Fang Yin

Regulation of drug sensitivity of gastric cancer cells by human calcyclin-binding protein (CacyBP)

BackgroundCalcyclin-binding protein (CacyBP) was previously identified as an upregulated gene in a multidrug-resistant gastric cancer cell line, SGC7901/ADR, compared to its parental cells, SGC7901, by subtractive hybridization. The aim of this study was to explore the role of CacyBP in multidrug resistance (MDR) in gastric cancer cells.MethodsThe cDNA encoding CacyBP was generated by reverse-transcription-polymerase chain reaction (RT-PCR), and mouse antisera against CacyBP was raised using recombinant CacyBP as the immunogen. The expression of CacyBP in gastric cancer cells was determined by Northern and Western blots. Sense and antisense vectors for CacyBP were introduced into SGC7901 and SGC7901/ADR cells, respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed to evaluate the drug sensitivity of gastric cancer cells. Flow cytometry was employed to determine adriamycin accumulation and retention in gastric cancer cells.ResultsNorthern and Western blots demonstrated upregulation of CacyBP in SGC7901/ADR cells compared to SGC7901 cells. SGC7901-CacyBP and SGC7901/ADR-anCacyBP cells were prepared, in which CacyBP was genetically increased and decreased, respectively. As compared with SGC7901, SGC7901-CacyBP cells exhibited significantly increased (P < 0.01) IC50 values for vincristine, adriamycin, and 5-fluorouracil. Meanwhile, as compared with SGC7901/ADR, SGC7901/ADR-anCacyBP cells exhibited significantly decreased (P < 0.01) IC50 values for these three drugs. SGC7901-CacyBP and SGC7901/ADR-anCacyBP cells displayed no obvious difference (P > 0.05) in intracellular adriamycin content compared to their corresponding parental cells.ConclusionsUpregulation of CacyBP is associated with MDR in gastric cancer cells. CacyBP could regulate the responses of gastric cancer cells to chemotherapy. But the underlying mechanisms of CacyBP-related MDR need further identification.

In vitro and in vivo characterization of silk fibroin/gelatin composite scaffolds for liver tissue engineering

OBJECTIVE:  To investigate the cytotoxicity of silk fibroin/gelatin (SF/G) composite scaffolds in vitro as well as their biocompatibility and degradation in vivo.

MAD2 as a key component of mitotic checkpoint: A probable prognostic factor for gastric cancer.

We studied the subcellular localization of MAD2 in normal human tissues and gastric cancers. MAD2 showed nuclear and cytoplasmic localization in normal tissues such as muscle, testis, thyroid gland, cerebrum, trachea, and skin; blood vessels in some organs were also MAD2+. In normal stomach, MAD2 was expressed mainly in cytoplasm but showed nuclear staining in the majority of gastric cancers. MAD2 was significantly overexpressed in gastric cancer compared with matched adjacent tissues (P < .001), and expression was related to differentiation and other clinical parameters of cancer (P < .001). The cancer/adjacent normal tissue (C/N) ratio of MAD2 expression was higher than 2 and more frequently observed in patients with lymph gland metastasis (P < .05) and related to cancer differentiation. Our findings suggest that the steady-state amount of MAD2 inside cells may serve as a molecular switch in mitotic checkpoint control and that the subcellular localizations of this spindle protein undergo a shift during malignant transformation. The change of MAD2 expression may be involved mainly in gastric carcinogenesis and associated with the prognosis of gastric cancer; a C/N of more than 2 may be associated with the worse prognosis for survival in gastric carcinoma.

Depression of MAD2 inhibits apoptosis and increases proliferation and multidrug resistance in gastric cancer cells by regulating the activation of phosphorylated survivin.

Mitotic arrest-deficient 2 (MAD2) is one of the essential mitotic spindle checkpoint regulators, and it can protect cells from aberrant chromosome segregation. The Mad2 gene is very rarely mutated in many kinds of human cancer, but aberrantly reduced expression of MAD2 has been correlated with defective mitotic checkpoints in several human cancers. We have previously found that the MAD2 expression level is also shown to be associated with the multidrug resistance of tumour cells. In this study, we constructed a small interfering RNA (siRNA) eukaryotic expression vector of MAD2 and downregulated MAD2 expression in the gastric cancer cell line SGC7901 by transfection of MAD2-siRNA. SGC7901 cells stably transfected with the MAD2-siRNA exhibited significantly increased expression of phosphorylated survivin protein and enhanced drug resistance. Furthermore, MAD2-siRNA suppressed the proliferation of SGC7901 cells and inhibited tumour formation in athymic nude mice. This study clearly reveals that downregulation of MAD2 could regulate the cell cycle, increase proliferation, and improve the drug resistance of gastric cancer cells by regulating the activation of phosphorylated survivin. It also suggests both that MAD2 might play an important role in the development of human gastric cancer and that silencing the MAD2 gene may help to deal with the multidrug resistance of gastric cancer cells.

