Background
Lung cancer remains one of the leading causes of cancer-related deaths worldwide. It can be divided into small-cell lung cancer (SCLC, 15%) and non-small cell lung cancer (NSCLC, 85%) according to the histologic features. In patients with advanced NSCLC who generally have a poor prognosis [
1], new strategies to improve survival are urgently required.
Aberrant signal transduction pathways often occur in tumorigenesis and progress. Studies demonstrated that autophagy and apoptosis play central roles during lung cancer initiation and progression [
2]. Fundamental cellular physiological activities such as apoptosis and autophagy are critical to control cell survival and cell death [
2]. Apoptosis is one form of programmed cell death with the function of removing damaged cells. Resistance to apoptosis is regarded as one of the hallmarks of cancer [
3], thus targeting apoptosis in cancer is a practicable therapy with the suggest of many studies [
4].
Autophagy is a self-degradation process to keep constant supply of cellular energy [
5]. The relationship between autophagy and cell death is subtle and intricate, and it may promote or inhibit cell death in different contexts. The role of autophagy in tumor initiation and progression is multifaceted and complicated. It has been reported that autophagy inhibits tumorigenesis in some circumstances but promotes carcinogenesis under most conditions [
6]. Through upregulating autophagy, cancer cells can survive, growth and become aggressive under pressured microenvironment [
6]. Therefore, it makes autophagy as an attractive therapeutic target for effective treatment of tumors including lung cancer [
7,
8].
Traditional Chinese Medicine has been used extensively to treat diseases from ancient time. The stem of
Marsdenia tenacissima (Roxb.) Wight et Arn. is mainly produced in Yunnan (China), and its medical use was firstly recorded in “Dian Nan Ben Cao”, a medical literature written by Mao Lan in Ming Dynasty with the activity of expectorant, diuresis, eliminating heat and purging fire, lactating.
M. tenacissima has long been used as a remedy to treat malignant diseases, tracheitis, and pneumonia in China [
9,
10].
There is a great number of studies demonstrated that the water extract of
M. tenacissima (MTE, trade name: Xiao-Ai-Ping injection) has anti-tumor effects in cell culture models, laboratory animal models and the clinics. (a) The cell culture models include gastric carcinoma cells (SGC-7901) [
11], non-small cell lung cancer cells (H1975, H292, H460) [
12], Burkitt lymphoma cells [
13], human umbilical vein endothelial cells (HUVECs) [
14,
15], hepatoma cells (HepG2) [
14], esophageal cancer cells (KYSE150 and Eca-109) [
16], etc. (b) Xenograft mouse models were generated from gastric cancer [
11], hepatocellular carcinoma [
17], lymphoma [
13] and the chick embryo chorioallantoic membrane [
14] etc. (c) The clinic trials were mainly conducted in advanced non-small cell lung cancer patients [
18,
19]. Mechanisms accounting for the anti-tumor activities of MTE comprise of anti-angiogenesis [
14], cell apoptosis induction [
20] and cell cycle arrest [
16]. However, the molecular mechanisms underlying the pharmacological action of MTE treatment resulting in cell death remains obscure and need further exploration.
Due to the vital role of apoptosis and autophagy in cell death, in the present study, we evaluated the influence of MTE on cell apoptosis and autophagy in NSCLC cell lines A549 and H1975. Meanwhile, the molecular mechanisms of MTE treatment shared by both apoptosis and autophagy were also explored and elucidated.
