Photochemoprevention of ultraviolet Beam Radiation-induced DNA damage in keratinocytes by topical delivery of nanoformulated Epigallocatechin-3-gallate.

Ultraviolet Beam (UVB) radiation is the main cause of skin cancer worldwide. Besides biocompatibility, the instability and limited skin permeability are the most challenging features of many effective photochemopreventive agents. (-)-Epigallocatechin-3-gallate (EGCG) is a natural polyphenolic compound extracted from Camellia sinensis that has been demonstrated to have antioxidant, anti-inflammatory, and anti-cancer properties. We evaluated the efficacy of three innovative EGCG nanoformulations in chemoprevention of UVB-induced DNA damage in keratinocytes. Results indicated that the EGCG nanoformulations reduced UVB-induced oxidative stress elevation and DNA damage. The nanoformulations also reduced the UVB-induced formation of pyrimidine and pyrimidone photoproducts in 2D human immortalized HaCaT keratinocytes and SKH-1 hairless mice through antioxidant effects and possibly through absorption of UVB radiation. In addition, EGCG nanoformulations inhibited UVB-induced chemokine/cytokine activation and promoted EGCG skin permeability and stability. Taken together, the results suggest the use of EGCG nanoformulations as potential natural chemopreventive agents during exposure to UVB radiation.

Fisetin, a 3,7,3',4'-Tetrahydroxyflavone Inhibits the PI3K/Akt/mTOR and MAPK Pathways and Ameliorates Psoriasis Pathology in 2D and 3D Organotypic Human Inflammatory Skin Models.

Psoriasis is a chronic immune-mediated skin disease that involves the interaction of immune and skin cells, and is characterized by cytokine-driven epidermal hyperplasia, deviant differentiation, inflammation, and angiogenesis. Because the available treatments for psoriasis have significant limitations, dietary products are potential natural sources of therapeutic molecules, which can repair the molecular defects associated with psoriasis and could possibly be developed for its management. Fisetin (3,7,3',4'-tetrahydroxyflavone), a phytochemical naturally found in pigmented fruits and vegetables, has demonstrated proapoptotic and antioxidant effects in several malignancies. This study utilized biochemical, cellular, pharmacological, and tissue engineering tools to characterize the effects of fisetin on normal human epidermal keratinocytes (NHEKs), peripheral blood mononuclear cells (PBMC), and CD4+ T lymphocytes in 2D and 3D psoriasis-like disease models. Fisetin treatment of NHEKs dose- and time-dependently induced differentiation and inhibited interleukin-22-induced proliferation, as well as activation of the PI3K/Akt/mTOR pathway. Fisetin treatment of TNF-α stimulated NHEKs also significantly inhibited the activation of p38 and JNK, but had enhanced effect on ERK1/2 (MAPK). In addition, fisetin treatment significantly decreased the secretion of Th1/Th-17 pro-inflammatory cytokines, particularly IFN-γ and IL-17A by 12-O-tetradecanolylphorbol 13-acetate (TPA)-stimulated NHEKs and anti-CD3/CD28-activated human PBMCs. Furthermore, we established the in vivo relevance of fisetin functions, using a 3D full-thickness human skin model of psoriasis (FTRHSP) that closely mimics in vivo human psoriatic skin lesions. Herein, fisetin significantly ameliorated psoriasis-like disease features, and decreased the production of IL-17 by CD4+ T lymphocytes co-cultured with FTRHSP. Collectively, our data identify the prodifferentiative, antiproliferative, and anti-inflammatory effects of fisetin, via modulation of the PI3K-Akt-mTOR and p38/JNK pathways and the production of cytokines in 2D and 3D human skin models of psoriasis. These results suggest that fisetin has a great potential to be developed as an effective and inexpensive agent for the treatment of psoriasis and other related inflammatory skin disorders.

Chitosan-based nanoformulated (-)-epigallocatechin-3-gallate (EGCG) modulates human keratinocyte-induced responses and alleviates imiquimod-induced murine psoriasiform dermatitis.

