A pilot study of perillyl alcohol in pancreatic cancer.

BACKGROUND: Chemotherapy has been largely unsuccessful in pancreatic cancer. Measurement of cell-specific biological endpoints may clarify the evaluation of a newer generation of compounds. Perillyl alcohol has shown chemotherapeutic activity in preclinical systems through enhancing apoptosis. AIMS: To pilot a new trial template for testing novel agents in pancreatic cancer and to assess the biological activity of perillyl alcohol in patients with resectable pancreatic cancer. METHODS: Apoptosis was quantified with ApopTag in situ, Bak staining, and light microscopy. Tumor size, serum CA 19-9 level, and survival were also measured. RESULTS: Eight patients enrolled. Toxicity was mild and perillyl alcohol was generally well tolerated. Tumor size and CA 19-9 level were unchanged with perillyl alcohol treatment. Survival time was longer in patients who received full perillyl alcohol treatment (288 +/- 32 days) compared to those who did not (204 +/- 96 days), but this result did not achieve statistical significance (P = 0.2). There was a trend toward greater apoptosis in patients receiving perillyl alcohol compared to fresh operative controls; there was also a suggestion of greater apoptosis in tumor compared to normal pancreatic tissue in the same patient. CONCLUSIONS: Incorporation of cell-specific biological endpoints is challenging but feasible and should be used in clinical studies of pancreatic cancer treatment. Our pilot study suggests that perillyl alcohol may indeed have effects on biological endpoints. This study will serve as a useful template for examining cell-specific biological endpoints in the testing of future agents that are thought to induce apoptosis in pancreatic cancer.

Cell cycle effects of nonsteroidal anti-inflammatory drugs and enhanced growth inhibition in combination with gemcitabine in pancreatic carcinoma cells.

Increased cyclooxygenase-2 (COX-2) expression in human pancreatic adenocarcinomas, as well as the growth-inhibitory effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in vitro, suggests that NSAIDs may be an effective treatment for pancreatic cancer. Gemcitabine is currently the most effective chemotherapeutic drug available for patients with pancreatic cancer, but is only minimally effective against this aggressive disease. Clearly, other treatment options must be identified. To design successful therapeutic strategies involving compounds either alone or in combination with others, it is necessary to understand their mechanism of action. In the present study, we evaluated the effects of three NSAIDs (sulindac, indomethacin, and NS-398) or gemcitabine in two human pancreatic carcinoma cell lines, BxPC-3 (COX-2-positive) and PaCa-2 (COX-2-negative), previously shown to be growth-inhibited by these NSAIDs. Effects on cell cycle and apoptosis were investigated by flow cytometry or Western blotting. Treatment with NSAIDs or gemcitabine altered the cell cycle phase distribution as well as the expression of multiple cell cycle regulatory proteins in both cell lines, but did not induce substantial levels of apoptosis. Furthermore, we demonstrated that the combination of the NSAID sulindac or NS-398 with gemcitabine inhibited cell growth to a greater degree than either compound alone. These results indicate that the antiproliferative effects of NSAIDs and gemcitabine in pancreatic tumor cells are primarily due to inhibition of cell cycle progression rather than direct induction of apoptotic cell death, regardless of COX-2 expression. In addition, NSAIDs in combination with gemcitabine may hold promise in the clinic for the treatment of pancreatic cancer.

Cyclooxygenase-2 expression in human pancreatic adenocarcinomas.

