Chemical constituents and anticancer activity of Curcuma zedoaria Roscoe essential oil against non-small cell lung carcinoma cells in vitro and in vivo
Chien-chang Chen1, Yuhsin Chen2, Yi-Ting Hsi1, Chih-Sheng Chang2, Li-Fen Huang3,
Chi-Tang Ho4, Tzong-Der Way1,5,6** and Jung-Yie Kao1*
1Institute of Biochemistry, College of Life Science, National Chung Hsing University,
Taichung, Taiwan
2Taichung District Agricultural Research and Extension Station, Council of
Agriculture, Taichung, Taiwan
3Graduate School of Biotechnology and Bioengineering, Yuan Ze University,
Taoyuan, Taiwan
4Department of Food Science, Rutgers University, New Brunswick, New Jersey, USA
5Department of Biological Science and Technology, College of Life Sciences, China
Medical University, Taichung, Taiwan
6Department of Health and Nutrition Biotechnology, College of Health Science, Asia
University, Taichung, Taiwan 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
*Corresponding author: Dr. Jung-Yie Kao,
Institute of Biochemistry, College of Life Science, National Chung Hsing University, Taichung, Taiwan Tel: (886)-4-22840468 ext: 222 Fax: (886)-4-2285-3487 E-mail: jykao@dragon.nchu.edu.tw **Co-correspondence author: Dr. Tzong-Der Way,
Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan
No.91 Hsueh-Shih Road, Taichung, Taiwan 40402 Tel: +886-4-2205-3366 ext: 2509
Fax: +886-4-2203-1075
E-mail: tdway@mail.cmu.edu.tw
ABSTRACT
In this study, we report that the essential oil obtained from Curcuma zedoaria 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Rosc, known as zedoary, possessed efficient cytotoxic effects on non-small cell lung carcinoma (NSCLC) cells and caused cell apoptosis. Zedoary essential oil increased sub-G1 population and annexin-V binding, induced cleavage and activation of caspases 3, 8, 9, and poly(ADPribose) polymerase (PARP). Decrease in Bcl-2 and Bcl-xL and elevation of the Bax/Bcl-2 ratio were also observed following zedoary essential oil treatment. Notably, zedoary essential oil led to the release of AIF, Endo G, and cytochrome c into the cytosol and increase p53 levels in H1299 cells. Our results indicated that zedoary essential oil slightly inhibited the phosphorylation of ERK1/2 and increased the phosphorylation of JNK1/2 and p38. Zedoary essential oil also inhibited AKT/NF-κB signaling pathways in H1299 cells. Moreover, intraperitoneal administration of zedoary essential oil significantly suppressed the growth of H1299 cells in vivo. In addition, potential active compounds were detected using gas chromatography-mass (GC-MS), 8,9-dehydro-9-formyl-cycloisolongifolene, 6-ethenyl-4,5,6,7-tetrahydro-3,6-dimethyl-5-isopropenyl-trans-benzofuran, eucalyptol and -elemene were found in zedoary essential oil. In summary, our findings provide insight into the molecular mechanisms underlying zedoary essential oil-induced apoptosis in NSCLC cells and worthy of continued study. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
Key words: Curcuma zedoaria Rosc; Apoptosis; p53; MAPK; AKT/NF-κB
ABBREVIATIONS
DISC, death-inducing signaling complex; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; DRs, death receptors; GC-MS, gas chromatography-mass; LPS, lipopolysaccharides; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NSCLC, non-small cell lung carcinoma; PARP, poly(ADPribose) polymerase; PI, propidium-iodide; PS, phosphatidylserine
INTRODUCTION
Curcuma zedoaria (Christm.) Roscoe known as zedoary is a perpetual plant
found in tropical countries. Zedoary is used as spice, natural flavor and herb in India, Indonesia and China. In China, it is traditionally for the treatment of flatulence, 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84
dyspepsia, menstrual disorders, dyspepsia, cough and fever.1 Zedoary extract has been
tested anti-allergic,2 antitumor,3 presenting analgesic,4 and antimicrobial activity.5
Lung cancer is the leading cause of cancer deaths in both men and women. Non-small cell lung carcinoma (NSCLC) accounts for approximately 80-85% of all lung cancer patients. NSCLC is an aggressive tumor with poor prognosis and a 5 year survival rate of only 16%.6,7 The high prevalence and mortality rate make it an urgent
