214 Original Article
Authors
Z. Zhang, S. Guo, X. Liu, X. Gao
Affiliation
Department of Oncology, First affiliated Hospital of Henan University of Science and Technology, Luoyang, China
Key words
Abstract
▶ paclitaxel ● ▶ non-small-cell lung cancer ● ▶ synergy ● ▶ α-pinene ● ▶ anticancer activity ●
▼
The objective of the present research work was to evaluate the synergistic interactions between Paclitaxel (PAC) with α-pinene and β-pinene using isobolographic method against non-smallcell lung cancer cells (NSCLC). This type of interaction between an established drug and a new compound is expected to enhance the efficacy of paclitaxel in combination as compared in isolation. Further, cell cycle analysis was carried out using flow cytometric analysis. Phase contrast microscopy was used to assess the effect of paclitaxel, α-pinene and β-pinene alone and in combination with each other in order to evaluate the effect of combination on cell apoptosis. Further, mitochondrial membrane potential was monitored in non-small-cell lung cancer cells (NSCLC) when treated with paclitaxel, α-pinene and β-pinene alone and in combination. The
results revealed that the combination of PAC with α-pinene or with β-pinene showed a plotted curve below the straight line, generating a substantial synergistic effect. The effects of the following combinations were examined utilizing isobolograms: PAC and α-pinene and PAC and β-pinene. The combination of PAC and α-pinene as well as of PAC and β-pinene actually generated a synergistic effect. We also examined the effects of these compounds on the cell cycle distributions of A549 cells by flow cytometric analysis. The percentage of sub-G0/G1-phase cells was decreased on the addition of α-pinene to PAC, while the population of G0/G1 cells was increased. The morphological changes characteristic of apoptosis like chromatin condensation and fragmentation of the nucleus were seen in PAC + α-pinene and PAC + β-pinene treated NSCLC cells.
received 28.04.2014 accepted 16.05.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1377025 Published online: September 4, 2014 Drug Res 2015; 65: 214–218 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. Guo Department of Oncology First affiliated Hospital of Henan University of Science and Technology No.24, Jing Hua Road Luoyang Henan 471003 China Tel.: + 86/379/64923 362 Fax: + 86/379/64923 362 [email protected]
Introduction
▼
Lung cancer is the most frequently diagnosed cancer responsible for almost 1.38 million deaths every year worldwide [1, 2]. In China, lung cancer has replaced liver cancer as the leading cause of death among people with malignant tumors in 2008. According to the statistics from the National office on tumor cure and prevention, about 600 000 people die of lung cancer each year in China. Lung cancer can be classified into 2 types, small cell lung carcinoma (SCLC) and nonsmall-cell lung carcinoma (NSCLC). NSCLC accounts for more than 80 % of all lung cancer patients. SCLC usually responds better to chemotherapy and radiotherapy than NSCLC. NSCLC is highly aggressive and metastatic cancer with limited treatment options. Most NSCLC patients show locally advanced (37 %) or metastatic (38 %) state when diagnosed [3–6]. The most frequently used therapies for NSCLC are chemotherapy,
Zhang Z et al. Synergy Effect of Pinenes … Drug Res 2015; 65: 214–218
radiotherapy and surgical treatments. Generally NSCLC is treated by surgical operation followed by chemotherapy to avoid recurrence. Unfortunately, 2/3 of the patients are inoperable when diagnosed. Among the patients who undergo surgical operation with overall 5-year survival rates of about 40 %, nearly 40 % belong to stage ΙΙΙA and a few to stage ΙΙΙB with a 5-year survival rate of about 20 %. About 50 % of the NSCLS patients receive chemotherapy with paclitaxel and carboplatin. However, these chemotherapeutic drugs produce serious side-effects such as leukopenia, hepatic dysfunction, disorder in renal function, nausea and vomiting. These sideeffects are most commonly attributed to tissue necrosis, apoptosis and inflammation caused by the chemotherapy treatment [7–10]. The toxic side-effects of these anticancer drugs can be minimized by using non-toxic potentiating agent or synergistic compound in combination with anticancer drugs which may possibly potentiate
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Synergistic Antitumor Effect of α-pinene and β-pinene with Paclitaxel against Non-small-cell Lung Carcinoma (NSCLC)
Original Article 215
onto standard 96-well microtiter plates. Following overnight culture, serially diluted samples (PAC, α-pinene, β-pinene or their various combinations) were added into the wells. After 3-day culture, the cell growth rates were evaluated by the assay of WST-1 and the IC50 values were calculated.
