Moammir H. Aziz

Resveratrol-caused apoptosis of human prostate carcinoma LNCaP cells is mediated via modulation of phosphatidylinositol 3′-kinase/Akt pathway and Bcl-2 family proteins

Prostate cancer is a major health problem in the U.S. and the available treatment and surgical options have proven to be inadequate in controlling the mortality and morbidity associated with this disease. It is therefore necessary to intensify our efforts to better understand this disease and develop novel approaches for its prevention and treatment. This study was conducted to evaluate the chemopreventive/antiproliferative potential of resveratrol (trans-3,4′,5,-trihydroxystilbene) against prostate cancer and its mechanism of action. Treatment with resveratrol (0–50 μmol/L for 24 hours) resulted in a significant (a) decrease in cell viability, (b) decrease of clonogenic cell survival, (c) inhibition of androgen (R1881)-stimulated growth, and (d) induction of apoptosis in androgen-responsive human prostate carcinoma (LNCaP) cells. Interestingly, at similar concentrations, resveratrol treatment did not affect the viability or rate of apoptosis in normal human prostate epithelial cells. Furthermore, our data showed that resveratrol-treatment resulted in significant dose-dependent inhibition in the constitutive expression of phosphatidylinositol 3′-kinase and phosphorylated (active) Akt in LNCaP cells. Resveratrol treatment for LNCaP cells was also found to result in a significant (a) loss of mitochondrial membrane potential, (b) inhibition in the protein level of antiapoptotic Bcl-2, and (c) increase in proapoptotic members of the Bcl-2 family, i.e., Bax, Bak, Bid, and Bad. Taken together, our data suggested that resveratrol causes an inhibition of phosphatidylinositol 3′-kinase/Akt activation that, in turn, results in modulations in Bcl-2 family proteins in such a way that the apoptosis of LNCaP cells is promoted. Based on these studies, we suggest that resveratrol could be developed as an agent for the management of prostate cancer. [Mol Cancer Ther 2006;5(5):1335–41]

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.

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.

Plumbagin, a Medicinal Plant–Derived Naphthoquinone, Is a Novel Inhibitor of the Growth and Invasion of Hormone-Refractory Prostate Cancer

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men. Hormone-refractory invasive PCa is the end stage and accounts for the majority of PCa patient deaths. We present here that plumbagin (PL), a quinoid constituent isolated from the root of the medicinal plant Plumbago zeylanica L., may be a potential novel agent in the control of hormone-refractory PCa. Specific observations are the findings that PL inhibited PCa cell invasion and selectively induced apoptosis in PCa cells but not in immortalized nontumorigenic prostate epithelial RWPE-1 cells. In addition, i.p. administration of PL (2 mg/kg body weight), beginning 3 days after ectopic implantation of hormone-refractory DU145 PCa cells, delayed tumor growth by 3 weeks and reduced both tumor weight and volume by 90%. Discontinuation of PL treatment in PL-treated mice for as long as 4 weeks did not result in progression of tumor growth. PL, at concentrations as low as 5 micromol/L, inhibited in both cultured PCa cells and DU145 xenografts (a) the expression of protein kinase Cepsilon (PKCepsilon), phosphatidylinositol 3-kinase, phosphorylated AKT, phosphorylated Janus-activated kinase-2, and phosphorylated signal transducer and activator of transcription 3 (Stat3); (b) the DNA-binding activity of transcription factors activator protein-1, nuclear factor-kappaB, and Stat3; and (c) Bcl-xL, cdc25A, and cyclooxygenase-2 expression. The results indicate for the first time, using both in vitro and in vivo preclinical models, that PL inhibits the growth and invasion of PCa. PL inhibits multiple molecular targets including PKCepsilon, a predictive biomarker of PCa aggressiveness. PL may be a novel agent for therapy of hormone-refractory PCa.

Prevention of Ultraviolet-B Radiation Damage by Resveratrol in Mouse Skin Is Mediated via Modulation in Survivin¶

