory has reported that wild-type BRCA1 transcriptionally regulates ß-hCG in triple negative breast cancers (TNBCs). These factors culminated into analysis of BRCA1 status in GTD, which would emanate into elucidation of BRCA1- ß-hCG relationship and unraveling etio-pathology of GTD. BRCA1 level in GTD is down-regulated due to the over-expression of DNMT3b and subsequent promoter hypermethylation, when compared to the normal placentae accompanied with its shift in localization. There is an inverse correlation of serum ß-hCG levels with BRCA1 mRNA expression. The effects of methotrexate (MTX), which is the first-line chemotherapeutic used for GTD treatment, when analyzed in comparison with plumbagin (PB) revealed that PB alone is efficient than MTX alone or MTX-PB in combination, in showing selective cytotoxicity against GTD. Interestingly, PB increases BRCA1 levels post-treatment, altering DNMT3b levels and resultant BRCA1 promoter methylation. Also, cohort study analyzed the incidence of GTD at Sree Avittom Thirunal (SAT) Hospital, Thiruvananthapuram, which points out that 11.5% of gestational trophoblastic neoplasia (GTN) cases were referred to Regional Cancer Centre, Thiruvananthapuram, for examination of breast lumps. This has lend clues to supervene the risk of GTD patients towards BRCA1-associated diseases and unveil novel therapeutic for GTD, a plant-derived naphthoquinone, PB, already reported as selectively cytotoxic against BRCA1 defective tumors.

ß-hCG-induced mutant BRCA1 ignites drug resistance in susceptible breast tissue.

ß-hCG expression in breast cancer is highly controversial with reports supporting both protective and tumorigenic effects. It has also been reported that risk of breast cancer at an early age is increased with full-term pregnancies if a woman is a BRCA1 mutation carrier. We have already demonstrated that BRCA1-defective cells express high levels of ß-hCG and that when BRCA1 is restored, ß-hCG level is reduced. Also, BRCA1 can bind to the promoter and reduce the levels of ß-hCG. ß-hCG induces tumorigenicity in BRCA1-defective cells by directly binding to TGFBRII and induces TGFBRII-mediated cell proliferation. In this study, we analyzed the mechanism of action of ß-hCG on BRCA1 expression and its influence on drug sensitivity in breast cancer cells. We demonstrate that ß-hCG induces mutant BRCA1 protein expression in BRCA1 mutant cells; however, in BRCA1 wild-type cells, ß-hCG reduced wild-type BRCA1 protein expression. Transcriptionally, ß-hCG could induce Slug/LSD1-mediated repression of wild-type and mutant BRCA1 messenger RNA levels. However, ß-hCG induces HSP90-mediated stabilization of mutant BRCA1 and hence the overexpression of mutant BRCA1 protein, resulting in partial restoration of homologous recombination repair of damaged DNA. This contributes to drug resistance to HSP90 inhibitor 17AAG in BRCA1-defective cancer cells. A combination of HSP90 inhibitor and TGFBRII inhibitor has shown to sensitize ß-hCG expressing BRCA1-defective breast cancers to cell death. Targeting the ß-hCG-HSP90-TGFBRII axis could prove an effective treatment strategy for BRCA1-mutated breast tumors.

Proteomic Profiling of ß-hCG-Induced Spheres in BRCA1 Defective Triple Negative Breast Cancer Cells.

