Clinical Cancer Research Meeting Calendar Metabolism
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 14, 1840, March 15, 2008. doi: 10.1158/1078-0432.CCR-07-1811
© 2008 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gu, X.
Right arrow Articles by Califano, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gu, X.
Right arrow Articles by Califano, J. A.

Cancer Therapy: Preclinical

Vitamin E Succinate Induces Ceramide-Mediated Apoptosis in Head and Neck Squamous Cell Carcinoma In vitro and In vivo

Xinbin Gu1,4, Xiaodong Song5,6, Yongheng Dong7, Hui Cai1, Eric Walters2, Renshu Zhang3,4, Xiaowu Pang1, Tianpei Xie5, Yinhan Guo7, Rajagopalan Sridhar3,4 and Joseph A. Califano8

Authors' Affiliations: Departments of 1 Oral Diagnostic Service, 2 Biochemistry and Molecular Biology, and 3 Radiation Oncology, and 4 Cancer Center, Howard University, Washington, District of Columbia; 5 Shanghai TenGen Biomedical Co. Ltd., Shanghai, China; 6 Experiment Center, Binzhou Medical College, Shandong, China; 7 Oriental TenGen Tech Development Co. Ltd., Beijing, China; and 8 Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, Maryland

Requests for reprints: Xinbin Gu, Department of Oral Diagnostic Service, College of Dentistry, Howard University, 600 W Street, NW, Washington, DC 20059. Phone: 202-806-0345; Fax: 202-806-0446. E-mail: xgu{at}howard.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Vitamin E succinate ({alpha}-TOS) inhibits the growth of cancer cells without unacceptable side effects. Therefore, the mechanisms associated with the anticancer action of {alpha}-TOS, including ceramide-mediated apoptosis, were investigated using head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo.

Experimental Design: Five different human HNSCC cell lines (JHU-011, JHU-013, JHU-019, JHU-022, and JHU-029) were treated with {alpha}-TOS, and its effects on cell proliferation, cell cycle progression, ceramide-mediated apoptosis, and ceramide metabolism were evaluated. The anticancer effect of {alpha}-TOS was also examined on JHU-022 solid tumor xenograft growth in immunodeficient mice.

Results: {alpha}-TOS inhibited the growth of all the HNSCC cell lines in vitro in a dose- and time-dependent manner. Thus, JHU-013 and JHU-022 cell lines were more sensitive to {alpha}-TOS than the other cell lines. Cellular levels of ceramide, sphingomyelinase activity, caspase-3, and p53 were elevated with increasing time of exposure to {alpha}-TOS. The degradation of poly(ADP-ribose) polymerase protein in JHU-022 cells treated with {alpha}-TOS provided evidence for apoptosis. The amounts of nuclear factor {kappa}B, Bcl-2, and Bcl-XL proteins were reduced in the cells treated with {alpha}-TOS for 6 hours. The levels of caspase-9, murine double minute-2, and I{kappa}B-{alpha} proteins were unchanged after {alpha}-TOS treatment. I.p. administration of {alpha}-TOS slowed tumor growth in immunodeficient mice.

Conclusions: {alpha}-TOS showed promising anticancer effects to inhibit HNSCC growth and viability in vivo and in vitro. The induction of enzymes involved in ceramide metabolism by {alpha}-TOS suggests that ceramide-mediated apoptosis may expand therapeutic strategies in the treatment of carcinoma.

Each year, ~31,000 Americans are diagnosed with head and neck cancer and ~7,400 die from the disease (1). Overall survival rates of head and neck cancers have only marginally improved over the last three decades (2). In addition, the incidence of oral cancer in African American men has been approximately twice higher than in Caucasian men, and the 5-year relative survival is lower for African Americans (1). Therefore, novel therapeutic and preventive approaches are warranted.

Accumulating evidence suggested that an esterified derivative of RRR-{alpha}-tocopherol ({alpha}-TOH), RRR-{alpha}-tocopheryl succinate ({alpha}-TOS), is a vitamin E analogue, which inhibits tumor growth (3–7). It induces apoptosis, inhibits tumor cell proliferation and differentiation, arrests DNA synthesis, and blocks cell cycle progression in various cancer cell lines and animal models of breast, colon, head and neck, prostate, and lung cancers (8–18). In addition, {alpha}-TOS selectively kills tumor cells without toxic effects on normal cells and tissues (4, 7, 18). The parent compound of vitamin E, {alpha}-TOH, is a free radical–scavenging antioxidant, which protects polyunsaturated fats from peroxidation in human body but does not induce cancer cell apoptosis (19). In contrast to {alpha}-TOH, {alpha}-TOS is a redox-inactive molecule, which has a charged side group when it exists at physiologic pH. {alpha}-TOS can be converted to {alpha}-TOH by cellular esterase (20). All the reports thus far indicate that {alpha}-TOS causes an increase in cancer cell apoptosis (3–7), but the mechanism(s) of {alpha}-TOS–induced cancer cell apoptosis and inhibition of cancer cell growth are not fully understood.

In previous studies, we found that {alpha}-TOS induced apoptosis in a hamster cheek pouch carcinoma cell line (HCPC-1) and altered sphingolipid metabolism (11). Moreover, cell viability was significantly reduced in cultures treated with {alpha}-TOS. A critical finding in our study was that {alpha}-TOS interacts with cell membrane to shift phospholipids and sphingolipid in the lipid bilayer of the abnormal plasma membrane. This makes sphingolipids more accessible to hydrolases and then to generate ceramide. Ceramide is a central molecule in sphingolipid metabolism, having a significant role in the apoptotic response of various cancer cells (21–27). For example, androgen ablation in LNCaP prostate cancer cells results in selective accumulation of de novo generated C16-ceramide. This accumulation of ceramide leads to G0-G1 arrest followed by apoptosis (24). Mice deficient in acid sphingomyelinase lose the ability to accumulate ceramide and acquire resistance to radiation-induced apoptosis (25). Interruption of sphingomyelin synthesis or hydrolysis of sphingomyelin by sphingomyelinase can increase cellular levels of ceramide. Increased levels of ceramide can damage mitochondria and induce apoptosis (27, 28). Experiments designed to reveal the relationship between {alpha}-TOS and ceramide-mediated apoptosis in cancer cells, especially in head and neck cancer cells, are warranted.

