[Effect of γδ T cells on the Proliferation, Apoptosis and Autophagy of Multiple Myeloma Cells].

AbstractObjective: To investigate the effect of γδ T cells on the proliferation, apoptosis and autophagy of multiple myeloma cells. METHODS: Peripheral blood mononuclear cells (PBMNC) were isolated from healthy volunteers, and stimulated with zoledronic acid (Zol) in combination with rhIL-2. Flow cytometry analysis was used to detected the purity of γδ T cells. γδ T cells were collected and co-cultured with RPMI-8226 or U-266 cells at different effector target ratios. The proliferation of RPMI-8226 or U-266 cell lines were detected by CCK-8. Cell cycle and cell apoptosis were detected by flow cytometry and Western blot．The expressions of autophagy-related proteins were detected by Western blot. RESULTS: γδ T cells can be expanded in vitro. γδ T cells could inhibit the proliferation of RPMI-8226 or U-266 cells, induced cell cycle arrest and promoted apoptosis in an effector target-dependent manner. In addition, γδ T cells could induce autophagy of myeloma cells, inhibited the expression of autophagy-related PI3K, P-AKT and P-mTOR, while increased the expression of AMPK and Beclin-1. CONCLUSION: γδ T cells can inhibit the proliferation of RPMI-8226 and U-266 myeloma cells, induce cell cycle arrest, promote apoptosis, and enhance autophagy in vitro. The mechanism may be related to inhibition of PI3K/AKT/mTOR signaling pathway and/or activation of AMPK/Beclin-1 signaling pathway.

[Research Progress on Regulating Autophagy in the Treatment of Multiple Myeloma --Review].

Abstract　　Autophagy is a process in which cells in eukaryotes degrades abnormal proteins and organelles, thus it possesses important effects on the survival of normal cells and tumor cells. The related studies have shown that autophagy is widely present in the life activities of myeloma cells, which not only protects myeloma cells, but also induces death, and plays an important role in survival, proliferation, invasion and migration of myeloma cells and the treatment of multiple myeloma. This review focuses on the progress of regulating autophagy in the treatment of multiple myeloma.

[Effects of Autophagy Regulating Drugs on Proliferation, Apoptosis and Autophagy of Multiple Myeloma Cells].

OBJECTIVE: To investigate the effects of autophagy activator (rapamycin, RAPA) and autophagy inhibitor (hydroxychloroquine, HCQ and 3-methyl adenine, 3-MA) on the proliferation, apoptosis and autophagy of multiple myeloma cell line of RPMI8226. METHODS: RPMI8226 cells were treated with autophagy regulating drugs of different concentrations. The proliferation and apoptosis of cells were determined by CCK-8 and flow cytometry, respectively. The expressions of apoptosis-related proteins BCL-2, caspase-3 and PARP protein were assessed by Western blot. Autophagy was detected by monodansylcadaverine staining. Autophagic protein (LC-3b) and apoptosis-related proteins (caspase-3, PARP and BCL-2) were analyzed by Western blot. RESULTS: RAPA and HCQ inhibited the proliferation of RPMI8226 in a concentration- and time-dependent manner, and increased the apoptosis. However, 3-MA did not show significantly inhibitory effect on the proliferation and apoptosis of RPMI8226. MDC staining showed that the more autophagic vacuoles could be detected in the higher concentration of RAPA, but the less autophagic vacuoles in the higher concentration of HCQ and 3-MA. Western blot showed that RAPA increased the expression of LC3-II/LC3-I, caspase-3 and PARP, but inhibited the expression of BCL-2. HCQ inhibited the expression of LC3-II/LC3-I and BCL-2, but increased the expression of caspase-3 and PARP. 3-MA inhibited the expression of LC3-II/LC3-I, but had no effect on the expression of caspase-3, PARP or BCL-2. CONCLUSION: Rapamycin can inhibit the proliferation, induce apoptosis and autophagy of RPMI 8226, the hydroxychloroquine can inhibit autophagy and proliferation of RPMI 8226, and induce apoptosis, the 3-MA can inhibit autophagy of RPMI 8226, but hardly has any effects on proliferation and apoptosis of RPMI 8226 cells.

[Effect of Complicated Hemophagocytic Syndrome on Clinical Prognosis of Patients with Non-Hodgkin's Lymphoma and Analysis of Its Affecting Factors].

