Curcumin inhibits colon cancer progression through direct interaction with SIRT1

Abstract

Colorectal cancer (CRC) is the third most common cancer diagnosed and the third leading cause of cancer-related death in both males and females in the United States. It has been widely believed that frequent consumption of Western diet containing red meat and saturated fats or alcohol, lack of physical activity and obesity are the risk factors for CRC. Thus, the incidence rates of CRC have been gradually increased in recent years in the South Korea, which is partly attributable to Westernization of dietary habits. Even though the colonoscopy for detection of colorectal polyps has been commonly used as a diagnostic method for CRC, recent studies have paid attention to usability of tumor-specific biomarkers as diagnostic and prognostic factors for CRC. <br/> The SIRT protein family is an NAD+-dependent class Ⅲ HDAC which can deacetylate non-histone proteins as well as histones and consists of seven isoforms (SIRT1-7). Among these isoforms, SIRT1 was first reported to extend the life span of budding yeast in 1999. Subsequent studies demonstrated diverse physiological actions of SIRT1, such as metabolic regulation, differentiation and stress response. However, the role of SIRT1 in tumorigenesis is still controversial. Recent studies have revealed that SIRT1 is abnormally overexpressed in CRC tissues. Notably, the elevated levels of SIRT1 are significantly associated with not only tumor invasion and lymph node metastasis, but higher grades of malignancy and mutations of KRAS and BRAF, suggesting the prognostic utility of SIRT1. In the current study, I also confirmed that 27 out of 35 samples (77.14%) showed upregulation of SIRT1 in human colon tumor tissues compared with that of corresponding adjacent normal colon tissues as measured by immunofluorescent analysis. When SIRT1 was silenced in human CRC cell lines (HCT-116, HCT-15, DLD-1 and SW480) by transfection with SIRT1-specific small interfering RNA (siRNA), cell viability, migration, growth and invasion decreased. A xenograft mouse model revealed that the tumor volume and the tumor weight decreased in SIRT1-silenced xenograft tumor compared with those observed in control xenograft tumor. These results suggest the oncogenic function of SIRT1 in the progression of CRC.<br/> The use of natural compounds derived from edible plants has been considered as an attractive strategy to control tumor-promoting proteins. Interestingly, according to data published by World Health Organization (WHO) in 2003, the incidence of CRC is 10 times lower in India than in United states. Epidemiologic studies suggest that dietary phytochemicals, such as curcumin abundant in Indian food, may be responsible for the low risk of intestinal cancer in India. Curcumin, a yellow pigment present in the rhizome of turmeric (Curcuma longa L., Zingiberaceae) used in preparing the curry powder, is one of the most extensively investigated phytochemicals in regard to chemopreventive and antitumorigenic potential. It modulates the expression or activity of multiple proteins involved in cellular signal transduction pathways. It has been reported that curcumin can exert preventive effects against pathogenesis of several disorders by activating SIRT1 in cells or tissues of noncancerous origin. However, the relationship between curcumin and SIRT1 in CRC cells remains largely unknown. Thus, the purpose of this study was to investigate the effect of curcumin on the expression of SIRT1 and underlying molecular mechanisms in the context of inhibition of CRC progression. <br/> When HCT-116 cells were treated with curcumin, the protein expression of SIRT1 was reduced while its mRNA transcript level was not changed, suggesting that SIRT1 is posttranslationally regulated by curcumin. Moreover, treatment of cells with curcumin significantly shortened the half-life of SIRT1, suggesting that curcumin destabilizes SIRT1 by enhancing its degradation rather than inhibiting de novo synthesis. Notably, the inhibitory effect of curcumin on SIRT1 protein was blunted by a proteasome-specific inhibitor MG-132, indicating that proteasomes are required for curcumin-induced degradation of SIRT1. This breakdown of SIRT1 was mediated through its ubiquitination both at endogenous and exogenous levels as measured by immunoprecipitation assay. These findings suggest that curcumin stimulates ubiquitin-dependent proteasomal degradation of SIRT1, thereby decreasing the protein stability.