Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.     

Resveratrol decreases breast cancer cell viability and glucose metabolism by inhibiting 6-phosphofructo-1-kinase

Cancer cells are highly dependent on glycolysis to supply the energy and intermediates required for cell growth and proliferation. The enzyme 6-phosphofructo-1-kinase (PFK) is critical for glycolysis, and its activity is directly correlated with cellular glucose consumption. Resveratrol is a potential anti-tumoral drug that decreases glucose metabolism and viability in cancer cells. However, the mechanism involved in resveratrol-mediated anti-tumor activity is not entirely clear. In this work, it is demonstrated that resveratrol decreases viability, glucose consumption and ATP content in the human breast cancer cell line MCF-7. These effects are directly correlated with PFK inhibition by resveratrol in these cells. Moreover, resveratrol directly inhibits purified PFK, promoting the dissociation of the enzyme from fully active tetramers into less active dimers. This effect is exacerbated by known negative regulators of the enzyme, such as ATP and citrate. On the other hand, positive modulators that stabilize the tetrameric form of the enzyme, such as fructose-2,6-bisphosphate and ADP, prevent the inhibition of PFK activity by resveratrol, an effect not observed with increased pH. In summary, our results provide evidence that resveratrol directly inhibits PFK activity, therefore disrupting glucose metabolism and reducing viability in cancer cells.     

Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells via JAK2/STAT3 Pathway Inhibition

Natural killer (NK) cell malignancies, particularly aggressive NK cell leukaemias and lymphomas, have poor prognoses. Although recent regimens with L-asparaginase substantially improved outcomes, novel therapeutic approaches are still needed to enhance clinical response. Resveratrol, a naturally occurring polyphenol, has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities. In this study, we investigated the potential anti-tumour activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92 and NK-YS. Resveratrol induced robust G0/G1 cell cycle arrest, significantly suppressed cell proliferation and induced apoptosis in a dose- and time-dependent manner for all four cell lines. In addition, resveratrol suppressed constitutively active STAT3 in all the cell lines and inhibited JAK2 phosphorylation but had no effect on other upstream mediators of STAT3 activation, such as PTEN, TYK2, and JAK1. Resveratrol also induced downregulation of the anti-apoptotic proteins MCL1 and survivin, two downstream effectors of the STAT3 pathway. Finally, resveratrol induced synergistic effect on the apoptotic and antiproliferative activities of L-asparaginase against KHYG-1, NKL and NK-92 cells. These results suggest that resveratrol may have therapeutic potential against NK cell malignancies. Furthermore, our finding that resveratrol is a bonafide JAK2 inhibitor extends its potential benefits to other diseases with dysregulated JAK2 signaling.     

Pro-apoptotic versus anti-apoptotic properties of dietary resveratrol on tumoral and normal cardiac cells

Resveratrol is a natural dietary polyphenol found in grape skin, red wine, and various other food products. Resveratrol has proved to be an effective chemopreventive agent for different malignant tumors. It has also been shown to prevent vascular alterations such as atherosclerosis and inflammatory-associated events. In view of these observations, we investigated the anti-proliferative and pro-apoptotic activities of resveratrol on a tumoral cardiac cell line (HL-1 NB) derived from mouse tumoral atrial cardiac myocytes. These effects were compared with those found on normal neonatal mouse cardiomyocytes. HL-1 NB cells and neonatal cardiomyocytes were treated with resveratrol (5, 30, and/or 100 μM) for different times of culture (24, 48, and/or 72 h). Resveratrol effects were determined by various microscopical and flow cytometric methods. After resveratrol treatment, a strong inhibition of tumoral cardiac HL1-NB cell growth associated with a loss of cell adhesion was observed. This cell proliferation arrest was associated with an apoptotic process revealed by an increased percentage of cells with fragmented and/or condensed nuclei (characteristic of apoptotic cells) identified after staining with Hoechst 33342 and by the presence of cells in subG1. At the opposite, on normal cardiomyocytes, no cytotoxic effects of resveratrol were observed, and a protective effect of resveratrol against norepinephrine-induced apoptosis was found on normal cardiomyocytes. Altogether, the present data demonstrate that resveratrol (1) induces apoptosis of tumoral cardiac HL1-NB cells, (2) does not induce cell death on normal cardiomyocytes, and (3) prevents norepinephrine-induced apoptosis on normal cardiomyocytes.     

