Construction of Porcine CCK pDNA and Its Expression in COS-7 Cells

CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The con- structed plasmid was transfected into COS-7 cells by lepofectamineTM2000-mediated transfer method. The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected. And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells trans- fected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells, which lays a foundation for further research on the relationship between CCK and tumor.

Expression of Recombinant Protein Bovine Prion pCIp264 in COS-7 Cells and Its Detection

Bovine spongiform encephalopathy （BSE） is thought to be caused by prions initially derived from sheep scrapie, and epidemiological studies suggest that new viriant form of CJD manifest in Great Britain may be caused by BSE prions. PrP^Sc is thought to pathogenic factor of transmissible spongiform encephalopathy （TSE）, which invariably involve a post-translational modification process of PrPc encoded by the host euchromosome PrP gene during the period it converted into the pathogenic form （PrP^Sc）, PrP is nomal cellular protein which has been found in both neuronal and nonneuronal tissues. Since the crucial infectious event in protein-transmitted diseases is an induced misfolding of prion proteins （PrP^c） catalyzed by already misfolded PrP^Sc, it is of high importance that such collisions are enhanced by two-dimensional diffusion in cell membranes is of high importance compared to three-dimensional diffusion in solution. The level of PrP mRNA in brain is higher than other tissue, but purification of PrPc from rodent has been difficult. To understand the formation of PrP^Sc, it seemed useful to develop a system for produced a large quantities of PrPc since there is no nature source of PrP^c. The pCI-neo mammalian expression vector contains the neomycin phosphtransferase gene which serves as a marker for the selection of stable transfected cells with G418. COS-7 cells constitutively express simian viruse 40 （SV40） T-antigen and support replication of expression plasmids containing the SV40 origin of replication, amplifying the introduced expression cassettes, now become important routine of expression a large number of heterologous gene products. In this paper, the authors used pCI-neo vector to construct a recombnant pCIp264 （cotains mPrP, N-signalpeptide and C-GPI anchor） plasmid to express it in the COS-7 cells and meanwhile detect the expression fusion using IN-ELISA, IN-IFA and western blot, and obtain some approximative nature PrPc.

Dodecylamine Derivative of Hydroxocobalamin Acts as a Potent Inhibitor of Cobalamin-Dependent Methionine Synthase in Mammalian Cultured COS-7 Cells

We evaluated whether the dodecylamine derivative of hydroxocobalamin acts as a potent inhibitor of cobalamin-dependent enzymes in an African green monkey kidney cell, COS-7. When the dodecylamine derivative (1.0 μmol/L) did not show any cytotoxicity in the cultured cells, the derivative could not affect methylmalonyl-CoA mutase (holo-enzyme) activity, but significantly inhibit methionine synthase (holo-enzyme) activity in the cell homogenates of COS-7 grown in 1.0 μmol/L hydroxocobalamin-supplemented medium. An immunoblot analysis indicated that the dodecylamine derivative could not decrease the protein level of methionine synthase, but significantly inhibit the enzyme activity.

Cone-rod homeobox transcriptionally activates TCF7 to promote the proliferation of retinal pigment epithelial and retinoblastoma cells in vitro

