Perillyl Alcohol Treatment Research Articles

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

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-kappaB (NF-kappaB), 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-kappaB 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-kappaB because its prior activation abolished the cell killing effects of perillyl alcohol treatment. Our findings suggest that perillyl alcohol can inhibit NF-kappaB 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-kappaB pathway as a molecular target of perillyl alcohol activity in different cancer cell types.

Perillyl alcohol is an angiogenesis inhibitor.

Aberrant angiogenesis is essential for the progression of solid tumors and hematological malignancies. Thus, antiangiogenic therapy is one of the most promising approaches to control cancer. In the present work, we examined the ability of perillyl alcohol (POH), a dietary monoterpene with well-established tumor chemopreventive and chemotherapeutic activity, to interfere with the process of angiogenesis. POH remarkably prevented new blood vessel growth in the in vivo chicken embryo chorioallantoic membrane assay and proved to be effective in inhibiting the morphogenic differentiation of cultured endothelial cells into capillary-like networks both in collagen gel and Matrigel models. In addition, POH reduced the cell number in a proliferation assay and induced apoptosis of endothelial cells as indicated by the POH-mediated increase of caspase-3 activity and DNA fragmentation. Consistent with the observed antisurvival effect, POH treatment resulted in a significant inhibition of Akt phosphorylation in endothelial cells. Finally, POH was able to differentially modulate the release of two important angiogenic regulators: vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2). POH decreased the release of VEGF from cancer cells but stimulated the expression of Ang2 by endothelial cells, indicating that it might suppress neovascularization and induce vessel regression. Overall, these data underscore the antiangiogenic potential of POH and suggest that POH, in addition to its anticancer activity, may be an effective agent in the treatment of angiogenesis-dependent diseases.

Perillyl alcohol-mediated inhibition of lung cancer cell line proliferation: potential mechanisms for its chemotherapeutic effects

Perillyl alcohol (POH) is currently being tested in clinical trials as an anticancer agent, though its mechanism of action has not been definitively established. We treated two human lung cancer cell lines, H322 and H838, with POH to determine its antitumor properties. A sulforhodamine B (SRB) cell proliferation assay was used to determine the effects of POH after 1 and 5 days of treatment with 0.25, 0.5, 0.75, 1.0, and 1.5 mM POH. After 1 day of treatment, little difference could be seen between the lowest and highest concentrations of POH. However, after 5 days, both cell lines showed a dose-dependent decrease in cell proliferation that ranged from 15% to 83%. A clonogenic assay confirmed these results—while there was no significant effect of POH after 1 day of exposure, a dose-dependent decrease in colony formation, ranging from 15% to 100%, was seen after 5 days of treatment. Time-lapse video microscopy revealed that apoptotic cells were evident within 24–48 h of treatment with 1.5 mM POH. The appearance of apoptotic cells was preceded by increased caspase-3 activity and cleavage of poly (ADP-ribose) polymerase (PARP) as POH activated caspase-3 activity 3–6-fold. Nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) confirmed the classical characteristics of apoptosis in POH-treated cells. DNA microarray expression analysis was performed following 8 and 24 (H322) or 8 and 48 (H838) h of treatment with 1.5 mM POH. While a large number of genes were up- or downregulated in the two cell lines at various times after POH treatment, the levels of expression of only eight genes were up- or down-related in both cell lines at both of the time points examined. The significance of these genes as potential mediators of POH action is still uncertain, but the limited number of commonly up- or downregulated genes detected by microarray expression analysis suggests that POH may mediate its effects via posttranscriptional mechanisms. Our results suggest that POH may have potential use as an anticancer drug that stimulates or sensitizes lung tumor cells to apoptosis, and this effect may depend on genetic lesions present in tumor cells.