Isobolographic analysis of the interaction between 2 substances in A549 cells The IC50 values of the respective substances were as follows: 36.0 μM (α-pinene); 0.21 mg/mL (β-pinene); 11.5 nM (PAC). Then, IC50 values of PAC was measured on the addition of the respective concentrations of α-pinene (2.5, 5.0, 10.0 and 20.0 μM) or β-pinene (0.0125, 0.025, 0.05, 0.1, 0.2 and 0.4 mg/ ml). The IC50 value of PAC is expressed as 1.00 on the Y axis of the isobologram, while that of α-pinene or β-pinene alone is expressed as 1.00 on the X axis. The IC50 values thus obtained were plotted to make a line graph in the same way as above.
Examination of cell cycle regulation by flow cytometry A549 cells (1.0 × 105/well) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were plated onto a 60 mm dish. After incubation for 48 h, the cells were treated in the same way as described previously. The cells were incubated with the following substances for 48 h: control (0.5 % DMSO); α-pinene (80 μM); [PAC (80 nM)] [PAC (80 nM)] + α-pinene (80 μM). The adherent cells were treated with 0.25 % trypsin (Invitrogen Life Technologies) and combined with the floating cells. All the cells were treated with a CycletestTM Plus DNA Reagent Kit. Results of flow cytometry were obtained by FACScan flow cytometry (Becton Dickanson, San Jose, CA) at a wavelength of 488 nm.
Materials and Methods
Morphological changes in A549 cells after drug treatment
Cell lines and chemicals
The cells were cultured on 6 well plates and treated with PACand combinations of PAC with α-pinene and β-pinene for 48 h. After treatment, cells were fixed with methanol for 20 min, washed with phosphate-buffered saline (PBS), and then stained with 40 μg/ml of propidium iodide (PI) in the presence of 50 μg/ml RNase A. The morphology of nuclear chromatin was defined by the fluorescence of DNA-binding dye, PI, under a fluorescence microscope (Nikon, Japan).
▼
A-549 and H 460 non-small-cell lung cancer cell lines were purchased from the Shanghai Institute for Biological Sciences, CAS). The cells were maintained in RMPI-1640 medium (Gibco) with 10 % heat-inactivated fetal bovine serum (Gibco) under a humidified atmosphere of 5 % CO2 at 37 °C. α-pinene ( > 95 % purity by HPLC) was purchased from Sigma Chemical Company (St. Louis. Co). Paclitaxel was purchased from Hainanhaiyao Co., Ltd., China. Both paclitaxel and α-pinene were dissolved in dimethyl sulfoxide (DMSO). The caspase assay kit was purchased from G-Biosciences.
MTT viability assay Inhibitory effects of variable dosages of paclitaxel, α-pinene and β-pinene were measured using MTT cell viability assay. Cells were seeded in a 96-well plate at 1 × 105 cells per well overnight. Cells were treated with either paclitaxel, or α-pinene, or the combination, and incubated for 24 h at 37 °C. MTT (0.8 mg/ml, purchased from Sigma) was added to each well and incubated for 3 h at 37 °C, and the medium was replaced with 150 ml 0.01 N HCl and 10 % SDS mixture and incubated for 30 min at room temperature. Absorbance was measured with a microplate reader at 655 nm wavelength.
Inhibition against the growth of A549 and H460 cell lines
Disruption of mitochondrial membrane potential (ΛΨm) in lung cancer cells Mitochondrial membrane potential (ΛΨm) was measured by Rhodamine-123 dye. A-549 cells (5 × 105) were treated with PAC and combinations of PAC with α-pinene and β-pinene for 48 h and (ΛΨm) was measured by flow-cytometry. Rhodamine-123 (2 mM) was added 2 h before the end of experiment. After it, the cells were washed with PBS and incubated with PI (10 μg/ml) for 20 min the cells were analyzed by a FACScan flow cytometer.
Statistical analysis Statistical analysis was performed with SPSS16.0 software system. Data was analyzed statistically by using one-way ANOVA and Student’s t test. Values are shown as mean ± SD. All of the experiments were repeated 3 times. p < 0.05 was considered significant.
A549 or H460 cells (2 × 104/well) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were inoculated
Zhang Z et al. Synergy Effect of Pinenes … Drug Res 2015; 65: 214–218
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their effectiveness while minimizing their toxicity by reducing the dose. Such non-toxic potentiating agents which can possibly solve many problems of the clinical cancer treatment include low molecular weight terpenoids present in essential oils [11–14]. Synergy means working together of 2 or more substances to produce an effect greater than the sum of their individual effects. Synergism occurs when 2 or more herbal ingredients mutually enhance each other’s effect more significantly than the simple sum of these ingredients. Synergy represents a form of interaction as opposed to a simple addition response. The elucidation of drug combination interaction (i. e., whether additive, synergistic, or antagonistic) is complex. The most common methods are median effect, isobologram, and combination index (CI) analyses. These 3 methods take into account the sigmoidal shape of the dose-response relationships, as typically seen in cytotoxicity experiments. α-pinene has been reported to exhibit anticancer activity against human ovarian cancer cell lines and human hepatocellular liver carcinoma cell lines [15, 16]. α-pinene has also been reported to show antioxidant, anticancer and genotoxic properties on N2a neuroblastoma cells [17]. In the present study, we investigated the synergistic potentiation of the anticancer activity of paclitaxel against NSCLC in presence of α-pinene using combination index method and isobologram analysis. We also studied the mechanism of action by which α-pinene enhances the efficacy of paclitaxel in combination. To the best of our knowledge, this kind of study is the first such report on paclitaxel on NSCLC cell lines using α-pinene as a bioenhancer.
