YC-1 inhibits proliferation of human hepatoma cells through a cyclic GMP-independent pathway

673 3-(5’-Hydroxymethyl-2’-furyl)-1-benzyl indazole (YC-1), a novel type of soluble guanylyl cyclase (sGC) activator, is useful in investigating the signaling of cGMP and may provide a new approach for treating cardiovascular diseases. Recently, YC-1 has been shown to inhibit the proliferation of vascular smooth muscle cells and endothelial cells, and blocks tumor angiogenesis by suppressing hypoxia-inducible factor-1 (HIF-1) activity. In the current study, we investigated whether YC-1 can directly affect cancer cell survival and growth and YC-1’s anticancer mechanism in human hepatoma. Our data showed that YC-1 inhibited the growth of HA22T human hepatoma cells in a concentration-dependent manner by SRB and [ 3 H]thymidine incorporation assay. DAPI staining and trypan blue exclusion tests indicated that the YC-1-induced antiproliferative effect was not through direct cytotoxicity, but through the inhibition of cell growth. Flow cytometry showed that YC-1 induced a concentration-dependent increase in G1 phase with a concomitant decrease in the S and G2-M phases of the cell cycle. The role of cGMP in YC-1-induced growth inhibition in HA22T cells was examined. ODQ (a selective sGC inhibitor), and KT-5823 (a selective inhibitor of cGMP-dependent protein kinase) did not prevent the inhibition of cell growth that was induced by YC-1. SNP (an NO donor), IBMX (an non-specific phosphodiesterase inhibitor) did not significantly potentiate YC-1-mediated growth inhibition. Moreover, intracelluar cGMP levels were also not increased by YC-1 in HA22T cells. These data suggest that the sGC/cGMP signaling pathway is not involved in YC-1-induced inhibition of HA22T proliferation. Western blotting showed that YC-1 did induce a profound elevation in p21 CIP1/WAF1 expression, and the formation of CDK2-p21 complexes, but not other CDK-CKI complexes, was increased by YC-1. The mRNA level of p21 CIP1/WAF1 was analyzed in YC-1-treated cells using RT-PCR. Immunohistochemical analysis revealed that the nuclear localization signal of p21 CIP1/WAF1 was increased in YC-1-treated regressed tumor tissue. On the basis of our findings, we conclude that YC-1 induces cell cycle arrest and inhibits tumor growth both in vitro and in vivo via the up-regulation of p21 CIP1/WAP1 expression. This YC-1-induced antiproliferation effect in human hepatoma cells is through a cyclic GMP-independent pathway.