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. 2020 Sep 24;5(39):25156–25163. doi: 10.1021/acsomega.0c02748

C21 Fraction Refined from Marsdenia tenacissima-Induced Apoptosis is Enhanced by Suppression of Autophagy in Human Gastric Cell Lines

Kaiqiang Li †, Ke Hao †, Yu Zhang †, Aibo Xu †, Qianni Wang ‡, Yaoqiang Du †, Lingling Wu ‡, Bingyu Chen †, Wei Zhang †,*, Zhen Wang ‡,*
PMCID: PMC7542599  PMID: 33043194

Abstract

graphic file with name ao0c02748_0008.jpg

C21 steroidal glycosides have been extensively reported for treating several types of cancer and are widely found in Marsdenia tenacissima. In this study, a C21 fraction was synthesized from M. tenacissima, and its anti-cancer potency was assessed against in vitro gastric cell lines BGC-823, SGC-7901, and AGS. Significant growth inhibition and cell cycle arrest were observed in C21 fraction-treated gastric cancer cells. The results of apoptotic staining techniques in C21 fraction-treated gastric cells were confirmed with excess reactive oxygen species generation. Moreover, SOD and H2O2 levels were increased by C21 fraction, especially when combined with chloroquine (CQ). The apoptotic inducing potential of C21 fraction was also evidenced by upregulation of proapoptotic proteins cleaved-PARP and BAX and downregulation of antiapoptotic proteins Bcl-2 and p-AKT by western blot, especially in the presence of the autophagy inhibitor CQ. The results showed that the apoptosis of gastric cancer cells caused by C21 fraction was enhanced by inhibiting autophagy. The current findings reveal a new mechanism for the antitumor activity of C21 fraction on gastric cancer.

Introduction

Gastric cancer remains one of the leading causes of cancer-related mortality worldwide, and more than half of patients’ deaths are attributable to the lack of effective treatment options.1,2 Surgical resection and chemotherapy are normally offered to patients with gastric cancer, but they have limited therapeutic effects.3 Thus, the development of new therapeutic regimes to improve patients’ prognoses is urgently needed.

Autophagy is a conserved eukaryotic catabolic reaction that catabolizes cytoplasmic proteins and organelles.4 As a cytoprotective process, it helps to maintain cancer cell homeostasis and serves as an alternative energy source under harsh environments. This suggests that autophagy may have a protective function in tumor development.5 Recent research indicates that key autophagy genes, including autophagy-related genes (Atg), could be induced by cell reactive oxygen species (ROS) damages via cell death.6 Cully et al. have found that regulating the expression of the suppressor gene PTEN and p53 could positively regulate autophagy.7 It is also known that oncogene products like BCL2 antiapoptotic protein interact with the evolutionarily conserved autophagy protein, and binding with Beclin-1 may help maintain autophagy at levels that are compatible with cell survival.8 With regard to gastric cancers, it has recently been shown that autophagy has a dual impact on the disease.9 Xu et al. showed that the anti-cancer drug Akebia saponin PA promotes the apoptosis of AGS cells by activating autophagy and apoptosis.10 Conversely, another study reported that the use of autophagy inhibitors enhances the cytotoxicity of anticancer drugs in the gastric cancer cell line SGC-7901.11 Therefore, autophagy might be a potential therapeutic target for the treatment of gastric cancers.12

Marsdenia tenacissima is an asclepiadaceous plant that is widely produced in China. Its liquid extracts (trade name: Xiao Ai Ping) have been approved to treat esophageal cancer, lung cancer, leukemia, hepatocellular carcinoma, and gastric cancer.13,14 We found that C21 steroidal glycosides are widely found in MTE, and this study’s authors’ previous work has shown that treatment with C21 fraction from M. tenacissima could induce cell apoptosis through the PTEN-AKT-mTOR signal pathway.15 However, the potential efficacy of C21 fraction on autophagy and whether autophagy inhibition can promote the anticancer effects of C21 has not yet been investigated.

Consequently, this study will examine whether autophagy could be induced by C21 fraction and whether autophagy inhibition can facilitate the anticancer effects of C21. It also explores the potential molecular mechanisms of gastric cancer treatment.

Results

C21 Fraction Stimulation Does Inhibit Gastric Cancer Cell Proliferation

The effect of C21 fraction on BGC-823, SGC-7901, and AGS cell viability was assessed using real-time cell analyzer (RTCA) assay and MTS assay. Growth curves show that C21 fraction greatly inhibits cell proliferation in BGC-823, SGC-7901, and AGS cells (Figure 1A–C) in a concentrated and time-dependent way. As shown in Figure 1D, this study also examined the IC50 in BGC-823, SGC-7901, and AGS using MTS assay. The results showed that C21 fraction significantly inhibited gastric cancer cell proliferation in a dose-dependent manner. The IC50 value of BGC-823 cells was 80.57 μg/mL. The IC50 value of SGC-7901 cells was 146.3 μg/mL. The IC50 value of AGS cells was 102.8 μg/mL. Such findings show that C21 fraction stimulation did significantly inhibit the proliferation of BGC-823, SGC-7901, and AGS cells.

