Seema Kumari

Therapeutic Potentials of CD151 shRNA in Targeting Metastasis of TripleNegative Breast Cancer Cell Line MDA-MB-231

Objective: CD151 is a master regulator of cell adhesion signalling and acts as a promoter in tumour progression. Induction of RNAi through shRNA expression holds great prospective in biomedical research. Bioinformatics approach used to predict potential shRNAs that knockdown CD151. The aim of present study is to investigate the role of CD151 in metastasis of triple negative breast cancer cells using shRNA. Methods: Triple negative breast cancer line, MDA-MB-231 obtained from NCCS Pune, India and expression of CD151 determined using western blot and RT-PCR analysis. Small hairpin RNA (shRNA) was constructed using pSilencer 2.1-U6 puro vector to knockdown CD151 expression. The role of CD151 in proliferation, apoptosis, migration, invasion and cell adhesion was evaluated by silencing CD151 gene using CD151shRNA. Results: RNA interference technology (RNAi) used to silence CD151 gene expression in MDA-MB-231 cells. Delivery of specific shRNA targeting human endogenous CD151 showed significant growth inhibition of MDA- MB-231 cells. The gene expression study by RT-PCR analysis showed that expression of CD151 at mRNA level reduced six fold with CD151 gene knockdown. CD151 gene silencing for 48h using shRNA decreased proliferation by 62.7%. CD151 knockdown also lead to the significant inhibition of metastasis and induced apoptosis. Conclusion: CD151 over expression is essential for tumour progression and our study shows that shRNA mediated gene silencing of CD151 decreases the metastasis, thus emphasizing CD151 as a prognostic marker and help in developing new therapeutics for treatment of triple negative breast cancer.

Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells

Purpose Lipid rafts are cholesterol enriched microdomains that colocalize signaling pathways involved in cell proliferation, metastasis, and angiogenesis. We examined the effect of methyl-β-cyclodextrin (MβCD)-mediated cholesterol extraction on the proliferation, adhesion, invasion, and angiogenesis of triple negative breast cancer (TNBC) cells. Methods We measured cholesterol and estimated cell toxicity. Detergent resistant membrane (DRM) and non-DRM fractions were separated using the OptiPrep gradient method. Cell cycles stages were analyzed by flow cytometry, apoptosis was assessed using the TdT-mediated dUTP nick end-labeling assay, and metastasis was determined using a Matrigel invasion assay. Neo-vessel pattern and levels of angiogenic modulators were determined using an in vitro angiogenesis assay and an angiogenesis array, respectively. Results The present study found that the cholesterol-depleting agent MβCD, efficiently depleted membrane cholesterol and caused concentration dependent (0.1–0.5 mM) cytotoxicity compared to nystatin and filipin III in TNBC cell lines, MDA-MB 231 and MDA-MB 468. A reduced proportion of caveolin-1 found in DRM fractions indicated a cholesterol extraction-induced disruption of lipid raft integrity. MβCD inhibited 52% of MDA-MB 231 cell adhesion on fibronectin and 56% of MDA-MB 468 cell adhesion on vitronectin, while invasiveness of these cells was decreased by 48% and 52% respectively, following MβCD treatment (48 hours). MβCD also caused cell cycle arrest at the G2M phase and apoptosis in MDA-MB 231 cells (25% and 58% cells, respectively) and in MDA-MB 468 cells (30% and 38% cells, respectively). We found that MβCD treated cells caused a 52% and 58% depletion of neovessel formation in both MDA-MB 231 and MDA-MB 468 cell lines, respectively. This study also demonstrated that MβCD treatment caused a respective 2.6- and 2.5-fold depletion of tyrosine protein kinase receptor (TEK) receptor tyrosine kinase levels in both TNBC cell lines. Conclusion MβCD-induced cholesterol removal enhances alterations in lipid raft integrity, which reduces TNBC cell survival.

Reactive Oxygen Species: A Key Constituent in Cancer Survival:

Background: Cancer is one of the major heterogeneous disease with high morbidity and mortality with poor prognosis. Elevated levels of reactive oxygen species (ROS), alteration in redox balance, and deregulated redox signaling are common hallmarks of cancer progression and resistance to treatment. Mitochondria contribute mainly in the generation of ROS during oxidative phosphorylation. Elevated levels of ROS have been detected in cancers cells due to high metabolic activity, cellular signaling, peroxisomal activity, mitochondrial dysfunction, activation of oncogene, and increased enzymatic activity of oxidases, cyclooxygenases, lipoxygenases, and thymidine phosphorylases. Cells maintain intracellular homeostasis by developing an immense antioxidant system including catalase, superoxide dismutase, and glutathione peroxidase. Besides these enzymes exist an important antioxidant glutathione and transcription factor Nrf2 which contribute in balancing oxidative stress. Reactive oxygen species–mediated signaling pathways activate pro-oncogenic signaling which eases in cancer progression, angiogenesis, and survival. Concomitantly, to maintain ROS homeostasis and evade cancer cell death, an increased level of antioxidant capacity is associated with cancer cells. Conclusions: This review focuses the role of ROS in cancer survival pathways and importance of targeting the ROS signal involved in cancer development, which is a new strategy in cancer treatment.