AIM:To investigate the proliferation regulatory effect of cone-rod homeobox(CRX)in retinal pigment epithelium(RPE)and retinoblastoma(RB)cells to explore the potential application and side effect(oncogenic potential)of CRXbased gene therapy in RPE-based retinopathies.METHODS:Adult human retinal pigment epithelial(ARPE)-19 and human retinal pigment epithelial(RPE)-1 cells and Y79 RB cell were used in the study.Genetic manipulation was performed by lentivirus-based technology.The cell proliferation was determined by a CellTiter-Glo Reagent.The mRNA and protein levels were determined by quantitative real-time polymerase chain reaction(qPCR)and Western blot assay.The transcriptional activity of the promoter was determined by luciferase reporter gene assay.The bindings between CRX and transcription factor 7(TCF7)promoter as well as TCF7 and the promoters of TCF7 target genes were examined by chromatin immunoprecipitation(ChIP)assay.The transcription of the TCF7 was determined by a modified nuclear run-on assay.RESULTS:CRX overexpression and knockdown significantly increased(n=3,P<0.05 in all the cells)and decreased(n=3,P<0.01 in all the cells)the proliferation of RPE and RB cells.CRX overexpression and knockdown significantly increased and deceased the mRNA levels of Wnt signaling target genes[including MYC proto-oncogene(MYC),JUN,FOS like 1(FOSL1),CCND1,cyclin D2(CCND2),cyclin D3(CCND3),cellular communication network factor 4(CCN4),peroxisome proliferator activated receptor delta(PPARD),and matrix metallopeptidase 7(MMP7)]and the luciferase activity driven by the Wnt signaling transcription factor(TCF7).TCF7 overexpression and knockdown significantly increased and decreased the proliferation of RPE and RB cells and depletion of TCF7 significantly abolished the stimulatory effect of CRX on the proliferation of RPE and RB cells.CRX overexpression and knockdown significantly increased and decreased the mRNA level of TCF7 and the promoter of TCF7 was significantly immunoprecipitated by CRX antibody.CONCLUSION:CRX transcriptionally activates TCF7 to promote the proliferation of RPE and RB cells in vitro.CRX is a potential target for RPE-based regenerative medicine.The potential risk of this strategy,tumorigenic potential,should be considered.

Hesperidin as a preventive resistance agent in MCF-7 breast cancer cells line resistance to doxorubicin

Objective:To evaluate of hesperidin to overcome resistance of doxorubicin in MCF-7 resistant doxorubicin cells(MCF-7/Dox)in cytotoxicity apoptosis and P-glycoprotein(Pgp)expression in combination with doxorubicin.Methods:The cytotoxic properties.50%inhibition concentration(IC_(50))and its combination with doxorubicin in MCF-7 cell lines resistant to doxorubicin(MCF-7/Dox)cells were determined using MTT assay.Apoptosis induction was examined by double staining assay using ethidium bromide-acridine orange.Immunocytochemistry assay was performed to determine the level and localization of Pgp.Results:Single treatment of hesperidin showed cytotoxic activity on MCF-7/Dox cells with IC_(50)value of 11μmol/L.Thus,combination treatment from hesperidin and doxorubicin showed addictive and antagonist effect(CI>1.0).Hesperidin did not increase the apoptotic induction,but decreased the Pgp expressions level when combined with doxorubicin in low concentration.Conclusions:Hesperidin has cytotoxic effect on MCF-7/Dox cells with IC_(50)of 11μmol/L.Hesperidin did not increased the apoptotic induction combined with doxorubicin.Cochemotherapy application of doxorubicin and hesperidin on MCF-7/Dox cells showed synergism effect through inhibition of Pgp expression.

Triterpenoid of avocado (Persea americana) seed and its cytotoxic activity toward breast MCF-7 and liver HepG2 cancer cells

Objective:To determine the structure of triterpenoid isolated from avocado seeds and the cytotoxic effect on MCF-7 and Hep G2 cells.Methods:The powder sample was macerated with ethanol,followed with separation of the extract by column chromatography.The target compound was monitored on thin layer chromatography plate and reagent Lieberman–Buchard.The isolated compound was characterized by spectral analysis,mainly ultraviolet,infrared,and liquid chromatographymass spectroscopy and their spectroscopic data with those reported in literature were compared.In vitro cytotoxic activity was investigated against Vero,MCF-7,and Hep G2 cell lines using MTT assay.Results:A triterpenoid compound was isolated from ethanol extract.The extracts,fraction(F3),and the isolated compound showed a significant cytotoxic activity against all investigated cell lines.MTT assay showed that the triterpenoid isolate inhibited cell proliferation of MCF-7 and Hep G2 cell line with the IC50 values of 62 mg/m L and 12 mg/m L,respectively,and was safe to normal cells.Conclusions:The results of the present study reveal that triterpenoid from avocado seeds have the potential for further development as anticancer agents.

