17-Allylamino-17-demethoxygeldanamycin activity against thyroid cancer cell lines correlates with heat shock protein 90 levels

Heat shock protein 90 (Hsp90) is a molecular chaperone that stabilizes growth factor receptors and signaling molecules. Disruption of this action inhibits the MAPK and phosphatidylinositol-3 kinase cascades and can induce cancer cell death. The goal of this study was to determine whether thyroid cancer cells are sensitive to the cytotoxic effects of 17-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor in clinical trials, and to determine predictors of this response. Papillary (NPA), follicular (WRO), and anaplastic (ARO) thyroid cancers were incubated with 17-AAG in vitro. Surprisingly, the ARO cells were most sensitive to the cytotoxic effects of this agent. Conversely, all cell lines displayed similar responses to specific blockers of phosphatidylinositol-3 kinase and MAPK kinase (LY294002 and U0126, respectively). Western blot demonstrated that the NPA cells that were most resistant to 17AAG-induced cytotoxicity had the lowest levels of Hsp90 and were the only cells with persistent levels of Akt protein. Interestingly, even the WRO and ARO cell lines that were sensitive to 17-AAG-induced cell death did not undergo apoptosis. These data suggest that sensitivity of thyroid cancer cells to 17-AAG-induced cytotoxicity relates to Hsp90 levels rather than histological subtype and that thyroid cancer cells have a reduced apoptotic response to 17-AAG.     

β-榄香烯抑制甲状腺癌细胞增殖及其作用机制体外实验研究

甲状腺癌是常见的内分泌肿瘤,目前主要靠手术切除和术后<'131>Ⅰ治疗.甲状腺癌对化疗不敏感,目前尚缺乏对该肿瘤有效的化疗药物.β-榄香烯(1-甲基-1-乙烯基-2,4-二异丙烯基.环己胺)是我国自行研制的新型抗肿瘤药物,已经证实β-榄香烯对脑肿瘤、乳腺癌、非小细胞肺癌等组织的肿瘤细胞具有抑制作用[1～2].本研究拟进一步验证β-榄香烯对甲状腺癌细胞的抑制作用,为治疗甲状腺癌寻找新的化疗药物.     

热休克蛋白90在肺癌诊治中的研究进展

肺癌的发病率和病死率在中国居各类肿瘤之首,热休克蛋白90 (heat shock protein 90,Hsp90)作为重要的分子伴侣参与调控和维持细胞内多种肿瘤相关蛋白的构象和功能,通过抑制Hsp90能抑制实体瘤如肺癌、乳腺癌、前列腺癌等的侵袭和转移.因此,Hsp90抑制剂作为治疗晚期非小细胞肺癌的抗肿瘤药物目前已进入美国临床试验,在单药治疗、联合治疗以及对EGFR-TKI/Crizotinib耐药的非小细胞肺癌患者Ⅰ、Ⅱ期临床试验中取得了良好的效果.而血清中的Hsp90α亦可作为肺癌诊断、治疗和判断预后的良好监测指标.本文将对Hsp90在肺癌的诊治方面的现状和研究进展进行综述.     

Re-expression of ABI3-binding protein suppresses thyroid tumor growth by promoting senescence and inhibiting invasion

Loss of ABI gene family member 3-binding protein (ABI3BP) expression may be functionally involved in the pathogenesis of cancer. Previous reports have indicated a loss of expression in lung cancer and a presumed role in inducing cellular senescence. We show here that ABI3BP expression is significantly decreased in most malignant thyroid tumors of all types. To better understand ABI3BP's role, we created a model by re-expressing ABI3BP in two thyroid cancer cell lines. Re-expression of ABI3BP in thyroid cells resulted in a decrease in transforming activity, cell growth, cell viability, migration, invasion, and tumor growth in nude mice. ABI3BP re-expression appears to trigger cellular senescence through the p21 pathway. Additionally, ABI3BP induced formation of heterochromatin 1-binding protein gamma-positive senescence-associated (SA) heterochromatin foci and accumulation of SA beta-galactosidase. The combination of a decrease in cell growth, invasion, and other effects upon ABI3BP re-expression in vitro helps to explain the large reduction in tumor growth that we observed in nude mice. Together, our data provide evidence that the loss of ABI3BP expression could play a functional role in thyroid tumorigenesis. Activation of ABI3BP or its pathway may represent a possible basis for targeted therapy of certain cancers.     

