Cancer Research Aziza Shad EMT and Cancer Progression and Treatment
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
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 Email this article to a friend
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 Berchtold, C. M.
Right arrow Articles by Gould, M. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Berchtold, C. M.
Right arrow Articles by Gould, M. N.
Related Collections
Right arrow Preclinical Intervention
Right arrow Preclinical Intervention: In Vitro: Drugs, Mechanisms
[Cancer Research 65, 8558-8566, September 15, 2005]
© 2005 American Association for Cancer Research

Epidemiology and Prevention

Perillyl Alcohol Inhibits a Calcium-Dependent Constitutive Nuclear Factor-{kappa}B Pathway

Craig M. Berchtold1,2,4, Kai-Shun Chen1,3, Shigeki Miyamoto2,4 and Michael N. Gould1,3

Departments of 1 Oncology and 2 Pharmacology; 3 McArdle Laboratory for Cancer Research; and 4 301 SMI, University of Wisconsin-Madison, Madison, Wisconsin

Requests for reprints: Michael N. Gould, McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Avenue, Room 506, Madison, WI 53706. Phone: 608-263-6615; E-mail: gould{at}oncology.wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cell death induced by the monoterpene anticancer agent perillyl alcohol correlates to the increased expression of certain proapoptotic genes known to influence cell survival. Whereas sequence-specific DNA-binding factors dictate the expression patterns of genes through transcriptional regulation, those transcriptional factors influencing constitutive cell survival with perillyl alcohol treatment are not well studied. Here, we investigated whether the monoterpenes can regulate the activity of nuclear factor-{kappa}B (NF-{kappa}B), a calcium-dependent transcription factor necessary for survival in the WEHI-231 B-lymphoma cells. Unique among the monoterpenes, perillyl alcohol short-term treatment induced a persistent decrease of calcium levels, whereas other various monoterpenes caused transient reductions in calcium levels. Perillyl alcohol treatment also rapidly elicited reductions of NF-{kappa}B DNA-binding activity and target gene induction, which was associated with an increase in apoptosis in these B-lymphoma cells. This apoptosis was directly due to NF-{kappa}B because its prior activation abolished the cell killing effects of perillyl alcohol treatment. Our findings suggest that perillyl alcohol can inhibit NF-{kappa}B function to modulate gene expression patterns and cell survival of certain B-lymphoma cells. The effects of perillyl alcohol were not limited to these B-lymphoma cells but were also observed in MDA-MB 468 cells, an estrogen receptor–negative breast cancer cell line. These results identify a calcium-dependent NF-{kappa}B pathway as a molecular target of perillyl alcohol activity in different cancer cell types.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The monoterpenes isolated from the essential oils of plants elicit multiple chemotherapeutic and chemopreventive effects in diverse models of cancer (1). Anticancer activities associated with regressing mammary tumors include the induction of cytostasis and apoptosis upon monoterpene application. This antitumor activity correlates with the differential expression of growth and apoptotic genes necessary for tumor proliferation (2). The sequence-specific DNA binding of transcription factors often dictates the expression of genes (3). However, the transcription factors that regulate the anticancer apoptotic response to perillyl alcohol treatment are not well understood.

The monoterpene menthol exhibits calcium-related molecular actions not associated with anticancer activity. Studies in situ have shown that menthol is a direct inhibitor of L-type calcium channels (LTCC) expressed on the plasma membrane of normal tissues (4). Interestingly, these studies mechanistically explain the mild to moderate gastroesophageal reflux or "heartburn" observed in clinical trials evaluating similarly structured monoterpenes for the treatment of different diseases, including cancer (5, 6). LTCC antagonists, such as the dihydropyridines, also cause the same disorder by inhibiting the calcium channel activity in smooth muscle, resulting in the relaxation of the esophageal stomach sphincter (7). LTCCs are specific receptors for the dihydropyridines and are also called dihydropyridine-sensitive calcium channels (8).

Lymphoma cells originate from cell types (9) that express dihydropyridine-sensitive calcium channels (10, 11). WEHI-231 B-lymphoma cells constitutively express calcium-dependent nuclear factor-{kappa}B (NF-{kappa}B) DNA-binding activity responsible for the transcription of antiapoptotic genes permissive for cell survival (12, 13). LTCCs regulate calcium-dependent cell survival pathways independent of NF-{kappa}B transcription in neuronal cells (14). However, an association between the regulation of dihydropyridine-sensitive calcium channels and constitutive NF-{kappa}B–mediated antiapoptotic pathways in these B-lymphoma cells are not well established. Therefore, we asked whether the NF-{kappa}B antiapoptotic pathway in the WEHI-231 cells responded to monoterpene treatment. Our findings suggest how a rapidly targeted calcium-dependent decrease of NF-{kappa}B in B-lymphoma cells due to monoterpene treatment can induce apoptosis. Consequently, we investigated whether perillyl alcohol treatment reduced constitutive NF-{kappa}B activity through a similar mechanism in certain estrogen receptor–negative/independent (ER–) breast cancer cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and materials. Propidium iodide (PI), 2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole trihydrochloride (Hoechst 33342), S-(–)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester [BayK8644(–)], and (R)-(+)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester [BayK8644(+)] were purchased from Sigma-Aldrich (St. Louis, MO). Aldrich Chemical Company (Milwaukee, WI) supplied the monoterpenes except for perillic acid, which was synthesized at the University of Wisconsin-Madison. All other chemicals were acquired from Calbiochem (San Diego, CA), including 1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tretraacetic acid pentaacetoxymethyl ester (INDO-1/AM) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM). The CD40 monoclonal antibody was purchased from BD Biosciences PharMingen (San Diego, CA).

Statistical analysis. Sigma Stat 3.0 software (SYSTAT Software, Inc., Richmond, CA) was used for the statistical analysis.

Cell culture. The American Type Culture Collection (Manassas, VA) supplied the cell lines used in our studies. The WEHI Bcl-XL cells have been described (15). All cell lines, including the WEHI-231, Ramos, MCF-7, T47D, MDA-MB 231, and MDA-MB 468 cells, were cultured in RPMI medium with 10% FCS (Sigma), 2 mmol/L L-glutamine, and penicillin/streptomycin. The WEHI-231 and WEHI Bcl-XL cells were also grown with 50 µmol/L ß-mercaptoethanol. Lymphocyte density medium was purchased from Mediatech (Herndon, VA). All cell culture materials unless specified were from Invitrogen (Carlsbad, CA). Solubilization of the solid monoterpenes perillic acid and menthol used ethanol as the vehicle. The final concentration of ethanol in the media for the solid monoterpenes was ≤0.07%. The dihydropyridines were suspended in 100% ethanol with a working stock concentration of 25 or 30 mmol/L. The concentration of ethanol in the media with 50 µmol/L of dihydropyridine was 0.2%.

Cell culture experiments. WEHI-231, WEHI Bcl-XL, and Ramos cells were seeded at 1 x 106 and 3 x 105 to 5 x 105 cells/mL for the 4- and 24-hour assays, respectively. The MDA-MB 468 cells were seeded at 2 x 105 cells/well in a six-well plate (BD Bioscience, Bedford, MA) and allowed to attach overnight before treatment.

