Advanced prostate cancer cells are typically hormone independent, resistant to apoptosis and do not respond to chemotherapeutic agents. The ability of methyl jasmonate (MJ) and cis-jasmone (CJ) to inhibit growth in hormone independent prostate cancer cell lines, PC-3 and DU-145, was evaluated. CJ and MJ inhibited cell growth, induced cell cycle arrest and apoptosis. Detailed studies with the PC-3 cell line revealed that 2 mM CJ or MJ treatment resulted in caspase 3 activation and Tumor Necrosis Factor Receptor 1 (TNFR1) activation, all hallmarks of apoptosis. These phytochemicals could be useful in the management of advanced prostate cancer.

Plant products such as perillyl alcohol have been reported to possess anti-tumor activities against a number of human cancers though the mechanism of action has not yet been elucidated. The effects of perillyl alcohol (POH) and its metabolite perillic acid (PA) on the proliferation of non small cell lung cancer (NSCLC, A549, and H520) cells were investigated. Both POH and PA elicited dose-dependent cytotoxicity, induced cell cycle arrest and apoptosis with increasing expression of bax, p21 and caspase-3 activity in both the cell lines. Combination studies revealed that exposing the cells to an IC50 concentration of POH or PA sensitized the cells to cisplatin and radiation in a dose-dependent manner. These results indicate that POH and PA in combination therapy may have chemotherapeutic value against NSCLC.

Methyl jasmonate--a plant stress hormone with striking resemblance to lipoxygenase products have been reported to induce apoptosis in several cancers. However, 5-HETE--a product of the lipoxygenase pathway has been implicated in human prostate cancer progression and yet possible interaction between methyl jasmonate and the lipoxygenase pathway has not been reported, thus, leaving some unanswered questions on the mechanism(s) of action by methyl jasmonate. Using cytotoxicity and flow cytometry assays (BrdU assay) as well as fluorescence microscopy, we investigated the effects of the methyl jasmonate on the proliferation of human prostate adenocarcinoma cell lines (DU-145, PC-3) in vitro and the potential interaction between methyl jasmonate and the lipoxygenase pathway. Methyl jasmonate (MJ) significantly (p = 0.01) inhibited the proliferation of human prostate carcinoma cells in dose- and kinetic-dependent manners and showed specific interaction with 5-lipoxygenase (5-LOX) enzyme pathway. Flow cytometric analyses and fluorescence microscopy confirmed that the inhibition of proliferation was via the induction of apoptosis. Based on our findings, it can be proposed that the interaction of methyl jasmonate with 5-lipoxygenase pathway may participate in the observed anticarcinogenic property.

The jasmonates, cis-jasmone (CJ) and methyl jasmonate (MJ), were investigated for their effects against NSCLC cell lines A549 and H520. CJ or MJ inhibited the proliferation of both cell lines in a dose-dependent manner as well as induced cell cycle arrest in the G2/M phase. Apoptosis was observed following treatment with CJ or MJ as indicated by Hoechst staining and confirmed by dual annexin V-fluorescein isothiocyanate (FITC)/prodium iodide (PI) and DAPI (4',6-diamidine-2'-phenylindole dihydrochloride) staining. p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was observed with increased expression of bax, p21, and caspase-3 activity. These observations indicate that jasmonates may have a therapeutic value in the treatment of lung cancer.

1933 Drugs and food supplements used for the treatment of various diseases originated from natural plant products. Research on plants used crude in developing countries for various medicinal purposes is increasing. In this research, we studied the inhibitory effects of crude soluble extracts from the leaves and seeds of three tropical plants, Piper guinenese, Tapinathus bangnensis and Xylopia aethiopicum on cancer cell lines. The cancer cell lines studied were: Prostate (PC-3), Head & Neck (SCC-25) and Lung (HTB-182).The preliminary anticarcinogenic screening of the plants extract on the cancer cell lines was carried out using Alamar blue and Mitochondria dehydrogenase (MTT) cytotoxicity assays with exposures of 24, 48 and 72hrs. Alamar blue cytotoxicity assay showed that 100ug/ml of the crude soluble extract from the plant; Tapinathus bangnensis had inhibitory effects of 40 and 35 percent on the proliferation of PC-3 and SCC-25 cells after 72 hours exposure. Piper guinenese extract had similar inhibitory effects on the two cell lines. Whereas the inhibition of proliferation from Xylopia aethiopicum on the two cell lines was 35 and 80 percent respectively. However all the three plants extract had minimal inhihitory effects on HTB-182. DNA studies using flow cytometry analyses of the treated cancer cells showed cell cycle arrest at the G 2 /M phase after 24, 48 and 72 hours. This was followed by combined Propidium Iodide staining and Annexin V FITC assay to confirm the induction of apoptosis. Results show that Xylopia aethiopicum induced a time dependent apoptosis of 75 and 90 percent in PC-3 and SCC-25 cell lines at 72 hours respectively. Whereas Tapinathus bangnensis extracts gave an induction of apoptosis of 85 percent on PC-3 cell lines after 72hrs exposure. There was induction of apoptosis by the three different plants extract on HTB-182 cell line at 48 and 72 hours but not comparable to the other cell lines. The extracts from Piper guinenses induced apoptosis minimally in PC-3, but had an apoptotic effect of 70 and 75 percent at 48 and 72 hour exposures in SCC-25 cells. Anticarcinogenic agents in natural plant products should compare well to the synthetic chemotherapeutic agents used in cancer treatments because they are already incorporated naturally in food and are not toxic to normal cells. It is hoped that the consumption of these natural products as part of normal food will help to prevent and reduce the incidence of cancer diseases.