We hypothesized that {alpha}-TOS may activate the accumulation of ceramide in the cancer cells of head and neck squamous cell carcinoma (HNSCC), and that ceramide buildup triggers apoptotic events. In this report, we tested this hypothesis in five different human HNSCC cell lines, including JHU-011, JHU-013, JHU-019, JHU-022, and JHU-029 cell lines, using in vitro and in vivo systems.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemical reagents. Chemicals were of the highest available grade. Ceramide (N-octadecanoyl-D-erythro-sphingosine), {alpha}-TOS, and phosphatidylcholine were obtained from Sigma Chemical Company. Escherichia coli sn-1,2-diacylglycerol kinase (specific activity >2 units/mg protein) and octyl-β-D-glucopyranoside were obtained from Calbiochem, and [{gamma}-32P]ATP (3,000 Ci/mmol) was obtained from Amersham.

{alpha}-TOS-liposome preparation. Two types of small unilamellar vesicles (SUV), vehicle-SUV and {alpha}-TOS encapsulated SUV, were made for in vitro experiments based on the previously reported method (11). The vehicle-SUV was a suspension of phosphatidylcholine liposomes in PBS, and {alpha}-TOS-SUV consisted of phosphatidylcholine liposomes encapsulating 1 mmol/L {alpha}-TOS. In brief, 0.2 g/mL phosphatidylcholine lipid stock solution in 1:1 mixture of chloroform/methanol was dried under nitrogen and then mixed with an isotonic solution of physiologic saline (0.9% NaCl) along with the anionic detergent sodium cholate. The resulting mixed micellar solution was then dialyzed in a Mini Lipoprep dialyzer (5,000 MW cutoff; Amika Corporation) for 4 h against an 8-liter reservoir of 0.9% NaCl solution. To encapsulate {alpha}-TOS into the SUV, {alpha}-TOS was dissolved along with phosphatidylcholine using the mixture of chloroform and methanol (1:1). This was then processed as described above for the preparation of SUV.

Cell lines and culture. HNSCC cell lines including JHU-011 (larynx, p53 mutated), JHU-013 (neck node metastasis, p53 mutated), JHU-019 (tongue), JHU-022 (larynx, wild type of p53), and JHU-029 (wild type of p53) were established by Johns Hopkins University. The HNSCC cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and antibiotic-antimycotic mixture (100 IU/mL penicillin and 100 µg/mL streptomycin; Cellgro). Cells were grown in 5% CO2 at 37°C and were subcultured at an initial density of 1 x 105/mL every 3 to 4 d. Cell density was determined with a hemocytometer and a phase-contrast microscope. Trypan blue dye exclusion assay (Sigma-Aldrich) detected 0.1% of dead cells in the untreated culture. All experiments were done with cells in logarithmic phase of growth.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. HNSCC cells were seeded in flat-bottomed 96-well cell culture plate (Costar) at a density of 5,000 per well and allowed to attach overnight. The cells were then treated with {alpha}-TOS-SUV at various concentrations (5-80 µmol/L) for 12 h. Twenty microliters of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (Sigma) were added to each well and then the plate was incubated in a humidified CO2 incubator at 37°C for 5 h. After removing the media, 200 µL of DMSO were added to each well and mixed for 30 min at room temperature to dissolve crystals. The plate was placed inside a 37°C incubator for 5 min. Finally, the plate was transferred to a microplate reader (Bio-Rad) and absorbance at 550 nm was measured.

Colony formation assay. HNSCC cells were seeded at a density of 200 per well in BD Falcon six-well tissue culture plate. The cells were allowed to attach overnight. The cultures were treated with {alpha}-TOS-SUV (20 µmol/L) for 3 to 24 h. Control cultures were treated with the same volume of medium with vehicle (SUV). The medium was removed from each well and the cells were washed with PBS. Fresh drug-free culture medium (5 mL) was added to the cultures, which were then placed back into the incubator to form colonies in 10 d. Any colony containing >50 cells was considered to represent a viable clonogenic cell. The colonies in the wells were counted after staining with 0.1% methylene blue in 50% ethanol. The experiment was done thrice for each treatment.

Flow cytometry assay. HNSCC cells (5 x 104) were seeded in six-well, flat-bottomed plates and were exposed to 20 µmol/L {alpha}-TOS-SUV for 6, 12, and 24 h. The cells were collected, washed, and suspended in cold PBS. The cells were fixed in chilled 75% methanol and then incubated in the dark for 15 min at room temperature in a solution containing Annexin V-FITC (Clontech Laboratories, Inc.) and propidium iodide (5 µg/mL). The expression of Annexin V and cell cycle status were analyzed by FACStar flow cytometer (Becton Dickinson & Co.). Ten thousand cells per sample were analyzed.

Human tumor xenograft in mice. Four-week-old, female, athymic nude mice (Nu/Nu) were obtained from Harlan Sprague-Dawley, Inc. They were given Harlan Teklad #2018 Global 18% Protein Rodent Diet containing 101 mg/kg {alpha}-tocopherol and water ad libitum in the animal facility for 3 wk before use. Mice were housed in temperature-controlled rooms (74 ± 2°F) with a 12-h alternating light-dark cycle. The mice were separated into three groups (three to five per group): {alpha}-TOS treatment group, DMSO vehicle control group, and untreated control group. The body weight and food/water intake were measured twice a week. The mice in the {alpha}-TOS group received a 3-wk pretreatment with {alpha}-TOS (1.0 mg dissolved in 0.1-mL DMSO/mouse by i.p. injection) every other day before injecting JHU-022 cells. The mice in the DMSO group received 0.1-mL DMSO by i.p. injection. JHU-022 cancer cells (2 x 106/100 µL/mouse) were injected s.c. into the lower back of the mice using a 25-gauge needle on day 21. I.p. injections of {alpha}-TOS were continued on alternate days till day 55. Thus, each mouse in the {alpha}-TOS group received a total of 25 mg of {alpha}-TOS via i.p. injection for the 55-d treatment period. Tumor volume was determined from either caliper measurements (Tvol = length x width x depth x 0.5236) or directly by weighing the tumor. Guidelines for the humane treatment of animals were followed as approved by the Howard University Animal Care and Use Committee.