BACKGROUND: Psoriasis is a chronic and currently incurable inflammatory skin disease characterized by hyperproliferation, aberrant differentiation, and inflammation, leading to disrupted skin barrier function. The use of natural agents that can abrogate these effects could be useful for the treatment of psoriasis. Earlier studies have shown that treatment of keratinocytes and mouse skin with the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) mitigated inflammation and increased the expression of caspase-14 while promoting epidermal differentiation and cornification. However, bioavailability issues have restricted the development of EGCG for the treatment of psoriasis. MATERIALS AND METHODS: To overcome these limitations, we employed a chitosan-based polymeric nanoparticle formulation of EGCG (CHI-EGCG-NPs, hereafter termed nanoEGCG) suitable for topical delivery for treating psoriasis. We investigated and compared the efficacy of nanoEGCG versus native or free EGCG in vitro and in an in vivo imiquimod (IMQ)-induced murine psoriasis-like dermatitis model. The in vivo relevance and efficacy of nanoEGCG formulation (48 µg/mouse) were assessed in an IMQ-induced mouse psoriasis-like skin lesion model compared to free EGCG (1 mg/mouse). RESULTS: Like free EGCG, nanoEGCG treatment induced differentiation, and decreased proliferation and inflammatory responses in cultured keratinocytes, but with a 4-fold dose advantage. Topically applied nanoEGCG elicited a significant (p<0.01) amelioration of psoriasiform pathological markers in IMQ-induced mouse skin lesions, including reductions in ear and skin thickness, erythema and scales, proliferation (Ki-67), infiltratory immune cells (mast cells, neutrophils, macrophages, and CD4+ T cells), and angiogenesis (CD31). We also observed increases in the protein expression of caspase-14, early (keratin-10) and late (filaggrin and loricrin) markers of differentiation, and the activator protein-1 factor (JunB). Importantly, a significant modulation of several psoriasis-related inflammatory cytokines and chemokines was observed compared to the high dose of free EGCG (p<0.05). Taken together, topically applied nanoEGCG displayed a >20-fold dose advantage over free EGCG. CONCLUSION: Based on these observations, our nanoEGCG formulation represents a promising drug-delivery strategy for treating psoriasis and possibly other inflammatory skin diseases.

Nummularic acid, a triterpenoid, from the medicinal plant Fraxinus xanthoxyloides, induces energy crisis to suppress growth of prostate cancer cells.

We recently identified and characterized nummularic acid (NA) as a major chemical constituent of Fraxinus xanthoxyloides, a medicinal plant used for over hundred years in traditional medicine. In this study, we describe its potential anti-cancer activity using prostate cancer (PCa) cells as a model. We found that NA treatment (5-60 µM) significantly reduced the proliferation and colony formation capabilities of PCa DU145 and C4-2 cells in a time and dose dependent manner, reduced the migratory and invasive properties and increased apoptotic cell population. Mechanistically, we found that NA treatment to PCa cells resulted in a sustained activation of adenosine monophosphate-activated protein kinase (AMPK). NA simultaneously increased acetyl CoA carboxylase phosphorylation and decreased pS6 phosphorylation, the two major substrates of AMPK. Further, NA treatment significantly elevated the cellular ADP/ATP ratio and altered glycolytic rate. We further observed a reversible decrease in oxygen consumption rate in NA treated cells when compared to the control. Finally, we performed global untargeted metabolomics which showed that NA treatment alters PCa cell metabolism at multiple sites including glycolysis, tricarboxylic acid, and glutamine metabolism which supported our observation of a possible AMPK activation. In summary, we report NA as a novel small molecule activator of AMPK that alters cellular metabolism to induce energy crisis and ultimately cancer cell death. Because of its unique mechanism NA could be potentially applicable against other cancer types.

Targeted nanoparticles encapsulating (-)-epigallocatechin-3-gallate for prostate cancer prevention and therapy.

