In this study, we have for the first time demonstrated that Ach enhanced cell migration and invasion but suppressed apoptosis in HCC cells. These results suggest that Ach, as a classical transmitter, plays an important role in cancer metastasis and apoptosis of HCC cells. Sexual hormone receptors, such as AR and estrogen receptor, have been suggested to become therapeutic targets in sexual organ 
cancers, including prostate cancer and breast cancer. However, their implications in non-sexual organ cancers remain elusive. According to the epidemiological and experimental studies, both androgen and AR could contribute to gender disparity of HCC, but molecular mechanisms of androgen and AR are still largely unknown. It has been reported that higher activity of androgen pathway functions promotes tumor formation in male hepatocarcinogenesis. In hepatocyte AR-knockout mice, AR inhibits HCC metastasis through modulation of cell migration and anoikis. We have previously found that activation of AR promotes HCC cell migration and invasion. In the AbMole Succinylsulfathiazole present study, we demonstrate that AR is present in numerous HCC cell lines, reflecting that AR is involved in HCC. Our results, together with other studies, indicate that AR indeed plays potential roles in HCC metastasis. However, the up- and down-stream targets of AR in regulating HCC cell fate determinations remain largely unknown. Systemic deregulation has been regarded as important basis of cancer onset and development. Endocrine system, nervous system, and immune system interact globally in carcinogenesis. In this study, we sought to explore relationship of neurotransmitters and hormones by investigating effects of Ach on AR mRNA and protein expression in HCC cells. We found that Ach enhances both mRNA and protein expression of AR in SNU-449 cells. In addition, luciferase assay shows that Ach activates AR in SNU-449 cells. Taken together, Ach not only enhances AR expression but activates AR in HCC cells. We also found that AR agonist R1881 promoted the migration and invasion but reduced the apoptosis of SNU-449 cells, whereas AR antagonist casodex inhibited the migration and invasion but stimulated the apoptosis of SNU449 cells. Collectively, these data illustrate that Ach acts via AR to control the migration, invasion, and apoptosis of HCC cells. Notably, using AR knockdown and overexpression in HCC cells, we further demonstrate that AR is essential for Ach to promote the migration and invasion but inhibit the apoptosis of HCC cells. AR is a ligand-activated transcription factor that mediates the biological responses of androgen. Nevertheless, non-androgenic pathways have also been shown to activate the AR. It has been reported that AR can be activated by other molecules such as IL-6 and HER2/Neu signals. Two signaling transduction pathways, namely PI3K/AKT and JAK/STAT3 pathways, have been shown to activate AR in prostate cancer cells. Activation of STAT3 and ERK1/2 by nicotine modulates cell proliferation in human bladder cancer cells. In this study, we found that phosphorylation of AKT and STAT3 was elevated by Ach, implicating that Ach activates AKT and STAT3 signaling pathways in HCC cells. Moreover, we have previously demonstrated the existence of a non-neuronal cholinergic autocrine/ paracrine system in normal human hepatocytes and its dysregulation in HCC. The present findings further illustrate that the neurotransmitter Ach can regulate the expression of AR. Thus, our studies implicate that there may exist a systemic regulatory system with neural/non-neural neurotransmitters, endocrine, and immune systems as the core elements, and its dysregulation might be critical in hepatocarcinogenesis and HCC progression. In summary, we have for the first time demonstrated that Ach activates STAT3 and AKT pathways and it acts on AR to promote the migration and invasion but inhibit the apoptosis of SNU-449 cells.