Prohibitin 1 inhibits cell proliferation and induces apoptosis via the p53-mediated mitochondrial pathway in vitro

BACKGROUND Prohibitin 1(PHB1)has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed,and it participates in a variety of essential cellular functions,including apoptosis,cell cycle regulation,prolifera-tion,and survival.Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma(HCC).However,the role of PHB1 in HCC is controversial.AIM To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro.METHODS HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria;then,PHB1 levels in the sera and liver tissues of these participates were determined using ELISA,RT-PCR,and immunohistoche-mistry.Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA(shRNA-PHB1)for 24-72 h.Cell prolif-eration was analysed with an MTT assay.Cell cycle progression and apoptosis were analysed using flow cytometry(FACS).The mRNA and protein expression levels of the cell cycle-related molecules p21,Cyclin A2,Cyclin E1,and CDK2 and the cell apoptosis-related molecules cytochrome C(Cyt C),p53,Bcl-2,Bax,caspase 3,and caspase 9 were measured by real-time PCR and Western blot,respectively.RESULTS Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals,and decreased PHB1 was positively correlated with low differentiation,TNM stage III-IV,and alpha-fetoprotein≥400μg/L.Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner.FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis.The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells.The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41%±1.06%,which was significantly greater than that of apoptotic control cells(3.65%±0.85%,P<0.01)and empty vector-transfected cells(4.21%±0.52%,P<0.01).Similar results were obtained with SMMC-7721 cells.Furthermore,the mRNA and protein expression levels of p53,p21,Bax,caspase 3,and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2,Cy-clin E1,CDK2,and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells.However,when PHB1 was upregulated in human HCC cells,Cyt C expression levels were increased in the cytosol and decreased in the mitochondria,which indicated that Cyt C had been released into the cytosol.Conversely,these effects were reversed when PHB1 was knocked down.CONCLUSION PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.

Molecular mechanisms of apoptosis in hepatocellular carcinoma cells induced by ethanol extracts of Solanum lyratum Thumb through the mitochondrial pathway

AIM To explore the induction effects and mechanism of Solanum lyratum Thumb(ST) on human hepatocellularcarcinoma SMMC-7721 cells through the mitochondrial pathway.METHODS The experiments were conducted on three groups: an experimental group (with ST ethanol extracts' concentration being 2.5, 5 and 10 mg/L), a negative control group (with only nutrient solution, 0 mg/L ST ethanol extracts), and a positive control group (2.5 mg/L DDP). The inhibition rate of cell proliferation was checked by using the methyl thiazolyl tetrazolium method, and cell apoptosis was tested by TUNEL method. Furthermore, RT-PCR was used to examine m RNA expression of Fas, Fas L, caspase-8, caspase-3, p53 and Bcl-2 genes.RESULTS Compared with the negative control group, the inhibition and apoptosis rates of the experimental group with different concentrations of ST extracts on human hepatocellular carcinoma SMMC-7721 cells significantly increased(P<0.05). Besides, the m RNA expression of Fas L and Bcl-2 significantly decreased(P<0.05) while the m RNA expression of Fas, caspase-8, caspase-3 and p53 increased significantly. When compared with the positive control group, the experimental groups with 5 mg/L ST ethanol extracts showed effects similar to the positive control group.CONCLUSION ST ethanol extracts induced the apoptosis of hepatocellular carcinoma SMMC-7721 cells through up-regulated Fas, caspase-8, caspse-3 and p53, and down-regulated Fas L and Bcl-2 in the mitochondrial pathway.

Small interfering RNA targeting HMGN5 induces apoptosis via modulation of a mitochondrial pathway and Bcl-2 family proteins in prostate cancer cells