Fucoidan Induces G_1 Phase Arrest and Apoptosis through Caspases-dependent Pathway and ROS Induction in Human Breast Cancer MCF-7 Cells

Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D 1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, cas- pase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS pro- duction was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce GI phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.

8-bromo-7-methoxychrysin inhibits properties of liver cancer stem cells via downregulation of β-catenin

AIM:To evaluate whether 8-bromo-7-methoxychrysin(BrMC),a synthetic analogue of chrysin,inhibits the properties of cancer stem cells derived from the human liver cancer MHCC97 cell line and to determine the potential mechanisms.METHODS:CD133+cells were sorted from the MHCC97 cell line by magnetic activated cell sorting,and amplified in stem cell-conditioned medium to obtain the enriched CD133+sphere forming cells(SFCs).The stem cell properties of CD133+SFCs were validated by the tumorsphere formation assay in vitro and the xenograft nude mouse model in vivo,and termed liver cancer stem cells(LCSCs).The effects of BrMC on LCSCs in vitro were evaluated by MTT assay,tumorsphere formation assay and transwell chamber assay.The effects of BrMC on LCSCs in vivo were determined using a primary and secondary xenograft model in Balb/c-nu mice.Expressions of the stem cell markers,epithelialmesenchymal transition(EMT)markers andβ-catenin protein were analyzed by western blotting or immunohistochemical analysis.RESULTS:CD133+SFCs exhibited stem-like cell properties of tumorsphere formation and tumorigenesis capacity in contrast to the parental MHCC97 cells.We found that BrMC preferentially inhibited proliferation and self-renewal of LCSCs(P<0.05).Furthermore,BrMC significantly suppressed EMT and invasion of LCSCs.Moreover,BrMC could efficaciously eliminate LCSCs in vivo.Interestingly,we showed that BrMC decreased the expression ofβ-catenin in LCSCs.Silencing ofβ-catenin by small interfering RNA could synergize the inhibition of self-renewal of LCSCs induced by BrMC,while Wnt3a treatment antagonized the inhibitory effects of BrMC.CONCLUSION:BrMC can inhibit the functions and characteristics of LCSCs derived from the liver cancer MHCC97 cell line through downregulation ofβ-catenin expression.

B7-H1 expression is associated with expansion of regulatory T cells in colorectal carcinoma

AIM： To investigate the expression of B7-H1 in human colorectal carcinoma （CRC） to define its regulating ef- fects on T cells in tumor microenvironment.

Dietary Daidzein Enhances Antiapoptotic Effect of 17β-Estradiol (E_2) on Breast Cancer MCF-7 Cells

Objective： To investigate whether dietary daidzein interact with endogenous 17β-Estradiol （E2） to give rise to additive or inhibitory effects on proliferation and apoptosis in breast cancer cells. Methods： Cell cycle distribution and apoptosis induction were analyzed by using flow cytometry when breast cancer cell lines MCF-7 were cotreated with daidzein （1, 5 μmol/L） and E2 （0.1-10 nmol/L） for 5 days. Whether daidzein could alter E2-modulated mRNA expression of estrogen receptor alpha （ERα）, estrogen receptor beta （ERI3） and ERβ-estrogen response element （ERE） dependent transcription was investigated by RT-PCR and luciferase induction assays. The effects of daidzein on E2-modulated expression of proapoptotic p53, bax and antiapoptotic bcl-2 at both mRNA and protein levels were also investigated by RT-PCR and Western blot. Results： Daidzein enhanced the antiapoptotic effect in an Ea dose-dependent manner, but had no effect on E2-induced proliferation. Daidzein antagonized E2-induced ERβ mRNA expression and ERβ-ERE dependent transcription. In addition, daidzein only antagonized E2-upregulated expression of p53 and bax, but had no effect on E2-upregulated expression of bcl-2. Conclusion： Daidzein enhances the antiapoptotic effect of E2 on breast cancer cells by inhibiting E2-mediated p53-bax proapoptotic pathway. These results suggest that dietary daidzein may enhance deleterious effect of endogenous E2 in hormone-dependent breast cancer.