requirement for functional diagnosis methods and useful medical drug development. The most efficient strategy for developing anti-cancer drugs is to induce apoptosis in cancer cells. Characteristics of apoptosis include membrane blebbing, translocation of phosphatidylserine (PS) of the plasma membrane, chromatin condensation, DNA fragmentation, apoptotic bodies, and the caspase cascade of cell death signaling. Apoptosis is triggered through two major pathways, the extrinsic and intrinsic pathways. The extrinsic pathway can induce activation of the intrinsic pathway via the caspase-8 activation. Once activated, caspase-8 cleaves Bid to a truncated Bid (tBid), thus, apoptotic signals initiated by death receptors (DRs) can be linked to mitochondria-mediated intrinsic pathway. The mitochondrial-mediated intrinsic apoptosis is initiated by an alteration in mitochondrial permeability, causing the release of apoptogenic factors, such as AIF, cytochrome c, and endonuclease G from the inter-membranal space. These apoptogenic factors complexe with apoptotic 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103
protease activating factor-1 (Apaf-1) to form apoptosome activates caspase-9 and other effector caspases that cleave different substrates.8,9
Pharmacological studies and clinical trials demonstrate that zedoary essential oil exhibits a lot of therapeutic activities, such as antioxidant, anticancer hepatoprotection, and anti-bacterial action.1,10,12 The main bioactive components in
zedoary essential oil include β-elemene, curdione, neocurdione, curzerene, germacrone, and furanodiene.11 In our study, the extract of zedoary essential oil was
analyzed by gas chromatography-mass (GC-MS). From database searching, we identified the components of zedoary essential oil. We also identified zedoary essential oil antitumor ability by cell toxicity and animal study. In our study, we have showed that the extract of zedoary essential oil has got an effective antitumor activity.
MATERIALS AND METHODS
Chemicals and reagents. Anexin V, MTT (3-(4,5 dimethylthiaxol-2-yl)-2,5-diphenyl tetrazolium bromide), Propidium-iodide (PI), and antibodies for caspase 9, caspase 3, Bax, Bcl-2, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), and β-Actin were purchased from Sigma (St. Louis, MO, USA). The caspase inhibitor Z-104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
VAD-FMK was obtained from Promega Corporation (Madison, WI, USA). Antibodies for AKT, p-AKT, ERK1/2, p-JNK1/2, JNK1/2, AIF, Endo G, cytochrome
c, Cox4, and p53 were purchased from Cell Signaling Technology (Beverly, MA,
USA). Antibodies for p38, p-ERK1/2, and p-p38 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for rabbit and mouse conjugated with horseradish peroxidase (HRP) were purchased from Chemicon (Temecula, CA, USA). Chemiluminescent HRP substrate was purchased from Millipore Corporation (Billerica, MA, USA).
Plant material, isolation and fractionation of zedoary essential oil. Zedoary was harvested at 5 months after planting in Taichung District Agricultural Research and Extension Station in 2012. The Rhizomes of zedoary were powdered and extracted at room temperature. A 200 g of the powder was mixed with distilled water and boiled for 3 h. The zedoary essential oil was collected and then dried under sodium sulfate. The yield of zedoary essential oil was 1.6% (mL/g).
GC-MS analysis of zedoary essential oil. Zedoary essential oil components were conducted on a GC-MS system (Shimadzu GCMS-QP2010, Japan) with capillary column (DB-5, 30 m × 0.250 mm, J&W Scientific, Folsom, CA, USA) using helium as a carrier gas. The flow rate of helium was 1.0 mL/min. The injection temperature was set at 60 oC, and then programmed at 5 oC /min to 120 oC held for 13
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min, then at 25 oC /min to 145 oC held for 20 min, finally at 30 oC /min to 280 oC. The
MS was set in electron-impact (EI) mode; the ionization energy was 70eV and scan rate 0.34 s per scan.
Cell culture. H1299, A549, and H23 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Carlsbad, CA, USA) in a humidified incubator at 37 oC with 5% CO
2.
