Chemopreventive action of Lygodium flexuosum extract in human hepatoma PLC/PRF/5 and Hep 3B cells

Ethnopharmacological relevance

Lygodium flexuosum (Lygodiaceae), a medicinal fern used in Indian traditional medicine against liver disorders.

Aim of the study

The rationale of the study was to examine whether the n-hexane extract from plant Lygodium flexuosum affects apoptosis on human hepatoma PLC/PRF/5 and Hep 3B cells.

Materials and methods

Chemopreventive activity of the Lygodium flexuosum extract was determined by MTT assay, annexin-V FITC binding to phosphatidyl serine and cleavage of PARP. Subdiploid condition of cells treated with Lygodium flexuosum was analyzed by flow cytometry. Further, used transiently transfected NF-κB reporter in PLC/PRF/5 cells to evaluate the inhibitive effect of Lygodium flexuosum extract.

Results

Lygodium flexuosum extract inhibited the cell viability and induced apoptosis in hepatoma cells in a concentration dependent manner as evidenced by apoptotic changes such as flipping of phosphatidyl serine, cleavage of PARP. Cell cycle analysis showed the subG1 apoptotic population in cells treated with higher concentrations of the extract. When activated with exogenous TNF-α in transfected hepatoma cells it was observed that NF-κB dependent gene expression was inhibited by treatment with Lygodium flexuosum extract in PLC/PRF/5 cells dose-dependently.

Conclusions

This investigation suggests that the Lygodium flexuosum extract has antiproliferative and apoptotic activity in both cancer cells and has inhibitive role in TNF-α induced NF-κB activation in PLC/PRF/5 cells confirms the potential of the extract as a chemopreventive agent.     

Cytotoxic and apoptotic activity of Cheilanthes farinosa (Forsk.) Kaulf. against human hepatoma,Hep3B cells

Aim of the study

Cheilanthes farinosa (Forsk.) Kaulf., family: Adianthaceae, is a fern of immense medicinal properties used in ethno-medicine. The Gaddis tribe of Himachal Pradesh, India, has been using this fern to treat liver damage. Aim of the current study was to determine the apoptosis inducing and cytotoxic activity, if any, of this fern towards hepatic cancer cells.

Materials and methods

Water extract of the plant was used in the study. MTT assay was performed in hepatocellular carcinoma cell line, Hep3B as well as murine macrophage cell line, RAW264.7 to analyze the cytotoxic activity of the plant. Further, the apoptosis inducing action of water extract of the plant was evaluated using comet assay, DNA fragmentation analysis, DAPI staining of chromatin and Annexin V-FITC staining.

Results

This plant was found to produce considerable cytotoxicity in hepatoma cell line, Hep3B without inducing substantial damage to non-cancerous cell line RAW264.7. In addition, this plant was found to induce apoptosis in Hep3B cells. This was substantiated by comet assay, DNA fragmentation analysis, DAPI staining of chromatin and Annexin V-FITC staining for detecting early stage of apoptosis.

Conclusions

This investigation shows that the water extract of Cheilanthes farinosa has antiproliferative and apoptotic activity in human liver cancer cells and is not deleterious towards non-cancerous macrophage cell line.     

Rhizoma Paridis ethanol extract selectively inhibits the proliferation of HUVECs comparing to Lovo cells and shows anti-angiogenesis effects in a mouse model

Ethnopharmacological relevance

Rhizoma Paridis, a traditional Chinese Medicine, was identified to be cytotoxic to cancer cells. The present study was designed to investigate the potential anti-angiogenic and antitumor effect of the ethanol extract of Rhizoma Paridis (RPE) in vitro and in vivo.

Materials and methods

The cytotoxic effect of RPE against human colon cancer Lovo cells and human umbilical vein endothelial cells (HUVECs) was examined using MTT assay. We also tested the effect of RPE on tube formation, migration, apoptosis and cell cycle of HUVECs. Moreover, Lovo subcutaneous xenograft was applied to study the antitumor and anti-angiogenesis effect of RPE in vivo.

Results

RPE exerted a higher inhibition effect on the proliferation of HUVECs than Lovo cells. The tube formation and cell migration were also significantly inhibited in the presence of RPE in a concentration-dependent manner though the significant inhibition effects were observed at the cytotoxic dose. RPE induced cell apoptosis and G0–G1 cell cycle arrest of HUVECs. In vivo, significant tumor growth inhibition was observed in human colon cancer xenografts established by Lovo cells, accompanying by a marked decrease in MVD.