BIRB 796 enhances cytotoxicity triggered by bortezomib, heat shock protein (Hsp) 90 inhibitor, and dexamethasone via inhibition of p38 mitogen-activated protein kinase/Hsp27 pathway in multiple myeloma cell lines and inhibits paracrine tumour growth

We have previously shown that heat shock protein (Hsp) 27 or its upstream activator p38 mitogen-activated protein kinase (MAPK) confers resistance to bortezomib and dexamethasone (Dex) in multiple myeloma (MM) cells. This study examined anti-MM activity of a novel p38 MAPK inhibitor, BIRB 796, alone and in combination with conventional and novel therapeutic agents. BIRB 796 blocked baseline and bortezomib-triggered upregulation of p38 MAPK and Hsp27 phosphorylation, thereby enhancing cytotoxicity and caspase activation. The Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17-AAG) upregulated protein expression and phosphorylation of Hsp27; conversely, BIRB 796 inhibited this phosphorylation and enhanced 17-AAG-induced cytotoxicity. Importantly, BIRB 796 inhibited Hsp27 phosphorylation induced by 17-AAG plus bortezomib, thereby enhancing cytotoxicity. In bone marrow stromal cells (BMSC), BIRB 796 inhibited phosphorylation of p38 MAPK and secretion of interleukin-6 (IL-6) and vascular endothelial growth factor triggered by either tumour necrosis factor-alpha or tumour growth factor-beta1. BIRB 796 also inhibited IL-6 secretion induced in BMSCs by adherence to MM cells, thereby inhibiting tumour cell proliferation. These studies therefore suggest that BIRB 796 overcomes drug-resistance in the BM microenvironment, providing the framework for clinical trials of a p38 MAPK inhibitor, alone and in combination with bortezomib, Hsp90 inhibitor, or Dex, to improve patient outcome in MM.     

外源性PTEN对胰腺癌细胞ASPC-1增殖、侵袭及转移的影响

目的 研究第10染色体同源丢失性磷酸酶-张力蛋白基因(PTEN)对胰腺癌细胞系ASPC-1细胞周期、细胞增殖、血管内皮生长因子(VEGF)和表皮生长因子受体(EGFR)蛋白表达、裸鼠成瘤及转移能力的影响.方法 将含PTEN基因的质粒pE-PTEN转染ASPC-1细胞,以空质粒pE转染为对照,分别获得ASPC-1-pE-PTEN (A-pE-P)细胞及ASPC-1-pE (A-pE)细胞.应用RT-PCR检测细胞PTEN mRNA表达;免疫细胞化学法检测PTEN、VEGF、EGFR蛋白表达;克隆形成实验观察克隆形成数;Transwell小室观察细胞侵袭能力;裸鼠皮下注射癌细胞法观察成瘤情况.结果 与ASPC-1细胞比较,A-pE-P细胞的PTEN mRNA表达增加179.3％,PTEN蛋白表达明显增加;VEGF蛋白表达明显减少;EGFR蛋白表达无明显变化;G2/M期细胞数明显增加[(31.5±1.76)％比(26.81±1.03)％,P＜0.05];克隆形成数降低[(24.0±3.9)比(33.3 ±3.4),F=4.283,P＜0.01],克隆形成率降低28％;穿膜细胞数明显减少[每高倍视野(46.3±6.6)比(63.8±7.5)个,F=2.476,P＜0.05];种植瘤体积明显缩小[(142.4±30.9)比(202.7±43.6)mm3,t=4.834,P＜0.01],抑瘤率达42.4％;远处转移灶明显减少[(2.0±0.7)比(5.0±1.3)个,t =0.451,P＜0.01].结论 PTEN基因转染后ASPC-1细胞VEGF表达减少,细胞生长被阻滞在G2/M期,增殖及转移被抑制.     

