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mTOR axis in human colorectal carcinoma HCT116 cells
Author’s Accepted Manuscript Pogostone Induces Autophagy and Apoptosis Involving PI3K/Akt/mTOR Axis in Human Colorectal Carcinoma HCT116 cells Zhi-Xing Cao, Yu-Ting Yang, Si Yu, Yu-Zhi Li, Wen-Wen Wang, Jing Huang, Xiao-Fang Xie, Liang Xiong, Song Lei, Cheng Peng www.elsevier.com/locate/jep
PII: DOI: Reference:
S0378-8741(16)30453-6 http://dx.doi.org/10.1016/j.jep.2016.07.028 JEP10293
To appear in: Journal of Ethnopharmacology Received date: 28 April 2016 Revised date: 7 July 2016 Accepted date: 8 July 2016 Cite this article as: Zhi-Xing Cao, Yu-Ting Yang, Si Yu, Yu-Zhi Li, Wen-Wen Wang, Jing Huang, Xiao-Fang Xie, Liang Xiong, Song Lei and Cheng Peng, Pogostone Induces Autophagy and Apoptosis Involving PI3K/Akt/mTOR Axis in Human Colorectal Carcinoma HCT116 cells, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.07.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pogostone Induces Autophagy and Apoptosis Involving PI3K/Akt/mTOR Axis in Human Colorectal Carcinoma HCT116 cells
Zhi-Xing Caoa,1, Yu-TingYanga,1, Si Yua,1, Yu-Zhi Lia, Wen-Wen Wanga,c, Jing Huanga,c, Xiao-Fang Xiea, Liang Xionga, Song Leib, Cheng Penga*
a
Pharmacy College，Chengdu University of Traditional Chinese Medicine；The
Ministry of Education Key Laboratory of Standardization of Chinese Herbal Medicine；Key Laboratory of Systematic Research，Development and Utilization of Chinese Medicine Resources in Sichuan Province—Key Laboratory Breeding Base of Co-founded by Sichuan Province and MOST b
Department of pathology, West China Hospital
c
School of Medical Technology, Chengdu University of Traditional Chinese Medicine,
*Corresponding authors at: Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China. Tel./fax: +86 28 61800018. E-mail: [email protected]
1
These three authors contributed equally to this work.
ABSTRACT Ethnophamacological relevance: Pogostemon cablin is a medicinal herb widely used to treat gastrointestinal diseases in many Asian countries. Pogostone is an important constituent of Pogostemon cablin, and possesses various bioactivitys. In this study, we performed to investigate the anti-colorectal tumor property of Pogostone by inducing aurophagy and apoptosis in human colorectal cancer cells, and to define the potential molecular mechanisms. Materials and methods: In vitro,The anti-tumor activity of pogostone was assessed using MTT assay. Autophagy was monitored by transmission electron microscopy observation and mRFP-GFP-LC3 fluorescence analysis in colorectal tumor cell line. Apoptosis was measured by flow cytometry and annexinV-FITC/PI staining. The protein expressions or activition of LC3-Ⅱ, AKT, mTOR, caspase-3 and caspase-7 were detected through western blotting. In vivo, the
anti-tumor effect of pogostone was tested with HCT116 colorectal tumor cells transplantation tumor model. The expression of Ki-67 was determed by Immunohistochemistry staining and the apoptosis was evaluated using TUNEL assay. Results: In vitro, pogostone exhibits significant anti-tumor activity against human cancer cell lines, especially for HCT116 (18.7±1.93 μg/ml). Transmission electron microscopy observation, mRFP-GFP-LC3 fluorescence analysis, flow cytometryand assay and western blotting detection revealed that the anti-colorectal tumor activity of pogostone was dependent on inducing autophagy and apoptosis through up-regulating the expression of LC3-Ⅱ, cleaved caspase-7 and caspase-3, and decreasing the phosphorylation of AKT/mTOR. In vivo, 150 mg/kg pogostone inhibited the HCT116 tumor growth in immunodeficient mice with an inhibitory rate of 43.3%, decreased the expression of Ki67, and induced apoptosis in three days. Conclusion: Pogostone showed anti-colorectal tumor effects by inducing autophagy and apoptosis involving PI3K/Akt/mTOR axis. Thus, pogostone may be a promising lead compound to be further developed for cancer therapy.
Keywords: Pogostone; autophagy; apoptosis; Akt; mTOR
1． Introduction Human colorectal cancer is one of the leading causes of cancer-related lethality worldwide, with more than 600, 000 deaths each year (Globocan, 2012). Although the mortality has decreased significantly in the past 2 decades with the treatment of surgery, new chemotherapeutic and targeted therapies, recurrence and drug resistance frequently occurs with in a short time (Van et al.,2010; Terme et al., 2013). Therefore, the development of novel anti-colorectal cancer agents is urgently needed.
PI3K/Akt/mTOR signaling pathway is a major signal transduction cascade involved in the cellular proliferation, survival, and metabolism, and plays an important role in colorectal cancer (Shimizu et al., 2012;Ma et al., 2015). Up-regulation of PI3K resulting in inhibition of apoptosis in colon-cancer cells (Zhirnov and Klenk, 2007). Inhibitors of PI3K/Akt/mTOR signaling have been suggested as potential therapeutic agents in colorectal cancer (Roper et al., 2011; Ashok, 2013). Previous studies have shown that inhibiting the activition of Akt and its downstream target mTOR contribute to the initiation of autophagy, which is now recognised as a novel forms of
“programmed” cell death (Heras-Sandoval et al., 2014; Sun et al., 2014; Shi and Cao, 2014). Normally, autophagy is a survival mechanism that facilitates the maintenance of intracellular homeostasis by degrading long-lived proteins and damaged organelles and participating in many human diseases and physiological processes (Mizushima et al., 2008; Ravikumar et al., 2010). On the other hand, many anti-cancer agents, such as rapamycin and everolimus, the mTOR inhibitors have the ability to induce autophagic cell death in cancer cells, suggesting that autophagy might be useful in tumor treatment (O'Donnell et al., 2008; Chresta et al., 2010). Rencent studies have shown that autophagy could induce a non-selective bulk degradation process and cause apoptosis and cell death in many kinds of cancer cells (Arlia-Ciommo et al., 2015; Nagelkerke et al., 2014; Vijayaraghavan and Keyomarsi, 2015). Targeting critical regulators with the purpose to promote autophagy in cancer cells is an attractive new cancer therapeutic strategy.
Pogostemon Cablin(Blanco) Benth, commonly known as “Guanghuoxiang”, is a traditional Chinese medicinal herb widely used to treat gastrointestinal diseases in many Asian countries (Chen et al., 2015). Pogostone is one of the major constituent of Pogostemon cablin, and possesses various bioactivitys. It has been reported that pogostone possess potent anti-fungal (Mo et al., 2004;Li et al., 2012), antibacterial (Osawa et al., 1990; Peng et al., 2014) and pesticidal activities (Huang et al., 2013). New reports also reveal that pogostone exert anti-inflammatory activity on lipopolysaccharide-stimulation RAW264.7 model and Immunosuppressive activity on T cell (Li et al., 2014; Su et al., 2015). Studies of its pharmacokinetic properties show that pogostone was easily absorbed after oral administration with a high bioavailability (Li et al., 2014). The present study was performed to investigate if pogostone shown anti-cancer activity in human colorectal cancer cells. Finally, we found that pogostone could induce autophagy and apoptosis in colorectal cancer by inhibiting the activation of PI3K/Akt/mTOR axis.
