Apoptotic role of natural isothiocyanate from broccoli (Brassica oleracea italica) in experimental chemical lung carcinogenesis.

CONTEXT: Sulforaphane (SFN) [1-isothiocyanato-4-(methylsulfinyl)butane] is a naturally occurring isothiocyanate found in cruciferous vegetables such as broccoli [Brassica oleracea L. var. italica Plenck. (Brassicaceae)]. Since it is among the most potent bioactive components with antioxidant and antitumor properties, it has received intense attention in the recent years for its chemopreventive properties. OBJECTIVE: The present work determined the rehabilitating role in alleviating the oxidative damage caused by benzo(a)pyrene [B(a)P] to biomolecules and the apoptotic cascade mediated by orally administered isothiocyanate-SFN (9 µmol/mouse/day) against B(a)P (100 mg/kg body weight, i.p.) induced pulmonary carcinogenesis in Swiss albino mice. MATERIALS AND METHODS: Oxidative damage was assessed by measuring lipid peroxidation, 8-hydroxydeoxyguanosine, hydrogen peroxide (H2O2) production, glycoprotein components, protein carbonyl levels and DNA-protein crosslinks. DNA fragmentation by agarose gel electrophoresis and caspase-3 activity by ELISA proved apoptotic induction by SFN along with the protein expression of Bcl-2, Bax and Cyt c. RESULTS: SFN treatment was found to decrease the H2O2 production (p < 0.001) in cancer induced animals, proving its antioxidant potential. Apoptosis was induced by increasing the release of Cyt c (p < 0.001) from mitochondria, decreasing and increasing the expression of Bcl-2 (p < 0.01) and Bax (p < 0.001), respectively. Caspase-3 activity was also enhanced (p < 0.001) which leads to DNA fragmentation in SFN treated groups. CONCLUSION: Our results reflect the rehabilitating role of SFN in B(a)P induced lung carcinogenesis.

In vitro and in vivo studies on antitumor effects of gossypol on human stomach adenocarcinoma (AGS) cell line and MNNG induced experimental gastric cancer.

The present study has evaluated the chemopreventive effects of gossypol on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis and on human gastric adenocarcinoma (AGS) cell line. Gossypol, C(30)H(30)O(8), is a polyphenolic compound that has anti proliferative effect and induces apoptosis in various cancer cells. The aim of this work was to delineate in vivo and in vitro anti-initiating mechanisms of orally administered gossypol in target (stomach) tissues and in human gastric adenocarcinoma (AGS) cell line. In vitro results prove that gossypol has potent cytotoxic effect and inhibit the proliferation of adenocarcinoma (AGS) cell line. In vivo results prove gossypol to be successful in prolonging the survival of MNNG induced cancer bearing animals and in delaying the onset of tumor in animals administrated with gossypol and MNNG simultaneously. Examination of the target (stomach) tissues in sacrificed experimental animals shows that administration of gossypol significantly reduces the level of tumor marker enzyme (carcino embryonic antigen) and pepsin. The level of Nucleic acid contents (DNA and RNA) significantly reduces, and the membrane damage of glycoprotein subsides, in the target tissues of cancer bearing animals, with the administration of gossypol. These data suggest that gossypol may create a beneficial effect in patients with gastric cancer.

Protective role of sulforaphane against oxidative stress mediated mitochondrial dysfunction induced by benzo(a)pyrene in female Swiss albino mice.

