Sanjeev K. Srivastava

Notch1 regulates the functional contribution of RhoC to cervical carcinoma progression

Background:The role of Notch signalling in human epithelial cancers is of immense interest. In this study, we examine the interplay between Notch signalling and RhoC, a well-established molecular factor in metastasis. By linking the function of Notch and RhoC, we further strengthen the notion that there is a pro-oncogenic role of Notch signalling in human cervical cancers.Methods:RhoC protein expression in cervical carcinoma cell lines was assessed by western blotting. Using CaSki and SiHa cells (cervical carcinoma cells lines), we show that RhoC contributes to wound healing, invasion and migration, anoikis resistance, colony formation, in vitro tube formation and tumour formation. Immunohistochemical studies were carried out to assess the co-expression of RhoC, pAkt and Notch1 in clinical sections.Results:An assessment of the changes associated with epithelial-to-mesenchymal transition (EMT) shows that both Notch1 and RhoC have similar phenotypic contribution to EMT. Rho activity assessment on Notch1 inhibition with DAPT shows decreased RhoC activity. We further show that constitutively active RhoC rescues the phenotypic effect of Notch1 inactivation, and a comparison of Notch1 with RhoC expression shows an overlap between the two proteins in the same areas of the tissue.Conclusion:This study has provided evidence to suggest that RhoC is an effector of Notch1 in cervical carcinoma.

MicroRNA-345 induces apoptosis in pancreatic cancer cells through potentiation of caspase-dependent and -independent pathways.

Background:Previously, miR-345 was identified as one of the most significantly downregulated microRNAs in pancreatic cancer (PC); however, its functional significance remained unexplored.Methods:miR-345 was overexpressed in PC cells by stable transfection, and its effect on growth, apoptosis and mitochondrial-membrane potential was examined by WST-1, Hoechst-33342/Annexin-V, and JC-1 staining, respectively. Gene expression was examined by quantitative reverse-transcription-PCR and/or immunoblotting, and subcellular fractions prepared and caspase-3/7 activity determined by commercially available kits. miR-345 target validation was performed by mutational analysis and luciferase-reporter assay.Results:miR-345 is significantly downregulated in PC tissues and cell lines relative to normal pancreatic cells, and its expression decreases gradually in PC progression model cell lines. Forced expression of miR-345 results in reduced growth of PC cells because of the induction of apoptosis, accompanied by a loss in mitochondrial membrane potential, cytochrome-c release, caspases-3/7 activation, and PARP-1 cleavage, as well as mitochondrial-to-nuclear translocation of apoptosis-inducing factor. These effects could be reversed by the treatment of miR-345-overexpressing PC cells with anti-miR-345 oligonucleotides. BCL2 was characterised as a novel target of miR-345 and its forced-expression abrogated the effects of miR-345 in PC cells.Conclusions:miR-345 downregulation confers apoptosis resistance to PC cells, and its restoration could be exploited for therapeutic benefit.

Exosomes confer chemoresistance to pancreatic cancer cells by promoting ROS detoxification and miR-155-mediated suppression of key gemcitabine-metabolising enzyme, DCK.

Background:Chemoresistance is a significant clinical problem in pancreatic cancer (PC) and underlying molecular mechanisms still remain to be completely understood. Here we report a novel exosome-mediated mechanism of drug-induced acquired chemoresistance in PC cells.Methods:Differential ultracentrifugation was performed to isolate extracellular vesicles (EVs) based on their size from vehicle- or gemcitabine-treated PC cells. Extracellular vesicles size and subtypes were determined by dynamic light scattering and marker profiling, respectively. Gene expression was examined by qRT-PCR and/or immunoblot analyses, and direct targeting of DCK by miR-155 was confirmed by dual-luciferase 3′-UTR reporter assay. Flow cytometry was performed to examine the apoptosis indices and reactive oxygen species (ROS) levels in PC cells using specific dyes. Cell viability was determined using the WST-1 assay.Results:Conditioned media (CM) from gemcitabine-treated PC cells (Gem-CM) provided significant chemoprotection to subsequent gemcitabine toxicity and most of the chemoresistance conferred by Gem-CM resulted from its EVs fraction. Sub-fractionation grouped EVs into distinct subtypes based on size distribution and marker profiles, and exosome (Gem-Exo) was the only sub-fraction that imparted chemoresistance. Gene expression analyses demonstrated upregulation of SOD2 and CAT (ROS-detoxifying genes), and downregulation of DCK (gemcitabine-metabolising gene) in Gem-Exo-treated cells. SOD/CAT upregulation resulted, at least in part, from exosome-mediated transfer of their transcripts and they suppressed basal and gemcitabine-induced ROS production, and partly promoted chemoresistance. DCK downregulation occurred through exosome-delivered miR-155 and either the functional suppression of miR-155 or restoration of DCK led to marked abrogation of Gem-Exo-mediated chemoresistance.Conclusions:Together, these findings establish a novel role of exosomes in mediating the acquired chemoresistance of PC.

