Govindarajah Vinothini

The neem limonoids azadirachtin and nimbolide inhibit cell proliferation and induce apoptosis in an animal model of oral oncogenesis

SummaryLimonoids from the neem tree (Azadirachta indica) have attracted considerable research attention for their cytotoxicity against human cancer cell lines. However, the antiproliferative and apoptosis inducing effects of neem limonoids have not been tested in animal tumour models. The present study was therefore designed to evaluate the relative chemopreventive potential of the neem limonoids azadirachtin and nimbolide in the hamster buccal pouch (HBP) carcinogenesis model by analyzing the expression of proliferating cell nuclear antigen (PCNA), p21waf1, cyclin D1, glutathione S-transferase pi (GST-P), NF-κB, inhibitor of κB (IκB), p53, Fas, Bcl-2, Bax, Bid, Apaf-1, cytochrome C, survivin, caspases-3, −6, −8 and −9, and poly(ADP-ribose) polymerase (PARP) by RT-PCR, immunohistochemical, and Western blot analyses. The results provide compelling evidence that azadirachtin and nimbolide mediate their antiproliferative effects by downregulating proteins involved in cell cycle progression and transduce apoptosis by both the intrinsic and extrinsic pathways. On a comparative basis, nimbolide was found to be a more potent antiproliferative and apoptosis inducing agent and offers promise as a candidate agent in multitargeted prevention and treatment of cancer.

The Flavonoid Quercetin Modulates the Hallmark Capabilities of Hamster Buccal Pouch Tumors

Epidemiological studies have consistently demonstrated the protective effects of dietary phytochemicals against cancer risk. Quercetin, a ubiquitous dietary flavonoid, has attracted considerable attention owing to its potent antioxidant and antiproliferative activities. The present study was designed to investigate the chemopreventive as well as the therapeutic ability of quercetin to modulate the key hallmark capabilities of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinomas. We analyzed the expression of markers associated with cell proliferation and survival (PCNA, p21, p53, cyclin D1, GST-P), apoptosis (Fas, Fas-L, Bcl-2 family proteins, cytochrome-C, Apaf-1, caspases, PARP, survivin, cFLIP, API1), invasion (MMPs, TIMP-2, RECK), angiogenesis (PlGF, VEGF, VEGF receptors, HIF-1α), as well as the epigenetic markers (HDAC-1, DNMT1) by immunohistochemical, Western blot, and RT-PCR analyses. Simultaneous administration of quercetin to DMBA-painted hamsters reduced tumor incidence and tumor burden, while posttreatment of quercetin resulted in a significant tumor growth delay. In addition, quercetin administration induced cell cycle arrest and apoptosis and blocked invasion and angiogenesis. We found a positive correlation between the inhibition of HDAC-1 and DNMT1 by quercetin and its anticancer properties. A dietary phytochemical such as quercetin that modulates a plethora of molecules offers promise as an ideal candidate for multitargeted cancer prevention and therapy.

Eugenol inhibits cell proliferation via NF-κB suppression in a rat model of gastric carcinogenesis induced by MNNG

SummaryThe modulation of intracellular nuclear factor-kappaB (NF-κB) signaling pathway involved in the deregulated expression of cell proliferation and cell cycle regulatory molecules is a pragmatic approach for chemoprevention. Eugenol (4-allyl-1-hydroxy-2-methoxybenzene), a natural phenolic constituent of oils of cloves is known to possess attractive remedial features. In the present study, we investigated the modulatory effects of eugenol on NF-κB signaling in a rat model of gastric carcinogenesis induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) by analysing the expression of nuclear factor-kappaB (NF-κB) family members ((NF-κB (p50 and p65), inhibitor of kappaB alpha (IκBα), phosphorylated IκBα (p-IκBα), IκB kinase β (IKKβ)) and the NF-κB target genes that promote (e.g., cyclin D1, cyclin B and PCNA) or inhibit (e.g., p53, p21, and Gadd45) cell proliferation and cell survival. MNNG-induced gastric tumours were characterized by NF-κB activation that correlated with upregulation of IKKβ, and phosphorylation and degradation of IκBα. Furthermore, upregulation of cyclins and PCNA with downregulation of p21, p53, and Gadd45 suggested that the proliferative advantage in gastric carcinomas is dependent on elevated constitutive NF-κB activity. Administration of eugenol significantly reduced the incidence of MNNG-induced gastric tumours by suppressing NF-κB activation and modulating the expression of NF-κB target genes that regulate cell proliferation and cell survival. The targeting of NF-κB signaling pathway by eugenol may have a significant impact on chemopreventive and therapeutic approaches for cancer.

