Sreevidya Santha

α-Santalol, a derivative of sandalwood oil, induces apoptosis in human prostate cancer cells by causing caspase-3 activation

The anticancer effects of α-santalol, a major component of sandalwood oil, have been reported against the development of certain cancers such as skin cancer both in vitro and in vivo. The primary objectives of the current study were to investigate the cancer preventive properties of α-santalol on human prostate cancer cells PC-3 (androgen independent and P-53 null) and LNCaP (androgen dependent and P-53 wild-type), and determine the possible mechanisms of its action. The effect of α-santalol on cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was confirmed by analysis of cytoplasmic histone-associated DNA fragmentation using both an apoptotic ELISA kit and a DAPI fluorescence assay. Caspase-3 activity was determined using caspase-3 (active) ELISA kit. PARP cleavage was analyzed using immunoblotting. α-Santalol at 25-75 μM decreased cell viability in both cell lines in a concentration and time dependent manner. Treatment of prostate cancer cells with α-santalol resulted in induction of apoptosis as evidenced by DNA fragmentation and nuclear staining of apoptotic cells by DAPI. α-Santalol treatment also resulted in activation of caspase-3 activity and PARP cleavage. The α-santalol-induced apoptotic cell death and activation of caspase-3 was significantly attenuated in the presence of pharmacological inhibitors of caspase-8 and caspase-9. In conclusion, the present study reveals the apoptotic effects of α-santalol in inhibiting the growth of human prostate cancer cells.

Antineoplastic Effects of α-Santalol on Estrogen Receptor-Positive and Estrogen Receptor-Negative Breast Cancer Cells through Cell Cycle Arrest at G2/M Phase and Induction of Apoptosis

Anticancer efficacy and the mechanism of action of α-santalol, a terpenoid isolated from sandalwood oil, were investigated in human breast cancer cells by using p53 wild-type MCF-7 cells as a model for estrogen receptor(ER)-positive and p53 mutated MDA-MB-231 cells as a model for ER-negative breast cancer. α-Santalol inhibited cell viability and proliferation in a concentration and time-dependent manner in both cells regardless of their ER and/or p53 status. However, α-santalol produced relatively less toxic effect on normal breast epithelial cell line, MCF-10A. It induced G2/M cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. Cell cycle arrest induced by α-santalol was associated with changes in the protein levels of BRCA1, Chk1, G2/M regulatory cyclins, Cyclin dependent kinases (CDKs), Cell division cycle 25B (Cdc25B), Cdc25C and Ser-216 phosphorylation of Cdc25C. An up-regulated expression of CDK inhibitor p21 along with suppressed expression of mutated p53 was observed in MDA-MB-231 cells treated with α-santalol. On the contrary, α-santalol did not increase the expression of wild-type p53 and p21 in MCF-7 cells. In addition, α-santalol induced extrinsic and intrinsic pathways of apoptosis in both cells with activation of caspase-8 and caspase-9. It led to the activation of the executioner caspase-6 and caspase-7 in α-santalol-treated MCF-7 cells and caspase-3 and caspase-6 in MDA-MB-231 cells along with strong cleavage of poly(ADP-ribose) polymerase (PARP) in both cells. Taken together, this study for the first time identified strong anti-neoplastic effects of α-santalol against both ER-positive and ER-negative breast cancer cells.

α‐Santalol, A Skin Cancer Chemopreventive Agent with Potential to Target Various Pathways Involved in Photocarcinogenesis

This study is designed to investigate the chemopreventive effect and molecular mechanisms of α‐santalol on UVB‐induced skin tumor development in SKH‐1 hairless mouse, a widely used model for human photocarcinogenesis. A dose of UVB radiation (30 mJ cm−2 day−1) that is in the range of human sunlight exposure was used for the initiation and promotion of tumor. Topical treatment of mice with α‐santalol (10%, wt/vol in acetone) caused reduction in tumor incidence, multiplicity and volume. In our study, the anticarcinogenic action of α‐santalol against UVB‐induced photocarcinogenesis was found to be associated with inhibition of inflammation and epidermal cell proliferation, cell cycle arrest and induction of apoptosis. α‐Santalol pretreatment strongly inhibited UVB‐induced epidermal hyperplasia and thickness of the epidermis, expression of proliferation and inflammation markers proliferating cell nuclear antigen (PCNA), Ki‐67 and cyclooxygenase 2 (Cox‐2). Significant decrease in the expression of cyclins A, B1, D1 and D2 and cyclin‐dependent kinases (Cdk)s Cdk1 (Cdc2), Cdk2, Cdk4 and Cdk6 and an upregulated expression of cyclin‐dependent kinase (CDK) inhibitor Cip1/p21 were found in α‐santalol pretreated group. Furthermore, an elevated level of cleaved caspase 3 and cleaved poly (ADP‐ribose) polymerase (PARP) were observed in α‐santalol‐treated group. Our data suggested that α‐santalol is a safer and promising skin cancer chemopreventive agent with potential to target various pathways involved in photocarcinogenesis.