Cox7a2 mediates steroidogenesis in TM3 mouse Leydig cells

Aim： To investigate the regulatory function of Cox7a2 on steroidogenesis and the mechanism involved in TM3 mouse Leydig cells. Methods： The cDNA of Cox7a2 was cloned from TM3 mouse Leydig cells. It was subcloned to pDsRed- Express-N 1 and transfected back into TM3 mouse Leydig cells for Cox7a2 overexpression by transient gene transfection. Steroidogenesis affected by overexpressed Cox7a2 was studied by ELISA. To elicit the mechanism of this effect, expression of steroidogenic acute regulatory （STAR） protein and reactive oxygen species （ROS） were examined by Western blot and fluorometer, respectively. Results： The cDNA of Cox7a2 （249 bp） was cloned from Leydig cells and confirmed by DNA sequencing. After constructed pDsRed-Express-N1-Cox7a2 was transfected back into TM3 mouse Leydig cells, Cox7a2 inhibited not only luteinizing hormone （LH）-induced secretion of testosterone but also the expression of StAR protein. At the same time, Cox7a2 increased the activity of ROS in TM3 mouse Leydig cells. Conclusion： Cox7a2 inhibited LH-induced StAR protein expression, and consequent testosterone production, at least in part, by increasing ROS activity in TM3 mouse Leydig cells.

Roles of Smad3 and Smad7 in rat pancreatic stellate cells activated by transforming growth factor-beta 1

BACKGROUND: Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor beta 1 (TGF-beta 1) is a critical mediator of this process. This study aimed to determine the expression of the Smad3 and Smad7 genes in the process of PSC activation, and explore the mechanisms of chronic pancreatitis. METHODS: The expressions of Smad3 and Smad7 in PSCs before and after TGF-beta 1 treatment were detected by reverse transcription-polymerase chain reaction and Western blotting analysis. Smad3 expression was detected in PSCs after treatment with 5 ng/ml of TGF-beta 1 for 24 hours. RESULTS: Smad7 expression was decreased in TGF-beta 1 -activated PSCs (P<0.05) in a dose-dependent manner. When TGF-beta 1 concentration reached 10 ng/ml, the expression of p-Smad3, Smad3, and Smad7 was inhibited (P<0.05). CONCLUSIONS: TGF-beta 1 promotes the expression of Smad3 and inhibits the expression of Smad7 during the activation of PSCs. In contrast, high-dose TGF-beta 1 downregulates the expression of Smad3 in completely activated PSCs.

Insulin-like growth factor binding protein-7 induces activation and transdifferentiation of hepatic stellate cells in vitro

AIM:To investigate the role of insulin-like growth factor binding protein-7 (IGFBP-7) in the activation and transdifferentiation of hepatic stellate cells (HSC) in vitro.METHODS:Rat HSC-T6 cells were cultured in separate dishes and treated with various concentration of transforming growth factor (TGF)-β1,IGFBP-7 or antiIGFBP-7 antibody for 24 h.The supernatant or a cytoplasm suspension was obtained from cultured HSC,followed by transfer of cells to form cell-coated dishes.Immunocytochemistry and Western blotting were used to analyze the expression of IGFBP-7 induced by TGF-β1 and the level of fibronectin,collagen and α-smooth muscle actin (SMA).The pro-apoptotic effect of antiIGFBP-7 antibody was determined by flow cytometry.RESULTS:Immunocytochemistry and Western blotting revealed that the expression of IGFBP-7 in TGF-β1 treated HSC was significantly up-regulated compared to that in the control group.In addition,fibronectin,collagen and α-SMA also showed enhanced expression in accordance with the transdifferentiation process in a dose-dependent manner to some extent.Moreover,flow cytometry suggested that anti-IGFBP-7 antibody induced apoptosis of activated HSC,which is responsible for the development of liver fibrosis,and may represent a novel pathway and target for therapeutic intervention.CONCLUSION:IGFBP-7 showed increased expression in activated HSC and played an important role in the activation and transdifferentiation process of HSC.AntiIGFBP-7 antibody may ameliorate liver fibrogenesis.

