The Potential of Ganoderma applanatum Polysaccharides Extracts on Apoptosis, Necrosis, TNF-α, and IL-6 in Diethylnitrosamine-Induced Mice
2Magister Program in Biology, Faculty of Science and Technology,
Universitas Airlangga, Surabaya, 60115, Indonesia.
4Faculty of Medicine, Universitas Airlangga, Surabaya, 60115, Indonesia.
6Department of Biology, Faculty of Science and Technology,
Universitas Airlangga, Surabaya, 60115, Indonesia.
*Corresponding Author E-mail: windarmanto@fst.unair.ac.id
ABSTRACT:
Carcinogenic Diethylnitrosamine (DEN) compounds are a class of nitrosamine compounds that have a negative impact on the body, often found in food preservatives. The increasing amount of DEN will damage the spleen organ so that the functional spleen organ decreases. The damage can include degeneration, intracellular accumulation, necrosis, and inflammation. Pro-inflammatory cytokines including TNF-α and IL-6 have an effect on tumor growth, IL-6 cytokines function as trophic factors for malignancy of cancer cells, TNF-α plays a role in cancer cell inflammation. Polysaccharides contained in G. applanatum such as ß-glucan have immunomodulating properties that can be developed as a strong biological response for cancer therapy. This study will examine the potential of G. applanatum polysaccharide extract on the percentage of apoptosis, necrosis, TNF-α and IL-6 levels in DEN-induced mice. Female DDY mice were randomly divided into 4 groups (n=6). The research method was carried out by comparing the normal control group (KN) given water; negative control (K-) given DEN 100mg/kg BW; positive control (K+) given DEN 100mg/kg BW and Doxorubicin 10 mg/kg BW; and treatment (P) given DEN 100mg/kg BW and G. applanatum extract 150mg/kg BW. G. applanatum polysaccharide extract showed a significant increase in the percentage of apoptosis and necrosis. G. applanatum polysaccharide extract 150mg/kg BW showed a significant decrease in TNF-α and IL-6 levels. In summary, the polysaccharides extract of G. applanatum are promising potential anticancer treatment.
KEYWORDS: ß-glucan, Inflammation, Apoptosis, Necrosis, Immunomodulatory.
INTRODUCTION:
Diethylnitrosamine (DEN) compounds are a class of nitrosamine compounds that result from the reaction of nitrite with secondary amines and are carcinogenic. This compound is commonly found in food preservatives such as meat, sausages, corned beef and salted fish. The increasing amount of DEN will damage the spleen organ so that its function decreases. These free radicals cause damage to cell tissue and cell organelles in the form of degeneration, intracellular accumulation, necrosis, and inflammation. Free radicals can be believed to cause cell membrane lipid peroxidation, DNA damage, and apoptosis1,2,3.
There are two types of cell death, namely apoptosis and necrosis. Programmed apoptosis serves to eliminate harmful, damaged cells without the need to cause an inflammatory response or tissue damage. Apoptosis happens through two pathways, the extrinsic pathway or death receptor (DR) pathway engaging the immune system and the intrinsic pathway that involves the mitochondria. Necrosis causes damage to the cell membrane resulting in a failure of homeostasis causing cell swelling, organelle damage, and rupture of the cell membrane, so that all cell content escapes into the extracellular fluid. This widespread tissue damage in necrosis causes inflammation4.
Inflammation can alter cell growth dynamics. Previous studies have pointed to a link between inflammation and cancer as demonstrated in inflammatory bowel disease (IBD). IBD patients show overexpression of inflammatory genes, like nuclear factor kappa B (NF-κB). Activation of NF-κB promotes overexpression of cell cycle genes, apoptosis inhibitors and proteases5. Interleukin 6 and Tumor necrosis factor-alpha play an essential role in the clinical and immunopathological manifestations of IBD. IL-6 inhibits apoptosis, resulting in faster cell proliferation causing a very strong immune response, functioning as a trophic factor for malignancy in the epithelium. Research on the immunostimulatory effects of Okra Raw Polysaccharide Extract (ORPE) on the production of proinflammatory cytokine TNF-α. TNF-α is involved in cancer inflammation. The levels of TNF-α significantly increased in the ORPE group at doses of 50, 100, and 200 mg/kg BW, compared to normal control and negative control6.
