Inhibition of mTOR suppresses human gallbladder carcinoma cell proliferation and enhances the cytotoxicity of 5-fluorouracil by downregulating MDR1 expression.

OBJECTIVE: Although 5-fluorouracil (5-FU) is widely used in the treatment of various cancers, drug resistance remains a limitation for its anti-cancer activity. Mammalian target of rapamycin (mTOR) is deregulated in diverse human cancers, including gallbladder carcinoma and mTOR inhibitors show promising anti-cancer activities with proliferation inhibitory effects. This study aims to clarify the benefit of the combination of 5-FU and the mTOR inhibitor, OSI-027, on gallbladder carcinoma cell proliferation. MATERIALS AND METHODS: Two gallbladder carcinoma cell lines and two agents (5-FU and OSI-027) were used in the present study. Cell counting kit-8 assays and EdU staining were performed to examine the proliferation of cancer cells. The expression of MDR1 protein was determined by western blot analysis. RESULTS: The combination of OSI-027 with 5-FU showed a synergistic anti-proliferative effect on the gallbladder cancer cells, RBE and GBC-SD cells. Upon 5-FU treatment, MDR1 expression was upregulated and OSI-027 could reverse 5-FU-induced MDR1 upregulation. Moreover, MDR1 depletion sensitized gallbladder carcinoma cells to 5-FU stimulation and attenuated the synergistic effect of OSI-027 and 5-FU. Finally, we determined that OSI-027 downregulated MDR1 expression by suppressing its synthesis rather than by promoting its degradation. CONCLUSIONS: Dual mTORC1/mTORC2 inhibitors such as OSI-027 are promising therapeutic agents in combination with 5-FU for the treatment of human gallbladder cancer.

Immortal human pancreatic duct epithelial cell lines with near normal genotype and phenotype.

Immortal epithelial cell lines were previously established after transduction of the HPV16-E6E7 genes into primary cultures of normal pancreatic duct epithelial cells. Single clones were isolated that demonstrated near normal genotype and phenotype. The proliferation of HPDE6-E6E7c7 and c11 cells is anchorage-dependent, and they were nontumorigenic in SCID mice. The cell lines demonstrated many phenotypes of normal pancreatic duct epithelium, including mRNA expression of carbonic anhydrase II, MUC-1, and cytokeratins 7, 8, 18, and 19. These cells have normal Ki-ras, p53, c-myc, and p16(INK4A) genotypes. Cytogenetic studies demonstrated losses of 3p, 10p12, and 13q14, the latter included the Rb1 gene. The wild-type p53 protein was detectable at very low levels consistent with the presence of E6 gene product, and the lack of functional p53 pathway was confirmed by the inability for gamma-irradiation to up-regulate p53 and p21waf1/cip1 protein. The p110/Rb protein level was also not detectable consistent with the expression of E7 protein and haploid loss of Rb1 gene. Despite this, the proliferation of both c7 and c11 cells were markedly inhibited by transforming growth factor-beta1. This was associated with up-regulation of p21cip1/waf1 but not p27kip1. Further studies showed that p130/Rb2 and cyclin D3 were expressed, suggesting that p130/Rb2 may have partially assumed the maintenance of G(1) cell cycle checkpoint regulation. These results indicate that except for the loss of p53 functional pathway, the two clones of HPDE6-E6E7 cells demonstrated a near normal genotype and phenotype of pancreatic duct epithelial cells. These cell lines will be useful for future studies on the molecular basis of pancreatic duct cell carcinogenesis and islet cell differentiation.

Autotaxin expression in non-small-cell lung cancer.

Autotaxin (ATX) is one of the newly discovered autocrine motility-stimulating factors with peptide sequences identical to those of the brain-type phosphodiesterase I (PD-Ialpha). Although ATX/PD-Ialpha is believed to play a role in tumor progression, its expression in various human cancers has not been extensively studied. We have studied the expression of ATX messenger RNA (mRNA) in normal human bronchial epithelial cell (HBEC) and non-small-cell lung cancer (NSCLC) cell lines, and in primary NSCLC with their corresponding normal lung tissues, using reverse transcription-polymerase chain reaction, Northern blot analysis, and in situ hybridization. ATX mRNA was commonly expressed in these cell lines and tissues. The predominantly expressed mRNA species corresponded to the ATX complementary DNA isolated from a human teratocarcinoma cell line. Overexpression of ATX mRNA was detected in seven of 12 (58%) tumor cell lines; however, there was no correlation between the levels of expression of ATX mRNA and the spontaneous motility of these cells. In situ hybridization localized ATX mRNA expression to the basal cells of normal bronchial epithelium, stromal B lymphocytes, and tumor cells. An overexpression of ATX mRNA as compared with its expression in normal bronchial epithelium was mainly found in poorly differentiated carcinomas. Our findings suggest that ATX may have roles additional to its motility-stimulating function in undifferentiated NSCLC.