Ananda Mukherjee

In Vitro Structural and Functional Evaluation of Gold Nanoparticles Conjugated Antibiotics

Bactericidal efficacy of gold nanoparticles conjugated with ampicillin, streptomycin and kanamycin were evaluated. Gold nanoparticles (Gnps) were conjugated with the antibiotics during the synthesis of nanoparticles utilizing the combined reducing property of antibiotics and sodium borohydride. The conjugation of nanoparticles was confirmed by dynamic light scattering (DLS) and electron microscopic (EM) studies. Such Gnps conjugated antibiotics showed greater bactericidal activity in standard agar well diffusion assay. The minimal inhibitory concentration (MIC) values of all the three antibiotics along with their Gnps conjugated forms were determined in three bacterial strains,Escherichia coli DH5α,Micrococcus luteus andStaphylococcus aureus. Among them, streptomycin and kanamycin showed significant reduction in MIC values in their Gnps conjugated form whereas; Gnps conjugated ampicillin showed slight decrement in the MIC value compared to its free form. On the other hand, all of them showed more heat stability in their Gnps conjugated forms. Thus, our findings indicated that Gnps conjugated antibiotics are more efficient and might have significant therapeutic implications.

Attenuation of PTEN perturbs genomic stability via activation of Akt and down-regulation of Rad51 in human embryonic kidney cells†

To address the involvement of PTEN/Akt signaling in DNA repair and genomic stability, we developed a shRNA‐mediated PTEN knockdown cell line from HEK293T cells and evaluated its response to etoposide by analyzing γH2AX and Rad51 foci formation, cell cycle analysis, and chromosome damage. HEK PTEN knockdown cells were impaired in DNA repair associated with loss of G2/M checkpoint and reduced Rad51 foci formation. Furthermore, inhibition of Akt did not restore etoposide‐induced G2/M arrest in PTEN knockdown cells, suggesting that loss of G2/M checkpoint in PTEN knockdown cells is Akt‐independent. On the other hand, these cells become sensitive to etoposide when Akt was inhibited. Thus, loss of G2/M checkpoint and reduction of Rad51‐mediated homologous recombination is responsible for the genomic instability of PTEN knockdown cells where activated Akt additionally contribute to strong survival signal.

Alprazolam Intercalates into DNA

Abstract In vitro interaction of a benzodiazepine group of drugs Alprazolam (Alp), a hypnotic and tranquilizer, with DNA was studied by various methods. Absorption spectrophotometric study showed that Alp binds strongly with supercoiled pUC 19 DNA and the calculated binding constant is 8.245 × 103 M−1 in 10 mM Tris-Cl buffer, pH 7.4. Spectrofluorometric study showed that ethidium bromide induced DNA fluorescence intensity was reduced completely after addition of Alp. But Alp did not interfere with the interaction of Hoechst 33258, a DNA minor groove binder, with plasmid DNA. Circular dichroic spectroscopic study showed that with the gradual increase in Alp concentrations, both the positive and the negative peaks of DNA were gradually decreased and at higher concentrations of Alp (60 μM and 80 μM), the negative peaks became positive indicating the intercalation and the conformational change in the DNA. Binding of Alp with DNA increased the thermal stability of DNA by 6 °C with respect to the mock treated sample. Gel electrophoresis study of supercoiled pUC 19 DNA showed more compact structure as a result of Alp binding. Transmission electron microscopic observations also confirmed this compactness. Thus, our observations suggest the strong interaction of Alp with DNA, which may raise serious questions about the random uses of Alprazolam.

Inactivation of PTEN is responsible for the survival of Hep G2 cells in response to etoposide-induced damage