OBJECTIVE: To investigate the effect of complicatal hemophagocytic syndrome on clinical prognosis of patients with non-Hodgkin's lymphoma (NHL) and analyze its factors affecting prognosis. METHODS: Ninety cases of NHL were selected and divided into 2 groups: 61 cases of NHL without hemophagocytic syndrome as group A and 29 cases of NHL with hemophagocytic syndrame as group B. The survival analysis of Kaplan-Meter method and the Cox regression model were used for univariate and multivariate analyses of related factors. RESULTS: The patients in group B were more likely to start with fever, moreover, the hemophagocytes could be found in bone marrow samples of 89.66% (26/29) patients; the levels of total bilirubin, triglycerides, serum ferritin, serum soluble CD25, DNA copies of epstein-barr virus (EBV) and lactate dehydrogenase level in the group B were significantly higher than those in the group A(P<0.05). And the patients in group B had worse physical state, later disease stage, worse disease status and lower overall prognosis as compared with patients in the group A. The complicased hemophagocytic syndrome, incomplete improvemant of deseases state after treatment and EBV infection were the independent risk factors for the poor prognosis of patients with NHL. CONCLUSION: The complicated hemophagocytic syndrome can increase the severity of NHL, there fore significantly influences the clinical prognosis of patients, while the complicated hemophagocytic syndrome, poor therapatic efficacy for patients and EBV infection are independent risk factors affecting the prognosis of NHL patients.

Knockdown of DEPTOR inhibits cell proliferation and increases chemosensitivity to melphalan in human multiple myeloma RPMI-8226 cells via inhibiting PI3K/AKT activity.

OBJECTIVE: The present study determined the role of DEP domain containing mTOR-interacting protein (DEPTOR) in the proliferation, apoptosis and chemosensitivity of RPMI-8226 multiple myeloma cells, using small hairpin RNA (shRNA) to knock down DEPTOR gene expression in vitro. METHODS: DEPTOR mRNA and protein levels in RPMI-8226 cells treated with DEPTOR-specific shRNA were evaluated by reverse transcription-polymerase chain reaction and Western blotting. Expression of apoptosis-associated proteins (including cleaved caspase-3 and cleaved poly-ADP ribose polymerase [PARP]) and activation of the phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homologue 1 (AKT) signalling pathway were detected by Western blotting. RESULTS: Transfection of DEPTOR-specific shRNA successfully knocked down DEPTOR gene expression in transfected RPMI-8226 cells. These transfected cells, together with control RPMI-8226 cells, were treated with 20 µmol/l melphalan for 24 h. Knockdown of DEPTOR exacerbated melphalan-induced growth inhibition and apoptosis, increased levels of cleaved caspase-3 and cleaved PARP, and reduced levels of phosphor-AKT. CONCLUSION: Downregulation of DEPTOR inhibited proliferation and increased chemosensitivity to melphalan in human multiple myeloma RPMI-8226 cells via inhibiting the PI3K/AKT pathway.

Dietary restriction suppresses tumor growth, reduces angiogenesis, and improves tumor microenvironment in human non-small-cell lung cancer xenografts.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide; however, only limited therapeutic treatments are available. The aim of present study was to elucidate the therapeutic effect of dietary restriction in human NSCLC xenografts. Adult female nude mice were injected subcutaneously in the right dorsal flank with NSCLC cell line A549 cells. 5 days after tumor implantation, animals were randomly divided into ad libitum-fed group (AL, 95% of average diary intake) or dietary-restriction-fed group (DR, 70% average diary intake). 24 days after implantation, it was found that DR inhibited tumor growth marked by lower tumor volume and weight. DR suppressed tumor proliferation marked by reduced proliferating cell nuclear antigen (PCNA) expression and activated mitochondria-mediated apoptosis. DR decreased microvessel density marked by decreased CD31 immunostaining and promoted vessel maturation marked by increased alpha-smooth muscle actin (α-SMA) and reduced Factor VIII expression. DR reduced intratumoral interstitial fluid pressure and attenuated tumor hypoxia detected by EF5 immunostaining. In addition, DR suppressed NFκB signaling pathway and downregulated its downstream proteins expression including cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). DR suppressed phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. In conclusion, dietary restriction suppresses tumor growth, reduces angiogenesis, and improves tumor microenvironment in human non-small-cell lung cancer xenografts. Dietary restriction could thus be envisaged as a nutritional countermeasure against non-small-cell lung cancer.

[Effect of DAPT on proliferation and apoptosis of human multiple myeloma cell line RPMI8226].

