Bufothionine, a possible effective component in cinobufocini injection for hepatocellular carcinoma

Ethnopharmacological relevance

Cinobufacini has been traditionally used in china for the treatment of tumor since hundreds years ago. For recent years, its modern preparation,cinobifucini injection has also obtained satisfactory therapeutic functions for cancer.

Materials and Methods

High Performance Liquid Chromatography (HPLC) analysis was applied to determine the content of cinobufagin, resibufogenin and bufothionine in cinobufacin extract liquid and injection; MTT assay and flow cytometric analysis were also respectively used to study the effect of cinobufacini extract liquid, injection and three chemical structures on cells and cell cycles.

Results

HPLC results demonstrated that in cinobufacini extract liquid three ingredients (cinobufagin, resibufogenin and bufothionine) were all monitored while in cinofacini injection only bufothinone was detected; MTT assays showed bufothionine could obviously inhibit the proliferation of human hepatocellular carcinoma cell lines such as SMMC-7721 and BEL-7402 in a dose- and time-dependent manner as well as cinobufagin and resibufogenin; further flow cytometric analysis indicated obvious increases in G2/M phase and decrease in G0/G1 phase when SMMC-7721 cell line exposure to bufothionine (480 μg/ml).

Conclusions

These results suggested bufothionine could be involved in treatment of human cancer for cinobufacini injection and the mechanism might be relative to induce G2/M phase cell cycle arrest.     

Evaluation of Samjunghwan,a traditional medicine,for neuroprotection against damage by amyloid-beta in rat cortical neurons

Aim of the study

Samjunghwan (SJH) is a multi-herbal traditional medicine composed of Mori Fructus, Lycii Radicis Cortex, and Atractylodis Rhizoma Alba and it is clinically applied as an anti-aging agent in neurodegenerative disorders, to promote longevity. In the present study, we evaluated the neuroprotective effect of SJH in Alzheimer's disease induced by amyloid-beta (Abeta) and examined the related pathways.

Materials and methods

To evaluate the protective effect of SJH, we conducted thiazolyl blue tetrazolium bromide, lactate dehydrogenase, and MAP-2 staining assays of primary cultured rat cortical neurons stressed by Abeta_25–35. To investigate the possible mechanism of action, we examined the Bcl-2/Bax expression ratio, mitochondrial membrane potential (Δψ_M), cytochrome C release, and caspase-3 activation, focusing on the mitochondria-mediated apoptotic pathways.

Results

SJH at concentrations of 10 and 100 μg/ml provided significant protection of rat cortical neurons from Abeta_25–35 neurotoxicity. At the maximum effective dose of 100 μg/ml, SJH significantly increased the anti-apoptotic protein (Bcl-2)/pro-apoptotic protein (Bax) ratio and inhibited Δψ_M depolarization, cytosolic cytochrome C release, and caspase-3 activation.

Conclusion

SJH appears to provide neuroprotection against mitochondria-mediated apoptotic pathways in this Abeta_25–35-induced Alzheimer's disease model.     

Anticancer potential of aqueous extract of alocasia macrorrhiza against hepatic cancer in vitro and in vivo

Ethnopharmacological relevance

Alocasia macrorrhiza has been used as a folk medicine for cancer treatment in the Southwest of China.

Aim of the study

The purpose of this study is to confirm the anticancer activity of aqueous extract of alocasia macrorrhiza against hepatic cancer and to elucidate its mechanism of action.

Materials and methods

Human normal liver cells and hepatocellular carcinoma cells were tested in vitro for cytotoxicity, colony formation inhibition, EdU incorporation, AO/EB staining apoptotic cells, apoptotic DNA fragmentation, and cell cycle distribution in response to alocasia macrorrhiza extract. The mRNA and protein expressions of PPARγ, Cyclin D1, Rb, P21, Bax, Bcl-2 and caspase-3 were detected through RT-PCR and Western blotting; the tumor growth inhibition in vivo was tested by oral administration of the extract.

Results

Alocasia macrorrhiza aqueous extract exhibited proliferation inhibition and apoptosis effects on human hepatocellular carcinoma cells in vitro, inhibited hepatoma growth in vivo.

Conclusion

Alocasia macrorrhiza extract has potential cytotoxic and apoptotic effect on human hepatocellular carcinoma cells and inhibits hepatoma growth in vivo, its mechanism of action might be associated with the inhibition of DNA synthesis, cell cycle (G₀/G₁) arrest, apoptosis induction through up-regulation the mRNA and protein expressions of PPARγ, Rb, Bax and capase-3genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes.     

