Perillyl Alcohol Inhibits a Calcium-Dependent Constitutive Nuclear Factor-KB Pathway

Research Article

Craig M. Berchtold,1,2,4 Kai-Shun Chen,1,3 Shigeki Miyamoto,2,4 and Michael N. Gould1,3

Departments of 1Oncology and 2Pharmacology; 3McArdle Laboratory for Cancer Research; and 4301 SMI, University of Wisconsin-Madison, Madison, Wisconsin

Abstract shown that menthol is a direct inhibitor of L-type calcium channels The cell death induced by the monoterpene anticancer agent (LTCC) expressed on the plasma membrane of normal tissues (4). perillyl alcohol correlates to the increased expression of Interestingly, these studies mechanistically explain the mild to certain proapoptotic genes known to influence cell survival. moderate gastroesophageal reflux or ‘‘heartburn’’ observed in Whereas sequence-specific DNA-binding factors dictate the clinical trials evaluating similarly structured monoterpenes for expression patterns of genes through transcriptional regula- the treatment of different diseases, including cancer (5, 6). LTCC tion, those transcriptional factors influencing constitutive cell antagonists, such as the dihydropyridines, also cause the same survival with perillyl alcohol treatment are not well studied. disorder by inhibiting the calcium channel activity in smooth Here, we investigated whether the monoterpenes can regu- muscle, resulting in the relaxation of the esophageal stomach sphincter (7). LTCCs are specific receptors for the dihydropyridines late the activity of nuclear factor-KB (NF-KB), a calcium- dependent transcription factor necessary for survival in the and are also called dihydropyridine-sensitive calcium channels (8). WEHI-231 B-lymphoma cells. Unique among the monoter- Lymphoma cells originate from cell types (9) that express penes, perillyl alcohol short-term treatment induced a per- dihydropyridine-sensitive calcium channels (10, 11). WEHI-231 B- lymphoma cells constitutively express calcium-dependent nuclear sistent decrease of calcium levels, whereas other various n n monoterpenes caused transient reductions in calcium levels. factor- B(NF-B) DNA-binding activity responsible for the Perillyl alcohol treatment also rapidly elicited reductions of transcription of antiapoptotic genes permissive for cell survival K (12, 13). LTCCs regulate calcium-dependent cell survival pathways NF- B DNA-binding activity and target gene induction, n which was associated with an increase in apoptosis in these independent of NF- B transcription in neuronal cells (14). However, B-lymphoma cells. This apoptosis was directly due to NF-KB an association between the regulation of dihydropyridine-sensitive calcium channels and constitutive NF-nB–mediated antiapoptotic because its prior activation abolished the cell killing effects pathways in these B-lymphoma cells are not well established. of perillyl alcohol treatment. Our findings suggest that n perillyl alcohol can inhibit NF-KB function to modulate gene Therefore, we asked whether the NF- B antiapoptotic pathway in expression patterns and cell survival of certain B-lymphoma the WEHI-231 cells responded to monoterpene treatment. Our findings suggest how a rapidly targeted calcium-dependent cells. The effects of perillyl alcohol were not limited to these n B-lymphoma cells but were also observed in MDA-MB 468 decrease of NF- B in B-lymphoma cells due to monoterpene treatment can induce apoptosis. Consequently, we investigated cells, an estrogen receptor–negative breast cancer cell line. n These results identify a calcium-dependent NF-KB pathway whether perillyl alcohol treatment reduced constitutive NF- B as a molecular target of perillyl alcohol activity in different activity through a similar mechanism in certain estrogen receptor– cancer cell types. (Cancer Res 2005; 65(18): 8558-66) negative/independent (ERÀ) breast cancer cell lines.

Introduction Materials and Methods The monoterpenes isolated from the essential oils of plants elicit Chemicals and materials. Propidium iodide (PI), 2V-(4-ethoxyphenyl)-5- multiple chemotherapeutic and chemopreventive effects in diverse (4-methyl-1-piperazinyl)-2,5V-bi-1H -benzimidazole trihydrochloride models of cancer (1). Anticancer activities associated with (Hoechst 33342), S-(À)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluorome- regressing mammary tumors include the induction of cytostasis thyl)phenyl]-3-pyridine carboxylic acid methyl ester [BayK8644(À)], and (R)-(+)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyr- and apoptosis upon monoterpene application. This antitumor idine carboxylic acid methyl ester [BayK8644(+)] were purchased from activity correlates with the differential expression of growth and Sigma-Aldrich (St. Louis, MO). Aldrich Chemical Company (Milwaukee, WI) apoptotic genes necessary for tumor proliferation (2). The supplied the monoterpenes except for perillic acid, which was synthesized sequence-specific DNA binding of transcription factors often at the University of Wisconsin-Madison. All other chemicals were acquired dictates the expression of genes (3). However, the transcription from Calbiochem (San Diego, CA), including 1-[2-amino-5-(6-carboxyindol- factors that regulate the anticancer apoptotic response to perillyl 2-yl)phenoxy]-2-(2V-amino-5V-methylphenoxy)ethane-N,N,NV,NV-tretraacetic alcohol treatment are not well understood. acid pentaacetoxymethyl ester (INDO-1/AM) and 1,2-bis(o-aminophenoxy)- The monoterpene menthol exhibits calcium-related molecular ethane-N,N,NV,NV-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM). actions not associated with anticancer activity. Studies in situ have 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. Requests for reprints: Michael N. Gould, McArdle Laboratory for Cancer Cell culture. The American Type Culture Collection (Manassas, VA) Research, University of Wisconsin-Madison, 1400 University Avenue, Room 506, supplied the cell lines used in our studies. The WEHI Bcl-X cells have Madison, WI 53706. Phone: 608-263-6615; E-mail: [email protected]. L I2005 American Association for Cancer Research. been described (15). All cell lines, including the WEHI-231, Ramos, MCF- doi:10.1158/0008-5472.CAN-04-4072 7, T47D, MDA-MB 231, and MDA-MB 468 cells, were cultured in RPMI

