In America and Western Europe, prostate cancer is the second leading cause of death in men. Emerging evidence suggests that chronic inflammation is a major risk factor for the development and metastatic progression of prostate cancer. We previously reported that the chemopreventive polyphenol curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast cancer metastases. In this study, we analyze the effects of curcumin on prostate carcinoma growth, apoptosis and metastasis. We show that curcumin inhibits translocation of NFκB to the nucleus through the inhibition of the IκB-kinase (IKKβ, leading to stabilization of the inhibitor of NFκB, IκBα, in PC-3 prostate carcinoma cells. Inhibition of NFκB activity reduces expression of CXCL1 and -2 and abolishes the autocrine/paracrine loop that links the two chemokines to NFκB. The combination of curcumin with the synthetic IKKβ inhibitor, SC-541, shows no additive or synergistic effects indicating that the two compounds share the target. Treatment of the cells with curcumin and siRNA-based knockdown of CXCL1 and -2 induce apoptosis, inhibit proliferation and downregulate several important metastasis-promoting factors like COX2, SPARC and EFEMP. In an orthotopic mouse model of hematogenous metastasis, treatment with curcumin inhibits statistically significantly formation of lung metastases. In conclusion, chronic inflammation can induce a metastasis prone phenotype in prostate cancer cells by maintaining a positive proinflammatory and prometastatic feedback loop between NFκB and CXCL1/-2. Curcumin disrupts this feedback loop by the inhibition of NFκB signaling leading to reduced metastasis formation in vivo.

Curcumin Breast cancer Inflammatory cytokines microRNAs A B S T R A C TChronic inflammation is a major risk factor for the development and metastatic progression of cancer. We have previously reported that the chemopreventive polyphenol Curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast and prostate cancer metastases. In the present study, we have analyzed the effects of Curcumin on miRNA expression and its correlation to the anti-tumorigenic properties of this natural occurring polyphenol.Using microarray miRNA expression analyses, we show here that Curcumin modulates the expression of a series of miRNAs, including miR181b, in metastatic breast cancer cells.Interestingly, we found that miR181b down-modulates CXCL1 and -2 through a direct binding to their 3 0 -UTR. Overexpression or inhibition of miR181b in metastatic breast cancer cells has a significant impact on CXCL1 and -2 and is required for the effect of Curcumin on these two cytokines. miR181b also mediates the effects of Curcumin on inhibition of proliferation and invasion as well as induction of apoptosis. Importantly, over-

BackgroundCurcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma.ResultsWe show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NFκB signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NFκB dependant manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NFκB. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression.ConclusionResistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker.

Breast cancer is the most common cancer and the second leading cause of cancer death in industrialized countries. Systemic treatment of breast cancer is effective at the beginning of therapy. However, after a variable period of time, progression occurs due to therapy resistance. Artesunate, clinically used as anti-malarial agent, has recently revealed remarkable anti-tumor activity offering a role as novel candidate for cancer chemotherapy. We analyzed the anti-tumor effects of artesunate in metastasizing breast carcinoma in vitro and in vivo. Unlike as expected, artesunate induced resistance in highly metastatic human breast cancer cells MDA-MB-231. Likewise acquired resistance led to abolishment of apoptosis and cytotoxicity in pre-treated MDA-MB-231 cells. In contrast, artesunate was more cytotoxic towards the less tumorigenic MDA-MB-468 cells without showing resistance. Unraveling the underlying molecular mechanisms, we found that resistance was induced due to activation of the tumor progression related transcription factors NFκB and AP-1. Thereby transcription, expression and activity of the matrix-degrading enzyme MMP-1, whose function is correlated with increased invasion and metastasis, was up-regulated upon acquisition of resistance. Additionally, activation of the apoptosis-related factor NFκB lead to increased expression of ant-apoptotic bcl2 and reduced expression of pro-apoptotic bax. Application of artesunate in vivo in a model of xenografted breast cancer showed, that tumors growth was not efficiently abolished as compared to the control drug doxorubicin. Taken together our in vitro and in vivo results correlate well showing for the first time that artesunate induces resistance in highly metastatic breast tumors.

Background: Curcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma. Results: We show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NFkappaB signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NFkappaB dependant manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NFkappaB. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression. Conclusion: Resistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3539.

Curcumin is a polyphenol, derived from the plant Curcuma longa that has promising anticancer properties in breast, colon, pancreas and some other cancer types. We recently demonstrated that the metastatic potential of breast cancer cells is strongly reduced upon treatment with Curcumin in a murine model of hematogenous metastasis: 68% of the mice treated had no or few metastases as opposed to 17% of the untreated mice. In order to elucidate its anti-tumoral effects in prostate cancer, we used three human prostate cancer cell lines of clear defined and graduated tumorigenicity (LnCaP<DU145>PC3) for our studies. In a first approach, we intended to obtain a complete picture of Curcumin-responsive genes and therefore we performed gene expression profiling (GeneChip Arrays Affymetrix) of Curcumin-treated cells versus non-treated cells followed by statistical analysis. Thereby we found 71 genes whose expression was significantly altered among which 8 genes were down-regulated. Treatment of prostate cancer cells with Curcumin reduced tumor cell proliferation (BrdU assay) and invasion (Matrigel assay) in a time and dose dependent manner. Furthermore expression of matrix-degrading enzymes (MMPs) which is a prerequisite for matrix degradation was significantly down-regulated on transcript as well as on protein level. The underlying regulation mechanism involves the survival associated NFγB signaling pathway: Fluorescent translocation assays clearly demonstrated that Curcumin inhibits p65 translocation into the nucleus of the tumor cells, which in consequence leads to diminished transcription and expression of tumor progression related genes and gene products resulting in the induction of apoptosis. Taken together, these features make Curcumin a promising drug for cancer chemoprevention and antitumor therapy of prostate cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A147.

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Breast cancer is the most common cancer and the second leading cause of cancer death in industrialized countries. Systemic treatment of breast cancer is effective at the beginning of therapy. However, after a variable period of time, progression occurs due to therapy resistance. Artesunate (ART), clinically used as anti-malarial agent, has recently revealed remarkable anti-tumor activity offering a role as novel candidate for cancer chemotherapy. We analyzed the anti-tumor effects of ART in metastasizing breast carcinoma in vitro and in vivo. Unlike as expected, ART induced resistance in highly metastatic human breast cancer cells MDA-MB-231. Likewise acquired resistance led to abolishment of apoptosis and cytotoxicity in pre-treated MDA-MB-231 cells. In contrast, ART was more cytotoxic towards the less tumorigenic MDA-MB-468 cells without showing resistance. Unraveling the underlying molecular mechanisms, we found that resistance was induced due to activation of the tumor progression related transcription factors NFκB and AP 1. Thereby transcription, expression and activity of the matrix-degrading enzyme MMP-1, whose function is correlated with increased invasion and metastasis, was up-regulated upon acquisition of resistance. Additionally, activation of the apoptosis-related factor NFκB lead to increased expression of ant-apoptotic bcl2 and reduced expression of pro-apoptotic bax. Application of ART in vivo in a model of xenografted breast cancer showed, that tumors growth was not efficiently abolished as compared to the control drug doxorubicin. Taken together our in vitro and in vivo results correlate well showing for the first time that ART induces resistance in highly metastatic breast tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 709. doi:10.1158/1538-7445.AM2011-709