Melatonin, a novel Sirt1 inhibitor, imparts antiproliferative effects against prostate cancer in vitro in culture and in vivo in TRAMP model.

We recently demonstrated that Sirt1, a NAD(+) -dependent histone deacetylase, was overexpressed in prostate cancer (PCa) and its inhibition resulted in a significant antiproliferative response in human PCa cells. Studies have suggested a link between Sirt1 and circadian rhythms, the disruption of which has been linked to cancer. Interestingly, a decreased production of the pineal melatonin has been shown to deregulate the circadian rhythm machinery and increase cancer risk. Furthermore, disruption in melatonin production and circadian rhythmicity has been associated with aging. Here, we challenged our hypothesis that melatonin will impart antiproliferative response against PCa via inhibiting Sirt1. We demonstrated that melatonin significantly inhibited Sirt1 protein and activity in vitro in multiple human PCa cell lines, and melatonin-mediated Sirt1 inhibition was accompanied with a significant decrease in the proliferative potential of PCa cells, but not of normal cells. Forced overexpression of Sirt1 partially rescued the PCa cells from melatonin's antiproliferative effects, suggesting that Sirt1 is a direct target of melatonin. Employing transgenic adenocarcinoma of mouse prostate (TRAMP) mice, we also demonstrated that oral administration of melatonin, at human-achievable doses, significantly inhibited PCa tumorigenesis as shown by decreases in (i) prostate and genitourinary weight, (ii) serum insulin-like growth factor-1 (IGF-1)/IGF-binding protein-3 (IGFBP3) ratio, (iii) mRNA and protein levels of the proliferation markers (PCNA, Ki-67). This anti-PCa response was accompanied with a significant decrease in Sirt1 in TRAMP prostate. Our data identified melatonin as a novel inhibitor of Sirt1 and suggest that melatonin can inhibit PCa growth via Sirt1 inhibition.

Antiproliferative effects of apple peel extract against cancer cells.

Studies have shown an inverse relationship between the consumption of apples and the risk of several cancers. The peels of apple, which have been shown to possess exceptionally high concentrations of antioxidants, are often discarded. In this study, we evaluated the antiproliferative effects of apple peel extract (APE) in variety of cancer cell types. Our data demonstrated that APE, obtained from organic Gala apples, imparted significant reduction in the viability of a variety of cancer cell lines. Further, our data showed a significant decrease in growth and clonogenic survival of human prostate carcinoma CWR22Rnu1 and DU145 cells and breast carcinoma Mcf-7 and Mcf-7:Her18 cells. Also, the antiproliferative effects of APE were found to be accompanied by a G0-G1 phase arrest of prostate and breast cancer cells. Furthermore, APE treatment resulted in a marked concentration-dependent decrease in the protein levels of proliferative cell nuclear antigen, a marker for proliferation. In addition, APE treatment resulted in a marked increase in maspin, a tumor suppressor protein that negatively regulates cell invasion, metastasis, and angiogenesis. Our data suggested that APE possesses strong antiproliferative effects against cancer cells, and apple peels should not be discarded from the diet. Detailed mechanistic studies, especially in appropriate in vivo animal models, are needed to further examine the antiproliferative and preventive effects of APE against cancer.

Combination of vitamin E and selenium causes an induction of apoptosis of human prostate cancer cells by enhancing Bax/Bcl-2 ratio.

