Perillyl alcohol and perillaldehyde induced cell cycle arrest and cell death in BroTo and A549 cells cultured in vitro
Introduction
Monoterpenes are naturally occurring plant compounds that are synthesized via the mevalonate pathway. Physiologically, they function as chemoattractants or chemorepellents (McGarvey and Croteau, 1995) and are responsible for the distinctive fragrance of many plants. The parent monoterpene compound, d-limonene, is formed by the cyclization of geranylpyrophosphate and serves as precursor to a host of other oxygenated monoterpenes including perillyl alcohol, perillaldehyde, carveol, carvone, menthol and others McGarvey and Croteau, 1995, Karp et al., 1990. Many of the monoterpenes are components of plants such as citrus (especially in the peel oils), caraway, dill, cherry, spearmint, lemongrass and herbal teas, most of which are found in or used for human foods.
The anti-carcinogenic actions of limonene and perillyl alcohol in several animal tumor models such as breast, liver, colon, and prostate have been reported Elegbede et al., 1986, Haag and Gould, 1994, Mills et al., 1995, Kelloff et al., 1996. In vitro, perillyl alcohol induced cell cycle arrest and apoptosis (Mills et al., 1995), inhibited the isoprenylation of small G proteins (21–26 kDa) involved in signal transduction (Crowell et al., 1994), and affected differential gene regulation (Jirtle et al., 1993). Both limonene and perillyl alcohol are being evaluated in human clinical trials Ripple et al., 2000, Vigushin et al., 1998. To our knowledge, there has been no prior study of the effects of perillyl alcohol and perillaldehyde on either human head and neck squamous cell carcinoma or lung adenocarcinoma cell lines. In this study, we investigated the effects of perillyl alcohol (POH) and perillaldehyde (PALD) on human squamous cell carcinoma of the tongue (BroTo) and human lung adenocarcinoma (A549) cell lines cultured in vitro.
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Chemicals and reagents
POH and PALD were obtained from Aldrich Chemical Co. (Milwaukee, WI). Propidium iodide (PI) and ribonuclease A (RNase A) were purchased from Sigma (St. Louis, MO). Hams F12-K and RPMI media, phosphate buffered saline (PBS, lacking Ca2+ and Mg2+), penicillin (100 units/ml) and 100 μg/ml streptomycin (P/S) were obtained from Invitrogen Corporation (Grand Island, NY). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT).
Cells and cell culture
Human lung adenocarcinoma cell line A549 was obtained
Cytotoxicity
The effects of POH and PALD on cell proliferation were assessed using the mitochondrial dehydrogenase (MTT) assay (Mosmann, 1983). Both POH and PALD inhibited proliferation of BroTo and A549 cell lines in a dose-dependent fashion (Fig. 1). The concentrations that elicited 50% inhibition of proliferation (IC50) after 24-hr exposure were determined. The IC50 values for POH and PALD respectively were 1.0 and 3.2 mM in BroTo cells and 1.2 and 3.0 mM in A549 cells (Fig. 1).
Dose-response and kinetic
Discussion
POH and PALD inhibited the proliferation of A549 and BroTo cells cultured in vitro. POH (IC50 = 1 mM) was more effective at inhibiting proliferation than PALD (IC50 = 3 mM) (Fig. 1). Both compounds inhibited colony formation in both cell lines. POH elicited a dose-dependent reduction in colony formation compared with Controls (Fig. 2).
When cells were treated with PALD for 12 hr, a dose-dependent effect was observed in A549 cells while no colony was formed in treated BroTo cells, compared to its
Acknowledgements
The authors wish to thank Kathy Schell, University of Wisconsin-Madison Flow Cytometry Section for DNA data acquisition and analysis, and Dr. Steve Carper, UNLV Cancer Institute/Chemistry Department, for his helpful comments and suggestions during the preparation of the manuscript. This work was supported in part by grants from UNLV New Investigator Award (NIA) to JAE and from the Western Alliance to Expand Student Opportunities (WAESO).
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