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Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma
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Available online at
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Original Article
Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma
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Q1 Bingling
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Dai, Wenjie Wang, Rui Liu, Hongying Wang, Yanmin Zhang *
School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an 710061, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 January 2015 Accepted 23 January 2015
Taspine is an active compound in anticancer agent development. 12k was synthesized with taspine as lead compound bearing biphenyl scaffold and showed potent anticancer activity. Here, we investigated the effect of taspine derivative 12k on A549 lung cells. We showed that 12k not only decreased significantly A549 cell viability, A549 cell colony formation but also impaired A549 cell migration. Moreover, 12k treatment blocked cell cycle progression by increasing cell number in S phase to 42.80% for 6 mmol/L vs. 28.86% for control while decreasing cell number in G1 phase. Accordingly, this was associated with an increase protein expression of cyclin E and a decrease protein expression of cyclin D1, cyclin B1 and its associated CDK1 (cdc2). Meanwhile, we found that 12k induced A549 cell apoptosis, which was closely associated with the effect of the Bcl-2 family. Increase of Bad, Bak and Bax expression levels, decrease of Bcl-2 and Mcl-1 expression levels were observed. SiRNA knockdown of c-myc in A549 cells significantly attenuated tumor inhibition effects of 12k. In conclusion, our results demonstrate that 12k has an inhibitory effect on growth of A549 cell by inducing cell cycle arrest and apoptosis. ß 2015 Published by Elsevier Masson SAS.
Keywords: 12k A549 cells Cell cycle Apoptosis
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1. Introduction Q2
Uncontrolled cell proliferation is the hallmark of cancer, and tumor cell proliferation is normally restrained through control of the cell division cycle, therefore induction of cell cycle arrest is an effective method of controlling tumor cell proliferation [1]. The cell cycle is often divided into four sequential phases, G1 phase, S phase, G2 phase and M phase. Meanwhile, G0 phase is known as quiescence, into which cells may into G1 phase, also may reversibly exit from G1 when it lacks of appropriate growthpromoting signals. Transition from one phase to the next is regulated by various cyclins, cyclin-dependent kinases (Cdks), Cdk inhibitor and certain tumor suppressor gene products [2]. Cdk family of serine/threonine kinases is the core of the cell division cycle, and it functions by associating with a regulatory subunit known as a cyclin. So the expression of cyclin tends to be cell cycle dependent to ensure a specific Cdk have full activity. Both cyclin D and cyclin E are activators of the G1/S transition, by combining with Cdk4, Cdk6 and Cdk2 respectively. Cdk4/cyclin D, Cdk6/cyclin D and Cdk2/cyclin E complex play an * Corresponding author at: School of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76, Yanta West Street, #54, Xi’an, Shaanxi Province 710061, PR China. E-mail address: [email protected] (Y. Zhang).
important role in phosphorylate several proteins that function at specific cell cycle states [3,4]. Progression from G2 into mitosis requires Cdk1 (also known as Cdc2) associate with cyclin B, which is known to phosphorylate proteins regulated during mitosis [5–7]. A variety of anti-cancer drugs have been shown to mediate their therapeutic effect by triggering apoptosis [8,9]. The inhibition of apoptosis plays a vital role in the deterioration process of tumors [10]. Apoptosis is a highly controlled physiological process and a core signaling pathway, which requires specific protein and gene regulation [11]. Various evidences show that Bcl-2 protein family is the most common protein on relevant gene of cell apoptosis, including two kinds of protein with contradictory effect on cell apoptosis. Several proteins that inhibit apoptosis have been identified, including bcl-2, mcl-1, bcl-xL and the IAPs. The others function as pro-apoptotic regulators including Bax, Bad and Bak [12,13]. In 2010, lung cancer is the 1st most common cause of cancerrelated death across the world, and its incidence continues to rise [14]. The main reason for the unfavorable prognosis of lung tumors is its propensity to metastasize early and develop resistance to a wide range of functionally unrelated anticancer agents. Chemotherapy can be used to improve quality of life and gain a modest survival benefit of pancreatic cancer [15]. Meanwhile, the understanding of the molecular mechanisms involved in the development of lung
http://dx.doi.org/10.1016/j.biopha.2015.01.024 0753-3322/ß 2015 Published by Elsevier Masson SAS.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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cancer has provided many hopes for the discovery of new chemotherapeutical agents in lung cancer therapy. Taspine, isolated from Radix et Rhizoma Leonticsi, had been reported to have antiviral, anti-inflammatory as well as antitumor activity [16–19]. 12k (Fig. 1A) was synthesized with taspine as lead compound bearing biphenyl scaffold and showed potent anticancer activity [20]. Based on the previous promising results, in the present study, we further investigated the effect of 12k on tumor growth and migration of human lung cancer cell A549 and its related mechanism, while its apoptosis induction mechanism was evaluated.
RT Master Mix Perfect Real Time kit and SYBR1 Premix Ex Taq TM II were purchased from Takara biotechnology (Dalian, China). The RNA oligo was purchased from Gene Pharma (Shanghai, China).
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2.2. Cell culture
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Human lung cancer cell line A549, NCI-H1299, NCI-H460 were purchased from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 supplemented with 10% FBS. All cell lines were incubated at 37 8C in a 5% CO2 atmosphere.
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2. Materials and methods
2.3. Cell viability assay
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2.1. Chemicals and reagents
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Taspine derivate 12k (purity >98%) was synthesized in the Research and Engineering Center for Natural Medicine, Xi’an Jiaotong University. RPMI-1640, MTT, DMSO, RNase, PI and Hoechst 33258 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). Trypsin was obtained from Amresco (Solon, OH, USA). The penicillin was purchased from General Pharmaceutical Factory (Haerbin, China), and the streptomycin was purchased from North China Pharmaceutical (Shijiazhuang, China). Annexin V-FITC reagent kit was purchased from Neo Bioscience (Beijing, China). Crystal violet was purchased from Beijing Chemical Plant (Beijing, China). C-myc rabbit polyAb, mcl-1 rabbit polyAb, cyclin B1 rabbit mAb, cyclin D1 rabbit polyAb, cyclin E rabbit polyAb, cdc2 rabbit polyAb, Bax rabbit polyAb, BAD rabbit polyAb, BAK rabbit polyAb and Bcl-2 rabbit polyAb were from proteintech group (USA). P53 rabbit mAb was from epitomics (England). Rabbit anti-GAPDH, goat anti-rabbit IgG, BCA protein assay reagent kit and enhanced chemiluminescent (ECL) plus reagent kit were obtained from Pierce (Pierce Biotech, Rockford, IL, USA). Protease inhibitor cocktail was purchased from Roche Technology (Basle, Switzerland, USA). The RNA fast 200 kit was purchased from Fastagen (Shanghai, China) and lipofectamine 2000 reagent was purchased from Invitrogen (Carlsbad, CA, USA). Prime Script
Exponentially growing A549, NCI-H1299, and NCI-H460 cells were seeded to the 96-well plate and cultivated overnight. Cells were treated with various concentrations of 12k for 48 h. Then, the medium was replaced with 180 mL serum-free RPMI-1640 medium and 20 mL MTT solution (5 mg/mL). After 4 h incubation, the supernatants were removed, and the formazan crystals were dissolved with 150 mL DMSO. After shaking thoroughly for 15 min, the absorbance was measured at 490 nm in a microplate reader (Bio-RAD instruments, USA). Results were expressed as the percentage of cell viability ratio. Percentage of cell viability ratio = [1 (ODtreatment group ODblank group)/ (ODcontrol group ODblank group)] 100%. The experiment was performed in triplicate.