Perillyl Alcohol as a Radio-/Chemosensitizer in Malignant Glioma

The prognosis for patients with malignant glioma has not significantly changed in two decades, despite advances in surgery, radiation, and chemotherapy, emphasizing the growing need for novel approaches to glioma therapy. Perillyl alcohol (POH) is a naturally occurring monoterpene that has been shown to possess chemotherapeutic as well as chemopreventive activity in animal tumor models and is currently in Phase I and Phase II clinical trials. In the present study, we have demonstrated that POH is an effective radiosensitizer at clinically relevant doses of radiation using established glioma cell lines. POH caused a transient arrest in the G2/M phase of the cell cycle and induced apoptosis in glioma cells. POH treatment sensitized glioma cells to Fas-mediated apoptosis, which was further augmented in the presence of ionizing radiation and abrogated in the presence of antagonistic antibody. POH-induced radiosensitization was partially inhibited in glioma cells expressing dominant negative Fas-associated death domain and completely inhibited in glioma cells overexpressing the cytokine response modifier A. In addition, POH treatment resulted in a dose-dependent sensitization to cisplatin and doxorubicin induced cytotoxicity in glioma cells, highlighting its usefulness as a potent radio/chemosensitizer in the treatment of malignant glioma.

Perillyl alcohol mediated radiosensitization via augmentation of the Fas pathway in prostate cancer cells.

The management of hormone-insensitive locally advanced prostate cancer is difficult and complex and there is an urgent need for the development of effective chemotherapeutic agents intended for combination with currently available treatment modalities. The present paper demonstrates the effectiveness of the monoterpene perillyl alcohol (POH) as potent radiosensitizer on DU145 and PC3 cell lines by performing clonogenic survival assays, cycle analysis, and assays to detect viability, apoptosis, and Fas receptor/ligand by flow cytometry. POH pretreatment resulted in a dose dependent sensitization to kill cell by radiation. Furthermore, POH treatment induced a transient G(2)/M arrest, enhanced the expression of the membrane bound form of the Fas ligand and sensitized the cells to Fas mediated apoptosis. The unique manner of radiosensitization in addition to its low toxicity profile makes POH a promising new agent for preclinical evaluation as a potential radiosensitizer in the treatment of prostate cancer.

Induction of cytostasis in mammary carcinoma cells treated with the anticancer agent perillyl alcohol.

The monoterpene perillyl alcohol (POH) has preventive and therapeutic effects in a wide variety of pre-clinical tumor models, including those for breast cancers, and is currently being tested in human phase I clinical trials. POH causes both cytostasis and apoptosis in rat mammary carcinomas. In vitro, POH inhibits cellular proliferation in a variety of mammalian cell lines. Here we investigated the mechanisms underlying cytostasis by studying the effects of POH on the cell cycle in vitro using the murine mammary transformed cell line TM6. In TM6 cells, POH causes an early G(1) cell-cycle block and slows the G(2)-M transition. An increase in pRB in its hypophosphorylated state is associated with the early G(1) block caused by POH. POH treatment inhibits two important targets in the cells during the G(1)-S transition: cyclin D1- and cyclin E-associated kinase. POH treatment leads to a reduction in cyclin D1 RNA and protein levels and prevents the formation of active cyclin D1-associated kinase complexes in synchronous cells during the exit of G(0) and entry into the cell cycle. In addition, POH treatment induces an increased association of p21(WAF1) with cyclin E-Cdk2 complexes, and inhibits the activating phosphorylation of Cdk2. All these effects of POH may contribute to the inhibition of the transition out of the G(1) phase of the cell cycle.

Failure to demonstrate chemoprevention by the monoterpene perillyl alcohol during early rat hepatocarcinogenesis: a cautionary note.

