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

Abstract

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.

Introduction

Most cancer chemotherapy regimens make use of highly cytotoxic drugs that target proliferating cell populations (Kaufman and Chabner, 1996). The nondiscriminatory nature of these agents leads to severe side effects in normal cells with a high proliferative index, such as those of the gastrointestinal tract and bone marrow, thus limiting the effective dose of anticancer drug that can be administered. Studies conducted over the past two decades have examined tumors from both patients and experimental animal model systems in an attempt to identify molecular lesions responsible for the malignant phenotype. One of the goals of this molecular approach has been to identify aberrantly functioning gene products in tumors that could be used as molecular targets for novel anticancer drugs.

Monoterpenes, which are naturally occurring plant compounds, have been proposed as potential anti-neoplastic agents that could specifically target deficiencies in signal transduction pathways found in many tumors. Several studies have shown that monoterpenes can prevent in vivo tumor growth in a variety of organ systems in animal models, including skin Barthelman et al., 1998, Homburger et al., 1971, breast Elegbede et al., 1984, Haag and Gould, 1994, Haag et al., 1992, Maltzman et al., 1989, forestomach Wattenberg and Coccia, 1991, Wattenberg et al., 1989, lung Wattenberg and Coccia, 1991, Wattenberg et al., 1989, pancreas (Stark et al., 1995), liver (Mills et al., 1995), and colon Kawamori et al., 1996, Reddy et al., 1997. While monoterpenes have been shown to have chemopreventive effects and inhibit the appearance of tumors in several of these animal model systems Elegbede et al., 1984, Kawamori et al., 1996, Maltzman et al., 1989, Reddy et al., 1997, Wattenberg and Coccia, 1991, Wattenberg et al., 1989, many studies have also shown that perillyl alcohol (POH) and limonene have potent antitumor activity, resulting in the regression of tumors at different organ sites Haag and Gould, 1994, Haag et al., 1992, Mills et al., 1995, Stark et al., 1995, particularly in the mammary gland. As a result of studies demonstrating the potent chemopreventive and chemotherapeutic effects of these compounds, limonene and POH are currently undergoing clinical trials for their antitumor effects Bailey et al., 2002, Chow et al., 2002. Because of the current interest in POH as a potential antitumor agent and its use in clinical trials, it is important to determine the spectrum of tumors against which this agent may be effective and gain a better mechanistic understanding of the effects of this agent in vivo (Fig. 1).

The mechanism of action of these compounds is still uncertain as monoterpenes have been shown to inhibit tumors by a variety of pathways that include: (1) the inhibition of G-protein prenylation by farnesyltransferase, including members of the ras and rho gene families Crowell et al., 1994, Gelb et al., 1995, Gould et al., 1994, Lluria-Prevatt et al., 2002, Ren and Gould, 1998, Ruch and Sigler, 1994. Although initial studies attributed the chemotherapeutic activities of these compounds to their ability to inhibit RAS protein prenylation, it has since become evident that monoterpenes can inhibit tumor growth regardless of the presence or absence of mutated RAS protein Gould et al., 1994, Ruch and Sigler, 1994, Waddick and Uckun, 1998; (2) alterations in cell cycle genes that prevent formation of cyclin D1 complexes Alexandrow and Moses, 1995, Bardon et al., 1998, Bardon et al., 2002, Kamb, 1995, Sherr and Roberts, 1995; (3) cytotoxicity, demonstrated in pancreatic and liver cells, resulting from increased activity of proapoptotic pathways, such as TGFβ and BAK protein Jirtle et al., 1993, Mills et al., 1995, Stayrook et al., 1997; and (4) a cytostatic mechanism as a result of remodeling or redifferentiation of the tumor tissue Alexandrow and Moses, 1995, Haag and Gould, 1994, Shi and Gould, 1995. Taken together, these studies suggest that POH probably mediates its effects by a variety of mechanisms that may depend on the cell type and or molecular lesions driving tumor cell growth.

Although POH has been shown in animal studies to be an effective antitumor agent against a variety of tumor types, particularly liver and breast Crowell and Gould, 1994, Gould, 1995, little information is available on its effects in lung tumors, despite the fact that pulmonary tumors are the leading cause of cancer-related deaths in the United States (Jemal et al., 2003). Thus, we have investigated the chemotherapeutic effects of POH on two human lung cancer cell lines, H838 non-small cell lung cancer (NSCLC) cells which were derived from an adenocarcinoma and H322 bronchioloalveolar carcinoma cells. We examined the possible mechanism of action of POH on lung tumor cell growth inhibition and provide data demonstrating the antiproliferative effects of POH on lung tumor cell lines. Our data suggest that POH may mediate its antiproliferative effects on lung tumor cells through induction of apoptosis via the caspase-3-mediated pathway, and may have the potential for use as an anti-neoplastic agent in the treatment of NSCLC.