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