Conclusions

Our current study exhibited that RPE has a selective cytotoxity against HUVECs comparing to Lovo cells and also demonstrated significant anti-angiogenic effect in vivo.     

Antitumor effect of Croton lechleri Mull. Arg. (Euphorbiaceae)

Ethnopharmagological relevance

Medicinal plants are an important source of antitumor compounds. This study evaluated the acute toxicity in mice, as well as the cytotoxic and antitumoral effects of methanolic extracts of Croton lechleri leaves (CLE).

Materials and methods

The cytotoxicity of CLE on human cancer cell lines and human non-cancerous cells was evaluated using the MTT and apoptosis assays. Apoptosis induced by CLE on human cancer cell lines was determined using flow cytometry with annexin-Alexa 488/propidium iodide. The acute toxicity in mice was performed according to the Lorke procedure. Different doses of CLE were injected intraperitoneally daily into athymic mice bearing tumor during 18 days. The growth and weight of tumors was measured.

Results

CLE showed low IC₅₀ values on HeLa (17 μg/ml) cells but lack toxic effects against human normal cells. Induction of cell death in HeLa cells by CLE was confirmed by an increase of apoptosis (Annexin/PI) by 30% compared to untreated cells. The LD₅₀ was 356 mg/kg by intraperitoneal route (i.p.) and 500 mg/kg by oral route. CLE administrated at 1, 10 and 50 mg/kg i.p. inhibited the tumor growth by 38%, 48% and 59%, respectively, in mice bearing HeLa tumor.

Conclusion

Croton lechleri shows moderate toxic effects in vivo, exerts cytotoxic effects on HeLa cells and has antitumor effects in mice bearing HeLa tumor.     

Antiproliferative and apoptosis inducing activity of Markhamia tomentosa leaf extract on HeLa cells

Ethnopharmacological relevance

Markhamia tomentosa (Benth) K. Schum ex. Engl. (Bignoniaceae), a tree widely dispersed in West Tropical Africa, is used traditionally to treat various diseases as it possesses antimicrobial, antioxidant, analgesic, anticancer and anti-inflammatory activities.

Materials and methods

This study evaluates the cytotoxic effect and underlying mechanisms of the ethanolic extract of Markhamia tomentosa on HeLa and MCF-7 cancer cell lines and non-cancerous Vero cell line. Brine shrimp lethality test was used for preliminary screening. Cytotoxicity was determined using the MTT assay and IC₅₀ was calculated. Effect of Markhamia tomentosa on the cell cycle was monitored by flow cytometry and the apoptosis-induction capability confirmed by exposure of phosphatidylserine to the outer leaflet of the plasma membrane. Loss of mitochondrial membrane potential was analysed by flow cytometry using JC-1.

Results

Markhamia tomentosa was toxic to brine shrimps with LD₅₀ of 31.62 µg/ml. Cell viability and growth of HeLa cells was inhibited by the extract with an IC₅₀ of 189.1±1.76 µg/ml at 24 h post treatment. However, no cytotoxic effect was observed in MCF-7 and Vero cell lines. The extract induced cell cycle arrest in HeLa cells in the G₀/G₁ phase resulting in cell death after 24 h exposure. Induction of apoptosis in HeLa cells was substantiated by Annexin V-FITC/PI double staining showing phosphatidylserine translocation and depolarisation of the mitochondrial membrane potential by flow cytometry of JC-1 stained cells.

Conclusion

The ethanolic extract of Markhamia tomentosa induces G₀/G₁ in HeLa cells followed by induction of the intrinsic pathway of apoptosis.     

In vitro cytotoxic activity of nine plants used in Mayan traditional medicine

Ethnopharmacological relevance

Plants have been used in folk medicine by Mayan ancient people from the Yucatan Peninsula, Mexico, to treat some diseases considered as cancer diseases such as chronic wounds or tumors.

Aim of the study

We collected a selection of nine plants in order to investigate their cytotoxic activity against cancer cell lines.

Materials and methods

Methanolic extracts were tested for their cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on four cancer cell lines; nasopharynx carcinoma (KB), laryngeal carcinoma (Hep-2), cervix adenocarcinoma (HeLa), and cervix squamous carcinoma cells (SiHa) and one normal cell line; canine kidney (MDCK).