Figure 1.

Figure 1

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C21 fraction suppressed gastric cancer proliferation in vitro. (A–C) Gastric cancer cells were treated with C21 fraction (0, 40, 80, and 160 μg/mL), and the RTCA results were analyzed by GraphPad’s Prism software. (D) Gastric cancer cells were treated with C21 fraction (0, 20, 40, 80, 160, and 320 μg/mL), and the MTS results were analyzed by GraphPad’s Prism software. *P < 0.05, vs NC.

Cell Cycle Arrest and Apoptosis-Inducing Effect of C21 Fraction on BGC-823 and AGS Cells

This study examined the cell cycle phase distribution of treated cells using flow cytometric analysis; the results showed that 160 μg/mL of C21 fraction significantly arrested cell cycle progression in BGC-823 and AGS cells by decreasing the percentage of cells in the G0/G1 phase. It also increased the percentage of cells in the G2/M phase (Figure 2A,B). In addition, flow cytometry analysis was performed to determine whether cell growth inhibition by C21 fractionation was through apoptosis. As shown in Figure 2C,D, gastric cancer cells treated with various doses of C21 fraction for 24 h caused cell apoptosis in a dose-dependent manner, especially in late-stage apoptosis. Next, proteins related to apoptosis were examined using western blot analysis. Treatment of C21 fraction for 24 h increased cleaved PARP in both cell lines. This data suggests that apoptosis may contribute to cell growth inhibition by C21 fraction in gastric cancer cells (Figure 2E,F).

Figure 2.

Figure 2

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C21 fraction treatment-induced cycle arrest and apoptosis in gastric cells. BGC-823 and AGS cells were treated with 0, 80, and 160 μg/mL of C21 fraction for 24 h. (A) After being stained by staining buffer A and reagent B, the cell count (%) was determined by cytometry. (B) Cell cycle phase distribution of treated cells was quantified. (C) After being stained with Annexin-V-fluorescein isothiocyanate (FITC) and PI, the apoptosis rate was determined with flow cytometry. Lower left shows a normal rate, lower right shows early apoptosis, and upper right shows late apoptosis. (D) Percentage of apoptotic cells was quantified. (E) Protein levels of PARP were determined by western blot analysis and β-actin was used as the loading control. (F) Expression ratio of cleaved PARP was quantified: *P < 0.05, vs NC.

C21 Fraction Treatment Disrupted Autophagic Flux in Gastric Cells

To assess the recruitment of LC3-II into autophagosomes in response to C21 fraction treatment, GFP-LC3 plasmid was transiently transfected into gastric cancer cells. As shown in Figure 3A, a punctate LC3 pattern was observed in C21 fraction-treated cells in the cytoplasm, while the untreated control cells showed diffuse and weak LC3 punctate spots. The conversion of the lipidated form of LC3 from LC3-I to LC3-II is a characteristic of autophagy. As indicated in Figure 3B,C, LC3-II accumulated in gastric cancer cell lines after exposure to C21 fraction. In addition, cotreatment with autophagy inhibitor chloroquine (CQ), which blocks the final step of autophagy degradation, enhanced C21 fraction-induced LC3-II accumulation.

Figure 3.

Figure 3

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C21 fraction treatment disrupted autophagic flux in gastric cells. (A) After being treated with C21 fraction (120 μg/mL) in the absence or presence of CQ (20 μM) for 24 h, colocalization of GFP-LC3 (green) was observed using a confocal microscope. (B) After the cells were exposed to C21 fraction (120 μg/mL) in the absence or presence of CQ (20 μM) for 24 h, the protein levels of LC3 were determined by western blot analysis, and GAPDH was used as the loading control. (C) Expression ratio of LC3-II was quantified. *P < 0.05, vs NC.

CQ Promotes C21 Fraction-Induced Gastric Cancer Cell Apoptosis

Cell apoptosis assays show that CQ promotes C21 fraction-induced gastric cancer cell apoptosis. As shown in Figure 4A,B, compared with the control group, the percentages of BGC-823 and AGS cells undergoing apoptosis increased by exposure to C21 fraction, especially enhanced by exposure to C21 fraction combined with CQ. In addition, after 24 h of treatment with C21 fraction alone or in combination with CQ, intracellular ROS production was measured in gastric cancer cells. As shown in Figures 4C and S2, the production of intracellular ROS increased during treatment compared to control cells. As presented in Figure 4D,E, we found that C21 fraction extracted from MTE promoted the levels of superoxide dismutase (SOD) and hydrogen peroxide (H2O2) in gastric cancer cell line BGC823 and AGS, compared to that in control cells.

