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Objective: Human breast cancer is among one major health concerns with high prevalence and mortality among women worldwide. Various cellular signaling pathways are implicated in carcinogenesis. One of the major pathways that affect the downstream cellular growth cascades is Mevalonate pathway (MVA). The inhibition of MVA is therapeutically beneficial for various cancers. Pamidronate (PAM) (MVA inhibitor), a nitrogen-containing bisphosphosphonate, is an antiresorptive FDAapproved drug. The objective of our study was to explore adjuvant therapy using a combination of PAM and an alkylating agent, Temozolomide (TMZ) against breast cancer. Methods: We have examined the differential gene and protein expression in response to the combination treatment strategy. For gene expression analysis RT-qPCR and for proteomic study, twodimensional gel electrophoresis and mass spectrometry techniques were utilized. Results: Combination treatment (PAM+TMZ) showed more pronounced cytotoxic effect as compared to single agent treatment. Our results indicate that MVA pathway regulatory genes (FDFT1, FDPS, KRAS) are significantly (p Conclusion: The present study reveals that a combination of PAM and TMZ produces an effective anti-cancerous effect on breast cancer cells. Therefore, this novel therapeutic regimen is likely to provide a better treatment strategy for breast cancer.

Background: Hepatocellular carcinoma is a primary liver cancer and 6th most common cancer globally. Inefficient diagnostic strategies and the limited availability of treatments are the foremost reasons. Variable factors directly impact the disease burden, among them, molecular alterations have been found to play a significant role. In liver, arginosuccinate synthase-1 is a center of arginine metabolism and rate limiting enzyme of urea cycle. It also triggers multiple mechanisms that lead to HCC pathogenesis. Objectives: The aim of this study is to analyze the ASS1 gene expression, its polymorphic genotype and microsatellite instability among HCC patients from our Pakistani population. Method: Blood samples were collected from disease and healthy control individuals. Allele-Specific PCR was performed for SNP analysis. MSI of tri and tetra nucleotide repeats were analyzed by PCR. The differential expression of ASS1 gene was also investigated. Furthermore, the reactome database and STRING software were utilized for finding correlations between ASS1 gene with other associated gene/proteins. Results: The GG wild-type genotype was more prevailed in the disease group as compared to the control. Significant downregulation in ASS1 and NOS2 genes was observed. Bioinformatics analysis reveals the correlation between ASS1 polymorphism and HCC development appears to be linked with the EMT pathway and polyamine production. Furthermore, MSI significantly resided in the disease group. Results were analyzed statistically to calculate the significance of obtained results. Conclusion: Study concludes that the insight of HCC mechanism through population-specific genetic mutations and altered gene expression of ASS1 might be helpful in early diagnostic and therapeutic purposes.

Background — The mental health of medical students has long been a concern. Symptoms of depression and burnout are common among medical students, but the overlap of symptoms of both diseases with risk factors remains unclear. The goal of this study was to determine the prevalence of burnout and depression symptoms’ overlap in medical students. Methods — This cross-sectional study was conducted among students of the clinical course (3rd, 4th and 5th years). A multistage stratified random sampling design was used to select 284students that were offered a validated and structured questionnaire that included a modified Oldenburg Burnout Inventory and Patient Health Questionnaire-9 to assess their emotional burnout and depression, respectively. Chi-square and multinomial regression analyses were done to identify overlapping symptoms of burnout and depression with association of risk factors with dependent variable. Results — Out of 284 students, 13% had symptoms of severe depression, 24% had high burnout symptoms, and 32% had overlapping symptoms of burnout and depression. In regression analysis, higher academic year more than 2 times (5th year, OR 2.03, 95% CI 1.42-7.96, p-value 0.004) and living at dormitory more than 3 times (OR 3.97, 95% CI 1.97-5.01, p-value 0.003) were more likely associated with more extensive overlap of burnout and depression symptoms. Conclusion — There is a high prevalence of burnout and depression symptoms among medical students, with a strong association between the two disorders. Early burnout detection and psychiatric therapies given to affected students and these disorders management training at medical school, may help reduce negative consequences of these conditions.

