Effects of different time points of bortezomib on the pathway of NF-κB in drug-resistant K562 cells

Objective To study the effects of bortezomib on the expression of NF-κB, IκB and P-gp of drug-resistant K562 cells induced by daunorubicin (K562/DNR), to explore the molecular mechanism of drug-resistant reverse. Methods The expression of NF-κB, IκB and P-gp in K562/DNR cells were detected when the cells had been treated with 100 μg/ml DNR only or together with 4 μg/L bortezomib for 12 h, 24 h and 36 h. The apoptosis rates were detected in each group respectively and the activity of NF-κB was detected by ELISA method. Results Compared with the control group, the expressions of NF-κB and P-gp in K562/DNR could be increased and IκB was decreased after being treated with DNR. When K562/DNR were cultured with bortezomib, the expressions of NF-κB and P-gp induced by DNR were significantly suppressed and IκB was increased. The activity of NF-κB were detected in different time points: (15.3±1.87) %[(23.8± 2.27) % in DNR group] at 12 h, (10.2±1.69) % [(25.4±1.98) % in DNR group] at 24 h, (6.08±2.53) % [(26.9±2.58) % in DNR group] at 36 h. There were a significant differences between DNR group and DNR+PS-341group. The apoptosis rates were increased in DNR+PS-341 group at different time points than those in DNRgroup, (35.23±5.15) % [(15.56±4.12) % in DNR group] at 12 h, (40.26±6.89) % [(17.25±2.89) % in DNR group] at 24 h, (43.58±7.69) % [(22.47±4.58) % in DNR group] at 36 h. The effccts showed the character of time-dependent pattern. Conclusion Bortezomib could downregulate the expressions of NF-κB and P-gp in K562/DNR, reverse the drug resistance and up-regulate the apoptotic rates in K562/DNR cells.     

Effects of different time points of bortezomib on the pathway of NF-κB in drug-resistant K562 cells

Objective To study the effects of bortezomib on the expression of NF-κB, IκB and P-gp of drug-resistant K562 cells induced by daunorubicin (K562/DNR), to explore the molecular mechanism of drug-resistant reverse. Methods The expression of NF-κB, IκB and P-gp in K562/DNR cells were detected when the cells had been treated with 100 μg/ml DNR only or together with 4 μg/L bortezomib for 12 h, 24 h and 36 h. The apoptosis rates were detected in each group respectively and the activity of NF-κB was detected by ELISA method. Results Compared with the control group, the expressions of NF-κB and P-gp in K562/DNR could be increased and IκB was decreased after being treated with DNR. When K562/DNR were cultured with bortezomib, the expressions of NF-κB and P-gp induced by DNR were significantly suppressed and IκB was increased. The activity of NF-κB were detected in different time points: (15.3±1.87) %[(23.8± 2.27) % in DNR group] at 12 h, (10.2±1.69) % [(25.4±1.98) % in DNR group] at 24 h, (6.08±2.53) % [(26.9±2.58) % in DNR group] at 36 h. There were a significant differences between DNR group and DNR+PS-341group. The apoptosis rates were increased in DNR+PS-341 group at different time points than those in DNRgroup, (35.23±5.15) % [(15.56±4.12) % in DNR group] at 12 h, (40.26±6.89) % [(17.25±2.89) % in DNR group] at 24 h, (43.58±7.69) % [(22.47±4.58) % in DNR group] at 36 h. The effccts showed the character of time-dependent pattern. Conclusion Bortezomib could downregulate the expressions of NF-κB and P-gp in K562/DNR, reverse the drug resistance and up-regulate the apoptotic rates in K562/DNR cells.     