Previously, we identified that ß-hCG is expressed by BRCA1 mutated but not wild type breast cancers in vitro/in vivo and exhibited a novel event in ß-hCG overexpressing BRCA1 mutated HCC1937 cells where the cells were able to form spheres (HCC1937 ß spheres) in adherent cell culture plates even in the absence of any growth factors. These spheres express stem cell and EMT markers. In the present study, we carried out the total proteomic profiling of these HCC1937 ß spheres obtained from BRCA1 defective ß-hCG expressing stable breast cancer cells to analyze the cell signaling pathways that are active in these cells. Functional annotation revealed proteins (164 cellular and 97 secretory) predominantly involved in oxygen binding, nucleosome assembly, cytoskeleton organization, protein folding, etc. Many of the proteins identified from HCC1937 ß spheres in this study are also up regulated in breast cancers, which are directly linked with poor prognosis in human cancer samples as analyzed using TCGA data set. Survival analysis shows that ß-hCG expressing cancer patients are linked with poor survival rate. Interestingly, hemoglobins were identified at both cellular and secretory level in HCC1937 ß spheres and experiments after treating with ROS inducers revealed that ß-hCG induces hemoglobin and protects the cancer cells during oxidative stress. Our proteomic data strongly propose ß-hCG as an oncogenic molecule associated with BRCA1 mutation, and hence, targeting ß-hCG could be a strategy to treat BRCA1 defective breast cancers.

Molecular trail for the anticancer behavior of a novel copper carbohydrazone complex in BRCA1 mutated breast cancer.

Novel chelated metal complexes were synthesized from carbohydrazones to thiocarbohydrazones using metal-based drug designing platforms and their combination effect with Pb, a naphthaquinone were analyzed for anticancer activity in breast cancer cell lines. A panel of BRCA1 wild-type and mutated breast cancer cells: MCF-7 (BRCA1+ /ER+ ), MDA-MB-231 (BRCA1+ /ERα- ), HCC-1937 (BRCA1- /ERα- ), HCC1937/wt BRCA1, MX1 (BRCA1- /ERα- ), and MDA-MB-436 (BRCA1- /ERα- ) were screened for anti-cancer activity. Cu2 (HL)(HSO4 ) · H2 O]SO4 · 6 H2 O (CS2) is the most potent anticancer agent among the copper carbohydrazone and thiocarbohydrazone complexes analyzed in this study. It can be suggested that the presence of sulphate, as pharmacologically active centre, can induce cytotoxicity more effectively when compared to chlorine, bromine, perchlorate, and methanol. This is the first report demonstrating that CS2 can bind to DNA by hindering BamH1 activity and could induce DNA double strand breaks as evidenced by γ-H2AX expression. In addition to this, CS2 could also act as a Topo II inhibitor at a much lower concentration than etoposide and induce apoptosis, making it a potent anticancer agent. In combination with Pb, a potent ROS inducer, CS2 could induce synergistic anti-cancer activity in HR/ BRCA1 defective breast cancer cells. This is the first study reporting the mechanism involved in the induction of apoptosis for a metal chelated copper carbohydrazone complex and its combination effects with Pb in HR defective, BRCA1 mutated breast cancer cells.

Selective mode of action of plumbagin through BRCA1 deficient breast cancer stem cells.

BACKGROUND: Studies over the past decade and half have identified cancer stem cells (CSCs) to be responsible for tumorigenesis, invasion, sustenance of metastatic disease, radio- and chemo-resistance and tumor relapse. Recent reports have described the plasticity of breast CSCs (BCSCs) to shift between the epithelial and mesenchymal phenotypes via Epithelial-Mesenchymal Transition (EMT) and Mesenchymal-Epithelial Transition (MET) states as the reason for their invasive capabilities. Additionally, BRCA1 has been found to be a mammary stem cell fate determinant. However, it is not clear what would be the best marker that can be used for identifying CSCs in BRCA1 mutated cancers. Also, anticancer agents that can reduce CSC population in a BRCA1 defective condition have not been addressed so far. METHODS: Putative BCSCs were identified based on Hoechst exclusion, CD44(+)/24(-/low) expression and Aldehyde Dehydrogenase 1 (ALDH1) positivity using flow cytometry. The 'stemness' of the isolated ALDH1+ cells were analysed by immunofluorescence, western blotting for stem cell and EMT markers as well as in vitro mammosphere assays. Induction of Reactive Oxygen Species (ROS) by Plumbagin (PB) in BCSCs was assayed by Dichloro-dihydro-fluorescein diacetate (DCF-DA) staining. Ovarian cancer xenografts treated with PB were subjected to immunohistochemical analysis to study the ability of PB to target CSCs. RESULTS: We have confirmed that ALDH1 positivity is the best marker for the identification of BCSCs in BRCA1-defective breast cancer cell lines when compared to the CD marker profile and Side Population (SP) analysis. BRCA1 status was observed to be a determinant of the abundance of epithelial-like (ALDH1+) or mesenchymal-like (CD44(+)/24(-/low)) BCSCs, and the reconstitution of a full length, wild type BRCA1 in HCC1937 breast cancer cells possessing a mutated BRCA1, transforms them from 'stem-like' to more 'mesenchymal'. For the first time we have shown that Plumbagin (PB), a naturally occurring naphthoquinone which is predominantly a ROS inducer, could reduce BCSCs specifically in BRCA1-defective, basal-like cancer cells. CONCLUSIONS: The best marker for identifying BCSCs in BRCA1 defective condition could be ALDH1 and that BRCA1 mutated BCSCs would be mostly 'stem like' than 'mesenchymal'. Also ROS inducers like PB could reduce BCSCs in BRCA1 defective cancers.

