School Of Basic And Applied Sciences
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Item ANALYSIS OF MICRORNA SIGNATURES AS BIOMARKER TO INVESTIGATE INTERLINK BETWEEN TYPE 2 DIABETES AND BREAST CANCER(Central University of Punjab, 2018) Sharma, Prateek; Kumar, SanjeevType 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.Item Host-Guest Interaction of calix[4]arene derivatives with MetforminAC(Central University of Punjab, 2018) Gupta, Harshita; BabuJ. NagendraCalix[4]arene was lower rim functionalized to furnish receptor 2-3 and 4-6, containing propyl and cyanomethoxy moieties, respectively. The receptors were characterized by FTIR, 1H and EI-MS. UV-visible studies were performed with receptor 2-6 cone (1'10-4M) upon addition of metformin (1'10-5 - 1'10-3M). Upon addition of metformin the UV-Visible absorption showed a hyperchromic shift.The 1:1 Host-Guest complexation studied by Benesi-Hildebrand equation showed a stability constant in the range 2'104 -10'104 M-1 .Item Effect of metform on insuln mediated proliferation of lung cancer cell lines(Central University of Punjab, 2012) Seema; Bast, FelixBackground: The incidence of cancer in diabetes mellitus (DM) patients is a matter of concern. Lung cancer is the most commonly diagnosed cancer and leading cause of death in males. Smoking is the main risk factor contributing to lung cancer progression. The occurrence of cancer is more with the type 2 DM. Besides, hyperglycemia and endogenous insulinaemia exist together for a long duration as a result of insulin resistance. As a consequence of this, the mitogenic activity of insulin is amplified. Moreover, other growth factors, and hormones are activated under the influence of insulin that further enhances this effect. It is also related to obesity, central fat accumulation, physical inactivity and smoking. The nicotine of smoke induces oxidative stress and endothelial malfunction creating metabolic abnormalities in lung cancer. In this perspective, role of insulin sensitizing drug, metformin in inhibiting the growth proliferation of lung cancer cells is hereby explored. Objective: The present study was aimed to evaluate the growth proliferation effect of insulin on non small cell lung carcinoma cell lines. It also proposed to evaluate role of metformin in preventing insulin mediated proliferation in p53 and liver kinase B1 (LKB1) mutant and wild type cell lines. Materials and methods: Two non small cell lung carcinoma cell lines, A549 and H1299 (p53 and LKB1 wild type and mutant) were used to analyze the mitogenic role of insulin by incubating for 24 hours with human recombinant insulin at a range of concentrations from 1nM to 10'M. This was followed by the metformin (concentrations from 1'M to 50mM) treatment for 24 hours along with insulin (500'M for A549 and 1mM for H1299). The proliferations were assessed by MTT dye reduction test and the percentage of the survival of the treated cells was compared with the control. One way ANOVA was used for the data analysis and the proliferation between cell lines were evaluated by student's t-test and two way analysis of variance (Two way ANOV). Results: Both the cell lines exhibited a significant proliferation (p<0.001) with the concentrations of insulin. Insulin stimulated the proliferation approximately by two fold and 1.78 times for A549 cells and H1299 cell line respectively compared to control cells. The growth of two lung carcinoma cell lines were significantly (p<0.001) inhibited by metformin treatment for 24 hours. The maximum reduction in growth was 73% and 67% for A549 and H1299 respectively for a concentration of 50mM of metformin compared to the control. The results followed a dose dependant response pattern for insulin as well as metformin treatment. Concentration at which 50% inhibition of growth observed (IC50) was comparable for both the cell lines. Conclusions: Insulin in high circulating concentrations can augment the growth proliferation of lung cancer cells. Metformin can inhibit this insulin mediated proliferation of lung cancer cells in a multifaceted way. The mechanism of action is independent of p53 and liver kinase B1.Item To Study the Dose and Time Dependent Effect of Human Insulin and Metformin on the Growth of Breast Cancer Cells(Central University of Punjab, 2012) Cholia, Ravi Prakash; Thakur, SanjeevCancer and diabetes, both are leading causes of mortality globally. Both the diseases are multifactorial and share number of common risk factors. Hyperglycemia and hyperinsulinemia which are the characteristic features of diabetes influences the growth rate and proliferation of tumor cells directly or indirectly. Type 2 diabetes shows stronger association with various cancers. Breast cancer is one of the malignancy affecting