216 Original Article
▼
Determination of cytotoxicity of paclitaxel, α-pinene and β-pinene using the MTT assay In order to assess the combination effects of the chemotherapy drugs with α-pinene or β-pinene against the growth of A549 cells, isobolograms were prepared according to the procedure ▶ Fig. 1, the indicated in Materials and Methods. As shown in ● combination of PAC with α-pinene or with β-pinene showed a plotted curve below the straight line, generating a substantial synergistic effect. The effects of the following combinations were examined utilizing isobolograms: PAC and α-pinene and PAC and β-pinene. The combination of PAC and α-pinene as well as of PAC and β-pinene actually generated a synergistic effect ▶ Fig. 1). It was thus demonstrated that PAC was responsible for (●
the synergistic effect with α-pinene or with β-pinene. These results were also the case for H460 cells.
Cell cycle analysis after combination drug treatment We also examined the effects of these compounds on the cell cycle distributions of A549 cells by flow cytometric analysis. In accordance with the aforementioned findings, the percentage of sub-G0/G1-phase cells was decreased on the addition of α-pinene to PAC, while the population of G0/G1 cells was ▶ Fig. 2. The growth of the NSCLC cell increased as illustrated in ● lines A549 and H460 was synergistically inhibited by the treatment of the combination of PAC with α-pinene. These results can be explained by assuming that the cytostatic agent α-pinene caused the G1 cell cycle arrest of A-549 and H460 cells, which
Fig. 1 IC50 isobolograms of PAC + α-pinene and PAC + β-pinene against A549 cells. The IC50 value of PTC alone is expressed as 1.0 on the Y axis of the isobologram, while that of α-pinene or β-pinene alone is expressed as 1.0 on the X axis.
Fig. 2 Cell cycle analyses of A549 cells. Cells were treated with 0.5 % DMSO, α-pinene, PTC + α-pinene or PTC + β-pinene for 48 h, as described in Materials and Methods. The percentages in the graphs represent the rates of cells of each phase to the total cells.
Zhang Z et al. Synergy Effect of Pinenes … Drug Res 2015; 65: 214–218
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Results
Original Article 217
Fig. 4 Effect of α-pinene, β-pinene, PAC + α-pinene and PAC + β-pinene on the mitochondrial membrane potential in non-small-cell lung cancer cells (NSCLS) treated for 48 h. A representative experiment of 3 repeats is shown.
protected the cells from initiation of the death pathways induced by PAC.
Drug combinations of PAC with α-pinene and β-pinene induced mitochondrial membrane potential loss in NSCLC cells
Morphological changes in NSCLC cells after single and combination drug treatment
One important and indicative stage in the intrinsic apoptosis pathway is the depolarization of the mitochondrial membrane and the subsequent leakage of the outer membrane by pore formation. This is accompanied by release of pro-apoptotic molecules and cytochrome C. The fluorescent dye, rhodamine-123 (Rh-123) is a specific probe for the detection of alterations in mitochondrial membrane potential in living cells. Our observations revealed that drug combinations of PAC with α-pinene and β-pinene induced a progressive reduction in the number of cells with intact ΛΨm and increased the number of cells with low ΛΨm after 48 h incubation. α-pinene and β-pinene also induced mitochondrial membrane potential loss individually, but the
The morphological changes characteristic of apoptosis like chromatin condensation and fragmentation of the nucleus were seen in PAC + α-pinene and PAC + β-pinene treated NSCLC cells. ▶ Fig. 3 shows the morphological changes in these cells using ● phase-contrast microscopy. The PAC + α-pinene combination produced significant cytotoxic and apoptotic effects as revealed ▶ Fig. 3). The other by nuclear condensation and cell shrinkage (● combination of PAC + β-pinene did not produce as significant effects as was produced by the PAC + α-pinene combination.
Zhang Z et al. Synergy Effect of Pinenes … Drug Res 2015; 65: 214–218
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Fig. 3 Phase-contrast micrographs of NSCLC cells treated with α-pinene, β-pinene, PTC + α-pinene or PTC + β-pinene for 48 h. A representative experiment of 3 repeats is shown.