Chemoprevention of rat mammary carcinogenesis by Azadirachta indica leaf fractions: Modulation of hormone status, xenobiotic-metabolizing enzymes, oxidative stress, cell proliferation and apoptosis

We evaluated the chemopreventive potential of the ethyl acetate fraction (EAF) and methanolic fraction (MF) of Azadirachta indica (neem) leaf on 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinogenesis. Estradiol and estrogen receptor status, xenobiotic-metabolizing enzyme activities, redox status, DNA and protein modifications, and the expression of cell proliferation, and apoptosis related proteins in the mammary gland and liver were used as biomarkers of chemoprevention. Administration of both EAF and MF at a dose of 10mg/kg bw effectively suppressed tumour incidence. Chemoprevention by neem leaf fractions was associated with modulation of hormone and receptor status, xenobiotic-metabolising enzymes, and lipid and protein oxidation, with upregulation of antioxidants, inhibition of oxidative DNA damage, protein modification, and cell proliferation, and induction of apoptosis. However EAF rich in constituent phytochemicals was more effective than MF in modulating multiple molecular targets. These results provide evidence for the chemopreventive efficacy of neem leaf fractions in the rat mammary tumour model.

Correlation of xenobiotic-metabolizing enzymes, oxidative stress and NFκB signaling with histological grade and menopausal status in patients with adenocarcinoma of the breast

BACKGROUND Adenocarcinoma of the breast is the most common cancer worldwide and accounts for the highest morbidity and mortality. The increasing global incidence of breast cancer emphasizes the need to understand the molecular mechanisms of breast tumorigenesis. The present study was designed to correlate changes in xenobiotic-metabolizing enzymes (XME), oxidative stress and NFkappaB signaling with histological grading and menopausal status in breast cancer patients. METHOD Sixty breast cancer patients histologically categorized as grades I, II and III, and as pre- and postmenopausal were chosen for the study. We analyzed phase I and phase II XME activities as well as the expression of the CYP isoforms CYP1A1 and CYP1B1, oxidative stress markers, and the expression of NFkappaB family members in tumor and adjacent tissues by immunohistochemical localization and Western blot analyses. RESULTS The breast tumors analyzed in the present study were characterized by increased activities of xenobiotic-metabolizing enzymes and enhanced oxidative damage to lipids, proteins, and DNA associated with variations in the expression of NFkappaB family members. The magnitude of the changes was however more pronounced in premenopausal patients and in grade III breast tumors. CONCLUSION The present study delineates the correlation between XME-mediated oxidative stress and NFkappaB signaling that leads to the development of breast cancer.

Evaluation of molecular markers in a rat model of mammary carcinogenesis.