Recent focus of cancer chemoprevention is on intermediate biomarkers capable of detecting early changes that can be correlated with inhibition of carcinogenic initiation and progression. The present study aimed to delineate in vivo anti-initiating mechanisms of orally administered sulforaphane (SFN) with special reference to mitochondrial dysfunction in target (lungs) and non-target (liver) tissues employing benzo(a)pyrene [B(a)P] as the model carcinogen. The recent revival of interest in the study of mitochondria has been stimulated by the evidence that genetic and/or metabolic alterations in this organelle lead to a variety of human diseases including cancer. These alterations could be either causative or contributing factors. Hence the activities of mitochondrial specific enzymes of TCA cycle and the electron transport complexes were analyzed in the experimental groups to assess the ATP production. Short-term tests such as the assessment of antioxidant status and reactive oxygen species (ROS)-induced lipid peroxidation, are widely used in the detection and evaluation of anticarcinogens. The assessment of mitochondrial lipid peroxidation along with the antioxidant status proved worthy for the correlation to the degree of damage. The induction of cancer was confirmed by the immunohistochemistry for Bcl-2. The results prove sulforaphane to be very successful in combating the oxidative stress mediated mitochondrial dysfunction in experimental lung carcinogenesis induced by benzo(a)pyrene.

Anti-cancer effect of Cassia auriculata leaf extract in vitro through cell cycle arrest and induction of apoptosis in human breast and larynx cancer cell lines.

The in vitro anti-cancer effect of Cassia auriculata leaf extract (CALE) was evaluated in human breast adenocarcinoma MCF-7 and human larynx carcinoma Hep-2 cell lines. CALE preferentially inhibited the growth of both the cell lines in a dose-dependent manner with IC(50) values of 400 and 500 microg for MCF-7 and Hep-2 cells, respectively. The results showed the anti-cancer action is due to nuclear fragmentation and condensation, associated with the appearance of A(0) peak in cell cycle analysis that is indicative of apoptosis. In addition, CALE treated MCF-7 and Hep-2 cells had decreased expression of anti-apoptotic Bcl-2 protein and increased expression of pro-apoptotic Bax protein, eventually leading a decrease in the Bcl-2/Bax ratio. These results demonstrated that CALE inhibits the proliferation of MCF-7 and Hep-2 cells through induction of apoptosis, making CALE a candidate as new anti-cancer drug.

Effect of mangiferin on benzo(a)pyrene induced lung carcinogenesis in experimental Swiss albino mice.

The present study is an effort to identify a potent chemopreventive agent against cancer, in which oxidative stress plays an important causative role. The modulatory effect of mangiferin on mitochondrial lipid peroxidation (LPO), tricarboxylic acid (TCA) cycle key enzymes and electron transport chain complexes was investigated against lung carcinogenesis induced by benzo(a)pyrene (50 mg kg(-1) b/w orally) in Swiss albino mice. Decreased activities of electron transport chain complexes and TCA cycle key enzymes such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH), in lung cancer bearing animals were observed. Pre- and post-treatment with mangiferin (100 mg kg(-1) b/w orally) for 18 weeks, prevented the above biochemical changes, which were inclined towards normal control animal values. This study further confirms the chemopreventive and chemotherapeutic effect of mangiferin and these results are consistent with our hypothesis that mangiferin is a promising chemopreventive agent.

Preliminary studies on induction of apoptosis by genistein on HepG2 cell line.

Consumption of soy products has been linked to lower the incidence of number of cancers. Genistein, one of the principal soy isoflavones, has been shown to inhibit the growth of a number of tumor cell lines in vitro. In this study, we investigate the effects of genistein on cell growth and apoptosis in human hepatocellular carcinoma HepG2 cell by looking for the formation of nuclear apoptotic bodies and DNA ladder formation. Additionally, flow cytometry analysis with propidium iodide staining has been conducted to detect the apoptotic cells. We found inhibition of cell growth and apoptotic nuclei, DNA fragmentation and increased apoptotic cells after treatment with genistein, indicating apoptotic cell deaths. From these results we observed that genistein inhibits the growth of HepG2 cells and induce apoptosis, however, further definitive studies are needed. These results may support the potentially effective chemopreventive and/or chemotherapeutic of genistein against liver cancer.

Modulation of TCA cycle enzymes and electron transport chain systems in experimental lung cancer.

