Gowhar Shafi

Induction of apoptosis in HeLa cells by chloroform fraction of seed extracts of Nigella sativa

BackgroundCancer remains one of the most dreaded diseases causing an astonishingly high death rate, second only to cardiac arrest. The fact that conventional and newly emerging treatment procedures like chemotherapy, catalytic therapy, photodynamic therapy and radiotherapy have not succeeded in reverting the outcome of the disease to any drastic extent, has made researchers investigate alternative treatment options. The extensive repertoire of traditional medicinal knowledge systems from various parts of the world are being re-investigated for their healing properties. This study progresses in the direction of identifying component(s) from Nigella sativa with anti cancer acitivity. In the present study we investigated the efficacy of Organic extracts of Nigella sativa seed powder for its clonogenic inhibition and induction of apoptosis in HeLa cancer cell.ResultsMethanolic, n-Hexane and chloroform extracts of Nigella sativa seedz effectively killed HeLa cells. The IC50 values of methanolic, n-hexane, and chloroform extracts of Nigella sativa were 2.28 μg/ml, 2.20 μg/ml and 0.41 ng/ml, respectively. All three extracts induced apoptosis in HeLa cells. Apoptosis was confirmed by DNA fragmentation, western blot and terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL) assay.ConclusionWestern Blot and TUNEL results suggested that Nigella sativa seed extracts regulated the expression of pro- and anti- apoptotic genes, indicating its possible development as a potential therapeutic agent for cervical cancer upon further investigation.

Luminescent mesoporous LaVO4:Eu3+ core-shell nanoparticles: synthesis, characterization, biocompatibility and their cytotoxicity

A general and facile method was used for preparation of water-soluble silica coated LaVO4:Eu3+ core-shell nanoparticles. In the present study, we have discussed and compared the cytotoxicity characteristics of the synthesized LaVO4:Eu3+ and silica coated LaVO4:Eu3+ core-shell nanoparticles. X-ray diffraction (XRD), field-emission transmission electron microscopy (FE-TEM), energy dispersive X-ray analysis (EDX), Fourier-transform infrared spectroscopy (FTIR), UV/Vis absorption spectroscopy and photoluminescence spectroscopic techniques were employed to characterize the structure and morphology of the prepared products. To obtain high aqueous solubility, luminescent LaVO4:Eu3+ nanoparticles were encapsulated with silica groups, giving the nanoparticles a negatively charged surface at physiological pH. The results of XRD confirm the formation of a well-crystallized LaVO4:Eu3+ phase with a tetragonal zircon structure. Optical absorption spectra show that the optical properties of silica-coated LaVO4:Eu3+ core-shell nanoparticles are related to their sizes and shapes. Further, in order to assess cytotoxicity, we investigated whether the LaVO4:Eu3+ nanoparticles could alter biological samples once they enter human H522 and peripheral blood mono nuclear cells (PBMCs). An MTT assay was performed to measure the mitochondrial activity that reflects the number of viable cells. Silica coated LaVO4:Eu3+ core-shell nanoparticles exhibited no significant effect on the viability of both types of cells up to 24 h after exposure. Strikingly, no dose effects were detected, even at highest concentrations.

Differential Expression Profile and Genetic Variants of MicroRNAs Sequences in Breast Cancer Patients

The technology available for cancer diagnosis and prognosis is not yet satisfactory at the molecular level, and requires further improvements. Micro RNAs (miRNAs) have been recently reported as useful biomarkers in diseases including cancer. We performed a miRNA expression profiling study using peripheral blood from breast cancer patients to detect and identify characteristic patterns. A total of 100 breast cancer patients and 89 healthy patients were recruited for miRNA genotyping and expression profiling. We found that hs-miR-196a2 in premenopausal patients, and hs-miR-499, hs-miR-146a and hs-miR-196a2 in postmenopausal patients, may discriminate breast cancer patients from healthy individuals. In addition, we found a significant association between two microRNA polymorphisms (hs-miR-196a2 and hs-miR-499) and breast cancer risk. However, no significant association between the hs-miR-146a gene and breast cancer risk was found. In summary, the study demonstrates that peripheral blood miRNAs and their expression and genotypic profiles can be developed as biomarkers for early diagnosis and prognosis of breast cancer.

