Reactive oxygen species-mediated PKC and integrin signaling promotes tumor progression of human hepatoma HepG2.

The poor prognosis and recurrence of HCC are majorly caused by intrahepatic metastasis. Delineating the molecular pathways mediating these processes may benefit developing effective targeting therapies. Using human hepatoma HepG2 as a model, we have found reactive oxygen species (ROS) may cooperate with protein kinase C (PKC) for sustained ERK phosphorylation and migration of HepG2 induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We further investigated whether integrin signaling is involved. Various antagonists of integrin signaling prevented TPA-induced activation of ERK and PKC, ROS generation and migration of HepG2. On the other hand, TPA-induced phosphorylation of integrin signaling components including focal adhesion kinase (FAK), Src (Tyr416) and paxillin (Tyr31 and Ser178) can be prevented by PKC inhibitor Bisindolylmaleimides (BIS) and antioxidant dithiotheritol (DTT). HepG2 overexpressing PKCα contained elevated phosphorylated paxillin. Also, ROS generator phenazine methosulfate and tert-Butyl hydroperoxide may induce phosphorylation of paxillin and activation of PKC. Taken together, ROS mediated cross talk of PKC and integrin for migration of HepG2 induced by TPA. Furthermore, TPA induced intrahepatic metastasis of HepG2 in SCID mice, which was prevented by BIS or (BIS plus DTT). Elevated phosphorylation of paxillin was observed in tumor of mice treated with TPA as compared with those co-treated with TPA/BIS. In summary, the signal pathways for tumor progression of hepatoma induced by TPA can be established both in vitro and in vivo.

Snail associates with EGR-1 and SP-1 to upregulate transcriptional activation of p15INK4b.

Snail is a multifunctional transcriptional factor that has been described as a repressor in many different contexts. It is also proposed as an activator in a few cases relevant to tumor progression and cell-cycle arrest. This study investigated the detailed mechanisms by which Snail upregulates gene expression of the CDK inhibitor p15(INK4b) in HepG2 induced by the tumor promoter tetradecanoyl phorbol acetate (TPA). Using deletion mapping, the TPA-responsive element on the p15(INK4b) promoter was located between 77 and 228 bp upstream of the transcriptional initiation site, within which the putative binding regions of early growth response gene 1 (EGR-1) and stimulatory protein 1 (SP-1) were found. Gene expression of EGR-1, Snail and SP-1 can be induced by TPA within 0.5-6 h. In addition, basal levels of SP-1, but not of the other two transcriptional factors, were observed. Blockade of TPA-induced gene expression of Snail, EGR-1 or SP-1 suppressed activation of the p15-pro228 reporter plasmid harboring the TPA-responsive element. More detailed deletion mapping and site-directed mutagenesis further concluded that the overlapping EGR-1/SP-1-binding site was required for TPA-induced p15-pro228 activation. In an EMSA, a DNA-protein complex was elevated by TPA, which can be blocked by antibodies against EGR-1, SP-1 or Snail at 6 h. Immunoprecipitation/western blotting demonstrated that TPA could trigger the association of EGR-1 with Snail or SP-1. Furthermore, a double chromatin immunoprecipitation assay verified that EGR-1 could form a complex with Snail or SP-1 on the TPA-responsive element after treatment with TPA for 2-6 h. Finally, we demonstrated a novel Snail-target region which could be bound by Snail and was also required for TPA-induced p15-pro228 activation. In conclusion, Snail associates with EGR-1 and SP-1 to mediate TPA-induced transcriptional upregulation of p15(INK4b) in HepG2.

The transcriptional factor Snail simultaneously triggers cell cycle arrest and migration of human hepatoma HepG2.

Snail was recently highlighted as a critical transcriptional factor for tumor metastasis. Real time RT/PCR and Western blot analysis demonstrated that Snail mRNA and protein, respectively, were induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in hepatoma cell HepG2. Blockade of gene expression of Snail by antisense oligodeoxynucleotide and/or siRNA technique can prevent not only the TPA-triggered EMT/cell migration and growth inhibition of HepG2 but also TPA-induced down-regulation of E-cadherin and up-regulation of p15(INK4b). Moreover, the TPA-triggered promoter activation of p15(INK4b) was also prevented. On the other hand, two of the HepG2 clone over-expressing Snail, namely S7 and S15, had a scattered fibroblastic morphology and acquired higher motility than parental HepG2. Also, the proportion of G0/G1 phase of S7 and S15 was higher than that of parental HepG2, consistent with the longer doubling time of both cells. Semiquantitative RT/PCR analysis demonstrated a greatly elevated gene expression of Snail accompanied with decreased E-cadherin and increased p15(INK4b) in both Snail-overexpressing cells. On the transcriptional level, p15(INK4b) promoter activity was 2.6-fold higher in S7 as compared with parental HepG2. Furthermore, electrophoretic mobility of DNA fragments encompassing proximal p15(INK4b) promoter can be retarded by incubation of nuclear extract of S7. Our results demonstrated that Snail play diverse trans-regulatory roles in HepG2. Notably, we suggested that Snail may upregulate p15(INK4b) gene expression by directly activating its promoter.