Cancer Res 2005; 65: (18). September 15, 2005 8558 www.aacrjournals.org

medium with 10% FCS (Sigma), 2 mmol/L L-glutamine, and penicillin/ either the consensus NF-nBorOct-1(5V-TGTCGAATGCAAATCACTAGAA-3V; streptomycin. The WEHI-231 and WEHI Bcl-XL cells were also grown with Promega, Madison, WI) binding sequence. The paired samples were then 50 Amol/L h-mercaptoethanol. Lymphocyte density medium was run on the same 4% nondenaturing polyacrylamide gel and analyzed by purchased from Mediatech (Herndon, VA). All cell culture materials a PhosphorImager (Amersham Biosciences, Piscataway, NJ). Densitometry unless specified were from Invitrogen (Carlsbad, CA). Solubilization of the values of NF-nB (c-rel/p50) were normalized to paired Oct-1 and Oct-2 solid monoterpenes perillic acid and menthol used ethanol as the vehicle. values from the same sample and then to the nontreated control values The final concentration of ethanol in the media for the solid at the indicated time point. Supershift analysis was done with Oct-1 and monoterpenes was V0.07%. The dihydropyridines were suspended in Oct-2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). The 100% ethanol with a working stock concentration of 25 or 30 mmol/L. breast cancer studies used total protein extracts (6 Ag) for EMSA The concentration of ethanol in the media with 50 Amol/L of analysis (26). dihydropyridine was 0.2%.

Cell culture experiments. WEHI-231, WEHI Bcl-XL, and Ramos cells were seeded at 1 Â 106 and 3 Â 105 to 5 Â 105 cells/mL for the 4- and 24- Results 5 hour assays, respectively. The MDA-MB 468 cells were seeded at 2 Â 10 Perillyl alcohol induces apoptosis of WEHI-231 B-lymphoma cells/well in a six-well plate (BD Bioscience, Bedford, MA) and allowed to cells. The monoterpenes are known inducers of apoptosis in vivo attach overnight before treatment. (2); however, their induction of programmed cell death in cell Apoptosis assays. The PI/viability assay with the WEHI-231, WEHI Bcl- culture is not well characterized. To determine the sensitivity of the XL, and Ramos cells used the addition of 30 Ag/mL of PI to the cells 5 minutes before analysis by FACScan flow cytometry (Becton Dickinson, San WEHI-231 cells to monoterpene treatment for 24 hours, PI staining Jose, CA; ref. 16). Examination of the cells (17) was done with Cell Quest was used to examine cell death. This method readily detects software (Becton Dickinson). The DNA laddering assay was carried out as apoptosis in nonfixed cells with flow cytometric analysis (16). previously described (18). Perillyl alcohol–treated Ramos cells (another B-lymphoma cell line) Calcium analysis. The calcium analysis used cells (5 Â 105/mL) helped to validate the incidence of apoptosis in B-lymphoma cells. preloaded with INDO-1/AM (2 Amol/L) in Buffer A [25 mmol/L HEPES, 5.4 The sorted Ramos cells showed cell populations with normal (R1), mmol/L KCl, 0.8 mmol/L MgCl2, 1.8 mmol/L CaCl2, 121 mmol/L NaCl, 5.5 apoptotic (R2), and secondary necrotic (R3) morphologies (17) A h mmol/L glucose, 6 mmol/L NaHCO3 (pH 7.3), 50 mol/L -mercaptoe- after monoterpene treatment (Fig. 1A). The perillyl alcohol–treated thanol, and 1% FCS] at 25jC for 30 minutes. Cells were isolated, WEHI-231 cells also showed the fragmented nuclear morphology resuspended in Buffer A with 10% FCS, and placed at 25jC for 20 minutes to allow deesterification of INDO-1/AM. PI (1 Ag/mL) was added to the characteristic of apoptosis (data not shown). Furthermore, the sample to gate live cells and the calcium analysis was carried out with flow isolated R3 (secondary necrotic gate) population of treated WEHI- cytometry at 37jC. A 325 to 360 nm wavelength excites the ratiometric 231 cells displayed oligonucleosomal laddering indicative of INDO-1 fluorophore into emitting light at 405 nm when bound to calcium apoptosis (Fig. 1B; ref. 18). Further examination of the WEHI-231 and 520 nm when free of calcium. To estimate cytoplasmic calcium cells treated with perillyl alcohol for 24 hours showed a marked concentrations, a procedure according to Eastman (19) was incorporated. reduction in viability with increasing concentrations of perillyl Digitonin (20 Amol/L) was applied to the cells to examine the alcohol. Collectively, these results show that the B-lymphoma cell compartmentalization of INDO-1 into organelles. The application of lines undergo apoptosis upon perillyl alcohol treatment. The WEHI A ionomycin (20 mol/L) and manganese (1 mmol/L) to the cells enabled Bcl-X cells, which highly express an exogenous antiapoptotic the estimation of background unmetabolized INDO-1/AM contributing to L Bcl-X gene, did not show a decrease in cell viability with perillyl the 405/520 reading (20). L RNA analysis. Total RNA was isolated with RNAzol B reagent (Tel-Test, alcohol treatment (Fig. 1C). Friendswood, TX). The RNase Protection Assay kit was used according to Studies in WEHI-231 cells showed varying cell death–inducing the instructions of the manufacturer (PharMingen). The normalizing activities with monoterpene treatment for 24 hours (Fig. 1D). control transcripts consisted of both glyceraldehyde-3-phosphate dehydro- Perillyl alcohol application induced a >40-fold increase in cell genase (GAPDH) and L32. L32 mRNA is a rRNA regulated posttranscrip- death, whereas limonene and perillic acid applications elicited a tionally (21). minimum increase in cell death at f2.5- and 3.0-fold, respectively. Reverse transcription-PCR amplification of mouse L-type calcium Menthol did not induce cell death in these studies. Therefore, A channel 1-subunit cDNA. First-strand cDNA synthesis from total RNA perillyl alcohol is the most potent, whereas limonene and perillic was done with oligo(dT)-primed reverse transcription using the Thermo- acid are the least potent, inducers of cell death of the script reverse transcription-PCR (RT-PCR) kit (Invitrogen). The University of monoterpenes tested in our studies. Wisconsin-Biotechnology Center synthesized oligonucleotide primers Perillyl alcohol inhibits constitutive nuclear factor-KB corresponding to conserved regions in the mouse LTCC a1-subunit cDNA (Genbank accession number L01776; ref. 22). PCR amplification of the 920 activity in WEHI-231 cells. As part of the regulation of bp fragment was done with the Herculase enzyme (Stratagene, La Jolla, CA) transcriptional factor activity by the 10-carbon cyclic monoter- and gel electrophoresis was carried out to the isolate the PCR product. The penes (Fig. 2A), we ask the question if treatment would rapidly Taq DNA polymerase–modified PCR product was subcloned into a pCRII TA reduce the calcium-mediated constitutive NF-nB DNA-binding vector (Invitrogen) and transformed into bacteria. Sequencing of multiple activity in the WEHI-231 cells. The NF-nB c-rel/p50 heterodimer, bacterial clones validated the expression of the Cav1.3 (a1D) LTCC a1- generally regarded as a transcriptional activator, has a trans- subunit in the WEHI-231 cells. activation domain that is absent in the transcriptionally repressive A Reverse transcription-PCR amplification of human Cav1.3 ( 1D) p50 homodimer complex (27). The WEHI-231 cells were treated L-type calcium channel A -subunit cDNA. The sequence of the primers 1 with perillyl alcohol in 4-hour time course studies because the cells and conditions used to amplify the LTCC gene from human breast cancer lines have been published (23, 24). The sequencing of the resulting PCR did not exhibit apoptotic morphology at this early time point with products was as described above. PI viability analysis (data not shown). Nuclear proteins were Electrophoretic mobility shift assay. The electrophoretic mobility isolated for EMSA using a consensus nB oligomer. Perillyl alcohol shift assay (EMSA) analysis was done as described (25). Briefly, nuclear application to the WEHI-231 cells showed a marked down- protein extracts from a single reaction were divided into duplicate regulation of NF-nB DNA-binding activity at 4 hours as detected samples and incubated with a 32P-radiolabeled oligonucleotide containing by the reduction of the NF-nB band (c-rel/p50) intensity compared www.aacrjournals.org 8559 Cancer Res 2005; 65: (18). September 15, 2005

