Ganoderma lucidum Spore Polysaccharide Inhibits the Growth of Hepatocellular Carcinoma Cells by Altering Macrophage Polarity and Induction of Apoptosis

Abstract

Background. Ganoderma lucidum has certain components with known pharmacological effects, including strengthening immunity and anti-inflammatory activity. G. lucidum seeds inherit all its biological characteristics. G. lucidum spore polysaccharide (GLSP) is the main active ingredient to enhance these effects. However, its specific biological mechanisms are not exact. Our research is aimed at revealing the specific biological mechanism of GLSP to enhance immunity and inhibit the growth of H22 hepatocellular carcinoma cells. Methods. We extracted primary macrophages (Mø) from BALB/c mice and treated them with GLSP (800 μg/mL, 400 μg/mL, and 200 μg/mL) to observe its effects on macrophage polarization and cytokine secretion. We used GLSP and GLSP-intervened macrophage supernatant to treat H22 tumor cells and observed their effects using MTT and flow cytometry. Moreover, real-time fluorescent quantitative PCR and western blotting were used to observe the effect of GLSP-intervened macrophage supernatant on the PI3K/AKT and mitochondrial apoptosis pathways. Results. In this study, GLSP promoted the polarization of primary macrophages to M1 type and the upregulation of some cytokines such as TNF-α, IL-1β, IL-6, and TGF-β1. The MTT assay revealed that GLSP+Mø at 400 μg/mL and 800 μg/mL significantly inhibited H22 cell proliferation in a dose-dependent manner. Flow cytometry analysis revealed that GLSP+Mø induced apoptosis and cell cycle arrest at the G2/M phase, associated with the expression of critical genes and proteins (PI3K, p-AKT, BCL-2, BAX, and caspase-9) that regulate the PI3K/AKT pathway and apoptosis. GLSP reshapes the tumor microenvironment by activating macrophages, promotes the polarization of primary macrophages to M1 type, and promotes the secretion of various inflammatory factors and cytokines. Conclusion. Therefore, as a natural nutrient, GLSP is a potential agent in hepatocellular carcinoma cell treatment and induction of apoptosis.

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1. Introduction

Hepatocellular carcinoma (HCC) is the most life-threatening disease worldwide, having high mortality and poor prognosis and an incidence of more than one million cases per year [1]. At present, the treatments for liver cancer are surgery, radiotherapy, and chemotherapy [2]. Its occurrence and development are closely related to various molecular mechanisms in the cell. Recently, an increasing number of chemical drugs and new targeted drugs have been developed. However, some patients are still resistant to drugs. Therefore, the development of new natural medicines is expected to become another strategy for treating liver cancer. Active extracts of various natural medicinal plants have been tested for cancer treatment and have shown good antitumor efficacy [3].

Nowadays, researches have focused on the immunomodulation and antitumor activity of natural products, and this has become the focus of emerging research [4]. Naturally sourced antitumor drugs have been shown to exhibit therapeutic effects and few adverse reactions in tumor therapy. They can repair the body’s immune system and even cure tumors [5]. Ganoderma lucidum, which is also called “Lingzhi” has been used medicinally for more than 2000 years [6] and has been regarded as an effective medicinal compound, reinforcing healthy qi to restore normal function and prolong life and has almost no toxic side effects [7]. G. lucidum spores are microscopic and are ejected from the cap during growth and maturation. These germ cells have all of G. lucidum genetically active substances [8]. Modern pharmacological studies have shown that G. lucidum spores have antitumor effects, increase immune regulation, lower blood sugar and lipid, increase anti-inflammatory and antihypoxia ability, and scavenge free radicals [9].

Macrophages (Mø) play an essential role in humoral and cellular immunity and in maintaining tissue homeostasis [10]. Related studies have found that macrophages are incredibly plastic and can be activated into a series of continuously adjustable functional states under the stimulation of different environments or drugs [11]. Classically activated (M1 type) macrophages and alternatively activated (M2 type) macrophages are the two extremes of this state. The process by which naive (Mφ type) macrophages are stimulated by exogenous factors in specific tissues to differentiate into M1 or M2 macrophages is called macrophage polarization [12]. The dynamic balance between M1 and M2 is vital for maintaining homeostasis. Once the balance is broken, the human body faces a variety of diseases that can sometimes be treated with drugs to regulate these macrophages. The transformation of M1 and M2 macrophages is a dynamic and reversible process. Directional polarization may provide new methods for cancer treatment [13, 14].

Tumor-associated macrophages (TAMs) are similar to the function of immune cells in the tumor microenvironment and mainly infiltrate the tumor matrix to mediate inflammation [15]. The secretion of cytokines, chemokines, growth factors, and proteases and the regulation of intracellular signaling pathways play a vital role in modulating the function of TAMs and tumor cells. The tumor microenvironment combines chronic inflammation, low oxygen levels, nutritional deficiencies, and acidosis, creating extremely complex dynamic systems [16] that regulate tumor growth, proliferation, metastasis, and immune escape. Therefore, we think that the treatment of tumors by reducing the stress state of the tumor’s internal environment and then feeding it back to the tumor cells may promote tumor cell apoptosis or autophagy [17, 18]. Supernatant transfer of various cell cocultures in vitro has been used to mimic the tumor microenvironment [19].