Significance of expression of heat shock protein90α in human gastric cancer

AIM: To evaluate the significance of hsp90α expression in human gastric cancer tissues.METHODS: Immunohistochemical staining was used in clinical specimens from 33 cases of gastric cancer and 33 cases of gastritis with rabbit anti-human hsp90α multi-clonal antibody in order to explore the relationship between the expression of hsp90α in gastric carcinoma tissue and gastritis tissue as well as in mucous membrane adjacent to cancer and lymph node metastasis.RESULTS: Hsp90α was detected in 88 % of gastric carcinoma cases and 55 % of gastritis cases. The hsp90α positive rate in gastric cancer group was significantly higher than that in gastritis group (P＜0.01, P=0.005). The hsp90α positive rate in gastric cancer and in mucous membrane adjacent to cancer was 88 % and 55 % respectively (P＜0.01,P=0.005). The hsp90α positive rate in lymph node metastasis group and non-lymph node metastasis group was 100 % and 60 %respectively, and a significant correlation between hsp90α expression and lymph node metastasis was shown (P＜0.01,P=0.005).CONCLUSION: The hsp90α expression rate in gastric cancer group was significantly higher than that in gastritis group as well as that in the group of mucous membrane adjacent to cancer. The hsp90α expression in lymphatic node metastasis group was higher than that in non-lymphatic node metastasis group. The results indicate that increased hsp90α expression has a close relationship with occurrence and lymph node metastasis of gastric cancer.     

Significance of expression of heat shock protein90α in human gastric cancer

AIM: To evaluate the significance of hsp90α expression in human gastric cancer tissues. METHODS: Immunohistochemical staining was used in clinical specimens from 33 cases of gastric cancer and 33 cases of gastritis with rabbit anti-human hsp90α multi-clonal antibody in order to explore the relationship between the expression of hsp90α in gastric carcinoma tissue and gastritis tissue as well as in mucous membrane adjacent to cancer and lymph node metastasis.RESULTS: Hsp90α was detected in 88% of gastric carcinoma cases and 55 % of gastritis cases. The hsp90α positive rate in gastric cancer group was significantly higher than that in gastritis group (P&lt;0.01, P=0.005). The hsp90α positive rate in gastric cancer and in mucous membrane adjacent to cancer was 88% and 55 % respectively (P&lt;0.01,P=0.005). The hsp90α positive rate in lymph node metastasis group and non-lymph node metastasis group was 100% and 60% respectively, and a significant correlation between hsp90α expression and lymph node metastasis was shown (P&lt;0.01,P=0.005). CONCLUSION: The hsp90α expression rate in gastric cancer group was significantly higher than that in gastritis group as well as that in the group of mucous membrane adjacent tocancer. The hsp90α expression in lymphatic node metastasis group was higher than that in non-lymphatic node metastasisgroup. The results indicate that increased hsp90α expression has a close relationship with occurrence and lymph node metastasis of gastric cancer.     

The PARP inhibitor PJ34 modifies proliferation,NIS expression and epigenetic marks in thyroid cancer cell lines

Since PARP-1 is supposed to be part of a multimeric repressor of sodium iodide symporter (NIS) expression, in this study the effect of the PARP inhibitor PJ34 on several properties of thyroid cancer cell lines was investigated. In TPC1, BCPAP, FRO, WRO cell lines PJ34 induced a strong increase in NIS mRNA levels. In BCPAP and TPC1 cells also significant increase of radio-iodine uptake was induced. Accordingly, in transfection experiments performed in TPC1 cells, treatment with PJ34 increased NIS promoter activity without affecting PARP-1 binding to the promoter sequence. We also investigated the epigenetic status of NIS promoter after PJ34 treatment in TPC1 cell line: in addition to an increase of histone modification activation marks (H3K9K14ac, H3K4me3), surprisingly we observed also an increase of H3K27me3, a classical repressive mark.     