Cytotoxicity assay. The effect of zedoary essential oil on cell cytotoxicity was determined by MTT assay.13 Cells were dispersed evenly in culture medium and
seeded in a 96-well plate for 1×104 cell number/well. After 37 oC and 5% CO
2 for 24
h, the culture medium was replaced by different concentrations of zedoary essential oil. Positive controls were treated with 1% DMSO for the same duration. After treatment with zedoary essential oil at various concentrations, and incubated at 24, 48 and 72 h intervals, 40 μL MTT solution were add into each well and incubated at 37
oC and 5% CO
2 incubator for 1 h. The supernatant was aspirated and MTT-formazan
crystals were dissolved by 110 μL of DMSO. Finally, the absorbance was determined and recorded by a microplate reader at a wavelength of 570 nm.
Analysis for cell cycle distribution. The cell cycle state was determined by 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161
using PI stainingas reported previously.14 H1299 cells (5×105) were cultured in 6-cm
cell culture dish and treated with 110 μg/mL zedoary essential oil for 12, 24, 48, and 72 h, respectively. Then cells from each dish were harvested individually by centrifugation and were measured the cell cycle distribution by flow cytometeric assay. Briefly, isolated cells were fixed gently by 2 mL of 100 % ethanol at -20 oC
overnight and then re-suspended in PBS containing 50 μg/ml PI and 0.1 mg/ml RNase A and 0.1% Triton X-100. The mixture was allowed to stand on ice for 30 min and analyzed with a FAC-Scan cytometry (BD Biosciences, San Jose, CA, USA).
Apoptosis detection. Cell undergoing apoptosis was detected by PI and annexin V staining. H1299 cells were treated with 110 μg/ml zedoary essential oil for 24, 48, and 72 h, respectively. 5×105 H1299 cells were harvested and washed with PBS, then
resuspended in 500 μL binding buffer. Each of 5 μL PI and annexin V FITC were added and incubated in dark room for 30 min and analyzed with a FAC-Scan cytometry (BD Biosciences, San Jose, CA, USA).
DNA fragmentation assay. Fresh H1299 cell DNA was extracted from 2×106
cultured cells using a Sigma genElute Mammalian Genomic DNA Purification Kit following the manufacturer’s instructions. The DNA fragmentation is screened by 1% agarose gel electrophoresis. The DNA ladder was visualized with ethidium bromide staining. 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180
Western blotting analysis. Cells (1 × 106 per dish) were placed in 10-cm cell
culture dish and were treated with various agents as indicated in legends. After treatment with zedoary essential oil, cells were harvested and lysed with ice-cold lysis buffer. Equivalent amounts of proteins in each treatment were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% (w/v) nonfat milk, and immunoblotted as described previously.15
Reactive oxygen species (ROS) production. H1299 cells (2 × 105 per well)
placed in 12-well plates were treated with 110 g/mL of zedoary essential oil for 0, 0.5, 1, 2, and 4 h to determine the level of ROS. H1299 cells were harvested and suspended in 500 L of 10 M dichloro-dihydro-fluorescein diacetate (DCFH-DA) for ROS measurement. Finally, all samples were incubated at 37 °C for 30 min and analyzed with a FAC-Scan cytometry (BD Biosciences, San Jose, CA, USA).
Cytochrome c, AIF, and Endo G release. The cytosolic and mitochondrial fractions were prepared by resuspending H1299 cells in ice-cold buffer A (1 mM EDTA, 20 mM HEPES, 250 mM sucrose, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1
mM EGTA, 17 mg/mL PMSF, 2 mg/mL leupeptin, and 8 mg/mL aprotinin (pH 7.4)). H1299 cells were passed 10 times through a 27G-needle. Nuclei and nnlysed cells were pelleted by centrifugation at 750 g for 10 min, and the supernatant was then centrifuged at 100,000 g for 15 min. The pellet, representing the mitochondrial 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199
fraction, was resuspended in ice-cold buffer A. The supernatant was centrifuged again at 100,000 g for 1 h, and the supernatant from the final centrifugation represents the cytosolic fraction.
In vivo studies. In vivo tumorigenesis inhibition was determined by subcutaneous injection of 1 × 105 H1299 cells in the back side of 4-7 week-old
BALB/c (nu/nu) nude mice, according to Institutional Animal Care and Use Committee (IACUC) standard protocols. After two weeks, tumor volumes larger than 100 mm3 were selected for next step inhibition study. Four animals were used for each
set of different concentrated zedoary essential oil solution. Zedoary essential oil was dissolved in 10% ethanol-PBS solution with different concentrations. The zedoary essential oil solution was intraperitoneal (i.p.) injected to mice according to standard protocols. Tumor inhibition were determined by measuring tumor size twice a week and tumor volume was calculated by standard formula with (tumor width)2 × (tumor
length)/2.