Cyclooxygenase-2 (COX-2) expression is up-regulated in several types of human cancers and has also been directly linked to carcinogenesis. To investigate the role of COX-2 in pancreatic cancer, we evaluated COX-2 protein expression in primary human pancreatic adenocarcinomas (n = 23) and matched normal adjacent tissue (n = 11) by immunoblot analysis. COX-2 expression was found to be significantly elevated in the pancreatic tumor specimens compared with normal pancreatic tissue. To examine whether the elevated levels of COX-2 protein observed in pancreatic tumors correlated with the presence of oncogenic K-ras, we determined the K-ras mutation status in a subset of the tumors and corresponding normal tissues. The presence of oncogenic K-ras did not correlate with the level of COX-2 protein expressed in the pancreatic adenocarcinomas analyzed. These observations were also confirmed in a panel of human pancreatic tumor cell lines. Furthermore, in the pancreatic tumor cell line expressing the highest level of COX-2 (BxPC-3), COX-2 expression was demonstrated to be independent of Erk1/2 activation. The lack of correlation between COX-2 and oncogenic K-ras expression suggests that Ras activation may not be sufficient to induce COX-2 expression in pancreatic tumor cells and that the aberrant activation of signaling pathways other than Ras may be required for up-regulating COX-2 expression. We also report that the COX inhibitors sulindac, indomethacin and NS-398 inhibit cell growth in both COX-2-positive (BxPC-3) and COX-2-negative (PaCa-2) pancreatic tumor cell lines. However, suppression of cell growth by indomethacin and NS-398 was significantly greater in the BxPC-3 cell line compared with the PaCa-2 cell line (P = 0.004 and P < 0.001, respectively). In addition, the three COX inhibitors reduce prostaglandin E(2) levels in the BxPC-3 cell line. Taken together, our data suggest that COX-2 may play an important role in pancreatic tumorigenesis and therefore be a promising chemotherapeutic target for the treatment of pancreatic cancer.

Prevention and therapy of cancer by dietary monoterpenes.

Monoterpenes are nonnutritive dietary components found in the essential oils of citrus fruits and other plants. A number of these dietary monoterpenes have antitumor activity. For example, d-limonene, which comprises >90% of orange peel oil, has chemopreventive activity against rodent mammary, skin, liver, lung and forestomach cancers. Similarly, other dietary monoterpenes have chemopreventive activity against rat mammary, lung and forestomach cancers when fed during the initiation phase. In addition, perillyl alcohol has promotion phase chemopreventive activity against rat liver cancer, and geraniol has in vivo antitumor activity against murine leukemia cells. Perillyl alcohol and d-limonene also have chemotherapeutic activity against rodent mammary and pancreatic tumors. As a result, their cancer chemotherapeutic activities are under evaluation in Phase I clinical trials. Several mechanisms of action may account for the antitumor activities of monoterpenes. The blocking chemopreventive effects of limonene and other monoterpenes during the initiation phase of mammary carcinogenesis are likely due to the induction of Phase II carcinogen-metabolizing enzymes, resulting in carcinogen detoxification. The post-initiation phase, tumor suppressive chemopreventive activity of monoterpenes may be due to the induction of apoptosis and/or to inhibition of the post-translational isoprenylation of cell growth-regulating proteins. Chemotherapy of chemically induced mammary tumors with monoterpenes results in tumor redifferentiation concomitant with increased expression of the mannose-6-phosphate/insulin-like growth factor II receptor and transforming growth factor beta1. Thus, monoterpenes would appear to act through multiple mechanisms in the chemoprevention and chemotherapy of cancer.

Effects of the antitumor agent perillyl alcohol on H-Ras vs. K-Ras farnesylation and signal transduction in pancreatic cells.

BACKGROUND: Perillyl alcohol has chemotherapeutic activity against pancreas cancers that have a K-ras oncogene, and it inhibits the prenylation of Ras and other proteins in many cell types. MATERIALS AND METHODS: We tested the hypothesis that perillyl alcohol would impair Ras farnesylation and Ras signal transduction pathways in pancreatic tumor cells. RESULTS: In B12/13 pancreatic tumor cells that had a K-ras oncogene, perillyl alcohol inhibited total protein prenylation and decreased Ras farnesylation. However, the decrease in Ras farnesylation was not sufficient to affect Ras GTP/GDP ratios or MAP kinase phosphorylation. We then investigated the effects of perillyl alcohol on H-Ras vs. K-Ras. Interestingly, H-Ras, but not K-Ras, farnesylation was inhibited by perillyl alcohol, and perillyl alcohol inhibited MAP kinase phosphorylation in H-ras but not K-ras oncogene-transformed pancreatic cells. CONCLUSIONS: The antitumor activity of perillyl alcohol against pancreatic cancers may stem from its ability to inhibit the prenylation of growth-regulatory proteins other than K-Ras, including H-Ras.

