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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 12  |  Page : 942-947

Gambogic acid-induced autophagy in nonsmall cell lung cancer NCI-H441 cells through a reactive oxygen species pathway


Department of Respiratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China

Date of Web Publication11-Dec-2018

Correspondence Address:
Shujun Wang
Department of Respiratory, The First Affiliated Hospital of Zhengzhou University, No. 1, Jianshedong Road, Zhengzhou 450052
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.206866

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 > Abstract 


Aim of the Study: Garcinia hanburyi is a traditional herbal medicine with activities of anti-inflammation and hemostasis used by people in South Asia. Gambogic acid (GA) is the main active component extracted from it, which has anticancer and anti-inflammatory effects. The aim of the current study is to investigate the molecular mechanisms of GA's effective anticancer activity.
Materials and Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to measure cell proliferation. Apoptosis induced by GA was analyzed by flow cytometry. In addition, monodansylcadaverine (MDC) and 2',7'-dichlorofluorescein diacetate were used to evaluate autophagy and reactive oxygen species (ROS) generation, respectively.
Results: GA could significantly inhibit nonsmall cell lung cancer (NSCLC) NCI-H441 cell growth. In addition, GA induced NCI-H441 cells autophagy, confirmed by MDC staining, upregulation of Beclin 1 (initiation factor for autophagosome formation), and conversion of LC3 I to LC3 II (autophagosome marker). Moreover, generated ROS was induced by GA in NCI-H441 cells and the ROS scavenger N-acetylcysteine reversed GA-induced autophagy and restored the cell survival, which indicated GA-induced autophagy in NCI-H441 cells through an ROS-dependent pathway. In addition, in vivo results further indicated that GA significantly inhibited the growth of NCI-H441 xenografts.
Conclusions: The results shed new light on the interaction between ROS generation and autophagy in NSCLC cells and provide theoretical support for the usage of GA in clinical treatment.

Keywords: Autophagy, gambogic acid, NCI-H441, nonsmall cell lung cancer, reactive oxygen species


How to cite this article:
Ye L, Zhou J, Zhao W, Jiao P, Ren G, Wang S. Gambogic acid-induced autophagy in nonsmall cell lung cancer NCI-H441 cells through a reactive oxygen species pathway. J Can Res Ther 2018;14, Suppl S5:942-7

How to cite this URL:
Ye L, Zhou J, Zhao W, Jiao P, Ren G, Wang S. Gambogic acid-induced autophagy in nonsmall cell lung cancer NCI-H441 cells through a reactive oxygen species pathway. J Can Res Ther [serial online] 2018 [cited 2019 Sep 15];14:942-7. Available from: http://www.cancerjournal.net/text.asp?2018/14/12/942/206866




 > Introduction Top


Autophagy is a physiological cellular mechanism that degrades and recycles cellular components to maintain an adequate amino acid level during nutritional starvation to protect cells from death.[1] Autophagosome nucleation introduced by the PI3 kinase type III-Atg6/Beclin 1 complex, and the elongation monitored by Atg12-Atg5 and Atg8/LC3-phosphatidylethanolamine conjugate systems, which are two key characteristics of autophagy.[2] Other studies have pointed out that autophagy is also induced in the processes of many anticancer therapies and is considered to be a major tumor cell intrinsic resistance mechanism.[3]

Reactive oxygen species (ROS) consisting of hydroxyl radicals, superoxide anions, singlet oxygen, and hydrogen peroxide are highly reactive resulted by the possession of their unpaired valence shell electrons. ROS is a natural by-product generated in the process of cellular metabolism, primarily in the mitochondria. Cancer cells of tumors in advanced stage usually display a high oxidative stress, demonstrating that increased levels of ROS are required for tumor progression and also engender cancer cells with a lower tolerance for ROS. Accumulation of ROS has been reported to associate with the initiation of autophagy and be invariably involved in the outcome of autophagy.[4]

Garcinia hanburyi Hook. f., a small tree of Guttiferae family, is distributed throughout Thailand, Cambodia, India, as well as throughout the Southern part of China. Its resin is used as a dye and also a folk medicine for its potent purgative effects, and for the treatment effects of infected wounds. Besides it was reported that in the 1970s, G. hanburyi Hook resin was already regarded as an antitumor drug via intravenous injection in China for clinical testing.[5] Gambogic acid (GA), a natural product isolated from the resin of G. hanburyi trees in Southeastern Asia, is a caged polyprenylated xanthone.[6] There is accumulated evidence proving that GA has antitumor effects both in vitro and in vivo. GA exhibits remarkable inhibition of proliferation, induction of apoptosis, reversion of multidrug resistance and anti-angiogenesis. GA also constrains nuclear factor kappa B signaling pathway and potentiate apoptosis through interactions with the transferrin receptor.[7] In addition, GA induces ROS accumulation and triggers mitochondrial signaling pathway in human hepatoma SMMC-7721 cells.[8]

Lung cancer is the leading cause of cancer death global, and about 85–90% of lung cancers are nonsmall cell lung cancer (NSCLC). The outcome of the patients with advanced lung cancer remains poor, and new treatment strategy is much needed.[9] In the current study, our findings indicate GA has significant antitumor effect on NSCLC cell growth. However, the molecular mechanisms of GA's effective anticancer activity stay uncertain and wait for further exploration.


