School Of Basic And Applied Sciences

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End date: 2020Subject: search.filters.subject.Breast cancer

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    In silico identification of natural anticancer product and their efficacy in breast cancer cells and cancer stem like cells
    (Central University of Punjab, 2020) Kushwaha, Prem Prakash; Kumar, Shashank
    Breast cancer is the most commonly diagnosed lethal cancer in women worldwide. Notch signaling pathway is directly linked to breast cancer recurrence and aggressiveness. Natural remedies are becoming a prime choice to overcome against cancer due to lesser side effect and cost-effectiveness. Literature survey and in silico study identified Bulbine frutescens (Asphodelaceae), Kurarinone (KU) and 3-O-(E)-p- coumaroylbetulinic acid (CB) as lead plant product/phytochemicals. Methanolic and hexane extract of B. frutescens (BME and BHE respectively), KU and CB were studied for their anticancer activity and notch signaling pathway inhibitory potential in breast cancer cells. Moreover, KU and CB were also studied for their effect in mammosphere. Literature-based identification of methanol soluble phytochemicals of B. frutescens and in silico docking study revealed Bulbineloneside D as a potent notch signaling inhibitor (ϒ-secretase). In silico docking potential of KU and CB were equal to standard gamma secretase inhibitor DAPT (-8.74 kcal/mol). KU-gamma secretase complex showed lower RMSD value, marginal fluctuation in Radius of gyration (Rg), more number of inter hydrogen bonding, and stable secondary structure of the protein which indicates KU as candidate gamma secretase inhibitor (GSI). B. frutescens extracts (IC50 4.8– 28.4 μg/ml), Kurarinone (IC50 0.43-3.42 µM) and CB (IC50 0.99-5.88 µM) significantly decreased cell viability in MDA-MB-231 and T47D cells in time dependent manner. B. frutescens, KU and CB induced cell cycle arrest at G1 phase in MDA-MB-231 and T47D cells. RT-PCR analysis of cell cycle (cyclin D1, CDK4, and p21) and apoptosis modulating genes (caspase 3, Bcl2 and survivin) revealed upexpression of p21, and caspase 3, and down expression of cyclin D1, CDK4, Bcl2 and survivin genes in test extract/phytochemicals treated breast cancer cells. Western Blot analysis showed reduced expression of cyclin D1 and increased procaspase 3 protein expression in extract/phytochemicals treated breast cancer cells in time dependent manner. Fluorescence spectrophotometry and confocal microscopy showed extract/phytochemicals induced nuclear morphology and mitochondrial integrity disruption, and increased reactive oxygen species production in MDA-MB-231 and T47D cells at IC50 and sub IC50 concentration. Flow cytometric apoptosis analysis of extract/phytochemicals treated MDA-MB-231 cells showed significant increase in early apoptotic population in comparison to non-treated cells at IC50 and sub IC50 (half of the IC50) concentration. Dual-Luciferase Reporter assay confirmed notch promoter inhibitory activity of B. frutescens, Kurarinone and CB in HEK293 transfected cells at IC50 concentration. Moreover, RT-PCR analysis showed down regulation of notch responsive genes (Hes1 and Hey1) at transcription levels in extract/phytochemical treated breast cancer cells in time dependent manner. Western Blot analysis showed reduced notch responsive protein (Hes1, Hey1 and E-cadherin) expression in extract/phytochemical treated breast cancer cells. KU and CB treatment decreased the mammosphere formation ability in MCF-7 cells at IC50 concentration by lowering the notch signaling target proteins (Hes1, Hey1, and E-cadherin) and proteins involved in cancer cell self-renewal (c-Myc, SOX-2, CD44). In conclusion, extract/phytochemicals have cell cycle arrest, ROS production, apoptosis induction, and mitochondria membrane potential disruption efficacy in breast cancer cells. KU and CB have the ability to downregulate the notch signaling pathway in breast cancer and cancer stem like cells.