BACKGROUND: The Selenium and Vitamin E Chemoprevention Trial (SELECT) is aimed at determining the usefulness of a combination of vitamin E and selenium for Prostate cancer (PCa) prevention in humans. The aim of this study is to evaluate the efficacy and mechanistic basis of this combination. METHODS: We determined the effect of vitamin E (+-alpha-tocopheryl succinate, VES) and selenium (methylselenic acid, MSA), alone and in combination, on the proliferation of LNCaP, DU145, and PC-3 cells as well as normal prostate PrEC cells. We also determined the involvement of Bcl-2 family proteins as a mechanism of the biological effects of vitamin E and selenium combination. RESULTS: VES or MSA alone led to a modest inhibition in the viability and growth of PCa cells. However, a combination of these two agents resulted in a dramatic increase in growth inhibition of PCa cells. Interestingly, VES and/or MSA were not found to have any effect on the growth or viability of normal PrEC cells. VES and MSA treatment to human PCa cells resulted in (i) induction of apoptosis, (ii) increase in Bax, Bak, and Bid proteins, and (iii) decrease in Bcl-2 protein. Furthermore, Bax knockdown via shRNA and Bcl-2 overexpression via Bcl-2 plasmid resulted in a rescue of PCa cells from apoptosis. CONCLUSIONS: Our study suggested that vitamin E and selenium combination may be more effective than either of these agents alone. Further, our data demonstrated a causal connection between Bax and Bcl-2 modulation and induction of apoptosis by VES and MSA combination.

Resveratrol imparts photoprotection of normal cells and enhances the efficacy of radiation therapy in cancer cells.

Solar radiation spans a whole range of electromagnetic spectrum including UV radiation, which are potentially harmful to normal cells as well as ionizing radiations which are therapeutically beneficial towards the killing of cancer cells. UV radiation is an established cause of a majority of skin cancers as well as precancerous conditions such as actinic keratosis. However, despite efforts to educate people about the use of sunscreens and protective clothing as preventive strategies, the incidence of skin cancer and other skin-related disorders are on the rise. This has generated an enormous interest towards finding alternative approaches for management of UV-mediated damages. Chemoprevention via nontoxic agents, especially botanical antioxidants, is one such approach that is being considered as a plausible strategy for prevention of photodamages including photocarcinogenesis. In this review, we have discussed the photoprotective effects of resveratrol, an antioxidant found in grapes and red wine, against UVB exposure-mediated damages in vitro and in vivo. In addition, we have also discussed studies showing that resveratrol can act as a sensitizer to enhance the therapeutic effects of ionizing radiation against cancer cells. Based on available literature, we suggest that resveratrol may be useful for (1) prevention of UVB-mediated damages including skin cancer and (2) enhancing the response of radiation therapies against hyperproliferative, precancerous and neoplastic conditions.

Dose translation from animal to human studies revisited.

As new drugs are developed, it is essential to appropriately translate the drug dosage from one animal species to another. A misunderstanding appears to exist regarding the appropriate method for allometric dose translations, especially when starting new animal or clinical studies. The need for education regarding appropriate translation is evident from the media response regarding some recent studies where authors have shown that resveratrol, a compound found in grapes and red wine, improves the health and life span of mice. Immediately after the online publication of these papers, the scientific community and popular press voiced concerns regarding the relevance of the dose of resveratrol used by the authors. The animal dose should not be extrapolated to a human equivalent dose (HED) by a simple conversion based on body weight, as was reported. For the more appropriate conversion of drug doses from animal studies to human studies, we suggest using the body surface area (BSA) normalization method. BSA correlates well across several mammalian species with several parameters of biology, including oxygen utilization, caloric expenditure, basal metabolism, blood volume, circulating plasma proteins, and renal function. We advocate the use of BSA as a factor when converting a dose for translation from animals to humans, especially for phase I and phase II clinical trials.

Role of GLI2 transcription factor in growth and tumorigenicity of prostate cells.

Aberrant activation of the Hedgehog (Hh) signaling pathway has been reported in various cancer types including prostate cancer. The GLI2 transcription factor is a primary mediator of Hh signaling. However, its relative contribution to development of prostate tumors is poorly understood. To establish the role of GLI2 in maintaining the tumorigenic properties of prostate cancer cells, we developed GLI2-specific small hairpin RNA. Knockdown of GLI2 in these cells resulted in significant down-regulation of the Hh signaling pathway, followed by inhibition of colony formation, anchorage-independent growth, and growth of xenografts in vivo. Conversely, ectopic expression of Gli2 in nontumorigenic prostate epithelial cells resulted in accelerated cell cycle progression, especially transition through G(2)-M, and augmented proliferation. Altogether, our findings suggest that GLI2 plays a critical role in the malignant phenotype of prostate cancer cells, and GLI2 may potentially become an attractive therapeutic target for the treatment of prostate cancer.