Apoptosis assays. The PI/viability assay with the WEHI-231, WEHI Bcl-XL, and Ramos cells used the addition of 30 µg/mL of PI to the cells 5 minutes before analysis by FACScan flow cytometry (Becton Dickinson, San Jose, CA; ref. 16). Examination of the cells (17) was done with Cell Quest software (Becton Dickinson). The DNA laddering assay was carried out as previously described (18).

Calcium analysis. The calcium analysis used cells (5 x 105/mL) preloaded with INDO-1/AM (2 µmol/L) in Buffer A [25 mmol/L HEPES, 5.4 mmol/L KCl, 0.8 mmol/L MgCl2, 1.8 mmol/L CaCl2, 121 mmol/L NaCl, 5.5 mmol/L glucose, 6 mmol/L NaHCO3 (pH 7.3), 50 µmol/L ß-mercaptoethanol, and 1% FCS] at 25°C for 30 minutes. Cells were isolated, resuspended in Buffer A with 10% FCS, and placed at 25°C for 20 minutes to allow deesterification of INDO-1/AM. PI (1 µg/mL) was added to the sample to gate live cells and the calcium analysis was carried out with flow cytometry at 37°C. A 325 to 360 nm wavelength excites the ratiometric INDO-1 fluorophore into emitting light at 405 nm when bound to calcium and 520 nm when free of calcium. To estimate cytoplasmic calcium concentrations, a procedure according to Eastman (19) was incorporated. Digitonin (20 µmol/L) was applied to the cells to examine the compartmentalization of INDO-1 into organelles. The application of ionomycin (20 µmol/L) and manganese (1 mmol/L) to the cells enabled the estimation of background unmetabolized INDO-1/AM contributing to the 405/520 reading (20).

RNA analysis. Total RNA was isolated with RNAzol B reagent (Tel-Test, Friendswood, TX). The RNase Protection Assay kit was used according to the instructions of the manufacturer (PharMingen). The normalizing control transcripts consisted of both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L32. L32 mRNA is a rRNA regulated posttranscriptionally (21).

Reverse transcription-PCR amplification of mouse L-type calcium channel {alpha}1-subunit cDNA. First-strand cDNA synthesis from total RNA was done with oligo(dT)-primed reverse transcription using the Thermoscript reverse transcription-PCR (RT-PCR) kit (Invitrogen). The University of Wisconsin-Biotechnology Center synthesized oligonucleotide primers corresponding to conserved regions in the mouse LTCC {alpha}1-subunit cDNA (Genbank accession number L01776; ref. 22). PCR amplification of the 920 bp fragment was done with the Herculase enzyme (Stratagene, La Jolla, CA) and gel electrophoresis was carried out to the isolate the PCR product. The Taq DNA polymerase–modified PCR product was subcloned into a pCRII TA vector (Invitrogen) and transformed into bacteria. Sequencing of multiple bacterial clones validated the expression of the Cav1.3 ({alpha}1D) LTCC {alpha}1-subunit in the WEHI-231 cells.

Reverse transcription-PCR amplification of human Cav1.3 ({alpha}1D) L-type calcium channel {alpha}1-subunit cDNA. The sequence of the primers and conditions used to amplify the LTCC gene from human breast cancer lines have been published (23, 24). The sequencing of the resulting PCR products was as described above.

Electrophoretic mobility shift assay. The electrophoretic mobility shift assay (EMSA) analysis was done as described (25). Briefly, nuclear protein extracts from a single reaction were divided into duplicate samples and incubated with a 32P-radiolabeled oligonucleotide containing either the consensus NF-{kappa}B or Oct-1 (5'-TGTCGAATGCAAATCACTAGAA-3'; Promega, Madison, WI) binding sequence. The paired samples were then run on the same 4% nondenaturing polyacrylamide gel and analyzed by a PhosphorImager (Amersham Biosciences, Piscataway, NJ). Densitometry values of NF-{kappa}B (c-rel/p50) were normalized to paired Oct-1 and Oct-2 values from the same sample and then to the nontreated control values at the indicated time point. Supershift analysis was done with Oct-1 and Oct-2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). The breast cancer studies used total protein extracts (6 µg) for EMSA analysis (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Perillyl alcohol induces apoptosis of WEHI-231 B-lymphoma cells. The monoterpenes are known inducers of apoptosis in vivo (2); however, their induction of programmed cell death in cell culture is not well characterized. To determine the sensitivity of the WEHI-231 cells to monoterpene treatment for 24 hours, PI staining was used to examine cell death. This method readily detects apoptosis in nonfixed cells with flow cytometric analysis (16). Perillyl alcohol–treated Ramos cells (another B-lymphoma cell line) helped to validate the incidence of apoptosis in B-lymphoma cells. The sorted Ramos cells showed cell populations with normal (R1), apoptotic (R2), and secondary necrotic (R3) morphologies (17) after monoterpene treatment (Fig. 1A). The perillyl alcohol–treated WEHI-231 cells also showed the fragmented nuclear morphology characteristic of apoptosis (data not shown). Furthermore, the isolated R3 (secondary necrotic gate) population of treated WEHI-231 cells displayed oligonucleosomal laddering indicative of apoptosis (Fig. 1B; ref. 18). Further examination of the WEHI-231 cells treated with perillyl alcohol for 24 hours showed a marked reduction in viability with increasing concentrations of perillyl alcohol. Collectively, these results show that the B-lymphoma cell lines undergo apoptosis upon perillyl alcohol treatment. The WEHI Bcl-XL cells, which highly express an exogenous antiapoptotic Bcl-XL gene, did not show a decrease in cell viability with perillyl alcohol treatment (Fig. 1C).



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Cell viability and apoptosis analysis in cultured B-lymphoma cells with agent treatment for 24 hours. A, perillyl alcohol (0.7 mmol/L) treatment for 24 hours induces apoptosis in the Ramos cells. Live cells stained with PI (30 µg/mL) were sorted and isolated by flow cytometry. The isolated cells from the three gates shown (R1, R2, and R3) were stained with Hoechst 33342 and analyzed by UV light microscopy. B, perillyl alcohol (0.7 mmol/L) treatment induces oligonucleosomal laddering consistent with apoptosis in the WEHI-231 cells. WEHI-231 cells stained with PI (30 µg/mL) were isolated as in (A). The lanes (R1, R2, and R3) on the ethidium bromide–stained gel show the migration of the DNA isolated from these cells. C, perillyl alcohol (POH) treatment reduces the cell viability of the WEHI-231 cells but not WEHI Bcl-XL cells. Cells were treated for 24 hours with perillyl alcohol (0.1-0.7 mmol/L), stained with PI, and analyzed by flow cytometry. The data are expressed as the percentage of viable treated cells divided by the percentage of viable nontreated control cells and multiplied by 100. Points, average of three independent experiments. D, monoterpene induction of apoptosis in the WEHI-231 cells. Cells treated with four different monoterpenes [perillyl alcohol, limonene (LIM), methanol (MET), and perillic acid (PA)] for 24 hours were stained with PI and analyzed by flow cytometry. The data are expressed as the ratio of the percentage of apoptotic, treated cells divided by the percentage of apoptotic, nontreated control or ethanol-vehicle cells. Column, average from one experiment done in triplicate; bars, SD.