females worldwide. This study demonstrates that glucose not only acts as energy source in tumor cell but also acts as mitogen. Insulin not only regulates the blood glucose level but also induces growth and proliferation in MCF 7 and MDA MB 231 breast cancer cell lines independently and in combination with glucose. Metformin inhibit proliferation of MCF 7 and MDA MB 231 breast cancer cell lines independently and also in presence of glucose and insulin, but shows more inhibitory effect in presence of insulin as compare to glucose. Recently discovered insulin receptor antagonist S961 did not showed any significant response in breast cancer cell lines MCF 7 and MDA MB 231. The ineffectiveness is probably due to blocking effect of higher insulin dose. So with this investigation it can be concluded that metabolic alteration leads to proliferation of breast cancer cell lines.Item Metformin inhibits human breast cancer cell growth by promoting apoptosis via a ROS-independent pathway involving mitochondrial dysfunction: pivotal role of superoxide dismutase (SOD)(Springer, 2018) Sharma, Prateek; Kumar, SanjeevPurpose Despite a growing body of evidence indicating a potential efficacy of the anti-diabetic metformin as anti-cancer agent, the exact mechanism underlying this efficacy has remained largely unknown. Here, we aimed at assessing putative mechanisms associated with the ability of metformin to reduce the proliferation and migration of breast cancer cells. Methods A battery of in vitro assays including MTT, colony formation, NBT and scratch wound healing assays were performed to assess the viability, proliferation, anti-oxidative potential and migration of breast cancer-derived MCF-7, MDA-MB-231 and T47D cells, respectively. Reactive oxygen species (ROS) assays along with fluorescence microscopy were used to assess apoptotic parameters. Quantification of SOD, Bcl-2, Bax, MMPs, miR-21 and miR-155 expression was performed using qRT-PCR. Results We found that metformin inhibited the growth, proliferation and clonogenic potential of the breast cancer-derived cells tested. ROS levels were found to be significantly reduced by metformin and, concomitantly, superoxide dismutase (SOD) isoforms were found to be upregulated. Mitochondrial dysfunction was observed in metformin treated cells, indicating apoptosis. In metastatic MDA-MB-231 cells, migration was found to be suppressed by metformin through deregulation of the matrix metalloproteinases MMP-2 and MMP-9. The oncogenic microRNAs miR-21 and miR-155 were found to be downregulated by metformin, which may be correlated with the suppression of cell proliferation and/or migration. Conclusions Our data indicate that metformin may play a pivotal role in modulating the anti-oxidant system, including the SOD machinery, in breast cancer-derived cells. Our observations were validated by in silico analyses, indicating a close interaction between SOD and metformin. We also found that metformin may inhibit breast cancer-derived cell proliferation through apoptosis induction via the mitochondrial pathway. Finally, we found that metformin may modulate the pro-apoptotic Bax, anti-apoptotic Bcl-2, MMP-2, MMP-9, miR-21 and miR-155 expression levels. These findings may be instrumental for the clinical management and/or (targeted) treatment of breast cancer.Item INFLUENCE OF INSULIN AND METFORMIN ON PROSTATE CANCER(Nova Science Publishers, 2017) Singh, Pushpendra; Bast, Felix; Kumar, Shashank; Saini, Khem ChandDietary habit and hormonal factor play a significant role in prostate cancer deregulation in addition to genetic and environmental factor. Nonandrogenic growth factor like insulin and insulin growth factor are influences the prostate cancer initiation and progression. Insulin and Insulin-like growth factor regulate various metabolic pathways, cell growth, cellular proliferation and apoptosis. Various epidemiological results point out that insulin not only increased the risk of cardiovascular, macrovascular, and microvascular complications but also at significantly increase the risk of various cancers. The use of metformin, the usually approved drug for type 2 diabetes, was continually linked with the decreased risk of the incidence of a variety of cancers. More than 60 clinical trials of metformin being tested as a treatment for various types of cancer, including breast, colon, prostate, endometrial, and pancreatic cancer. The ability of metformin to lower circulating insulin may be predominantly imperative for the treatment of cancers. Moreover, metformin inhibiting mammalian target of rapamycin promoted cell growth signaling. In this chapter, the confirmation behind a role for metformin in cancer therapy and its prospective molecular mechanisms of action are discussed.