We sought to evaluate the molecular markers involved in breast tumorigenesis in a rat model that mimics many essential elements of human breast cancer. Female Sprague-Dawley rats were divided into two groups. Animals in group 1 were given a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) (20 mg/rat) dissolved in 1 ml of sesame oil by intragastric intubation. Group 2 animals received basal diet and served as control. We analyzed DMBA-induced changes in the expression of CYP isoforms (CYP1A1 and 1B1) involved in DMBA metabolism, markers of oxidative stress (4HNE, HEL, and 8-OHdG), cell survival and proliferation (PCNA, NF-kappaB-p50, NF-kappaB-p65, GST-P, and p53), apoptosis (Bcl-2, Bax, caspases, Apaf-1, cytochrome C, and Fas), invasion (uPA, MMP-2, MMP-9, TIMP-2, and RECK), and angiogenesis (VEGF, VEGF-R1, HIF-1alpha, and PLGF) by immunohistochemical localization, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The present study demonstrates increased carcinogen metabolism, oxidative stress, cell proliferation, together with apoptosis evasion, invasion, metastasis, and neovascularization that may confer a selective growth advantage to DMBA-induced mammary tumors. Aberrant expression of multiple molecules in key signaling pathways in Sprague-Dawley rat mammary tumors renders this model as an important tool for monitoring carcinogenic progression and chemointervention.

Mitochondria-mediated apoptosis in patients with adenocarcinoma of the breast: Correlation with histological grade and menopausal status

The present study was designed to investigate the abnormalities in the expression of apoptosis-associated proteins that lead to the progression of breast cancer. Sixty breast cancer patients histologically categorized as grade I, II and III, and as pre- and post-menopausal were chosen for the study. We analyzed the expression of the anti-apoptotic and pro-apoptotic Bcl-2 family proteins as well as cytochrome C, Apaf-1 and caspases in tumour and adjacent tissues by immunohistochemical and Western blot analyses. The breast tumours analyzed in the present study were characterized by increased expression of Bcl-2, Bcl-xL and Mcl-1, associated with downregulation in the expression of Bax, cytosolic cytochrome C, Apaf-1 and caspases. The magnitude of the changes was however more pronounced in premenopausal patients and in grade III tumours. The results of the present study confirm that differential expression patterns of Bcl-2 family proteins and caspases are involved in evasion of apoptosis and in the progression of breast cancer.

Investigation of the chemopreventive potential of neem leaf subfractions in the hamster buccal pouch model and phytochemical characterization

Chemoprevention by medicinal plants has evolved as a practical strategy to control the incidence of cancer. Azadirachta indica (neem) containing various bioactive components is a promising candidate for chemoprevention. The present study was undertaken to evaluate the chemopreventive efficacy of the bioactive subfractions ethyl acetate chloroform insoluble fraction (ECIF) and the methanol ethyl acetate insoluble fraction (MEIF) following activity-guided fractionation of neem leaf extract. Analysis of the mechanism of chemoprevention revealed multitargeted mode of action that involved modulation of xenobiotic-metabolizing enzymes, inhibition of cell proliferation, induction of mitochondrial apoptosis, and abrogation of NF-κB signaling. HP-TLC, GC-MS and LC-MS analyses indicated the presence of several polar phytochemical entities in the neem leaf subfractions that might be responsible for their potent chemopreventive efficacy.

Correlation of matrix metalloproteinases and their inhibitors with hypoxia and angiogenesis in premenopausal patients with adenocarcinoma of the breast

OBJECTIVES The present study was designed to correlate the expression of proteins regulating invasion and angiogenesis in patients with adenocarcinoma of the breast. DESIGN AND METHOD Seventy-five premenopausal breast cancer patients histologically categorized as grades I, II and III were chosen for the study. We analyzed the expression of MMP-2, and -9 and their inhibitors TIMP-2 and RECK together with HIF-1α and VEGF in tumor, adjacent tissues and serum samples by immunohistochemical and Western blot analysis. RESULTS The breast tumors analyzed in the present study were characterized by increased expression of MMP-2, -9, HIF-1α and VEGF with differential expression patterns of TIMP-2 and downregulation of RECK. CONCLUSIONS The simultaneous analysis of the expression of these molecular markers is important to understand the intricate network between key molecules involved in invasion and angiogenesis that eventually determines the clinical course of the disease.