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Anticancer activities of harmine by inducing a pro-death autophagy and apoptosis in human gastric cancer cells
Accepted Manuscript
Anticancer activities of harmine by inducing a pro-death autophagy and apoptosis in human gastric cancer cells Chuan Li , Yihai Wang , Chunhua Wang , Xiaomin Yi , Mingya Li , He Xiangjiu PII: DOI: Reference:
S0944-7113(17)30039-9 10.1016/j.phymed.2017.02.008 PHYMED 52169
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
28 August 2016 10 January 2017 24 February 2017
Please cite this article as: Chuan Li , Yihai Wang , Chunhua Wang , Xiaomin Yi , Mingya Li , He Xiangjiu , Anticancer activities of harmine by inducing a pro-death autophagy and apoptosis in human gastric cancer cells, Phytomedicine (2017), doi: 10.1016/j.phymed.2017.02.008
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Anticancer activities of harmine by inducing a pro-death autophagy and apoptosis in human gastric cancer cells
Chuan Li
a, b
, Yihai Wang
a, b
, Chunhua Wang
a, b
, Xiaomin Yi
a, b
, Mingya Li a,*
a
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[email protected], Xiangjiu He a, b,* [email protected]
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006,
b
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China
Guangdong Engineering Research Center for Lead Compounds & Drug Discovery,
*
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Guangzhou 510006, China
Corresponding authors at:School of Pharmacy, Guangdong Pharmaceutical
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University, Guangzhou 510006, China.
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Graphical abstract
ABSTRACT 1
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Background: Harmine, a β-carboline alkaloid from Peganum harmala, has multiple anti-tumor activities, especially for its folk therapy for digestive system neoplasm. However, the underlying mechanism of harmine on gastric cancer remains unclear. Purpose: To illuminate the potential anti-tumor activity and mechanism of harmine
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against gastric cancer cells. Methods/Study designs: The anti-proliferative activity of harmine in vitro was evaluated by MTT assay. The autophagic activity induced by harmine was assessed
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using GFP-LC3 transfection. FITC/PI double staining was applied for the apoptosis inspection. The mitochondrial membrane potential was detected by JC-1 fluorescence probe. The potential mechanisms for proteins level in autophagy and apoptosis were
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analyzed by Western blot.
Results: Harmine exhibited potent effects on both autophagy and apoptosis. Treatment
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with harmine could enhance dots of GFP-LC3 in cells. Meanwhile, the process had
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connection with Beclin-1, LC3-II, and p62 by the inhibition of Akt/mTOR/p70S6K signaling. However, high concentration of harmine led to apoptosis characterized by
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the propidium/Annexin V-positive cell pollution, cell shrunk and the collapse of
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mitochondrial membrane potential. The regulation of Bcl-2, Bax and the gathering of cleaved-PARP, cleaved-caspase 3 and cleaved-caspase 9 contributed to the induction of apoptosis. In addition, 10 μM LY294002 (a specific inhibitor of PI3K/Akt) combination with 40 μM harmine significantly increased the cytotoxicity to the gastric cancer cells and up-regulated both the apoptosis-related protein (cleaved-PARP, cleaved-caspase-3) and autophagy-related protein (Beclin-1, LC3-II, and p62). 2
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Adding the inhibitor of autophagy, 3-MA or BafA1, increased the viability of harmine-exposured gastric cancer cells, which confirmed the role of autophagy played in the gastric cancer cell death induced by harmine. Conclusion: Harmine might be a potent inducer of apoptosis and autophagy, which
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offered evidences to therapy of harmine in gastric carcinoma in the folk medicine.
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Keywords: Harmine, Gastric cancer, Autophagy, Apoptosis, mTOR
Abbreviations: Akt, protein kinase B; AMPK, adenosine 5-monophosphate-activated protein kinase; BafA1, bafilomycin A1; FBS, fetal bovine serum; HM, harmine; LC3,
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the light chain 3; 3-MA, 3-methyladenine; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; mTOR,
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mammalian target of rapamycin; mTORC1, mTOR complex 1; mTORC2, mTOR
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complex 2; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; PARP,
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poly-ADP-ribose polymerase; cccp, carbonyl cyanide 3-chlorophenylhydrazone.
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Introduction
Harmine (HM, Fig.1) is a natural β-carboline alkaloid, isolated from the seeds of
Peganum harmala L. (Zygophyllaceae family). In China, P. harmala mainly distributes in Xinjiang, Inner Mongolia and other places in Northwest desert areas, and it has been used extensively for digestive system neoplasm in traditional Chinese medicine and Uygur medicine (Frost et al., 2011). Previous research showed that HM 3
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presented remarkable therapeutic effect on various diseases, which include apoplexia, asthma, jaundice and lumbago (Wang et al., 2011). Besides, HM possesses anti-tumor, anti-Alzheimer, anti-inflammatory, antimycotic and anti-viral activities regulated by several signaling pathways such as mitochondrial-mediated signaling pathways,
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PI3K/Akt pathway and kinase (Wang et al., 2015; Hamsa et al., 2015; Wang et al., 2015).
In the perspective of human cancer, autophagy (type II cell death) may be a
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potential method apart from apoptosis (the type I programmed cell death) for interventions in pharmacology. Autophagy is a crucial mechanism of cell death in the condition of cellular growth factor’s absence, lower nutrient and other analogous,
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which is highly conserved in mammalian, characterized the turnover of cellular with endolysosome, where autolysosome recycles breakdown contents (for instance,
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macromolecules and long-lived proteins) using the hydrolases and discarded
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organelles (Codogno et al., 2005; Kroemer et al., 2005; Levine et al., 2004). Autophagy is a stepwise of digesting its own intracellular contents depended on
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fusion. Intriguingly, the “self-digestion” manner plays not merely a protective role for
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cell survival but also a pro-apoptotic role for cell death in response to extrinsic stress (Hara et al., 2006). It is initiated with a biosynthesis for double-membrane structure regulated by autophagy related protein, such as Beclin-1, LC3. Several signaling pathways make crucial effects on autophagy process. mTOR (mammalian target of rapamycin) is an extremely important mediated factor in the regulation of autophagy. PI3K/Akt pathway plays crucial role in regulating cell cycle, cell growth, translation, 4
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apoptosis, autophagy, DNA repair. Studies showed that the mitogen-activated protein kinase (MAPK) signaling pathway and adenosine 5-monophosphate-activated protein kinase (AMPK) signaling pathway were significant for up-regulation of autophagy, apoptosis, cell growth and development. Autophagy prevented DNA damage caused
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by ROS (Bester et al., 2011). Autophagy also could maintain tumor development and survival by recycling nutrient during extrinsic damage and cancer.
Type I programmed cell death, apoptosis, is characterized as a condensation in
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nuclear chromatin, DNA fragmentation, cell shrinkage, apoptotic body formation proceeds in a high level as a response (Maiuri et al., 2007). The classical pathway involved in apoptosis could be subdivided into extrinsic way and intrinsic way. The extrinsic way refers to the death receptor-mediated way. Moreover, the
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mitochondrial-regulated apoptotic way is the intrinsic way. Both of the two ways’
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execution depends on the cascade reaction of caspases (Soldatenkov and Smulson,
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2000).
In recent years, reports have revealed the alkaloid from P. harmala, harmol and the
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derivatives of HM are a potent multi-target medicine, could intervene in both
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apoptosis and autophagy (Li et al., 2015). Therefore, the alkaloids from P. harmala have fair chance to exert an antitumor action in multiple tumor cells. The current study tried to clarify the antiproliferative action of HM in gastric cancer
cell (MGC-803 and SGC-7901), and elucidate the underlying molecular mechanisms. Our team innovated searching in the present study for autophagy (Akt/mTOR/p70S6K signaling pathway and AMPK pathway), apoptotic (Akt/mTOR/p70S6K pathway and 5
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mitochondrial-mediated signaling pathway) traits in HM-exposed gastric cancer cells.