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 Ag/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 Ag/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.

with controls (Fig. 2B). The multiple bands of the Oct-1 probe were because they are associated with neoplasia (29, 30). However, these composed of the constitutive Oct-1 and Oct-2 transcriptional mRNAs were not highly expressed and probably not mediated by factors (data not shown; ref. 28) and used as a calcium- the high constitutive NF-nB nuclear levels in the WEHI-231 cells independent control with agent treatment. Further EMSA studies (Fig. 3A and B). Perillic acid was included as the no-effect control included the analysis of the different monoterpenes with varying because this monoterpene did not alter NF-nB DNA-binding levels anticancer activities. Menthol and limonene moderately decreased compared with controls (Fig. 2B). Moreover, perillic acid treatment NF-nB DNA-binding activity, whereas perillic acid treatment was did not consistently reduce mRNA levels either (Fig. 3A and B). Bax not effective (Fig. 2C). was analyzed because steady-state mRNA levels of this cell death EMSA analysis was also done after 24 hours to examine if the transcript increased with perillyl alcohol treatment of regressing NF-nB DNA-binding levels correlated with the apoptosis observed mammary tumors (2). However, Bax mRNA levels seemed to be after 24 hours. These data suggested that over time, limonene, relatively unaltered (Fig. 3A and B), whereas InBa mRNA perillic acid, and menthol actually increased nuclear NF-nB levels expression decreased below control levels with perillyl alcohol compared with Oct-1 levels in the WEHI-231 cells (data not treatment. In accord with this outcome, NF-nB is a known positive shown). The apoptosis-resistant Bcl-XL cells were next treated for regulator of the IjBa gene in the WEHI-231 cells (31). Furthermore, 24 hours with perillyl alcohol to ask if the monoterpene prolonged perillyl alcohol (0.7 mmol/L) application induced a reduction of the the decrease of NF-nB activity without the interference from Bcl-2 gene family mRNA transcript, Bfl-1/A-1, to f50% of controls apoptotic cell death. The NF-nB DNA-binding activity in these cells levels. Thus, a rapid decrease of NF-nB nuclear levels at 4 hours is considered to be regulated similarly to the parental WEHI-231 was associated with a drop in the known NF-nB–regulated cells (15). Perillyl alcohol (0.7 mmol/L) administration maintained antiapoptotic Bfl-l/A1 (30) and IjBa genes in WEHI-231 cells with a marked reduction of NF-nB levels at 4 and 24 hours in the perillyl alcohol treatment. K WEHI-231 cells (Fig. 2B and D) and Bcl-XL cells at 24 hours. Thus, Forced activation of nuclear factor- B can reverse perillyl perillyl alcohol treatment of the WEHI-231 cells for 24 hours show alcohol–induced apoptosis of WEHI-231 cells. The activated a persistent decrease of the prosurvival NF-nB DNA-binding CD40 receptor induces a tumor necrosis–associated factor– activity. dependent NF-nB–mediated antiapoptotic pathway in the WEHI- Expression of certain nuclear factor-KB target genes is 231 cells (32, 33). Therefore, CD40 stimulation was used to validate repressed in perillyl alcohol–treated WEHI-231 cells. To that the persistent reduction of the constitutive calcium-dependent determine if NF-nB DNA-binding activity leads to a reduction in NF-nB antiapoptotic pathway is a plausible mechanism of cell death target gene expression, RNase protection analysis examined with perillyl alcohol treatment (Fig. 1). Cells were initially exposed whether a decrease in the mRNA levels of candidate genes known to transient CD40 antibody pretreatment followed by perillyl to be positively regulated by NF-nB correlated with the reduction of alcohol application for 18 hours. A density gradient was used to the DNA-binding activity of the transcriptional factor following isolate viable cells for gel shift analysis. EMSA analysis showed that monoterpene treatment (Fig. 2). Known NF-nB transcriptionally perillyl alcohol treatment did not inhibit the NF-nB DNA-binding regulated genes, such as notch, jagged, and bcl-2, were analyzed activity from the CD40 antibody pretreated cells compared with

