Structural Elucidation And Antitumor Mechanisms Of Polysaccharides Isolated From Lentinus Edodes

Lentinus edodes, which belongs to fungus of basidiomycotina and agaricales, is famous as a source of medicine and food. Lentinan was considered the most effective biological active ingredient isolated from the fruitbodies of Lentinus edodes with the properties of immunomodulatory, antitumor, antioxidation, antivirus and regulating blood lipid. However, lentinan was limited in clinical application and was just be used for the adjuvant therapy of cancer as an immunomodulatory agent due to its large molecular weight, poor solubility, complicated spatial structure and uncertain anti-tumor mechanisms. Moreover, there was lack study of structure and activity of lentinan. Besides the immunomodulation function, previously, we fortunately found that inducing tumor cell apoptosis by polysaccharide from L. edodes might provide an attractive strategy for antitumor drug development and less frequent appearing side effects of clinical chemotherapy drugs. Therefore, excavating the medicinal value of mushroom fully and searching for antitumor polysaccharide with low toxicity and high efficiency, and further exploring the direct antitumor mechanisms of lentinan was of great significance. In this study, we extracted the crude polysaccharides by water extraction-alcohol precipitation and alkaline extraction-alcohol precipitation from Lentinus edodes. After decolorization, removing proteins and ultrafiltration, five purified polysaccharide fractions, which named SLNT1、SLNT2、 JLNT1、JLNT2and JLNT3respectively, are obtained. We used a combination of chemical analyses, spectral analyses and modern instrumental analysis techniques to identify the purity, primary chemical structures and the carbohydrate chain conformation. Experiments in vitro were employed to investigate the antitumor activities of five kinds of polysaccharide fractions against different tumor cells. Moreover, we discussed the antitumor activity in vivo and the possible molecular mechanisms of inducing tumor cell apoptosis through H22tumor-bearing mice model. This study would lay a solid foundation for the preparations of new dosage forms of lentinan and the application in the field of medicine, food and health care. Part I The refined polysaccharide fractions of SLNT1, SLNT2, JLNT1, JLNT2and JLNT3We extracted crude polysaccharides SLNT and JLNT by water extraction-alcohol precipitation and alkaline extraction-alcohol precipitation from the fruitbodies of lentinus edodes, which was purchased from xixia, Henan province. After decolorization and ultrafiltration, five different and refined polysaccharide fractions with uniform molecular weight that named SLNT1、SLNT2、JLNT1. JLNT2and JLNT3were obtained. The sugar content, UV scanning and determination of molecular weight were used to identify the samples’purity. The sugar contents of the five polysacchfaride fractions were96.89%、98.64%、95.12%、97.32%and101.56%by phenol-sulfuric acid method. Based on the results of HPGPC, it was found that the relative molecular weight of the five components were6.17×105Da,9.76×104Da,6.39×105Da,2.74×105Da and1.51X105Da respectively. UV scanning showed that they didn’t contain nucleic acids and proteins. In short, results indicated that the five refined components obtained not only have good traits and high purity, but also meet the requirements of the follow-up experiments.Part II Study on the primary structure and carbohydrate chain conformation of Lentinus edodes polysaccharideSpectral analyses and chemical analyses were employed to investigate the primary structure and carbohydrate chain conformation of the five polysaccharides. Infrared spectroscopy (IR) scanning showed the characteristic absorption peaks and the sugar residues’configuration of each purified polysaccharide. Complete acid hydrolysis was used to determine the monosaccharide composition. Periodic acid oxidation and Smith degradation analyzed the type and proportion of their sugar residues. Methylation analysis further confirmed the sugar residues’ ratio. Compared the changes of the maximum absorption wavelength λmax that contributed by the complexes formed between the five fractions with congo red respectively under the condition of different concentrations of NaOH solutions through congo red experiments to determine the carbohydrate chain conformation of the five polysaccharides. IR scanning indicated the characteristic absorption peaks of polysaccharide and the structure was (β-D-pyranose. Results demonstrated that SLNT1, SLNT2, JLNT1, JLNT2and JLNT3were composed of glucose, and chemical analysis indicated that each of them contains four types of sugar residue. The proportion (1-3and1-3-6:1-6and end) of them were about3.48:1,0.84:1,2.85:1,2.69:1and2.92:1. According to results above, we inferred that SLNT1, JLNT1, JLNT2and JLNT3mainly consist of (1→3)-glucose main chains and (1→6)-glucose side chains, and they had similar structures. Nevertheless, SLNT2was