Ganoderic acid DM induces autophagic apoptosis in non-small cell lung cancer cells by inhibiting the PI3K/Akt/mTOR activity

https://doi.org/10.1016/j.cbi.2019.108932Get rights and content

Highlights

  • Ganoderic acid DM, a natural compound, induced apoptosis of A549 and NCI–H460 cells.

  • Ganoderic acid DM promoted autophagic flux, which contributed to it induced cell death in NSCLC.

  • Ganoderic acid DM increased autophagy and apoptosis through inhibition of Akt/mTOR pathway.

Abstract

The incidence and mortality of lung cancer are the highest among cancer-related deaths. However, the long-term use of currently available cytotoxic drugs can increase genetic alterations in cancer cells and cause drug-resistance, which significantly limits their usage. Since current systemic treatment options are limited, effective chemotherapeutic agents are urgently needed for non-small cell lung cancer (NSCLC) treatment. In this study, we demonstrated that ganoderic acid DM (GA-DM) could increase apoptosis in A549 and NCI–H460 NSCLC cells. GA-DM treatment decreased the protein expression levels of Bcl-2 and increased the expression levels of Bax, cleaved caspase-3 and cleaved PRAP. Furthermore, GA-DM could promote autophagic flux, and the cytotoxic effect against cancer cells of GA-DM was significantly inhibited by targeted suppression of autophagy, suggesting that autophagy contributed to GA-DM-induced cell death in NSCLC. Moreover, GA-DM clearly induced autophagy by inactivating the PI3K/Akt/mTOR pathway. When overexpression of Akt reactivated Akt/mTOR pathway in A549 or NCI–H460 cells, the increase of autophagy related marker LC3B-II and apoptosis related protein cleaved PARP and cleaved caspase 3 and the ration of apoptotic cells by GA-DM was reversed, suggesting that GA-DM promoted autophagy and apoptosis by inhibiting Akt/mTOR pathway-mediated autophagy induction. In conclusion, our study indicated that GA-DM can induce autophagic apoptosis in NSCLC by inhibiting Akt/mTOR activity. (209 words).

Introduction

According to global cancer data, lung cancer accounts for approximately 13% of all new cancers and was the leading cause of cancer-related mortality in 2012 [1]. Although the incidence and mortality of lung cancer have stabilized, it remains the most prevalent cause of cancer-related death in American patients. At least 27% of cancer-related deaths were due to lung cancer in 2015 [2]. In China, lung cancer has always ranked first in the mortality rate of malignant tumours, accounting for 24.41% of the total number of malignant tumour deaths, and its mortality rate has also shown an increasing trend [3]. In 2015, the annual incidence of lung cancer in Chinese men and women was 50.9 per 100,000 people and 22.4 per 100,000 people, respectively, which is the most important cause of death among cancer patients [4]. Approximately 85% of patients with lung cancer have been diagnosed with non-small cell lung cancer (NSCLC), and the majority of patients are diagnosed at advanced stages [5]. For many years, cytotoxic drugs such as platinum-based antineoplastic paclitaxel, docetaxel and gemcitabine were used for the treatment of NSCLC patients [[6], [7], [8]]. However, the long-term use of these cytotoxic drugs can increase the genetic alterations in cancer cells and induce drug-resistance, which significantly limit their usage [9,10]. Since current systemic treatment options are limited, effective chemotherapy agents are urgently needed for NSCLC treatment.

Natural products extracted from herbs are one of the important original sources for the development of anticancer drugs. Ganoderma lucidum (G. lucidum) is one an important Asian fungi that is known as the reishi mushroom in Japan and Ling Zhi in China and Korea [11]. Although G. lucidum has been used to improve health and promote longevity in traditional medicine, its potential therapeutic effects, including anti-tumour, anti-HIV, anti-myocardial ischaemia, regulation of blood lipids, hypoglycaemia, sedation and liver protection, were discovered for the treatment of a variety of diseases [12,13]. Ganoderic acid DM (GA-DM) is a type of ganoderic acid and is the main anticancer component in G. lucidum. It has been reported to have biological activity against many kinds of tumours, such as prostate cancer, melanoma and breast cancer [[14], [15], [16]]. However, an anti-NSCLC effect has not been reported to date. Moreover, its anti-tumour mechanism is not clear.