We investigated the importance of HMGN5, a nuclear protein that binds to nucleosomes, unfolds chromatin, and affects transcription, in the LNCaP prostate cancer cell line. We also examined the molecular mechanisms that promote apoptosis of LNCaP cells after infection with small interfering RNA （siRNA） targeting HMGN5 （siRNA-HMGN5）. The androgen-dependent LNCaP human prostate cancer cells were infected with siRNA-HMGN5. Apoptosis was detected using the Annexin V-PE/7-AAD double staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL） assays. Mitochondrial membrane potential was measured by JC-1 staining. HMGN5and GAPDHmRNA expression were determined using real-time PCR. Bcl-2 and other apoptosis-related protein levels were determined by Western blot analysis. Caspase activity was measured by cleavage of the caspase substrate. Infection with siRNA targeting HMGN5 efficiently and specifically reduced the HMGN5 expression in LNCaP cells. The downregulation of HMGN5 induced remarkable apoptosis of LNCaP cells and resulted in the reduction of mitochondrial membrane potential. The induction of cell apoptosis was accompanied by the upregulation of Bax, the Bax/Bcl-2 ratio and the activation of caspase3. The HMGN5-targeted siRNA was effective in downregulating the expression of HMGN5 in androgen-dependent prostate cancer cells and inducing cell apoptosis via the regulation of a caspase-related mitochondrial pathway and Bcl-2 family proteins. This study suggests that HMGN5 may be a potential molecular target with therapeutic relevance for the treatment of prostate cancer.

Essential Oil from Siegesbeckia pubescens Induces Apoptosis through the Mitochondrial Pathway in Human HepG2 Cells

Siegesbeckia pubescens（SP） has been used as a traditional medicine for the treatment of and inflammatory diseases. However, the activities of SP against hepatocellular carcinoma and the related mechanisms remain unclear. The present study aimed to examine the effects of the essential oil of SP（SPEO） on the proliferation of hepatocellular carcinoma cells and the possible mechanisms. The growth inhibition of Hep G2 cells was analyzed by MTT assay. Hoechst 33258 and fluorescence microscopy were utilized to observe the nuclear morphological changes of apoptotic cells. Flow cytometry was used to detect cell apoptosis and cell cycle. The expressions of the target proteins were detected by Western blotting. The results showed that SPEO obviously inhibited the proliferation of Hep G2 cells in a dose-dependent manner. SPEO activated a series of apoptotic proteins in Hep G2 cells, increasing expression levels of Bax, caspase-3 and caspase-9, and decreasing the bcl-2 expression level. SPEO displayed promising anti-hepatocellular carcinoma activities in vitro, partly by inducing apoptosis in Hep G2 cells through activating the mitochondrial pathway.

Electroacupuncture improves cognitive function in a rat model of mild traumatic brain injury by regulating the SIRT-1/PGC-1α/mitochondrial pathway

Background:Mild traumatic brain injury(mTBI)is a common neurological trauma that can lead to cognitive impairment.The sirtuin-1(SIRT-1)/peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α)pathway has been reported to have neuroprotective effects in rats with craniocerebral injury.We evaluated potential mechanisms underlying electroacupuncture-mediated recovery of cognitive function after mTBI,focusing on the SIRT-1/PGC-1α/mitochondrial pathway.Methods:We included forty 6-week-old male Sprague-Dawley rats in this study.Rats were randomly divided into four groups:controlled cortical impactor(CCI,n=10),sham operation(sham,n=10),electroacupuncture-treated CCI(CCI+EA,n=10),and electroacupuncture-treated sham(sham+EA,n=10)group.Randomization was performed by assigning a random number to each rat and using a random number table.The mTBI rat model was established using a controllable cortical impactor.Electroacupuncture therapy was performed on the back of rats,by inserting acupuncture needles to the specific acupoints and setting appropriate parameters for treatment.We evaluated spatial learning and memory functions with the Morris water maze test.We performed quantitative real-time polymerase chain reaction(qRT-PCR),western blotting,adenosine triphosphate(ATP)determination,and mitochondrial respiratory chain complex I(MRCC I)determination on rat hippocampal tissue.We analyzed SIRT-1/PGC-1α expression levels and the results of mitochondrial function assays,and compared differences between groups using bilateral Student’s t-tests.Results:Compared with the sham group,SIRT-1/PGC-1α expression was downregulated in the hippocampus of CCI group(P<0.01).Although this expression was upregulated following electroacupuncture,it did not reach the levels observed in the sham group(P<0.05).Compared with the sham group,MRCC I and ATP levels in the CCI group were significantly reduced,and increased after electroacupuncture(P<0.01).In the Morris water maze,electroacupuncture reduced the incubation period of rats and increased average speed and number of crossing platforms(P<0.05).Conclusion:Electroacupuncture may improve cognitive function in the mTBI rat model by regulating the SIRT-1/PGC-1α/mitochondrial pathway.

