Myeloid Deletion of Cdc42 Protects Liver From Hepatic Ischemia-Reperfusion Injury via Inhibiting Macrophage-Mediated Inflammation in Mice.

BACKGROUND & AIMS: Hepatic ischemia-reperfusion injury (HIRI) often occurs in liver surgery, such as partial hepatectomy and liver transplantation, in which myeloid macrophage-mediated inflammation plays a critical role. Cell division cycle 42 (Cdc42) regulates cell migration, cytoskeleton rearrangement, and cell polarity. In this study, we explore the role of myeloid Cdc42 in HIRI. METHODS: Mouse HIRI models were established with 1-hour ischemia followed by 12-hour reperfusion in myeloid Cdc42 knockout (Cdc42mye) and Cdc42flox mice. Myeloid-derived macrophages were traced with RosamTmG fluorescent reporter under LyzCre-mediated excision. The experiments for serum or hepatic enzymic activities, histologic and immunologic analysis, gene expressions, flow cytometry analysis, and cytokine antibody array were performed. RESULTS: Myeloid deletion of Cdc42 significantly alleviated hepatic damages with the reduction of hepatic necrosis and inflammation, and reserved hepatic functions following HIRI in mice. Myeloid Cdc42 deficiency suppressed the infiltration of myeloid macrophages, reduced the secretion of proinflammatory cytokines, restrained M1 polarization, and promoted M2 polarization of myeloid macrophages in livers. In addition, inactivation of Cdc42 promoted M2 polarization via suppressing the phosphorylation of STAT1 and promoting phosphorylation of STAT3 and STAT6 in myeloid macrophages. Furthermore, pretreatment with Cdc42 inhibitor, ML141, also protected mice from hepatic ischemia-reperfusion injury. CONCLUSIONS: Inhibition or deletion of myeloid Cdc42 protects liver from HIRI via restraining the infiltration of myeloid macrophages, suppressing proinflammatory response, and promoting M2 polarization in macrophages.

Ischemic stroke in a patient with EGFR-mutated non-small-cell lung cancer after treatment with ramucirumab.

Vascular endothelial growth factor (VEGF) inhibitors have been widely investigated in the last 10 years, with particular attention paid to their adverse effects because of their efficacy in improving cancer patient survival. Previous research primarily focused on the monoclonal anti-vascular endothelial growth factor antibody bevacizumab and its adverse outcomes. Reports show a higher risk of ischemic stroke, one of the most concerning clinically relevant events, after treatment with bevacizumab. However, few studies have examined the relationship between anti-VEGF receptor 2 monoclonal antibody ramucirumab and its adverse events. This article presents the case of a non-small-cell lung cancer patient who experienced a new ischemic stroke after treatment with ramucirumab. The findings suggest that further studies may be necessary to investigate the relationship between ramucirumab and the risk of ischemic stroke.

Antimicrobial lanostane triterpenoids from the fruiting bodies of Ganoderma applanatum.

A chemical investigation on the 90% EtOH extract of the fruiting bodies of Ganoderma applanatum led to the isolation of three new lanostane triterpenoids, designated as 25-methoxy-11-oxo-ganoderiol D (1), 3-oxo-25-methoxy-24,26-dihydroxy-lanosta-7,9(11)-diene (2), and 3ß-acetyloxy-lucidone H (3). Structural elucidation of all the compounds were performed by spectral methods such as 1 D and 2 D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy. All the triterpenoids were in vitro evaluated for their antimicrobial activities against six pathogenic microorganisms. Compounds 1 and 2 exhibited some activities against three Gram positive bacteria with MIC values less than 60 µg/ml.