Materials and methods
Cell cultures and reagents
Human lung carcinoma cell lines A549 and H1975 (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI-1640 (GIBCO, Thermo Fisher, Hudson, NH, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified incubator at 37 °C under 5% CO2/95% air. The reagents used in this study were: Earle’s balanced salt solution (EBSS, Solarbio, H2020, Beijing, China), Bafilomycin A1 (Baf A1, B1793, Sigma-Aldrich, St. Louis, MO, USA), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, M2128, Sigma-Aldrich), 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, D9542, Sigma-Aldrich), LysoTracker Red (L7528, Thermo Fisher), U0126 (S1102, Selleckchem, Houston, TX, USA). Antibodies of anti-LC3-II (L7543) was purchased from Sigma-Aldrich; anti-p62 (ab56416), anti-Cathepsin B (ab33538), and anti-Caspase 3 (ab32351) antibodies were purchased from Abcam (Cambridge, UK); anti-Poly (ADP-ribose) polymerase (PARP) (9542), anti-Lysosome-associated membrane protein 1 (LAMP1) (9091), anti-p-ERK1/2 (Thr202/Tyr204) (9101), anti- ERK1/2 (9102), anti-Bcl-2 alpha (2876) and anti-Bax (5023) were obtained from Cell Signal Technology (Beverly, MA, USA). Anti-β-actin (TDY041) was obtained from TDYbio (Beijing, China). Secondary antibodies including peroxidase-conjugated goat anti-mouse IgG (ZB2305), peroxidase-conjugated goat anti-rabbit IgG (ZB2301), and fluorescein-conjugated affiniPure goat anti-mouse IgG (ZF0312) were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd.
MTE (
M. tenacissima extract, trade name: Xiao-Ai-Ping injection) (1 g crude/ml) was obtained from SanHome Pharmaceutical Co., Ltd (Nanjing, China). The stem of
M. tenacissima was collected from Yunnan, China. A voucher specimen (200907-T009-05) was deposited in the herbarium of SanHome Pharmaceutical Co., Ltd (NanJing, China) and was identified by Professor De-Kang Wu (Nanjing University of Chinese Medicine). The preparation of MTE is described as previously [
21]. 1 kg powder of the stem of
M. tenacissima was extracted with water for three times which is 1.5 h, 1 h and 0.8 h, respectively. The combined extracts were filtered, concentrated, and then precipitated with 8 times 85% ethanol at 4 °C for 24 h. The ethanol was recovered and new 85% ethanol was added to cause further precipitation. The ethanol in extract was recovered thoroughly and the insoluble precipitate was removed by filtration. Finally, the extract was concentrating to 200 ml. This condensed extract was dilute with water for injection, added 0.3% polysorbate 80, and adjust pH to 5.5–6.0 to get Xiao-Ai-Ping injection following the standard of State Food and Drug Administration (SFDA) of China.
Cell viability assays
A549 or H1975 cells were suspended in complete RPMI-1640 medium and plated at a density of 5 × 103 cells/well in 96-well culture dishes (Costar, Cambridge, MA, USA). Following 24 h of culture, the medium was replaced with complete culture medium supplemented with various concentrations of drugs. On the collection time points, cells were incubated with MTT at 37 °C for 4 h, and the precipitate was dissolved in DMSO. Subsequently, the absorbance (optical density, OD) at 570 nm was measured using a microplate reader (Model 680; Bio-Rad Laboratories, Hercules, CA, USA) and cell viability was calculated according to the following formula: (ODsample − ODblank)/(ODcontrol − ODblank) × 100%.
Western blot analysis
For immunoblot analysis, cells were harvested and lysed in RIPA lysis buffer (WB0002, TDYBio). Protein concentrations were determined using the BCA protein assay kit (Thermo Fisher). Protein samples (20 μg per lane) were separated on the 8–15% SDS–polyacrylamide gel electrophoresis (PAGE) and blotted onto polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA, USA). Following transfer, the membranes were blocked in 5% nonfat milk or bovine serum albumin (BSA) (for phosphorylated proteins) in phosphate-buffered saline (PBS) with 0.1% Tween-20, probed with primary antibodies overnight at 4 °C. After washing, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies. Visualization of the protein bands was accomplished using an Immobilon Western Chemiluminescent HRP substrate (Millipore). Image J software was used to calculate the expression of each protein, which was normalized by β-actin.
Apoptosis analysis
Cell apoptosis was assayed by Annexin V and PI staining (AD10, Dojindo, Kumamoto, Japan). Cells were treated with different concentrations of MTE for 24 h, without or with MEK/ERK inhibitor U0126 (50 µM for A549, 20 µM for H1975). Then cells were collected and incubated with the buffer containing FITC-conjugated Annexin V and PI for 15 min at room temperature, and then analyzed by FACScan flow cytometry (Bection Dikinson, USA). Quantification of early apoptotic cells (Annexin V+/PI− cells) and late apoptotic cells (Annexin V+/PI+ cells) was calculated by CellQuest software.