Abstract Nonmelanoma skin cancer is the most frequently diagnosed malignancy in the United States, and multiple exposures to solar ultraviolet (UV) radiation (particularly its UV-B component, 290–320 nm) is its major cause. “Chemoprevention” by naturally occurring agents is being appreciated as a newer dimension in the management of neoplasia including skin cancer. We recently demonstrated that resveratrol (trans-3,5,4-trihydroxystilbene), an antioxidant found in grapes, red wines and a variety of nuts and berries, imparts protection from acute UV-B–mediated cutaneous damages in SKH-1 hairless mice. Understanding the mechanism of resveratrol-mediated protection of UV responses is important. We earlier demonstrated that resveratrol imparts chemopreventive effects against multiple UV-exposure–mediated modulations in (1) cki-cyclin-cdk network and (2) mitogen activated protein kinase (MAPK)-pathway. This study was conducted to assess the involvement of inhibitor of apoptosis protein family protein Survivin during resveratrol-mediated protection from multiple exposures of UV-B (180 mJ/cm2; on alternate days; for a total of seven exposures) radiations in the SKH-1 hairless mouse skin. Our data demonstrated that topical pretreatment of resveratrol (10 μmol in 200 μL acetone/mouse) resulted in significant inhibition of UV-B exposure–mediated increases in (1) cellular proliferations (Ki-67 immunostaining), (2) protein levels of epidermal cyclooxygenase-2 and ornithine decarboxylase, established markers of tumor promotion, (3) protein and messenger RNA levels of Survivin, and (4) phosphorylation of survivin in the skin of SKH-1 hairless mouse. Resveratrol pretreatment also resulted in (1) reversal of UV-B–mediated decrease of Smac/DIABLO and (2) enhancement of UV-B–mediated induction of apoptosis, in mouse skin. Taken together, our study suggested that resveratrol imparts chemopreventive effects against UV-B exposure–mediated damages in SKH-1 hairless mouse skin via inhibiting Survivin and the associated events.

Protein Kinase Cε Interacts with Signal Transducers and Activators of Transcription 3 (Stat3), Phosphorylates Stat3Ser727, and Regulates Its Constitutive Activation in Prostate Cancer

Prostate cancer is the most common type of cancer in men and ranks second only to lung cancer in cancer-related deaths. The management of locally advanced prostate cancer is difficult because the cancer often becomes hormone insensitive and unresponsive to current chemotherapeutic agents. Knowledge about the regulatory molecules involved in the transformation to androgen-independent prostate cancer is essential for the rational design of agents to prevent and treat prostate cancer. Protein kinase Cepsilon (PKCepsilon), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKCepsilon expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKCepsilon mediates progression to prostate cancer remains elusive. We present here for the first time that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, including prostate cancer, interacts with PKCepsilon. The interaction of PKCepsilon with Stat3 was observed in human prostate cancer, human prostate cancer cell lines (LNCaP, DU145, PC3, and CW22rv1), and prostate cancer that developed in transgenic adenocarcinoma of mouse prostate mice. In reciprocal immunoprecipitation/blotting experiments, prostatic Stat3 coimmunoprecipitated with PKCepsilon. Localization of PKCepsilon with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKCepsilon with Stat3 was PKCepsilon isoform specific. Inhibition of PKCepsilon protein expression in DU145 cells using specific PKCepsilon small interfering RNA (a) inhibited Stat3Ser727 phosphorylation, (b) decreased both Stat3 DNA-binding and transcriptional activity, and (c) decreased DU145 cell invasion. These results indicate that PKCepsilon activation is essential for constitutive activation of Stat3 and prostate cancer progression.

Protein kinase Cvarepsilon mediates Stat3Ser727 phosphorylation, Stat3-regulated gene expression, and cell invasion in various human cancer cell lines through integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2).

Protein kinase C epsilon (PKCɛ), a novel calcium-independent PKC isoform, has been shown to be a transforming oncogene. PKCɛ-mediated oncogenic activity is linked to its ability to promote cell survival. However, the mechanisms by which PKCɛ signals cell survival remain elusive. We found that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, is a protein partner of PKCɛ. Stat3 has two conserved amino-acid (Tyr705 and Ser727) residues, which are phosphorylated during Stat3 activation. PKCɛ interacts with Stat3α isoform, which has Ser727, and not with Stat3β isoform, which lacks Ser727. PKCɛ–Stat3 interaction and Stat3Ser727 phosphorylation was initially observed during induction of squamous cell carcinomas and in prostate cancer. Now we present that (1) PKCɛ physically interacts with Stat3α isoform in various human cancer cells: skin melanomas (MeWo and WM266-4), gliomas (T98G and MO59K), bladder (RT-4 and UM-UC-3), colon (Caco-2), lung (H1650), pancreatic (PANC-1), and breast (MCF-7 and MDA:MB-231); (2) inhibition of PKCɛ expression using specific siRNA inhibits Stat3Ser727 phosphorylation, Stat3-DNA binding, Stat3-regulated gene expression as well as cell invasion; and (3) PKCɛ mediates Stat3Ser727 phosphorylation through integration with the MAPK cascade (RAF-1, MEK1/2, and ERK1/2). The results indicate that PKCɛ-mediated Stat3Ser727 phosphorylation is essential for constitutive activation of Stat3 and cell invasion in various human cancers.