Figure 4.

Figure 4

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C21 fraction treatment induced apoptosis enhanced by CQ. (A) After treated with C21 fraction (120 μg/mL) in the absence or presence of CQ (20 μM) for 24 h, cells were stained with Annexin-V- FITC and PI. Then, the apoptosis rate was determined with flow cytometry. Lower left shows a normal rate, lower right shows early apoptosis, and upper right shows late apoptosis. (B) Percentage of apoptotic cells was quantified. (C) After being stained with 2’-7’dichlorofluorescin diacetate (DCFH-DA), the ROS levels were quantified. (D) SOD was analyzed by a microplate reader at 450 nm. (E) Level of H2O2 was analyzed at 560 nm using a microplate reader.

The levels of apoptosis-related proteins in gastric cancer cells were examined using western blot analysis. As Figure 5 shows, C21 fraction treatment promoted the protein levels of cleaved PARP and BAX compared with the control group in both cell lines. The expression of BCL-2 was significantly decreased. All results indicated that the inhibition of autophagy could promote apoptosis.

Figure 5.

Figure 5

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C21 fraction treatment influenced apoptosis protein expression by CQ. (A,B) After the cells were exposed to C21 fraction (120 μg/mL) in the absence or presence of CQ (20 μM) for 24 h, the protein associated with autophagy was determined by western blot analysis, and GAPDH was used as the loading control. (C) Expression ratio of cleaved-PARP was quantified. (D) Expression ratio of BCL-2 was quantified. (E) Expression ratio of BAX was quantified. *P < 0.05, vs NC.

C21 Fraction Treatment Promotes the Occurrence of Autophagy Pathways through AKT Activation

Next, we determined whether C21 fraction protects cell oxidative damage by modulating the activities of AKT. The levels of autophagy-related proteins in gastric cancer cells were examined using western blot analysis. As Figure 6 shows, CQ decreased C21 fraction-induced accumulation of Beclin-1 and ATG-5 proteins. In contrast, the expression of p-AKT was significantly decreased. This phenomenon is consistent in both gastric cancer cells. The apoptosis of gastric cancer cells caused by C21 fraction which was enhanced by inhibiting autophagy can partly ascribe to AKT activation.

Figure 6.

Figure 6

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C21 fraction disrupted autophagy via AKT signaling pathway. (A,B) After the cells were exposed to C21 fraction (120 μg/mL) in the absence or presence of CQ (20 μM) for 24 h, the protein associated with apoptosis was determined by western blot analysis, and GAPDH was used as the loading control. (C) Expression ratio of Beclin-1 was quantified. (D) Expression ratio of ATG-5 was quantified. (E) Expression ratio of p-AKT was quantified. *P < 0.05, vs NC.

Discussion

Gastric cancer is the third most common cancer worldwide and is one of the leading causes of cancer-related deaths.16 Lack of effective treatments has resulted in a less than 5-year survival rate and a poor quality of life for cancer patients.17 As a traditional herbal medicine, M. tenacissima extract (MTE) has been proved to have a significant impact on cancers.18−20 C21 fraction is separated from MTE and has been proven to inhibit proliferation and induce apoptosis of hepatoma carcinoma cells.15 Recent research found that MTE, which is a water extract, could inhibit tumor activity by suppressing autophagy in lung cancer cells.21 Whether this antitumor effect, which is caused by C21 fraction extracted from MTE, applies to gastric cancer had not been reported prior to this study. This study demonstrates that C21 fraction has the capacity to suppress the proliferation and promote the apoptosis of gastric cancer cells by suppressing autophagy.

Herbal medicines are often given to cancer patients due to their negligible side effects. M. tenacissima has played an important role in the treatment of patients with tumors,22 while MTE has long been used for cancer therapy due to the bioactive constituents of its polyoxypregnane glycosides.23,24 Our previous research has shown that C21 fraction as an important component of MTE also has obvious cytotoxic effects in hepatic carcinoma cells by apoptosis through the PTEN/AKT/mTOR signaling pathway.15 Our results also showed that C21 fraction greatly inhibits cell proliferation in gastric cancer cells (Figure 1), and the reasons may be through cell cycle arrest and the obvious enhancement of apoptosis (Figure 2A,C). At the same time, cleaved PARP was observed in BGC-823 and AGS cell lines (Figure 2E). The above results indicate that inducing apoptosis using C21 fraction may contribute to the death of gastric cancer cells.