Earlier we introduced the concept of 'nanochemoprevention' i.e. the use of nanotechnology to improve the outcome of cancer chemoprevention. Here, we extended our work and developed polymeric EGCG-encapsulated nanoparticles (NPs) targeted with small molecular entities, able to bind to prostate specific membrane antigen (PSMA), a transmembrane protein that is overexpressed in prostate cancer (PCa), and evaluated their efficacy in preclinical studies. First, we performed a molecular recognition of DCL- and AG-PEGylation on ligand binding on PSMA active site. Next, the biocompatible polymers PLGA-PEG-A were synthesized and used as base to conjugate DCL or AG to obtain the respective copolymers, needed for the preparation of targeted NPs. The resulting EGCG encapsulating NPs led to an enhanced anti-proliferative activity in PCa cell lines compared to the free EGCG. The behavior of EGCG encapsulated in NPs in modulating apoptosis and cell-cycle, was also determined. Then, in vivo experiments, in mouse xenograft model of prostatic tumor, using EGCG-loaded NPs, with a model of targeted nanosystems, were conducted. The obtained data supported our hypothesis of target-specific enhanced bioavailability and limited unwanted toxicity, thus leading to a significant potential for probable clinical outcome.

Dual Inhibition of PI3K/Akt and mTOR by the Dietary Antioxidant, Delphinidin, Ameliorates Psoriatic Features In Vitro and in an Imiquimod-Induced Psoriasis-Like Disease in Mice.

AIM: The treatment of psoriasis remains elusive, underscoring the need for identifying novel disease targets and mechanism-based therapeutic approaches. We recently reported that the PI3K/Akt/mTOR pathway that is frequently deregulated in many malignancies is also clinically relevant for psoriasis. We also provided rationale for developing delphinidin (Del), a dietary antioxidant for the management of psoriasis. This study utilized high-throughput biophysical and biochemical approaches and in vitro and in vivo models to identify molecular targets regulated by Del in psoriasis. RESULTS: A kinome-level screen and Kds analyses against a panel of 102 human kinase targets showed that Del binds to three lipid (PIK3CG, PIK3C2B, and PIK3CA) and six serine/threonine (PIM1, PIM3, mTOR, S6K1, PLK2, and AURKB) kinases, five of which belong to the PI3K/Akt/mTOR pathway. Surface plasmon resonance and in silico molecular modeling corroborated Del's direct interactions with three PI3Ks (α/c2ß/γ), mTOR, and p70S6K. Del treatment of interleukin-22 or TPA-stimulated normal human epidermal keratinocytes (NHEKs) significantly inhibited proliferation, activation of PI3K/Akt/mTOR components, and secretion of proinflammatory cytokines and chemokines. To establish the in vivo relevance of these findings, an imiquimod (IMQ)-induced Balb/c mouse psoriasis-like skin model was employed. Topical treatment of Del significantly decreased (i) hyperproliferation and epidermal thickness, (ii) skin infiltration by immune cells, (iii) psoriasis-related cytokines/chemokines, (iv) PI3K/Akt/mTOR pathway activation, and (v) increased differentiation when compared with controls. Innovation and Conclusion: Our observation that Del inhibits key kinases involved in psoriasis pathogenesis and alleviates IMQ-induced murine psoriasis-like disease suggests a novel PI3K/AKT/mTOR pathway modulator that could be developed to treat psoriasis. Antioxid. Redox Signal. 26, 49-69.

Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis.