Materials and methods Experimental materials
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MGC-803 and SGC-7901 cells were acquired from KeyGen Biotech (Nanjing, China). RPMI 1640 medium, fetal bovine serum (FBS), and Lipofectamine 2000 were products of Thermo Scientific (Waltham, MA USA). 3-MA, BafA1 and protease
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inhibitor cocktail were obtained from Sigma-Aldrich (St. Louis, MO, USA). LY294002 was purchased from Selleck (Houston, USA). LC3-II, Beclin-1, p62 (SQSTM1), mTOR, p-mTOR, Akt, p-Akt, p70S6K, p-p70S6K, caspase 3, caspase 9, PARP-1, Bax, Bcl-2 primary antibody were obtained from Cell Signaling Technology
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(Danvers, MA, USA). β-Actin was obtained from Proteintech Group, Inc. (Chicago,
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IL, USA). FITC-Annexin V apoptosis detection kit I was product of BD biosciences
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(Franklin Lakes, NJ, USA). RIPA cell lysis buffer and mitochondrial membrane potential assay kit with JC-1 were obtained from Beyotime biotechnology (Shanghai,
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China). Hyperfilm ECL was product of GE Healthcare Life Sciences (Uppsala,
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Sweden). Harmine was isolated and purified from the seeds of P. harmala in our lab. The purity is above 98% by HPLC analysis and its structure was identified through MS, IR and NMR spectra.
Cell culture and HM treatment Human gastric cancer cell lines (MGC-803 and SGC-7901) were cultured in 6
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RPMI-1640 medium with 10% FBS and 1% 100 U/ml penicillin, 100 mg/ml streptomycin sulfate at 37 C in humidified atmosphere containing 5% CO2 and 95% air. HM was dissolved in dimethyl sulfoxide (DMSO) to be the stock solution (20 mM) and further diluted to final concentrations with culture medium. For the time-dosage
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experiments, the cells were treated with HM of 10, 20, 40, 80 μM for 24, 48, and 72 h.
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Cell viability (MTT assay)
The cell viability was evaluated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide (MTT) assay. The cells were seeded in 96 well-plates with the density of 5 × 104 cells/ml at 37 C in the incubator. Incubated for 12 hours, the
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cells were treated with HM of 10, 20, 40, 80 μM for 24, 48 and 72 h. For the
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inhibition experiments, cells were treated with 2.5 mM 3-MA, 100 nM BafA1 or 10
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μM LY294002 for 1 h before HM added. Thereafter, 5 mg/ml MTT solution was added to each well for 4 h in the incubator. Subsequently, 150 μl DMSO dissolves the
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formazan crystal. The absorbance was determined at 492 nm with a microplate reader.
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Cell viability was presented based on proportion of the untreated cells.
Western blot analysis After treated with HM, cells were extracted with Radio Immunoprecipitation Assay containing 1% (v/v) protease inhibitor cocktail (Sigma, USA), and the concentration of the protein was measured using a BCA protein assay kit. Subsequently, the equal 7
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proteins (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide Gel electrophoresis (Bio-Rad, Hercules, CA, USA) and electrotransferred to a polyvinylidene fluoride membrane (0.45 μm). After the transferring, the nonspecific binding blots were blocked in 5% no-fat milk dissolved in TBST including 0.1% v/v
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Tween-20 for 1 h at room temperature with a gently shaking. Then, the specific primary antibodies were incubated for overnight at 4 C followed by incubation for 1 h with the respective second antibody at room temperature. The protein signals were
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visualized using ECL chemiluminescence detection reagents. β-Actin was performed as a loading control.
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Plasmid transfection
The GFP-LC3 plasmid was transfected to the MGC-803 and SGC-7901 cells with
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Lipofectamine 2000 according to the manufacture’s protocol. Briefly, 1 × 106
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cells/well were seeded onto 6 cm dish for overnight at 37 C, and transfected with the GFP-LC3 plasmid in no-serum medium for 4 h. The treated cells were visualized with
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an inverted fluorescence microscope.
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Apoptosis assay
Cell apoptosis was determined using FITC-Annexin V apoptosis detection kit I.
Cells were trypsinized at 37 C, 1 × 105 cells were labeled with the fluorochrome (5 μl Annexin V and 5 μl propidium iodide) and the cells were gently vortexed, then incubated with the reagent for 15 minutes at room temperature in the dark. At the end of incubation, the samples were analyzed by a fluorescence-activated cell sorter 8
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within 1 h.
Mitochondrial membrane potential(△Ψm) detection Measurement of the mitochondrial membrane potential (MMP) with JC-1 serves as
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a specific detection of mitochondrial function. JC-1 presents a fluorescent probe with an extremely sensitivity to the change in mitochondrial membrane potential. Once the MMP collapse, JC-1 emerges from red fluorescence to green fluorescence. Thereafter,
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the ratio of the red/green fluorescence suggests the collapse of MMP. The gastric cancer cells were seeded in 6-well plates for HM-treatment. After the 24h treatment, the supernatant was discarded and 300 μl of 5 μg/ml JC-1 was added to each well for
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20 min in the incubator. Cccp (10 µM) was added for the cells to be a positive control, which significant changes the mitochondrial membrane potential. After the incubation,
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JC-1 buffer solution washed each well for 300 μl/ml for twice. The treated cells were
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visualized with an inverted fluorescence microscope.
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Statistical analysis
All values were presented as means ± SD. One-way ANOVA was used to evaluate
the data statistic differences using Prism 5 software for P-value less than 0.05 was expected to denote statistical significance. At least two independent tests were performed for all assay condition.
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Results HM exhibited anti-proliferative activity against MGC-803 and SGC-7901 cells To evaluate the anti-proliferative activity of HM against human gastric carcinoma cells, MGC-803 and SGC-7901 cells were exposed to different concentration of HM
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for 24, 48, and 72 h, respectively. As shown in Table 1 and Fig. 2, the IC50 value against MGC-803 cell proliferation ranged from 7.50 to 58.84 μM, and it ranged from 1.77 to 40.94 μM for the SGC-7901 cell. It demonstrated that the cytotoxicity induced
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by HM was in a dose- and time-dependent manner.
HM induced autophagy in the gastric carcinoma cells
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To ascertain the HM-activated autophagy in MGC-803 and SGC-7901 cells, the gastric cancer cells were treated with HM. As shown in Fig. 3A, 3B, the formation of
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autophagosome was determined by the GFP-LC3 puncta localization after treated
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with HM for 12 h. The results indicated that autophagy presented a rising trend. When autophagy was induced, the formation of autophagosome was triggered with
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membrane of autophagosome elongation. LC3-I, the soluble type of autophagy,
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involved in the elongation via lipid conjugation to converse to the LC3-II, the vesicle-affined form, which is generally supposed to a specific marker protein of autophagy. The transfection experiment showed an increase in the GFP-LC3 dots in the cytoplasm. Furthermore, the expression of LC3-II (microtubule-associated protein 1 light chain 3), was dramatically accumulated in a dosage- and time- manners according to the Western blot analysis (Fig. 3C, 3D). Concordant with the increase of 10
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the LC3 lipidation, treated with HM also increased the level of Beclin-1, an indispensable protein for the formation of autophagosome membrane in the two gastric cancer cells. These results supplied evidence that HM had effect on autophagy
HM induced apoptosis in MGC-803 and SGC-7901 cells
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in MGC-803 and SGC-7901 cells.
MGC-803 and SGC-7901 cells were exposed to HM (10, 20, 40, 80 μM) for
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respective time, in addition to the occurrence of autophagy, the morphologic change of apoptotic features were also observed, such as cellular rounded, cell shrinkage, apoptosis bodies as visualized using an optical inverted microscope (data was not
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shown). To further ascertain whether the apoptosis cell death contributed to the cytotoxicity on the gastric cancer cell, flow cytometry was performed to detect the
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apoptosis manner with Annexin V and PI staining. According to the results (Fig. 4A),
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the apoptosis ratio increased from 7.55 % to 54.36 % in a time-dependent manner by the treatment of HM (60 μM) in the MGC-803 cells. Similarly, the apoptosis ratio
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increased from 3.63 % to 52.90% in the SGC-7901 cells (Fig. 4B). Meanwhile, HM
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was significantly increased apoptotic-associated protein (caspase families) and decreased apoptosis-suppressed protein (Bcl-2) in MGC-803 cells. As shown in Fig. 5A, cleaved-PARP was evoked when HM (40 μM) treated MGC-803 and SGC-7901 cells for 24 h. Prolonging the treatment to 48 h, PARP cleavage slightly decreased （Fig. 5B）. The changes of the mitochondrial membrane potential (△Ψm) was detected by JC-1. As shown in the Fig. 5C and 5D, non-treatment MGC-803 and 11
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SGC-7901 cells emitted strong red fluorescence. In the high dose group (80 μM HM), cells emitted a green fluorescence strongly same as the cccp-treatment group reflecting low mitochondrial membrane potential. The red fluorescence turned to green, which showed that HM could decrease the mitochondrial membrane potential.