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2.4. Colony survival assay
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Exponentially growing A549 cells were seeded to the 12-well plate (200 cells/well). Treated with 12k at 0, 1.5, 3, 6 mmol/L, the plates were incubated in a CO2 incubator until the colonies were clearly visible and countable. Then the colonies were fixed with methanol for 15 min and stained with crystal violet for 15 min. Images were photographed under the enhanced chemiluminescence reagent and the inverted fluorescence microscope. Survival was plotted as percentage of the cells surviving on the untreated control.
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Fig. 1. 12k inhibited lung cancer cell proliferation and colony formation. (A) Chemical structure of 12k. (B) Effect of 12k on viability of lung cancer cells. Cells were cultured without or with 12k at indicated concentration for 48 h. Cell viability was determined by MTT assay. (C) Effect of 12k on colony formation of A549 cells. The top row was colony formation and the bottom row was the individual colony. (D) Quantitation data of (C). Values were presented as means SEM (n = 3). *P < 0.05, **P < 0.01 vs. the control group.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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2.5. Wound healing assay
2.10. Western blot analyses
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Exponentially growing A549 cells were seeded to the 12-well plate and cultivated to grow approximately 80% confluence overnight. Wounds were made by scratching the cells with pipette tips (100–200 mL) the following day. Then 12k at 0, 1.5, 3 and 6 mmol/L were added to allow cells migrate into the scratched area for different time. The migration of cells was visualized at time 0 h (after the wound was scratched), 24 h and 48 h after 12k treatment.
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2.6. Transwell migration assay
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Exponentially growing A549 cells were seeded to the transwell chambers (1 105 cells/well). After 24 h, 12k at 0, 1.5, 3 and 6 mmol/L was added to the chamber and incubated for 48 h. Then the medium in the chamber was replaced with serum-free medium and 30% FBS contained medium was added to the 24well plate as chemoattractant. The chamber was incubated for 24 h at incubator. After incubation, the cells in the upper surface of the chamber were carefully removed with a cotton swab and the cells migrated through the chamber to the lower surface were fixed with methanol for 15 min and stained with crystal violet for 15 min. The number of migrated cells was photographed under microscopy (200).
A549 cells exposed to 12k (0, 1.5, 3, 6 mmol/L) for 48 h were lysed with cell RIPA buffer containing protease inhibitor cocktail on ice for 30 min. The insoluble protein lysate was harvested and centrifuged at 12,000 rpm for 10 min at 4 8C. Protein concentration was determined by the BCA Protein Quantification kit according to manufacturer’s instructions. The cell lysates were denatured by boiling with 5 reducing sample buffer for 5 min and run on SDSPAGE gel. After electrophoresis, separated proteins were transferred to PVDF membranes and blocked with 5% non-fat milk in TBST buffer for 2 h at room temperature with continuous agitation. Membranes were then incubated with specific primary antibodies overnight at 4 8C followed by washing and incubating with secondary antibodies at dilution of 1:20,000 in TBST buffer for 2 h at 37 8C. The membranes were then developed with enhanced chemiluminescence (ECL) kit.
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2.11. Statistical analysis
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Data were expressed as mean SEM. Statistical analysis was performed using the statistical software SPSS18.0 and ANOVA was used to analyze statistical differences between groups under different conditions. A P-value <0.05 was considered statistically significant.
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3. Results
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2.7. Analyses of the cell cycle and cell apoptosis
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Exponentially growing A549 cells were cultured in 6-well culture plates. Cells were treated with 12k (0, 1.5, 3 and 6 mmol/L) for 48 h after serum starved for 24 h. Then the cells were washed with PBS and suspended in 70% ice-cold ethanol solution and incubated at 20 8C overnight. After fixation, the cells were washed thrice with PBS and incubated with 1 mL RNase (50 mg/ mL) and 1 mL PI (60 mg/mL) for 30 min in the dark at room temperature Cells treated with 12k (0, 1.5, 3 and 6 mmol/L) for 48 h were harvest and washed with PBS. After centrifugation, cells were suspended in binding buffer and incubated with 5 mL AnnexinVFITC. Three minutes later, 10 mL PI (20 mg/mL) was added and incubated in the dark at room temperature for 10 min. All stained cells were analyzed by FACS (Becton Dickinson, Mountain View, CA, USA). The obtained data were analyzed with Modfit LT software.
3.1. 12k suppressed lung cancer cells proliferation and A549 cells colony formation
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To assess the effect of 12k on lung cancer cell growth, lung cancer cells including A549, NCI-H1299 and NCI-H460 cells were treated with 12k at different concentrations. The results showed that 12k inhibited the cell viability of the 3 types of cancer cells, the IC50 values of 12k on A549 cells, NCI-H1299 and NCI-H460 cells at 48 h were 7.89, 13.35, 10.25 mmol/L respectively. 12k showed more suppressive effect in A549 cells than in NCI-H1299 cells and NCI-H460 cells (Fig. 1B). So A549 cells were used for the subsequent experiment. We also investigated the effect on A549 cells colony formation by 12k. The results showed that 12k significantly inhibited the anchorage-dependent growth of A549 cells in a dose-dependent manner (Fig. 1C and D). These findings indicate that 12k has potential anti-tumor properties in lung cancer cells.
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2.8. Hoechst staining assay
3.2. 12k inhibited A549 cells migration
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Exponentially growing A549 cells were seeded to the 24-well plate and then treated with 12k for 48 h. Cells were washed by PBS and fixed with 4% paraformaldehyde for 10 min. Then cells were washed by PBS and stained with Hoechst 33258 in the dark at room temperature for 20 min. Images were photographed under the inverted fluorescence microscope.
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2.9. siRNA transfection
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Specific knockdown was achieved by using siRNAs against c-myc and control siRNA. A double-stranded siRNA against cmyc and control siRNA were obtained from Shanghai Genec Pharma Co., Ltd. A549 cells were seeded in 6-well plate and transfected with siRNA against c-myc (at a final concentration of 80 nmol/L) for 24 h. Transfection with control siRNA was served as a negative control. Transfected cells were subjected to RT-PCT and western blotting to detect relative gene expression and protein expression. The transfected cells were used for cell proliferation assay.