We and others have shown previously that fisetin, a plant flavonoid, has therapeutic potential against many cancer types. Here, we examined the probable mechanism of its action in prostate cancer (PCa) using a global metabolomics approach. HPLC-ESI-MS analysis of tumor xenografts from fisetin-treated animals identified several metabolic targets with hyaluronan (HA) as the most affected. Efficacy of fisetin on HA was then evaluated in vitro and also in vivo in the transgenic TRAMP mouse model of PCa. Size exclusion chromatography-multiangle laser light scattering (SEC-MALS) was performed to analyze the molar mass (Mw) distribution of HA. Fisetin treatment downregulated intracellular and secreted HA levels both in vitro and in vivo Fisetin inhibited HA synthesis and degradation enzymes, which led to cessation of HA synthesis and also repressed the degradation of the available high-molecular-mass (HMM)-HA. SEC-MALS analysis of intact HA fragment size revealed that cells and animals have more abundance of HMM-HA and less of low-molecular-mass (LMM)-HA upon fisetin treatment. Elevated HA levels have been shown to be associated with disease progression in certain cancer types. Biological responses triggered by HA mainly depend on the HA polymer length where HMM-HA represses mitogenic signaling and has anti-inflammatory properties whereas LMM-HA promotes proliferation and inflammation. Similarly, Mw analysis of secreted HA fragment size revealed less HMM-HA is secreted that allowed more HMM-HA to be retained within the cells and tissues. Our findings establish that fisetin is an effective, non-toxic, potent HA synthesis inhibitor, which increases abundance of antiangiogenic HMM-HA and could be used for the management of PCa.

The pentacyclic triterpenoid, plectranthoic acid, a novel activator of AMPK induces apoptotic death in prostate cancer cells.

Epidemiologic studies indicated that diabetics treated with metformin had a lower incidence of cancer than those taking other anti-diabetes drugs. This led to a surge in the efforts for identification of safer and more effective metformin mimetic compounds. The plant Ficus microcarpa is widely used for the treatment of type 2 diabetes in traditional medicine in South Asia. We obtained extracts from this plant and identified a small molecule, plectranthoic acid (PA), with potent 5'AMP-activated kinase (AMPK) activating properties far superior than metformin. AMPK is the central hub of metabolic regulation and a well-studied therapeutic target for metabolic syndrome, type-2 diabetes and cancer. We observed that treatment of prostate cancer (PCa) cells with PA inhibited proliferation and induced G0/G1 phase cell cycle arrest that was associated with up-regulation of cyclin kinase inhibitors p21/CIP1 and p27/KIP1. PA treatment suppressed mTOR/S6K signaling and induced apoptosis in PCa cells in an AMPK-dependent manner. Interestingly, PA-induced autophagy in PCa cells was found to be independent of AMPK activation. Combination studies of PA and metformin demonstrated that metformin had an inhibitory effect on PA-induced AMPK activation and suppressed PA-mediated apoptosis. Given the anti-proliferative role of PA in cancer and its potent anti-hyperglycemic activity, we suggest that PA should be explored further as a novel activator of AMPK for its ultimate use for the prevention of cancers and treatment of type 2 diabetes.

Dietary polyphenols in prevention and treatment of prostate cancer.

Prostate cancer is the most prevalent disease affecting males in many Western countries, with an estimated 29,480 deaths in 2014 in the US alone. Incidence rates for prostate cancer deaths have been decreasing since the early 1990s in men of all races/ethnicities, though they remain about 60% higher in African Americans than in any other group. The relationship between dietary polyphenols and the prevention of prostate cancer has been examined previously. Although results are sometimes inconsistent and variable, there is a general agreement that polyphenols hold great promise for the future management of prostate cancer. Various dietary components, including polyphenols, have been shown to possess anti-cancer properties. Generally considered as non-toxic, dietary polyphenols act as key modulators of signaling pathways and are therefore considered ideal chemopreventive agents. Besides possessing various anti-tumor properties, dietary polyphenols also contribute to epigenetic changes associated with the fate of cancer cells and have emerged as potential drugs for therapeutic intervention. Polyphenols have also been shown to affect post-translational modifications and microRNA expressions. This article provides a systematic review of the health benefits of selected dietary polyphenols in prostate cancer, especially focusing on the subclasses of polyphenols, which have a great effect on disease prevention and treatment.