An improved MLVF method and its comparison with traditional MLVF, spa typing, MLST/SCCmec and PFGE for the typing of methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important nosocomial pathogen, causing considerable morbidity and mortality. During the last 20 years, a variety of genotyping methods have been introduced for screening the prevalence of MRSA. In this study, we developed and evaluated an improved approach capillary gel electrophoresis based multilocus variable-number tandem-repeat fingerprinting (CGE/MLVF) for rapid MRSA typing. A total of 42 well-characterized strains and 116 non-repetitive clinical MRSA isolates collected from six hospitals in northeast China between 2009 and 2010 were tested. The results obtained by CGE/MLVF against clinical isolates were compared with traditional MLVF, spa typing, Multilocus sequence typing/ staphylococcal cassette chromosome mec (MLST/SCCmec) and pulse field gel electrophoresis (PFGE). The discriminatory power estimated by Simpson's index of diversity was 0.855 (28 types), 0.855 (28 patterns), 0.623 (11 types), 0.517 (8 types) and 0.854 (28 patterns) for CGE/MLVF, traditional MLVF, spa typing, MLST/SCCmec and PFGE, respectively. All methods tested showed a satisfied concordance in clonal complex level calculated by adjusted Rand's coefficient. CGE/MLVF showed better reproducibility and accuracy than traditional MLVF and PFGE methods. In addition, the CGE/MLVF has potential to produce portable results. In conclusion, CGE/MLVF is a rapid and easy to use MRSA typing method with lower cost, good reproducibility and high discriminatory power for monitoring the outbreak and clonal spread of MRSA isolates.

Icariin potentiates the antitumor activity of gemcitabine in gallbladder cancer by suppressing NF-κB.

AIM: Gemcitabine has been increasingly prescribed for the treatment of gallbladder cancer. However, the response rate is low. The aim of this study is to determine whether icariin, a flavonoid isolated from Epimedi herba, could potentiate the antitumor activity of gemcitabine in gallbladder cancer. METHODS: Human gallbladder carcinoma cell lines GBC-SD and SGC-996 were tested. Cell proliferation and apoptosis were analyzed using MTT assay and flow cytometry, respectively. The expression of apoptosis- and proliferation-related molecules was detected with Western blotting. Caspase-3 activity was analyzed using colorimetric assay, and NF-κB activity was measured with ELISA. A gallbladder cancer xenograft model was established in female BALB/c (nu/nu) mice. The mice were intraperitoneally administered gemcitabine (125 mg/kg) in combination with icariin (40 mg/kg) for 2 weeks. RESULTS: Icariin (40-160 µg/mL) dose-dependently suppressed cell proliferation and induced apoptosis in both GBC-SD and SGC-996 cells, with SGC-996 cells being less sensitive to the drug. Icariin (40 µg/mL) significantly enhanced the antitumor activity of gemcitabine (0.5 µmol/L) in both GBC-SD and SGC-996 cells. The mice bearing gallbladder cancer xenograft treated with gemcitabine in combination with icariin exhibited significantly smaller tumor size than the mice treated with either drug alone. In GBC-SD cells, icariin significantly inhibited both the constitutive and gemcitabine-induced NF-κB activity, enhanced caspase-3 activity, induced G(0)-G(1) phase arrest, and suppressed the expression of Bcl-2, Bcl-xL and surviving proteins. CONCLUSION: Icariin, by suppressing NF-κB activity, exerts antitumor activity, and potentiates the antitumor activity of gemcitabine in gallbladder cancer. Combined administration of gemcitabine and icariin may offer a better therapeutic option for the patients with gallbladder cancer.

Surgical outcomes for gastric cancer of a single institute in southeast China.

BACKGROUND: In recent years, with social and economic development and lifestyle changes, the incidence of gastric cancer as well as the surgical results and prognoses of patients with gastric cancer have changed significantly in southeast China. METHODS: A total of 1,451 patients were divided into 2 groups according to admission time periods. Trends in clinicopathologic characteristics and operative outcomes of these patients were analyzed retrospectively. RESULTS: The numbers of old and young patients were significantly increased in period 2 compared with period 1. Tumors located in the proximal stomach increased from 20.26% to 36.83%. The incidence of early gastric cancer was significantly increased from period 1 to period 2. Lymph node metastasis was seen more prevalently in period 2 than in period 1. The rate of operation-related major complications decreased from 5.23% to 1.43%. Operative mortality was .49% in period 1 and .24% in period 2. The 5-year survival rate increased from 38.40% to 53.99%. CONCLUSIONS: Early diagnosis, standardized surgical treatment including pertinent lymph node dissection, and better perioperative care notably improve the outcomes of patients with gastric cancer.