 
Studies in WEHI-231 cells showed varying cell death–inducing activities with monoterpene treatment for 24 hours (Fig. 1D). Perillyl alcohol application induced a >40-fold increase in cell death, whereas limonene and perillic acid applications elicited a minimum increase in cell death at ~2.5- and 3.0-fold, respectively. Menthol did not induce cell death in these studies. Therefore, perillyl alcohol is the most potent, whereas limonene and perillic acid are the least potent, inducers of cell death of the monoterpenes tested in our studies.

Perillyl alcohol inhibits constitutive nuclear factor-{kappa}B activity in WEHI-231 cells. As part of the regulation of transcriptional factor activity by the 10-carbon cyclic monoterpenes (Fig. 2A), we ask the question if treatment would rapidly reduce the calcium-mediated constitutive NF-{kappa}B DNA-binding activity in the WEHI-231 cells. The NF-{kappa}B c-rel/p50 heterodimer, generally regarded as a transcriptional activator, has a transactivation domain that is absent in the transcriptionally repressive p50 homodimer complex (27). The WEHI-231 cells were treated with perillyl alcohol in 4-hour time course studies because the cells did not exhibit apoptotic morphology at this early time point with PI viability analysis (data not shown). Nuclear proteins were isolated for EMSA using a consensus {kappa}B oligomer. Perillyl alcohol application to the WEHI-231 cells showed a marked down-regulation of NF-{kappa}B DNA-binding activity at 4 hours as detected by the reduction of the NF-{kappa}B band (c-rel/p50) intensity compared with controls (Fig. 2B). The multiple bands of the Oct-1 probe were composed of the constitutive Oct-1 and Oct-2 transcriptional factors (data not shown; ref. 28) and used as a calcium-independent control with agent treatment. Further EMSA studies included the analysis of the different monoterpenes with varying anticancer activities. Menthol and limonene moderately decreased NF-{kappa}B DNA-binding activity, whereas perillic acid treatment was not effective (Fig. 2C).



View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. NF-{kappa}B DNA-binding activity in the WEHI-231 cells with monoterpene treatment. A, chemical structure of the monoterpenes. B, NF-{kappa}B DNA-binding activity after a 4-hour treatment with perillyl alcohol was determined by EMSA as described in Materials and Methods. Perillyl alcohol treatment reduced NF-{kappa}B levels (0.3 mmol/L, 38 ± 16%; 0.5 mmol/L, 43 ± 15%; 0.7 mmol/L, 34 ± 21%, n = 3) compared with control values at 4 hours. C, WEHI-231 cells were treated with perillic acid (0.7 mmol/L), limonene (0.7 mmol/L), and menthol (0.7 mmol/L) for 4 hours and were analyzed as in (B). The gels shown represent one of two independent experiments. The NF-{kappa}B levels from both experiments (n = 2) are as follows: menthol: 40%, 41%; limonene: 12%, 39%; and perillic acid: –5%, 11%. D, WEHI Bcl-XL cells were treated with perillyl alcohol (0.3-0.7 mmol/L) for 24 hours and analyzed as in (B). The 100x designation represents the addition of the cold oligomer 100 times in excess of the radiolabeled oligomer during the incubation period. The gels shown represent one of three independent experiments. The data are normalized to the control (white columns); columns, average of the three experiments; bars, SD.

 
EMSA analysis was also done after 24 hours to examine if the NF-{kappa}B DNA-binding levels correlated with the apoptosis observed after 24 hours. These data suggested that over time, limonene, perillic acid, and menthol actually increased nuclear NF-{kappa}B levels compared with Oct-1 levels in the WEHI-231 cells (data not shown). The apoptosis-resistant Bcl-XL cells were next treated for 24 hours with perillyl alcohol to ask if the monoterpene prolonged the decrease of NF-{kappa}B activity without the interference from apoptotic cell death. The NF-{kappa}B DNA-binding activity in these cells is considered to be regulated similarly to the parental WEHI-231 cells (15). Perillyl alcohol (0.7 mmol/L) administration maintained a marked reduction of NF-{kappa}B levels at 4 and 24 hours in the WEHI-231 cells (Fig. 2B and D) and Bcl-XL cells at 24 hours. Thus, perillyl alcohol treatment of the WEHI-231 cells for 24 hours show a persistent decrease of the prosurvival NF-{kappa}B DNA-binding activity.

Expression of certain nuclear factor-{kappa}B target genes is repressed in perillyl alcohol–treated WEHI-231 cells. To determine if NF-{kappa}B DNA-binding activity leads to a reduction in target gene expression, RNase protection analysis examined whether a decrease in the mRNA levels of candidate genes known to be positively regulated by NF-{kappa}B correlated with the reduction of the DNA-binding activity of the transcriptional factor following monoterpene treatment (Fig. 2). Known NF-{kappa}B transcriptionally regulated genes, such as notch, jagged, and bcl-2, were analyzed because they are associated with neoplasia (29, 30). However, these mRNAs were not highly expressed and probably not mediated by the high constitutive NF-{kappa}B nuclear levels in the WEHI-231 cells (Fig. 3A and B). Perillic acid was included as the no-effect control because this monoterpene did not alter NF-{kappa}B DNA-binding levels compared with controls (Fig. 2B). Moreover, perillic acid treatment did not consistently reduce mRNA levels either (Fig. 3A and B). Bax was analyzed because steady-state mRNA levels of this cell death transcript increased with perillyl alcohol treatment of regressing mammary tumors (2). However, Bax mRNA levels seemed to be relatively unaltered (Fig. 3A and B), whereas I{kappa}B{alpha} mRNA expression decreased below control levels with perillyl alcohol treatment. In accord with this outcome, NF-{kappa}B is a known positive regulator of the I{kappa}B{alpha} gene in the WEHI-231 cells (31). Furthermore, perillyl alcohol (0.7 mmol/L) application induced a reduction of the Bcl-2 gene family mRNA transcript, Bfl-1/A-1, to ~50% of controls levels. Thus, a rapid decrease of NF-{kappa}B nuclear levels at 4 hours was associated with a drop in the known NF-{kappa}B–regulated antiapoptotic Bfl-l/A1 (30) and I{kappa}B{alpha} genes in WEHI-231 cells with perillyl alcohol treatment.



View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. RNase protection analysis of candidate NF-{kappa}B–regulated genes with perillyl alcohol treatment. A, WEHI-231 cells were treated with the monoterpenes for 6 hours and total RNA was isolated and analyzed by RNase Protection Assay for mRNA levels of I{kappa}B{alpha} and the antiapoptotic gene Bfl-1/A1. B, mRNA levels. Densitometry values of the Bfl-1/A1, I{kappa}B{alpha}, and Bax complexes are normalized to the sum of the values of both control genes (L32 and GAPDH) within each sample and then to control values at 6 hours. Columns, average of three independent experiments; bars, SD.