Protein kinase Cε and development of squamous cell carcinoma, the nonmelanoma human skin cancer

Protein kinase C (PKC) represents a large family of phosphatidylserine (PS)‐dependent serine/threonine protein kinases. At least five PKC isoforms (α, δ, ε, η, and ζ) are expressed in epidermal keratinocytes. PKC isoforms are differentially expressed in proliferative (basal layer) and nonproliferative compartments (spinous, granular, cornified layers), which exhibit divergence in their roles in the regulation of epidermal cell proliferation, differentiation, and apoptosis. Immunocytochemical localization of PKC isoforms indicate that PKCα is found in the membranes of suprabasal cells in the spinous and granular layers. PKCε is mostly localized in the proliferative basal layers. PKCη is localized exclusively in the granular layer. PKCδ is detected throughout the epidermis. PKC isozymes exhibit specificities in their signals to the development of skin cancer. PKCε, a calcium‐insensitive PKC isoform mediates the induction of squamous cell carcinoma (SCC) elicited either by the DMBA‐TPA protocol or by repeated exposures to ultraviolet radiation (UVR). PKCε overexpression, which sensitizes skin to UVR‐induced carcinogenesis, suppresses UVR‐induced sunburn (apoptotic) cell formation, and enhances both UVR‐induced levels of TNFα and hyperplasia. UVR‐induced sunburn cell formation is mediated by Fas/Fas‐L and TNFα NFR1 extrinsic apoptotic pathways. The death adaptor protein termed Fas‐associated death domain (FADD) is a common adaptor protein for both of these apoptotic pathways. PKCε inhibits UVR‐induced expression of FADD leading to the inhibition of both apoptotic pathways. It appears that PKCε sensitizes skin to the development of SCC by UVR by transducting signals, which inhibit apoptosis on one hand, and enhances proliferation of preneoplastic cells on the other hand.

Protein kinase Cε interacts with Stat3 and regulates its activation that is essential for the development of skin cancer

Protein kinase C (PKC) represents a large family of phosphatidylserine (PS)‐dependent serine/threonine protein kinases. At least six PKC isoforms (α, δ, ε, η, µ, and ζ) are expressed in epidermis. PKC is a major intracellular receptor for 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and is also activated by a variety of stress factors including ultraviolet radiation (UVR). PKC isozymes (α, δ, ε, and η), exhibit specificities to the development of skin cancer. PKCε, a calcium‐insensitive PKC isoform, is linked to the development of squamous cell carcinoma (SCC) elicited either by the 7,12‐Dimethylbenzanthracene (DMBA)‐TPA protocol or by repeated exposures to UVR. PKCε overexpressing transgenic mice, when treated either with TPA or exposed to UVR, elicit similar responses such as inhibition of apoptosis, promotion of cell survival, and development of SCC. PKCε overexpression increases Stat3 activation after either TPA treatment or UVR exposure. Both PKCε and signal transducers and activators of transcription‐3 (Stat3) are implicated in the development of SCC. However, the link between PKCε and Stat3 remains elusive. We found that PKCε interacts with Stat3. PKCε interaction with Stat3 was dependent upon UVR treatment. In reciprocal immunoprecipitation/blotting experiments, Stat3 coimmunoprecipitated with PKCε. Colocalization of PKCε with Stat3 was confirmed by double immunofluorescence staining. PKCε interaction with Stat3 was PKCε isoform specific and was not observed with other protein kinases. As observed in vitro with immunocomplex kinase assay with immunopurified PKCε and Stat3, PKCε phosphorylated Stat3 at the serine 727 residue. PKCε depletion prevented Stat3Ser727 phosphorylation, Stat3 DNA binding, and transcriptional activity. The results presented indicate that PKCε mediates Stat3 activation.

Ultraviolet-B Radiation Causes an Upregulation of Survivin in Human Keratinocytes and Mouse Skin¶

Abstract Understanding of the mechanism of ultraviolet (UV)–mediated cutaneous damages is far from complete. The cancer-specific expression of Survivin, a member of the inhibitor of apoptosis family of proteins, coupled with its importance in inhibiting cell death and in regulating cell division, makes it a target for cancer treatment. This study was designed to investigate the modulation of Survivin during UV response, both in vitro and in vivo. We used UV-B–mediated damages in normal human epidermal keratinocytes (NHEK) cells as an in vitro model and SKH-1 hairless mouse model for the in vivo studies. For in vitro studies, NHEK were treated with UV-B and samples were processed at 5, 15, 30 min, 1, 3, 6, 12 and 24 h after treatment. Our data demonstrated that UV-B exposure (50 mJ/cm2) to NHEK resulted in a significant upregulation in Survivin messenger RNA (mRNA) and protein levels. We also observed that UV-B exposure to NHEK resulted in significant (1) decrease in Smac/DIABLO and (2) increase in p53. For in vivo studies, the SKH-1 hairless mice were subjected to a single exposure of UV-B (180 mJ/cm2), and samples were processed at 3, 6, 12 and 24 h after UV-B exposure. UV-B treatment resulted in a significant increase in protein or mRNA levels (or both) of Survivin, phospho-Survivin and p53 and a concomitant decrease in Smac/DIABLO in mouse skin. This study demonstrated, for the first time, the involvement of Survivin (and the associated events) in UV-B response in vitro and in vivo in experimental models regarded to have relevance to human situations.