Immunohistochemistry. Tumor tissues were fixed in 10% formalin, embedded in paraffin, and cut into 5-µm-thick sections. The immunostaining was done using the Universal DAKO LSAB2 system (DAKO Corporation). Briefly, the tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol/water rinses. Antigen retrieval was done by heating the slides in a steamer to 95°C to 99°C in Target Retrieval Solution (DAKO) for 20 min. At room temperature, the slides were then treated with 3% hydrogen peroxide for 10 min, followed by primary antibody, monoclonal ceramide antibody (Alexis USA), used at a 1:100 dilution, for 1 h. Visualization with streptavidin-horseradish peroxidase (Biogenex) and 3-amino-9-ethylcarbazole was followed by a hematoxylin counterstain (Biomeda).

{alpha}-TOS analysis. Blood was collected from the mouse before the termination of the experiment on day 55. {alpha}-TOS was measured by using reverse-phase high-performance liquid chromatography. Each plasma sample (100 µL) was extracted thrice in hexane/ethanol 3:1 with SDS. The lipid fraction was evaporated to dryness under a nitrogen stream. The samples were then resuspended in methanol and injected into Agilent 1100 high-performance liquid chromatography. The mobile phase consisted of 99.4% methanol and 0.6% glacial acetic acid. Samples were separated on an Elite hypersil ODS2 5 µm (250 x 4.6 mm) column (Elite). HPLC detector was set at 284 mm for all detection. Quantitation of the separated compounds was done based on {alpha}-TOS standard pattern and Agilent software for data analyses.

Diacylglycerol kinase assay. Ceramide levels were determined with the diacylglycerol kinase assay as described in the previous study (11). In brief, monolayer cultures of HNSCC cells (106) were treated with {alpha}-TOS-SUV (20 µmol/L) or SUV for 30 min to 9 h. Cellular lipids were extracted from the treated cells using a mixture of chloroform/methanol/1 N HCl at a ratio of 100:100:1 (v/v), and then hydrolyzed with 0.1 N methanolic KOH for 1 h at 37 °C to remove glycerophospholipids. Ceramide containing samples were resuspended in 100 µL of reaction mixture containing 150 µg cardiolipin, 280 µmol/L diethylenetriaminepentaacetic acid, 51 µmol/L octyl-β-D-glucopyranoside (Calbiochem), 1 mmol/L ATP, 10 µCi [{gamma}-32P]ATP (DuPont New England Nuclear), and 35 µg/mL E. coli diacylglycerol kinase, pH 6.5 (Calbiochem). After 60 min at room temperature, the reaction was stopped by extraction of lipids with 1 mL of solvent mixture of chloroform/methanol/1 N HCl (100:100:1, v/v). Ceramide 1-phosphate was separated on TLC plate using a solvent system of chloroform/methanol/acetic acid (65:15:5, v/v) and detected by autoradiography. The incorporated 32P was quantified by liquid scintillation counting. Ceramide was determined by comparison with standard samples containing known amounts of ceramide.

In vivo sphingomyelinase assay. Sphingomyelinase assay was modified based on Cifone's method (29). In brief, the monolayer culture of HNSCC cells with 5% serum was labeled for 48 h with [N-methyl-14C]-choline (NEN Life Science Products, Inc.). The cells were washed thrice with Ca- and Mg-free PBS and then treated with {alpha}-TOS-SUV (20 µmol/L) or SUV for 30 min to 6 h. The cells were harvested from cultures by trypsinization and kept at 4°C to avoid protein degradation. Cellular lipids were extracted with a mixture of ice-cold methanol/chloroform/water at a ratio of 250:125:100 (v/v), and the samples corresponding to equal amounts of proteins were loaded onto individual lanes on the TLC plate. The lipids were separated by developing the thin layer chromatogram using a mixed solvent system composed of chloroform/methanol/acetic acid/water at a ratio of 100:60:20:5 (v/v). The radioactive spots were visualized by autoradiography and scraped along with silica gel from the plate and transferred to a scintillation vial containing 1 mL of scintillation cocktail of water and aquasol for determination of radioactivity.

Apopain assay. Caspase-3 activity measurement was done with FluorAce Apopain Kit (Bio-Rad Laboratories) according to the manufacturer's instructions. In brief, the {alpha}-TOS-SUV–treated (20 µmol/L) or SUV-treated cells (1 x 106) were lysed with apopain cell lysis buffer and the supernatant was incubated with fluorogenic peptide substrate Ac-DEVD-AFC, 55 µmol/L, and reaction buffer at 37°C. At the same time, to distinguish caspase-3 activity from nonspecific protease activity, the sample reactions were carried out after adding relatively nonselective caspase inhibitor z-VAD-fmk (50 µmol/L) or caspase-3 inhibitor z-DEVD-fmk (50 µmol/L; Clontech). The fluorescence readings were recorded after 30 and 60 min of incubation using a VersaFluor fluorometer (Bio-Rad), with excitation and emission wavelengths of 390 and 520 nm, respectively. Results were expressed as units of Apopain per 0.1 milligram of protein, according to the formula provided with the FluorAce Apopain Assay Kit. Protein concentration was measured using a DC Protein Assay kit (Bio-Rad Laboratories).

Western immunoblotting. The treated and untreated HNSCC cells were harvested and washed twice in PBS and then suspended in lysis buffer [50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, phenylmethylsulfonyl fluoride at 100 µg/mL, aprotinin at 2 µg/mL, pepstatin at 1 µg/mL, and leupetin at 10 µg/mL]. This mixture was placed on ice for 30 min. After centrifugation at 15,000 x g for 15 min at 4°C, the supernatant was collected. Protein concentrations were quantitated by using the Bio-Rad protein assay (Bio-Rad Laboratories). Whole-cell lysates (30 µg) were separated by 8% SDS-PAGE gel, transferred onto a polyvinylidene difluoride membrane (Immobilon; Amersham Corp.), and then probed sequentially with antibodies against the following proteins: caspase-3, caspase-9, poly(ADP-ribose) polymerase (PARP), Bcl-2, Bcl-xL, p53, murine double minute-2, nuclear factor {kappa}B, I{kappa}B-{alpha}, and β-actin (Sigma). Blots were washed thrice for 10 min with PBS + 0.1% Tween 20 and incubated with horseradish peroxidase–conjugated antirabbit, antimouse, or antigoat antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Blots were developed by a peroxidase reaction using the enhanced chemiluminescence detection system (Bio-Rad).

