Treatment of Staphylococcus aureus-induced chronic osteomyelitis with bone-like hydroxyapatite/poly amino acid loaded with rifapentine microspheres.

PURPOSE: The purpose of this study was to investigate the curative effect of bone-like hydroxyapatite/poly amino acid (BHA/PAA) as a carrier for poly(lactic-co-glycolic acid)-coated rifapentine microsphere (RPM) in the treatment of rabbit chronic osteomyelitis induced by Staphylococcus aureus. METHODS: RPM was prepared through an oil-in-water emulsion solvent evaporation method, and RPM was combined with BHA/PAA to obtain drug-loaded, slow-releasing materials. Twenty-six New Zealand white rabbits were induced to establish the animal model of chronic osteomyelitis. After debridement, the animals were randomly divided into three groups (n=8): the experimental group (with RPM-loaded BHA/PAA), the control group (with BHA/PAA), and the blank group. The RPM-loaded BHA/PAA was evaluated for antibacterial activity, dynamics of drug release, and osteogenic ability through in vitro and in vivo experiments. RESULTS: In vitro, RPM-loaded BHA/PAA released the antibiotics slowly, inhibiting the bacterial growth of S. aureus for up to 5 weeks. In vivo, at week 4, the bacterial colony count was significantly lower in the experimental group than in the control and blank groups (P<0.01). At week 12, the chronic osteomyelitis was cured and the bone defect was repaired in the experimental group, whereas the infection and bone defect persisted in the control and blank groups. CONCLUSION: In vitro and in vivo experiments demonstrated that RPM-loaded BHA/PAA effectively cured S. aureus-induced chronic osteomyelitis. Therefore, BHA/PAA has potential value as a slow-releasing material in clinical setting. Further investigation is needed to determine the optimal dosage for loading rifapentine.

P115 promotes growth of gastric cancer through interaction with macrophage migration inhibitory factor.

AIM: To investigate the role of P115 in the proliferation of gastric cancer cells and the mechanism involved. METHODS: The RNA and protein level of P115 and macrophage migration inhibitory factor (MIF) in gastric cancer and normal gastric tissue/cells were measured and the effect of P115 on cell proliferation was assessed. The role of P115 in cell cycle checkpoints was investigated and the related proteins and signaling pathways, such as cyclin D1, Mcm2, p53, PCNA as well as the MAPK signaling pathway were determined. The interaction between P115 and MIF and the effect of P115 on MIF secretion were examined. The data were analyzed via one-way ANOVA comparisons between groups and P < 0.05 was considered significant. RESULTS: P115 and MIF were both specifically expressed in gastric cancer tissues compared with normal gastric mucosa (both P < 0.01). The mRNA and protein levels of P115 and MIF in gastric cancer cell lines MKN-28 and BGC-823 were higher than in the human gastric epithelial cell line GES-1 (both P < 0.01). In MKN-28 and BGC-823 cell lines, P115 promoted cell proliferation and G0-G1 to S phase transition. In addition, several cell cycle-related regulators, including cyclin D1, Mcm2, PCNA, pERK1/2 and p53 were up-regulated by P115. Furthermore, the interaction between P115 and MIF was confirmed by co-immunoprecipitation assay. ELISA showed that P115 stimulated the secretion of MIF into the culture supernatant (P < 0.01) and the compensative expression of MIF in cells was observed by Western blotting. CONCLUSION: P115 promotes proliferation of gastric cancer cells through an interaction with MIF.

[Comparison of the Golgi proteome of hepatocellular carcinoma with that of the adjacent non-tumor tissues].

OBJECTIVE: To compare the Golgi proteome of hepatocellular carcinoma (HCC) with that of the adjacent non-tumor tissues. METHODS: Hepatocellular carcinoma and adjacent non-tumor tissues were obtained from HCC patients. The protein expression maps in Golgi were obtained by two-dimensional gel electrophoresis (2-DE), and the differentially expressed protein spots were analyzed by PD-Quest software. Peptide mass fingerprint (PMF) of differential protein spots was obtained with MALD-TOT-MS. RESULTS: According to 2-DE maps, the average numbers of protein spots were (1153+/-49) and (1086+/-37) in hepatocellular carcinoma and the adjacent non-tumor tissues. Compared to the adjacent non-tumor tissues, 27 proteins were upregulated, and 20 proteins were downregulated in HCC Golgi. CONCLUSIONS: The Golgi proteome in HCC tissues is different from that in the adjacent non-tumor tissues, and the differential expression proteins are involved in energy metabolism, tumor metastasis, and cell cycle regulation.

[Construction of a eukaryotic vector expressing MASPIN gene and its effect on cell apoptosis in gastric cancer cell line SGC7901].

