tBHQ delayed replicative senescence by activating the proteasome system of BMSCs

AIM: To investigate the effect of tert-butylhydroquinone(tBHQ) on the replicative senescence of bone marrow mesenchymal stem cells(BMSCs).METHODS: Late stage BMSCs were continuously treated with tBHQ at concentration of 30 μmol/L for 4 weeks and the cells were used for the following assays immediately. The proteasomal activity was determined by chemiluminescence method. The samples were subjected to CCK-8 assay and BrdU incorporation as well as flow cytometry analysis for analyzing the cell vitality and proliferation. Percentage of senescent cells was detected by senescence-associated β-galactosidase(SA-β-Gal) staining. The expression of P53 was measured by Western blot.RESULTS: After the continuous treatment of tBHQ(30 μmol/L) for 4 weeks, the proteasomal activity of late stage BMSCs increased by 21.96%±1.98%(P<0.05). The cell vitality and survival were significantly increased with the increases in tBHQ doses till 40 μmol/L, and no cytotoxicity reaction with the increased dose of tBHQ till 120 μmol/L was observed. BrdU-positive cells, which represented the cell proliferation, were significantly increased(P<0.05). The proliferation index was also significantly increased by flow cytometry analysis(P<0.05). The SA-β-Gal positive cells and the expression of P53 were decreased(P<0.05).CONCLUSION: tBHQ delays the proteasome dysfunction associated senescence progress of BMSCs by increasing the proteasomal activity.     

Dysfunction of proteasome inhibits the proliferation of hBMSCs via activation of P53

AIM: To investigate the role of P53 in decreased cell proliferation and proteasomal activity during culture expansion of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: The proteasomal activity and expression level of P53 in early-passage (passage 3~4) and late-passage (passage≥14) hBMSCs were observed. Early-passage hBMSCs were divided into DMSO control group, MG132 group (treated with 10 μmol/L MG132 for 4 h) and pifithrin-α(PET-α) + MG132 group (pretreatment with 20 μmol/L PFT-α for 1 h then exposure to MG132 for 4 h). The proliferation and cell cycle distribution of the cells were measured by BrdU incorporation assay and PI staining. To further confirm the effect of P53 inhibitor on late-passage hBMSCs, the cells were incubated with 20 μmol/L PFT-α and the BrdU-positive cells were counted. RESULTS: Accompanied by reduced proteasomal activity, the expression level of P53 in late-passage hBMSCs was up-regulated by 16.89%±4.44% compared with early-passage cells. Application of the proteasome inhibitor MG132 reduced the proliferation of early-passage hBMSCs, and the percentage of BrdU-positive cells dropped to 33.36%±2.24%. However, MG132-induced decrease in cell proliferation was partially reversed by pretreatment with PFT-α. BrdU-positive cells in PFT-α + MG132 group were increased to 49.23%±2.67%. The cell cycle distribution had no significant difference between PFT-α + MG132 group and DMSO group, and the higher proliferation index was found in PFT-α + MG132 group but not in MG132 group. Inhibition of P53 activity in late-passage hBMSCs by PFT-α promoted the cell proliferation, and the percentage of BrdU-positive cells was higher than that in control group. CONCLUSION: Long-term expansion of hBMSCs in vitro reduces proteasomal activity, which in turn activates P53 to suppress cell proliferation through blocking cell cycle progression.     

Effect of RelB on proteasome inhibitor-induced maspin expression

AIM: To investigate the effects of RelB on proteasome inhibitor-induced maspin expression in prostate cancer cells. METHODS: Western blotting analysis was performed to examine endogenous and proteasome inhibitor(MG-132)-induced expression of RelA, RelB and maspin in prostate cancer cells. The expression profiles of RelB and maspin in human prostate cancer tissues were obtained by immunohistochemistry assay. RNA interference targeting RelB was performed in DU145 cells. The effects of RelB-silencing on maspin expression induced by MG-132 were detected by Western blotting. The cell viability was determined by PI staining and FACS analysis. RESULTS: RelB expression was increased in androgen-independent prostate cancer cell line DU145, while maspin expression was minimally detected. Among 10 tissue samples tested, a strong nuclear RelB staining and an absence of maspin expression were found in high-grade specimens (Gleason scores 4-5). RelB expression was reduced upon treatment with MG-132 for 24 h, which was coincided with the induction of maspin expression. RelB-silencing in DU145 cells by siRNA didn't influence the proteasome inhibitor-induced maspin expression. CONCLUSION: The expression of RelB is inversely correlated to maspin expression in androgen-independent prostate cancer cells and prostate cancer tissues. RelB expression is critical to the proteasome inhibitor-induced maspin expression.     