Statistical Analysis. The independent Student’s t-test was used to compare the continuous variables between two groups, a probability of p <0.05 being considered significant. 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218
RESULTS
Zedoary essential oil inhibited NSCLC cell proliferation. The anti-tumor effect of zedoary essential oil has been successfully reported in a wide range of cancers.1,16 To evaluate the anti-tumor effect of zedoary essential oil against NSCLC
cells, the H1299, A549 and H23 cells were treated with various concentrations of zedoary essential oil (Figure 1) for 24, 48, and 72 h, respectively and examined for cell viability by MTT assay. Zedoary essential oil caused a time- and concentration-dependent inhibition of NSCLC cell proliferation. Zedoary essential oil with IC50
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values ranging from 80 to 170 g/mL in H1299 cells, 80 to 250 g/mL in A549 cells, and 180 to 185 g/mL in A549 cells, respectively. Compared to other NSCLC cells, zedoary essential oil demonstrated the most efficient cytotoxic effects on H1299 cells (Figure 1). These results show that zedoary essential oil exhibits high potency in inhibiting cell proliferation in NSCLC cells.
Zedoary essential oil induced cell death in H1299 cells. To determine whether zedoary essential oil altered cell cycle progression or induced apoptosis, we performed flow cytometry by using PI staining. H1299 cells were treated with 110 μg/mL zedoary essential oil (the half maximal inhibitory concentration as assessed by MTT assay) and then harvested at 12, 24, 48, and 72 h, respectively. As shown in Figure 2A, zedoary essential oil induced cell death (sub-G1 phase) in a time-dependent manner. The proportion of sub-G1 phase cells was raised from 11.4% to 53.3% in 72 h (Figure 2B).
Zedoary essential oil induced apoptosis in H1299 cells. The increased sub-G1 phase cell number in flow cytometry is suggestive of apoptosis. To further determine whether zedoary essential oil induced apoptosis in H1299 cells, we performed flow cytometry using PI and FITC-conjugated annexin V staining. As shown in Figure 3A, zedoary essential oil induced cell apoptosis in a time-dependent manner. The highest value was observed at 72 h exposure, being for 41.6% apoptosis. In comparison with 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259
the control, zedoary essential oil treatment resulted in DNA fragmentations, a hallmark of cell apoptosis in a time-dependent manner (Figure 3B).
Zedoary essential oil induced cleavage and activation of caspase in H1299 cells. Caspase activation plays a important role in the initiation and success of apoptosis.17 To monitor the enzymatic activity of caspases during zedoary essential oil
-induced apoptosis, we carried out western blotting. Our result found that cleavage patterns of caspase-3 were observed in H1299 cells in a concentration-dependent (Figure 4A) and time-dependent (Figure 4B) manner. Cleavage of PARP was also observed in a concentration-dependent (Figure 4A) and time-dependent (Figure 4B) manner. We next determined levels of other caspases associated with apoptosis. Cleavage of caspase-8 and caspase-9 was detected after zedoary essential oil treatment in a concentration-dependent (Figure 4C) and time-dependent (Figure 4D) manner. Agreeing with these finding, the general caspase inhibitor Z-VAD-FMK significantly inhibited the zedoary essential oil-induced apoptosis in a dose-dependent manner (Figure 4E).
Zedoary essential oil affected ROS production in H1299 cells. We next tested whether zedoary essential oil-induced apoptosis is accompanied with the production of ROS. The results showed in Figure 5A indicated that zedoary essential oil promoted the production of ROS in a time-responded manner.
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Effect of zedoary essential oil on the expression of Bcl-2 family in H1299 cells. One of the major mechanisms underlying the ultimate fate of cells in the apoptotic process is the imbalance of expression of anti- and proapoptotic proteins.18
We examined the expression of antiapoptotic proteins, Bcl-xL and Bcl-2, after various concentrations zedoary essential oil treatment. The exposure of H1299 cells to zedoary essential oil resulted in down-regulation of Bcl-xL and Bcl-2 in a concentration-dependent manner (Figure 5B). However, data in Figure 5C showed that zedoary essential oil increased Bax in a concentration-dependent manner. After treatment with zedoary essential oil, optical densitometric analysis revealed an increase in the ratio of Bax to Bcl-2 (data not shown). Moreover, as shown in Figure 5D, zedoary essential oil caused an increase of p53 protein level.