 
Forced activation of nuclear factor-{kappa}B can reverse perillyl alcohol–induced apoptosis of WEHI-231 cells. The activated CD40 receptor induces a tumor necrosis–associated factor–dependent NF-{kappa}B–mediated antiapoptotic pathway in the WEHI-231 cells (32, 33). Therefore, CD40 stimulation was used to validate that the persistent reduction of the constitutive calcium-dependent NF-{kappa}B antiapoptotic pathway is a plausible mechanism of cell death with perillyl alcohol treatment (Fig. 1). Cells were initially exposed to transient CD40 antibody pretreatment followed by perillyl alcohol application for 18 hours. A density gradient was used to isolate viable cells for gel shift analysis. EMSA analysis showed that perillyl alcohol treatment did not inhibit the NF-{kappa}B DNA-binding activity from the CD40 antibody pretreated cells compared with controls (P = 0.760; Fig. 4A). However, consistent with our previous results (Fig. 2A and D), perillyl alcohol application induced a statistically significant (P < 0.05) reduction in the NF-{kappa}B DNA-binding activity compared with controls (Fig. 4B). Cellular viability analysis showed a statistically significant (P < 0.05) protective effect with CD40 stimulation followed by perillyl alcohol application compared with the perillyl alcohol–only treated WEHI-231 cells at 24 hours (Fig. 4C). We infer that the prolonged reduction of constitutive NF-{kappa}B DNA-binding activity (Fig. 2A and D) is responsible for a significant percentage (~80%) of the cell death in the WEHI-231 cells (Fig. 1C and D) with perillyl alcohol treatment. These results show the specificity of the targeted reduction of a calcium-dependent NF-{kappa}B cell survival pathway without interfering with a calcium-independent CD40 induced NF-{kappa}B cell survival pathway with perillyl alcohol treatment.



View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. CD40 stimulation of NF-{kappa}B DNA-binding activity blocked the anticancer effects of perillyl alcohol treatment. A, WEHI-231 cells transiently treated with CD40 (10 µg/mL) for ~4 hours were exposed to perillyl alcohol for an additional 18 hours. Nonapoptotic cells in treated and controls (CON) were isolated using a lymphocyte density gradient and subjected to EMSA analysis. B, NF-{kappa}B DNA-binding activity. Columns, average of three independent experiments; bars, SD. Densitometry values of NF-{kappa}B (c-rel/p50) were analyzed by Sigma Stat 3.0 software. The differences of the NF-{kappa}B (c-rel/p50) densitometry values are statistically significant among the different treatment groups (one-way ANOVA, P = 0.001). Multiple comparison analysis used Tukey's test. Control values are significantly different from perillyl alcohol (P < 0.05) values but not CD40 and CD40 + perillyl alcohol treatments. Perillyl alcohol values (*) are significantly different from control (P < 0.05), CD40 (P < 0.005), and CD40 + perillyl alcohol (P < 0.005) treatments. C, cells treated as in (A) were exposed to perillyl alcohol for 24 hours. The results from PI viability staining are represented as the percentage of control viability. Columns, average of three independent experiments each done in triplicate; bars, SD. The results were normalized to the controls from each experiment and logarithmically transformed before the statistical analysis. The unpaired t test determined a statistically significant difference (**P < 0.05) between CD40 + perillyl alcohol and perillyl alcohol only–treated cells.

 
Perillyl alcohol treatment reduces the estimated steady-state calcium levels compared with controls. To answer the question of why the monoterpenes decreased the calcium-dependent NF-{kappa}B pathway in the WEHI-231 cells, steady-state calcium concentrations were estimated at relatively early time points after monoterpene treatment. Perillic acid application did not reduce calcium levels within 60 minutes relative to controls (Fig. 5A). Treatment with the monoterpenes (perillyl alcohol, limonene, menthol) seemed to decrease the calcium concentrations at 30 minutes compared with control levels, whereas the transient limonene– and menthol-mediated decreases approached controls at 60 minutes (Fig. 5B). However, perillyl alcohol administration elicited a consistent and statistically significant (P < 0.05) reduction of the estimated calcium concentrations at both time points relative to the controls (Fig. 5C).



View larger version (8K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Short-term perillyl alcohol treatment persistently decreases the estimated steady-state calcium levels relative to controls. A, analysis of calcium levels in WEHI-231 cells after perillic acid (0.7 mmol/L) treatment was done as in Materials and Methods. The data are expressed as the estimated calcium concentration of treated cells (nmol/L) divided by the estimated calcium concentration (nmol/L) of nontreated control cells multiplied by 100. Columns, average of one experiment done in triplicate; bars, SD. B, monoterpenes perillyl alcohol, limonene, and menthol (0.7 mmol/L) treatment effects on steady-state calcium levels were analyzed and expressed as in (A). Columns, average of one experiment done in duplicate. C, effect of perillyl alcohol (0.7 mmol/L) treatment on steady-state calcium levels compared with controls were analyzed and expressed as in (A). Columns, average of three independent experiments done in triplicate at 30 minutes (control: 181 ± 22 nmol/L; perillyl alcohol: 110 ± 33 nmol/L, n = 3) and four independent experiments done in triplicate at 60 minutes (control: 248 ± 95 nmol/L; perillyl alcohol: 121 ± 30 nmol/L, n = 4); bars, SD. An unpaired t test was used to determine statistical significance (*, **, P < 0.05) with perillyl alcohol treatment compared with controls at both time points.

 
L-type calcium channel antagonist decreases the calcium-dependent nuclear factor-{kappa}B pathway. Further studies characterized the LTCC channel expression in the WEHI-231 cells as a potential mechanism of how the monoterpenes reduced intracellular calcium levels in the WEHI-231 cells. The characterization of the LTCC {alpha}1-subunit or calcium pore is indicative of a dihydropyridine-sensitive calcium channel (34). To confirm the expression of a dihydropyridine-sensitive calcium channel in the WEHI-231 cells, RNA was isolated to screen for multiple LTCC gene families by semiquantitative PCR. The PCR analysis showed the expected product, which when cloned and sequenced showed 100% homology to the Cav1.3 or {alpha}1D gene family of LTCCs (Fig. 6A, lane 2; ref. 22). An increase of calcium in cells treated with the LTCC agonist BayK8644(–) functionally shows a constitutively opened LTCC (34). BayK8644(–) treatment of INDO-1/AM-loaded WEHI-231 cells caused an increase in calcium levels from ~70 to ~120 nmol/L over 4 minutes using flow cytometric analysis (Fig. 6B). However, application of the BayK8644(+) LTCC antagonist did not seem to alter calcium levels over time. Therefore, these results suggest that the WEHI-231 cells express a constitutively active dihydropyridine-sensitive calcium channel that can directly influence steady-state calcium levels.



