Resveratrol

Resveratrol, 3, 5, 4'-trihydroxy stilbene, is a polyphenol found in numerous plants, especially the skin of grapes and peanuts 1.

Research in resveratrol (RSV) began when it was noticed that the French had a lower risk of heart disease than would be expected from their high fat diet, this became known as the “French Paradox” 2. It was suggested that this is due to the high consumption of red wine, which is the richest source of resveratrol.

Since then resveratrol has been studied for its possible anti-carcinogenic effects, cardiovascular protective effects and role in life extension. Research on the compound is still young and the mechanisms are not totally understood, so there is still investigation to be done on the full effects of resveratrol on the cell.

Resveratrol and Life extension

Calorie reduction (CR) is a promising method of reducing the risk factor of aging 3 [. It is defined as reducing the energy intake of the body considerably without damaging the overall health of the living organism. CR enhances the activity of a group of proteins (deactylases and ADP-transferases) , SIRT1-7 4, While has been proven true by experiments on mice and it is not practical. Resveratrol (RV) is looked upon as a possible substitute for CR as it also activates SIRT1 . A study by Pearson K.J et al., which was referenced multiple times in other studies, used two groups of lab mice where one group was kept on a lean diet while the other was kept on a fat diet. The lean mouse was given resveratrol to see whether the same effects of CR would be observed. The fat mice were fed with and without resveratrol to see whether it could prevent or reverse the effects of obesity. Resveratrol improved the health of the mice on the fat diet while no improvements were observed in the lean mice. Resveratrol was found to activate an enzyme (AMP-activated protein kinase – AMPK) that was found to control cellular mechanisms and cellular homeostasis 5. Also, a study done by Margie. T. Borra et al. revealed that although SIRT1 activation was independent of protein structure but needed a fluorophore-containing substance to be initiated . Biding of resveratrol to SIRT1 then induced a conformational change in the enzyme which allowed a tighter bond with the fluorophore substance. They have also speculated that resveratrol may help to up-regulate the SIRT1 or it may help the activated SIRT1 to demonstrate a higher affinity for its substrate which could result in the longevity properties thought to be characteristic of resveratrol. In another study by Meilian Liu et al., it was found that resveratrol inhibits mTOR signalling, through a mechanism that is still unclear. mTOR is a PI 3-kinase related protein kinase. Resveratrol inhibits insulin- and leucine- stimulated mTOR as well. Resveratrol binds directly to the binding site of the PI3-kinase enzyme, therefore functioning as a class IAB13K inihibtor. Activation is necessary for the insulin-stimulation mTOR t ooccur 6,7. It is possible that resveratrol inhibits mTOR signalling by inhibiting PI3K signalling pathways. mTORCI, which is one of the two distinct mTOR complexes which is papamycin-sensitive. It is possible that resveratrol inhibits mTOR by targeting TSC1/2. TSC1/2 are tumour suppressor tuberous sclerosis complexes that occupy the role of negative regulation of Rheb which in turn is a small GTP-ase that activates mTOR 7,8. Also, DEPTOR is a negative regulator of mTOR. By providing conditions where DEPTOR can bind to mTOR, therefire inhibiting mTOR 9,10, the interaction between mTOR and DEPTOR which would help provide a mechanism by which resveratrol inhibits the mTORCI signal pathway.

Cardiovascular protection

Pre-conditioning of heart

Today, cardio benefits are being associated with moderate red wine consumption due to the presence of RSV 11 which exerts cardio protection during ischemic cardiac disease due to insufficient oxygen . This is through the NO-mediated pre-conditioning of the heart 12 with a protective mechanism of alternating ischemia and reperfusion periods. An NO blocker, aminoguanine abolishes this protection, indicating the role of NO in RSV preconditioning. RSV elicits I-R injury resistance at reperfusion by NO elevation. Post ischemic ventricular functioning, pressure and aortic flow are enhanced.

RSV dose response is biphasic. Low dosage facilitates stimulation through anti-apoptotic and -oxidant properties while high dosage favors inhibition. At 10µM, the optimal dose for preconditioning according to Hattori et al 12, RSV protection is based on activating the survival signal through Adenosine A3 receptor or PI3 kinase-Akt-BCl2 signaling pathways. Above 10µM the benefit is reduced 13. Low dosage lowers the infarct size by reducing both necrosis and apoptosis 12 and elicits an adaptive stress response during preconditioning. This survival mechanism defends against environmental stressors. Cardio-protective gene is expressed and anti oxidative proteins are formed.14

Thrombus formation results in blood supply blockage and ultimately tissue death. RSV prevents this by elevating eNO synthase, the primary controller of smooth muscle tone. NO-cGMP pathway 15 and lowered vasoconstrictor Endothelin-1 16 regulate endothelial dysfunction. NO stimulates smooth muscle relaxation through IC cGMP increase, K+ channel activation and myosin light chain dephosphorylation. Flow mediated dialation is enhanced. High [NO] also opens the mitochondrial permeability transitional pore 17 at the beginning of reperfusion but not during ischemia 18. This suggests a RSV-protection possibility during the first few minutes of reperfusion by modulating the mPTP opening 19.