Antineoplastic effects of honokiol on melanoma

Honokiol, a plant lignan has been shown to have antineoplastic effects against nonmelanoma skin cancer developments in mice. In this study, antineoplastic effects of honokiol were investigated in malignant melanoma models. In vitro effects of honokiol treatment on SKMEL-2 and UACC-62 melanoma cells were evaluated by measuring the cell viability, proliferation, apoptosis, cell cycle analysis, and expressions of various proteins associated with cell cycle progression and apoptosis. For the in vivo study, male nude mice inoculated with SKMEL-2 or UACC-62 cells received injections of sesame oil or honokiol for two to seven weeks. In vitro honokiol treatment caused significant decrease in cell viability, proliferation, cell cycle arrest, increased apoptosis, and modulation of apoptotic and cell cycle regulatory proteins. Honokiol caused an accumulation of cells in the G2/M phase of the cell cycle in SKMEL-2 and G0/G1 phase in UACC-62 cells. An elevated level of caspases and PARP were observed in both cell lines treated with honokiol. A decrease in the expression of various cell cycle regulatory proteins was also observed in honokiol treated cells. Honokiol caused a significant reduction of tumor growth in SKMEL-2 and UACC-62 melanoma xenografts. These findings suggest that honokiol is a good candidate for further studies as a possible treatment for malignant melanoma.

Abstract 2152: Akt/survivin pathway inhibition synergizes the apoptotic cell death induced by A-santalol in prostate cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA α-santalol, a major component of sandalwood oil inhibits growth of cultured prostate cancer cells in vitro by causing apoptosis. The present study was undertaken to investigate the mechanistic details associated with the induction of apoptosis by α-santalol in cultured prostate cancer cells (LNCaP and PC-3). Expressions of major proteins studied in the present investigation were determined using standard Western blot protocol and analyzed by LICOR-Odyssey infra-red scanner. Activity of survivin was confirmed employing survivin ELISA kit. The cell viability was determined by trypan blue dye exclusion assay and caspase-3 activity was confirmed using caspase-3 (active) ELISA kit. Treatment of prostate cancer cells with α-santalol (20, 40 µM) resulted in the down regulation of survivin, XIAP and p-AKT (s-473) levels. Furthermore, α-santalol significantly reduced the activity of survivin as evidenced by survivin ELISA assay. Inhibition of PI3K/Akt by pharmacological inhibitor LY294002 synergized the apoptotic cell death induced by α-santalol as determined by cell viability, active caspase-3 activity and expression of cleaved PARP, caspase-3 levels. In conclusion, the present study reveals that α-santalol downregulates activity and expression of survivin and that inhibition of PI3K/Akt pathway synergize the apoptotic cell death by downregulating survivin. This study was supported by Mentoring Task Force, Type I grant by Wilkes University and by Translational Cancer Research Center funded by South Dakota Governors Office of Economic Development. Citation Format: Ajay Bommareddy, Lauren Lockus, Jonathan Seward, William Eggelston, Stacy Prelewicz, Andrea Antal, Sarah Fillman, Christian Castro, Adam L. VanWert, Sreevidya Santha, Chandradhar Dwivedi. Akt/survivin pathway inhibition synergizes the apoptotic cell death induced by A-santalol in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2152. doi:10.1158/1538-7445.AM2014-2152