Resveratrol suppresses the proliferation of breast cancer cells by inhibiting fatty acid synthase signaling pathway

In breast cancer cells, overexpression of human epidermal growth factor receptor 2 (HER2) increases the translation of fatty acid synthase (FASN) by altering the activity of PI3K/Akt signaling pathways. Cancer chemotherapy causes major side effects and is not effective enough in slowing down the progression of the disease. Earlier studies showed a role for resveratrol in the inhibition of FASN, but the molecular mechanisms of resveratrol-induced inhibition are not known. In the present study, we examined the novel mechanism of resveratrol on Her2-overexpressed breast cancer cells.The effect of resveratrol on the growth of breast cancer cells was assessed as percent cell viability by cytotoxicity-based MTT assay and the induction of apoptosis was determined by cell-death detection ELISA and flow cytometric analysis of Annexin-V–PI binding. Western immunobloting was used to detect signaling events in human breast cancer (SKBR-3) cells.Data showed that resveratrol-mediated down-regulation of FASN and HER2 genes synergistically induced apoptotic death in SKBR-3 cells. This concurrently caused a prominent up-regulation of PEA3, leads to down-regulation of HER2 genes. Resveratrol also alleviated the PI3K/Akt/mTOR signaling by down-regulation of Akt phosphorylation and up-regulation of PTEN expression.These findings suggest that resveratrol alters the cell cycle progression and induce cell death via FASN inhibition in HER2 positive breast cancer.     

Combination Treatment with Resveratrol and Sulforaphane Induces Apoptosis in Human U251 Glioma Cells

Resveratrol is a naturally occurring polyphenolic compound highly enriched in grapes, peanuts, red wine, and a variety of food sources. Sulforaphane belongs to the family of isothiocyanates and is highly enriched in cruciferous vegetables. Our previous study showed that resveratrol, when used at high concentrations, inhibited cell proliferation, caused the cell cycle arrest and induced apoptotic cell death in glioma cells. In the current study, we tested the effect of combination treatment with resveratrol and sulforaphane, when both were used at low concentrations, on cell proliferation, migration and death in human U251 glioma cells. Our study shows that combination treatment with resveratrol and sulforaphane inhibits cell proliferation and migration, reduces cell viability, induces lactate dehydrogenase release, decreases pro-survival Akt phosphorylation and increases caspase-3 activation. The use of combination of bioactive food components, such as resveratrol and sulforaphane, may be a viable approach for the treatment of glioma.     

Resveratrol and 4-hydroxynonenal act in concert to increase glutamate cysteine ligase expression and glutathione in human bronchial epithelial cells

Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and glutamate cysteine ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined effects.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

Purinergic signaling as potential target of thiamethoxam-induced neurotoxicity using silver catfish (<Emphasis Type="Italic">Rhamdia quelen</Emphasis>) as experimental model

Resveratrol is a polyphenolic compound in many edible foods including grapes, peanuts, and berries. Several studies have revealed the beneficial effects of resveratrol against various diseases such as heart disease, diabetes, obesity, neurological disorders, and cancer. A recent study showed that resveratrol inhibits the proliferation of HCT116 human colorectal cancer cells in three-dimensional culture (3DC) via induction of luminal apoptosis in HCT116 cell spheroids. In this study, we showed that a novel compound, caffeic acid-adducted resveratrol, has a stronger inhibitory effect on the growth of HCT116 cell spheroids in 3DC than resveratrol. It showed almost the same inhibitory efficacy as 5-fluorouracil, a conventional anticancer drug. We further showed that the resveratrol derivative did not affect the growth of HKe3 cell spheroids derived from HCT116 cells by disruption of the activating mutant KRAS gene. These results suggest that the resveratrol derivative inhibits the growth of HCT116 cell spheroids via inhibition of an oncogenic KRAS-mediated signaling pathway.     