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The results suggested that prolonging treatment with HM might induce apoptosis cell death in MGC-803 and SGC-7901 cells via mitochondrial pathway.
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The PI3K/Akt/mTOR signaling pathway involved in apoptosis as well as autophagy and AMPK signaling pathway modulated autophagy in MGC-803 and SGC-7901 cells
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Considerable evidences showed several signaling pathways have involved in autophagy, for instance, phosphatidylinositol 3-kinase/protein kinase B/mammalian
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target of rapamycin (PI3K/Akt/mTOR) and adenosine 5-monophosphate-activated
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protein kinase (AMPK) signaling pathways. In particular, mTOR plays a central role in regulating autophagy, apoptosis, metabolism and resistance in tumor cells. mTOR
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is a critical regulated protein in protein synthesis and belongs to the PI3K family.
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PI3K-related kinase has a close connection to mTOR and has a close association with the formation of autophagosome membrane (Gozuacik and Kimchi, 2004). mTOR can be divided in two protein complexes via interacting with certain proteins, named mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) (Guertin and Sabatini, 2007). Protein synthesis effects achieved by the mTORC1, which could phosphorylate 4E-BP1 (the eukaryotic initiated factor 4E-binding protein 1) and 12
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p70S6K (p70 ribosomal S6 kinase 1) to expedite mRNA translation. Studies have shown the mTOR pathway contributes to autophagy and plays a negative effect on autophagy (Codogno and Meijer, 2005). In consequence, to further illuminate the mechanism
of
the
autophagy
induced
by
HM,
we
evaluated
whether
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Akt/mTOR/p70S6K signaling involves in the autophagy. As shown in Fig. 6A and 6B, compared with non-treated group, treatment with HM (10, 20, 40, 80 μM) for 24 h exerted a decrease of the p-Akt/Akt and p-mTOR/mTOR expression. Similarly, the
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downstream protein of mTOR and p-p70S6K/p70S6K also performed a negative effect either in MGC-803 or in SGC-7901 cells. To determine the effect of Akt/mTOR/p70S6K pathway on apoptosis and autophagy, LY294002, a PI3K/Akt
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inhibitor was exposured with HM for 24 h, the expression of autophagy related protein (LC3-II, Beclin-1 and p62) was markedly increased compared to the control
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group (Fig. 6C). Beyond that, as shown in Fig.6D, the addition of LY294002 notably
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increased the expression of cleaved-caspase-3 and the cleaved-PARP. These data indicated that HM was capable of inhibiting Akt/mTOR/p70S6K pathway, thereby
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induced apoptosis and autophagy simultaneously.
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In addition, AMPK regulates cell metabolism, energy equilibrium, and cell development via multiple channels. Of which, AMPK involves in autophagy by inhibiting activity of mTOR and directly phosphorylating ULK1 (Lee et al., 2010; Alers et al., 2012). Therefore, the effect of HM on the expression of p-AMPK α was further tested by Western blot. The expression of phosphorylation form of AMPK α exhibited in a dosage- and time-independent manner (Fig. 7A and 7B). Consequently, 13
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it suggested that HM inhibited the PI3K/Akt/mTOR signaling pathway and AMPK pathway to strengthen autophagy.
Autophagy involves in the HM-induced cell death in MGC-803 and SGC-7901 cells
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To clarify whether the occurrence of autophagy affected the HM-induced apoptosis, cell viability assay was used to examine the effect of the blocking of autophagy via pharmacological method on the cells exposed to HM. The 3-MA, served as a specific
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inhibitor for autophagy, which could significantly inhibit class III PI3K (Vps34)/Beclin-1 complex, interfering in the formation of autophagosome, was applied to treat with HM (40 μM) in the concentration of 2.5 mM. Pretreated for 1 h
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to inhibit the formation of LC3-II, 3-MA succeeded in preventing the expression of LC3-II (Fig. 8A and 8B). Beclin-1 presented a significant decrease with the 3-MA
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addition. Moreover, the autophagy substrate, p62, was increased as expected in both
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MGC-803 and SGC-7901 cells. Herein, in the MTT assay, the addition of 3-MA could notably suppressed harmine-induced cytotoxicity in the two gastric cancer cells.
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In order to effectively explain the impact of the autophagy blockage on the cell
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viability, we evaluated the survival cells via MTT assay with another autophagy inhibitor, BafA1 (inhibiting the degradation of LC3-II). MTT results (Fig.8C and 8D) was similar to the pre-treatment with 3-MA. It indicated that the survival cells increased via co-treatment with BafA1. To clarify the effect of 3-MA on apoptosis further, cleaved-caspase 3 and PARP cleavage expression was detected and presented a mild decline comparison to HM-treatment. It reminded that the autophagy for 40 14
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μM HM treatment gave assistance to the apoptosis to suppress the growth of gastric cancer cells (Fig. 8E and 8F). As a whole, the block of autophagy, the addition of 3-MA significantly heightened the viability of both MGC-803 and SGC-7901 indicated autophagy might play an
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analogous role as apoptosis.
Discussion
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HM is a natural alkaloid isolated from the seeds of P. harmala, which served as the dominant ingredient for the folk anticancer therapy. Studies had shown that HM and its several derivatives exhibited effects on the apoptotic cell death via increasing p21,
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diminishing ERK/Bad pathway, and activating the Caspase-8/Bid cleavage. Simultaneously, conspicuous G2 phase arrest was observed in human gastric cancer
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cell (MGC-803) and human hepatoma cell (SMMC-7721) (Li et al., 2015; Zhang et al.
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2016). Harmol, another critical -carboline alkaloid from P. harmala, was reported to induce not merely Akt pathway involved autophagy but also apoptosis in human
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glioma cell (U251MG) by downregulating the survivin (Abe and Kokuba, 2013). In
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the present study, HM-induced gastric cancer cell death was remarkable, and the cytotoxicity was in a time- and dosage- manner (Fig. 2). The present research preliminarily clarified the cell death caused by HM might due to apoptosis via mitochondrial damage and the inhibition of Akt/mTOR pathway. Meanwhile, the inhibition of Akt/mTOR pathway simultaneously triggered autophagic cell death. In addition, AMPK signaling also involved in regulating the autophagy process. 15
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There are three key ways contributed to the cell death: apoptosis (Type I programmed cell death), autophagy (Type II programmed cell death) and necrosis. Therefore, we have investigated the autophagy induced by HM using the GFP-LC3 plasmid (the biomarker protein) and Western blot for autophagy-related proteins (Fig.