Autophagy is a homeostatic and catabolic process, which has been proved to play different roles in cancer occurrence and progression.25−27 Several chemotherapeutic agents have demonstrated that anticancer drugs can induce tumor cell autophagy to survive under stressful environments.28 The inhibition of autophagy could therefore induce the development of various cancers including hepatic carcinoma, lung cancer, and gastric cancer.29 One study reported that celastrol, a quinine methide triterpenoid, could inhibit gastric cancer cell proliferation induced by autophagy through the AKT/mTOR signaling pathway.30 Liu et al. proved that Celecoxib regulates apoptosis and autophagy via down-regulation of PI3K/AKT expression in the SGC-7901 gastric cancer cell line.31 Therefore, the inhibition of autophagy is considered as a potential therapeutic strategy for cancer treatment. In this study, a significant increase of LC3-II was observed in stimulated BGC-823 and AGS, suggesting that C21 fraction induces autophagy in gastric cancer cells (Figure 3).

To investigate the role of autophagy induced by C21 fraction in gastric cancer cells, this study used an inhibitor of autophagic degradation (CQ) combined with C21 fraction.1 Cooper et al. found that ROS can induce autophagy and in turn, ROS can be reduced by consuming damaged mitochondria.32 The level of ROS was significantly increased by C21 fraction, and this study hypothesizes that ROS may be a positive regulator in the process of C21 fraction-induced autophagy (Figures 4C and S2). In addition, we also found that C21 fraction extracted from MTE promoted the levels of SOD and H2O2 in gastric cancer cell lines BGC823 and AGS, especially enhanced by C21 fraction combined with CQ. Importantly, the apoptosis rate of BGC-823 cells and AGS cells increased after they were both treated with C21 fraction combined with CQ. CQ also promotes changes in the C21 fraction-induced apoptosis-related protein Bcl-2 (Figure 5). This suggests that C21 fraction can promote apoptosis by inhibiting autophagy in gastric cancer cells.

In order to elucidate molecular mechanisms, the expression of apoptosis-related proteins was investigated in Figure 5. The activation of the PI3K/Akt pathway has been proved in many types of tumors; it contributes to the proliferation and survival of tumor cells.33,34 In this study, the decrease in p-AKT in BGC-823 and AGS cells indicates that C21 fraction may induce cell apoptosis through the PTEN-AKT signal pathway. The authors’ previous research has confirmed that C21 fraction could induce cell apoptosis through the PTEN/AKT/mTOR signaling pathway.14,15 The PI3K/Akt/mTOR pathway has been proven to play a negative role in regulating autophagy; its decline will actually activate autophagy.5,35 Moreover, studies have also found that autophagy-related proteins (Atg) play a special function in regulating the formation of autophagosomes encoded by the autophagy-related genes.36,37 In this study, the autophagy-related proteins Atg5 and Beclin-1 increased when autophagy was induced by C21 fraction and decreased when autophagy inhibition occurred (Figure 6), further indicating that C21 fraction could activate autophagy in gastric cancer cells. This study hypothesizes that C21 fraction could induce protective autophagy in gastric cancer cells by inhibiting the PI3K/Akt/mTOR signaling pathway.

In conclusion, this study demonstrated that C21 fraction could lead to the inhibition of cell proliferation and the induction of apoptosis and autophagy in gastric cancer cells. The apoptosis of gastric cancer cells caused by C21 fraction which was enhanced by inhibiting autophagy can partly ascribe to AKT activation. Taken together, our study suggests that C21 fraction may be a novel candidate derived from natural compounds for the treatment of gastric cancer.

Material and Methods

Chemicals and Reagents

Fetal bovine serum (FBS) and Roswell Park Memorial Institute medium 1640 (RPMI 1640) were purchased from Hyclone. C21 fraction from MTE (names: 99318-1, which has been checked with http://www.theplantlist.org/) was provided by the Pharmaceutical School Laboratory at the Zhejiang University of Technology.15 GFP-LC3 plasmid was provided by the Clinical Laboratory of Zhejiang Provincial People’s Hospital. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) was purchased from Promega. CQ diphosphate salt was purchased from Sangon Biotech.

Cell Culture and C21 Fraction Treatment

BGC-823, SGC-7901, and AGS cells—human gastric cancer cells—were obtained from the Clinical Laboratory of Zhejiang Provincial People’s Hospital. All cells were cultured in Roswell Park Memorial Institute medium 1640 (RPMI 1640, Hyclone) supplemented with 10% FBS (Hyclone). All cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. Then, they were either left untreated or were treated with C21 fraction.

Cell Proliferation Assay

50μl of culture medium was added to each well of E-Plate 96 (Roche Applied Science) to obtain equilibrium. Then, 2000 cells were seeded in E-Plate 96 and incubated for 24 h. Then, they were treated with C21 fraction (0, 40, 80, and 160 μg/mL). E-Plate 96 was locked in a RTCA-multiplate device at 37 °C with 5% CO2. Measured changes in electrical impedance were recorded that directly reflected cellular proliferation on biocompatible microelectrode-coated surfaces. Cell index was read automatically every 2 min.

