Alan Young

Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action

BackgroundMagnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development.MethodsUVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle.ResultsMagnolol pretreated groups (30, 60 μ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice.Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells.ConclusionsMagnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer.

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.

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.

Microtubule S-glutathionylation as a potential approach for antimitotic agents

BackgroundMicrotubules have been one of the most effective targets for the development of anticancer agents. Cancer cells treated by these agents are characterized by cell arrest at G2/M phase. Microtubule-targeting drugs are, therefore, referred to as antimitotic agents. However, the clinical application of the current antimitotic drugs is hampered by emerging drug resistance which is the major cause of cancer treatment failure. The clinical success of antimitotic drugs and emerging drug resistance has prompted a search for new antimitotic agents, especially those with novel mechanisms of action. The aim of this study was to determine whether microtubules can be S-glutathionylated in cancer cells and whether the glutathionylation will lead to microtubule dysfunction and cell growth inhibition. The study will determine whether microtubule S-glutathionylation can be a novel approach for antimitotic agents.Methods2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenyl carbamoylsulfanyl]propionic acid (2-AAPA) was used as a tool to induce microtubule S-glutathionylation. UACC-62 cells, a human melanoma cell line, were used as a cancer cell model. A pull-down assay with glutathione S-transferase (GST)-agarose beads followed by Western blot analysis was employed to confirm microtubule S-glutathionylation. Immunofluorescence microscopy using a mouse monoclonal anti-α-tubulin-FITC was used to study the effect of the S-glutathionylation on microtubule function; mainly polymerization and depolymerization. Flow cytometry was employed to examine the effect of the S-glutathionylation on cell cycle distribution and apoptosis. Cell morphological change was followed through the use of a Zeiss AXIO Observer A1 microscope. Cancer cell growth inhibition by 2-AAPA was investigated with ten human cancer cell lines.ResultsOur investigation demonstrated that cell morphology was changed and microtubules were S-glutathionylated in the presence of 2-AAPA in UACC-62 cells. Accordingly, microtubules were found depolymerized and cells were arrested at G2/M phase. The affected cells were found to undergo apoptosis. Cancer growth inhibition experiments demonstrated that the concentrations of 2-AAPA required to produce the effects on microtubules were compatible to the concentrations producing cancer cell growth inhibition.ConclusionsThe data from this investigation confirms that microtubule S-glutathionylation leads to microtubule dysfunction and cell growth inhibition and can be a novel approach for developing antimitotic agents.

Design, synthesis, and biological evaluation of N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine and its analogs as a novel class of anticancer agents

N-Acetyl-S-(p-chlorophenylcarbamoyl)cysteine (NACC) was identified as a metabolite of sulofenur. Sulofenur was demonstrated to have broad activity against solid tumors in preclinical studies but exhibited disappointing clinical responses due to its high protein binding related adverse effects. NACC exhibited low protein binding and excellent activity against a sulofenur sensitive human colon cancer cell line. In this study, analogs of NACC were synthesized and evaluated with four human cancer cell lines. Two of the NACC analogs showed excellent activity against two human melanoma cell lines, while NACC remains the most potent of the series. All three compounds were more potent than dacarbazine, which is used extensively in treating melanoma. NACC was shown to induce apoptosis without affecting the cell cycle. Further, NACC exhibited low toxicity against monkey kidney cells. The selective anticancer activity, low toxicity, an unknown yet but unique anticancer mechanism and ready obtainability through synthesis make NACC and its analogs promising anticancer agents.