2. Materials and methods 2. 1 Chemicals and reagents DMSO,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT),
3-Methyladenine (3-MA) was purchased from Sigma(NY, USA). Cell count kit-8 (CCK-8) was purchased from Tokyo, Japan. Trypsin, Cell culture media, DMEM, RPMI-1640, IMDM and fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA), Antibodies against Akt, p-AKT, mTOR, p-mTOR, Caspase-3 and (-actin were purchased from Cell Signaling Technology(Danvers, MA). Annexin V-FITC and propidium iodide(PI) Apoptosis Detection Kit and BCA protein assay kit were purchased from Beyotime (Shanghai, China). 2. 2 Extraction and isolation of pogostone pogostone was isolated from P. cablin as previously described[15]. The extraction and isolation procedure was as follows. Briefly, the dried leaves(15kg) of P. cablin were exhaustively extracted through water-steam distillation. Essential oil (300g) was obtained from P. cablin and dissolved in diethyl ether(1500ml) and partitioned with 2%NaOH( 1500 ml) five times. The five alkaline extracts were pooled and diluted HCl was added to adjust the PH to 1-2. After acidification, the solution was partitioned with diethyl ether(2000ml) five times; then, the ether phase was evaporated under reduced pressure to give a residue, which was further purified by recrystallization with n-hexane at -4℃to yield pogostone. The purity of pogostone was determined to be 99. 8% by gas chromatograph-mass spectroscopy(Agilent Technologies, USA) with an Agilent 7890A/5975C instrument(Agilent Technologies, USA). In addition, the structure of pogostone was further confirmed by spectroscopic data analysis(1H-nuclear magnetic resonance spectra(1H-NMR), 13C-NMR, and mass spectrometry(MS)). In the in vitro experiments, pogostone was dissolved in dimethyl sulfoxide(DMSO) and diluted with culture medium. The final concentration of DMSO in the test solutions was less than 0. 1%. In animal experiments, pogostone was suspended in 0. 5% Cellulose CM sodium salt(CMC-Na) at 37℃.
2. 3 Cell lines and cultures The human colorectal carcinoma cell line HCT116、SW620 , human lung cancer cell line A549 and H1975, human mammary carcinoma cell line MDA-MB-231, MCF-7 and MDA-MB-435, human melanoma cell line A375, human esophageal squamous cell carcinoma cell line KYSE-510R, Human ovarian carcinoma cell line SKOV3, human hepatocellular carcinoma cell line HepG-2, Human neuroblastoma cell SH-SY5Y, human embryonic kidney 293A cell, human acute myeloid leukemia cell line MV4-11 and THP-1, were obtained from American Type Culture Collection(Manassas, VA, USA). Cells were grown in RPMI-1640, DMEM or IMDM culture mediumcontaining 10% fetal bovine serum in 5% CO2 at 37℃. 2.4 Cell viability assay MTT assay Briefly, exponentially growing tumor cells were seeded into 96-well microplates for 24hours. Afterwards, equal volume of medium containing various concentrations of pogostone ranging from 3.12 µg/mL to 200.0 µg/mL were added to each well. At the end of the incubation period (72 hours at 37℃), 20μL of 5 mg/mL MTT reagent was added per well for 2~4-hour of incubation, and 50 μL of 20% acidified SDS per well was used to lyse the cells. Finally, the light absorption (OD) of the dissolved cells was measured at 570 nm using a SpectraMAX M5 microplate spectrophotometer (Molecular Devices). All experiments were performed in triplicate. The percentage of viability was calculated and compared with that of the control cells treated with DMSO (0.1%). CKK-8 assay HCT116 cells in 96-well microplates were divided into four groups: control, 3-MA, pogostone, and pogostone plus 3-MA groups. After passage 24 h, cells were changed to the equal volume of medium containing 25, 50 and 100 µg/mL pogostone with or without 10 mM 3-MA, and added 1‰ DMSO or 10 mM 3-MA alone as control group. After the culture period (24 hours at 37℃), the supernatant fluid was removed and medium contains 10% CCK- 8 reagent was added for 1-3 h incubation at 37° C. The light absorptions (OD) were measured at 450 nm with SpectraMAX M5
microplate spectrophotometer 2. 5 Autophagy detection 2.5.1 Evaluation of fluorescent LC3 puncta LC3 puncta were indicated by mRFP-GFP-LC3 adenovirus (Hanbio Co., LTD, China). Briefly, HCT116 cells were transfected with mRFP-GFP-LC3 for 24 h before receiving pogostone treatments. After 20-hours pogostone treatment, cells were observed under a confocal fluorescence microscopy (Olympus FV1200, Japan). Autophagic cells, which contained five or more mRFP-GFP-LC3 dots were recorded and the images were acquired. 2.5.2 Transmission Electron microscopy observation For transmission electron microscopy(TEM) observation, after being treated with 50 μg/mL pogostone for 20h, the cells were collected and fixed in ice-cold glutaraldehyde(2.5% in 0.1 mol/L cacodylate buffer, PH 7. 4) overnight and postfixed in osmium tetroxide. After dehydration with a graded series of alcohol concentrations, the samples were rinsed in propylene oxide and impregnated with epoxy resins. The ultrathin sections were contrasted with uranyl acetate and lead citrate for electron microscopy. Electron micrographs were observed through a transmission electron microscope(H7650;Hitachi, Tokyo, Japan) ,according to a method previously described by Wang et al., 2013. 2. 6 Flow cytometric analysis of cell cycle and apoptosis A flow cytometric assay was used for cell cycle and apoptosis assays. Briefly, after treatment with increasing concentrations of pogostone for 30 hours, HCT116 cells were harvested and washed with PBS. For cell cycle assays, cells were stained with hypotonic 50 μg/mL propidium iodide solution containing 0.1% sodium citrate and 0.1% Triton X-100, and the DNA content was immediately analyzed using flow cytometry (Coulter Episs XL, Beckman Coulter) immediately. For apoptosis assays, cells were gently resuspended in annexin-V binding buffer and incubated with annexinV-FITC/PI at room temperature in the dark for 15 min and analyzed by flow cytometry using cell quest software (BC Epics XL, MIAMI, Florida, USA)
2.7 Western Blot Analysis After treatment with a series of concentrations of pogostone for 20(autophagy) or 30 (apoptosis) hours at 37°C, HCT116 cells were harvested, washed in ice-cold PBS, and lysed with RIPA buffer (10 mM Tris-HCl (pH 7.8), 1% NP40, 0.15 M NaCl, 1 mM EDTA, 10 µM aprotinin, 1 mM NaF and 1 mM Na3VO4). Protein concentrations were determined using a modified Lowry protein assay kit and equalized before loading. Equal amounts of protein (40 μg/sample) was separated electrophoretically by 10%–15% SDS-PAGE and blotted onto PVDF membranes(Bio-Rad, Hercules, CA). For immunodetection, membranes were probed with specific antibodies (Cell Signaling Technology) including phospho-Akt, Akt, phospho-mTOR, mTOR, LC3-Ⅱ, caspase-3,
β-actin.
Blots
were
developed
with
horseradish
peroxidase
(HRP)-conjugated secondary antibodies and chemiluminescent substrate on Kodak X-ray films 2.8 Effect of pogostone on Tumor Growth In Vivo Six-week-old female athymic (nu/nu) mice were obtained from Chinese Academy of Medical Science (Beijing, China). HCT116 tumors were established by injection of 5×106 cells (100 μL) into the hind flank subcutaneously. When tumors grew up to approximately 100 mm3 , the mice were randomly divided into different groups (6 mice each group) and dosed intraperitoneally with pogostone (100 mg/kg and 150 mg/kg, respectively) or vehicle (1% CMC-Na) once daily for 21 days. Besides, Doorubicin (5 mg/kg) was applied to treat mice as a positive control. Tumor growth and body weight was measured every 3 days using Vernier calipers for the duration of the treatment. The volume was calculated as follows: tumor volume = a×b2/2 (a, long diameter; b, short diameter). 2.9 Histological analysis for tumor tissue Female BALB/c nude mice were implanted with HCT116 cells as described above. After 3 days administration of pogostone (150 mg/kg/day, i.p.), mice were sacrificed and tumors were fixed with formalin and embedded in paraffin. Then, paraffin sections of tumor tissue (4~8μm) were prepared and detected using immunostaining with the Ki67 antibody (Thermo Fisher Scientific, Fremont, CA). Apoptosis was
determined using transferase-mediated dUTP nick-end labeling (TUNEL) staining (Roche Applied Science). Finally, images were captured with an Olympus digital camera attached to a light microscope. 2. 10 Statistical Analysis All in vitro experiments were performed in triplicate and repeated 3 times. Differences in the therapeutic effects were analyzed using the variance (ANOVA) method. Finally, the significance was determined using a two-tailed t-test, and differences were considered to be statistically significant when P <0.05.