Statistical analyses. Results are presented relative to untreated controls. Values represent mean ± SD of three or more replicate tests. Data were analyzed by Duncan test following the ANOVA procedure when multiple comparisons were made. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effect of {alpha}-TOS on the proliferation and viability of HNSCC cell lines in vitro. We first confirmed the inhibitory effects of {alpha}-TOS on cell proliferation and viability in five different human HNSCC cell lines (JHU-011, JHU-013, JHU-019, JHU-022, and JHU-029) in vitro by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. {alpha}-TOS inhibited cell proliferation in all five cell lines in a dose-dependent manner when the cells were exposed to graded doses (5-80 µmol/L) for 12 hours. This inhibition was most pronounced in JHU-013 and JHU-022 cell lines (Fig. 1A ). Because 20 µmol/L {alpha}-TOS-SUV was the half maximal inhibitory concentration (IC50) in JHU-013 and JHU-022 cell lines, this dose of {alpha}-TOS-SUV was used for further in vitro studies. The potency of {alpha}-TOS was determined by using colony formation assay to measure the reproductive integrity of JHU-022 and JHU-029 cells treated with 20 µmol/L {alpha}-TOS-SUV from 0 to 24 hours (Fig. 1B). As expected, clonogenic capacities of JHU-022 and JHU-029 cell lines decreased with increasing exposure time to 20 µmol/L {alpha}-TOS-SUV (Fig. 1B). As compared with untreated cancer cells, the relative survival of JHU-022 cells decreased to 31.9% and 8.7%, whereas survival of JHU-029 cells decreased to 43.3% and 20.4% after treatment with {alpha}-TOS-SUV for 12 and 24 hours, respectively (Fig. 1B). The effects of {alpha}-TOS-SUV treatment of the different cell lines were explored further by flow cytometry analysis for cell cycle distribution and estimation of apoptosis. As summarized in Table 1 , flow cytometry data indicated that a 24-hour exposure to {alpha}-TOS induced S-phase arrest and reduced the proportion of cells in G1 phase in all five HNSCC cell lines after treatment. Moreover, {alpha}-TOS induced a significant proportion of apoptosis in JHU-011 (57.87%), JHU-013 (58.02%), JHU-019 (54.87%), JHU-022 (49.39%), and JHU-029 (48.23%) cell lines after a 24-hour exposure (Table 1). However, there was <1% apoptotic cells in JHU-011 cells treated for 12 hours with {alpha}-TOS-SUV, vehicle (SUV), and untreated control cells (Fig. 1C). There were no significant differences in the cell cycle distribution patterns of untreated cultures and control cultures treated with SUV alone.


Figure 1
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Cytotoxicity of {alpha}-TOS toward HNSCC cell lines. A, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the viability of cells in five different HNSCC cell lines treated with graded doses (0-80 µmol/L) of {alpha}-TOS-SUV for 12 h. B, cell survival was determined on the basis of colony formation assays after incubation of JHU-022 and JHU-029 cells with {alpha}-TOS-SUV (20 µmol/L) for 0 to 24 h. C, flow cytometry profiles of JHU-011 cells treated with SUV (vehicle alone) or {alpha}-TOS-SUV (20 µmol/L) for 12 and 24 h. Columns, mean cell viability of two independent experiments with triplicate dishes; bars, SD.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Comparison of cell cycle in {alpha}-TOS–treated cell lines

 
Antitumor effect of {alpha}-TOS against JHU-022 human and head neck cancer cells in vivo. We further examined the antitumor effects of {alpha}-TOS on JHU-022 cell growth as solid tumor xenografts in immunodeficient mice. The experiment is outlined in Fig. 2A . Tumor growth occurred in all JHU-022 inoculated mice. All mice were euthanized on day 55 and tumors were dissected and weighed. The mice in the {alpha}-TOS–treated group showed suppression of tumor growth as compared with untreated and DMSO control groups (Fig. 2B and C). Average tumor weight was significantly lower in the {alpha}-TOS–treated group (0.24 g) than in the untreated (0.39 g) and DMSO-treated (0.36 g) groups (Fig. 2C). The body weight increased slightly in all mice during the 55-day experiment, with no significant differences among {alpha}-TOS–treated group and the two control groups (Fig. 2D). In addition, there were no differences in food and water intake among the groups. The blood samples were also collected on the last day of the experiment and the plasma {alpha}-TOS levels were determined by high-performance liquid chromatography. Plasma {alpha}-TOS was only detected in the {alpha}-TOS–treated group and was not detectable in the control and DMSO-treated groups. The plasma {alpha}-TOS level in the {alpha}-TOS–treated group was in an average range of 5 µg {alpha}-TOS/100 µL plasma (P < 0.001).


Figure 2
View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Inhibition of JHU-022 tumor xenograft growth in mice by {alpha}-TOS. A, schematic representation of the in vivo animal study. There were three groups of mice that included untreated control group, {alpha}-TOS–treated group, and DMSO vehicle control group. The experiment start date is indicated as day 1, when {alpha}-TOS and DMSO (vehicle) treatment was initiated. JHU-022 cells were injected s.c. on day 21, and the mice were sacrificed 34 d later on day 55. B, left, picture of mice with tumor. The circled region represents the location of tumor. Right, dissected tumors from the untreated group (top), DMSO group (middle), and {alpha}-TOS group (bottom). C, scatter plot of tumor weights from the different groups. The average tumor mass is indicated as a bold bar in each scatter group; P < 0.001. D, the mouse body weight was measured periodically at different days during the 55-d experimental period.