The cells were seeded in 96-well plates with 5000 cells/well in 200 μL of RPIM 1640 medium supplemented with 10% FBS at 37 °C in a humidified atmosphere of 5% CO2. Then, they were either left untreated or were treated with C21 fraction (0, 20, 40, 80, 160, and 320 μg/mL). Following this, they were incubated for 24 h. 40μl of MTS solution was added to each well and then incubated for another 2 h. Absorbance was measured at 490 nm with a microplate reader.

Cell Cycle Analysis and Quantification of Apoptosis by Flow Cytometry

Cells were seeded into 12-well plates that were incubated at 37 °C for 24 h. Then, the cells were treated with C21 fraction (0, 80, and 160 μg/mL) and incubated at 37 °C for 24 h. Afterward, whole cells were harvested and treated with 500 μL of staining buffer A and 5 μL of reagent B (Lianke Biotechnology). These cells were incubated for 30 min at room temperature in the dark, and their cell cycle was analyzed using flow cytometry.

The percentage of apoptosis cells was examined by double staining them with Annexin-V-FITC and PI (Lianke Biotechnology). They were then treated in the same way as the cell cultures used in cell cycle analysis. Whole cells were harvested and treated with 5 μL of Annexin V-FITC and 10 μL of PI. They were then incubated for 5 min in the dark at room temperature, and fluorescence-activated cell sorting was analyzed using flow cytometry.

ROS Measurement

Cells were incubated for 24 h in the presence or absence of C21 fraction alone or with CQ. After harvesting and resuspending them in 500 μL of PBS, the cells were stained with DCFH-DA (5 mM) from ROS assay for 30 min according to the manufacturer’s instructions (Beyotime). The DCFH-DA signal was then analyzed using flow cytometry.

SOD and H2O2 Measurements

SOD was analyzed by a commercial kit (Beyotime). Briefly, 20 μL of protein sample was mixed with 160 μL of WST-8/enzyme working solution (mixing 151 μL of SOD assay buffer, 8 μL of WST-8, and 1 μL of enzyme solution) and 20 μL of the diluted reaction started working solution (1 μL original reaction started working solution diluted with 39 μL SOD buffer) and then incubated at 37 °C for 30 min. The absorbance of the sample was measured at 450 nm. The level of H2O2 was analyzed by a commercial kit (Beyotime) and the absorbance was measured at 560 nm using a microplate reader (BioTek, USA). Quantitative measurements were obtained against a standard curve of H2O2 consisting of a gradient of concentrations and then normalized with respect to the cell counts.

Confocal Microscopy

Cells adhered to slides were transfected with GFP-LC3 plasmid using the transfection reagent (Yeasen), according to the manufacturer’s protocol. After transfection for 24 h, the cells were treated with C21 fraction, alone or with CQ, and then incubated for 24 h. Finally, the cells were stained with DAPI and an anti-quenching agent (1:1000, Beyotime) and observed using confocal microscopy (Leica).

Western Blot Analysis

Cells that had been treated with C21 fraction were seeded into 12-well plates. Then, they were incubated for 24 h. A total cell protein extraction kit (RIPA, Beyotime) was used to extract total protein. An equivalent amount of protein from each sample was electrophoresed by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After being blocked, the membranes were incubated with anti-PTEN (1:1000; Cell Signaling), anti-Bax (1:1000; Cell Signaling), anti-Bcl-2 (1:1000; Cell Signaling), anti-AKT (1:1000; Cell Signaling), anti-PARP (1:1000; Cell Signaling), anti-LC3 (1:1000; Cell Signaling), and anti-Beclin-1 (1:1000; Cell Signaling) overnight at 4 °C. Then, the membranes were washed three times with PBS/0.1% Tween-20 for 10 min. They were then incubated with a corresponding secondary antibody (1:2000, HuaBio) for 1 h at room temperature before again being washed three times. The membranes were detected using a chemical luminescence ECL kit (FUDE Biological Technology).

Analysis

All assays were performed in at least three independent experiments. The results from western blot analysis were analyzed using ImageJ software. All data was analyzed using GraphPad’s prism software and all values were expressed as mean ± SD. Statistical differences were assessed by an independent t-test. The significance of the difference was set at p < 0.05.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (81772664), Zhejiang Medical and Health Science and Technology Project (2019KY017). Key projects jointly constructed by the Ministry and the province of Zhejiang Medical and Health Science and Technology Project (WKJ-ZJ-2019). Key and Major Projects of Traditional Chinese Medicine Scientific Research Foundation of Zhejiang Province (2019ZZ001, 2018ZY001).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02748.

  • C21 fraction extracted from MTE promoted the expression of autophagy-related mRNA in BGS-823 cell line and C21 fraction extracted from MTE promoted the levels of intracellular ROS in BGC-823 and AGS cell lines (PDF)

Author Contributions

Co-first author: K.H. and Y.Z.