 > Materials and Methods Top


Reagents

GA, N-acetylcysteine (NAC), monodansylcadaverine (MDC), 2',7'-dichlorofluorescein diacetate (DCF-DA), 3-methyladenine (3-MA), and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). The purity of GA was 98% (high-performance liquid chromatography), which dissolved in dimethylsulfoxide (DMSO). All drugs were dissolved in sterile DMSO and a 10 mM working solution was prepared and stored in aliquots at −22°C. Beclin 1 and LC3 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other chemicals used in this study were of analytical reagent grade.

Cell growth inhibition assay

NCI-H441 human NSCLC cell line was purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 105 U/L penicillin, and 100 mg/L streptomycin (Hyclone, Logan, UT, USA) at 37°C in an atmosphere containing 5% CO2. The cells were dispensed in 96-well, flat-bottomed microtiter plates (NUNC, Roskilde, Denmark) at a density of 1.5 × 104 cells per well. After 24 h incubation, the cells were treated with the tested agents for the indicated time periods. A volume of 20 μL aliquot of MTT solution (5.0 mg/mL) was added to each well followed by 4 h incubation, and the optical density was measured using an ELISA reader (Tecan Spectra, Wetzlar, Germany).

Measurement of reactive oxygen species generation and autophagy

NCI-H441 cells were treated and then incubated with 10 mM DCF-DA at 37°C for 30 min. The intracellular ROS mediated the oxidation of DCF-DA into the fluorescent compound DCF. Subsequently, the cells were harvested and cell pellets were suspended in 1 mL phosphate-buffered saline (PBS). Samples were taken and analyzed by an FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The fluorescent compound MDC was used as a tracer for autophagic vacuoles. Following incubation with GA for the indicated periods, the cells were cultured with 0.05 mM MDC at 37°C for 1 h and then analyzed by flow cytometry.

Western blotting test

The expressions of Beclin 1 and LC3 in tumor cells were examined by Western blotting. Cells were harvested and lysed in a lysis buffer: 50 mM Tris-HCl, pH 8.0, 0.25 M NaCl, 0.5% NP-40, 1 mM PMSF, 1 ng/mL aprotinin (Boehringer, Mannheim, Germany), 1 ng/mL leupeptin (Boehringer, Mannheim), and 20 ng/mL TPCK (Boehringer, Mannheim). The lysates were centrifuged at 13,000 rpm for 20 min at 4°C and the supernatants were stored at −80°C. Extracts equivalent to 50 μg of total protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% acrylamide) and transferred onto nitrocellulose membranes (Hybond TM-C super, Amersham, UK). The membranes were blocked in Tris-buffered saline-Tween (TBST) (0.2 M NaCl, 10 mM Tris, pH 7.4, 0.2% Tween-20) containing 5% nonfat dry milk and 0.02% NaN3 for 1 h, and then incubated with the first antibodies in TBST containing 5% nonfat dry milk. The membranes were then incubated with the sheep anti-mouse or rabbit Ig (Amersham) in TBST containing 5% nonfat dry milk. Membranes were then washed with 1× PBS-Tween three times and visualized with enhanced chemiluminescence (Amersham, GE Healthcare, Little Chalfont, UK).

Apoptosis assay

Apoptotic cells were analyzed using Annexin V-FITC/PI double-staining methods. In brief, NCI-H441 cells were treated with desired concentrations of GA for 72 h. Cells were digested into single-cell suspension with ethylenediaminetetraacetic acid-free trypsin and stained according to the manufacturer's instruction of Annexin V-FITC/PI Apoptosis Detection kit (Pierce Biotechnology, Rockford, IL, USA). As soon as possible, stained cells were analyzed by flow cytometry.

Animal study

Female Balb/c nude mice of 6–8-weeks-old were purchased from Vital River (Beijing, China). Each nude mouse was subcutaneously implanted with 5 × 106 NCI-H441 cells. When tumor volumes reach 100–200 mm3 in size, tumor-bearing mice were randomized into vehicle (normal saline), 25 or 50 mg/kg GA treatment groups with a cohort of 8 per group. The mice were administrated with vehicle or GA treatments orally once a day for 3 weeks, and the predetermined endpoint was a tumor volume of 1500 mm3. To observe for adverse effects, animals were weighed twice a week. Tumors were measured across two perpendicular diameters, and volumes were calculated using the formula: Tumor size = Length × Width2/2. All animal experiments were performed in accordance with protocols approved by the Zhengzhou University Experimental Animal Care and Use Committee.