GB7 acetate,a galbulimima alkaloid from Galbulimima belgraveana,possesses anticancer effects in colorectal cancer cells

GB7 acetate is a galbulimima alkaloid obtained from Galbulimima belgraveana.However,information regarding its structure,biological activities,and related mechanisms is not entirely available.A series of spectroscopic analyses,structural degradation,interconversion,and crystallography were performed to identify the structure of GB7 acetate.The MTT assay was applied to measure cell proliferation on human colorectal cancer HCT 116 cells.The expressions of the related proteins were measured by Western blotting.Transmission electron microscopy(TEM),acridine orange(AO)and monodansylcadaverine(MDC)staining were used to detect the presence of autophagic vesicles and autolysosomes.A transwell assay was performed to demonstrate metastatic capabilities.Oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)assays were performed to determine the mitochondrial oxidative phosphorylation(OXPHOS)and glycolysis activity of HCT 116 cells.The data showed that GB7 acetate suppressed the proliferation and colony-forming ability of HCT 116 cells.Pretreatment with GB7 acetate significantly induced the formation of autophagic vesicles and autolysosomes.GB7 acetate upregulated the expressions of LC3 and Thr172 phosphorylated adenosine 5'-monophosphate(AMP)-activated protein kinase a(p-AMPKa),which are key elements of autophagy.In addition,GB7 acetate suppressed the metastatic capabilities of HCT 116 cells.Additionally,the production of matrix metallo-proteinase-2(MMP-2)and MMP-9 was reduced,whereas the expression of E-cadherin(E-cad)was upregulated.Furthermore,GB7 acetate significantly reduced mitochondrial OXPHOS and glycolysis.In conclusion,the structure of the novel Galbulimima alkaloid GB7 acetate was identified.GB7 acetate was shown to have anti-proliferative,pro-autophagic,anti-metastatic,and anti-metabolite capabilities in HCT 116 cells.This study might provide new insights into cancer treatment efficacy and cancer chemoprevention.

miR-34c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene

MicroRNAs(miRNAs) are implicated in swine spermatogenesis via their regulations of cell proliferation, apoptosis, and differentiation. Recent studies indicated that miR-34 c is indispensable in the late steps of spermatogenesis. However, whether miR-34 c plays similar important roles in immature porcine Sertoli cells remain unknown. In the present study, we conducted two experiments using a completely randomised design to study the function roles of miR-34 c. The results from experiment I demonstrated that the relative expression level of miR-34 c in swine testicular tissues increased(P=0.0017) quadratically with increasing age, while the relative expression level of SMAD family member 7(SMAD7) decreased(P=0.0009) with curve. Furthermore, miR-34 c expression levels showed a significant negative correlation(P=0.013) with SMAD7 gene expression levels. The results from experiment II indicated that miR-34 c directly targets the SMAD7 gene using a luciferase reporter assay, and suppresses(P<0.05) SMAD7 mRNA and protein expressions in immature porcine Sertoli cells. Overexpression of miR-34 c inhibited(P<0.05) proliferation and enhanced(P<0.05) apoptosis in the immature porcine Sertoli cells, which was supported by the results from the Cell Counting Kit-8(CCK-8) assay, the 5-Ethynyl-2′-deoxyuridine(EdU) assay, and the Annexin V-FITC/PI staining assay. Furthermore, knockdown of SMAD7 via small interfering RNA(siR NA) gave a similar result. It is concluded that miR-34 c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene.

