Vijayashree Nayak

Evaluation of the antineoplastic activity of guduchi (Tinospora cordifolia) in cultured HeLa cells

Exposure of HeLa cells to 0, 5, 10, 25, 50 and 100 microg/ml of guduchi extracts (methanol, aqueous and methylene chloride) resulted in a dose-dependent but significant increase in cell killing, when compared to non-drug-treated controls. The effects of methanol and aqueous extracts were almost identical. However, methylene chloride extract enhanced the cell killing effect by 2.8- and 6.8-fold when compared either to methanol or aqueous extract at 50 and 100 microg/ml, respectively. Conversely, the frequency of micronuclei increased in a concentration-dependent manner in guduchi-treated groups and this increase in the frequency of micronuclei was significantly higher than the non-drug-treated control cultures and also with respect to 5 microg/ml guduchi extract-treated cultures, at the rest of the concentrations evaluated. Furthermore, the micronuclei formation was higher in the methylene chloride extract-treated group than in the other two groups. The dose response relationship for all three extracts evaluated was linear quadratic. The effect of guduchi extracts was comparable or better than doxorubicin treatment. The micronuclei induction was correlated with the surviving fraction of cells and the correlation between cell survival and micronuclei induction was found to be linear quadratic. Our results demonstrate that guduchi killed the cells very effectively in vitro and deserves attention as an antineoplastic agent.

Proliferation of chondrocytes on a 3-d modelled macroporous poly(hydroxyethyl methacrylate)-gelatin cryogel.

Tissue-engineering constructs should be designed to mimic the native tissue environment for cells, the scaffold matching to stiffness and strength of the tissues while maintaining an interconnected porous network and a reasonable porosity. This study presents a new single-step protocol for synthesis of a poly(hydroxyethyl methacrylate)–poly(ethylene glycol) diacrylate–gelatin (HPG) macroporous polymeric scaffold with well-controlled porous structure and good mechanical strength. The pore size of these matrices lies in the range of 30 to 100 μm with an average pore diameter of 80 μm and with an interconnected pore structure as analyzed by scanning electron microscopy. Further, interconnectivity was also confirmed by high solvent uptake capacity, as the cryogel reached its equilibrium within 2 min. The gels also showed substantial mechanical integrity, i.e., the average compressive modulus was 32.73 ± 2.36 kPa at 15% compression of their original length. The degree of weight loss of these cryogels was found to be approx. 88% within 8 weeks of incubation in PBS (pH 7.4) at 37°C. Physio-chemically optimized cryogel was further evaluated for in vitro growth and proliferation of isolated primary goat chondrocytes up to 3 weeks. The cell adherence on cryogel was examined by SEM analysis, while cell–matrix interaction was examined by 4-6-diamidino-2-phenylindole and propidium iodide staining. Furthermore, the cell compatibility and proliferation was evaluated using the MTT assay. Increase in total cellular metabolic activity was observed as shown by continuous increase in glycosaminoglycan and collagen contents with time. Collagen type-I and type-II gene expression analysed for over 3 weeks by RT-PCR showed the prominent expression of collagen type-II. These results suggest the use of synthesised cryogel scaffold as a matrix for chondrocyte attachment and proliferation in 3-D environment and as a delivery system in cartilage-tissue engineering.

Effect of Doxorubicin on Cell Survival and Micronuclei Formation in HeLa Cells Exposed to Different Doses of Gamma-Radiation