The authors declare no competing financial interest.

Supplementary Material

ao0c02748_si_001.pdf (137.7KB, pdf)

References

  1. Zhao Z.; Han F.; Yang S.; Wu J.; Zhan W. Oxamate-mediated inhibition of lactate dehydrogenase induces protective autophagy in gastric cancer cells: Involvement of the Akt–mTOR signaling pathway. Cancer Lett. 2015, 358, 17–26. 10.1016/j.canlet.2014.11.046. [DOI] [PubMed] [Google Scholar]
  2. Lee H.-W.; Jang K. S. B.; Choi H. J.; Jo A.; Cheong J.-H.; Chun K.-H. Celastrol inhibits gastric cancer growth by induction of apoptosis and autophagy. BMB Rep. 2014, 47, 697–702. 10.5483/bmbrep.2014.47.12.069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chuang W.-L.; Su C.-C.; Lin P.-Y.; Lin C.-C.; Chen Y.-L. Sann-Joong-Kuey-Jian-Tang induces autophagy in HepG2 cells via regulation of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and p38 mitogen-activated protein kinase pathways. Mol. Med. Rep. 2015, 12, 1677–1684. 10.3892/mmr.2015.3573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Yu L.; Mcphee C. K.; Zheng L.; Mardones G. A.; Rong Y.; Peng J.; Mi N.; Zhao Y.; Liu Z.; Wan F.; Hailey D. W.; Oorschot V.; Klumperman J.; Baehrecke E. H.; Lenardo M. J. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 2010, 465, 942–946. 10.1038/nature09076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Liang X. H.; Kleeman L. K.; Jiang H. H.; Gordon G.; Goldman J. E.; Berry G.; Herman B.; Levine B. Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J. Virol. 1998, 72, 8586–8596. 10.1128/jvi.72.11.8586-8596.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Li L.; Tan J.; Miao Y.; Lei P.; Zhang Q. ROS and Autophagy: Interactions and Molecular Regulatory Mechanisms. Cell. Mol. Neurobiol. 2015, 35, 615–621. 10.1007/s10571-015-0166-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cully M.; You H.; Levine A. J.; Mak T. W. Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat. Rev. Cancer 2006, 6, 184–192. 10.1038/nrc1819. [DOI] [PubMed] [Google Scholar]
  8. Pattingre S.; Tassa A.; Qu X.; Garuti R.; Liang X. H.; Mizushima N.; Packer M.; Schneider M. D.; Levine B. Bcl-2 Antiapoptotic Proteins Inhibit Beclin 1-Dependent Autophagy. Cell 2005, 122, 927–939. 10.1016/j.cell.2005.07.002. [DOI] [PubMed] [Google Scholar]
  9. Qian H. R.; Yang Y. Functional role of autophagy in gastric cancer. Oncotarget 2016, 7, 17641. 10.18632/oncotarget.7508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Xu M.-Y.; Lee D. H.; Joo E. J.; Son K. H.; Kim Y. S. Akebia saponin PA induces autophagic and apoptotic cell death in AGS human gastric cancer cells. Food Chem. Toxicol. 2013, 59, 703–708. 10.1016/j.fct.2013.06.059. [DOI] [PubMed] [Google Scholar]
  11. Zhang H.-Q.; He B.; Fang N.; Lu S.; Liao Y.-Q.; Wan Y.-Y. Autophagy Inhibition Sensitizes Cisplatin Cytotoxicity in Human Gastric Cancer Cell Line Sgc7901. Asian Pac. J. Cancer Prev. 2013, 14, 4685–4688. 10.7314/apjcp.2013.14.8.4685. [DOI] [PubMed] [Google Scholar]
  12. Choi P. R.; Kang Y. J.; Sung B.; Kim J. H.; Moon H. R.; Chung H. Y.; Kim S. E.; Park M. I.; Park S. J.; Kim N. D. MHY218-induced apoptotic cell death is enhanced by the inhibition of autophagy in AGS human gastric cancer cells. Int. J. Oncol. 2015, 47, 563–572. 10.3892/ijo.2015.3031. [DOI] [PubMed] [Google Scholar]