MAGE-A, mMage-b, and MAGE-C proteins form complexes with KAP1 and suppress p53-dependent apoptosis in MAGE-positive cell lines.

The MAGE-A, MAGE-B, and MAGE-C protein families comprise the class-I MAGE/cancer testes antigens, a group of highly homologous proteins whose expression is suppressed in all normal tissues except developing sperm. Aberrant expression of class I MAGE proteins occurs in melanomas and many other malignancies, and MAGE proteins have long been recognized as tumor-specific targets; however, their functions have largely been unknown. Here, we show that suppression of class I MAGE proteins induces apoptosis in the Hs-294T, A375, and S91 MAGE-positive melanoma cell lines and that members of all three families of MAGE class I proteins form complexes with KAP1, a scaffolding protein that is known as a corepressor of p53 expression and function. In addition to inducing apoptosis, MAGE suppression decreases KAP1 complexing with p53, increases immunoreactive and acetylated p53, and activates a p53 responsive reporter gene. Suppression of class I MAGE proteins also induces apoptosis in MAGE-A-positive, p53wt/wt parental HCT 116 colon cancer cells but not in a MAGE-A-positive HCT 116 p53-/- variant, indicating that MAGE suppression of apoptosis requires p53. Finally, treatment with MAGE-specific small interfering RNA suppresses S91 melanoma growth in vivo, in syngenic DBA2 mice. Thus, class I MAGE protein expression may suppress apoptosis by suppressing p53 and may actively contribute to the development of malignancies and by promoting tumor survival. Because the expression of class I MAGE proteins is limited in normal tissues, inhibition of MAGE antigen expression or function represents a novel and specific treatment for melanoma and diverse malignancies.

Protective effect of sanguinarine on ultraviolet B-mediated damages in SKH-1 hairless mouse skin: implications for prevention of skin cancer.

Excessive exposure of solar ultraviolet (UV) radiation, particularly its UVB component (280-320 nm), to human skin is the major cause of skin cancers. UV exposure also leads to the development of precancerous conditions such as actinic keratosis and elicits a variety of other adverse effects such as sunburn, inflammation, hyperplasia, immunosuppression and skin aging. Therefore, there is a need to intensify our efforts towards the development of novel mechanism-based approaches/agents for the protection of UVB-mediated damages. Chemoprevention is being investigated as a potential approach for the management of UV damages including skin cancer. We have earlier shown that sanguinarine, a benzophenanthridine alkaloid, inhibits UVB exposure-mediated damages in HaCaT keratinocytes. In this study, to determine the relevance of our in vitro findings to in vivo situations, we assessed the effects of sanguinarine on UVB-mediated damages in SKH-1 hairless mice. Our data demonstrated that a topical application of sanguinarine (5 micromol 0.3 mL(-1) ethanol per mouse), either as a pretreatment (30 min prior to UVB) or posttreatment (5 min after UVB), resulted in a significant decrease in UVB-mediated increases in skin edema, skin hyperplasia and infiltration of leukocytes. Further, sanguinarine treatments (pre and post) also resulted in a significant decrease in UVB mediated (1) generation of H2O2 and (2) increases in the protein levels of markers of tumor promotion/proliferation viz. ornithine decarboxylase (ODC), proliferating cell nuclear antigen (PCNA) and Kiel antigen-67. Based on this data, we suggest that sanguinarine could be developed as an agent for the management of conditions elicited by UV exposure including skin cancer. However, further detailed studies are needed to support this suggestion.