<i>Vernonia amygdalina</i>—Induced Growth Arrest and Apoptosis of Breast Cancer (MCF-7) Cells

Breast cancer is the second leading cause of cancer-related deaths of women in the United States. Fortunately, the mortality rate from breast cancer has decreased in recent years due to an increased emphasis on early detection and more effective treatments. Although great advancements have been made in the treatment and control of cancer progression, significant deficiencies and room for improvement remain. The central objective of this research was to further determine the in vitro mechanisms of Vernonia amygdalina (VA) leaf extracts as an anticancer candidate for the treatment of breast cancer. To achieve our objective, MCF-7 cells were treated with different concentrations of VA for 24 hand 48 h. Cell viability, live and dead cells were determined by the means of trypan blue exclusion test. Live and dead cells were further evaluated by propidium iodine (PI) assay using the Cellometer Vision. Cell apoptosis was measured by flow cytometry assessment using annexin V/PI kit. Data obtained from the trypan blue test demonstrated that VA treatment reduces cell viability in a concentration- and time-dependent manner. Result of the PI assay showed a gradual increase in the population of necrotic cells (fluorescence positive cells) in VA-treated cells compared to the control cells (fluorescence negative cells). Treatment of these cancer cells (MCF-7) for 48 h at concentrations ranging from 250 μg/mL to 1000 μg/mL caused early signs of apoptosis resulting from phosphatidylserine externalization as judged by annexin V assay. We observed a strong concentration-response relationship with regard to VA exposure and annexin V/PI positive cells. In summary, our finding demonstrates that VA-induced cytotoxicity and apoptosis in MCF-7 cells involve phosphatidylserine externalization accompanied by secondary necrotic cell death. With previous findings in our laboratory, the data generated in the present study confirms that VA is a valuable botanical therapeutic agent for the treatment of breast cancer.

EFFECT OF CIS-9, TRANS-11-CONJUGATED LINOLEIC ACID ON CELL CYCLE OF MAMMARY ADENOCARCINOMA CELLS(MCF-7)

Objective: To determine the effect of cis-9, trans-1 1-conjugated linoleic acid on the cell cycle of mammary cancer cells (MCF-7) and the possible mechanism of the inhibitory effect of c9,t11-CLA. Methods: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B1, D1, p16ink4a and p21cip/waf1 of MCF-7 cells at various c9,t11-CLA concentrations (25μM, 50μM, 100μM and 200μM), at 24h and 48h. 96% ethand was used as negative control. Results: The cell growth and DNA synthesis of MCF-7 cells were inhibited by c9,t11-CLA. After treatment with various doses of c9,t11-CLA mentioned above for 8 days, the inhibition frequency was 27.18%, 35.43%, 91.05%, and 92.86%, respectively. Inhibitory effect of c9,t11-CLA on DNA synthesis (except for 25μM, 24h) was demonstrated by significantly less incorporation of 3H-TdR than the negative control (P<0.05 and P<0.01). To further investigate the influence of the cell cycle progression, we found that c9,t11-CLA may arrest the cell cycle of MCF-7 cells. Immunocytochemical staining demonstrated that incubation with different concentration of c9,t11-CLA at various times significantly decreased the expression of PCNA, Cyclin A, B1, D1 in MCF-7 cells compared to the negative control (P<0.01), whereas the expression of p16ink4a and p21cip/waf1, cyclin-dependent kinases inhibitors (CDKI), were increased. Conclusions: The cell growth and proliferation of MCF-7 cells is inhibited by c9,t11-CLA via blocking cell cycle, accompanying reduced expression of cyclin A, B1, D1 and enhanced expression of CDKI (p16ink4a and p21cip/waf1).

Acid Water-ground Nano-realgar Is Superior to Crude Realgar in Promoting Apoptosis of MCF-7 Breast Cancer Cells

