Cytotoxicity of chlorhexidine and neem extract on cultured human gingival fibroblasts through fluorescence-activated cell sorting analysis : An in-vitro study.

OBJECTIVE: To assess the influence of chlorhexidine (CHX), neem vehicle control (NVC), and neem extract (NE) on cultured human gingival fibroblasts (hGFs) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and fluorescence-activated cell sorting (FACS) analysis. MATERIALS AND METHODS: Fibroblasts were derived from healthy gingival biopsy specimens harvested aseptically. The effects of CHX, NVC, and NE were evaluated on cultured hGFs through FACS and MTT assay. RESULTS: MTT assay with hGFs indicated altered morphology with maximum cell death at 10% CHX, while NVC and NE showed similar results at a concentration of 75% and above. On FACS analysis, beyond 1%, CHX adversely affected the cell cycle phase distribution whereas NE exerted a detrimental effect only at 100%. Moreover, both with NVC and NE cells were well differentiated in all the three phases of the cell cycle, with distinction getting lost at 50% to finally causing cell death at 100%. CONCLUSIONS: CHX beyond 1% concentration exhibited a toxic effect on hGFs at 1, 5, and 15 min time exposure. However, NE did not adversely affect the fibroblasts even up to 50% concentration showing a less toxic effect in comparison with CHX on these cells. The cytoprotective and oral friendly quality of NE emphaisze the superiority of NE over CHX.

Neem Seed Oil Induces Apoptosis in MCF-7 and MDA MB-231 Human Breast Cancer Cells

Background: In traditional Indian medicine, azadirachta indica (neem) is known for its wide range of medicinal properties. Various parts of neem tree including its fruit, seed, bark, leaves, and root have been shown to possess antiseptic, antiviral, antipyretic, anti-inflammatory, antiulcer, antimalarial, antifungal and anticancer activity. Materials and Methods: MCF-7 and MDA MB-231 cells were exposed to various concentrations of 2% ethanolic solution of NSO (1-30 µl/ml) and further processed for cell viability, cell cycle and apoptosis analysis. In addition, cells were analyzed for alteration in Mitochondrial Membrane Potential (MMP) and generation of Reactive Oxygen Species (ROS) using JC-1 and DCFDA staining respectively. Results: NSO give 50% inhibition at 10 µl/ml and 20 µl/ml concentration in MCF-7 and MDA MB-231 cells respectively and, arrests cells at G0/G1 phase in both the cell types. There was a significant alteration in mitochondrial membrane potential that leads to the generation of ROS and induction of apoptosis in NSO treated MCF-7 and MDA MB-231 cells. Conclusion: The results showed that NSO inhibits the growth of human breast cancer cells via induction of apoptosis and G1 phase arrest. Collectively these results suggest that NSO could potentially be used in the management of breast cancer.

Polyphenols sensitization potentiates susceptibility of MCF-7 and MDA MB-231 cells to Centchroman.

Polyphenols as "sensitizers" together with cytotoxic drugs as "inducers" cooperate to trigger apoptosis in various cancer cells. Hence, their combination having similar mode of mechanism may be a novel approach to enhance the efficacy of inducers. Additionally, this will also enable to achieve the physiological concentrations facilitating significant increase in the activity at concentrations which the compound can individually provide. Here we propose that polyphenols (Resveratrol (RES) and Curcumin (CUR)) pre-treatment may sensitize MCF-7/MDA MB-231 (Human Breast Cancer Cells, HBCCs) to Centchroman (CC, antineoplastic agent). 6 h pre-treated cells with 10 µM RES/CUR and 100 µM RES/30 µM CUR doses, followed by 10 µM CC for 18 h were investigated for Ser-167 ER-phosphorylation, cell cycle arrest, redox homeostasis, stress activated protein kinase (SAPKs: JNK and p38 MAPK) pathways and downstream apoptosis effectors. Low dose RES/CUR enhances the CC action through ROS mediated JNK/p38 as well as mitochondrial pathway in MCF-7 cells. However, RES/CUR sensitization enhanced apoptosis in p53 mutant MDA MB-231 cells without/with involvement of ROS mediated JNK/p38 adjunct to Caspase-9. Contrarily, through high dose sensitization in CC treated cells, the parameters remained unaltered as in polyphenols alone. We conclude that differential sensitization of HBCCs with low dose polyphenol augments apoptotic efficacy of CC. This may offer a novel approach to achieve enhanced action of CC with concomitant reduction of side effects enabling improved management of hormone-dependent breast cancer.