The modulatory effect of Withania somnifera along with paclitaxel on tricarboxylic acid (TCA) cycle key enzymes and electron transport chain complexes were investigated against lung cancer induced by benzo(a)pyrene in Swiss albino mice. Decreased activities of TCA cycle key enzymes such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) in lung cancer bearing animals were observed. Upon W. somnifera along with paclitaxel administration the above biochemical changes were inclined towards normal control animal values. Activities of mitochondrial enzymes and electron transport complexes were analyzed in the experimental groups to determine the efficiency of energy production. This study further confirms the chemotherapeutic effect of W. somnifera along with paclitaxel which is found to be more effective in the treatment of lung cancer. Thus these results are consistent with our hypothesis that the combination chemotherapy of W. somnifera along with paclitaxel as a promising chemotherapeutic agent.

Chemoprevention and cytotoxic effect of Bauhinia variegata against N-nitrosodiethylamine induced liver tumors and human cancer cell lines.

The chemopreventive and cytotoxic effect of ethanol extract of Bauhinia variegata (EBV) was evaluated in N-nitrosodiethylamine (DEN, 200 mg/kg) induced experimental liver tumor in rats and human cancer cell lines. Oral administration of ethanol extract of Bauhinia variegata (250 mg/kg) effectively suppressed liver tumor induced by DEN as revealed by decrease in DEN induced elevated levels of serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), total bilirubin, gamma glutamate transpeptidase (GGTP), lipid peroxidase (LPO), glutathione peroxidase (GPx) and glutathione S-transferase (GST). The extract produced an increase in enzymatic antioxidant (superoxide dismutase and catalase) levels and total proteins when compared to those in liver tumor bearing rats. The histopathological changes of liver samples were compared with respective controls. EBV was found to be cytotoxic against human epithelial larynx cancer (HEp2) and human breast cancer (HBL-100) cells. These results show a significant chemopreventive and cytotoxic effect of ethanol extract of Bauhinia variegata against DEN induced liver tumor and human cancer cell lines.

Preliminary study on inhibition of genotoxicity by piperine in mice.

A significant suppression (33.9-66.5%) in the micronuclei formation induced by benzo(a)pyrene and cyclophosphamide was reduced following oral administration of piperine at doses of 25, 50 and 75 mg/kg in mice.

Immunomodulatory effect of tungstate on streptozotocin-induced experimental diabetes in rats.

Immune dysfunction has been found to be associated with diabetes mellitus. Tungstate treatment restored the number and function of immune cells as well as the immunoglobulin level in STZ diabetic rats. This indicated the immunomodulatory effect of tungstate in diabetics and would be effective in inhibiting diabetic-induced alterations.

Stabilization of membrane bound enzyme profiles by sodium selenite in N-nitrosodiethylamine induced and phenobarbital promoted hepatocarcinogenesis in rats.

As part of a substantial effort to curtail the adverse health effects posed by hepatoma, studies have been conducted to elucidate the possible mechanism for the anticarcinogenic action of sodium selenite against N-nitrosodiethylamine induced hepatocarcinogenesis. Sodium selenite administered through drinking water at a dose of 4 ppm before initiation, or during initiation and/or during the promotion period of carcinogenesis exerted an in vivo stabilizing effect on cell membranes in rat hepatoma. This was demonstrated in normal rats and in animals whose biomembranes were rendered fragile by induction of hepatoma with N-nitrosodiethylamine and subsequent treatment with sodium selenite. The obtained results have shown a significant decrease in the activities of Na(+)/K(+)-ATPases, Mg(2+)-ATPases and Ca(2+)-ATPases (P < 0.001) in erythrocyte membrane; hepatoma and surrounding liver tissue and also erythrocyte membrane are more susceptible to lysis in cancer-bearing animals. The selenite administration reversed these adverse changes to near normal in selenite-treated animals. Such stabilization of biomembranes by selenite has a beneficial effect in the treatment of hepatoma and other cancers involving abnormal fragility of cell membrane. Previous evidence from this laboratory with respect to the anticancer potency of selenite against N-nitrosodiethylamine-induced hepatoma together with the present results suggests that potentially effective therapeutic protection can be achieved by pre-supplementation of selenite.