Cancer Res 2005; 65: (18). September 15, 2005 8560 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Perillyl Alcohol Exploits a Calcium-Specific NF-kB Pathway 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-nB DNA- binding activity compared with controls (Fig. 4B). Cellular viability analysis showed a statistically significant (P < 0.05) protective effect

Figure 3. RNase protection analysis of candidate NF-nB–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 InBa and the antiapoptotic gene Bfl-1/A1. B, mRNA levels. Densitometry values of the Bfl-1/A1, InBa, 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.

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-nB DNA-binding activity (Fig. 2A and D)is responsible for a significant percentage (f80%) 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-nB cell survival pathway without interfering with a calcium-independent CD40 induced n Figure 2. NF-nB DNA-binding activity in the WEHI-231 cells with monoterpene NF- B cell survival pathway with perillyl alcohol treatment. treatment. A, chemical structure of the monoterpenes. B, NF-nB DNA-binding Perillyl alcohol treatment reduces the estimated steady- activity after a 4-hour treatment with perillyl alcohol was determined by EMSA state calcium levels compared with controls. To answer the as described in Materials and Methods. Perillyl alcohol treatment reduced NF-nB levels (0.3 mmol/L, 38 F 16%; 0.5 mmol/L, 43 F 15%; 0.7 mmol/L, question of why the monoterpenes decreased the calcium- 34 F 21%, n = 3) compared with control values at 4 hours. C, WEHI-231 dependent NF-nB pathway in the WEHI-231 cells, steady-state 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 calcium concentrations were estimated at relatively early time represent one of two independent experiments. The NF-nB levels from both points after monoterpene treatment. Perillic acid application did experiments (n = 2) are as follows: menthol: 40%, 41%; limonene: 12%, 39%; not reduce calcium levels within 60 minutes relative to controls and perillic acid: À5%, 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 100Â (Fig. 5A). Treatment with the monoterpenes (perillyl alcohol, designation represents the addition of the cold oligomer 100 times in excess of limonene, menthol) seemed to decrease the calcium concentra- the radiolabeled oligomer during the incubation period. The gels shown represent one of three independent experiments. The data are normalized to the control tions at 30 minutes compared with control levels, whereas the (white columns); columns, average of the three experiments; bars, SD. transient limonene– and menthol-mediated decreases approached www.aacrjournals.org 8561 Cancer Res 2005; 65: (18). September 15, 2005

L-type calcium channel antagonist decreases the calcium- dependent nuclear factor-KB 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 a1-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 a1D gene family of LTCCs (Fig. 6A, lane 2; ref. 22). An increase of calcium in cells treated with the LTCC agonist BayK8644(À) functionally shows a constitutively opened LTCC (34). BayK8644(À) treatment of INDO-1/AM-loaded WEHI- 231 cells caused an increase in calcium levels from f70 to f120 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 Figure 4. CD40 stimulation of NF-nB DNA-binding activity blocked the suggest that the WEHI-231 cells express a constitutively active anticancer effects of perillyl alcohol treatment. A, WEHI-231 cells transiently dihydropyridine-sensitive calcium channel that can directly treated with CD40 (10 Ag/mL) for f4 hours were exposed to perillyl alcohol influence steady-state calcium levels. for an additional 18 hours. Nonapoptotic cells in treated and controls (CON) were isolated using a lymphocyte density gradient and subjected to EMSA analysis. The dihydropyridine antagonists nitrendipine and nifedipine B, NF-nB DNA-binding activity. Columns, average of three independent were used to test whether targeting the dihydropyridine-sensitive n experiments; bars, SD. Densitometry values of NF- B (c-rel/p50) were analyzed calcium channel would initiate the reduction of the calcium- by Sigma Stat 3.0 software. The differences of the NF-nB (c-rel/p50) densitometry values are statistically significant among the different treatment mediated constitutive NF-nB DNA-binding activity in the WEHI- groups (one-way ANOVA, P = 0.001). Multiple comparison analysis used 231 cells. Nitrendipine decreased NF-nB DNA-binding activity at 4 Tukey’s test. Control values are significantly different from perillyl alcohol (P < 0.05) values but not CD40 and CD40 + perillyl alcohol treatments. Perillyl hours (Fig. 6C), whereas nifedipine did not (Fig. 6D). The prolonged alcohol values (*) are significantly different from control (P < 0.05), CD40 treatment of nitrendipine in the Bcl-XL cells persistently decreased (P < 0.005), and CD40 + perillyl alcohol (P < 0.005) treatments. C, cells treated NF-nB DNA-binding levels, whereas Oct-1 remained relatively 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, unchanged (Fig. 6E). Nifedipine treatment was not effective in this average of three independent experiments each done in triplicate; bars, SD. assay. Furthermore, nitrendipine treatment also induced cell death The results were normalized to the controls from each experiment and in the WEHI-231 cells over 24 hours, whereas nifedipine was not logarithmically transformed before the statistical analysis. The unpaired t test determined a statistically significant difference (**P < 0.05) between CD40 + effective (Fig. 6F). Neither dihydropyridine compound induced perillyl alcohol and perillyl alcohol only–treated cells. apoptosis in the Bcl-XL cells with 24 hours of treatment. Thus, the reduction of the constitutive antiapoptotic NF-nB levels for 24 controls at 60 minutes (Fig. 5B). However, perillyl alcohol hours is associated with apoptosis at 24 hours with the administration elicited a consistent and statistically significant dihydropyridine antagonist compound nitrendipine. These results (P < 0.05) reduction of the estimated calcium concentrations at suggest that perillyl alcohol maybe similar to nitrendipine in its both time points relative to the controls (Fig. 5C). mechanism of action.