View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. The dihydropyridine antagonist nitrendipine reduces NF-{kappa}B levels in the dihydropyridine-sensitive calcium channel expressing WEHI-231 cells. A, expression of the {alpha}1-subunit mRNA in WEHI-231 cells. Agarose gel electrophoresis depicts the 920 bp RT-PCR product (arrow; lane 2), with the molecular weight marker (lane 1). B, INDO-l/AM (2 µmol/L) loaded WEHI-231 cells were analyzed for intracellular calcium concentration ([Ca2+]) by flow cytometry and treated with BayK8644(–) (10 µmol/L). BayK8644(+), 10 µmol/L, was used as the negative control. Each 40 seconds analyzed is the average of ~6,000 counts. C, cells were treated with nitrendipine (20 and 30 µmol/L) for 4 hours and analyzed as in Fig. 2. The values from the analysis for each experiment (n = 2) are 24% and 44%. D, WEHI-231 cells were treated with nifedipine (30 µmol/L) for 4 hours and analyzed as in Fig. 2. Neither of two experiments showed any change in the level of NF-{kappa}B. E, WEHI Bcl-XL cells were treated with nitrendipine (50 µmol/L), nifedipine (50 µmol/L), ethanol-vehicle, and control for 24 hours and were analyzed for NF-{kappa}B DNA-binding activity as in Fig. 2. The results shown represent one of three independent experiments. F, WEHI-231 and WEHI Bcl-XL cells were treated for 24 hours with dihydropyridine and analyzed as in Fig. 1D. The results show one experiment done in triplicate.

 
The dihydropyridine antagonists nitrendipine and nifedipine were used to test whether targeting the dihydropyridine-sensitive calcium channel would initiate the reduction of the calcium-mediated constitutive NF-{kappa}B DNA-binding activity in the WEHI-231 cells. Nitrendipine decreased NF-{kappa}B DNA-binding activity at 4 hours (Fig. 6C), whereas nifedipine did not (Fig. 6D). The prolonged treatment of nitrendipine in the Bcl-XL cells persistently decreased NF-{kappa}B DNA-binding levels, whereas Oct-1 remained relatively unchanged (Fig. 6E). Nifedipine treatment was not effective in this assay. Furthermore, nitrendipine treatment also induced cell death in the WEHI-231 cells over 24 hours, whereas nifedipine was not effective (Fig. 6F). Neither dihydropyridine compound induced apoptosis in the Bcl-XL cells with 24 hours of treatment. Thus, the reduction of the constitutive antiapoptotic NF-{kappa}B levels for 24 hours is associated with apoptosis at 24 hours with the dihydropyridine antagonist compound nitrendipine. These results suggest that perillyl alcohol maybe similar to nitrendipine in its mechanism of action.

Perillyl alcohol and different regulators of calcium decrease constitutive nuclear factor-{kappa}B levels in a human breast cancer cell line. We have previously characterized perillyl alcohol–induced cytostasis (0.3-1.0 mmol/L) and the induction of apoptosis (1.0 mmol/L) in human breast cancer cell lines with perillyl alcohol treatment for 3 days (35). However, the effects of perillyl alcohol on NF-{kappa}B DNA-binding activity are unknown in these breast cancer cell lines. Generally, ER– breast cancer cell lines (MDA-MB 468, MDA-MB 231) exhibit higher levels of the NF-{kappa}B DNA-binding complex consisting of the p65/p50 dimer than the estrogen receptor–positive/dependent (ER+) breast cancer cell lines (MCF-7, T47D; ref. 36). Because our current study suggested that perillyl alcohol treatment reduced calcium-dependent NF-{kappa}B levels in cells that express the Cav1.3 {alpha}1-subunit (Fig. 6A), we first assayed for the expression of this human LTCC subunit. RT-PCR analysis of total mRNA and sequencing of the resulting gel-purified product showed that the MCF-7, T47D, and MDA-MB 468 cells express the Cav1.3 gene (Fig. 7A, lanes 2, 3, and 5, arrow; refs. 23, 24). The MDA-MB 231 cells (Fig. 7A, lane 4) also generated an uncharacterized faint band migrating at a similar molecular weight as the other Cav1.3 gene bands.



View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 7. NF-{kappa}B DNA-binding activity in the ER– human breast cancer cells with perillyl alcohol treatment. A, expression of the Cav1.3 {alpha}1-subunit gene in various human breast cancer cell lines. Agarose gel electrophoresis depicts the ~900 bp RT-PCR product (arrow; lanes 2, 3, 4, and 5), with the molecular weight marker (lane 1). The bands from the MCF-7, T47D, and MDA-MB 468 lanes were isolated, sequenced, and determined to be 99% homologous to the human Cav1.3 {alpha}1-subunit gene. The MDA-MB 231 lane (lane 4) depicts a reproducible RT-PCR product that migrated with the validated Cav1.3 {alpha}1-subunit gene. B, MDA-MB 468 cells were grown to near confluence and treated with BAPTA-AM (10 and 30 µmol/L) for 2 hours. The majority of the cells (~90%) were adherent at this time point. The adherent cells were isolated and total proteins were processed for EMSA analysis. The gel shift assay for NF-{kappa}B and Oct-1 DNA binding was carried out as in Fig. 2, with all experiments (n = 3) run on the same gel with the same 32P-radiolabeled probe. The resulting densitometry values of NF-{kappa}B and Oct-1 were analyzed as in Fig. 4B. The results shown represent one of three independent experiments for each cell line. NS, a nonspecific band as shown by supershift analysis (data not shown). The densitometry values from the treated cells were normalized to the untreated controls values for each probe and expressed as the average ± SD from three independent experiments. NF-{kappa}B levels with BAPTA-AM treatment are as follows: 10 µmol/L: 6.0 ± 2.2% and 30.0 µmol/L: 6.2 ± 1.3%. Oct-1 levels with BAPTA-AM treatment are: 10 µmol/L: 95 ± 40% and 30.0 µmol/L: 76 ± 23%. C, perillyl alcohol treatment of the ER– MDA-MB 468 cells and EMSA analysis of constitutive NF-{kappa}B levels at 24 and 48 hours. Only the adherent cells were harvested and processed for EMSA gel shift analysis as in (B). The results shown represent one of three independent experiments. The densitometry values are normalized and expressed as in (B). The unpaired t test was used to assess the statistical significance between the control and perillyl alcohol densitometry values. NF-{kappa}B levels with perillyl alcohol treatment are as follows: 24 hours: 23 ± 12% and 48 hours: 16 ± 13%. Oct-1 levels with perillyl alcohol treatment are as follows: 24 hours: 96 ± 37% and 48 hours: 93 ± 14%. D, ER– MDA-MB 468 cells were treated with nitrendipine (50 µmol/L), nifedipine (50 µmol/L), ethanol-vehicle, and control for 24 hours and tested for NF-{kappa}B DNA-binding activity. Only the adherent cells were harvested and processed for EMSA gel shift analysis as in (B). The results shown represent one of three independent experiments. The resulting densitometry values of NF-{kappa}B and Oct-1 were analyzed as in Fig. 4B. The densitometry values are normalized and expressed as in (B). NF-{kappa}B levels with treatment are as follows: vehicle: 97 ± 41%; nifedipine: 103 ± 31%; and nitrendipine: 35 ± 13%. Oct-1 levels with perillyl alcohol treatment are as follows: vehicle: 105 ± 27%; nifedipine: 113 ± 45%; and nitrendipine: 260 ± 50%.