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3). The physical level of autophagy plays a crucial role in maintaining normal cells’ steady state. Once excessive autophagy or interference in the process of autophagy appearing, it is harmful to cells. Multiple signals contribute to the autophagy. Recent
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studies have shown down-regulation of the mTOR pathway is an inducement to autophagy. Several studies showed that Akt/mTOR pathway played a negative role in regulating autophagy. Inhibiting the phosphorylation of Akt and its downstream
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signaling protein, mTOR, contributed to the initiation of autophagy (Rubinsztein et al., 2007; Iwamaru et al., 2007; Kim et al., 2006; Easton and Houghton, 2006). In
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MGC-803 cells and SGC-7901 cells, HM exposure inhibited the phosphorylating of
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Akt and downstream protein, mTOR (Fig. 6). Moreover, the downstream of mTOR, p70S6K, was detected to be an analogous suppression of the phosphorylation of
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mTOR (Fig. 6). Generally, a triple inhibition of mTORC1 affected by AMPK, which
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is capable of phosphorylating TSC2 at the site of Thr-1227 and Ser-1345. Additionally, AMPK increased the inhibition effect of the TSC1/2 complex on mTORC1 (Inoki et al., 2003). mTOR could be directly inactivated by AMPK via the phosphorylation of the Thr-2446 site, resulting in an inhibition of mTOR (Cheng et al., 2004). AMPK could also phosphorylate mTOR-binding partner Raptor, resulting in the inhibition of mTORC1 (Gwinn et al., 2008). Therefore, AMPK, serving as a critical regulated 16
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protein to mTOR, was detected to proceeding Western blot, presenting an escalating trend with prolonging the HM treatment (Fig. 7). Thus, a crucial AMPK pathway may involve in the autophagy induced by HM, contributing to AMPK activation. Moreover, the occurrence of autophagy with HM-prolonging was lethal for
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MGC-803 and SGC-7901cells, for the melioration of cytotoxicity when autophagy was block by 3-MA or BafA1. In accordance with reported studies on tumor cells (Yang et al., 2011), the results demonstrated that MGC-803 and SGC-7901 cells had
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sensitivity to autophagy retardant for the accumulation of LC3-II dramatically receding via co-treatment with HM and 3-MA (Fig. 8), suggesting that HM induced autophagosome gathering via the increase of autophagic flux. What is more, the
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3-MA addition dramatically declined the expression of Beclin-1 and sequestosome 1, commonly known as p62/SQSTM1 (an ubiquitin-combination scaffold protein),
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combining to LC3 using a designated sequence and serving as a specific substrate for
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autophagic flux via linking ubiquitin-like proteins to the autophagic-related proteins to accomplish degradation in the lysosome (Pankiv et al., 2007). These results
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confirmed the inhibition effect on autophagy of 3-MA, whether alone or in
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combination with HM. The addition of the low-toxic inhibitor, 3-MA or BafA1 ascertained that autophagy involved in the mechanism of HM-induced cell death. Apoptosis is another vital pathway regulating the cell death. In present study, we
also observed and researched the apoptosis in the gastric cancer cells with the HM treatment. PARP cleavage can assist both the DNA repair enzymes and transcription factors to chromatin to undergo apoptosis. Present study has examined the 17
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apoptotic-related protein expression in HM-treated gastric tumor cells. It showed an increase in PARP1 cleavage and an increase ratio of Bax/Bcl-2. The cleavage of caspase 3 and caspase 9 was accumulated in the course. To figure out the relationship between apoptosis and autophagy, PARP and caspase-3 level were examined for HM
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co-treat with 3-MA. The data showed apoptotic-related protein mildly decline when the 3-MA added. Autophagy in the gastric cancer cell could cooperate with apoptosis. In spite of the crucial role in cellular homeostasis, Autophagy behaves as a
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double-edged sword. Several apoptotic-related signal pathways induce autophagy. Autophagy is suppressed by several anti-apoptotic signals, such as the PI3K/Akt pathway. Thus, similar inducing factor can trigger apoptosis or autophagy or both.
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Finding the overlapping regulatory molecules for the two type of cell death is of avail to this issue.
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In summary, it is worth that a novel mechanism of HM-induced gastric cancer cells
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death has been disclosed, i.e. the inhibition of Akt/mTOR/p70S6K and the activation of AMPK pathway and mitochondrial pathway. Enhancing the autophagy induced by
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rapamycin may be an analogous factor to apoptosis, indicating HM co-treatment with
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inducer of autophagy may be a latent strategy to enhance the chemical drug sensitivity during the chemotherapy of gastric cancer patient. Although we cannot conclude the accurate molecular mechanisms of the interassociation between the two types of cell death induced by HM. More in-depth investigation involves in the two major pathway participating in the cell death in HM-exposured gastric cancer cells remains to be advanced. 18
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Conflict of interest None.
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Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. U1203103), the project of Guangdong provincial department of science
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and technology (No. 2015A020211027) and the project of Department of education of
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Guangdong province (No. 2014KZDXM054).
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human leukemia cell lines from the toxic plant Peganum harmala, Toxins 7,
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Wang Z.T., Wang C.H., Zhang Z.J., Cheng X.M., Zhang L., Zheng X.Y., 2011. Identification of acetylcholinesterase inhibitors from seeds of plants of genus Peganum by thin-layer chromatography-bioautography, JPC-J. Planar Chromat. 24, 470-474.
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Yang S., Wang X., Contino G., Liesa M., Sahin E., Ying H., et al., 2011. Pancreatic cancers require autophagy for tumor growth, Genes Dev. 25, 717-729.
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Zhang P., Huang C.R., Wang W., Zhang X.K., Chen J.J., Wang J.J., et al. 2016. Harmine hydrochloride triggers G2 phase arrest and apoptosis in MGC-803 cells
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and SMMC-7721 cells by upregulating p21, activating caspase-8/Bid, and
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downregulating ERK/Bad pathway, Phytother. Res. 30, 31-40.
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Table 1. The IC50 value of HM against the proliferation of human gastric carcinoma cells.
IC50 (μM) 24 h
48 h
72 h
MGC-803
58.84±11.34
19.97±3.98
7.50±1.64
SGC-7901
40.94±5.65
12.29±2.49
1.77±0.55
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Fig. 1．Structure of harmine (HM).
Fig. 2. Antiproliferative activity of the MGC-803 and SGC-7901 cells induced by
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HM.
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Cells were exposed to different concentrations of HM for 24, 48, and 72 h. The cell
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viability was measured by MTT assay. The data were presented as mean ± SD.
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Fig. 3．HM induced autophagy in the gastric carcinoma cells. MGC-803 (3A) and SGC-7901 cells (3B) were transfected with GFP-LC3 plasmid,
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then the cells were treated with 40 μM HM for 12 h. To determine the autophagic
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response, cells were inspected at 40× magnification for numbers of GFP-LC3 puncta. GFP-LC3 dots gathered in the presence of HM. MGC-803 (3C) and SGC-7901 cells (3D) were exposed to different concentrations (0, 10, 20, 40, 80 μM) of HM for indicated time. The LC3-II level and the Beclin-1 was examined by immunoblotting. Statistical analysis of autophagy related-proteins (Beclin-1, LC3-II) were analyzed as histogram. Data were presented as the means ± SD of three independent tests. * p < 24
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0.05, ** p < 0.01, *** p < 0.001 vs. control. β-Actin was used as an internal standard.
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B
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Fig. 4. HM increased the percentage of apoptosis cells in MGC-803 and SGC-7901 cells.
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MGC-803 (4A) and SGC-7901 cells (4B) were incubated with 60 μM HM for 0, 6,
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12, 24, 36, and 48 h, then subjected to apoptotic analysis by AnnexinV-FITC/PI
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staining combined with flow cytometry.
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Fig. 5. HM mediated the apoptosis via mitochondrial signals in MGC-803 and
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SGC-7901 cells.
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MGC-803 and SGC-7901 cells were exposure to HM (0, 10, 20, 40, 80 μM) for 24 h. (5A) The two gastric cancer cells were treated with HM (0, 10, 20, 40, 80 μM) for 24 h, subjected to analysis of the expression of caspase 3, caspase 9, Bcl-2, Bax and PARP by Western blot analysis. (5B) 40 μM HM treated to MGC-803 and SGC-7901cells for 0, 6, 12, 24, 36, and 48 h, then subjected to analysis of the expression of caspase 3, caspase 9, Bcl-2, Bax and PARP by western blotting. β-Actin 27
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was used as an internal standard. Mitochondrial membrane potential(△Ψm) detection with JC-1. MGC-803cells (5C) and SGC-7901 cells (5D) were exposure to HM (40,
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60, 80 μM）and cccp (positive group).
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Fig. 6. The PI3K/Akt/mTOR Signaling Pathway involves in apoptosis as well as autophagy in MGC-803 and SGC-7901 cells
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(6A) MGC-803 and SGC-7901 cells were treated with HM (0, 10, 20, 40, 80 μM)
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for 24 h before subjected to analysis of the expression of the phosphorylated levels of Akt, mTOR and p70S6K by Western blot analysis. (6B) 40 μM HM treated to
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MGC-803 and SGC-7901cells for 0, 6, 12, 24, 36, and 48 h, then the expression of the phosphorylated levels of Akt, mTOR and p70S6K were determined by Western blot analysis. (6C) and (6D) cells were exposure with 10 μM LY294002 (PI3K/Akt inhibitor) in the absence or presence of 40 μM HM for 24 h. The expression of autophagy marker (LC3-II, Beclin-1 and p62) and apoptosis related protein (PARP and caspase-3) was detected by western blot. β-Actin was used as an internal 28
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standard.
Fig. 7. AMPK signaling pathway involved in the autophagy induced by HM. (7A) MGC-803 and SGC-7901 cells were treated with HM (0, 10, 20, 40, 80 μM)
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for 24 h before subjected to analysis of the expression of the phosphorylated levels of
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AMPK α by Western blot analysis. (7B) 40 μM HM treated to MGC-803 and SGC-7901 cells for 0, 6, 12, 24, 36, and 48 h, then the expression of the
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phosphorylated level of AMPK α was determined by Western blot analysis. β-Actin
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was used as an internal standard.