The aim of this study was to explore the effect of DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester) on proliferation in vitro of human multiple myeloma cell line RPMI8226 and its underlying mechanism. The proliferation of RPMI8226 cells was detected by CCK-8 method; flow cytometry was employed to assay the cell apoptosis rate;the expressions of Notch1 and Hes1 proteins were detected by Western blot. The results indicated that the proliferation of human RPMI8226 cells significantly decreased after treatment with DAPT 0.5 - 5.0 µmol/L for 24 - 72 h (P < 0.05) in a concentration- and time-dependent manner. DAPT significantly induced apoptosis of RPMI8226 cells (P < 0.05). The expressions of Notch1 and Hes1 proteins were gradually downregulated with the increase of DAPT concentration. It is concluded that the DAPT can inhibit the proliferation of RPMI8226 cells, which may be related with the down-regulation of the protein expression of Notchl and Hes1.

MicroRNA-98 and microRNA-214 post-transcriptionally regulate enhancer of zeste homolog 2 and inhibit migration and invasion in human esophageal squamous cell carcinoma.

BACKGROUND: The enhancer of zeste homolog 2 (EZH2) was found to be overexpressed and associated with tumor metastasis in esophageal squamous cell carcinoma (ESCC). On the other hand, it was reported that miR-26a, miR-98, miR-101, miR-124, miR-138 and miR-214 could inhibit the expression of EZH2 in some tumors. However, the role of miRNAs in the regulation of EZH2 expression in human ESCC has not been documented. The aim of this study was to determine the role of these miRNAs in the regulation of tumor metastasis via EZH2 overexpression in human ESCC. METHODS AND RESULTS: The expression of these miRNAs and EZH2 mRNA were examined by qPCR and the expression of EZH2 protein was detected by western blot. The role of these miRNAs in migration and invasion was studied in ESCC cell line (Eca109) transfected with miRNA mimics or cotransfected with miRNA mimics and pcDNA-EZH2 plasmid (without the 3'-UTR of EZH2). Through clinical investigation, we found that miR-98 and miR-214 expression was significantly lower in ESCC tissues than in matched normal tissues, and the expression level of miR-98 and miR-214 was inversely correlated to EZH2 protein expression and the clinical features such as pathological grade, tumor stage and lymph node metastasis in ESCC. In Eca109 cells, overexpression of miR-98 and miR-214 significantly inhibited the migration and invasion of ESCC cells, which was reversed by transfection of EZH2. CONCLUSIONS: These findings suggest that decreased expression of miR-98 and miR-214 might promote metastasis of human ESCC by inducing accumulation of EZH2 protein.

MicroRNA-203 inhibits cell proliferation by repressing ΔNp63 expression in human esophageal squamous cell carcinoma.

BACKGROUND: This study was performed to investigate the effect of microRNA-203 (miR-203) and ΔNp63 on cell proliferation and the functional connection between miR-203 and ΔNp63 in ESCC. METHODS: We employed 2 human ESCC cell lines, Eca109 and TE-1, as the model system. The effect of miR-203 and ΔNp63 on cell proliferation was determined in cells transfected with miR-203 mimic and ΔNp63 small interfering RNA (siRNA), respectively. The regulation of ΔNp63 expression in ESCC cells by miR-203 was studied by luciferase reporter assay, RT-PCR and western blot analysis in cells transfected with miR-203. The effect of ΔNp63 re-expression on miR-203 induced inhibition of cell proliferation was studied by cell proliferation assay in cells cotransfected with miR-203 and pcDNA-ΔNp63 plasmid (without the 3'-UTR of ΔNp63). RESULTS: We found that both miR-203 and ΔNp63 siRNA signicantly inhibited cell proliferation in ESCC. MiR-203 could down-regulate endogenous ΔNp63 expression at the posttranscriptional level. Moreover, re-expression of ΔNp63 in cells transfected with miR-203 significantly attenuated the miR-203 induced inhibition of cell proliferation. CONCLUSIONS: Our data implied that miR-203 could inhibit cell proliferation in human ESCC through ΔNp63-mediated signal pathway. Therefore, we propose that miR-203 might be used as a therapeutic agent for human ESCC.

[Expression of Aurora-B in non-small cell lung cancer and its clinical significance].

