Anand Ramteke

Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules

Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer‐associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2) or normoxic (21% O2) conditions, and exosomes secreted under hypoxic (ExoHypoxic) and normoxic (ExoNormoxic) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that ExoHypoxic have smaller average size as compared to ExoNormoxic. Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in ExoHypoxic compared to ExoNormoxic. Co‐culturing with ExoHypoxic increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. ExoHypoxic also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α‐SMA (a CAF biomarker) expression in PrSC. Compared to ExoNormoxic, ExoHypoxic showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF‐β2, TNF1α, IL6, TSG101, Akt, ILK1, and β‐catenin). Furthermore, proteome analysis revealed a higher number of proteins in ExoHypoxic (160 proteins) compared to ExoNormoxic (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, ExoHypoxic targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that ExoHypoxic are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness.

SNAI1 is critical for the aggressiveness of prostate cancer cells with low E-cadherin.

BackgroundA better molecular understanding of prostate carcinogenesis is warranted to devise novel targeted preventive and therapeutic strategies against prostate cancer (PCA), a major cause of mortality among men. Here, we examined the role of two epithelial-to-mesenchymal transition (EMT) regulators, the adherens junction protein E-cadherin and its transcriptional repressor SNAI1, in regulating the aggressiveness of PCA cells.MethodsThe growth rate of human prostate carcinoma PC3 cells with stable knock-down of E-cadherin (ShEC-PC3) and respective control cells (Sh-PC3) was compared in MTT and clonogenic assays in cell culture and in nude mouse xenograft model in vivo. Stemness of ShEC-PC3 and Sh-PC3 cells was analyzed in prostasphere assay. Western blotting and immunohistochemistry (IHC) were used to study protein expression changes following E-cadherin and SNAI1 knock-down. Small interfering RNA (siRNA) technique was employed to knock- down SNAI1 protein expression in ShEC-PC3 cells.ResultsShEC-PC3 cells exerted higher proliferation rate both in cell culture and in athymic nude mice compared to Sh-PC3 cells. ShEC-PC3 cells also formed larger and a significantly higher number of prostaspheres suggesting an increase in the stem cell-like population with E-cadherin knock-down. Also, ShEC-PC3 prostaspheres disintegration, in the presence of serum and attachment, generated a bigger mass of proliferating cells as compared to Sh-PC3 prostaspheres. Immunoblotting/IHC analyses showed that E-cadherin knock-down increases the expression of regulators/biomarkers for stemness (CD44, cleaved Notch1 and Egr-1) and EMT (Vimentin, pSrc-tyr416, Integrin β3, β-catenin, and NF-κB) in cell culture and xenograft tissues. The expression of several bone metastasis related molecules namely CXCR4, uPA, RANKL and RunX2 was also increased in ShEC-PC3 cells. Importantly, we observed a remarkable increase in SNAI1 expression in cytoplasmic and nuclear fractions, prostaspheres and xenograft tissues of ShEC-PC3 cells. Furthermore, SNAI1 knock-down by specific siRNA strongly inhibited the prostasphere formation, clonogenicity and invasiveness, and decreased the level of pSrc-tyr416, total Src and CD44 in ShEC-PC3 cells. Characterization of RWPE-1, WPE1-NA22, WPE1-NB14 and DU-145 cells further confirmed that low E-cadherin is associated with higher SNAI1 expression and prostasphere formation.ConclusionsTogether, these results suggest that E-cadherin loss promotes SNAI1 expression that controls the aggressiveness of PCA cells.

Preparation and evaluation of the effect of particle size on the properties of chitosan-montmorillonite nanoparticles loaded with isoniazid

In this report, efforts have been made to develop isoniazid loaded chitosan-montmorillonite nanoparticles by ionic gelation of chitosan with pentasodium tripolyphosphate. The nanoparticles have been characterized by FTIR, XRD, SEM and TEM. The effect of surfactant and particle size on chitosan nanoparticles have been assessed with regard to swelling, encapsulation efficiency and release of isoniazid in different mediums. Swelling experiments provide important information on drug diffusion properties, which indicates that the chitosan nanoparticles are highly sensitive to the pH environment. The drug release mechanism has been studied during different time periods using a UV-visible spectrophotometer. Cytotoxicity has been assessed by MTT assay analysis. Mucoadhesion properties have been appraised by an in vitro wash off test and an ex vivo mucoadhesion test. The results imply that chitosan-montmorillonite nanoparticles can be exploited as potential drug carriers for controlled-release applications.