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 F 22 nmol/L; perillyl alcohol: 110 F 33 nmol/L, n = 3) and four independent experiments done in triplicate at 60 minutes (control: 248 F 95 nmol/L; perillyl alcohol: 121 F 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.

Cancer Res 2005; 65: (18). September 15, 2005 8562 www.aacrjournals.org

Perillyl alcohol and different regulators of calcium decrease constitutive nuclear factor-KB 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-nB DNA-binding activity are unknown in these breast cancer cell lines. Generally, ERÀ breast cancer cell lines (MDA-MB 468, MDA-MB 231) exhibit higher levels of the NF-nB 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-nB levels in cells that express the Cav1.3 a1-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. To characterize the potential calcium-dependent regulation of constitutive NF-nB 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-nB levels in the ER+ cell lines and thus focused on the regulation of the high NF-nB levels in the MDA-MB 468 cells (data not shown). BAPTA-AM treatment at both concentrations (10 and 30 Amol/L) significantly reduced NF-nB 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-nB activity. Because perillyl alcohol and nitrendipine treatment persistently reduced long-term NF-nB 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-nB 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-nB 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-nBor Oct-1 in these studies. Therefore, perillyl alcohol and nitrendipine treatment elicited a significant decrease in a plausible calcium- Figure 6. The dihydropyridine antagonist nitrendipine reduces NF-nB levels dependent NF-nB activity in an ERÀ breast cancer cell line. in the dihydropyridine-sensitive calcium channel expressing WEHI-231 cells. A, expression of the a1-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 Amol/L) loaded WEHI-231 Discussion cells were analyzed for intracellular calcium concentration ([Ca2+]) by flow cytometry and treated with BayK8644(À) (10 Amol/L). BayK8644(+), 10 Amol/L, Diverse groups of chemicals with varying molecular structures was used as the negative control. Each 40 seconds analyzed is the average bind directly to LTCCs (37). However, we observed widely varied f of 6,000 counts. C, cells were treated with nitrendipine (20 and 30 Amol/L) outcomes in the persistent decrease of constitutive calcium- 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 dependent NF-nB DNA-binding activity using multiple compounds nifedipine (30 Amol/L) for 4 hours and analyzed as in Fig. 2. Neither of two with known LTCC antagonistic activity in the WEHI-231 cells. In experiments showed any change in the level of NF-nB. E, WEHI Bcl-XL cells were treated with nitrendipine (50 Amol/L), nifedipine (50 Amol/L), agreement with studies characterizing menthol as an LTCC ethanol-vehicle, and control for 24 hours and were analyzed for NF-nB antagonist (4), the monoterpenes (perillyl alcohol, menthol, DNA-binding activity as in Fig. 2. The results shown represent one of three limonene) exhibited at least a temporary reduction in steady-state 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 calcium levels in our studies in the WEHI-231 cells. These results one experiment done in triplicate. suggest that the dihydropyridine-sensitive calcium channel is a www.aacrjournals.org 8563 Cancer Res 2005; 65: (18). September 15, 2005

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research plausible monoterpene-mediated target in certain cancer cells data supports the hypothesis that the different outcomes with expressing calcium-dependent NF-nB levels. In support of this monoterpene and dihydropyridine treatments are due to the agent- conclusion, perillyl alcohol mediated a significant reduction of specific regulation of the dihydropyridine-sensitive calcium chan- calcium-sensitive NF-nB levels in the MDA-MB 468 cell line shown nel in the WEHI 231 cells. to express a LTCC. The concentrations of the dihydropyridines used in our studies The calcium reducing activity of monoterpene treatment did not are equal to or less than the concentrations to examine LTCC- generally correlate with the most lipophillic monoterpene applied mediated differentiation and signal transduction pathways (40, 41). in our studies. Limonene, the most lipophillic monoterpene used Thus, the relative comparisons made between the monoterpenes (38), did not reduce calcium levels as markedly or persistently as and the dihydropyridines with the concentrations used in the perillyl alcohol relative to controls with short-term treatment. The current study are rationally based. Furthermore, the monoterpenes control dihydropyridines (nitrendipine, nifedipine) applied in our (perillyl alcohol, limonene, perillic acid) elicit cytostatic activities studies bind to the dihydropyridine receptor with approximately in mammalian fibroblast and carcinoma cells in vitro with the similar affinities (39). Reasonably increasing the concentration concentrations used in the present paper (<1.0 mmol/L; refs. 42–44). (30-50 Amol/L) of the lower affinity dihydropyridine (nifedipine) in The apoptotic activity of monoterpene (limonene and perillyl the WEHI-231 cells did not have an effect on NF-nB DNA-binding alcohol) treatment in vivo in regressing rat mammary tumors is activity or apoptosis. Therefore, the efficacy of nitrendipine treat- associated with high serum concentrations (f0.8 mmol/L) of ment in reducing constitutive calcium-dependent NF-nB activity metabolites, including perillic acid (1, 2, 45). In the present study, was plausibly dependent on the binding and subsequent regulation however, perillic acid and limonene were noneffective inducers of of the dihydropyridine-sensitive calcium channel. Alternatively, apoptosis in the WEHI-231 cells compared with perillyl alcohol. other sites of action could be involved in eliciting this unreported Therefore, the molecular mechanisms associated with the mono- anticancer activity of the well-studied dihydropyridines. However, terpene-induced apoptosis in mammary cancer either do not perillyl alcohol treatment seemed to reduce calcium levels longer involve the reduction of NF-nB antiapoptotic activity or are not than the other monoterpenes. Thus, perillyl alcohol may regulate manifested within the duration of our experiments in the WEHI-231 the dihydropyridine-sensitive channel differently from the other cells. monoterpenes. Furthermore, the calcium-independent constitutive A constitutive calcium-dependent process targets the protea- DNA-binding activity of the Oct transcriptional factors in the some-independent degradation of InBa, releasing NF-nB to the WEHI-231 cells was unaffected with agent treatment, showing nucleus in the WEHI-231 cells (12). This unique degradation relative specificity toward calcium-dependent NF-nB activity. Our pathway seems to be saturated because calcium ionophore (46)

