Curcumin, a natural, biologically active compound extracted from rhizomes of Curcuma species, has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. The mechanism by which curcumin initiates apoptosis remains poorly understood. In the present report we investigated the effect of curcumin on the activation of the apoptotic pathway in human renal Caki cells. Treatment of Caki cells with 50 microM curcumin resulted in the activation of caspase 3, cleavage of phospholipase C-gamma1 and DNA fragmentation. Curcumin-induced apoptosis is mediated through the activation of caspase, which is specifically inhibited by the caspase inhibitor, benzyloxycarbony-Val-Ala-Asp-fluoromethyl ketone. Curcumin causes dose-dependent apoptosis and DNA fragmentation of Caki cells, which is preceded by the sequential dephosphorylation of Akt, down-regulation of the anti-apoptotic Bcl-2, Bcl-XL and IAP proteins, release of cytochrome c and activation of caspase 3. Cyclosporin A, as well as caspase inhibitor, specifically inhibit curcumin-induced apoptosis in Caki cells. Pre-treatment with N-acetyl-cysteine, markedly prevented dephosphorylation of Akt, and cytochrome c release, and cell death, suggesting a role for reactive oxygen species in this process. The data indicate that curcumin can cause cell damage by inactivating the Akt-related cell survival pathway and release of cytochrome c, providing a new mechanism for curcumin-induced cytotoxicity.

Purpose: Most breast cancers have chromosomal instability that seems related to defective mitotic spindle checkpoints. Because the molecular basis of this defect is unknown, we evaluated breast cancer cell lines and tissues for possible defects involving the major mitotic checkpoint genes responsible for maintaining chromosomal stability. Experimental Design:We analyzed sequences and expression levels (RNA and protein) of eight major spindle checkpoint genes (MAD1L1, MAD2L1, MAD2L2, BUB1, BUB1B, BUB3, CDC20, and TTK) in a panel of 12 breast cancer cell lines, most with established genetic instability and defective spindle damage checkpoint response. mRNA levels of these genes were also measured in primary tumor samples, and immunohistochemical staining was used to evaluate BUB1B protein levels in a panel of 270 additional cases of breast cancer. Results: No functionally significant sequence variations were found for any of the eight genes in the breast cancer cell lines with chromosomal instability. More surprisingly, the mRNA and protein levels for these checkpoint genes are significantly higher in the genetically unstable breast cancer cell lines and in high-grade primary breast cancer tissues than in the stable (and checkpoint proficient) MCF-10A and normal mammary epithelial cells, or in normal breast tissues. In fact, overexpression of the BUB1B protein is a marker that recognizes nearly 80% of breast cancers in paraffin-embedded tissues. Conclusions: Defective mitotic spindle checkpoints in breast cancer are most likely not caused by low expression or mutations of these eight checkpoint genes. High levels of these particular transcripts could represent a cellular compensation for defects in other molecular components of the mitotic spindle damage checkpoint, and increased expression of these genes might be markers of breast cancers with chromosomal instability.Most breast cancers have significantly aberrant genomic structure, including abnormal numbers of chromosomes. We have previously shown that in breast cancer cell lines, chromosome numbers are both variable and unstable (1), a phenomenon that seems related to defective mitotic spindle checkpoint controls. This chromosomal instability in breast cancer is similar to what has been observed in other cancers, where defective mitotic spindle checkpoint genes have been implicated as a basis of chromosomal instability. For example, mutations in the BUB1 mitotic checkpoint gene were initially reported in a small number of colorectal cancers with chromosomal instability (2, 3), Subsequently, mutations of BUB1 were reported in colon and pancreatic cancers (3, 4), and a truncating mutation of the MAD2L1 gene was reported for a single breast cancer cell line (4).Further analyses of the BUB1, BUB1B, and MAD2L1 checkpoint genes, however, have revealed that these mutations are relatively uncommon in cancer. Supporting a possible alternative mechanism for reducing the activity of checkpoint proteins, several studies have provided evidence that low expression ...