Differentiation of bel-7402 human hepatocarcinoma cells induced by aqueous extracts of fresh gecko (AG) and its anti-tumor activity in vivo

Ethnopharmacological relevance

Gecko, a kind of reptile, has been widely used as a traditional Chinese medicine to treat various diseases including cancer in China for thousands of years. The aim of this study was to investigate the anti-tumor effect of AG (aqueous extracts of fresh gecko) on human hepatocellular carcinoma cell Bel-7402 in vitro and mouse H22 hepatocellular in vivo. Further to underlie the molecular mechanism of AG inducing the differentiation of Bel-7402 cells.

Materials and methods

AG was obtained by water extracting method and qualitatively analyzed through High Performance Liquid Chromatography. The total protein concentration of AG was measured by BCA (bicinchoninic acid disodium) assay. The anti-tumor activities in vivo were analyzed through H22 (mouse hepatocellular carcinoma cell line H22) tumor xenografts mice. The cytotoxic activity of AG on Bel-7402 cells was evaluated by MTT assays. AFP (alpha fetoprotein) was detected by radioimmunoassay. ALB (albumin), ALP (alkaline phosphatase) and γ-GT (γ-glutamyl transpeptidase) were detected by biochemical methods with commercial kits. While morphological changes were observed through an inverted microscope. Moreover, the expression level of the proteins involved in MAPK (mitogen-activated protein kinase) signal pathway which was closely related to cellular differentiation was assessed by Western blot.

Results

AG showed obviously anti-tumor activity in vivo and anti-proliferative activity on Bel-7402 cells in vitro both dose-dependently. The number of clones of Bel-7402 cells treated with AG reduced and the cells were displaying differentiation state such as relatively bigger size and dispersed growth. The biochemical function markers of the cells were significantly changed after being treated with AG. The data showed that AFP secretion of the cells decreased 42.5%, ALB secretion increased 58.9%, the activity of ALP and γ-GT markedly decreased 67.0% and 48.5% separately when the concentration of AG was 10 μl/ml, and those effects were all in a dose-dependent manner. The major original and phosphorylated signal proteins (ERK1/2 (extracellular sigal-regualted kinase 1/2), P38 (p38 MAPK) and JNK1/2 (c-Jun N-terminal kinase 1/2)) involved in MAPK signal pathway were measured and the results showed that AG activated the ERK1/2 of Bel-7402 cells.

Conclusions

AG has anti-tumor activity in vivo and inhibits Bel-7402 cell proliferation in vitro through inducing cell differentiation, and the mechanism involves the activation of ERK1/2.     

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.     

Isolation and characterization of an anticancer catechol compound from Semecarpus anacardium

Ethnopharmacological relevance

The fruits and seeds of Semecarpus anacardium are used widely for the treatment of human cancers and other diseases in the Ayurvedic and Sidda systems of medicine in India.

Aim of the study

The principal aim of this investigation was to isolate and characterize the anticancer compound from the kernel of Semecarpus anacardium nut.

Materials and methods

The bioactivity-tailored isolation and detailed chemical characterization were used to identify the active compound. Cytotoxicity, apoptosis, cell cycle arrest as well as synergism between the identified anticancer compound and doxorubicin in human tumor cell lines were analyzed.

Results

GC/MS, IR, proton NMR, carbon NMR and collisionally induced dissociation (CID) spectra analysis showed that the isolated active compound is 3-(8′(Z),11′(Z)-pentadecadienyl) catechol (SA-3C). SA-3C is cytotoxic to tumor cell lines with IC₅₀ values lower than doxorubicin and even multidrug resistant tumor cell lines were equally sensitive to SA-3C. SA-3C induced apoptosis in human leukemia cell lines in a dose-dependent manner and showed synergistic cytotoxicity with doxorubicin. The cell cycle arrest induced by SA-3C at S- and G₂/M-phases correlated with inhibition of checkpoint kinases.

Conclusion

SA-3C isolated from the kernel of Semecarpus anacardium can be developed as an important anticancer agent for single agent and/or multiagent cancer therapy.     

Chrysanthemum morifolium Ramat (CM) extract protects human neuroblastoma SH-SY5Y cells against MPP-induced cytotoxicity

Ethnopharmacological relevance

Chrysanthemum morifolium Ramat (Asteraceae) has (CM) long been used in Korean and Chinese traditional herbal medicines with numerous therapeutic applications.