Zedoary essential oil-induced cytochrome c, Endo G, and AIF release. The release of cytochrome c, Endo G, and AIF from the mitochondria is the main gate in turning on apoptosis. We next tested whether zedoary essential oil induced AIF, Endo G, and cytochrome c release. Figure 5E, 5F showed that zedoary essential oil led to the release of cytochrome c, Endo G, and AIF into the cytosol in a concentration-dependent manner.
Effects of zedoary essential oil on ERK1/2, p38, and JNK1/2 activation in H1299 cells. To identify whether zedoary essential oil affects mitogen-activated 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297
protein kinases (MAPK) activation, H1299 cells were exposed to zedoary essential oil for various incubation times. Using antibodies specific for phosphorylated ERK1/2, JNK1/2 and p38, Figure 6A indicated that zedoary essential oil slightly inhibited the phosphorylation of ERK1/2 but increased the phosphorylation of p38 and JNK1/2 (Figure 6A).
Zedoary essential oil inhibited AKT/NF-kB signaling pathways in H1299 cells. A prominent mechanism linking AKT/NF-κB signaling to NSCLC cancer progression is the abrogation of apoptosis.19,20 We next identified the effect of zedoary
essential oil on the phosphorylation of AKT and IκB. Figure 6B found that zedoary essential oil inhibited phosphorylation of AKT and IκB in a time-dependent manner.
Antitumor activity of zedoary essential oil in NSCLC xenografts. To evaluate the in vivo antitumor activity of zedoary essential oil, BALB/c (nu/nu) nude mice was implanted s.c. with H1299 cells. The control group was treated with 10% ethanol-PBS and the treated groups were given i.p. five times a week with zedoary essential oil (2.4, 12, 60, and 240 mg/kg). Our result showed that i.p. administration of zedoary essential oil induced a dose-dependent inhibition of H1299 tumor volume (Figure 7A) and reduction of tumor weight (Figure 7B). During the experiment duration, animals did not lose weight (Figure 7B) and no pathologic signs were seen.
Profile of constituents in zedoary essential oil extracts. To analyze the 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316
involved compositions of the zedoary essential oil, MS was performed. The GC-MS profile of the zedoary essential oil is shown in Supplementary Material Figure 8A. Two major compounds identified in zedoary essential oil were 8,9-dehydro-9-formyl-cycloisolongifolene (60%), 6-ethenyl-4,5,6,7-tetrahydro-3,6-dimethyl-5-isopropenyl--trans-benzofuran (12%) (Structures shown in Figure 8B and 8C). Other compounds such as eucalyptol and -elemene were identified as minor compounds.
DISCUSSION
Recent research suggests that zedoary essential possesses anticancer properties21,22. Zedoary essential oil could induce apoptosis in hepatic stellate cells.23
The hexane extract of zedoary essential oil has also been reported to have significant cytotoxicity on HepG224, SNU-1, SiHa, and HL-60 cells12. However, its function on
NSCLC cells has not been examined. In our study, zedoary essential oil was used to test its cytotoxicity on NSCLC cells. From MTT assay, tumor survival rate was decreased with the increasing zedoary essential oil dosage and expose time. The PI and annexin V staining result showed that the cell apoptosis had happened and the cell 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335
fragmentation confirmed apoptosis.
Cell death plays an important role in the efficacy of chemotherapy. Apoptosis is regulated by programmed cellular signaling pathways that control normal cell homeostasis and thought to be the principal mechanism by which anti-cancer drugs kill cancer cells. Importantly, dysregulation of apoptosis is a hallmark of cancer, with both the gain of anti-apoptotic mechanisms and the loss of pro-apoptotic signals contributing to tumorigenesis. The induction of apoptosis in many cancer cells can be divided into the extrinsic and intrinsic pathways. The extrinsic pathway is initiated by the interaction between specific ligands and death receptor and their downstream molecules are caspases-8. The intrinsic pathway is initiated by the mitochondria-mediated pathway and their downstream molecules are caspases-9.25 However,
cross-talk between the extrinsic and intrinsic pathways also exists.26 In this study, we
observed that zedoary essential oil induced the activition of caspase-8, -9, and -3 in H1299 cells. Interestingly, zedoary essential oil changed the extrinsic and intrinsic pathways-associated proteins, which subsequently promoted caspase-8 and caspase-9 activation and then activated the downstream effector caspase-3 in H1299 cells.
