Phosphoproteome of signaling by ErbB2 in ovarian cancer cells.

The gene for receptor tyrosine kinase ErbB2 is amplified in breast and ovarian tumours. The linear pathway by which signals are transduced through ErbB2 are well known. However, second generation questions that address spatial aspects of signaling remain. To address this, we have undertaken a mass spectrometry approach to identify phosphoproteins specific for ErbB2 using the inhibitors Lapatinib and CP724714 in ovarian cancer cells. The ErbB2 specific proteins identified in SKOV-3 cells were Myristoylated alanine-rich C-kinase substrate, Protein capicua homolog, Protein peptidyl isomerase G, Protein PRRC2C, Chromobox homolog1 and PRP4 homolog. We have evaluated three phosphoproteins PKM2, Aldose reductase and MARCKS in SKOV-3 cells. We observed that PKM2 was phosphorylated by EGF but was not inhibited by Lapatinib and CP724714. The activity of aldose reductase in reducing NADPH as a substrate was significantly higher in EGF stimulated cells which was inhibited by Lapatinib and CP724714 but not by Geftinib (EGFR inhibitor). MARCKS was phosphorylated on stimulation of SKOV-3 cells with EGF that was inhibited by Lapatinib and CP724714 which was dependent on the kinase activity of ErbB2. These results have identified phosphoproteins that are specific to ErbB2 which have not been previously reported and sets the basis for future experiments.

Induced pluripotent stem cells: A new strategy to model human cancer.

Induced pluripotent stem cells are derived from adult somatic cells by ectopic expression of stem cell factors OCT4, SOX2, MYC and KLF4. These cells have characteristic features similar to embryonic stem cells. Although there exists in vitro and in vivo models of cancer, recapitulating the earliest events in the pathogenesis remain challenging. More recently, induced pluripotent stem cells have been generated to model human disease and cancer. There are advantages in the cancer models derived from these cells as compared to existing conventional approaches. Induced pluripotent stem cells have been generated from cancer cell lines, primary tumours and from those with an inherited predisposition to develop cancer. In addition, these cells provide a valuable tool in understanding the pathogenesis of familial cancer in its earliest stages, and to identify additional genetic alterations that are required to develop cancer. Furthermore, these cells can serve as a resource in drug screening and developing new therapies.

Spontaneous Medial Cribriform CSF Leak: Endoscopic Surgical Repair with Free Mucosal Graft-Our Experience.

Cribriform plate is the commonest site of spontaneous CSF leak, the fragility of the plate and juxtaposition of arachnoid's investment to the bone, where the olfactory nerve pierces the skull made this area, a vulnerable site for CSF leak. Transnasal endoscopic approach has gained popularity for CSF leak repair over the years. To describe the 5 year experience of spontaneous medial cribriform CSF leak repair with free mucosal graft in a tertiary medical centre. All patients who underwent transnasal endoscopic repair with free mucosal graft for spontaneous medial cribriform CSF leak in our institution between 2011 and 2016 were reviewed. Twelve patients were identified, all were women with a mean age of 44.5 years. The defect was localised by preoperative computed tomography scans with 1 mm cuts and MR cisternography. Via medial approach, the mucosa surrounding the entire defect was denuded and the defect was closed with free mucosal graft harvested either from the middle turbinate or from the nasal septum and middle turbinate was finally sutured with septum to stabilise the repair. The overall success rate was 100% with the first attempt with no recurrence or postoperative complications. Follow up ranged from 1 to 5 years. The endoscopic transnasal technique with free mucosal graft for the repair of spontaneous medial cribriform CSF rhinorrhoea is associated with a very high success rate and it should be considered for majority of cases.

In Vitro Evaluation of the Antioxidant, 3,5-Dihydroxy-4-ethyl-trans-stilbene (DETS) Isolated from Bacillus cereus as a Potent Candidate against Malignant Melanoma.

