Impact of High ABCA3 Expression in Nasopharyngeal Carcinoma Stem Cells on theResistance to Cisplatin / 医药导报

Objective To investigate the role of ATP￣binding cassette ( ABC ) family on the resistance of nasopharyngeal carcinoma (NPC) stem cells (CSCs) to cisplatin. Methods We compared the differences between the drug extravasation capability of CNE￣2 and CNE￣2S by using Rhodamine￣123 efflux assay. We determined the mRNA and protein expression levels of ABC transport family members, including ABCA3,ABCB1,ABCB5,ABCC1,ABCC2 and ABCG2,after 48 h being treated with 1 μmol.L-1 cisplatin by RT￣PC and Western blotting.Rhoamine￣123 efflux and apoptosis by cisplatin in two kinds of cells was examined by ABCA3 gene silencing with specific small￣interfering RNA. Results The IC50 of cisplatin on CNE￣2S was 4.1 fold to that on CNE￣2(P<0.05).For the relative drug effluent activity and Na+K+ ATPase activity,CNE￣2S was 4.8 fold to CNE￣2(P<0.05),suggested that CNE￣2S expressed more ABCA3,ABCB1,ABCC1 and ABCG2 in comparison to CNE￣2(P<0.05).After 48 h treatment with 1 μmol.L-1 cisplatin,ABCA3 specifically highly expressed in CNE￣2S (P<0.05), and knocking down of ABCA3 resulted in reduction of rhodamine￣123 efflux and increase of apoptosis. Conclusion The cisplatin resistance of NPC CSCs is associated with enhanced expression of ABCA3,ABCC1 and ABCG2, suppression of ABCA3 could reverse the resistance of NPC CSCs to cisplatin.

Nasopharyngeal carcinoma stem cells develop resistant against Cisplatin through up-regulating SOD / 天津医药

Objective To investigate the way that nasopharyngeal carcinoma (NPC) and NPC stem cells develops resistance to cisplatin through anti-reactive oxygen species mechanism. Methods Using CCK-8 cell counting kit, we measured the half inhibitory concentration of cisplatin against NPC cellsCNE-2and NPC stem cellsCNE-2S, and compared their resistant index. We examined the differences in the reactive oxygen species (ROS) levels, total glutathi?one (GSH) levels, and total superoxide dismutase (SOD) levels between CNE-2 and CNE-2S at different concentrations of cisplatin administration (0.1,0.5 and 1.0μmol·L-1). Using q-PCR, we determined the mRNA expression level of GSS, GCLC, GCLM, SOD1 and SOD2 after 48 hours administration of cisplatin at 1 μmol · L-1. Protein expression level of SOD2 was also tested using Western Blot after 48 hours administration of cisplatin at 1μmol · L-1. Upon silencing the SOD2 in NPC cell through siRNA, Trypan blue was used to analyze cell survival after cisplatin was administrated at 1μmol · L-1. Results The inhibition concentration of cisplatin against CNE-2 was higher than that against CNE-2S (μmol · L-1:9.8 ± 1.1 vs 2.4 ± 0.6,P<0.05). ROS levels in CNE-2 and CNE-2S both rise with cisplatin administration, but ROS levels of CNE-2 before and after cisplatin treatment were both higher than those in CNE-2S (P<0.05). The total gluta?thione levels in CNE-2 and CNE-2S were both increased after 1μmol·L-1 cisplatin treatment but there is no significant dif?ference in levels of glutathione between these two cell lines. After treated with cisplatin, SOD level were increased in both CNE-2S and CNE-2, but it is higher in CNE-2S than that in CNE-2 (P<0.05). The mRNA levels of GSS, GCLC, GCLM, and SOD1 were not different significantly between in CNE-2 and in CNE-2S with or without cisplatin treatment. However, SOD2 in CNE-2S were higher than that in CNE-2 on both mRNA and protein levels (P<0.05). Silenced SOD2 disrupted the resistance of cisplatin in CNE-2S. Conclusion These data suggest that NPC stem cells (CNE-2S) enhance its drug re?sistance to cisplatin through highly expression of SOD2 which posed anti-ROS capacity.