Synergistic effects of coralyne and paclitaxel on cell migration and proliferation of breast cancer cells lines

Breast cancer is one of the most frequently diagnosed cancer in woman. Triple-negative breast cancer (TNBC) is most aggressive form of breast cancer. There is a growing interest in the use of natural products in combinational chemotherapy to improve the effectiveness in combating proliferation of cancer cells. Here, we hypothesized that coralyne in combination with paclitaxel may exhibit synergistic effect on inhibition of proliferation, migration and induction of apoptosis in MCF-7 and MDA-MB-231 breast cancer cell lines. MTT and BrdU incorporation assays were performed to study the effect of drugs alone and in combination on cell cytotoxicity and proliferation of the breast cancer cell lines, respectively. Adhesion and wound healing assays were performed to study the cell and extracellular matrix interactions. In addition, expression of proliferation marker ki-67 and apoptotic markers Bax and Bcl-2 was determined to study the effect of coralyne in combination with paclitaxel by reverse transcriptase PCR and confirmed by Western blot. The results indicated the synergism between coralyne and paclitaxel on proliferation and migration of breast cancer cell lines. This study also showed that combinational drug treatment decreased the expression of ki-67 and there was an increase in pro apoptotic factor Bax with decreased in expression of anti-apoptotic factor Bcl-2 in breast cancer cell lines with negligible effect on normal breast cell line. Overall, our data described the promising therapeutic potential of coralyne in combination with paclitaxel in treating breast cancer at lower effective dose.

Coralyne Targets Proteases Involved in Cancer Progression: An In Silico Study

Molecular docking has a significant application in finding the targets involved in cancer meta stasis. A positive correlation between the vigourness of tumor and expression of various proteases has been established, which includes serine proteases like furin and uPA, matrix metalloproteinases such as membrane type 1 MMP and tissue inhibitor of metalloproteinases, cysteine proteases such as cathepsin B & S and aspartate protease like cathepsin D. Virtual screening based on structure and post-screening analysis are routinely used in search of novel lead compound and its optimization. In the present study, the binding energy of coralyne with various metastatic proteases was analyzed using in silico docking tools such as iGEMDOCK v2.1, hex v6.3 and patch dock. The analysis of results indicates that coralyne exhibited significantly good binding affinity with furin and uPA predicting the possibility of coralyne in regulating cancer invasion and metastasis. Further, protein-protein network was analyzed using STRING version 10 based on KEGG pathways and clustered into groups using on MCL and k-mean algorithms to unfold its interacting partners proteins in cancer metastasis.

A study on the immunisation status and the factors responsible for incomplete immunization amongst children of age group 0-12 months coming to a tertiary care hospital (IGIMS)

The health of children has been of vital importance to all societies because children are the basic resources of future mankind. Immunization is one of the best indicators to evaluate the health outcomes and services distributed across social and economic groups. It is also one of the most cost-effective interventions to prevent a series of major illnesses, particularly in environments where children are undernourished and die from preventable diseases. Vaccines ensure that all children, no matter their circumstances, have a shot at a healthy life children in India continue to lose their lives to vaccine-preventable diseases such as measles, which remains the bigger killer. Tetanus in newborn also remains a problem. Diarrhea remains the second major cause of death among children, after respiratory-tract infections. Unhygienic practices and unsafe drinking water are some of its main causes. Immunization saves more than 3 million lives worldwide each year, and it saves millions more from suffering illness and lifelong disability (WHO estimate, 2009). ABSTRACT

Lipid rafts disruption induces apoptosis by attenuating expression of LRP6 and survivin in triple negative breast cancer