In-vitro cyto-toxicity, geno-toxicity, and bio-imaging evaluation of one-pot synthesized luminescent functionalized mesoporous SiO2@Eu(OH)(3) core-shell microspheres

UNLABELLED Luminescent functionalized mesoporous SiO2@Eu(OH)3 core-shell microspheres (LFMCSMs) were prepared by coating of europium hydroxide (Eu(OH)3) shell on mesoporous silica (SiO2) nanospheres via a facile one-pot process at low temperature. The FETEM images revealed that a well-defined luminescent europium hydroxide shell was successfully grafted on the surface of mesoporous silica nanospheres. These experimental results showed that the LFMCSM has a typical diameter of ca. 392 nm consisting of the silica core with about 230 nm in diameter and europium hydroxide shell with an average thickness of about 162 nm. LFMCSMs exhibited strong red emission peak upon irradiation with ultraviolet light, which originated from the electric-dipole transition (5)D0 → (7)F2 (614 nm) of Eu(3+) ion. The biocompatibility of the synthesized LFMCSMs was evaluated in vitro by assessing their cytotoxic and genotoxic effect on human hepatoblastoma (HepG2) cells using MTT, TUNEL, fluorescent staining, DNA ladder and Gene expression assays respectively. FROM THE CLINICAL EDITOR This paper describes the development of a one-pot synthesis of luminescent mesoporous SiO2@Eu(OH)3 core-shell microspheres and evaluates their favorable in vitro cyto-toxicity and geno-toxicity, and their applications in bio-imaging of these particles that emit bright red signal under UV exposure.

MicroRNA Signatures in Neurological Disorders

A class of small, non-coding transcripts called microRNAs (miRNAs) that play a major role in post-transcriptional gene regulation has recently emerged and become the focus of intense research. MicroRNAs are abundant in the nervous system, where they have key roles in development and are likely to be important mediators of plasticity. A highly conserved pathway of miRNA biogenesis is closely linked to the transport and translatability of mRNAs in neurons. MicroRNAs have been shown to modulate programmed cell death during development. Although there are nearly 750 known human miRNA sequences, each of only approximately 20-25 nucleotides in length that bind to multiple mRNA targets, the accurate prediction of miRNA targets seems to lie just beyond our grasp. Nevertheless, the identification of such targets promises to provide new insights into many facets of neuronal function. In this review, we briefly describe miRNA biogenesis and the principle approaches for studying the function of miRNAs and potential application of miRNAs as biomarkers, diagnostic targets, and potential therapeutic tools of human diseases in general and neurological disorders in particular.

Phosphodiesterase 4D (PDE4D) gene variants and the risk of ischemic stroke in a South Indian population.

Stroke is the third largest cause of death and a major cause of adult disability and mortality worldwide. Experimental evidence suggests that genetic determinants do contribute a large part to stroke risk. The identification of phosphodiesterase 4D gene as a risk factor for stroke caused a great deal of interest in stroke genetics. Many of the studies of PDE4D gene have focused on the original Icelandic findings but the association between specific SNPs and haplotypes has been inconsistent. The aim of the present study was to investigate the association of three SNPs 32 (rs 456009), 83 (rs 966221) and 87 (rs 2910829), originally described by deCODE group; with stroke in a South Indian population from Andhra Pradesh. Two hundred and fifty ischemic stroke patients and two hundred and fifty controls were included in the study. The stroke patients were sub typed according to TOAST classification. SNP 83 showed significant association with stroke in the population under study while SNPs 87 and 32 were monomorphic. Further SNP 83 was found to be significantly associated with two stroke subtypes, intracranial large artery atherosclerosis (the most frequent subtype in the population) and small artery occlusion. The association with other subtypes was found to be insignificant. Further, SNP 83 was found to be associated significantly with some conventional stroke risk factors like diabetes and smoking.

Mechanism of cadmium induced apoptosis in human peripheral blood lymphocytes: The role of p53, Fas and Caspase-3

Cadmium (Cd) is a major pollutant of environment. It can be fatal to human. In spite of bulk of research and literatures, the mechanism of a fatality against human is still not understood completely. Toxic and carcinogenic effects of Cd in rodents and humans are well known. However, effects of Cd on induction of apoptosis are still elusive. This study indicates immunosuppression and immunotoxicity due to Cd exposure. Present study was undertaken to determine the mechanism of cell death in vitro in human peripheral blood lymphocytes induced by Cd. Our findings suggest the toxicity due to Cd is attributed to programmed cell death-apoptosis. IC₅₀ was calculated at 21.74 μM. A significant increase of expression of the pro-apoptotic genep53, Fas and Caspase-3 in human lymphocytes was found. Cd induced p53-dependent apoptosis through cooperation between Bak upregulation without changing the Bcl-2 and Bax expression. Data of this study compel to speculate that apoptosis may also be attributed to CD95/Fas complex formation, and p53 direct apoptogenic potential at mitochondria. It was confirmed by the increased expression of Caspase-3. Although, this work does not address all the questions regarding the mechanism of Cd induced apoptosis, but these findings establish an important role of p53 and mitochondrial function during apoptosis in human lymphocyte. Moreover, based upon our findings, the role of Fas in Cd induced apoptosis is also undeniable. Hence further investigations are required to understand the different mechanism involved into apoptosis of lymphocytes due to Cd exposure.