Effects of baicalin on CA46 cell proliferation inhibition and apoptosis induction

AIM: To investigate the effects of baicalin on proliferation inhibition and apoptosis induction in human Burkitt lymphoma cell line CA46 and to explore its underlying mechanisms. METHODS: CA46 cells were exposed to baicalin at different dosages and its proliferation inhibition was detected by MTT assay. The ability of baicalin to induce CA46 cell apoptosis was examined by Annexin V-FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation. The mRNA expressions of c-myc and bcl-2 were detected by RT-PCR, and the protein expressions of c-Myc, Bcl-2, caspase-3 precursor (procaspase-3) and poly ADP-ribose polymerase (PARP) were detected by Western blotting. RESULTS: Baicalin remarkably inhibited the CA46 cell proliferation, with an IC50 value of 10 μmol/L. Apoptosis was remarkably induced by baicalin in a dose-dependent manner, and its earlier and later stages were detected by annexin V-FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation, respectively. Furthermore, RT-PCR showed that the mRNA expressions of c-myc and bcl-2 in treated CA46 cells decreased in a time-dependent manner. Western blotting showed that the protein expressions of c-Myc, Bcl-2, procaspase-3 and PARP (116 kD) in baicalin treated CA46 cells were down-regulated in a time-dependent manner, while the expression of PARP(85 kD) was up-regulated. CONCLUSION: Baicalin efficiently induces proliferation inhibition and apoptosis in CA46 cells, which may be related with the down-regulation of c-Myc and Bcl-2 expressions, as well as the up-regulation of caspase-3 activity.     

Roles of maspin in biological behaviors of PC-3 prostate cancer cells

AIM: To investigate the roles of maspin in the biological behaviors of prostate cancer cells. METHODS: Specific shRNA targeting maspin gene was designed. The plasmid targeting maspin gene was constructed and lentiviral expression system was used for transfection. qRT-PCR and Western blotting were performed to identify the stable maspin-shRNA-transfected PC-3 cells. The expression of apoptosis-related genes was analyzed by qRT-PCR. Dynamic observation of cell growth and doubling time were conducted by an xCELLigence system. The cell death upon proteasome inhibitor treatment was determined by flow cytometry analysis. The expression levels of RelA and RelB were detected by Western blotting. RESULTS: The recombinant plasmid containing maspin-shRNA was successfully constructed. Limited dilution was performed to obtain monoclonal PC-3-siMaspin cells. The doubling time of PC-3-siMaspin cells was 26.83 h while that of PC-3-control cells was 37.95 h. The mRNA expression of bcl-2 and A20 in PC-3-siMaspin cells was increased, while that of bax and bim was down-regulated. The cell death rates of PC-3-control cells and PC-3-siMaspin cells after treated with MG-132 were 27.1% ?5.6% and 7.5% ?2.3% at 8 h, 24.2% ?3.7% and 8.2% ?2.5% at 24 h, and 28.7% ?3.7% and 7.6%?2.5% at 36 h after treatment,respectively. RelA expression was decreased in PC-3-control cells treated with MG-132 while that in PC-3-siMaspin cells stayed unchanged. CONCLUSION: Maspin expression is increased in androgen-independent prostate cancer PC-3 cells. Maspin silencing significantly reduces the doubling time and accelerates the cell growth. Maspin silencing markedly reduces the sensitivity of PC-3 cells to proteasome inhibitor, which may be linked to the abolishment of RelA degradation.     