Endogenous antioxidants can fight free radicals but the amount is very limited. Therefore, additional exogenous antioxidants are needed to strengthen endogenous antioxidants. Natural ingredients as a source of exogenous antioxidants are appropriate to overcome various diseases related to free radicals. Ganoderma applanatum is a fungus that grows on wood with a yellow or brown fan-like shape on the inner layer. Treatment using mushrooms has been the focus of several studies this year7,8,9. Ganoderma acts as antihepatitis, antihypertensive, anti-aging, antidiabetic, anti-inflammatory, immunomodulatory and antioxidant10. Mice given G. applanatum extract did not experience signs of toxicity even though the dose given reached 2,000 mg/kg BW11. Another study showed that the administration of G. applanatum extract 2,000 mg/kg BW did not cause death in experimental animals, indicating that G. applanatum extract is non-toxic12.
The ß-glucan-type polysaccharides extracted from the basidiocarp and mycelium of G. applanatum function as antioxidants and reduce oxidative damage due to direct exposure to free radicals by triggering increased immunity13. These polysaccharides can inhibit the release of histamine and are anti-inflammatory in nature which plays a role in reducing the levels of TNF-α and IL-6 as proinflammatory cytokines. Low levels of these cytokines can prevent cell necrosis. Previous research on the hepatoprotective effect of crude extract of G. applanatum polysaccharides on hepatic fibrosis of mice induced by carbon tetrachloride. Giving various doses of crude extract of G. applanatum polysaccharides can avoid an increase in the percentage of necrosis, collagen density, and improve the histological picture of mice hepatic fibrosis14. This research is a development of this research by observing the potential of G. applanatum on the percentage of apoptosis, necrosis, TNF-α and IL-6 levels in mice induced by DEN. Thus, results of this study are expected to be one of the first steps in efforts to provide safe and affordable nutraceutical supplements as a companion to chemotherapy.
Preparation of crude extract G. applanatum:
The part used as polysaccharide material is the basidiocarp. G. applanatum mushroom was washed and cut into pieces. Then, the mushrooms were sun-dried at 300°C- 500°C for 14 days. The pieces of G. applanatum mushroom ground into powder. G. applanatum powder was weighed and boiled in water at 600°C - 650°C for 6 hours. The ratio of G. applanatum powder to water was 1:6, then G. applanatum decoction was filtered and the precipitate obtained was re-extracted at the same time and temperature. This process was carried out twice. The filtrate obtained from each extraction was mixed and then heated at 250°C- 300°C for 24hours. Then, the supernatant was freeze dried.
Measurement of apoptosis and necrosis percentages according to previous research15. The centrifugation results will separate the supernatant and pellet which is the cell. Then, supernatant was decanted and the pellet in each microtube was washed twice with BioLegend's Cell Staining Buffer. Cells were centrifuged at 1,500 rpm at 4°C. Afterward, cells were resuspended in Annexin V Binding Buffer at a concentration of 0.25-1.0 x 10cells/mL. Aliquots of 100μL of cell suspension was transferred into a 5mL test tube, followed by the addition of 5μL FITC Annexin V and 10μL Propidium Iodide Solution, and the mixture was vortexed. The samples were incubated for 15 minutes at room temperature (25°C) in dark conditions. Subsequently, 400μL Annexin V binding buffer was added to each test tube and the cells were analyzed by using a BD FACS Verse flowcytometer (Becton Dickinson Bioscience).
The upper layer containing serum was collected by centrifuging whole blood at 2000-3000rpm for 20 minutes. TNF-α and IL-6 cytokine was analysis using an ELISA kit (BTLab, Shanghai, China). Following the manufacturer’s protocol, approximately 40μl of blood serum was placed into the wells and 50μl of streptavidin-HRP and 10μl of antibodies (anti-TNF-α; anti-Il-6) were added to each well. The mixture was incubated at 37°C for 60 minutes. Afterward, the plate was washed five times with wash buffer and 50μl of substrate solution A and B was added to each well. The plate covered with a new seal was incubated at 37°C for 10minutes in the dark. Then, 50μl of stop solution was added to each well, the previously blue color will immediately turn into yellow. The optical density (OD) value was measured using a microplate reader at a wavelength of 450 nm after the stop solution was added.