Statistical analysis of the data

All results and data were confirmed in at least three separate experiments. Data are expressed as means ± standard deviation and were analyzed by Student's t-test using Statistical Package for Social Sciences (SPSS) software (version 13.0; SPSS, Chicago, IL, USA). Differences were considered to be statistically significant when P < 0.05.


 > Results Top


Gambogic acid dose-dependently inhibited NCI-H441 cell growth in vitro

GA-inhibited NCI-H441 cell growth was shown in a concentration-dependent manner. GA from 10 to 50 μM exerted a potent inhibitory effect on NCI-H441 cell growth. By 72 h after treatment with 40 μM GA, cell death rate reached to almost 90%. The IC50 for 72 h GA treatment was 26.8 μM [Figure 1]a. A significant decrease in the number of cells and morphologic changes, including cell shrinkage and membrane blebbing nuclear, was observed [Figure 1]b.
Figure 1: Inhibitory effects of gambogic acid on NCI-H441 cell growth in vitro. The cells were cultured for 24 h, and then incubated with different concentrations of gambogic acid for 72 h (a). The morphologic changes of cells after treatment with gambogic acid were observed (b). Mean ± standard deviation, n = 5. **P < 0.01 and *P < 0.05

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Gambogic acid-induced autophagy in NCI-H441 cells

MDC is commonly used as a selective fluorescent marker for tracking autophagic vacuoles. Compared with the control group at 72 h, GA-treated group had higher fluorescent density and more MDC-labeled particles in NCI-H441 cells [Figure 2]a, which indicated that GA induced an increased MDC recruitment of autophagosomes in the cytoplasm of the cells. In addition, the specific autophagy inhibitor 3-MA could reduce the MDC-positive cells in GA-treatment groups. Once autophagy initiated, phosphatidylethanolamine will covalently bind to the cytosolic protein LC3 I to yield LC3 II, and this conversion is often used as a marker for autophagy.[10] To further verify the autophagy-enhancing effect of GA, Western blot analysis was conducted to reveal the expression of Beclin 1 and the conversion of LC3 proteins [Figure 2]b. This result also showed that GA was able to provoke autophagy in NCI-H441 cells.
Figure 2: Gambogic acid induced autophagy in NCI-H441 cells. NCI-H441 cells were treated with gambogic acid at the indicated concentration for 72 h, and then the cells were suffered to quantitative analysis detected a positive ratio of monodansylcadaverine staining by flow-cytometric (a), or Western blot analysis (b). Mean ± standard deviation, n = 5. *P < 0.05 versus control. **P < 0.01 and *P < 0.05

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Gambogic acid induced reactive oxygen species generation in NCI-H441 cells

The production of ROS was evaluated by a flow cytometric analysis with the ROS fluorescent probe DCF-DA. In GA-treated NCI-H441 cells, the DAF-2T-positive cell ratio increased over time. These data indicated that GA could induce ROS production, which was obviously reduced by an ROS scavenger, NAC. In addition, inhibition of autophagy had no effect on ROS generation [Figure 3]a. Meanwhile, the result of MTT assay showed that NAC could reverse GA-induced cell growth inhibition in NCI-H441 cells [Figure 3]b. In addition, the result of flow cytometry indicated that NAC treatment decreased the MDC-positive ratio [Figure 3]c.
Figure 3: Gambogic acid-induced autophagy through reactive oxygen species generation in NCI-H441 cells. Reactive oxygen species generation was detected by a flow-cytometric staining with 2',7'-dichlorofluorescein diacetate (a). NCI-H441 cells were incubated with different concentrations of gambogic acid or co-incubated with N-acetylcysteine, and the cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (b). The monodansylcadaverine fluorescent intensity of treated cells was analyzed by flow cytometry (c). **P < 0.01 and *P < 0.05

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Gambogic acid could not induce apoptosis in NCI-H441 cells

Annexin V-FITC/PI double staining methods were performed to investigate whether GA would induce apoptosis in NCI-H441 cells. The results showed that GA could not induce apoptosis in NCI-H441 cells [Figure 4]a and [Figure 4]b.
Figure 4: Gambogic acid could not induce apoptosis in NCI-H441 cells. NCI-H441 cells were treated with the indicated dose of gambogic acid for 72 h, and then cells were suffered to Annexin V-FITC/PI double staining methods (a). Quantification of the results of Annexin V-FITC/PI double staining (b). Mean ± standard deviation, n = 5