  13. Wang Z.; Ying Y.-M.; Li K.-Q.; Zhang Y.; Chen B.-Y.; Zeng J.-J.; He X.-J.; Jiang M.-M.; Chen B.-X.; Wang Y.; Xu X.-D.; Hao K.; Zhu M.-H.; Zhang W. Marsdeniae tenacissima extract-induced growth inhibition and apoptosis in hepatoma carcinoma cells is mediated through the p53/nuclear factor-κB signaling pathway. Exp. Ther. Med. 2017, 14, 2477–2484. 10.3892/etm.2017.4833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Wang Y.; Chen B. Y.; Wang Z.; Zhang W.; Hao K.; Chen Y.; Li K. Q.; Wang T. T.; Xie Y. W.; Huang Z. H.; Tong X. M. Marsdenia tenacissimae extraction (MTE) inhibits the proliferation and induces the apoptosis of human acute T cell leukemia cells through inactivating PI3K/AKT/mTOR signaling pathway via PTEN enhancement. Oncotarget 2016, 7, 82851. 10.18632/oncotarget.12654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Zhang Y.; Li K. Q.; Ying Y. M.; Chen B. Y.; Hao K.; Chen B. X.; Zheng Y.; Lyu J. X.; Tong X. M.; Chen X. P.; Wan Y.; Zhan Z. J.; Zhang W.; Wang Z. C21 steroid-enriched fraction refined from Marsdenia tenacissima inhibits hepatocellular carcinoma through the coordination of Hippo-Yap and PTEN-PI3K/AKT signaling pathways. Oncotarget 2017, 8, 110576. 10.18632/oncotarget.22833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ferlay J.; Soerjomataram I.; Dikshit R.; Eser S.; Mathers C.; Rebelo M.; Parkin D. M.; Forman D.; Bray F. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 2014, 136, E359 10.1002/ijc.29210. [DOI] [PubMed] [Google Scholar]
  17. Kunz P. L.; Gubens M.; Fisher G. A.; Ford J. M.; Lichtensztajn D. Y.; Clarke C. A. Long-Term Survivors of Gastric Cancer: A California Population-Based Study. J. Clin. Oncol. 2012, 30, 3507–3515. 10.1200/jco.2011.35.8028. [DOI] [PubMed] [Google Scholar]
  18. Chen B.-Y.; Chen D.; Lyu J.-X.; Li K.-Q.; Jiang M.-M.; Zeng J.-J.; He X.-J.; Hao K.; Tao H.-Q.; Mou X.-Z.; Ying Y.-M.; Zhang W.; Zhu M.-H.; Wang Z. Marsdeniae tenacissimae extract (MTE) suppresses cell proliferation by attenuating VEGF/VEGFR2 interactions and promotes apoptosis through regulating PKC pathway in human umbilical vein endothelial cells. Chin. J. Nat. Med. 2016, 14, 922–930. 10.1016/s1875-5364(17)30017-1. [DOI] [PubMed] [Google Scholar]
  19. Han S.-Y.; Zhao W.; Sun H.; Zhou N.; Zhou F.; An G.; Li P.-P. Marsdenia tenacissima extract enhances gefitinib efficacy in non-small cell lung cancer xenografts. Phytomedicine 2015, 22, 560–567. 10.1016/j.phymed.2015.03.001. [DOI] [PubMed] [Google Scholar]
  20. Li D.; Li C.; Song Y.; Zhou M.; Sun X.; Zhu X.; Zhang F.; Zhou C.; Huan Y.; Xia S.; Zhuo X.; Dong P.; Sui X.; Liao H.; Yang Z.-F. Marsdenia tenacssimaextract and its functional components inhibits proliferation and induces apoptosis of human Burkitt leukemia/lymphoma cellsin vitroandin vivo. Leuk. Lymphoma 2015, 57, 419–428. 10.3109/10428194.2015.1043546. [DOI] [PubMed] [Google Scholar]
  21. Jiao Y. N.; Wu L. N.; Xue D.; Liu X. J.; Tian Z. H.; Jiang S. T.; Han S. Y.; Li P. P. Marsdenia tenacissima extract induces apoptosis and suppresses autophagy through ERK activation in lung cancer cells. Cancer Cell Int. 2018, 18, 149. 10.1186/s12935-018-0646-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fan W.; Sun L.; Zhou J.-Q.; Zhang C.; Qin S.; Tang Y.; Liu Y.; Lin S.-S.; Yuan S.-T. Marsdenia tenacissima extract induces G0/G1 cell cycle arrest in human esophageal carcinoma cells by inhibiting mitogen-activated protein kinase (MAPK) signaling pathway. Chin. J. Nat. Med. 2015, 13, 428–437. 10.1016/s1875-5364(15)30036-4. [DOI] [PubMed] [Google Scholar]
  23. Han S. Y.; Ding H. R.; Zhao W.; Teng F.; Li P. P. Enhancement of gefitinib-induced growth inhibition by Marsdenia tenacissima extract in non-small cell lung cancer cells expressing wild or mutant EGFR. BMC Complementary Altern. Med. 2014, 14, 165. 10.1186/1472-6882-14-165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Han S.-Y.; Zhao M.-B.; Zhuang G.-B.; Li P.-P. Marsdenia tenacissima extract restored gefitinib sensitivity in resistant non-small cell lung cancer cells. Lung Cancer 2012, 75, 30–37. 10.1016/j.lungcan.2011.06.001. [DOI] [PubMed] [Google Scholar]
