G. Sharmila

Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway.

We aimed to investigate the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human breast cancer cells. The molecular mechanisms involved in the apoptotic activity exerted by nimbolide were studied on the estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. The growth inhibitory effect of nimbolide was assessed by MTT assay. Apoptosis induction by nimbolide treatment was determined by JC-1 mitochondrial membrane potential staining, cytochrome c release, caspase activation, cleavage of PARP and AO/EtBr dual staining. The modulation of apoptotic proteins (intrinsic pathway: Bax, bad, Bcl-2, Bcl-xL, Mcl-1, XIAP-1 and caspase-3, 9; extrinsic pathway: TRAIL, FasL, FADDR and Caspase-8) were studied by western blot and real time PCR analysis. Treatment with nimbolide resulted in dose and time-dependent inhibition of growth of MCF-7 and MDA-MB-231 cells. The occurrence of apoptosis in these cells was indicated by JC-1 staining, modulation of both intrinsic and extrinsic apoptotic signaling molecules expression and further apoptosis was confirmed by AO/EtBr dual staining. These events were associated with: increased levels of proapoptotic proteins Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c and reduced levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP-1. Nimbolide induces the cleavage of pro-caspase-8, pro-caspase-3 and PARP. The above data suggest that nimbolide induces apoptosis by both the intrinsic and extrinsic pathways. With evidence of above data it is suggested that nimbolide exhibit anticancer effect through its apoptosis-inducing property. Thus, nimbolide raises new hope for its use in anticancer therapy.

Quercetin inhibits invasion, migration and signalling molecules involved in cell survival and proliferation of prostate cancer cell line (PC-3)

Urokinase‐type plasminogen activator (uPA) is a serine protease that is involved in cancer progression, especially invasion and metastasis including prostate cancer. uPA activation is mediated by transactivation of uPAR and epidermal growth factor receptor (EGF‐R) in prostate cancer progression. Prostate cancer (PC‐3) cells have highly invasive capacity and they express uPA and uPAR gene. PC‐3 cells are treated with quercetin, which inhibits invasion and migration of PC‐3 cells. Quercetin downregulates uPA, uPAR and EGF, EGF‐R mRNA expressions. Quercetin inhibits cell survival factor β‐catenin, NF‐κB and also proliferative signalling molecules such as p‐EGF‐R, N‐Ras, Raf‐1, c.Fos c.Jun and p‐c.Jun protein expressions. But quercetin increased p38 mitogen‐activated protein kinase protein expression. Our results suggest that quercetin inhibit migration and invasion of prostate cancer cells. It shows the value for treatment of invasive and metastasis type of prostate cancer. Copyright

Quercetin reverses EGF-induced epithelial to mesenchymal transition and invasiveness in prostate cancer (PC-3) cell line via EGFR/PI3K/Akt pathway

Epidermal growth factor (EGF) plays an important role in metastasis and tumorigenesis of prostate cancer. Epithelial-mesenchymal transition (EMT) is a process in tumor progression during which cancer cells undergo dramatic changes acquiring highly invasive properties. The purpose of this study was to determine the effect of quercetin on EGF-induced EMT in prostate cancer (PC-3) cell line. Quercetin, a plant flavonoid, prevented EGF-induced invasion and migration of PC-3 cells. The protein and mRNA expressions of E-cadherin and N-cadherin were studied by immunocytochemistry, Western blotting and real-time polymerase chain reaction. Quercetin prevented EGF-induced expression of N-cadherin and vimentin and increased the expression of E-cadherin in PC-3 cells, therefore preventing EGF-induced EMT. EGF-induced cell adhesion proteins, intercellular adhesion molecule and vascular cell adhesion molecule were significantly decreased by quercetin treatment. Furthermore, mRNA and protein expressions of Snail, Slug and Twist showed that quercetin significantly decreased EGF-induced expressions of Snail, Slug and Twist. The protein expressions of epidermal growth factor receptor (EGFR)/phosphatidylinositide 3-kinases (PI3K)/Akt/extracellular signal-regulated kinase (ERK)1/2 pathway showed that quercetin prevents EGF-induced EMT via EGFR/PI3k/Akt/ERK1/2 pathway and by suppressing transcriptional repressors Snail, Slug and Twist in PC-3 cells. Thus, it is concluded from the present study that quercetin may prevent cancer metastasis by targeting EMT.