Induction of the apoptosis-promoting protein Bak by perillyl alcohol in pancreatic ductal adenocarcinoma relative to untransformed ductal epithelial cells.

Perillyl alcohol has antitumor activity toward pancreas and other cancers with low toxicity. Here, we have investigated the mechanism of action responsible for the differential sensitivity of malignant versus non-malignant pancreatic cells to the drug. We report that the rate of apoptosis is over 6-fold higher in perillyl alcohol-treated pancreatic adenocarcinoma cells than in untreated cells, and that the effect of perillyl alcohol on pancreatic tumor cells is significantly greater than its effect on non-malignant pancreatic ductal cells. Moreover, the perillyl alcohol-induced increase in apoptosis in all of the pancreatic tumor cells is associated with a 2- to 8-fold increase in the expression of the proapoptotic protein Bak, but Bak expression is not affected by perillyl alcohol in non-malignant cells. Thus, the antitumor activity of perillyl alcohol toward pancreatic cancers may be due to preferential stimulation of Bak-induced apoptosis in malignant versus normal cells. Bak may, therefore, be a useful biomarker for the chemopreventive and therapeutic effects of perillyl alcohol.

Inhibition of pancreatic cancer growth by the dietary isoprenoids farnesol and geraniol.

Fruits and vegetables have protective effects against many human cancers, including pancreatic cancer. Isoprenoids are one class of phytochemicals which have antitumor activity, but little is known about their effects on cancer of the pancreas. We tested the hypothesis that isoprenoids would inhibit the growth of pancreatic tumor cells. Significant (60-90%) inhibition of the anchorage-independent growth of human MIA PaCa2 pancreatic tumor cells was attained with 25 microM farnesol, 25 microM geranylgeraniol, 100 microM perillyl amine, 100 microM geraniol, or 300 microM perillyl alcohol. We then tested the relative in vivo antitumor activities of dietary farnesol, geraniol, and perillyl alcohol against transplanted PC-1 hamster pancreatic adenocarcinomas. Syrian Golden hamsters fed geraniol or farnesol at 20 g/kg diet exhibited complete inhibition of PC-1 pancreatic tumor growth. Both farnesol and geraniol were more potent than perillyl alcohol, which inhibited tumor growth by 50% at 40 g/kg diet. Neither body weights nor plasma cholesterol levels of animals consuming isoprenoid diets were significantly different from those of pair-fed controls. Thus, farnesol, geraniol, and perillyl alcohol suppress pancreatic tumor growth without significantly affecting blood cholesterol levels. These dietary isoprenoids warrant further investigation for pancreatic cancer prevention and treatment.

Monoterpenes in breast cancer chemoprevention.

A number of dietary monoterpenes have chemopreventive activity against rat mammary cancer. For example, d-limonene, which comprises over 90% of orange peel oil, has chemopreventive activity against rodent mammary cancer during the initiation phase as well as the promotion/progression phase. Similarly, the monoterpenoids carveol, uroterpenol, and sobrerol have chemopreventive activity against mammary cancer when fed during the initiation phase. d-limonene and perillyl alcohol, a more potent analog of limonene, also have chemotherapeutic activity against rodent mammary and pancreatic tumors. As a result, their cancer chemotherapeutic activities are under evaluation in Phase I clinical trials. Several mechanisms of action may account for the antitumor activities of monoterpenes. The blocking chemopreventive effects of limonene and other monoterpenes during the initiation phase of mammary carcinogenesis are due to the induction of Phase II carcinogen-metabolizing enzymes, resulting in carcinogen detoxification. The post-initiation phase chemopreventive and chemotherapeutic activities of monoterpenes may be due to the induction of tumor cell apoptosis, tumor redifferentiation, and/or inhibition of the post-translational isoprenylation of cell growth-regulating proteins. Thus, monoterpenes act through multiple mechanisms in the chemoprevention of mammary and other cancers.

Prenylation of oncogenic human PTP(CAAX) protein tyrosine phosphatases.

