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Pyropolyporus fomentarius (L. ex Fr.) Teng is a unique woody mushroom due to its medicinal value with numerous pharmacological activities. This study presented the potential antitumor and immunomodulatory properties of ethanol extract of P. fomentarius. The results showed that P. fomentarius extract inhibited the viability of murine leukemia L1210 cells in a dose-dependent manner with IC50 value of 69.35 μg/ml. Flow cytometry analysis demonstrated that the extract induced apoptosis in L1210 cells. Additionally, the decline of mitochondrial membrane potential was observed as well as the changes of caspase-3, caspase-9, Bcl-2, and Bax, suggesting that proapoptosis effect of the extract involved mitochondria-related pathway. Simultaneously, the P. fomentarius extract significantly enhanced the proliferation and activation of splenic lymphocytes in a dose-dependent manner. This P. fomentarius extract has potential applications as a natural antitumor agent with immunomodulatory activity.

Ethnopharmacological Relevance: Polytrichum commune L.ex Hedw (PCLH), a moss of Bryopsida, has been used as a traditional Chinese medicine and shown to possess anticancer activities. Previous studies have indicated its anti-leukemia effect but the potential mechanisms have not been fully explained., Aim of the Study: The present study aimed to further investigate the efficacy of PCLH ethyl acetate fraction (PC-EEF) and the associated mechanisms in human leukemia cells., Materials and Methods: Phytochemical analysis of PC-EEF was performed by spectrophotometry and HPLC. MTT analysis and trypan blue exclusion assay were adopted to examine its cytotoxicity on a panel of leukemia cells (K562, U937, HL-60 and K562/DOX cells) and non-cancerous cells (human PBMCs). Anti-proliferative effect was monitored by colony formation assay and EdU incorporation assay. Ultrastructural alterations on K562 cell membrane surface were observed by scanning electron microscopy. Changes on plasma membrane integrity, cell membrane potential, mitochondrial membrane potential and apoptosis were analyzed by flow cytometry. Fluorescence microscope was performed to assess [Ca(2+)]i level, mitochondrial injury and cytochrome c release. Apoptosis-associated protein expression was analyzed by western blot. The role of Ca(2+) in PC-EEF-induced cell death was investigated by Ca(2+) chelating reagent BAPTA-AM., Results: PC-EEF possessed relatively high flavonoid content (about 88.84 ± 0.89%) and showed significant cytotoxicity to human leukemia cells. PC-EEF could cause obvious cell morphological deformation, membrane integrity loss and membrane depolarization. Meanwhile, PC-EEF treatment could dramatically potentiate perturbations in cellular Ca(2+) homeostasis. Subsequently, mitochondrial membrane potential (MMP) collapse, cytochrome c release and Bcl-2/Bax down-regulation were all observed. Consistent with these results, PC-EEF treatment resulted in significant activation of caspase 3, poly (ADP-ribose) polymerase (PARP) degradation and apoptosis. Moreover, PC-EEF-caused cytotoxicity, membrane damage, mitochondrial injury and apoptosis were remarkably reversed by BAPTA-AM., Conclusions: PC-EEF damaged the membrane system and triggered Ca(2+)-dependent mitochondrial apoptosis, which may provide some new insights into its efficacy against human leukemia cells., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Fomitopsis pinicola (Sw. Ex Fr.m) Karst (FPK) which belongs to the Basidiomycota fungal class is one of the most popular medical fungi in China. It has been used for many diseases: cancer, heart diseases, diabetes and so on. However, little study on the pro-apoptotic effect and migration inhibition of FPK chloroform extract (FPKc) has been reported and the possible involved mechanism has not been illuminated., Methodology/principal Findings: Chemical analysis was performed by HPLC which showed ergosterol (ES) concentration was 105 µg/mg. MTT assay revealed that FPKc could selectively inhibit SW-480 cells viability with the IC50 of 190.28 µg/ml. Wound healing and transwell assay indicated that FPKc