Triple negative breast cancer is a clinically challenging subtype due to lack of biomarker for rational targeted therapy. Lipid rafts are cholesterol enriched rigid platforms, which colocalize signalling molecules of cancer progression. This study explores the effect of lipid rafts disruption by cholesterol depleting agent, MβCD on induction of apoptosis and expression of WNT receptor LRP6, survivin and common apoptotic markers in TNBC cell lines. The in vitro effect of lipid rafts disruption on viability, single cell reproductive ability, proliferation and migration were evaluated by MTT, clonogenic, BrdU incorporation and wound scratch assays, respectively. The morphological changes were assessed by tryphan blue, Wright and Giemsa staining; nuclear changes by Hoechst staining. The induction of apoptosis was evaluated by AO/EtBr staining, DNA damage and DNA fragmentation assays. Expression of Caveolin-1, LRP6, β-Catenin, Survivin, Bcl2, BAX, Caspase-3, Ki67 and c-myc were analyzed by PCR and Western blotting techniques. The lipid raft disruption resulted in reduction of the proliferation of MDA-MB 231 and MDA-MB 468 cells by 56.3 and 42.0%; survival fraction by 54.7 and 59.4%; migration by 44.3 and 48.4%, respectively. It also induced apoptosis by causing cell shrinkage, membrane blebbing, nuclear condensation, chromatin cleavage, oligonucleotide fragmentation with an apoptotic index of 59.1 and 46.6% in MDA-MB 231 and 468 cells, respectively. Further, it downregulated the expression of caveolin-1, LRP6, β-catenin, survivin, Bcl2, ki67, c-myc and upregulated BAX, caspase-3. The cholesterol supplementation enhanced the clonogenic potential and upregulated the expression of caveolin-1 and LRP6. The results underline a potential effect of lipid rafts disruption on induction of apoptosis in TNBC cells.

Exosomal tetraspanins as regulators of cancer progression and metastasis and novel diagnostic markers

Exosomes are cell–cell communicators emerging as a new paradigm for noninvasive diagnosis and prognosis of treatment response. Exosomal tetraspanin proteins like CD63, CD9 and CD81 play a critical role in sorting, selective recruitment of biomolecules, target selection, cell‐specific entry, capturing, angiogenesis and vasculogenesis. These tetraspanins are being used as markers for oral, colorectal and colon cancers and glioblastoma. However, exosomal markers with robust specificity for early detection of carcinomas are the furthest along. EXO CARTA database shows the presence of CD151 in exosomes of colorectal, melanoma, ovarian and prostate cancers. CD151 preferentially targets exosomes to lung, lymph node and stroma cells. The present review discussed the possible role of tetraspanins in the formation, cargo selection, target selection and uptake of exosomes and suggests exciting new directions for future research.

Knockdown of CD151 Gene Expression Reduces Survival of Estrogen Receptor Positive Breast Cancer Cells

Tetraspnin CD151 is involved in proliferation, motility, and invasion. Yet, the role of CD151 in estrogen receptor positive breast cancer is not reported. In the present study, the role of CD151 in survival of ER positive cell line and the underlying molecular partners was reported. CD151 shRNA expression vector was transfected into MCF-7 cells and its efficacy was evaluated by RT-PCR. The capacity of proliferation, migration, and invasion, cell adhesion, angiogenesis of MCF-7 cells was diminished by the knockdown of CD151 via CD151 specific shRNA. It arrested the cell cycle at G2/M phase and induced the apoptosis. The expressions of c-myc, α3β1 integrin, IL-8, Ras, FAK and VEGF were reduced by knockdown of CD151. The results conclude that CD151 gene silencing affects the expression of its network partner’s associate with survival of MCF-7 cells. Therefore, CD151 may be a potential target of luminal and basal subtypes.

C-glycosyl Flavone from Urginea indica Inhibits Growth and Dissemination of Ehrlich Ascites Carcinoma Cells in Mice

BACKGROUND C-glycosyl flavone, a phytochemical constituent in U.indica bulb, has been reported to possess cytotoxic activity. OBJECTIVE The present study aims to investigate the toxicity and anticancer potentials of C-glycosyl flavone against Ehrlich ascites carcinoma mice model. METHOD In present study, acute and chronic toxicity along with antitumor activity of C-glycosyl flavone isolated from U.indica bulb were Performed using in vitro and in vivo methods. Acute and chronic toxicity of C-glycosyl flavone was evaluated using Swiss albino mice. The effect of C-glycosyl flavone on proliferation of Ehrlich ascites carcinoma (EAC) cells was determined. Further, growth inhibition and dissemination were studied using EAC induced mice model. RESULTS C-glycosyl flavone showed significant therapeutic potency against EAC cells in terms of reduced viability, cell cycle arrest, induction of apoptosis, inhibition of capillary formation, reduced VEGF levels. Moreover, there was reduction in body weight, tumor volume, viable tumor cells, increased survival of EAC induced mice upon C-glycosyl flavone treatment. Treatment also reduced dissemination of EAC cells into heart, kidney, liver and brain and diminished the pathological alterations induced by EAC cells in mice. In addition, there was an improvement in hemoglobin levels and counts of RBC, neutrophils, lymphocytes and monocytes in C-glycosyl flavone-treated mice with tumor. An enhancement of antioxidant status in C-glycosyl flavone treated EAC-bearing mice which appeared in terms of decreased serum thiobarbituric acid reactive substance and lipid peroxidation, increased GSH, SOD, Catalase and GPX. These results were comparable to a standard 5- fluorouracil treatment. C-glycosyl flavone exhibited safety profile in toxicity studies. CONCLUSION Our study confirms the therapeutic potency of C-glycosyl flavone against EAC in inhibition of dissemination and growth of EAC in mice.