Many isoprenylated proteins are known to participate in signal transduction, but not all have been identified. Using an in vitro prenylation screen, two human cDNAs (PTP(CAAXI) and PTP(CAAX2)) homologous to the rat PRL-1 and human OV-1 protein tyrosine phosphatase genes were identified. PTP(CAAXI) and PTP(CAAX2) were farnesylated in vitro by mammalian farnesyl:protein transferase, and epitope-tagged PTP(CAAX2) was prenylated in epithelial cells. Overexpression of PTP(CAAXI) and PTP(CAAX2) in epithelial cells caused a transformed phenotype in culture and tumor growth in nude mice. Thus, PTP(CAAXI) and PTP(CAAX2) represent a novel class of isoprenylated, oncogenic protein tyrosine phosphatases.

Antitumorigenic effects of limonene and perillyl alcohol against pancreatic and breast cancer.

Perillyl alcohol is a natural product from cherries and other edible plants. Perillyl alcohol and d-limonene, a closely related dietary monoterpene, have chemotherapeutic activity against pancreatic, mammary, and prostatic tumors. In addition, perillyl alcohol, limonene, and other dietary monoterpenes have chemopreventive activity. Several mechanisms may account for the antitumorigenic effects of monoterpenes. For example, many monoterpenes inhibit the post-translational isoprenylation of cell growth-regulatory proteins such as Ras. Perillyl alcohol induces apoptosis without affecting the rate of DNA synthesis in both liver and pancreatic tumor cells. In addition, monoterpene-treated, regressing rat mammary tumors exhibit increased expression of transforming growth factor beta concomitant with tumor remodeling/redifferentiation to a more benign phenotype. Monoterpenes are effective, nontoxic dietary antitumor agents which act through a variety of mechanisms of action and hold promise as a novel class of antitumor drugs for human cancer.

Chemotherapy of pancreatic cancer with the monoterpene perillyl alcohol.

Perillyl alcohol has antitumor activity against rat mammary and liver cancer. Here, we report the chemotherapeutic effects of perillyl alcohol on pancreatic cancer. Perillyl alcohol reduced the growth of hamster pancreatic tumors to less than half that of controls (P < 0.025). Moreover, 16% of perillyl alcohol-treated pancreatic tumors completely regressed whereas no control tumors regressed (P < 0.05). Perillyl alcohol induced contact inhibition in cultured human pancreatic carcinoma cells and inhibited their anchorage-independent growth (P < 0.001). Thus, perillyl alcohol has antitumor activity against pancreatic carcinomas at non-toxic doses, and may be an effective chemotherapeutic agent for human pancreatic cancer.

Structure-activity relationships among monoterpene inhibitors of protein isoprenylation and cell proliferation.

The monoterpene d-limonene inhibits the post-translational isoprenylation of p21ras and other small G proteins, a mechanism that may contribute to its efficacy in the chemoprevention and therapy of chemically induced rodent cancers. In the present study, the relative abilities of 26 limonene-like monoterpenes to inhibit protein isoprenylation and cell proliferation were determined. Many monoterpenes were found to be more potent than limonene as inhibitors of small G protein isoprenylation and cell proliferation. The relative potency of limonene-derived monoterpenes was found to be: monohydroxyl = ester = aldehyde > thiol > acid = diol = epoxide > triol = unsubstituted. All monoterpenes that inhibited protein isoprenylation did so in a selective manner, such that 21-26 kDa proteins were preferentially affected. Perillyl alcohol, one of the most potent terpenes, reduced 21-26 kDa protein isoprenylation to 50% of the control level at a concentration of 1 mM, but had no effect on the isoprenylation of 67, 47 or 17 kDa proteins. In particular, p21ras farnesylation was inhibited 40% by 1 mM perillyl alcohol. At the same concentration, perillyl alcohol completely inhibited the proliferation of human HT-29 colon carcinoma cells. The structure-activity relationships observed among the monoterpene isoprenylation inhibitors support a role for small G proteins in cell proliferation, and suggest that many limonene-derived monoterpenes warrant further investigation as antitumor agents.