Effect of dietary selenite on N-nitrosodiethylamine-induced and phenobarbital promoted multistage hepatocarcinogenesis in rat: reflection in some minerals.

Selenium (Se), a dietary micronutrient, plays a vital role in cancer chemotherapy in many organs including the liver. We have studied the relationship between some minerals, which are essential in normal functioning of cells and anticancer effect of Se in N-nitrosodiethylamine (DEN) induced and phenobarbital (PB) promoted multistage hepatocarcinogenesis. Se (4 ppm through drinking water; as sodium selenite) was given to animals throughout the study, before initiation and during promotion phase of hepatocarcinogenesis, in a defined experimental protocol. Se, sodium, potassium, calcium and iron were measured either in hepatoma, or surrounding liver tissue or whole liver tissue and serum of experimental animals. DEN and PB treatment significantly (P < 0.001) increased potassium, calcium and iron levels in serum, while it decreased (P < 0.001) the Se and sodium levels when compared with control rats. We have also observed significantly increased (P < 0.001) sodium, calcium and iron levels in hepatoma and surrounding liver tissue, whereas, Se, and potassium level was found to be decreased (P < 0.001) when compared with control rats. Supplementation of selenite throughout the study, before initiation and during promotion stage significantly alters the above mineral content. Results showed that the most significant beneficial effect of selenium during hepatocarcinogenesis was exerted potentially in long-term continuous and/or before the initiation phase of carcinogenicity, rather than in the promotion phase. The present and previous results from our laboratory suggest that sub-optimal intake of a single trace mineral can have broad effects on chemotherapy, providing a framework for understanding the multiple beneficial effects of selenium in cancer chemoprevention.

Chemopreventive effect of piperine on modulating lipid peroxidation and membrane bound enzymes in benzo(a)pyrene induced lung carcinogenesis.

The current study was designed to evaluate the effects of oral supplementation of the piperine on lung tumour initiation by orally applied benzo(a)pyrene (B(a)p). To evaluate the effects of orally supplemented piperine on lung tumour initiation by B(a)p, its effects on ATPase enzymes were first evaluated. Lung cancer bearing mice showed an increase in erythrocyte membrane and tissues ATPase enzymes (Na(+)/K(+)-ATPases, Mg(2+)-ATPases and Ca(2+)-ATPases). Na(+) K-ATPase and Mg-ATPase enzyme activities were decreased and calcium ATPase increased (P < 0.05) in erythrocyte membrane and tissues of lung cancer bearing animals compared with control groups. The elevation of these enzyme activities in membrane and tissues were indicative of the persistent deteriorating effect of B(a)p in cancer bearing animals. These enzyme activities were reversed to near normal control values in animals treated with piperine (50 mg/kg body weight). It is apparent that the beneficial effect of piperine is primarily exerted on the during initiation phase and post-initiation stage of B(a)p induced lung carcinogenesis. Overall, these data indicative that piperine has chemopreventive effects when administered orally on lung cancer bearing animals.

Antioxidant-associated chemoprevention by sodium selenite in N-nitrosodiethylamine-induced and phenobarbital-promoted hepatocarcinogenesis in rats.