[Effect of spleen tyrosine kinase expression re-activation by inhibition of DNA methylation on oncogenesis growth of gastric cancer].

OBJECTIVE: To explore the effects of 5-Aza-2'-deoxycytidine on spleen tyrosine kinase (Syk) expression by inhibition of DNA methylation and the effect of re-activation of Syk on oncogenesis of gastric cancer. METHODS: Syk mRNA of SGC7901, MGC803, MKN28 and MKN45 cell lines were analyzed by RT-PCR, and Syk methylation were detected by MSP. 5-aza-CDR was used to incubate with human gastric cancer cell line SGC7901, Methylation of Syk promoter region was detected by MSP and RT-PCR technique was used to detected Syk gene in the methylated and silenced Syk gene in the cell line SGC7901. Meanwhile, cell lines were inoculated into subcutaneous tissue of nude mice. RESULTS: No Syk mRNA were found in SGC7901 and MKN45 gastric cancer cell lines, but methylation of Syk were detected in those cell lines. No methylation of Syk promoter region was found and Syk gene was detected in the Syk-negative cell line SGC7901 after incubated with 5-aza-CDR. Of 10 nude mice which were inoculated SGC7901(Syk(+)), 3 were observed macroscopic tumor 8 weeks after the injection. On contrast, tumors were found in 10 nude mice which were inoculated SGC7901 (Syk(-)) 8 weeks after the injection, a significant difference was noted between the two groups (chi (2)=7.91, P<0.05). CONCLUSION: Syk gene is re-expressed in the cell line SGC7901 by demethylation with 5-aza-CDR. Syk gene re-expression suppress the malignant oncogenesis and growth of human gastric cancer.

CD44v6 in peripheral blood and bone marrow of patients with gastric cancer as micro-metastasis.

AIM: To detect the expression of CD44 correlated with the ability of micro-metastasis in peripheral blood and bone marrow of patients with gastric cancer and to deduce its clinical significance. METHODS: Preoperative peripheral blood and bone marrow specimens from 46 patients with gastric cancer and 6 controls were studied by semi-quantitative RT-PCR amplification of CD44v6mRNA. Preoperative and postoperative peripheral blood specimens from 40 patients with gastric cancer and 14 controls were studied by quantitative RT-PCR amplification of CD44v6mRNA in the corresponding period. RESULTS: Semi-quantitative RT-PCR amplification showed that CD44v6mRNA expression of peripheral blood and bone marrow was positive in 39 (84.8%) and 40 (86.9%) of 46 patients with gastric cancer, respectively. In peripheral blood, CD44v6mRNA expression was positive for diffuse type in 30 (93.8%) of 32 patients and for intestinal type in 9 (64.3%) of 14 patients. On the other hand, in bone marrow, CD44v6mRNA expression was positive for diffuse type in 31 (96.9%) of 32 patients and for intestinal type in 10 (71.4%) of 14 patients. There was a significant difference between the diffuse type and intestinal type. Quantitative RT-PCR amplification demonstrated that CD44v6mRNA was not expressed in the peripheral blood of controls and CD44v6mRNA expression was positive for preoperative peripheral blood in 40 patients with gastric cancer, the expression levels being from 4.9 x 10(8) - 3.2 x 10(11) copies/g RNA. The average expression level of CD44v6mRNA in peripheral blood was 3.9 x 10(10) copies/g RNA. The expression levels of CD44v6mRNA in peripheral blood in gastric cancer patients after curative operation increased from 5.5 x 10(6) to 7.6 x 10(9) copies/g RNA and the average level was 2.4 x 10(8) copies/g RNA (Figure 3B) (P = 0.00496). After curative operation, the expression level decreased markedly. CONCLUSION: Semi-quantitative and quantitative RT-PCR amplification for CD44v6mRNA is a sensitive and specific method for the detection of micro-metastasis in peripheral blood and bone marrow, which might be used as an indicator of tumor burden and therapeutic effect.

Mutation in D-loop region of mitochondrial DNA in gastric cancer and its significance.

