Inositol hexafosfato e câncer de próstata: a importância do p21/cip1 e p27?Kip1

Inositol hexaphosphate (IP6) causes G(1) arrest and increases cyclin-dependent kinase inhibitors p21/Cip1 and p27/Kip1 protein levels in human prostate cancer (PCa) DU145 cells lacking functional p53. However, whether cyclin-dependent kinase inhibitor I induction by IP6 plays any role in its antitumor efficacy is unknown. Herein, we observed that either p21 or p27 knockdown by small interfering RNA has no considerable effect on IP6-induced G(1) arrest, growth inhibition, and death in DU145 cells; however, the simultaneous knockdown of both p21 and p27 reversed the effects of IP6. To further confirm these findings both in vitro and in vivo, we generated DU145 cell variants with knockdown levels of p21 (DU-p21), p27 (DU-p27), or both (DU-p21+p27) via retroviral transduction of respective short hairpin RNAs. Knocking down p21 or p27 individually did not alter IP6-caused cell growth inhibition and G(1) arrest; however, their simultaneous ablation completely reversed the effects of IP6. In tumor xenograft studies, IP6 (2% w/v, in drinking water) caused a comparable reduction in tumor volume (40-46%) and tumor cell proliferation (26-28%) in DU-EV (control), DU-p21, and DU-p27 tumors but lost most of its effect in DU-p21+p27 tumors. IP6-caused apoptosis also occurred in a Cip/Kip-dependent manner because DU-p21+p27 cells were completely resistant to IP6-induced apoptosis both in cell culture and xenograft. Together, these results provide evidence, for the first time, of the critical role of p21 and p27 in mediating the anticancer efficacy of IP6, and suggest their redundant role in the antiproliferative and proapoptotic effects of IP6 in p53-lacking human PCa cells, both in vitro and in vivo.PMID: 19176374 Introdution Prostate cancer (PCa) accounts for ∼25% (186,320) of cancer incidence and is the second leading cause of cancer-related deaths in the male population in the United States ( 1). The disease is initially responsive to androgen ablation therapy, but it eventually progresses to the androgen-independent form which is relatively more invasive and metastatic ( 2). Usually, PCa is a disease of elderly males and the time elapsed between the onset of preneoplastic lesions and detectable malignancy often ranges in decades, thus allowing for a sufficiently large time window for preventive intervention. However, the use of synthetic agents in long-term chemopreventive strategies is intrinsically associated with safety and toxicity problems ( 3, 4). This has led to the increasing enthusiasm in diet-related cancer chemoprevention strategies, which are also supported by epidemiologic and case-control studies indicating an important role of dietary agents in the etiology as well as the prevention of cancer ( 5– 7). Low-fat and high-fiber (especially soluble fibers present in oat bran, legumes, etc.) diets have been suggested to protect against various forms of cancers including PCa ( 8). Inositol hexaphosphate (IP6) is a polyphosphorylated carbohydrate present in abundant amounts in high-fiber–containing food sources such as legumes, nuts, cereals, etc. IP6 and its lower phosphate forms (IP1–IP5) are also present in mammalian cells, in which they govern various cellular functions, including signal transduction, growth, and differentiation ( 9). IP6 is available as a dietary supplement and has long been known for its beneficial effects, including strengthening the immune system, preventing kidney stone formation and lowering serum cholesterol levels ( 9– 11). Shamsuddin and coworkers first established the cancer-preventive efficacy of IP6 in carcinogen-induced colon cancer models ( 12), following which there has been an increasing interest in investigating the antineoplastic potential of this phytonutrient. IP6 has shown significant promise against a broad spectrum of cancer models including prostate, colon, pancreas, liver, and breast ( 13– 18). Moreover, in breast cancer models, when used in a combination regimen with Adriamycin or tamoxifen, IP6 increases the activity of these conventional chemotherapeutic agents and helps to overcome drug resistance ( 19). Over the past few years, our laboratory has focused on establishing the chemopreventive efficacy of IP6 in cell culture and animal models of PCa. Initial studies showed that IP6 induces prominent G1 arrest as well as apoptosis in both androgen-dependent and androgen-independent PCa cell lines ( 20, 21). In tumor xenografts of DU145 cells, IP6 significantly reduced tumor volume, tumor weight, cell proliferation and angiogenesis, and induced apoptosis ( 22). Furthermore, in the transgenic adenocarcinoma of mouse prostate model, which is a reliable murine model for studying the multistage events of prostate carcinogenesis, IP6 treatment showed a significant reduction in tumor grade and cell proliferation, and an induction of apoptosis in prostate tumor tissues ( 23). There is no report, thus far, of any considerable toxicity of IP6 in animal studies ( 16, 17, 22, 23). The promising anticancer efficacy, along with the lack of toxicity associated with IP6 administration in preclinical models, underscores the chemopreventive potential of IP6 as well as warranting further investigation of its molecular mechanism of action. Threshold kinase activity of cyclin-dependent kinases (CDK) is a crucial determinant of the cell cycle progression, and thus, the agents which inhibit CDK activity directly or indirectly, by up-regulating CDK inhibitor (CDKI) expression, represent a rational approach to intervene with the uncontrolled proliferation of carcinoma cells ( 24). Moreover, prolonged cell cycle arrest resulting in apoptosis is also an effective strategy in reducing the burden of cancer cells. The key features of IP6 efficacy, as established in various PCa cell lines, are cell cycle arrest, apoptosis, and concurrent Cip/Kip protein induction ( 20, 21). In order to address the question of whether the biological effects of IP6 are mediated in a Cip/Kip-dependent manner, we generated DU145 cells with stable knockdown levels of p21 and/or p27 proteins and examined the effect of IP6 on these cell variants for cell proliferation, growth arrest, apoptosis, and tumorigenicity. Our studies reveal that induction of p21 and p27 protein expression is an essential event for mediating the antitumor, antiproliferative, and apoptotic effects of IP6. Discussion The novel and central finding in the present study is that p21 and p27, the Cip/Kip family proteins, are indispensable in causing growth arrest and apoptotic death of advanced human PCa DU145 cells by IP6, both in vitro and in vivo. Furthermore, the simultaneous down-regulation of both p21 and p27 levels results in DU145 cells with a more aggressive phenotype. In this study, we found that the more aggressive DU145 cell variant with knockdown levels of both p21 and p27 was also resistant to IP6-induced cell growth inhibition and apoptosis under both in vitro and in vivo conditions, thus establishing p21 and p27 as the primary molecular targets of IP6 for its antitumor efficacy against PCa. Cell cycle progression is governed by the well-orchestrated activation and inactivation of CDKs ( 29, 30). G1 to S phase transition plays a crucial role in maintaining the genomic integrity because this phase is critically linked to external stimuli and also commits the cells to DNA replication and subsequent mitosis ( 31, 32). G1 checkpoint abrogation is a common phenomenon in carcinogenesis which endows the tumor cells with limitless replicative potential ( 17, 24). p21 and p27 physically interact with CDK via their amino terminal domain and inhibit CDK kinase activity ( 29, 30). Thus, activating the G1 checkpoint by up-regulating the expression of p21 and p27 is a ca-0182.pdf http://www.medicinacomplementar.com.br/convertido/ca-0182.htm