Neetu Kalra

Resveratrol induces apoptosis involving mitochondrial pathways in mouse skin tumorigenesis

Resveratrol, a plant constituent enriched in the skin of grapes, is one of the most promising agents for chemoprevention. In the present study, resveratrol-induced apoptosis in 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA) promoted, mouse skin tumors. The chemopreventive effects of resveratrol in terms of delayed onset of tumorigenesis, cumulative number of tumors and average number of tumors/mouse were recorded. Resveratrol treatment resulted in regression of tumors (28%) after withdrawal of the TPA treatment. Induction of apoptosis by resveratrol in DMBA-TPA induced skin tumors was recorded by the appearance of a sub-G1 fraction (30%) using flow cytometry and an increase in the number of apoptotic cells by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay. Western blot analysis combined with multivariable flow cytometry, showed that resveratrol application induces the expression of the p53 and pro-apoptotic Bax, with concomitant decrease in anti-apoptotic protein Bcl-2. Alteration in Bax/Bcl2 ratio by resveratrol treatment resulted in apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1(APAF-1). Further, this effect was found to result in cleaved fragments of caspase-9,-3, and poly (ADP-ribose) polymerase (PARP). These findings demonstrate for the first time that resveratrol induces apoptosis through activation of p53 activity in mouse skin tumors, thereupon suggesting its chemopreventive activity, through the modulation of proteins involved in mitochondrial pathway of apoptosis.

Theaflavins induced apoptosis of LNCaP cells is mediated through induction of p53, down-regulation of NF-kappa B and mitogen-activated protein kinases pathways

Prostate cancer (PCA), the most frequently diagnosed malignancy in men, represents an excellent candidate disease for chemoprevention studies because of its particularly long latency period, high rate of mortality and morbidity. Infusion of black tea and its polyphenolic constituents have been shown to possess antineoplastic effects in androgen dependent PCA in both in vivo and in vitro models including transgenic animals. In the present study, we report that black tea polyphenol, Theaflavins (TF)-induced apoptosis in human prostate carcinoma, LNCaP cells is mediated via modulation of two related pathways: up-regulation of p53 and down-regulation of NF-kappa B activity, causing a change in the ratio of pro-and antiapoptotic proteins leading to apoptosis. The altered expression of Bcl-2 family member proteins triggered the release of cytochrome-C and activation of initiator capsase 9 followed by activation of effector caspase 3. Furthermore, TF also affected the protein expression of mitogen activated protein kinases (MAPK) pathways. Our results demonstrated that TF treatment resulted in down-regulation of phospho-extracellular signal-regulated protein kinase (Erk1/2) and phospho-p38 MAPK expressions. We conclude that TF induces apoptosis in LNCaP cells by shifting the balance between pro-and antiapoptotic proteins and down-regulation of cell survival pathways leading to apoptosis. Further extending this work, we also showed that TF induces apoptosis in androgen independent PCA cell line, PC-3 through caspases and MAPKs mediated pathways. Thus, effect of TF on PCA cell lines seems to be irrespective of their androgen status.

Regulation of signaling pathways involved in lupeol induced inhibition of proliferation and induction of apoptosis in human prostate cancer cells

Prostate cancer (PCa) is the most frequently diagnosed noncutaneous cancer and the leading cause of cancer related deaths in men in the United States and many other Asian countries. Dietary factors are considered as a strategic agent to control the risk of PCa. Lupeol, a triterpene, present in fruits and medicinal plants, has been shown to possess many pharmacological properties including anticancer effects. Here, effect of lupeol on cell proliferation and cell death was evaluated using human PCa cells, PC-3. In MTT assay, lupeol inhibited the cell proliferation (12-71%) in dose (50-800 microM) and time dependent manner. Flow-cytometric analysis of cell-cycle revealed that an antiproliferative effect of lupeol (400-600 microM) is associated with an increase in G(2)/M-phase arrest (34-58%). RT-PCR analysis showed that lupeol-induced G2/M-phase arrest was mediated through the inhibition of cyclin regulated signaling pathway. Lupeol inhibited the expression of cyclin B, cdc25C, and plk1 but induced the expression of 14-3-3sigma genes. However no changes were observed in the expression of gadd45, p21(waf1/cip1) and cdc2 genes. Results of western blot showed that lupeol regulates the phosphorylation of cdc2 (Tyr15) and cdc25C (Ser198). Further, on increase of lupeol exposure to PC-3 cells an induction of apoptosis was recorded, which was associated with upregulation of bax, caspase-3, -9, and apaf1 genes and down regulation of antiapoptotic bcl-2 gene. The role of caspase-induced apoptosis was confirmed by increase in reactive oxygen species, loss of mitochondrial membrane potential followed by DNA fragmentation. Thus, our study suggests that lupeol possess novel antiproliferative and apoptotic potential against PCa.