3,5-dihydroxy Q1 -4-ethyl-trans-stilbene (DETS) is a natural stilbene, which was first identified as bioactive bacterial secondary metabolite isolated from Bacillus cereus associated with a rhabditid entomopathogenic nematode. The present study was intended to investigate the antioxidant and anticancer activity of this compound in vitro. Antioxidant activity was investigated by assaying DPPH free radical scavenging, superoxide radical-(O2..) scavenging, hydroxyl radical scavenging and metal chelating activity, which proved that the compound is a powerful antioxidant. The metal chelating activity of DETS was higher than butylated hydroxyanisol (BHA) and gallic acid, two well-known antioxidants. As the molecule exhibited strong antioxidant potential, it was further evaluated for cytotoxic activity toward five cancer cells of various origins. Since the compound has a strong structural similarity with resveratrol (trans- 3,4,5-trihydroxystilbene), a well-studied chemopreventive polyphenolic antioxidant, its anticancer activity was compared with that of resveratrol. Among the five cancer cells studied, the compound showed maximum cytotoxicity toward the human melanoma cell line, [A375, IC50: 24.01 µM] followed by cervical [HeLa-46.17 µM], colon [SW480- 47.28 µM], liver [HepG2- 69.56 µM] and breast [MCF-7- 84.31 µM] cancer cells. A375 was much more sensitive to DETS compared to the non-melanoma cell line, A431, in which the IC50 of the compound was more than double (49.60 µM). In the present study, the anticancer activity of DETS against melanoma was confirmed by various apoptosis assays. We also observed that DETS, like resveratrol, down-regulates the expression status of major molecules contributing to melanoma progression, such as BRAF, ß-catenin and Brn-2, all of which converge in MITF-M, the master regulator of melanoma signaling. The regulatory role of MITF-M in DETS-induced cytotoxicity in melanoma cells was confirmed by comparing the cytotoxicity of DETS in A375 cells (IC50-24.01 µM), with that in SK-MEL-2 (IC50-67.6 µM), another melanoma cells which highly over-express MITF-M. The compound arrests the cells at S-G2 transition state of the cell cycle, as resveratrol. Our results indicate that DETS is a powerful antioxidant, having anticancer efficacy comparable with that of resveratrol, and is a potential candidate to be explored by in vivo studies and in-depth mechanistic evaluation. To our knowledge, this is the first report on the antioxidant and anticancer properties of DETS.

DW-F5: A novel formulation against malignant melanoma from Wrightia tinctoria.

Wrightia tinctoria is a constituent of several ayurvedic preparations against skin disorders including psoriasis and herpes, though not yet has been explored for anticancer potential. Herein, for the first time, we report the significant anticancer properties of a semi-purified fraction, DW-F5, from the dichloromethane extract of W. tinctoria leaves against malignant melanoma. DW-F5 exhibited anti-melanoma activities, preventing metastasis and angiogenesis in NOD-SCID mice, while being non-toxic in vivo. The major pathways in melanoma signaling mediated through BRAF, WNT/ß-catenin and Akt-NF-κB converging in MITF-M, the master regulator of melanomagenesis, were inhibited by DW-F5, leading to complete abolition of MITF-M. Purification of DW-F5 led to the isolation of two cytotoxic components, one being tryptanthrin and the other being an unidentified aliphatic fraction. The overall study predicts Wrightia tinctoria as a candidate plant to be further explored for anticancer properties and DW-F5 as a forthcoming drug formulation to be evaluated as a chemotherapeutic agent against malignant melanoma.

Tolerance of ARPE 19 cells to organophosphorus pesticide chlorpyrifos is limited to concentration and time of exposure.

Age related macular degeneration is a blinding disease common in elder adults. The prevalence of age related macular degeneration has been found to be 1.8% in the Indian population. Organophosphates are widely used insecticides with well documented neurological effects, and the persistent nature of these compounds in the body results in long term health effects. Farmers exposed to organophosphorus pesticides in USA had an earlier onset of age related macular degeneration when compared to unexposed controls. A recent study found significant levels of an organophosphate, termed chlorpyrifos, in the blood samples of Indian farmers. Therefore, in understanding the link between age related macular degeneration and chlorpyrifos, the need for investigation is important. Our data show that ARPE-19 (retinal pigment epithelial cells) exhibit a cytoprotective response to chlorpyrifos as measured by viability, mitochondrial membrane potential, superoxide dismutase activity, and increased levels of glutathione peroxidase and reduced glutathione, after 24 h exposure to chlorpyrifos. However, this cytoprotective response was absent in ARPE-19 cells exposed to the same range of concentrations of chlorpyrifos for 48 h. These results have physiological significance, since HPLC analysis showed that effects of chlorpyrifos were mediated through its entry into ARPE-19 cells. HPLC analysis also showed that chlorpyrifos remained stable, as we recovered up to 80% of the chlorpyrifos added to 6 different ocular tissues.