Anti-atherosclerotic effect

RSV decreases the hardening and narrowing of arteries due to plaque accumulation by inhibiting LDL oxidation, platelet aggregation, and vascular proliferation of smooth muscle.

• It blocks TXB2 synthesis, the stable degradation product of coagulative factor TXA2 20 so platelet adhesion is blocked.

• Procoagulant tissue factor in monocytes, a component that may cause cardiovascular disease is suppressed 21. Methyl-RSV derivatives are more efficient in tissue factor inhibiton so structural modification may help thrombosis reduction.

• Anti inflammatory property of RSV inhibits plaque formation 22 by reducing intima area of lesions. High [LDL] aides endothelial inflammation. RSV, given adequate time prevents peroxidative degradation and uptake of LDL in vascular wall.

Anti-oxidant effect

The anti-oxidant power of polyphenolic RSV is greater than Vitamin E 11 and C 23. It acts against oxidative stress by inducing autophagy in damaged or aged cells and as a free radical scavenger 24 i.e. RSV binds toxic peroxynitrite, the product of NO and super oxide.

RSV as a phytoestrogen

RSV is structurally similar to synthetic estrogen diethyl stilbestrol and was hypothised to have similar cardiac effects to estrogen 25. It reduces LDL in menopausal women by cholesterol-bile acid conversion and endothelial NO release. Underlying smooth muscle will dialate 26.

Resveratrol and Cancer

RSV has been shown to be effective as a treatment in many types of cancers, including breast1, prostate2 and pancreatic3 cancer. It appears to influence multiple signaling pathways related to cell cycle, proliferation and apoptosis. RSV induces caspase-dependent apoptosis in ovarian4, prostate and breast5 cancer cells. The caspases are a family of proteases that are important in cell death. Gagoda et al. suggest that RSV targets mitochondria in the cell, leading to permeabilization of the outer michondrial membrane5. This causes the release of cytochrome c, an intermediate of apoptosis. Release of cytochrome c from the mitochondria activates caspase-9, an initiator, which in turn activates caspases -3 and -7, the “executioner” caspases which destroy the cell.

In colon cancer cells, RSV was shown to inhibit cell proliferation and arrest the cell cycle through its effect on the IGF-1R/AKT/Wnt-signaling pathway6. Vanamal et al. demonstrated that IGF-1 increased cell proliferation by 87%, showing it's effect on growth of cancer cells. Insulin like growth factor (IGF) has been linked with obesity related cancers and is thought to stimulate growth of existing cancer cells. Before exposure to IGF-1, cancer cells were treated with RSV and cell proliferation was inhibited by up to 95%. When cells were incubated with IGF-1 and then treated with RSV, cell proliferation was still inhibited by up to 94%. RSV's ability to inhibit proliferation in the presence of IGF-1 appears to be due to it having multiple targets, one of which Vanamal et al. suggest is IGF-1R. Suppressing this receptor negated the effect of IGF-1 on growth of the cells. Meanwhile, RSV also activated tumor suppressor p53. The combined effect is high inhibition of cell proliferation, even with the presence of IGF-1.

In the same study, they found RSV arrested cells in the G0/G1-S phase of the cell cycle, possibly by increasing levels of FoxO3a. The forkhead transcriptional factors of the O subclass (FoxO) are proteins involved in suppressing tumor growth. FoxO3a's activitiy occurs downstream of the PI3K/AKT pathway, an anti-apoptotic pathway. Roy et al. suggest that RSV regulates the PI3K/AKT pathway, by inhibiting AKT activity, while also influencing the target genes of FoxO3a3. Removing FoxO genes stopped RSV's effect on the cell cycle and apoptosis, suggesting that RSV's effects are dependent on the presence of FoxO transcription factors.

In 2012, Iqbal et al. claimed to show the first example of RSV effecting the metabolism of cancer cells7. Cancer cells use glucose for macromolecule synthesis of their daughter cells, which is promoted by pyruvate kinase M2. PKM2 is expressed mostly in the S phase of proliferating tumor cells. Treatment with RSV arrested cancer cells in G0/G1 phase, suggesting that PKM2 was downregulated by RSV. They believe that RSV targeted PKM2 through mTOR inhibition. The mammalian target of rapamyacin (mTOR) is a protein involved in regulating protein synthesis and is found to be dysfunctional in diseases such as cancer.

Gliomas are tumors arising from glial cells in the brain or spine. High grade gliomas are the most common and aggressive form of brain tumor. There is evidence to suggest that there exist a sort of cancer stem cell (CSC), at least in gliomas, that behave similarly to normal stem cells and are responsible for tumor formation and metastasis8. Filippi-Chiela et al. showed that in glioma cells, RSV arrested the cell cycle in S-G2/M phase9. They also suggest that RSV may have an effect on CSCs, indicating a possibility for it as a treatment for gliomas. They believe that current cancer therapies, like chemotherapy, kill differentiatied or differentiating cancer cells, but not CSCs themselves.