Centchroman mediated apoptosis involves cross-talk between extrinsic/intrinsic pathways and oxidative regulation.

AIMS: Centchroman (CC) has been established as a potent antineoplastic agent in MCF-7 (ER+ve) and MDA MB-231 (ER-ve) Human Breast Cancer Cells (HBCCs) previously by us. To elucidate its antineoplastic action, we investigated the factors involved in cell-cycle progression and apoptosis. MAIN METHODS: Tamoxifen (TAM), a widely used antiestrogen was employed as a positive control. Role of Cycloheximide (CHX), Actinomycin-D (Act-D) and caspases were explored using specific inhibitors. Involvement of cell-cycle and apoptosis related factors were explored using western blotting and immunoprecipitation. KEY FINDINGS: Metabolic inhibitors viz. CHX, Act-D and pan-Caspase inhibitor, Z-VAD-FMK attenuated CC-induced apoptosis. The upregulation of both p21(Waf1/Cip1) and p27(Kip1) along with p21-CDK6 (Cyclin Dependent Kinase 6) and p21-PCNA (Proliferating Cell Nuclear Antigen) interaction suggests their role in CC-induced cell-cycle arrest. The downregulation of Cyclin-D(1) and -E levels further confirms the antiestrogenic profile of CC. Unlike MDA MB-231, in MCF-7 cells, CC upregulates the level of phospho-p53 (Ser-15) and FasL, suggesting the involvement of extrinsic pathway. CC altered the intracytosolic balance of members of Bcl-2 family along with the cleavage of Poly (ADP-ribose) polymerase (PARP), Bcl-X(L), Bid and AIF (Apoptosis Inducing Factor). The evaluation of Mitogen Activated Protein Kinases (MAPKs) using specific inhibitors and Western blotting confirms CC-induced the upregulation of phospho-c-Jun and phospho-p38. Additionally elevated SOD (Superoxide Dismutase) and unaltered CAT (Catalase) expression further suggest the involvement of oxidative stress. SIGNIFICANCE: These results confirm that the antineoplasticity of CC in MCF-7 and MDA MB-231 cells involves the extrinsic and intrinsic pathways of apoptosis along with oxidative stress.

Caspase mediated enhanced apoptotic action of cyclophosphamide- and resveratrol-treated MCF-7 cells.

Cyclophosphamide (CPA) is a widely used chemotherapeutic drug for neoplasias. It is a DNA and protein alkylating agent having a broad spectrum of activity against a variety of neoplasms including breast cancer. The therapeutic effectiveness of CPA is limited by the high-dose hematopoietic, renal, and cardiac toxicity that accompanies the systemic distribution of liver-derived activated drug metabolites. The present study examines the potential of combining resveratrol (RES) with CPA and aims to increase the understanding of the mechanism of cell killing. Interestingly, we found that RES significantly enhances the caspase-mediated cytotoxic activity of CPA on MCF-7 cells in vitro. RES at 50 microM decreases the IC(50) value of CPA from 10 to 5 mM. FACS data reveals CPA or RES alone mediated G0/G1 and S phase arrest, while the combination of these drugs released both the arrests and results in an increase in the sub G0/G1 peak. Additional analyses indicated the significant up-regulation (P = 0.001) of tumor suppressor p53 and p53-regulated pro-apoptotic Bax and Fas in MCF-7 cells following CPA treatment in combination with RES, which may contribute to the enhancement of the antitumor effect of CPA. Furthermore, downregulation of anti-apoptotic Bcl-2 (P = 0.001) was observed in MCF-7 cells treated with CPA with or without RES when compared to untreated MCF-7. These results suggest the possibility of a new combination chemotherapeutic regimen leading to improvements in the treatment of breast cancer.

Diagnostic and prognostic values of S-phase fraction and aneuploidy in patients with bone marrow aplasia.