The role of Forkhead-box Class O (FoxO) transcription factors in cancer: a target for the management of cancer.

Human Forkhead-Box Class O (FoxO) transcription factors are primarily regulated through the phosphoinositide-3-kinase (PI3k)-Akt pathway via phosphorylation and nuclear exclusion. Acetylation and ubiquitination represent another level of regulation for FoxO proteins and FoxO-regulated gene expression. FoxO factors can act as tumor suppressors; however, the loss of FoxO function leads to increased cellular survival and a predisposition to neoplasia, especially of epithelial cancers. Based on the critical role of FoxO signaling, this family of transcription factors appears to be a promising target for future drug discovery for epithelial cancers. This review describes mechanism of the regulation of FoxO proteins and their role in epithelial cancers. Based on the current knowledge and studies in the past decade, we suggest that the development of novel agents which specifically activate FoxO members could be useful in the prevention as well as treatment of cancer in general and epithelial cancers in particular.

Select cancer testes antigens of the MAGE-A, -B, and -C families are expressed in mast cell lines and promote cell viability in vitro and in vivo.

MAGE antigens are proteins that are normally expressed only in gametes but are often aberrantly expressed in melanomas, hematopoietic malignancies, and other "cancers". The functions of most MAGE proteins are unknown. Data have accumulated suggesting expression of MAGE proteins by malignant cells may contribute to advanced disease or resistance to chemotherapy, but direct evidence supporting this hypothesis is lacking. We show here that small interfering RNA (siRNA) suppression of MAGE-A, -B, and -C gene expression slows proliferation and induces caspase independent apoptosis in human and murine mast cell lines. Furthermore, treatment with MAGE specific siRNA suppresses growth of malignant cells in an in vivo murine model of mastocytosis. These observations demonstrate that MAGE protein expression can contribute to the development of tumors by permitting proliferation and prolonging the survival of malignant cells. We suggest a shift of the current clinical paradigm from one that envisions MAGE proteins solely as targets for immunologic attack to one in which MAGE genes and proteins are also targets for functional manipulation.

Sanguinarine induces apoptosis of human pancreatic carcinoma AsPC-1 and BxPC-3 cells via modulations in Bcl-2 family proteins.

Pancreatic cancer is associated with low responsiveness to conventional chemotherapies and its incidence nearly equals its death rate. This warrants the development of novel mechanism-based approaches for the management of pancreatic cancer. This study was designed to determine the potential of sanguinarine, a plant alkaloid known to possess strong antimicrobial, anti-inflammatory, and antioxidant activities, against human pancreatic carcinoma cells. Employing human pancreatic carcinoma AsPC-1 and BxPC-3 cells, we specifically evaluated the pro-apoptotic and cell cycle deregulatory effects of sanguinarine and evaluated the involvement of Bcl-2 family proteins and p53 as the mechanism of the biological effects of sanguinarine. Our data demonstrated that sanguinarine (at low concentrations of 0.1-10 microM; for 24 h) treatment to AsPC-1 and BxPC-3 cells resulted in a dose dependent (i) inhibition of viability and growth, (ii) colony formation ability, (iii) induction of apoptosis, and (iv) G0-G1 phase cell cycle arrest. Further, sanguinarine-treatment to AsPC-1 and BxPC-3 cells resulted in a dose dependent (i) increase in pro-apoptotic Bax, Bid and Bak proteins; (ii) decrease in anti-apoptotic Bcl-2 and Bcl-X(L) proteins; and (iii) decrease in p53 with an increase in its phosphorylation. Based on our study, we suggest that sanguinarine may be developed as an agent for the management of pancreatic cancer. Indeed, more in depth studies both in vitro as well as in vivo in appropriate relevant animal models are needed to strengthen this suggestion.

S100A4 accelerates tumorigenesis and invasion of human prostate cancer through the transcriptional regulation of matrix metalloproteinase 9.