Recent study shows that AKT is constitutively active in >90% of NSCLC and contributes to chemotherapeutic resistance and led this cancer cells to evade apoptosis.19 The phosphorylation on Ser473 has been used as an indicator of AKT
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activity and have been found to correlate with poor prognosis.20 In our study, zedoary
essential oil inhibited Ser473 phosphorylation of AKT and the inhibition was an earlier event which subsequently followed with a reduced phosphorylation of other downstream members in the AKT signaling pathway. Activated AKT can phosphorylate pro-apoptotic Bcl-2 family member Bad. The Ser136 phosphorylation of Bad promotes its interaction with 14-3-3 proteins in the cytosol, blocking its interaction with Bcl-XL at the mitochondrial level.27,28 The present study indicates that
zedoary essential oil-mediated inactivation of AKT is associated with reduced Ser136 phosphorylation of Bad thus allowed Bad to interact with Bax to aggregate on mitochondrial membrane resulting release of cytochrome c to the cytosol. Once cytochrome c is released, it binds to APAF1 and leads to the assembly of apoptosome which triggered the pathway for caspase cascade in NSCLC cells. The present study observed that the pan broad spectrum caspase inhibitor Z-VAD-FMK could only partially alleviate apoptosis. We therefore concluded that zedoary essential oil might induce apoptosis both through caspase-dependent and –independent mechanisms.
Excessive production of mitochondria-derived ROS is important regulator of apoptosis. Our study showed that zedoary essential oil induced NSCLC cells apoptosis through overproduction of intracellular ROS. ROS have been showed to induce apoptosis by activation of the MAPK pathways.29 Three classic MAPK include
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ERK1/2, p38, and JNK1/2 have been identified in mammalian cells. The involvement of MAPK pathways during zedoary essential oil-induced NSCLC cells apoptosis has not been reported so far. Herein, we attempted to verify whether the MAPK pathways were involved in the process. We found that zedoary essential oil induced JNK1/2 and p38 phosphorylation. Continuous phosphorylation of JNK1/2 plays a crucial role in apoptosis.30 Based on our findings, it was hypothesized that zedoary essential oil
induce intracellular ROS generation and induced JNK1/2 and p38 phosphorylation, which contribute to NSCLC cells apoptosis. However, future studies are necessary to fully understand the mechanism for zedoary essential oil induced JNK1/2 and p38 phosphorylation.
The in vivo tumor inhibition study in this study has shown that after 3 weeks injection the essential oil as an edible drug can functionally inhibit tumor proliferation. The tumor inhibition rate in high dose injected mouse could reach forty percent. This result emphasizes that the zedoary essential oil has a great potential to become a new anti-tumor candidate.
The main components of zedoary essential oil including neocurdione, curdione, germacrone, curzerene, furanodiene and -elemene have been reported. However, in our study, we found that the content of an interesting sesquiterpene, 8,9-dehydro-9-formyl-cycloisolongifolene reached about 60% in essential oil. The effectually tumor 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393
inhibition ability in our result indicated that the essential oil might contain potential compounds for novel anti-tumor drug discovery. Further study to find out the active compound in this essential is needed.
Acknowledgment
This study was supported by the Taichung District Agricultural Research and Extension Station, Council of Agriculture grant 101AS-9.2.1-CS-D4.
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Figure legends
Figure 1. The proliferation-inhibitory effect of zedoary essential oil on NSCLC 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535
cells. H1299, A549, and H23 cells were treated with various concentrations of zedoary essential oil at 37 oC for 24, 48, and 72 h, respectively. The effect on cell
growth was examined by MTT assay, and the percentage of cell proliferation was calculated by defining the absorption of cells without zedoary essential oil as 100%. This experiment was repeated three times. Bar represents the SEM. Values significantly were different from the control group. *, P < 0.05.