could inhibit the migration of SW-480 cells obviously, FPKc could also dramatically decreased the matrix metalloproteinases-2, 9 (MMP-2 and MMP-9) expression. Annexin V-FITC/PI staining, nuclear Hoechst 33342 staining and DNA fragmentation analysis revealed that FPKc and ES could induce SW-480 cells apoptosis. The apoptosis process closely involved in ROS accumulation and depletion of GSH, activation of caspase 3, poly (ADP-ribose) polymerase (PARP) degradation. FPKc could also up-regulate P53 expression and thus lead to G1 phase arrest. When SW-480 cells were pretreated with N-acetylcysteine (NAC), the ROS generation, cell viability and apoptotic ratio were partially declined, which indicated that ROS was vertical in the pro-apoptosis process induced by FPKc. Moreover, in the whole process, ES which has been previously found in FPKc had the similar effect to FPKc. Thus we could conclude that ES, as one of the highest abundant components in FPKc, might also be one of the active constituents., Conclusion/significance: FPKc could inhibit the migration of SW-480 cells, induce SW-480 cells G1 phase arrest and cause ROS-mediated apoptosis effect. And ES might be one of the effective constituents in the whole process.

Ethnopharmacological Relevance: Polytrichum commune L.ex Hedw is a traditional Chinese herb for treatment of fever, hemostatic, uterine prolapse and especially for lymphocytic leukemia, but the antitumor effect and its potential mechanism remains unclear., Aims of the Study: The present study was to investigate the possible anti-proliferative activity of ethanolic extract and the organic fractions from P. commune on murine leukemia L1210 cells., Materials and Methods: The content of ethanolic extract and its fractions was performed on HPLC analysis with gradient elution. L1210 cells were treated with different concentrations of ethanolic extract and its fractions at different time intervals. Cell viability was evaluated using MTT assay. Apoptotic cell death was monitored by nuclear condensation and confirmed by exposure of phosphatidylserine to outer leaflet of plasma membrane. Loss of mitochondrial membrane potential was analyzed by flow cytometry using rhodamine 123 staining., Results: The obtained results showed that the cell viability of L1210 cells was reduced by ethanolic extract of P. commune in a concentration-dependent manner, and the IC(50) value was about 77.22 μg/ml at 24h post treatment. The ethylacetate fraction displayed higher anti-tumor effect than that of chloroform and butanol fractions with 32.29 μg/ml (IC(50) value, 48 h). Microscopy studies revealed that ethanolic extract and ethylacetate fraction treated cells showed morphological characteristics of apoptosis such as chromatin condensation and DNA aggregation. Further, Annexin V-PE/ 7-AAD double staining showing the out leaflet of phosphatidylserine and the decline of mitochondrial membrane potential by flow cytometry confirmed that the extracts do, in fact, induce apoptosis in L1210 cells., Conclusion: This is the first report on anti-tumor and pro-apoptotic effect of P. commune in cultured leukemia cells, which provides scientific basis for its usefulness as traditional medicine. Further studies are needed to confirm the precise mechanism not only the crude extract but as well the monomeric chemical substances of Polytrichum commune L.ex Hedw., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: Fomitopsis pinicola (Sw. Ex Fr.m) Karst (FPK) which belongs to the Basidiomycota fungal class is one of the most popular medical fungi in China. It has been used for many diseases: cancer, heart diseases, diabetes and so on. However, little study on the pro-apoptotic effect and migration inhibition of FPK chloroform extract (FPKc) has been reported and the possible involved mechanism has not been illuminated. Methodology/Principal Findings: Chemical analysis was performed by HPLC which showed ergosterol (ES) concentration was 105 µg/mg. MTT assay revealed that FPKc could selectively inhibit SW-480 cells viability with the IC50 of 190.28 µg/ml. Wound healing and transwell assay indicated that FPKc could inhibit the migration of