 
Effect of {alpha}-TOS on ceramide metabolism in JHU-022 cancer cells. The effects of {alpha}-TOS on the ceramide expression and sphingomyelinase activity in JHU-022 cells were studied. The amount of ceramide product gradually increased with time in JHU-022 cell cultures treated with 20 µmol/L {alpha}-TOS-SUV for up to 6 hours. The levels of ceramide were maintained nearly at this elevated level in cultures treated with {alpha}-TOS-SUV for 6 and 9 hours (Fig. 3A ). There was an ~2.5-fold increase in the ceramide level of cells treated with {alpha}-TOS-SUV for 6 hours compared with control cells treated with vehicle (SUV) alone (Fig. 3A). The level of sphingomyelinase activity was significantly increased in the first hour of treatment of cells with {alpha}-TOS-SUV. There was a 3.3-fold and a 3.7-fold increase of the sphingomyelinase activity in cells after treatment with {alpha}-TOS-SUV for 30 minutes and 1 hour, respectively. After that, the enzyme activity gradually diminished to ~2.5-fold higher level relative to DMSO-treated cells and remained at this level throughout the treatment period (Fig. 3B). The ceramide level in tumor specimens was examined by immunohistologic methods. Tumors from mice treated with {alpha}-TOS exhibited a significant degree of immune staining of ceramide within the cellular membranous regions, whereas the untreated group or DMSO-vehicle control group displayed little staining with ceramide antibody (Fig. 3C).


Figure 3
View larger version (57K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Increased levels of ceramide in JHU-022 cells treated with {alpha}-TOS. A, ceramide levels in JHU-022 cells treated with {alpha}-TOS-SUV (20 µmol/L) for 0 to 9 h. Top right inset, TLC bands of ceramide from cells treated with {alpha}-TOS-SUV (20 µmol/L) for 3, 6, and 9 h. B, acid-sphingomyelinase (SMase) activity relative to untreated control in JHU-022 cells treated with {alpha}-TOS-SUV (20 µmol/L) or vehicle (SUV) for various exposure times from 0 to 6 h. Points, mean ceramide levels and relative sphingomyelinase activity of two independent experiments with triplicate dishes; bars, SD. C, immunohistochemical staining of ceramide in JHU-022 tumor specimens from mice, which were untreated or treated with DMSO (vehicle) or {alpha}-TOS as described in Materials and Methods. At the end of the experiment, JHU-022 tumors were removed, fixed in formalin, and stained with H&E and ceramide monoclonal antibody. Arrows, ceramide specific staining. Magnification, x400.

 
Effects of {alpha}-TOS on caspase-3, caspase-9, and PARP proteins. Caspase-3 activities were nearly 8-fold higher in JHU-022 cells treated with {alpha}-TOS for 12 hours and JHU-029 treated similarly for 6 hours, compared with control cells treated with the vehicle (DMSO) alone (Fig. 4A ). Caspase-3 activities were lower in cultures treated with {alpha}-TOS for longer periods. Coadministration of the selective caspase-3 inhibitor z-DEVD-fmk or nonselective caspase inhibitor z-VAD-fmk significantly reduced {alpha}-TOS induced caspase-3 activities with a range of 70% to 84% inhibition in both JHU-022 (Fig. 4A) and JHU-029 (Fig. 4B) cell lines. In addition, both caspase inhibitors showed an ability to partially protect against {alpha}-TOS–induced cell death in JHU-22 cell cultures (Fig. 4C). The expression levels of caspase-3 protein and related molecules such as caspase-9 and PARP proteins were also analyzed by Western blot (Fig. 4A and C). A qualitative assessment of apoptotic markers in JHU-022 cell cultures treated with {alpha}-TOS for 3 to 24 hours showed an increase in caspase-3, but not caspase-9, protein (Fig. 4A). Treatment of JHU-022 cells with {alpha}-TOS for ≥3 hours resulted in apoptosis as indicated by PARP cleavage to produce 85-kDa fragments (4 D).


Figure 4
View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Effect of {alpha}-TOS on activity of caspase-3 and related markers in JHU-022 cells. A, caspase-3 activities (apopain levels) were measured in JHU-022 or JHU-029 cells treated with {alpha}-TOS-SUV (20 µmol/L) for 0, 3, 6, 12, and 24 h. The caspase-3 inhibitors such as z-DEVD-fmk and z-VAD-fmk were used as enzyme-specific controls. Columns, mean enzyme activity of two independent experiments with triplicate dishes; bars, SD. The protein levels of caspase-3 and caspase-9 in {alpha}-TOS-SUV–treated JHU-022 cells were also analyzed by Western blot (top right). The cell lysates were harvested after 6, 12, and 24 h for Western blot, which was probed sequentially for expression of caspase-3, caspase-9, and β-actin. Control cells were treated with vehicle alone. B, JHU-022 cells were pretreated with z-VAD-fmk and z-DEVD-fmk for 30 min, followed by treatment with {alpha}-TOS-SUV (20 µmol/L) for 6 or 12 h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. C, the JHU-022 cells were treated with {alpha}-TOS-SUV (20 µmol/L) for 30 min and 1, 2, 3, 4, and 5 h. PARP expression and degradation were analyzed by Western blotting with antibodies directed against PARP protein. The intact form of PARP is 116 kDa and the cleaved form is 85 kDa. The amount of protein was normalized to β-actin content.

 
Effects of {alpha}-TOS on the expression of ceramide-related apoptotic markers. The role of ceramide-mediated apoptosis was examined in {alpha}-TOS–treated JHU-022 and JHU-029 cells. First, Annexin V-FITC antibody was applied to track the cells through apoptosis when exposed to {alpha}-TOS (Fig. 5A ). The cells were labeled with Annexin V-FITC antibody and propidium iodide and analyzed by flow cytometry. The data indicated that 41.30%, 44.39%, and 64.82% of the JHU-022 cells underwent early apoptosis after exposure to {alpha}-TOS-SUV for 6, 12, and 24 hours, respectively. Thirty-eight percent of JHU-022 cells were no longer viable (late apoptosis) after 12-hour exposure to {alpha}-TOS. Higher proportion (24.24%) of late apoptosis was found in JHU-029 cell cultures treated with {alpha}-TOS for 12 hours. An increase in the proportion (16.23 to 34.18%) of early apoptosis was seen in JHU-029 cells when {alpha}-TOS treatment was increased from 6 to 24 hours (Fig. 5A).


