Curcumol Induces Apoptosis in SPCA-1 Human Lung Adenocarcinoma Cells and Displays Antineoplastic Effects in Tumor Bearing Mice

Curcuma rhizomes are herbal remedies commonly used in oriental medicines such as Ayurveda and traditional Chinese medicine (Shishodia et al., 2005; Xia et al., 2005). During the past twenty years, curcumin, one of the key active substances of curcuma rhizomes attracted great interests in biomedical research (Shishodia et al., 2005; Anand et al., 2007). So far, thousands of research papers have been published with curcumin. The research interests have been extended to other active substances of curcuma rhizomes recently. Curcumol, a sesquiterpene is another active substance of curcuma rhizomes (Carey et al., 2013; Chen et al., 2014). Similar to curcumin, curcumol also displayed a variety of beneficial pharmacological activities in preclinical studies, including anti-inflammation (Chen et al., 2014), anti-rheumatism (Wang et al., 2012; Wang et al., 2014; Yu et al., 2014) and anti-seizure (Ding et al., 2014) and hepato-protection (Chen et al., 2014). Moreover, the anti-neoplastic effects of curcumol have been well reported in various cell culture models (Wang et al., 2011; Zhang et al., 2011; Tian et al., 2012; Jing et al., 2013; Guo et al., 2014). However, the in vivo anti-cancer efficacy of curcumol remained unclear. To


Introduction
Curcuma rhizomes are herbal remedies commonly used in oriental medicines such as Ayurveda and traditional Chinese medicine (Shishodia et al., 2005;Xia et al., 2005).During the past twenty years, curcumin, one of the key active substances of curcuma rhizomes attracted great interests in biomedical research (Shishodia et al., 2005;Anand et al., 2007).So far, thousands of research papers have been published with curcumin.The research interests have been extended to other active substances of curcuma rhizomes recently.

Curcumol Induces Apoptosis in SPC-A-1 Human Lung Adenocarcinoma Cells and Displays Anti-neoplastic Effects in Tumor Bearing Mice
Qi-Ling Tang 1, 5& , Ji-Quan Guo 2& , Qi-You Wang 3& , Hai-Shu Lin 4 , Zhou-Ping Yang 5 , Tong Peng 1 , Xue-Diao Pan 5 , Bing Liu 5 , Su-Jun Wang 5 , Lin-Quan Zang 5 * further examine the medicinal potential of curcumol, its anti-proliferative and apoptosis-inductive activities were assessed in SPC-A-1 human lung adenocarcinoma cells while the anti-cancer effects were examined in tumor bearing mice.To test whether the suppressive effects of curcumol are more specific to cancerous cells, the impact of curcumol on the growth of non-transformed cells was also attempted usingMRC-5 human embryonic lung fibroblasts as a model.To the authors' knowledge, this is the first report on the in vivo anti-neoplastic efficacy of curcumol in lung cancer model.Findings from this study will be useful to evaluate the application of curcumol as a chemotherapeutic agent.

Instruments
Plate reader (BIO-RAD, California, USA) was used for the MTT assays.The apoptosis were quantified with a flow cytometer (Bechman-Coulter, California, USA), and the morphological analysis of cells was obtained with an inverted fluorescence microscope (Leica, Germany).

Animals
The in vivo study was carried out with compliance to the Guide for the Care and Use of Laboratory Animals (National Academy Press, 2011).The animal handling protocol was reviewed and approved by the Institutional Animal Care and Utilization Committee (IACUU) of the Experimental Animal Center of Guangdong Pharmaceutical University (approval no.SYXK (Yue) 2012-0125).Five-week-old BALB/c nude mice were obtained from Animal Experimental Center of Guangdong Province (Experimental Animals Certificate: SCXK (Yue) 2013-0002) (Guangdong, China).The animals were maintained in specific pathogen free animal facility with a 12h/12h light-dark cycle at 22℃-24℃ and 50% humidity.The animals had free access to food and water.