By comparing the content and composition of GLSP and G. lucidum polysaccharides (GLP), we found that the overall structure is similar, but there are still many differences. At present, more than 200 kinds of substances have been separated, of which the largest is β-glucan and a few are α-glucan [20]. Although there have been many studies on GLP, because the shell of G. lucidum spores is hard and difficult to remove completely, we apply a brand-new removal wall technology that makes it possible to extract GLSP with higher purity. GLSP has better physical and chemical properties than GLP, and its application prospects are broader [21, 22]. Besides, GLSP plays a vital role in nourishing and protecting the liver, resisting radiation, resisting gene mutations, and resisting inflammation. Such effects have not been confirmed in GLP-related studies.

In our previous experiments, we found that the G. lucidum spore water extract had no inhibitory effect on H22 liver cancer cells and no cytotoxicity. However, when added to macrophages, it had a significant inhibitory effect on H22 liver tumor cells. To clarify the antitumor mechanism of G. lucidum spores, we studied the antitumor activity of G. lucidum spore polysaccharides (GLSP). We speculate that it is one of the targets of liver cancer, as shown in Figure 1.

[figure omitted; refer to PDF]

The monocyte-macrophage system is an essential part of innate immunity [35, 36]. During inflammation or infection, monocytes in the blood are recruited into the tissue and differentiate into mature macrophages, a group of highly heterogeneous cells. Depending on the microenvironment, macrophages can polarize into different functional phenotypes [37]. According to their different activation states, they are mainly divided into classically activated macrophages (M1 type) and alternatively activated macrophages (M2 type) [38]. The polarization of phagocytic cells is affected by various cytokines in the microenvironment [39]. When the epithelial barrier is destroyed and pathogenic microorganisms invade, a large number of circulating monocytes are recruited under the action of chemokines and differentiate into proinflammatory cells, namely, M1 macrophages, induced by local cytokines [40]. M1 macrophages have potent cytotoxicity; are highly sensitive to LPS; secrete many inflammatory factors and reactive oxygen products, such as IL-6, IL23, and TNF-α; promote inflammation cascades and tissue damage; activate Th1/Th17 adaptive immunity; and promote the elimination of pathogenic microorganisms. M2 type macrophages also increase during the disease, inhibit the inflammatory response, avoid excessive damage to the tissue, and, at the same time, remove pathogenic bacteria and cell debris in the process of inflammation subsiding. They also promote tissue repair and immune balance. The immune balance of the intestine depends on the two types of macrophages working together and coordinating with each other. Therefore, regulating the balance between M1 and M2 macrophages is essential for the occurrence and development of cancer [41].

TAMs are derived from monocytes in the blood system and enter tumor tissues under the action of chemokines [42]. The colony-stimulating factor secreted by tumor cells can prolong the survival time of TAMs. When TAMs are moderately activated in the tumor environment (M1 type), they exert antitumor immune function, which can kill tumor cells and destroy vascular endothelium, thereby inhibiting tumor development. However, if this stimulus is not suppressed in a short time, TAMs will be polarized into M2 type under the action of various cytokines secreted by tumor cells, which is why most TAMs in tumor tissues are M2 type [38]. In contrast to the M1 type, M2 TAMs can secrete growth factors, angiogenesis factors, and proteases, thereby stimulating tumor cell proliferation, promoting angiogenesis and tumor cell invasion and migration, and escaping the surveillance of antitumor immunity [43]. Therefore, the induction of secondary polarization of TAMs in tumor tissues and the transformation of M2 TAMs to M1 have become an essential target for tumor therapy in recent years [44]. Previous research has found that glycopeptide derived from G. lucidum (Gl-PS) could promote polarization of M1 macrophage vs. M2 macrophages [45]. In our macrophage typing experiments, GLSP can increase the number of CD86+ cells, which is an M1 macrophage marker. When H22 tumor cells were cocultured with macrophages, we found that GLSP decreased the number of CD206+ macrophages, an M2 type marker. Overall, when H22 tumor cells are cocultured with macrophages in the TME, GLSP increases the ratio of M1/M2 macrophages. Therefore, we can speculate that GLSP has a regulatory effect on M1 and M2 macrophages in the tumor microenvironment.

Currently, chemotherapy is one of the most common cancer treatments, but it has noticeable side effects [46]. In contrast, “nutritional drugs” are known for their low toxicity. “Nutrition” is a concept that has attracted much attention to prevent and treat diseases [47]. Traditional Chinese medicine (TCM) is a rich source of nutritional medicine that has been used for thousands of years [48]. It has an excellent effect on treating many chronic diseases. Additionally, TCM can also be used as part of a daily diet. TCM is a safe and effective way to prevent and treat diseases. GLSP is one of the foremost effective ingredients in TCM as it has a wide range of therapeutic effects and relatively low toxicity. It is a promising nutritional drug and has attracted wide attention in biomedicine in recent years [49, 50]. The above studies revealed that GLSP activates macrophages to induce apoptosis of H22 hepatocellular carcinoma cell in vitro and the biological mechanism. Next, we will continue to verify the biological activity of GLSP to enhance immunity and antitumor in vivo.

5. Conclusions

In summary, GLSP reshapes the tumor microenvironment by activating macrophages, regulating the polarization of macrophages, and promoting the secretion of various inflammatory factors and cytokines. Moreover, we found that GLSP can block H22 tumor cells in the G2/M phase by activating macrophages and can activate PI3K/AKT signaling pathways to affect the mitochondrial apoptotic pathway and promote tumor cell apoptosis. Therefore, as a natural nutrient, GLSP can alter macrophage polarity and has potential to reshape the tumor microenvironment activity.

Acknowledgments

This work was supported by the National Key R & D Program of China (2018YFC1706800).

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