 
To characterize the potential calcium-dependent regulation of constitutive NF-{kappa}B levels in human breast cancer cells expressing the Cav1.3 gene family, the calcium chelator BAPTA-AM was applied in short-term studies (2 hours). We did not detect constitutive NF-{kappa}B levels in the ER+ cell lines and thus focused on the regulation of the high NF-{kappa}B levels in the MDA-MB 468 cells (data not shown). BAPTA-AM treatment at both concentrations (10 and 30 µmol/L) significantly reduced NF-{kappa}B levels in the MDA-MB 468 cells (P < 0.001; Fig. 7B). BAPTA-AM treatment did not affect Oct-1 DNA binding. Thus, the MDA-MB 468 cells seem to express calcium-dependent NF-{kappa}B activity.

Because perillyl alcohol and nitrendipine treatment persistently reduced long-term NF-{kappa}B levels in the WEHI-231 cells (Figs. 2 and 6), we applied these same agents to the MDA-MB 468 cells. Perillyl alcohol treatment elicited a significant decrease in the NF-{kappa}B DNA-binding levels in the MDA-MB 468 cells at 24 (P < 0.05) and 48 hours (P < 0.005; Fig. 7C). Oct-1 levels were not significantly affected. Application of nitrendipine also significantly decreased NF-{kappa}B levels at 24 hours (Fig. 7D) whereas nifedipine did not (P < 0.01). Nitrendipine treatment significantly increased Oct-1 DNA binding in the MDA-MB 468 cells, suggesting that Oct-1 maybe regulated differently in these cells with nitrendipine (P < 0.001; Fig. 7D). Moreover, vehicle and nifedipine treatments were not significantly different from control values with either NF-{kappa}B or Oct-1 in these studies. Therefore, perillyl alcohol and nitrendipine treatment elicited a significant decrease in a plausible calcium-dependent NF-{kappa}B activity in an ER– breast cancer cell line.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Diverse groups of chemicals with varying molecular structures bind directly to LTCCs (37). However, we observed widely varied outcomes in the persistent decrease of constitutive calcium-dependent NF-{kappa}B DNA-binding activity using multiple compounds with known LTCC antagonistic activity in the WEHI-231 cells. In agreement with studies characterizing menthol as an LTCC antagonist (4), the monoterpenes (perillyl alcohol, menthol, limonene) exhibited at least a temporary reduction in steady-state calcium levels in our studies in the WEHI-231 cells. These results suggest that the dihydropyridine-sensitive calcium channel is a plausible monoterpene-mediated target in certain cancer cells expressing calcium-dependent NF-{kappa}B levels. In support of this conclusion, perillyl alcohol mediated a significant reduction of calcium-sensitive NF-{kappa}B levels in the MDA-MB 468 cell line shown to express a LTCC.

The calcium reducing activity of monoterpene treatment did not generally correlate with the most lipophillic monoterpene applied in our studies. Limonene, the most lipophillic monoterpene used (38), did not reduce calcium levels as markedly or persistently as perillyl alcohol relative to controls with short-term treatment. The control dihydropyridines (nitrendipine, nifedipine) applied in our studies bind to the dihydropyridine receptor with approximately similar affinities (39). Reasonably increasing the concentration (30-50 µmol/L) of the lower affinity dihydropyridine (nifedipine) in the WEHI-231 cells did not have an effect on NF-{kappa}B DNA-binding activity or apoptosis. Therefore, the efficacy of nitrendipine treatment in reducing constitutive calcium-dependent NF-{kappa}B activity was plausibly dependent on the binding and subsequent regulation of the dihydropyridine-sensitive calcium channel. Alternatively, other sites of action could be involved in eliciting this unreported anticancer activity of the well-studied dihydropyridines. However, perillyl alcohol treatment seemed to reduce calcium levels longer than the other monoterpenes. Thus, perillyl alcohol may regulate the dihydropyridine-sensitive channel differently from the other monoterpenes. Furthermore, the calcium-independent constitutive DNA-binding activity of the Oct transcriptional factors in the WEHI-231 cells was unaffected with agent treatment, showing relative specificity toward calcium-dependent NF-{kappa}B activity. Our data supports the hypothesis that the different outcomes with monoterpene and dihydropyridine treatments are due to the agent-specific regulation of the dihydropyridine-sensitive calcium channel in the WEHI 231 cells.

The concentrations of the dihydropyridines used in our studies are equal to or less than the concentrations to examine LTCC-mediated differentiation and signal transduction pathways (40, 41). Thus, the relative comparisons made between the monoterpenes and the dihydropyridines with the concentrations used in the current study are rationally based. Furthermore, the monoterpenes (perillyl alcohol, limonene, perillic acid) elicit cytostatic activities in mammalian fibroblast and carcinoma cells in vitro with the concentrations used in the present paper (<1.0 mmol/L; refs. 42–44). The apoptotic activity of monoterpene (limonene and perillyl alcohol) treatment in vivo in regressing rat mammary tumors is associated with high serum concentrations (~0.8 mmol/L) of metabolites, including perillic acid (1, 2, 45). In the present study, however, perillic acid and limonene were noneffective inducers of apoptosis in the WEHI-231 cells compared with perillyl alcohol. Therefore, the molecular mechanisms associated with the monoterpene-induced apoptosis in mammary cancer either do not involve the reduction of NF-{kappa}B antiapoptotic activity or are not manifested within the duration of our experiments in the WEHI-231 cells.

A constitutive calcium-dependent process targets the proteasome-independent degradation of I{kappa}B{alpha}, releasing NF-{kappa}B to the nucleus in the WEHI-231 cells (12). This unique degradation pathway seems to be saturated because calcium ionophore (46) and BayK8644(–) treatment of the WEHI-231 cells did not augment NF-{kappa}B DNA-binding activity (data not shown). Furthermore, stimulation of calcium-independent NF-{kappa}B DNA-binding activity through the CD40 receptor abrogated the effects of perillyl alcohol treatment. Our previous work implicated the involvement of the calcium effector protein calmodulin in the constitutive pathway (15). Calmodulin inhibition transiently decreased I{kappa}B{alpha} degradation, resulting in the concomitant reduction of NF-{kappa}B DNA-binding activity. The modulation of the dihydropyridine-sensitive calcium channel upon administration of several monoterpenes in the present study resulted in kinetically similar transient reductions of NF-{kappa}B DNA-binding activity. Previous studies with calmodulin inhibitors indicate that a prolonged reduction in constitutive NF-{kappa}B activity is required to cause apoptosis in these cells (15). Similarly, in the current study, the induction of apoptosis with perillyl alcohol and nitrendipine treatments associated with a prolonged decrease of NF-{kappa}B activity.

Agents targeting dihydropyridine-sensitive calcium channels are effective for treatment of cardiovascular diseases (37). However, a knowledge gap exists on whether the rapidly stimulated reduction of steady-state calcium levels in neoplastic tissues is a desirable end point in chemotherapy and/or chemoprevention. Interestingly, this premise was useful in elucidating a novel calcium-dependent proteasome inhibitor–resistant (PIR) pathway of constitutive NF-{kappa}B activity (12, 47). Studies in certain mammary tumor models suggest that HER-2/neu overexpression can induce proteasome-independent NF-{kappa}B activity (48). However, the relationship between HER-2/neu overexpression and PIR constitutive NF-{kappa}B activation remains undefined. Nevertheless, our study shows that certain compounds (perillyl alcohol and nitrendipine) from two different families of agents known to bind to LTCCs can significantly reduce NF-{kappa}B in a widely used ER– human breast cancer cell line. The outcomes of the inhibition of NF-{kappa}B activity probably varies in different cancer cells because this transcription factor regulates multiple target genes ranging from those affecting proliferation and tumor promotion to cell survival (49). We conclude that the dihydropyridine-sensitive calcium channel is a candidate receptor for monoterpene regulation of NF-{kappa}B levels in certain cancers.