Immunosuppressive effect of dihydroartemisinin on murine T lymphocytes

AIM: To investigate the effect of dihydroartemisinin (DHA) on the proliferation of murine T lymphocytes stimulated by Con A in vitro and its related immunosuppressive mechanism. METHODS: Murine T lymphocytes were stimulated by Con A and treated with different concentrations of DHA. Cell proliferation was measured by carboxyl fluoresce in diacetate succinmidyl ester (CFDA-SE) staining. The expression of CD69, CD25 and CD71,which was the marker of early, middle, later activation of CD3+ T lymphocytes, was measured by flow cytometry (FCM) combined with two-color immunofluorescent staining of cell surface antigen. Fluorescence calcium indicator fluo-4/AM was used to measure the change of the intracellular calcium concentration (［Ca2+］i) of murine T lymphocytes. The distribution of the cell cycle was analyzed by PI staining. The expression of CD69, the early activation antigen on CD4+CD25high Treg was also measured by FCM combined with three-color immunofluorescent staining. RESULTS: The result of CFDA-SE staining showed that DHA efficiently inhibited the Con A-induced proliferation of T-lymphocytes in a time-and dose-dependent manners. DHA showed modestly increased proportions of CD69 and CD25 on Con A-stimulated CD3+T cells, but inhibited the expression of CD25 in a dose dependent manner. DHA with Con A, but not DHA alone, caused an increase in intracellular calcium concentration of T cells. The results of FCM analysis with PI staining showed that DHA imposed a total cell cycle arrest in G0/G1 and prevented cells entering S phase and G2/M phase. Furthermore, DHA reduced the expression of CD69 on CD4+CD25high Treg. CONCLUSION: DHA, which exhibits immunosuppressive effect on the proliferation of murine T-lymphocytes, is promising to be developed as an immunosuppressive reagent.     

OX1R expression in chronic ischemic brain tissue of rats

AIM: To study the orexin-1 receptor (OX1R) expression in chronic ischemic brain tissue of rats and the change following the ischemic process.METHODS: The cerebral ischemic model was established by ligating double carotid arteries in rats. The behavior of the models was evaluated by water maze. The OX1R expression was determined by immunohistochemical technique, and the location of OX1R expression was further confirmed by double immunofluorescent staining.RESULTS: The intelligence of rats 15 d after ligating double carotid arteries was impaired obviously, and improved in 1 month and 2 months compared with the model in 15 d. At the same time, the OX1R expression increased obviously from acute phase until 15 d of cerebral ischemia and decreased notably in 1 month compared to the model in 15 d, and then increased significantly for the second time in 2 months of model. From the histology, partial neurons of 15 d model rats got atrophy, and most neurons in 1 month model rats got cytomorphosis and atrophy, however partial neurons in 2-month model rats recovered normally. The OX1R expression was confirmed in neurons definitely by double immunofluorescent staining.CONCLUSION: During the pathologic process of chronic ischemic injury, orexin system has two-way regulatory functions through.     

Upregulation of proteasome activity by 18α-GA promotes proliferation of late-passage BMSCs in vitro

AIM: To investigate the effect of 18 alpha-glycyrrhetinic acid (18α-GA) on delaying the senescent progress and promoting the proliferation in late-passage bone marrow mesenchymal stem cells (BMSCs). METHODS: Late-passage BMSCs were incubated with 2.0 mg /L 18α-GA or the same volume of DMSO for 30 d, and the cells were harvested to determine the proteasome activity. The expression of senescence-related proteins p53, p21 and p16 was detected by senescence-associated β-galactosidase (SA-β-Gal) staining and Western blot. The cell proliferation, the expression level of cell cycle-related proteins and cell cycle distribution of the cells were measured by CCK-8 assay, BrdU incorporation, Western blot and flow cytometry. RESULTS: Compared with DMSO group, the proteasome activity in 18α-GA group increased significantly by about 0.2 times (P<0.01). SA-β-Gal-positive cells in 18α-GA group decreased, and cell staining was lighter. The contents of p53 and p21 in 18α-GA group were decreased (P<0.05). The results of CCK-8 assay showed that the A value in 18α-GA group was 0.3 times higher than that in DMSO group (P<0.01). BrdU incorporation showed the increased proliferation in 18α-GA group compared with DMSO group (P<0.05). The cells in G₁ phase in 18α-GA group decreased significantly compared with DMSO group, while the cells in S phase increased significantly (P<0.05). The expression level of cyclin D1 in 18α-GA group was 2.8 times higher than that in DMSO group (P<0.01), and the CDK4 level was 1.4 times higher than that in DMSO group (P<0.05). CONCLUSION: Activation of the proteasome activity by 18α-GA delays the aging process in the BMSCs and promotes the cell proliferation via up-regulation of the cell cycle-related proteins.     