Quercetin regulates insulin like growth factor signaling and induces intrinsic and extrinsic pathway mediated apoptosis in androgen independent prostate cancer cells (PC-3)

Progression of prostate cancer is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. To investigate the effect of quercetin on insulin-like growth factor signaling and apoptosis in androgen independent prostate cancer cells (PC-3), IGF-IR, PI-3K, p-Akt, Akt, cyclin D1, Bad, cytochrome c, PARP, caspases-9 and 10 protein levels were assessed by western blot analysis. Mitochondrial membrane potency was detected by rhodamine-123 staining. Quercetin induced caspase-3 activity assay was performed for activation of apoptosis. Further, RT-PCR was also performed for Bad, IGF-I, II, IR, and IGFBP-3 mRNA expression. Quercetin significantly increases the proapoptotic mRNA levels of Bad, IGFBP-3 and protein levels of Bad, cytochrome C, cleaved caspase-9, caspase-10, cleaved PARP and caspase-3 activity in PC-3 cells. IGF-IRβ, PI3K, p-Akt, and cyclin D1 protein expression and mRNA levels of IGF-I, II and IGF-IR were decreased significantly. Further, treatment with PI3K inhibitor (LY294002) and quercetin showed decreased p-Akt levels. Apoptosis is confirmed by loss of mitochondrial membrane potential in quercetin treated PC-3 cells. This study suggests that quercetin decreases the survival of androgen independent prostate cancer cells by modulating the expression of insulin-like growth factors (IGF) system components, signaling molecules and induces apoptosis, which could be very useful for the androgen independent prostate cancer treatment.

Chemopreventive effect of quercetin, a natural dietary flavonoid on prostate cancer in in vivo model

BACKGROUND & AIM Prostate cancer is one of the frequently diagnosed cancers in men. Increased Growth factor IGF-1/IGF-1R axis activation mediated by both PI3K/Akt or RAF/MEK/ERK system and AR expression remains important in the development and progression of prostate cancer. Targeting such system by dietary agents quercetin in vivo model could aid its application in both treatment as well as prevention of prostate cancer. METHODS In our study the rats were divided into four groups; Group I: control (propylene glycol-vehicle), Group II: cancer-induced (MNU and Testosterone treated) rats, Group III: cancer-induced + Quercetin (200 mg/kg body wt/orally) and Group IV: Quercetin (200 mg/kg body wt) thrice a week. After the treatment period rats were sacrificed and the ventral and dorsolateral prostate lobes were dissected. RESULTS Antioxidant enzymes and apoptotic proteins were significantly decreased in cancer-induced animal and upon quercetin supplement its level was increased. The IGFIR, AKT, AR, cell proliferative and anti-apoptotic proteins were increased in cancer-induced group whereas supplement of quercetin decreased its expression. CONCLUSIONS Quercetin down regulates the cell survival, proliferative and anti-apoptotic proteins thereby prevents prostate cancer, by acting as a chemopreventive agent in preclinical model.

Anti-proliferative and apoptosis inducing effect of nimbolide by altering molecules involved in apoptosis and IGF signalling via PI3K/Akt in prostate cancer (PC-3) cell line.

Prostate cancer is responsible for major deaths globally after lung cancer. Nimbolide is an important constituent of neem, and it acts as a potent inhibitor for many cancer cells. The present study was designed to evaluate the effects of nimbolide on apoptosis and insulin‐like growth factor (IGF) signalling molecules in androgen‐independent prostate cancer (PC‐3) cells line. Nimbolide (0.5–2 μM) treatment resulted in 50% inhibition at a dose of 2 μM in the PC‐3 cell line. The mRNA expression of Fas ligand, Fas‐associated death domain receptor (FADDR), Bcl‐2‐associated X protein (Bax), Bcl‐2‐associated death promoter (Bad), phosphatidylinositide 3‐kinases (PI3K), Akt, IGF1, IGF1 receptor (IGF1R) and IGF binding protein 3 were quantified by reverse transcription polymerase chain reaction and protein expression of Bax, cytochrome c, X‐linked inhibitor of apoptosis protein (XIAP), B‐Cell Lymphoma 2 (Bcl‐2), caspases −8, −9, −10 and −3, poly(ADP‐ribose) polymerase (PARP), cleaved PARP, IGF1R, PI3K, Akt, p‐Akt was determined by western blot analysis, in nimbolide‐treated PC‐3 cell line. Nimbolide‐induced apoptosis by activating DNA fragmentation in PC‐3 cells. Nimbolide treatment increased the mRNA of Fas ligand, FADDR, Bax, Bad and IGF binding protein 3, decreased PI3K, Akt, IGF1 and IGF1R, increased protein expression of caspases 8, 3, 10, 9, Bax and cytochrome c and decreased the expression of XIAP, Bcl2, cleaved PARP, p‐Akt and IGF1R. The results suggest that nimbolide acts as a potent anti‐cancer agent by inducing apoptosis and inhibiting cell proliferation via PI3K/Akt pathway in PC‐3 cells. Copyright

Effect of diallyl disulfide on insulin-like growth factor signaling molecules involved in cell survival and proliferation of human prostate cancer cells in vitro and in silico approach through docking analysis