Background Hepatocellular carcinoma (HCC) has a high incidence and mortality that epitomizes one of the prominent causes of cancer-related death globally. Novel therapeutic approaches are therefore required. Reactive oxygen species (ROS) are necessary for maintaining cell cycle. Although ROS is involved in HCC progression, hydrogen peroxide (H2O2) has anti-proliferative effect on HCC. Method HCC Huh-7 cells were cultured and incubated with various concentrations of H2O2. Paraoxonase activity, levels of malondialdehyde, glutathione and protein oxidation were measured in treated and untreated Huh-7 cells. Furthermore, untreated and treated Huh-7 cells were subjected to two dimensional gel electrophoresis and identified protein spots which were differentially expressed by LC-MS/MS analysis. qRT-PCR was performed to validate the identified proteins. Results H2O2 depleted glutathione (GSH) with the concomitant up-regulation of GSTP1 and Prx2. H2O2 also increased malondialdehyde and protein oxidation, decreased the activity of paraoxonase in Huh-7 cells. Conclusion H2O2 could be used as a novel therapeutic agent that might be beneficial in inducing cell cytotoxicity and hence suppress HCC proliferation.

Background: Oral Squamous Cell Carcinoma (OSCC) is a major sub-type of oral cancer that shares 90% proportion of oral cavity cancers. It is declared as the sixth most frequent cancer among all cancer types throughout the world. Higher morbidity in Asian countries is reported due to frequent use of Smokeless Tobacco (SLT) products besides exposure to other risk factors. Hyperactivation of epidermal growth factor receptors is a molecular event in many solid tumors including oral cancer making them potential therapeutic targets. Objective: Current study was designed to explore the effect of varlitinib, a pan-HER inhibitor, on oral cancer cell line. We investigated key regulatory genes in downstream pathway in response to drug treatment. Furthermore, we also examined expression profile of these genes in malignant and healthy oral tissue. Method: Gene expression pattern in drug treated and untreated cancer cell line along with OSCC tumor samples (n=45) and adjacent normal tissues was studied using real time PCR. Results: In response to varlitinib treatment, significant suppression of oncogenes (IGF1R, MAPK1, SFN and CDK2) was observed. Interestingly, mRNA expression level of CDKN1A and Akt1 was found to be the opposite of what was expected. In case of malignant tissue, over expression of oncogenes (IGF1R, Akt1, MAPK1, SFN and CDK2) with simultaneous down expression of tumor suppressor genes (Tp53 and CDKN1A) was noted. STRING analysis indicated a strong association among differentially expressed genes suggesting their combined role in carcinogenesis. Conclusion: In summary, our results indicate that varlitinib can be considered as a potential therapeutic agent in oral cancer due to its antitumor potential.

Receptor-ligand complex mediated signaling significantly contributesin cellular activities such as growth, proliferation, differentiation, and survival. However, augmented expression of signal transducing receptors and ligands is the most frequent molecular event and major hallmark of oral carcinogenesis. Among these receptors, Epidermal Growth Factor Receptor (EGFR) with intracellular tyrosine kinase activity is the most frequently overexpressed molecule by Squamous epithelial cells of oral cavity. Aberrated EGFR mediated signaling has laid the foundation of targeted therapy thus providing rationale for the conducted study. We have selected EGFR pathway as targeted intracellular signaling cascade inOral squamous cell carcinoma (OSCC). Deactivating EGFR by blocking the binding sites is likely to result in prevention of intracellular downstream signaling. In this context, Tyrosine Kinase Inhibitors (TKIs) have come into play. Quinazolines (aromatic heterocyclic compounds) and their derivatives have shown promising clinical outcomes. Present study focused to investigate anti-EGFR potential of quinazoline derivative, varlitinib-a pan-EGFR inhibitor on oral squamous epithelial cell lines. We performed proteomic analyses to identify differential expression pattern of proteins in SCC-25 cells in response to varlitinib treatment. Identified proteins include Binding Immunoglobulin Protein (BiP), Heat Shock Protein 7C (HSP7C), Protein Disulfide Isomerase 1 A (PDIA1), Vimentin (VIME), Keratin type I Cytoskeletal 14 (K1C14), and β-Actin (ACTB). Among these, five proteinswere found to be downregulated upon varlitinib treatment whereas only Keratin type I Cytoskeletal 14 was upregulated. Differential expression of proteins and possible role of varlitinib as potential antitumor drug in oral carcinoma is discussed.