We first investigated the effect of 12k on A549 cells migration by a wound healing assay. The results showed that in the absence of 12k, the cells migrated within 48 h to fill the scratched area, but the treatment of 12k significantly prevented this migration of A549 cells in a dose-dependent manner at 24 h and 48 h (Fig. 2A). Meanwhile, we adopted millicell chamber to explore the inhibitory effect of 12k on A549 cells migration. The results showed that after treatment with 12k for 48 h, the cell number on the lower surface of the membrane decreased and it showed in a dose-independent manner (Fig. 2B). Taken together, both wound healing assay and transwell migration assay validate that 12k impairs A549 cells migration.
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3.3. 12k induced S-phase arrest in A549 cells and regulated cell cycle regulatory molecules
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To understand the mechanisms responsible for 12k mediated A549 cell growth inhibition, we examined their effects on cell cycle distribution. The effect of 12k on A549 cells cycle profile was analyzed by PI staining and flow cytometry analysis. Cells were
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Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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Fig. 2. 12k suppressed A549 migration. (A) Photographs of A549 cells migrated by scratched wound at 24 h and 48 h. (B) Photographs of A549 cells (treated with 12k of different concentrations) migrated through the polycarbonate membrane stained with 0.2% crystal violet.
Fig. 3. 12k arrested A549 cell cycle at S phase and regulated cell cyclin protein levels. (A) Effect of 12k on A549 cell cycle. Cells were treated with 12k for 48 h and stained with PI. The cell cycle progression was evaluated by FACS. (B) Effect of 12k on cell cycle protein expression of cyclin D1, cyclin E, cyclin B1 and cdc2 by western blot analysis. (C) Results were quantified by densitometry analysis of the bands form (B). Values were presented as means SEM (n = 3). *P < 0.05, **P < 0.01 vs. the control group.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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treated without 12k or with 12k at 1.5, 3, 6 mmol/L for 48 h and stained with propidium iodide. As shown in Fig. 3A, treatment of 12k induced an accumulation of A549 cells in the S phase of the cell cycle. The population of untreated cells in S phase was 28.86%, whereas in cells treated with 12k at 1.5, 3, 6 mmol/L the population of cells in S phase showed a dose-dependent increase and was 33.60%, 35.81% to 42.80% respectively. A decrease in the population of cells in the G0-G1 phase was also observed, and changed from 71.14% to 65.90%, 64.19% and 57.20%, respectively. Meanwhile, sub-G0 phase cells were observed in cell cycle analysis. To elucidate the specific cell cycle regulatory proteins responsible for the cell cycle arrest mediated, we explored the effect of 12k on cell cycle regulatory protein molecules including cyclin D1, cyclin E, and cyclin B1 which are cyclins required for advance from G1 to S, S to G2, G2 to G1 respectively. Meanwhile, we investigated the effect of ESEE on cyclin-dependent kinases cdc2. As shown in Fig. 3B and C, the levels of cyclin D1, cyclin B1 and cdc2 proteins gradually decreased following treatment with 12k at the concentration of 1.5, 3, 6 mmol/L. In contrast, the amount of cyclin E was significantly up-regulated in 12k treated A549 cells. These data suggest that these cell cycle regulatory
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molecules are involved in 12k-induced changes in cell cycle progression.
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3.4. 12k induced A549 cells apoptosis and regulated cell apoptosis regulatory molecules
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Based on the observation of sub-G0 phase cells, the apoptotic effect of 12k in A549 cells was explored by flow cytometry analysis and Hoechst 33258 staining assay. As shown in Fig. 4A, the FACS results showed that the early apoptotic cells (lower right) in the control group were 6.30%, after treatment with 12k (0, 1.5, 3, 6 mmol/L), early apoptotic cells increased evidently and the percentage were 8.11%, 26.17%, 42.51% respectively. While there was little change of the late apoptotic cells (upper right) and the percentage of late apoptotic cells were 8.13%, 7.19%, 10.51%, 5.04%, respectively in the control group and 12k treated group. Consistent with this result, Hoechst staining results indicated that 12k induced condensed bright blue apoptotic nuclei in A549 cells compared with the untreated cells (Fig. 4B). Our data suggest that 12k could induce apoptosis in A549 cells.
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Fig. 4. 12k induced A549 cell apoptosis. (A) Annexin V-PI staining for apoptosis in A549 cells treated with 12k. (B) Apoptosis level after treatment of 12k by Hoechst 33258 staining. (C) Western blot analysis of Bad, Bak, Bax, Bcl-2 and Mcl-1 expression in A549 cells treated by 12k. (D) Results were quantified by densitometry analysis of the bands form (C). Values were presented as means SD (n = 3). *P < 0.05, **P < 0.01 vs. the control group.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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Since 12k induced A549 cells apoptosis was observed, we examined the expression level of Bad, Bak, Bax, Bcl-2 and Mcl-1 of cell apoptosis molecules in Bcl-2 family. The results showed that Bad, Bak and Bax protein expression gradually increased following treatment with 12k at the concentration of 1.5, 3, 6 mmol/L. Meanwhile, the amount of Bcl-2 and Mcl-1 were significantly down-regulated in 12k treated A549 cells (Fig. 4C and D). These data suggest that 12k up-regulated Bad, Bak and Bax protein level, and down-regulated Bcl-2 and Mcl-1 level in A549 cells.
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3.5. Effect of 12k on cell growth signaling pathway and siRNA of c-myc
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In order to clarify the inhibition and mechanism on the A549 growth by 12k, we assessed the effect on c-myc expression of growth signaling pathway. Western blot results showed that cmyc protein expressions were strongly decreased in a dosedependent manner (Fig. 5A and B). Based on the c-myc, the most significant growth protein molecule was inhibited by 12k. We carried out the siRNA assay on the c-myc gene in A549 cells so that we could further evaluate the important role of c-myc. As shown in Fig. 5C–E, c-myc was selectively knocked down in A549 cells. To identify the impact of cmyc on the biological effect induced by 12k, normal A549 cells and knockdown of c-myc in A549 cells were treated with 12k at different concentrations. The results showed that there was obvious difference in the two groups, the inhibitory effect of A549 cells (wild type) was more prominent than that of A549 cells (c-myc knockdown) (Fig. 5F). It suggested that siRNA knockdown of c-myc in A549 cells significantly attenuated the inhibitory effects of 12k on proliferation. It indicates that c-myc is a key target and could affect cell growth by 12k.