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Gambogic acid inhibited tumor growth in NCI-H441 xenografts

Since the significant inhibitory effect of GA on NCI-H441 cells was observed in vitro, we next investigated if GA has in vivo inhibitory action. The mice were randomized into three groups: control, 25 or 50 mg/kg GA. We then balance the average tumor volume and average body weight in each group. The results indicated GA inhibited NCI-H441 xenografts tumor growth dose-dependently [Figure 5]a. At the end of in vivo study, the mice were sacrificed and the tumors were isolated [Figure 5]b and [Figure 5]c. These in vivo results consist with in vitro findings and further verified antitumor effect of GA on NCI-H441 cancer.
Figure 5: Gambogic acid inhibited tumor growth in NCI-H441 xenografts. Mice bearing NCI-H441 tumors were either treated with vehicle 25 or 50 mg/kg gambogic acid (orally once a day) (a). Tumors were harvested and weighted at the end of in vivo study (b and c). Mean ± standard deviation, n = 8. **P < 0.01 and *P < 0.05

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 > Discussion Top


GA was reported to exert significant antiproliferative and proapoptotic effects on a variety of human cancer cell lines in vitro and in vivo.[7] Although its anticancer mechanisms are not clear yet, GA is already eligible for clinical trials in China.[11] In this study, we found that GA is capable of initiating autophagy in NCI-H441 cells in a dose-dependent manner. Autophagy, a highly regulated biological process responsible for clearing damaged or long-lived proteins and organelles, plays essential roles in tissue homeostasis, development, and disease.[12] Luo et al. reported that inhibition of autophagy enhances the growth inhibition and pro-apoptotic effect of GA in glioblastoma cells.[13] Nevertheless, we found that inhibition of autophagy reduced GA-induced cell growth inhibition in the current study. The novel finding of the current study is that GA-induced autophagy in NCI-H441 was mediated by ROS generation. Our finding is different from Pandey's report; the possible reason would be due to different cell type.

In recent years, GA-induced ROS accumulation was found in human hepatoma SMMC-7721 cells, ovarian cancer cell line (SKOV-3), and also in multiple myeloma RPMI-8226 cells, resulted in apoptosis.[8] Likewise, ROS was also involved in GA-induced autophagy. As the first step, we examined intracellular ROS levels of NSCLC cells treated with and without GA. And then to further evaluate the potential contribution of ROS in GA-induced autophagy, NAC was applied to block intracellular ROS generation. The results demonstrated that NAC treatment completely inhibited GA-induced ROS production; meanwhile, GA-induced autophagy was attenuated significantly. All these results indicated that ROS generation was a requirement for GA-induced autophagy in NCI-H441.

Luo et al. also reported autophagy inhibition promotes GA-induced suppression of growth and apoptosis in glioblastoma cells.[13] Although autophagy and apoptosis are two different cellular processes often with opposing outcomes, they could still be induced by same stimuli and are extensively interconnected through various crosstalk mechanisms. However, our study showed that GA could not provoke apoptosis in NCI-H441 cells. The detailed mechanisms between ROS, autophagy, and apoptosis in this case are still unclear and needed further investigation. Previous studies have different views as for the role of autophagy in cancer treatment. There are two types of autophagy: the first one acts as a protective mechanism against apoptosis, which is called protective autophagy; the other is autophagic cell death that can induce cancer cell death.[14] In the current study, GA-induced autophagy in NCI-H441 cells belong to the latter type of autophagy. Meanwhile,in vivo results indicated GA dose-dependently inhibited NCI-H441 xenografts tumor growth, which further verified antitumor effect of GA on NCI-H441 cancer. To the best of our knowledge, this is the first study to show the role of ROS and autophagy during the inhibitory action of GA on NSCLC.


 > Conclusion Top


Our study revealed GA-induced autophagy in NCI-H441 NSCLC cells in vitro and in vivo, and the inhibition of ROS generation reduced the anticancer, as well as autophagy effects induced by GA in the cells, which will shed new light on cancer treatment using GA in clinical practice.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Pao W, Girard N. New driver mutations in non-small-cell lung cancer. Lancet Oncol 2011;12:175-80.  Back to cited text no. 9
    
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Zhou Z, Wang J. Phase I human tolerability trial of gambogic acid. Chin J New Drugs 2007;16:79.  Back to cited text no. 11
    
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Luo GX, Cai J, Lin JZ, Luo WS, Luo HS, Jiang YY, et al. Autophagy inhibition promotes gambogic acid-induced suppression of growth and apoptosis in glioblastoma cells. Asian Pac J Cancer Prev 2012;13:6211-6.  Back to cited text no. 13
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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