Shannon Reagan-Shaw

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 allomet‐ric 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.—Reagan‐Shaw S., Nihal, M., Ahmad N. Dose translation from animal to human studies revisited. FASEB J. 22, 659–661 (2007)

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.

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

According to the World Cancer Report, skin cancer constitutes ∼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 ∼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.

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.

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 ∼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.

Modulations of critical cell cycle regulatory events during chemoprevention of ultraviolet B-mediated responses by resveratrol in SKH-1 hairless mouse skin

Multiple exposures to solar ultraviolet (UV) radiation cause critical damages that may lead to the development of several cutaneous disorders including skin cancer, the most frequently diagnosed malignancy in the USA. Therefore, efforts are needed to: (i) study the mechanism(s) of UV-mediated cutaneous damages, and (ii) design novel approaches for the management of skin cancer. ‘Chemoprevention’ via plant-based agents may be a useful approach for the management of neoplasia. Here, we evaluated the involvement of cell cycle regulatory molecules during resveratrol-mediated protection from multiple exposures of UVB (180 mJ/cm2; on alternate days × 7 exposures) radiations in the SKH-1 hairless mouse skin. Resveratrol was topically applied on the skin of SKH-1 hairless mice at a dose of 10 μmol/mouse (in 0.2 ml acetone; 30 min prior to each UVB exposure). Studies were performed at 24 h following the last UVB exposure. Topical application of resveratrol resulted in significant decrease in UVB-induced bi-fold skin thickness, hyperplasia, and infiltration of leukocytes. The data from immunoblot and/or immunohistochemical analyses revealed that multiple exposure to UVB radiations causes significant upregulation in: (i) proliferating cell nuclear antigen (PCNA), a marker of cellular proliferation, and (ii) cyclin-dependent kinase (cdk)-2, -4 and -6, cyclin-D1, and cyclin-D2. Resveratrol treatment resulted in significant downregulation in UV-mediated increases in these critical cell cycle regulatory proteins. An interesting observation of this study was that resveratrol treatment resulted in a further stimulation of UVB-mediated increases in cyclin kinase inhibitor WAF1/p21 and tumor suppressor p53. Further, resveratrol was also found to cause significant decreases in UVB-mediated upregulation of: (i) the mitogen-activated protein kinase kinase, and (ii) the 42 kDa isotype of mitogen-activated protein kinase (MAPK). Thus, our data suggested that the antiproliferative effects of resveratrol might be mediated via modulation in the expression and function of cell cycle regulatory proteins cyclin-D1 and -D2, cdk-2, -4 and -6, and WAF1/p21. Our data further suggest that the modulation of cki–cyclin–cdk network by resveratrol may be associated with inhibition of the MAPK pathway. We suggest that resveratrol may be useful for the prevention of UVB-mediated cutaneous damages including skin cancer.

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, CWR22Rν1, 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. (Cancer Epidemiol Biomarkers Prev 2006;15(2):217–27)

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

Abstract:  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.

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.

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

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.