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4. Discussion
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In the present study, the anti-tumor effect of 12k on lung carcinoma cell A549 was investigated. 12k reduced A549 cell proliferation and colony formation in a dose-dependent manner, while inhibited A549 cell migration. Moreover, 12k blocked A549 cells at the S stage and induced A549 cells apoptosis. SiRNA knockdown of c-myc in A549 cells significantly impaired tumor
inhibition effects of 12k. The molecular mechanism, which 12k affected cell cycle and apoptosis, involves the regulation of cycle and apoptosis proteins. All the results demonstrated that 12k inhibited A549 cell growth as results of cell cycle arrest in S phase and induction of cell apoptosis. Lung cancer is the most common diagnosed cancers worldwide and is the leading cause of cancer-related death [14]. By far, it is an aggressive tumor with a 5-year survival rate of less than 15% indicating that current therapy is still inefficient [21]. Therefore, it is urgent to search for better chemotherapeutic agents with advanced activity against lung cancer. To explore novel taspine derivative 12k as potential chemotherapeutics for treatment of lung cancers, our initial study was performed to explore the effect of 12k on growth of human lung cancer cells using MTT assay. We found an obvious difference in the anticancer activity of 12k. Results showed that 12k had more effective on A549 cells than NCI-H1299 and NCI-H460 cells. Meanwhile, we found that 12k could suppress A549 cells colony formation. Moreover, cell migration is important during both the early invasion and later metastasis of a tumor, it is responsible for most cancer deaths [22]. In this paper, it was confirmed by our experiments that 12k effectively inhibited A549 cells migration in a time- and dose-dependent manner. Compounds that suppress tumor growth by induction of cell cycle arrest and apoptosis are desired strategy in cancer therapy [8,23]. Cell cycle arrest is one of the main signals that inhibit cell proliferation [24]. To determine whether 12k inhibited A549 cell growth as a result of induction of cell cycle arrest, we examined its effect on cell cycle progression. Flow cytometry analysis revealed that A549 cell growth was arrested at S phase. At the same time, in order to analyze the molecular mechanism by which 12k led to S phase arrest in A549 cells, the status of key factors to regulate cell cycle progression was evaluated. Cyclin D1 and cyclin E which are identified as candidate biomarkers used as surrogate intermediate. Cdc2 plays an important role by uniquely associating with Cyclin B1 which is a key molecule for G2-M phase transition. Western blot results indicated that cyclin E was significantly up-regulated and the levels of cyclin D1, cyclin B1 and cdc2 proteins were gradually decreased by 12k in a dose-dependent manner. Taken together, our results suggested that the growth inhibition by 12k in A549 cells by arresting cell cycle caused by regulating cyclin D1, cyclin E,
Fig. 5. Effect of 12k on c-myc protein. (A) 12k downregulated c-myc protein expression. (B) Quantitation data of (A). (C) C-myc mRNA expression in A549 cells transfection with 80 nM c-myc siRNA using Lipofectamine 2000 reagent and A549 cells (wild type) were determined by RT-PCR analysis. (D) C-myc protein expression in A549 cells transfection with 80 nM c-myc siRNA. (E) Quantitation data of (D). (F) Effect of 12k on cell proliferation in A549 cells (wild type) and knockdown cells. Values were presented as means SEM (n = 3). *P < 0.05, **P < 0.01 vs. the control group.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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cyclin B1 and cdc2 expression. Moreover, sub-G0 cells were observed. This implied that 12k could induce A549 cells apoptosis. Apoptosis induction is one of the main mechanisms that impede cancer growth and is considered as criteria for the identification on screening for a new cancer chemotherapy agent [25]. In our study, flow cytometry analysis results demonstrated that 12k triggered A549 cells apoptosis. Besides, apoptotic cells were visualized by nuclear morphological changes in Hoechst 33258 staining. To further clarify the underlying mechanisms that 12k inducing apoptosis, we detected protein changes of Bad, Bak, Bax, Bcl-2 and Mcl-1, which were known to have an important function in the regulation of cell survival/apoptosis. Western blot results showed that Bad, Bak and Bax were upregulated, Bcl-2 and Mcl-1 were downregulated. It indicated that induction of A549 cells by 12k is significantly associated with the effect on the Bcl-2 family. C-myc is a multifuctional, nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation. It functions as a transcription factor that regulates transcription of specific target genes [26]. Western blot results indicated that 12k significantly decreased the levels of c-myc. Meanwhile, the inhibitory effect on A549 cells (wild type) was much greater than that of A549 cells (c-myc knockdown). In other words, siRNA knockdown of c-myc in A549 cells significantly attenuated 12k inhibitory effects, which suggested that c-myc was an important target for 12k. In conclusion, our present findings showed that taspine derivative 12k showed effective in inhibition of human lung cancer A549 cell growth. Moreover, the anticancer activity of 12k was correlated with a cell cycle arrest at S phase and induction of cell apoptosis. These results suggest 12k may be promising molecule for treatment of lung cancer.
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Disclosure of interest
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The authors declare that they have no conflicts of interest concerning this article.
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Acknowledgments
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This study was supported by the National Natural Science Foundation of China (grant nos. 81370088 and 81227802), the Fundamental Research Funds for the Central Universities of Zhuizong, the Project of Shaanxi Star of Science and Technology (grant no. 2012kJXX-06) and the Supporting Plan of Education Ministry’s New Century Excellent Talents (grant no. NCET-130467).
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[3] Harbour JW, Dean DC. The Rb/E2F pathway: expanding roles and emerging paradigms. Genes Dev 2000;14:2393–409. [4] Harbour JW, Luo RX, Dei Santi A, Postigo AA, Dean DC. Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1. Cell 1999;98:859–69. [5] Kariya K, Koyama S, Nakashima S, Oshiro T, Morinaka K, Kikuchi A. Regulation of complex formation of POB1/epsin/adaptor protein complex 2 by mitotic phosphorylation. J Biol Chem 2000;275:18399–406. [6] Heix J, Vente A, Voit R, Budde A, Michaelidis TM, Grummt I. Mitotic silencing of human rRNA synthesis: inactivation of the promoter selectivity factor SL1 by cdc2 cyclin B-mediated phosphorylation. EMBO J 1998;17: 7373–81. [7] Blangy A, Lane HA, dHerin P, Harper M, Kress M, Nigg EA. Phosphorylation by p34(cdc2) regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell 1995;83:1159–69. [8] Los M, Burek CJ, Stroh C, Benedyk K, Hug H, Mackiewicz A. Anticancer drugs of tomorrow: apoptotic pathways as target for drug design. Drug Discov Today 2003;8:67–77. [9] Roy C, Brown DL, Little JE, Valentine BK, Walker PR, Sikorska M, et al. The topoisomerase-II inhibitor teniposide (VM-26) induces apoptosis in unstimulated mature murine lymphocytes. Exp Cell Res 1992;200:416–24. [10] Kerr JFR, Wyllie AH, Currie AR. Apoptosis-basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239–45. [11] Kohl NE, Mosser SD, Desolms SJ, Giuliani EA, Pompliano DL, Graham SL, et al. Selective inhibition of ras-dependent transformation by a farnesyl transferase inhibitor. Science 1993;260:1934–7. [12] Adida C, Crotty PL, McGrath J, Berrebi D, Diebold J, Altieri DC. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. Am J Pathol 1998;152:43–9. [13] Nomura M, Shimizu S, Ito T, Narita M, Matsuda H, Tsujimoto Y. Apoptotic cytosol facilitates Bax translocation to mitochondria that involves cytosolic factor regulated by Bcl-2. Cancer Res 1999;59:5542–8. [14] Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA-Cancer J Clin 2010;60:277–300. [15] Takahashi K, Saito H, Hasegawa Y, Ando M, Yamamoto M, Kojima E, et al. Firstline gefitinib therapy for elderly patients with non-small cell lung cancer harboring EGFR mutation: Central Japan Lung Study Group 0901. Cancer Chemother Pharmacol 2014;74:721–7. [16] Persinosperdue G, Blomster RN, Blake DA, Farnsworth NR. South-American plants 2: taspine isolation and anti-inflammatory activity. J Pharm Sci 1979;68:124–6. [17] Rollinger JM, Schuster D, Baier E, Ellmerer EP, Langer T, Stuppner H. Taspine: bioactivity-guided isolation and molecular ligand-target insight of a potent acetylcholinesterase inhibitor from Magnolia soulangiana. J Nat Prod 2006;69:1341–6. [18] Zhang YM, He LC, Meng L, Luo WJ. Taspine isolated from Radix et Rhizoma Leonticis inhibits proliferation and migration of endothelial cells as well as chicken chorioallantoic membrane neovascularisation. Vascul Pharmacol 2008;48:129–37. [19] Zhang YM, He LC, Wang HY. Inhibitory effect of taspine on mouse S180 sarcoma and its mechanism. China J Chin Mater Med 2007;32:27–30. [20] Gao HP, Su P, Shi YL, Shen XX, Zhang YM, Dong JY, et al. Discovery of novel VEGFR-2 inhibitors. Part II: Biphenyl urea incorporated with salicylaldoxime. Eur J Med Chem 2015;90:232–40. [21] Dempke WCM, Suto T, Reck M. Targeted therapies for non-small cell lung cancer. Lung Cancer 2010;67:257–74. [22] Lou LQ, Ye WW, Chen YX, Wu S, Jin LZ, He JK, et al. Ardipusilloside inhibits survival, invasion and metastasis of human hepatocellular carcinoma cells. Phytomedicine 2012;19:603–8. [23] Schultz DR, Harrington WJ. Apoptosis: programmed cell death at molecular level. Semin Arthritis Rheum 2003;32:345–69. [24] Qiu J, Zhao BB, Shen Y, Chen W, Ma YD, Shen YM. A novel p-terphenyl derivative inducing cell-cycle arrest and apoptosis in MDA-MB-435 cells through topoisomerase inhibition. Eur J Med Chem 2013;68:192–202. [25] Cotter TG. Apoptosis and cancer: the genesis of a research field. Nat Rev Cancer 2009;9:501–7. [26] Soucek L, Whitfield J, Martins CP, Finch AJ, Murphy DJ, Sodir NM, et al. Modelling Myc inhibition as a cancer therapy. Nature 2008;455:679–83.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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Available online at
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Original Article
Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma
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Q1 Bingling
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Dai, Wenjie Wang, Rui Liu, Hongying Wang, Yanmin Zhang *
School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an 710061, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 January 2015 Accepted 23 January 2015
Taspine is an active compound in anticancer agent development. 12k was synthesized with taspine as lead compound bearing biphenyl scaffold and showed potent anticancer activity. Here, we investigated the effect of taspine derivative 12k on A549 lung cells. We showed that 12k not only decreased significantly A549 cell viability, A549 cell colony formation but also impaired A549 cell migration. Moreover, 12k treatment blocked cell cycle progression by increasing cell number in S phase to 42.80% for 6 mmol/L vs. 28.86% for control while decreasing cell number in G1 phase. Accordingly, this was associated with an increase protein expression of cyclin E and a decrease protein expression of cyclin D1, cyclin B1 and its associated CDK1 (cdc2). Meanwhile, we found that 12k induced A549 cell apoptosis, which was closely associated with the effect of the Bcl-2 family. Increase of Bad, Bak and Bax expression levels, decrease of Bcl-2 and Mcl-1 expression levels were observed. SiRNA knockdown of c-myc in A549 cells significantly attenuated tumor inhibition effects of 12k. In conclusion, our results demonstrate that 12k has an inhibitory effect on growth of A549 cell by inducing cell cycle arrest and apoptosis. ß 2015 Published by Elsevier Masson SAS.
Keywords: 12k A549 cells Cell cycle Apoptosis
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1. Introduction Q2
Uncontrolled cell proliferation is the hallmark of cancer, and tumor cell proliferation is normally restrained through control of the cell division cycle, therefore induction of cell cycle arrest is an effective method of controlling tumor cell proliferation [1]. The cell cycle is often divided into four sequential phases, G1 phase, S phase, G2 phase and M phase. Meanwhile, G0 phase is known as quiescence, into which cells may into G1 phase, also may reversibly exit from G1 when it lacks of appropriate growthpromoting signals. Transition from one phase to the next is regulated by various cyclins, cyclin-dependent kinases (Cdks), Cdk inhibitor and certain tumor suppressor gene products [2]. Cdk family of serine/threonine kinases is the core of the cell division cycle, and it functions by associating with a regulatory subunit known as a cyclin. So the expression of cyclin tends to be cell cycle dependent to ensure a specific Cdk have full activity. Both cyclin D and cyclin E are activators of the G1/S transition, by combining with Cdk4, Cdk6 and Cdk2 respectively. Cdk4/cyclin D, Cdk6/cyclin D and Cdk2/cyclin E complex play an * Corresponding author at: School of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76, Yanta West Street, #54, Xi’an, Shaanxi Province 710061, PR China. E-mail address: [email protected] (Y. Zhang).
important role in phosphorylate several proteins that function at specific cell cycle states [3,4]. Progression from G2 into mitosis requires Cdk1 (also known as Cdc2) associate with cyclin B, which is known to phosphorylate proteins regulated during mitosis [5–7]. A variety of anti-cancer drugs have been shown to mediate their therapeutic effect by triggering apoptosis [8,9]. The inhibition of apoptosis plays a vital role in the deterioration process of tumors [10]. Apoptosis is a highly controlled physiological process and a core signaling pathway, which requires specific protein and gene regulation [11]. Various evidences show that Bcl-2 protein family is the most common protein on relevant gene of cell apoptosis, including two kinds of protein with contradictory effect on cell apoptosis. Several proteins that inhibit apoptosis have been identified, including bcl-2, mcl-1, bcl-xL and the IAPs. The others function as pro-apoptotic regulators including Bax, Bad and Bak [12,13]. In 2010, lung cancer is the 1st most common cause of cancerrelated death across the world, and its incidence continues to rise [14]. The main reason for the unfavorable prognosis of lung tumors is its propensity to metastasize early and develop resistance to a wide range of functionally unrelated anticancer agents. Chemotherapy can be used to improve quality of life and gain a modest survival benefit of pancreatic cancer [15]. Meanwhile, the understanding of the molecular mechanisms involved in the development of lung
http://dx.doi.org/10.1016/j.biopha.2015.01.024 0753-3322/ß 2015 Published by Elsevier Masson SAS.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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cancer has provided many hopes for the discovery of new chemotherapeutical agents in lung cancer therapy. Taspine, isolated from Radix et Rhizoma Leonticsi, had been reported to have antiviral, anti-inflammatory as well as antitumor activity [16–19]. 12k (Fig. 1A) was synthesized with taspine as lead compound bearing biphenyl scaffold and showed potent anticancer activity [20]. Based on the previous promising results, in the present study, we further investigated the effect of 12k on tumor growth and migration of human lung cancer cell A549 and its related mechanism, while its apoptosis induction mechanism was evaluated.