Cells culture and MTT assay
Human lung cancer cell line SPC-A-1 and human embryonic lung fibroblast cell line MRC-5 was obtained from Culture Biology of the Chinese Academy of Sciences (Shanghai, China).SPC-A-1 cells was cultured in RPMI-1640 medium while MRC-5 cells in DMEM medium, both of them was supplemented with 10% fetal bovine serum.The cells were subcultured in a humidified atmosohere with 5% CO 2 at 37℃ till reaching logarithmic growth phase.
Cells were seeded in a 96-well culture plate and treated with various concentrations of curcumol once for 24, 48 and 72h, respectively.After that, MTT assay was carried out using standard protocol and optical density (OD) was measured at 570 nm by using anenzyme-labeling instrument (BIO-RAD, USA).Cell proliferation was calculated, and the experiment was repeated 3 times.

Apoptosis assay by hoechst 33258
The SPC-A-1 cells and MRC-5 cells in logarithmic growth phase were seed in 24-well culture plate, and the cells were exposed to curcumol (6. 25, 12.5, 25, 50, 100μM) for 48h.Then SPC-A-1 cells were stained by Hoechst 33258, and the changes in the nuclei of cells were examined and photographed by using a fluorescence microscope (Leica, Germany).

Determination of cell apoptosis by annexin V/PI double staining
Logarithmic growth phase of the SPCA-1 cells were seeded in 6-wells plate with density of 1×10 5 cells/ml.After cells were treated with various concentrations (0, 6. 25, 12.5, 25, 50, 100μM) of curcumol for 48h, the cells were doubled stained by Annexin V/PI apoptosis kit following to the instructions.Then the cell apoptosis rate was detected in a flow cytometer (Bechman-Coulter, USA).

Determination of cell cycle by PI staining
Logarithmic growth phase of the SPC-A-1 cells were seeded in 6-wells plate and treated with various concentrations (0, 6. 25, 12.5, 25, 50, 100μM) curcumol for 48h.Then the cells were exposed in RNase (20 μg/ml), and incubated with PI (50 μg/ml).Cell cycle distribution was analyzed using flow cytometry.The ModFit 3.1 program was used to determine the percentage of cells stalled at each phase of the cell cycle.

Antitumor activity study of curcumol in vivo
All the mice were injected in the right flank subcutaneously with SPC-A-1 cells (2×10 6 cells in 100 μl per site) at right forelimb armpit.When the tumors grew to approximate 4-5 mm in diameter, the tumor bearing mice were randomly allocated to 5 groups.
All the groups were observed for 13 days, and the tumor sizes were measured every two days.Tumor diameters were determined by using a Vernier caliper, Volume= (width 2 ×length)/2.The mice were sacrificed on day 13, the tumors were isolated and weighted.And the tumors tissues were collected for Hematoxylin-eosin staining.

Pathological observation of tumor tissue by hematoxylineosin staining
Each tumor of nude mice in different groups was fixed in 4% formaldehyde, dehydrated, paraffin-embedded, sectioned, and stained with Hematoxylin-eosin to observe changes in pathological cell morphology using a light microscope.

Data analysis and statistics
All experimental data are presented as Mean±Standard Error of the Mean (SEM).The value of 50% inhibitory concentration (IC 50 ) was calculated with GraphPad Prism 6.05 (GraphPad Software, Inc., La Jolla, CA, USA).Two-tailed independent sample t-test was carried out with GraphPad InStat version 3.10 (GraphPad Software, Inc.).A p value less than 0.05 was considered to be statistically significant.

Anti-proliferative activities of curcumol
The growth-inhibitory effects of curcumol were examined in SPC-A-1human lung adenocarcinoma cells using cisplatin (25 µM), a common chemotherapeutic agent for lung cancer as a positive control (Figure 1 (a), (b) & (c)).Clearly, curcumol displayed time-and concentration-dependent anti-proliferative activities in SPC-A-1 cells.After 72h incubation, the 50% inhibitory concentration (IC 50 ) on proliferation was found to be 32.7 µM.Cisplatin at 25 µM possessed stronger growth inhibitory effect.
To find out whether the anti-proliferative activities of curcumol are more specific to cancerous cells, the DOI:http://dx.doi.org/10.7314/APJCP.2015.16.6.2307Curcumol Induces Apoptosis in SPC-A-1 Human Lung AC Cells and Anti-neoplastic Effects in Tumor Bearing Mice impact of curcumol on the growth of MRC-5 human embryonic lung fibroblasts (non-transformed cells)was also attempted (Figure 1 (d), (e) & (f)).Interestingly, curcumol did not have much growth inhibitory effect in MRC-5 and incubation with curcumol at 100 µM for 72h only led to less than 20% inhibition in comparison to the control.Clearly, the anti-proliferative activities of curcumol appeared to be more specific to cancerous cells.