Figure 5
View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Effect of {alpha}-TOS on the induction of apoptosis in JHU-022 cells. A, JHU-022 and JHU-029 cells were cultured either with {alpha}-TOS-SUV (20 µmol/L) or vehicle for 0, 3, 6, 12, and 24 h, followed by measurement of Annexin V protein in the cell membrane by flow cytometry using FITC-conjugated Annexin V antibody. The cells were also stained with propidium iodide. Vehicle-treated cells served as controls. The pixels in the bottom right quadrant of each panel display V-FITC-positive and propidium iodide (PI)–negative cells, indicating that the cells were in an early stage of apoptosis. Pixels in the top right quadrant represent cells stained with both propidium iodide and Annexin V, indicating that the cells were in late apoptosis or no longer viable. B, JHU-022 cells were treated with {alpha}-TOS-SUV (20 µmol/L) for 0, 3, 6, 12, and 24 h, and the expression levels of apoptotic markers such as Bcl-2, Bcl-XL, p53, murine double minute-2 (MDM-2), nuclear factor {kappa}B (NF-{kappa}B; p65), and I{kappa}B-{alpha} were evaluated by Western blot with appropriate antibody probes. The amount of protein was normalized to β-actin.

 
We also examined the expression levels of apoptosis associated proteins such as Bcl-2, Bcl-XL, p53, murine double minute-2, nuclear factor {kappa}B, and I{kappa}B-{alpha} by Western blot analysis (Fig. 5B and C). The expression levels of Bcl-2 and Bcl-XL were dramatically decreased in JHU-022 cells treated with {alpha}-TOS for 6 and 12 hours as compared with control groups (Fig. 5B). A slight recovery of expression of Bcl-2 and Bcl-XL proteins was seen in cultures treated for 24 hours with {alpha}-TOS (Fig. 5B). The level of p53 increased with prolonged exposure of JHU-022 cells to {alpha}-TOS. In contrast, the level of nuclear factor {kappa}B was lower in JHU-022 cells treated with {alpha}-TOS for 12 hours. There was a very slight change in murine double minute-2 protein level of cells treated with {alpha}-TOS. There was no change in the level of I{kappa}B-{alpha} in the {alpha}-TOS–treated and control groups (Fig. 5C).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The key findings from the present study are that {alpha}-TOS inhibits the growth and viability of human HNSCC, and that this effect may be a consequence of ceramide-mediated apoptosis. In vitro studies showed dose- and time-dependent cytotoxicity of {alpha}-TOS. In vivo studies showed that i.p. injection of low-dose {alpha}-TOS on alternating days significantly decreased primary tumor burden. Regarding the safety of {alpha}-TOS, our limited studies in mice suggest that it is relatively nontoxic for up to 55 experimental days. The {alpha}-TOS treatment did not produce any overt signs of toxicity such as weight loss or observable changes in behavior. Clearly, more studies are needed for establishing the safety of {alpha}-TOS for potential use in cancer chemoprevention or treatment.

Our data showed that {alpha}-TOS induced sphingomyelinase activity and enhanced ceramide levels in cultured cells and tumor specimens. The importance of ceramide lies in its ability to modulate the biochemical and cellular processes that lead to apoptosis (30, 31). The accumulation of ceramide in cells could be influenced through one or more routes of ceramide generation and/or clearance. Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to ceramide and phosphocholine. Mechanism for generation of ceramide may also involve the activation of acid sphingomyelinase, which participates in regulating apoptotic responses. Neutral sphingomyelinase is also implicated in the regulation of apoptosis in response to a range of stimuli, including tumor necrosis factor {alpha} in breast cancer cells, ethanol in HepG2 hepatoma cells, and the Alzheimer proapoptotic amyloid-β peptide in neuronal cells (24, 32, 33). Our analysis of sphingomyelinase activity included neutral and acidic contributions toward ceramide formation.

The results also show that ceramide-mediated apoptotic signaling seems to affect activation of caspase and Bcl-2 family proteins. One effect of {alpha}-TOS is the activation of caspase-3 activity and expression. Caspase-3 is a member of the caspase family of aspartate-specific cysteine proteases that play a central role in the execution of the apoptotic program, and is primarily responsible for the cleavage of PARP during cell death. The sequence at which caspase-3 cleaves PARP is very well conserved in the PARP protein, indicating the potential importance of PARP cleavage in apoptosis (34–36). In {alpha}-TOS–treated JHU-022 cells, caspase-3–like activity, measured with a specific apopain enzymatic assay in vitro, peaks at 12 hours after exposure to {alpha}-TOS, concomitant with the onset of apoptosis. The expression level of caspase-3 was significantly increased after exposure to {alpha}-TOS, whereas the amount of caspase-9 remained constant. In addition, pretreatment with caspase-3 inhibitor or nonselective caspase inhibitor blocks {alpha}-TOS–mediated cell death, suggesting that caspase-3 activity is involved in ceramide-mediated apoptosis in HNSCC cells. We have also examined the time course of PARP protein cleavage during apoptosis in these cells by Western blot analysis. PARP cleavage occurred at an early stage of induced apoptosis in JHU-022 cells. Further work will be required to discern whether {alpha}-TOS–induced ceramide-mediated apoptosis is truly caspase-9 independent or involves other regulation such as phosphorylation of caspase-9 protein. We should note that the immunoblotting experiments reflect a limited assessment of relative differences of apoptotic markers within and between samples, and more detailed quantitation of the apoptotic profiles under {alpha}-TOS treatment is warranted. Furthermore, the variability observed when using specific and nonspecific caspase inhibitors may be a consequence of using a single concentration of inhibitor, culture conditions, or the degree of sensitivity of the cell lines. Caspase-9 has been linked to ceramide-induced neuronal death (37). The regulation of alternative processing of pre-mRNA of both caspase-9 and Bcl-XL was reported in response to the proapoptotic action of ceramide (38). A collective interpretation of our data suggests that the rapid response of sphingomyelinase activity and ceramide accumulation in cells exposed to {alpha}-TOS (Fig. 3A and B) precede the maximal caspase expression profiles and earliest detection of PARP cleavage (Fig. 4A and C). This is consistent with the hypothesis that caspase-mediated apoptosis is a consequence of ceramide accumulation within HNSCC treated with {alpha}-TOS.