5902 Natural plant products such as perillyl alcohol and their metabolites have been reported to possess anti-tumor and drug related activities against a number of human cancers though the mechanism of action of these compounds is not yet fully known. The p53 tumor suppressor protein is a regulator of cell cycle progression. The P53 gene is mutated in most lung cancer cells resulting in the over- or under-expression of the mutant p53 protein. We studied the effects of the perillyl alcohol metabolite, perillic acid (PA), and methyl jasmonate (MJ), a naturally occurring plant compound, on the proliferation of human lung adenocarcinoma (A549) and squamous cell carcinoma of the lung (NCI-H520 and NCI-H2170) cell lines using cytotoxicity, flow cytometry, enzyme linked immunosorbent assay (ELISA) and western blotting techniques. The expression levels of p53 protein in NCI-H520 and NCI-H2170 cell lines were studied using ELISA and western blotting techniques. Both PA and MJ elicited dose- and kinetic-dependent cytotoxicity against the cell lines. Decreases in the viability of A549, NCI-H520 and NCI-H2170 were observed with the following 72-hr IC50 values: PA (2.8, >3, 2.8 mM) and MJ (1.8, 2.2, 1.25 mM) respectively. DNA content analyses using flow cytometry showed that both PA and MJ caused cell cycle arrest at G0/G1 in A549 cell line, S phase arrest in NCI-H520 cell line and G2-M phase arrest in NCI-H2170 cell line, respectively. The cell cycle arrests were accompanied by significant (p

2990 The jasmonates, consisting of cis-jasmone (CJ), methyl jasmonate (MJ), and jasmonic acid (JA) are a family of plant stress hormones. In plants, these compounds activate cellular responses to diverse conditions, including cell death, to protect plants that come under biotic stress such as insect attack or wounding. Human prostate cancer is a hormone-dependent cancer. We studied the effects of the jasmonates on the proliferation of human prostate adenocarcinoma cell lines (PC-3, HTB-81) and the human fibroblast cell line (HFL-1) using the mitochondrial dehydrogenase activity (MTT) and the colony formation (CFA) assays. The mechanism of inhibition of proliferation by these agents was studied using flow cytometry and western blotting techniques. Cytotoxicity assays showed that the jasmonates inhibited proliferation of the cells in both dose- and kinetic-dependent fashions. The relative efficacy, as measured by the concentration of agent that inhibited proliferation by 50% (IC50) was MJ>>CJ>JA. Flow cytometric and other analyses confirmed that the inhibition of proliferation by these agents was as a result of induction of cell death via apoptosis. Our results indicate a potential application of the jasmonates in the treatment of human prostate cancer. [The work was supported in part by grants from ACS #IRG-103719 and UNLV 2002-2003 Planning Initiative Award (to JAE)].

BACKGROUND:Perillyl alcohol (POH) elicited anticarcinogenic effects in a number of cancer models and pharmacokinetic studies in humans revealed that PA is the major circulating metabolite following POH administration.MATERIALS AND METHODS:Effects of PA or POH alone, or in combination with radiation, on human head and neck squamous cell carcinoma cell lines (HNSCC), were investigated using cytotoxicity and flow cytometry assays. HNSCC cells were pretreated with 1.0 mM PA or 0.5 mM POH for 72 h before exposure to 1 or 2Gy dose of radiation.RESULTS:Pretreatment of the cells with 1.0 mM PA or 0.5 mM POH prior to irradiation, caused the following growth inhibition: HTB-43 (50% or 71%), SCC-25 (55% or 68%), and BroTo (18% or 53%). PA and POH induced cell-cycle arrest and apoptosis.CONCLUSION:PA and POH have potential for use as radiosensitizers in chemo-radiation therapy of head and neck cancers and should be further studied.