AIM: To investigate the mutation in D-loop region of mitochondrial DNA in gastric cancer and its influence on the changes of reactive oxygen species (ROS) and cell cycle. METHODS: The D-loop region was amplified by PCR and sequenced. Reactive oxygen species and cell cycle were detected by flow cytometry in 20 specimens from gastric cancer and adjacent normal tissues. According to the sequence results, gastric cancer tissue was divided into mutation group and control group. Reactive oxygen species, apoptosis and proliferation in the two groups were compared. RESULTS: Among the 20 gastric cancer specimens, 18 mutations were identified in 7 patients, the mutation rate being 35%. There were four microsatellite instabilities in the mutations. No mutation was found in the adjacent tissues. Reactive oxygen species, apoptosis, and proliferation in the mutation group were all significantly higher than those in control group. CONCLUSION: Mutation in D-loop region plays a role in the genesis and development of gastric cancer.

Detection of micrometastasis in peripheral blood by multi-sampling in patients with colorectal cancer.

AIM: To evaluate the reverse transcriptase-PCR assay and multiple sampling for detection of cytokeratin-positive cells in peripheral blood of colorectal carcinoma patients and to investigate the clinical significance of micrometastasis in peripheral blood. METHODS: The expression of CK20 mRNA by RT-PCR was investigated in bone marrow, portal vein and peripheral blood in 58 colorectal cancer patients and 12 controls without known cancer. The peripheral blood was sampled twice at intervals of 3 d before operation. All the patients were followed up for one year. RESULTS: There was no positive expression of CK20mRNA in 12 volunteers. The positive expression of CK20mRNA was 77.6% (45/58) in bone marrow, and that in portal vein was 74.1% (43/58) of colorectal carcinoma patients. The positive expression of CK20mRNA cells in peripheral blood rose from 44.8% (26/58) to 69.0% (40/58) (P<0.01). The total positivity of CK20mRNA expression in peripheral blood was similar to the positivity of CK20mRNA in bone marrow and portal vein. The positive rates became higher in later clinical stages than in early stages. The CK20mRNA positive patients had a higher relapse rate within one year than the CK20mRNA negative patients. CONCLUSION: Multiple blood sampling can increase the detection of tumor cells in peripheral blood by RT-PCR for CK20mRNA in colorectal carcinoma patients and it is as sensitive and specific as that of bone marrow and portal vein. This technique may be reliable and convenient to diagnose micrometastasis of colorectal carcinoma and has an important significance in determining the prognosis of cancer patients.

Hypermethylation of Syk gene in promoter region associated with oncogenesis and metastasis of gastric carcinoma.

AIM: To investigate the relationship between methylation of Syk (spleen tyrosine kinase) gene in promoter region and oncogenesis, metastasis of gastric carcinoma. The relation between silencing of the Syk gene and methylation of Syk promoter region was also studied. METHODS: By using methylation-specific PCR (MSP) technique, the methylation of Syk promoter region in specimens from 61 gastric cancer patients (tumor tissues and adjacent normal tissues) was detected. Meanwhile, RT-PCR was used to analyse syk expression exclusively. RESULTS: The expression of the Syk gene was detected in all normal gastric tissues. Syk expression in gastric carcinoma was lower in 14 out of 61 gastric cancer samples than in adjacent normal tissues (chi(2)=72.3, P<0.05). No methylation of Syk promoter was found in adjacent normal tissues. hypermethylation of Syk gene in promoter was detected 21 cases in 61 gastric carcinoma patients. The rate of methylation of Syk promoter in gastric carcinoma was higher than that in adjacent normal tissues (chi(2)=25.1, P<0.05). In 31 patients with lymph node metastasis, 17 were found with Syk promoter methylation. A significant difference was noted between two groups (chi(2)=11.4,P<0.05). CONCLUSION: Hypermethylation leads to silencing of the Syk gene in human gastric carcinoma. Methylation of Syk promoter is correlated to oncogenesis and metastasis of gastric carcinoma. Syk is considered to be a potential tumor suppressor and anti-metastasis gene in human gastric cancer.

Detection of micrometastasis of gastric carcinoma in peripheral blood circulation.