Background: The increasing incidence and mortality rate of HCC is a major concern, especially for developing countries of the world. Hence, extensive research is being carried out in order to explore new approaches for developing successful therapeutic strategies for HCC. The controversial role of oxidative stress in the prognosis and treatment of various diseases such as cancer has become an area of great interest and intrigue for many scientists throughout the world. Objective: We aim to investigate the role of induced oxidative stress on the suppression of HCC Huh-7 cancerous cells as a therapeutic approach. Methods: Induction of oxidative stress via H2O2 treatment produced cell cytotoxicity in a dose dependent manner and also led to the overexpression of GSTP-1 and PRX-2. The expression of GSTP- 1 and PRX-2 was compared in HCC Huh-7 treated, untreated cells and normal hepatocytes using immunocytochemistry. Furthermore, the effects of oxidative stress on cell cycle arrest were also studied through flow cytometry. Results: Our study demonstrated the inhibition of cancer cell proliferation as a result of H2O2 induction by arresting the cell cycle at the G2 phase. Conclusion: The induction of oxidative stress could be a potential therapeutic approach for treating HCC in the future. GSTP-1 and PRX-2 can serve as substantial therapeutic targets for the treatment of HCC.

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers and has been listed as sixth most common human cancer. Due to late diagnosis and insufficient therapeutic response among patients, the survival rate remains very low accentuating the importance of early diagnostic markers. The study aimed to identify differentially expressed proteins in search for putative serum biomarkers and drug targets. Serum samples (n = 45) were depleted and resolved on two dimensional gel electrophoresis. Among differentially expressed proteins, two were identified using MALDI-TOF mass spectrometry. Gene expression levels of identified proteins were quantified in malignant and normal tissue using RT-qPCR. To validate serum Rabl3 expression, sandwich ELISA was performed. Proteomics analysis revealed two proteins which were found to be associated with oral cancer. The expression of GIMAP7 was found to be down regulated in serum of patients suffering from oral cancer while the expression of Rabl3 was found to be up-regulated. Gene expression analysis in malignant tissue and adjacent normal tissue revealed the same pattern. Quantitative ELISA was used to validate expression of Rabl3 in serum from oral cancer patients and healthy subjects which demonstrated significant up-regulation in cancer patients. Findings in current study demonstrate differential expression of novel putative biomarkers GIMAP7 and Rabl3 in oral cancer which suggests their potential role in oral cancer pathology and can be considered as predictive biomarkers.

Oral squamous cell carcinoma (OSCC) is the eight most common malignancy worldwide with an incidence rate of 40% in south-east Asia. Lack of effective diagnostic tools at early stage and disease recurrence despite extensive treatments are main reasons for high mortality and low survival rates. The aim of current study was to identify differentially expressed proteins to explore potential candidate biomarkers having diagnostic significance. We performed comparative proteomic analysis of paired protein samples (cancerous buccal mucosa and adjacent normal tissue) from OSCC patients using a combination of two dimensional gel electrophoresis and Mass spectrometric analysis. On the basis of spot intensity, seventeen proteins were found to be consistently differentially expressed among most of the samples which were identified through mass spectrometry. For validation of identified proteins, expression level of stratifin was determined using immuno-histochemistry and Western blot analysis. All identified proteins were analyzed by STRING to explore their interaction. Among uniquely identified proteins in this study, at least two candidate markers (Ig Kappa chain C region and Isoform 2 of fructose bisphosphate aldolase A) were found to be novel with respect to OSCC which can be explored further. Results presented in current study are likely to contribute in understanding the involvement of these molecules in carcinogenesis apart from their plausible role as diagnostic/prognostic markers.