Regulation of cell proliferation and malignant potential by irisin in endometrial,colon, thyroid and esophageal cancer cell lines

Objective

Irisin is a novel hormone that has been proposed to mediate the beneficial effects of exercise on metabolism, including body weight regulation and insulin resistance. No previous studies have evaluated whether irisin may regulate cell proliferation and malignant potential of obesity-related cancer cell lines.

Materials/Methods

Cell proliferation and malignant potential i.e. cell adhesion and colony formation were studied in vitro using human and mouse obesity-related cancer cell lines i.e. endometrial (KLE and RL95-2), colon (HT29 and MCA38), thyroid (SW579 and BHP7) and esophageal (OE13 and OE33).

Results

We observed that, in contrast to metformin, cell proliferation is not regulated by irisin in a dose-dependent manner in human and mouse obesity-related cancer cell lines. Specifically, physiological (5 to 10 nmol/L) and high physiological/pharmacological (50 to 100 nmol/L) concentrations of irisin had no effect on cell proliferation when compared to control in human and mouse endometrial, colon, thyroid and esophageal cancer cell lines. Also, we observed that, in contrast to metformin, neither physiological nor high physiological/pharmacological concentrations of irisin regulate cell adhesion and/or colony formation in human and mouse endometrial, colon, thyroid and esophageal cancer cell lines.

Conclusions

Our data suggest that irisin, in physiological and high physiological/pharmacological concentrations, has no in vitro effect on cell proliferation and malignant potential of obesity-related cancer cell lines. Future work is needed to determine the regulation of irisin levels and any physiological effects it may have on obesity-related cancers in vivo in animals and humans.     

Cortistatin-14 inhibits cell proliferation of human thyroid carcinoma cell lines of both follicular and parafollicular origin

Cortistatin (CST-14, Pro-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys]-Lys-NH2), a neuropeptide member of the SRIH family, binds to all 5 SRIH receptor (sst) subtypes, but also possesses a significant binding affinity to GH secretagogue receptors (GHS-R), which have been reported to mediate the antiproliferative activity of GHS on thyroid cancer cells. The effect of CST-14 on cell proliferation was studied in 3 different human thyroid carcinoma cell lines of follicular origin (N-PAP, WRO, ARO) and in one thyroid medullary carcinoma cell line (TT). CST-14 1 pM determined a significant inhibition of cell proliferation in TT, N-PAP and WRO cells and this effect was dose-dependent and more pronounced than that displayed by SRIH-14 (Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys]-OH) treatment. To a minor extent, CST-14, but not SRIH-14, also temporary inhibited ARO cell proliferation. By immunofluorescence, sst2, sst3 and sst5 have been demonstrated in TT cells, whereas types 3 and 5 only were expressed in N-PAP and WRO cells, and no sst subtype was found in ARO cells. The presence of both GHS-Rla and lb mRNA has been studied and demonstrated in the TT medullary carcinoma cell line, whereas follicular derived cell lines were already known to express GHS binding sites. Addition of EP-80874 (D-Mrp-c[D-Cyspyridilalanyl3-D-Trp-Lys-Val-Cys]-Mrp-NH2), a synthetic peptide that binds to SRIH and GHS-R, completely abolished the antiproliferative effects of CST-14 or SRIH-14 on sst/GHS-R positive thyroid carcinoma cell lines (WRO, N-PAP and TT). EP-80874 was also able to antagonize the inhibitory activity of CST-14 on the growth of cells (ARO) expressing GHS-R but not sst. Taken together, these data firstly demonstrate that EP-80874 has a mixed SRIH/CST antagonist activity and suggest that the oncostatic effect of CST-14 on thyroid cancer cells could be mediated by both sst and/or GHS-R.     