Necroptosis, apoptosis and autophagic cell death are the three types of programmed cell death. Once apoptosis or necroptosis is initiated, the final destiny of cells is death [17]. However, autophagy exhibits bidirectional roles in cell destiny determination depending on the duration and intensity of inducers [18]. Autophagy is a conservative eukaryotic cell stress system characterized by increased production of autophagic vesicles to remove longevity proteins and damaged organelles that are eventually digested in lysosomes. Moderate and controlled autophagy can help cells to adapt to stress stimuli such as nutrient deficiency or reactive oxygen species accumulation and consequently promote cell survival. However, excessive autophagy can impair necessary cellular processes, thereby activating apoptosis or necroptosis and ultimately leading to cell death, which is commonly referred to as autophagic death [19]. Several studies have reported autophagic cell death as the mechanism of many anticancer reagents [[20], [21], [22], [23]].

In this study, we verified the effect of GA-DM on apoptosis induction in NSCLC cells and further demonstrated that apoptosis led to growth inhibition in NSCLC cells under GA-DM treatment. GA-DM could also induce autophagy, which may contribute to the apoptosis observed in NSCLC cells. Moreover, we found that GA-DM could activate autophagic apoptosis in an Akt/mTOR-dependent manner.

Section snippets

Reagents

GA-DM was purchased from Shanghai U-sea Bio-tech co., Ltd. (Shanghai, China). 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and ethidium bromide were purchased from Keygen Biotech (Nanjing, China). Anti-Bcl-2, anti-Bax, anti-caspase 3, anti-PRAR, anti-LC3B, anti-p-Akt (Ser473), anti-Akt 1/2/3, anti-p-mTOR (Ser2448), anti-mTOR, anti-p-PI3K (Try 458), anti-PI3K, anti-BECN1 and anti-β-Actin antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA). The eukaryotic

GM-DM inhibits the proliferation of NSCLC cells

MTT assays were performed to determine the anti-proliferative effect of GA-DM on NSCLC cells (A549 and NCI–H460 cell lines). As shown in Fig. 1A and B, GA-DM inhibited the cell viability of both cell lines in a concentration and time-dependent manner. When the cells were treated with GA-DM (40 μM) for 24, 48 and 72 h, the cell viability of A549 cells was 66.0 ± 1.2%, 47.3 ± 1.6% and 24.9 ± 1.8%, respectively, while NCI–H460 cells showed 77.5 ± 4.2%, 43.9 ± 5.6% and 20.2 ± 8.8% viability,

Discussion

As an herbal medicine, G. lucidum is widely used for the treatment of multiple diseases because of its anti-inflammatory and antioxidant activities against inflammation-associated diseases, cancers, and cardiovascular and cerebrovascular diseases [26]. The main category of biologically active compounds produced by G. lucidum are triterpenoids, which are known as ganoderic acids. GA-DM is extracted from the G. lucidum mushroom and is a potential therapeutic candidate for the treatment of a

Conclusion

In this study, we indicated that GA-DM activates autophagic apoptosis in NSCLC cells by inhibiting the Akt/mTOR pathway. Inhibition of autophagy leads to apoptosis resistance in GA-DM-treated NSCLC cells, revealing a novel anti-tumour mechanism of GA-DM and providing a theoretical foundation for the clinical application of GA-DM.

CRediT authorship contribution statement

Junbo Xia: Formal analysis, Writing - original draft. Jing Zhu: Conceptualization, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Medical Health Science and Technology Project of Zhejiang Province (Grant No.2019RC069), the Program of Zhejiang University of Traditional Chinese Medicine (Grant No.2018ZY24), and the Science and Technology Development Programme of Nanjing Medical University (Grant No. 2017NJMU086). The authors alone are responsible for the content and writing of the paper.

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