Objective:Realgar is a traditional mineral Chinese medicine with antitumor effects,but it has high toxicity and low efficacy in its crude form.The purpose of this study was to optimize realgar to increase its efficacy and therapeutic potential.Methods:Crude realgar(CR)was mechanically ground to obtain nano-realgar(NR),and then nano-realgar processed products(NRPPs)were obtained using three different traditional Chinese medicine processing methods:grinding in water,acid water,and alkali water,respectively.Results:By analyzing the size distribution of nanoparticles and the content of arsenic trioxide(As_(2)O_(3);ATO),we found that acid water-ground NRPPs had the characteristics of high purity and low toxicity.The effects of CR,NR,and NRPPs on proliferation,cell cycle,and apoptosis of MCF-7 cells were detected,and the ability of NRPPs to induce apoptosis in MCF-7 cells was analyzed.The results showed that the average particle size of acid water-ground NRPPs was 137.7 nm,and the content of ATO was 2.83 mg/g.Acid water-ground NRPPs showed better effects on inhibiting proliferation,cell cycle,and apoptosis of MCF-7 cells than CR and NR.Western blot assays further confirmed that acid water-ground NRPPs upregulated the protein expression of TP53,Bax,cytochrome c,caspase-9,and caspase-3 in MCF-7 cells(P<0.05)and inhibited the expression of Bcl-2(P<0.05).Conclusion:These results suggest that acid water-ground NRPPs can induce apoptosis of MCF-7 cells through regulating mitochondrial-mediated apoptosis,providing evidence for the clinical application of realgar.

Contralateral C7 transfer combined with acellular nerve allografts seeded with differentiated adipose stem cells for repairing upper brachial plexus injury in rats

Nerve grafting has always been necessary when the contralateral C7 nerve root is transferred to treat brachial plexus injury. Acellular nerve allograft is a promising alternative for the treatment of nerve defects, and results were improved by grafts laden with differentiated adipose stem cells. However, use of these tissue-engineered nerve grafts has not been reported for the treatment of brachial plexus injury. The aim of the present study was to evaluate the outcome of acellular nerve allografts seeded with differentiated adipose stem cells to improve nerve regeneration in a rat model in which the contralateral C7 nerve was transferred to repair an upper brachial plexus injury. Differentiated adipose stem cells were obtained from Sprague-Dawley rats and transdifferentiated into a Schwann cell-like phenotype. Acellular nerve allografts were prepared from 15-mm bilateral sections of rat sciatic nerves. Rats were randomly divided into three groups: acellular nerve allograft, acellular nerve allograft + differentiated adipose stem cells, and autograft. The upper brachial plexus injury model was established by traction applied away from the intervertebral foramen with micro-hemostat forceps. Acellular nerve allografts with or without seeded cells were used to bridge the gap between the contralateral C7 nerve root and C5–6 nerve. Histological staining, electrophysiology, and neurological function tests were used to evaluate the effect of nerve repair 16 weeks after surgery. Results showed that the onset of discernible functional recovery occurred earlier in the autograft group first, followed by the acellular nerve allograft + differentiated adipose stem cells group, and then the acellular nerve allograft group;moreover, there was a significant difference between autograft and acellular nerve allograft groups. Compared with the acellular nerve allograft group, compound muscle action potential, motor conduction velocity, positivity for neurofilament and S100, diameter of regenerating axons, myelin sheath thickness, and density of myelinated fibers were remarkably increased in autograft and acellular nerve allograft + differentiated adipose stem cells groups. These findings confirm that acellular nerve allografts seeded with differentiated adipose stem cells effectively promoted nerve repair after brachial plexus injuries, and the effect was better than that of acellular nerve repair alone. This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University of China(approval No. 2016-150) in June 2016.

Identification of TRPM7 channels in human intestinal interstitial cells of Cajal

AIM： To investigate the characteristics of slow electrical waves and the presence of transient receptor potential melastatin-type 7 （TRPM7） in the human gastrointestinal （GI） tract. METHODS： Conventional microelectrode techniques were used to record intracellular electrical responses from human GI smooth muscle tissue. Immunohistochemistry was used to identify TRPM7 channels in interstitial cells of Cajat （ICCs）. RESULTS： The human GI tract generated slow electrical waves and had ICCs which functioned as pacemak er cells. Flufenamic acid, a nonselective cation channel blocker, and 2-APB （2-aminoethoxydiphenyl borate） and La3＋, TRPM7 channel blockers, inhibited the slowwaves. Also, TRPM7 channels were expressed in ICCs in human tissue. CONCLUSION： These results suggest that the human GI tract generates slow waves and that TRPM7 channels expressed in the ICCs may be involved in the gen- eration of the slow waves.