   Acknowledgments
 
Grant support: NIH grants CA38128 (M.N. Gould) and CA81065 (S. Miyamoto).

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.

We thank J. Haag and Dr. Laurie Shepel for reading the manuscript and Kathy Schell (University of Wisconsin Flow Cytometry Facility) for support.

Received 11/15/04. Revised 6/27/05. Accepted 7/ 6/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gould MN. Cancer chemoprevention and therapy by monoterpenes. Environ Health Perspect 1997;105:977–9.
  2. Ariazi EA, Satomi Y, Ellis MJ, et al. Activation of the TGF-ß signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol. Cancer Res 1999;59:1917–28.[Abstract/Free Full Text]
  3. Karin M, Hunter T. Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr Biol 1995;5:747–57.
  4. Hawthorn M, Ferrante J, Luchowski E, et al. The actions of peppermint oil and menthol on calcium channel dependent processes in intestinal, neuronal and cardiac preparations. Aliment Pharmacol Ther 1988;2:101–18.[Medline]
  5. Ripple GH, Gould MN, Stewart JA, et al. Phase I clinical trial of perillyl alcohol administered daily. Clin Cancer Res 1998;4:1159–64.[Abstract]
  6. Mascher H, Kikuta C, Schiel H. Pharmacokinetics of menthol and carvone after administration of an enteric coated formulation containing peppermint oil and caraway oil. Arzneimittelforschung 2001;51:465–9.[Medline]
  7. Vaughan TL, Farrow DC, Hansten PD, et al. Risk of esophageal and gastric adenocarcinomas in relation to use of calcium channel blockers, asthma drugs, and other medications that promote gastroesophageal reflux. Cancer Epidemiol Biomarkers Prev 1998;7:749–56.[Abstract]
  8. Kenakin T. Drugs and receptors. An overview of the current state of knowledge. Drugs 1990;40:666–87.[Medline]
  9. Lukes RJ, Collins RD. Immunologic characterization of human malignant lymphomas. Cancer 1974;34 Suppl:1488–503.
  10. Sadighi Akha AA, Willmott NJ, Brickley K, Dolphin AC, Galione A, Hunt SV. Anti-Ig-induced calcium influx in rat B lymphocytes mediated by cGMP through a dihydropyridine-sensitive channel. J Biol Chem 1996;271:7297–300.[Abstract/Free Full Text]
  11. Savignac M, Badou A, Moreau M, et al. Protein kinase C-mediated calcium entry dependent upon dihydropyridine-sensitive channels: a T cell receptor-coupled signaling pathway involved in IL-4 synthesis. FASEB J 2001;15:1577–9.[Free Full Text]
  12. Miyamoto S, Seufzer BJ, Shumway SD. Novel I{kappa}B{alpha} proteolytic pathway in WEHI-231 immature B cells. Mol Cell Biol 1998;18:19–29.[Abstract/Free Full Text]
  13. Wu M, Lee H, Bellas RE, et al. Inhibition of NF-{kappa}B/Rel induces apoptosis of murine B cells. EMBO J 1996;15:4682–90.[Medline]
  14. Dolmetsch RE, Pajvani U, Fife K, Spotts JM, Greenberg ME. Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. Science 2001;294:333–9.[Abstract/Free Full Text]
  15. Shumway SD, Berchtold CM, Gould MN, Miyamoto S. Evidence for unique calmodulin-dependent NF-{kappa}B regulation in WEHI-231 B cells. Mol Pharmacol 2002;61:177–85.[Abstract/Free Full Text]
  16. Zamai L, Falcieri E, Marhefka G, Vitale M. Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation. Cytometry 1996;23:303–11.[CrossRef][Medline]
  17. Hirsch T, Marchetti P, Susin SA, et al. The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 1997;15:1573–81.[CrossRef][Medline]
  18. Smith CA, Williams GT, Kingston R, Jenkinson EJ, Owen JJ. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 1989;337:181–4.[CrossRef][Medline]
  19. Eastman A. Assays for DNA fragmentation, endonucleases, and intracellular pH and calcium associated with apoptosis. Methods Cell Biol 1995;46:41–55.[Medline]
  20. Kao JP. Practical aspects of measuring calcium with fluorescent indicators. Methods Cell Biol 1994;40:155–81.[Medline]
  21. Kaspar RL, Kakegawa T, Cranston H, Morris DR, White MW. A regulatory cis element and a specific binding factor involved in the mitogenic control of murine ribosomal protein L32 translation. J Biol Chem 1992;267:508–14.[Abstract/Free Full Text]
  22. Perez-Reyes E, Wei XY, Castellano A, Birnbaumer L. Molecular diversity of L-type calcium channels. Evidence for alternative splicing of the transcripts of three non-allelic genes. J Biol Chem 1990;265:20430–6.[Abstract/Free Full Text]
  23. Agoston A, Kunz L, Krieger A, Mayerhofer A. Two types of calcium channels in human ovarian endocrine cells: involvement in steroidogenesis. J Clin Endocrinol Metab 2004;89:4503–12.[Abstract/Free Full Text]
  24. Heubach JF, Graf EM, Leutheuser J, et al. Electrophysiological properties of human mesenchymal stem cells. J Physiol 2004;554:659–72.[Abstract/Free Full Text]
  25. Miyamoto S, Schmitt MJ, Verma IM. Qualitative changes in the subunit composition of {kappa}B-binding complexes during murine B cell differentiation. Proc Natl Acad Sci U S A 1994;91:5056–60.[Abstract/Free Full Text]
  26. Huang TT, Wuerzberger-Davis SM, Seufzer BJ, et al. NF-{kappa}B activation by camptothecin: a linkage between nuclear DNA damage and cytoplasmic signaling events. J Biol Chem 2000;275:9501–9.[Abstract/Free Full Text]
  27. Lernbecher T, Muller U, Wirth T. Distinct NF-{kappa}B/Rel transcription factors are responsible for tissue-specific and inducible gene activation. Nature 1993;365:767–70.[CrossRef][Medline]
  28. Feldhaus AL, Klug CA, Arvin KL, Singh H. Targeted disruption of the Oct-2 locus in a B cell provides genetic evidence for two distinct cell type-specific pathways of octamer element-mediated gene activation. EMBO J 1993;12:2763–72.[Medline]
  29. Fitzgerald K, Harrington A, Leder P. Ras pathway signals are required for notch-mediated oncogenesis. Oncogene 2000;19:4191–8.[CrossRef][Medline]
  30. Wang CY, Guttridge DC, Mayo MW, Baldwin AS. NF-{kappa}B induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Mol Cell Biol 1999;19:5923–9.[Abstract/Free Full Text]
  31. Chiao PJ, Miyamoto S, Verma IM. Autoregulation of I{kappa}B{alpha} activity. Proc Natl Acad Sci U S A 1994;91:28–32.[Abstract/Free Full Text]