Electroacupuncture alleviates ciliary muscle cell apoptosis in lens-induced myopic guinea pigs through inhibiting the mitochondrial signaling pathway

AIM:To investigate the effect of electroacupuncture(EA)on the mitochondria-dependent apoptotic signaling pathway in the ciliary muscle of guinea pigs with negative lensinduced myopia(LIM).METHODS:Guinea pigs were randomly divided into normal control(NC)group,LIM group,LIM+SHAM acupoint(LIM+SHAM)group,and LIM+EA group.Animals in the NC group received no intervention,while those in other three groups were covered with-6.0 diopter(D)lenses on right eyes.Meanwhile,animals in the LIM+EA group received EA at Hegu(LI4)combined with Taiyang(EX-HN5)acupoints,while those in the LIM+SHAM group were treated at sham points.After treatments for 1,2,and 4wk,morphological changes in ciliary muscles were observed with hematoxylin and eosin(H&E)staining and nick end labeling(TUNEL),and the expression of the mitochondrial apoptotic signaling pathway-related molecules in ciliary muscles was measured by real-time quantitative polymerase chain reaction(qPCR)and Western blot.Additionally,the adenosine triphosphate(ATP)contents were also determined in ciliary muscles.RESULTS:Axial length increased significantly in the LIM and LIM+SHAM groups and decreased in the LIM+EA group.The ciliary muscle fibers were broken and destroyed in both LIM and LIM+SHAM groups,whereas those in the LIM+EA group improved significantly.TUNEL assay showed the number of apoptotic cells increased in the LIM and LIM+SHAM groups,whereas reduced in the LIM+EA group.ATP contents showed a significant decrease in the LIM and LIM+SHAM groups,whereas increased after EA treatment.Compared with the NC group,the dynamin-related protein 1(DRP1),Caspase3,and apoptotic protease activator 1(APAF1)levels were significantly increased in the LIM group and decreased in the LIM+EA group.CONCLUSION:The results provide evidence of EA inhibiting the development of myopia by regulating the mitochondrial apoptotic signaling pathway.

Euphorbia factor L2 induces apoptosis in A549 cells through the mitochondrial pathway

Euphorbia factor L2, a lathyrane diterpenoid isolated from caper euphorbia seed(the seeds of Euphorbia lathyris L.), has been traditionally applied to treat cancer. This article focuses on the cytotoxic activity of Euphorbia factor L2 against lung carcinoma A549 cells and the mechanism by which apoptosis is induced. We analyzed the cytotoxicity and related mechanism of Euphorbia factor L2 with an MTT assay, an annexin V-FITC/PI test, a colorimetric assay, and immunoblotting. Euphorbia factor L2 showed potent cytotoxicity to A549 cells. Euphorbia factor L2 led to an increase in reactive oxygen species(ROS) generation,a loss of mitochondrial electrochemical potential, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase, suggesting that Euphorbia factor L2 induced apoptosis through a mitochondrial pathway. The cytotoxic activity of Euphorbia factor L2 in A549 cells and the related mechanisms of apoptotic induction provide support for the further investigation of caper euphorbia seeds.

Study on Norcantharidin-induced Apoptosis in SMMC-7721 Cells through Mitochondrial Pathways

Objective： To investigate the mechanism of norcantharidin （NCTD）-induced SMMC-7721 hepatoma cell apoptosis. Methods： SMMC-7721 cell growth inhibition was measured by the MTT method. Apoptosis was detected by Annexin Ⅴ/propidium iodide staining. The mitochondrial membrane potential was measured by flow cytometry. Western blot analysis was used to evaluate the level of cytochrome c, caspase-3, AIF, Bcl-2 and Bax expression. Results： NCTD inhibited SMMC-7721 cell growth in a time-and dose-dependent manner. The cells treated with NCTD showed the loss of mitochondrial membrane potential. The activities of caspase-3, cytochrome c, AIF, and Bax were up-regulated after NCTD treatment at different doses. The expression of Bcl-2 was decreased after treatment with NCTD. Conclusions： NCTD could induce SMMC-7721 cell apoptosis. The activation of the mitochondrial pathway was involved in the process of NCTD-induced SMMC-7721 cell apoptosis.

