The Effect of Ras Homolog C/Rho-Associated Coiled-Protein Kinase (Rho/ROCK) Signaling Pathways on Proliferation and Apoptosis of Human Myeloma Cells.

BACKGROUND The aim of this study was to explore the impact of Ras homolog C/Rho-associated coiled-protein kinase (Rho/ROCK) signaling pathways intervention on biological characteristics of the human multiple myeloma cell lines RPMI-8226 and U266 cells, and to investigate the expression of RhoC, ROCK1, and ROCK2 in RPMI-8226 and U266 cells. MATERIAL AND METHODS RPMI8226 and U266 cell lines were treated by 5-aza-2-deoxycytidine (5-Aza-Dc), trichostatin A (TSA), RhoA inhibitor CCG-1423, Rac1 inhibitor NSC23766, and ROCK inhibitor fasudil. Cell proliferation was examined by Cell Counting Kit-8 (CCK-8) assay and clone formation. Cell apoptosis was examined by flow cytometry and TUNEL assay. The mRNA and protein expressions of RhoC, ROCK1, and ROCK2 were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot, respectively. RESULTS CCG-1423, NSC23766, and fasudil could significantly inhibit the proliferation of RPMI8226 and U266 cells. The inhibitory effect was dose- and time-dependent within a certain concentration range (P<0.05). After treatment with CCG-1423, NSC23766, and fasudil for 24 hours, the apoptosis rates of RPMI8226 and U266 cells were significantly higher than those of the control group, which were dose-dependent (P<0.05). Compared with the control group, the mRNA and protein expressions of RhoC, ROCK1, and ROCK2 in RPMI8226 and U266 cells were significantly decreased with single 5-Aza-Dc or TSA treatment. However, the effects were obviously stronger after combined treatment of 5-Aza-CdR and TSA (P<0.05). CONCLUSIONS We found that 5-Aza-Dc and TSA can effectively decrease the mRNA and protein expressions of RhoC, ROCK1, and ROCK2. Furthermore, Rho and ROCK inhibitors significantly inhibit cell growth and induce cell apoptosis in the human multiple myeloma cell lines RPMI-8226 and U266.

Vertebral artery dissection associated with viral meningitis.

BACKGROUND: Vertebral artery dissection (VAD) is often associated with trauma or occurs spontaneously, inevitably causing some neurological deficits. Even though acute infection can be related to the development of spontaneous VAD (sVAD), VAD associated with viral meningitis has never been reported in the literature. CASE PRESENTATION: A 42-year-old man with fever, sore throat, and runny nose developed sudden onset of occipital headache, vertigo, transient confusion, diplopia, and ataxia. Brain stem encephalitis was diagnosed initially because the cerebrospinal fluid (CSF) study showed inflammatory changes. However, subsequent diffusion-weighted (DWI) magnetic resonance imaging of his brain demonstrated left lateral medullary infarction, and the digital subtraction angiography (DSA) confirmed VAD involving left V4 segment of the artery. Consequently, the patient was diagnosed as VAD accompanied by viral meningitis. CONCLUSION: This case suggests that viral meningitis might lead to inflammatory injury of the vertebral arterial wall, even sVAD with multiple neurological symptoms.

[High Risk Factors for Transformation into Acute myeloid Leukemia in Patients with Intermediate and High Risk Myelodysplastic syndrome].

OBJECTIVE: To study the high risk factors for the transformation into acute myeloid leukemia(AML) in patients with intermediate and high risk myelodysplastic syndrome(MDS) treated by decitabine-based regimen. METHODS: The clinical characterstics of 60 intermediate and high risk MDS patients and the factors of its transformed into AML were retrospectively analyzed. RESULTS: The overall response rate(ORR) of the patients suffered from intermediate and high risk MDS treated by decitabine-based regimen was 65.0％(39/60), among the 60 cases 17 achieved complete remission(CR), 5 achieved morrow complete remission(mCR), 4 achieved partial remission(PR) and 13 achieved hematologic improvement(HI). Twenty-one cases(35.0%) were transformed into AML among 60 cases of intermediate and high risk MDS treated by decitabine-based regimen. The median time of transformation from intermediate and high risk MDS into AML was 10.0 months(1.6-32.0). χ2 or Fisher's exact test showed that 2016 WHO MDS diagnostic subgrouping, myeloid hyperplasia markedly active, delayed interval of decitabine-based treatment associated with the transformation from intermediate to high risk MDS into AML (χ2=9.878，P=0.031；χ2=4.319，P=0.038；χ2=6406，P=0.011); Univariate analysis of Kaplan-Meier test showed that 2016 WHO MDS diagnostic subgroups, bone marrow blast cell ratio, bone marrow dysplasia coefficients, prolonged interval of decitabine-based treatment associated with the transformation from intermediate and high risk MDS into AML (P=0.015，P=0.008，P=0.012，P=0.032); multivariate analysis showed the bone marrow blast cell ratio and the bone marrow dysplasia coefficients were independent risk factors for the transformation from intermediate to high risk MDS into AML (P=0.022，P=0.018). CONCLUSION: The bone marrow blast cell ratio and the bone marrow dysplasia coefficients are independent risk factors of transformation into AML in the patients with intermediate and high risk MDS treated by decitabine-based regimen. The regular interval of dicitabine treatment is beneficial to maintain the stability of patients conditions.