Development and Characterization of a Novel in vitro Progression Model for UVB-Induced Skin Carcinogenesis

Epidemiological studies suggest ultraviolet B (UVB) component (290–320 nm) of sun light is the most prevalent etiologic factor for skin carcinogenesis- a disease accounting for more than two million new cases each year in the USA alone. Development of UVB-induced skin carcinoma is a multistep and complex process. The molecular events that occur during UVB-induced skin carcinogenesis are poorly understood largely due to the lack of an appropriate cellular model system. Therefore, to make a progress in this area, we have developed an in vitro model for UVB-induced skin cancer using immortalized human epidermal keratinocyte (HaCaT) cells through repetitive exposure to UVB radiation. We demonstrate that UVB-transformed HaCaT cells gain enhanced proliferation rate, apoptosis-resistance, and colony- and sphere-forming abilities in a progressive manner. Moreover, these cells exhibit increased aggressiveness with enhanced migration and invasive potential and mesenchymal phenotypes. Furthermore, these derived cells are able to form aggressive squamous cell carcinoma upon inoculation into the nude mice, while parental HaCaT cells remain non-tumorigenic. Together, these novel, UVB-transformed progression model cell lines can be very helpful in gaining valuable mechanistic insight into UVB-induced skin carcinogenesis, identification of novel molecular targets of diagnostic and therapeutic significance, and in vitro screening for novel preventive and therapeutic agents.

Gemcitabine triggers angiogenesis-promoting molecular signals in pancreatic cancer cells: Therapeutic implications

Pancreatic tumor microenvironment (TME) is characterized by poor tumor-vasculature and extensive desmoplasia that together contribute to poor response to chemotherapy. It was recently shown that targeting of TME to inhibit desmoplasiatic reaction in a preclinical model resulted in increased microvessel-density and intratumoral drug concentration, leading to improved therapeutic response. This approach; however, failed to generate a favorable response in clinical trial. In that regard, we have previously demonstrated a role of gemcitabine-induced CXCR4 signaling as a counter-defense mechanism, which also promoted invasiveness of pancreatic cancer (PC) cells. Here, we investigated the effect of gemcitabine on endothelial cell phenotype. Gemcitabine-treatment of human-umbilical-vein-endothelial-cells (HUVECs) did not promote the growth of HUVECs; however, it was induced when treated with conditioned media from gemcitabine-treated (Gem-CM) PC cells due to increased cell-cycle progression and apoptotic-resistance. Moreover, treatment of HUVECs with Gem-CM resulted in capillary-like structure (CLS) formation and promoted their ability to migrate and invade through extracellular-matrix. Gemcitabine-treatment of PC cells induced expression of various growth factors/cytokines, including IL-8, which exhibited greatest upregulation. Further, IL-8 depletion in Gem-CM diminished its potency to promote angiogenic phenotypes. Together, these findings suggest an indirect effect of gemcitabine on angiogenesis, which, in light of our previous observations, may hold important clinical significance.

Ion velocity, charge state and substrate dependent electronic sputtering of fullerene

Abstract In order to understand electronic sputtering phenomenon, a systematic study is performed by bombarding fullerene (C 60 ) thin films deposited on Si and glass substrates with Au and Ag ions of different energies. The incident ion velocity, charge state and substrate dependent electronic sputtering yield (sputtered atoms/ion) of C is measured on-line by elastic recoil detection analysis (ERDA) technique in the three different sets of studies. The salient features observed in these experiments are that the yield is quite high (∼10 3 atoms/ion and above), which is influenced by the above-mentioned three physical parameters. Slower ion having same electronic energy deposition ( S e ) as compared to its high velocity counterpart erodes more. C 60 films deposited on more insulating substrate (electrically and thermally: glass) shows higher sputtering yield as compared to those deposited on Si substrate. However, no charge state effect was observed in the electronic sputtering yield within the detection limit of the set up.

Conversion Electron Mössbauer Study of Mixing Induced by Swift Heavy Ions at the Fe/Si Interface

Conversion Electron Mössbauer Spectroscopy (CEMS) was employed in order to study the mixing induced by swift Au and Ag ions at the Fe/Si interface in the Fe/Fe57/Si system. An increase in the amount of mixing with ion fluence and electronic energy loss (Se) has been observed. Atomic Force Microscopy (AFM) results provided indirect evidence to support the above observations.