Proteolytic degradation of the extracellular matrix and tumor metastasis correlate with the expression of endopeptidases known as matrix metalloproteinases (MMPs). The expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate (PMA). We found that resveratrol, a phytoalexin present in grapes, significantly inhibits the PMA-induced increase in MMP-9 expression and activity. These effects of resveratrol are dose dependent and correlate with the suppression of MMP-9 mRNA expression levels. PMA caused about a 23-fold increase in MMP-9 promoter activity, which was suppressed by resveratrol. Transient transfection utilizing MMP-9 constructs, in which specific transcriptional factors were mutagenized, indicated that the effects of PMA and resveratrol were mediated via an activator protein-1 and nuclear factor-kappaB response element. Resveratrol inhibited PMA-mediated activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)-delta activation. Therefore, we conclude that the MMP-9 inhibition activity of resveratrol and its inhibition of JNK and PKC-delta may have a therapeutic potential, given that a novel means of controlling growth and invasiveness of tumors.

Most human cancers are aneuploid and have chromosomal instability, which contrasts to the inability of human cells to normally tolerate aneuploidy. Noting that aneuploidy in human breast cancer correlates with increased expression levels of the Mps1 checkpoint gene, we investigated whether these high levels of Mps1 contribute to the ability of breast cancer cells to tolerate this aneuploidy. Reducing Mps1 levels in cultured human breast cancer cells by RNAi resulted in aberrant mitoses, induction of apoptosis, and decreased ability of human breast cancer cells to grow as xenografts in nude mice. Remarkably, breast cancer cells that survive reductions in levels of Mps1 have relatively less aneuploidy, as measured by copies of specific chromosomes, compared with cells that have constitutively high levels of Mps1. Thus, high levels of Mps1 in breast cancer cells likely contribute to these cells tolerating aneuploidy.

A pharmaceutical grade extract of Coix lachryma-jobi seeds is currently the most commonly used treatment for cancer in China. Although clinical data support the use of this preparation of a Traditional Chinese Medicine for cancer treatment, biological basis for the activity of this preparation has not been previously established. To address this issue, we first evaluated the anti-neoplastic activity of a Coix extract emulsion in xenografts of MDA-MB-231 breast cancer cells and found that the extract significantly inhibits growth of MDA-MB-231 xenografts in athymic nude mice. Using oligonucleotide microarrays, we determined that Coix seed extract also significantly affects gene expression in these cells, including downregulation of genes (such as COX-2 and matrixmetalloproteinases) that are considered to be important in neoplasia. The specific gene expression changes noted after Coix seed extract treatment are characteristic of inhibition of NFkB-dependent transcription, leading us to evaluate how the treatment affects that pathway. An NFkBdependent reporter assay demonstrated dose-dependant inhibition of NFkB signaling by treatment of cultures with the extract, and immunofluorescent microscopy found that these effects are associated with reduced translocation of the Rel-A/p65 subunit of NFkB to the nucleus. Coix extract also inhibits activity of protein kinase C, a major mediator of signal transduction and activator of NFkB. Thus, this Traditional Chinese Medicine-based cancer treatment affects cellular pathways of recognized importance in neoplasia.

Protein kinase CKII (CKII) plays a critical role in cell growth and proliferation. In this study, we examine how CKII activity is regulated during cellular senescence. Our results demonstrate that CKII activity apparently decreases during both replicative and H 2 O 2 -induced senescence in human diploid fibroblast IMR-90 cells. The mRNA and protein levels of CKIIa decreases significantly during replicative and H 2 O 2 -induced senescence, while only slight reduction in those of CKIIb is observed during replicative senescence. Treatment of IMR-90 cells with CKII inhibitors 5,6-dichloro-1-b-D D-ribofuranosylbenzimidazole and apigenin led cells to acquire a senescent phenotype as judged by the senescence-associated b-galactosidase marker and overexpression of p53 and p21 Waf-1 . Knockdown of CKIIa in IMR-90 cells by RNA interference also dramatically induced the senescent phenotype. In parallel, CKII activity was transcriptional downregulated in rat liver and testis with advancing age. Taken together, these results suggest that downregulation of CKII activity is tightly associated not only with cellular senescence but also with organism aging.