Results

All species exhibited some degree of cytotoxic activity. The root bark extract of Hamelia patens exhibited the highest cytotoxic activity on HeLa cells with a CC₅₀ of 13 μg/mL and selectivity index of 13.3, higher than docetaxel. Gossypium schottii and Dioon spinulosum showed similar good cytotoxic activity and selectivity index on HeLa and Hep-2 cells, respectively.

Conclusions

Hamelia patens, Dioon spinulosum and Gossypium schottii demonstrated promising cytotoxic activity and have been selected for future bio-guided fractionation and isolation of active cytotoxic compounds.     

Panaxatriol saponins extracted from Panax notoginseng induces thioredoxin-1 and prevents 1-methyl-4-phenylpyridinium ion-induced neurotoxicity

Aim of the study

Thioredoxin-1 has various biologic activities, including the control of redox balance and the inhibition of apoptosis. The current study was designed to examine the effects of panaxatriol saponins (PTS) extracted from Panax notoginseng on thioredoxin-1 expression and 1-methyl-4-phenylpyridinium ion-induced injury.

Materials and methods

Using PC12 cells and Kunming mice, we test thioredoxin-1 expression after PTS treatment by Western blot. The protective effect of PTS against 1-methyl-4-phenylpyridinium ion-induced injury was assessed by MTT assay and LDH release assay.

Results

PTS induced thioredoxin-1 expression in vitro and in vivo, and attenuated 1-methyl-4-phenylpyridinium ion-induced cell death of PC12 cells.

Conclusions

PTS is a new inducer of thioredoxin-1 and has a possible potential as a therapeutic agent for neurodegenerative diseases including Parkinson's disease.     

Inhibitory effects of Agaricus blazei extracts on human myeloid leukemia cells

Ethnopharmacological relevance

Agaricus blazei has been used as an adjuvant in cancer chemotherapy and is found to inhibit the growth of various types of tumor cells.

Aim of the study

Our study has adopted a systematic and bioassay-guided approach to optimize the extraction of Agaricus blazei for anti-leukemic bioactive components. The tumor-selective growth inhibitory activity of the extracts on leukemic cell lines was evaluated in vitro and in vivo using tumor-bearing nude mice.

Materials and methods

Agaricus blazei extracts were prepared using different methods. MTT and tritiated thymidine incorporation assays were used to evaluate the in vitro anti-leukemic effects. The most potent extract was further investigated using NB-4 cells-bearing nude mice and mechanistic studies using DNA fragmentation assay and cell death detection ELISA.

Results

The JAB80E70 extract showed the most potent tumor-selective growth inhibitory activity against human leukemia NB-4 and K-562 cells. This is the first report of anti-leukemic activity of JAB80E70 in athymic nude mice bearing NB-4 cells. Using DNA fragmentation assays and cell death detection ELISA, JAB80E70 was found to induce apoptosis in NB-4 cells. However, the polysaccharide enriched fractions failed to show significant cytotoxicity on NB-4 cells in vitro.

Conclusions

The JAB80E70 extract exhibited potent anti-leukemic effect in vitro and in vivo. The effect can be attributed, at least in part, to the induction of apoptosis. Besides, polysaccharides in Agaricus blazei may not possess direct anti-leukemic activity in vitro.     

Total alkaloids of Tripterygium hypoglaucum (levl.) Hutch inhibits tumor growth both in vitro and in vivo

Ethnopharmacological relevance

Tripterygium hypoglaucum (levl.) Hutch (Celastraceae) (THH) root is a traditional Chinese medicinal herb commonly used for treating autoimmune diseases and cancer. Alkaloid is one of the most bioactive components of THH extract. To evaluate the in vitro and in vivo antitumor properties of the total alkaloids of THH (THHta).

Materials and methods

THHta was extracted in pilot-scale. HCT116 cells were chose to establish human colon cancer xenograft model. The in vitro anti-tumor activity of THHta was tested by Cell malignant transformation test, Soft agar colony formation assay and MTT assay. The in vivo anti-tumor effect of THHta was confirmed by xenograft mouse model. THHta-induced apoptosis was examined by flow cytometry. The levels of apoptosis-related proteins were investigated by Western blot.