BACKGROUND & OBJECTIVE: MASPIN gene is closely associated with carcinogengsis and plays a key role in cell proliferation, adhesion, migration and apoptosis. This study was to construct a recombinant eukaryotic vector expressing MASPIN, and explore the effect of MASPIN overexpression on the apoptosis in human gastric carcinoma cell line SGC7901. METHODS: A eukaryotic expression vector MASIPIN/PCR2.1 was constructed and transfected into SGC7901 cells. RT-PCR and Western blot were used to detect the expression changes of MASPIN and Bax/Bcl-2. TNF-related apoptosis inducing ligand (TRAIL) (50ng/ml) was used to induce apoptosis at different time courses. DNA apoptotic ladders were determined using agarose gel electrophoresis (AGE). Cell apoptosis was measured by flow cytometry (FCM). RESULTS: Recombinant plasmid MASIPIN/PCR2.1 was successfully constructed and transfected into SGC7901 cells. The mRNA and protein levels of MASPIN were significantly higher in the MASPIN/PCR2.1 group (33.6+/-1.2, 23.4+/-1.6) than in the PCR2.1(15.0+/-1.5, 12.3+/-1.5)and the untreated group (13.7+/-2.0, 12.0+/-1.3) (P<0.05). After transfection of MASIPIN/PCR2.1, DNA apoptotic ladders appeared in SGC7901 cells and the induction of apoptosis was in a time-dependent manner. The apoptosis rates were 8.0%, 16.3% and 25.8%in the MASPIN/PCR2.1 plus TRAIL group, 3.0%, 8.2%, 14.4% in the MASPIN/PCR2.1 group, and 4.1%, 9.8%,15.9% in the TRAIL group at 12, 24, and 48 h(P<0.05). The expression levels of Bax mRNA and protein at 48 h after MASIPIN/PCR2.1 transfection were significantly higher in MASPIN/PCR2.1 plus TRAIL group(55.3+/-2.1, 75.4+/-1.3) than in the PCR2.1 group (34.3+/-1.2, 40.7+/-1.8) and the TRAIL group (43.2 +/-1.8,36.2+/-1.3)(P<0.05). The expression of Bcl-2 mRNA in the MASPIN/PCR2.1 plus TRAIL group, PCR2.1 group and TRAIL group were 28.3+/-2.5, 34.3+/-1.2, 32.8+/-2.1, respectively (P<0.05), and those of Bcl-2 protein were 17.4+/-1.5, 45.1+/-2.1, 42.8+/-1.5 in the three groups, respectively (P<0.05). CONCLUSIONS: upregulation of MASPIN/PCR2.1 can significantly enhance the sensibility of gastric cancer cell line SGC7901 to the apoptosis inducer. This maybe related to the upregulation of Bax and downregulation of Bcl-2.

Expression of mucins and E-cadherin in gastric carcinoma and their clinical significance.

AIM: To investigate the expression of three types of mucin (MUC1, MUC2, MUC5AC) and E-cadherin in human gastric carcinomas and their clinical significance. METHODS: Ninety-four gastric cancer specimens were classified according to WHO criteria and detected by immunohistochemical assay of expression of mucins and E-cadherin. RESULTS: The positive expression rates of MUC1, MUC2, MUC5AC and E-cadherin were 82% (77/94), 84% (79/94), 40% (38/94) and 56% (53/94) respectively. MUC1 expression was significantly correlated with the types of cancer (the positive rates of MUC1 in well and moderately differentiated tubular adenocarcinoma, poorly differentiated adenocarcinoma, signet-ring cell carcinoma and mucinous carcinoma were 91%, 87%, 71%, 71%, respectively, P<0.05), age of patients (the positive rates of it among the people who are younger than 40 years, between 40-60 years and over 60 year were 74%, 81%, 89%, P<0.05), lymph nodes involvement (the positive rates in the non-interfered group and the interfered group were 78%, 85%, P<0.05) and tumor size (the positive rates in the tumors with the size less than 3 cm, 3-6 cm and larger than 6 cm were 69%, 92%, 69%, P<0.05); MUC2 expression was significantly associated with types of cancers and had the strongest expression in mucinous carcinomas(the positive rates of MUC2 in well and moderately differentiated tubular adenocarcinoma, poorly differentiated adenocarcinoma, signet-ring cell carcinoma and mucinous carcinoma were 94%, 70%, 81%, 100%, P<0.05), but it had no obvious relation to age, gender, tumor location, lymph nodes involvement, depth of invasion and metastasis to extra-gastric organs (P>0.05); MUC5AC expression was not related to any of the characteristics investigated except that it had relation to gender, whereas MUC5AC showed the tendency to higher expression in less invasive lesions and lower expression in advanced stage cancers (P>0.05); No significant difference was found for E-cadherin expression. There were strong positive relationships between the expression of MUC1 and E-cadherin, MUC2 and E-cadherin, MUC1 and MUC2 (R = 0.33, R = 0.22, R = 0.32, respectively, P<0.05). According to the COX proportional hazards model, older patients, involvement of lymph nodes, different types of gastric cancer and MUC2 expression were significantly associated with poorer outcome of gastric carcinoma patients (beta = 0.08, beta = 3.94, beta = 1.33, beta = 0.75, respectively, P<0.05). CONCLUSION: MUC1 and MUC2 are good markers of different types of gastric cancer. MUC2 is especially a good marker of mucinous carcinoma. MUC1, MUC2 may interfere with the function of E-cadherin in gastric carcinomas, and have synergic effect on progression of gastric cancers.