OBJECTIVE: To study the expression of Aurora-B in non-small cell lung cancer (NSCLC) tissues and NSCLC cell lines. METHOD: Aurora-B expression was examined using immunohistochemical SP method in 91 stage I and 69 stage II-III NSCLC tissues and 40 adjacent tissues. The mRNA and protein expressions of Aurora-B in NSCLC cell lines (A549, H460 and H1299) were examined by RT-PCR and Western blotting, respectively. RESULTS: The protein expression of Aurora-B was detected in 77.7% (94/121) of the tumor tissues and 9.8% (4/41) of the adjacent tissues, showing a significant difference between them (P<0.01). The positivity rate of Aurora-B protein was not related with the gender and age of NSCLC patients, but with lymph node metastasis, differentiation and histological type of NSCLC (P<0.05). Aurora-B was expressed in all the NSCLC cell lines (A549, H460 and H1299) at both mRNA and protein levels. A549 cells showed the highest expression of Aurora-B. CONCLUSION: Aurora-B protein is highly expressed in NSCLC tissues and cell lines, and may play a crucial role in the invasion, metastasis and development of NSCLC. The mRNA and protein expression levels of Aurora-B differ significantly between different NSCLC cell lines.

[Observation on the long-term complications after esophageal replacement with colon].

OBJECTIVE: To study the etiology and preventive measures of the long-term postoperative complication after esophageal replacement with colon for esophageal benign disease. METHODS: To review the clinical data of 577 patients with esophageal replacement with colon our department, including 123 cases of esophageal benign disease. Of all, there were 25 cases-time for 11 cases following with severe complication: redundancy and dilated colon 12 cases-time, severe stricture of stoma 4, macrocyst esophagus 2, colon-stomach stoma expansion 4, mechanical obstruction of colon 3. The etiology included iatrogenic and functionality. The therapy included stricture form or resection, redundancy segment resection, obstructed segment solution and stoma resection and form. RESULTS: Eight cases underwent once operation, 2 case twice, 1 case three times. After operation, 9 cases took food normally, 2 improved symptoms obviously. CONCLUSIONS: The iatrogenic and functionality factor contributed to severe complication after esophageal replacement with colon for esophageal benign disease. The preventive measure is followed during operation: cervical esophageal-colon anastomosis exceed 2.5 centimeter, abdominal colon-stomach anastomosis reflux, channel width of colon passage, intestinal canal lay up straight. Re-operation is best choice to for local stricture, colon expansion, redundancy and dilated colon.

[Risk factors for postoperative pulmonary complications after gastroduodenal operation].

OBJECTIVE: To investigate the risk factors for postoperative pulmonary complications (PPC) after gastroduodenal operation. METHODS: From December 1999 to December 2003, clinical data of 508 patients undergoing gastroduodenal operation were analyzed retrospectively. Risk factors for PPC were screened. RESULTS: The complication rate of PPC was 25.8% (131/508). Multivariate logistic regression analysis revealed that age (OR=1.052), history of respiratory diseases (OR=2.915), serum albumin level (OR=0.995), length of intratracheal intubation (OR=1.005), length of nasogastric intubation (OR=1.059) and length of postoperative mechanical ventilation (OR=1.367) were risk factors for PPC. CONCLUSION: Patients with old age, lower serum albumin level, intraoperative or postoperative nasogastric intubation, intratracheal intubation or long-term mechanical ventilation were more prone to develop PPC.

[The effect of recombinant adenovirus encoding antisense MAT1 on cell cycle of human pancreatic cancer cells BxPC-3].

OBJECTIVE: To investigate the regulatory effect of the human MAT1 gene on the cell cycle G(1)/S transition of human pancreatic cancer BxPC-3 cells. METHODS: To construct the replication deficient recombinant adenovirus of antisense MAT1 gene using homologous recombination by AdEasy system. Cell growth assay was carried out by counting alive cells after trypan blue exclusion. The protein expressions of MAT1, cyclin D1, cyclin E, and Rb were detected by western blotting. The cell cycle status was determined by flow cytometry. RESULTS: The recombinant adenovirus encoding antisense MAT1 fragment Ad-MAT1AS was obtained with the titer of expression was 5 x 10(10) pfu/ml. The expression of MAT1 of BxPC-3 was significantly reduced after Ad-MAT1AS infection. In this case BxPC-3 cell cycle was arrested in G(1) phase. The estimated proportion of G(0)/G(1) phase cells in the control for blank and vector cultures ranged from 40% to 44%. In contrast, 79% of the Ad-MAT1AS cells were in G(0)/G(1) phase. Cyclin E and pRb gene expression changes were observed in the infected cells. CONCLUSION: The results suggest that MAT1 gene may play an important role in the regulation of cell cycle G(1)-->S transition of BxPC-3 cells.