The anticarcinogenic/antioxidant potential of sodium selenite (Se), a micronutrient, was evaluated on liver tumourigenesis induced by N-nitrosodiethylamine (DEN) and promoted by phenobarbital (PB; 0.05% in diet). Male, albino rats of the Wistar strain were exposed intravenously to a single dose of DEN (200 mg x kg(-1) body weight). Se (4 ppm in drinking water) was supplemented before initiation, or during initiation and/or during the promotion period of carcinogenesis. At the end of 16 weeks (after DEN administration) nodular incidence, the total number of nodules and non-enzymic antioxidants such as vitamin E, vitamin C, total thiol, protein thiol and non-protein thiol contents were measured in hepatoma, surrounding tissue and kidney tissue of control and experimental groups. In hepatoma-bearing animals the above biochemical changes were decreased when compared with normal control animals. On Se treatment throughout the study, (20 weeks) the above biochemical changes reverted to normal levels. Pre- and post-treatment with Se also shows a tendency to reverse the above changes. The results indicate that prior application of Se significantly reverses the adverse changes produced during the tumourigenesis. Furthermore, prior applications of Se significantly reduced the cumulative number of tumours per tumour-bearing animals. The present study reveals the antitumour potential of Se against DEN-induced liver carcinogenesis.

Protective effect of Emblica officinalis ethanolic extract against 7,12-dimethylbenz(a) anthracene (DMBA) induced genotoxicity in Swiss albino mice.

Ethanolic extract of Emblica officinalis (EO) fruit extract was evaluated for protection against genotoxicity induced by the rodent carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA). Oral administration of EO fruit extract in various concentrations (100, 250, 500 mg/kg b.wt) for seven consecutive days prior to a single intraperitoneal injection of DMBA decreased the frequency of bone marrow micronuclei induced in Swiss albino mice. Significant increases in the liver antioxidants, such as glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR) and detoxifying enzyme glutathione-S-transferase (GST), were found in the fruit extract treated group. The extract also reduced the hepatic levels of the activating enzymes cytochrome (Cyt) P450 and Cyt b5. These increased in the carcinogen treated group, which emphasizes its protective effect against the carcinogen. There was a dose-dependent effect of the extract against the genotoxin with the maximum effect at 500 mg/kg b.wt. The protection afforded by EO may be associated with its antioxidant capacity and through its modulatory effect on hepatic activation and detoxifying enzymes.

Cytoprotective effect of piperine against benzo[a]pyrene induced lung cancer with reference to lipid peroxidation and antioxidant system in Swiss albino mice.

The cytoprotective effect of piperine on benzo[a]pyrene (B[a]P) induced experimental lung cancer was investigated in male Swiss albino mice. Oral administration of piperine (100 mg/kg body wt.) effectively suppressed lung cancer initiated with B[a]P as revealed by the decrease in the extent of lipid peroxidation with concomitant increase in the activities of enzymatic antioxidants (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic antioxidant (reduced glutathione, vitamin E and vitamin C) levels when compared to lung cancer bearing animals. Our data suggest that piperine may extend its chemopreventive effect by modulating lipid peroxidation and augmenting antioxidant defense system.

In vitro studies on the selective cytotoxic effect of crocetin and quercetin.

Crocetin (5-20 microg/ml), quercetin (10-40 microg/ml), and cisplatin (60-180 microg/ml) used as a positive control drug, were tested against human rhabdomyosarcoma (RD) cells and African green monkey kidney (Vero) cells. The cell viability, morphological changes, and lactate dehydrogenase activity were assessed. RD cell growth was found to be inhibited dose dependently by the three tested compounds. Morphological observation by phase contrast microscopy revealed that both crocetin and quercetin caused intense damage only on the malignant (RD) cells, whereas mild toxic effect was seen with cisplatin also on normal (Vero) cells.

Role of sulforaphane in the anti-initiating mechanism of lung carcinogenesis in vivo by modulating the metabolic activation and detoxification of benzo(a)pyrene.