The effect of RNA interference induced by inhibition of hTERT and Bi-1 gene expression / 重庆医学

Objective In this study ,we constructed a series of recombinant plasmids carriers expressing shRNA targeting hTERT and Bi‐1 gene .These recombinant plasmids carriers were transfected into CNE‐2Z cell lines using Lip and continuously in‐duced the expression of shRNAs .Furthermore ,the shRNAs caused the degradation of mRNAs homologous in sequence with the target genes ,which lead to a sequence‐specific gene silencing .Methods The CNE‐2Z cells was divided into untreated group ,pEG‐FP‐N1 group and pEGFP‐N1/Lip group .Flow cytometry(FCM ) was applied to determine the transfection efficiency .The changes of hTERT and Bi‐1 gene expression were detected by Real‐time RT‐PCR and Western blotting .Results The best transfection effi‐ciency between plasmid and Lip was 2 .5 μg plasmid and 6 .25μL Lip .Conclusion We constructed several shRNA recombinant eu‐karyotic expression plasmids successfully .The recombinant plasmid can inhibit the expression of hTERT and Bi‐1 gene specifically and effectively .

Isolation and characterization of cancer stem cell-like sphere-forming cell subpopulation from the human nasopharyngeal cell line CNE-2 / 中国肿瘤临床

Objective: The present study aimed to isolate and characterize a cancer stem cell-like sphere-forming cell subpopula-tion. Methods: By using a spheroid culture stem cell-conditioned medium, we isolated a subgroup of cancer stem-like cells from naso-pharyngeal cancer cell lines. Chemotherapy resistance was analyzed by using methyl thiazolyl tetrazolium, and clone-forming capabili-ty was determined by using softer agar clone formation assay. Finally, we verified the expression of the stemness-specific gene andβ-catenin by using immunocytochemistry staining and RT-PCR. Results: The lower-differentiated nasopharyngeal cancer lines con-tained more sphere-forming cells, whereas sphere-forming cells were not observed in the high differentiated nasopharyngeal cancer line CNE-1. Compared with CNE-2, CNE-2S (sphere-forming cells derived from CNE-2) exhibited higher chemotherapy resistance and clone-forming ability. Interestingly, the stemness genes BMI-1, ABCG2, and ALDH1 exhibited higher expression in CNE-2S than in CNE-2. β-catenin, a vital transcription factor of the WNT pathway related to stem cells, exhibited higher expression in CNE-2s cellular nucleus and plasma than in CNE-2. Conclusion: We isolated a subgroup of stem-like nasopharyngeal cancer sphere-forming cells. This discovery paves the way for the development of therapeutic strategies aimed at eradicating tumorigenic subpopulations in nasopharyn-geal cancer.

Inhibitory effect of arsenic trioxide combined with cisplatin on human nasopharyngeal carcinoma xenograft and DAPK in nude mice / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To study the inhibitory effect of Arsenic Trioxide (As2O3) combined with diamminedichloroplatinum (DDP) on the growth of human nasopharyngeal carcinoma cell strain CNE-2Z xenograft in nude mice, and to explore the possible effect mechanisms of the antitumor.@*METHOD@#The models of human poorly differentiated nasopharyngeal carcinoma in nude mice were established and randomly divided into four groups, control group, As2O3 group, DDP group and As2O3 + DDP group. The effect of antitumor on each group was studied. The specimen obtained from the mice were detected by optical microscope and tdt-mediated dutp rock end labeling (tunel) method. Expression of DAPK was detected by real time-PCR and immunohistochemistry.@*RESULT@#As2O3 group and AS2O3 + DDP group could obviously inhibit the growth of tumor, induce the apoptosis of human naso pharyngeal carcinoma cell and up-regulate the expression of RASSF1A.@*CONCLUSION@#As2O3 can greatly inhibit the growth of human nasopharyngeal carcinoma cell strain CNE-2Z xenograft in nude mice, which were related to the induced apoptosis of human nasopharyngeal carcinoma cell and up-regulated expression of DAPK Combination of As2O3 with DDP seem to be more effective.