Human metabolism of the experimental cancer therapeutic agent d-limonene.

d-Limonene has efficacy in preclinical models of breast cancer, causing > 80% of carcinomas to regress with little host toxicity. We performed a pilot study on healthy human volunteers to identify plasma metabolites of limonene and to assess the toxicity of supradietary quantities of d-limonene. Seven subjects ingested 100 mg/kg limonene in a custard. Blood was drawn at 0 and 24 h for chemistry-panel analysis and at 0, 4, and 24 h for limonene-metabolite analysis. On-line capillary gas chromatography/mass spectrometry (GC/MS) analysis indicated that at least five compounds were present at 4 h that were not present at time zero. Two major peaks were identified as the rat limonene metabolites dihydroperillic acid and perillic acid, and two minor peaks were found to be the respective methyl esters of these acids. A third major peak was identified as limonene-1,2-diol. Limonene was a minor component. At a dose of 100 mg/kg, limonene caused no gradable toxicity. Limonene is metabolized by humans and rats in a similar manner. These observations and the high therapeutic ratio of limonene in the chemotherapy of rodent cancers suggest that limonene may be an efficacious chemotherapeutic agent for human malignancies.

Chemoprevention and therapy of cancer by d-limonene.

The naturally occurring monoterpene d-limonene has chemopreventive and chemotherapeutic activity against many rodent solid tumor types. The chemopreventive activity of limonene during initiation can be attributed to the induction of phase I and phase II enzymes, with resulting carcinogen detoxification. The chemopreventive activity of limonene during promotion/progression may be due in part to inhibition of the posttranslational isoprenylation of growth-controlling small G proteins, such as p21ras. The complete regression of mammary carcinomas by limonene appears to involve tissue redifferentiation. The multiple antitumorigenic effects of limonene are attainable at a high therapeutic ratio, suggesting that limonene and related monoterpenes may be efficacious in the chemoprevention and chemotherapy of human malignancies.

Chemoprevention of mammary carcinogenesis by hydroxylated derivatives of d-limonene.

The monoterpene d-limonene has been shown to an effective, non-toxic chemopreventive agent in mammary and other rodent tumor models. The studies reported here investigated structure-activity relationships among limonene and three hydroxylated derivatives in the prevention of dimethylbenz[a]anthracene (DMBA)-induced mammary cancer. Rats were fed control or 1% limonene, carveol, uroterpenol or sobrerol diets from 2 weeks before to one week after carcinogen administration. Carveol, uroterpenol and sobrerol significantly prolonged tumor latency and decreased tumor yield. Sobrerol was the most potent of the monoterpenes tested, decreasing tumor yield to half that of the control, a level previously achieved with 5% limonene diets. Excretion of radioactivity from [3H]DMBA was doubled in rats fed 5% limonene and nearly tripled in rats fed 1% sobrerol. Sobrerol is thus 5-fold more potent than limonene in both enhancing carcinogen excretion and in preventing tumor formation. These data demonstrate that hydroxylation of monoterpenes affects chemopreventive potential, with 2 hydroxyl groups greater than 1 greater than 0. Sobrerol, carveol and uroterpenol are novel cancer chemopreventive agents with little or no toxicity.

Identification of metabolites of the antitumor agent d-limonene capable of inhibiting protein isoprenylation and cell growth.

Limonene has been shown to be an effective, nontoxic chemopreventive and chemotherapeutic agent in chemically induced rat mammary-cancer models. The present study characterized circulating metabolites of limonene in female rats and determined their effects on cell growth. Metabolism of limonene was analyzed in plasma extracts by gas chromatography. Rapid conversion of limonene to two major metabolites was detected. These metabolites comprised more than 80% of the circulating limonene-derived material at 1 h after administration and thereafter, whereas limonene itself accounted for only 15%. The metabolites were characterized by mass spectroscopy and infrared spectroscopy. The probable structures were synthesized, and identities were confirmed by comparison of retention times and mass spectra. The two major circulating metabolites of limonene were found to be perillic acid and dihydroperillic acid. We have previously reported that limonene, perillic acid, and dihydroperillic acid inhibit the posttranslational isoprenylation of p21ras and other 21- to 26-kDa cell-growth-associated proteins in NIH3T3 cells and in mammary epithelial cells. In the present study, perillic acid was found to inhibit cell growth in a dose-dependent manner. Thus, perillic acid and dihydroperillic acid, the two major circulating metabolites of limonene in the rat, are more potent inhibitors of protein isoprenylation than is limonene, and perillic acid is also a more potent inhibitor of cell growth. These data raise the possibility that the antitumor effects of limonene in vivo may be mediated via perillic acid and, perhaps, other metabolites.

