The molecular mechanism of protein kinase C inhibitors on anti-metastasis

我们研究了蛋白激酶C（PKC）抑制剂抗转移的机制。PKC412（4′- n -苯甲酰基staurosporine）是一种传统的PKC（α， β和γ）抑制剂，当与细胞在体外培养1小时时，可降低小鼠恶性黑色素瘤（B16-BL6）细胞在小鼠体内形成肺部菌落的能力，并减少细胞外基质的侵袭。此外，PKC412（200mg/kg/天，连续4周，p.o）显著延长了自发转移小鼠模型的存活时间，该模型是在肿瘤接种2周后，将B16-BL6细胞（1×10^6细胞）皮下接种到原发肿瘤切除的C57BL/6Cr小鼠的右足部产生的。为了阐明PKC412抗侵袭作用的机制，我们测量了细胞运动、细胞分泌的基质金属蛋白酶（MMP）活性和细胞中整合素β 1蛋白的表达。因此，PKC412以剂量依赖的方式降低了B16-BL6细胞中整合素β 1蛋白的表达。然而，PKC412不能抑制黑色素瘤细胞的细胞运动和MMP活性。这些结果表明PKC412通过降低整合素β 1的表达抑制黑色素瘤细胞转移途径的侵袭过程。最后，我们使用特异性PKC抑制剂rottlerin检查了与转移相关的PKC亚型。由于rotlerin不能阻断B16-BL6细胞小鼠的血源性肺转移，因此我们认为传统的PKC异构体可以诱导肿瘤转移，而delta异构体则没有。

We investigated the mechanism of protein kinase C(PKC) inhibitors on anti-metastasis. PKC412 (4'-N-benzoyl staurosporine), a conventional PKC(alpha, beta and gamma) inhibitor, reduced the ability of mouse malignant melanoma(B16-BL6) cells to form lung colonies in mice and reduced invasion of the extracellular matrix in vitro when pie-incubated with the cells for 1 hour. Further, PKC412(200mg/kg/day for 4 weeks, p.o.) significantly prolonged survival time in a spontaneous metastatic mouse model, produced by subcutaneous inoculation of B16-BL6 cells(1×10^6 cells) into the right footpad of C57BL/6Cr mice Mowed by surgical amputation of the primary tumor 2 weeks after tumor inoculation. To elucidate mechanisms of anti-invasive action for PKC412, we measured cell motility, matrix metalloproteinase(MMP) activity secreted from cells and expression of integrin beta 1 protein in cells. As a result, PKC412 decreased the expression of integrin beta 1 protein of B16-BL6 cells in a dose-dependent manner. However, PKC412 could not inhibit cell motility and MMP activity of the melanoma cells. These results suggest that PKC412 inhibits the process of invasion in the metastatic pathway of melanoma cells by attenuation of integrin beta 1 expression. Finally, we examined metastasis-related PKC isoforms using rottlerin, a specific PKC delta inhibitor. Since rottlerin could not block the hematogenic lung metastasis in mice using B16-BL6 cells, conventional PKC isoforms were considered to induce tumor metastasis, while the delta isoform did not.