3. Results 3.1 Cell viability assay We investigated the growth inhibitory potencies of pogostone against various cell lines, and the results are presented in Table 1. A comparison between the cell lines examined showed that HCT116 cell was by far the most sensitive to pogostone treatment than the other cell lines with an IC50 value of 18.7±1.93 µg/ml, and show strikingly lower cytotoxicity on normal human embryonic kidney cell 293A (IC50: 95.13±19.44 µg/ml) and endothelial cell HUVEC (IC50: 112±20.77 µg/ml) （Table 1 and Figure 1）
Fig.1. Effect of pogostone on the viability of HCT116, 293A and HUVEC cells. The cells were incubated with different concentrations (0–200 ug/ml) of pogostone for 72h. Cell viability was measured with an MTT assay. All data are representative of three independent experiments
Table 1 Antiproliferative profile of pogostone on various cell lines IC50 g/mL meanSE
Tumor type HCT116
Human colorectal cancer cell
18.71.93
SW620
Human colorectal cancer cell
21.392.61
MV4-11
Human leukemia cell
22.623.74
MOLM-13
Human acute myeloid leukemia cell
22.214.27
A375
Human melanoma cell
31.325.83
KYSE-510R
Human esophageal squamous cell
38.25.29
MCF-7
Human breast cancer
49.3911.27
H1975
Human Lung cancer
49.647.29
SKOV3
Human ovarian carcinoma cell
53.209.77
MDA-MB-435
Human breast cancer
55.2118.32
THP-1
Human leukemia cell
69.5621.48
HEPG-2
Human Hepatocellular carcinoma cell
72.5219.52
A549
Human Lung cancer
74.5713.84
SH-SY5Y
Human neuroblastoma cell
89.5914.85
293A
Human embryonic kidney cell
95.1319.44
HUVEC
Human endothelial cell
11220.77
3.2 Pogostone induces HCT116 cell autophagy Recently, autophagy has been argued to be another way to die for cells as a novel form of programmed cell death (Tsujimoto and Shimizu, 2005). Inducing autophagic cell death in cancer cells might be useful in tumor treatment（Chresta et al., 2010）. The
TEM
detection
and
the
formation
of
mRFP-GFP-LC3
puncta
are
well-characterized methods to image autophagosomes. We employed these two methods to visualize autophagy. As shown in Figure 2A, pogostone can induced massive flow of autophagy and large number of autophagy body (green) and autophagy-lysosome (yellow) in HCT116 cells. By using transmission electron microscopy, we monitored a large number of of autophagy body and autophagy-lysosome in HCT116 cells treated by pogostone (Figure 2B). Next, we
measured the expression of LC3-II, the membrane-bound form of LC3 which is an autophagy marker. Finally, a dose-dependent increase in the level of LC3-II was observed for pogostone-treated HCT116 cells(Figure 4C). These results indicate that pogostone induces autophagy in HCT116 cells. Next, the changes of HCT116 cell death were examined using pogostone with or without 3-MA (an inhibitor of autophagy). In the combination group (pogostone plus 10mM 3-MA), the viability of HCT116 decreased more slowly than in the pogostone group. After 24h treatment, the combination drove 54.5%, 25.7% and 10.8% of the cells to death when the concentration of pogostone was 100, 50 and 25ug/ml, and exhibited 20.5%, 13.5%, 16.3% decrease compared with the rate of cell death in the pogostone alone group (Figure 2C). These results indicate that autophagy induced by pogostone is responsible for the cell death.
Fig.2. Pogostone induced autophagy in HCT116 cells. A, After 20-hours pogostone treatment, HCT116
cells transfected with mRFP-GFP-LC3 adenovirus were observed under a confocal fluorescence microscopy. Images are presented to indicate the cellular localization patterns of the mRFP-GFP-LC3 fusion protein. B, TEM was used to observe autophagy. a large number of autophagy body (thick arrows) and autophagy-lysosome (thin arrows) were monitored in HCT116 cells treated for 20-hours by pogostone. C, CCK-8 assay was used to examine the changes of HCT116 cell death induced by pogostone with or without 3-MA (an inhibitor of autophagy). The combination groups exhibited 20.5%, 13.5%, 16.3% decrease compared with the rate of cell death in the pogostone alone groups when the concentration of pogostone was 100, 50 and 25ug/ml respectively（*, P<0.05; **, P<0.01）.
3. 3 Pogostone induced cell cycle arrest and apoptosis of HCT116 Cell cycle and apoptosis assays were performed using flow cytometry. After treatment with a series of concentrations of pogostone for 30 hours, HCT116 cells exhibited a dose-dependent increase in the percentage of G0/G1 cells and a dose-dependent decrease in the percentage of S, G2/M phase cells (Figure 3A and Figure 3C), indicating cell cycle arrest in G0/G1. By Two-color flow cytometry analysis of AnnexinV-FITC/PI double-staining, we also found that the percentage of apoptosis was slightly increased when HCT116 cells were treated with pogostone for 30 h. After 30 h of 25, 50 and 100 µg/ml pogostone treatment, cells exhibited 28.3%, 58.3% and 61.4% apoptosis by accumulating both in AnnexinV-positive/PI-negative (early apoptosis) and annexin V-positive/PI-positive (late apoptosis) quadrant(Figure 3B and Figure 3D). The levels of cleaved caspase-3 and caspase-7 were also measured. After a 30 h treatment with increasing concentrations of pogostone, HCT116 cells were harvested and lysed for an immunoblot assay. A dose-dependent decrease in the level of pro–caspase-3 and a dose-dependent increase in the level of the cleaved caspase-3 and caspase-7 fragment were observed for pogostone-treated HCT116 cells at the concentration of 25~100 µg/ml (Figure 4B). These results indicate that pogostone could induce cell cycle arrest in G0/G1 and could eventually cause apoptosis regulated by cleaved caspase-3 and caspase-7 fragment at concentrations above the IC50 value for HCT116 cells.
Fig.3. Cell cycle arrest and apoptosis of HCT116 cells induced by pogostone. HCT116 cells were analyzed by flow cytometry after treatment with pogostone for 30 hours. A and C, The cell cycle was assessed by PI staining. pogostone induced G0-G1 phase arrest. the ratio of HCT116 tumor cells in G0/G1 phase increased significantly, while the ratio of cells in S and G2/M phase decreased.B and D, Apoptosis induction of the pogostone on HCT116 cells. Apoptitic cells were defined as Annexin V+/PI- plus Annexin V+/PI+ cells. the ratio of apoptosis increased from 0.8% to 28.3%(25 µg/ml), 58.3%(50 µg/ml) and 61.4%(100 µg/ml) for 30h.
3. 4 Pogostone inhibits Akt/mTOR signaling pathway in HCT116 cells Akt/mTOR signaling pathway leads to the activation of various downstream signaling substrates that are responsible for tumor cell metabolism, proliferation, antiapoptosis and regulating autophagy. To investigate whether pogostone inhibited Akt/mTOR signaling, we assessed the ability of pogostone to inhibit the phosphorylation of Akt and mTOR in HCT116 cells using western blot analysis. After a 20 h treatment with increasing concentrations of pogostone, HCT116 cells were harvested and lysed for an immunoblot assay. As shown in Figure 4A,
pogostone inhibited Akt and mTOR phosphorylation in a dose-dependent manner. Meanwhile, we also observed that pogostone did not modulate the expression of these proteins during the drug treatment period. The expression of Ki67 a nuclear protein used as a cell mitotic index was also determined by western blot assay. Pogostone significantly inhibited the expression of Ki67 at the concentration of 25~100 µg/ml(Figure 4A). Previous studies have shown that inhibiting the activition of Akt and its downstream target mTOR contribute to the initiation of autophagy and apoptosis (Heras-Sandoval et al., 2014; Sun et al., 2014; Shi and Cao, 2014). These data indicate that pogostone inducing autophagy and apoptosis depended on the inhibition of Akt and mTOR phosphorylation.
Fig.4. Pogostone inhibited Akt/mTOR signal and regulated the expressions of autophagy and apoptosis related protein. A, After a 20-hour treatment with pogostone, the phosphorylation statuses of Akt and mTOR and the expression of Ki67 in HCT116 cells were detected by immunoblot. B, The cells lysate of HCT116 cells treated with pogostone for 30 hours was analyzed using caspase-3 and caspase-7 antibody, and the results show a significant decrease in pro-caspase-3 levels and an increase in cleaved caspase-3 and caspase-7 levels with increasing dose of pogostone. C, After 20h treatment of pogostone, a dose-dependent increase in the level of LC3-II was observed in HCT116 cells.