Inhibitory effects of a specific phage-displayed peptide on high peritoneal metastasis of gastric cancer

Peritoneal dissemination in gastric cancer is the most frequent cause of the noncurative resection and recurrence after curative resection. We, therefore, evaluated the feasibility of a peptide, which was obtained by screening a random phage display library, in the treatment of peritoneal metastases of gastric cancer. In this study, a novel cell line, GC9811-P, with a high potential peritoneal metastasis of gastric cancer derived from its parental cell line, GC9811, was established. Using a phage display library, we isolated a specific peptide that selectively bound to GC9811-P cells rather than its parental GC9811cells. The isolated phage-displaying peptide, SMSIASPYIALE (named peptide PIII), was obtained after four rounds of selection, showing a tendency to preferentially bind to GC9811-P cells compared with a panel of other gastric cancer cell lines, and preferentially accumulate in peritoneal metastasis tumor tissue in comparison with control organs, peritoneum, liver, pancreas, spleen, lung, and kidney. Further study showed that synthetic peptide PIII could significantly inhibit adhesive and invasional ability of GC9811-P cells and could effectively block the corresponding phage binding to the GC9811-P cells, whereas, exposure of the cells to various concentrations of peptide PIII showed no obvious cell growth inhibition. Furthermore, a highly reproducible animal experimental model of gastric cancer with peritoneal dissemination was established in nude mice by injecting a suspension of the cell line into the gastric wall of nude mice. Animals intraperitoneally treated with peptide PIII in this model or another animal model of gastric cancer with peritoneal dissemination established using MKN45 cells showed suppressed tumor metastasis to peritoneum and significantly prolonged survival. In conclusion, the selected peptide PIII was a biologically active peptide and could effectively inhibit peritoneal dissemination of gastric cancer.

High frequency occurrence of 1‐OPRD variant of PRNP gene in gastric cancer cell lines and Chinese population with gastric cancer

The prion protein gene PRNP encodes PrPc and PrPsc, causing a number of neurological disorders. Approximately 10–15% of human prion disease is inherited and more than 20 pathogenic mutations have been found. Most of the genetic alterations are point mutations, with the exception of genetic insertions of one to nine extra octapeptide repeats occurring in the important octapeptide‐coding region. Our previous work showed that PrPc was overexpressed in gastric cancer. We wondered whether mutations of PrPc existed in human gastric cancer. DNA sequencing and gel electrophoresis were used to determine the possible mutation of PrPc in patients and cell lines of gastric cancer. We found that 1‐OPRD (one octapeptide‐repeat deletion) homozygosity or heterozygosity exists in several gastric cancer cell lines, e.g. MKN28 and KatoIII are homozygous for 1‐OPRD, and SGC7901 and BGC‐823 are heterozygous for 1‐OPRD. The mutation frequency in tissues of gastric cancer cases is significantly higher than that in the common population (p < 0.05). All positive cases in gastric cancer were found to be heterozygous for 1‐OPRD. Further study of the variant may be helpful in understanding the mechanisms of occurrence and development of clinical gastric carcinoma as well as the biology of the mysterious gene PRNP.

Hypoxia induced HIF-1 accumulation and VEGF expression in gastric epithelial mucosa cells: Involvement of ERK1/2 and PI3K/Akt

