Anticancer Activity of Linalool Terpenoid: Apoptosis Induction and Cell Cycle Arrest in Prostate Cancer Cells

Purpose: To evaluate the anticancer activity of linalool against human prostate cancer (DU145) cells. Methods: The anticancer activity of linalool against DU145 cancer cells was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry, using propidium iodide and Annexin V-FITC, was applied to study apoptosis and cell cycle phase distribution. Inverted light microscopy was used to study the effect of linalool on cell morphology and apoptotic body formation in DU145 cells while gel electrophoresis was employed to evaluate the effect of linalool on DNA fragmentation. Results: Linalool induced a dose-dependent as well as time-dependent growth inhibitory effect on DU145 prostate cancer cells. It induced sub-G1 phase growth arrest which led to increase in sub-G0/G1 cell population after treatment with increasing doses of linalool. DNA ladder appeared to be more evident with increasing linalool concentration. However, no DNA fragments were observed in the control groups. It was observed that 4.36, 11.54, 21.88 and 15.54 % of the cells underwent early apoptosis after treatment with 0 (no linalool treatment), 20, 40, and 80 µM of linalool, respectively. Compared to control cells, linalool treatment resulted in the appearance of cell shrinkage along with membrane blebbing which are characteristic features of cell apoptosis. Conclusion: The findings of this study indicate that linalool can be developed as a plant-based chemotherapeutic agent against prostate cancer


INTRODUCTION
Prostate cancer (PC) is the principal cause of new cancer cases and the second most common cause of cancer-related deaths in men in United States of America [1]. Prevalence rate for clinical prostate cancer in Western men are 35 -45 times higher than those for Asian men. In the United States, prostate cancer accounts for about 30 % of all cancer cases in men whereas in China, prostate cancer accounts for less than 2 % of all cancers in men. The survival rate for advanced prostate cancer has not improved much during the past 10 years [2,3].
The standard treatment options for prostate cancer include surgery, radiotherapy and hormonal therapy, but these treatment options are not without serious side-effects like urinary incontinence and sexual dysfunction among others.
Traditionally, chemotherapy and radiotherapy have not provided important survival benefits to patients with advanced prostate cancer and most treatment options available are only palliative. Recent studies on taxane derivatives alone and in combination with other chemotherapeutic agents have revealed some limited benefit [4], but the need for more effective and less toxic means to target and/or thwart this disease still exists. Complementary and alternative medicine may provide better treatment options by inhibiting or slowing down disease progression. According to a recent survey, around 30 -40 % of patients suffering from prostate cancer use complementary and alternative medicine as an additional therapy [5][6][7].
Keeping in view the limited treatment options available for prostate cancer along with the sideeffects of known treatment strategies, the aim of this study was to identify a plant-based drug molecule (linalool, a monoterpene) that will capable of inhibiting prostate cancer cell growth.

EXPERIMENTAL Chemicals and biological reagents
Linalool was purchased from Sigma Chemical Company (St Louis, MO, USA). The cells were cultured in Dulbecco's modified Eagle's media supplemented with 10 % fetal bovine serum (Lonza Biologics, Singapore) and 100 U/mL penicillin and 100 μg/mL streptomycin (Vega Pharma Limited, Zhejiang, China). MTT kit was obtained from Roche (USA). Annexin V-FITC-Propidium Iodide Apoptosis Detection Kit was purchased from Bestbio, Shanghai, China. All other chemicals and solvents used were of the highest purity grade. Cell culture plastic ware was bought from BD Falcon (USA).

Cell line and cell viability assay
Human prostate carcinoma (DU145) cells were procured at the China Center for Type Culture Collection (Wuhan, China) and grown in a humidified 5 % CO 2 atmosphere at 37 o C in an incubator, and cultured in RPMI-1640 medium supplemented with 10 % heat-inactivated newborn calf serum, 100 IU/mL penicillin and 100 μg/mL streptomycin. MTT assay (3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) was used to measure the inhibition of cell proliferation. MTT was added in cells exposed to different concentrations (5,10,20,40 and 80 µM) of linalool for 24 and 48 h. Three hours later, the formazan precipitate was dissolved in 100 μL dimethyl sulfoxide, and then the absorbance was measured in a microplate reader (Bio-Rad).The cell viability was calculated as in Eq 1.

DNA fragmentation analysis
After subjecting DU145 cells to linalool treatment at various concentrations (0, 20, 40 and 80 µM), both adherent and floating cells were collected and washed with PBS. Pellets were then lysed with DNA lysis buffer (20 mM EDTA, 100 mM Tris, pH 8.0, 0.8 % SDS) at room temperature for 25 min. After centrifugation for 10 min at 12 000 × g, the supernatants were collected and treated with RNase A (final concentration, 500 μg/mL) for 20 min at 37 o C, followed by digestion with proteinase K (final concentration 500 μg/ml) for 3.0 h at 55 o C. The DNA was extracted using the phenol/chloroform/isoamylol (25:24:1), precipitated with ethanol, dissolved in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA), and subjected to 2 % agarose gel electrophoresis for DNA fragmentation analysis.