Association of the −344C/T aldosterone synthase (CYP11B2) gene variant with hypertension and stroke

Stroke is a complex disease caused by combination of multiple risk factors. Recent findings have suggested that stroke has a significant genetic component. Various types of genetic polymorphisms have been suggested to contribute to the risk of stroke. Gene polymorphisms of renin-angiontensin aldosterone system (RAAS) have been suggested to be risk factors for hypertension, cardiovascular diseases and stroke. In the present case-control study we investigated the association of -344C/T (rs1799998) [corrected] polymorphism in the promoter region of the human aldosterone (CYP11B2) gene with genetic predisposition to hypertension, ischemic stroke and stroke subtypes classified according to TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification. Four hundred and three stroke patients (hypertensives:normotensives=219:184) and three hundred and ninety four, sex and age matched healthy controls (hypertensives:normotensives=118:276) were involved in the study. The region of interest in the CYP11B2 gene was amplified by polymerase chain reaction and genotypes determined by subjecting the PCR products to restriction digestion by the enzyme HaeIII. Significant difference was observed in the genotypic distribution and allelic frequency between the stroke patients and healthy controls. TT genotype and T allele associated significantly with hypertension and stroke (p<0.000 in hypertension and p=0.000 in case of stroke). A stepwise logistic regression analysis confirmed these findings. To establish that this polymorphism is associated with stroke independent of hypertension, we compared stroke patients without hypertension with normotensive controls. Significant difference was observed in genotypic distribution and allelic frequency between the two groups (p=0.000). Further evaluating the association of this polymorphism with stroke subtypes we found significant associations with intracranial large artery atherosclerosis, lacunar stroke and cardioembolic stroke (p=0.000 in each case). In conclusion our study suggests that -344T allele of CYP11B2 gene is an important risk factor for hypertension and ischemic stroke. However, this is a preliminary study and the results need to be confirmed in a larger cohort.

VNTR polymorphism in intron 4 of the eNOS gene and the risk of ischemic stroke in a South Indian population.

Ischemic stroke is a leading cause of death throughout the world. An increasing number of studies have suggested that genetic factors are important in the stroke risk. The aim of our study was to investigate whether the Variable Number of Tandem Repeats (VNTR) polymorphism in intron 4 of the endothelial nitric oxide synthase (eNOS) gene is associated with ischemic stroke in a South Indian population. 357 patients and 283 controls were enrolled in this case-control study. The ischemic stroke patients were classified according to TOAST classification. The eNOS gene polymorphism was determined by polymerase chain reaction-polyacrylamide gel electrophoresis. The genotypes were confirmed by sequencing the PCR products. There were significant differences in the genotype and allele frequencies of eNOS polymorphism between the patients with ischemic stroke and healthy controls (p=0.000). Multiple logistic regression analysis with forward stepwise selection using the potential confounders (sex, age, diabetes, hypertension, smoking and alcoholism) and eNOS gene variant revealed that the VNTR polymorphism in intron 4 of the eNOS gene is significantly [adjusted odds ratio=6.23, 95%CI (4.30-9.29), p=0.000] associated with ischemic stroke in the South Indian population from Andhra Pradesh. We did not find significant association of this polymorphism with any specific stroke subtype. Further hypertensives bearing 4a allele in high frequency are more predisposed to stroke.

Association of genetic variants of fibrinolytic system with stroke and stroke subtypes.

Genetic variants of tPA (PLAT) and PAI-1 genes have been suggested to be the risk factors for stroke. In the present case-control study we investigated the association of -7351C/T polymorphism (rs2020918) and I/D polymorphism of tPA gene and Insertion/deletion polymorphism (4G/5G) of PAI-1 gene with genetic predisposition to ischemic stroke. 516 stroke patients and 513, sex and age matched healthy controls were involved in the study. We did not find a significant association of tPA -7351C/T polymorphism and PAI-1 4G/5G polymorphism with stroke. However, in case of I/D polymorphism significant difference was observed in the genotypic distribution and allelic frequency between the stroke patients and healthy controls. DD genotype and D allele associated significantly with stroke (p=0.002 and <0.001 respectively). We also found significant association of I/D polymorphism with intracranial large artery atherosclerosis and stroke of undetermined etiology. Exploring the association between gene-gene interaction (26 combinations including the three variants) and stroke, we found that individuals with CC+4G4G+DD, CC+5G5G+ID, CT+4G5G+ID, CT+5G5G+II, CT+5G5G+ID and TT+4G5G+II had a significantly higher risk of stroke. The results of this study suggest that -7351C/T polymorphism of tPA and 4G/5G polymorphism of PAI-1 are not associated with stroke, while as DD genotype and D allele of tPA gene are important risk factors for ischemic stroke. Further we found that the subjects with different tPA and PAI genotype combinations displayed a significantly high risk for overall ischemic stroke suggesting that gene-gene interaction involving more variants may change the susceptibility of particular subjects to the disease.