This study also provides evidence that inhibition of Bcl-XL/Bcl-2 function represents a major pathway whereby {alpha}-TOS induces ceramide-mediated apoptosis in head and neck cancer cells. The data indicate that {alpha}-TOS can markedly decrease the levels of Bcl-XL and Bcl-2 in the first 12 hours of treatment. The tendency of Bcl-2 and Bcl-XL to recover during later time points could reflect metabolic degradation of {alpha}-TOS on prolonged incubation with cells. Bcl-2 and Bcl-XL are antiapoptotic paralogues that inhibit apoptosis elicited by a wide variety of stimuli, and play critical roles in cancer development and resistance to treatment. Many clinical studies have indicated that expression of these antiapoptotic proteins in tumors is associated with poor prognosis (39). In addition, the nuclear factor-{kappa}B expression level seemed to decrease after the exposure of cells to {alpha}-TOS. Nuclear factor {kappa}B is an important molecular target that has a role in carcinogenesis and cancer progression (40).

In summary, our data show that the vitamin E analogue {alpha}-TOS decreases primary tumor burden in a human tumor xenograft model. The findings presented here strongly suggest that antitumor activity of {alpha}-TOS is initiated through a ceramide-mediated apoptotic pathway without any overt toxic effects in vivo.


   Acknowledgments
 
We thank Dan Zhang for her excellent technical assistance in the animal experiments, Dr. Liang Shan for pathologic analysis, Dr. Yanfei Zhou for statistical analysis, and Hongli Zhu for high-performance liquid chromatography analysis of {alpha}-TOS in plasma.