Reversion of multidrug-resistance by proteasome inhibitor bortezomib in K562/DNR cell line

Objective:To observe the reversion of multi-drug resistance by proteasome inhibitor bortezomib in K562/DNR cell line and to analyze the possible mechanism of reversion of multidrug-resistance.Methods:MTT method was used to determine the drug resistance of K562/DNR cells and the cellular toxicity of bortezomib.K562/DNR cells were cultured for 12 hours,24 hours and 36 hours with 100 μg/ml DNR only or plus 4 μg/L bortezomib.The expressions of NF-κB,IκB and P-gp of K562/DNR were detected with Western blot method,the activity of NF-κB was tested by ELISA method and the apoptosis rate was observed in each group respectively.Results:The IC50 of DNR on cells of K562/S and K562/DNR groups were 1.16 μg/ml and 50.43 μg/mL,respectively.The drug-resistant fold was 43.47.The IC10 of PS-341 on Cell strain K562/DNR was 4 μg/L.Therefore,4 μg/L was selected as the concentration for PS-341 to reverse drug-resistance in this study.DNR induced down-regulation of IκB expression,up-regulation of NF-κB and P-gp expression.After treatment with PS-341,a proteasome inhibitor,the IκB degradation was inhibited,IκB expression increased,NF-κB and P-gp expression decreased in a time dependent manner.Compared to DNR group,the NF-κB p65 activity of DNR+PS-341 group was decreased.Compared to corresponding DNR group,DNR induced apoptosis rate increases after addition of PS-341 in a time dependent manner.Conclusion:Proteasome inhibitor bortezomib can convert the leukemia cell drug resistance.The mechanism may be that bortezomib decreases the degradation of IκB and the expression of NF-κB and P-gp,therefore induces the apoptosis of multi-drug resistant cells.     

硼替佐米作用不同时间对K562耐药细胞株核因子-κB途径的影响

 目的　观察硼替佐米（商品名：万珂,PS-341）对柔红霉素（DNR）诱导的K562耐药细胞株（K562／DNR）核因子-κB（NF-κB）、抑制蛋白κB（IκB）及P-糖蛋白（P-gp）表达的影响,探讨PS-341逆转耐药的分子机制。方法　以100 μg／ml DNR单用或联合应用4 μg／L PS-341分别作用于K562／DNR 12、24及36 h,检测不同时间点各组NF-κB、IκB及P-gp表达情况,同时测定NF-κB p65活性,检测各组细胞凋亡率。结果　Western blot结果显示：与阴性对照组相比,DNR可诱导NF-κB表达上调及活性增强、IκB表达下调、P-gp表达上调;加用PS-341可显著抑制 DNR诱导的NF-κB及P-gp表达,使IκB表达增加。加用PS-341后,NF-κB活性12 h为（15.3±1.87）%[DNR组为（23.8±2.27）%],24 h为（10.2±1.69）%[DNR组为（25.4±1.98）%],36 h为（6.08±2.53）%[DNR组为（26.9±2.58）%],与相应单用DNR组相比均有明显下降,差异有统计学意义（P值均＜0.05）。DNR与PS-341联用后,细胞凋亡率12 h为（35.23±5.15）%[DNR组为（15.56±4.12）%],24 h为（40.26±6.89）%[DNR组为（17.25±2.89）%],36 h为（43.58±7.69）%[DNR组为（22.47±4.58）%],与DNR组相比,细胞凋亡率均明显增加,差异具有统计学意义（P值均＜0.05）。上述作用呈时间依赖性。结论　PS-341可减少K562／DNR细胞NF-κB的活化,降低P-gp表达,逆转细胞耐药,促进细胞凋亡。     