Hypoxia is a common environmental stress that influences signaling pathways and cell function, which through initiates intracellular signaling pathways and hence leads to the activation of the transcription factor hypoxia-inducible factor 1 (HIF-1). In this study, we initially confirm that hypoxia activates HIF-1α protein expression in a time-dependent manner with a maximum reached at 60 min in vitro and 4h in vivo in gastric mucosa epithelial cells. The expression of HIF-1α is correlated with the activation of HIF-1 DNA binding and transcriptional activity. Hypoxia does not affect HIF-1α mRNA transcription but regulates HIF-1α protein expression through a translation-dependent pathway to regulate protein synthesis. Hypoxia could induce phosphorylation of Akt, MAPK (ERK), and a target of p70S6K1. PI3K and MAPK inhibitor and LY294002 and U0126 could inhibit hypoxia-induced HIF-1 and VEGF expression. We also investigated the role of reactive oxygen species (ROS) involved in HIF-1 and VEGF expression. Exogenous addition of H2O2 was sufficient to activate Akt and ERK, scavengers of H2O2 significantly inhibited hypoxia-induced Akt and ERK, and subsequent HIF-1α expression and transcriptional activity. In conclusion, our data suggested that hypoxia-PI3K signaling through Akt and ERK kinases regulated ROS-dependent, hypoxia-induced HIF-1 activation and VEGF expression in gastric mucosa epithelial cells.

Function of PrPC (1-OPRD) in biological activities of gastric cancer cell lines.

Approximately 10–15% of the human prion disease is inherited and one of the important genetic mutations occurs in the octapeptide repeat region of prion protein gene. One of the variants, one octapeptide repeat deletion (1‐OPRD), existed in several gastric cancer cell lines and its mutation frequency was higher in gastric cancer cases. However, the biological functions of it remain unknown. Wild‐type and mutation forms of PrPC were cloned and transfected into gastric cancer cells. Cell apoptosis, adhesion, invasion, multidrug resistance (MDR) and proliferation were, respectively, investigated. Different expressed genes were screened by gene array and proved by PT‐PCR. Further, luciferase report assay was used to explore the transcriptional activation of target genes. Forced overexpression PrPC (1‐OPRD) could promote the gastric cancer cells SGC7901 growth through facilitating G1‐ to S‐phase transition in the cell cycle. PrPC (1‐OPRD) could also inhibit apoptosis, and promote adhesion, invasion and MDR in SGC7901. However, it exhibited no significant difference between wild‐type PrPC (1‐OPRD) and PrPC on apoptosis, invasion or MDR effects. Further experiments indicated that PrPC (1‐OPRD) could trigger the transactivation of cyclinD3 besides cyclinD1 to promote cell transition and proliferation. Overexpression of PrPC (1‐OPRD) might promote the proliferation of gastric cancer cells at least partially through transcriptional activation of cyclinD3 to accelerate the G1‐/S‐phase transition. The promoting proliferation effect of PrPC (1‐OPRD) was more than that of wild‐type PrPC. However, they showed no difference on apoptosis, adhesion, invasion or MDR effects of gastric cancer cells.

Forty-eight-week retrospective study of telbivudine and lamivudine treatment in patients with hepatitis B-related cirrhosis

The aim of this study was to evaluate the efficacy and safety of telbivudine 600 mg/day compared with lamivudine 100 mg/day for 48 weeks of treatment in patients with hepatitis B‐related cirrhosis. Data were reviewed retrospectively from 165 hepatitis B‐related cirrhotic patients (55 compensated patients and 110 decompensated) who received antiviral therapy with telbivudine or lamivudine. Serum alanine aminotransferase (ALT) and hepatitis B virus (HBV) DNA levels, hepatitis B e antigen (HBeAg) loss and seroconversion, histological improvement and various adverse events (AEs) were evaluated. Baseline characteristics were comparable. ALT levels declined but showed no significant difference in treatment with telbivudine or lamivudine (P > 0.05). Reduction in serum HBV DNA levels was evident by week 4 in compensated HBV‐related cirrhosis patients (telbivudine, 2.34 log10 copies/mL; lamivudine, 2.07 log10 copies/mL; P = 0.02) and persisted by week 8. Patients administrated with telbivudine had slightly greater HBeAg loss and seroconversion than patients with lamivudine, but the difference was not statistically significant (P > 0.05). Accumulative HBeAg loss was seen at week 48 (25.0% vs 25.0% and 13.3% vs 10.0% for telbivudine vs lamivudine in compensated and decompensated cirrhotic groups, respectively), as well as HBeAg seroconversion (15.0% vs 8.3% and 8.9% vs 6.7%). Mean Knodell Histologic Activity Index scores decreased in both compensated and decompensated cirrhotic patients (3.92 vs 3.64, 3.85 vs 3.73, for telbivudine vs lamivudine). Telbivudine and lamivudine were both well tolerated with minor AEs. The results of this study support telbivudine as an effective therapy for patients with both compensated and decompensated HBV‐related cirrhosis.