Hepatocellular carcinoma (HCC) or hepatoma is the top one cause of death in Taiwan based on the Cause of Death Statistics from the Department of Health, Executive Yuan, Taiwan, for many years. To identify any reliable HCC markers and further applied with the AFP measurement to improve the early diagnosis of HCCs is the most important thing. A high expression level of S-phase protein kinase associated protein 2 (SKP2) protein and its cofactor CDC28 protein kinase regulatory subunit 1B (CKS1B) involved in ubiquitination of some cyclin-dependent kinase (Cdk) inhibitors has been reported in various carcinoma. In this study, we examined the expression of CKS1B in HCC tissues and cell lines, and tested the cell cycle effect caused by specific small interference RNA (siRNA) of CKS1B in SK-hep1 cell line. Up-regulated CKS1B mRNAs in HCC cell lines and tissues were identified in our study, when comparing to the normal liver tissues. But we also found lack of up-regulated CKS1B proteins in our HCC tissues at the same time, indicated that CKS1B proteins might be unstable in HCCs. Down regulation of the Cdk inhibitors p27 was only partially associated with HCCs, and the expressions of CKS1B and p27 were not correlated to each other in HCCs, suggesting other pathway(s) might involve in the regulation(s) of CKS1B and p27 proteins in the HCCs. Down-regulation of the p21 proteins was also found to be not significantly associated with HCCs tissues, this result strongly suggested a post-translational stabilization way might regulate(s) the p21 protein levels in HCCs tissues. On the other hands, in time course experiment, disruption of CKS1B mRNA by si-CKS1B up-regulated the expressions of p27 and SKP2 protein levels and down-regulated the p21 protein level in the SK-hep1 hepatoma cell lines for 24 hrs later. But the mRNA expression level of p21 and p27 were actually both up-regulated for 48 hrs after transfected with si-CKS1B. We also tested the mRNA expression level of many cell cycle regulatory factors for 48 hrs after transfected with si-CKS1B. The results exhibited almost all of the factors (excepted p21, p27 and Cyclin D2) were down-regulated. Furthermore, we saw the apoptosis appearance of SK-hep1 cell after transfected with si-CKS1B for 48 hrs, suggesting the abnormal cell proliferation and tumorigenesis were controlled by siRNA transfection. Taken together, these results suggest that SCFSKP2-CKS1B pathway might not direct involved in ubiquitination of Cdk inhibitors. Another pathway(s), either known or novel, in addition to APC/CCHD1 (G0-G1 phase) and SCFSKP2-CKS1B (G1-S phase) regulation pathways, might regulate the tumorigenesis of HCCs.

Our previous study identified both high p27kip1 and low CKS1B protein levels were independent prognosis markers in hepatocelular carcinomas (HCC), however, CKS1B overexpression implicated clinical aggressiveness of HCC but not p27Kip1 protein turnover. In this study, we demonstrated a cytotoxic compound, goniothalamin (GTN), that stabilized p27kip1 protein and downregulated CKS1B mRNA as well as protein levels in HCC-derived cell lines. The IC50 of TP53-negative Hep-3B and TP53-positive SK-Hep1 after treatment with GTN for 48 h were detected as 25 and 10 £gM, respectively. The GTN induced DNA damages, formation of £^H2AFX foci, and upregulation of £^H2AFX protein levels in dose-dependent manners in both cell lines. In addition, GTN arrested Hep-3B and SK-Hep1 cells in G2/M and G1 phases, respectively. Protein levels of several cell cycle regulators, including p27Kip1 in Hep-3B and, p21Cip1 and CKS1B in SK-Hep1 cells have been noticeable upregulated after treatment with GTN. In Hep-3B cells, GTN induced the configuration of p27Kip1/CCND1, p27Kip1/CDK2; p27Kip1/CCNB1 and p27Kip1/CDK1, however, repressed the p27Kip1/STMN1 complexes. On the other hand, GTN evidently induced the configuration of p21Cip1/CDK2 and p21Cip1/CCNE1, but repressed the p21Cip1/SKP2 and p21Cip1/CKS1B complexes, in SK-Hep1 cells. The CKS1B protein abundance that was induced by GTN was stemmed from the CKS1B transactivity. However, GTN-induced p27Kip protein profusion in Hep-3B cell line was, instead, due to the stabilization of p27Kip1 protein, mainly in the cytosol. Both GTN and the proteasome inhibitor, MG132, induced p27Kip1 ubiquitination and p27Kip1 protein profusion with an additive effect, regardless of CKS1B protein level, suggesting that the p27Kip1 protein might be regulated by GTN through an alternative ubiquitin-complex in Hep-3B cells. In addition to the quantitative reverse transcription -polymerase chain reaction, GTN-induced p21Cip1 protein level in SK-Hep1 cells could be traced back to mRNA level, by evidence of quantitative immunoprecipitation/chromatin immunoprecipitation assay with primer sets specific to two regions of the p21Cip1 proximal promoter. Taken together, GTN is able to induce the cell cycle arrest via stabilization of p27Kip1 protein and downregulation of CSK1B mRNA and subsequent protein levels in TP53-negative Hep-3B and TP53-positive SK-Hep1 cells, respectively. The GTN-repressed CKS1B transcription and thereafter, cell cycle arrest, was irrelevant to the p27Kip1 protein stability in Hep-3B cells.

Hepatocellular carcinoma (HCC) or hepatoma is the fifth most common cancer and the third most common cause of cancer-related deaths worldwide. Early detection of HCC still remains challenge. Patients with late-stage HCCs have very limited therapeutic options and relatively poor prognosis. Therefore, recognition of useful tumor marker for the diagnosis of HCC may aid in the management with this lethal malignancy, and designing effective therapies. In our previous study, through in silico data mining of numerous available cDNA microarray databases, the CDC28 protein kinase regulatory subunit 1B (CKS1B) transcript was found to be frequently upregulated in HCCs and the gene is strongly associated in clinical aggressiveness. Moreover, gain of chromosome 1q copy is one of the most frequently detected alterations in HCC and 1q21 is the most frequent minimal amplifying region. Mammalian CKS1B gene, a member of the highly conserved cyclin kinase subunit 1 protein family, mapped to 1q21.2-q22, which interacts with cyclin-dependent kinases and frequently up-regulated in human HCC specimens, and this gene also has been reported to play oncogenic roles in other human cancers. Furthermore, this gene was identified as an essential cofactor of S-phase kinase-associated protein 2 (SKP2) in the SKP2-containing SKP1-Cullin1-F-box ubiquitin ligase complex¡Vmediated ubiquitination of the cell cycle inhibitor protein cyclin-dependent kinases 1A (p27kip1) that leads to the proteasomal degradation of p27kip1. In the present study, our results indicate that CKS1B overexpression may contribute to hepatocarcinogenesis by enhancing the cellular tumorigenic potential. In addition, CKS1B was correlated with the HCC-derived cells proliferation, and CKS1B knockdown significantly inhibited the tumorigenic potential of HCC-derived cells. In this study, we examined that CKS1B knockdown and overexpressed in HCC cells significantly changed the expression of genes known to participate in various cellular processes, including, apoptosis, cell cycle, proliferation, viability, invasion, and migration, implying that CKS1B may play considerable and diverse roles in hepatocarcinogenesis.