Resveratrol induces cell‐cycle disruption and apoptosis in chemoresistant B16 melanoma

Resveratrol, a naturally occurring polyphenol, has been shown to possess chemopreventive activities. In this study, we show that resveratrol (0–500 µM) inhibits the growth of a doxorubicin‐resistant B16 melanoma cell subline (B16/DOX) (IC₅₀ = 25 µM after 72 h, P < 0.05). This was accomplished by imposing an artificial checkpoint at the G₁–S phase transition, as demonstrated by cell‐cycle analysis and down‐regulation of cyclin D1/cdk4 and increased of p53 expression level. The G₁‐phase arrest of cell cycle in resveratrol‐treated (10–100 µM) B16/DOX cells was followed by the induction of apoptosis, which was revealed by pyknotic nuclei and fragmented DNA. Resveratrol also potentiated at subtoxic dose (25 µM for 24 h) doxorubicin cytotoxicity in the chemoresistant B16 melanoma (P < 0.01). When administered to mice, resveratrol (12.5 mg/kg) reduced the growth of an established B16/DOX melanoma and prolonged survival (32% compared to untreated mice). All these data support a potential use of resveratrol alone or in combination with other chemotherapeutic agents in the management of chemoresistant tumors. J. Cell. Biochem. 110: 893–902, 2010. © 2010 Wiley‐Liss, Inc.     

Resveratrol Alleviates Endotoxin-Induced Myocardial Toxicity via the Nrf2 Transcription Factor

Background/Aims

Septic cardiomyopathy is a severe condition that remains a challenge for clinical management. This study investigated whether the natural polyphenolic compound resveratrol could be used as a prophylactic treatment to alleviate sepsis-related myocardial injury; the underlying molecular mechanisms were deciphered by both in vitro and in vivo experiments.

Methods

A mouse model of endotoxin-induced cardiomyopathy was developed by intraperitoneal injection of LPS, and resveratrol was administered prophylatically to the animals. Serum LDH and CK activities were measured to detect myocardial injury, and echocardiography was performed to monitor cardiac structure and function. Various cytokines/chemokines and the Nrf2 antioxidant defense system were examined in the heart tissue. The effects of resveratrol on LPS-induced Nrf2 activation, ROS generation, and apoptotic cell death were further investigated in cultured primary human cardiomyocytes. An Nrf2 specific siRNA was used to define its role in resveratrol-mediated cardiomyocyte protective effect.

Results

Resveratrol pretreatment significantly attenuated LPS-induced myocardial injury in mice, which was associated with suppressed proinflammatory cytokine production and enhanced Nrf2 activation in the heart. In cultured primary human cardiomyocytes, resveratrol activated Nrf2, inhibited LPS-induced ROS generation, and effectively protected the cells from LPS-induced apoptotic cell death. Knockdown of Nrf2 abrogated resveratrol-mediated protection of the cells from LPS-induced cell death.

Conclusion

Resveratrol effectively alleviates endotoxin-induced cardiac toxicity through mechanisms that involve the Nrf2 antioxidant defense pathway. Our data suggest that resveratrol might be developed as a useful prophylactic management for septic cardiomyopathy.     

Resveratrol modulates gene expression associated with apoptosis, proliferation and cell cycle in cells with mutated human c-Ha-Ras, but does not alter c-Ha-Ras mRNA or protein expression