Radix Kansui Stir-Fried with Vinegar Reduces Radix Kansui-Related Hepatotoxicity in Mice via Mitochondrial Pathway

Objective To investigate the mechanism of Radix Kansui(RK)stir-fried with vinegar(VRK)decreased hepatotoxicity in mice.Methods According to a random number table,40 mice were randomly divided into negative control group(0.5%carboxymethylcellulose sodium,20 mL/kg),positive control group(0.1%mixture of carbon tetrachloride in soybean oil,20 mL/kg),RK group(the ethyl acetate extracts of RK,250 g crude drug/kg)and VRK group(the ethyl acetate extracts of VRK,250 g crude drug/kg)with 10 mice per group.All mice were administered orally by gavage daily for 7 continuous days.The morphology of liver tissues was examined to assess the liver injury by a transmission electron microscope.Hepatocyte apoptosis in vivo was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nickend labeling(TUNEL)assay.Immunohistochemical technique was adopted to detect the expression of particular antiapoptotic and proapoptotic proteins in the mitochondrial pathways,including B-cell lymphoma(Bcl-2)and caspase-3,as well as the expression of inflammatory mediators,including nuclear factor kappa B(NF-κB)and intercellular adhesion molecule-1(ICAM-1).Results Liver injury and hepatocyte apoptosis were observed in RK mice,and the liver injury were significantly reduced in VRK-treated mice.In immunohistochemistry study,compared with the negative control group,RK inhibited dramatically the Bcl-2 protein expression and significantly increased the expression of caspase-3,NF-κB and ICAM-1(all P<0.01).Compared with the RK group,VRK group induced significant increase on Bcl-2 protein expression,and decreased the caspase-3,NF-κB and ICAM-1 protein expression(P<0.05 or <0.01).Conclusion The mechanism of reduced hepatotoxicity of VRK may be associated with the reduced inflammation,regulation of antiapoptotic and proapoptotic mediators in the mitochondrial pathway.

Research on Xijiao Dihuang Decoction suppressing platelet apoptosis in immune-mediated aplastic anemia based on mitochondrial mediated pathway

The aplastic anemia mice model was established in this study.BALB/c mice shall be treated with whole body irradiation with 60 Co-γradiation(5.5Gy,1.1Gy/min×5 min).then within 4 h,DBA/2 mice were injected lymphocyte suspension 1×106 cells/mouse through caudal vein.Grouping of testing animals:Normal control mice were healthy C57BL/6 mice without AA modeling.AA control group mice were exposed to radiation and cell transfusion and had no treatment with either CSA or XDD.CSA group mice received daily lavage with 0.027g/kg(0.1ml/10g)of CSA whereas XDD group mice received daily lavage with 19.5g/kg of XDD.The experimental result indicated CSA and XDD lavage mice had significantly higher platelet count andΔΨm than AA mice(P<0.05).Levels of Cyt C,PS and Ca2+were significantly lower in CSA and XDD groups compare with AA group.More specifically,compared to CSA group,XDD group also had lower level ofΔΨm and higher level of Cyt C and Ca2+(both P<0.05).Both CSA and XDD treatment reduced Bak and Bax levels significantly compared to AA mice.However,XDD treatment still showed higher expressions than CSA(P<0.05).CSA and XDD treatment increased the levels of Pro-apoptotic protein expressions(caspase-8,caspase-3,caspase-9).XDD showed less potent effect than CSA in increasing these protein expressions(all P<0.05).Therefore,we hypothesized XDD was possible to increase platelet number and prevent its apoptosis in immune-induced AA via the mitochondrial pathway.

