Montcourrier P, Silver I, Farnoud R, Bird I, Rochefort H
Unité Hormones et Cancer (U 148) INSERM, Montpellier, France.
Clin Exp Metastasis. 1997 Jul;15(4):382-92. doi: 10.1023/a:1018446104071.
The extracellular pH in malignant tumors is known to be lower than in normal tissues and may therefore facilitate extracellular activation of secreted lysosomal cathepsins. We have tested the capability of human mammary cells (continuous cell lines and primary culture) to acidify their extracellular environment, using two techniques. By measuring pH changes through alterations of phenolsulfone phthaleine absorbance, we found that the more aggressive MDA-MB-231 human breast cancer cells were more active in acidifying a non-buffered balanced salt solution than the estrogen receptor positive MCF7 and ZR75 cell lines and than normal mammary epithelial cells in primary culture. Metastatic breast cancer cells from pleural effusions were up to 200-fold more active in acidifying their extracellular milieu than non-malignant mammary cells cultured in the same conditions, strongly suggesting that this difference also occurs in vivo. The use of inhibitors in the presence or absence of glucose showed that both lactate and an ATP-driven proton pump sharing some characteristics of the vacuolar H+ pump were involved. Bafilomycin A1, a specific inhibitor of the vacuolar (V-type) ATP-H+ pump inhibited part of the acidification by MCF7 cells, but not by MDA-MB-231 cells. We also used microelectrodes to measure extracellular pH, in close contact to the MCF7 breast cancer cells. The pH at the free surface of MCF7 cells was lower by 0.33 +/- 0.14 unit than that of the surrounding medium, while insertion of the microelectrode tip beneath the attached surface of the cells showed a greater lowering of pH from 0.3 to 1.7 pH unit as long as cell attachment on the substrate prevented H+ diffusion. We conclude that breast carcinoma cells have a higher capacity for acidifying their extracellular milieu than normal mammary cells, and that both a plasma membrane H(+)-ATPase, and lactic acid production are involved in this acidification. It is therefore possible that the aspartyl and cysteinyl pro-cathepsins secreted in excess by tumor cells may be activated extracellularly in vivo close to the basement membrane.
已知恶性肿瘤中的细胞外pH低于正常组织，因此可能促进分泌型溶酶体组织蛋白酶的细胞外激活。我们使用两种技术测试了人乳腺细胞（连续细胞系和原代培养细胞）酸化其细胞外环境的能力。通过测量酚红吸光度变化引起的pH变化，我们发现侵袭性更强的MDA-MB-231人乳腺癌细胞在酸化非缓冲平衡盐溶液方面比雌激素受体阳性的MCF7和ZR75细胞系以及原代培养的正常乳腺上皮细胞更活跃。来自胸腔积液的转移性乳腺癌细胞在酸化其细胞外环境方面的活性比在相同条件下培养的非恶性乳腺细胞高200倍，强烈表明这种差异在体内也会出现。在有或没有葡萄糖的情况下使用抑制剂表明，乳酸和具有液泡H⁺泵某些特征的ATP驱动质子泵都参与其中。巴弗洛霉素A1是液泡（V型）ATP-H⁺泵的特异性抑制剂，它抑制了MCF7细胞的部分酸化作用，但对MDA-MB-231细胞无效。我们还使用微电极测量与MCF7乳腺癌细胞紧密接触的细胞外pH。MCF7细胞自由表面的pH比周围培养基低0.33±0.14个单位，而将微电极尖端插入细胞附着表面下方时，只要细胞在底物上的附着阻止了H⁺扩散，pH就会有更大程度的降低，从0.3到1.7个pH单位。我们得出结论，乳腺癌细胞比正常乳腺细胞具有更高的酸化其细胞外环境的能力，并且质膜H⁺-ATP酶和乳酸产生都参与了这种酸化过程。因此，肿瘤细胞过量分泌的天冬氨酸和半胱氨酸组织蛋白酶原可能在体内靠近基底膜的位置被细胞外激活。