Prodifferentiation, anti-inflammatory and antiproliferative effects of delphinidin, a dietary anthocyanidin, in a full-thickness three-dimensional reconstituted human skin model of psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory disorder of skin and joints for which conventional treatments that are effective in clearing the moderate-to-severe disease are limited due to long-term safety issues. This necessitates exploring the usefulness of botanical agents for treating psoriasis. We previously showed that delphinidin, a diet-derived anthocyanidin endowed with antioxidant and anti-inflammatory properties, induces normal epidermal keratinocyte differentiation and suggested its possible usefulness for the treatment of psoriasis [1]. OBJECTIVES: To investigate the effect of delphinidin (0-20 µM; 2-5 days) on psoriatic epidermal keratinocyte differentiation, proliferation and inflammation using a three-dimensional reconstructed human psoriatic skin equivalent (PSE) model. METHODS: PSEs and normal skin equivalents (NSEs) established on fibroblast-contracted collagen gels with respective psoriatic and normal keratinocytes and treated with/without delphinidin were analyzed for histology, expression of markers of differentiation, proliferation and inflammation using histomorphometry, immunoblotting, immunochemistry, qPCR and cultured supernatants for cytokine with a Multi-Analyte ELISArray Kit. RESULTS: Our data show that treatment of PSE with delphinidin induced (1) cornification without affecting apoptosis and (2) the mRNA and protein expression of markers of differentiation (caspase-14, filaggrin, loricrin, involucrin). It also decreased the expression of markers of proliferation (Ki67 and proliferating cell nuclear antigen) and inflammation (inducible nitric oxide synthase and antimicrobial peptides S100A7-psoriasin and S100A15-koebnerisin, which are often induced in psoriatic skin). ELISArray showed increased release of psoriasis-associated keratinocyte-derived proinflammatory cytokines in supernatants of the PSE cultures, and this increase was significantly suppressed by delphinidin. CONCLUSIONS: These observations provide a rationale for developing delphinidin for the management of psoriasis.

Excellent anti-proliferative and pro-apoptotic effects of (-)-epigallocatechin-3-gallate encapsulated in chitosan nanoparticles on human melanoma cell growth both in vitro and in vivo.

Earlier we demonstrated the anti-proliferative and pro-apoptotic effects of green tea polyphenol epigallocatechin-3-gallate (EGCG) on human melanoma cells (Int J Cancer. 2005; 114(4): 513-21). The doses used in this study were not physiologically attainable and for chemoprevention the preferred route of administration is oral consumption. To overcome these shortcomings, and taking advantage of our novel concept of nanochemoprevention (Cancer Res. 2009;69(5):1712-6), we developed a nanotechnology based oral delivery system to encapsulate EGCG. Here, using human melanoma Mel 928 cells we demonstrate 8-fold dose advantage of this nanoformulation over native EGCG. Further, nano-EGCG treated cells showed marked induction of apoptosis and cell cycle inhibition along with the growth of Mel 928 tumor xenograft. Nano-EGCG also inhibited proliferation (Ki-67 and PCNA) and induced apoptosis (Bax, PARP) in tumors harvested from the treated mice. These observations warrant further in vivo efficacy studies of nano-EGCG in robust animal models of human melanoma. FROM THE CLINICAL EDITOR: This team of investigators developed a nanotechnology based oral delivery system to encapsulate EGCG, a green tea-derived polyphenol in chitosan nanoparticles. Using human melanoma cells, an eight-fold dose advantage was demonstrated over native EGCG, leading to measurable apoptosis induction and proliferation inhibition, warranting further in vivo investigations.

Fisetin inhibits human melanoma cell growth through direct binding to p70S6K and mTOR: findings from 3-D melanoma skin equivalents and computational modeling.