  32. Schauer SL, Wang Z, Sonenshein GE, Rothstein TL. Maintenance of NF-{kappa}B/Rel and c-myc expression during CD40 ligand rescue of WEHI 231 early B cells from receptor-mediated apoptosis through modulation of I{kappa}B{alpha}. J Immunol 1996;157:81–6.[Abstract]
  33. Sutherland CL, Krebs DL, Gold MR. An 11-amino acid sequence in the cytoplasmic domain of CD40 is sufficient for activation of c-Jun N-terminal kinase, activation of MAPKAP kinase-2, phosphorylation of I{kappa}B{alpha}, and protection of WEHI-231 cells from anti-IgM-induced growth arrest. J Immunol 1999;162:4720–30.[Abstract/Free Full Text]
  34. Hockerman GH, Peterson BZ, Johnson BD, Catterall WA. Molecular determinants of drug binding and action on L-type calcium channels. Annu Rev Pharmacol Toxicol 1997;37:361–96.[CrossRef][Medline]
  35. Berchtold CM, Ariazi EA, Gould MN. Perillyl alcohol mediated induction of apoptosis in human breast cancer cells in vitro. Proc Annu Meet Am Assoc Cancer Res 2000;40:487.
  36. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ, Sledge GW. Constitutive activation of NF-{kappa}B during progression of breast cancer to hormone-independent growth. Mol Cell Biol 1997;17:3629–39.[Abstract]
  37. Mitterdorfer J, Grabner M, Kraus RL, et al. Molecular basis of drug interaction with L-type calcium channels. J Bioenerg Biomembr 1998;30:319–34.[CrossRef][Medline]
  38. Griffin S, Wyllie SG, Markham J. Determination of octanol-water partition coefficient for terpenoids using reversed-phase high-performance liquid chromatography. J Chromatogr A 1999;864:221–8.[CrossRef][Medline]
  39. Ferry DR, Goll A, Glossmann H. Putative calcium channel molecular weight determination by target size analysis. Naunyn Schmiedebergs Arch Pharmacol 1983;323:292–7.[CrossRef][Medline]
  40. Porter GA, Makuck RF, Rivkees SA. Reduction in intracellular calcium levels inhibits myoblast differentiation. J Biol Chem 2002;277:28942–7.[Abstract/Free Full Text]
  41. Kotturi MF, Carlow DA, Lee JC, Ziltener HJ, Jefferies WA. Identification and functional characterization of voltage-dependent calcium channels in T lymphocytes. J Biol Chem 2003;278:46949–60.[Abstract/Free Full Text]
  42. Crowell PL, Chang RR, Ren ZB, Elson CE, Gould MN. Selective inhibition of isoprenylation of 21-26-kDa proteins by the anticarcinogen D-limonene and its metabolites. J Biol Chem 1991;266:17679–5.[Abstract/Free Full Text]
  43. Crowell PL, Ren Z, Lin S, Vedejs E, Gould MN. Structure-activity relationships among monoterpene inhibitors of protein isoprenylation and cell proliferation. Biochem Pharmacol 1994;47:1405–15.[CrossRef][Medline]
  44. Shi W, Gould MN. Induction of cytostasis in mammary carcinoma cells treated with the anticancer agent perillyl alcohol. Carcinogenesis 2002;23:131–42.[Abstract/Free Full Text]
  45. Ariazi EA, Gould MN. Identifying differential gene expression in monoterpene-treated mammary carcinomas using subtractive display. J Biol Chem 1996;271:29286–94.[Abstract/Free Full Text]
  46. Fields ER, Seufzer BJ, Oltz EM, Miyamoto SA. Switch in distinct I{kappa}B{alpha} degradation mechanisms mediates constitutive NF-{kappa}B activation in mature B cells. J Immunol 2000;164:4762–7.[Abstract/Free Full Text]
  47. O'Connor S, Shumway SD, Amanna IJ, Hayes CE, Miyamoto S. Regulation of constitutive p50/c-rel activity via proteasome inhibitor-resistant I{kappa}B{alpha} degradation in B cells. Mol Cell Biol 2004;24:4895–908.[Abstract/Free Full Text]
  48. Pianetti S, Arsura M, Romieu-Mourez R, Coffey RJ, Sonenshein GE. Her-2/neu overexpression induces NF-{kappa}B via a PI3-kinase/Akt pathway involving calpain-mediated degradation of I{kappa}B{alpha} that can be inhibited by the tumor suppressor PTEN. Oncogene 2001;20:1287–99.[CrossRef][Medline]
  49. Aggarwal BB. NF-{kappa}B: The enemy within. Cancer Cell 2004;6:203–28.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 GeneticsHome page
M. N. Gould
The Utility of Comparative Genetics to Inform Breast Cancer Prevention Strategies
Genetics, October 1, 2009; 183(2): 409 - 412.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
B. B. Aggarwal, M. E. Van Kuiken, L. H. Iyer, K. B. Harikumar, and B. Sung
Molecular Targets of Nutraceuticals Derived from Dietary Spices: Potential Role in Suppression of Inflammation and Tumorigenesis
Experimental Biology and Medicine, August 1, 2009; 234(8): 825 - 849.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
I. V. Lebedeva, Z.-z. Su, N. Vozhilla, L. Chatman, D. Sarkar, P. Dent, M. Athar, and P. B. Fisher
Mechanism of In vitro Pancreatic Cancer Cell Growth Inhibition by Melanoma Differentiation-Associated Gene-7/Interleukin-24 and Perillyl Alcohol
Cancer Res., September 15, 2008; 68(18): 7439 - 7447.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
S. Markovina, N. S. Callander, S. L. O'Connor, J. Kim, J. E. Werndli, M. Raschko, C. P. Leith, B. S. Kahl, K. Kim, and S. Miyamoto
Bortezomib-Resistant Nuclear Factor-{kappa}B Activity in Multiple Myeloma Cells
Mol. Cancer Res., August 1, 2008; 6(8): 1356 - 1364.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
I. V. Lebedeva, Z.-z. Su, N. Vozhilla, L. Chatman, D. Sarkar, P. Dent, M. Athar, and P. B. Fisher
Chemoprevention by perillyl alcohol coupled with viral gene therapy reduces pancreatic cancer pathogenesis
Mol. Cancer Ther., July 1, 2008; 7(7): 2042 - 2050.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
C.M. Payne, C. Weber, C. Crowley-Skillicorn, K. Dvorak, H. Bernstein, C. Bernstein, H. Holubec, B. Dvorakova, and H. Garewal
Deoxycholate induces mitochondrial oxidative stress and activates NF-{kappa}B through multiple mechanisms in HCT-116 colon epithelial cells
Carcinogenesis, January 1, 2007; 28(1): 215 - 222.
[Abstract] [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 Email this article to a friend
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 Berchtold, C. M.
Right arrow Articles by Gould, M. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Berchtold, C. M.
Right arrow Articles by Gould, M. N.
Related Collections
Right arrow Preclinical Intervention
Right arrow Preclinical Intervention: In Vitro: Drugs, Mechanisms

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