An accumulating body of evidence suggests that resveratrol can inhibit carcinogenesis through antiproliferative and apoptotic effects. One proposed mechanism for this is the modulation of genes, for example, Ras and p53, frequently associated with human cancer. To test the effect of resveratrol on gene expression, we used the WR-21 cell line because it contains a mutated human c-Ha-ras gene. Cells at > or =70% confluency were incubated with media alone or with increasing concentrations of trans-resveratrol (0.1-1000 microM) for 24 h. Resveratrol (30-100 microM) decreased cellular proliferation by 80% (bromodeoxyuridine incorporation) and increased apoptosis by 60% (TUNEL). Cells were then treated with media alone or with 50-microM resveratrol for 24 h. RNA was isolated for nylon-based macroarray analyses and protein for immunoblotting. Resveratrol increased (+) and decreased (-) gene expression associated with apoptosis (Birc5+, Cash+, Mcl-1+, Mdm2+, Rpa-like+), cellular proliferation (Ctsd+, Mdm2+, Egr1+, ODC+) and cell cycle (cyclin D+, cyclin g+, Gadd45a-, Mad2l-, Mdm2+). Resveratrol consistently increased by > or =6-fold Mdm2 expression and other downstream p53 effectors, but not p53 itself at 24 h. Subsequent cell cycle analysis indicated a significant accumulation of cells in G2/M, and a decrease in G1/G0 suggesting a G2/M blockade. Further RT-PCR and Western blot analyses indicated no differential changes in Ras mRNA expression or p21(ras) protein levels, respectively. These results suggest that resveratrol potently inhibits cellular proliferation, increases apoptosis, alters cell cycle dynamics and modulates associated gene expression. Furthermore, these effects appear mediated, in part, by p53 without direct modulation of mutant c-Ha-ras expression.     

Resveratrol suppresses the epithelial-to-mesenchymal transition in PC-3 cells by down-regulating the PI3K/AKT signaling pathway

Resveratrol possesses a wide spectrum of pharmacological properties and has been an ideal alternative drug for the treatment of different cancers, including prostate cancer. However, the mechanisms by which resveratrol inhibits the growth of prostate cancer are still not fully elucidated. To understand the effect of resveratrol on the apoptosis and the epithelial-to-mesenchymal transition (EMT) of prostate cancer as well as its related mechanism, we investigated the potential use of resveratrol in PC-3 prostate cancer cells in vitro using real-time PCR, fluorescence-activated cell sorting, Western blotting, etc. Resveratrol suppresses the PC-3 prostate cancer cell growth and induces apoptosis. Resveratrol also influences the expression of EMT-related proteins (increased E-cadherin and decreased Vimentin expression). Finally, resveratrol also suppressed Akt phosphorylation in PC-3 cells. This study indicates that resveratrol may be a potential anti-cancer treatment for prostate cancer; moreover, it provides new evidence that resveratrol suppresses prostate cancer growth and metastasis.     

Resveratrol Inhibits the Growth of Gastric Cancer by Inducing G1 Phase Arrest and Senescence in a Sirt1-Dependent Manner

Resveratrol, a naturally occurring polyphenolic compound, has been reported to exert anticancer activity by affecting diverse molecular targets. In this study, we examined the effects and the underlying mechanisms of resveratrol on gastric cancer. We found that resveratrol inhibited the proliferation of gastric cancer cells in a dose-dependent manner. At the concentration of 25 and 50 µM, resveratrol inhibited the cell viability and diminished the clonogenic potential of gastric cancer cells. Resveratrol treatment arrested gastric cancer cells in the G1 phase and led to senescence instead of apoptosis. Regulators of the cell cycle and senescence pathways, including cyclin D1, cyclin-dependent kinase (CDK4 and 6), p21 and p16, were dysregulated by resveratrol treatment. The inhibitory effects of resveratrol on gastric cancer were also verified in vivo using a nude mice xenograft model. Resveratrol (40 mg/kg/d) exerted inhibitory activities on gastric cancer development and significantly decreased the fractions of Ki67-positive cells in the tumor specimens from the nude mice. After resveratrol treatment, the induction of senescence and the changes in the expression of the regulators involved in the cell cycle and senescence pathways were similar to what we observed in vitro. However, the depletion of Sirtuin (Sirt)1 reversed the above-described effects of resveratrol both in vitro and in vivo. Our data suggest that resveratrol inhibits gastric cancer in a Sirt1-dependent manner and provide detailed evidence for the possibility of applying resveratrol in gastric cancer prevention and therapy.