3. 5 In vivo effects of pogostone against HCT116 tumor xenografts To study the antitumor activity of pogostone in vivo, HCT116 xenograft-bearing BALB/c nude mice were treated with pogostone at the doses of 100 mg/kg and 150 mg/kg or with vehicle alone. Doxorubicin at dose of 5 mg/kg/w was used as a positive control. After 24 days treatment, Pogostone exhibited a significant antitumor activity in inhibiting tumor progress compared with vehicle. Pogostone significantly inhibited the HCT116 tumor growth with an anti-tumor rate of 43.9% at a dose of 150 mg/kg. Doxorubic ininhibited the HCT116 tumor growth with an anti-tumor rate of 61.3% (Figure 5A). Meanwhile, the body weight of mice was monitored once every 3 days throughout the whole experiment. As shown in Figure 5B, no significant differences was observed among the pogostone treatment and vehicle groups, but the body weight of mice treated by doxorubicin decreased fastly after being dosed. No adverse effects in other gross measures such as skin ulcerations or toxic death were observed in pogostone group. These data determined that the inhibition of tumor growth was not attributable to systemic toxicity. Ki-67 is a nuclear protein expressed in proliferating cells and has been used as a cell mitotic index. We evaluated the effects of pogostone on the expression of Ki67 in tissue tumor cells using immunohistochemical method. After treatment for 3 days, tumors were collected and analyzed. Tumor tissues from the vehicle group stained strongly with Ki67, indicating a large number of highly proliferative cells (Figure 5C). Conversely, the tumor tissues treated by 150 mg/kg pogostone showed a significantly fewer Ki67-positive cells (Figure 5D). Furthermore, we analyzed the apoptosis induced by pogostone in vivo using TUNEL assay. As seen in Figure 5C, the percentage of TUNEL-positive cells in the tumors from the 150 mg/kg pogostone-treated group was increased compared with vehicle group (Figure 5D), suggesting that pogostone induced apoptosis in tissue tumor cells.
Fig.5. In vivo effects of pogostone against s.c. HCT116 tumor xenografts. HCT116 cells (5×106/mouse) were s.c. injected into Balb/C null mice, and treatment with pogostone was initiated when the tumors grew to 100 mm3. A, pogostone significantly inhibited the HCT116 tumor growth at a dosages of 150 mg/kg/d, and doxorubicin exhibited more activity as positive control. B, After 24 days treatment, the mice body weight among the groups were statisticed. there were no significant difference were found between pogostone and vehicle groups, but the mice body weight treated by doxorubicin decreased significantly. C and D, After 3-day of pogostone treatment, the HCT116 tumors were collected separately (three per group). Ki67 and TUNEL detection show that pogostone significantly inhibits the proliferation and induces the apoptosis of the HCT116 cells in vivo（*, P<0.05; **, P<0.01）.
4. Discussion Malignant tumor is the abnormal growth of tumor cells in our bodies that can lead to death. Every year, millions of people are diagnosed with malignant tumor (Marusyk et al., 2012). According to the American Cancer Society, malignant tumor kills about 3.5 million people annually all over the world (Cragg and Newman, 2005;
Fouche et al., 2008). Although the mortality has decreased significantly in the past 2 decades with the treatment of surgery, new chemotherapeutic and targeted therapies, recurrence and drug resistance frequently occurs within a short time (Holohan et al., 2013; Simard et al., 2013). Recently, an attempt has been made to explore the potential of newly discovered anticancer compounds, from medicinal plants, as a lead for anticancer drug development （Assaf et al., 2013）. Plants have been used for treating various diseases of human beings and animals since time immemorial. More than 50% of all modern drugs in clinical use are of natural product origins, many of which have the ability to control cancer cells (Newman and Cragg, 2012). The aerial part of Pogostemon cablin, or Guang Huo Xiang in Chinese, has been used in Chinese medicine for centuries (Lu et al., 2011). Several studies indicated that pogostone, an naturally occurring product isolated from this medicinal plant, possesses pharmacological properties such as anti-inflammatory, anti-bacterial, anti-fungal and Immunosuppressive activity. However, the anti-tumor activities and the potencial anti-cancer mechanisms of pogostone have not been elucidated. In this study, we aimed to evaluate the anti-tumor activity of pogostone and clarify the mechanism, which is associated with promoting autophagy and inducing apoptosis involving PI3K/Akt/mTOR signaling pathway in human colorectal cancer cells. in vitro, pogostone exhibits potent anti-proliferative activities against multiple human tumor cell lines, especially potency in inhibiting human colorectal cell line HCT116 (IC50=18.7±1.93 µg/mL), and exhibited low toxic effects in normal cells (IC50=112±20.77 µg/mL). In vivo, pogostone inhibited the growth of HCT116 tumor,
reduced
the
tumor
volume
significantly
with
well
tolerated.
Immunohistochemical detection showed an obvious decrease the cell number stained with Ki67 and an increase in the percentage of apoptotic cells. Our findings suggested that pogostone might have potential anti-tumor activity on HCT116. Apoptosis is recognized as an efficient strategy for cancer chemotherapy and a useful indicator for cancer treatment and prevention (Ma et al., 2012). FCM assay indicated that pogostone could induce cell cycle arrest in G0/G1 and increase the
percentage of apoptosis cells at concentrations above the IC50 value for HCT116 cells(Fig.3). After 30h treatment, pogostone lead to the cleavage of the apoptosis markers caspase-7 and caspase-3 in HCT116 cells. Inducing autophagy also became a potencial useful strategy in anti-tumor chemotherapy (Levine et al., 2015). We applied HCT116 cells transfected with mRFP-GFP-LC3 adenovirus to detect the autophagy induced by pogostone. Green GFP-LC3 punta and red mRFP-LC3 punta were observed in cells treated by pogostone, under fluorescence microscopic examination. To further confirm that pogostone induced autophagy, we detected autophagosome under TEM. Next, we discovered the dose-dependent nature of pogostone-induced autophagy. 50 and 100 µg/ml pogostone treatment for 20h caused the maximum expression of LC3-Ⅱ.Our dates demonstrated that pogostone induces the accumulation of LC3-Ⅱ and in a concentration-dependent manner. The relationship between autophagy and apoptosis is intricate. Many researchers consider that the accumulation of autophagy could induce a non-selective bulk degradation process and lead to apoptosis finally (Chresta et al., 2010; Gordy and He, 2012). Once apoptosis occurs irreversibly, autophagy will be negatively regulated by initiation of apoptosis (Gong et al., 2014). In the process of our study, we found that pogostone treatment for 16 or 20 h could initiate the formation of autophagosome and increase the expression of LC3-Ⅱ without inducing apoptosis. After 30h treatment, pogostone activate apoptotic pathways to inhibit autophagy, so we can not detect the autophagosome after 30 h pogostone treatment(data not shown). However, apoptosis appearance might block autophagy. Akt and mTOR are well-known major regulatory signaling pathways that modulate cell proliferation, metabolism and survival in cancer cells. Therefore, several inhibitors, such as everolimus, have been developed and used for treatment to induce apoptosis in cancer cells. To inhibit the mTOR signaling pathway, many researchers have used rapamycin. However, rapamycin only inhibits mTOR complex (TORC) 1, and it consequently induces Akt phosphorylation via feedback activation (Radhakrishnan et al., 2013). These actions reduce the anti-cancer effect of rapamycin. Our data showed that pogostone could not only inhibit the
phosphorylation of mTOR, but also inhibit the activition of Akt; thus, pogostone will exhibit more anti-tumor activity by inducing apoptosis and autophagy. 5. Conclusions In summary, due to its ability to inhibit tumor growth by inducing apoptosis and autophagy, pogostone can be a leading compounds for anti-tumor drug discovery, particularly in the treatment of Human Colorectal cancer. Our finding suggest that pogostone efficiently accelerates cell death is largly dependent on initiating the accumulation of autophay and inducing apoptosis. Furthermore, we provide evidence that pogostone activates multiple signaling pathways related on autophagy and apoptosis, inducing Akt/mTOR inhibition. Our study indicated that the pogostone may be a promising compound to be further developed for cancer therapy.
Conflict of interest The authors declare no conflicts of interests.