Purpose: The present study was undertaken to obtain an insight into the combined effects of doxorubicin with radiation on the cell survival and micronuclei induction in HeLa cells. Material and Methods: HeLa S3 cells were allowed to grow till they reached plateau phase, inoculated with 10 μg/ml doxorubicin hydrohloride and then exposed to 0, 0.5, 1, 2 and 3 Gy γ-radiation. Clonogenicity of cells was measured using the colony forming assay, micronuclei formation using the micronucleus assay. Results: The treatment of HeLa cells with doxorubicin (adriamycin) for 2 hours before exposure to different doses of γ-radiation resulted in a significant and dose-dependent decline in the cell survival and cell proliferation when compared to the PGS + irratiation group. Conversely, the frequency of micronuclei increased in a dose-related manner in both the PBS + irradiation and doxorubicin + irradiation groups. The pretreatment of HeLa cells with doxorubicin before irradiation to various doses of γ-rays resulted in a significant elevation in the frequency of micronuclei when compared with the concurrent PBS + irradiation group. The dose-resonse relationship for both PBS + irradiation and doxorubicin + irradiation groups was linear. The correlation between cell survival and micronuclei induction was also determined for PBS or doxorubicin + irradiation group, where the clonogenicity of cells declined with the increase in micronuclei formation. The correlation between cell survival and micronuclei induction was linear quadratic for both PBS + irradiation and doxorubicin + irradiation groups. Conclusion: From our study it can be concluded that combination treatment with doxorubicin and radiation increased the genotoxic effect of the either treatment given alone.Hintergrund: Der kombinierte Effekt einer Doxorubicingabe mit Bestrahlung auf das Zellüberleben und die Mikronukleiinduktion wurde an HeLa-Zellen untersucht. Material und Methoden: He-La-S3-Zellen in der Plateauphase wurden mit 10 μg/ml Doxorubicinhydrochlorid inokuliert und dann einer γ-Bestrahlung von 0, 0,5, 1, 2 und 3 Gy ausgesetzt. Die Konogenität der Zellen wurde mit dem Koloniebildungstest, die Bildung von Mikronuklei mit Hilfe des Mikronuleuassays untersucht. Ergebnisse: Die Behandlung von HeLa-Zellen mit Doxorubicin für zwei Stunden vor einer γ-Bestrahlung mit verschiedenen Dosen resultierte in einer signifikanten und dosisabhängigen Abnahme des Zellüberlebens und der Zellproliferation im Vergleich zu einer nur bestrahlten Kontrollgruppe. Die Häufigkeit der Mikronukleusbildung hingegen nahm in einer dosisabhängigen Weise sowohl für allein bestrahlte wie mit Doxorubicin und Bestrahlung behandelte Gruppen zu. Die Vorbehandlung von HeLa-Zellen mit Doxorubicin vor Bestrahlung mit verschiedenen Dosen ergab einen signifikanten Anstieg der Häufigkeit der Mikronuklei im Vergleich zu der nur bestrahlten Gruppe. Die Dosis-Wirkungs-Beziehung sowohl für die nur bestrahlte als auch für die mit Doxorubicin und Bestrahlung behandelte Gruppe war linear. Die Korrelation zwischen Zellüberleben und Mikronukleusinduktion wurde ebenso für die allein bestrahlte wie für die mit Doxorubicin und Bestrahlung behandelte Gruppe bestimmt; die Klonogenität der Zellen nahm dabei mit ansteigender Mikronukleusformation ab. Die Korrelation zwischen Zellüberleben und Mikronukleusinduktion war linear quadratisch sowohl für die nur bestrahlte als auch für die mit Doxorubicin und Bestrahlung behandelte Gruppe. Schlussfolgerung: Aus unserer Studie kann geschlossen werden, dass die Kombination aus Doxorubicin und Bestrahlung die genotoxische Wirkung der Einzelmodalitäten erhöht.

Three-Dimensional Supermacroporous Carrageenan-Gelatin Cryogel Matrix for Tissue Engineering Applications

A tissue-engineered polymeric scaffold should provide suitable macroporous structure similar to that of extracellular matrix which can induce cellular activities and guide tissue regeneration. Cryogelation is a technique in which appropriate monomers or polymeric precursors frozen at sub-zero temperature leads to the formation of supermacroporous cryogel matrices. In this study carrageenan-gelatin (natural polymers) cryogels were synthesized by using glutaraldehyde and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride and N-hydroxysuccinimide (EDC-NHS) as crosslinking agent at optimum concentrations. Matrices showed large and interconnected pores which were in the range of 60–100 μm diameter. Unconfined compression analysis showed elasticity and physical integrity of all cryogels, as these matrices regained their original length after 90% compressing from the original size. Moreover Youngs modulus was found to be in the range of 4–11 kPa for the dry cryogel sections. These cryogels also exhibited good in vitro degradation capacity at 37 °C within 4 weeks of incubation. Supermacroporous carrageenan-gelatin cryogels showed efficient cell adherence and proliferation of Cos-7 cells which was examined by SEM. PI nuclear stain was used to observe cell-matrix interaction. Cytotoxicity of the scaffolds was checked by MTT assay which showed that cryogels are biocompatible and act as a potential material for tissue engineering and regenerative medicine.