Cadmium Activates Reactive Oxygen Species-dependent AKT/mT OR and Mitochondrial Apoptotic Pathways in Neuronal Cells

Objective To examine the role of Cd-induced reactive oxygen species（ROS） generation in the apoptosis of neuronal cells. Methods Neuronal cells（primary rat cerebral cortical neurons and PC12 cells） were incubated with or without Cd post-pretreatment with rapamycin（Rap） or N-acetyl-L-cysteine（NAC）. Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3’-kinase/protein kinase B（Akt）/mammalian target of rapamycin（m TOR） and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays. Results Cd-induced activation of Akt/m TOR signaling, including Akt, m TOR, p70 S6 kinase（p70 S6K）, and eukaryotic initiation factor 4E binding protein 1（4E-BP1）. Rap, an m TOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/m TOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein（Bcl-2/Bax） ratio, release of cytochrome c, cleavage of caspase-3 and poly（ADP-ribose） polymerase（PARP）, and nuclear translocation of apoptosis-inducing factor（AIF） and endonuclease G（Endo G）. Conclusion Cd-induced ROS generation activates Akt/m TOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that m TOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.

Yi Qi Jie Du Decoction Inhibits Proliferation and Induces Apoptosis of Nasopharyngeal Carcinoma Stem Cells Through Mitochondrial Apoptosis Pathway

Objective To assess the effects of Yi Qi Jie Du Decoction(YQJDD)on nasopharyngeal carcinoma stem cells(NPC-SCs)and investigate the underlying mechanism.Methods NPC-SCs were collected in serum-free culture system and identified by the sphere formation assay.The effect of YQJDD on the proliferation of NPC-SCs was detected using the Cell Counting Kit-8 assay.The change of mitochondrial membrane potential(ΔΨm)in NPC-SCs following treatment with YQJDD was investigated by JC-1 staining.The levels of apoptosisassociated proteins were examined using western blot analysis.Results YQJDD significantly inhibited the proliferation of NPCSCs in dose-and time-dependent manners.JC-1 staining revealed that YQJDD lowered theΔΨm of NPC-SCs in a timedependent manner.After treatment with YQJDD,the expression of cleaved caspase-3,-7 and-9,cleaved poly-ADP ribose polymerase,P21 and P53 were increased,while the expression of Survivin in NPC-SCs was decreased.However,the expression of cleaved caspase-8 remained nearly unchanged.Conclusions YQJDD inhibits the growth and induces apoptosis of NPC-SCs via an intrinsic apoptosis pathway.

Apoptosis of colon cancer CT-26 cells induced polysaccharide from Cyclocarya paliurus and its phosphorylated derivative via intrinsic mitochondrial passway

In this study,the antitumor properties and the possible molecular mechanisms of Cyclocarya paliurus polysaccharide(CP)and its phosphorylated derivative(P-CP)on CT-26 mouse colon carcinoma cells were investigated.Results found that CP had high inhibition ratio against CT-26 cells.The flow cytometry results found that CP treatment could cause the intracellular acidification,arrest the cell cycle in the S phase and increase reactive oxygen species generation.Additionally,CP treatment triggered mitochondrial membrane potential depolarization and Ca^(2+)overloading,and broke down the balance of antioxidant system,Na^(+)/K^(+)-ATPase and Ca^(2+)-ATPase.Further analysis found CP induced cell apoptosis through improving the activities of caspase-3 and caspase-9,and increasing the level of cytochrome C.Furthermore,the comparative study of antitumor effect on CT-26 cells displayed that the phosphorylation enhanced antitumor activities of polysaccharides.These results suggest CP is a potential natural therapeutic agent for colon cancer and phosphorylation represents an effective method of enhancing the antitumor activity of CP.

The study on DNA methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma

Objective： To study the methylation status of several genes on p53-Bax mitochondrial apoptosis pathway and clinical significance in cholangiocarcinoma. Methods： Promoter hypermethylation of DAPK, p14 and ASC genes were detected by methylation-specific PCR. p53 gene status （exon 5-8） were examined by automated sequencing, combined with the clinical documents of patients by statistics analysis. Results： （1） We found 66.7% of 36 cases cholangiocarcinoma had methylation of at least one tumor suppressor gene. The frequency of tumor suppressor gene methylation in cholangiocarcinoma was： p14 （24%）, DAPK （30.6%）, TMSI/ASC （36.1%）. The frequency of tumor suppressor gene methylation in tissues near cancer was： DAPK （5.6%）, TMS1/ASC （8.3%）. （2） p53 gene mutations were found in 22 of 36 patients （61.1%）. （3） There were no statistically relationship among the methylation of DAPK, p14 and ASC genes. There were negative relationship differences between the methylation of p14 and p53 gene mutation （P 〈 0.05）. （4） p53 gene mutation combined with the methylation of tumor suppressor were 14 cases （38.9%）. There were statistically differences on extent of pathologic biology, differentiation and invasion （P 〈 0.05）. Conclusion： Our study indicated that methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma was a common epigenetic event. Although the methylation of ASC, DAPK genes was low, it might be significance for early diagnosis, p53 gene mutation combined with the methylation of tumor suppressor might be relationship with pathologic biology, it trended to more malignancy.