Caspase 3 activity assay
The activity of Caspase 3 was determined using a kit from Beyotime Institute of Biotechnology (C1116, Beijing, China). The activity of Caspase 3 was based on its ability to change acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) into a yellow formazan product (p-nitroaniline (pNA)). An increase in absorbance at 405 nm was used to quantify Caspase 3 activity. After 24 h exposure, cells with various designated treatments were collected and rinsed with cold PBS, and then lysed by lysis buffer (60 μL) for 15 min on ice, respectively. Cell lysates were centrifuged at 16,000×g for 15 min at 4 °C. The detail analysis procedure was described in the manufacturer’s protocol. The Caspase 3 activity was shown as fold change of enzyme activity compared to control. All the experiments were carried out in triplicates.
Immunofluorescence, fluorescence, and confocal microscopy
Cells were seeded to cover glasses in 24-well plates and treated as indicated, fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 (ST795, Beyotime). The cells were then blocked with 5% FBS for 1 h and exposed to anti-LC3-II (PM036, MBL, Nagoya, Japan) antibody overnight at 4 °C. After washing three times with PBS, cells were incubated with FITC-conjugated secondary antibody solution. After staining nuclei with DAPI, cells were observed under a confocal microscope (Leica, Welzler, Germany). For each group, the number of endogenous LC3-II puncta per cell was assessed in 100 cells, and statistical data were obtained from three independent experiments.
For LysoTracker staining, A549 or H1975 cells with stable expression of GFP-LC3 were cultured in confocal dishes and incubated for 90 min in complete RPMI-1640 medium supplemented with 500 nM LysoTracker Red. The colocalization of LC3 and LysoTracker was analyzed by the confocal microscopy.
Acridine orange (AO) staining
Autophagy is a lysosomal degradation pathway for cytoplasmic material and organelles. The acidic intracellular compartments were visualized by supravital AO staining. After the treatment with MTE (0, 20, 40 mg/ml) for 6 h, cells were washed with PBS and stained with 1 μg/ml AO (318337, Sigma-Aldrich) for 20 min at 37 °C. Subsequently, cells were analyzed under the confocal microscopy.
Statistical analysis
All experiments were repeated at least three times and values are expressed as the mean ± standard error of mean (SEM). Student’s t-test was used to determine the difference between two independent groups. All data were analyzed using SPSS statistical software 16.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant difference between values.
Discussion
Studies showed C21 steroidal glycosides are major components in
M. tenacissima. Compounds such as Tenacigenoside A [
13], 11α-
O-benzoyl-12β-
O-acetyl tenacigenin B [
27], tenacissoside C, tenacissoside B, tenacissoside C, Tenacissoside I and marsdenoside K [
28] etc. are demonstrated to possess anti-cancer activity. According to our previous HPLC–MS analysis, 13 compounds including most of compounds mentioned above were identified from MTE by HPLC–MS analysis [
12]. In recent years,
M. tenacissima has attracted extensive interest in cancer research area with multiple effects, such as inhibiting tumor growth and angiogenesis, reversing anti-tumor drug resistance [
12,
14,
17]. However, the reasons why MTE treatment resulted in the inhibition on cancer cell growth still remain largely unknown. In the present study, we found MTE significantly induced cell apoptosis, suppressed cell autophagy through impairing lysosomes function in A549 and H1975 NSCLC cells. Our results also indicated that ERK may mediate autophagy inhibition and apoptosis induction effect of MTE in NSCLC cells.
Programmed forms of cell death pathway at least include apoptosis and autophagy. Apoptosis is a physiological process to eliminate damaged, mutant or aged cells to maintain cellular homeostasis in normal tissue [
29]. The inhibition of apoptosis is regarded as one of the hallmarks of cancer [
3], and apoptosis-inducing has been exploited as an indispensable anticancer therapeutic strategy. Approaches targeting apoptotic pathway can result in cancer cell death, increasing sensitivity to current treatments or reversing drug resistance, thus may bring promising clinical benefits. Till now, different apoptosis targeted therapies have entered clinical trials for efficacy evaluation in various tumor types including lung cancer [
30]. In the present study, MTE induced significant cell apoptosis in both A549 and H1975 cells along with Caspase 3 activation. Although cleaved PARP was not observed in A549 cells with MTE treatment, remarkable apoptotic cells presented after stained with Annexin V-FITC for flow cytometry analysis. The above results indicated that apoptosis-inducing may contribute to the cell death caused by MTE treatment.