Morphological analysis of curcumol treated SPC-A-1 cells and MRC-5 cells
The morphology of curcumol treated SPC-A-1 cells and MRC-5 cells was observed after Hoechst 33258 staining.The nuclei of SPC-A-1 cells in control group (0 µM curcumol) emitted quite homogenous blue fluorescence (Figure 2 (a)), which could be explained by an even distribution of the chromatin in the nuclei.On the other hand, for some cells treated with curcumol, nuclear condensation, which emitting bright fluorescence was observed (Figure 2 (b), (c), (d), (e) & (f)).Moreover, the cells treated with higher levels of curcumol, i.e. 25, 50 and 100 µM had higher percentage of condensation.However, apoptotic bodies did not observed in MRC-5 cells treated by curcumol (Figure 3 (a), (b), (c), (d), (e), (f)).Clearly, curcumol treatment could induce apoptosis in SPC-A-1 cells, while did not display apoptosis in MRC-5 cells.

Curcumol induced phase-arrest and apoptosis SPC-A-1 cells
The anti-neoplastic effects of curcumol were subsequently examined with flow cytometry.The cell cycle distribution was profiled after propidium iodide (PI) staining while the percentage of cells undergone apoptosis was quantified with Annexin V/PI double staining.The results are summarized in Table 1, the apoptosis of cells were observed in Figure 4.
Clearly, curcumol treatment led to cell cycle phase arrest at G0/G1 phase.Even at a concentration as low as 6.25 µM, curcumol increased the percentage of G0/ G1 phase SPC-A-1 cells.Interestingly, further increases in curcumol concentration up to 50 µM did not lead to much change in the of phase distribution.In agree with the morphological observation, the flow cytometry analysis using Annexin V/PI double staining also confirmed that curcumol treatment induced apoptosis in SPC-A-1 cells.The apoptosis-inductive effect appeared to be concentration-dependent.

Curcumol displayed in vivo anti-cancer activities in tumor bearing mice
As curcumol displayed promising anti-neoplastic effects in in vitro studies, its in vivo anti-cancer efficacy  was subsequently attempted in tumor bearing mice using cisplatin (1.5 mg/kg) as a positive control.Tumors were introduced through subcutaneous injection of SPC-A-1 cells (2×10 6 cells) at right forelimb armpit.Drug treatment (curcumol or cisplatin) was started when the tumors diameter were about 4-5 mm and the drug treatment last for 12 days.At the end of the experiments, the mice were sacrificed and the tumors were isolated for further examination.The in vivo anti-cancer efficacy of curcumol is shown in Figure 6.This mouse xenograft tumor model appeared to be fairly aggressive.For the mice in vehicle group, as they were not treated with anti-cancer agent (curcumol or cisplatin), the tumors progressed rapidly and the volumes increased by a few folds within 13 days (Figure 6 (a)).
Moreover, all tumor bearing mice suffered from severe weight loss (Figure 6 (c)).As a positive control, cisplatin at the dose of 1.5 mg/kg displayed some anti-tumor effects.Although it decreased tumor volume and weight after 12 daily doses (p<0.05vs vehicle group), it did not have protective effect in weight loss.As a chemotherapeutic agent, curcumol exhibited dose-dependent anti-cancer activities.Although the difference was statistically insignificant, the mice received low and intermediate doses of curcumol (6.67 and 20 mg/kg) tended to have smaller tumors and suffered less from weight loss in comparison to the control group.When the dose of curcumol further increased to 60 mg/kg, the in vivo anti-cancer cancer efficacy (using tumor volume, tumor weight and loss in body weight as endpoints) could be confirmed by statistical test (p<0.05vs vehicle group).Interestingly, the anti-tumor activities of curcumol appeared to fast-acting.After 4 daily doses (60 mg/kg), curcumol was able to reduced tumor volume.The disease modifying effects of curcumol (60 mg/kg) was much faster than cisplatin (1.5 mg/kg).
The pathology of the isolated tumors was also examined.The histological observations are shown in Figure 7.The tumor cells isolated from mice received vehicle treatment are oval, large and polygonal; these cells also have hyperchromatic nuclei.On the other hand, the tumor cells isolated from mice treated with curcumol displayed nuclear condensation, chromatin gathered and