PURPOSE Diallyl Disulfide (DADS) is one of the major components of garlic, which inhibits the proliferation of various cancer cells. Our previous studies showed that DADS inhibits cell growth and induces apoptosis on prostate cancer cells. Insulin like growth factor signaling pathway plays a significant role on prostate cancer cell growth and survival and its over expression also identified in human prostate cancers. The molecular mechanism of IGF mediated PI3K/Akt signaling remains to be elucidated. The present study was designed to evaluate the effects of diallyl disulfide on IGF signaling in androgen independent prostate cancer cells (PC-3). METHODS DADS (10-50 μM) caused dose-dependent inhibition of PC-3 cells, were analyzed by MTT, IC50 value of PC-3 cells was 40 μM for 24h. Interestingly, DADS also altered the mRNA and protein expressions of IGF signaling and apoptotic molecules which were confirmed by semi quantitative PCR and western blot method. Further the docking study of DADS with IGF receptor was carried out by Ligand Fit of Discovery studio. Accord Excel Package was used for the prediction of ADME properties of the compound. RESULTS The results suggests that DADS decreases the survival rate of androgen independent prostate cancer cells by modulating the expression of IGF system, which leads to inhibition of phosphorylation of Akt, thereby inhibits cell cycle progression and survival by lowering the expression of cyclin D1, NFkB and anti-apoptotic Bcl-2 molecule and increasing the level of pro-apoptotic (Bad and Bax) signaling molecules which leads to apoptosis. CONCLUSION The present investigation showed downregulation of Akt and a concomitant increase in apoptosis in DADS treated prostate cancer cells. Since inhibition of this Akt pathway by DADS leads to inhibition in cancer cell progression, it is highly suggested that DADS has the potential use as a therapy for prostate cancer.

Nimbolide inhibits IGF‐I‐mediated PI3K/Akt and MAPK signalling in human breast cancer cell lines (MCF‐7 and MDA‐MB‐231)

The insulin‐like growth factor I (IGF‐I) signalling pathway contributes a major role on various cancer cell proliferation, survival and cell cycle. The present study was aimed to investigate the effect of nimbolide on IGF signalling and cell cycle arrest in MCF‐7 and MDA‐MB‐231 breast cancer cell lines. The protein expression of IGF signalling molecules and cell cycle protein levels was assessed by western blot analysis. In order to study the interaction of nimbolide on IGF‐1 signalling pathway, IGF‐I and phosphoinositide 3‐kinase (PI3K) inhibitor (LY294002) were used to treat MCF‐7 and MDA‐MB‐231 cells. Further, the cell cycle arrest was analysed by flow cytometry. The protein expression of IGF signalling molecules was significantly decreased in nimbolide‐treated breast cancer cells. PI3K inhibitor and IGF‐I with nimbolide treatment notably inhibited phosphorylated Akt. The cell cycle arrest was observed at the G0/G1 phase, and accumulation of apoptotic cells was observed in nimbolide‐treated breast cancer cell lines. Nimbolide also increased the protein expression of p21 and decreased the cyclins in both the cell lines. Nimbolide decreases the proliferation of breast cancer cells by modulating the IGF signalling molecules, which could be very useful for the breast cancer treatment. Copyright

Chemopreventive Effect of Quercetin in MNU and Testosterone Induced Prostate Cancer of Sprague-Dawley Rats

Prostate cancer becomes an ideal target for chemoprevention because of its high incidence and extended natural history. The consumption of quercetin (plant flavonoid) in diet is associated with decreased risk of disease and many cancers but then this was not elucidated in prostate malignancy. Hence, a study in which the male Sprague-Dawley rats were induced prostate cancer by hormone (testosterone) and carcinogen (MNU) and simultaneously supplemented with quercetin (200 mg/Kg body weight) thrice a week, was conducted. After the treatment period, rats were killed; ventral and dorsolateral lobes of the prostate were dissected. Histology and oxidative stress markers LPO, H2O2, and antioxidant GSH level were measured in both lobes. The lipid peroxidation, H2O2, in (MNU+T) treated rats were increased and GSH level was decreased, whereas simultaneous quercetin-treated rats reverted back to normal level in both ventral and dorsolateral regions. The different patterns of PIN were observed with associated hyperplasia and dysplasia; changes in these regions and the occurrence of this lesion were reduced in simultaneous quercetin-treated rats. The study concluded that dietary quercetin prevented MNU + T-induced prostate carcinogenesis on both ventral and dorsolateral lobes of Sprague-Dawley rats.

Nimbolide inhibits invasion and migration, and down‐regulates uPAR chemokine gene expression, in two breast cancer cell lines

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in women, worldwide. Urokinase type plasminogen activator (uPA) is a serine protease that is involved in cancer progression, especially invasion and metastasis of breast cancer. Nimbolide is a potent cytotoxic limnoid isolated from Azadirachta indica. Our previous studies have shown that nimbolide elicits pleiotropic effects on breast cancer cells; however, its roles in invasion and migration have not previously been fully elucidated.