composed of (1→6)-glucose main chains and (1→3)-glucose side chains due to its less β-(1→3)-linkages. It could be speculated preliminarily that all of the five polysaccharides had triple-helix conformation through Congo red experiment.Part Ⅲ Study on the antitumor activity of Lentinus edodes polysaccharide both in vivo and in vitroWe examined the growth inhibition effects of polysaccharides SLNT1, SLNT2, JLNT1, JLNT2and JLNT3on H22, HepG2, SMMC-7721and MKN45cells in vitro by MTT colorimetric method. Results indicated that SLNT1, JLNT1, JLNT2and JLNT3had direct inhibitory effects on the four kinds of tumor cells’ proliferation in a dose-dependent tendency. SLNT2just exhibited antitumor activity against H22and HepG2cells because of its structural differences, suggesting that the inhibitory effect of them had some selectivity for different tumor cells and was not a broad-spectrum antitumor drug. The inhibitory rates of high molecular weight polysaccharides (SLNT1and JLNT1) were higher than that of low molecular weight polysaccharides (SLNT2, JLNT2and JLNT3).SLNT1and JLNT1exhibiting the strongest antitumor activities in vitro were chosen to further test their antitumor activities in vivo in H22-bearing mice. H22tumor cells were subcutaneously injected into the right axillary of BALB/c mice to establish H22-bearing mice model. Normal mice didn’t make any treatment as blank control group. H22-bearing mice were randomly divided into five groups with10mice per group as follows:negative control group, mice were intraperitoneally injected with the same volume of0.9%normal saline (NS); positive control group, mice were intraperitoneally injected with cytoxan (CTX, 25mg/kg) dissolved in0.9%normal saline; experimental groups, mice were intraperitoneally injected with different concentrations of SLNT1and JLNT1at50,100and200mg/kg once daily for10days. All mice were killed by cervical dislocation on the next day after the last treatment, and then the tumors and spleens were removed and weighed. The levels of IL-2and TNF-a in serum were detected by ELISA and we also observed the H&E staining morphologic changes of the tumor tissues. The inhibition ratios of SLNT1were23.31%,39.85%, and65.41%at various doses of50,100, and200mg/kg in a dose-dependent manner, while the inhibition ratios of JLNT1were38.17%,61.07%and29.01%. Compared with the negative control, SLNT1and JLNT1treatment significantly increased the spleen index and the levels of both IL-2and TNF-a in the serum, but there was no obvious difference in thymus index, demonstrating that SLNT1and JLNT1could stimulate the proliferation and differentiation of host’s immune tissue to enhance immunity. Additionally, the levels of IL-2and TNF-a were significantly reduced down by CTX treatment, indicating that CTX was immunesuppressive. After SLNT1, JLNT1, and CTX treatment, marked morphological changes in nucleus morphology such as karyorrhexis, nucleus fragmentation, and irregular arrangement of karyomorphism, which were typical characteristics of apoptosis cells, were observed. Based on the results above, we could speculate that the antitumor activity of SLNT1and JLNT1might be ascribed to combining enhancing immunity with promoting apoptosis.Combining results of our study and literature, we preliminarily explored the relationships between the main chain structure, molecular weight, helical structure and the antitumor activity. We have found that polysaccharide with (1→3)-glucose main chains and (1→6)-glucose side chains had stronger antitumor activity than that of polysaccharide with (1→6)-glucose main chains and (1→3)-glucose side chains, and the inhibiton ratios of high molecular weight polysaccharides is higher than that of low molecular weight polysaccharides which are extracted by the same method, shows as SLNT1>SLNT2, JLNT1>JLNT2>JLNT3. Furthermore, once the triple helical conformation of lentinan denaturated into single helical structure, the antitumor activity would weaken, indicating the triple helical conformation is a key factor to affect the antitumor effect. Part IV Molecular mechanism of SLNT1inducing tumor cell apoptosisAccording to results of animal study in vivo, SLNT1with good solubility and adequate absorption was selected to investigate the molecular mechansism of inducing tumor cell apoptosis. The apoptosis rates of SLNT1on H22, HepG2and MCF-7cells were detected through Annexin V-FITC/PI staining. Furthermore, Hoechst33258, AO/EB, Annexin V-FITC/PI staining and scanning electron microscope (SEM) were used to observe the morphological changes of H22apoptotic cells. Both the percentages of early apoptotic cells and late apoptotic cells observed by flow cytometry were increased with different concentrations of SLNT1in a dose-dependent manner. Changes in the percentages of total apoptotic cells were more obvious with an increase in SLNT1concentration, and the apoptosis rates of H22, HepG2and MCF-7cells at the highest concentration of SLNT1at1500μg/mL were24.0%,24.7%and17.5%, respectively, which had