Abstract 4860: Chemopreventive effects of α-santalol on ultraviolet B induced skin tumorigenesis by modulating cell cycle regulators.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Previous studies from our laboratory have shown chemopreventive effects of 5% α-santalol, a sequiterpene isolated from the sandalwood oil in ultraviolet-B induced skin tumor multiplicity in SKH-1 hairless mice. However, this dosage did not have significant effect on tumor incidence. The objective of this study was to investigate the effects of α-santalol (10%, w/v in acetone) on UVB-induced skin tumor incidence in SKH-1 hairless mouse model and to identify the molecular targets in α-santalol mediated protection. Tumor initiation and promotion were carried out by UVB radiation (30 mJ/cm2/day for 5 days a week up to 30 week) that is in the range of human exposure to sunlight that can cause skin cancer. Topical treatment of mice with α-santalol causes a significant reduction in tumor multiplicity, tumor volume and tumor incidence, and also delayed tumor development. Histopathological analysis of hematoxylin-eosin stained skin sections showed that α-santalol pretreatment strongly inhibited UVB-induced epidermal hyperplasia and total thickness of the epidermis. The proliferation potential of skin and tumor tissue sections from UVB-induced carcinogenesis protocol was evaluated by immunohistochemical expression of proliferation marker PCNA. Both intensity of brown staining as well as the number of positively stained nuclei were reduced in skin tumors and adjacent skin tissues from α-santalol treated group when compared to control group. Western blot analysis of skin lysates showed that α-santalol decreases the level of different cyclin-dependent kinases and associated cyclins, Cdc25 phosphatases (Cdc25B and Cdc25C) together with an up-regulation of CDK inhibitor Cip1/p21. However, the level of Kip1/p27 and p53 did not show a significant difference. Our data suggested that α-santalol (10%, topical) is a safer and promising skin cancer chemopreventive agent through targeting different cell cycle regulators. (This work is supported by a Translational Cancer Center Research Grant, funded by the State of South Dakota). Citation Format: Sreevidya Santha, Chandradhar Dwivedi. Chemopreventive effects of α-santalol on ultraviolet B induced skin tumorigenesis by modulating cell cycle regulators. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4860. doi:10.1158/1538-7445.AM2013-4860

Abstract 5555: Honokiol inhibits tumor growth in a xenograft melanoma mice model and activates the apoptotic signaling pathway in vivo.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Melanoma is the deadliest type of skin cancer. For adults 25-29 years old, melanoma is the most common cancer, while it is the second most common cancer for 15-29 years old. Limited therapies are available for advanced stages; therefore, there is a need for development of new treatments for melanoma. Honokiol is a plant lignan extracted from the magnolia bark tree. Studies from our laboratory showed its chemopreventive effects in a UVB-induced skin cancer model, as well as in vitro pro-apoptotic effects in A431 squamous cancer cells, UACC-62 and SKMEL-2 human melanoma cell lines. In this study, the antineoplastic effects of honokiol were evaluated in vivo against the malignant melanoma cell lines UACC-62 that harbors mutated BRAF, CDK4i, PTEN and SKMEL-2 cells that presents NRAF, and TP53 mutated genes. The present study investigated the mechanism of honokiol-induced antitumoral effects in human melanoma UACC-62 and SKMEL-2 cells in two xenograft melanoma models in mice. UACC-62 and SKMEL-2 melanoma cells were subcutaneously grafted in nude mice, treatment groups received honokiol 50 mg/kg thrice a week and tumor growth was followed. After five to seven weeks, animals were euthanized and tumor tissue collected for gene profiling and western blots. The genetic expression of apoptotic biomarkers was assessed by qRT-PCR by using PCR arrays for the apoptotic pathway (SABiosciences, CA). After subcutaneous grafting of UACC-62 or SKMEL-2 human melanoma cells in the nude mice, the intraperitoneal administration of honokiol at 50 mg/kg body weight for five to seven weeks caused a significant reduction of tumor growth. Honokiol activated the apoptotic pathway in UACC-62 cells xenograft tumors in mice, as attested by the significant up-regulation of BAK1, BAX, CARD8, CASP10, CASP2, CASP6, CASP7, FADD, TNFRSF10B, TNFRSF25, TP53 and TP53BP2 transcripts. In the western blots from the UACC-62 cells tumors, there was an increase in the expression of CASP3, CASP7, CASP8, CASP6, cleaved CASP6 and CASP10 proteins in the honokiol treated group. In the tumors originated from the SKMEL-2 melanoma cells, a significant up-regulation of BAD, BAK1, BAX, NOD1, CARD6, CASP7, FADD, TNFRSF21 and TRAF3 transcripts was observed. The activation of the apoptotic pathway in the SKMEL-2 cells tumors by honokiol was further confirmed by western blots in the tumor lysates, where it was observed an increase in the activation of caspases and cleavage of PARP in the honokiol treated animals. Honokiol treatment exerted significant antitumor effects, including the activation of the apoptotic signaling pathway in UACC-62 and SKMEL-2 human melanoma cells in xenograft tumors in mice. These findings suggest that honokiol is a good candidate for further studies as a treatment for malignant melanoma. This work is supported by a Translational Cancer Center Research Grant, funded as 2010 Research Initiative Center by the State of South Dakota. Citation Format: Ruth F. Guillermo, Sreevidya Santha, Jonathan Stevens, Emily Coughlin, Pious Patel, Radhey S. Kaushik, Chandradhar Dwivedi. Honokiol inhibits tumor growth in a xenograft melanoma mice model and activates the apoptotic signaling pathway in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5555. doi:10.1158/1538-7445.AM2013-5555