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Fig. 8. HM-mediated autophagy involved in the HM-induced gastric cancer cell death MGC-803 (8A) and SGC-7901 cells (8B) were treated with an autophagy inhibitor
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3-MA (2.5 mM) for 1 h before incubation with 40 μM HM for 24 h, and subjected to
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analysis of the expression of the hallmark of autophagy: LC3-II, Beclin-1 and p62. MGC-803 (8C) and SGC-7901 (8D) cells viabilities were determined by MTT assay.
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(E) and (F) The expression of apoptosis related protein (PARP and caspase-3) was
detected by western blot for the addition of 2.5 mM 3-MA in the absence or presence of 40 μM HM for 24 h. The data were presented as the means ± SD of three independent tests. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.
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Anticancer activities of harmine by inducing a pro-death autophagy and apoptosis in human gastric cancer cells Chuan Li , Yihai Wang , Chunhua Wang , Xiaomin Yi , Mingya Li , He Xiangjiu PII: DOI: Reference:
S0944-7113(17)30039-9 10.1016/j.phymed.2017.02.008 PHYMED 52169
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
28 August 2016 10 January 2017 24 February 2017
Please cite this article as: Chuan Li , Yihai Wang , Chunhua Wang , Xiaomin Yi , Mingya Li , He Xiangjiu , Anticancer activities of harmine by inducing a pro-death autophagy and apoptosis in human gastric cancer cells, Phytomedicine (2017), doi: 10.1016/j.phymed.2017.02.008
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Anticancer activities of harmine by inducing a pro-death autophagy and apoptosis in human gastric cancer cells
Chuan Li
a, b
, Yihai Wang
a, b
, Chunhua Wang
a, b
, Xiaomin Yi
a, b
, Mingya Li a,*
a
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[email protected], Xiangjiu He a, b,* [email protected]
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006,
b
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China
Guangdong Engineering Research Center for Lead Compounds & Drug Discovery,
*
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Guangzhou 510006, China
Corresponding authors at:School of Pharmacy, Guangdong Pharmaceutical
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University, Guangzhou 510006, China.
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Graphical abstract
ABSTRACT 1
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Background: Harmine, a β-carboline alkaloid from Peganum harmala, has multiple anti-tumor activities, especially for its folk therapy for digestive system neoplasm. However, the underlying mechanism of harmine on gastric cancer remains unclear. Purpose: To illuminate the potential anti-tumor activity and mechanism of harmine
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against gastric cancer cells. Methods/Study designs: The anti-proliferative activity of harmine in vitro was evaluated by MTT assay. The autophagic activity induced by harmine was assessed
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using GFP-LC3 transfection. FITC/PI double staining was applied for the apoptosis inspection. The mitochondrial membrane potential was detected by JC-1 fluorescence probe. The potential mechanisms for proteins level in autophagy and apoptosis were
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analyzed by Western blot.
Results: Harmine exhibited potent effects on both autophagy and apoptosis. Treatment
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with harmine could enhance dots of GFP-LC3 in cells. Meanwhile, the process had
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connection with Beclin-1, LC3-II, and p62 by the inhibition of Akt/mTOR/p70S6K signaling. However, high concentration of harmine led to apoptosis characterized by
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the propidium/Annexin V-positive cell pollution, cell shrunk and the collapse of
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mitochondrial membrane potential. The regulation of Bcl-2, Bax and the gathering of cleaved-PARP, cleaved-caspase 3 and cleaved-caspase 9 contributed to the induction of apoptosis. In addition, 10 μM LY294002 (a specific inhibitor of PI3K/Akt) combination with 40 μM harmine significantly increased the cytotoxicity to the gastric cancer cells and up-regulated both the apoptosis-related protein (cleaved-PARP, cleaved-caspase-3) and autophagy-related protein (Beclin-1, LC3-II, and p62). 2
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Adding the inhibitor of autophagy, 3-MA or BafA1, increased the viability of harmine-exposured gastric cancer cells, which confirmed the role of autophagy played in the gastric cancer cell death induced by harmine. Conclusion: Harmine might be a potent inducer of apoptosis and autophagy, which
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offered evidences to therapy of harmine in gastric carcinoma in the folk medicine.
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Keywords: Harmine, Gastric cancer, Autophagy, Apoptosis, mTOR
Abbreviations: Akt, protein kinase B; AMPK, adenosine 5-monophosphate-activated protein kinase; BafA1, bafilomycin A1; FBS, fetal bovine serum; HM, harmine; LC3,
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the light chain 3; 3-MA, 3-methyladenine; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; mTOR,
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mammalian target of rapamycin; mTORC1, mTOR complex 1; mTORC2, mTOR
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complex 2; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; PARP,
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poly-ADP-ribose polymerase; cccp, carbonyl cyanide 3-chlorophenylhydrazone.
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Introduction
Harmine (HM, Fig.1) is a natural β-carboline alkaloid, isolated from the seeds of
Peganum harmala L. (Zygophyllaceae family). In China, P. harmala mainly distributes in Xinjiang, Inner Mongolia and other places in Northwest desert areas, and it has been used extensively for digestive system neoplasm in traditional Chinese medicine and Uygur medicine (Frost et al., 2011). Previous research showed that HM 3
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presented remarkable therapeutic effect on various diseases, which include apoplexia, asthma, jaundice and lumbago (Wang et al., 2011). Besides, HM possesses anti-tumor, anti-Alzheimer, anti-inflammatory, antimycotic and anti-viral activities regulated by several signaling pathways such as mitochondrial-mediated signaling pathways,
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PI3K/Akt pathway and kinase (Wang et al., 2015; Hamsa et al., 2015; Wang et al., 2015).
In the perspective of human cancer, autophagy (type II cell death) may be a
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potential method apart from apoptosis (the type I programmed cell death) for interventions in pharmacology. Autophagy is a crucial mechanism of cell death in the condition of cellular growth factor’s absence, lower nutrient and other analogous,
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which is highly conserved in mammalian, characterized the turnover of cellular with endolysosome, where autolysosome recycles breakdown contents (for instance,
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macromolecules and long-lived proteins) using the hydrolases and discarded
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organelles (Codogno et al., 2005; Kroemer et al., 2005; Levine et al., 2004). Autophagy is a stepwise of digesting its own intracellular contents depended on
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fusion. Intriguingly, the “self-digestion” manner plays not merely a protective role for
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cell survival but also a pro-apoptotic role for cell death in response to extrinsic stress (Hara et al., 2006). It is initiated with a biosynthesis for double-membrane structure regulated by autophagy related protein, such as Beclin-1, LC3. Several signaling pathways make crucial effects on autophagy process. mTOR (mammalian target of rapamycin) is an extremely important mediated factor in the regulation of autophagy. PI3K/Akt pathway plays crucial role in regulating cell cycle, cell growth, translation, 4
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apoptosis, autophagy, DNA repair. Studies showed that the mitogen-activated protein kinase (MAPK) signaling pathway and adenosine 5-monophosphate-activated protein kinase (AMPK) signaling pathway were significant for up-regulation of autophagy, apoptosis, cell growth and development. Autophagy prevented DNA damage caused
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by ROS (Bester et al., 2011). Autophagy also could maintain tumor development and survival by recycling nutrient during extrinsic damage and cancer.
Type I programmed cell death, apoptosis, is characterized as a condensation in
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nuclear chromatin, DNA fragmentation, cell shrinkage, apoptotic body formation proceeds in a high level as a response (Maiuri et al., 2007). The classical pathway involved in apoptosis could be subdivided into extrinsic way and intrinsic way. The extrinsic way refers to the death receptor-mediated way. Moreover, the
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mitochondrial-regulated apoptotic way is the intrinsic way. Both of the two ways’
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execution depends on the cascade reaction of caspases (Soldatenkov and Smulson,
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2000).
In recent years, reports have revealed the alkaloid from P. harmala, harmol and the
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derivatives of HM are a potent multi-target medicine, could intervene in both
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apoptosis and autophagy (Li et al., 2015). Therefore, the alkaloids from P. harmala have fair chance to exert an antitumor action in multiple tumor cells. The current study tried to clarify the antiproliferative action of HM in gastric cancer
cell (MGC-803 and SGC-7901), and elucidate the underlying molecular mechanisms. Our team innovated searching in the present study for autophagy (Akt/mTOR/p70S6K signaling pathway and AMPK pathway), apoptotic (Akt/mTOR/p70S6K pathway and 5
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mitochondrial-mediated signaling pathway) traits in HM-exposed gastric cancer cells.