The mechanism of neuronal injury and repair after focal cerebral ischemia

AIM:To explore the mechanism of neuronal injury and repair by investigating the expression of caspase-3 and apurinic/apyrimidinic endonuclease (APE/Ref-1) after focal cerebral ischemia. METHODS:A model of middle cerebral artery occlusion in rats was performed. The expression of caspase-3P₂₀ and APE/Ref-1 was examined by immunohistochemistry staining, TUNEL was applied to detected DNA damage, and double labeling with TUNEL and APE/Ref-1 was used to determine the relationship between APE/Ref-1 and DNA damage. RESULTS:The active subunit P₂₀ of caspase-3 was predominantly expressed within ischemic penumbra. The peak time of caspase-3P₂₀ positive cells preceded the appearance of TUNEL. With aggravation of cerebral ischemia, APE/Ref-1 immunoreactive cells in penumbra were significantly decreased. CONCLUSION:The activation of caspase enzymatic cascade following cerebral ischemia leads to degradation in DNA, meanwhile, decrease in DNA repair molecules or the failure of DNA repair may deteriorate the course.     

Effect of trastuzumab (herceptin) on the apoptosis induced by ZD1839 (iressa) in lung cancer A549 cells

AIM: ZD1839 and trastuzumab are reported to improve the therapeutic efficacy of treatment for non-smallcell lung cancer (NSCLC) and breast cancer, respectively, although the effectiveness of either drug alone is not satisfactory. NSCLC cells often express both EGFR and HER2. We therefore investigated whether a combination of ZD1839 and trastuzumab had an additive or synergistic antitumor effect. METHODS: MTT was used to measure the inhibitory effects of ZD1839 (iressa) and trastuzumab (herceptin) on the growth of A549 cells. The cell apoptosis was studied by DAPI staining, and Annexin V/PI double labeling. RESULTS: The inhibitory action of cell growth was seen in A549 cells dealing with ZD1839 and trastuzumab. They inhibited the growth of the human lung cancer cell line A549 in a concentration and time dependent manners. Compared with either ZD1839 or trastuzumab alone, combination with curcumin respectively increased the growth inhibition rate and increased apoptosis of A549 cells (P<0.05) significantly, suggesting the synergistic actions of the two drugs. CONCLUSION: The results suggest that combination treatment with ZD1839 and trastuzumab might have improved therapeutic efficacy against NSCLC cells expressing both EGFR and HER2.     

Effects of human growth arrest-specific homeobox gene delivery on proliferation,migration and cell cycle distribution of vascular smooth muscle cells

AIM: To explore a new gene therapeutic strategy for vein graft restenosis by investigating the effects of adenovirus-mediated human growth arrest-specific homeobox (Ad5-hGax) gene delivery on the proliferation, migration and cell cycle distribution of serum-induced rabbit vascular smooth muscle cells (VSMCs). METHODS: The recombinant adenovirus vector containing hGax gene was constructed and transfected into rabbit VSMCs. The expression of hGax in VSMCs was detected by RT-PCR and immunofluorescent staining. Methyl thiazolyl tetrazolium (MTT) assay was used to assess the effect of hGax over-expression on serum-induced proliferation of VSMCs. Wound healing method was applied to examine the distance of serum-induce VSMCs migration. Flow cytometry was employed to analyze the cell cycle distribution. RESULTS: The recombinant adenovirus vector Ad5-hGax was successfully constructed. The results of RT-PCR and immunofluorescent staining revealed that the hGax -transfected cells contained a 174 bp specific fragment of hGax gene and target protein 48 h after transfection. The proliferation of serum-induced VSMCs was significantly inhibited by overexpression of hGax gene as compared with control group. The migration of serum-induced VSMCs was inhibited after hGax gene delivery. Flow cytometry showed that 72 h after serum induction, the cells in G₀/G₁ phase in Ad5-hGax group were significantly increased, whereas the cells in G₂/M+S phase were significantly decreased. CONCLUSION: Overexpression of hGax gene inhibits the proliferation and migration of serum-induced rabbit VSMCs, and arrests the cells in G₀/G₁ phase. It is likely that hGax gene is a potential target for the gene therapy of vein graft restenosis.     