Figure 2. Effect of zedoary essential oil on cell cycle progression in H1299 cells. (A) H1299 cells were treated with 110 μg/mL zedoary essential oil for 12, 24, 48, and 72 h, respectively and analyzed for PI-stained DNA content by flow cytometry. (B) The indicated percentages are the mean of three independent experiments, each in duplicate. The sub-G1 phase H1299 cells increased with time.
Figure 3. Zedoary essential oil induces H1299 cells apoptosis. (A) H1299 cells were treated with 110 μg/ml zedoary essential oil for 24, 48, and 72 h, respectively. Cell apoptosis percentages were determinate by flow cytometry with annexix V/PI staining. (B) H1299 cells were treated with various concentrations of zedoary essential oil at 37 oC for 48 h. Zedoary essential oil treatment resulted in typical DNA
fragmentation as indicated by DNA laddering. M, 100bp DNA marker. D, 1% DMSO treatment controls.
Figure 4. Effect of zedoary essential oil on caspases activity in H1299 cells. (A) 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554
H1299 cells were treated with various concentrations of zedoary essential oil for 48 h. Cells were then harvested and lysed for the detection of cleaved caspase 3, cleaved PARP and -actin protein expression. (B) H1299 cells were treated with 110 μg/mL zedoary essential oil for 6, 24, 48, and 72 h, respectively. Cells were then harvested and lysed for the detection of pro-caspase 3, cleaved caspase 3, cleaved PARP and -actin protein expression. (C) H1299 cells were treated with various concentrations of zedoary essential oil for 48 h. Cells were then harvested and lysed for the detection of cleaved caspase 9, cleaved caspase 8 and -actin protein expression. (D) H1299 cells were treated with 110 μg/mL zedoary essential oil for 6, 24, 48, and 72 h, respectively. Cells were then harvested and lysed for the detection of pro-caspase 9, cleaved caspase 9, pro-caspase 8, cleaved caspase 8 and -actin protein expression. Western blot data presented are representative of those obtained in at least three separate experiments. (E) Z-VAD-FMK or the vehicle (DMSO) was added to the medium at 1 h before the 110 μg/mL zedoary essential oil treatment. After the 72 h incubation, the H1299 cell viability was determined using MTT assay. This experiment was repeated three times. Bar represents the SEM. Values significantly were different from the control group. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Figure 5. Effect of zedoary essential oil on apoptosis related proteins in H1299 cells. (A) Cells were treated with 110 g/mL of zedoary essential oil for 0, 0.5, 1, 2, 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573
and 4 h for the production of ROS. All samples were analyzed by flow cytometric assay as described in Materials and Methods. H1299 cells were treated with various concentrations of zedoary essential oil for 48 h. Cells were then harvested and lysed for the detection of (B) Bcl-2, Bcl-xL and actin (C) Bax and actin (D) p53 and β-actin protein expression. H1299 cells were incubated with 110 g/mL of zedoary essential oil for indicated duration. Levels of AIF, Endo G, and cytochrome c in the (E) cytosolic and (F) mitochondrial fraction were analyzed by immunoblotting. Western blot data presented are representative of those obtained in at least three separate experiments.
Figure 6. Effect of zedoary essential oil on the MAPK and AKT/NF-κB signaling pathways. H1299 cells were treated with a vehicle (DMSO) or zedoary essential oil (110 µg/mL) for the indicated time. Cells were then harvested and lysed for the detection of (A) ERK1/2, phospho-ERK1/2, p38, phospho-p38, JNK, phospho-JNK, and β-actin (B) AKT, phospho-AKT, IB, phospho-IB, and β-actin protein expression. Western blot data presented are representative of those obtained in at least three separate experiments.
Figure 7. Effect of zedoary essential oil on anti-tumor activity. H1299 cells were used to establish xenografts in male BALB/c nude mice. Animals (six mices/group) were given control, zedoary essential oil (2.4, 12, 60, 240 mg/kg, respectively) by 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592
given i.p. injection 5 times/weekly. (A) tumor volume (mm3), (B) tumor weight (g),
and body weight (g).
Figure 8. The profile of constituents in zedoary essential oil extracts. (A) The GC-MS profile of the zedoary essential oil. (B) Chemical structure of 8,9-dehydro-9-formyl-cycloisolongifolene. (C) Chemical structure of 6-ethenyl-4,5,6,7-tetrahydro-3,6-dimethyl-5-isopropenyl-trans-benzofuran. 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610
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