Effect of crosslinker on drug delivery properties of curcumin loaded starch coated iron oxide nanoparticles

Aminated starch coated iron oxide magnetic nanoparticles loaded with curcumin were synthesized via coprecipitation technique. The nanoparticles were crosslinked by using three different crosslinkers: glutaraldehyde, genipin and citric acid and the effect of crosslinking on different properties of the nanoparticles was evaluated. Characterisation of the nanoparticles was done with FTIR (Fourier Transform Infrared spectroscopy) and XRD (X-Ray Diffraction). Magnetic property study using VSM (Vibrating Sample Magnetometer) showed their superparamagnetic nature. Morphology of the nanoparticles was studied by SEM (Scanning Electron Microscopy) and TEM (Transmission Electron Microscopy). Zeta potential values showed that crosslinking imparted stability to the system. Crosslinking also enhanced drug loading and encapsulation efficiency of the system. Swelling and in vitro studies of the nanoparticles showed that the release of drug was dependent on time, crosslinker nature, crosslinker concentration and pH of the medium. The aminated starch coated nanoparticles also showed good mucoadhesive character. The cell viability assessment by MTT study revealed their compatibility with human lymphocytes cells and their considerable cell growth inhibiting properties with MCF7 and HepG2 cells. The nanoparticles showed good internalization in HepG2 cells along with considerable ROS formation.

Study on crosslinked gelatin-montmorillonite nanoparticles for controlled drug delivery applications

Gelatin, because of its biodegradability and ecofriendly nature, has been the best choice for controlled release applications. Montmorillonite (MMT) clay shows a very important role in controlling drug delivery. Hence, an attempt was made in this work to prepare gelatin–MMT nanoparticles by desolvation method using acetone as precipitating agent, glutaraldehyde (GA) as crosslinking agent, and water as reaction media for controlled delivery of isoniazid, a drug for tuberculosis. Characterization of the MMT and isoniazid-loaded gelatin–MMT nanoparticles was carried out using Fourier transform infrared spectroscopy, X-ray diffraction study, scanning electron microscopy study, and transmission electron microscopy study. The effect of MMT on gelatin nanoparticles was evaluated in terms of water uptake studies, and subsequently to the release of isoniazid drug in buffer solution at pH 1.2 (gastric pH) and pH 7.4 (intestinal pH). Swelling experiment indicated that the gelatin nanoparticles were very sensitive to the pH environment. The release profile of drug was studied by a UV–Visible spectrophotometer. Cytotoxicity study revealed that MMT-containing nanoparticles showed less cytotoxicity than MMT-free nanoparticles.

Soy flour nanoparticles for controlled drug delivery: effect of crosslinker and montmorillonite (MMT)

Soy flour (SF)–Montmorillonite (MMT) nanoparticles crosslinked with glutaraldehyde (GA) have been made and used as a carrier for isoniazid. The nanoparticles have been characterized by fourier transmission infra-red spectroscopy (FTIR), X-ray diffractometry (XRD), scanning electron microscopy (SEM) and transmission emission microscopy (TEM). The effects of MMT and glutaraldehyde on the nanoparticles have been assessed with regard to swelling, encapsulation efficiency and consequently, the release of isoniazid in different mediums. The drug release mechanism has been studied for different time periods by UV-Vis spectroscopy. Cytotoxicity testing has been performed by MTT assay analysis. The results imply that the nanoparticles can be exploited as a potential drug carrier for controlled release applications.

Silibinin inhibits hypoxia‐induced HIF‐1α‐mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics

Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia‐induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis, and metabolic changes in human PCa, LNCaP, and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity, and endothelial cells tube formation by hypoxic (1% O2) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl‐Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia‐induced HIF‐1α expression in PCa cells together with a strong reduction in hypoxia‐induced NADPH oxidase (NOX) activity. HIF‐1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF‐1α expression, lipid levels, clonogenicity, and NOX activity even in HIF‐1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast‐enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion‐weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG‐PET; however, reduced the lipid synthesis measured by quantitative 1H‐NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF‐1α, FASN, and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia‐induced signaling.