Discussion
According to World Health Organization, lung cancer is the leading cancer killer and it leads to 1.59 million deaths annually.As more than 80% of lung cancer cases are non-small cell lung cancer (NSCLC), we tested the anti-cancer activities of curcumol in SPC-1-A cell lines, a model for non-small cell lung cancer.Since cytotoxic combination chemotherapy, the first-line treatment for stage IV NSCLC includes platinum (cisplatin or carboplatin), we used cisplatin as the positive control.
For cytotoxic chemotherapy, the clinical toxicity and tolerability are one of the key issues.Therefore, it is of great interest to identify innovative chemotherapeutic agents that displayed selective cytotoxic effects to the malignant cells.Such selective chemotherapeutic agents may have better tolerability and safety profiles.The anti-proliferative effect of curcumol appeared to be more selective to cancer cells.For example, at 100 µM, curcumol did not possess much growth suppression in MRC-5 human embryonic lung fibroblasts (nontransformed cells); however, it induced substantial apoptosis SPC-1-A cells.The selectivity of curcumol might lead to fewer side effects.Moreover, as curcuma rhizomes are commonly used as spices in cooking and curcumol is present in curcuma rhizomes at high level, the toxicity of curcumol would be limited.Such postulation was supported by pre-clinical observation in dogs (Zhang et al., 2007).Relatively low toxicity appeared to be a major advantage of curcumol and it may lead to better clinical tolerability/safety.
Pharmacokinetics plays an important role in drug development.The pre-clinical pharmacokinetics of curcumol has been attempted in dogs and rats after intravenous administration (Zhang et al., 2007;Zhao et al., 2010).Curcumol had moderate clearance in rats (~30 ml/min/kg) but relatively rapid clearance in dogs (300~400ml/min/kg).As a cancer chemotherapeutic agent, curcumol could be delivered through prolonged intravenous infusion.Therefore, a relatively rapid clearance is not a barrier to its clinical application.Moreover, its half-life was not too short (rats: ~40 min, dogs: ~2h).So, its pharmacokinetic s was unlikely a barrier to its clinical application.
The anti-tumor activities of curcumol were examined in tumor-bearing mice in this study.To our knowledge, this is the first report on its in vivo anti-cancer efficacy in NSCLC model.Curcumol displayed rapid onset diseasemodifying effects and such effects could be observed as early as after 4 daily treatments.The fast-acting antineoplastic effects appear to be another advantage of curcumol.
The remedies used in Traditional Chinese Medicine (TCM) have emerged as an important source for anticancer drug discovery.Various new anti-neoplastic drugs, e.g.Huachansu (water extract of Chinese toad skin) and Kanglaite (oily extract of coix seeds) have been developed and approved for the application in oncology.These TCM originated anti-cancer drugs sometimes potentiate the anti-neoplastic effects of some standard chemotherapeutic agents and therefore offer a superior clinical anti-cancer efficacy (Zhan et al., 2012;Xie et al., 2013).Currently, cytotoxic combination chemotherapy is used as the firstline treatment for stage IV NSCLC.As curcumol worked fast and might have good safety profile, it is of great interest to test curcumol in combination together with other standard chemotherapeutic agent (s) in NSCLC.Cisplatin / carboplatin plus curcumol could be the initial attempt in the future study.
In summary, curcumol exhibited time-and concentration-dependent anti-proliferative and apoptosis-inductive activities in SPC-A-1human lung adenocarcinoma cells and displayed anti-cancer effects in tumor-bearing mice.In conclusion, as its growthsuppressive effects were more specific to cancer cells and it might have good safety profile, curcumol appears as favorable anti-cancer candidate for further development.

Figure 6 .Figure 7 .
Figure 6.In Vivo Anti-cancer Efficacy of Curcumol.(a) tumor volume; (b) tumor weight; (c) loss in body weight.The anti-cancer efficacy of curcumol was examined tumor bearing mice.Tumor volume was measured every two days.On day 13, the mice were sacrificed; the tumors were isolated and weighted.Symbols or columns represent mean values while error bars represent SEM (n = 8, except n = 7 in the group of mice received curcumol at 60 mg/kg); *p<0.05;**p<0.01