Abstract 611: Alpha-santalol, a chemopreventive agent induces apoptosis in human prostate cancer cells by causing caspase-3 activation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The anticancer effects of α-santalol, a major component of sandalwood oil, have been reported against the development of certain cancers such as skin cancer both in vitro and in vivo. The primary objectives of the current study were to investigate the cancer preventive properties of α-santalol on human prostate cancer cells PC-3 (androgen independent and P-53 null) and LNCaP (androgen positive and P-53 wild-type), and determine the possible mechanisms of its action. The effect of α-santalol on cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was confirmed by analysis of cytoplasmic histone-associated DNA fragmentation using both an apoptotic ELISA kit and a DAPI fluorescence assay. Caspase-3 activity was determined using caspase-3 (active) ELISA kit. PARP cleavage was analyzed using Immunoblotting. α-santalol at 25-75 µM decreased cell viability in both cell lines in a concentration and time dependent manner. Treatment of prostate cancer cells with α-santalol resulted in induction of apoptosis as evidenced by DNA fragmentation and nuclear staining of apoptotic cells by DAPI. α-santalol treatment also resulted in activation of caspase-3 activity and PARP cleavage. The α-santalol-induced apoptotic cell death and activation of caspase-3 was significantly attenuated in the presence of pharmacological inhibitors of caspas-8 and caspase-9. In conclusion, the present study reveals the apoptotic effect of α-santalol in inhibiting the growth of human prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 611. doi:1538-7445.AM2012-611

Abstract 574: α-santalol induces caspase-dependent apoptosis and G2/M phase cell cycle arrest in human breast cancer cells

Alpha-santalol, an active component of sandalwood oil, has been shown to have chemopreventive effects on skin cancer both in vitro and in vivo. However, effects of alpha-santalol on other types of cancer have not been studied. In this study, we examine the effects and mechanisms of action of alpha-santalol on human breast cancer cells using estrogen receptor (ER)- positive (MCF-7) and estrogen receptor- negative (MDA-MB-231) breast cancer cell lines. MTT and BrdU cell proliferation ELISA results showed inhibition of cell viability and proliferation in a time and dose-dependent manner in both cell lines. Alpha-santalol at 25-150 µM concentration decreased cell viability after 12h treatment. In addition, TUNEL assay and flow cytometry results revealed that alpha-santalol significantly induced apoptosis in MCF-7 and MDA-MB-231 cells. Further, immunoblotting and caspase activity assays showed involvement of caspase-3, caspase-8 and caspase-9 in apoptotic cell death. Induction of apoptosis by alpha-santalol was further confirmed by detecting the cleavage of Poly (ADP-ribose) Polymerase (PARP) through western blotting. Cell cycle distribution of alpha-santalol treated MFC-7 and MDA-MB-231 cells were analyzed by fluorescence- activated cell sorting (FACS) analysis of propidium iodide staining. We observed that alpha-santalol treatment arrests cell cycle at G2/M phase at 25µM −75µM concentration in both cell lines. Taken together, our studies reveal a potential mechanism for the chemopreventive effect of alpha-santalol on ER- positive and ER- negative breast cancer cells through induction of apoptosis and cell cycle arrest at G2/M phase. Alpha-santalol could be effective for the prevention and treatment of breast cancer. (Supported by Translational Cancer Research Center funded by South Dakota, Governors Office of Economic Development) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 574. doi:1538-7445.AM2012-574