INHIBITION OF NF-(B ACTIVITY ENHANCED CYTOSINE ARABINOSIDE INDUCED APOPTOSIS IN LEUKEMIC CELL LINE HL60-N

Objective: To explore the effects of dexamethasone (DXM) and vincristine (VCR) on cytosine arabinoside (Ara-C) induced apoptosis and activation of nuclear factor-κ-gene binding (NF-κB) in leukemic cell line HL60-n. Methods: Apoptosis of HL60-n cells was analysed by TdT-mediated X-dUTP nick and end labeling (TUNEL) and DNA electrophoresis. NF-κB activity of HL60-n cells was detected by electrophoretic mobility shift assay (EMSA). Results: There was slight activation of NF-κB in HL60-n cells without drug induction. Ara-C at 1 μmol/L significantly enhanced the activation of NF-κB in HL60-n cells. The level of NF-κB activation induced by DXM at 1 μmol/L or VCR at 0.1 μmol/L had no significant difference compared with that of the control group. However, in HL60-n cells pre-treated with 1 μmol/L of DXM or 0.1 μmol/L of VCR, the activation of NF-κB induced by 1 μmol/L of Ara-C was significantly suppressed with inhibition rates of 31.0% and 47.0%, respectively. The apoptosis rates of HL60-n cells induced by 1.0 μmol/L, 10 μmol/L and 100 μmot/L Ara-C were 45.00±3.16%, 61.88±3.40% and 77.62±4.75%, respectively. The apoptotic rates of HL60-n cells induced by DXM at 1 μmol/L or VCR at 0.1 μmol/L were similar to that of the control group. However, either DXM at 1 μmol/L or VCR at 0.l μmol/L could enhance the apoptosis of HL60-n cells induced by Ara-C at 1 μmol/L with rates of 39.1% and 59.2%, respectively. Conclusion: Ara-C can induce apoptosis and activation of NF-κB in HL60-n cells. The mechanism of increased apoptosis of HL60-n cells by DXM or VCR may be related to suppression of NF-κB activation.     

The Proteasomal Inhibitor MG132 Potentiates Apoptosis of Triptolide-Treated K562 Cells by Regulating the NF-KB Signal Pathway

OBJECTIVE To explore the anticancer mechanism of triptolide in human leukemia K562 cells,and to further determine whether the proteasomal inhibitor,MG132,can potentiate apoptosis in triptolide-treated K562 cells.METHODS Apoptosis was assessed via annexin V/PI doublelabeled cytometry.The expressions of the IκBα and NF-κB/p65 proteins in K562 cells was investigated using Western blotting.RESULTS The inhibitory rates of K562 cells treated by triptolide gradually increased in a dose-and time-dependent manner,and treatment with triptolide plus MG132 potentiated the apoptotic rate.Triptolide inhibited the degradation of the IκBα protein and the nuclear localization of NF-κB/p65 proteins induced by TNF-α,and MG132 potentiated the effect of triptolide.Triptolide plus MG132 almost completely blocked the NF-κB activation induced by TNF-α.CONCLUSION The anti-proliferative activities of triptolide and MG132 were related to the NF-κB signal pathway.     

The Proteasomal Inhibitor MG132 Potentiates Apoptosis of Triptolide-Treated K562 Cells by Regulating the NF-κB Signal Pathway

OBJECTIVE To explore the anticancer mechanism of triptolide in human leukemia K562 cells,and to further determine whether the proteasomal inhibitor,MG132,can potentiate apoptosis in triptolide-treated K562 cells.METHODS Apoptosis was assessed via annexin V/PI double-labeled cytometry.The expressions of the IκBα and NF-κB/p65 proteins in K562 cells was investigated using Western blo ing.RESULTS The inhibitory rates of K562 cells treated by triptolide gradually increased in a dose-and time-dependent manner,and treatment with triptolide plus MG132 potentiated the apoptotic rate.Triptolide inhibited the degradation of the IκBα protein and the nuclear localization of NF-κB/p65 proteins induced by TNF-α,and MG132 potentiated the effect of triptolide.Triptolide plus MG132 almost completely blocked the NF-κB activation induced by TNF-α.CONCLUSION The anti-proliferative activities of triptolide and MG132 were related to the NF-κB signal pathway.     

Down-Regulation of Notchl and NF-κB by Curcumin in Breast Cancer Cells MDA-MB-231

Objective:To test whether the down-regulation of Notch1 gene expression by curcumin could inhibit cell growth and induce apoptosis,which may be associated mechanistically with the down-regulation of NF-κB in breast cancer cells. Methods:Breast cancer cell lines MDA-MB-231 were cultured in vitro and treated with different dosages of curcumin(0,1.25,5.0,20.0μmol/L)for dose-dependent assay and different time(0,24,48,72 h)at the dosage of 5.0μmol/L for time course assay.The changes of the mRNA and protein expression of Notch1 and NF-κB were measured by RT-PCR and Western Blot,and MTT assay was used to measure the change of proliferation. Results:The mRNA and protein levels of Notch 1 and NF-κB were decreased significantly in human breast cancer cell line with the increase of dosage of curcumin(P＜0.05),and with the extension of time course(P＜0.05).These changes suggested a dose- and time-dependent manner.The proliferation rate of cells also was significantly inhibited(P＜0.05). Conclusion:The current results show that the Notch-1 signaling pathway is associated mechanistically with NF-κB activity during curcumin-induced cell growth inhibition and apoptosis of breast cancer cells.These results suggest that the down-regulation of Notch signaling by curcumin may be a novel strategy for the treatment of patients with breast cancer.     

