Perillyl alcohol and perillic acid induced cell cycle arrest and apoptosis in non small cell lung cancer cells
Introduction
Lung cancer is the leading cause of cancer deaths in the US among both men and women. Non small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancer cases and has an overall survival at 5 years of less than 15% [1], [2]. Current treatment options for lung cancer include surgery, chemotherapy, and radiation therapy. Surgery is recommended but complete surgical resection is not always possible because most patients present with advanced disease [3]. Hence, there is a need to develop effective chemotherapy against NSCLC.
Perillyl alcohol (POH) is a naturally occurring monoterpene found in essential oils of mints, cherries, lavenders, lemongrass, sage, cranberries, perilla, wild bergamot, gingergrass, savin, caraway, and celery seeds [4], [5], [6]. POH is a hydroxylated product of d-limonene formed by the condensation of two isoprene units. POH is 5–10 times more lethal than limonene and has shown chemo preventive and therapeutic activity in DMBA-induced mammary cancer [7]. POH inhibited 4-(methyl-nitrosoamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumorigenisis, prevented the azoxymethane (AOM)-induced rat colon tumor models, inhibited diethyl-nitrosamine (DEN)-induced rat liver tumors and showed anti-tumor activity against pancreatic tumor models [8], [9], [10], [11], [12], [13]. POH also inhibited the growth of human breast, colon, lung, murine glial, and hamster pancreatic ductal adenocarcinoma cells in vitro[14], [15], [16], [17], [18], [19].
POH readily metabolizes to perillic acid (PA) and dihydroperillic acid (DHPA) in animals, whereas in humans, PA is the major circulating metabolite [20], [21], [22]. POH and PA inhibit the protein prenylating enzymes, protein farnesyl transferase and geranylgeranyl transferase. PA and POH also cause cell cycle arrest at the G0/G1 phase [23], [24]. POH has been reported to regulate gene expression in lung cancer cells. POH also increased the expression of bax, bid and p21waf1, induced apoptosis and cell cycle arrest as well as decreased cyclinD and cdk2 expression without changing the levels of p53 [16]. PA has been shown to inhibit the proliferation of lung cancer cells H322 and H838 with an increase in caspase-3 activity and cleavage of PARP. DNA microarray studies showed that a large number of genes changed expression in these two cell lines, but analysis suggested that POH may mediate its effects via posttranslational modifications [16]. Pretreatment of prostate as well as head and neck cancer cells with POH sensitized the cells to radiation [25], [26].
Apoptosis is a process regulated by specific genes and occurs through two major pathways, extrinsic and intrinsic, in mammalian cells [27]. The extrinsic pathway involves signals transduced through death receptors while the intrinsic pathway involves proteins of the Bcl2 family. Bax is an apoptotis inducing protein of the Bcl2 family and its homodimerization or heterodimerization with Bcl2 determines whether or not apoptosis will occur [28]. Direct activation of bax on the outer mitochondrial membrane renders the membrane permeable and results in release of cytochrome c. Cytochrome c forms an apoptosome complex with apaf-1 and caspase-9 in the cytosol which activates procaspase-3 to caspase-3. The protein p21 is a universal inhibitor of cyclin dependent kinases (CDKs) that are involved in cell cycle progression along with specific cyclins (B or D or E) [29], [30], [31], [32], [33].
POH and PA have been reported to inhibit the proliferation of lung cancer cells [14], [15], [16]; however, PA’s mechanism of action remains unknown. We studied the effects of PA and POH on non small cell lung cancer (NSCLC) cells and demonstrated that POH and PA inhibited the growth of lung cancer cells. Cells treated with PA and POH showed long-term proliferation inhibition and increased sensitivity to cisplatin and radiation. POH and PA induced cell cycle arrest and apoptosis in lung cancer cells. We observed increased expression of Bcl2, bax, p21, and caspase-3 activity in cells treated with POH and PA. We conclude that POH and PA induced apoptosis via a caspase-3 pathway.
Section snippets
Chemicals and reagents
Cell culture media (RPMI 1640 and Ham’s F-12K) was obtained from ATCC (Manassas, VA). Fetal bovine serum (FBS), penicillin (1000 U/ml) and streptomycin (1000 μg/ml) (P/S) were purchased from Hyclone Laboratories (Logan, UT). Perillic acid (PA), perillyl alcohol (POH), proteinase inhibitors, propidium iodide (PI), and ribonuclease A (RNase A) were obtained from Sigma Chemical Company (St. Louis, MO). Phosphate buffered saline (PBS, lacking Ca2+ and Mg2+) was purchased from Invitrogen Corporation
POH and PA induce cell death in vitro
POH inhibited the proliferation of A549 and H520 lung cancer cells in a dose-dependent manner while PA did not (Fig. 1). The concentration of POH at which 50% of cell proliferation was inhibited following a 24 h exposure was 1.7 mM in H520 cells (Fig. 1b). The concentrations of POH at 1.0 mM and above were all statistically significant from the control values in both the A549 (p = 0.01) and H520 (p = 0.004) cell lines. The long-term proliferation inhibition of A549 cells evaluated by a survival assay
Discussion and conclusion
Monoterpenes have chemotherapeutic and chemopreventive activities against cancer cells. In vitro studies reported that POH inhibited the growth of pancreatic, breast, and lung cancer cells [14], [15], [16], [17], [18], [19]. To determine the chemotherapeutic effects of POH and PA, proliferation and apoptotic assays were used. This study showed that POH caused growth inhibition in a dose-dependent manner in NSCLC cells. PA and POH showed long-term proliferation inhibition in A549 cells in vitro.
Acknowledgments
The authors thank Casey Hall, Shirley Shen, John Adebayo, and Nicole Stevens for their technical assistance. We also thank Dr. Enitan Bababunmi for first suggesting jasmonates as anticancer agents to study and Becky Hess for her editorial assistance. This research was supported in part by ACS Grant No. IRG-103719 (to J.A.E.) and in part by NIH Grant No. 2P20RR016464 from the INBRE Program of the National Center for Research Resources.
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