Hyaluronan Induces Tumor Lymphangiogenesis In Xenografts And Differential Phosphorylation Of P38 Induced By Apoptotic And Anti-apoptotic Stimuli In Murine Hepatocytes

Hyaluronan (HA) is a high-molecular-weight glycosaminoglycan in extracellularmatrix (ECM). In these studies, we investigated the effect of HA on tumorlymphangiogenesis, angiogenesis, tumor growth, as well as tumor lymph nodemetastasis and its potential mechanism. In addition, we investigated the differentialphosphorylation of p38 MAP kinase (Mitogen-activated Protein kinase) induced bypro-apoptotic Transforming Growth Factor-β1 (TGF-β1) and anti-apoptotic EpidermalGrowth Factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA).1. We reported for the first time that HA, which is one the most importantcomponents in ECM and elevated in malignant tumor stroma, is able to induce tumorlymphangiogenesis. Previously, we have established a model that mouse hepatomaascites Hca-F cells selectivaly metastasis to the lymph node in C3H/Hej mice. Here,we examined lymphangiogenesis and angiogenesis in Hca-F xenografts byimmunostaining and RT-PCR of lymphatic marker LYVE-1 and vascular markerCD31 respectively. The data showed that there are a few lymphatic vessels in theperitumoral region and no intratumoral lymphatic vessels can be detected inxenografts, which have displayed lymph node metastasis. This result indicated thattumor lymangiognesis does not occur in Hca-F xenografts and implied that metastasisto lymph node of Hca-F cells are independent of tumor lymphangiognesis. However,when HA and Hca-F cells were co-injected subcutaneously into C3H/Hej mice,numerous intratumoral lymphatic vessels can be detected in Hca-F xenografts.Besides large and strong LYVE-1-possitive lymphatic vessels, small and weakLYVE-1-possitive vas was also detected, which can be organized by only severallymphatic endothelial cells. The latter may represent the developing lymphatic vessels.The results demonstrated that HA is able to induce tumor lyphangiogenesis in Hca-Fxenografts. We further found that HA-induced lymphangiogenesis is associated withpromoted intralymphatic tumor growth in both subcutaneous and newbornintratomoral lymphatic vessels. However, HA is not sufficient to promote theincrease the rate of tumor lymph node metastasis. These results suggested that tumorlymphangiogenesis might play an important role in the processes of lymph nodemetastasis, for example the invasion of tumor cells to lymphatic vessels, although itcould play a minor role in the lymph node metastasis of Hca-F cells. Furthermore, thedata showed that HA treatment increases the expression of lymphangiogenic growthfactor VEGF-D but not angiogeneic VEGF-A and VEGF-B in Hca-F cells in vitro,suggesting VEGF-D might be involved, at least partly, in HA-induced tumorlymphangiogenesis.2. We investigated the differential phosphorylation of p38 MAP kinase inducedby pro-and anti-apoptotic factors. The results showed that EGF and TPA can suppressTGF-β1-induced apoptosis. However, all these factors can induce the phosphorylationof p38 MAP kinase. The selective p38 inhibitor SB202190 is able to block the EGFand TPA-induced p38 phosphorylation but has no effect on that induced by TGF-β1.These results suggested that the pro-apoptotic TGF-β1 and anti-apoptotic EGF andTPA induce the phosphorylation and activation of p38 through differentialmechanisms: while the intrinsic p38 kinase activity is not necessary forTGF-β1-induced p38 phosphorylation, EGF and TPA can promote this event throughan autophosphorylation-dependent mechanism.