核转录因子-κ B在胰腺癌中的表达及其与上皮细胞间质转化的关系

目的 研究核转录因子-κ B(NF-κ B)在人类胰腺癌组织中的表达及与上皮细胞间质转化(EMT)标志物上皮性钙黏附蛋白(E-cad)和波形蛋白(Vimentin)的关系,探讨其与胰腺癌恶性生物学行为的关系.方法 应用免疫组织化学法检测62例人类胰腺癌组织中NF-κ B、上皮源性标志物E-cad蛋白、间质源性标志物Vimentin蛋白的表达,并分析它们相互间及其与临床病理因素间的关系.结果 胰腺癌组织中NF-κ B和Vimentin蛋白阳性表达率分别为81%(50/62)和61%(38/62),E-cad蛋白的表达缺失率为55%(34/62).在胰腺癌组织中NF-κ B的表达和Vimentin蛋白的阳性表达、E-cad蛋白的缺失表达均呈正相关(r值分别为0.343、0.352,均P＜0.05),并且NF-κ B的阳性表达与胰腺癌的淋巴结转移(x2=11.761,P＜0.05)、远处转移(x2=9.225,P＜0.05)相关,而与患者性别、年龄、肿瘤部位、肿瘤类型及分化程度无关(P＞0.05).E-cad蛋白的表达缺失、Vimentin蛋白的阳性表达与胰腺癌的淋巴结转移、远处转移均有相关性(x2值分别为6.914、4.984;7.753、5.144,均P＜0.05).结论 NF-κ B在胰腺癌中表达增高,同时可能通过诱导胰腺癌组织的EMT而促进胰腺癌的浸润和转移.

Abstract：

Objective To investigate the expression of NF-κB and epithelial-mesenchymal transition (EMT) related markers E-cadherin and Vimentin proteins in human pancreatic cancer tissues and its relation with the malignant features. Methods The expression of NF-κB、E-cadherin and Vimentin proteins in 62 cases of pancreatic cancer tissues were detected by using immunohistochemistry and compared with the clinicopathological data of pancreatic cancer. Results The positive expression rate of NF-κB was 81% (50/62), Vimentin protein increased of expression was 61% (38/62), and E-cadherin protein loss of expression was 55 % (34/62) in pancreatic cancer. The positive expression rate of NF-κB was significantly related with the lymph node metastasis (x2=11.761, P＜0.05), distant metastasis (x2=9.225, P＜0.05), the absent expression of epithelial marker E-cadherin protein (r =0.352, P ＜0.05) and the positive expression of mesenchymal marker Vimentin protein (r=0.343, P ＜0.05), but there was no relation with the patients gender,age, tumor location, tumor type and tumor differentiation (P ＞0.05). In addition, the significant correlation of E-cadherin expression loss and Vimentin expression with tumor lymph node metastasis and distant metastasis was found (x 2= 6.914, 4.984, 7.753, 5.144, P ＜0.05). Conclusion The overexpression of NF-κB in pancreatic cancer may accelerate invasion and metastasis of pancreatic cancer through inducing EMT.     