The incidence of melanoma continues to rise. Inspite of treatment advances, the prognosis remains grim once the disease has metastasized, emphasizing the need to explore additional therapeutic strategies. One such approach is through the use of mechanism-based dietary intervention. We previously showed that the flavonoid fisetin inhibits melanoma cell proliferation, in vitro and in vivo. Here, we studied fisetin-mediated regulation of kinases involved in melanoma growth and progression. Time-course analysis in 3-D melanoma constructs that transitioned from radial to vertical growth showed that fisetin treatment resulted in significant decrease in melanocytic lesions in contrast to untreated controls that showed large tumor nests and invading disseminated cells. Further studies in melanoma cultures and mouse xenografts showed that fisetin-mediated growth inhibition was associated with dephosphorylation of AKT, mTOR and p70S6K proteins. In silico modeling indicated direct interaction of fisetin with mTOR and p70S6K with favorable free energy values. These findings were validated by cell-free competition assays that established binding of fisetin to p70S6K and mTOR while little affinity was detected with AKT. Kinase activity studies reflected similar trend with % inhibition observed for p70S6K and mTOR at lower doses than AKT. Our studies characterized, for the first time, the differential interactions of any botanical agent with kinases involved in melanoma growth and demonstrate that fisetin inhibits mTOR and p70S6K through direct binding while the observed inhibitory effect of fisetin on AKT is mediated indirectly, through targeting interrelated pathways.

Oral administration of naturally occurring chitosan-based nanoformulated green tea polyphenol EGCG effectively inhibits prostate cancer cell growth in a xenograft model.

In preclinical animal models, several phytochemicals have shown excellent potential to be used as effective agents in preventing and treating many cancers. However, the limited bioavailability of active agents could be one reason for their restricted usefulness for human consumption. To overcome this limitation, we recently introduced the concept of nanochemoprevention by encapsulating useful bioactive food components for their slow and sustained release. Here, we report the synthesis, characterization and efficacy assessment of a nanotechnology-based oral formulation of chitosan nanoparticles encapsulating epigallocatechin-3-gallate (Chit-nanoEGCG) for the treatment of prostate cancer (PCa) in a preclinical setting. Chit-nanoEGCG with a size of <200nm diameter and encapsulating EGCG as determined by dynamic light scattering and transmission electron microscope showed slow release of EGCG in simulated gastric juice acidic pH and faster release in simulated intestinal fluid. The antitumor efficacy of Chit-nanoEGCG was assessed in subcutaneously implanted 22Rν1 tumor xenografts in athymic nude mice. Treatment with Chit-nanoEGCG resulted in significant inhibition of tumor growth and secreted prostate-specific antigen levels compared with EGCG and control groups. In tumor tissues of mice treated with Chit-nanoEGCG, compared with groups treated with EGCG and controls, there was significant (i) induction of poly (ADP-ribose) polymerases cleavage, (ii) increase in the protein expression of Bax with concomitant decrease in Bcl-2, (iii) activation of caspases and (iv) reduction in Ki-67 and proliferating cell nuclear antigen. Through this study, we propose a novel preventive and therapeutic modality for PCa using EGCG that addresses issues related to bioavailability.

The S100A4 Oncoprotein Promotes Prostate Tumorigenesis in a Transgenic Mouse Model: Regulating NFκB through the RAGE Receptor.