Honokiol Suppresses Pancreatic Tumor Growth, Metastasis and Desmoplasia by Interfering with Tumor-Stromal Cross-Talk

The poor clinical outcome of pancreatic cancer (PC) is largely attributed to its aggressive nature and refractoriness to currently available therapeutic modalities. We previously reported antitumor efficacy of honokiol (HNK), a phytochemical isolated from various parts of Magnolia plant, against PC cells in short-term in vitro growth assays. Here, we report that HNK reduces plating efficiency and anchorage-independent growth of PC cells and suppresses their migration and invasiveness. Furthermore, significant inhibition of pancreatic tumor growth by HNK is observed in orthotopic mouse model along with complete-blockage of distant metastases. Histological examination suggests reduced desmoplasia in tumors from HNK-treated mice, later confirmed by immunohistochemical analyses of myofibroblast and extracellular matrix marker proteins (α-SMA and collagen I, respectively). At the molecular level, HNK treatment leads to decreased expression of sonic hedgehog (SHH) and CXCR4, two established mediators of bidirectional tumor-stromal cross-talk, both in vitro and in vivo . We also show that the conditioned media (CM) from HNK-treated PC cells have little growth-inducing effect on pancreatic stellate cells (PSCs) that could be regained by the addition of exogenous recombinant SHH. Moreover, pretreatment of CM of vehicle-treated PC cells with SHH-neutralizing antibody abolishes their growth-inducing potential on PSCs. Likewise, HNK-treated PC cells respond poorly to CM from PSCs due to decreased CXCR4 expression. Lastly, we show that the transfection of PC cells with constitutively active IKKβ mutant reverses the suppressive effect of HNK on nuclear factor-kappaB activation and partially restores CXCR4 and SHH expression. Taken together, these findings suggest that HNK interferes with tumor-stromal cross-talk via downregulation of CXCR4 and SHH and decreases pancreatic tumor growth and metastasis.

Electronic structure and magnetic properties of 3d impurities in antiferromagnetic Cr

Using the full-potential linearized augmented plane wave (FLAPW) method based on the density function theory (DFT) we have performed ab initio calculations of magnetic properties for dilute Cr–X alloys with 3d transition metals. We present here our results for the density of states, local magnetic moments and hyperfine fields. The paramagnetic local density of states (LDOS) analyzed with the Stoner model suggests the occurrence of local moments for Mn, Fe, Co and Ni consistent with results obtained from spin polarized calculations. In addition, we find a large magnetic moment for the non-magnetic V atom in Cr. The impurity moments of Sc, Ti, V and Mn are found to be antiparallel with respect to the near neighbor Cr moment while, they show ferromagnetic coupling for Fe, Co, and Ni. The observed sign change for the exchange coupling is also reflected in our calculated hyperfine field results. The trend observed for the variation of the impurity moment and hyperfine fields of 3d impurities in antiferromagnetic Cr show remarkable similarity with the results reported for ferromagnetic Fe, Co and Ni hosts. The results presented here would be helpful for a common understanding of local magnetic properties of transition metal impurities in ferro and antiferromagnetic hosts.

Deep sequencing and in silico analyses identify MYB-regulated gene networks and signaling pathways in pancreatic cancer

We have recently demonstrated that the transcription factor MYB can modulate several cancer-associated phenotypes in pancreatic cancer. In order to understand the molecular basis of these MYB-associated changes, we conducted deep-sequencing of transcriptome of MYB-overexpressing and -silenced pancreatic cancer cells, followed by in silico pathway analysis. We identified significant modulation of 774 genes upon MYB-silencing (p < 0.05) that were assigned to 25 gene networks by in silico analysis. Further analyses placed genes in our RNA sequencing-generated dataset to several canonical signalling pathways, such as cell-cycle control, DNA-damage and -repair responses, p53 and HIF1α. Importantly, we observed downregulation of the pancreatic adenocarcinoma signaling pathway in MYB-silenced pancreatic cancer cells exhibiting suppression of EGFR and NF-κB. Decreased expression of EGFR and RELA was validated by both qPCR and immunoblotting and they were both shown to be under direct transcriptional control of MYB. These observations were further confirmed in a converse approach wherein MYB was overexpressed ectopically in a MYB-null pancreatic cancer cell line. Our findings thus suggest that MYB potentially regulates growth and genomic stability of pancreatic cancer cells via targeting complex gene networks and signaling pathways. Further in-depth functional studies are warranted to fully understand MYB signaling in pancreatic cancer.