Role of divalent metal transporter 1 in degeneration of dopaminergic neuron

AIM: To observe the expression of divalent metal transporter 1(DMT1) in SH-SY5Y cells with lactacystin-induced injury, and to investigate the possible role of DMT1 in the degeneration of dopaminergic neuron in Parkinson disease(PD). METHODS: An in vitro model of cell injury was established in SH-SY5Y cells induced by lactacystin. The protein expression of DMT1 was detected by immunofluorescence and Western blotting. Under the environment of high iron level, the cellular oxidative stress was observed by fluorescent probe DCFH-DA. The level of α-synuclein was determined by immunohistochemistry and Western blotting. RESULTS: The cell viability rate was reduced by lactacystin in a concentration-dependent pattern. Compared with the control, the protein level of DMT1 was obviously increased in lactacystin-treated cells. The arrangements of the changes from high to low in decreased cell viability, increased intracellular oxidative stress and increased aggregation of α-synuclein(43-55 kD) were Fe²⁺ treatment group > lactacystin treatment group > control group(P<0.05). CONCLUSION: Lactacystin up-regulates the protein expression of DMT1. By this way, the increased function of DMT1-mediated iron uptake may play an important role in iron or iron-catalyzed oxidative reactions to enhance α-synuclein aggregation, leading to the degeneration of neurons in PD.     

Relation between astroglial activation state and ischemic tolerance in the gerbil hippocampus

AIM:To observe the relation between astroglial activation state and ischemic tolerance in the gerbil hippocampus. METHOD: Bilateral occlusion of common carotid arteries and immunofluorescent methods in the gerbil hippocampal tissue. Slices were used. The morphological changes of the neurons were observed by light microscopy.RESULTS:Pretreatment with 2 minute bilateral carotid artery occlusion produced protective effects of CA₁ neurons. Glial fibrillary acidic protein (GFAP) staining showed weak positive cells in control group. Most of GFAP positive cells showed weak and middle positive cells after recirculation 1d and 2d following ischemic 3.5 min and preconditioning of the brain with sublethal ischemia respectively.CONCLUSION:Astroglial played an important role in cerebral ischemia. It is possible that state of astroglial activation related to neuronal survival in ischemic tolerance.     

Dynamic expression of CD30 on peripheral blood lymphocytes in mice after a cecal ligation and puncture

AIM: To investigate the details of Th2 cell differentiation in septic mice. METHODS: Experimental septic mice were induced by cecal ligation and puncture (CLP). The exression of CD30 on CD4⁺T cells at different time after CLP were estimated by flow cytometry following three-color immunofluorescent staining.RESULTS: CD30 expression on CD4⁺T cell was different at each time point. The highest expression was showed at 38 h after CLP and declined later, which matched the changes in mortality of the animals. CONCLUSION: During sepsis, differentiation of Th2 cell changed with the development of sepsis and might be associated with the severity of the disease.     

Involvement of 26S proteasome LMP2 subunit in development of tolerance against oxidative stress in vascular endothelial cells