The heat shock protein 90 inhibitor 17-AAG induces cell cycle arrest and apoptosis in mantle cell lymphoma cell lines by depleting cyclin D1, Akt, Bid and activating caspase 9

Mantle cell lymphoma (MCL), a distinct type of non-Hodgkin lymphoma, is characterised by the overexpression of cyclin D1. Heat shock protein 90 (HSP90) is a molecular chaperon to proteins that regulate cell cycle and survival. 17-allylamino-17-demethoxy-geldanamycin (17-AAG), a HSP90 small molecule inhibitor, induced G(0/1) cell cycle arrest and cell death in a dose- and time-dependent manner in MCL cell lines. This effect was associated with the downregulation of cyclin D1, cdk4 and Akt, depletion of Bid, and activation of the intrinsic/mitochondrial caspase pathway. These data suggest that 17-AAG may have a potential therapeutic value in patients with MCL.     

Heat shock protein 70 antisense oligonucleotide inhibits cell growth and induces apoptosis in human gastric cancer cell line SGC-7901

AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer. METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting. RESULTS: The number of viable cells decreased in a dose-and time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10μmol/L for 48 h or 8μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G_1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h. CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions.     

罗格列酮对人胃癌细胞系迁移侵袭的抑制及其机制

目的:探讨罗格列酮对人胃癌SGC7901、SGC7901/VCR细胞侵袭转移能力及LIMK1基因蛋白表达的影响.方法:罗格列酮(40 mg/L)作用SGC7901和SGC7901/VCR细胞24 h后,采用划痕实验和Transwell实验分别观察罗格列酮对细胞的迁移和侵袭能力.RT-PCR检测LIMKl mRNA和co...     

帕瑞昔布对人胰腺癌细胞株BxPC-3、AsPC-1增殖凋亡的影响及其机制

目的:研究帕瑞昔布(parecoxib,PCB)对人胰腺癌细胞株BxPC-3和AsPC-1的增殖、凋亡及其可能的分子机制.方法:BxPC-3、AsPC-1细胞用不同浓度PCB的培养液孵育后,利用MTT法测定细胞活性,计算IC50值,TUNEL法检测处理后细胞的凋亡情况,RT-PCR验证相关蛋白的变化表达.结果:PCB对两种细胞生长呈时间和计量依赖性抑制;PCB处理后BxPC-3、AsPC-1两种细胞IC50值为:400.98μmol/L±10.78μmol/L、256.3μmol/L±2.98μmol/L;TUNEL法检测证明凋亡率增加;RT-PCR显示COX-2表达明显降低.结论:PCB可以抑制胰腺癌细胞增殖,并诱导其凋亡生长,其可能机制是通过抑制COX-2表达来实现的.     

Inhibition by protein kinase-C inhibitor and cycloheximide of phorbol ester- and epidermal growth factor-induced arachidonic acid metabolism in cultured porcine thyroid cells

In an attempt to elucidate possible mechanism(s) for stimulated arachidonic acid metabolism by phorbol 12-myristate 13-acetate (PMA) and epidermal growth factor (EGF) in porcine thyroid cells, we examined the effects of protein kinase inhibitors, isoquinolinesulfonamide derivatives (H-7 and HA-1004), and cycloheximide. The production of PGE2 stimulated by either PMA or EGF was strongly inhibited by H-7, with an ID50 value of approximately 20 to 25 mumol/L in each case, as well as by cycloheximide, with an ID50 value of less than 0.5 micrograms/mL in each case. In contrast, 100 mumol/L of HA-1004 showed less inhibition of PGE2 production provocated by either PMA or EGF. On the other hand, PGE2 production in basal or stimulated condition by exogenously added arachidonic acid, was inhibited to an even lesser extent by both H-7 and cycloheximide. The EGF- and PMA-stimulated release of 3H-arachidonic acid from the cells was also strongly inhibited by H-7 and cycloheximide. These results suggest an induction of synthesis of some proteins responsible for the release of arachidonic acid, which might be attributed to protein kinase-C activation in arachidonic acid metabolism stimulated by PMA or EGF. Moreover, PGE2 production was potently induced by PMA and slightly by EGF in the cyclooxygenase-inactivated cells by acetyl salicylate pretreatment, which also suggests that both agents might induce the synthesis of cyclooxygenase in cultured porcine thyroid cells, although we did not measure its activity.