Resveratrol enhances ultraviolet B-induced cell death through nuclear factor-κB pathway in human epidermoid carcinoma A431 cells

Resveratrol has been reported to suppress cancer progression in several in vivo and in vitro models, whereas ultraviolet B (UVB), a major risk for skin cancer, is known to induce cell death in cancerous cells. Here, we investigated whether resveratrol can sensitize A431 human epidermoid carcinoma cells to UVB-induced cell death. We examined the combined effect of UVB (30 mJ/cm(2)) and resveratrol (60 microM) on A431 cells. Exposure of A431 carcinoma cells to UVB radiation or resveratrol can inhibit cell proliferation and induce apoptosis. However, the combination of resveratrol and UVB exposure was associated with increased proliferation inhibition of A431 cells compared with either agent alone. Furthermore, results showed that resveratrol and UVB treatment of A431 cells disrupted the nuclear factor-kappaB (NF-kappaB) pathway by blocking phosphorylation of serine 536 and inactivating NF-kappaB and subsequent degradation of IkappaBalpha, which regulates the expression of survivin. Resveratrol and UVB treatment also decreased the phosphorylation of tyrosine 701 of the important transcription factor signal transducer activator of transcription (STAT1), which in turn inhibited translocation of phospho-STAT1 to the nucleus. Moreover, resveratrol/UVB also inhibited the metastatic protein LIMK1, which reduced the motility of A431 cells. In conclusion, our study demonstrates that the combination of resveratrol and UVB act synergistically against skin cancer cells. Thus, resveratrol is a potential chemotherapeutic agent against skin carcinogenesis.

Induction of Apoptosis by Lupeol and Mango Extract in Mouse Prostate and LNCaP Cells

Prostate cancer (PCA) is one of the most invasive malignancy and second leading cause of cancer related deaths in United States and some other countries. Long latency period makes PCA an ideal disease for pharmacologic or nutritional chemoprevention. Lupeol, a triterpene present in mango and other fruits, has shown to possess anticancer properties in in vivo and in vitro assays. Here, we recorded the apoptogenic activity in mouse prostate by lupeol and mango pulp extract (MPE). Testosterone was injected subcutaneously (5 mg/kg body weight) for 14 consecutive days to male Swiss albino mice. Lupeol/MPE supplementation resulted in arrest of prostate enlargement in testosterone-treated animals. In mouse prostate tissue, lupeol and MPE supplementation resulted in a significantly high percentage of apoptotic cells in the hypodiploid region. The induction of apoptosis in mouse prostate cells was preceded by the loss of mitochondrial transmembrane potential and DNA laddering. In testosterone-induced mouse prostate, upregulation of antiapoptotic B-cell non-Hodgkin lymphoma-2 and downregulation of proapoptotic Bcl-2-associated X protein and caspase-3 were also recorded. We further observed apoptogenic activities of lupeol in an in vitro model using human prostate cancer cells [lymph node carcinoma of the prostate (LNCaP)]. The apoptogenic response of lupeol-induced changes in LNCaP cells can be summarized as early increase of reactive oxygen species followed by induction of mitochondrial pathway leading to cell death. Thus, the results of this study demonstrate that lupeol/MPE is effective in combating testosterone–induced changes in mouse prostate as well as causing apoptosis by modulating cell-growth regulators.

Regulation of oxidative stress–mediated apoptosis by diallyl sulfide in DMBA-exposed Swiss mice:

Diallyl sulfide, a sulfur-containing volatile compound present in garlic (Allium sativum), exerts anticarcinogenic activity in various rodent tumor models. In the present study, apoptosis-inhibiting effects of diallyl sulfide against a carcinogenic polycyclic aromatic hydrocarbon, 7,12-dimethyl benz(a)anthracene (DMBA), in Swiss albino mice were observed. The animals were given either 250 μg/mouse or 500 μg/mouse of diallyl sulfide for 1 week after a single intragastric dose of 7,12-dimethyl benz(a)anthracene (50 mg/kg body weight). Results showed that diallyl sulfide supplementation effectively protects against 7,12-dimethyl benz(a)anthracene—induced oxidative stress, characterized by restored antioxidant enzyme levels (up to 64%) and lipid peroxidation (up to 25%). Flow cytometric analysis showed a reduction in apoptotic cell population in hypodiploid region in diallyl sulfide–supplemented animals. Inhibition of apoptosis was preceded by decrease in reactive oxygen species levels and restoration of mitochondrial transmembrane potential followed by decreased DNA fragmentation. In 7,12-dimethyl benz(a)anthracene–exposed animals, downregulation (~30%) of antiapoptotic Bcl-2 and upregulation (~60%) of pro-apoptotic Bax proteins were observed. These alterations were restored significantly by diallyl sulfide supplementation, indicating inhibition of apoptosis. Thus, these results show that diallyl sulfide provides protection against oxidative damage induced by 7,12-dimethyl benz(a)anthracene in mouse liver and may be an effective chemopreventive and therapeutic agent by modulating expression of cell-growth regulatory proteins.