AIM: To observe the expression of 26S proteasome LMP2 subunit in vascular endothelial cells (VECs) under oxidative stress, and to evaluate its role in the development of tolerance against oxidative stress in VECs. METHODS: The cell model of H₂O₂ preconditioning-induced oxidative tolerance was established in VECs. The expression of LMP2 was detected by cellular immunofluorescent labeling and Western blotting. The LMP2 anti-sense and sense oligonucleotides were transfected into VECs by Lipofectamine^TM 2000. The damages of VECs were evaluated by detecting the activity of lactate dehydrogenase (LDH) and the concentration of malondialdehyde (MDA) in the culture medium. RESULTS: H₂O₂ (500 μmol/L for 3 h) induced oxidative stress in VECss in a dose- and the activity of time-dependent manner, characterized by the increase in the concentration of MDA and LDH in the culture medium. Pretreatment with H₂O₂ (10 μmol/L for 24 h) up-regulated the expression of LMP2. Meanwhile, the capacity of cellular tolerance against oxidative stress induced by H₂O₂ was increased as the concentration of MDA and the activity of LDH in the culture medium significantly decreased. Compared with H₂O₂ group, up-regulation of LMP2 by IFN-γ pretreatment (20 μg/L for 48 h) increased the tolerance of VECs against H₂O₂ injury, and the MDA conentration and the activity of LDH in the culture medium also significantly decreased. Transfection with LMP2 antisense oligonucleotide partly inhibited the increased expression of LMP2 induced by IFN-γ in VECs and abolished the tolerance against H₂O₂. CONCLUSION: The 26S proteasome LMP2 subunit is associated with the development of the tolerance against H₂O₂-induced oxidative stress in VECs.     

Genotype evaluation,muscle histopathology and ultrastructure in a Duchenne muscular dystrophy animal model

AIM: To evaluate the genotype , muscle histopathology and ultrastructure in dko mice. METHODS: Dystrophin/Utrophin-deficient double knockouts (dko) mice were obtained from university of Oxford, UK. Genotype of filial generation of heterozygote was evaluated by PCR-SSP. HE staining and fluorescent immunohistochemistry by SABC-Cy3 were used to detect striated muscle of dko mouse, and the muscle ultrastructure was observed by transmission electron microscope(TEM). RESULTS: In 112 filial generation mice, there were 28 mdx (25.0%), 26 dko (23.2%) and 58 heterozygote (51.8%), which coincided with the law of Mendelian inheritance. HE staining showed that the myocytes were not very uniform, there were phenomenon of round outline, centrally nucleated fibers, widening interspace, inflammatory cell infiltration and connective tissue proliferation in dko mice. There were no any immunofluorescent expression of dystrophin and utrophin in sarcolemma in dko mice. TEM showed sarcolemma breakage, separation and edema, and loose myofibril texture, inflammatory cell infiltration and connective tissue proliferation in dko mice. CONCLUSION: PCR-SSP is a very quick and accurate way for genotype evaluation of filial generation. The pathophysiology of dko mouse was very similar to Duchenne muscular dystrophy (DMD), and dko mouse is an ideal animal model for study of DMD clinical therapy.     

Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial BEAS-2B cells

AIM: To explore the effect of hexokinase 2 (HK2) on lipopolysaccharide (LPS)-induced apoptosis of human lung epithelial BEAS-2B cells and the underlying mechanisms.METHODS: BEAS-2B cells were treated with LPS to induce cell injury, and the cell viability was examined by CCK-8 assay. Hoechst 33342 staining and Annexin V/PI double staining were used to analyze the apoptosis. The apoptotic pathway was identified by the specific inhibitor for caspase-8 or caspase-9. The releases of key mediators in mitochondrial apoptosis pathway were examined by Western blot. The effects of HK2 in these process were confirmed by HK2 over-expression followed by LPS treatment.RESULTS: CCK-8 assay showed that LPS treatment decreased the viability of BEAS-2B cells in a dose/time-dependent manner (P<0.01). The apoptosis of BEAS-2B cells was manifested by Hoechst 33342 and Annexin V/PI double staining. Pretreatment with z-LEHD-fmk, but not z-IETD-fmk, reversed the decreased cell viability under LPS stimulation. HK2 down-regulation was involved in LPS-induced apoptosis of the BEAS-2B cells. After HK2 over-expression, the cell viability was increased after LPS treatment. Releases of cytochrome C and apoptosis-inducing factor from mitochondrion to cytoplasm during apoptosis were also inhibited by HK2 over-expression.CONCLUSION: Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial cells.     