The role of the monoterpenes, especially limonene and perillyl alcohol, in the treatment of certain cancers is currently being evaluated in clinical trials. In this study, the effects of perillyl alcohol (POH) and its analog, perillaldehyde (PALD), on human carcinoma cell lines (BroTo and A549) cultured in vitro were investigated using proliferation assays (MTT and colony formation) and DNA content analysis by flow cytometry. POH and PALD elicited dose- and time-dependent inhibition of proliferation in both cell lines. Concentrations of POH and PALD that inhibited cell proliferation by 50% (IC50) in 24 hr were 1 and 3 mM, respectively. DNA content analysis revealed that 1 mM of either POH or PALD caused cell cycle arrest in the G1 phase in both cell lines while POH alone caused increased hypodiploid and annexin V-positive populations in both BroTo and A549 cells. POH induced apoptosis and was more effective than PALD at inhibiting the proliferation of BroTo and A549 cells cultured in vitro.

Perillyl alcohol and d-limonene are naturally occurring plant compounds that exhibited anti-carcinogenic activities in mammary tumor models. The effects of these monoterpenes at the initiation stage of aflatoxin B1-induced hepatocarcinogenesis were investigated. Male F344 rats were fed Control or treatment diets throughout the study and exposed to aflatoxin for 5 days. Three days after the last aflatoxin dose, blood and liver samples were obtained. Analysis of liver samples showed that both limonene and perillyl alcohol significantly inhibited (p<0.05) aflatoxin-DNA adducts formation in hepatocytes. The monoterpenes may have potential for use as chemopreventive agent against aflatoxin-induced liver cancer. Keywords: Aflatoxin B1, hepatocarcinogenesis, monoterpenes, chemoprevention.

In hematopoietic cells, the Bcr/Abl tyrosine kinase that is encoded by the Philadelphia chromosome translocation both stimulates proliferation and activates an anti-apoptotic program that is associated with a G2/M delay upon exposure to various apoptotic stimuli. We recently reported that the monocyclic monoterpene, perillyl alcohol (POH) selectively induces in Bcr/Abl transformed cells, G0/G1 arrest and apoptosis. Therefore, POH activates anti-proliferative and apoptotic pathways against which the Bcr/Abl kinase does not protect. In this report, we show that in Bcr/Abl-transformed cells, POH induces cytoplasmic acidification, redistribution of phosphatidylserine in the plasma membrane along with DNA fragmentation, all of which can be prevented by the phorbol ester, TPA. The ability of TPA to protect against POH-induced cytotoxicity was blocked by inhibitors of protein kinase C (PKC) and the Na+/H+ antiport. In contrast, TPA does not protect the cells from POH-mediated G0/G1 arrest. While POH inhibits a distal step in the mevalonate biosynthesis pathway, lovastatin, also a potential anticancer agent, inhibits the initial step in this pathway. Not surprisingly, lovastatin also induces G0/G1 arrest and apoptosis in Bcr/Abl-transformed cells, however, TPA protects cells from both apoptosis and G0/G1 arrest caused by lovastatin. Thus, in Bcr/Abl-transformed cells, POH and lovastatin cause growth arrest by different mechanisms. Together, these observations demonstrate that POH-mediated cell cycle arrest precedes apoptosis and raises the possibility that that the primary effect of POH is to induce G0/G1 arrest with apoptosis being a consequence of the growth arrest.

Okadaic acid (OA), a tumor promoter in the mouse skin carcinogenesis model, has been shown to induce apoptosis in tumor cell lines that harbor H-ras mutations. We examined the effects of OA on mouse keratinocytes with (308) and without (C50) H-ras mutation in vitro and in an in vivo system. Following exposure to varying concentrations of OA over time, the effects of OA in vitro were assessed using microscopic, biochemical and flow cytometric techniques. OA effects on the cells included incorporation of propidium iodide, externalization of phosphatidylserine, and development of hypodiploidy. 308 cells demonstrated typical DNA ladder formation, rapid chromatin and nuclear condensation, while C50 cells demonstrated delayed chromatin condensation and nuclear fragmentation, but no DNA ladder formation. In vivo, OA elicited delayed papilloma formation and reduced tumor multiplicity. Though its mechanism of action is not fully known, we found that OA-induced inhibition of the clonal expansion of initiated cells may be related to the presence or absence of H-ras mutation.

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.