Aim of the study

To evaluate the neuroprotective activities of Chrysanthemum morifolium (CM) extract against 1-methyl-4-phenylpridinium ions (MPP⁺), Parkinsonian toxin through oxidative stress and impaired energy metabolism, in human SH-SY5Y neuroblastoma cells and the underlying mechanisms.

Materials and methods

The effects of CM against MPP⁺-induced cytotoxicity and neuronal cell viability, oxidative damage, the expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase (PARP) proteolysis were evaluated by using SH-SY5Y neuroblastoma cells.

Results

CM effectively inhibited the cytotoxicity and improved cell viability. CM also attenuated the elevation of reactive oxygen species (ROS) level, increase in Bax/Bcl-2 ratio, cleavage of caspase-3 and PARP proteolysis.

Conclusion

These results demonstrate that CM possesses potent neuroprotective activity and therefore, might be a potential candidate in neurodegenerative diseases such as Parkinson's disease.     

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.     

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.     

Valeriana amurensis improves Amyloid-beta 1-42 induced cognitive deficit by enhancing cerebral cholinergic function and protecting the brain neurons from apoptosis in mice

Ethnophamacological relevance

Valeriana amurensis, a perennial medicinal herb, has been widely used as anxiolytic, antidepressant, antispasmodic, and sedative in traditional Chinese medicines (TCMs). Moreover, it has been used to treat dementia in Mongolia preparations. In our previous study, we reported that AD-effective fraction of Valeriana amurensis (AD-EFV) has protective effect on Aβ-induced toxicity in PC12 cells. Up to now, however, the therapeutic effect of Valeriana amurensis on Alzheimer disease (AD) has not been explored. This study was designed to determine whether the AD-EFV could improve the Amyloid-beta (Aβ)-induced cognitive deficit and to explore the mechanism of AD-EFV improves cognitive deficit in intact animals.

Materials and methods

The constituents of AD-EFV were isolated with silica gel, octadecyl silica gel (ODS) column chromatography (CC) and preparative HPLC. The structures of compounds were determined by detailed NMR and ESI–MS data analyses. AD mice model was established by injecting Aβ_1-42 (1 μL, 200 μmol) into the bilateral ventricle. Cognitive performance was evaluated by the Morris water maze (MWM) test. The level of cerebral acetylcholine (ACh), the activities of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) were investigated using Enzyme-linked immunoassay (ELISA) kits. Brain sections were processed and neuronal apoptosis in hippocampus were evaluated by Hematoxylin and Eosin (HE), Nissl, and Tunel stainings. The analyses of p-ERK/ERK and Bcl-2/Bax protein expression by western blot assay were used to explore the anti-neuronal apoptosis mechanism of AD-EFV.

Results

Seventeen compounds (15 lignans and two iridoids) were isolated from AD-EFV. A significant improvement in cognitive function was observed in administrated AD-EFV AD model mice. AD-EFV increased the ACh level by enhancing the ChAT activity but has no effect on AChE activity in the cerebral cortex and hippocampus in mice. Moreover, the histological injury in hippocampus CA1 induced by Aβ_1-42 was inhibited following administration of the AD-EFV. As well as the expression ratios of Bcl-2 to Bax and p-ERK to ERK were increased significantly in the mice which were administrated AD-EFV.

Conclusion

These findings suggest that AD-EFV could ameliorate Aβ induced cognitive dysfunction through two underlying mechanisms: AD-EFV enhances the cerebral cholinergic function by increasing the secretion of ACh and enhancing the ChAT activity, and AD-EFV protects the brain neurons from Aβ induced apoptosis via activating the p-ERK and Bcl-2 signaling and suppressing the Bax pathways. Besides, the main constituents of AD-EFV are lignans which might be responsible for the AD-activity of Valeriana amurensis.     

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.     

Cordyceps pruinosa extracts induce apoptosis of HeLa cells by a caspase dependent pathway

Aim of the study

Cordyceps is a parasitic fungus and has long been used as a traditional Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and cancer. In this study, we reveal the mechanisms underlying apoptosis induced by Cordyceps pruinosa butanol fraction (CPBF) in the human cervical adenocarcinoma cell line, HeLa.

Materials and methods

Proliferation and apoptosis of cells were examined by MTT assay, DNA fragmentation, phosphatidyl serine distribution assay, Western blot analysis, and immunocytochemistry. To determine the association between CPBF related apoptosis and ROS, electron spin resonance (ESR) trapping experiments were used.