Effects of the grub extract on apoptosis of MCF-7 human breast cancer cell line

AIM: To investigate the apoptotic pathway of MCF-7 breast cancer induced by the grub extract in vitro.METHODS: MTT assay was used to determine the effect of the grub extract on proliferation of MCF-7 human breast cancer cell line and cell toxicity. Morphological changes of the apoptosis in cancer cells were observed by HE staining through invert microscope, light microscope, AO/EB double fluorescent staining under fluorescent microscope. FCM was used to assay the change of apoptotic rate. The expression of Bcl-2, Fas, caspase-9, caspase-3 in apoptotic pathway was detected with immunocytochemical method before and after exposure to the grub extract, and the effect of that on apoptotic pathway was explored.RESULTS: (1) The MTT test showed that the growth of MCF-7 human breast cancer cell line was significantly inhibited by the grub extract in dose and time dependent manners. The inhibitory rate in exposure group was significantly different from that in control group (P<0.01). (2) Morphological changes of apoptosis including nuclear condensation, fragment and apoptosis body formation were observed by invert microscope. (3) The MCF-7 human breast cancer cells in experimental group by HE staining showed nuclear condensation and blue-black, cytoplasm slight red, nuclear chromatin condensation and fragment shape, apoptosis body formations. (4) Apoptosis in the experimental group was observed by AO/EB double fluorescent staining under fluorescent microscope. (5) FCM assay indicated that apoptotic rate increased significantly in time dependent manner in experimental group. (6) The expression of Bcl-2 was down-regulated, while that of Fas, caspase-3, caspase-9 was up-regulated, compared with control group (P<0.01).CONCLUSION: (1) The proliferation of MCF-7 human breast cancer cell line can be inhibited significantly by the grub extract in vitro. (2)The mechanism of effect of the grub extract on MCF-7 human breast cancer cell line might be mediated by down-regulation of Bcl-2 and up-regulation of Fas, caspase-3, caspase-9. This type of apoptosis starting and performing is through death receptor pathway and mitochondrial pathway.     

Inhibitory effects of tumor associated mitochondrial protein 12 on HepG2 cell apoptosis

AIM: To explore the inhibitory effects of tumor associated mitochondrial protein 12 (TAMP12) on tumor cell apoptosis. METHODS: (1) A retrovirus expression vector was recombinated and transfected into the packaging cell line PA317. The virus particles were obtained to infect the target cell line HepG2 low expressing of TAMP12. The expression of TAMP12 mRNA was detected by RT-PCR. The subcellular localization and quantification of TAMP12 protein labeled with double fluorescein were observed under confocal laser scanning microscope (CLSM). (2) Hoechst33258 staining and flow cytometry (FACS) were used to analysis the apoptosis of HepG2 cells treated with 5-fluorouracil (5-FU). RESULTS: (1) The CLSM observation showed that TAMP12 protein was mainly expressed in mitochondria of HepG2 cells. The expressions of TAMP12 gene and protein were stable and high in transfected HepG2 cells. (2) Upon treatment with 5-FU, the transfected HepG2 cells showed a fairly integrated nucelus while the control HepG2 cells exhibited chromatin condensation, marginalization and karyorhexix. Moreover, the apoptosis rate of transfeced HepG2 cells was significantly lower than that in control HepG2 cells (P<0.05 or P<0.01) by FACS. CONCLUSION: TAMP 12 is an anti-apoptotic element in 5-FU induced HepG2 cell apoptosis.     

Biological functions of α-synuclein and its role in Parkinson’s disease

α-Synuclein is identified as a constant component of dopaminergic neuronal pale eosinophilic inclusions "the Lewy bodies" in Parkinson’s disease. In fact, normal α-synuclein has lots of biological functions, which regulates synaptic plasticity, integrates presynaptic signaling, regulates dopamine level in the synapse, modulates microglial activation and lipid metabolism, and exerts heat shock protein-like function. However, abnormal α-synuclein leads to pathological lesion. Many studies show that pathological α-synuclein levels are elevated under the condition of its gene mutation, certain posttranslational modifications, dysfunction of molecular chaperones or ubiquitin proteasome system. The pathological α-synuclein plays an important role in neurodegeneration, in particular, Parkinson’s disease because it interrupts integrity of synaptic vesicles, ER-Golgi traffic and axonal transport, inhibits histone acetylation and chaperone-mediated autophagy, stabilizes itself against proteasomal degradation, and leads to neuroinflammation and abnormal phosphorylation of tau.