RhoA和核因子κB在胃癌中的表达及其临床意义

目的 探讨RhoA和核因子κB(NF-κB)在胃癌组织中的表达,分析其与胃癌患者临床病理特征及预后的关系.方法 利用免疫组化、组织芯片技术检测189例胃癌、54例相应癌旁及32例正常胃黏膜组织中RhoA蛋白和NF-κB蛋白的表达.用Kaplan-Meier法行单因素生存分析,应用Cox回归模型进行多因素生存分析.结果 RhoA蛋白在胃癌、癌旁和正常胃黏膜组织中的表达阳性率分别是84.7%、68.5%和65.6%,胃癌与癌旁及正常胃黏膜组织的阳性率差异有统计学意义(P＜0.05).NF-κB蛋白在胃癌、癌旁和正常胃黏膜组织中的表达阳性率分别是75.1%、42.6%和15.6%,且三者的阳性率相互比较,差异均有统计学意义(P＜0.05).RhoA和NF-κB在胃癌组织中的蛋白表达呈正相关(r=0.203,P=0.005).RhoA的蛋白表达与胃癌的浸润深度有关(P＜0.05).NF-κB的蛋白表达与胃癌的浸润深度和有无淋巴结转移有关(P＜0.05).单因素分析结果显示,肿瘤大小、有无淋巴结转移、浸润深度和NF-κB的蛋白表达影响胃癌患者术后的生存(P＜0.05);多因素回归分析结果显示,NF-κB蛋白表达、有无淋巴结转移和浸润深度为影响胃癌患者预后的独立因素(均P＜0.05).结论 RhoA和NF-κB参与胃癌的发生、发展,并在胃癌的浸润和转移中起重要作用;NF-κB的蛋白表达水平、有无淋巴结转移和浸润深度是影响胃癌患者预后的独立因素.

Abstract：

Objective To investigate the expression of RhoA and NF-κB in gastric carcinoma and their correlation with clinicopathological fearures.To determine the effective prognostic factors of long-term suivival of gastric carcinoma patients.Methods The role of RhoA and NF-κB in gastric carcinoma was assessed by tissue array technology and the levels of RhoA and NF-κB expression in paraffin-embedded tissues was quantified by immunohistochemistry from 189 cases of gastric carcinoma, 54 cases of their adjacent tissues, and 32 cases of normal gastric mucosa.The prognosis of gastric carcinoma was evaluated by Kaplan-Meier survival analysis and Cox multivariate regression analysis.Results The positive rates of RhoA expression were 84.7%, 68.5% and 65.6% in gastric carcinoma, adjacent tissues and normal mucosa, respectively.The expression of RhoA in gasric carcinoma was significantly higher than that in adjacent tissues and normal mucosa ( P ＜ 0.05 ).The positive rates of NF-κB expression were 75.1%,42.6% and 15.6%% in gastric carcinoma, adjacent tissues and normal mucosa, respectively.The expression of NF-κB in gasric carcinoma was significantly higher than that in adjacent tissues and normal mucosa (P ＜ 0.05 ).RhoA was positively linked with NF-κB ( r = 0.203, P = 0.005 ).In gastric carcinoma, the expression of RhoA was related with depth of invasion (P ＜ 0.05), and the expression of NF-κB was related with depth of invasion and lymph node metastasis ( P ＜ 0.05 ).The Kaplan-Meier survival analysis showed that the tumor size, lymph node metastasis, depth of invasion, expression of RhoA and NF-κB can shorten the cumulative survival rate.With these paramaters entering the Cox multivariate regression analysis mode, it was revealed that expression of NF-κB, lymph node metastasis and depth of invasion are independent prognostic factors.Conclusions The overexpression of RhoA and NF-κB is involved in the occurrence and development of gastric carcinoma.RhoA is positively linked with NF-κB.They are correlated with the invasion and metastasis of gastric carcinoma.The expression of NF-κB, lymph node metastasis, depth of invasion are independent prognostic factors playing an important role in prediction of the clinical outcome after radical resection of gastric carcinoma.     