Resveratrol chemosensitizes HER-2-overexpressing breast cancer cells to docetaxel chemoresistance by inhibiting docetaxel-mediated activation of HER-2-Akt axis.

As breast cancer cells often develop chemoresistance, better therapeutic options are in search to circumvent it. Here we demonstrate that human epidermal growth factor receptor-2 (HER-2)-overexpressing breast cancer cells resist docetaxel-induced cytotoxicity by upregulating HER-2 and its activity downstream, through Akt and mitogen-activated protein kinase (MAPK) pathways. We observed that introducing resveratrol as a chemosensitizer in docetaxel chemotherapy blocks upregulation and activation of HER-2 in addition to blocking downstream signaling pathways such as Akt. Resveratrol and docetaxel combination results in the synergistic induction of cell death in HER-2-overexpressing SK-BR-3 cells, whereas introduction of wild-type HER-2 in MDA-MD-231 cells increased the resistance to docetaxel. Dominant-negative HER-2 sensitizes SK-BR-3 cells to docetaxel. Our study identified a new synergistic therapeutic combination that targets HER-2-induced breast cancer resistance and might help to overcome therapeutic resistance during breast cancer therapy. The synergism of docetaxel and resveratrol was maximum in SK-BR-3, which is unique among the cell lines studied, due to its high expression status of HER-2, a receptor known to dictate the signaling environment of breast cancer cells. Docetaxel could further induce HER-2 activity in these cells, which was downregulated on resveratrol treatment. Transfection of DN-HER-2 in SK-BR-3 cells inhibits the synergism as the transfection itself sensitizes these cells to docetaxel, leaving no role for resveratrol, whereas ectopic expression of HER-2 introduces the synergism in MDA-MB-231, the triple-negative cell line, in which the synergism was minimum, attesting the crucial role of HER-2 in suppressing the sensitivity to docetaxel. Single-agent docetaxel induced HER-2-mediated resistance to cell death, which was blocked by resveratrol. Resveratrol also downregulated docetaxel-induced activation of MAPK and Akt, survival signaling pathways downstream of HER-2. In short, this study, for the first time, establishes the role of HER-2-Akt signaling axis in regulating the synergistic effect of docetaxel and resveratrol in breast cancer cells overexpressing HER-2.

Indigenously produced polyhydroxyalkanoate based co-polymer as cellular supportive biomaterial.

The focus of this study was to evaluate the growth of the cells on a scaffold based on novel polyhydroxyalkanoate (PHA) (Polyhydroxy propionate copoly hydroxy ocatadecanoate copolymer), derived from a mutant strain of Pseudomonas sp. Naive PHA was also blended with several biodegradable polymeric materials (PEG, PLA, and MMT) to improve the scaffold properties. Protein adsorption study was done to evaluate the capability of scaffolds for cellular interaction. PHA:PEG blended scaffold showed better adsorption than others. 3T3 fibroblast cultures on various polymers were equally viable when compared with control culture except for the blend PHA:MMT by CCK 8 kit. MTT assay, performed with the continuous cultures HeLa, HEp-2, Vero, and McCoy on the polymer blends, supported the above finding. Among the blends PHA:PEG showed increased viability and was selected for further studies. Cell proliferation assay with colorimetric BrdU ELISA kit showed increase in cell proliferation over the matrix PHA:PEG than that of control. There were no observable morphological changes of continuous cells grown over matrix PHA:PEG when observed by phase contrast microscopy. HEp-2 cells were enclosed within the matrix when analyzed by SEM. The current study states that the scaffold prepared by using the indigenous PHA in combination with PEG supports cell growth better than the conventional plastic surface. PHA:PEG would be a promising material for tissue engineering.