The chemo-resistance character of human hepatocellular carcinoma cells is well known but the anomalies associated with such resistance character are not completely understood. In this study, etoposide-induced signaling events in human hepatocellular carcinoma cell line, Hep G2 has been compared with Chang Liver cells, a normal human liver cell line. Hep G2 cells are resistant to etoposide when compared with Chang Liver cells. Etoposide-induced γH2AX foci in Hep G2 cells are persisted for a longer time without affecting cell cycle, indicating that Hep G2 cells are able to maintain its growth with damaged DNA. Further, Akt signaling pathway is deregulated in Hep G2 cells. The upstream negative regulator of Akt, PTEN remains inactive, as it is hyperphosphorylated in Hep G2 cells. Inhibition of PI-3K pathway by wortmannin partially reverses the etoposide-resistance character of Hep G2 cells. Either Hep G2 or Chang Liver cells when transfected with plasmid carrying active Akt (myr-Akt) become resistance towards etoposide compared to the cells transfected with empty vectors or kinase defective Akt. Transient transfection of wild type PTEN in Hep G2 cells does not change its response towards etoposide whereas Chang Liver cells become sensitive after transfection with same plasmid. These results suggest that inactivation of PTEN, which renders activation of Akt, may contribute largely for the etoposide-resistance character of Hep G2 cells.

Multinucleation regulated by the Akt/PTEN signaling pathway is a survival strategy for HepG2 cells

Hepatocellular carcinoma (HCC) is non-responsive to many chemotherapeutic agents including etoposide. The aim of this study was to examine the survival strategy of the HCC cell line HepG2 after etoposide treatment. Here we analyzed and compared spontaneous and etoposide-induced DNA damage in HepG2 (α-fetoprotein (AFP)-positive) and Chang Liver (AFP-negative) cell lines. Compared to Chang Liver cells, HepG2 cells exhibited a significantly higher degree of micronucleation and a higher nuclear division index, as determined by the cytokinesis-block micronucleus assay, following exposure to etoposide. HepG2 cells were also more resistant to etoposide-induced cytotoxicity compared to Chang Liver cells. We also establish that increased etoposide-induced multinucleation in HepG2 cells is dependent on the catalytic activity of Akt, as phosphatidylinositol-3-kinase inhibitors as well as the overexpression of kinase-defective Akt reversed this phenotype. Moreover, ectopic expression of wild type PTEN reduced the frequency of etoposide-induced multinucleated HepG2 cells, and restored HepG2 etoposide sensitivity. Taken together, these results implicate the Akt/PTEN cellular axis as a major determinant of the etoposide resistance of HCC cells.

Comparative study of the physico-chemical parameters of the coastal waters in rivers Matla and Saptamukhi: Impacts of coastal water coastal pollution

A qualitative and quantitative research has been performed on the physico-chemical parameters of the neighboring coastal waters of the rivers Matla and Saptamukhi in the Sunderban district, West Bengal. The distribution pattern of eight physico-chemical parameters, namely pH, salinity, dissolved oxygen, suspended solids, nitrogen and phosphorus concentrations (inorganic and total) has been graphed and compared for the two rivers over a time period of ten years (’90s decade). A statistical analysis has been carried out and the correlation data between these parameters has been rationalized based on both natural and man-made activities during that time. This has pointed to various causes behind coastal pollution of river waters. The changes in water quality have been related to flood impacts, storm surge, eutrophication, domestic sewage, agricultural and industrial wastes. In order to avoid coastal degradation and maintain environmental balance, it is very important to understand the impact of these parameters on coastal zones.

Caffeine augments Alprazolam induced cytotoxicity in human cell lines

Combined effects of alprazolam (Alp), a member of benzodiazepine group of drugs and caffeine on human cell lines, HeLa and THP1 were investigated in this study. Alp mediated cytotoxicity was enhanced while caffeine was present. The cell death was confirmed by observing morphological changes, LDH assay and membrane anisotropic study. Also such combined effects induced elevated level of ROS and depletion of GSH. The mechanism of cell death induced by simultaneous treatment of Alp and caffeine was associated with the calcium-mediated activation of mu-calpain, release of lysosomal protease cathepsin B, activation of PARP and cleavage of caspase 3. Our results indicate that, Alp alone induces apoptosis in human cells but in the presence of caffeine it augments necrosis in a well-regulated pathway. Thus our observations strongly suggest that, alprazolam and caffeine together produce severe cytotoxicity in human cell lines.

Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts

ABSTRACT Induction of pluripotency in differentiated cells through the exogenous expression of the transcription factors Oct4, Sox2, Klf4 and cellular Myc involves reprogramming at the epigenetic level. Histones and their metabolism governed by histone chaperones constitute an important regulator of epigenetic control. We hypothesized that histone chaperones facilitate or inhibit the course of reprogramming. For the first time, we report here that the downregulation of histone chaperone Aprataxin PNK-like factor (APLF) promotes reprogramming by augmenting the expression of E-cadherin (Cdh1), which is implicated in the mesenchymal-to-epithelial transition (MET) involved in the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Downregulation of APLF in MEFs expedites the loss of the repressive MacroH2A.1 (encoded by H2afy) histone variant from the Cdh1 promoter and enhances the incorporation of active histone H3me2K4 marks at the promoters of the pluripotency genes Nanog and Klf4, thereby accelerating the process of cellular reprogramming and increasing the efficiency of iPSC generation. We demonstrate a new histone chaperone (APLF)–MET–histone modification cohort that functions in the induction of pluripotency in fibroblasts. This regulatory axis might provide new mechanistic insights into perspectives of epigenetic regulation involved in cancer metastasis. Summary: This study reveals, for the first time, how a histone chaperone, which is also associated with DNA repair machinery, could influence cellular transition and pluripotency and thereby regulate reprogramming.

Phosphorylation of PTEN at STT motif is associated with DNA damage response

Phosphatase and tensin homolog deleted on chromosome Ten (PTEN), a tumor suppressor protein participates in multiple cellular activities including DNA repair. In this work we found a relationship between phosphorylation of carboxy (C)-terminal STT motif of PTEN and DNA damage response. Ectopic expression of C-terminal phospho-mutants of PTEN, in PTEN deficient human glioblastoma cells, U87MG, resulted in reduced viability and DNA repair after etoposide induced DNA damage compared to cells expressing wild type PTEN. Also, after etoposide treatment phosphorylation of PTEN increased at C-terminal serine 380 and threonine 382/383 residues in PTEN positive HEK293T cells and wild type PTEN transfected U87MG cells. One-step further, DNA damage induced phosphorylation of PTEN was confirmed by immunoprecipitation of total PTEN from cellular extract followed by immunobloting with phospho-specific PTEN antibodies. Additionally, phospho-PTEN translocated to nucleus after etoposide treatment as revealed by indirect immunolabeling. Further, phosphorylation dependent nuclear foci formation of PTEN was observed after ionizing radiation or etoposide treatment which colocalized with γH2AX. Additionally, etoposide induced γH2AX, Mre11 and Ku70 foci persisted for a longer period of times in U87MG cells after ectopic expression of PTEN C-terminal phospho-mutant constructs compared to wild type PTEN expressing cells. Thus, our findings strongly suggest that DNA damage induced phosphorylation of C-terminal STT motif of PTEN is necessary for DNA repair.

A novel Cu(II)–mal–picoline complex induces mitotic catastrophe mediated by deacetylation of histones and α-tubulin leading to apoptosis in human cell lines

In this study, we investigated mitotic catastrophe followed by apoptosis induced in human cell lines [HeLa, HepG2 and THP1] by a novel Cu(II) complex having malonate as the primary ligand and protonated 2-amino-4-picoline as the counter ion; whose in vitro DNA binding ability was demonstrated previously (B. Saha et al., J. Phys. Chem. B, 2010, 114(17), 5851–5861). Using the auto-fluorescence property of the complex, it was observed that the complex entered into the cells within 15 min after the exposure and was able to kill cells as determined by clonogenic survivability and MTT assay in a dose and time dependent manner. While dissecting the cell killing mechanisms, it was found that initially the complex induced multinucleated cells by inhibiting acetylation of a histone acetyl transferase (HAT) domain of CBP/p300, although histone deacetylase 6 (HDAC6) expression did not change much. As a result, histone proteins, H3 and H2AX, along with a non-histone protein, α-tubulin, were mostly deacetylated after 48 h of the treatment. This eventually led to mitotic catastrophe (MC), as histone acetylation–deacetylation dynamics is essential for the successful mitosis. DNA damage-induced γH2AX and 53BP1 foci in the treated cells were also observed after 72 h of treatment, as abnormal mitosis with decondensed chromosomes are prone to nucleolytic attack. These molecular phenomena ultimately rendered apoptosis. Taken together, our results provided evidence that the said complex perturbed the signaling events associated with mitosis and consequently induced cell death.