Rita Ghosh

Induced resistance in cells exposed to repeated low doses of H2O2 involves enhanced activity of antioxidant enzymes

We have derived cells from the Chinese hamster V79 cell line by conditioning them with repeated low doses of hydrogen peroxide (H2O2). This mimics the physiological condition where cells are repeatedly exposed to low levels of oxidants. In an attempt to characterize such cells, we have exposed both conditioned cells (V79C) and the parental V79 cells (V79P) to different types of cytotoxic agents and compared their sensitivity to cell killing. The V79C cells were found to be stably resistant to killing by agents that produced toxicity through oxidative stress, e.g. H2O2 and cisplatin. It was also found that the lipid peroxidation produced by these agents were considerably lower in the V79C cells. Thus, the difference in sensitivity could be due to lesser extent of damage to these cells. V79C cells had greater antioxidant defense through higher GSH content and greater activity of enzymes such as Cu—Zn superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which provided protection from damage. Enzyme activities were also assayed at different times after treatment with various cytotoxic agents; there was a relatively large increase in SOD activity which perhaps plays a key role in determining the resistance of the V79C cells to killing.

9-Phenyl acridine exhibits antitumour activity by inducing apoptosis in A375 cells

Several acridine derivatives have been screened for their therapeutic potential and some are already established as antiprotozoan, antibacterial or anticancer agents. However, phenyl derivative at C-9 position of acridine had remained unexplored for their biological activity so far. In this report, we present our findings with 9-phenyl acridine (ACPH) as an anticancer agent. Both A375 and HeLa, two human cancer cell lines, were more sensitive to ACPH than normal cells namely human lymphocytes and also Chinese hamster V79 cells. ACPH also led to regression of tumour volume in mice. In A375 cells, we have shown that it caused DNA damage and blocked cell cycle progression at G2-M phase. Treatment with ACPH led to lowering of mitochondrial potential, upregulation of bax, release of cytochrome C and activation of caspase 3. As a new agent showing lower toxicity to normal cells and greater sensitivity towards cancerous cell line, ACPH shows promise as an effective cancer chemotherapeutic agent. ACPH treatment resulted in apoptotic death of cells through mitochondria-mediated caspase-dependent pathway.

A Fluorescent Guanosine Dinucleoside as a Selective Switch‐On Sensor for c‐myc G‐Quadruplex DNA with Potent Anticancer Activities

Like likes like! A novel fluorescent C2 -symmetric guanosine-based dinucleoside has been engineered by chemical ligation of two guanosine units with a biocompatible dansyl tag. The nucleoside exhibits high selectivity for c-myc G-quadruplex DNA through fluorescence enhancement over duplex DNA and other promoter G-quadruplexes (see scheme). It stains the nucleus preferentially, arrests the cell cycle at the G2/M phase, inhibits cell growth, and induces apoptosis in A375 cancer cells.

Effect of radiofrequency radiation in cultured mammalian cells: A review

ABSTRACT The use of mobile phone related technologies will continue to increase in the foreseeable future worldwide. This has drawn attention to the probable interaction of radiofrequency electromagnetic radiation with different biological targets. Studies have been conducted on various organisms to evaluate the alleged ill-effect on health. We have therefore attempted to review those work limited to in vitro cultured cells where irradiation conditions were well controlled. Different investigators have studied varied endpoints like DNA damage, cell cycle arrest, reactive oxygen species (ROS) formation, cellular morphology and viability to weigh the genotoxic effect of such radiation by utilizing different frequencies and dose rates under various irradiation conditions that include continuous or pulsed exposures and also amplitude- or frequency-modulated waves. Cells adapt to change in their intra and extracellular environment from different chemical and physical stimuli through organized alterations in gene or protein expression that result in the induction of stress responses. Many studies have focused on such effects for risk estimations. Though the effects of microwave radiation on cells are often not pronounced, some investigators have therefore combined radiofrequency radiation with other physical or chemical agents to observe whether the effects of such agents were augmented or not. Such reports in cultured cellular systems have also included in this review. The findings from different workers have revealed that, effects were dependent on cell type and the endpoint selection. However, contradictory findings were also observed in same cell types with same assay, in such cases the specific absorption rate (SAR) values were significant.

Characterisation of methotrexate-resistant clones

Three methotrexate (MTX)-resistant clones M1, M2 and M3 have been isolated from Chinese hamster V79 cells and characterised for aneuploidy, chromosomal aberrations, sister-chromatid exchange (SCE) mutation and transfection. Amplification of the dihydrofolate reductase (DHFR) gene in these clones has been established from (a) direct measurement of DHFR activity, (b) existence of double minute chromosomes and (c) homogeneously staining region (HSR) in chromosome number 2 by G-banding technique. Clone M1 on further exposure to gradually increasing concentrations of MTX gave rise to two more clones M4 and M5, resistant to 1200 nM and 2400 nM MTX, respectively. The levels of folate reductase activity in clones M4 and M5 were 21.90 units per 10(7) cells and 33.30 units per 10(7) cells, whereas the value was 13.90 units per 10(7) cells in clone M1 and 1.1 units per 10(7) cells in normal V79 cells. Increased chromosomal aberrations were observed in each of these clones in comparison to those in normal V79 cells. There was a correlation between the increase in DHFR activity and the increase in the level of background SCE as well as the increase in aneuploidy. However, background mutation frequency at the HGPRT locus remained unaffected though transfection frequency decreased.

Antioxidant enzymes and the mechanism of the bystander effect induced by ultraviolet C irradiation of A375 human melanoma cells.