Acknowledgments This study was supported by the National Natural Science Foundation of China(81300437, 81403149), Youth Scientific Research Fund of CDUTCM (ZRQN1450, CGPY1402), Sichuan Province Youth Science and technology innovation research team project (2014TD0007, 2016TD0006)
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Graphical abstract
PII: DOI: Reference:
S0378-8741(16)30453-6 http://dx.doi.org/10.1016/j.jep.2016.07.028 JEP10293
To appear in: Journal of Ethnopharmacology Received date: 28 April 2016 Revised date: 7 July 2016 Accepted date: 8 July 2016 Cite this article as: Zhi-Xing Cao, Yu-Ting Yang, Si Yu, Yu-Zhi Li, Wen-Wen Wang, Jing Huang, Xiao-Fang Xie, Liang Xiong, Song Lei and Cheng Peng, Pogostone Induces Autophagy and Apoptosis Involving PI3K/Akt/mTOR Axis in Human Colorectal Carcinoma HCT116 cells, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.07.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pogostone Induces Autophagy and Apoptosis Involving PI3K/Akt/mTOR Axis in Human Colorectal Carcinoma HCT116 cells
Zhi-Xing Caoa,1, Yu-TingYanga,1, Si Yua,1, Yu-Zhi Lia, Wen-Wen Wanga,c, Jing Huanga,c, Xiao-Fang Xiea, Liang Xionga, Song Leib, Cheng Penga*
a
Pharmacy College，Chengdu University of Traditional Chinese Medicine；The
Ministry of Education Key Laboratory of Standardization of Chinese Herbal Medicine；Key Laboratory of Systematic Research，Development and Utilization of Chinese Medicine Resources in Sichuan Province—Key Laboratory Breeding Base of Co-founded by Sichuan Province and MOST b
Department of pathology, West China Hospital
c
School of Medical Technology, Chengdu University of Traditional Chinese Medicine,
*Corresponding authors at: Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China. Tel./fax: +86 28 61800018. E-mail: [email protected]
1
These three authors contributed equally to this work.
ABSTRACT Ethnophamacological relevance: Pogostemon cablin is a medicinal herb widely used to treat gastrointestinal diseases in many Asian countries. Pogostone is an important constituent of Pogostemon cablin, and possesses various bioactivitys. In this study, we performed to investigate the anti-colorectal tumor property of Pogostone by inducing aurophagy and apoptosis in human colorectal cancer cells, and to define the potential molecular mechanisms. Materials and methods: In vitro,The anti-tumor activity of pogostone was assessed using MTT assay. Autophagy was monitored by transmission electron microscopy observation and mRFP-GFP-LC3 fluorescence analysis in colorectal tumor cell line. Apoptosis was measured by flow cytometry and annexinV-FITC/PI staining. The protein expressions or activition of LC3-Ⅱ, AKT, mTOR, caspase-3 and caspase-7 were detected through western blotting. In vivo, the
anti-tumor effect of pogostone was tested with HCT116 colorectal tumor cells transplantation tumor model. The expression of Ki-67 was determed by Immunohistochemistry staining and the apoptosis was evaluated using TUNEL assay. Results: In vitro, pogostone exhibits significant anti-tumor activity against human cancer cell lines, especially for HCT116 (18.7±1.93 μg/ml). Transmission electron microscopy observation, mRFP-GFP-LC3 fluorescence analysis, flow cytometryand assay and western blotting detection revealed that the anti-colorectal tumor activity of pogostone was dependent on inducing autophagy and apoptosis through up-regulating the expression of LC3-Ⅱ, cleaved caspase-7 and caspase-3, and decreasing the phosphorylation of AKT/mTOR. In vivo, 150 mg/kg pogostone inhibited the HCT116 tumor growth in immunodeficient mice with an inhibitory rate of 43.3%, decreased the expression of Ki67, and induced apoptosis in three days. Conclusion: Pogostone showed anti-colorectal tumor effects by inducing autophagy and apoptosis involving PI3K/Akt/mTOR axis. Thus, pogostone may be a promising lead compound to be further developed for cancer therapy.
Keywords: Pogostone; autophagy; apoptosis; Akt; mTOR
1． Introduction Human colorectal cancer is one of the leading causes of cancer-related lethality worldwide, with more than 600, 000 deaths each year (Globocan, 2012). Although the mortality has decreased significantly in the past 2 decades with the treatment of surgery, new chemotherapeutic and targeted therapies, recurrence and drug resistance frequently occurs with in a short time (Van et al.,2010; Terme et al., 2013). Therefore, the development of novel anti-colorectal cancer agents is urgently needed.
PI3K/Akt/mTOR signaling pathway is a major signal transduction cascade involved in the cellular proliferation, survival, and metabolism, and plays an important role in colorectal cancer (Shimizu et al., 2012;Ma et al., 2015). Up-regulation of PI3K resulting in inhibition of apoptosis in colon-cancer cells (Zhirnov and Klenk, 2007). Inhibitors of PI3K/Akt/mTOR signaling have been suggested as potential therapeutic agents in colorectal cancer (Roper et al., 2011; Ashok, 2013). Previous studies have shown that inhibiting the activition of Akt and its downstream target mTOR contribute to the initiation of autophagy, which is now recognised as a novel forms of
“programmed” cell death (Heras-Sandoval et al., 2014; Sun et al., 2014; Shi and Cao, 2014). Normally, autophagy is a survival mechanism that facilitates the maintenance of intracellular homeostasis by degrading long-lived proteins and damaged organelles and participating in many human diseases and physiological processes (Mizushima et al., 2008; Ravikumar et al., 2010). On the other hand, many anti-cancer agents, such as rapamycin and everolimus, the mTOR inhibitors have the ability to induce autophagic cell death in cancer cells, suggesting that autophagy might be useful in tumor treatment (O'Donnell et al., 2008; Chresta et al., 2010). Rencent studies have shown that autophagy could induce a non-selective bulk degradation process and cause apoptosis and cell death in many kinds of cancer cells (Arlia-Ciommo et al., 2015; Nagelkerke et al., 2014; Vijayaraghavan and Keyomarsi, 2015). Targeting critical regulators with the purpose to promote autophagy in cancer cells is an attractive new cancer therapeutic strategy.
Pogostemon Cablin(Blanco) Benth, commonly known as “Guanghuoxiang”, is a traditional Chinese medicinal herb widely used to treat gastrointestinal diseases in many Asian countries (Chen et al., 2015). Pogostone is one of the major constituent of Pogostemon cablin, and possesses various bioactivitys. It has been reported that pogostone possess potent anti-fungal (Mo et al., 2004;Li et al., 2012), antibacterial (Osawa et al., 1990; Peng et al., 2014) and pesticidal activities (Huang et al., 2013). New reports also reveal that pogostone exert anti-inflammatory activity on lipopolysaccharide-stimulation RAW264.7 model and Immunosuppressive activity on T cell (Li et al., 2014; Su et al., 2015). Studies of its pharmacokinetic properties show that pogostone was easily absorbed after oral administration with a high bioavailability (Li et al., 2014). The present study was performed to investigate if pogostone shown anti-cancer activity in human colorectal cancer cells. Finally, we found that pogostone could induce autophagy and apoptosis in colorectal cancer by inhibiting the activation of PI3K/Akt/mTOR axis.
2. Materials and methods 2. 1 Chemicals and reagents DMSO,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT),
3-Methyladenine (3-MA) was purchased from Sigma(NY, USA). Cell count kit-8 (CCK-8) was purchased from Tokyo, Japan. Trypsin, Cell culture media, DMEM, RPMI-1640, IMDM and fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA), Antibodies against Akt, p-AKT, mTOR, p-mTOR, Caspase-3 and (-actin were purchased from Cell Signaling Technology(Danvers, MA). Annexin V-FITC and propidium iodide(PI) Apoptosis Detection Kit and BCA protein assay kit were purchased from Beyotime (Shanghai, China). 2. 2 Extraction and isolation of pogostone pogostone was isolated from P. cablin as previously described[15]. The extraction and isolation procedure was as follows. Briefly, the dried leaves(15kg) of P. cablin were exhaustively extracted through water-steam distillation. Essential oil (300g) was obtained from P. cablin and dissolved in diethyl ether(1500ml) and partitioned with 2%NaOH( 1500 ml) five times. The five alkaline extracts were pooled and diluted HCl was added to adjust the PH to 1-2. After acidification, the solution was partitioned with diethyl ether(2000ml) five times; then, the ether phase was evaporated under reduced pressure to give a residue, which was further purified by recrystallization with n-hexane at -4℃to yield pogostone. The purity of pogostone was determined to be 99. 8% by gas chromatograph-mass spectroscopy(Agilent Technologies, USA) with an Agilent 7890A/5975C instrument(Agilent Technologies, USA). In addition, the structure of pogostone was further confirmed by spectroscopic data analysis(1H-nuclear magnetic resonance spectra(1H-NMR), 13C-NMR, and mass spectrometry(MS)). In the in vitro experiments, pogostone was dissolved in dimethyl sulfoxide(DMSO) and diluted with culture medium. The final concentration of DMSO in the test solutions was less than 0. 1%. In animal experiments, pogostone was suspended in 0. 5% Cellulose CM sodium salt(CMC-Na) at 37℃.
