吡咯烷二硫代氨基甲酸盐对肾癌细胞系具有抗增殖和促凋亡作用。

Pyrrolidine dithiocarbamate exerts anti-proliferative and pro-apoptotic effects in renal cell carcinoma cell lines.

作者信息

Morais Christudas, Pat Betty, Gobe Glenda, Johnson David W, Healy Helen

机构信息

Conjoint Renal Laboratory, Bancroft Centre, Queensland Health Pathology Service, Royal Brisbane and Women's Hospital, 300 Herston Road, Brisbane, Queensland 4006, Australia.

出版信息

Nephrol Dial Transplant. 2006 Dec;21(12):3377-88. doi: 10.1093/ndt/gfl543. Epub 2006 Sep 23.

DOI:10.1093/ndt/gfl543

PMID:16998220

Abstract

BACKGROUND

The activation of nuclear factor-kappaB (NF-kappaB) has been implicated in the development, progression and metastasis of renal cell carcinoma (RCC). This study investigates the effect of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, on two metastatic human RCC cell lines, ACHN and SN12K1.

METHODS

RCC cell lines and normal cells were exposed to 25 or 50 microM of PDTC. Apoptosis was measured by flow cytometry and TdT-mediated nick end labelling methods. Cell viability and proliferation were measured by MTT and BrdU assays, respectively. Expression of NF-kappaB subunits, IkappaBs, IkappaB Kinase (IKK) complex and apoptotic regulatory proteins were analysed by western blotting and/or immunofluorescence. DNA-binding activity of NF-kappaB subunits were measured by ELISA.

RESULTS

RCC cell lines had a higher basal level expression of all the five subunits of NF-kappaB than normal primary cultures of human proximal tubular epithelial cells or HK-2 cells. PDTC decreased the viability and proliferation of RCC, but not normal cells. Of the two RCC cell lines, ACHN had a higher basal level expression of all the five NF-kappaB subunits than SN12K1 and was more resistant to PDTC. While PDTC induced an overall decrease in expression of all the five NF-kappaB subunits in both RCC cell lines, unexpectedly, it increased the nuclear expression of NF-kappaB in ACHN, but not in SN12K1. PDTC reduced the DNA-binding activity of all the NF-kappaB subunits and the expression of the IKK complex (IKK-alpha, IKK-beta and IKK-gamma) and the inhibitory units IkappaB-alpha and IkappaB-beta. PDTC induced a significant increase in apoptosis in both RCC cell lines. This was associated with a decrease in expression of the anti-apoptotic proteins, Bcl-2 and Bcl-(XL), without marked changes in the pro-apoptotic protein Bax.

CONCLUSION

These data suggest that PDTC has the potential to be an anticancer agent in some forms of RCC.

摘要

背景

核因子-κB（NF-κB）的激活与肾细胞癌（RCC）的发生、发展和转移有关。本研究调查了NF-κB抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）对两种转移性人RCC细胞系ACHN和SN12K1的影响。

方法

将RCC细胞系和正常细胞暴露于25或50微摩尔的PDTC中。通过流式细胞术和TdT介导的缺口末端标记法检测细胞凋亡。分别通过MTT和BrdU测定法检测细胞活力和增殖。通过蛋白质免疫印迹和/或免疫荧光分析NF-κB亚基、IκB、IκB激酶（IKK）复合物和凋亡调节蛋白的表达。通过ELISA测定NF-κB亚基的DNA结合活性。

结果

RCC细胞系中NF-κB的所有五个亚基的基础水平表达均高于人近端肾小管上皮细胞或HK-2细胞的正常原代培养物。PDTC降低了RCC细胞的活力和增殖，但对正常细胞无此作用。在两种RCC细胞系中，ACHN的所有五个NF-κB亚基的基础水平表达均高于SN12K1，且对PDTC的耐药性更强。虽然PDTC导致两种RCC细胞系中所有五个NF-κB亚基的表达总体下降，但出乎意料的是，它增加了ACHN中NF-κB的核表达，而在SN12K1中未增加。PDTC降低了所有NF-κB亚基的DNA结合活性以及IKK复合物（IKK-α、IKK-β和IKK-γ）和抑制单位IκB-α和IκB-β的表达。PDTC诱导两种RCC细胞系中的细胞凋亡显著增加。这与抗凋亡蛋白Bcl-2和Bcl-(XL)的表达降低有关，而促凋亡蛋白Bax无明显变化。