Decitabine: An effective and safe treatment for myelodysplastic syndrome and acute myeloid leukemia.

OBJECTIVE: Decitabine is reported to be valuable in treating multiple malignant blood diseases. However, the application of decitabine in myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML) has not been fully examined. Thus, our study aimed to investigate the clinical efficacy and safety of decitabine in treating such patients. MATERIALS AND METHODS: Clinical data of MDS or AML patients treated with decitabine were retrospectively analyzed. All the patients were regularly followed up, and the risk factors affecting clinical efficacy were also detected. RESULTS: A total of 36 patients (MDS, n = 27; AML, n = 9) were included in the study. The response rate of MDS patients was 55%, and there were three cases (15%) of complete remission (CR), three cases (15%) of marrow CR, and five cases (15%) of hematologic improvement. It was about three cycles to achieve the best efficiencies. Gender, age, percentage of blasts in bone marrow, International Prognostic Scoring System risk group, and cytogenetic factors were not associated with response rate. The median overall survival of MDS patients was 8 (1-44) months. Agranulocytosis (P = 0.037) and severe anemia (P = 0.044) were the independent factors for prognosis. The complete response rate of AML was 33.3%. From the investigation, infection was the most common complication in our cohort, especially lung infection with the incidence of 27.8%. CONCLUSIONS: Our data demonstrated that decitabine was effective and relatively safe in treating MDS and AML. Patients with agranulocytosis and severe anemia were prone to have poor survival, which should be monitored in clinical practice.

Effect of 5-aza-2'-deoxycytidine combined with trichostatin A on RPMI-8226 cell proliferation, apoptosis and DLC-1 gene expression.

This study was aimed to investigate the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA) on DLC-1 gene transcription regulation and molecular biological behaviours in the human multiple myeloma RPMI-8226 cells. The cells were treated respectively with 5-Aza-CdR and TSA alone, or the both combination; the cell proliferation and apoptosis, DLC-1 expression, the protein expression of Ras homolog family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) were examined by CCK-8 method, RT-PCR and ELISA, respectively. The results showed that the 5-Aza-CdR and TSA had cell growth inhibitory and apoptosis-inducing effects in dose-dependent manner (P < 0.05). Compared with a single drug (5-Aza-CdR or TSA alone), the effects were significantly enhanced after treatment with the combination of 5-Aza-CdR and TSA (P < 0.05). DLC-1 was weakly expressed in the control group; the treatment with 5-Aza-CdR alone enhanced its re-expression dose-dependently (P < 0.05). Compared with 5-Aza-CdR alone, 5-Aza-CdR plus TSA enhanced DLC-1 re-expression significantly.Compared with the control, 5-Aza-CdR and TSA significantly decreased RhoA and Rac1 protein expression (P < 0.05). It is concluded that 5-Aza-CdR and TSA can effectively reverse DLC-1 expression of RPMI-8226 cells; TSA has a synergistic effect on its re-expression. 5-Aza-CdR and TSA have significant cell growth inhibitory and apoptosis-inducing effects on RPMI-8226 cells. These effects may be related to the inhibition of Rho/Rho kinase signalling pathway.

CD59 underlines the antiatherosclerotic effects of C-phycocyanin on mice.

The effects of C-phycocyanin (C-PC) on atherosclerosis and the regulatory effects of CD59 gene on anti-atherosclerotic roles of C-PC were investigated. Apolipoprotein E knockout (ApoE(-/-)) mice were randomly divided into four groups: control group, C-PC treatment group, CD59 transfection group and C-PC+CD59 synergy group. The mice were fed with high-fat-diet and treated with drug intervention at the same time. Results showed the atherosclerotic mouse model was successfully established. CD59 was over-expressed in blood and tissue cells. Single CD59 or C-PC could reduce blood lipid levels and promote the expression of anti-apoptotic Bcl-2 but inhibit pro-apoptotic Fas proteins in endothelial cells. The expression levels of cell cycle protein D1 (Cyclin D1) and mRNA levels of cyclin dependent protein kinase 4 (CDK4) in smooth muscle cells were restrained by CD59 and C-PC. CD59 or C-PC alone could inhibit the formation of atherosclerotic plaque by suppressing MMP-2 protein expression. In addition, C-PC could promote CD59 expression. So both CD59 and C-PC could inhibit the progress of atherosclerosis, and the anti-atherosclerotic effects of C-PC might be fulfilled by promoting CD59 expression, preventing smooth muscle cell proliferation and the apoptosis of endothelial cells, reducing blood fat levels, and at last inhibiting the development of atherosclerosis.