Apoptosis and necrosis percentages:
Spleen cells were isolated and stained using Annexin-V and PI antibodies. The flow cytometry’s results (Figure 1) showed that cells expressing Annexin-V-PI+ (upper left quadrant) showed necrotizing cells. The lower right quadrant, showing Annexin-V+PI-, represents cells in the early stage of apoptosis, while the upper right quadrant, with Annexin-V+PI+, indicates cells in the late stage of apoptosis. The total number of apoptotic cells is calculated by combining the cells in both early and late apoptotic phase. In Fig.1 the percentage of apoptosis in the KN cell group was 13.43%, the K- group 9.06%, the K+ group 10.42%, and the P group 12.87%. Meanwhile, the flow cytometry results for the necrosis phase in the KN group amounted to 6.79%, the K- group 4.11%, the K+ group 7.03%, and the P group 6.99%.
Fig. 2. Apoptosis percentages (%) after treatment. KN: normal control group (water), K-: negative control group (DEN 100mg/kg BW), K+: positive control group (DEN 100mg/kg BW and doxorubicin 10 mg/kg BW); P: treatment group (DEN 100 mg/kg BW and G. applanatum extract 150mg/kg BW). The different letter indicated a significant difference based on Duncan’s Test (α<0.05).
Effect of G. applanatum extract on TNF-α and IL-6 level:
Based on measurement of TNF-α and IL-6 levels in serum, there were differences after treatment with G. applanatum polysaccharide extract. It can be seen in Figure 4 and Figure 5, the administration of G. applanatum polysaccharide extract can reduce TNF-α and IL-6 level significantly (p<0.05) when compared to the negative group. Compared to the doxorubicin group, the administration of DEN 100mg/kg BW+G. applanatum polysaccharide extract at a dose of 150 mg/kg BW was not able to significantly reduce TNF-α and IL-6 level.
Fig. 5. IL-6 level after treatment. KN: normal control group (water), K-:negative control group (DEN 100mg/kg BW), K+: positive control group (DEN 100mg/kg BW and doxorubicin 10 mg/kg BW); P: treatment group (DEN 100mg/kg BW and G. applanatum extract 150mg/kg BW). The different letter indicated a significant difference based on Duncan’s Test (α<0.05).
Diethylnitrosamine (DEN) is a carcinogen nitrosamine compound16. DEN administration causes mitochondria to generate Reactive Oxygen Species (ROS), which triggers the release of cytochrome c and initiates apoptosis. If the activity of apoptosis continues to occur, the spleen cells will experience necrosis. The spleen (lien) is the site of lymphocyte cells and the first place an adaptive immune response takes form17. The spleen can produce antibodies and macrophages as a form of defense against infiltrating antigen. Splenocytes, the immune cells in the spleen, include T lymphocytes, B lymphocytes, macrophages, NK cells, and dendritic cells18. Lymphocyte activation occurs due to the immune response, aided by macrophages and NK cells, through the production of cytokines like IFN-γ and TNF-α19. The flow cytometry results in Fig. 1 show a decrease the number of apoptotic and necrotic cells in the K- compared to the KN group after treatment with 100 mg/kg BW of DEN. The percentage of apoptosis and necrosis can be observed in Fig. 2 and 3. This situation occurs because the exposure to DEN that enters the bloodstream will damage the structure and function of proteins in cells, so the number of necrosis cells due to DEN is greater than the normal group. Other research states that necrosis can occur due to chemical compounds that cause cell membrane damage and failure of homeostasis, followed by the entry of water and ions around the cell which causes cell swelling, organelle damage, and rupture of the cell membrane, thus the entire cell content comes out into the extracellular fluid4.
