Possible roles of a tumor suppressor gene PIG11 in hepatocarcinogenesis and As2O3-induced apoptosis in liver cancer cells.

BACKGROUND: Our previous studies demonstrated that p53-induced gene 11 (PIG11) was involved in arsenic trioxide (As(2)O(3))-induced apoptosis in human gastric cancer MGC-803 cells. Here, we studied further PIG11 expression in human hepatocellular carcinoma (HCC) tissues and cell lines and compared the sensitivity to As(2)O(3)-induced cell apoptosis in HepG2 and L-02 cells. METHODS: PIG11 expression in human normal liver tissues, HCC tissues, and cell lines was determined by immunohistochemistry and immunocytochemistry methods, using an anti-human PIG11 antibody. Cell viability was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diplenyltetrazolium bromide (MTT) assay. Cell apoptosis was determined by flow cytometry. Reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to analyze PIG11 mRNA and protein expression in cells. Protein intensity was calculated by comparison with the intensity of beta-actin, using densitometry. PIG11 was knocked down using small interfering RNA (siRNA). RESULTS: We found that PIG11 expression was significantly downregulated in HCC tissue and the cell lines (Bel-7402, SMMC-7721, HepG2 cells). Further, HepG2 cells were more sensitive to As(2)O(3)-induced apoptosis than L-02 cells. The expression of PIG11 mRNA and protein was upregulated to a greater extent in HepG2 than in L-02 cells. In the presence of actinomycin D or cycloheximide, the amount of PIG11 protein expression did not increase. Likewise, the inhibition of PIG11 by siRNA decreased As(2)O(3)-induced PIG11 protein expression by more than 85% and partially prevented As(2)O(3)-induced apoptosis in both HepG2 and L-02 cells. CONCLUSION: The above results demonstrated that the PIG11 gene may be involved in As(2)O(3)-induced apoptosis in HepG2 cells and suggested that the adaptive response of PIG11 expression is one of the important factors in enhancing cell sensitivity to As(2)O(3)-induced apoptosis.

A P53 target gene, PIG11, contributes to chemosensitivity of cells to arsenic trioxide.

The tumor suppressor p53 regulates the expression of various genes that promote apoptosis. PIG11 (P53-induced gene 11), also referred to as TP53I11 (tumor protein p53 inducible protein 11), is a direct p53 target gene. Recent data demonstrated that PIG11 was up-regulated markedly in arsenic trioxide induced apoptosis by DDRT-PCR, suggesting a new class of p53 target genes that sensitize cells to the effects of chemotherapeutic agents. In this study, through the construction of a recombinant GFP-PIG11 expression vector and transfection of HEK293 cells with GFP or GFP-PIG11, the role of PIG11 in apoptosis was analyzed. Results demonstrated that the percentage (11.38%) of apoptotic cells with GFP-PIG11 transfection was higher than that (7.28%) of with only GFP transfection (P<0.05). At 24 h after 1 microM of arsenic trioxide treatment, apoptotic cells exhibited a significant increase in the expression of GFP-PIG11 (36.67%+/-2.78), in contrast, 10.50%+/-2.03 only GFP and 5.25%+/-0.96 vehicle control (P<0.01). In addition, we showed that intracellular content of reactive oxygen species (ROS) was 9.66+/-0.52 in GFP-PIG11 transfection, higher than 5.21+/-0.08 in GFP only and 5.99+/-0.45 in vehicle control (P<0.01). The above results suggest that overexpression of PIG11 could induce cell apoptosis in the low levels and enhanced the apoptotic effects of arsenic trioxide. The process could be involved in intracellular generation of ROS.

Increasing sensitivity to arsenic trioxide-induced apoptosis by altered telomere state.