Micronuclei-induction and its correlation to cell survival in HeLa cells treated with different doses of adriamycin

The treatment of HeLa cells with different concentrations of adriamycin (0, 5, 10, 25, 50 and 100 microg/ml) resulted in a significant and dose-dependent decline in the cell survival. Conversely, the frequency of micronuclei increased in a concentration-dependent manner. The micronuclei-induction and cell survival were found to be inversely related.

Efficacy of supermacroporous poly(ethylene glycol)-gelatin cryogel matrix for soft tissue engineering applications.

Three dimensional scaffolds synthesized using natural or synthetic polymers act as an artificial niche for cell adherence and proliferation. In this study, we have fabricated cryogels employing blend of poly (ethylene glycol) (PEG) and gelatin using two different crosslinkers like, glutaraldehyde and EDC-NHS by cryogelation technique. Synthesized matrices possessed interconnected porous structure in the range of 60-100 μm diameter and regained their original length after 90% compression without deformation. Visco-elastic behavior was studied by rheology and unconfined compression analysis, elastic modulus of these cryogels was observed to be >10(5)Pa which showed their elasticity and mechanical strength. TGA and DSC also showed the stability of these cryogels at different temperatures. In vitro degradation capacity was analyzed for 4 weeks at 37°C. IMR-32, C2C12 and Cos-7 cells proliferation and ECM secretion on PEG-gelatin cryogels were observed by SEM and fluorescent analysis. In vitro biocompatibility was analyzed by MTT assay for the period of 15 days. Furthermore, cell proliferation efficiency, metabolic activity and functionality of IMR-32 cells were analyzed by neurotransmitter assay and DNA quantification. The cell-matrix interaction, elasticity, mechanical strength, stability at different temperatures, biocompatible, degradable nature showed the potentiality of these cryogels towards soft tissue engineering such as neural, cardiac and skin.

Effect of Coccinia indica on Blood Glucose, Insulin and Key Hepatic Enzymes in Experimental Diabetes

The radiosensitizing effect of various guduchi (Tinospora cordifolia Miers, Menispermaceae) extracts, i.e., pretreatment with 10 µg/ml of methanol (Met), aqueous (Aqu) or methylene chloride (Mch) extracts, was studied in the HeLa cells exposed to 0, 0.5, 1, 2, or 3Gy of ? -radiation. The irradiation of cells resulted in a dose-dependent decline in the clonogenicity of cells expressed as reduction in cell survival. Treatment of HeLa cells with Met, Aqu or Mch extract before exposure to different doses of ? -radiation resulted in a significant decline in the cell survival when compared with the phosphate-buffered saline (PBS) + irradiation group. The highest loss in the clonogenicity was observed in Mch + irradiation group, where the cell survival was lower than doxorubicin (Dox) + irradiation group. Conversely, the frequency of micronuclei increased in a dose-dependent manner in all the groups and a significant elevation in micronuclei frequency was observed in Met + irradiation, Aqu + irradiation, Mch + irradiation and Dox + irradiation groups relative to the PBS + irradiation group. The dose enhancement factor for micronuclei-induction varied between 1.4 to 1.97, depending on the type of extract and dose of irradiation. The dose response for all the groups was linear quadratic. The biological response was determined by plotting the surviving fraction of cells versus micronuclei frequency, respectively, for all the groups. The correlation between cell survival and micronuclei-induction was found to be linear quadratic for all the groups. In spite of a good correlation between micronuclei frequency and cell survival, the frequency of micronuclei was lower in Mch + irradiation group than in the other two groups (i.e., Met and Aqu), owing to the greater cell kill as evidenced by the higher decline in the cell survival in this group when compared to the other groups. However, the statistical difference among all guduchi extracts pretreated groups was not significant. The present study demonstrates that all the guduchi extracts were able to enhance the effect of radiation significantly. However, the most effective extract was Mch, where the reduction in the surviving fraction of cells was higher than either Aqu + irradiation, Met + irradiation or Dox + irradiation group.