RT Master Mix Perfect Real Time kit and SYBR1 Premix Ex Taq TM II were purchased from Takara biotechnology (Dalian, China). The RNA oligo was purchased from Gene Pharma (Shanghai, China).
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2.2. Cell culture
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Human lung cancer cell line A549, NCI-H1299, NCI-H460 were purchased from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 supplemented with 10% FBS. All cell lines were incubated at 37 8C in a 5% CO2 atmosphere.
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2. Materials and methods
2.3. Cell viability assay
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2.1. Chemicals and reagents
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Taspine derivate 12k (purity >98%) was synthesized in the Research and Engineering Center for Natural Medicine, Xi’an Jiaotong University. RPMI-1640, MTT, DMSO, RNase, PI and Hoechst 33258 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). Trypsin was obtained from Amresco (Solon, OH, USA). The penicillin was purchased from General Pharmaceutical Factory (Haerbin, China), and the streptomycin was purchased from North China Pharmaceutical (Shijiazhuang, China). Annexin V-FITC reagent kit was purchased from Neo Bioscience (Beijing, China). Crystal violet was purchased from Beijing Chemical Plant (Beijing, China). C-myc rabbit polyAb, mcl-1 rabbit polyAb, cyclin B1 rabbit mAb, cyclin D1 rabbit polyAb, cyclin E rabbit polyAb, cdc2 rabbit polyAb, Bax rabbit polyAb, BAD rabbit polyAb, BAK rabbit polyAb and Bcl-2 rabbit polyAb were from proteintech group (USA). P53 rabbit mAb was from epitomics (England). Rabbit anti-GAPDH, goat anti-rabbit IgG, BCA protein assay reagent kit and enhanced chemiluminescent (ECL) plus reagent kit were obtained from Pierce (Pierce Biotech, Rockford, IL, USA). Protease inhibitor cocktail was purchased from Roche Technology (Basle, Switzerland, USA). The RNA fast 200 kit was purchased from Fastagen (Shanghai, China) and lipofectamine 2000 reagent was purchased from Invitrogen (Carlsbad, CA, USA). Prime Script
Exponentially growing A549, NCI-H1299, and NCI-H460 cells were seeded to the 96-well plate and cultivated overnight. Cells were treated with various concentrations of 12k for 48 h. Then, the medium was replaced with 180 mL serum-free RPMI-1640 medium and 20 mL MTT solution (5 mg/mL). After 4 h incubation, the supernatants were removed, and the formazan crystals were dissolved with 150 mL DMSO. After shaking thoroughly for 15 min, the absorbance was measured at 490 nm in a microplate reader (Bio-RAD instruments, USA). Results were expressed as the percentage of cell viability ratio. Percentage of cell viability ratio = [1 (ODtreatment group ODblank group)/ (ODcontrol group ODblank group)] 100%. The experiment was performed in triplicate.
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2.4. Colony survival assay
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Exponentially growing A549 cells were seeded to the 12-well plate (200 cells/well). Treated with 12k at 0, 1.5, 3, 6 mmol/L, the plates were incubated in a CO2 incubator until the colonies were clearly visible and countable. Then the colonies were fixed with methanol for 15 min and stained with crystal violet for 15 min. Images were photographed under the enhanced chemiluminescence reagent and the inverted fluorescence microscope. Survival was plotted as percentage of the cells surviving on the untreated control.
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Fig. 1. 12k inhibited lung cancer cell proliferation and colony formation. (A) Chemical structure of 12k. (B) Effect of 12k on viability of lung cancer cells. Cells were cultured without or with 12k at indicated concentration for 48 h. Cell viability was determined by MTT assay. (C) Effect of 12k on colony formation of A549 cells. The top row was colony formation and the bottom row was the individual colony. (D) Quantitation data of (C). Values were presented as means SEM (n = 3). *P < 0.05, **P < 0.01 vs. the control group.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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2.5. Wound healing assay
2.10. Western blot analyses
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Exponentially growing A549 cells were seeded to the 12-well plate and cultivated to grow approximately 80% confluence overnight. Wounds were made by scratching the cells with pipette tips (100–200 mL) the following day. Then 12k at 0, 1.5, 3 and 6 mmol/L were added to allow cells migrate into the scratched area for different time. The migration of cells was visualized at time 0 h (after the wound was scratched), 24 h and 48 h after 12k treatment.
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2.6. Transwell migration assay
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Exponentially growing A549 cells were seeded to the transwell chambers (1 105 cells/well). After 24 h, 12k at 0, 1.5, 3 and 6 mmol/L was added to the chamber and incubated for 48 h. Then the medium in the chamber was replaced with serum-free medium and 30% FBS contained medium was added to the 24well plate as chemoattractant. The chamber was incubated for 24 h at incubator. After incubation, the cells in the upper surface of the chamber were carefully removed with a cotton swab and the cells migrated through the chamber to the lower surface were fixed with methanol for 15 min and stained with crystal violet for 15 min. The number of migrated cells was photographed under microscopy (200).
A549 cells exposed to 12k (0, 1.5, 3, 6 mmol/L) for 48 h were lysed with cell RIPA buffer containing protease inhibitor cocktail on ice for 30 min. The insoluble protein lysate was harvested and centrifuged at 12,000 rpm for 10 min at 4 8C. Protein concentration was determined by the BCA Protein Quantification kit according to manufacturer’s instructions. The cell lysates were denatured by boiling with 5 reducing sample buffer for 5 min and run on SDSPAGE gel. After electrophoresis, separated proteins were transferred to PVDF membranes and blocked with 5% non-fat milk in TBST buffer for 2 h at room temperature with continuous agitation. Membranes were then incubated with specific primary antibodies overnight at 4 8C followed by washing and incubating with secondary antibodies at dilution of 1:20,000 in TBST buffer for 2 h at 37 8C. The membranes were then developed with enhanced chemiluminescence (ECL) kit.
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2.11. Statistical analysis
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Data were expressed as mean SEM. Statistical analysis was performed using the statistical software SPSS18.0 and ANOVA was used to analyze statistical differences between groups under different conditions. A P-value <0.05 was considered statistically significant.
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3. Results
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2.7. Analyses of the cell cycle and cell apoptosis
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Exponentially growing A549 cells were cultured in 6-well culture plates. Cells were treated with 12k (0, 1.5, 3 and 6 mmol/L) for 48 h after serum starved for 24 h. Then the cells were washed with PBS and suspended in 70% ice-cold ethanol solution and incubated at 20 8C overnight. After fixation, the cells were washed thrice with PBS and incubated with 1 mL RNase (50 mg/ mL) and 1 mL PI (60 mg/mL) for 30 min in the dark at room temperature Cells treated with 12k (0, 1.5, 3 and 6 mmol/L) for 48 h were harvest and washed with PBS. After centrifugation, cells were suspended in binding buffer and incubated with 5 mL AnnexinVFITC. Three minutes later, 10 mL PI (20 mg/mL) was added and incubated in the dark at room temperature for 10 min. All stained cells were analyzed by FACS (Becton Dickinson, Mountain View, CA, USA). The obtained data were analyzed with Modfit LT software.