In this work, we investigated the synergic effects between low-dose arsenic trioxide and diethyloxadicarbocyanine (DODC), a telomerase inhibitor, on cell apoptosis. Results revealed that low-dose arsenic could block cell cycle arrest at the G2/M phase and induce apoptosis, whereas DODC could block cell cycle arrest at the G0/G1 phase but not induce apoptosis. However, cells pretreated with DODC showed greater sensitivity to arsenic than untreated cells. The percentage of apoptosis produced by combination treatment with the two agents increased and that was similar to the effect of high-dose arsenic treatment alone. Further studies showed that DODC alone could induce hairpin G-quadruplex formation and inhibit telomerase activity in a dose-dependent manner. Compared with HT1080 cells, 293 cells were more sensitive to cell growth inhibition and apoptosis and were less sensitivity to telomerase activity. These results indicate that DODC can synergistically enhance the apoptosis induced by arsenic, suggesting the increased cell senescence in response to arsenic is induced by an altered telomere state rather than by a loss of telomerase. Thus clinical application of combination treatment with arsenic and telomerase inhibitor may have potential in cancer therapy.

Expression of p21(WAF1) and p53 and polymorphism of p21(WAF1) gene in gastric carcinoma.

AIM: To investigate the relationship between expression of p21(WAF1) and p53 gene, and to evaluate the deletion and polymorphism of p21(WAF1) gene in gastric carcinoma (GC). METHODS: Expression of p21 and p53 proteins, and deletion and polymorphism of p21 gene in GC were examined by streptavidin-peroxidase conjugated method (SP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) respectively. RESULTS: The expression of p21 and p53 was found in 100% (20/20) and 0% (0/20) of normal gastric mucosae(NGM), 92.5% (37/40) and 15.0% (6/40) of dysplasia (DP) and 39.8% (43/108) and 56.5% (61/108) of GC, respectively. The positive rate of p21 in GC was lower than that in NGM and DP (P<0.05), while the positive rate of p53 in GC was higher than that in NGM and DP (P<0.05). p21 and p53 were significantly expressed in 63.3% (19/30) and 36.7% (11/30), 35.0% (14/40) and 77.5% (31/40), 26.7% (4/15) and 80.0% (12/15), 30.8% (4/13) and 30.8% (4/13), and 20.0% (2/10) and 30.0% (3/10) of well-differentiated, poorly-differentiated, undifferentiated carcinomas, mucoid carcinomas and signet ring cell carcinomas. The expression of p21 in well-differentiated carcinomas was significantly higher than that in poorly-differentiated, un-differentiated, mucoid carcinomas and signet ring cell carcinomas (P<0.05). Contrarily, The expression of p53 was increased from well-differentiated to poorly-differentiated and un-differentiated carcinomas (P<0.05). The expression of p21 and p53 in paired primary and metastatic GC (35.3% and 70.6%) was different from non-metastatic GC (62.5% and 42.5%) markedly (P<0.05). The expression of p21 in invasive superficial muscle (60.0%) was higher than that in invasive deep muscle or total layer (35.2%) (P<0.05) and was higher in TNM stages I (60.0%) and II (56.2%) than in stages III (27.9%) and IV (22.2%) (P<0.05), whereas the expression of p53 did not correlate to invasion depth or TNM staging (P>0.05). The exoression patterns of p53+/p21-, and of p53-/p21+ were found in 5.0% and 82.5% of DP. There was a significant correlation between expression of p21 and p53 (P<0.05). But there was no significant correlation between expression of both in GC (P>0.05). There was no deletion in exon 2 of p21 gene in 30 cases of GC and 45 cases of non-GC, but polymorphism of p21 gene at exon 2 was found in 26.7% (8/30) of GC and 8.9% (4/45) of non-GC, a significant difference was found between GC and non-GC (P<0.05). There was no significant relation between p21 expression of polymorphism (37.5%, 3/8) and non-polymorphism (45.5%, 10/22) in GC (P>0.05). CONCLUSION: The loss of p21 protein and abnormal expression of p53 are related to carcinogenesis, differentiation and metastasis of GC. The expression of p21 is related to invasion and clinical staging in GC intimately. The expression of p21 protein depends on p53 protein largely in NGM and DP, but not in GC. No deletion of p21 gene in exon 2 can be found in GC. The polymorphism of p21 gene might be involved in gastric carcinogenesis.There is no significant association between polymorphism of p21 gene and expression of p21 protein.

