Gudiseva Chandrasekher

Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

Backgroundα-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action.MethodsMTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells.Resultsα-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G2/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G2/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells.ConclusionsThis study for the first time identifies effects of α-santalol in G2/M phase arrest and describes detailed mechanisms of G2/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models.

Honokiol, a chemopreventive agent against skin cancer, induces cell cycle arrest and apoptosis in human epidermoid A431 cells

Honokiol is a plant lignan isolated from bark and seed cones of Magnolia officinalis. Recent studies from our laboratory indicated that honokiol pretreatment decreased ultraviolet B-induced skin cancer development in SKH-1 mice. The aim of the present investigation was to study the effects of honokiol on human epidermoid squamous carcinoma A431 cells and to elucidate possible mechanisms involved in preventing skin cancer. A431 cells were pretreated with different concentrations of honokiol for a specific time period and investigated for effects on apoptosis and cell cycle analysis. Treatment with honokiol significantly decreased cell viability and cell proliferation in a concentration- and time-dependent manner. Honokiol pretreatment at 50 μmol/L concentration induced G0/G1 cell cycle arrest significantly (P < 0.05) and decreased the percentage of cells in the S and G2/M phase. Honokiol down-regulated the expression of cyclin D1, cyclin D2, Cdk2, Cdk4 and Cdk6 proteins and up-regulated the expression of Cdks inhibitor proteins p21 and p27. Pretreatment of A431 cells with honokiol leads to induction of apoptosis and DNA fragmentation. These findings indicate that honokiol provides its effects in squamous carcinoma cells by inducing cell cycle arrest at G0/G1 phase and apoptosis.

Regulation of Cdc42 expression and signaling is critical for promoting corneal epithelial wound healing.

PURPOSE Cdc42, a member of Rho GTPases (guanosine triphosphatases), participates in cytokine- and growth factor-controlled biological functions in mammalian tissues. Here, we examined Cdc42 role in corneal epithelial wound healing and the influence of hepatocyte, keratinocyte, and epidermal growth factor (HGF, KGF, and EGF)-mediated signaling on Cdc42. METHODS Epithelial wounds were created on the corneas of live rabbits by complete debridement and in rabbit corneal epithelial primary cultures through scratch injury. Cdc42 expression in cultures was suppressed using Cdc42 siRNA. Cdc42 activation was determined by pull-down assays with PAK-agarose beads. Cdc42 expression was analyzed by immunoblotting and immunofluorescence. Association of Cdc42 with cell-cycle proteins was identified by immunoprecipitation. RESULTS In rabbit corneas, significant increase in Cdc42 expression that occurred 2 to 4 days after the injury coincided with wound closure, and by 8 days the expression reached near basal levels. Silencing of Cdc42 expression in cultures caused inhibition of wound closure as a result of 60% to 75% decrease in epithelial migration and growth. HGF, KGF, and EGF increased Cdc2 expression, activation, and its phosphorylation on ser71. Inhibition of growth factor-mediated PI-3K signaling resulted in the downregulation of Cdc42 expression and its phosphorylation. Increased association of cell-cycle proteins p27(kip) and cyclin-dependent kinase 4 (CDK4) with Cdc42; and phosphorylated Cdc42 with plasma membrane leading edges was also observed in the presence of growth factors. CONCLUSIONS Cdc42 is an important regulator of corneal epithelial wound repair. To promote healing, Cdc42 may interact with receptor tyrosine kinase-activated signaling cascades that participate in cell migration and cell-cycle progression.

Abstract 1083: Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53 mutated human epidermoid carcinoma A431 cells, and p53 wild-type human melanoma UACC-62 cells

α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer development in murine models by either chemical- or UVB-induced skin carcinogenesis. α-Santalol has shown induction of apoptosis in both murine models and epidermoid carcinoma A431 cells. However, to the best of our knowledge, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate the effects of α-santalol on cell cycle progression and protein components involved in the regulation of cell cycle in both p53 mutated human epidermoid carcinoma A431 cells and p53 wide-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action. MTT assay was used to determine cell viability in A431 cells, UACC-62 cells and monkey kidney CV-1 cells; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells, and Western immunoblotting was used for determining the expression of various proteins involved in the cell cycle progression. Results showed that α-santalol at concentration of 25 to 75 μM resulted in a concentration-and time-dependent decrease in cell viability from 24 h treatment in both A431 and UACC-62 cells. Cell cycle analysis by flow cytometry showed that α-santalol at 50 μM and 75 μM induced G 2 /M phase cell cycle arrest starting from 6 hour treatment in both UACC-62 and A431 cells. Furthermore, Western immunoblotting data showed that α-santalol altered the expressions of cell cycle proteins such as cyclin A, cyclin B1, cell division cycle 2 (cdc2), cdc25c and cyclin-dependent kinase 2 (Cdk2) in A431 cells which are required for G 2 /M transition. α-Santalol treatment upregulated the expression of p21 CIP1/WAF1 and suppressed the expression of mutated p53 in A431 cells; whereas, α-santalol treatment increased the expressions of wide-type p53 in UACC-62 cells. Taken together, this study for the first time identifies the effects of α-santalol in G 2 /M phase arrest and describes the detailed mechanisms of G 2 /M phase arrest by this agent in both p53 mutated A431 cells and p53 wide-type UACC-62 cells, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models in addition to induction of apoptosis by α-santalol. (Supported by 2010 Research Initiative Grant from the State of South Dakota funded as Translational Cancer Research Center). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1083.

Abstract 4136: Effects of magnolol on cell cycle, STAT 3, NF-kappa B and AKT signaling

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Magnolol is plant lignan isolated from bark and seed cones of Magnolia glaucan has been investigated for its anticancer effects. Previous studies from our laboratory have shown magnolol to be chemopreventive against UVB-caused skin carcinogenesis by inducing apoptosis. The objective of present study is to investigate other mechanisms involved for its chemopreventive effects. For this investigation, A431 human epidermoid carcinoma cells were used. Effects of magnolol on cell cycle were determined by flow cytometry. Expressions of various proteins involved in cell cycle were determined by Western blotting using appropriate antibodies. A431 human epidermoid carcinoma cells were incubated with different concentrations at different time periods. We further investigated the effects of magnolol on signal transducer and activator of transcription (STAT 3), nuclear factor-kappa B (NF-kappa B/p65) and AKT signaling pathways that play an important role in cell proliferation and survival. Magnolol at 100 μM and 125 µM caused G0/G1 cell cycle arrest by decreasing cyclin D1, cyclin D2, and CDK 4 and by increasing CDK inhibitors p21 and p27 at 24 and 48 h. We further found that magnolol inhibited expressions of STAT 3, NF-kappa B and AKT by inhibiting their phosphorylation at 24 and 48 h. These results indicate that chemopreventive effects of magnolol are mediated through multiple mechanisms. This study is supported by Translational Cancer Research Center funded as one of the 2010 Research Initiative Center by the State of South Dakota. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4136.