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    Design, Synthesis and Evaluation of Indole Based Compounds as Putative Anticancer Agents
    (Central University of Punjab, 2018) Singla, Ramit; Jaitak, Vikas
    In the course of efforts to develop new chemotherapeutic agent for targeting breast cancer, indole-benzimidazole, indole-xanthendione, indole-chromene carbonitrile and indole-dihydropyridine derivatives were computationally designed and synthesized. All the compounds were first analyzed for antiproliferative activity using ER-α responsive T47D breast cancer cells line and cytotoxicity using hPBMC. Further, all the synthesized compounds were also evaluated for ER-α binding affinity. Lead compounds 5f and 8f of series 1 and 2; 10e and 10f of series 3, 11c and 12d of series 4 and 5 were found to be most active at both cellular and receptor level hence were biologically evaluated for gene expression studies for targeting ER-α. Cell imaging experiment clearly suggest that compounds were able to cross cell membrane and accumulate thus causing cytotoxicity. Semiquantitative RT-PCR and Western blotting experiments further supported that lead compounds altered the expression of mRNA and protein of ER-α, thereby preventing the further transactivation and signaling pathway in T47D cells line. Structural investigation from induced fit simulation study suggest that lead compounds binds in a conformation similar to bazedoxifene by extensive hydrogen bonding and Van der Waals forces. All these results indicate that compounds 5f, 8f, 10e, 10f, 11c and 12d represents new putative anticancer agents and can be proved promising in the discovery of antiestrogens for the management of breast cancer.
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    ANALYSIS OF MICRORNA SIGNATURES AS BIOMARKER TO INVESTIGATE INTERLINK BETWEEN TYPE 2 DIABETES AND BREAST CANCER
    (Central University of Punjab, 2018) Sharma, Prateek; Kumar, Sanjeev
    Type 2 diabetes and breast cancer are two heterogeneous, multifactorial, chronic health problems involving several overlapping risk factors. Studies have suggested that type 2 diabetes is associated with 10-20% excessive relative risk of breast cancer. Evidence indicates link between type 2 diabetes and breast cancer, through insulin resistance and hyperinsulinemia. Numerous substantial evidence pointing towards the potential efficacy of antidiabetic metformin as anticancer therapeutics. MicroRNAs are endogenous, small non-coding RNA molecules regulating protein-coding gene expression and participate in nearly all the events of life. These small RNA molecules can have diagnostic or prognostic value, as microRNA expression profiles reflect disease origin, stage and other pathological factors. We hypothesized that there might be several microRNAs which commonly function in the “origin of type 2 diabetes to progression towards breast cancer.” Such common microRNAs can act via the related signalling pathways which may provide the critical insight into the better understanding of these diseases. The present study is aimed to investigate the interlinking between type 2 diabetes and breast cancer through microRNA signatures. Methods: In vitro cell experiments (using breast cancer cell lines MCF-7, MDA-MB-231, & T47D and pancreatic beta insulinoma cell lines MIN6 and RIN-5F) referred as MTT proliferation, trypan blue exclusion test, NBT assay, colony formation analysis, and scratch assay. Reactive oxygen species (ROS) assays (DCFH-DA and DHE) along with fluorescence microscopy (DAPI staining, Acridine orange + Ethidium bromide dual staining, JC1 staining) were used for apoptotic parameters. Insulin release in pancreatic beta cell lines was measured by ELISA. mRNA expression levels of Bax, Bcl-2, MMP-2, MMP-9, SOD 1, SOD 2, SOD 3, were quantified by qRT-PCR. Four common microRNAs- let 7a, miR-21, miR-155, miR-375 expression profiling in both breast cancer cell lines and pancreatic cell lines was performed by relative quantification real time analysis. Results: Insulin acts as a potential mitogenic factor accelerating the proliferation of breast cancer cells. On the other hand, metformin inhibits growth, proliferation and v clonogenic potential of breast carcinoma cells. ROS levels in breast cancer cells were significantly reduced by metformin by up-regulating SOD isoforms expression. Insulin increased the ROS to a very small limit. Metformin activates apoptosis by inducing mitochondrial dysfunction, upregulating Bax and downregulating Bcl-2. Migration is strongly suppressed by metformin by regulating matrix metalloproteinase (MMP-2 and MMP-9). Oncogenic miR-21 and miR-155 were downregulated by metformin, significantly correlated with reduced metastasis. The results of our study suggest that both MIN6 and RIN-5F cells show a significant differential pattern of proliferation, insulin secretion, and microRNA expression pattern. RIN-5F beta cells were found to be highly refractory to glucose-stimulated insulin secretion. However, metformin negatively regulates glucose-stimulated insulin release in both MIN6 and RIN-5F. In MIN6 cells, levels of microRNA-375 and let-7a were significantly up- & down-regulated by metformin at normal-glucose and high glucose culture conditions respectively whereas in RIN-5F both were significantly down-regulated. Conclusions: Our data supports that metformin plays a pivotal role in the modulation of the antioxidant system including SOD machinery. Our results indicate that metformin inhibit breast cancer cell proliferation by inducing apoptosis via mitochondrial signalling. Furthermore, emerging view from this study is that microRNAs (let-7a, mir-21, miR-155 and miR- 375) are involved in the process of disease (type 2 diabetes and breast cancer) development, and there is the potential utility of microRNAs as effective biomarker for diagnostic and prognostic application in type 2 diabetes and breast cancer.