Study on the anti-inflammatory effect of proanthocyanidins and its mechanism / 国际检验医学杂志

Objective To investigate the anti-inflammatory effect of proanthocyanidins and its mechanisms.Methods Inflammation models such as dimethylbenzene-indueed ear swelling and carrageenan-induced hind paw edema in mice and rats were prepared.The contents of PGE2 in exudate from edema paws of rats were measured by ultraviolet spectrophotography and the protein expression of COX-2 in edema paws of rats by Western-blot and immunohistoehemistry(IHC)assay.Results Pro-anthocyanidins remarkably inhibited the ear edema induced by dimethylbenzene in mice at the dose of10 mg/kg and 20 mg/kg;paw edema of rats induced with carrageenan was significantly inhibited byproanthocyanidins 5,20 mg/kg ip from 2 to 5 h;proanthoeyanidins 5,20 mg/kg ip reduced the contents of PGE2 in exudate from edema paws of rats induced by carrageenan;proanthocyanidins 5,20 mg/kg ip inhibited the protein expression of COX-2.Conclusion Proanthocyanidins has an anti-inflammatory effect in vivo which may be related to inhibition of protein expression of COX-2 and downregutation of PGE2 biosynthesis.

Construction of the Key Laboratory for Teaching Efficiency of Biochemistry and Molecular Biology / 中华医学教育探索杂志

This paper describes the construction and practical experience of the key laboratory for teaching of biochemistry and molecular biology,and indicates that the laboratory promotes the development of teaching and scientific research.It is proved to be a suitable measure for sharing teaching resource,improving teaching quality and raising teacher' academic level.

c-FLIP:The Regulator of Extrinsic Apoptotic Pathway / 生物化学与生物物理进展

Cellular FADD-like interleukin-1-? converting enzyme inhibitory protein (c-FLIP) is a kind of inhibitory protein of caspase with the death effector domain (DED), naturally existing in many species such as virus, eukaryote and mammal inclusively. Recently, it has been discovered that c-FLIP participates the regulation of apoptosis. Overexpression of c-FLIP may inhibits the apoptosis induced by the death receptor of Fas and TRAIL-R. With the development on the mechanism of action and molecular regulation of c-FLIP, its multi-biology function has been found, and also it is associated with the nosogenesis and progression of many diseases.

Effects of homoharringtonine (HHT) on expression of caspase-3 protein and mRNA in nasopharyrgeal carcinoma cells CNE-2Z / 中国药理学通报

AIM To investigate the effects of homoharringtonine (HHT) on the expression of caspase-3 pro-tein and mRNA in nasopharyngeal carcinoma cells CNE-2Z. METHODS After CNE-2Z cells were treated with HHT [0(control),0.125,0.25,0.5,1 mg?L -1] for 8 h, the expression of pro-caspase-3 protein was analyzed by Western Blot and the expression of caspase-3 mRNA was detected by semi-quantitative RT-PCR. RESULTS As HHT-concentration increased, the expression of pro-caspase-3 protein decreased significantly (P

Quantitative analysis of the 5-lipoxygenase mRNA in the rat peritoneal macrophages cultured in vitro by capillary electrophoresis / 中华检验医学杂志

Objective To quantify the mRNA of 5-lipoxygenase (5-LO) in the rat peritoneal macrophages cultured in vitro by capillary electrophoresis (CE). Methods The rat peritoneal macrophages were isolated and cultured for indicated time. The mRNA of 5-LO was detected by RT-PCR, and the products of RT-PCR were quantified by CE. Results The DNA fragments in the 100 bp DNA marker and the products of the RT-PCR were separated successfully by CE with the sieving buffer containing 1.8% hydroxypropylmethylcellulose (HPMC). It proved that there were no significant changes on the expressions of 5-LO mRNA when the cells were cultured for 72 h quantified by CE. The mRNA of 5-LO significantly decreased by almost 80% by CE with the cells cultured for 120 h in vitro.Conclusions The products of RT-PCR could be separated and quantified by CE directly.The 5-LO mRNA could express normally in the rat peritoneal macrophages for 72 h in vitro.

bcl-xs mediated sensitization of human nasopharyngeal carcinoma cells to camptothecin-induced apoptosis / 中国耳鼻咽喉头颈外科

Objective: In order to detect the effect of bcl-xs on camptothecin-induced apoptosis in human nasopharyngeal carcinoma CNE-2Z cells in vitro.Methods:bcl-xs gene-bearing mammalian expression vector(pcDNA3xs)was transfected into CNE-2Z cells using LipofectAmine.The expression of bcl-xs was determined with western blot.Cells which were transfected with native pcDNA3 vector were used as control.Apoptotic cells were detected with flow cytometry after exposure to camptothecin for 24h.Results:Cell clone(CNE-2Zxs)with stable expression of bcl-xs was obtained as confirmed with western blot.Results from flow cytometry analysis showed a significant increase of apoptotic cells in CNE-2Zxs as compared with CNE-2Zneo after treatment with the same dose of camptothecin.Conclusion:Exogenous bcl-xs expression sensitized nasopharyngeal carcinoma CNE-2Z cells to camptothecin-induced apoptosis.