<br/> It has been reported that increased protein expression of SIRT1 in CRC and hepatocellular carcinoma is attributable to enhanced protein stability rather than elevated mRNA expression of SIRT1, suggesting that abnormally stabilized SIRT1 could contribute to tumorigenesis. Nonetheless, much less is known about regulation of SIRT1 protein stability. Curcumin has two electrophilic α,β-unsaturated carbonyl groups, and hence can act as a Michael reaction acceptor. Considering tetrahydrocurcumin, a non-electrophilic analogue of curcumin, fails to exert the inhibitory effect on SIRT1 protein, structural feature of curcumin, namely, the α,β-unsaturated carbonyl group plays an important role in reducing the stability of SIRT1. There are several reports suggesting that curcumin can directly modify cysteine thiols of cellular proteins by Michael addition, thereby modulating their functions. Based on these reports, recombinant human SIRT1 was incubated with curcumin followed by nano-LC-ESI-MS/MS analysis to investigate the modifiable residues of SIRT1 by curcumin. The data revealed modification of a single cysteine residue at 67 on SIRT1 by curcumin. Indeed, replacement of cysteine 67 of SIRT1 with alanine abolished the binding of curcumin to SIRT1. Moreover, the protein stability and clonogenicity of a mutant SIRT1 in which cysteine 67 was substituted by alanine were unaffected by curcumin. Taken together, these observations suggest that curcumin facilitates the proteasomal degradation of oncogenic SIRT1 through covalent modification of SIRT1, presumably at the cysteine 67 residue.<br/> It has been suggested that the protein levels of SIRT1 and phospho-SIRT1 are higher in colorectal cancer tissues than adjacent normal tissues. Phospho-SIRT1 is positively correlated with a cellular proliferation marker, Ki67, indicating distinctive role of phospho-SIRT1 independently of total SIRT1 in the tumorigenesis of colon. It has also been reported that c-Jun N-terminal kinases 2 (JNK2) phosphorylates SIRT1 at serine 27 residue, thereby enhancing the protein stability of SIRT1 in human colon cancer (HCT-116) cells. Based on these reports, I hypothesized a possible involvement of dephosphorylation of SIRT1 in its destabilization induced by curcumin. When HCT-116 cells were exposed to curcumin, phosphorylation level of SIRT1 at the serine 27 residue was reduced, together with the decrease in total level of SIRT1. Given the fact that phosphorylation defective mutant SIRT1 (SIRT1-S27A) had shortened half-life of SIRT1, and phosphomimetic mutant SIRT1 (SIRT1-S27D) was resistant against curcumin compared to wild-type of SIRT1, decreased phosphorylation level of SIRT1 was not due to its reduced total level of SIRT1. Immunoblot analysis showed that SP600125, a selective inhibitor of JNK, reduced the levels of phospho- and total SIRT1, but not the mRNA level, suggesting that JNK posttranslationally regulates SIRT1. Indeed, inhibition of JNK by SP600125 provoked ubiquitination-dependent degradation of SIRT1, resulting in its decreased stability. In parallel with these findings, anisomycin-induced activation of JNK prevented curcumin-mediated suppression of migration and growth of HCT-116 cells as well as SIRT1 by attenuating its ubiquitination. These data suggest that phosphorylation of serine 27 residue protects SIRT1 from curcumin-induced breakdown, which contribute to the maintenance of oncogenicity of HCT-116 cells against curcumin.<br/> Taken together, curcumin can covalently modify SIRT1, preferentially at the cysteine 67 residue. Likewise, it can induce dephosphorylation of SIRT1 at the serine 27 residue through inhibition of JNK. These direct and indirect modifications of SIRT1 by curcumin can collaboratively lead to proteasomal degradation of oncogenic SIRT1, which accounts for anti-cancer effects of curcumin in terms of suppression of cell migration and growth.<br/> <br/> Key words<br/> Colorectal cancer