Plumbagin, a naphthaquinone derivative induces apoptosis in BRCA 1/2 defective castrate resistant prostate cancer cells as well as prostate cancer stem-like cells.

Eventhough the role of BRCA1/2 in hereditary prostatic cancer is being unleashed at a rapid rate; their optimal clinical management remains undefined. Cancer stem cells are thought to be responsible for cancer chemoresistance and relapse, thus they represent a significant concern for cancer prognosis and therapy. In this study, we have analyzed the effect of Plumbagin (PB) and structurally related naphthaquinones on BRCA1/2 silenced prostate cancer cells and the ability of PB to target stem cells. Our cell proliferation studies showed that both PC-3 and DU145 cells were more sensitive to PB, though all the compounds induced mitochondrial potential loss, DNA fragmentation and morphological changes which are indicative of apoptosis. Both BRCA1/2 siRNA transfected PC-3 and DU145 cells exhibited increased sensitivity to PB. Gene expression profiling post PB treatment in BRCA1/2 silenced cells revealed that PB has a putative role in tumor suppression in BRCA defective cancers. Using flow cytometric analysis we have proved that PB has the putative ability to directly target CSCs. Overall studies suggest that PB's antitumour mechanisms holds promise for novel therapeutic approaches against BRCA mutated cancers as well as CSCs.

Phenethyl caffeate benzoxanthene lignan is a derivative of caffeic acid phenethyl ester that induces bystander autophagy in WiDr cells.

We recently reported that Phenethyl caffeate benzoxanthene lignan (PCBL), a semisynthetic compound derived from Caffeic Acid Phenethyl Ester (CAPE), induces DNA damage and apoptosis in tumor cells. In this study, we further investigated whether PCBL induces autophagy in WiDr cells. We also analyzed the pathways regulating autophagy and the role of autophagy in PCBL-induced cell death. Our acridine orange staining and LC3 II expression results suggest that PCBL induces autophagosomes in WiDr cells. The levels of LC3 II expression we observed after co-treatment of PCBL with bafilomycin A1 and the reductions in p62 expression we observed after PCBL treatment in WiDr cells demonstrate increased autophagic flux, a reliable indicator of autophagic induction. The increased Beclin 1 expression in PCBL-treated cells and the incapacity of PCBL to induce LC3 II in 3-methyladenine (3-MA)-treated cells we observed suggests that PCBL-induced autophagy is class III PI3-kinase dependent. PCBL did not alter phosphorylation of the mTOR substrate p70 S6 kinase, indicating that PCBL-induced autophagy was not mTOR regulated. Two autophagy related proteins, Atg5 and Atg12, also remained uninduced during PCBL treatment. The increased caspase activity and expression levels of LC3 II and p62 we observed in response to PCBL treatment in primary glioma cells demonstrates that PCBL-induced apoptosis and autophagy were not cell line specific. Pharmacological inhibition of autophagy did not alter the antitumor efficacy of PCBL in WiDr cells. This attests to the bystander nature of PCBL-induced autophagy (in terms of cell death). In toto, these data suggest that PCBL induces a class III kinase dependent, but mTOR independent, bystander mode of autophagy in WiDr cells.