218 Original Article
Discussion
▼
The concept of “new uses for old drugs” provides an efficient and novel route to rediscover new uses for existing drugs with well known pharmacokinetics, pharmacodynamics and safety profiles. Paclitaxel (PAC) is a chemotherapeutic anticancer drug which acts as a microtubule stabilizing agent leading to cell cycle growth arrest in G2/M phase of the cell cycle eventually leading to apoptosis. As one of the most commonly used anticancer agents, PAC has exhibited strong efficacy against different range of tumors including head, neck, breast, ovary, and nonsmall cell lung cancers as well as Kaposi’s sarcoma [18–20]. Despite the success of PAC in treating a range of malignancies, the emergence of drug-resistance coupled with a broad range of harmful side-effects limit the use of PAC in cancer chemotherapy. As a consequence, several recent studies have focused their attention on the paclitaxel synergistic therapy in which a second chemical compound or drug is used in combination with PAC to find an effective and long-lasting solution for overcoming the PAC resistance problem and minimizing toxicity induced by PAC without compromising the drug efficiency [21–25]. In the current study, we have reported that α-pinene and β-pinene, which are well known monoterpenes mostly found in essential oils and other aroma mixtures, could synergistically enhance the growth-inhibitory effect of PAC in non-small-cell lung cancer cells. α-pinene and β-pinene separately did not produce such significant effects on these cell lines, but in combination with PAC, the cytotoxic effects of PAC were much more pronounced as indicated by cell cycle analysis, phase contrast microscopy as well as effects on mitochondrial membrane potential monitored by flow cytometry. α-pinene and β-pinene enhanced the PAC-induced mitotic cell cycle arrest as well as apoptosis. In addition, combination of PAC with α-pinene and β-pinene significantly increased effects on mitochondrial membrane loss. In conclusion, our results showed that the co-treatment of α-pinene and β-pinene in combination with PAC could exert strong synergistic cytotoxicity in NSCLC to curb cancer cell growth in vitro. The current data indicate that α-pinene and β-pinene in combination with PAC could be a new chemotherapeutic strategy in combating PAC-resistant cancers. Such a combination could reduce severe side-effects by minimizing PAC dose without compromising its anticancer efficacy.
Conflict of Interest
▼
The authors of the manuscript declare that there is no conflict of interest to reveal.
Zhang Z et al. Synergy Effect of Pinenes … Drug Res 2015; 65: 214–218
References 1 Alberg AJ, Brock MV, Stuart JM. Epidemiology of lung cancer: Looking to the future. Journal of Clinical Oncology 2005; 23: 3175–3185 2 American Cancer Society. Cancer Facts & Figures 2014. Atlanta, Ga: American Cancer Society, 2014 3 Posther KE, Harpole DH. The surgical management of lung cancer. Cancer Investigation 2006; 24: 56–67 4 Quoix E, Ramlau R, Westeel V et al. Therapeutic vaccination with TG4010 and first-line chemotherapy in advanced non-small-cell lung cancer: A controlled phase 2B trial. The Lancet Oncology 2011; 12: 1125–1133 5 Huang GJ, Fang DK, Cheng GY et al. Surgical therapeutic strategy for non-small cell lung cancer with (N2) mediastinal lymph node metastasis. Zhonghua Zhong Liu Za Zhi 2006; 28: 62–64 6 Li Y, Li H, Hu Y et al. Clinical study of lymph node metastasis in lung cancer. Chinese Journal of Thoracic Cardiovascular Surgery 2000; 16: 10–12 7 Ukena D. Chemotherapy in stage I + II non-small cell lung cancer. Lung Cancer 2001; 33: S25–S28 8 Waller DA. Neoadjuvant chemotherapy in non small cell lung cancer – the UK experience. Lung Cancer 2001; 34: S31–S33 9 Azim HA, Ganti AK. Treatment options for relapsed small-cell lung cancer. Anticancer drugs 2007; 18: 255–261 10 MacCallum C, Gillenwater HH. Second-line treatment of small-cell lung cancer. Current Oncology Reports 2006; 8: 258–264 11 Eid SY, El-Readi MZ, Wink M. Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters. Phytomedicine 2012; 19: 977–987 12 Bardon S, Picard K, Martel P. Monoterpenes inhibit cell growth, cell cycle progression, and cyclin D1 gene expression in human breast cancer cell lines. Nutrition Cancer 1998; 32: 1–7 13 Gómez-Contreras PC, Hernández-Flores G, Ortiz-Lazareno PC et al. In vitro induction of apoptosis in U937 cells by perillyl alcohol with sensitization by pentoxifylline: Increased Bcl-2 and Bax protein expression. Chemotherapy 2006; 52: 308–315 14 Rotem R, Heyfets A, Fingrut O et al. Jasmonates: novel anticancer agents acting directly and selectively on human cancer cell mitochondria. Cancer Research 2005; 65: 1984–1993 15 Wang W, Wu N, Zu YG et al. Antioxidative activity of Rosmarinus officinalis L. essential oil compared to its main components. Food Chemistry 2008; 108: 1019–1022 16 Cock IE. The phytochemistry and chemotherapeutic potential of Tasmannia lanceolata (Tasmanian pepper): A review. Pharmacognosy Communications 2013; 3: 17 Elanur A, Hasan T, Fatime G. Antioxidative, anticancer and genotoxic properties of α-pinene on N2a neuroblastoma cells. Biologia 2013; 68: 1004–1009 18 Jordan MA, Toso RJ, Thrower D et al. Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations. Proceeding of the National Academy of Sciences of the United States of America 1993; 90: 9552–9556 19 Milross CG, Mason KA, Hunter NR et al. Relationship of mitotic arrest and apoptosis to antitumor effect of paclitaxel. Journal of the National Cancer Institute 1996; 88: 1308–1314 20 Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nature Reviews Cancer 2004; 4: 253–265 21 Rodriguez-Antona C. Pharmacogenomics of paclitaxel. Pharmacogenomics 2010; 11: 621–623 22 Gottesman MM. Mechanisms of cancer drug resistance. Annual Review of Medicine 2002; 53: 615–627 23 Aoki D, Oda Y, Hattori S et al. Overexpression of class III beta-tubulin predicts good response to taxane-based chemotherapy in ovarian clear cell adenocarcinoma. Clinical Cancer Research 2009; 15: 1473–1480 24 Qi Y, Fu X, Xiong Z et al. Methylseleninic acid enhances paclitaxel efficacy for the treatment of triple-negative breast cancer. PLoS One 2012; 7: e31539 25 Le XF, Mao W, He G et al. The role of p27(Kip1) in dasatinib-enhanced paclitaxel cytotoxicity in human ovarian cancer cells. Journal of the National Cancer Institute 2011; 103: 1403–1422
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effect was much more significant when they were used in com▶ Fig. 4). Staurosporine (0.5 μM), which is a bination with PAC (● known agent causing loss of ΛΨm was used as a positive control.
Authors
Z. Zhang, S. Guo, X. Liu, X. Gao
Affiliation
Department of Oncology, First affiliated Hospital of Henan University of Science and Technology, Luoyang, China
Key words
Abstract
▶ paclitaxel ● ▶ non-small-cell lung cancer ● ▶ synergy ● ▶ α-pinene ● ▶ anticancer activity ●
▼
The objective of the present research work was to evaluate the synergistic interactions between Paclitaxel (PAC) with α-pinene and β-pinene using isobolographic method against non-smallcell lung cancer cells (NSCLC). This type of interaction between an established drug and a new compound is expected to enhance the efficacy of paclitaxel in combination as compared in isolation. Further, cell cycle analysis was carried out using flow cytometric analysis. Phase contrast microscopy was used to assess the effect of paclitaxel, α-pinene and β-pinene alone and in combination with each other in order to evaluate the effect of combination on cell apoptosis. Further, mitochondrial membrane potential was monitored in non-small-cell lung cancer cells (NSCLC) when treated with paclitaxel, α-pinene and β-pinene alone and in combination. The
results revealed that the combination of PAC with α-pinene or with β-pinene showed a plotted curve below the straight line, generating a substantial synergistic effect. The effects of the following combinations were examined utilizing isobolograms: PAC and α-pinene and PAC and β-pinene. The combination of PAC and α-pinene as well as of PAC and β-pinene actually generated a synergistic effect. We also examined the effects of these compounds on the cell cycle distributions of A549 cells by flow cytometric analysis. The percentage of sub-G0/G1-phase cells was decreased on the addition of α-pinene to PAC, while the population of G0/G1 cells was increased. The morphological changes characteristic of apoptosis like chromatin condensation and fragmentation of the nucleus were seen in PAC + α-pinene and PAC + β-pinene treated NSCLC cells.
