Elzarrad M Khair, Haroon Abu, Willecke Klaus, Dobrowolski Radoslaw, Gillespie Mark N, Al-Mehdi Abu-Bakr
Department of Pharmacology and Center for Lung Biology, University of South Alabama, North University Boulevard, Mobile, AL 36688, USA.
BMC Med. 2008 Jul 22;6:20. doi: 10.1186/1741-7015-6-20.
The modulation of gap junctional communication between tumor cells and between tumor and vascular endothelial cells during tumorigenesis and metastasis is complex. The notion of a role for loss of gap junctional intercellular communication in tumorigenesis and metastasis has been controversial. While some of the stages of tumorigenesis and metastasis, such as uncontrolled cell division and cellular detachment, would necessitate the loss of intercellular junctions, other stages, such as intravasation, endothelial attachment, and vascularization, likely require increased cell-cell contact. We hypothesized that, in this multi-stage scheme, connexin-43 is centrally involved as a cell adhesion molecule mediating metastatic tumor attachment to the pulmonary endothelium.
Tumor cell attachment to pulmonary vasculature, tumor growth, and connexin-43 expression was studied in metastatic lung tumor sections obtained after tail-vein injection into nude mice of syngeneic breast cancer cell lines, overexpressing wild type connexin-43 or dominant-negatively mutated connexin-43 proteins. High-resolution immunofluorescence microscopy and Western blot analysis was performed using a connexin-43 monoclonal antibody. Calcein Orange Red AM dye transfer by fluorescence imaging was used to evaluate the gap junction function.
Adhesion of breast cancer cells to the pulmonary endothelium increased with cancer cells overexpressing connexin-43 and markedly decreased with cells expressing dominant-negative connexin-43. Upregulation of connexin-43 was observed in tumor cell-endothelial cell contact areas in vitro and in vivo, and in areas of intratumor blood vessels and in micrometastatic foci.
Connexin-43 facilitates metastatic 'homing' by increasing adhesion of cancer cells to the lung endothelial cells. The marked upregulation of connexin-43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, suggests that connexin-43 can serve as a potential marker of micrometastases and tumor vasculature and that it may play a role in the early incorporation of endothelial cells into small tumors as seeds for vasculogenesis.
在肿瘤发生和转移过程中，肿瘤细胞之间以及肿瘤细胞与血管内皮细胞之间的间隙连接通讯调节十分复杂。间隙连接细胞间通讯丧失在肿瘤发生和转移中所起作用的观点一直存在争议。虽然肿瘤发生和转移的某些阶段，如不受控制的细胞分裂和细胞脱离，必然需要细胞间连接的丧失，但其他阶段，如血管内渗、内皮细胞附着和血管生成，可能需要增加细胞间接触。我们推测，在这个多阶段过程中，连接蛋白43作为一种细胞粘附分子，在介导转移性肿瘤附着于肺内皮细胞方面起着核心作用。
将同基因乳腺癌细胞系尾静脉注射到裸鼠体内后，获取转移性肺肿瘤切片，研究肿瘤细胞对肺血管的附着、肿瘤生长及连接蛋白43的表达情况，这些细胞系分别过表达野生型连接蛋白43或显性负性突变的连接蛋白43蛋白。使用连接蛋白43单克隆抗体进行高分辨率免疫荧光显微镜检查和蛋白质印迹分析。通过荧光成像观察钙黄绿素橙红AM染料转移情况，以评估间隙连接功能。
过表达连接蛋白43的癌细胞对肺内皮细胞的粘附增加，而表达显性负性连接蛋白43的细胞粘附明显减少。在体外和体内的肿瘤细胞 - 内皮细胞接触区域、肿瘤内血管区域和微转移灶中均观察到连接蛋白43的上调。
连接蛋白43通过增加癌细胞与肺内皮细胞的粘附促进转移性“归巢”。在肿瘤细胞 - 内皮细胞接触区域，无论是在预先存在的“归巢”血管还是新形成的肿瘤血管中，连接蛋白43的显著上调表明，连接蛋白43可作为微转移和肿瘤血管的潜在标志物，并且它可能在早期将内皮细胞纳入小肿瘤作为血管生成种子的过程中发挥作用。