黄芪总苷诱导人白血病NB4细胞凋亡的机制

目的 研究黄芪总苷(TA)对体外培养的人早幼粒细胞白血病细胞株NB4增殖及凋亡机制的影响.方法 将不同浓度的TA与NB4细胞共培养48 h,采用细胞增殖及细胞毒性检测试剂盒8(CCK-8)检测细胞生长抑制率,以流式细胞仪检测细胞凋亡率及凋亡过程中细胞内核因子κB(NF-κB)和蛋白激酶B(PKB,又称Akt)蛋白浓度的变化.结果 200、400、600、800 ms/L TA均可抑制NB4细胞生长,抑制率分别为(14.54±3.20)%、(24.79±3.98)%、(57.28±4.71)%和(88.28±4.65)%,呈浓度依赖性增加(P<0.05).200、400、600、800 mg/L TA组的细胞凋亡率分别为(10.03±3.31)%、(14.87±3.65)%、(23.45±1.90)%和(25.26 4-2.07)%,与对照组[(1.80±1.24)%]相比,差异均有统计学意义(P<0.05),且200、400、600 ms/L TA组的细胞凋亡率呈浓度依赖性增加(P<0.05);800 mg/L TA组的细胞凋亡率增加不明显,与600 ms/L TA组相比,差异无统计学意义(P>0.05),但出现了大量的坏死细胞,坏死率达(45.65±3.16)%.细胞凋亡过程中伴随NF-κB蛋白表达下降,不同浓度TA组与对照组差异均有统计学意义(P<0.05),而Akt蛋白的表达无明显变化(P>0.05).结论 黄芪总苷可以抑制NB4细胞增殖,并可以通过不依赖Akt的NF-κB信号通路来诱导NB4细胞凋亡.

Abstract：

Objective To investigate the effect of total astragalosides (TA) on proliferation and apoptosis in human leukemia NB4 cells in vitro. Methods The NB4 cells were treated with TA at different concentrations for 48 h in culture. Growth inhibition rates were measured by CCK-8 method. Flow cytometry was used to explore the cell apoptosis and the activity of NF-κB and Akt during apoptosis. Results TA at different concentrations (200, 400, 600, 800 mg/L) inhibited proliferation of NB4 cells in a dose-dependent manner ( P < 0. 05), and the inhibitory rates of TA on NB4 cells were (14. 54 ± 3. 20) % , (24.79 ±3.98)%, (57.28 ±4.71)% and (88.28 ±4.65)% , respectively. In terms of the induction of apoptosis, there was a significant difference between the TA group and blank control [(1.80±1.24)%, P<0.05]. At TA doses of 200, 400 and 600 mg/L, the apoptotic rates of NB4 cells were ( 10. 03 ± 3.31)% , (14.87 ±3.65)% , (23.45 ± 1.90) % , respectively. Besides, TA induced apoptosis of NB4 cells in a dose-dependent manner in the groups of 200 mg/L, 400 mg/L, 600 mg/L (P<0.05). But there was no significant difference in apoptotic rates between the groups of 800 mg/L and 600 mg/L [(23.45 ±1.90)%, P> 0.05]. In the group of 800 mg/L, the necrotic cells increased highly and the necrotic rate reached (45.65 ± 3.16)%. After TA treatment of NB4 cells at different concentrations (200, 400, 600 mg/L), the expression of NF-kB protein was significantly decreased compared with that of the blank control (9. 79 ±0. 95, P<0.05), while Akt protein was not significantly decreased (P>0.05). Conclusion TA can inhibit the growth of NB4 cells and induce apoptosis in NB4 cells through an Akt-independent NF-kB signaling pathway.     

Lipopolysaccharide enhances the inhibition of NF-κB expression in NNK-mediated peritoneal macrophages

Objective： The aim of the study was to investigate the effect of lipopolysaccharide （LPS） on the expression of nuclear factor kappa B （NF-κB） in 4-（methylitrosamino）-1-（3-pyridyl）-1-butanone （NNK）-mediated primary mouse peritoneal macrophages in vitro. Methods： The activity of peritoneal rnacrophages treated with different concentrations of LPS was detected by MTT assay in rider to find the optimal concentration. Peritoneal macrophages were also treated with NNK （100-500 μM）, with or without LPS for 9 h. The expression of NF-κB was demonstrated via immunocytochemistry （ICC） and Western- blot, respectively. Results： The concentration of LPS at 25 μg/mL was found to be the optimal concentration to improve the activity of peritoneal macrophages （P 〈 0.01）. Simultaneously, LPS （25 μg/mL） increased the expression of NF-κB in both the nucleus and cytoplasm and facilitated transfer of NF-κB to the nucleus. NNK treatment significantly inhibited the expression of NF-κB in a concentration-dependent manner, among the LPS-stimulated or unstimulated peritoneal macrophages, especially when cotreated with LPS （25 μg/mL, P 〈 0.01 ）. Furthermore, NNK treatment （500 μM） with LPS yielded a significant decrease in NF-κB translocation to nucleus and inhibited the expression of NF-κB （P 〈 0.005）. Conclusion： LPS enhances the suppression of NF-κB expression in NNK-mediated mouse peritoneal macrophages, which may provide a theoretical basis for the inhibition of cancer.     