Environmental hypoxia induces apoptosis in large yellow croaker Larimichthys crocea via both intrinsic and extrinsic pathways

Hypoxia has become an unfavorable factor affecting the sustainable development of the large yellow croaker Larimichthys crocea,an economically important mariculture fish in China.Apoptosis is a consequence of hypoxia on fish.However,the effects of hypoxia stress on apoptosis in L.crocea remain largely unknown.We investigated the effect of environmental hypoxia on apoptosis in L.crocea.Results show that hypoxia induced apoptosis in L.crocea both in vivo and in vitro.The mitochondrial membrane potential was significantly reduced in large yellow croaker fry(LYCF)cells.The expression levels of Bcell lymphoma/leukemia-2(Bcl-2)m RNA and protein were also significantly decreased in the liver and LYCF cells during 96 h and 48 h of hypoxia stress,respectively,whereas the expression level of Bcl-2 associated X(Bax)mRNA,Casp3 mRNA,and activity of caspase-3/7/9 were significantly increased,indicating that hypoxia induced caspase-dependent intrinsic apoptosis in L.crocea.The expression level of the apoptosis-inducing factor(AIF)protein was significantly increased in the liver and LYCF cells.The level of AIF protein was significantly decreased in the cytoplasm but increased in the nuclei of L.crocea,demonstrating that hypoxia induced the AIF-mediated caspase-independent intrinsic apoptosis.In addition,the activity of caspase-8 was significantly increased,indicating that hypoxia stress induced extrinsic apoptosis in L.crocea.Therefore,hypoxia induced apoptosis in L.crocea through both the intrinsic and extrinsic pathways.The present study accumulated basic biological information to help elucidate the mechanism of hypoxia response in marine fish.

Essential oil from Saussurea costus inhibits proliferation and migration of Eca109 cells via mitochondrial apoptosis and STAT3 signaling

Objective:To investigate the effect and its underlying molecular mechanisms of essential oil from Saussurea costus in esophageal cancer cell line Eca109.Methods:The chemical composition of essential oil from Saussurea costus was investigated by gas chromatography-mass spectrometry(GC-MS).The anti-proliferative,anti-migrative,and apoptotic effects of essential oil from Saussurea costus against Eca109 cells were analyzed.Moreover,the expression of proteins associated with cell cycle,metastasis,and apoptosis was determined.Results:GC-MS analysis showed that essential oil from Saussurea costus was predominantly comprised of sesquiterpenes.Saussurea costus essential oil inhibited the viability of Eca109 cells in a dose-and time-dependent manner with IC_(50) values of(24.29±1.49),(19.16±2.27)and(6.97±0.86)μg/mL at 12,24,and 48 h,respectively.The expression levels of target proteins in the cell cycle(phase G_(1)/S),including cyclin D1,p21,and p53,were affected by Saussurea costus essential oil.The essential oil also downregulated the expression of metastasis-related proteins MMP-9 and MMP-2.Moreover,it induced apoptosis of Eca109 cells through the mitochondrial pathway,as well as inhibition of STAT3 phosphorylation.Conclusions:The essential oil from Saussurea costus exhibited anti-proliferative,anti-migrative,and apoptotic effects on Eca109 cells,and could be further explored as a potential anti-esophageal cancer agent.