Xenoestrogen and Its Interaction with Human Genes and Cellular Proteins: An In-Silico Study.

BACKGROUND: Breast cancer represents one of the leading causes of death worldwide. Apart from genetic factors, the sex hormone estrogen plays a pivotal role in breast cancer development. We are exposed to a plethora of estrogen mimics on a daily basis via various routes. Nevertheless, how xenoestrogens, the exogenous estrogen mimics, modulate cancer-associated signaling pathways and interact with specific genes is still underexplored. Hence, this study aims to explore the direct or indirect binding partners of xenoestrogens and their expression upon exposure to these estrogenic compounds. METHODS: The collection of genes linked to the xenoestrogens Octylphenol, Nonylphenol, Bisphenol-A, and 2,2-bis(4-hydroxyphenyl)-1,1,1-trichloroethane were gathered from the Comparative Toxicogenomics Database. Venny 2.1 was utilized to pinpoint the genes shared by these xenoestrogens. Subsequently, the shared genes underwent Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis using the Database for Annotation, Visualization, and Integrated Discovery bioinformatics resource. A xenoestrogen-protein interaction network was constructed using Search Tool for Interactions of Chemicals. The expressions of common genes were studied with the microarray dataset GSE5200 from the Gene Expression Omnibus database. Also, the expression of a common gene set within different breast cancer subtypes was identified using the University of California, Santa Cruz Xena. RESULTS: The genes linked to xenoestrogens were identified, and 13 genes were found to interact with all four xenoestrogens. Through DAVID analysis, the genes chosen are found to be enriched for various functions and pathways, including pathways in cancer, chemical carcinogenesis-receptor activation, and estrogen signaling pathways. The results of the Comparative Toxicogenomics Database and the chemical-protein interaction network derived from STITCH were similar. Microarray data analysis showed significantly high expression of all 13 genes in another study, with Bisphenol-A and Nonylphenol treated MCF-7 cells, most of the genes are expressed in luminal A or basal breast cancer subtype. CONCLUSION: In summary, the genes associated with the four xenoestrogens were mostly linked to pathways related to tumorigenesis, and the expression of these genes was found to be higher in breast cancer.

Plumbagin inhibits tumorigenesis and angiogenesis of ovarian cancer cells in vivo.

Angiogenesis is a hallmark of tumor development and metastatic progression, and anti-angiogenic drugs targeting the VEGF pathway have shown to decrease the disease progression in cancer patients. In this study, we have analyzed the anti-proliferative and anti-angiogenic property of plumbagin in cisplatin sensitive, BRCA2 deficient, PEO-1 and cisplatin resistant, BRCA2 proficient PEO-4 ovarian cancer cells. Both PEO-1 and PEO-4 ovarian cancer cells are sensitive to plumbagin irrespective of BRCA2 status in both normoxia and hypoxia. Importantly, plumbagin treatment effectively inhibits VEGF-A and Glut-1 in PEO-1 and PEO-4 ovarian cancer cells. We have also analyzed the p53 mutant, cisplatin resistant, and BRCA2 proficient OVCAR-5 cells. Plumbagin challenge also restricts the VEGF induced pro-angiogenic signaling in HUVECs and subsequently endothelial cell proliferation. In addition, we observe a significant effect on tumor regression among OVCAR-5 tumor-bearing mice treated with plumbagin, which is associated with significant inhibition of Ki67 and vWF expressions. Plumbagin also significantly reduces CD31 expression in an ear angiogenesis assay. Collectively, our studies indicate that plumbagin, as an anti-cancer agent disrupts growth of ovarian cancer cells through the inhibition of proliferation as well as angiogenesis.

