Small hairpin RNA-mediated Krüppel-like factor 8 gene knockdown inhibits invasion of nasopharyngeal carcinoma.

The present study aimed to characterize the expression of Krüppel-like factor 8 (KLF8) in nasopahryngeal carcinoma (NPC) cell lines and determine its effect on tumor development and invasion following KLF8 gene knockdown by small hairpin RNA (shRNA). KLF8 expression in four NPC cell lines was examined by quantitative polymerase chain reaction (qPCR) and western blotting. KLF8 was knocked down in the SUNE1-5-8F/Sh-KLF8 cell line using shRNA, and the resulting stable cell line SUNE1-5-8F-sh-KLF8 was transplanted into nude mice in order to observe tumor formation and invasion. The results obtained from qPCR and western blotting revealed that, of the four NPC cell lines, KLF8 expression was lowest in the CNE-1 cells and highest in the SUNE1-5-8F cells. The tumor xenograft mouse models revealed that SUNE1-5-8F/Sh-KLF8 cells had a reduced ability for tumor formation and invasion compared with the control group. These results demonstrated for the first time that KLF8 modulates the formation and invasive ability of nasopharyngeal carcinoma.

Identification of PAQR3 as a new candidate tumor suppressor in hepatocellular carcinoma.

Progestin and adipoQ receptor family member III (PAQR3) is a regulator that negatively modulates the Ras/Raf/MEK/ERK signaling cascade and the GPCR Gßγ subunit signaling pathway. The role of PAQR3 in hepatocellular carcinoma (HCC) has not been elucidated. The present study investigated the expression of PAQR3 and its prognostic value in primary HCC patients. Furthermore, the functional aspects of PAQR3 were also studied using an in vitro cell model. PAQR3 expression was examined in paired HCC and adjacent noncancerous tissues using real-time quantitative RT-PCR (62 pairs) and western blotting (26 pairs). We also analyzed PAQR3 expression in 132 additional HCC samples by immunohistochemistry. The functional impact of PAQR3 on the proliferation and colony formation of an HCC cell line was analyzed by transfecting cells with a full-length PAQR3 expression vector or siRNA targeting PAQR3. The expression of PAQR3 was significantly decreased in the cancer tissues. Clinicopathological analyses showed that the expression of PAQR3 was significantly correlated with expression of serum α-fetoprotein (AFP), mitotic count, tumor size, histological grade and recurrence. Notably, Kaplan-Meier survival curves revealed a correlation between decreased expression of PAQR3 and the poor prognosis of HCC patients. Multivariate analyses showed that PAQR3 expression is an independent prognostic marker for overall and disease-free survival of HCC patients. Furthermore, restoring PAQR3 expression in HCC cells significantly diminished Hep3B cell proliferation and colony formation. Silencing PAQR3 expression in hepatic normal cell line LO2 significantly enhanced cell growth. PAQR3 may play an important role in the progression of HCC and serve as a potential candidate for the targeted therapy of HCC.

Targeting mTOR to overcome epidermal growth factor receptor tyrosine kinase inhibitor resistance in non-small cell lung cancer cells.

AIMS: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells. METHODS: We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry. RESULTS: In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC50 concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels. CONCLUSION: Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.

[Improvement of refolding efficiency of HLA-A2-peptide complex in vitro by preformed disulfide bonds in heavy chain].

AIM: To improve the refolding efficiency of soluble HLA-A2-peptide complex in vitro. METHODS: The heavy chain (HC) of MHC class I was extracted from bacteria under denaturing and non-reducing conditions. Anion-exchange and (NH4)2SO4 precipitation were applied to purify the HC. Then the purified HC, beta2m and an antigenic peptide (N-YMDGTMSQV-COOH of Try(369-377)) were refolded to form an HLA-A2-peptide complex by dilution method in the buffer of pH 6.6. The refolded products were detected by Western blot and ELISA with W6/32 and anti-human beta2m antibody. RESULTS: The refolded products consisted of HLA-A2-peptide complex, beta2m, and a little amount of HC polymer. The refolding efficiency was 2.5 fold higher than that of the conventional method. CONCLUSION: This study confirmed that the refolding efficiency of the method reported in this paper is higher as compared with the conventional method, which is of importance to the preparation of HLA-peptide tetramers and artificial antigen presenting cells.