Pamela L. Crowell

Prenylation of oncogenic human PTPCAAX 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.

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 μM farnesol, 25 μM geranylgeraniol, 100 μM perillyl amine, 100 μM geraniol, or 300 μM perillyl alcohol. We then tested the relative in vivo antitumor activities of dietary farnesol, geraniol, and perilyl 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.

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.

Cell Cycle Arrest by the Isoprenoids Perillyl Alcohol, Geraniol, and Farnesol Is Mediated by p21Cip1 and p27Kip1 in Human Pancreatic Adenocarcinoma Cells

Pancreatic cancer, the fourth leading cause of cancer-associated mortality in the United States, usually presents in an advanced stage and is generally refractory to chemotherapy. As such, there is a great need for novel therapies for this disease. The naturally derived isoprenoids perillyl alcohol, farnesol, and geraniol have chemotherapeutic potential in pancreatic and other tumor types. However, their mechanisms of action in these systems are not completely defined. In this study, we investigated isoprenoid effects on the cell cycle and observed a similar antiproliferative mechanism of action among the three compounds. First, when given in combination, the isoprenoids exhibited an additive antiproliferative effect against MIA PaCa-2 human pancreatic cancer cells. Furthermore, all three compounds induced a G0/G1 cell cycle arrest that coincided with an increase in the expression of the cyclin kinase inhibitor proteins p21Cip1 and p27Kip1 and a reduction in cyclin A, cyclin B1, and cyclin-dependent kinase (Cdk) 2 protein levels. Immunoprecipitation studies demonstrated increased association of both p21Cip1 and p27Kip1 with Cdk2 as well as diminished Cdk2 kinase activity after isoprenoid exposure, indicating a cell cycle-inhibitory role for p21Cip1 and p27Kip1 in pancreatic adenocarcinoma cells. When siRNA was used to inhibit expression of p21Cip1 and p27Kip1 proteins in MIA PaCa-2 cells, conditional resistance to all three isoprenoid compounds was evident. Given similar findings in this cell line and in BxPC-3 human pancreatic adenocarcinoma cells, we conclude that the chemotherapeutic isoprenoid compounds perillyl alcohol, farnesol, and geraniol invoke a p21Cip1- and p27Kip1-dependent antiproliferative mechanism in human pancreatic adenocarcinoma cells.

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.

Enhanced cell cycle progression and down regulation of p21Cip1/Waf1 by PRL tyrosine phosphatases

Human PRL-1, PRL-2, and PRL-3 tyrosine phosphatases induce the malignant transformation of epithelial cells. We tested the hypothesis that the oncogenic effects of PRL occur by increasing cellular proliferation. Cells stably transfected with PRL-1 or PRL-2 exhibited 2.7-3.3-fold increases over control cells in the rate of DNA synthesis and the proportion of cells in S-phase, and they progressed more rapidly from G1 into S. In addition, cells overexpressing either PRL-1 or PRL-2 exhibited enhanced cyclin-dependent kinase 2 (CDK2) activity and significantly lower p21(Cip1/Waf1) protein levels, and PRL-1 overexpressing cells had higher cyclin A protein levels than control cells. We conclude that PRL phosphatases increase cell proliferation by stimulating progression from G1 into S phase, and this process may be dependent on the down regulation of the cyclin dependent kinase inhibitor p21(Cip1/Waf1).

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.

Cellular Localization of PRL-1 and PRL-2 Gene Expression in Normal Adult Human Tissues

Recent evidence suggests that the PRL-1 and −2 phosphatases may be multifunctional enzymes with diverse roles in a variety of tissue and cell types. Northern blotting has previously shown widespread expression of both transcripts; however, little is known about the cell type-specific expression of either gene, especially in human tissues. Therefore, we investigated expression patterns for PRL-1 and −2 genes in multiple normal, adult human tissues using in situ hybridization. Although both transcripts were ubiquitously expressed, they exhibited strikingly different patterns of expression. PRL-2 was expressed heavily in almost every tissue and cell type examined, whereas PRL-1 expression levels varied considerably both between tissue types and between individuals. Widespread expression of PRL-1 and −2 in multiple organ systems suggests an important functional role for these enzymes in normal tissue homeostasis. In addition, the variable patterns of expression for these genes may provide distinct activities in each tissue or cell type.

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.

Abstract 4415: Differential effects of biochanin A on cell proliferation and ROS-dependent pathways in estrogen receptor positive and HER-2 positive breast cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The naturally occurring isoflavone biochanin A has been reported to have chemo-preventative properties towards cancer. Biochanin A exhibits antioxidant properties. Because of its antioxidant properties, biochanin A may be used to target breast cancer cells with high intracellular levels of reactive oxygen species (ROS). In this study, we investigated the effects of biochanin A on two cell lines — an estrogen receptor (ER) positive and a HER-2 positive breast cancer cell line. Biochanin A is implicated to affect both hormonal receptors and receptor tyrosine kinases. Overstimulation of ER is a basis for tumor progression due to the disruption of the cell cycle and apoptosis in estrogen positive breast cancer cells. Activation of HER-2 initiates cell growth, survival and invasion promoting cell signaling events in HER-2 positive breast cancer cells. MCF-7 (estrogen positive) and SK-BR-3 (HER-2 positive) cells were treated with biochanin A (2-100 μM) for 72 hours. Then MTT (cell survival) assay was carried out to determine the effects of biochanin A on cell survival. Our results suggest that biochanin A induces a dose-related inhibition of MCF-7 and SK-BR-3 cell viability at 50 μM. MCF-7 and SK-BR-3 cells were also treated with biochanin A (50 μM) for 12 hours and subsequently treated with hydrogen peroxide (H2O2)(2 μM) for 30 minutes: then western blotting was carried out to determine their expression of ERK and transcription factor NFκB. We found that biochanin A inhibits phospho-ERK pathway in SK-BR-3 but not in MCF-7 cells. However, expression of the transcription factor NFκB was decreases in both SK-BR-3 and MCF-7 cells. Thus, biochanin A may be an effective chemotherapeutic agent for breast cancer because of its ability to affect both hormonal receptors and receptor tyrosine kinases and to decrease intracellular ROS levels. (Supported by NIH INBRE P20RR016454, URC and MSTMRI) Citation Format: Lindsy Dickerson, Vikas Sehdev, Pamela L. Crowell, James C.K. Lai, Alok Bhushan. Differential effects of biochanin A on cell proliferation and ROS-dependent pathways in estrogen receptor positive and HER-2 positive breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4415. doi:10.1158/1538-7445.AM2013-4415