Results

TPA-induced cell transformation was significantly inhibited by THHta in JB6 Cl41 cells. THHta inhibits the growth of colon cancer cells in vitro in a significant dose-dependent manner. Compared to the control set, i.p. administration of THHta to xenograft mice signi?cantly reduced both tumor weight and volume. Apoptosis induction of THHta was mediated by activation of caspase-3, PARP and inhibiting of Bcl-2, Bcl-xL and XIAP.

Conclusion

THHta was effective in inhibiting tumor growth both in vitro and in vivo at less toxic concentrations by inducing apoptosis which suggested it could be developed as a potential anticancer agent.     

Identification of bioactive constituents of Ziziphus jujube fruit extracts exerting antiproliferative and apoptotic effects in human breast cancer cells

Ethnopharmacological relevance

Ziziphus extracts have been used in Traditional Chinese Medicine for the treatment of cancer.

Aim of the study

In the present study we have investigated the effects of Ziziphus jujube extracts (ZEs) on breast cancer.

Materials and methods

We evaluated the effects of increasing concentrations of ZEs on ERα positive MCF-7 and ERα negative SKBR3 breast cancer cell proliferation using MTT assays. Apoptosis was analyzed by evaluating the involvement of some pro-apoptotic proteins, including Bax, Bad, Bid and PARP cleavage by immunoblotting analysis. Moreover, the effects of ZEs treatment on apoptosis were tested by both DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. By using chromatographic techniques, we identified the constituents of the effective extracts.

Results

ZE1, ZE2, and ZE4 exerted significant antiproliferative effects on estrogen receptor alpha (ERα) positive MCF-7 (IC₅₀ values of 14.42, 7.64, 1.69 μg/mL) and ERα negative SKBR3 (IC₅₀ values of 14.06, 6.21, 3.70 μg/mL) human breast cancer cells. Remarkably, ZEs did not affect cell viability of both normal human fibroblasts BJ1-hTERT and nonmalignant breast epithelial MCF-10A cells. Treatment with ZEs induced cell death by apoptosis in both malignant breast cells. We found that the most effective extracts ZE2 and ZE4 shared a number of triterpenic acids, already known for their anticancer activities.

Conclusions

Our data provide a rational base for the use of Ziziphus extracts in the treatment of breast cancer in Traditional Chinese Medicine.     

Standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal induces apoptosis via mitochondrial pathway in human cervical cancer HeLa cells

Ethnopharmacological relevance

Artemisia princeps Pampanini is widely used in Eastern traditional medicine for the treatment of circulatory disorders, such as, dysmenorrhea, hematuria, hemorrhoids, and inflammation, and is also used to treat chronic conditions, such as, cancers, ulcers, and digestive disorders.

Aim of the study

The purpose of this study is to investigate the effect of a standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal (FRAP) on the induction of apoptosis and the molecular mechanism involved in human cervical cancer HeLa cells.

Materials and methods

Human cervical cancer HeLa cells were treated with FRAP and apoptosis was detected by cell morphologic observation, annexin-V-PI staning and western blot analysis on the expression of protein associated with cell death.

Results

FRAP led to the cleavages of caspase-3, -8, and -9 and the cleavage of poly (ADP-ribose) polymerase (PARP) in HeLa cells. Caspase-3 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD), and broad caspase inhibitor (z-VAD-fmk) significantly suppressed the FRAP-induced accumulation of annexin V positive cells. Furthermore, it was found that FRAP caused a loss of mitochondrial membrane potential (MMP) and the release of cytochrome c to the cytosol. Furthermore, the overexpression of Bcl-xL significantly prevented FRAP-induced apoptosis, MMP changes, and the activations of caspase-3, -8, and -9. Interestingly, pretreatment with caspase-8 inhibitor significantly reduced the FRAP-induced activation of caspase-3 but not that of caspase-9, whereas the caspase-3 inhibitor, z-DEVD-fmk, markedly attenuated the FRAP-induced activation of caspase-8. In BALB/c^nu/nu mice bearing a HeLa xenograft, FRAP dosed at 25 or 50 mg/kg significantly inhibited tumor growth.

Conclusion

Our results indicate caspase-mediated activation of the mitochondrial death pathway plays a critical role in the FRAP-induced apoptosis of HeLa cells and that FRAP inhibits the in vivo tumor growth of HeLa xenograft mice.     