AIM: It is often difficult and challenging task to differentiate aplastic anemia (AA) from hypoplastic myelodysplastic syndrome (HMDS) among the patients with bone marrow aplasia. This is possibly because of the considerable clinical, cytological and histological similarities between these two disorders. As prognostic and therapeutic approach to AA and HMDS are different, it is imperative to differentiate them at the time of initial diagnosis. Various attempts have been made in the past to differentiate AA from HMDS. In the present study, we explored the value of certain new parameters i.e. S-phase fraction (SPF) and aneuploidy that could be used for this purpose. MATERIALS AND METHODS: The study included 46 consecutive patients with aplastic anemia, 15 patients with HMDS along with 32 age and sex-matched control subjects. S-phase fraction and aneuploidy analysis was carried out by flow cytometry using Mod Fit-LT V3.0 software. RESULTS: The mean SPF value was significantly lower (p=0.02) in patients with AA and higher (p=0.01) in HMDS as compared to that of the control. Aneuploidy was present in 15.2% patients with AA and in 33.3% HMDS cases. During follow-up, 4 patients with AA developed MDS, out of these, three patients had aneuploidy as well as high SPF value at the time of diagnosis. Two patients with HMDS who had aneuploidy and high SPF, converted into AML. Eleven patients died during the study, in whom 8 had aneuploidy and high SPF value. CONCLUSION: We conclude that high SPF value and presence of aneuploidy favour the diagnosis of HMDS rather than AA. SPF and aneuploidy may be important parameters in patients with AA to predict their propensity to evolve into myelodysplastic syndrome and acute myeloid leukemia. SPF value may also be useful in the early diagnosis of HMDS before morphologically evidence of dysplasia is apparent.

Stimulatory effect of Ceriops tagal on hexose uptake in L6 muscle cells in culture.

The ethanolic extracts of a mangrove plant Ceriops tagal (Family Rhizophoraceae) and its sequential fractions thereof were studied for their effect on 3H-2-deoxyglucose uptake by L6 rat muscle cells cultured to the myotube stage. Among these, the n-hexane soluble fraction of ethanolic extract of the leaves of C. tagal enhanced 3H-2-deoxyglucose uptake even at 2 microg mL(-1) concentration with half maximum activity at 10 microg mL(-1), comparable with insulin (1 microM) and metformin (400 microM). This enhancement in glucose uptake was found to be insulin independent and in contrast to insulin, its effect was also prevalent in undifferentiated myoblasts. It may be concluded from the results that n-hexane soluble fraction of ethanolic extract of C. tagal have the property to stimulate the glucose uptake, which might be a useful source for the isolation of new antihyperglycemic compounds.

DNA aneuploidy study for early detection of chromosomal abnormality in patients with aplastic anemia: prognostic and therapeutic implications.

BACKGROUND: Undetected aneuploidy exists in a large percentage of patients with aplastic anemia at the time of diagnosis, which may not be identified by conventional cytogenetics. The presence of aneuploidy at any time in the clinical course implies poor prognosis in such patients. This warrants a need for the early detection of chromosomal abnormalities for prognosis and delineation of therapeutic modalities. PATIENTS AND METHODS: Fifty patients with aplastic anemia and 30 controls were studied with an aim to determine the role of aneuploidy as an indicator of chromosomal abnormalities. DNA aneuploidy analysis was carried out by flow cytometry using Mod Fit-LT V3.0 software, whereas chromosomal analysis was performed by conventional cytogenetics. RESULTS: DNA aneuploidy was present in 14% of cases and chromosomal abnormalities were found in 4% of cases of aplastic anemia at the time of diagnosis before therapy. Overall, DNA aneuploidy was detected in 36% of cases by flow cytometry, whereas the cytogenetic method revealed chromosomal abnormalities in 14% of cases of aplastic anemia. Flow cytometric analysis showed hypodiploidy in one patient at the time of diagnosis who developed monosomy 7 during follow-up. All patients with hypodiploidy had short survival and they did not respond to therapy. CONCLUSION: The present study demonstrates the role of flow cytometry in the early detection of chromosomal abnormalities in patients at a time when they remain undetected by conventional cytogenetics. The presence of DNA aneuploidy in patients with aplastic anemia may be an early indicator of subsequent overt cytogenetic abnormality, associated with poor response to immunosuppressive therapy and a lower survival.