Breast cancer has high incidence in women from both developed and developing countries. Approximately 2 million women are diagnosed with breast cancer in 2018. In Asia, unfortunately Pakistan leads the highest number of breast cancer patients. Various treatment strategies are present but they are not well developed. There is a great need to develop effective methods for early detection and treatment of the disease. For cancer treatment chemotherapeutic interventions have always been a method of choice. One of the mechanisms involved in cancerous cell proliferation is Mevalonate (MVA) pathway. It is hypothesized that arresting MVA pathway leads to cell death hence cancer cell growth is suppressed. Various inhibitors of MVA pathway have been studied that can suppress cell proliferation. Nitrogen containing bisphosphonates are MVA pathway inhibitor and clinically used for treatment of bone diseases. Their anticancer efficacy is also reported. Current study focuses on alendronate, a nitrogen containing bisphosphonate to examine their anticancer effect on breast cancer cell line. Results of this study may help in addition of new anticancer drug for breast cancer.

Aim: This study aims to perform differential protein expression analysis of serum samples from Oral Squamous Cell Carcinoma (OSCC) patients and healthy controls in search of potential diagnostic and/or prognostic biomarker(s). Objective: OSCC is usually diagnosed late, which results in poor survival and high mortality. Identification of non-invasive prognostic biomarkers is of utmost importance for early diagnosis and proper management of the disease; hence we used a proteomic approach to identify potential biomarkers from serum. Methods: Serum samples (OSCC n=45 and control n=30) were depleted, and proteins were separated using 2-D gel electrophoresis followed by identification by mass spectrometric analysis. Gene expression analysis of identified proteins in malignant and normal tissue was also performed to complement proteomics studies. Results: Among differentially expressed proteins, up-regulation of heat shock protein alpha (HSP90α) from the serum of oral cancer patients was observed. We also observed elevated levels of Haptoglobin (HP) along with downregulation of Type II keratin cytoskeletal 1(KRT1) and serum albumin (ALB) in oral cancer patients. Gene expression studies on identified proteins in malignant and normal tissue revealed a similar pattern with the exception of KRT1. We believe that elevated levels of serum HSP90 alpha might be used as a potential biomarker. Conclusion: Our findings suggest a contribution of HSP90 alpha and other identified proteins in oral pathology as pro/anti-apoptotic modulators, thus considering their potential as predictive biomarkers.

Mesenchymal stem cells (MSCs) have multi-lineage differentiation potential which make them an excellent source for cell-based therapies. Histone modification is one of the major epigenetic regulations that play central role in stem cell differentiation. Keeping in view their ability to maintain gene expression essential for successful differentiation, it was interesting to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, in the hepatic differentiation of MSCs within the 3D scaffold. MSCs were treated with the optimized concentration of VPA in the 3D collagen scaffold. Analyses of hepatic differentiation potential of treated MSCs were performed by qPCR, immunostaining and periodic acid Schiff assay. Our results demonstrate that MSCs differentiate into hepatic-like cells when treated with 5 mM VPA for 24 h. The VPA-treated MSCs have shown significant upregulation in the expression of hepatic genes, CK-18 (P