Materials and methods Experimental materials
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MGC-803 and SGC-7901 cells were acquired from KeyGen Biotech (Nanjing, China). RPMI 1640 medium, fetal bovine serum (FBS), and Lipofectamine 2000 were products of Thermo Scientific (Waltham, MA USA). 3-MA, BafA1 and protease
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inhibitor cocktail were obtained from Sigma-Aldrich (St. Louis, MO, USA). LY294002 was purchased from Selleck (Houston, USA). LC3-II, Beclin-1, p62 (SQSTM1), mTOR, p-mTOR, Akt, p-Akt, p70S6K, p-p70S6K, caspase 3, caspase 9, PARP-1, Bax, Bcl-2 primary antibody were obtained from Cell Signaling Technology
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(Danvers, MA, USA). β-Actin was obtained from Proteintech Group, Inc. (Chicago,
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IL, USA). FITC-Annexin V apoptosis detection kit I was product of BD biosciences
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(Franklin Lakes, NJ, USA). RIPA cell lysis buffer and mitochondrial membrane potential assay kit with JC-1 were obtained from Beyotime biotechnology (Shanghai,
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China). Hyperfilm ECL was product of GE Healthcare Life Sciences (Uppsala,
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Sweden). Harmine was isolated and purified from the seeds of P. harmala in our lab. The purity is above 98% by HPLC analysis and its structure was identified through MS, IR and NMR spectra.
Cell culture and HM treatment Human gastric cancer cell lines (MGC-803 and SGC-7901) were cultured in 6
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RPMI-1640 medium with 10% FBS and 1% 100 U/ml penicillin, 100 mg/ml streptomycin sulfate at 37 C in humidified atmosphere containing 5% CO2 and 95% air. HM was dissolved in dimethyl sulfoxide (DMSO) to be the stock solution (20 mM) and further diluted to final concentrations with culture medium. For the time-dosage
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experiments, the cells were treated with HM of 10, 20, 40, 80 μM for 24, 48, and 72 h.
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Cell viability (MTT assay)
The cell viability was evaluated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide (MTT) assay. The cells were seeded in 96 well-plates with the density of 5 × 104 cells/ml at 37 C in the incubator. Incubated for 12 hours, the
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cells were treated with HM of 10, 20, 40, 80 μM for 24, 48 and 72 h. For the
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inhibition experiments, cells were treated with 2.5 mM 3-MA, 100 nM BafA1 or 10
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μM LY294002 for 1 h before HM added. Thereafter, 5 mg/ml MTT solution was added to each well for 4 h in the incubator. Subsequently, 150 μl DMSO dissolves the
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formazan crystal. The absorbance was determined at 492 nm with a microplate reader.
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Cell viability was presented based on proportion of the untreated cells.
Western blot analysis After treated with HM, cells were extracted with Radio Immunoprecipitation Assay containing 1% (v/v) protease inhibitor cocktail (Sigma, USA), and the concentration of the protein was measured using a BCA protein assay kit. Subsequently, the equal 7
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proteins (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide Gel electrophoresis (Bio-Rad, Hercules, CA, USA) and electrotransferred to a polyvinylidene fluoride membrane (0.45 μm). After the transferring, the nonspecific binding blots were blocked in 5% no-fat milk dissolved in TBST including 0.1% v/v
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Tween-20 for 1 h at room temperature with a gently shaking. Then, the specific primary antibodies were incubated for overnight at 4 C followed by incubation for 1 h with the respective second antibody at room temperature. The protein signals were
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visualized using ECL chemiluminescence detection reagents. β-Actin was performed as a loading control.
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Plasmid transfection
The GFP-LC3 plasmid was transfected to the MGC-803 and SGC-7901 cells with
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Lipofectamine 2000 according to the manufacture’s protocol. Briefly, 1 × 106
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cells/well were seeded onto 6 cm dish for overnight at 37 C, and transfected with the GFP-LC3 plasmid in no-serum medium for 4 h. The treated cells were visualized with
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an inverted fluorescence microscope.
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Apoptosis assay
Cell apoptosis was determined using FITC-Annexin V apoptosis detection kit I.
Cells were trypsinized at 37 C, 1 × 105 cells were labeled with the fluorochrome (5 μl Annexin V and 5 μl propidium iodide) and the cells were gently vortexed, then incubated with the reagent for 15 minutes at room temperature in the dark. At the end of incubation, the samples were analyzed by a fluorescence-activated cell sorter 8
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within 1 h.
Mitochondrial membrane potential(△Ψm) detection Measurement of the mitochondrial membrane potential (MMP) with JC-1 serves as
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a specific detection of mitochondrial function. JC-1 presents a fluorescent probe with an extremely sensitivity to the change in mitochondrial membrane potential. Once the MMP collapse, JC-1 emerges from red fluorescence to green fluorescence. Thereafter,
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the ratio of the red/green fluorescence suggests the collapse of MMP. The gastric cancer cells were seeded in 6-well plates for HM-treatment. After the 24h treatment, the supernatant was discarded and 300 μl of 5 μg/ml JC-1 was added to each well for
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20 min in the incubator. Cccp (10 µM) was added for the cells to be a positive control, which significant changes the mitochondrial membrane potential. After the incubation,
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JC-1 buffer solution washed each well for 300 μl/ml for twice. The treated cells were
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visualized with an inverted fluorescence microscope.
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Statistical analysis
All values were presented as means ± SD. One-way ANOVA was used to evaluate
the data statistic differences using Prism 5 software for P-value less than 0.05 was expected to denote statistical significance. At least two independent tests were performed for all assay condition.
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Results HM exhibited anti-proliferative activity against MGC-803 and SGC-7901 cells To evaluate the anti-proliferative activity of HM against human gastric carcinoma cells, MGC-803 and SGC-7901 cells were exposed to different concentration of HM
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for 24, 48, and 72 h, respectively. As shown in Table 1 and Fig. 2, the IC50 value against MGC-803 cell proliferation ranged from 7.50 to 58.84 μM, and it ranged from 1.77 to 40.94 μM for the SGC-7901 cell. It demonstrated that the cytotoxicity induced
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by HM was in a dose- and time-dependent manner.
HM induced autophagy in the gastric carcinoma cells
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To ascertain the HM-activated autophagy in MGC-803 and SGC-7901 cells, the gastric cancer cells were treated with HM. As shown in Fig. 3A, 3B, the formation of
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autophagosome was determined by the GFP-LC3 puncta localization after treated
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with HM for 12 h. The results indicated that autophagy presented a rising trend. When autophagy was induced, the formation of autophagosome was triggered with
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membrane of autophagosome elongation. LC3-I, the soluble type of autophagy,
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involved in the elongation via lipid conjugation to converse to the LC3-II, the vesicle-affined form, which is generally supposed to a specific marker protein of autophagy. The transfection experiment showed an increase in the GFP-LC3 dots in the cytoplasm. Furthermore, the expression of LC3-II (microtubule-associated protein 1 light chain 3), was dramatically accumulated in a dosage- and time- manners according to the Western blot analysis (Fig. 3C, 3D). Concordant with the increase of 10
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the LC3 lipidation, treated with HM also increased the level of Beclin-1, an indispensable protein for the formation of autophagosome membrane in the two gastric cancer cells. These results supplied evidence that HM had effect on autophagy
HM induced apoptosis in MGC-803 and SGC-7901 cells
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in MGC-803 and SGC-7901 cells.
MGC-803 and SGC-7901 cells were exposed to HM (10, 20, 40, 80 μM) for
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respective time, in addition to the occurrence of autophagy, the morphologic change of apoptotic features were also observed, such as cellular rounded, cell shrinkage, apoptosis bodies as visualized using an optical inverted microscope (data was not
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shown). To further ascertain whether the apoptosis cell death contributed to the cytotoxicity on the gastric cancer cell, flow cytometry was performed to detect the
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apoptosis manner with Annexin V and PI staining. According to the results (Fig. 4A),
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the apoptosis ratio increased from 7.55 % to 54.36 % in a time-dependent manner by the treatment of HM (60 μM) in the MGC-803 cells. Similarly, the apoptosis ratio
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increased from 3.63 % to 52.90% in the SGC-7901 cells (Fig. 4B). Meanwhile, HM
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was significantly increased apoptotic-associated protein (caspase families) and decreased apoptosis-suppressed protein (Bcl-2) in MGC-803 cells. As shown in Fig. 5A, cleaved-PARP was evoked when HM (40 μM) treated MGC-803 and SGC-7901 cells for 24 h. Prolonging the treatment to 48 h, PARP cleavage slightly decreased （Fig. 5B）. The changes of the mitochondrial membrane potential (△Ψm) was detected by JC-1. As shown in the Fig. 5C and 5D, non-treatment MGC-803 and 11
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SGC-7901 cells emitted strong red fluorescence. In the high dose group (80 μM HM), cells emitted a green fluorescence strongly same as the cccp-treatment group reflecting low mitochondrial membrane potential. The red fluorescence turned to green, which showed that HM could decrease the mitochondrial membrane potential.