We previously showed that the calcium-binding protein S100A4 is overexpressed during the progression of prostate cancer (CaP) in humans and in the TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model. We tested a hypothesis that the S100A4 gene plays a role in the invasiveness of human CaP and may be associated with its metastatic spread. We observed that siRNA-mediated suppression of the S100A4 gene significantly reduced the proliferative and invasive capability of the highly invasive CaP cells PC-3. We evaluated the mechanism through which the S100A4 gene controls invasiveness of cells by using a macroarray containing 96 well characterized metastatic genes. We found that matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1) were highly responsive to S100A4 gene suppression. Furthermore, S100A4 suppression significantly reduced the expression and proteolytic activity of MMP-9. By employing an MMP-9-promoter reporter, we observed a significant reduction in the transcriptional activation of the MMP-9 gene in S100A4-siRNA-transfected cells. Cells overexpressing the S100A4 gene (when transfected with pcDNA3.1-S100A4 plasmid) also significantly expressed MMP-9 and TIMP-1 genes with increased proteolytic activity of MMP-9 concomitant to increased transcriptional activation of the MMP-9 gene. S100A4-siRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. Our data demonstrate that the S100A4 gene controls the invasive potential of human CaP cells through regulation of MMP-9 and that this association may contribute to metastasis of CaP cells. We suggest that S100A4 could be used as a biomarker for CaP progression and a novel therapeutic or chemopreventive target for human CaP treatment.

Enhancement of UVB radiation-mediated apoptosis by sanguinarine in HaCaT human immortalized keratinocytes.

In this article, we studied the chemopreventive effects of sanguinarine on UVB-mediated responses in human HaCaT immortalized keratinocytes. For our studies, HaCaT cells were treated with a low dose (50 nmol/L) of sanguinarine for 24 hours followed by irradiation with UVB (15 or 30 mJ/cm2). Our data showed that UVB exposure, at both doses, resulted in decreased cell viability and increased apoptosis. Interestingly, pretreatment of the cells with sanguinarine caused a significant enhancement in the antiproliferative response of UVB. These responses on UVB and/or sanguinarine treatments were associated with (a) decrease in Bcl-2 and Bcl-X(L) and (b) increase in Bax, Bid, and Bak protein levels. Bax knockdown and Bcl-2 overexpression resulted in a rescue of HaCaT cells from sanguinarine-mediated apoptosis. DNA cell cycle analysis revealed that UVB treatment resulted in an accumulation of cells in the G2-M phase of the cell cycle, whereas pretreatment of sanguinarine resulted in a significant shift of cells in the S phase at a low UVB dose and a further accumulation of cells in the G2-M phase at a higher UVB dose. These effects on cell cycle were accompanied with modulations in the protein levels of cyclin (B1, E, and A) and cdc2 and cyclin-dependent kinase 1. Furthermore, sanguinarine treatment was found to result in significant modulations in p53, p66Shc, MsrA, and superoxide dismutase levels. Based on our data, we suggest the sanguinarine may protect skin cells from UVB-mediated damages via apoptotic elimination of damaged cells that escape programmed cell death and therefore possess a potential of clonal expansion.

A novel biomarker for staging human prostate adenocarcinoma: overexpression of matriptase with concomitant loss of its inhibitor, hepatocyte growth factor activator inhibitor-1.