AIM: To detect the micrometastasis of gastric carcinoma in peripheral blood circulation using immunomagnetic beads sorting technique and RT-PCR technique, and to discuss its significance and the difference between the two methods. METHODS: Density gradient centrifugation was used to isolate mononuclear cells from peripheral blood, immunomagnetic beads sorting technique and RT-PCR technique were used to detect the disseminated carcinoma cells. HE, immunocytochemical and immunofluorescence staining were also used to identify the characteristics of the cells separated with immunomagnetic beads sorting technique. RESULTS: Cells expressing cytokeratin were separated and enriched from the peripheral blood specimens of patients suffering from gastric carcinoma or chronic gastritis. After HE staining, two kinds of cells with little cytoplasm were found. Majority of these cells had small and round nuclei, even chromatins and the thickness of nuclear membrane was normal. Immunohistochemical staining indicated that there were CD34 and CD45 expression on the cell membrane of this kind of cells and these cells also showed expressed human telomerase reverse transcriptase by immunofluorescence staining, but the expression of carcinoembryonic antigen was absent. So, these cells might hematopoiesis precursors. Another kind of cells had larger and abnormal nuclei with thicker nuclear membranes. Massed chromatins and poly-nucleoli were found in the nuclei. These cells expressed human telomerase reverse transcriptase and carcinoembryonic antigen, but CD34 and CD45 were not found on the cell membrane. So, these cells were considered as gastric carcinoma cells escaping from the original focuses and existing in the peripheral blood circulation. Carcinoma cells were found in 25 of 60(41.7%) specimens of peripheral blood from patients with gastric carcinoma, while there were no such cells separated from the blood specimens of chronic gastritis patients. The difference of positive rates of disseminated carcinoma cells between two groups was markedly significant (P<0.005). The expressions of CK20 mRNA in peripheral blood specimens were examinated with RT-PCR. CK20 mRNA was detected from 32 of 60(53.3%) peripheral blood specimens in the group of gastric carcinoma patients, while none of the specimens from patients suffering from chronic gastritis had CK20 mRNA. Significant difference was also found between two groups (P<0.005). Statistic analyses also showed that there was a significant difference between the positive rates of two methods in detecting the disseminated carcinoma cells from the peripheral blood circulation of gastric carcinoma patients (P<0.05). CONCLUSION: The results demonstrated that there were disseminated carcinoma cells in the peripheral blood circulation of some patients with gastric carcinoma. Disseminated carcinoma cells can be detected from the peripheral blood samples with immunomagnetic beads sorting technique and RT-PCR technique. The positive rate of RT-PCR technique is higher than that of immunomagnetic beads sorting technique in detecting micrometastasis.

Correlation of tumor-positive ratio and number of perigastric lymph nodes with prognosis of patients with surgically-removed gastric carcinoma.

AIM: To evaluate the tumor-positive ratio and number of perigastric lymph nodes as prognostic factors of gastric carcinoma in surgically-treated patients. METHODS: The postoperative survival of 169 patients with gastric cancer who were performed D2 curative gastrectomy was analyzed with regard to its lymph node metastasis ratio and number. Meanwhile correlation of tumor-positive ratio and number of perigastric lymph nodes with pathological parameters of these patients was studied. RESULTS: The overall 5-year survival rate of all the patients studied was 29.6%. The 5-year cumulative survival rate in patients with 1%-20% and more than 20% of tumor-positive lymph nodes was 70.6% and 12.0% respectively, and 46.6% and 17.4% in those with 1-5 and more than 5 of tumor-positive lymph nodes respectively, which were significantly decreased with the increment of involved lymph nodes assessed by either numbers or ratio (P<0.05). Multiple stepwise regression analysis showed that both the positive ratio and number of tumor-involved lymph nodes were sensitive prognostic factors in these surgically-treated patients, which were also significantly correlated with tumor size and depth of submucosal invasion (P<0.05). CONCLUSION: Tumor-positive ratio and number of perigastric lymph nodes are associated with cancer progression and five-year survival rate, and may serve as valuable prognostic factors of gastric cancer in surgically-treated patients.

Association of VCAM-1 overexpression with oncogenesis, tumor angiogenesis and metastasis of gastric carcinoma.