3.1. 12k suppressed lung cancer cells proliferation and A549 cells colony formation
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To assess the effect of 12k on lung cancer cell growth, lung cancer cells including A549, NCI-H1299 and NCI-H460 cells were treated with 12k at different concentrations. The results showed that 12k inhibited the cell viability of the 3 types of cancer cells, the IC50 values of 12k on A549 cells, NCI-H1299 and NCI-H460 cells at 48 h were 7.89, 13.35, 10.25 mmol/L respectively. 12k showed more suppressive effect in A549 cells than in NCI-H1299 cells and NCI-H460 cells (Fig. 1B). So A549 cells were used for the subsequent experiment. We also investigated the effect on A549 cells colony formation by 12k. The results showed that 12k significantly inhibited the anchorage-dependent growth of A549 cells in a dose-dependent manner (Fig. 1C and D). These findings indicate that 12k has potential anti-tumor properties in lung cancer cells.
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3.2. 12k inhibited A549 cells migration
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Exponentially growing A549 cells were seeded to the 24-well plate and then treated with 12k for 48 h. Cells were washed by PBS and fixed with 4% paraformaldehyde for 10 min. Then cells were washed by PBS and stained with Hoechst 33258 in the dark at room temperature for 20 min. Images were photographed under the inverted fluorescence microscope.
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2.9. siRNA transfection
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Specific knockdown was achieved by using siRNAs against c-myc and control siRNA. A double-stranded siRNA against cmyc and control siRNA were obtained from Shanghai Genec Pharma Co., Ltd. A549 cells were seeded in 6-well plate and transfected with siRNA against c-myc (at a final concentration of 80 nmol/L) for 24 h. Transfection with control siRNA was served as a negative control. Transfected cells were subjected to RT-PCT and western blotting to detect relative gene expression and protein expression. The transfected cells were used for cell proliferation assay.
We first investigated the effect of 12k on A549 cells migration by a wound healing assay. The results showed that in the absence of 12k, the cells migrated within 48 h to fill the scratched area, but the treatment of 12k significantly prevented this migration of A549 cells in a dose-dependent manner at 24 h and 48 h (Fig. 2A). Meanwhile, we adopted millicell chamber to explore the inhibitory effect of 12k on A549 cells migration. The results showed that after treatment with 12k for 48 h, the cell number on the lower surface of the membrane decreased and it showed in a dose-independent manner (Fig. 2B). Taken together, both wound healing assay and transwell migration assay validate that 12k impairs A549 cells migration.
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3.3. 12k induced S-phase arrest in A549 cells and regulated cell cycle regulatory molecules
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To understand the mechanisms responsible for 12k mediated A549 cell growth inhibition, we examined their effects on cell cycle distribution. The effect of 12k on A549 cells cycle profile was analyzed by PI staining and flow cytometry analysis. Cells were
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Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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Fig. 2. 12k suppressed A549 migration. (A) Photographs of A549 cells migrated by scratched wound at 24 h and 48 h. (B) Photographs of A549 cells (treated with 12k of different concentrations) migrated through the polycarbonate membrane stained with 0.2% crystal violet.
Fig. 3. 12k arrested A549 cell cycle at S phase and regulated cell cyclin protein levels. (A) Effect of 12k on A549 cell cycle. Cells were treated with 12k for 48 h and stained with PI. The cell cycle progression was evaluated by FACS. (B) Effect of 12k on cell cycle protein expression of cyclin D1, cyclin E, cyclin B1 and cdc2 by western blot analysis. (C) Results were quantified by densitometry analysis of the bands form (B). Values were presented as means SEM (n = 3). *P < 0.05, **P < 0.01 vs. the control group.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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treated without 12k or with 12k at 1.5, 3, 6 mmol/L for 48 h and stained with propidium iodide. As shown in Fig. 3A, treatment of 12k induced an accumulation of A549 cells in the S phase of the cell cycle. The population of untreated cells in S phase was 28.86%, whereas in cells treated with 12k at 1.5, 3, 6 mmol/L the population of cells in S phase showed a dose-dependent increase and was 33.60%, 35.81% to 42.80% respectively. A decrease in the population of cells in the G0-G1 phase was also observed, and changed from 71.14% to 65.90%, 64.19% and 57.20%, respectively. Meanwhile, sub-G0 phase cells were observed in cell cycle analysis. To elucidate the specific cell cycle regulatory proteins responsible for the cell cycle arrest mediated, we explored the effect of 12k on cell cycle regulatory protein molecules including cyclin D1, cyclin E, and cyclin B1 which are cyclins required for advance from G1 to S, S to G2, G2 to G1 respectively. Meanwhile, we investigated the effect of ESEE on cyclin-dependent kinases cdc2. As shown in Fig. 3B and C, the levels of cyclin D1, cyclin B1 and cdc2 proteins gradually decreased following treatment with 12k at the concentration of 1.5, 3, 6 mmol/L. In contrast, the amount of cyclin E was significantly up-regulated in 12k treated A549 cells. These data suggest that these cell cycle regulatory
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3.4. 12k induced A549 cells apoptosis and regulated cell apoptosis regulatory molecules
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Based on the observation of sub-G0 phase cells, the apoptotic effect of 12k in A549 cells was explored by flow cytometry analysis and Hoechst 33258 staining assay. As shown in Fig. 4A, the FACS results showed that the early apoptotic cells (lower right) in the control group were 6.30%, after treatment with 12k (0, 1.5, 3, 6 mmol/L), early apoptotic cells increased evidently and the percentage were 8.11%, 26.17%, 42.51% respectively. While there was little change of the late apoptotic cells (upper right) and the percentage of late apoptotic cells were 8.13%, 7.19%, 10.51%, 5.04%, respectively in the control group and 12k treated group. Consistent with this result, Hoechst staining results indicated that 12k induced condensed bright blue apoptotic nuclei in A549 cells compared with the untreated cells (Fig. 4B). Our data suggest that 12k could induce apoptosis in A549 cells.
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Fig. 4. 12k induced A549 cell apoptosis. (A) Annexin V-PI staining for apoptosis in A549 cells treated with 12k. (B) Apoptosis level after treatment of 12k by Hoechst 33258 staining. (C) Western blot analysis of Bad, Bak, Bax, Bcl-2 and Mcl-1 expression in A549 cells treated by 12k. (D) Results were quantified by densitometry analysis of the bands form (C). Values were presented as means SD (n = 3). *P < 0.05, **P < 0.01 vs. the control group.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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Since 12k induced A549 cells apoptosis was observed, we examined the expression level of Bad, Bak, Bax, Bcl-2 and Mcl-1 of cell apoptosis molecules in Bcl-2 family. The results showed that Bad, Bak and Bax protein expression gradually increased following treatment with 12k at the concentration of 1.5, 3, 6 mmol/L. Meanwhile, the amount of Bcl-2 and Mcl-1 were significantly down-regulated in 12k treated A549 cells (Fig. 4C and D). These data suggest that 12k up-regulated Bad, Bak and Bax protein level, and down-regulated Bcl-2 and Mcl-1 level in A549 cells.