SW-480 cells obviously, FPKc could also dramatically decreased the matrix metalloproteinases-2, 9 (MMP-2 and MMP-9) expression. Annexin V–FITC/PI staining, nuclear Hoechst 33342 staining and DNA fragmentation analysis revealed that FPKc and ES could induce SW-480 cells apoptosis. The apoptosis process closely involved in ROS accumulation and depletion of GSH, activation of caspase 3, poly (ADP-ribose) polymerase (PARP) degradation. FPKc could also up-regulate P53 expression and thus lead to G1 phase arrest. When SW-480 cells were pretreated with N-acetylcysteine (NAC), the ROS generation, cell viability and apoptotic ratio were partially declined, which indicated that ROS was vertical in the pro-apoptosis process induced by FPKc. Moreover, in the whole process, ES which has been previously found in FPKc had the similar effect to FPKc. Thus we could conclude that ES, as one of the highest abundant components in FPKc, might also be one of the active constituents. Conclusion/Significance: FPKc could inhibit the migration of SW-480 cells, induce SW-480 cells G1 phase arrest and cause ROS-mediated apoptosis effect. And ES might be one of the effective constituents in the whole process. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Polytrichum commune L.ex Hedw is a traditional Chinese herb for treatment of fever, hemostatic, uterine prolapse and especially for leukemia. Previous studies indicated its anti-leukemia effect but the potential mechanisms have not been fully explained. Aims of the study: The present study was further to investigate the underlying mechanism of ethyl acetate extract of Polytrichum commune L.ex Hedw (EEF)-induced toxicity and apoptosis in L1210 cells. Materials and methods: Viability, DNA damage and apoptotic protein expressions of L1210 cells were analyzed by ViaCount, comet assay and western blot, respectively. At different times after EEF treatment, Bax redistribution in L1210 cells was examined using confocal microscopy; loss of mitochondrial membrane potential (MMP) was monitored by fluorescence microscope using rhodamine 123 staining; Intracellular reactive oxygen species (ROS) generation and DNA fragmentation were measured by flow cytometry using fluorescent dye, DCFH-DA and PI, respectively. Results: EEF significantly inhibited L1210 cell survival, promoted Bax translocation onto mitochondria, stimulated caspase-9 activation and subsequent DNA damage in L1210 cells. Abundant ROS was detected in L1210 cells after EEF treatment, and the ROS scavenger NAC significantly relieved EEF-induced cell viability decline, MMP loss, and DNA fragmentation. Conclusion: EEF could induce mitochondria-dependent cell apoptosis in L1210 cells, and ROS may play an important role in this action. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Equisetum hyemale has been used as a traditional herbal medicine to treat various diseases such as hypertension, inflammatory diseases, acute stroke, bleeding and cancer. The present study aimed to investigate the anti-proliferative effect and the underlying mechanisms of E.hyemale extract on murine leukemia L1210 cells. Materials and methods: The inhibitory effect of Ehyemale extract on L1210cells was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cell cycle distribution was evaluated with flow cytometry following PI (propidium iodide) staining. Apoptotic cell death was determined by Annexin V–FITC/PI and nuclear DAPI (4′-6-diamidino-2-phenylindole) staining. DNA damage and changes of mitochondrial membrane potential were also detected with flow cytometry analysis. Results: E.hyemale extract exerted significant antiproliferative effects on L1210 cells in a dose- and time-dependent manner. Flow cytometry analysis showed that E.hyemale extract induced cell cycle arrest at G2/M phase in L1210 cells. Phosphatidylserine exposure, chromatin condensation, DNA damage and loss of mitochondrial membrane potential were observed clearly after treatment with Ehyemale extract. Conclusion: The results in this study indicate that E.hyemale extract could inhibit L1210 cell proliferation through inducing G2/M arrest and cell apoptosis. [Copyright &y& Elsevier]