Selective inhibition of isoprenylation of 21-26-kDa proteins by the anticarcinogen d-limonene and its metabolites.

Limonene has chemotherapeutic activity against chemically induced rat mammary carcinomas, many of which contain activated ras genes. Given the recent discovery of the post-translational modification of p21ras and other cell growth-associated proteins by intermediates in the mevalonic acid pathway, and the common biochemical origins of limonene and these isoprene products, we investigated the effect of limonene on protein isoprenylation. NIH3T3 and human mammary epithelial cells were incubated with lovastatin and [2-14C]mevalonolactone in the absence and presence of limonene. Labeled proteins were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Limonene inhibited isoprenylation of a class of cellular proteins of 21-26 kDa, including p21ras and possibly other small GTP-binding proteins, in a dose-dependent manner in both cell lines. In contrast, limonene did not affect the isoprenylation of several other proteins, including nuclear lamins. Limonene is metabolized extensively in vivo but not in cultured cells. The two major rat serum metabolites of limonene, perillic acid and dihydroperillic acid, were more potent than limonene in the inhibition of isoprenylation. These results demonstrate that limonene selectively inhibits isoprenylation of 21-26-kDa proteins at a point in the mevalonic acid pathway distal to 3-hydroxy-3-methylglutaryl coenzyme A reductase, and they provide a plausible explanation for its chemotherapeutic activity. Inhibition of isoprenylation of proteins such as p21ras and other small GTP-binding proteins would alter their intracellular localization and, hence, disrupt their biological activity.

High branched-chain alpha-keto acid intake, branched-chain alpha-keto acid dehydrogenase activity, and plasma and brain amino acid and plasma keto acid concentrations in rats.

Diets containing high quantities of individual branched-chain alpha-keto acids (BCKAs) or a combination of BCKAs as used for treatment of renal disease were fed to rats. When the diet contained a single BCKA, its concentration was high in plasma and the concentration of its corresponding amino acid was high in plasma and brain. Liver BCKA dehydrogenase (BCKD) was 42% active in control rats. Consumption of diets containing 0.38 mol/kg diet of alpha-ketoisocaproate (KIC), alpha-keto-beta-methylvalerate (KMV), or alpha-ketoisovalerate (KIV) resulted in complete activation of liver BCKD. Consumption of the diet containing the combination of BCKAs increased basal BCKD activity of liver twofold. Muscle BCKD was activated after feeding the KIV diet (2-fold), the KIC diet (3-fold), and the KMV diet (15-fold). Total BCKD activity of liver and muscle was unaffected by dietary treatments. Activation of liver and muscle BCKD by dietary BCKA is consistent with their ability to inhibit BCKD kinase in vitro.

Activities of branched-chain amino acid--degrading enzymes in liver from rats fed different dietary levels of protein.

The relationships among dietary protein intake, plasma branched-chain amino acid (BCAA) and keto acid (BCKA) concentrations, and liver BCAA-degrading enzyme activities were investigated in rats fed, for 5 h/d for 2, 6 or 9 d, diets containing from 0 to 50% casein. Plasma, liver and muscle BCAA concentrations were proportional to protein intake over the entire range tested; plasma BCKA concentration, however, was proportional only in the range from 0 to 20% casein, after which a plateau was reached. By d 2, liver cytosolic BCAA aminotransferase activity had increased in rats fed 50% casein; by d 9, activity had increased in rats fed 0 or 5% casein as well. Liver mitochondrial BCAA aminotransferase activity was unresponsive to dietary treatment. Basal liver BCKA dehydrogenase activity and the percent active complex were proportional to protein intake on d 2 and 6. On d 2, total BCKA dehydrogenase activity was the same in all groups; by d 6, total activity had increased in rats fed 30 or 50% casein. We conclude that although the adaptive changes in BCAA-degrading enzyme activities are small, they are sufficient to compensate for excessively high or low protein intakes, so that tolerable concentrations of BCAA and BCKA are maintained.