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The results suggested that prolonging treatment with HM might induce apoptosis cell death in MGC-803 and SGC-7901 cells via mitochondrial pathway.
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The PI3K/Akt/mTOR signaling pathway involved in apoptosis as well as autophagy and AMPK signaling pathway modulated autophagy in MGC-803 and SGC-7901 cells
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Considerable evidences showed several signaling pathways have involved in autophagy, for instance, phosphatidylinositol 3-kinase/protein kinase B/mammalian
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target of rapamycin (PI3K/Akt/mTOR) and adenosine 5-monophosphate-activated
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protein kinase (AMPK) signaling pathways. In particular, mTOR plays a central role in regulating autophagy, apoptosis, metabolism and resistance in tumor cells. mTOR
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is a critical regulated protein in protein synthesis and belongs to the PI3K family.
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PI3K-related kinase has a close connection to mTOR and has a close association with the formation of autophagosome membrane (Gozuacik and Kimchi, 2004). mTOR can be divided in two protein complexes via interacting with certain proteins, named mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) (Guertin and Sabatini, 2007). Protein synthesis effects achieved by the mTORC1, which could phosphorylate 4E-BP1 (the eukaryotic initiated factor 4E-binding protein 1) and 12
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p70S6K (p70 ribosomal S6 kinase 1) to expedite mRNA translation. Studies have shown the mTOR pathway contributes to autophagy and plays a negative effect on autophagy (Codogno and Meijer, 2005). In consequence, to further illuminate the mechanism
of
the
autophagy
induced
by
HM,
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evaluated
whether
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Akt/mTOR/p70S6K signaling involves in the autophagy. As shown in Fig. 6A and 6B, compared with non-treated group, treatment with HM (10, 20, 40, 80 μM) for 24 h exerted a decrease of the p-Akt/Akt and p-mTOR/mTOR expression. Similarly, the
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downstream protein of mTOR and p-p70S6K/p70S6K also performed a negative effect either in MGC-803 or in SGC-7901 cells. To determine the effect of Akt/mTOR/p70S6K pathway on apoptosis and autophagy, LY294002, a PI3K/Akt
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inhibitor was exposured with HM for 24 h, the expression of autophagy related protein (LC3-II, Beclin-1 and p62) was markedly increased compared to the control
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group (Fig. 6C). Beyond that, as shown in Fig.6D, the addition of LY294002 notably
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increased the expression of cleaved-caspase-3 and the cleaved-PARP. These data indicated that HM was capable of inhibiting Akt/mTOR/p70S6K pathway, thereby
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induced apoptosis and autophagy simultaneously.
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In addition, AMPK regulates cell metabolism, energy equilibrium, and cell development via multiple channels. Of which, AMPK involves in autophagy by inhibiting activity of mTOR and directly phosphorylating ULK1 (Lee et al., 2010; Alers et al., 2012). Therefore, the effect of HM on the expression of p-AMPK α was further tested by Western blot. The expression of phosphorylation form of AMPK α exhibited in a dosage- and time-independent manner (Fig. 7A and 7B). Consequently, 13
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it suggested that HM inhibited the PI3K/Akt/mTOR signaling pathway and AMPK pathway to strengthen autophagy.
Autophagy involves in the HM-induced cell death in MGC-803 and SGC-7901 cells
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To clarify whether the occurrence of autophagy affected the HM-induced apoptosis, cell viability assay was used to examine the effect of the blocking of autophagy via pharmacological method on the cells exposed to HM. The 3-MA, served as a specific
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inhibitor for autophagy, which could significantly inhibit class III PI3K (Vps34)/Beclin-1 complex, interfering in the formation of autophagosome, was applied to treat with HM (40 μM) in the concentration of 2.5 mM. Pretreated for 1 h
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to inhibit the formation of LC3-II, 3-MA succeeded in preventing the expression of LC3-II (Fig. 8A and 8B). Beclin-1 presented a significant decrease with the 3-MA
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addition. Moreover, the autophagy substrate, p62, was increased as expected in both
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MGC-803 and SGC-7901 cells. Herein, in the MTT assay, the addition of 3-MA could notably suppressed harmine-induced cytotoxicity in the two gastric cancer cells.
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In order to effectively explain the impact of the autophagy blockage on the cell
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viability, we evaluated the survival cells via MTT assay with another autophagy inhibitor, BafA1 (inhibiting the degradation of LC3-II). MTT results (Fig.8C and 8D) was similar to the pre-treatment with 3-MA. It indicated that the survival cells increased via co-treatment with BafA1. To clarify the effect of 3-MA on apoptosis further, cleaved-caspase 3 and PARP cleavage expression was detected and presented a mild decline comparison to HM-treatment. It reminded that the autophagy for 40 14
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μM HM treatment gave assistance to the apoptosis to suppress the growth of gastric cancer cells (Fig. 8E and 8F). As a whole, the block of autophagy, the addition of 3-MA significantly heightened the viability of both MGC-803 and SGC-7901 indicated autophagy might play an
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analogous role as apoptosis.
Discussion
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HM is a natural alkaloid isolated from the seeds of P. harmala, which served as the dominant ingredient for the folk anticancer therapy. Studies had shown that HM and its several derivatives exhibited effects on the apoptotic cell death via increasing p21,
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diminishing ERK/Bad pathway, and activating the Caspase-8/Bid cleavage. Simultaneously, conspicuous G2 phase arrest was observed in human gastric cancer
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cell (MGC-803) and human hepatoma cell (SMMC-7721) (Li et al., 2015; Zhang et al.
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2016). Harmol, another critical -carboline alkaloid from P. harmala, was reported to induce not merely Akt pathway involved autophagy but also apoptosis in human
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glioma cell (U251MG) by downregulating the survivin (Abe and Kokuba, 2013). In
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the present study, HM-induced gastric cancer cell death was remarkable, and the cytotoxicity was in a time- and dosage- manner (Fig. 2). The present research preliminarily clarified the cell death caused by HM might due to apoptosis via mitochondrial damage and the inhibition of Akt/mTOR pathway. Meanwhile, the inhibition of Akt/mTOR pathway simultaneously triggered autophagic cell death. In addition, AMPK signaling also involved in regulating the autophagy process. 15
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There are three key ways contributed to the cell death: apoptosis (Type I programmed cell death), autophagy (Type II programmed cell death) and necrosis. Therefore, we have investigated the autophagy induced by HM using the GFP-LC3 plasmid (the biomarker protein) and Western blot for autophagy-related proteins (Fig.