S100A4, a calcium-binding protein, is known for its role in the metastatic spread of tumor cells, a late event of cancer disease. This is the first report showing that S100A4 is not merely a metastatic protein but also an oncoprotein that plays a critical role in the development of tumors. We earlier showed that S100A4 expression progressively increases in prostatic tissues with the advancement of prostate cancer (CaP) in TRAMP, an autochthonous mouse model. To study the functional significance of S100A4 in CaP, we generated a heterozygously deleted S100A4 (TRAMP/S100A4(+/-)) genotype by crossing TRAMP with S100A4(-/-) mice. TRAMP/S100A4(+/-) did not show a lethal phenotype, and transgenes were functional. As compared to age-matched TRAMP littermates, TRAMP/S100A4(+/-) mice exhibited 1) an increased tumor latency period (P < 0.001), 2) a 0% incidence of metastasis, and 3) reduced prostatic weights (P < 0.001). We generated S100A4-positive clones from S100A4-negative CaP cells and tested their potential. S100A4-positive tumors grew at a faster rate than S100A4-negative tumors in vitro and in a xenograft mouse model. The S100A4 protein exhibited growth factor-like properties in multimode (intracellular and extracellular) forms. We observed that 1) the growth-promoting effect of S100A4 is due to its activation of NFκB, 2) S100A4-deficient tumors exhibit reduced NFκB activity, 3) S100A4 regulates NFκB through the RAGE receptor, and 4) S100A4 and RAGE co-localize in prostatic tissues of mice. Keeping in view its growth-promoting role, we suggest that S100A4 qualifies as an excellent candidate to be exploited for therapeutic agents to treat CaP in humans.

The dietary terpene lupeol targets colorectal cancer cells with constitutively active Wnt/ß-catenin signaling.

SCOPE: Aberrant activation of the Wingless-type mouse mammary tumor virus integration site family (Wnt)/ß-catenin signaling pathway is the most common modification, and often considered, a hallmark of colorectal cancer (CRC). Typically in this pathway the ß-catenin translocates from the cytoplasm to the nucleus, where it functions as a transcription regulator of several genes that support tumor formation and progression. Thus, any agent that could attenuate the translocation of ß-catenin could be extremely valuable against CRC, especially the tumors that exhibit constitutively active Wnt/ß-catenin signaling. METHODS AND RESULTS: Using human CRC cells that exhibit differential expression of Wnt/ß-catenin signaling, we demonstrate that treatment of CRC cells with dietary triterpene lupeol results in a dose-dependent (i) decrease in cell viability, (ii) induction of apoptosis, (iii) decrease in colonogenic potential, (iv) decrease in ß-catenin transcriptional activity, and (v) decrease in the expression of Wnt target genes. Most importantly lupeol was observed to inhibit the translocation of ß-catenin from the cytoplasm to the nucleus. Importantly, all these effects of lupeol were restricted to cells that harbor constitutively active Wnt/ß-catenin signaling while negligible effects were observed in cells that lack constitutively active Wnt/ß-catenin signaling. Further, we also demonstrate that inhibition of Wnt signaling in cells with constitutive active Wnt/ß-catenin results in loss of lupeol efficacy while inducing Wnt signaling sensitizes the cells to inhibitory effects of lupeol. CONCLUSION: In summary, our data strongly advocate the efficacy of lupeol against CRC cells that exhibit constitutively active Wnt/ß-catenin signaling.

Delphinidin, a dietary antioxidant, induces human epidermal keratinocyte differentiation but not apoptosis: studies in submerged and three-dimensional epidermal equivalent models.

Delphinidin (Del), [3,5,7,3'-,4'-,5'-hexahydroxyflavylium], an anthocyanidin and a potent antioxidant abundantly found in pigmented fruits and vegetables exhibits proapoptotic effects in many cancer cells. Here, we determined the effect of Del on growth, apoptosis and differentiation of normal human epidermal keratinocytes (NHEKs) in vitro in submerged cultures and examined its effects in a three-dimensional (3D) epidermal equivalent (EE) model that permits complete differentiation reminiscent of in vivo skin. Treatment of NHEKs with Del (10-40 µm; 24-48 h) significantly enhanced keratinocyte differentiation. In Del-treated cells, there was marked increase in human involucrin (hINV) promoter activity with simultaneous increase in the mRNA and protein expressions of involucrin and other epidermal differentiation markers including procaspase-14 and transglutaminase-1 (TGM1), but without any effect on TGM2. Del treatment of NHEKs was associated with minimal decrease in cell viability, which was not associated with apoptosis as evident by lack of modulation of caspases, apoptosis-related proteins including Bcl-2 family of proteins and poly(ADP-ribose) polymerase cleavage. To establish the in vivo relevance of our observations in submerged cultures, we then validated these effects in a 3D EE model, where Del was found to significantly enhance cornification and increase the protein expression of cornification markers including caspase-14 and keratin 1. For the first time, we show that Del induces epidermal differentiation using an experimental system that closely mimics in vivo human skin. These observations suggest that Del could be a useful agent for dermatoses associated with epidermal barrier defects including aberrant keratinization, hyperproliferation or inflammation observed in skin diseases like psoriasis and ichthyoses.