Abstract 3809: Effects of honokiol on UACC-62 and SKMEL-2 melanoma cell lines

Honokiol is a plant lignan extracted from the magnolia bark tree, previous studies from our laboratory showed its chemopreventive effects in a UVB-induced skin cancer model, as well as pro-apoptotic effects in A431 squamous cancer cell line. Studies from other groups have reported antineoplastic effects in different cancer models as well. In this study, the antineoplastic effects of honokiol were evaluated in vivo and in vitro against UACC-62 cell line that harbors mutated BRAF, CDK4i, PTEN and against SKMEL-2 cell line that presents NRAF, and TP53 mutated genes. For the in vivo studies, nude male mice were injected subcutaneously with 5x106 UACC-62 or SKMEL-2 cells. The next day animals were randomized into control and treatment groups, each group containing 20 animals. The control group received 250 αl of sesame oil intraperitoneally (IP), the treatment groups received honokiol dissolved in sesame oil; 1.25 mg (IP) in the UACC-62 group and 50 mg/kg (IP) in the SKMEL-2 injected mice. Animals were treated three times a week, for 5-7 weeks. Tumors’ incidence, multiplicity and volume were recorded every three days, mice9s weights and external signs of toxicity were also closely monitored. Tumor tissues were collected for histopathology, Western blots, and PCR arrays. For the in vitro studies, both cell lines were evaluated for cell viability, proliferation, apoptosis by TUNEL assay, cell cycle arrest by propidium iodide staining, and Western blots for proteins expressions. Honokiol treatment significantly reduced (p 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 3809. doi:1538-7445.AM2012-3809

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

Abstract 5058: Effects of honokiol on UACC-62 malignant melanoma cell line

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Studies from our laboratory have indicated the chemopreventive effects of honokiol on UVB-induced squamous cell carcinoma in mouse skin. The purpose of this study is to investigate the effects of honokiol on UACC-62 malignant melanoma cell and elucidate the mechanisms of action. UACC-62 cells were grown in RPMI media in cell culture conditions. The effects of honokiol on cell viability and cell proliferation were studied by the MTT and BrdU assays. To investigate the effects of honokiol on cell cycle, UACC-62 cells were treated with honokiol, stained with propidium iodide and analyzed by flow cytometry. Cell lysates were prepared in similar conditions as the previous experiments to assess how honokiol affected the expressions of proteins involved in different pathways. The MTT assay showed a significant decrease in cell viability starting at 75 μM honokiol at 24 hours. The BrdU assay results indicated decreased cell proliferation as early as 12 hours and 50 μM honokiol. The cell cycle analysis indicated that honokiol caused G0/G1 phase arrest in melanoma cells (p < 0.05) at 24h and 50 μM. Western blots of the cell lysates prepared showed that honokiol decreased the expressions of cyclin E, CDK4, cyclin D2, NF-kβ, and p-MEK starting at 50 μM honokiol at 24 hours. The expressions of apoptotic proteins cleaved caspase-3, cleaved caspase-9, caspase 8, and cleaved PARP were increased by honokiol treatments. This data indicated that honokiol decreases cell proliferation and induces apoptosis of UACC-62 cells thus could be a candidate for pre-clinical studies as an agent for treating or preventing melanoma skin cancer. Supported by Translational Cancer Research Center funded as a 2010 Research Initiative Center by the State of South Dakota. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5058. doi:10.1158/1538-7445.AM2011-5058

Abstract 4422: 2-AAPA: A novel anticancer agent that induces microtubule depolymerization and apoptosis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC This study was aimed to investigate the anticancer activities and the mechanism of the anticancer activities of a novel anticancer agent - 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA). 2-AAPA was found to inhibit various human cancer cell lines with a narrow range of IC50 values (28-75 μM). These cell lines include lung (NCI-H226), prostate (PC-3), breast (MCF-7), skin (A431), melanoma (UACC-62 and SK-MEL-2), and renal (UO-31) cancers, as well as human ovarian cancers (OVCAR-3 and NCI/ADR-RES) which are resistant toward doxorubicin. Flow cytometric studies revealed that 2-AAPA induced cell cycle arrest at G2/M phase and produced significant cell apoptosis in UACC-62 cells. Through immunofluorescence studies, 2-AAPA was found to depolymerize microtubules and cause cell morphological change. An HPLC (high performance liquid chromatography) assay demonstrated that proteins were glutathionylated in both OVCAR-3 and UACC-62 cells when treated with 2-AAPA. A Western blot analysis also showed that a band corresponding to the molecular weight of tubulins was significantly glutathionylated in cell lysate obtained from UACC-62 cells treated with 2-AAPA. Considering the rich thiol content of tubulin proteins and the nature of protein glutathionylation, it is likely that microtubule function might be affected through microtubule glutathionylation resulting in cell cycle arrest at G2/M phase. Immunoprecipitation is being employed to confirm that tubulins are glutathionylated in cells treated with 2-AAPA. Together, our findings could lead to an antimitotic agent with a novel mechanism which is different than those of current antimitotic drugs. 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 4422.