IL-17 Induces MPTP Opening through ERK2 and P53 Signaling Pathway in Human Platelets

The opening of mitochondrial permeability transition pore（MPTP） plays a critical role in platelet activation. However,the potential trigger of the MPTP opening in platelet activation remains unknown. Inflammation is the crucial trigger of platelet activation. In this study,we aimed to explore whether and how the important inflammatory cytokine IL-17 is associated with MPTP opening in platelets activation by using MPTP inhibitor cyclosporine-A（Cs A）. The mitochondrial membrane potential（Δψm） was detected to reflect MPTP opening levels. And the platelet aggregation,activation,and the primary signaling pathway were also tested. The results showed that the MPTP opening levels were increased and Δψm reduced in platelets administrated with IL-17. Moreover,the levels of aggregation,CD62 P,PAC-1,P53 and the phosphorylation of ERK2 were enhanced along with the MPTP opening in platelets pre-stimulated with IL-17. However,Cs A attenuated these effects triggered by IL-17. It was suggested that IL-17 could induce MPTP opening through ERK2 and P53 signaling pathway in platelet activation and aggregation.

Emodin induces apoptosis in human prostate cancer cell LNCaP

Aim： To elucidate effects and mechanisms of emodin in prostate cancer cells. Methods： Viability of emodin-treated LNCaP cells and PC-3 cells was measured by MTT assay. Following emodin treatments, DNA fragmentation was assayed by agarose gel electrophoresis. Apoptosis rate and the expression of Fas and FasL were assayed by flow cytometric analysis. The mRNA expression levels of androgen receptor （AR）, prostate-specific antigen （PSA）, p53, p21, Bcl-2, Bax, caspase-3, -8, -9 and Fas were detected by RT-PCR, and the protein expression levels of AR, p53 and p21 were detected by Western blot analysis. Results： In contrast to PC-3, emodin caused a marked increase in apoptosis and a decrease in cell proliferation in LNCaP cells. The expression of AR and PSA was decreased and the expression of p53 and p21 was increased as the emodin concentrations were increased. In the same time, emodin induced apoptosis of LNCaP cells through the upregulation of caspase-3 and -9, as well as the increase of Bax/Bcl-2 ratio. However, it did not involve modulation of Fas or caspase-8 protein expression. Conclusion： In prostate cancer cell line, LNCaP, emodin inhibites the proliferation by AR and p53-p21 pathways, and induces apoptosis via the mitochondrial pathway.

Astragaloside Ⅳ protects RGC-5 cells against oxidative stress

Astragaloside Ⅳ is the main active compound of Astragalus membranaceus. Astragaloside Ⅳ has strong anti-oxidative activities and protective effects against progression of peripheral neuropathy. In this study, we determined whether astragaloside Ⅳ protects retinal ganglion cells（RGC） from oxidative stress injury using the rat RGC-5 cell line. Hydrogen peroxide（H_2O_2） was used to induce oxidative stress injury, with the protective effect of astragaloside Ⅳ examined. Cell Counting Kit-8 and 4′,6-diamidino-2-phenylindole staining showed that astragaloside Ⅳ increased cell survival rate and decreased apoptotic cell number. Flow cytometry showed that astragaloside Ⅳ decreased H_2O_2-induced reactive oxygen species levels. While laser confocal microscopy showed that astragaloside Ⅳ inhibited the H_2O_2-induced decrease of mitochondrial membrane potential. Western blot assay showed that astragaloside Ⅳ reduced cytochrome c release induced by H_2O_2, inhibited Bax and caspase-3 expression, and increased Bcl-2 expression. Altogether, these results indicate that astragaloside Ⅳ has potential protective effects against H_2O_2-induced oxidative stress in retinal ganglion cells.

Induction of mitochondrion-mediated apoptosis of CHO cells by tripchloro lide

Tripchlorolide (TC) is a potent antitumor reagent purified from a Chinese herb Tripterygium Wilfordii Hook. f.. However, its cellular effects and mechanism of action are unknown. We showed here that TC induced apoptosis of Chinese Hamster Ovary (CHO) cells in time- and dose-dependent manners. TC resulted in the degradation of Bcl-2, the translocation of Bax from the cytosol to mitochondria, and the release of cytochrome c from mitochondria. Stable overexpression of human Bcl-2 could reduce the apoptosis of TCtreated cells by blocking the translocation of Bax and the release of cytochrome c. These results indicate that TC induces apoptosis of CHO cell by activating the mitochondrion-mediated apoptotic pathway involving the proteins of Bcl-2 family and cytochrome c.