BACKGROUND: Matriptase, a type II transmembrane serine protease is involved in angiogenesis, degradation of extracellular matrix, and in the progression of some epithelial cancers. Here, we establish the clinical significance of matriptase and its inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1), during the progression of human prostate cancer (CaP). METHODS: The expression patterns of matriptase and HAI-1 were determined in primary cultures of normal human prostate epithelial (NHPE) cells, human CaP cells LNCaP, DU-145, CWR22Rnu1, and PC-3, and in tissue samples of 172 patients with normal prostate, benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and adenocarcinoma of different tumor grades. RESULTS: The protein and mRNA levels of matriptase were significantly higher in all carcinoma cells as compared with NHPE cells. Conversely, all CaP cells exhibited a reduced expression of HAI-1 as compared with NHPE cells. A progressive increase in the protein levels of matriptase was observed with increasing tumor grade in CaP specimens as compared with normal and BPH tissue specimens. Tissue samples of normal prostate exhibited a high constitutive protein level of HAI-1 compared with BPH and low-grade cancer with a progressive loss with increasing tumor grade. CONCLUSION: The increased expression of matriptase and loss of HAI-1 may be an important event during the progression of CaP in humans. We suggest that the ratio of these two gene products may serve as a promising biomarker for CaP progression and a potential marker for establishing the efficacy of therapeutic and chemopreventive interventions.

Inhibition of CWR22Rnu1 tumor growth and PSA secretion in athymic nude mice by green and black teas.

Cancer of the prostate gland (CaP), the most common invasive malignancy and a major cause of cancer related deaths in male population in the USA, is an ideal candidate disease for chemoprevention because it is typically detected in elderly population with a relatively slower rate of growth and progression. Many dietary phytochemicals are showing promising chemopreventive effects, at-least in pre-clinical models of CaP. Our published data in cell culture and animal studies, supported by the work from other laboratories, as well as epidemiological observations and case-control studies, suggest that polyphenols present in green tea possess CaP chemopreventive and possibly therapeutic effects. This present study was designed to compare CaP cancer chemopreventive effects of green tea polyphenols (GTP), water extract of black tea, and their major constituents epigallocatechin-3-gallate and theaflavins, respectively, in athymic nude mice implanted with androgen-sensitive human CaP CWR22Rnu1 cells. Our data demonstrated that the treatment with all the tea ingredients resulted in (i) significant inhibition in growth of implanted prostate tumors, (ii) reduction in the level of serum prostate specific antigen, (iii) induction of apoptosis accompanied with upregulation in Bax and decrease in Bcl-2 proteins, and (iv) decrease in the levels of VEGF protein. Furthermore, we also found that GTP (0.01 or 0.05% w/v; given after establishment of CWR22Rnu1 tumor) causes a significant regression of tumors suggesting therapeutic effects of GTP at human achievable concentrations.

RNA interference-mediated depletion of phosphoinositide 3-kinase activates forkhead box class O transcription factors and induces cell cycle arrest and apoptosis in breast carcinoma cells.

Breast cancer is one of the most common malignancies affecting women in the Western world and one in seven women is predicted to develop invasive breast cancer in their lifetime. Breast cancer arises following the accumulation of a series of somatic changes often including deregulation of key signal transduction pathways. The phosphoinositide 3-kinase (PI3K) pathway has been shown to be activated in breast cancer and overexpression of PI3K is sufficient to confer a malignant phenotype. Activation of the PI3K pathway serves to repress forkhead box class O (FoxO) transcription factor-mediated growth arrest and apoptosis. In this study, we used small interfering RNA (siRNA) to knockdown PI3K in three breast cancer cell lines representing different stages of cancer development. Transfection of PI3K siRNA in breast cancer cells resulted in a significant decrease in cell viability and induction of apoptosis irrespective of their estrogen receptor alpha (ERalpha) or ErbB2 status. PI3K depletion also resulted in a significant G(1) phase cell cycle arrest in ERalpha-positive breast cancer cells. Further, our data showed that PI3K knockdown resulted in a significant activation of FoxO; interestingly, a simultaneous knockdown of FoxO1a rescued the cells from apoptosis. Furthermore, the downstream effects of FoxO activation were found to be inhibition of cyclin-dependent kinase 4, cyclin-dependent kinase 6, and cyclin D1, and accumulation of p27/Kip1. Thus, we suggest that (a) PI3K plays a critical role in breast cancer development and (b) gene therapeutic approaches aimed at PI3K or the pharmacologic inhibitors of PI3K could be developed for the management of breast cancer.