Figure 7. NF-nB DNA-binding activity in the ERÀ human breast cancer cells with perillyl alcohol treatment. A, expression of the Cav1.3 a1-subunit gene in various human breast cancer cell lines. Agarose gel electrophoresis depicts the f900 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 a1-subunit gene. The MDA-MB 231 lane (lane 4) depicts a reproducible RT-PCR product that migrated with the validated Cav1.3 a1-subunit gene. B, MDA-MB 468 cells were grown to near confluence and treated with BAPTA-AM (10 and 30 Amol/L) for 2 hours. The majority of the cells (f90%) 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-nBand 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-nB 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 F SD from three independent experiments. NF-nB levels with BAPTA-AM treatment are as follows: 10 Amol/L: 6.0 F 2.2% and 30.0 Amol/L: 6.2 F 1.3%. Oct-1 levels with BAPTA-AM treatment are: 10 Amol/L: 95 F 40% and 30.0 Amol/L: 76 F 23%. C, perillyl alcohol treatment of the ERÀ MDA-MB 468 cells and EMSA analysis of constitutive NF-nB 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-nB levels with perillyl alcohol treatment are as follows: 24 hours: 23 F 12% and 48 hours: 16 F 13%. Oct-1 levels with perillyl alcohol treatment are as follows: 24 hours: 96 F 37% and 48 hours: 93 F 14%. D, ERÀ MDA-MB 468 cells were treated with nitrendipine (50 Amol/L), nifedipine (50 Amol/L), ethanol-vehicle, and control for 24 hours and tested for NF-nB 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-nB and Oct-1 were analyzed as in Fig. 4B. The densitometry values are normalized and expressed as in (B). NF-nB levels with treatment are as follows: vehicle: 97 F 41%; nifedipine: 103 F 31%; and nitrendipine: 35 F 13%. Oct-1 levels with perillyl alcohol treatment are as follows: vehicle: 105 F 27%; nifedipine: 113 F 45%; and nitrendipine: 260 F 50%.

Cancer Res 2005; 65: (18). September 15, 2005 8564 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Perillyl Alcohol Exploits a Calcium-Specific NF-kB Pathway and BayK8644(À) treatment of the WEHI-231 cells did not proteasome inhibitor–resistant (PIR) pathway of constitutive NF- augment NF-nB DNA-binding activity (data not shown). Further- nB activity (12, 47). Studies in certain mammary tumor models more, stimulation of calcium-independent NF-nB DNA-binding suggest that HER-2/neu overexpression can induce proteasome- activity through the CD40 receptor abrogated the effects of perillyl independent NF-nB activity (48). However, the relationship between alcohol treatment. Our previous work implicated the involvement HER-2/neu overexpression and PIR constitutive NF-nB activation of the calcium effector protein calmodulin in the constitutive remains undefined. Nevertheless, our study shows that certain pathway (15). Calmodulin inhibition transiently decreased InBa compounds (perillyl alcohol and nitrendipine) from two different degradation, resulting in the concomitant reduction of NF-nB families of agents known to bind to LTCCs can significantly reduce DNA-binding activity. The modulation of the dihydropyridine- NF-nB in a widely used ERÀ human breast cancer cell line. The sensitive calcium channel upon administration of several mono- outcomes of the inhibition of NF-nBactivityprobablyvariesin terpenes in the present study resulted in kinetically similar different cancer cells because this transcription factor regulates transient reductions of NF-nB DNA-binding activity. Previous multiple target genes ranging from those affecting proliferation and studies with calmodulin inhibitors indicate that a prolonged tumor promotion to cell survival (49). We conclude that the reduction in constitutive NF-nB activity is required to cause dihydropyridine-sensitive calcium channel is a candidate receptor apoptosis in these cells (15). Similarly, in the current study, the for monoterpene regulation of NF-nB levels in certain cancers. induction of apoptosis with perillyl alcohol and nitrendipine n treatments associated with a prolonged decrease of NF- B activity. Acknowledgments Agents targeting dihydropyridine-sensitive calcium channels are Received 11/15/2004; revised 6/27/2005; accepted 7/6/2005. effective for treatment of cardiovascular diseases (37). However, a Grant support: NIH grants CA38128 (M.N. Gould) and CA81065 (S. Miyamoto). knowledge gap exists on whether the rapidly stimulated reduction of The costs of publication of this article were defrayed in part by the payment of page steady-state calcium levels in neoplastic tissues is a desirable end charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. point in chemotherapy and/or chemoprevention. Interestingly, this We thank J. Haag and Dr. Laurie Shepel for reading the manuscript and Kathy premise was useful in elucidating a novel calcium-dependent Schell (University of Wisconsin Flow Cytometry Facility) for support.