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    Recent development in indole derivatives as anticancer agents for breast cancer
    (Bentham Science Publishers, 2019) Kaur K.; Jaitak V.
    Background: Breast Cancer (BC) is the second most common cause of cancer related deaths in women. Due to severe side effects and multidrug resistance, current therapies like hormonal therapy, surgery, radiotherapy and chemotherapy become ineffective. Also, the existing drugs for BC treatment are associated with several drawbacks such as poor oral bioavailability, non-selectivity and poor pharmacodynamics properties. Therefore, there is an urgent need for the development of more effective and safer anti BC agents. Objective: This article explored in detail the possibilities of indole-based heterocyclic compounds as anticancer agents with breast cancer as their major target. Methods: Recent literature related to indole derivatives endowed with encouraging anti BC potential is reviewed. With special focus on BC, this review offers a detailed account of multiple mechanisms of action of various indole derivatives: aromatase inhibitor, tubulin inhibitor, microtubule inhibitor, targeting estrogen receptor, DNA-binding mechanism, induction of apoptosis, inhibition of PI3K/AkT/NFkB/mTOR, and HDAC inhibitors, by which these derivatives have shown promising anticancer potential. Results: Exhaustive literature survey indicated that indole derivatives are associated with properties of inducing apoptosis and disturbing tubulin assembly. Indoles are also associated with the inhibition of NFkB/mTOR/PI3K/AkT and regulation of estrogen-mediated activity. Furthermore, indole derivatives have been found to modulate critical targets such as topoisomerase and HDAC. These derivatives have shown significant activity against breast cancer cells. Conclusion: In BC, indole derivatives seem to be quite competent and act through various mechanisms that are well established in case of BC. This review has shown that indole derivatives can further be explored for the betterment of BC chemotherapy. A lot of potential is still hidden which demands to be discovered for upgrading BC chemotherapy.
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    Association of elevated levels of C-reactive protein with breast cancer, breast cancer subtypes, and poor outcome
    (Mosby Inc., 2019) Kaur R.P.; Rubal; Banipal R.P.S.; Vashistha R.; Dhiman M.; Munshi A.
    Background and Purpose: Inflammation and caner are linked in a bidirectional manner. C-reactive protein (CRP) is an important inflammatory marker. The aim of the study was to test whether the inflammatory marker, CRP at the time of diagnosis of breast cancer is associated with metastasis, recurrence, and death in breast cancer patients from Malwa region of Punjab where breast cancer is widely feared. Material and Methods: Two hundred and forty-two breast cancer patients and 242 age and sex matched controls were included in the study. CRP levels were estimated using fully automated bio analyzer Erba200. Follow up interviews were conducted at an interval of 3, 6, 9, 12, 15, 18, 21, 24, and 27 months to determine the outcome among breast cancer patients. Results: Elevated levels of CRP were found among the diseased in comparison with controls (P < 0.0001). Higher CRP levels associated significantly with poor outcome including metastasis and recurrence among breast cancer patients [P = 0.03; 95% confidence interval; odds ratio: 2.954 (0.9125-9.561)]. Conclusion: Elevated levels of CRP associated significantly with increased risk of breast cancer and poor outcome. CRP estimation may be a simple and inexpensive tool for the risk assessment and outcome of the disease in Malwa region of Punjab where incidence of breast cancer is reported to be very high.