When DEN enters the body through the blood, it can evoke organ damage. If the body is exposed to DEN, it will result in an increase in free radicals that cause cellular oxidative stress. Oxidative stress arises from an imbalance between prooxidants and antioxidants20. Oxidative stress can contribute to tissue damage, even damaging cell organelles such as mitochondria, nuclei and other organelles. Free radicals are known to lead to lipid peroxidation in cell membranes1,2,3. Oxidative damage to lipid compounds occurs when free radical compounds react with PUFA (Poly Unsaturated Fatty Acids) compounds. Lipid peroxidation can be identified by the detection of its byproducts, such as Malondialdehyde (MDA), lipid hydroperoxides, isoprostanes17. If the oxidant substance is more abundant due to the greater concentration of DEN, it causes the cell membrane to rupture so that large molecules can enter and irreversible necrosis occurs. In apoptosis, cell dissolution occurs which results in biochemical changes and cell morphology, intracellular material is not dispersed out of the cell. Several studies related to DEN carcinogenesis revealed that DEN induction with a concentration of 50mg/kg BW in rats for 16 weeks caused fibrosis, DNA became damaged but cell proliferation continued21. The effect of DEN exposure on colon organ function in experimental animals to understand the pathogenetic changes underlying colon cancer formation22. Another study was also conducted by the injection of DEN at a concentration of 200 mg/kgBB for 42 days resulting in significantly increased colon weight and the appearance of microscopic changes for studies treated in mice23.
In this study, serum levels of TNF-α and IL-6 (Fig. 4 and 5) showed a significant increase in the K- group compared to KN due to the effect of DEN. The presence of ROS due to DEN induction can also affect the activation of NF-κB, a transcription factor crucial for regulating innate immunity. The NF-κB pathway is one of the primary pathways activated in response to proinflammatory cytokines, including TNFα, IL-1β, IL-6 and IL-18, particularly through TLR activation by pathogen associated molecular patterns (PAMPS)24. Activation of these pathways are essential for inflammation, as they regulate genes that encode proinflammatory cytokines, adhesion molecules and chemokines. Inflamed cells geenrate ROS, which can lead to DNA damage and genomic instability, increasing the risk for cancer. Additionally, IL-6 promotes cell proliferation and inhibits apoptosis more rapidly than it induces cell death. Modulation of IL-6 expression has an effect on tumor growth and serves as a trophic factor for tumor malignancy. It is widely known that TNF-α induces hemorrhagic necrosis in a select group of tumor types25.
G. applanatum polysaccharide extract in mice can reduce the damage caused by DEN exposure, which of course must pay attention to the concentration or dose to be given. The concentration of G. applanatum polysaccharide extract used was 150 mg/kg BW. Fig. 2 and 3 indicate a significant increase in the percentage of apoptosis and necrosis in the treatment group compared to the K- group. Treatment with G. applanatum polysaccharide extract was also able to have an effect on TNF-α and IL-6 levels. The study results showed a significant reduction in TNF-α and IL-6 levels in serum compared to K-. Ganoderma has the ability as an anti-inflammatory, immunomodulator and antioxidant10. Ganoderma functions as an antioxidant and reduces oxidative damage due to direct exposure to free radicals by stimulating to enhance immunity13. In addition, it can repair cell damage and help cell regeneration in damaged tissues. G. applanatum polysaccharides are able to stimulate the spleen by producing antibodies and macrophages as a form of resistance to antigen26. The results of this study align with other research that demonstrated the effectiveness of G. applanatum polysaccharide treatment in reducing inflammation caused by CCl4 induction by lowering TNF-α and IL-6 levels in mice with kidney and liver fibrosis14,27.
Polysaccharide compounds derived from G. applanatum have the ability as anticancer with its role to induce apoptosis, necrosis, and as immunomodulatory. Further research can be carried out with several concentrations of G. applanatum polysaccharide extract in order to get optimal results on its effect on TNF-α and IL-6 levels.
The support of this study was granted by International Collaboration Research Fund from Universitas Airlangga (1136/UN3.14/LT/2019).
The authors state that there are no conflict of interest.
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Received on 25.01.2024 Revised on 13.07.2024 Accepted on 22.10.2024 Published on 28.01.2025 Available online from February 27, 2025 Research J. Pharmacy and Technology. 2025;18(2):809-814. DOI: 10.52711/0974-360X.2025.00119 © RJPT All right reserved
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