Rapid synthesis of 4-benzylidene and 4-[bis-(4-methoxyphenyl)-methylene-2-substituted phenyl-benzopyrans as potential selective estrogen receptor modulators (SERMs) using McMurry coupling reaction.

7-Methoxy-4-(4-methoxybenzylidene)-2-substituted phenyl-benzopyrans I and 4-[bis-(4-methoxyphenyl)-methylene-2-substituted phenyl-benzopyrans II carrying different alkylamino residues, designed as estrogen receptor (ER) binding ligands, were successfully synthesized through the McMurry coupling reaction of substituted benzaldehyde/substituted benzophenones and 2-hydroxyphenyl-7-methoxy-chroman-4-one in presence of lithium aluminum hydride and titanium (IV) chloride (LAH-TiCl(4)). Self-coupling of carbonyl reactants led to the formation of several side products. The prototypes were evaluated for their relative binding affinity (RBA), as well as their estrogenic and antiestrogenic activities. High order of estrogenic activity (>50% gain) observed with compounds 3, 7a, 7b, 7c, 8, and 10a and also their partial estrogen antagonistic activity (> or =15%) at the uterine level points toward successful designing of the compounds. Compounds 4, 7a, 7b, 7c, and 10a also possessed significant anticancer activity against human adenocarcinoma cell line (MCF-7 cell line) that may be related to their estrogen-dependent action.

Mechanism of 4-HPR-induced apoptosis in glioma cells: evidences suggesting role of mitochondrial-mediated pathway and endoplasmic reticulum stress.

N-(4-hydroxyphenyl)retinamide (4-HPR), a synthetic retinoid is under clinical evaluation as a therapeutic agent in a variety of cancers. Its mechanism(s) of action involves multiple overlapping pathways that still remain unclear. In glioma cells its mechanism of action is not well elucidated. Here, we show that 4-HPR and not all-trans retinoic acid and 9-cis retinoic acid effectively induce apoptosis in glioma cells. 4-HPR-induced apoptosis is associated with hydroperoxide production and loss of mitochondrial membrane potential (Delta Psi(m)). Ultrastructural changes further indicate 4-HPR-induced mitochondrial swelling, endoplasmic reticulum (ER) dilation as well as close proximity of mitochondria and ER. As suggested by dilated ER, 4-HPR treatment increased the free cytosolic Ca(2+) as well as mitochondrial Ca(2+). Chelation of extracellular Ca(2+) by EGTA did not prevent Ca(2+) elevation, thus suggesting involvement of intracellular calcium stores in the release. Buffering of intracellular calcium by BAPTA-AM did not prevent 4-HPR-induced apoptosis; however, blocking the release of Ca(2+) from ER by heparin inhibited apoptosis, indicating the role of depletion of Ca(2+) from ER stores in apoptosis. 4-HPR treatment also resulted in an increase in Bax levels along with its translocation to mitochondria that promote mitochondrial membrane permeabilization. 4-HPR-induced apoptosis was further associated with the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytosol and nucleus, respectively, along with caspase-3 and caspase-7 activation. However, AIF nuclear translocation, peripheral chromatin condensation and apoptosis were not completely prevented by general caspase inhibitors, thus suggesting involvement of a caspase-dependent and caspase-independent pathway in 4-HPR-induced apoptosis. Taken together, these results suggest the role of mitochondrial-mediated pathway and ER stress as a key event in 4-HPR-induced apoptosis in glioma cells.

Substituted phenanthrenes with basic amino side chains: a new series of anti-breast cancer agents.

In the course of our search for new anti-breast cancer agents, substituted phenanthrenes with basic amino side chains were synthesized and some of them showed remarkable antiproliferative activity against ER +ve MCF-7 cell line with IC(50) in the range of 3.53-22.25 microM. One of the compounds 15 ca showed anti-breast cancer activity in 7,12-dimethylbenz[a]anthracene (DMBA) induced hormone-dependent mammary tumor in rat and the activity was comparable to that shown by tamoxifen.