The monoterpene perillyl alcohol (PA) is being considered as a useful chemopreventive and therapeutic agent against human cancers. However, no data are available on the effects of PA in the first stages of hepatocarcinogenesis. To study such effects, putatively initiated cells and preneoplastic foci in hepatocarcinogenesis were used as a model. Male Wistar rats were treated with a single dose of N:-nitrosomorpholine (NNM). Between days 4 and 91 after NNM, subgroups of rats received either PA (1 g/kg body wt/day) or phenobarbital (PB) (50 mg/kg body wt/day) in the diet. Since PA treatment reduced food intake, one control group was fed ad libitum, while a second control was pair fed between days 4 and 91. In order to enhance any treatment effects, all groups, including the controls, were treated with the potent tumor promoter PB after day 91 until the end of the experiment at day 266. Rats were killed at multiple time points and putatively initiated cells and preneoplastic foci were identified by staining positively for placental glutathione S-transferase (G+). The following results were obtained. (i) A few days after NNM treatment single G+ cells emerged; a considerable portion of which developed into foci. (ii) Treatment with PB resulted in an increase in number and size of G+ foci. (iii) PA treatment failed to reduce the number of G+ cells; it somewhat lowered rates of apoptosis in G+ foci and clearly increased their average size. (iv) Eighty-seven days of PA revealed no protective effect on day 266, but, similar to PB treatment, increased the growth of foci. In conclusion, PA exerted no detectable chemopreventive effect in the early stages of rat hepatocarcinogenesis. It rather exerted a PB-like tumor promoting activity. These data argue against a recommendation of PA as a chemopreventive agent for healthy humans.

CHEMOPREVENTIVE EFFICACY OF PROMISING FARNESYLTRANSFERASE INHIBITORS

The studies presented were designed to test the efficacy of farnesyltransferase inhibitors (FTIs) as potential chemopreventive compounds in the mouse lung tumor model, and in tumor cell lines. The compounds included manumycin, gliotoxin, dihydroepiandrosterone (DHEA), perillyl alcohol (POH), and FTI-276. Each of these compounds had the potential, based on in vitro and limited in vivo evidence, to inhibit mouse lung tumorigenesis. In vitro studies were conducted with both K-ras-transformed NIH-3T3 cells and mouse lung tumor epithelial cell lines. Weutilized 2 primary mouse lung tumor models that reliably produce lung tumors with an oncogenic K-ras mutation when induced by 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Manumycin, gliotoxin, DHEA, and POH were administered 3 times per week peritoneally (IP), starting 1 week prior to carcinogen treatment, and throughout the test period (4.5 months). FTI-276 was delivered daily for 4 months by a time-release pellet method. Both the manumycin and gliotoxin treatment groups demonstrated 100% incidence and an increase in tumor multiplicity over control, of 66% and 58% increase respectively (P<.05). Although DHEA showed no significant chemopreventive effect, POH treatment demonstrated a 22% reduction in tumor incidence (P<.05) and a 58% reduction in tumor multiplicity (P<.05). Finally, FTI-276 reduced both the tumor multiplicity by 41.7% (P <.005), and the total tumor volume/burden per mouse by 79.4% (P<.0001). The apoptotic index in FTI-276-treated tumors showed an increase of 77% over control tumors (P<.05). In vitro, all compounds demonstrated growth inhibition at a dose-response manner; however, manumycin, gliotoxin, and DHEA demonstrated an initial increase in growth rate at lower doses. In summary, we have shown that POH and FTI-276 are chemopreventive in a primary mouse lung tumor model. In contrast, DHEA was not significantly chemopreventive at the dosage utilized, and treatment of an immunocompetent host with manumycin or gliotoxin demonstrated a significant increase in tumorigenicity over carcinogen control.

Enhancement of sterol synthesis by the monoterpene perillyl alcohol is unaffected by competitive 3-hydroxy-3-methylglutaryl-CoA reductase inhibition.