received 28.04.2014 accepted 16.05.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1377025 Published online: September 4, 2014 Drug Res 2015; 65: 214–218 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. Guo Department of Oncology First affiliated Hospital of Henan University of Science and Technology No.24, Jing Hua Road Luoyang Henan 471003 China Tel.: + 86/379/64923 362 Fax: + 86/379/64923 362 [email protected]
Introduction
▼
Lung cancer is the most frequently diagnosed cancer responsible for almost 1.38 million deaths every year worldwide [1, 2]. In China, lung cancer has replaced liver cancer as the leading cause of death among people with malignant tumors in 2008. According to the statistics from the National office on tumor cure and prevention, about 600 000 people die of lung cancer each year in China. Lung cancer can be classified into 2 types, small cell lung carcinoma (SCLC) and nonsmall-cell lung carcinoma (NSCLC). NSCLC accounts for more than 80 % of all lung cancer patients. SCLC usually responds better to chemotherapy and radiotherapy than NSCLC. NSCLC is highly aggressive and metastatic cancer with limited treatment options. Most NSCLC patients show locally advanced (37 %) or metastatic (38 %) state when diagnosed [3–6]. The most frequently used therapies for NSCLC are chemotherapy,
Zhang Z et al. Synergy Effect of Pinenes … Drug Res 2015; 65: 214–218
radiotherapy and surgical treatments. Generally NSCLC is treated by surgical operation followed by chemotherapy to avoid recurrence. Unfortunately, 2/3 of the patients are inoperable when diagnosed. Among the patients who undergo surgical operation with overall 5-year survival rates of about 40 %, nearly 40 % belong to stage ΙΙΙA and a few to stage ΙΙΙB with a 5-year survival rate of about 20 %. About 50 % of the NSCLS patients receive chemotherapy with paclitaxel and carboplatin. However, these chemotherapeutic drugs produce serious side-effects such as leukopenia, hepatic dysfunction, disorder in renal function, nausea and vomiting. These sideeffects are most commonly attributed to tissue necrosis, apoptosis and inflammation caused by the chemotherapy treatment [7–10]. The toxic side-effects of these anticancer drugs can be minimized by using non-toxic potentiating agent or synergistic compound in combination with anticancer drugs which may possibly potentiate
Downloaded by: University of Georgia Libraries. Copyrighted material.
Synergistic Antitumor Effect of α-pinene and β-pinene with Paclitaxel against Non-small-cell Lung Carcinoma (NSCLC)
Original Article 215
onto standard 96-well microtiter plates. Following overnight culture, serially diluted samples (PAC, α-pinene, β-pinene or their various combinations) were added into the wells. After 3-day culture, the cell growth rates were evaluated by the assay of WST-1 and the IC50 values were calculated.
Isobolographic analysis of the interaction between 2 substances in A549 cells The IC50 values of the respective substances were as follows: 36.0 μM (α-pinene); 0.21 mg/mL (β-pinene); 11.5 nM (PAC). Then, IC50 values of PAC was measured on the addition of the respective concentrations of α-pinene (2.5, 5.0, 10.0 and 20.0 μM) or β-pinene (0.0125, 0.025, 0.05, 0.1, 0.2 and 0.4 mg/ ml). The IC50 value of PAC is expressed as 1.00 on the Y axis of the isobologram, while that of α-pinene or β-pinene alone is expressed as 1.00 on the X axis. The IC50 values thus obtained were plotted to make a line graph in the same way as above.
Examination of cell cycle regulation by flow cytometry A549 cells (1.0 × 105/well) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were plated onto a 60 mm dish. After incubation for 48 h, the cells were treated in the same way as described previously. The cells were incubated with the following substances for 48 h: control (0.5 % DMSO); α-pinene (80 μM); [PAC (80 nM)] [PAC (80 nM)] + α-pinene (80 μM). The adherent cells were treated with 0.25 % trypsin (Invitrogen Life Technologies) and combined with the floating cells. All the cells were treated with a CycletestTM Plus DNA Reagent Kit. Results of flow cytometry were obtained by FACScan flow cytometry (Becton Dickanson, San Jose, CA) at a wavelength of 488 nm.
Materials and Methods
Morphological changes in A549 cells after drug treatment
Cell lines and chemicals
The cells were cultured on 6 well plates and treated with PACand combinations of PAC with α-pinene and β-pinene for 48 h. After treatment, cells were fixed with methanol for 20 min, washed with phosphate-buffered saline (PBS), and then stained with 40 μg/ml of propidium iodide (PI) in the presence of 50 μg/ml RNase A. The morphology of nuclear chromatin was defined by the fluorescence of DNA-binding dye, PI, under a fluorescence microscope (Nikon, Japan).
▼
A-549 and H 460 non-small-cell lung cancer cell lines were purchased from the Shanghai Institute for Biological Sciences, CAS). The cells were maintained in RMPI-1640 medium (Gibco) with 10 % heat-inactivated fetal bovine serum (Gibco) under a humidified atmosphere of 5 % CO2 at 37 °C. α-pinene ( > 95 % purity by HPLC) was purchased from Sigma Chemical Company (St. Louis. Co). Paclitaxel was purchased from Hainanhaiyao Co., Ltd., China. Both paclitaxel and α-pinene were dissolved in dimethyl sulfoxide (DMSO). The caspase assay kit was purchased from G-Biosciences.