Dentatin isolated from Clausena excavata induces apoptosis in MCF-7 cells through the intrinsic pathway with involvement of NF-κB signalling and G0/G1 cell cycle arrest: A bioassay-guided approach

Ethnopharmacological relevance

Clausena excavata Burm. f. has been used in folk medicines in eastern Thailand for the treatment of cancer.

Materials and methods

To investigate the apoptosis mechanism, we isolated dentatin (DTN) from this plant using a bioassay-guided approach. DTN-induced cytotoxicity was observed with the MTT assay. Acridine orange/propidium iodide staining was used to detect cells in early apoptosis and high content screening (HCS) to observe nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Apoptosis was confirmed with a clonogenic assay, DNA laddering and caspase 3/7 and 9 assays. Reactive oxygen species (ROS) formation, Bcl-2 and Bax expression, and cell cycle arrest were also investigated. The involvement of nuclear factor-kappa B (NF-κB) was analysed with the HCS assay.

Results

A significant increase in chromatin condensation in the cell nucleus was observed by fluorescence analysis. Apoptosis was confirmed by the reduced number of colonies in the clonogenic assay and the increased number of cellular DNA breaks in treated cells observed as a DNA ladder. Treatment of MCF-7 cells with DTN encouraged apoptosis with cell death-transducing signals that reduced MMP by down-regulation of Bcl-2 and up-regulation of Bax, triggering cytochrome c release from the mitochondria to the cytosol. The released cytochrome c triggered the activation of caspase 9 followed by the executioner caspase 3/7. DTN treatment significantly arrested MCF-7 cells at the G0/G1 phase (p<0.05) and ROS was significantly elevated. Moreover, DTN significantly blocked the induced translocation of NF-κB from cytoplasm to nucleus.

Conclusion

Together, the results demonstrated that the DTN isolated from Clausena excavata inhibited the proliferation of MCF-7 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway with involvement of the NF-κB signalling pathway.     

Induction of apoptosis in human hepatocarcinoma SMMC-7721 cells in vitro by flavonoids from Astragalus complanatus

Aim of the study

Flavonoids extracted from the seeds of Astragalus complanatus R.Br. reduce the proliferation of many cancer cells. The present study was carried out to evaluate the effects of these flavonoids from Astragalus complanatus (FAC) on human hepatocarcinoma cell viability and apoptosis and to investigate its mechanisms of action in SMMC-7721 cells.

Materials and methods

Cell viability was measured using the MTT assay. To detect apoptotic cells, SMMC-7721 cells treated with FAC were stained with Hoechst 33258 and subjected to agarose gel electrophoresis. Quantitative detection of apoptotic cells was performed by flow cytometry. The effects of FAC on apoptosis and cell cycle regulatory genes and proteins in SMMC-7721 cells were examined using an S series apoptosis and cell cycle gene array and Western blot analysis.

Results

The growth of SMMC-7721 and HepG2 cells was inhibited by treatment with FAC. Cell death induced by FAC was characterized by nuclear condensation and DNA fragmentation. Moreover, the cell cycle was arrested in the G0/G1 and S phases in FAC-treated SMMC-7721 cells. A sub-G1 peak with reduced DNA content was also formed. The activity of caspase-3 was significantly increased following FAC treatment. Microarray data indicated that the expression levels of 76 genes were changed in SMMC-7721 cells treated with FAC: 35 genes were up-regulated and 41 were down-regulated. Western blot analysis showed that caspase-3, caspase-8, Bax, P21, and P27 protein levels in SMMC-7721 cells were increased after 48 h of FAC treatment, while cyclinB1, cyclinD1, CDK1, and CDK4 protein levels were decreased.

Conclusions

These results suggest that FAC may play an important role in tumor growth suppression by inducing apoptosis in human hepatocarcinoma cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.     

Ganoderma lucidum induced apoptosis in NB4 human leukemia cells: Involvement of Akt and Erk

Aim of the study

The final goal of this work was to study the toxic and apoptosis effects induced by fractions from Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] on NB4 human leukemia cells.

Materials and methods

Two aqueous extracts and a methanol-extracted column-chromatography semipurified fraction were obtained from Ganoderma lucidum fruiting body. Flow cytometry analyses were used to measure cell viability, cell cycle and DNA fragmentation and to quantify apoptosis. Western-blot analyses were used to quantify changes in apoptosis proteins and intracellular kinases.