Two-Dimensional （Polyacrylamide） Gel Electrophoresis Analysis of Apoptosis Induced by Harringtonine in K562 Cells

OBJECTIVE The anti-tumor drug, harringtonine (HT), has been extensively used with satisfactory results in the treatment of acute or chronic myeloid leukemia. Previous studies have shown that the anti-tumor activity of the drug is related to induced apoptosis of tumor cells, but the molecular mechanism still remains unclear. The main purpose of this research was to analyze the protein profiles formed during HT-induced apoptosis in K562 cells and to screen the apoptotic-related proteins.METHODS Annexin V and Pi double staining was used in combination with flow cytometry to examine the early and the late stages of HT-induced apoptosis in K562 cells. In addition two-dimensional gel electrophoresis and computer-assisted image analysis were employed to separate and compare the HT-induced apoptotic proteins of the K562 cells and the controls.RESULTS When a concentration of 10 μg/ml HT was used to treat K562 cells,the percentage of the early-apoptotic cells (Annexin V /PI-) was found to be 28.3% and 18.1% at 5 and 24 h, respectively (P＜0.01), while the rate of lateapoptotic cells (Annexin V /PI ) was at a level of 9.1% and 20.2%,respectively (P＜0.01). Matching analysis of the proteome among the control group and the early- and late-apoptotic groups showed 1,300 ± 50 protein spots which were identified in the control K562 cells with a matching rate of 88.3 ± 2.0 % for the protein spots in the two treated groups. Ten protein spots showed overt and steady changes in both quality and quantity in the cells of the late-apoptotic group (P＜0.01), among which the level of expression for eight of the ten protein spots was up-regulated after apoptosis, one was down-regulated and one was merely expressed as in the control cells.CONCLUSION The proteins with differential expression might be important proteins involved in the process of apoptosis in K562 cells induced by HT.     

Role of toll-like receptor 4-dependent signaling pathway in lipopolysaccharide-induced human lung carcinoma cell proliferation北大核心

Objective: Our aim was to study the mechanism through which toll-like receptor 4 (TLR4) -dependent signaling pathway was involved in lipopolysaccharide (LPS) -induced the proliferation of human lung adenocarcinoma A549 cells. Methods: MTT assay and flow cytometry (FCM) were performed to examine the proliferation of A549 cells after stimulation with LPS. Immunocytochemistry staining was used to evaluate the TLR4, c-Jun, c-Fos and nuclear factor-kappa B (NF-κB) protein expressions in A549 cells after treatment with different concentrations of LPS. Results: LPS stimulated the proliferation of A549 cells, especially after 24 h treatment. The ratio of cells in G2/M phase in the 100 ng/mL LPS group was significantly higher than that in 10 ng/mL LPS group. The difference was significant (P<0.05). The expressions of TLR4, c-Jun, c-Fos, and NF-κB detected in A549 cells were gradually elevated with the increase in the concentrations of LPS. The expressions of TLR4, c-Jun, c-Fos and NF-κB were the highest in LPS (100 ng/mL) group compared with the control group (P <0.05). Conclusion: LPS promoted the proliferation of A549 cells. The mechanism may be explained as follows: binding with TLR4 receptor, stimulating TLR4 signaling pathways, and then activating AP-1 (heterodimer of c-Jun and c-Fos) and NF-κB , finally inducing persisted proliferation of human lung carcinoma cells.     