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3). The physical level of autophagy plays a crucial role in maintaining normal cells’ steady state. Once excessive autophagy or interference in the process of autophagy appearing, it is harmful to cells. Multiple signals contribute to the autophagy. Recent
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studies have shown down-regulation of the mTOR pathway is an inducement to autophagy. Several studies showed that Akt/mTOR pathway played a negative role in regulating autophagy. Inhibiting the phosphorylation of Akt and its downstream
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signaling protein, mTOR, contributed to the initiation of autophagy (Rubinsztein et al., 2007; Iwamaru et al., 2007; Kim et al., 2006; Easton and Houghton, 2006). In
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MGC-803 cells and SGC-7901 cells, HM exposure inhibited the phosphorylating of
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Akt and downstream protein, mTOR (Fig. 6). Moreover, the downstream of mTOR, p70S6K, was detected to be an analogous suppression of the phosphorylation of
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mTOR (Fig. 6). Generally, a triple inhibition of mTORC1 affected by AMPK, which
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is capable of phosphorylating TSC2 at the site of Thr-1227 and Ser-1345. Additionally, AMPK increased the inhibition effect of the TSC1/2 complex on mTORC1 (Inoki et al., 2003). mTOR could be directly inactivated by AMPK via the phosphorylation of the Thr-2446 site, resulting in an inhibition of mTOR (Cheng et al., 2004). AMPK could also phosphorylate mTOR-binding partner Raptor, resulting in the inhibition of mTORC1 (Gwinn et al., 2008). Therefore, AMPK, serving as a critical regulated 16
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protein to mTOR, was detected to proceeding Western blot, presenting an escalating trend with prolonging the HM treatment (Fig. 7). Thus, a crucial AMPK pathway may involve in the autophagy induced by HM, contributing to AMPK activation. Moreover, the occurrence of autophagy with HM-prolonging was lethal for
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MGC-803 and SGC-7901cells, for the melioration of cytotoxicity when autophagy was block by 3-MA or BafA1. In accordance with reported studies on tumor cells (Yang et al., 2011), the results demonstrated that MGC-803 and SGC-7901 cells had
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sensitivity to autophagy retardant for the accumulation of LC3-II dramatically receding via co-treatment with HM and 3-MA (Fig. 8), suggesting that HM induced autophagosome gathering via the increase of autophagic flux. What is more, the
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3-MA addition dramatically declined the expression of Beclin-1 and sequestosome 1, commonly known as p62/SQSTM1 (an ubiquitin-combination scaffold protein),
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combining to LC3 using a designated sequence and serving as a specific substrate for
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autophagic flux via linking ubiquitin-like proteins to the autophagic-related proteins to accomplish degradation in the lysosome (Pankiv et al., 2007). These results
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confirmed the inhibition effect on autophagy of 3-MA, whether alone or in
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combination with HM. The addition of the low-toxic inhibitor, 3-MA or BafA1 ascertained that autophagy involved in the mechanism of HM-induced cell death. Apoptosis is another vital pathway regulating the cell death. In present study, we
also observed and researched the apoptosis in the gastric cancer cells with the HM treatment. PARP cleavage can assist both the DNA repair enzymes and transcription factors to chromatin to undergo apoptosis. Present study has examined the 17
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apoptotic-related protein expression in HM-treated gastric tumor cells. It showed an increase in PARP1 cleavage and an increase ratio of Bax/Bcl-2. The cleavage of caspase 3 and caspase 9 was accumulated in the course. To figure out the relationship between apoptosis and autophagy, PARP and caspase-3 level were examined for HM
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co-treat with 3-MA. The data showed apoptotic-related protein mildly decline when the 3-MA added. Autophagy in the gastric cancer cell could cooperate with apoptosis. In spite of the crucial role in cellular homeostasis, Autophagy behaves as a
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double-edged sword. Several apoptotic-related signal pathways induce autophagy. Autophagy is suppressed by several anti-apoptotic signals, such as the PI3K/Akt pathway. Thus, similar inducing factor can trigger apoptosis or autophagy or both.
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Finding the overlapping regulatory molecules for the two type of cell death is of avail to this issue.
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In summary, it is worth that a novel mechanism of HM-induced gastric cancer cells
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death has been disclosed, i.e. the inhibition of Akt/mTOR/p70S6K and the activation of AMPK pathway and mitochondrial pathway. Enhancing the autophagy induced by
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rapamycin may be an analogous factor to apoptosis, indicating HM co-treatment with
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inducer of autophagy may be a latent strategy to enhance the chemical drug sensitivity during the chemotherapy of gastric cancer patient. Although we cannot conclude the accurate molecular mechanisms of the interassociation between the two types of cell death induced by HM. More in-depth investigation involves in the two major pathway participating in the cell death in HM-exposured gastric cancer cells remains to be advanced. 18
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Conflict of interest None.
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Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. U1203103), the project of Guangdong provincial department of science
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and technology (No. 2015A020211027) and the project of Department of education of
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Guangdong province (No. 2014KZDXM054).
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Table 1. The IC50 value of HM against the proliferation of human gastric carcinoma cells.
IC50 (μM) 24 h
48 h
72 h
MGC-803
58.84±11.34
19.97±3.98
7.50±1.64
SGC-7901
40.94±5.65
12.29±2.49
1.77±0.55
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Fig. 1．Structure of harmine (HM).
Fig. 2. Antiproliferative activity of the MGC-803 and SGC-7901 cells induced by
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HM.
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Cells were exposed to different concentrations of HM for 24, 48, and 72 h. The cell
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viability was measured by MTT assay. The data were presented as mean ± SD.
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Fig. 3．HM induced autophagy in the gastric carcinoma cells. MGC-803 (3A) and SGC-7901 cells (3B) were transfected with GFP-LC3 plasmid,
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then the cells were treated with 40 μM HM for 12 h. To determine the autophagic
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response, cells were inspected at 40× magnification for numbers of GFP-LC3 puncta. GFP-LC3 dots gathered in the presence of HM. MGC-803 (3C) and SGC-7901 cells (3D) were exposed to different concentrations (0, 10, 20, 40, 80 μM) of HM for indicated time. The LC3-II level and the Beclin-1 was examined by immunoblotting. Statistical analysis of autophagy related-proteins (Beclin-1, LC3-II) were analyzed as histogram. Data were presented as the means ± SD of three independent tests. * p < 24
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0.05, ** p < 0.01, *** p < 0.001 vs. control. β-Actin was used as an internal standard.
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B
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Fig. 4. HM increased the percentage of apoptosis cells in MGC-803 and SGC-7901 cells.
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MGC-803 (4A) and SGC-7901 cells (4B) were incubated with 60 μM HM for 0, 6,
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12, 24, 36, and 48 h, then subjected to apoptotic analysis by AnnexinV-FITC/PI
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staining combined with flow cytometry.
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Fig. 5. HM mediated the apoptosis via mitochondrial signals in MGC-803 and
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SGC-7901 cells.
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MGC-803 and SGC-7901 cells were exposure to HM (0, 10, 20, 40, 80 μM) for 24 h. (5A) The two gastric cancer cells were treated with HM (0, 10, 20, 40, 80 μM) for 24 h, subjected to analysis of the expression of caspase 3, caspase 9, Bcl-2, Bax and PARP by Western blot analysis. (5B) 40 μM HM treated to MGC-803 and SGC-7901cells for 0, 6, 12, 24, 36, and 48 h, then subjected to analysis of the expression of caspase 3, caspase 9, Bcl-2, Bax and PARP by western blotting. β-Actin 27
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was used as an internal standard. Mitochondrial membrane potential(△Ψm) detection with JC-1. MGC-803cells (5C) and SGC-7901 cells (5D) were exposure to HM (40,
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60, 80 μM）and cccp (positive group).
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Fig. 6. The PI3K/Akt/mTOR Signaling Pathway involves in apoptosis as well as autophagy in MGC-803 and SGC-7901 cells
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(6A) MGC-803 and SGC-7901 cells were treated with HM (0, 10, 20, 40, 80 μM)
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for 24 h before subjected to analysis of the expression of the phosphorylated levels of Akt, mTOR and p70S6K by Western blot analysis. (6B) 40 μM HM treated to
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MGC-803 and SGC-7901cells for 0, 6, 12, 24, 36, and 48 h, then the expression of the phosphorylated levels of Akt, mTOR and p70S6K were determined by Western blot analysis. (6C) and (6D) cells were exposure with 10 μM LY294002 (PI3K/Akt inhibitor) in the absence or presence of 40 μM HM for 24 h. The expression of autophagy marker (LC3-II, Beclin-1 and p62) and apoptosis related protein (PARP and caspase-3) was detected by western blot. β-Actin was used as an internal 28
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standard.
Fig. 7. AMPK signaling pathway involved in the autophagy induced by HM. (7A) MGC-803 and SGC-7901 cells were treated with HM (0, 10, 20, 40, 80 μM)
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for 24 h before subjected to analysis of the expression of the phosphorylated levels of
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AMPK α by Western blot analysis. (7B) 40 μM HM treated to MGC-803 and SGC-7901 cells for 0, 6, 12, 24, 36, and 48 h, then the expression of the
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phosphorylated level of AMPK α was determined by Western blot analysis. β-Actin
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was used as an internal standard.
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Fig. 8. HM-mediated autophagy involved in the HM-induced gastric cancer cell death MGC-803 (8A) and SGC-7901 cells (8B) were treated with an autophagy inhibitor
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3-MA (2.5 mM) for 1 h before incubation with 40 μM HM for 24 h, and subjected to
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analysis of the expression of the hallmark of autophagy: LC3-II, Beclin-1 and p62. MGC-803 (8C) and SGC-7901 (8D) cells viabilities were determined by MTT assay.
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(E) and (F) The expression of apoptosis related protein (PARP and caspase-3) was
detected by western blot for the addition of 2.5 mM 3-MA in the absence or presence of 40 μM HM for 24 h. The data were presented as the means ± SD of three independent tests. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.
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