Autophagy has complicated functions on cell death, as it may promote or inhibit cell death under certain circumstances. Although autophagy may offer tumor suppressive function in some conditions [
31], it is mainly a cytoprotective process to facilitate cancer cells survive under stressful environments [
32]. Studies showed that autophagy suppression in NSCLC cells resulted in cell proliferation suppression [
33] and cell apoptosis increase [
34]. In addition, constitutive activation of autophagy is also associated with anti-cancer therapeutic resistance [
35], and inhibiting autophagy may overcome drug resistance in tumors [
36]. Therefore, targeting autophagy is considered as a potential therapeutic strategy for cancer treatment.
LC3-II and p62 serve as marker of autophagic flux. The level of p62 increased when autophagy inhibition occurred; and decreased when autophagy is induced. In our study, MTE treatment caused accumulation of both LC3-II and p62, which means the substrate degradation was blocked and autophagic flux was impaired. The autophagy inhibitory effect of MTE was further confirmed by adding autophagy inhibitor Baf A1 and autophagy inducer EBSS. Next, GFP-LC3 stable NSCLC cells labeled with LysoTracker showed MTE suppressed the fusion of lysosomes with autophagosome at the late stage. This effect was further confirmed by detecting lysosomal marker LAMP1 and lysosomal protease Cathepsin B, indicating MTE impaired lysosomal function. Consistent with our results, other study demonstrated that inhibition of the fusion between lysosomes and autophagosomes leading to accumulated LC3-II and increased acidic vacuolar compartment [
37]. Our results demonstrated that MTE can target both apoptosis and autophagy leading to NSCLC cells death. In fact, the molecular connections exist between apoptosis and autophagy, and some regulators are shared to maintain a subtle and complicated balance with each other [
38‐
40]. ERK is a crucial molecule to control diverse cell responses including proliferation, migration, and differentiation [
25]. High levels of ERK has been found in many malignant tumors, but ERK activation is not always correlated with cell survival protection, it can also interplay with cell death including apoptosis, autophagy, and senescence [
25,
26]. Growing evidence demonstrated that activated ERK has positive contribution to cancer treatment, such as induced cell apoptosis and cell death [
41,
42]. BPIQ, a synthetic quinoline analog, upregulated ERK phosphorylation leading to H1299 cell death, and this can be abrogated by ERK inhibitor [
43]. In consistent with other studies, our results demonstrated that ERK activation plays important roles in drug-induced cancer cells apoptotic death.
Accumulated evidence demonstrated activated ERK is also involved in autophagic cell death [
26]. 8-CEPQ, a novel quercetin derivative, inhibited colon cancer cell growth by inducing autophagic cell death through ERK activation [
44]. Tan IIA induces autophagic cell death via activation of AMPK and ERK in KBM-5 cells, and ERK inhibitor PD184352 suppressed LC3-II expression induced by Tan IIA [
45]. Rhuscoriaria induced autophagic cell death through p38 and ERK1/2 activation in breast cancer cells [
46]. All the above studies link ERK activation with autophagic cell death. In our study, MEK/ERK inhibitor U0126 effectively abrogated the impaired autophagy flux caused by MTE. Taken together, our results revealed the effect of MTE on cell apoptosis-induction and autophagy-inhibition can partly ascribe to ERK activation.
Authors’ contributions
Study design (LNW, YNJ, DX, SYH, PPL); Biochemical experiments (YNJ, LNW, XJL, ZHT, STJ); Statistical analyses (YNJ, LNW, DX, XJL, ZHT, STJ, SYH); drafting the manuscript (LNW, YNJ, DX, SYH). All authors read and approved the final manuscript.