Polo-like kinase (Plk) 1 as a target for prostate cancer management.

Prostate cancer (PCa) is the most commonly occurring cancer in American men, next to skin cancer. Existing treatment options and surgical intervention are unable to effectively manage this cancer. Therefore, continuing efforts are ongoing to establish novel mechanism-based targets and strategies for its management. The serine/threonine kinases Polo-like kinase (Plk) 1 plays a key role in mitotic entry of proliferating cells and regulates many aspects of mitosis which are necessary for successful cytokinesis. Plk1 is over-expressed in many tumor types with aberrant elevation frequently constituting a prognostic indicator of poor disease outcome. This review discusses the studies which indicate that Plk1 could be an excellent target for the treatment as well as chemoprevention of prostate cancer.

Chemoprevention of skin cancer by grape constituent resveratrol: relevance to human disease?

According to the World Cancer Report, skin cancer constitutes approximately 30% of all newly diagnosed cancers in the world, and solar ultraviolet (UV) radiation (particularly, its UVB component; 290-320 nm) is an established cause of approximately 90% of skin cancers. The available options have proven to be inadequate for the management of skin cancers. Therefore, there is an urgent need to develop mechanism-based novel approaches for prevention/therapy of skin cancer. In this study, we evaluated the chemopreventive effects of resveratrol against UVB radiation-mediated skin tumorigenesis in the SKH-1 hairless mouse model. For our studies, we used a UVB initiation-promotion protocol in which the control mice were subjected to chronic UVB exposure (180 mJ/cm2, twice weekly, for 28 weeks). The experimental animals received either a pretreatment (30 min before each UVB) or post-treatment (5 min after UVB) of resveratrol (25 or 50 micro mole/0.2 ml acetone/mouse). The mice were followed for skin tumorigenesis and were killed at 24 h after the last UVB exposure, for further studies. The topical application of skin with resveratrol (both pre- and post- treatment) resulted in a highly significant 1) inhibition in tumor incidence, and 2) delay in the onset of tumorigenesis. Interestingly, the post-treatment of resveratrol was found to impart equal protection than the pretreatment; suggesting that resveratrol-mediated responses may not be sunscreen effects. Because Survivin is a critical regulator of survival/death of cells, and its overexpression has been implicated in several cancers, we evaluated its involvement in chemoprevention of UVB-mediated skin carcinogenesis by resveratrol. Our data demonstrated a significant 1) up-regulation of Survivin (both at protein- and mRNA- levels), 2) up-regulation of phospho-Survivin protein, and 3) down-regulation of proapoptotic Smac/DIABLO protein in skin tumors; whereas treatment with resveratrol resulted in the attenuation of these responses. Our study also suggests that resveratrol enhanced apoptosis in UVB-exposure-mediated skin tumors. Our study, for the first time, demonstrated that 1) resveratrol imparts strong chemopreventive effects against UVB exposure-mediated skin carcinogenesis (relevant to human skin cancers), and 2) the chemopreventive effects of resveratrol may, at least in part, be mediated via modulations in Survivin and other associated events. On the basis of our work, it is conceivable to design resveratrol-containing emollient or patch, as well as sunscreen and skin-care products for prevention of skin cancer and other conditions, which are believed to be caused by UV radiation.

Silencing of polo-like kinase (Plk) 1 via siRNA causes induction of apoptosis and impairment of mitosis machinery in human prostate cancer cells: implications for the treatment of prostate cancer.