  25. Guigon C. J.; Fozzatti L.; Lu C.; Willingham M. C.; Cheng S.-y. Inhibition of mTORC1 signaling reduces tumor growth but does not prevent cancer progression in a mouse model of thyroid cancer. Carcinogenesis 2010, 31, 1284–1291. 10.1093/carcin/bgq059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wei F.; Jiang X.; Gao H.-Y.; Gao S.-H. Liquiritin induces apoptosis and autophagy in cisplatin (DDP)-resistant gastric cancer cells in vitro and xenograft nude mice in vivo. Int. J. Oncol. 2017, 51, 1383–1394. 10.3892/ijo.2017.4134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. White E. Deconvoluting the context-dependent role for autophagy in cancer. Nat. Rev. Cancer 2012, 12, 401–410. 10.1038/nrc3262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sishi B. J. N.; Loos B.; van Rooyen J.; Engelbrecht A.-M. Autophagy upregulation promotes survival and attenuates doxorubicin-induced cardiotoxicity. Biochem. Pharmacol. 2013, 85, 124–134. 10.1016/j.bcp.2012.10.005. [DOI] [PubMed] [Google Scholar]
  29. Guo X.-l.; Li D.; Hu F.; Song J.-r.; Zhang S.-s.; Deng W.-j.; Sun K.; Zhao Q.-d.; Xie X.-q.; Song Y.-j.; Wu M.-c.; Wei L.-x. Targeting autophagy potentiates chemotherapy- induced apoptosis and proliferation inhibition in hepatocarcinoma cells. Cancer Lett. 2012, 320, 171–179. 10.1016/j.canlet.2012.03.002. [DOI] [PubMed] [Google Scholar]
  30. Hu W.; Chen S.-S.; Zhang J.-L.; Lou X.-E.; Zhou H.-J. Dihydroartemisinin induces autophagy by suppressing NF-κB activation. Cancer Lett. 2014, 343, 239–248. 10.1016/j.canlet.2013.09.035. [DOI] [PubMed] [Google Scholar]
  31. Liu M.; Li C.-M.; Chen Z.-F.; Ji R.; Guo Q.-H.; Li Q.; Zhang H.-L.; Zhou Y.-N. Celecoxib regulates apoptosis and autophagy via the PI3K/Akt signaling pathway in SGC-7901 gastric cancer cells. Int. J. Mol. Med. 2014, 33, 1451–1458. 10.3892/ijmm.2014.1713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Cooper K. F. Till Death Do Us Part: The Marriage of Autophagy and Apoptosis. Oxid. Med. Cell. Longevity 2018, 2018, 1–13. 10.1155/2018/4701275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Xu G.-y.; Tang X.-j. Troxerutin (TXN) potentiated 5-Fluorouracil (5-Fu) treatment of human gastric cancer through suppressing STAT3/NF-κB and Bcl-2 signaling pathways. Biomed. Pharmacother. 2017, 92, 95–107. 10.1016/j.biopha.2017.04.059. [DOI] [PubMed] [Google Scholar]
  34. Petrulea M. S.; Plantinga T. S.; Smit J. W.; Georgescu C. E.; Netea-Maier R. T. PI3K/Akt/mTOR: A promising therapeutic target for non-medullary thyroid carcinoma. Canc. Treat Rev. 2015, 41, 707–713. 10.1016/j.ctrv.2015.06.005. [DOI] [PubMed] [Google Scholar]
  35. Chen B. Y.; Jiang L. X.; Hao K.; Wang L.; Wang Y.; Xie Y. W.; Shen J.; Zhu M. H.; Tong X. M.; Li K. Q.; Wang Z. Protection of plasma transfusion against lipopolysaccharide/D-galactosamine-induced fulminant hepatic failure through inhibiting apoptosis of hepatic cells in mice[J]. J. Zhejiang Univ., Sci., B 2018, 19, 436–444. 10.1631/jzus.b1700277. [DOI] [Google Scholar]
  36. Klionsky D. J.; Cregg J. M.; Dunn W. A.; Emr S. D.; Sakai Y.; Sandoval I. V.; Sibirny A.; Subramani S.; Thumm M.; Veenhuis M.; Ohsumi Y. Unified Nomenclature for Yeast Autophagy-Related Genes. Dev. Cell 2003, 5, 539–545. 10.1016/s1534-5807(03)00296-x. [DOI] [PubMed] [Google Scholar]
  37. Thumm M.; Egner R.; Koch B.; Schlumpberger M.; Straub M.; Veenhuis M.; Wolf D. H. Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS Lett. 1994, 349, 275–280. 10.1016/0014-5793(94)00672-5. [DOI] [PubMed] [Google Scholar]

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