Molecular mechanism of inhibitory effects of CD59 gene on atherosclerosis in ApoE (-/-) mice.

BACKGROUND: How to find an effective gene locus resistant to atherosclerosis has become a hotspot of today's medicine. Membrane attack complex (MAC) has proved to be related with the occurrence and development of atherosclerosis. Complement regulatory protein CD59 is a key regulator of complement MAC assembly. So this study aimed at discussing the effects of CD59 gene on occurrence and development of atherosclerosis and relative mechanism. METHODS: Apolipoprotein E knockout (ApoE (-/-)) mice were randomly divided into four groups: control group, empty plasmid-treated group, 0.5 ml CD59-treated group and 1.0 ml CD59-treated group. At the end of the 12th week, CD59 mRNA levels in whole blood were determined by RT-PCR and CD59 protein expressions were detected by western blot. The biochemical indexes in blood serum were detected. The paraffin sections of aortic root of mice were made and the degrees of atherosclerotic plaques formation were observed by hematoxylin/eosin (HE) staining. The expressions of cell apoptosis-related proteins (Bcl-2 and Fas) and plaque stability related protein (MMP-2) were detected by immunohistochemistry. Then the cell apoptosis levels were detected by TUNEL, the expression of Cyclin D1 and the mRNA level of cyclin dependent protein kinase 4 (CDK4) were detected by immunofluorescence and in situ hybridization, respectively. RESULTS: Atherosclerotic mouse model was successfully established. CD59 gene was overexpressed in blood cells and tissue cells after liposome transfection. CD59 could reduce blood lipid levels, promote the expression of anti-apoptotic Bcl-2 protein and inhibit pro-apoptotic Fas proteins, so finally lead to degradation of apoptosis levels of endothelial cells. In addition, Cyclin D1 protein and CDK4 mRNA levels were restrained by CD59 so as to inhibit the proliferation of smooth muscle cells. CD59 could inhibit the formation of atherosclerotic vulnerable plaque by suppressing the MMP-2 expression, which was further confirmed by HE staining. The anti-atherosclerotic effects were enhanced with the increase of CD59 gene dose. CONCLUSIONS: CD59 could lower blood lipid levels, positively regulate cell cycle, maintain the stability of cell proliferation and apoptosis of aorta cells, slow down the development of atherosclerotic vulnerable plaque, and finally inhibit the progress of atherosclerosis. So CD59 gene might be a new genetic locus for the therapy of atherosclerosis.

[Expression and significance of melanoma antigen gene-3 in endoplasmic reticulum stress-induced apoptosis of K562 cells].

This study was aimed to explore the expression and significance of melanoma antigen gene-3 (MAGE-3) in endoplasmic reticulum stress-induced apoptosis. After the treatment of leukemia cell line K562 and its multidrug-resistant cell line K562/A02 by thapsigargin, intracellular calcium concentrations ([Ca(2+)]i) in K562 and K562/A02 were measured by fluorescence spectrophotometer with calcium sensitive fluorescence indicator Fura-2/AM; expression changes of glucose-regulated protein 78 (GRP78) were detected by Western blot; morphological change of apoptotic cell was investigated by AO/EB fluorescent staining under fluorescent microscope; apoptosis rate was determined by terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) staining; the expression of MAGE-3 gene mRNA was detected by RT-PCR. The results showed that (1) thapsigargin induced the enhancement of [Ca(2+)]i with different extent in K562 and K562/A02 cells, and the enhancement of [Ca(2+)]i was dose-dependent in experiment range. At the same time, thapsigargin induced upregulation of GRP78 protein expression and typical apoptotic changes of K562 and K562/A02 cells, apoptotic rate was also dose-dependent in experiment range. The [Ca(2+)]i in K562/A02 cells were higher than that in K562 cells. (2) in the course of endoplasmic reticulum stress-induced apoptosis by thapsigargin, the expression of MAGE-3 gene mRNA was remarkably downregulated. Moreover, the expression of MAGE-3 gene mRNA in K562/A02 cells was higher than that in K562 cells. It is concluded that (1) thapsigargin induces endoplasmic reticulum stress-induced apoptosis of K562 and K562/A02 cells in experiment range, and this may be associated with downregulation of MAGE-3 mRNA expression or MAGE-3 gene may participates in the regulation of endoplasmic reticulum stress-induced apoptosis. (2) MAGE-3 gene may possess anti-apoptotic activity, multidrug resistance in K562/A02 cells can be associated with [Ca(2+)]i increase and upregulation of MAGE-3 expression.