Irradiated cells generate dynamic responses in non-irradiated cells; this signaling phenomenon is known as the bystander effect (BE). Factors secreted by the irradiated cells communicate some of these signals. Conditioned medium from UVC-irradiated A375 human melanoma cells was used to study the BE. Exposure of cells to conditioned medium induce cell-cycle arrest at the G2/M transition. Although conditioned medium treatment, by itself, did not alter cell viability, treated cells were more resistant to the lethal action of UVC or H2O2. This protective effect of conditioned medium was lost within 8h. Apoptotic or autophagic cell death was not involved in this resistance. Exposure to conditioned medium did not influence the rate of DNA repair, as measured by NAD(+) depletion. The activities of catalase and superoxide dismutase were elevated in cells exposed to conditioned medium, but returned to normal levels by 8h post-treatment. These results indicate a close correlation between BE-stimulated antioxidant activity and cellular sensitivity. Cell-cycle arrest and stimulation of antioxidant activity may account for the resistance to killing that was observed in bystander cells exposed to UVC or H2O2 treatment and are consistent with the role of the BE as a natural defense function triggered by UVC irradiation.

Response of V79 cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment: inhibition of poly(ADP-ribose) and topoisomerase activity

MNNG-induced killing of V79 cells has been found to be enhanced on inhibition of topoisomerase II activity by nalidixic acid and poly(ADP-ribose) polymerase synthesis by benzamide. Using these 2 inhibitors in conjunction after MNNG treatment, some overlap in the functions of these 2 enzymes was observed. Nalidixic acid and benzamide were found to suppress the yields of mutations and SCEs induced by MNNG. Benzamide was more effective in suppressing the mutation yield whereas nalidixic acid was more effective in suppressing SCEs. A model based on the relative requirement of topoisomerase and poly(ADP-ribose) for the repair of different types of damage has been proposed to explain the results.

UV Released Factors Induce Antioxidant Defense in A375 Cells

UV light leads to release of different secretory factors from irradiated cells of which some of them have been characterized. We have reported earlier that cells exposed to the supernatant medium from irradiated cells were resistant to killing by some genotoxic agents. In this study, we present our finding that demonstrates DNA damage induced by UV or H2O2 is lowered on prior exposure to the UV released factors (UVRF). Production of ROS in cells and lipid peroxidation was also lowered. It was found that treatment of unexposed cells with UVRF present in the supernatant medium altered the antioxidant defense activity in cells. Significant was the increase in catalase (CAT) and Cu–Zn superoxide dismutase (SOD) activity, whereas glutathione peroxidase (GPx) and reduced glutathione (GSH) levels remained unaffected. Cells exposed to UVRF prior to UV or H2O2 treatment also experienced such upregulation; however, the remarkable increase in the GPx activity exhibited by these cells was not observed in cells exposed to H2O2 or UV alone. It appears that exposure to UVRF tinkered with antioxidant defense in cells to facilitate its proliferation upon assault by an agent that can produce oxidative damage.

Studies on the interaction of a synthetic nitro-flavone derivative with DNA: A multi-spectroscopic and molecular docking approach

Interaction of a ligand with DNA is often the basis of drug action of many molecules. Flavones are important in this regard as their structural features confer them the ability to bind to DNA. 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO) is an important biologically active synthetic flavone derivative. We are therefore interested in studying its interaction with DNA. Absorption spectroscopy studies included standard and reverse titration, effect of ionic strength on titration, determination of stoichiometry of binding and thermal denaturation. Spectrofluorimetry techniques included fluorimetric titration, quenching studies and fluorescence displacement assay. Assessment of relative viscosity and estimation of thermodynamic parameters from CD spectral studies were also undertaken. Furthermore, molecular docking analyses were also done with different short DNA sequences. The fluorescent flavone 4NCO reversibly interacted with DNA through partial intercalation as well as minor-groove binding. The binding constant and the number of binding sites were of the order 104 M-1 and 1 respectively. The binding stoichiometry with DNA was found to be 1:1. The nature of the interaction of 4NCO with DNA was hydrophobic in nature and the process of binding was spontaneous, endothermic and entropy-driven. The flavone also showed a preference for binding to GC rich sequences. The study presents a profile for structural and thermodynamic parameters, for the binding of 4NCO with DNA. DNA is an important target for ligands that are effective against cell proliferative disorders. In this regard, the molecule 4NCO is important since it can exert its biological activity through its DNA binding ability and can be a potential drug candidate.

Role of Proteases in Photo-aging of the Skin

Aging is an inevitable process in living organisms that results from molecular damage over time. The skin being the most exposed part of the body, time and environmental aggressor leave their indelible mark on the skin. So, skin aging consists of two clinically and biologically independent processes – the intrinsic chronological aging and the aging through extrinsic factors. While intrinsic aging process proceeds at a genetically determined pace due to buildup of damaging products from cellular metabolism, exposure to solar radiations produces biological damages to the cells, known as photo-aging. It adds up to the effects of chronological aging, and it is the most prominent and important among the extrinsic factors. The normal architecture of the skin is disrupted due to degradation of skin components like collagens, fibers, etc. Photo-aged skin presents fine and coarse wrinkles with blotchy pigmentation, increased fragility, and rough texture. It results from complex biological phenomena that lead to activation of several proteases; the most crucial among them are the matrix metalloproteases (MMPs). UV irradiation generates reactive oxygen species and activates a number of transcription factors like AP1, NF-κB, p53, and growth factor like TGFβ. These, in turn, stimulate the MMPs and other proteases. UV radiation also inhibits the expression of natural inhibitors of MMP (TIMP), thereby enhancing the activity of the MMPs. Understanding of the molecular basis of photo-aging is important for its prevention and effective recovery. Antioxidants and other compounds that inhibit the molecular pathways that result in expression of the proteases have proved to be useful in prevention/reversal of skin aging.