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    Bulbine frutescens phytochemical inhibits notch signaling pathway and induces apoptosis in triple negative and luminal breast cancer cells
    (Elsevier Inc., 2019) Kushwaha P.P.; Vardhan P.S.; Kapewangolo P.; Shuaib M.; Prajapati S.K.; Singh A.K.; Kumar S.
    Breast cancer (BCa) is the most commonly diagnosed lethal cancer in women worldwide. Notch signaling pathway is directly linked to BCa recurrence and aggressiveness. Natural remedies are becoming a prime choice to overcome against cancer due to lesser side effect and cost-effectiveness. Bulbine frutescens (Asphodelaceae), a traditional medicinal plant in South Africa possess bioactive flavonoids and terpenoids. Polar (methanol) and non-polar (hexane) B. frutescens plant extracts were prepared. GC–MS analysis revealed the differential presence of secondary metabolites in both methanolic and hexane extracts. We hereby first time evaluated the anticancer potential of B. frutescens methanolic and hexane extract in triple-negative and luminal BCa cells. B. frutescens extracts significantly decreased cell viability (IC50 4.8–28.4 μg/ml) and induced cell cycle arrest at G1 phase in MDA-MB-231 and T47D cells as confirmed by spectrophotometry and flow cytometry technique. RT-PCR analysis of cell cycle (cyclin D1, CDK4, and p21) and apoptosis modulating genes (caspase 3, Bcl2 and survivin) revealed upexpression of p21, and caspase 3, and down expression of cyclin D1, CDK4, Bcl2 and survivin genes in extract-treated BCa cells. Fluorescence spectrophotometry and confocal microscopy showed B. frutescens induced nuclear morphology and mitochondrial integrity disruption, and increased reactive oxygen species production in MDA-MB-231 and T47D cells. Flow cytometric apoptosis analysis of B. frutescens extracts treated MDA-MB-231 cells showed ≈13% increase in early apoptotic population in comparison to non-treated cells. Dual-Luciferase Reporter assay confirmed notch promoter inhibitory activity of B. frutescens extracts. Moreover, RTPCR analysis showed down regulation of notch responsive genes (Hes1 and Hey1) at transcription levels in extract-treated BCa cells. Western Blot analysis showed increased procaspase 3 protein expression in extract-treated BCa cells. In all the assays methanolic extract showed better anti-cancer properties. Literature-based identification of methanol soluble phytochemicals in B. frutescens and in silico docking study revealed Bulbineloneside D as a potent ϒ-secretase enzyme inhibitor. In comparison to standard notch inhibitor, lead phytochemical showed two additional hydrophobic interactions with Ala80 and Leu81 amino acids. In conclusion, B. frutescens phytochemicals have cell cycle arrest, ROS production, apoptosis induction, and mitochondria membrane potential disruption efficacy in breast cancer cells. B. frutescens phytochemicals have the ability to downregulate the notch signaling pathway in triple-negative and luminal breast cancer cells.
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    Drug Targeting Strategies in Breast Cancer Treatment
    (Bentham Science, 2014) Mayank; Jaitak, Vikas
    Breast cancer (BC) is the leading cause of death among women all over the world. Estrogen receptor (ER) based therapy is one of the major approaches to target BC and is associated with various problems such as primary as well as secondary resistance. ER signaling is a complex pathway as many factors are involved; including several types of ERs and their associated co-regulators. Increasing understanding of ER signals results in new approaches targeting towards BCs. In this context, ER co-regulators have been explored and many modulators of ER co-regulators have been found out. EGFR and mTOR pathways also have significant impact on BC endocrine therapy because of the complex crosstalk mechanism which is responsible for primary and secondary resistance. Triple negative breast cancer (TNBC) is majorly associated with BRCA mutations. Currently there is no approved targeted therapy available in such form of cancer. Although PARP inhibitors seem to be suitable candidates for it. The present review is focused on the current scenario of ER, EGFR, as well as mTOR signaling target therapy. We have also discussed the current status of PARP inhibitors in BC chemotherapy.
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    Identification of novel indole based heterocycles as selective estrogen receptor modulator.