Results

CPBF inhibited proliferation and induced apoptosis in HeLa cells in a dose-dependent manner using a MTT assay, DNA fragmentation, and a phosphatidyl serine distribution assay. Western blot analysis showed that apoptosis in HeLa cells was caspase-3- and -9-dependent. Proteolytic cleavage of PARP and the release of cytochrome c from the mitochondria into the cytosol were significantly increased and the Bcl-2/Bax protein ratio was decreased. Apoptosis induced by CPBF was not prevented by various antioxidants.

Conclusions

These results indicate that apoptotic effects of CPBF on HeLa cells are mediated by mitochondria-dependent death-signaling pathway independent of reactive oxygen species, suggesting that CPBF might be effective as an anti-proliferative agent for cancer.     

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.     

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.     

An in vitro study on antiproliferative and antiestrogenic effects of Boerhaavia diffusa L. extracts

Ethnopharmacological relevance

Boerhaavia diffusa L. (Nyctinaceae) is a plant of tropical region used in Indian traditional medicine for the treatment of human ailments including abdominal tumor, jaundice, dyspepsia, menstrual disorders, etc. This plant also has antilymphoproliferative, antimetastatic and immunomodulatory effects.

Aim of the study

This study aimed to assess the antiproliferative and antiestrogenic properties of methanol extract of Boerhaavia diffusa (BME) in MCF-7 breast cancer cell lines.

Materials and methods

The effective concentration range of BME on cell viability was analyzed using MTT assay. Hydroxylapatite assay (HAP) was carried out to confirm the competitive binding of BME to the estrogen receptor (ER). The effect of BME on the expression of a selected estrogen responsive gene pS2 was analyzed by RT-PCR. The ability of BME to alter the cell cycle phases and distributions were studied using FACS analysis.

Results

Treatment with varying concentrations of BME (20–320 μg/mL) resulted in moderate to very strong growth inhibition in MCF-7 cell lines. BME competed with [³H]-estradiol for binding to ER with IC₅₀ value of 320 ± 25 μg/mL. RT-PCR analysis revealed that BME reduced the mRNA expression of pS2 indicating the antiestrogenic action of BME. BME treatment for 48 h resulted in a remarkable increase in the number of MCF-7 cells in the G0-G1 fraction from 69.1% to 75.8%, with a reciprocal decrease of cells in all other phases indicating cell cycle arrest at G0-G1 phase.

Conclusions

The results demonstrate that Boerhaavia diffusa possess antiproliferative and antiestrogenic properties and suggest that it may have therapeutic potential in estrogen dependent breast cancers.     

Magnesium lithospermate B improves myocardial function and prevents simulated ischemia/reperfusion injury-induced H9c2 cardiomyocytes apoptosis through Akt-dependent pathway

Ethnopharmacological relevance

Magnesium lithospermate B (MLB), an active polyphenol acid of Radix Salviae Miltiorrhizae (Danshen), showed a wide range of pharmacological activities in cardiovascular diseases. However, its role in protection against ischemia/reperfusion injury in H9c2 cardiomyocytes has not been elucidated. This study was aimed to investigate the protective effect and potential molecular mechanisms of MLB on apoptosis in H9c2 cardiomyocytes in vitro.

Materials and methods

We tested cell viability, shortening amplitude, necrosis, apoptosis, and the expression levels of Akt, phosphorylated Akt, Bcl-2 and Bax after 2-h simulated ischemia and 24-h simulated reperfusion in H9c2 cardiomyocytes. We further observed the contractile function in hearts after they were subjected to global 30-min ischemia and 180-min reperfusion.

Results

Pretreatment with MLB markedly increased cell viability and while reducing evidence of necrosis and apoptosis in H9c2 cardiomyocytes. In addition, the expression of Bcl-2 and Bax protein was modulated. The results also showed that MLB significantly increased phosphorylation of Akt and that this phosphorylation can be partially inhibited by phosphoinositide 3-kinase/Akt inhibitor. Furthermore, MLB improved MI/R-induced myocardial contractile function.

Conclusion

Our results showed that MLB prevents I/R-induced myocardial damage by reducing necrosis and apoptosis in H9c2 cardiomyocytes and improving myocardial function in rat hearts.     

Protective effect of 5-hydroxymethylfurfural derived from processed Fructus Corni on human hepatocyte LO2 injured by hydrogen peroxide and its mechanism

Aim of the study

The aim of the present study was to evaluate the putative protective effect of 5-hydroxymethylfurfural (5-HMF) derived from processed Fructus Corni on human hepatocyte cell line (LO2) injured by hydrogen peroxide in vitro and the mechanism of its protection.