The monocyclic monoterpenoid compounds limonene and sobrerol have anticarcinogenic activity when fed during the initiation stage of dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinogenesis. Here we investigated the potential roles of hepatic glutathione-S-transferase (GST; EC 2.5.1.18) and uridine diphosphoglucuronosyl transferase (UDPGT; EC 2.4.1.17) in monoterpene-mediated chemoprevention. Diets containing the isoeffective anticarcinogenic terpenes, 5% limonene or 1% sobrerol, elevated hepatic GST activity > 2-fold when measured using the general substrate 1-chloro-2,4-dinitrobenzene and 3,4-dichloronitrobenzene for the GST dimer 3-3. However, there were no significant changes in hepatic GST activity when 1,2-epoxy-3-(p-nitrophenoxy)propane was used. We found that both terpene diets increased GST affinity-purified protein 1.5-fold and the HPLC subunit profile. Liver GST subunit 3 had the greatest increase followed by 1 and 4 with no change in subunit 2. Both terpene diets significantly increased the activity of the methylcholanthrene-inducible and the phenobarbital-inducible UDPGT isozymes. We propose that much of the anticarcinogenic activity of these monocyclic monoterpenes during the initiation phase of DMBA carcinogenesis is mediated through the induction of the hepatic detoxification enzymes GST and UDPGT.

Disruption of the integrity of tumor cellular membranes has been proposed as an initiating event in hyperthermic cell death. Thermosensitivity measured by the shift in the harmonic mean of tumor regrowth delay of CA755 mammary adenocarcinomas grown in the hind legs of male BDF1, mice increased 22% when the hosts were fed a diet enriched in polyunsaturated fatty acids. Although the diet elicited the anticipated increase in tumor membrane phospholipid polyunsaturated fatty acids, the proportion of total unsaturated fatty acids decreased and the proportion of membrane-rigidifying saturated fatty acids increased. Concomitantly, the concentrations of cholesterol and phospholipid phosphorus increased and the ratio of phosphatidylethanolamine to phosphatidylcholine decreased, presumably to counter the effect of the change in the fatty acid pattern. In host liver membranes, the diet-mediated increase in proportion of polyunsaturated fatty acids was not accompanied by an increase in the proportion of rigidifying saturated fatty acids. Instead, the homeoviscous adaptation consisted of decreases in monounsaturated fatty acids and cholesterol concentration and an increase in the phosphatidylethanolamine-phosphatidylcholine ratio. Addition of a natural inhibitor of cholesterol biosynthesis to the polyunsaturated fatty acid enriched-diet reversed the diet-mediated increase in the phosphatidylethanolamine-phosphatidylcholine ratio of host liver membranes. Tumor membrane lipids from hosts fed the combined dietary factors were characterized by the formentioned rigidifying increase in saturated fatty acids and compensatory decrease in the phosphatidylethanolamine-phosphatidylcholine ratio. The inhibitor reversed the compensatory increases in cholesterol and phospholipid phosphorus concentrations. As a consequence the thermosensitivity of tumors bearing this perturbed membrane was increased.

Orange peel oil has previously been shown to be a promoter of mouse skin tumors. It has been assumed that this activity is due to its major (95%) constituent, d-limonene. We have tested both orange peel oil and purified d-limonene as skin tumor promoters in a two-stage skin carcinogenesis model in which tumors were initiated with 7,12-dimethylbenz[a]-anthracene. We confirmed that topically applied orange peel oil is a very weak promoter of both skin papillomas and carcinomas. However, this promotional activity could not be accounted for by topically applied d-limonene. We thus feel that one or more minor components of orange peel oil has promotional activity. Neither orange peel oil nor d-limonene had promotional activity when given via the diet.

The effects of d-limonene on rat mammary tumors were investigated. Following the appearance of mammary tumors induced by 7,12-dimethylbenz[a]anthracene in female (W/Fu X F344)F2 rats, animals were assigned to pairs in which 1 rat was fed a diet containing 10% d-limonene and the other was pair fed an isocaloric diet containing 10% cellulose. There was a highly significant increase in the regression of the first tumors in the rats fed d-limonene. In addition, d-limonene given at this time inhibited the formation of subsequent multiple tumors in these rats.

We investigated whether the addition of d-limonene to the diets of rats would modify the process of mammary tumor induction by 7,12-dimethylbenz[a]anthracene (DMBA). When diets containing 1000 or 10 000 p.p.m. d-limonene were fed to rats from one week before DMBA administration to the end of the experiment (27 weeks post DMBA treatment), a significant reduction in mammary carcinogenesis was observed at each level. The inhibition of carcinogenesis was mainly due to an increase in latency; however, major differences in incidence could be seen during the follow-up period. For example, rats fed 10 000 p.p.m. of d-limonene had a 72% reduction in mammary tumors when compared to controls at 18 weeks post DMBA treatment. In addition to inhibiting the appearance of mammary tumors, d-limonene was also found to cause the regression of frank mammary tumors. No toxicity was evident in these rats even at the highest d-limonene dose. Control and all treated rats gained weight at the same rate throughout the experiment. In addition, no differences were found in their peripheral blood counts (Hct, total white cell and differential count).