2. 3 Cell lines and cultures The human colorectal carcinoma cell line HCT116、SW620 , human lung cancer cell line A549 and H1975, human mammary carcinoma cell line MDA-MB-231, MCF-7 and MDA-MB-435, human melanoma cell line A375, human esophageal squamous cell carcinoma cell line KYSE-510R, Human ovarian carcinoma cell line SKOV3, human hepatocellular carcinoma cell line HepG-2, Human neuroblastoma cell SH-SY5Y, human embryonic kidney 293A cell, human acute myeloid leukemia cell line MV4-11 and THP-1, were obtained from American Type Culture Collection(Manassas, VA, USA). Cells were grown in RPMI-1640, DMEM or IMDM culture mediumcontaining 10% fetal bovine serum in 5% CO2 at 37℃. 2.4 Cell viability assay MTT assay Briefly, exponentially growing tumor cells were seeded into 96-well microplates for 24hours. Afterwards, equal volume of medium containing various concentrations of pogostone ranging from 3.12 µg/mL to 200.0 µg/mL were added to each well. At the end of the incubation period (72 hours at 37℃), 20μL of 5 mg/mL MTT reagent was added per well for 2~4-hour of incubation, and 50 μL of 20% acidified SDS per well was used to lyse the cells. Finally, the light absorption (OD) of the dissolved cells was measured at 570 nm using a SpectraMAX M5 microplate spectrophotometer (Molecular Devices). All experiments were performed in triplicate. The percentage of viability was calculated and compared with that of the control cells treated with DMSO (0.1%). CKK-8 assay HCT116 cells in 96-well microplates were divided into four groups: control, 3-MA, pogostone, and pogostone plus 3-MA groups. After passage 24 h, cells were changed to the equal volume of medium containing 25, 50 and 100 µg/mL pogostone with or without 10 mM 3-MA, and added 1‰ DMSO or 10 mM 3-MA alone as control group. After the culture period (24 hours at 37℃), the supernatant fluid was removed and medium contains 10% CCK- 8 reagent was added for 1-3 h incubation at 37° C. The light absorptions (OD) were measured at 450 nm with SpectraMAX M5
microplate spectrophotometer 2. 5 Autophagy detection 2.5.1 Evaluation of fluorescent LC3 puncta LC3 puncta were indicated by mRFP-GFP-LC3 adenovirus (Hanbio Co., LTD, China). Briefly, HCT116 cells were transfected with mRFP-GFP-LC3 for 24 h before receiving pogostone treatments. After 20-hours pogostone treatment, cells were observed under a confocal fluorescence microscopy (Olympus FV1200, Japan). Autophagic cells, which contained five or more mRFP-GFP-LC3 dots were recorded and the images were acquired. 2.5.2 Transmission Electron microscopy observation For transmission electron microscopy(TEM) observation, after being treated with 50 μg/mL pogostone for 20h, the cells were collected and fixed in ice-cold glutaraldehyde(2.5% in 0.1 mol/L cacodylate buffer, PH 7. 4) overnight and postfixed in osmium tetroxide. After dehydration with a graded series of alcohol concentrations, the samples were rinsed in propylene oxide and impregnated with epoxy resins. The ultrathin sections were contrasted with uranyl acetate and lead citrate for electron microscopy. Electron micrographs were observed through a transmission electron microscope(H7650;Hitachi, Tokyo, Japan) ,according to a method previously described by Wang et al., 2013. 2. 6 Flow cytometric analysis of cell cycle and apoptosis A flow cytometric assay was used for cell cycle and apoptosis assays. Briefly, after treatment with increasing concentrations of pogostone for 30 hours, HCT116 cells were harvested and washed with PBS. For cell cycle assays, cells were stained with hypotonic 50 μg/mL propidium iodide solution containing 0.1% sodium citrate and 0.1% Triton X-100, and the DNA content was immediately analyzed using flow cytometry (Coulter Episs XL, Beckman Coulter) immediately. For apoptosis assays, cells were gently resuspended in annexin-V binding buffer and incubated with annexinV-FITC/PI at room temperature in the dark for 15 min and analyzed by flow cytometry using cell quest software (BC Epics XL, MIAMI, Florida, USA)
2.7 Western Blot Analysis After treatment with a series of concentrations of pogostone for 20(autophagy) or 30 (apoptosis) hours at 37°C, HCT116 cells were harvested, washed in ice-cold PBS, and lysed with RIPA buffer (10 mM Tris-HCl (pH 7.8), 1% NP40, 0.15 M NaCl, 1 mM EDTA, 10 µM aprotinin, 1 mM NaF and 1 mM Na3VO4). Protein concentrations were determined using a modified Lowry protein assay kit and equalized before loading. Equal amounts of protein (40 μg/sample) was separated electrophoretically by 10%–15% SDS-PAGE and blotted onto PVDF membranes(Bio-Rad, Hercules, CA). For immunodetection, membranes were probed with specific antibodies (Cell Signaling Technology) including phospho-Akt, Akt, phospho-mTOR, mTOR, LC3-Ⅱ, caspase-3,
β-actin.
Blots
were
developed
with
horseradish
peroxidase
(HRP)-conjugated secondary antibodies and chemiluminescent substrate on Kodak X-ray films 2.8 Effect of pogostone on Tumor Growth In Vivo Six-week-old female athymic (nu/nu) mice were obtained from Chinese Academy of Medical Science (Beijing, China). HCT116 tumors were established by injection of 5×106 cells (100 μL) into the hind flank subcutaneously. When tumors grew up to approximately 100 mm3 , the mice were randomly divided into different groups (6 mice each group) and dosed intraperitoneally with pogostone (100 mg/kg and 150 mg/kg, respectively) or vehicle (1% CMC-Na) once daily for 21 days. Besides, Doorubicin (5 mg/kg) was applied to treat mice as a positive control. Tumor growth and body weight was measured every 3 days using Vernier calipers for the duration of the treatment. The volume was calculated as follows: tumor volume = a×b2/2 (a, long diameter; b, short diameter). 2.9 Histological analysis for tumor tissue Female BALB/c nude mice were implanted with HCT116 cells as described above. After 3 days administration of pogostone (150 mg/kg/day, i.p.), mice were sacrificed and tumors were fixed with formalin and embedded in paraffin. Then, paraffin sections of tumor tissue (4~8μm) were prepared and detected using immunostaining with the Ki67 antibody (Thermo Fisher Scientific, Fremont, CA). Apoptosis was
determined using transferase-mediated dUTP nick-end labeling (TUNEL) staining (Roche Applied Science). Finally, images were captured with an Olympus digital camera attached to a light microscope. 2. 10 Statistical Analysis All in vitro experiments were performed in triplicate and repeated 3 times. Differences in the therapeutic effects were analyzed using the variance (ANOVA) method. Finally, the significance was determined using a two-tailed t-test, and differences were considered to be statistically significant when P <0.05.