Overexpression of fatty acid synthase (FAS)4 is common in aggressive human cancers, and blocking FAS inhibits growth and leads to apoptosis in these cancer cells (1, 2). Although it is now well accepted that increased activity of this enzyme plays a key role in maintaining the metabolic stability of cancer cells, the molecular consequences of inhibiting FAS are still not well understood.One central molecular network commonly altered in cancer pathogenesis and also affected by cancer treatment is that regulated by the transcription factor NF-B. NF-B signaling is constitutively activated in many epithelial solid tumors and hematologic malignancies (3-5), and this activation appears to generally confer resistance to apoptosis as well as enhance growth properties of cancer cells. Consequently, numerous inhibitors of NF-B are being investigated for potential use in treating cancer (6, 7), and bortezomib, a proteosome inhibitor widely used for treating myeloma, is thought to act largely through inhibition NF-B by stabilization of the IB␣ subunit (8, 9). Interestingly, NF-B activity is also inhibited by a number of naturally occurring lipid compounds that have recognized anti-neoplastic properties, including curcumin, resveratol, and coix seed extract (10 -12), providing additional evidence to suggest that NF-B activity supports or promotes the malignant phenotype.NF-B activity does not uniformly contribute to malignancy, however, and in some situations, increased NF-B activity may actually suppress malignant characteristics of cells (13). For example, it has been shown that induction of p53 leads to activation of NF-B, correlating with the ability of p53 to induce apoptosis (14). Thus, at least in some cellular settings, inhibition or loss of NF-B activity abrogates p53-induced apoptosis, indicating that NF-B can be functional in p53-mediated cell death.The role of NF-B signaling in the response of cancer cells to chemotherapy also appears to depend on variables of the particular situation. In many circumstances, activation of NF-B by therapeutic agents appears to inhibit apoptosis and thus attenuates the response to these agents (15-17). However, activation of NF-B by cancer therapeutic agents appears to mediate cell death in other circumstances, including treatment with UV light (18), doxorubicin (19), and paclitaxel (20). In light of the general importance of NF-B to cellular physiology and response to stress and the expectation that manipulations of lipid metabolic pathways could affect NF-B signaling, we investigated the effects of inhibiting FAS on NF-B and the role of NF-B signaling in the response of lung cancer cells to this inhibition. 4 The abbreviations used are: FAS, fatty acid synthase; mIB␣, mutant IB␣; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PGE2, prostaglandin E2; PMA, phorbol 12-myristate 13-acetate; CE, cloning efficiency. EXPERIMENTAL PROCEDURES

Transforming growth factor-betas (TGF-betas) have significant effects on testis development. The pattern of TGF-beta expression in aging testis has not been established to date. We examined age-related changes in the expression of TGF-beta and its receptors in the testis using Western blot analysis. TGF-beta1 expression increased continuously in aging rat testis, whereas no age-associated changes were observed for TGF-beta3. Strong expression of TGF-beta2, as well as type I and II receptors was observed in 12-month-old testis, but following this time, expression decreased dramatically. Interestingly, TGF-beta2 and -beta3 displayed strong and similar expression patterns in liver, regardless of age, suggesting that the down-regulation of TGF-beta2 is testis-specific. We observed significant induction of p53 and p21WAF1 in 18-month-old testis that appeared to correspond with aging. Moreover, caloric restriction (CR) prevented age-related decrease in TGF-beta2 expression. Using immunohistochemistry, we showed that all TGF-beta1, -beta2, and -beta3 proteins are expressed primarily in interstitial cells, which are located in the space between adjoining seminiferous tubules. Our data collectively indicate that aging in the testis is regulated by differential expression of TGF-beta proteins, and decreased levels of TGF-beta2 contribute to the aging process.