Structure activity relationship of plumbagin in BRCA1 related cancer cells.

It has been shown earlier that plumbagin, a naturally occurring naphthaquinone has specific anticancer activity in BRCA1 blocked ovarian cancer cells. Plumbagin can induce estrogen dependent cell signaling and apoptosis in BRCA1 blocked ovarian cancer cells. Being a reactive oxygen species (ROS) generator and apoptosis inducing agent, plumbagin has immense potential as a promising anticancer agent. In this study we analyzed whether there would be increased anticancer activity if the positions of the functional groups on plumbagin were altered and further to analyze the detailed molecular mechanism of action of the lead molecule. Methods like MTT assay, apoptosis analysis by flow cytometry, assessment of mitochondrial membrane potential-Δψm , suppression subtractive hybridization, microarray, molecular docking and estrogen receptor-DNA binding activity by electrophoresis mobility shift assay (EMSA) were adopted for assessing the anticancer activity. Consequently we found that, plumbagin was the most potent anticancer agent when compared to structurally related compounds. The anti-cancer activities were in the order plumbagin > 1,4-naphthaquinone > juglone > lawsone > menadione. Molecular docking studies showed that plumbagin could be well docked in the receptor ligand complex of TRAIL-DR5 complexes to activate the extrinsic pathway of apoptosis. Since the antiproliferative activity of plumbagin could be reduced by inhibiting ERα, we speculated that plumbagin interferes with the binding of ERα to ERE and we confirmed this by EMSA. This study clearly indicates that plumbagin can induce multiple pathways of apoptosis and cell cycle arrest in BRCA1 blocked cells compared to unblocked cells.

Dry eye module - An application intelligence platform for diagnosing and monitoring dry eye disease.

Dry Eye Module (DEM), a software application, was developed to facilitate the streamlining of dry eye evaluation and documentation, to unify diagnostic jargon, and to analyze data input to generate a dry eye diagnostic report. This diagnostic report generated is based on the current understanding of dry eye diagnostic algorithms (Dry Eye Workshop 2 [DEWS2]/Asia Dry Eye Society [ADES]). Apart from its plausible role in aiding unprecedented multicentric dry eye demographic data collection, the application software can generate a customized referral letter to the rheumatologist, highlighting the salient ophthalmic features to be shared. DEM uses schematic illustrations to depict eyelid, conjunctival, and corneal parameters that impact the ocular surface in dry eyes that can be captured and compared during serial visits. Furthermore, DEM displays a symptom sign trend chart that graphically represents improvement/stability or worsening of the subjective and objective dry eye status. DEM can generate a curated prescription using preloaded advice templates. DEM includes facility for state-of-the-art advanced dry eye diagnostic reporting for super specialty use. The addition of DEM to the dry eye diagnostic armamentarium would help bridge the current unmet needs of dry eye evaluation. These are lack of uniform reporting, lack of multicentric data on a unified platform, the inability to ensure complete evaluation, inability to avoid lacunae during follow-up visits, and the lack of a simple patient-ophthalmologist and an ophthalmologist-rheumatologist interface.

Insights into the targeted elimination of BRCA1-defective cancer stem cells.

BRCA1 expression is involved in normal embryonic development, mammary stem cell differentiation, and prostate cancer development. Inactivation of BRCA1 plays an important role in cancer stem cells and also leads to the development of high-grade, basal-like tumor. This review discusses why BRCA1-defective cancer stem cells need a prospective analysis, how ER alpha could influence BRCA1-defective tumor progression, phenotype of BRCA1-defective cancers, role of BRCA1 in other cancers and finally at what perspective BRCA1-defective cancers can be targeted for better therapeutic outcome.

Nimbolide retards tumor cell migration, invasion, and angiogenesis by downregulating MMP-2/9 expression via inhibiting ERK1/2 and reducing DNA-binding activity of NF-κB in colon cancer cells.