While RSV has been suggested as a means to sensitize cancer cells to chemotherapy10, it has been found to have protective effects in leukemic cells that were being treated with proteasomal inhibitors11. Xiso-Fang et al. suggest that the cytotoxic effects were negated by the RSV, and rather than sensitizing the cancer cells to the therapy, RSV protected them. This shows that the full effects of the compound on different types of cells hasn't been realised yet and the suggestions for further understanding of RSV before it is used in the treatment of human cancers are warranted12.

Anti-diabetic effect

RSV stimulates both insulin dependent and independent glucose uptake in muscle tissue (11) by activating insulin and AMP-activated protein kinase signaling. Uptake by GLUT-4 transporter is insulin based but AMPka1 is not. Therefore RSV overcomes insulin resistance (1) and protects against diet-induced insulin resistance syndrome through AMPKa1(5). The maximum translocation of GLUT-4 against RSV dosage was quantitatively expressed by Minakawa et al (1). At 1µM the optimal value was reached in 5-10min, whereas at 100µM it took half that time and was maintained up to 40min (1). RSV has an insulin like effect in type-1 diabetes and reduces common diabetes mellitus symptoms but follows a different mechanism to that of insulin (6). It also stimulates uptake in insulin independent liver cells and enhances glycogen synthesis. In contrast RSV can also reduce hyper-insulinemia.(11)

Beneficial effect on early diabetic nephropathy

2 factors are involved in Renin-Angiotensin System of kidney and blood glucose level; an increase in Angiotensin 2 (7) and renin dependant activation of RAS by saccinate (9) directly link rennin and high glucose levels, by the energy supply to demand ratio of saccinate in tissues. Therefore blood pressure rises, indicating diabetes. RSV protects against oxidative stress due to hyperglycemia, exhibits concurrent inflammation and anti-inflammation and up-regulates AMPK activation in diabetic renal cells (10). Blood glucose level declines due to enhanced glucose uptake.

Glycation inhibition

RSV also has an inhibitory effect on the impairment of bio molecular functioning under diabetic state. A sugar binds to lipid or protein in the absence of enzymes producing Advanced Glycation End-products (AGEs). RSV protects B cells from AGE-induced oxidative stress (1) and –apoptosis by inhibitiing ECM protein accumulation in the mesangial interstitial space and mesangial cell proliferation respectively (12).

Resveratrol and Viruses

Resveratrol has been found to have different effects on different viruses. M. Nakamura et al. found that resveratrol (RV) is unsuitable to be used for treatment against the hepatitis C virus 1. Cells originally taken from a hepatome cell line were used which contained Hepatitis C virus RNA, and using their luciferase enzyme activity to determine the concentration at which the RV had any effect. The RV increased replication of the HCV RNA. This replication increased which increasing dosage. They also found that RV also reversed the anti-viral effect of interferon and riboflavin. The mechanisms of RV on hepatitis C virus, riboflavin and interferon are still unclear.

On the other hand, RV was also found to inhibit certain virus’ activity instead of accelerate it. RV inhibits replication of influenza virus, as found by A.T. Palmara et al. RV modifies the Raf/MEK/ERK cascade that Influenza A virus activates that causes the virus’ symptoms 2. It does this by interfering with the kinase activity and by inhibiting the MAPK (Mitogen-activated protein kinase) pathway.

It has also been proven that RV inhibits the Herpes Simplex virus activity (HSV) 3. John J. Docherty et al. used human lung cells and African green monkey cells to produce a cell line containing HSV-1 and HSV-2 inserted in them. When RV was added to these cells it proved to be the most effective against HSV symptoms once added after 1 hour of HSV infection. They found that a protein that is essential for transcriptional genes of most of the essential early and late genes. ICP-4, was produced at much lower amounts than without RV present, thus inhibiting HSV activity. They concluded that RV effects were reversible.

Anti-Inflammatory Effects of Resveratrol

Interest in the use of naturally occuring compounds for the treatment of inflammatory conditions is increasing12.

Resveratrol appears to have potential as a topical treatment for chronic rhinosinusitis3. In part by inhibiting production of Interleukin-8, a chemotaxic protein associated with inflammation, in nasosinal epithelium, it had a stronger anti-inflammatory effect than the steroidal, synthetic drugs that it was tested against.

Using a mouse model similar to human ulcerative colitis, Xiangli et al. found that adding resveratrol to the diet of mice decreased inflammatory markers in a dose-dependent way4. It decreased inflammatory cytokines and neutrophils in the colon, and showed no toxic side effects. As a side effect, they found treatment with resveratrol to reduce tumorigenisis associated with colitis.

Qureshi et al. found that resveratrol was a potent anti-inflammatory agent, due to it's ability to inhibit macrophage production of cytokines, such as interleukins, and NO, which can cause inflammation5. It appears that resveratrol's effects NF-kB, a protein which regulates DNA transcription and controls many inflammatory genes, and in this way is able to exert its anti-inflammatory effect.

Conclusion

Up until now research in resveratrol has predominantly been in vitro and in animal subjects, with human clinical trials still in the early stages. However, results so far have been promising, but due to the multiple targets of resveratrol, the effects can vary between cell types. So, further studies are needed to fully understand its mechanisms.

References

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