Results

Aqueous extracts slightly reduce cell viability and induce DNA fragmentation in NB4 cells. Methanol-extracted semipurified fraction at dilutions down to 15% or 40% of the initial fraction concentration reduced significantly the viability of these leukemia cells (treated for 19 h) with induction of DNA fragmentation and induction of apoptosis. Overmore, the dilution down to 15% of the initial E3 concentration induced a reduction of p53 levels, of the Bcl2/Bax relationship as well as reduced levels of both unphosphorylated and phosphorylated Akt (Protein kinase Akt, protein kinase B) and Erk (Erk1 and 2).

Conclusions

Induction of apoptosis and alterations in signal transduction kinases (Akt and Erk) are produced by active fractions from Ganoderma lucidum on human leukemia cells. These data could be of important relevance from the viewpoint of antitumor actions of compounds from Ganoderma lucidum. Eventual therapy applications in leukemia cells might be developed.     

Antitumor and immunomodulatory effects of Justicia spicigera Schltdl (Acanthaceae)

Ethnopharmacological relevance

Medicinal plants are an important source of antitumor compounds. This study evaluated the acute toxicity in vitro and in vivo, as well as the cytotoxic, antitumor and immunomodulatory effects of ethanolic extracts of Justicia spicigera leaves (JSE).

Materials and methods

The in vitro and in vivo toxicity of JSE was evaluated with comet assay in peripheral blood mononuclear cells (PBMC) and acute toxicity in mice, according to the Lorke procedure, respectively. The apoptotic effect of JSE on human cancer cells and human noncancerous cells was evaluated using flow cytometry with annexin-Alexa 488/propidium iodide. Also, different doses of JSE were injected intraperitoneally daily into athymic mice bearing tumors of HeLa cells during 18 days. The growth and weight of tumors were measured. The in vitro immunomodulatory effects of JSE were evaluated estimating the effects of JSE on the phagocytosis of the yeast Saccharomyces cerevisiae, NO production and H₂O₂ release in macrophages, as well as the proliferation of splenocytes and NK activity.

Results

The comet assay showed that only JSE tested at 200 and 1000 μg/ml induced a significantly DNA damage in PBMC, compared to untreated cells, whereas the LD₅₀ was >5000 mg/kg by intraperitoneal route (i.p.) and by oral route. JSE showed pro-apoptotic (Annexin/PI) effects by 35% against HeLa cells, but lack toxic effects against human normal cells.JSE administrated at 10, 50 and 100 mg/kg i.p. inhibited the tumor growth by 28%, 41% and 53%, respectively, in mice bearing HeLa tumor. JSE stimulated, in a concentration dependent manner, the phagocytosis of Saccharomyces cerevisiae yeasts, the NO production and H₂O₂ release by human differentiated macrophages. In addition, JSE stimulated the proliferation of murine splenocytes and induced the NK cell activity.

Conclusion

Justicia spicigera shows low toxic effects in vitro and in vivo, exerts apoptotic effects on HeLa cells, has antitumor effects in mice bearing HeLa tumor and induces immunomodulatory activities in vitro.     

The ethyl acetate fraction of Polytrichum commune L.ex Hedw induced cell apoptosis via reactive oxygen species in L1210 cells

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.     

Total glucosides of Paeonia lactiflora Pall inhibit vascular endothelial growth factor-induced angiogenesis

Objective

To evaluate the anti-angiogenesis effect of total glucosides of Paeonia lactiflora Pall.

Methods

In this study, we determined the effect of TGP on the proliferation of human vascular endothelial cells through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and fluorescence-activated cell sorting analysis. A migration assay and a tube formation assay were used to investigate the migration properties and tube formation abilities of human vascular endothelial cells after being treated with TGP. Furthermore, the in vivo anti-angiogenic ability of TGP was determined through a chick chorioallantoic membrane assay.

Results

TGP (12.5, 62.5, and 312.5 μg/ml) resulted in a dose-dependent reduction in the proliferation of endothelial cells. This inhibition effect began 6 h after treatment and lasted at least 24 h. Fluorescence-activated cell sorting analysis data showed an accumulation of cells in the G0/G1 phase of the cell cycle, which exhibited apoptotic features indicative of cell death. The migration properties and tube forming abilities of endothelial cells were dramatically inhibited by the TGP extract.