Expression of TRF1, TRF2, TIN2, TERT, KU70, and BRCA1 proteins is associated with telomere shortening and may contribute to multistage carcinogenesis of gastric cancer.

PURPOSE: Telomere dysfunction is believed to be a significant factor in carcinogenesis. To elucidate the carcinogenesis mechanism in gastric cancer, the expression of telomeric proteins and changes in telomere length were investigated during multistage carcinogenesis of gastric cancer. METHODS: Tissue samples were obtained during surgical operations from the normal gastric mucosa of 10 patients, the precancerous lesions of 15 patients, the gastric cancer tissues (GC) of 20 patients, and of tumors due to gastric cancer with lymph node metastasis (GCLM) from 5 patients. The expression of TRF1, TRF2, and TIN2 proteins was measured by Western blotting, while the expression of TERT, KU70, and BRCA1 proteins was detected using the immunohistochemical method. The mean telomere length was determined by Southern blotting. RESULTS: Compared with normal gastric mucosa tissues, the expression of TRF1, TRF2, and TIN2 proteins was significantly higher in precancerous lesions, GC, and GCLM (P < 0.01). The expression of TRF1, TRF2, and TIN2 proteins was significantly higher in GC and GCLM than in precancerous lesions (P < 0.01). The expression of TERT and Ku70 proteins in precancerous lesions and GC tissues was significantly higher than that in normal gastric mucosa tissues (P < 0.01). The expression of TERT and Ku70 proteins in GC tissues was significantly higher than in precancerous lesions (P < 0.01). In normal gastric mucosa, the BRCA1 protein was primarily located in the cell nucleus. In precancerous lesions and GC, the expression of the BRCA1 protein was apparent in the cell cytoplasm. The mean telomere length in precancerous lesions, GC, and GCLM was significantly shorter than that in normal gastric mucosa tissues (P < 0.05). The mean telomere length in GC and GCLM was significantly shorter than that in precancerous lesions (P < 0.05). The mean telomere length in all tissue samples was inversely correlated with the level of TRF1, TRF2, TIN2, TERT, and Ku70 proteins. CONCLUSIONS: Our results suggest that the over-expression of telomeric proteins, TRF1, TRF2, TIN2, TERT, and Ku70, and the transposition of the BRCA1 protein may work together to reduce the telomere length in precancerous lesions and gastric cancer, and could contribute to the multistage carcinogenesis of gastric cancer. These findings offer new insight into the mechanism of carcinogenesis in gastric cancer.

PIG11 is involved in hepatocellular carcinogenesis and its over-expression promotes Hepg2 cell apoptosis.

PIG11 (p53-induced gene 11) is a p53 target gene and candidate tumour suppressor gene. In this study, the expression of PIG11 protein was detected in human hepatocellular carcinoma (HCC) and normal liver tissues with an immunohistochemical method. Compared with expression in human normal liver tissues, the expression of PIG11 protein was significantly down-regulated in human HCC tissues. In addition, a recombinant pLXSN-PIG11 retroviral vector was constructed and transfected into HepG2 cells (human hepatocellular carcinoma cell line) and the role of PIG11 in apoptosis was analyzed. The percentage (18.60%) of apoptotic cells transfected with pLXSN-PIG11 was higher than that in cells transfected with pLXSN only (6.03%) or the vehicle control (3.81%) (P < 0.01). DNA gel electrophoresis showed a clear DNA ladder in pLXSN-PIG11-infected HepG2 cells. Our results suggested that the PIG11 gene is involved in carcinogenesis and development of hepatocarcinoma. Therefore, PIG11 is considered to be a new candidate liver tumour suppressor gene, and may play an important role in tumour suppression through promotion of cell apoptosis.