Objective To assess the frequency of anatomical variations of pancreatic duct through magnetic resonance cholangiography pancreatography. . Methods The cross-sectional prospective study was conducted from May 2011 to December 2012 at the Department of Anatomy, Institute of Basic Medical Sciences (IBMS), Dow University of Health Sciences, Karachi, in collaboration with the Department of Radiology, Aga Khan University Hospital, Karachi. The study comprised diagnosed cases of pancreato-biliary disease booked for magnetic resonance cholangiography pancreatography. Images were obtained using 1.5 Tesla magnetic resonance imaging scanner, and information of anatomical variations visualised on the imaging film was assessed and documented. Results Of the 377 subjects, 196(52%) were females and 181(48%) were males. Pancreas divisum was found in 21(5.6%) subjects; 13(62%) females and 8(38%) males. . Duct of Santorini was detected in 3(0.8%) subjects; 2(66.6%) females and 1(33.3%) male. Conclusions Variations in pancreatic duct could be identified by using the simple, non-invasive method of magnetic resonance cholangiography pancreatography.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer related deaths around the world. Due to late diagnosis and development of drug resistance in patients suffering from HCC, development of more effective therapeutic strategies is inevitable. The aim of this study was to evaluate the combined apoptotic effect of 5'-Azacytidine (5'-AzaC) and alendronate (ALN) on Huh-7 HCC cell line and to explore differential expression at genomics and proteomics level. Incubation of HCC cell line with 5'-AzaC alone showed cell death in a time and dose dependent manner while in combination with ALN, increased cytotoxicity was observed. Up-regulation of CASP7(Caspase7) and LZTS1 (leucine zipper, putative tumor suppressor 1) and down-regulation of DNMT1(DNA (cytosine-5-)-methyltransferase 1) was noted in treated cells. Proteomic studies on the treated cells revealed altered expression of different proteins including peroxiredoxin 2 (Prx2), Annexin 5 (Anx5), Rho GTPase activating protein (RhoGAP), Nuclear factor-kappa B (NF-kB), tumor necrosis factor alpha-induced protein (TNF), triosephosphate isomerase (TPI), Glutathione S transferase (GSTP1) and Heat shock protein60 (HSP60). Our study demonstrated the cytotoxic effect of 5'-AzaC and ALN drug combination on Huh-7 HCC cells suggesting such combinations may be explored as a possible therapeutic approach. Current study revealed that Huh-7 HCC cells are sensitive to 5'-AzaC and ALN drug combination and such combination approaches could lead to the development of new therapeutic strategies. Furthermore, we also report the expression of Anx5 exclusively in untreated cancerous cell line indicating the possibility of being used as a potential therapeutic target and biomarker.

Background An important product of mevalonate pathway is downstream synthesis of isoprenoid units that has long been implicated in development and progression of tumor. It has been speculated that inhibition of protein prenylation might be therapeutically beneficial. The objective of current study was to evaluate antitumor potential of a novel therapeutic combination of mevalonate pathway inhibitors, FTI-277 and alendronate. We also examined differentially expressed proteins in response to treatment using proteomics approach. Methods Huh-7 cells were incubated with different concentrations of FTI-277 alone and in combination with alendronate. Differential protein and gene expression was examined through two dimensional gel electrophoresis and real-time quantitative polymerase chain reaction (qPCR), respectively. Proteins were identified using tandem mass spectrometry analysis. Results Treatment of hepatocellular carcinoma (HCC) cell line with FTI-277 alone showed cell death in a time and dose dependent manner while in combination with alendronate, a synergistic apoptotic effect at 24 h was observed. Proteomic studies on the 20 µmol/L FTI-277 and 5 µmol/L alendronate +20 µmol/L FTI-277 treated cells revealed altered expression of different proteins including peroxiredoxin 2 (Prx2), glutathione S transferase 1 (GSTP1), Rho GTPase activating protein (RhoGAP), triosephosphate isomerase (TPI), and heat shock protein 60 (HSP60). Down-regulated expression of Prx2 and GSTP1 in treated cells was also confirmed by real-time qPCR analysis. Conclusions Combined treatment of FTI-277 and alendronate on Huh-7 HCC cells showed cell death suggesting their anticancer potential. Such treatment approaches are likely to offer new therapeutic strategies.

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that is believed to be involved in the protection against oxidative stress. There is evidence that paraoxonase activity is reduced in patients with diabetes and cataract. In the current study, we analyzed mRNA expression of PON1 as well as other members of the paraoxonase family, PON2 and PON3, in human cataractous lens samples. Our results indicate that only PON1 is expressed at the gene and protein levels in human lens tissues. We quantified MDA levels and measured PON1 (paraoxonase/arylesterase) enzymatic activities in subjects suffering from cataract due to aging and diabetes. Decreased PON1 activity was more pronounced in diabetic patients (p0.001) compared to senile subjects, which may be due to glycation and increased oxidative insult. To examine the structural alterations that occur in response to glycation, we constructed a three-dimensional model of PON1 and its glycated variant. Glycation at Lys70 and Lys75 is predicted to cause hindrance in binding of substrate to the active site of the enzyme.

Taxon specific lens crystallins in vertebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uromastyx hardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in U. hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure–function relationship of these proteins.