Study On Anti - Tumor Effect Of Betula Platyphylla And Its Mechanism

Cancer has become the leading cause of death worldwide. Most of the chemotherapeutic drugs induce serious multi-drug resistance and a series of side effects. Therefore, searching for novel anti-tumor agents from natural products with fewer adverse effects is highly important. The use of medicinal mushrooms in the fight against cancer has been known for a very long time. Mushrooms produce a variety of complex, low-molecular-weight compounds with diverse chemical compositions, and many have shown direct beneficial effects on cancer development by interfering with specific transduction pathways. A number of polypore fungus, such as Ganoderma lucidum (Reishi or Ling Zhi), Laetiporus sulphureus (Chicken-of-the-Woods), Trametes versicolor (Yun Zhi), Grifola umbellata (Zhu Lin), Inonotus obliquus (Chaga) and Wolfiporia cocos (Hoelen), have long been used in herbal medicine. Pyropolyporus fomentarius (L. ex Fr.) Teng (P. fomentarius) is a fungus of polypore, and has been proven to have a variety of medicinal and other uses. However, there are no studies available demonstrating the anti-tumor activities and the underlying mechanism.To fully understand the value of the anti-tumor fungi, and to provide a theoretical basis for the development of anti-tumor drugs, a lot of fundamental work have been done in this study.1. The aim of the present study was to evaluate anti-leukemia activity in vitro and liver toxicity in vivo of P. fomentarius ethanol extract. The selectively inhibitory effect of P. fomentarius extract (40-120μg/ml) on K562 cells was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. DNA fragmentation assay, nuclear DAPI (4’-6-diamidino-2-phenylindole) staining and comet assay were performed to show DNA damage in K562 cells. The in vivo liver toxicity of P. fomentarius was assessed in the blood of mice treated orally with the extract (100-500 mg/kg), using glutamate pyruvatey tansaminase (GPT) and glutaruic oxaloacetid transaminase (GOT) as liver function indicators. Liver morphology was assessed using hematoxylin and eosin (HE) staining of liver tissue. The results showed ethanol extract of P. fomentarius is able to selectively induce proliferative inhibition of K562 cells, in a dose- and time-dependent manner, and the possible mechanism related to DNA damage. Meanwhile, in vivo experiment showed ethanol extract of P. fomentarius has no hepatic toxicity.2. The present study aimed to investigate the antiproliferative effect and the underlying mechanisms of P. fomentarius ethyl acetate extract (PFEAF) in K562 human chronic myelogenous leukemia cells, meanwhile, to analysis the composition of the extract. The results showed PFEAF could inhibit the proliferation of K562 cells, and this inhibitory effect was related to S phase arrest and cell apoptosis. Besides, apoptosis was confirmed by morphological changes, annexin V-PE/7-AAD double staining, which evidenced by ROS generation, loss of △Ψm, the increasment of cleaved caspase-3 and PARP. Importantly, the cytotoxic effect of extract on K562 cells is selectivity, and PFEAF had no toxicity to normal cells. Component analysis showed that dibutyl phthalate may play an important role in the process of effect.3. The present study was conducted to investigate the compositions and cell growth inhibition effects of P. fomentarius chloroform fraction, and to clarify the possible mechanisms. Gas Chromatography-Mass Spectrometry (GC-MS) analysis was performed to investigate the composition of the Pyropolyporus fomentarius chloroform fraction. Cell viability was measured using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell membrane damage was evaluated with scanning electron microscope (SEM) and flow cytometry following propidium iodide (PI), Bis-(1,3-dibarbituric acid)-trimethine oxanol [DiBAC4(3)] staining. Apoptosis was analyzed using Annexin V-PE/7-amino-actinomycin D (Annexin V-PE/7-AAD) staining. Generation of intracellular Ca2+, reactive oxygen species (ROS) and changes of mitochondrial membrane potential (△Ψm) were detected by flow cytometry using Fluo 3-acetoxymethyl ester (Fluo 3-AM),2’,7’-dichlorofluorescin-diacetate (DCFH-DA) and Rhodamine 123 (Rhl23). Our obtained data indicate that Pyropolyporus fomentarius chloroform fraction could inhibit K562 cells proliferation depending on both the dosage and the incubation time, cause cell membrane damage, influence intracellular [Ca2+]i variation, promote the yield of ROS, decrease the level of △Ψm, and initiate the apoptotic response in K562 cells.4. The chemical compositions and anti-tumor activities of the petroleum ether fraction (PE), from mushroom Pyropolyporus fomentarius, were studied. Upon gas chromatography-mass spectrometry (GC-MS) analysis, nine major constituents were identified in the fraction. In vitro, the PE showed cytotoxic activity against murine sarcoma SI80 (S180) cells in a dose- and time-dependent manner, and the cytotoxic effects were associated with apoptosis. The mitochondrial membrane potential loss and the intracellular ROS generation were greatly increased in the Pyropolyporus fomentarius PE treated group, suggesting cell apoptosis, induced by the PE in S180 cells, might be mitochondria dependent and ROS mediated. Consistent with in vitro findings, the in vivo study showed that the Pyropolyporus fomentarius PE was also effective in inhibiting the tumor growth induced by S180 cells and had lower immune organ toxicity. We found that the Pyropolyporus fomentarius PE has significant anti-tumor activity and great potential in screening anti-tumor drugs.