Annexin V-FITC assay for cell apoptosis
Apoptosis detection was performed using the Annexin V-FITC and PI apoptosis kit (Bestbio, Shanghai, China). DU145 cells were plated at a density of 2 × 10 5 cells/well into 12-well plates and incubated overnight. Apoptosis was induced by treating cells with linalool at various concentrations (0, 20, 40 and 80 µM). Cells grown in media containing an equivalent amount of DMSO without any drug served as control. After 48 h of incubation, the cells with different treatments were harvested and the final samples measured on a FACS Calibur flow cytometry (Becton Dickinson, San Jose, CA, USA).

Morphological assessment
DU145 cells were seeded in 6-well plates at 1.5 × 105 cells per well in 3 mL of complete growth medium, incubated for 24 h and treated with linalool at various concentrations (0, 20, 40 and 80 μM). Control cells treated with 0.3 % DMSO alone were also included. The morphological changes, characteristic of apoptosis or necrosis, were observed and the images were captured under an inverted light microscope (Olympus, PA, USA) after 48 h. The same spot of cells was marked and captured.

Statistical analysis
The experiments were performed in triplicate. Data were expressed as the mean ± standard deviation (SD). Statistical correlation of data was checked for significance by ANOVA and Student's t test. P < 0.05 was taken to indicate statistically significant difference.

Linalool induced strong cytotoxicity in human prostate cancer (DU145) cells
The cancer cells showed considerable and dosedependent susceptibility to the treatment of different concentrations of linalool. The compound also showed time-dependent inhibition of the cancer cell growth as shown in Figure 1 at 24 h and 48 h time intervals. The number of viable human prostate cancer cells exposed to linalool treatment reduced considerably. The IC 50 value of the compound after 12 h and 24 h time intervals was found to be 28.3 and 10.5 µM respectively.

Effect of linalool on DU145 cell apoptosis
The results of Annexin V assay are shown in Figure 4A-D. The results of the flow cytometry study with Annexin V/FITC and PI showed that within 48 h of incubation, approximately 4.36, 11.54, 21.88 and 15.54 % of the cells underwent early apoptosis after treatment with 0 (no linalool treatment), 20, 40 and 80 µM of linalool respectively. Similarly, 6.07 %, 25.11 %, 29.34 % and 43.14 % of the cells underwent late apoptosis after treatment with 0 (no linalool treatment), 20, 40 and 80 µM of linalool respectively (Figure 4).

Morphological characteristics of apoptosis
Morphological study using inverted light microscopy revealed that linalool induced growth inhibition and apoptosis in DU145 prostate cancer cells. As shown in Figure 5A-D, the number of cells in the control and the ones treated with different concentrations of linalool increased from 0, 20, 40 and 80 µM dose. The cells with higher doses revealed that cellular shrinkage and blebbing occurred. This effect was shown to be related to linalool dose. The number of cells with shrinkage increased with linalool concentration.

DISCUSSION
Today, plant-based drugs continue to play an essential role in health care. It has been estimated by the World Health Organization that 80 % of the population of the world rely mainly on traditional medicines for their primary health care [8].
Natural products have contributed significantly to the development of anticancer drugs. According to a recent review, among the 79 FDA approved anticancer drugs and vaccines from 1983 -2002, 9 were directly from the isolation of natural products and 21 were natural product derivatives [9].
Linalool has been reported to exhibit good inhibitory effects against breast, colorectal and liver cancer cells [10]. Cell cycle analysis also confirmed that linalool can lead to apoptosis. By using flow cytometry, it was reported that treatment with linalool significantly increased the sub-G1 phase and that there were more cells concentrated in the G1 phase. Linalool has also been reported to stimulate IFN-γ, IL-13, IL-2, IL-21, IL-21R, IL-4, IL-6sR and TNF-α secretion [11,12]. Linalool has also been investigated for its ability to induce apoptosis and differentiation in human leukemia HL-60 cells [13,14] In this study, linalool was found to exert anticancer effects against prostate cancer cells by inducing apoptosis and sub-G1 cell cycle phase arrest. Linalool even induced potent DNA fragmentation in these cells. It caused both early and late apoptosis with cell shrinkage and membrane blebbing which are characteristic features of apoptosis.

CONCLUSION
The findings of this work indicate that linalool exhibits potent dose-dependent as well as timedependent anticancer activity in human prostate cancer (DU145) cells by inducing apoptosis, DNA fragmentation and cell cycle arrest. these findings may pave way for future research on linalool in the hunt for novel and less toxic chemotherapeutic agents for prostate cancer.