Treatment of mice with a novel antineoplastic agent taxol before irradiation increases the frequency of micronuclei in the bone marrow

The frequency of micronucleated polychromatic erythrocytes (MPCE) and the normochromatic erythrocytes (MNCE) and polychromatic and normochromatic erythrocyte ratio (P/N ratio) was studied at 12, 24 and 36 h postirradiation in the bone marrow of male mice treated or not with taxol before exposure to 0-4 Gy of 60Co gamma radiation. The frequency of MPCE increased with the increase in radiation dose in a dose-related manner in the irradiated control group. A peak frequency of MPCE was observed at 24 h postirradiation in irradiated control group. The pattern of increase in MNCE was similar to that of MPCE except that a highest number of MNCE was scored at 36 h postirradiation. Taxol administration to animals before irradiation resulted in a significant elevation in the frequency of MPCE and MNCE at all the postirradiation time periods studied. This increase was dose related as observed in the irradiated control group. Irradiation resulted in a dose-dependent decline in the P/N ratio at all the postirradiation time periods studied. The P/N ratio was significantly lower in the taxol + irradiated group compared to the irradiated control group at all postirradiation time periods. A maximum decline in P/N ratio was observed at 36 h postirradiation for both irradiated control and taxol + irradiated groups. The dose response for MPCE, MNCE and P/N ratio was linear quadratic for both the irradiated and taxol + irradiated groups.

Study of Different Delivery Modes of Chondroitin Sulfate Using Microspheres and Cryogel Scaffold for Application in Cartilage Tissue Engineering

This study focuses on the development of an efficient delivery modes designed for chondroitin sulfate (CS) for application in cartilage tissue engineering. Novel three-dimensional (3-D) scaffold fabricated from natural polymers such as chitosan and gelatin blended with chondroitin sulfate (CGC) were synthesized using cryogelation technology. Other methods to deliver CS were also tried, which included incorporation into microparticles for sustained release and embedding the CS loaded microparticles in CG (chitosan-gelatin) cryogel scaffold. Novel CGC scaffolds were characterized by rheology, scanning electron microscopy (SEM), and mechanical assay. Scaffolds exhibited compression modulus of 50 KPa confirming the utility of these scaffolds for cartilage tissue engineering. Primary goat chondrocytes were used for the in vitro testing of all the delivery modes. So this study shows that CS microparticles when given freely with matrix (chitosan–gelatin) or embedded into scaffold has potential to enhance chondrocyte proliferation together with improved matrix production than in control without microspheres.

Study on Salmonella Typhi occurrence in gallbladder of patients suffering from chronic cholelithiasis-a predisposing factor for carcinoma of gallbladder.

Cholelithiasis is frequently associated with carcinoma of gallbladder, and the presence of Salmonella Typhi in gallbladder of patients suffering from cholelithiasis is implicated as a predisposing factor for carcinogenesis. This study was conducted on patients suffering from chronic cholelithiasis from a region in North India-endemic area for enteric fever with high incidence of gallstones and gallbladder cancer. Since culture studies rarely reveal the chronic Salmonella Typhi persistence, we use PCR assay to specifically amplify the H1-d flagellin gene sequence homologous with Salmonella Typhi. Seven cases (17.5%), none of which were positive for culture, showed positive PCR results for Salmonella Typhi, 4 (10%) of which were tissue, 2 bile (5%), and 1 gallstone (2.5%). The chronic existence of Salmonella Typhi in gallbladder disease was confirmed. Thus, the study would indicate the importance of vaccination so as to prevent chronic infection and need for early diagnostic tools to prevent any further complications.