Prostate cancer (PCa) is one of the most common cancers in men. Each year approximately 543,000 new cases are reported worldwide, and the disease kills 200,000 (mostly older men) in developed countries. The existing treatment approaches and surgical intervention have not been able to effectively manage this dreaded cancer and, therefore, continuing efforts are ongoing to explore novel targets and strategies for the management of PCa. The activity of polo-like kinase 1 (Plk1) is elevated in tissues and cells with a high mitotic index, including cancer cells. An increasing body of evidence suggests that the level of Plk1 expression has prognostic value for predicting outcomes in patients with some cancers. A close correlation between Plk1 expression and carcinogenesis has been documented. However, the role of Plk1 in PCa is not known. We propagated a hypothesis that Plk1 inhibition will result in elimination of human PCa cells via a mitotic arrest followed by apoptosis (1). To define the role of Plk1 in PCa, we used the technique of RNA silencing via small interfering RNA (siRNA). First, using a series of human prostate carcinoma cells and normal human prostate epithelial (PrEC) cells, we assessed Plk1 levels in PCa. Immunoblot analyses clearly showed a significant expression of Plk1 in LNCaP, DU145, and PC3 human PCa cells. Interestingly, Plk1 was not detectable in normal PrEC cells. Next, we transfected the PCa cells with Plk 1 siRNA, which resulted in a significant inhibition in Plk1 protein in all PCa cells. Plk1 depletion resulted in a decrease in cell viability and induction of apoptosis in PCa cells but had no appreciable effect in normal PrEC cells. Our data also demonstrated that Plk1 siRNA transfection of PCa cells resulted in 1) a mitotic cell cycle arrest, 2) failure of cytokinesis, and 3) defects in centrosome integrity and maturation. Thus, our study suggested that 1) Plk1 plays a critical role in the process of PCa development and 2) gene therapeutic approaches aimed at Plk1 or the pharmacological inhibitors of Plk1 may be developed for the management of PCa.

Sanguinarine causes cell cycle blockade and apoptosis of human prostate carcinoma cells via modulation of cyclin kinase inhibitor-cyclin-cyclin-dependent kinase machinery.

Prostate cancer is the second leading cause of cancer-related deaths in males in the United States. This warrants the development of novel mechanism-based strategies for the prevention and/or treatment of prostate cancer. Several studies have shown that plant-derived alkaloids possess remarkable anticancer effects. Sanguinarine, an alkaloid derived from the bloodroot plant Sanguinaria canadensis, has been shown to possess antimicrobial, anti-inflammatory, and antioxidant properties. Previously, we have shown that sanguinarine possesses strong antiproliferative and proapoptotic properties against human epidermoid carcinoma A431 cells and immortalized human HaCaT keratinocytes. Here, employing androgen-responsive human prostate carcinoma LNCaP cells and androgen-unresponsive human prostate carcinoma DU145 cells, we studied the antiproliferative properties of sanguinarine against prostate cancer. Sanguinarine (0.1-2 micromol/L) treatment of LNCaP and DU145 cells for 24 hours resulted in dose-dependent (1) inhibition of cell growth [as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], (2) arrest of cells in G0-G1 phase of the cell cycle (as assessed by DNA cell cycle analysis), and (3) induction of apoptosis (as evaluated by DNA ladder formation and flow cytometry). To define the mechanism of antiproliferative effects of sanguinarine against prostate cancer, we studied the effect of sanguinarine on critical molecular events known to regulate the cell cycle and the apoptotic machinery. Immunoblot analysis showed that sanguinarine treatment of both LNCaP and DU145 cells resulted in significant (1) induction of cyclin kinase inhibitors p21/WAF1 and p27/KIP1; (2) down-regulation of cyclin E, D1, and D2; and (3) down-regulation of cyclin-dependent kinase 2, 4, and 6. A highlight of this study was the fact that sanguinarine induced growth inhibitory and antiproliferative effects in human prostate carcinoma cells irrespective of their androgen status. To our knowledge, this is the first study showing the involvement of cyclin kinase inhibitor-cyclin-cyclin-dependent kinase machinery during cell cycle arrest and apoptosis of prostate cancer cells by sanguinarine. These results suggest that sanguinarine may be developed as an agent for the management of prostate cancer.