3. Results 3.1 Cell viability assay We investigated the growth inhibitory potencies of pogostone against various cell lines, and the results are presented in Table 1. A comparison between the cell lines examined showed that HCT116 cell was by far the most sensitive to pogostone treatment than the other cell lines with an IC50 value of 18.7±1.93 µg/ml, and show strikingly lower cytotoxicity on normal human embryonic kidney cell 293A (IC50: 95.13±19.44 µg/ml) and endothelial cell HUVEC (IC50: 112±20.77 µg/ml) （Table 1 and Figure 1）
Fig.1. Effect of pogostone on the viability of HCT116, 293A and HUVEC cells. The cells were incubated with different concentrations (0–200 ug/ml) of pogostone for 72h. Cell viability was measured with an MTT assay. All data are representative of three independent experiments
Table 1 Antiproliferative profile of pogostone on various cell lines IC50 g/mL meanSE
Tumor type HCT116
Human colorectal cancer cell
18.71.93
SW620
Human colorectal cancer cell
21.392.61
MV4-11
Human leukemia cell
22.623.74
MOLM-13
Human acute myeloid leukemia cell
22.214.27
A375
Human melanoma cell
31.325.83
KYSE-510R
Human esophageal squamous cell
38.25.29
MCF-7
Human breast cancer
49.3911.27
H1975
Human Lung cancer
49.647.29
SKOV3
Human ovarian carcinoma cell
53.209.77
MDA-MB-435
Human breast cancer
55.2118.32
THP-1
Human leukemia cell
69.5621.48
HEPG-2
Human Hepatocellular carcinoma cell
72.5219.52
A549
Human Lung cancer
74.5713.84
SH-SY5Y
Human neuroblastoma cell
89.5914.85
293A
Human embryonic kidney cell
95.1319.44
HUVEC
Human endothelial cell
11220.77
3.2 Pogostone induces HCT116 cell autophagy Recently, autophagy has been argued to be another way to die for cells as a novel form of programmed cell death (Tsujimoto and Shimizu, 2005). Inducing autophagic cell death in cancer cells might be useful in tumor treatment（Chresta et al., 2010）. The
TEM
detection
and
the
formation
of
mRFP-GFP-LC3
puncta
are
well-characterized methods to image autophagosomes. We employed these two methods to visualize autophagy. As shown in Figure 2A, pogostone can induced massive flow of autophagy and large number of autophagy body (green) and autophagy-lysosome (yellow) in HCT116 cells. By using transmission electron microscopy, we monitored a large number of of autophagy body and autophagy-lysosome in HCT116 cells treated by pogostone (Figure 2B). Next, we
measured the expression of LC3-II, the membrane-bound form of LC3 which is an autophagy marker. Finally, a dose-dependent increase in the level of LC3-II was observed for pogostone-treated HCT116 cells(Figure 4C). These results indicate that pogostone induces autophagy in HCT116 cells. Next, the changes of HCT116 cell death were examined using pogostone with or without 3-MA (an inhibitor of autophagy). In the combination group (pogostone plus 10mM 3-MA), the viability of HCT116 decreased more slowly than in the pogostone group. After 24h treatment, the combination drove 54.5%, 25.7% and 10.8% of the cells to death when the concentration of pogostone was 100, 50 and 25ug/ml, and exhibited 20.5%, 13.5%, 16.3% decrease compared with the rate of cell death in the pogostone alone group (Figure 2C). These results indicate that autophagy induced by pogostone is responsible for the cell death.
Fig.2. Pogostone induced autophagy in HCT116 cells. A, After 20-hours pogostone treatment, HCT116
cells transfected with mRFP-GFP-LC3 adenovirus were observed under a confocal fluorescence microscopy. Images are presented to indicate the cellular localization patterns of the mRFP-GFP-LC3 fusion protein. B, TEM was used to observe autophagy. a large number of autophagy body (thick arrows) and autophagy-lysosome (thin arrows) were monitored in HCT116 cells treated for 20-hours by pogostone. C, CCK-8 assay was used to examine the changes of HCT116 cell death induced by pogostone with or without 3-MA (an inhibitor of autophagy). The combination groups exhibited 20.5%, 13.5%, 16.3% decrease compared with the rate of cell death in the pogostone alone groups when the concentration of pogostone was 100, 50 and 25ug/ml respectively（*, P<0.05; **, P<0.01）.
3. 3 Pogostone induced cell cycle arrest and apoptosis of HCT116 Cell cycle and apoptosis assays were performed using flow cytometry. After treatment with a series of concentrations of pogostone for 30 hours, HCT116 cells exhibited a dose-dependent increase in the percentage of G0/G1 cells and a dose-dependent decrease in the percentage of S, G2/M phase cells (Figure 3A and Figure 3C), indicating cell cycle arrest in G0/G1. By Two-color flow cytometry analysis of AnnexinV-FITC/PI double-staining, we also found that the percentage of apoptosis was slightly increased when HCT116 cells were treated with pogostone for 30 h. After 30 h of 25, 50 and 100 µg/ml pogostone treatment, cells exhibited 28.3%, 58.3% and 61.4% apoptosis by accumulating both in AnnexinV-positive/PI-negative (early apoptosis) and annexin V-positive/PI-positive (late apoptosis) quadrant(Figure 3B and Figure 3D). The levels of cleaved caspase-3 and caspase-7 were also measured. After a 30 h treatment with increasing concentrations of pogostone, HCT116 cells were harvested and lysed for an immunoblot assay. A dose-dependent decrease in the level of pro–caspase-3 and a dose-dependent increase in the level of the cleaved caspase-3 and caspase-7 fragment were observed for pogostone-treated HCT116 cells at the concentration of 25~100 µg/ml (Figure 4B). These results indicate that pogostone could induce cell cycle arrest in G0/G1 and could eventually cause apoptosis regulated by cleaved caspase-3 and caspase-7 fragment at concentrations above the IC50 value for HCT116 cells.
Fig.3. Cell cycle arrest and apoptosis of HCT116 cells induced by pogostone. HCT116 cells were analyzed by flow cytometry after treatment with pogostone for 30 hours. A and C, The cell cycle was assessed by PI staining. pogostone induced G0-G1 phase arrest. the ratio of HCT116 tumor cells in G0/G1 phase increased significantly, while the ratio of cells in S and G2/M phase decreased.B and D, Apoptosis induction of the pogostone on HCT116 cells. Apoptitic cells were defined as Annexin V+/PI- plus Annexin V+/PI+ cells. the ratio of apoptosis increased from 0.8% to 28.3%(25 µg/ml), 58.3%(50 µg/ml) and 61.4%(100 µg/ml) for 30h.
3. 4 Pogostone inhibits Akt/mTOR signaling pathway in HCT116 cells Akt/mTOR signaling pathway leads to the activation of various downstream signaling substrates that are responsible for tumor cell metabolism, proliferation, antiapoptosis and regulating autophagy. To investigate whether pogostone inhibited Akt/mTOR signaling, we assessed the ability of pogostone to inhibit the phosphorylation of Akt and mTOR in HCT116 cells using western blot analysis. After a 20 h treatment with increasing concentrations of pogostone, HCT116 cells were harvested and lysed for an immunoblot assay. As shown in Figure 4A,
pogostone inhibited Akt and mTOR phosphorylation in a dose-dependent manner. Meanwhile, we also observed that pogostone did not modulate the expression of these proteins during the drug treatment period. The expression of Ki67 a nuclear protein used as a cell mitotic index was also determined by western blot assay. Pogostone significantly inhibited the expression of Ki67 at the concentration of 25~100 µg/ml(Figure 4A). Previous studies have shown that inhibiting the activition of Akt and its downstream target mTOR contribute to the initiation of autophagy and apoptosis (Heras-Sandoval et al., 2014; Sun et al., 2014; Shi and Cao, 2014). These data indicate that pogostone inducing autophagy and apoptosis depended on the inhibition of Akt and mTOR phosphorylation.
Fig.4. Pogostone inhibited Akt/mTOR signal and regulated the expressions of autophagy and apoptosis related protein. A, After a 20-hour treatment with pogostone, the phosphorylation statuses of Akt and mTOR and the expression of Ki67 in HCT116 cells were detected by immunoblot. B, The cells lysate of HCT116 cells treated with pogostone for 30 hours was analyzed using caspase-3 and caspase-7 antibody, and the results show a significant decrease in pro-caspase-3 levels and an increase in cleaved caspase-3 and caspase-7 levels with increasing dose of pogostone. C, After 20h treatment of pogostone, a dose-dependent increase in the level of LC3-II was observed in HCT116 cells.