Inhibition of Akt/mTOR signaling by the dietary flavonoid fisetin.

Plants have long been providing mankind with remedies of different ailments. Flavonoids, a family of naturally occurring polyphenolic compounds are ubiquitous in plants. Development of polyphenol-based drugs has not attracted much attention by researchers and drug companies. Therefore, despite extensive studies on polyphenols, this vast group of compounds is underrepresented in clinical medicine. Fisetin (3,7,3',4'-tetrahydroxyflavone) belongs to the flavonol subgroup of flavonoids together with quercetin, myricetin and kaempferol and is found in several fruits and vegetables including strawberries, apples, persimmons and onions. Fisetin is showing promise as a useful natural agent against cancer and has been evaluated for its potential inhibitory role against cancer in several in vitro and in vivo studies. The Akt/mTOR pathway is known to play a central role in various cellular processes that contribute to the malignant phenotype. Accordingly, inhibition of this signaling cascade has been a focus of recent therapeutic studies. Novel inhibitors of PI3-K, Akt, and mTOR are now passing through early phase clinical trials. Herein, we review the effect of fisetin on the PI3- K/Akt/mTOR pathway as studied in different cancer cell models.

Pomegranate extracts and cancer prevention: molecular and cellular activities.

There is increased appreciation by the scientific community that dietary phytochemicals can be potential weapons in the fight against cancer. Emerging data has provided new insights into the molecular and cellular framework needed to establish novel mechanism-based strategies for cancer prevention by selective bioactive food components. The unique chemical composition of the pomegranate fruit, rich in antioxidant tannins and flavonoids has drawn the attention of many investigators. Polyphenol rich fractions derived from the pomegranate fruit have been studied for their potential chemopreventive and/or cancer therapeutic effects in several animal models. Although data from in vitro and in vivo studies look convincing, well designed clinical trials in humans are needed to ascertain whether pomegranate can become part of our armamentarium against cancer. This review summarizes the available literature on the effects of pomegranate against various cancers.

Progress towards genetic and pharmacological therapies for keratin genodermatoses: current perspective and future promise.

Hereditary keratin disorders of the skin and its appendages comprise a large group of clinically heterogeneous disfiguring blistering and ichthyotic diseases, primarily characterized by the loss of tissue integrity, blistering and hyperkeratosis in severely affected tissues. Pathogenic mutations in keratins cause these afflictions. Typically, these mutations in concert with characteristic features have formed the basis for improved disease diagnosis, prognosis and most recently therapy development. Examples include epidermolysis bullosa simplex, keratinopathic ichthyosis, pachyonychia congenita and several other tissue-specific hereditary keratinopathies. Understanding the molecular and genetic events underlying skin dysfunction has initiated alternative treatment approaches that may provide novel therapeutic opportunities for affected patients. Animal and in vitro disease modelling studies have shed more light on molecular pathogenesis, further defining the role of keratins in disease processes and promoting the translational development of new gene and pharmacological therapeutic strategies. Given that the molecular basis for these monogenic disorders is well established, gene therapy and drug discovery targeting pharmacological compounds with the ability to reinforce the compromised cytoskeleton may lead to promising new therapeutic strategies for treating hereditary keratinopathies. In this review, we will summarize and discuss recent advances in the preclinical and clinical modelling and development of gene, natural product, pharmacological and protein-based therapies for these disorders, highlighting the feasibility of new approaches for translational clinical therapy.