References 12. Miyamoto S, Seufzer BJ, Shumway SD. Novel InBa physiological properties of human mesenchymal stem proteolytic pathway in WEHI-231 immature B cells. Mol cells. J Physiol 2004;554:659–72. 1. Gould MN. Cancer chemoprevention and therapy Cell Biol 1998;18:19–29. 25. Miyamoto S, Schmitt MJ, Verma IM. Qualitative by monoterpenes. Environ Health Perspect 1997;105: 13. Wu M, Lee H, Bellas RE, et al. Inhibition of NF-nB/Rel changes in the subunit composition of nB-binding 977–9. induces apoptosis of murine B cells. EMBO J 1996;15: complexes during murine B cell differentiation. Proc 2. Ariazi EA, Satomi Y, Ellis MJ, et al. Activation of the 4682–90. Natl Acad Sci U S A 1994;91:5056–60. TGF-h signaling pathway and induction of cytostasis 14. DolmetschRE,PajvaniU,FifeK,SpottsJM, 26. Huang TT, Wuerzberger-Davis SM, Seufzer BJ, et al. and apoptosis in mammary carcinomas treated with the Greenberg ME. Signaling to the nucleus by an L-type NF-nB activation by camptothecin: a linkage between anticancer agent perillyl alcohol. Cancer Res 1999;59: calcium channel-calmodulin complex through the MAP nuclear DNA damage and cytoplasmic signaling events. 1917–28. kinase pathway. Science 2001;294:333–9. J Biol Chem 2000;275:9501–9. 3. Karin M, Hunter T. Transcriptional control by protein 15. Shumway SD, Berchtold CM, Gould MN, Miyamoto S. 27. Lernbecher T, Muller U, Wirth T. Distinct NF-nB/ phosphorylation: signal transmission from the cell Evidence for unique calmodulin-dependent NF-nB Rel transcription factors are responsible for tissue- surface to the nucleus. Curr Biol 1995;5:747–57. regulation in WEHI-231 B cells. Mol Pharmacol specific and inducible gene activation. Nature 1993;365: 4. Hawthorn M, Ferrante J, Luchowski E, et al. The 2002;61:177–85. 767–70. actions of peppermint oil and menthol on calcium 16. Zamai L, Falcieri E, Marhefka G, Vitale M. Supravital 28. Feldhaus AL, Klug CA, Arvin KL, Singh H. Targeted channel dependent processes in intestinal, neuronal and exposure to propidium iodide identifies apoptotic cells disruption of the Oct-2 locus in a B cell provides genetic cardiac preparations. Aliment Pharmacol Ther 1988;2: in the absence of nucleosomal DNA fragmentation. evidence for two distinct cell type-specific pathways of 101–18. Cytometry 1996;23:303–11. octamer element-mediated gene activation. EMBO J 5. Ripple GH, Gould MN, Stewart JA, et al. Phase I clinical 17. Hirsch T, Marchetti P, Susin SA, et al. The apoptosis- 1993;12:2763–72. trial of perillyl alcohol administered daily. Clin Cancer necrosis paradox. Apoptogenic proteases activated after 29. Fitzgerald K, Harrington A, Leder P. Ras pathway Res 1998;4:1159–64. mitochondrial permeability transition determine the signals are required for notch-mediated oncogenesis. 6. Mascher H, Kikuta C, Schiel H. Pharmacokinetics of mode of cell death. Oncogene 1997;15:1573–81. Oncogene 2000;19:4191–8. menthol and carvone after administration of an enteric 18. Smith CA, Williams GT, Kingston R, Jenkinson EJ, 30. Wang CY, Guttridge DC, Mayo MW, Baldwin AS. NF- coated formulation containing peppermint oil and Owen JJ. Antibodies to CD3/T-cell receptor complex nB induces expression of the Bcl-2 homologue A1/Bfl-1 caraway oil. Arzneimittelforschung 2001;51:465–9. induce death by apoptosis in immature T cells in thymic to preferentially suppress chemotherapy-induced apo- 7. Vaughan TL, Farrow DC, Hansten PD, et al. Risk of cultures. Nature 1989;337:181–4. ptosis. Mol Cell Biol 1999;19:5923–9. esophageal and gastric adenocarcinomas in relation 19. Eastman A. Assays for DNA fragmentation, endonu- 31. Chiao PJ, Miyamoto S, Verma IM. Autoregulation of to use of calcium channel blockers, asthma drugs, cleases, and intracellular pH and calcium associated InBa activity. Proc Natl Acad Sci U S A 1994;91:28–32. and other medications that promote gastroesophage- with apoptosis. Methods Cell Biol 1995;46:41–55. 32. Schauer SL, Wang Z, Sonenshein GE, Rothstein TL. al reflux. Cancer Epidemiol Biomarkers Prev 1998;7: 20. Kao JP. Practical aspects of measuring calcium Maintenance of NF-nB/Rel and c-myc expression during 749–56. with fluorescent indicators. Methods Cell Biol 1994;40: CD40 ligand rescue of WEHI 231 early B cells from 8. Kenakin T. Drugs and receptors. An overview of the 155–81. receptor-mediated apoptosis through modulation of current state of knowledge. Drugs 1990;40:666–87. 21. Kaspar RL, Kakegawa T, Cranston H, Morris DR, InBa. J Immunol 1996;157:81–6. 9. Lukes RJ, Collins RD. Immunologic characterization of White MW. A regulatory cis element and a specific 33. Sutherland CL, Krebs DL, Gold MR. An 11-amino human malignant lymphomas. Cancer 1974;34 Suppl: binding factor involved in the mitogenic control of acid sequence in the cytoplasmic domain of CD40 is 1488–503. murine ribosomal protein L32 translation. J Biol Chem sufficient for activation of c-Jun N-terminal kinase, 10. Sadighi Akha AA, Willmott NJ, Brickley K, Dolphin 1992;267:508–14. activation of MAPKAP kinase-2, phosphorylation of AC, Galione A, Hunt SV. Anti-Ig-induced calcium influx 22. Perez-Reyes E, Wei XY, Castellano A, Birnbaumer L. InBa, and protection of WEHI-231 cells from anti- in rat B lymphocytes mediated by cGMP through a Molecular diversity of L-type calcium channels. Evi- IgM-induced growth arrest. J Immunol 1999;162: dihydropyridine-sensitive channel. J Biol Chem 1996;271: dence for alternative splicing of the transcripts of three 4720–30. 7297–300. non-allelic genes. J Biol Chem 1990;265:20430–6. 34. Hockerman GH, Peterson BZ, Johnson BD, Catterall 11. Savignac M, Badou A, Moreau M, et al. Protein kinase 23. Agoston A, Kunz L, Krieger A, Mayerhofer A. Two WA. Molecular determinants of drug binding and action C-mediated calcium entry dependent upon dihydropyr- types of calcium channels in human ovarian endocrine on L-type calcium channels. Annu Rev Pharmacol idine-sensitive channels: a T cell receptor-coupled cells: involvement in steroidogenesis. J Clin Endocrinol Toxicol 1997;37:361–96. signaling pathway involved in IL-4 synthesis. FASEB J Metab 2004;89:4503–12. 35. Berchtold CM, Ariazi EA, Gould MN. Perillyl alcohol 2001;15:1577–9. 24. Heubach JF, Graf EM, Leutheuser J, et al. Electro- mediated induction of apoptosis in human breast