Materials and methods

The percentage of cell viability was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The hepatocyte cell apoptosis and cell cycle were detected by flow cytometric analysis. The content of nitric oxide and caspase-3 activity were quantified spectrophotometrically by enzyme-linked immunoassay.

Result

The study showed that incubation with 5-HMF caused significant increase in the viability of LO2 cell, decrease of cell apoptosis and recovery of cell cycle in LO2 cell injured by hydrogen peroxide, which was accompanied with the decreased nitric oxide level and caspase-3 activity.

Conclusions

Our data suggest that 5-HMF protects LO2 cell against damage induced by hydrogen peroxide through inhibiting effect of cell apoptosis caused by promoting S phase to G2/M phase and the decreased caspase-3 activity and nitric oxide level. 5-HMF is one of the active principles in processed Fructus Corni.     

Swietenia macrophylla King induces mitochondrial-mediated apoptosis through p53 upregulation in HCT116 colorectal carcinoma cells

Ethnopharmacological relevance

Swietenia macrophylla King is a traditional herb used to treat various diseases including hypertension, diabetes and cancer. Previous study demonstrated its anti-tumor effect but the potential mechanisms have not been clearly defined. The current study was to further investigate the underlying mechanism of ethyl acetate fraction of Swietenia macrophylla (SMEAF)-induced anti-proliferative effect and apoptosis in HCT116 colorectal carcinoma cell.

Materials and methods

Cell viability was evaluated in HCT116 cells by trypan blue exclusion assay. Apoptotic cell death was detected by Hoechst 33342/propidium iodide (PI) staining and intracellular reactive oxygen species (ROS) was analyzed by flow cytometry. The apoptotic gene and protein expression were determined by Real-time quantitative PCR (q-PCR) and immunofluorescence staining using flow cytometry, respectively.

Results

SMEAF significantly inhibited HCT116 cell viability and induced apoptosis in a dose-dependent manner. SMEAF-induced apoptosis was triggered by the activation of p53 and intracellular reactive oxygen species (ROS) production. Moreover, the significant increase in p53 was accompanied by a decrease murine double minute 2 (MDM2) expression. SMEAF significantly increased the expression of the Bax protein resulting in a markedly elevated Bax/Bcl-2 ratio which may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3/7 and caspase-9 activation.

Conclusion

These results suggested that SMEAF exerts its antitumor activity in HCT116 cells by activating proapoptotic signaling pathway through intracellular ROS formation triggering the mitochondrial-mediated pathway via p53 activation.     

Eucommia ulmoides Oliv. Bark. protects against hydrogen peroxide-induced neuronal cell death in SH-SY5Y cells

Ethnopharmacological relevance

Eucommia ulmoides Oliv. Bark. (EUE), has commonly been used to fortify the muscles and lungs, lower blood pressure, prevent miscarriage, improve the tone of liver and kidneys, and promote longevity the traditional tonic medicines of Korea, China, and Japan.

Aim of the study

In this study, we investigated that the neuroprotective activities and possible mechanisms of EUE aqueous extract in hydrogen peroxide (H₂O₂)-induced neuronal cell death in human SH-SY5Y neuroblastoma cells.

Material and method

We examined the effects of EUE against H₂O₂-induced cytotoxicity, DNA condensation, the production of reactive oxygen species (ROS), loss of mitochondria membrane potential (MMP), the proteolysis of cleaved poly-ADP-ribose polymerase (PARP), and the expression of Bcl-2, Bcl-xL, cleaved caspase-3, and release of cytochrome c. Moreover, we attempted to determine whether EUE suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK 1/2), and phosphoinositide 3-kinase (PI3K)/Akt.

Results

Pretreatment with EUE increased cell viability and inhibited cytotoxicity and DNA condensation. EUE also attenuated the increase in ROS production and MMP reduction. Western blot data revealed that EUE inhibited H₂O₂-induced up- or down-regulation of cleaved PARP, cleaved caspase-3, Bcl-2, and Bcl-xL. The EUE inhibited release of cytochrome c from mitochondria to the cytosol, and significantly attenuated H₂O₂-induced phosphorylation of JNK, p38 MAPK, ERK 1/2, and PI3K/Akt.

Conclusion

The potent neuroprotective capacity of EUE, shown in these experiments, may potentially be applied in the prevention or treatment of neurodegenerative diseases such as Alzheimer's disease (AD).