Difference of the migration ability between PTEN transfected breast cells and non-transfected cells / 肿瘤研究与临床

Objective To disclose the influence of PTEN on the migration of breast cancer cells ZR-75-1. Methods Wild-type PTEN gene was transtected into ZR-75-1 breast cancer cells which lack PTEN gene, tranfected cells were selected by puromycin and the protein encoded by PTEN gene was tested by Western-blot. The inhibition rate of invasion and adhesion of ZR-75-1 was tested on reconstituted basement membrane. Results Wild-type PTEN gene was successfully transfected into ZR-75-1 and expressed efficiently. The inhibition rate of invasion and adhesion is 70.4 % and 60.0 % respectively. Conclusion PTEN gene can restrict the migration of breast cancer cells in some degree, so whether PTEN gene is deleted or not can partly estimates the risk of migration of breast cancer cells.

Effects of resveratrol on proliferation and apoptosis of nasopharyngeal carcinoma cell line CNE-2Z / 中草药

Object To investigate the effects of resveratrol (Res) on the proliferation and apoptosis of nasopharyngeal carcinoma cell line CNE-2Z. Methods IC 50 values were detected by MTT assay. The apoptosis was analyzed by flow cytometry (FCM), Hoechst 33258/PI fluorescence staining, and DNA agarose gel electrophoresis. Results After CNE-2Z cells were treated with different concentrations of Res for 24, 48, and 72 h, their IC 50 values were (109.2?7.5), (83.6?6.0), and (54.3?2.8) ?mol/L respectively (P

Effects of homoharringtonine on inhibition of proliferation and induction of apoptosis of nasopharyngeal carcinoma cells CNE-2Z / 中国药理学通报

AIM To study the effects of homoharringtonine(HHT) on inhibition of proliferation and induction of apoptosis of nasopharyngeal carcinoma cells CNE 2Z. METHODS The inhibitory rates of the proliferation and IC 50 were detected by MTT method. The apoptosis was analyzed by flow cytometry (FCM), agarose gel electrophoresis and Hoechst 33258/PI fluorescence staining. RESULTS After cells were treated with HHT of different concentrations for 24, 48 and 72 h,respectively,the inhibitory rates of the proliferation rised with concentration and time. The IC 50 values of 24, 48 and 72 h were (0 629?0 039), (0 483?0 011) and (0 389?0 027) mg?L -1 , respectively. The difference among IC 50 values was obvious ( P

Ovarian serous cystadenocarcinoma cell apoptosis induced by inhibition of Bax inhibitor-1(bi-1) gene expression with RNA interference / 中国病理生理杂志

AIM: To study the inhibition of the Bax inhibitor-1(bi-1) gene expression induced with RNA interference in HO8910PM and HO8910 cell lines in addition to the apoptotic induction.METHODS: After transfection of recombinant plasmid which expresses bi-1shRNA into HO8910PM and HO8910 cells,bi-1 mRNA and protein levels were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting.Apoptotic HO8910PM and HO8910 cells were detected by Hoechst 33258/PI fluorescent staining and flow cytometry.RESULTS: Compared with the control group,the expressions of bi-1 gene at mRNA and protein levels were declined evidently in the cells transfected with four candidate siRNA plasmids(P

Activation of caspase-6 during resveratrol(Res)-induced apoptosis of nasopharyn- geal carcinoma cells CNE-2Z / 中国免疫学杂志

Objective:To study whether caspase-6 was activated during resveratrol(Res)- induced apoptosis of nasopharyngeal carcinoma cells CNE-2Z. Methods:The cleavage of pro-caspase-6 was analyzed by Western-blot. TTie changes of caspase-6 mKNA was detected by semi-quantitative RT-PCR. The changes of caspase-6 activity was determined by colorimetric assay. Results: After CNE-2Z cells were treated with 0 (control), 25, 50, 100, 200 pmol/L Res for 24 h, respectively, the expression of pro-caspase-6 was decreased with concentration increasing. Caspase-6 active fragment P20(20 kD) appeared at 100 fjtmol/L and was increased at 200 (junol/L.The expression of caspase-6 mR-NA was increased in a concentration-dependent manner ( P