Melatonin Enhancement Of Curcumin Antitumor Mechanism Study Of Bladder Cancer

Bladder cancer is one of the most malignant types of cancer,which ranks ninth of world cancers with an estimated 430,000 new cases diagnosed,and caused 165,000 deaths in2012 Although the radical cystectomy is introduced as the standard treatment for bladder cancer with neoadjuvant chemotherapy,the 5-year survival rate in patients with invasive and metastatic bladder cancer is still very low.Thus,it is urgent to find novel therapeutic strategies and more effective agents that prolong survival and improve quality of life,without damaging normal cells.Curcumin(1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)is a natural polyphenolic compound isolated from the rhizome of Curcuma longa(turmeric),and has commonly been used as a food additive or in many traditional medicine remedies for over 2000 years in many Asian countries.Previous studies have demonstrated that curcumin possess various physiological and pharmacological properties in vitro and in vivo,including antioxidant,antibacterial,anti-inflammatory,immunomodulatory,free radical scavenging and antidiabetic activities.In particular,curcumin shows potential inhibition of cancer cell proliferation,induction of apoptosis,cancer cell cycle arrest and suppression of angiogenesis in a wide variety of cancers through modulating all kinds of molecular targets and cell signaling pathways.Furthermore,curcumin has been shown to be able to induce apoptosis and cell cycle arrest and inhibit proliferation in bladder cancer cells.Although curcumin presents itself as a pharmacologically safe and effective potential candidate for anticancer therapy,its effectiveness is not powerful enough due to its poor absorption,rapid metabolism,and rapid systemic elimination,and the use of high doses of curcumin is limited by the emergence of side effects.Therefore,more information and studies concerning its combination with other antitumor reagents,especially natural antitumor compound,should be improved for curcumin,and the underlying molecular mechanisms of such combination should also deserve better investigation to achieve higher potency.Melatonin is a major secretory product of pineal gland in vertebrates,modulating circadian rhythms,sleep,mood,reproduction and other biological processes.In the last few decades,in vitro and in vivo studies have demonstrated that melatonin possess various physiological and pharmacological activities including anti-proliferation,anti-angiogenesis,anti-inflammatory,suppressing tumor metastasis and inducing cell apoptosis activities,through affecting multiple signaling pathways,including NF-κB.Based on its contribution to diverse physiological functions and its very few side-effects,more attempts deserve to be made to develop melatonin as a partner of other chemopreventive or chemotherapeutic agents form a better and novel treatment strategy for cancer and meanwhile reduce their side effects.Cyclooxygenase-2(COX-2),as the rate-limiting enzymes for the synthesis of prostaglandins from arachidonic acid,involves in inflammatory progression and is inducible in response to certain stimuli such as growth factors and cytokines,in the meanwhile,is causally linked to the carcinogenesis of many human cancers.Previous studies have indicated that COX-2 protein was highly expressed in a wide range of human cancers including bladder cancer,and has been associated with high tumor aggressiveness and poor patients ’ prognosis.COX-2 expression is strictly and transcriptionally controlled by the binding of transactivators such as nuclear factor kappa B(NF-κB)to the corresponding sites of its promoters.Therefore,inhibition of COX-2 expression might be an effective alternative approach to suppress glioma development.However,whether curcumin could regulate COX-2 expression and whether melatonin could sensitize or enhance such regulation to further inhibit bladder cancer cell growth remains unclear.In this study,we hypothesized that melatonin might play a role in potentiating or enhancing curcumin’s antitumor effect in human bladder cancer cells.To test this hypothesis,the combinational effects of curcumin and melatonin on tumor cell proliferation,migration,and apoptosis in melanoma cells were analyzed.Next,the changes of some key proteins involved were also analyzed to uncover the underlying molecular mechanisms of this combinational mode.Our study showed that melatonin could be used as a potential combinational agent to sensitize the antitumor effect of curcumin.Such sensitization was mediated through simultaneous modulation of the multiple signaling pathways,implying that such a combinational treatment might potentially become an effective way in colon cancer therapy.Objective Curcumin,a natural polyphenolic compound,has commonly been used as a food additive or in many traditional medicine remedies for over 2000 years in many Asian countries.Melatonin is a hormone secreted from pineal glands of mammals and possesses diverse physiological functions.Both curcumin and melatonin has the effective potential to inhibit proliferation of various types cancers,but they have never been combined altogether as an anti-bladder cancer treatment,and the underlying mechanism remains poorly understood.In this study,we investigated whether the combination of curcumin and melatonin leads to an enhanced inhibition of cell proliferative in bladder cancer cells.Methods In the present study,human bladder cancer T24,UMUC3 and 5637 cells were treated by different concentrations of Curcumin or Melatonin alone or together,and then the cell viabilities were detected by MTT assay.The biological functions of Curcumin or Melatonin were investigated by clonogenic cell survival,wound-healing and apoptosis assays in bladder cancer cells.In mechanism investigation,the nuclear localization and interaction between NF-κB p50/p65 and their binding to COX-2promoter were analyzed by confocal immunofluorescence,DNA-protein binding by streptavidin-agarose pulldown assay,after treatment with Curcumin or Melatonin alone or together.In order to find the target for Curcumin or Melatonin alone or together,Western blot and Quantitative detection of IKKβ kinase activity photometric method was used to detect the protein expression level and IKKβ kinase activity.T24 cell lines were inoculated into nude mice,and monitored xenografts growth every 3 days.Immunohistochemical staining was used to evaluate the expression of related proteins.Results We found that the combinational treatment enhanced the suppression of COX-2 expression through regulating the nuclear translocation of NF-κB and abrogating their binding on COX-2 promoter via inhibiting IKKβ activity.In addition,combined treatment with Curcumin and melatonin triggered the release of cytochrome c from the mitochondrial intermembrane space into the cytosol resulting apoptosis in bladder cancer.Conclusions These results,therefore,demonstrated that melatonin synergized the effect of curcumin to inhibit the growth of bladder cancer by enhancing the anti-proliferation,anti-migration,and pro-apoptotic activities,and provide strong evidences for such a combinational treatment might potentially to become an effective way in bladder cancer therapy.