Conclusion

Our results show that TGP can inhibit angiogenesis in vitro and in vivo.     

Cytotoxicity and antibacterial activity of Lindera strychnifolia essential oils and extracts

Aim of the study

Lindera strychnifolia (LS) is widely used in traditional Chinese medicine. In the present study, we investigated cytotoxicity and antibacterial activity of essential oils and various fractions of ethanol extract of LS to explore the active components of LS and their pharmacological effects.

Materials and methods

The in vitro cytotoxicities of essential oils and various solvent fractions of LS on three human cancer cell lines (A549, HeLa and Hep G2) and a non-cancerous cell line (HUVEC) were examined using a modified MTT assay. And by using agar disc diffusion and broth microdilution methods, the antibacterial activity of these samples was evaluated against 10 bacteria including 5 clinically isolated strains. The compositions of the essential oils from the leaves and roots of LS were also analyzed by GC and GC–MS.

Results

The leaf oil showed the strongest cytotoxicity on the cancer cell lines tested with the IC₅₀ values ranged from 22 to 24 μg/ml after 24 h of treatment. The most sensitive microbial strain to all the samples was Staphylococcus aureus ATCC 25923.

Conclusions

Our results showed that the essential oils of LS exhibited greater cytotoxicity and antibacterial activity than the solvent fractions of ethanol extract of LS.     

The anti-tumor activity of Antrodia salmonea in human promyelocytic leukemia (HL-60) cells is mediated via the induction of G1 cell-cycle arrest and apoptosis in vitro or in vivo

Ethnopharmacological relevance

The medicinal mushroom Antrodia salmonea has been used as a traditional Chinese medicine and has demonstrated antioxidant and anti-inflammatory effects.

Materials and methods

In the present study, we examined the anti-tumor activity of the fermented culture broth of Antrodia salmonea (AS) in vitro and in vivo and revealed its underlying molecular mechanism of action.

Results

Treatment of human promyelocytic leukemia (HL-60) cells with AS (50–150 μg/mL) significantly reduced cell viability and caused G₁ arrest via the inhibition of cell-cycle regulatory proteins, including cyclin D1, CDK4, cyclin E, cyclin A, and phosphorylated retinoblastoma protein (p-Rb). Furthermore, AS treatment induced apoptosis, which was associated with DNA fragmentation, followed by a sequence of events, including intracellular ROS generation; mitochondrial dysfunction; Fas ligand activation; cytochrome c release; caspase-3, -8, -9, and PARP activation; and Bcl-2/Bax dysregulation. The results of the in vitro study suggested that AS-induced apoptosis in HL-60 cells was mediated by both the mitochondrial and death receptor pathways. Furthermore, we found that AS treatment was effective in delaying tumor incidence in HL-60 xenografted nude mice and reducing tumor burden.

Conclusions

To the best of our knowledge, this is the first report confirming the anti-tumor activity of this potentially beneficial mushroom against human promyelocytic leukemia.     

Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3

Aim of the study

Matrine, an alkaloid purified from the chinese herb Sophora flavescens Ait, is well known to possess activities including anti-inflammation, anti-fibrotic and anticancer. In this study, the mechanism of matrine inducing the apoptosis of gastric carcinoma cells was investigated.

Materials and Methods

Proliferation of SGC-7901 cells was examined by MTT assay. Cellular morphology was observed under transmission electron microscope. Flow cytometry (FCM) was used to observe the apoptosis of SGC-7901 cells by staining with annexinV-FITC/PI. The expression levels of Fas/FasL in SGC-7901 cells were monitored by FCM analysis using an indirect immunoflurenscence method. Activity of caspase-3 enzyme was measured by spectrofluorometry.

Results

MTT assay showed that matrine inhibited SGC-7901 cells proliferation in a dose-dependent and time-dependent manner. Apoptosis induction was demonstrated by morphological changes under electron microscope and FCM analysis. Florescence intensity levels of Fas and FasL were found to be equally up-regulated after matrine treatment, which were both correlated with apoptosis rate. The activity of caspase-3 enzyme increased in matrine groups, positively correlated with apoptosis rate.

Conclusions

Matrine could inhibit cell proliferation and induce apoptosis of SGC-7901 cells in vitro. The apoptosis induction appears to proceed by up-regulating Fas/FasL expression and activating caspase-3 enzyme.