MTT viability assay Inhibitory effects of variable dosages of paclitaxel, α-pinene and β-pinene were measured using MTT cell viability assay. Cells were seeded in a 96-well plate at 1 × 105 cells per well overnight. Cells were treated with either paclitaxel, or α-pinene, or the combination, and incubated for 24 h at 37 °C. MTT (0.8 mg/ml, purchased from Sigma) was added to each well and incubated for 3 h at 37 °C, and the medium was replaced with 150 ml 0.01 N HCl and 10 % SDS mixture and incubated for 30 min at room temperature. Absorbance was measured with a microplate reader at 655 nm wavelength.
Inhibition against the growth of A549 and H460 cell lines
Disruption of mitochondrial membrane potential (ΛΨm) in lung cancer cells Mitochondrial membrane potential (ΛΨm) was measured by Rhodamine-123 dye. A-549 cells (5 × 105) were treated with PAC and combinations of PAC with α-pinene and β-pinene for 48 h and (ΛΨm) was measured by flow-cytometry. Rhodamine-123 (2 mM) was added 2 h before the end of experiment. After it, the cells were washed with PBS and incubated with PI (10 μg/ml) for 20 min the cells were analyzed by a FACScan flow cytometer.
Statistical analysis Statistical analysis was performed with SPSS16.0 software system. Data was analyzed statistically by using one-way ANOVA and Student’s t test. Values are shown as mean ± SD. All of the experiments were repeated 3 times. p < 0.05 was considered significant.
A549 or H460 cells (2 × 104/well) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were inoculated
Zhang Z et al. Synergy Effect of Pinenes … Drug Res 2015; 65: 214–218
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their effectiveness while minimizing their toxicity by reducing the dose. Such non-toxic potentiating agents which can possibly solve many problems of the clinical cancer treatment include low molecular weight terpenoids present in essential oils [11–14]. Synergy means working together of 2 or more substances to produce an effect greater than the sum of their individual effects. Synergism occurs when 2 or more herbal ingredients mutually enhance each other’s effect more significantly than the simple sum of these ingredients. Synergy represents a form of interaction as opposed to a simple addition response. The elucidation of drug combination interaction (i. e., whether additive, synergistic, or antagonistic) is complex. The most common methods are median effect, isobologram, and combination index (CI) analyses. These 3 methods take into account the sigmoidal shape of the dose-response relationships, as typically seen in cytotoxicity experiments. α-pinene has been reported to exhibit anticancer activity against human ovarian cancer cell lines and human hepatocellular liver carcinoma cell lines [15, 16]. α-pinene has also been reported to show antioxidant, anticancer and genotoxic properties on N2a neuroblastoma cells [17]. In the present study, we investigated the synergistic potentiation of the anticancer activity of paclitaxel against NSCLC in presence of α-pinene using combination index method and isobologram analysis. We also studied the mechanism of action by which α-pinene enhances the efficacy of paclitaxel in combination. To the best of our knowledge, this kind of study is the first such report on paclitaxel on NSCLC cell lines using α-pinene as a bioenhancer.
216 Original Article
▼
Determination of cytotoxicity of paclitaxel, α-pinene and β-pinene using the MTT assay In order to assess the combination effects of the chemotherapy drugs with α-pinene or β-pinene against the growth of A549 cells, isobolograms were prepared according to the procedure ▶ Fig. 1, the indicated in Materials and Methods. As shown in ● combination of PAC with α-pinene or with β-pinene showed a plotted curve below the straight line, generating a substantial synergistic effect. The effects of the following combinations were examined utilizing isobolograms: PAC and α-pinene and PAC and β-pinene. The combination of PAC and α-pinene as well as of PAC and β-pinene actually generated a synergistic effect ▶ Fig. 1). It was thus demonstrated that PAC was responsible for (●
the synergistic effect with α-pinene or with β-pinene. These results were also the case for H460 cells.
Cell cycle analysis after combination drug treatment We also examined the effects of these compounds on the cell cycle distributions of A549 cells by flow cytometric analysis. In accordance with the aforementioned findings, the percentage of sub-G0/G1-phase cells was decreased on the addition of α-pinene to PAC, while the population of G0/G1 cells was ▶ Fig. 2. The growth of the NSCLC cell increased as illustrated in ● lines A549 and H460 was synergistically inhibited by the treatment of the combination of PAC with α-pinene. These results can be explained by assuming that the cytostatic agent α-pinene caused the G1 cell cycle arrest of A-549 and H460 cells, which
Fig. 1 IC50 isobolograms of PAC + α-pinene and PAC + β-pinene against A549 cells. The IC50 value of PTC alone is expressed as 1.0 on the Y axis of the isobologram, while that of α-pinene or β-pinene alone is expressed as 1.0 on the X axis.