Nimbolide, a plant-derived limonoid has been shown to exert its antiproliferative effects in various cell lines. We demonstrate that nimbolide effectively inhibited proliferation of WiDr colon cancer cells through inhibition of cyclin A leading to S phase arrest. It also caused activation of caspase-mediated apoptosis through the inhibition of ERK1/2 and activation of p38 and JNK1/2. Further nimbolide effectively retarded tumor cell migration and invasion through inhibition of metalloproteinase-2/9 (MMP-2/9) expression, both at the mRNA and protein level. It was also a strong inhibitor of VEGF expression, promoter activity, and in vitro angiogenesis. Finally, nimbolide suppressed the nuclear translocation of p65/p50 and DNA binding of NF-κB, which is an important transcription factor for controlling MMP-2/9 and VEGF gene expression.

Aloe emodin induces G2/M cell cycle arrest and apoptosis via activation of caspase-6 in human colon cancer cells.

Aloe emodin (AE), a natural anthraquinone, is reported to have antiproliferative activity in various cancer cell lines. In this study, we analyzed the molecular mechanisms involved in the growth-inhibitory activity of this hydroxyanthraquinone in colon cancer cell, WiDr. In our observation AE inhibited cell proliferation by arresting the cell cycle at the G2/M phase and inhibiting cyclin B1. AE appreciably induced cell death specifically through the induction of apoptosis and by activating caspases 9/6. Apoptotic execution was found to be solely dependent on caspase-6 rather than caspase-3 or caspase-7. This is the first study indicating that the AE induces apoptosis specifically through the activation of caspase-6.

Modulation of BRCA1 mediated DNA damage repair by deregulated ER-α signaling in breast cancers.

BRCA1 mutation carriers have a greater risk of developing cancers in hormone-responsive tissues like breasts and ovaries. However, this tissue-specific incidence of BRCA1 related cancers remains elusive. The majority of the BRCA1 mutated breast cancers exhibit typical histopathological features of high-grade tumors, with basal epithelial phenotype, classified as triple-negative molecular subtype and have a higher percentage of DNA damage and chromosomal abnormality. Though there are many studies relating BRCA1 with ER-α (Estrogen receptor-α), it has not been reported whether E2 (Estrogen) -ER-α signaling can modulate the DNA repair activities of BRCA1. The present study analyzes whether deregulation of ER-α signaling, arising as a result of E2/ER-α deficiency, could impact the BRCA1 dependent DDR (DNA Damage Response) pathways, predominantly those of DNA-DSB (Double Strand break) repair and oxidative damage response. We demonstrate that E2/E2-stimulated ER-α can augment BRCA1 mediated high fidelity repairs like HRR (Homologous Recombination Repair) and BER (Base Excision Repair) in breast cancer cells. Conversely, a condition of ER-α deficiency itself or any interruption in ligand-dependent ER-α transactivation resulted in delayed DNA damage repair, leading to persistent activation of γH2AX and retention of unrepaired DNA lesions, thereby triggering tumor progression. ER-α deficiency not only limited the HRR in cells but also facilitated the DSB repair through error prone pathways like NHEJ (Non Homologous End Joining). ER-α deficiency associated persistence of DNA lesions and reduced expression of DDR proteins were validated in human mammary tumors.

Evaluation of genoprotection against malathion induced toxicity by Orthosiphon thymiflorus Sleesen.

