Evaluation And Mechanism Of Antitumor Activity Of Curcumin And IDO Inhibitor NLG-919

The focus of this dissertation is on the characterization of antitumor compounds with novel mechanisms.We investigated curcumin,a natural product with documented antitumor activity against a broad range of tumors in both laboratory and clinical trials,and NLG-919,a selective inhibitor against indoleamine-2,3-dioxygenase that is currently under clinical trial for its antitumor activity.For curcumin antitumor activity study,we focused on its metal ion chelation property.It has been reported that curcumin targets multiple signaling pathways,including cell survival and proliferation,caspase activation and oncogene expression,to exert its antitumor activity.We and other researchers have questioned how could a compound as simple as curcumin with so many selective targets.Curcumin contains a?-diketone structure moiety that tautomerizes to keto-enol with metal ion chelating capability.Since copper has an integral role in promoting tumor growth and angiogenesis,we decided to investigate whether curcumin functions as a biologically active copper chelator.The copper chelation ability of curcumin was validated by US/VIS spectrum.The antitumor activity and in vivo copper removal ability of curcumin was determined in a murine xenograft model.The effect of curcumin on copper-induced MAPK activation and cell proliferation was determined in cell culture system.We showed that administration of curcumin to tumor-bearing animals resulted in suppression of A549 xenograft growth,an effect that was also observed in animals treated with ammonium tetrathiomolybdate(TM),a metal chelator used clinically for removal of excess copper systemically.The inhibition on tumor growth was associated with reduction of copper in the serum.In cell culture studies,we showed that copper promoted cell proliferation through Erk/MAPK activation.Treatment with curcumin or U0126,a specific MAPK inhibitor,or suppression of copper cellular uptake by knockdown of copper transporter protein 1(CTR1)blocked copper-induced cell proliferation.This study therefore links curcumin antitumor effect to its copper chelation capability.These results also implicate copper chelation as a general mechanism for their biological activities of naturally occurring polyphenols like flavonoids.In the second part of this dissertation,we investigated NLG-919,an indole-2,3-dioxygenase(IDO)inhibitor,for its antitumor activity using a murine 4T1 breast carcinoma xenograft model.The incidence of breast cancer has been increasing each year for more than 30 years.The side effects currently used therapeutic approaches are obvious.Cancer immunotherapy has gained significant attention since the approach is effective against a broad category of tumors.Immune checkpoint molecules play critical role in maintaining a dynamic balance of the immune system by either turning up or tuning down a signal for tumor surveillance.Cancer cells are known to avoid checkpoint surveillance by evading the immune system.The IDO pathway mediates immunosuppressive effects through catabolization of tryptophan to kynurenine.Expression of the IDO 1 gene by tumor cells or host APCs can inhibit tumor-specific effector CD8+T cells and enhance the suppressor activity of Treg.Drugs that target IDO pathway hold a lot of promise as cancer treatments.NLG-919 is a potent IDO pathway inhibitor with desirable pharmacological properties,and is currently under phase I trials for treating melanoma.We developed enzymatic assays for the verification of NLG-919 as a selective inhibitor against IDO1.The activity was the confirmed against IFN-y induced tryptophan degradation in HeLa cell by detection of kynurenine production with HPLC.In mice bearing established 4T1 tumors,administration of NLG-919 markedly enhanced the antitumor responses and reduced lung metastasis.NLG-919 profoundly blocked IDO-induced T cell suppression.We also determined the percentage of CD8+T and Treg cells in draining lymph nodes(DLNs)using flow cytometry.We found that NLG-919 treatment resulted in a dramatic increase in CD8+T cells that was accompanied with significant decrease of Treg cells in the tumor tissues as well as the DLNs.Although detailed mechanisms that regulate NLG-919 inhibition of tumor cell lung metastasis remain to be delineated,these results nonetheless demonstrate that IDO inhibitor suppresses tumor cell growth likely by strengthening the body's own anti-tumor immune response.