Fig. 2 Cell cycle analyses of A549 cells. Cells were treated with 0.5 % DMSO, α-pinene, PTC + α-pinene or PTC + β-pinene for 48 h, as described in Materials and Methods. The percentages in the graphs represent the rates of cells of each phase to the total cells.
Zhang Z et al. Synergy Effect of Pinenes … Drug Res 2015; 65: 214–218
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Results
Original Article 217
Fig. 4 Effect of α-pinene, β-pinene, PAC + α-pinene and PAC + β-pinene on the mitochondrial membrane potential in non-small-cell lung cancer cells (NSCLS) treated for 48 h. A representative experiment of 3 repeats is shown.
protected the cells from initiation of the death pathways induced by PAC.
Drug combinations of PAC with α-pinene and β-pinene induced mitochondrial membrane potential loss in NSCLC cells
Morphological changes in NSCLC cells after single and combination drug treatment
One important and indicative stage in the intrinsic apoptosis pathway is the depolarization of the mitochondrial membrane and the subsequent leakage of the outer membrane by pore formation. This is accompanied by release of pro-apoptotic molecules and cytochrome C. The fluorescent dye, rhodamine-123 (Rh-123) is a specific probe for the detection of alterations in mitochondrial membrane potential in living cells. Our observations revealed that drug combinations of PAC with α-pinene and β-pinene induced a progressive reduction in the number of cells with intact ΛΨm and increased the number of cells with low ΛΨm after 48 h incubation. α-pinene and β-pinene also induced mitochondrial membrane potential loss individually, but the
The morphological changes characteristic of apoptosis like chromatin condensation and fragmentation of the nucleus were seen in PAC + α-pinene and PAC + β-pinene treated NSCLC cells. ▶ Fig. 3 shows the morphological changes in these cells using ● phase-contrast microscopy. The PAC + α-pinene combination produced significant cytotoxic and apoptotic effects as revealed ▶ Fig. 3). The other by nuclear condensation and cell shrinkage (● combination of PAC + β-pinene did not produce as significant effects as was produced by the PAC + α-pinene combination.
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Fig. 3 Phase-contrast micrographs of NSCLC cells treated with α-pinene, β-pinene, PTC + α-pinene or PTC + β-pinene for 48 h. A representative experiment of 3 repeats is shown.
218 Original Article
Discussion
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The concept of “new uses for old drugs” provides an efficient and novel route to rediscover new uses for existing drugs with well known pharmacokinetics, pharmacodynamics and safety profiles. Paclitaxel (PAC) is a chemotherapeutic anticancer drug which acts as a microtubule stabilizing agent leading to cell cycle growth arrest in G2/M phase of the cell cycle eventually leading to apoptosis. As one of the most commonly used anticancer agents, PAC has exhibited strong efficacy against different range of tumors including head, neck, breast, ovary, and nonsmall cell lung cancers as well as Kaposi’s sarcoma [18–20]. Despite the success of PAC in treating a range of malignancies, the emergence of drug-resistance coupled with a broad range of harmful side-effects limit the use of PAC in cancer chemotherapy. As a consequence, several recent studies have focused their attention on the paclitaxel synergistic therapy in which a second chemical compound or drug is used in combination with PAC to find an effective and long-lasting solution for overcoming the PAC resistance problem and minimizing toxicity induced by PAC without compromising the drug efficiency [21–25]. In the current study, we have reported that α-pinene and β-pinene, which are well known monoterpenes mostly found in essential oils and other aroma mixtures, could synergistically enhance the growth-inhibitory effect of PAC in non-small-cell lung cancer cells. α-pinene and β-pinene separately did not produce such significant effects on these cell lines, but in combination with PAC, the cytotoxic effects of PAC were much more pronounced as indicated by cell cycle analysis, phase contrast microscopy as well as effects on mitochondrial membrane potential monitored by flow cytometry. α-pinene and β-pinene enhanced the PAC-induced mitotic cell cycle arrest as well as apoptosis. In addition, combination of PAC with α-pinene and β-pinene significantly increased effects on mitochondrial membrane loss. In conclusion, our results showed that the co-treatment of α-pinene and β-pinene in combination with PAC could exert strong synergistic cytotoxicity in NSCLC to curb cancer cell growth in vitro. The current data indicate that α-pinene and β-pinene in combination with PAC could be a new chemotherapeutic strategy in combating PAC-resistant cancers. Such a combination could reduce severe side-effects by minimizing PAC dose without compromising its anticancer efficacy.
Conflict of Interest
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The authors of the manuscript declare that there is no conflict of interest to reveal.
Zhang Z et al. Synergy Effect of Pinenes … Drug Res 2015; 65: 214–218
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effect was much more significant when they were used in com▶ Fig. 4). Staurosporine (0.5 μM), which is a bination with PAC (● known agent causing loss of ΛΨm was used as a positive control.