3. 5 In vivo effects of pogostone against HCT116 tumor xenografts To study the antitumor activity of pogostone in vivo, HCT116 xenograft-bearing BALB/c nude mice were treated with pogostone at the doses of 100 mg/kg and 150 mg/kg or with vehicle alone. Doxorubicin at dose of 5 mg/kg/w was used as a positive control. After 24 days treatment, Pogostone exhibited a significant antitumor activity in inhibiting tumor progress compared with vehicle. Pogostone significantly inhibited the HCT116 tumor growth with an anti-tumor rate of 43.9% at a dose of 150 mg/kg. Doxorubic ininhibited the HCT116 tumor growth with an anti-tumor rate of 61.3% (Figure 5A). Meanwhile, the body weight of mice was monitored once every 3 days throughout the whole experiment. As shown in Figure 5B, no significant differences was observed among the pogostone treatment and vehicle groups, but the body weight of mice treated by doxorubicin decreased fastly after being dosed. No adverse effects in other gross measures such as skin ulcerations or toxic death were observed in pogostone group. These data determined that the inhibition of tumor growth was not attributable to systemic toxicity. Ki-67 is a nuclear protein expressed in proliferating cells and has been used as a cell mitotic index. We evaluated the effects of pogostone on the expression of Ki67 in tissue tumor cells using immunohistochemical method. After treatment for 3 days, tumors were collected and analyzed. Tumor tissues from the vehicle group stained strongly with Ki67, indicating a large number of highly proliferative cells (Figure 5C). Conversely, the tumor tissues treated by 150 mg/kg pogostone showed a significantly fewer Ki67-positive cells (Figure 5D). Furthermore, we analyzed the apoptosis induced by pogostone in vivo using TUNEL assay. As seen in Figure 5C, the percentage of TUNEL-positive cells in the tumors from the 150 mg/kg pogostone-treated group was increased compared with vehicle group (Figure 5D), suggesting that pogostone induced apoptosis in tissue tumor cells.
Fig.5. In vivo effects of pogostone against s.c. HCT116 tumor xenografts. HCT116 cells (5×106/mouse) were s.c. injected into Balb/C null mice, and treatment with pogostone was initiated when the tumors grew to 100 mm3. A, pogostone significantly inhibited the HCT116 tumor growth at a dosages of 150 mg/kg/d, and doxorubicin exhibited more activity as positive control. B, After 24 days treatment, the mice body weight among the groups were statisticed. there were no significant difference were found between pogostone and vehicle groups, but the mice body weight treated by doxorubicin decreased significantly. C and D, After 3-day of pogostone treatment, the HCT116 tumors were collected separately (three per group). Ki67 and TUNEL detection show that pogostone significantly inhibits the proliferation and induces the apoptosis of the HCT116 cells in vivo（*, P<0.05; **, P<0.01）.
4. Discussion Malignant tumor is the abnormal growth of tumor cells in our bodies that can lead to death. Every year, millions of people are diagnosed with malignant tumor (Marusyk et al., 2012). According to the American Cancer Society, malignant tumor kills about 3.5 million people annually all over the world (Cragg and Newman, 2005;
Fouche et al., 2008). Although the mortality has decreased significantly in the past 2 decades with the treatment of surgery, new chemotherapeutic and targeted therapies, recurrence and drug resistance frequently occurs within a short time (Holohan et al., 2013; Simard et al., 2013). Recently, an attempt has been made to explore the potential of newly discovered anticancer compounds, from medicinal plants, as a lead for anticancer drug development （Assaf et al., 2013）. Plants have been used for treating various diseases of human beings and animals since time immemorial. More than 50% of all modern drugs in clinical use are of natural product origins, many of which have the ability to control cancer cells (Newman and Cragg, 2012). The aerial part of Pogostemon cablin, or Guang Huo Xiang in Chinese, has been used in Chinese medicine for centuries (Lu et al., 2011). Several studies indicated that pogostone, an naturally occurring product isolated from this medicinal plant, possesses pharmacological properties such as anti-inflammatory, anti-bacterial, anti-fungal and Immunosuppressive activity. However, the anti-tumor activities and the potencial anti-cancer mechanisms of pogostone have not been elucidated. In this study, we aimed to evaluate the anti-tumor activity of pogostone and clarify the mechanism, which is associated with promoting autophagy and inducing apoptosis involving PI3K/Akt/mTOR signaling pathway in human colorectal cancer cells. in vitro, pogostone exhibits potent anti-proliferative activities against multiple human tumor cell lines, especially potency in inhibiting human colorectal cell line HCT116 (IC50=18.7±1.93 µg/mL), and exhibited low toxic effects in normal cells (IC50=112±20.77 µg/mL). In vivo, pogostone inhibited the growth of HCT116 tumor,
reduced
the
tumor
volume
significantly
with
well
tolerated.
Immunohistochemical detection showed an obvious decrease the cell number stained with Ki67 and an increase in the percentage of apoptotic cells. Our findings suggested that pogostone might have potential anti-tumor activity on HCT116. Apoptosis is recognized as an efficient strategy for cancer chemotherapy and a useful indicator for cancer treatment and prevention (Ma et al., 2012). FCM assay indicated that pogostone could induce cell cycle arrest in G0/G1 and increase the
percentage of apoptosis cells at concentrations above the IC50 value for HCT116 cells(Fig.3). After 30h treatment, pogostone lead to the cleavage of the apoptosis markers caspase-7 and caspase-3 in HCT116 cells. Inducing autophagy also became a potencial useful strategy in anti-tumor chemotherapy (Levine et al., 2015). We applied HCT116 cells transfected with mRFP-GFP-LC3 adenovirus to detect the autophagy induced by pogostone. Green GFP-LC3 punta and red mRFP-LC3 punta were observed in cells treated by pogostone, under fluorescence microscopic examination. To further confirm that pogostone induced autophagy, we detected autophagosome under TEM. Next, we discovered the dose-dependent nature of pogostone-induced autophagy. 50 and 100 µg/ml pogostone treatment for 20h caused the maximum expression of LC3-Ⅱ.Our dates demonstrated that pogostone induces the accumulation of LC3-Ⅱ and in a concentration-dependent manner. The relationship between autophagy and apoptosis is intricate. Many researchers consider that the accumulation of autophagy could induce a non-selective bulk degradation process and lead to apoptosis finally (Chresta et al., 2010; Gordy and He, 2012). Once apoptosis occurs irreversibly, autophagy will be negatively regulated by initiation of apoptosis (Gong et al., 2014). In the process of our study, we found that pogostone treatment for 16 or 20 h could initiate the formation of autophagosome and increase the expression of LC3-Ⅱ without inducing apoptosis. After 30h treatment, pogostone activate apoptotic pathways to inhibit autophagy, so we can not detect the autophagosome after 30 h pogostone treatment(data not shown). However, apoptosis appearance might block autophagy. Akt and mTOR are well-known major regulatory signaling pathways that modulate cell proliferation, metabolism and survival in cancer cells. Therefore, several inhibitors, such as everolimus, have been developed and used for treatment to induce apoptosis in cancer cells. To inhibit the mTOR signaling pathway, many researchers have used rapamycin. However, rapamycin only inhibits mTOR complex (TORC) 1, and it consequently induces Akt phosphorylation via feedback activation (Radhakrishnan et al., 2013). These actions reduce the anti-cancer effect of rapamycin. Our data showed that pogostone could not only inhibit the
phosphorylation of mTOR, but also inhibit the activition of Akt; thus, pogostone will exhibit more anti-tumor activity by inducing apoptosis and autophagy. 5. Conclusions In summary, due to its ability to inhibit tumor growth by inducing apoptosis and autophagy, pogostone can be a leading compounds for anti-tumor drug discovery, particularly in the treatment of Human Colorectal cancer. Our finding suggest that pogostone efficiently accelerates cell death is largly dependent on initiating the accumulation of autophay and inducing apoptosis. Furthermore, we provide evidence that pogostone activates multiple signaling pathways related on autophagy and apoptosis, inducing Akt/mTOR inhibition. Our study indicated that the pogostone may be a promising compound to be further developed for cancer therapy.
Conflict of interest The authors declare no conflicts of interests.
Acknowledgments This study was supported by the National Natural Science Foundation of China(81300437, 81403149), Youth Scientific Research Fund of CDUTCM (ZRQN1450, CGPY1402), Sichuan Province Youth Science and technology innovation research team project (2014TD0007, 2016TD0006)
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Graphical abstract