significant differences as compared to negative control cells, indicating the apoptosis mechanism of SLNT1. AO/EB staining demonstrated that SLNT1-treated H22cells were dyed orange with cells shrinking and nucleus fragmentation. Hoechst33258staining observed typical apoptotic nuclei changes of H22cells, such as nuclei with thick dense clumps of blue fluorescent and nucleus fragmentation. Early apoptotic cells were dyed green and late apoptotic cells were dyed red through Annexin V-FITC/PI staining. Extensive plasma membrane blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic bodies. Through SEM, some morphology changes of apoptosis, including loss of microvilli, decrease in cell volume, plasma membrane blebbing and formation of wrinkles on cell surface were observed obviously. Results furtherly illustrated that SLNT1could induce tumor cell apoptosis.To investigate the molecular mechanisms of SLNT1inducing tumor cell apoptosis, the cell cycle distribution of H22cells by SLNT1treatment were analyzed and reactive oxygen species (ROS) production was monitored by flow cytometry using DCFH-DA, an oxidation-sensitive fluorescent dye. H22tumor cells were subcutaneously injected into the right axillary of BALB/c mice to establish H22-bearing mice model. Normal mice didn’t make any treatment as blank control group. H22-bearing mice were randomly divided into five groups with10mice per group as follows:negative control group, mice were intraperitoneally injected with the same volume of0.9%normal saline (NS); positive control group, mice were intraperitoneally injected with cytoxan (CTX,25mg/kg) dissolved in0.9%normal saline; experimental groups, mice were intraperitoneally injected with different concentrations of SLNT1at50,100and200mg/kg once daily for10days. All mice were killed by cervical dislocation on the next day after the last treatment, and then the tumors were removed. Western blot assay detected the expression of signal transduction proteins in H22tumor tissues, including p-JNK, p-p38MAPK, p-Akt, p53, cyclin B1, cyclin Dl, CDK.4, Bcl-2, Bax and cleaved caspase-3. Results indicated that SLNT1could significantly arrest cell cycle of H22cells at G2/M phase and increase the intracellular ROS. As compared to negative control, SLNT1treatment upregulated the expressions of p-JNK, Bax, p53and cleaved caspase-3, while the levels of p-Akt, p-p38MAPK, Bcl-2and cyclin B1were reduced. According to the results, we could speculate that ROS might participate in SLNT1-induced apoptosis. The elevation of ROS might trigger apoptosis signal transduction pathways to induce tumor cell apoptosis. The possible major mechanisms were as follows:(1) SLNT1incresased p-JNK and p53expression, resulting in activation of caspase-3. On the other hand, SLNT1increased the ratio of Bax/Bcl-2and contributed to release of cytochrome c from the mitochondria into cytosol, and then triggered to caspase-3activation and apoptosis.(2) SLNT1suppressed p-p38MAPK expression and elevated p53level, resulting in two effects:①ctivating Bax and prompting cytochrome C release, leading to DNA degradation and inducing tumor cell apoptosis.②P53accumulation suppressed cyclin B1expression, resulting in tumor cell cycle arrest at G2/M phase to inhibit the growth of tumor.(3) SLNT1downregulated p-Akt expression, resulting in two effects:①increase the expression of p53and inhibit cyclin B1to destruct the process of cell cycle, making tumor cell cycle arrest at G2/M phase.②Elevation of the ratio of Bax/Bcl-2, promoting the release of cytochrome C and apoptosis inducing factor (AIF) to induce tumor cell apoptosis.The molecular mechanisms of SLNT1inducing tumor cell apoptosis were summarized in the figure: Secondly, we explored the effect of SLNT1on depolymerization-polymerization of tubulin in vitro. Tubulin would form microtubule through polymerization at37℃and then depolymerized into tubulin at4℃. Based on this feature, we established a polymerization-depolymerization balance system of tubulin to evaluate the effect of SLNT1and taxol on the tubulin polymerization. It was observed that treatment with taxol could promote tubulin polymerization. On the contrary, various concentrations of SLNT1might possibly induce tumor cells apoptosis by inhibiting tubulin polymerization. To further confirm whether SLNT1could exhibit apoptosis-induction activity via inhibitions of polymerization, SLNT1were incubated with MCF-7cells. Immunofluorescence study showed that treatment of SLNT1for24h caused specific cellular microtubule depolymerization in MCF-7cells. Microtubules were detected in only a diffuse punctuated pattern, and cells were shrinkaged and round-shaped, leading to cytoskeleton damaged. Additionally, typical morphological characteristics of MCF-7cells were observed with SLNT1treatment, such as nucleus pycnosis and crescent. Results showed that SLNT1 could prevent tumor cell mitosis through inhibiting tubulin polymerization, and therefore arresting tumor cell cycle at G2/M phase to exert antitumor property.