    (Elsevier, 2018) Singla, Ramit; Prakash, Kunal; Gupta Kunj Bihari; Upadhyay, Shishir; Dhiman, Monisha; Jaitak, Vikas
    In the present study, we have designed and synthesized indole derivatives by coalescing the indole nucleus with chromene carbonitrile and dihydropyridine nucleus. Two compounds 5c and 6d were selected from series I and II after sequential combinatorial library generation, docking, absorption, distribution, metabolism and excretion (ADME) filtering, anti-proliferative activity, cytotoxicity, and ER-α competitor assay kit by utilizing estrogen receptor-α (ER-α) dominant T47D BC cells line and PBMCs (Peripheral Blood Mononuclear Cells). Cell imaging experiment suggested that both the compounds successfully cross cellular biomembrane and accumulate in nuclear, cytoplasmic and plasma membrane region. Semiquantitative RT-PCR and Western blotting experiments further supported that both compounds reduced the expression of mRNA and receptor protein of ER-α, thereby preventing downstream transactivation and signaling pathway in T47D cells line. Current findings imply that 5cand 6d represent novel ER-α antagonists and may be used in the development of chemotherapy for the management of BC.
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    Design, synthesis and biological evaluation of novel indole-benzimidazole hybrids targeting estrogen receptor alpha (ER-?)
    (Elsevier Masson SAS, 2018) Singla R.; Gupta K.B.; Upadhyay S.; Dhiman, Monisha; Jaitak V.
    In the course of efforts to develop novel selective estrogen receptor modulators (SERMs), indole-benzimidazole hybrids were designed and synthesised by fusing the indole nucleus with benzimidazole. All the compounds were first inspected for anti-proliferative activity using ER-? responsive T47D breast cancer cell lines and ER-? binding assay. From this study, two representative bromo substituted compounds 5f and 8f were found to be most active and thus were escalated for gene expression studies for targeting ER-?. Cell imaging experiment clearly suggest that compounds were able to cross cell membrane and accumulate thus causing cytotoxicity. RT-PCR and Western blotting experiments further supported that both compounds altered the expression of mRNA and receptor protein of ER-?, thereby preventing the further transactivation and signalling pathway in T47D cells lines. Structural investigation from induced fit simulation study suggest that compound 5f and 8f bind in antagonistic conformation similar to bazedoxifene by extensive hydrogen bonding and Van der Waals forces. All these results strongly indicate that compound 5f and 8f represents a novel potent ER-? antagonist properties and will proved promising in the discovery of SERM for the management of breast cancer.
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    S961, a biosynthetic insulin receptor antagonist, downregulates insulin receptor expression & suppresses the growth of breast cancer cells
    (Indian Council of Medical Research (ICMR), 2018) Sharma, PrateeK; Kumar, Sanjeev
    Background & objectives: Insulin resistance associated with hyperinsulinaemia and overexpression of insulin receptors (IRs) have been intricately linked to the pathogenesis and treatment outcomes of the breast carcinoma. Studies have revealed that upregulated expression of IRs in breast cancer pathogenesis regulates several aspects of the malignant phenotype, including cell proliferation and metastasis. This study was aimed to investigate the pivotal role of an IR antagonist S961 on IR signalling and other biological parameters in MCF-7, MDA-MB-231 and T47D cell lines. Methods: The effect of human insulin and S961 on growth, proliferation rate and clonogenic potential of breast cancer cells was evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay and clonogenic assay. The mRNA expression of IR isoforms (IR-A and IR-B) was measured in the breast carcinoma cells using quantitative PCR. Results: The study revealed that breast cancer cells predominantly expressed IR-A isoform and showed extensive growth and proliferation owing to IR overexpression. It was found that S961 downregulated the IRs (IR-A and IR-B) with nanomolar dose and efficiently blocked expression of IRs even in the presence of insulin. IR mRNA expression levels were significantly downregulated in the continued presence of S961. S961 also inhibited cellular proliferation and colony formation in breast tumour cells. Interpretation & conclusions: IR antagonist, S961 showed distinct antagonism in vitro and appeared to be a powerful therapeutic modality that might provide insight into the pathogenesis of impaired IR signalling.