Carboxymethyl starch-chitosan-coated iron oxide magnetic nanoparticles for controlled delivery of isoniazid

Abstract Context: The coating material of magnetic nanoparticles plays a great role in drug delivery application. The coatings not only increase the stability of the nanoparticles but also improve the drug release pattern, biocompatibility and mucoadhesivity. Objective: Montmorillonite (MMT) containing magnetic iron oxide nanoparticles coated with polyelectrolyte complex (PEC) of carboxymethyl starch-chitosan were prepared for controlled release applications. Method: The PEC-coated nanoparticles were characterised by Fourier Transmission Infra-red spectroscopy and X-ray diffraction, scanning electron microscope, transmission electron microscope, and dynamic light scattering. Cytotoxicity study was performed by MTT assay analysis. Mucoadhesivity test was performed by using in vitro wash off and ex vivo method. Result: The coating of PEC showed good stability, biocompatibility and mucoadhesivity of the iron oxide magnetic nanoparticles. MMT addition enhanced the swelling, drug loading and release and also the cytotoxicity and mucoadhesivity of the nanoparticles. Conclusion: This study revealed that the MMT incorporated PEC of CMS-CS can be effectively used for coating of iron oxide nanoparticles.

Alcohol and Tobacco Increases Risk of High Risk HPV Infection in Head and Neck Cancer Patients: Study from North-East Region of India

Background Human papilloma virus (HPV) associated Head and Neck Cancers (HNCs) have generated significant amount of research interest in recent times. Due to high incidence of HNCs and lack of sufficient data on high-risk HPV (hr-HPV) infection from North -East region of India, this study was conceived to investigate hr-HPV infection, its types and its association with life style habits such as tobacco, alcohol consumption etc. Methods A total of one hundred and six primary HNC tumor biopsy specimens were collected. These samples were analyzed for hr-HPV DNA (13 HPV types) using hybrid capture 2 (HC2) assay and genotyping was done by E6 nested multiplex PCR (NMPCR). Results The presence of hr-HPV was confirmed in 31.13% (n = 33) and 24.52% (n = 26) of the HNC patients by nested multiplex PCR (NMPCR) and HC2 assay respectively. Among hr-HPV positive cases, out of thirteen hr- HPV types analyzed, only two prevalent genotypes, HPV-16 (81.81%) followed by HPV-18 (18.18%) were found. Significant association was observed between hr-HPV infection with alcohol consumption (p <0.001) and tobacco chewing (p = 0.02) in HNC cases. Compared to HPV-18 infection the HPV-16 was found to be significantly associated with tobacco chewing (p = 0.02) habit. Conclusions Our study demonstrated that tobacco chewing and alcohol consumption may act as risk factors for hr-HPV infection in HNCs from the North-East region of India. This was the first study from North-East India which also assessed the clinical applicability of HC2 assay in HNC patient specimens. We suggest that alcohol, tobacco and hr- HPV infection act synergistically or complement each other in the process of HNC development and progression in the present study population.

Physical and biophysical assessment of highly fluorescent, magnetic quantum dots of a wurtzite-phase manganese selenide system

Combining fluorescence and magnetic features in a non-iron based, select type of quantum dots (QDs) can have immense value in cellular imaging, tagging and other nano-bio interface applications, including targeted drug delivery. Herein, we report on the colloidal synthesis and physical and biophysical assessment of wurtzite-type manganese selenide (MnSe) QDs in cell culture media. Aiming to provide a suitable colloidal system of biological relevance, different concentrations of reactants and ligands (e.g., thioglycolic acid, TGA) have been considered. The average size of the QDs is ∼7 nm, which exhibited a quantum yield of ∼75% as compared to rhodamine 6 G dye(®). As revealed from time-resolved photoluminescence (TR-PL) response, the near band edge emission followed a bi-exponential decay feature with characteristic times of ∼0.64 ns and 3.04 ns. At room temperature, the QDs were found to exhibit paramagnetic features with coercivity and remanence impelled by TGA concentrations. With BSA as a dispersing agent, the QDs showed an improved optical stability in Dulbeccos Modified Eagle Media(®) (DMEM) and Minimum Essential Media(®) (MEM), as compared to the Roswell Park Memorial Institute(®) (RPMI-1640) media. Finally, the cell viability of lymphocytes was found to be strongly influenced by the concentration of MnSe QDs, and had a safe limit upto 0.5 μM. With BSA inclusion in cell media, the cellular uptake of MnSe QDs was observed to be more prominent, as revealed from fluorescence imaging. The fabrication of water soluble, nontoxic MnSe QDs would open up an alternative strategy in nanobiotechnology, while preserving their luminescent and magnetic properties intact.