[Inhibitory effect of MUC2 antisense oligodeoxynucleotide with lipofectin on human gastric cancer cell proliferation].

BACKGROUND & OBJECTIVE: Our previous study showed that the expression of MUC2 protein was related with the biological behavior of gastric carcinoma. The aim of the present study was to investigate the inhibitory effect in vitro of mucin gene MUC2 antisense oligodeoxynucleotide (ASODN) on its gene expression and cell proliferation on gastric cancer cells SGC7901. METHODS: Phosphorothioate MUC2 ASODN was synthesized and transfected to SGC7901 cells mediated by lipofectin. Its inhibitory effects on cell proliferation was determined by MTT method, light and electron microscopy and immunohistochemical method. RESULTS: The determination by MTT method demonstrated that MUC2 ASODN of varied concentration significantly inhibited the growth of SGC7901 cells while the control lipofectin and control N-ODN showed no such effect. The inhibitory effect was dose-dependent and time-dependent. The inhibition peaked at 48th hour after transfection, and the inhibition rate reached 55% when the MUC2 ASODN concentration was 0.5 micromol/L. After transfecting with MUC2 ASODN, SGC7901 cells showed decrease in number, volume, and karyokinesis, and increase in necroses under light microscopy. Mitochondrion swelling, increased liposomes, myelin figures, chromatin margination were found under electron microscopy. And the test by immunohistochemical method indicated that transfected MUC2 ASODN downregulated the expression levels of MUC2 protein, but upregulated the expression levels of p16 protein. CONCLUSION: MUC2 ASODN transfection could specifically inhibit SGC7901 cells proliferation.

[Expression and significance of MRP, GST-pi, Topo IIalpha, and LRP in gastric carcinoma].

BACKGROUND & OBJECTIVE: Multidrug resistance-associated protein (MRP), glutathione-S-transferase-pi(GST-pi), topoisomerase IIalpha(Topo IIalpha)and lung resistance protein (LRP) play important roles in the multidrug resistance(MDR)of tumor chemotherapy. There were few reports on combined determination of the expression of MRP, GST-pi, Topo IIalpha and LRP in gastric carcinoma. This study was designed to investigate the expression and significance of MRP, GST-pi, Topo IIalpha and LRP in gastric carcinomas. METHODS: Immunohistochemistry SP method was used to determine the expression of MRP, GST-pi, Topo IIalpha, and LRP in 90 tumor samples from the patients with gastric carcinoma. Chi-square test and Fisher exact test were used to analyze the significance of the expression. RESULTS: (1)The positive expression rates of MRP, GST-pi, Topo IIalpha and LRP in gastric carcinoma were 88.9%, 91.1%, 74.4%, and 87.7%, respectively. They were all significantly higher than those in normal stomach tissues (P< 0.05). (2)The expression levels of MRP, GST-pi, and LRP in well-moderated differentiation adenocarcinoma were significantly higher than those in poor differentiation adenocarcinoma. The expression of Topo IIin well-moderated differentiation adenocarcinoma was significantly lower than that in poor differentiation adenocarcinoma. There was no difference between the expression levels of them in different degree of invasion or with lymph nodes metastasis and without lymph nodes metastasis (P > 0.05). (3) There was no correlation in any two items among the expression levels of MRP,GST-pi, topo IIalpha,and LRP. CONCLUSION: MRP, GST-pi, topo IIalpha,and LRP play important roles in the primary MDR of gastric carcinoma. The expression of them are associated with the differentiation, but are not associated with the invasion degree and lymph node metastasis.

Arylsulfatase, betagalactosidase and lysozyme in gastric cancer cells and its relationship to invasion.

AIM:To study the distribution of arylsulfatase,beta-galactosidase and lysozyme in gastric cancer cells, and its relationship to differentiation and invasion of gastric cancer cells.METHODS: Histochemical, immunohistochemical and ruthenium red (RR) electrocytochemical technique for three types of hydrolases and proteoglycans in pericancerous matrix in 33 cases of gastric cancer were observed under light and electron microscopy.RESULTS:The expression intensities of arylsulfatase,beta-glactosidase and lysozyme in mucinous cell carcinomas were more intensive than those in well-differentiated and poorly-differentiated adenocar-cinomas (P < 0.05-0.01). The fibrous tissues smooth muscle and proteoglycans close to the cancer cells were degraded. They were found in the region far from the cancer cells. Expression of three enzymes mentioned above was low in adenocarcinoma cells, and fibrous tissues and RR granules were present and intact near the well-differentiated and poorly differentiated adenocarcinoma cells.CONCLUSION: Mucinous cell carcinoma may release various hydrolases into extra-cellular matrix, inducing degradation of pericancerous matrix and facilitating cancer cell invasion and metastasis.