BACKGROUND: Pesticide toxicity is considered to be one of the significant reason for increased incidence of cancer. Plants are treasure troves of active phytochemical compounds which are used as herbal medicine as well as nutraceuticals. OBJECTIVE: To evaluate the genoprotective potential of Orthosiphon thymiflorus (Roth) Sleesen, (Lamiaceae) against the toxicity induced by malathion by a battery of four in vivo assays in Swiss albino mice. MATERIALS AND METHODS: Micronucleus assay was performed for analyzing the micronuclei induction and ratio of polychromatic and normochromatic erythrocytes (PCE/NCE). Anticlastogenic and mito depressive effect of the methanol and hexane extracts of O.thymiflorus were evaluated by chromosome aberration assay. Alkali comet assay was performed to assess double strand DNA repair. DNA damage sensing ability of the bone marrow cells were assessed by γ-H2AX foci formation. Phytochemical screening of hexane and methanol extract was done by GC-MS analysis. RESULT: O. thymiflorus extracts showed a dose dependant protective effect in all assays. It significantly decreased the frequency of micronuclei and improved PCE/NCE value in post treated groups of animals. Malathion induced clastogenic aberrations were effectively attenuated by methanol and hexane extracts. DNA comet assay showed that malathion induced damage can be protected by O. thymiflorus extracts. Multiple foci formation in γ-H2AX assay confirmed the activation of DNA repair proteins in post treated animals. CONCLUSION: Genoprotective effect of O. thymiflorus against malathion induced toxicity was confirmed. This study would be helpful to initiate more research including clinical using O. thymiflorus extract against pesticide induced toxicity.

Deregulated estrogen receptor signaling and DNA damage response in breast tumorigenesis.

Carriers of BRCA1 mutations have a higher chance of developing cancers in hormone-responsive tissues like the breast, ovary and prostate, compared to other tissues. These tumors generally exhibit basal-like characters and do not express estrogen receptor (ER) or progesterone receptor (PR). Intriguingly, BRCA1 mutated breast cancers have a less favorable clinical outcome, as they will not respond to hormone therapy. BRCA1 has been reported to exhibit ligand dependent and independent transcriptional inhibition of ER-α; however, there exists a controversy on whether BRCA1 induces or inhibits ER-α expression. The mechanisms associated with resistance of BRCA1 mutated cancers to hormone therapy, as well as the tissue restriction exhibited by BRCA1 mutated tumors are still largely unknown. BRCA1 mutated tumors possess increased DNA damages and decreased genomic integrity, as BRCA1 plays a cardinal role in high fidelity DNA damage repair pathways, like homologous recombination (HR). The existence of cross regulatory signaling networks between ER-α and BRCA1 speculates a role of ER on BRCA1 dependent DDR pathways. Thus, the loss or haploinsufficiency of BRCA1 and the consequential deregulation of ER-α signaling may result in persistence of unrepaired DNA damages, eventually leading to tumorigenesis. Therefore, understanding of this cross-talk between ER-α and BRCA1, with regard to DDR, will provide critical insights to steer drug development and therapy for breast/ovarian cancers. This review discusses the mechanisms by which estrogen and ER signaling influence BRCA1 mediated DNA damage response and repair pathways in the mammalian system.

Perspectives on mechanistic implications of ROS inducers for targeting viral infections.

In this perspective, we propose to leverage reactive oxygen species (ROS) induction as a potential therapeutic measure against viral infections. Our rationale for targeting RNA viral infections by pro-oxidants is routed on the mechanistic hypothesis that ROS based treatment paradigm could impair RNA integrity faster than the other macromolecules. Though antiviral drugs with antioxidant properties confer potential abilities for preventing viral entry, those with pro-oxidant properties could induce the degradation of nascent viral RNA within the host cells, as RNAs are highly prone to ROS mediated degradation than DNA/proteins. We have previously established that Plumbagin is a highly potent ROS inducer, which acts through shifting of the host redox potential. Besides, it has been reported that Plumbagin treatment has the potential for interrupting viral RNA replication within the host cells. Since the on-going Corona Virus Disease - 2019 (COVID-19) global pandemic mediated by Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) exhibits high infectivity, the development of appropriate antiviral therapeutic strategies remains to be an urgent unmet race against time. Therefore, additional experimental validation is warranted to determine the appropriateness of repurposable drug candidates, possibly ROS inducers, for fighting the pandemic which could lead to saving many lives from being lost to COVID-19.