   Footnotes
 
Grant support: NIH grants CA118770 and CA91409 and the Tianjue Gu Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/24/07; revised 10/23/07; accepted 1/11/08.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2006. CA Cancer J Clin 2006;56:106–30.[Abstract/Free Full Text]
  2. Kelloff G, Lippman SM, Dannenberg AJ, et al. Progress in chemoprevention drug development: the promise of molecular biomarkers for prevention of intraepithelial neoplasia and cancer—a plan to move forward. Clin Cancer Res 2006;12:3661–97.[Abstract/Free Full Text]
  3. Hampton T. Vitamin E derivative packs anticancer punch. JAMA 2006;296:32.[Free Full Text]
  4. Weber T, Lu M, Andera L, et al. Vitamin E succinate is a potent novel antineoplastic agent with high selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) in vivo. Clin Cancer Res 2002;8:863–9.[Abstract/Free Full Text]
  5. Wang XF, Dong L, Zhao Y, Tomasetti M, Wu K, Neuzil J. Vitamin E analogues as anticancer agents: lessons from studies with {alpha}-tocopheryl succinate. Mol Nutr Food Res 2006;50:675–85.[CrossRef][Medline]
  6. Basu A, Imrhan V. Vitamin E and prostate cancer: is vitamin E succinate a superior chemopreventive agent? Nutr Rev 2005;63:247–51.[CrossRef][Medline]
  7. Prasad KN, Kumar B, Yan XD, Hanson AJ, Cole WC. {alpha}-tocopheryl succinate, the most effective form of vitamin E for adjuvant cancer treatment. J Am Coll Nutr 2003;22:108–17.[Abstract/Free Full Text]
  8. Kline K, Yu W, Sanders BG. Vitamin E and breast cancer. J Nutr 2004;134:3458–62S.
  9. Wang XF, Birringer M, Dong LF, et al. A peptide conjugate of vitamin E succinate targets breast cancer cells with high ErbB2 expression. Cancer Res 2007;67:3337–44.[Abstract/Free Full Text]
  10. Ramanathapuram LV, Hahn T, Dial SM, Akporiave ET. Chemo-immunotherapy of breast cancer using vesiculated {alpha}-tocopheryl succinate in combination with dendritic cell vaccination. Nutr Cancer 2005;53:177–93.[CrossRef][Medline]
  11. Gu X, Schwartz J, Pang X, Zhou Y, Sirois D, Sridhar R. Cytotoxicity of liposomal {alpha}-tocopheryl succinate towards hamster cheek pouch carcinoma (HCPC-1) cells in culture. Cancer Lett 2006;239:281–91.[CrossRef][Medline]
  12. Schwartz JL, Gu X. Kittles RA, Shklar G. Experimental oral carcinoma of the tongue and buccal mucosal: possible biologic markers linked to cancer at two anatomic sites. Eur J Oral Oncol 2000;36:225–35.[CrossRef]
  13. Shiau CW, Huang JW, Wang DS, et al. {alpha}-tocopheryl succinate induces apoptosis in prostate cancer cells in part through inhibition of Bcl-XL/Bcl-2 function. J Biol Chem 2006;281:11819–25.[Abstract/Free Full Text]
  14. Ni J, Chen M, Zhang Y, Li R, Huang J, Yeh S. Vitamin E succinate inhibits human prostate cancer cell growth via modulating cell cycle regulatory machinery. Biochem Biophys Res Commun 2003;300:357–63.[CrossRef][Medline]
  15. Stapelberg M, Gellert N, Swettenham E, et al. {alpha}-tocopheryl succinate inhibits malignant mesothelioma by disrupting the fibroblast growth factor autocrine loop: mechanism and the role of oxidative stress. J Biol Chem 2005;280:25369–76.[Abstract/Free Full Text]
  16. Lee E, Choi MK, Lee YJ, et al. {alpha}-tocopheryl succinate, in contrast to {alpha}-tocopherol and {alpha}-tocopheryl acetate, inhibits prostaglandin E2 production in human lung epithelial cells. Carcinogenesis 2006;27:2308–15.[Abstract/Free Full Text]
  17. Zhang Y, Ni J, Messing EM, Chang E, Yang CR, Yeh S. Vitamin E succinate inhibites the function of androgen receptor and the expression of prostate-specific antigen in prostate cancer cells. Proc Natl Acad Sci U S A 2002;99:7048–13.
  18. Neuzil J, Weber T, Gellert N, Weber C. Selective cancer cell killing by {alpha}-tocopheryl succinate. Br J Cancer 2001;84:87–9.[CrossRef][Medline]
  19. Barroso MP, Gomez-Diaz C, Lopez-Lluch G, Malagon MM, Crane FL, Navas P. Ascorbate and {alpha}-tocopherol prevent apoptosis induced by serum removal independent of Bcl-2. Arch Biochem Biophys 1997;343:243–8.[CrossRef][Medline]
  20. Neuzil J. {alpha}-tocopheryl succinate epitomizes a compound with a shift in biological activity due to pro-vitamin-to-vitamin conversion. Biochem Biophys Res Commun 2002;293:1309–13.[CrossRef][Medline]
  21. Gutierrez G, Mendoza C, Montano LF, Lopez-Marure R. Ceramide induces early and late apoptosis in human papilloma virus+ cervical cancer cells by inhibiting reactive oxygen species decay, diminishing the intracellular concentration of glutathione and increasing nuclear factor-{kappa}B translocation. Anticancer Drugs 2007;18:149–59.[CrossRef][Medline]
  22. Ogretmen B, Hannun YA. Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer 2004;4:604–16.[CrossRef][Medline]
  23. Pettus BJ, Chalfant CE, Hannun YA. Ceramide in apoptosis: an overview and current perspectives. Biochim Biophys Acta 2002;1585:114–25.[Medline]
  24. Eto M, Bennouna J, Hunter OC, Lotze MT, Amoscato AA. Importance of C16 ceramide accumulation during apoptosis in prostate cancer cells. Int J Urol 2006;13:148–56.[CrossRef][Medline]
  25. Santana P, Pena LA, Haimovitz-Friedman A, et al. Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 1996;86:189–99.[CrossRef][Medline]
  26. Luberto C, Hassler DF, Signorelli P, et al. Inhibition of tumor-necrosis factor-induced cell death in MCF7 by a novel inhibitor of neutral sphingomyelinase. J Biol Chem 2002;277:41128–39.[Abstract/Free Full Text]
  27. Weber T, Dalen H, Andera L, et al. Mitochondria play a central role in apoptosis induced by {alpha}-tocopheryl succinate, an agent with antineoplastic activity: comparison with receptor-mediated pro-apoptotic signaling. Biochemistry 2003;42:4277–91.[CrossRef][Medline]
  28. Jiang Q, Wong J, Fyrst H, Saba JD, Ames BN. {gamma}-Tocopherol or combinations of vitamin E forms induce cell death in human prostate cancer cells by interrupting sphingolipid synthesis. Proc Natl Acad Sci U S A 2004;101:17825–30.[Abstract/Free Full Text]
  29. Cifone MG, Roncaioli P, De Maria R, et al. Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential invovement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal. EMBO J 1995;14:5859–68.[Medline]
  30. Lin CF, Chen CL, Lin YS. Ceramide in apoptotic signaling and anticancer therapy. Curr Med Chem 2006;13:1609–16.[CrossRef][Medline]
  31. Hail N, Jr., Carter BZ, Konopleva M, Andreeff M. Apoptosis effector mechanisms: a requiem performed in different keys. Apoptosis 2006;11:889–904.[CrossRef][Medline]
  32. Lee JT, Xu J, Lee JM, et al. Amyloid-β-peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway. J Cell Biol 2004;164:123–31.[Abstract/Free Full Text]
  33. Liu JJ. Wang JY, Herteryig E, et al. Activation of neutral sphingomyelinase participates in ethanol-induced apoptosis in HepG2 cells. Alcohol 2000;35:569–73.
  34. Ratnam K, Low J. Current development of clinical inhibitors of poly(ADP-Ribose) polymerase in oncology. Clin Cancer Res 2007;13:1384–8.
  35. Deep G, Oberlies NH, Kroll DJ, Agarwal R. Isosilybin B and isosilybin A inhibit growth, induce G1 arrest and cause apoptosis in human prostate cancer LNCaP and 22Rv1 cells. Carcinogenesis 2007;28:1533–42.[Abstract/Free Full Text]
  36. Israel K, Yu W, Sanders BG, Kline K. Vitamin E succinate induces apoptosis in human prostate cancer cells: role for Fas in vitamin E succinate-triggered apoptosis. Nutr Cancer 2000;36:90–100.[CrossRef][Medline]
  37. Movsesyan VA, Yakovlev AG, Dabaghyan EA, Stoica BA, Faden AI. Ceramide induces neruronal apoptosis through the caspase-9/caspase-3 pathway. Biochem Biophys Res Commun 2002;299:201–7.[CrossRef][Medline]
  38. Chalfant CE, Rathmen K, Pinkerman RL, et al. De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1. J Biol Chem 2002;277:12587–95.[Abstract/Free Full Text]
  39. Thomadaki H, Scorilas A. BCL2 family of apoptosis-related genes: functions and clinical implications in cancer. Crit Rev Clin Lab Sci 2006;43:1–67.[CrossRef][Medline]
  40. Aggarwal BB. Nuclear factor-{kappa}B: the enemy within. Cancer Cell 2004;6:203–8.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 CarcinogenesisHome page
P.-H. Huang, D. Wang, H.-C. Chuang, S. Wei, S. K. Kulp, and C.-S. Chen
{alpha}-Tocopheryl succinate and derivatives mediate the transcriptional repression of androgen receptor in prostate cancer cells by targeting the PP2A-JNK-Sp1-signaling axis
Carcinogenesis, July 1, 2009; 30(7): 1125 - 1131.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Prevention ResearchHome page
K.-K. Wong
Oral-Specific Chemical Carcinogenesis in Mice: An Exciting Model for Cancer Prevention and Therapy
Cancer Prevention Research, January 1, 2009; 2(1): 10 - 13.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gu, X.
Right arrow Articles by Califano, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gu, X.
Right arrow Articles by Califano, J. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online