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3.5. Effect of 12k on cell growth signaling pathway and siRNA of c-myc
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In order to clarify the inhibition and mechanism on the A549 growth by 12k, we assessed the effect on c-myc expression of growth signaling pathway. Western blot results showed that cmyc protein expressions were strongly decreased in a dosedependent manner (Fig. 5A and B). Based on the c-myc, the most significant growth protein molecule was inhibited by 12k. We carried out the siRNA assay on the c-myc gene in A549 cells so that we could further evaluate the important role of c-myc. As shown in Fig. 5C–E, c-myc was selectively knocked down in A549 cells. To identify the impact of cmyc on the biological effect induced by 12k, normal A549 cells and knockdown of c-myc in A549 cells were treated with 12k at different concentrations. The results showed that there was obvious difference in the two groups, the inhibitory effect of A549 cells (wild type) was more prominent than that of A549 cells (c-myc knockdown) (Fig. 5F). It suggested that siRNA knockdown of c-myc in A549 cells significantly attenuated the inhibitory effects of 12k on proliferation. It indicates that c-myc is a key target and could affect cell growth by 12k.
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In the present study, the anti-tumor effect of 12k on lung carcinoma cell A549 was investigated. 12k reduced A549 cell proliferation and colony formation in a dose-dependent manner, while inhibited A549 cell migration. Moreover, 12k blocked A549 cells at the S stage and induced A549 cells apoptosis. SiRNA knockdown of c-myc in A549 cells significantly impaired tumor
inhibition effects of 12k. The molecular mechanism, which 12k affected cell cycle and apoptosis, involves the regulation of cycle and apoptosis proteins. All the results demonstrated that 12k inhibited A549 cell growth as results of cell cycle arrest in S phase and induction of cell apoptosis. Lung cancer is the most common diagnosed cancers worldwide and is the leading cause of cancer-related death [14]. By far, it is an aggressive tumor with a 5-year survival rate of less than 15% indicating that current therapy is still inefficient [21]. Therefore, it is urgent to search for better chemotherapeutic agents with advanced activity against lung cancer. To explore novel taspine derivative 12k as potential chemotherapeutics for treatment of lung cancers, our initial study was performed to explore the effect of 12k on growth of human lung cancer cells using MTT assay. We found an obvious difference in the anticancer activity of 12k. Results showed that 12k had more effective on A549 cells than NCI-H1299 and NCI-H460 cells. Meanwhile, we found that 12k could suppress A549 cells colony formation. Moreover, cell migration is important during both the early invasion and later metastasis of a tumor, it is responsible for most cancer deaths [22]. In this paper, it was confirmed by our experiments that 12k effectively inhibited A549 cells migration in a time- and dose-dependent manner. Compounds that suppress tumor growth by induction of cell cycle arrest and apoptosis are desired strategy in cancer therapy [8,23]. Cell cycle arrest is one of the main signals that inhibit cell proliferation [24]. To determine whether 12k inhibited A549 cell growth as a result of induction of cell cycle arrest, we examined its effect on cell cycle progression. Flow cytometry analysis revealed that A549 cell growth was arrested at S phase. At the same time, in order to analyze the molecular mechanism by which 12k led to S phase arrest in A549 cells, the status of key factors to regulate cell cycle progression was evaluated. Cyclin D1 and cyclin E which are identified as candidate biomarkers used as surrogate intermediate. Cdc2 plays an important role by uniquely associating with Cyclin B1 which is a key molecule for G2-M phase transition. Western blot results indicated that cyclin E was significantly up-regulated and the levels of cyclin D1, cyclin B1 and cdc2 proteins were gradually decreased by 12k in a dose-dependent manner. Taken together, our results suggested that the growth inhibition by 12k in A549 cells by arresting cell cycle caused by regulating cyclin D1, cyclin E,
Fig. 5. Effect of 12k on c-myc protein. (A) 12k downregulated c-myc protein expression. (B) Quantitation data of (A). (C) C-myc mRNA expression in A549 cells transfection with 80 nM c-myc siRNA using Lipofectamine 2000 reagent and A549 cells (wild type) were determined by RT-PCR analysis. (D) C-myc protein expression in A549 cells transfection with 80 nM c-myc siRNA. (E) Quantitation data of (D). (F) Effect of 12k on cell proliferation in A549 cells (wild type) and knockdown cells. Values were presented as means SEM (n = 3). *P < 0.05, **P < 0.01 vs. the control group.
Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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cyclin B1 and cdc2 expression. Moreover, sub-G0 cells were observed. This implied that 12k could induce A549 cells apoptosis. Apoptosis induction is one of the main mechanisms that impede cancer growth and is considered as criteria for the identification on screening for a new cancer chemotherapy agent [25]. In our study, flow cytometry analysis results demonstrated that 12k triggered A549 cells apoptosis. Besides, apoptotic cells were visualized by nuclear morphological changes in Hoechst 33258 staining. To further clarify the underlying mechanisms that 12k inducing apoptosis, we detected protein changes of Bad, Bak, Bax, Bcl-2 and Mcl-1, which were known to have an important function in the regulation of cell survival/apoptosis. Western blot results showed that Bad, Bak and Bax were upregulated, Bcl-2 and Mcl-1 were downregulated. It indicated that induction of A549 cells by 12k is significantly associated with the effect on the Bcl-2 family. C-myc is a multifuctional, nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation. It functions as a transcription factor that regulates transcription of specific target genes [26]. Western blot results indicated that 12k significantly decreased the levels of c-myc. Meanwhile, the inhibitory effect on A549 cells (wild type) was much greater than that of A549 cells (c-myc knockdown). In other words, siRNA knockdown of c-myc in A549 cells significantly attenuated 12k inhibitory effects, which suggested that c-myc was an important target for 12k. In conclusion, our present findings showed that taspine derivative 12k showed effective in inhibition of human lung cancer A549 cell growth. Moreover, the anticancer activity of 12k was correlated with a cell cycle arrest at S phase and induction of cell apoptosis. These results suggest 12k may be promising molecule for treatment of lung cancer.
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The authors declare that they have no conflicts of interest concerning this article.
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This study was supported by the National Natural Science Foundation of China (grant nos. 81370088 and 81227802), the Fundamental Research Funds for the Central Universities of Zhuizong, the Project of Shaanxi Star of Science and Technology (grant no. 2012kJXX-06) and the Supporting Plan of Education Ministry’s New Century Excellent Talents (grant no. NCET-130467).
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Please cite this article in press as: Dai B, et al. Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma. Biomed Pharmacother (2015), http://dx.doi.org/10.1016/j.biopha.2015.01.024
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