L-929 Cells Harboring Ectopically Expressed RelA Resist Curcumin-induced Apoptosis

Abstract

Curcumin (diferuloyl methane), the yellow pigment in turmeric (Curcuma longa), possesses anti-inflammatory and antioxidant activities (1, 2). It is also a potent chemopreventive agent inhibiting tumor promotion against skin, oral, intestinal, and colon carcinogenesis (3-5). Many chemopreventive agents induce programmed cell death or apoptosis, a potent mechanism by which they eliminate preneoplastic or cancer cells. Curcumin induces several characteristics of apoptosis such as cell shrinkage, chromatin condensation, and DNA fragmentation in cells from colon, kidney, blood, and liver of human origin and NIH3T3, erbB2-transformed NIH3T3, and fibrosarcoma cells of murine origin (6, 7). However, curcumin fails to induce apoptosis in some fibroblastic and leukemic cell lines, and inhibition of cell proliferation is not always associated with apoptosis (8 -10). Although the molecular mechanisms of antiproliferative and apoptotic effects of curcumin remain elusive, the resistance of various cell types to apoptosis following treatment with curcumin may be attributed to the abnormal expression of specific proteins that regulate apoptosis. Many cancer cells protect themselves against therapy (tumor necrosis factor, ionizing radiation, and chemotherapeutic compounds) by activating NF-B 1 /Rel, a family of dimeric transcription factors, that helps in cell survival through unknown anti-apoptotic mechanisms (11)(12)(13)(14). Interestingly, constitutive expression of NF-B has been reported in breast and pancreatic cancer cells and tumors (but not in their normal counterparts), contributing to their apoptosis resistance/survival and tumor progression (15, 16). The typically active NF-B/Rel is a heterodimer composed of a 50-kDa (p50) and a 65-kDa (p65 or RelA) subunit, although other homo/heterodimers of the mammalian Rel family (c-Rel, p52, and RelB) also exist. Knockout mice lacking the RelA subunit of NF-B die before birth and show massive degeneration of liver cells caused by apoptosis. NF-B proteins are sequestered in cytoplasm as inactive forms by associating with inhibitory proteins known as IBs. IB-␣ represents a prototype of several IB proteins and IB kinases, which phosphorylate them on specific serine residues upon getting a signal, have been cloned and characterized recently (17). Phosphorylation of IB-␣ leads to its ubiquitination and subsequent degradation eventually releasing the heterodimers of NF-B to translocate to the nucleus (18).Curcumin is known to inhibit the activation of NF-B induced by tumor necrosis factor-␣, phorbol esters, and hydrogen peroxide (19). Curcumin inhibits cytokine-mediated NF-B activation by blocking a signal leading to IB kinase activity (20). Presumably, curcumin-induced apoptosis is regulated, in part, by the extent of NF-B inactivation by curcumin and the level of expression of NF-B in cells. To understand the role of NF-B in curcumin-induced apoptosis, we stably transfected relA gene into L-929 cells (mouse fibrosarcoma), and upon treatment with curcumin, the relA-transfected cells ...

Inhibition of NF-κB Sensitizes A431 Cells to Epidermal Growth Factor-induced Apoptosis, whereas Its Activation by Ectopic Expression of RelA Confers Resistance

“…Transient and Stable Transfections-A431-IB-␣ cells were transiently transfected with relA in pMT2T vector (33,34) using the calcium-phosphate transfection kit (Invitrogen) according to the manufacturer's protocol. Stable transfections in A431 cells with relA in pMT2T vector (co-transfected with pcDNA3) or the empty vector pcDNA3 or pcDNA3-IB-␣ were carried out by the LipofectAMINE method (35).…”

“…NF-B directly causes increased expression of proteins that contribute to the survival of tumor cells such as inhibitors of apoptotic proteins (IAPs) (31,32). Results from our laboratory have shown earlier that ectopic expression of the RelA subunit of NF-B into murine fibrosarcoma cells protects them from curcumin-induced apoptosis (33).…”

Curcumin induces Apaf-1-dependent, p21-mediated caspase activation and apoptosis

“…Curcumin (diferuloylmethane), a polyphenolic extract of turmeric, induces cell death in multiple cancer cell types with minimal toxicity to normal cells in cell culture as well as in mouse xenograft models. [1][2][3][4] Clinical trials also suggest that curcumin, even at relatively high doses, is safe for humans. 5,6 The mechanisms underlying curcumin-induced cancer cell death have not been clearly defined, although available evidence suggests that curcumin downregulates NFκB signaling leading to suppression of proliferation and induction of apoptosis.…”

Curcumin Inhibits Activation of Vγ9Vδ2 T Cells by Phosphoantigens and Induces Apoptosis Involving Apoptosis-Inducing Factor and Large Scale DNA Fragmentation

“…In addition to its role as a spice, curcumin has also been used for centuries to treat inflammatory disorders (16). Although the exact mechanism of action for curcumin is not well understood, it has been shown to prevent inhibitory factor I B degradation, retaining NF-B in the cytoplasm in its inactive form and to inhibit activation of c-Jun N-terminal kinase (17)(18)(19). In addition to its anti-inflammatory properties, curcumin induces the growth arrest and apoptosis of a number of different tumor cell lines, also possibly through an interaction with the NF-B signaling pathway (18 -20).…”