Monoterpenes such as limonene and perillyl alcohol (PA) are currently under investigation for their chemotherapeutic properties which have been tied to their ability to affect protein isoprenylation. Because PA affects the synthesis of isoprenoids, such as ubiquinone, and cholesterol is the end product of the synthetic pathway from which this isoprenoid pathway branches, we investigated the effects of this compound upon cholesterol metabolism in the colonic adenocarcinoma cell line SW480. PA (1 mM) inhibited incorporation of 14C-mevalonate into 21-26 kDa proteins by 25% in SW480 cells. Cholesterol (CH) biosynthesis was assessed by measuring the incorporation of 14C-acetate and 14C-mevalonate into 27-carbon-sterols. Cells treated with PA (1 mM) exhibited a fourfold increase in the incorporation of 14C-acetate but not 14C-mevalonate into cholesterol. Mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase, at 2 microM concentration, inhibited CH synthesis from 14C-acetate by 80%. Surprisingly, concurrent addition of mevinolin and PA did not significantly alter the stimulatory effects of PA. As observed differences in 14C-acetate and 14C-mevalonate precursor labeling could indicate PA affects early pathway events, the effects of this monoterpene on HMG-CoA reductase activity were evaluated. Unexpectedly, 1 mM PA did not stimulate activity of this enzyme. Consistent with its action as a reversibly bound inhibitor, in washed microsomes, 2 microM mevinolin pretreatment increased reductase protein expression causing a 12.7 (+/- 2.4)-fold compensatory HMG-CoA reductase activity increase; concurrent treatment with 1 mM PA attenuated this to a 5.3 (+/- 0.03)-fold increase. Gas chromatographic analysis confirmed CH was the major lipid present in the measured thin-layer chromatography spot. Since 14C-acetate incorporation into free fatty acid and phospholipid pools was not significantly affected by PA treatment, nonspecific changes in whole acetate pool sizes were not indicated. Because increases in endogenous CH synthesis should result in compensatory changes in exogenous sterol utilization, the effects of PA upon low density lipoprotein (LDL) receptor activity were evaluated. Consistent with the observed increases in CH synthesis, 1 mM PA decreased 125I-LDL internalization to 50% of the fetal bovine serum control; concurrent addition of 2 microM mevinolin attenuated this effect to a reduction of 80% of the control value. Data suggest that in certain colonic tumor cells PA strongly affects cholesterol metabolism via a mechanism of action that is insensitive to the HMG-CoA reductase inhibitor mevinolin.

R-(+)-perillyl alcohol-induced cell cycle changes, altered actin cytoskeleton, and decreased ras and p34 cdc2 expression in colonic adenocarcinoma SW480 cells

Monoterpenes as S-(-)-perillyl alcohol (PA) have been shown to inhibit the isoprenylation of such growth regulatory proteins as ras. In this study, we investigated the effects of the R-(+) enantiomer of PA on cell cycle, signaling, and cytoskeletal control in the colonic adenocarcinoma cell line SW480, which carries a K- ras mutation. Cell cycle analysis by flow cytometry of SW480 cells treated with 1 mM PA for 24 hours demonstrated an increase in the number of cells in G0/G1 with a decrease in S phase, compared with untreated control cells. These cell cycle changes correlated with an inhibition of protein isoprenylation from 14C-mevalonate and decreased expression of the cell cycle regulatory kinase p34 cdc2. Additionally, PA-treated cells acquired a flattened morphology with a condensation of cytoskeletal actin spikes to the periphery. This was in contrast to treatment with 15 μM mevinolin (MVN), a direct mevalonate synthesis inhibitor, which imparted to SW480 cells a more rounded and spindly morphology, associated with the depolymerization of actin microfilaments. Together, these data suggest that fluctuations in mevalonate and isoprenoid pools may involve different morphologic phenomenon. Because ras mediated signaling is related to the organization of the actin cytoskeleton, we investigated the effects of PA on the isoprenylation of ras. Although MVN treatment inhibited ras farnesylation, PA treatment decreased the expression of total ras protein. In summary, R-(+)-PA-induced cell signaling events correlated with alterations in the organization of cytoskeletal actin and decreased protein expression of growth regulatory proteins, such as ras and cdc2 kinase. These effects may contribute to the growth inhibitory activity of R-(+)-PA.