AIM: To investigate the relationship between the expression of vascular cell adhesion molecule-1 (VCAM-1) and oncogenesis, tumor angiogenesis and metastasis in gastric carcinoma, and to evaluate the clinical significance of serum VCAM-1 levels in gastric cancer. METHODS: Specimens from 41 patients with gastric cancer, 8 patients with benign gastric ulcer, and 10 healthy subjects were detected for the expression of VCAM-1 by immunohistochemistry. Microvessel density (MVD) was measured by counting the endothelial cells immunostained with the monoclonal antibody CD34 at x200 magnification. Serum VCAM-1 concentrations were measured by an enzyme linked immunosorbent assay in the 41 gastric cancer patients before surgery, and at 7 days after surgery as well as in 25 healthy controls. The association between preoperative serum VCAM-1 levels and clinicopathological features, and their changes following surgery was evaluated. In addition, serum carcinoembryonic antigen (CEA) was also examined. RESULTS: Of the 41 gastric cancer tissues, 31 (75.6 %) were VCAM-1 positive. The VCAM-1 positive gastric cancers were more invasive and classified in the more advanced stage than the VCAM-1 negative ones. The VCAM-1 positive cancers were associated with more lymph node metastases than VCAM-1-negative ones (P<0.05). The expression of VCAM-1 was detected in tissues of two of the eight patients with gastric ulcer and two of the 10 healthy controls. The expression of VCAM-1 in gastric cancer patients was significantly more frequent than that in the healthy controls and ulcer group (both P<0.05). MVD in VCAM-1 expressing tissues was higher than that in VCAM-1 negative tissues (t=2.13,P<0.05). Serum VCAM-1 levels in gastric cancer patients were significantly higher than those in controls (t=3.4, P<0.05). There was a significant association between serum VCAM-1 levels and disease stage, as well as invasion depth of the tumor and the presence of distant metastases. The concentrations of serum CEA in gastric cancer were higher than normal controls. Both serum VCAM-1 and CEA levels decreased significantly after radical resection of the primary tumor (P<0.05). Furthermore, the serum levels of VCAM-1 were positively correlated with the expression of VCAM-1 in the tumor tissue (r=0.85, P<0.05). CONCLUSION: The expression of VCAM-1 is closely related to oncogenesis, tumor angiogenesis and metastasis in gastric carcinoma. Serum VCAM-1 level in gastric cancer patients is significantly increased compared with normal controls, which decreases significantly after radical resection of the primary tumor. The serum concentration of VCAM-1 may be considered as an effective marker of tumor burden of gastric cancer. Moreover, overexpression of VCAM-1 in gastric cancer tissue is likely a major source of serum VCAM-1.

[Significance of detecting disseminated tumor cells in peripheral blood of gastric and colorectal cancer patients].

OBJECTIVE: To evaluate the clinical significance of CK20 mRNA expression in detecting disseminated tumor cells in peripheral blood of gastric and colorectal cancer patients. METHODS: Expression of CK20 mRNA was investigated by RT-PCR in bone marrow, portal vein and peripheral blood in 47 gastric, 58 colorectal cancer patients and 6 non-cancer volunteers. All the patients were followed-up for one year. RESULTS: There was no positive expression of CK20 mRNA in 6 non-cancer volunteers. The positive rates of CK20 mRNA in bone marrow, portal vein were 87.2% (41/47) and 85.1% (40/47) in gastric cancer, and were 77.6% (45/58) and 74.1% (43/58) in colorectal cancer. The positive rates of CK20 mRNA in peripheral blood in gastric and colorectal cancer patients were 42.6% (20/47) and 44.8% (26/58) by one single test, and were 74.5% (35/47) and 69.0% (40/58) by two tests. The overall positive rate of CK20 mRNA in peripheral blood (two tests) was similar to that in bone marrow and portal vein. The overall positive rate of CK20 mRNA in peripheral blood was higher in two tests than in one single test (P < 0.05) and in advanced than early lesions. The relapse rate within one year was higher in CK20 mRNA positive patients than the negative ones (P < 0.05). CONCLUSION: Detection of cancer cells by RT-PCR for CK20 mRNA in peripheral blood, being as sensitive and specific as in bone marrow and portal vein, is reliable and convenient in diagnosing micrometastasis of gastric and colorectal cancer, which possesses clinical significance in assessing the prognosis and scheme of therapy.