www.aacrjournals.org 8565 Cancer Res 2005; 65: (18). September 15, 2005

cancer cells in vitro. Proc Annu Meet Am Assoc Cancer intracellular calcium levels inhibits myoblast differenti- expression in monoterpene-treated mammary carcino- Res 2000;40:487. ation. J Biol Chem 2002;277:28942–7. mas using subtractive display. J Biol Chem 1996;271: 36. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ, 41. Kotturi MF, Carlow DA, Lee JC, Ziltener HJ, Jefferies 29286–94. Sledge GW. Constitutive activation of NF-nB during WA. Identification and functional characterization of 46. Fields ER, Seufzer BJ, Oltz EM, Miyamoto SA. Switch progression of breast cancer to hormone-independent voltage-dependent calcium channels in T lymphocytes. in distinct InBa degradation mechanisms mediates growth. Mol Cell Biol 1997;17:3629–39. J Biol Chem 2003;278:46949–60. constitutive NF-nB activation in mature B cells. 37. Mitterdorfer J, Grabner M, Kraus RL, et al. Molecular 42. Crowell PL, Chang RR, Ren ZB, Elson CE, Gould MN. J Immunol 2000;164:4762–7. basis of drug interaction with L-type calcium channels. Selective inhibition of isoprenylation of 21-26-kDa 47. O’Connor S, Shumway SD, Amanna IJ, Hayes CE, J Bioenerg Biomembr 1998;30:319–34. proteins by the anticarcinogen D-limonene and its Miyamoto S. Regulation of constitutive p50/c-rel activity 38. Griffin S, Wyllie SG, Markham J. Determination of metabolites. J Biol Chem 1991;266:17679–5. via proteasome inhibitor-resistant InBa degradation in octanol-water partition coefficient for terpenoids using 43. Crowell PL, Ren Z, Lin S, Vedejs E, Gould MN. B cells. Mol Cell Biol 2004;24:4895–908. reversed-phase high-performance liquid chromatogra- Structure-activity relationships among monoterpene 48. Pianetti S, Arsura M, Romieu-Mourez R, Coffey RJ, phy. J Chromatogr A 1999;864:221–8. inhibitors of protein isoprenylation and cell prolifera- Sonenshein GE. Her-2/neu overexpression induces NF- 39. Ferry DR, Goll A, Glossmann H. Putative calcium tion. Biochem Pharmacol 1994;47:1405–15. nB via a PI3-kinase/Akt pathway involving calpain- channel molecular weight determination by target size 44. ShiW,GouldMN.Inductionofcytostasisin mediated degradation of InBa that can be inhibited by analysis. Naunyn Schmiedebergs Arch Pharmacol mammary carcinoma cells treated with the anticancer the tumor suppressor PTEN. Oncogene 2001;20:1287–99. 1983;323:292–7. agent perillyl alcohol. Carcinogenesis 2002;23:131–42. 49. Aggarwal BB. NF-nB: The enemy within. Cancer Cell 40. Porter GA, Makuck RF, Rivkees SA. Reduction in 45. Ariazi EA, Gould MN. Identifying differential gene 2004;6:203–28.

Cancer Res 2005; 65: (18). September 15, 2005 8566 www.aacrjournals.org

Craig M. Berchtold, Kai-Shun Chen, Shigeki Miyamoto, et al.

Cancer Res 2005;65:8558-8566.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/65/18/8558

Cited articles This article cites 46 articles, 21 of which you can access for free at: http://cancerres.aacrjournals.org/content/65/18/8558.full#ref-list-1

Citing articles This article has been cited by 6 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/65/18/8558.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/65/18/8558. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.