Biomarkers are central to the molecular epidemiology approach. Since scientific research progress within this standard, a more complete biological understanding of the specific events underlying the multistage carcinogenesis model is essential. Hence the present investigation was designed to assess the anti-initiating potential of Sulforaphane (SFN) against benzo(a)pyrene [B(a)P] induced lung carcinogenesis in female Swiss Albino Mice by evaluating the activities of xenobiotic markers, and the balance between phase I and phase II carcinogen/drug metabolizing enzymes. We sought to institute whether orally administered SFN reaches the lung tissue and increases functional capacity of detoxification enzymes in this tissue and compare the biochemical changes associated with the initiation of cancer. We demonstrated the inhibitory effects of orally administered sulforaphane on B[a]P-induced aryl hydrocarbon receptor (AHR) activation which subsequently resulted in decreased Phase-I enzyme activities in vivo. The study also highlights that treatment with sulforaphane enhanced the Nuclear factor erythroid 2-related factor 2 (Nrf2) transcription which reflects its nuclear accumulation and DNA binding in mice, together with the induction of phase II enzymes as evident from our results. These modulations by sulforaphane further result in decreased carcinogen-induced stress. By and large, the results suggest an anti-initiating role of sulforaphane in pre- and post-initiation phase of experimentally induced lung carcinogenesis in female Swiss albino mice.

Chemopreventive role of sulforaphane by upholding the GSH redox cycle in pre- and post-initiation phases of experimental lung carcinogenesis.

Sulforaphane (SFN) is a natural, biologically active compound extracted from cruciferous vegetables such as broccoli and cabbage with anti-inflammatory and anti-cancer properties. The present study was carried to assess cytoprotective potential in alleviating oxidative stress, to influence the initiation and subsequent carcinogenesis caused by benzo(a)pyrene [B(a)P] administration in the pre- and post-initiation phases of carcinogenesis in Swiss albino mice. Sulforaphane, supplemented orally at a dose of 9µmoles /mouse/day was found to greatly lessen the damaging effects of B(a)P in mice by increasing the availability of reducing equivalents to fulfil the futile GSH redox cycle and replenish GSH biosynthesis, stabilizing the thiol status. Activity of superoxide dismutase and catalase in native gel prove their differential activities in cancer induced and treated animals. SFN was also found to prevent formation of leaky membranes by boosting the antioxidant status leading to maintenance of ATPase activity in B(a)P treated animals. Histopathological analysis confirmed reduction of carcinogen-associated morphological changes in the lung tissue. The results suggest that SFN has potential as a chemopreventive phytochemical against B(a)P induced lung damage in the processes of carcinogenesis.

Combination therapeutic effect of cisplatin along with Solanum trilobatum on benzo(a)pyrene induced experimental lung carcinogenesis.

Lung cancer is one of the leading causes of cancer death in the world and is notoriously difficult to treat effectively. In the present study, male Swiss albino mice were divided into five groups of six animals each: group I animals received corn oil orally and served as a control; group II cancer-induced animals received benzo(a)pyrene (B[a]P) (50 mg kg(-1) bodyweight dissolved in corn oil, orally) twice weekly for four successive weeks; group III cancer-bearing animals (after 12 weeks of induction) were treated with cisplatin (6 mg kg(-1) bodyweight, i.p.) once weekly for 4 weeks; group IV cancer-bearing animals were treated with cisplatin along with Solanum trilobatum (300 mg kg(-1) bodyweight) orally once weekly for 4 weeks; and group V animals constituted the drug control treated with cisplatin along with S. trilobatum. The serum, lung and liver were investigated biochemically for aryl hydrocarbon hydroxylase, gamma-glutamyl transpeptidase, 5'-nucleotidase, lactate dehydrogenase (LDH) and protein-bound carbohydrate components (hexose, hexosamine and sialic acid). These enzyme activities were increased significantly in cancer-bearing animals compared with control animals. The elevation of these in cancer-bearing animals was indicative of the persistent deteriorating effect of B[a]P in cancer-bearing animals. Our data suggest that cisplatin, administered with S. trilobatum, may extend its chemotherapeutic effect through modulating protein-bound carbohydrate levels and marker enzymes, as they are indicators of cancer. The combination of cisplatin with S. trilobatum could effectively treat the B[a]P-induced lung cancer in mice by offering protection from reactive oxygen species damage and also by suppressing cell proliferation.