低氧對腫瘤細胞引起的血管新生及對其表面粘著分子表現之改變及效應

Abstract

血管新生對於固態腫瘤的生長及轉移過程中是必要的。很多生長因子包括血管內皮生長因子(VEGF)、鹼性纖維母細胞生長因(bFGF)、腫瘤壞死因子-α (TNF-α)，上皮生長因子(EGF)是強的血管新生誘導劑，能促進血管內皮細胞的增生及移行。在很多的固態腫瘤中，這些血管因子是與高血管密度、淋巴節的轉移與不好的預後相關。處在低氧環境的腫瘤細胞會增加VEGF的表現。本篇研究的目的是想探討由低氧引起腫瘤細胞釋放之conditioned medium (CM)的可溶物質是否會促進血管新生。本文所探討的腫瘤包括小鼠黑色素腫瘤細胞及自發性神經母細胞瘤。與正常氧壓組相比，加入從低氧腫瘤細胞取得的CM，可促進HUVEC增生。當在低氧B16F10細胞取得的CM中加入neutralizing VEGF抗體後，這個促進增生的作用會被抑制。但在Neuro-2A細胞組，低氧與正常情況下都會被VEGF抗體所抑制。我們也發現低氧能促進B16F10細胞釋放VEGF。因此我們認為由低氧誘導之VEGF表現增加能促進內皮細胞的增生。接下來我們想探討低氧對於B16F10和Neuro-2A細胞表面integrin受體之表現及其附著在細胞外基質的作用為何。結果發現處在低氧下的B16F10細胞能增加β1 integrin的表現，而進一步增加對於fibronectin的附著，但對於collagen type I的附著卻是減少的。另外從流式細胞儀分析，低氧不會促進αv和β3 integrin的表現，但從附著實驗中，卻看到低氧會促進B16F10細胞對vitronectin的附著。另一方面，我們沒有觀察到低氧改變Neuro-2A細胞表面的integrin表現的作用。我們也利用基因微陣列分析來廣泛觀察低氧對B16F10細胞基因表現的影響，並藉由這個工具來驗證上述結果。這些結果顯示低氧可能藉由與β1有關的integrin來改變B16F10細胞對不同細胞外基質的附著。至於是哪些特定的integrin、調控腫瘤細胞附著及其他與腫瘤進程有關之行為的機轉仍有待進一步的研究。

Previous studies have shown that angiogenesis is required for growth and metastasis of solid tumors. Many growth factors including vascular endothelial growth factors (VEGFs), basic fibroblast growth factor (bFGF), tumor necrosis factor-α (TNF-α) and epidermal growth factor (EGF) are potent angiogenesis inducers through stimulating the proliferation and migration of capillary endothelial cells. In many solid tumors, expression of these factors correlates with high vascularity, lymph node metastasis, and poor clinical prognosis. Tumor cells exposed to hypoxia have been shown to up-regulate the expression of VEGF. The purpose of the present study was to investigate whether hypoxia-induced upregulation of soluble mediators that are released into tumor cell conditioned medium (CM) can result in an induction of angiogenesis. The tumors studied were Neuro-2A neuroblastoma from strain A albino mice and B16F10 melanoma from C57BL/6J mice. A significant increase in cell proliferation was seen at 48 hr when HUVEC were cultured with the CM from hypoxic tumor cells as compared with normoxic counterpart. This increased proliferation was abrogated by preincubation of neutralizing VEGF antibodies with CM from hypoxic B16F10 cells. However, for Neuro-2A cells cultured under hypoxic and normoxic conditions, the increase of HUVEC proliferation was inhibited by adding neutralizing VEGF antibodies to both CMs. We also detected that hypoxia enhanced VEGF release from B16F10 cells. Therefore, we suggest that the increased production of VEGF induced by hypoxia stimulates endothelial cell proliferation. The effects of hypoxia on integrin expression and adhesion to extracellular matrix (ECM) proteins were investigated. Exposure of B16F10 cells to hypoxia caused a significant upregulation of β1 integrin and an associated increase in cell adhesion to fibronectin, but intriguingly, a decrease in adhesion to collagen type I. Through flow cytometric analysis, we did not find any prominent upregulation of αv and β3 integrin expression on B16F10 cell surface under hypoxic condition. However, we found an increase in cell adhesion to vitronectin. On the other hand, we did not find any significant alteration of integrin expression of Neuro-2A cells under hypoxic condition. Using microarray analysis, we also extensively observed the effect of hypoxia on the gene expression level of B16f10 cells and use this tool to confirm the above results. These results demonstrate that hypoxia may alter adhesion properties of B16F10 cells to ECM proteins through the interaction of β1–associated integrin(s). However, the identity of the specific integrin and its mechanism of regulating B16F10 cell adhesion to ECM or other behaviors related to tumor progression still need to be investigated.