核因子-κBp65、IκB-a在肝炎相关性肝细胞癌中的表达及临床意义

目的研究乙型(B)肝炎和(或)丙型(C)肝炎相关性肝细胞癌(HCC)中NF-κBp65、IκB-a的表达及意义.方法采用免疫组化方法检测48例B型肝炎相关性HCC,20例C型肝炎相关性HCC,15例BC混合型肝炎相关性HCC,21例癌旁组织以及9例正常肝脏组织中NF-κ Bp65、IκB-a的表达.结果 NF-κ Bp65在B、C、BC组中的阳性表达率分别为60.4%(29/48)、85.0%(17/20)和93.3%(14/15),C组、BC组均高于B组,差异均有显著性(P<0.05);IκB-a在肝炎相关性HCC及癌旁组织中的阳性表达率分别为21.7%(18/83)和57.1%(12/21),差异有显著性(P<0.05).NF-κ Bp65、IκB-a在9例正常肝脏组织中无表达.结论 NF-κ Bp65高表达而IκB-a低表达在肝炎相关性HCC的发生过程中可能起重要作用.     

NF-κB抑制对宫颈癌HeLa细胞放疗敏感性影响的观察

目的:探讨抑制核转录因子κB(NF-κB)表达对宫颈癌HeLa细胞放疗敏感性的影响。方法:通过对HeLa细胞加入NF-κB的抑制剂100μmol/LPDTC或2μmol/L PS-341,均作用2 h来抑制NF-κB的表达。将HeLa细胞分为PDTC组、PS-341组及对照组加入等量生理盐水。各组均给予直线加速器照射,剂量分别为0、2、4和6 Gy。用蛋白质印迹法来观察细胞核内NF-κB含量的变化,用TUNEL法来检测细胞的凋亡,用MTT法检测细胞增殖。结果:辐射可以使HeLa细胞内NF-κB的表达增多,PDTC及PS-341均可以抑制由射线介导NF-κB的增加。在0 Gy条件下对照组与PDTC组及PS-341组HeLa细胞的凋亡指数分别为3%、12%和15%,两实验组的凋亡指数均高于对照组,P<0.01;6 Gy条件下实验组细胞的凋亡指数都高于同条件下对照组,P<0.01。结论:在宫颈癌HeLa细胞中,PDTC及PS-341均可抑制NF-κB的表达从而增加了由射线介导的细胞凋亡,增加HeLa细胞的放疗敏感性。     

冻融治疗后肝癌库普弗细胞分泌功能的变化

Objective To explore the effect of Kupffer cells (KCs) secretion on HCC cells after cryoablation and its mechanism. Methods Sixteen groups were divided: 37°C control group, three hypothermia groups(0°C, 5°C, 10°C), freeze-thaw necrotic substances group, three combined stimulation group and 8 parallel groups for each group. In the 8 parallel groups, pyrrolidine dithiocarbamate(PDTC) was added, and the final concentration was 100 µmol/ml. The concentration of TNF-α, IL-1β and INF-γ in 16 groups of KCs supernatant were determined by Elisa method. The expression of NF-κB protein in each group was detected by Western blot. Results The secretion of inflammatory factors TNF-α, IL-1β and INF-γ increased after combined stimulation of KCs with hypothermia and freeze-thaw necrotic substances (P<0.01). The combined stimulation of low temperature (0°C, 5°C, 10°C) and freeze-thaw necrotic substances in the three groups showed superposition effect. Hypothermia stimulation had no significant effect on the expression of NF- κB protein (P>0.05), but the expression of NF-κB protein in the combined stimulation group and freezethaw necrosis substances group were significantly up-regulated (P<0.05). Treated with NF-κB inhibitor, the expression of NF-κB protein and the secretion level of inflammatory factors did not change significantly among 8 parallel groups (P>0.05). The concentrations of TNF-α, IL-1β and INF-γ were positively correlated with the expression of NF-κB protein in the culture medium of KCs (r≥0.870, P=0.000). Conclusion Freeze-thaw therapy could enhance the function of KCs secretion cytokines, and to a certain extent, it could induce inflammatory response and eliminate tumor cells. The secretion function of KCs may play a role through NF-κB signaling pathway. © 2020, CHINA RESEARCH ON PREVENTION AND TREATMENT. All rights reserved.