Nitrites play multiple characteristic functions in invasion and metastasis of hepatic cancer cells, but the exact mechanism is not yet known. Cancer cells can maintain the malignant characteristics via clearance of excess mitochondria by mitophagy. The purpose of this article was to determine the roles of nitrite, reactive oxygen species (ROS) and hypoxia inducing factor 1 alpha (HIF-1 α) in mitophagy of hepatic cancer cells. After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen, the maximal cell vitality was increased by 16 mg x (-1) sodium nitrite. In addition, the potentials of migration and invasion for SMMC-7721 cells were increased significantly at the same time. Furthermore, sodium nitrite exposure displayed an increase of stress fibers, lamellipodum and perinuclear mitochondrial distribution by cell staining with Actin-Tracker Green and Mito-Tracker Red, which was reversed by N-acetylcysteine (NAC, a reactive oxygen scavenger). DCFH-DA staining with fluorescent microscopy showed that the intracellular level of ROS concentration was increased by the sodium nitrite treatment. LC3 immunostaining and Western blot results showed that sodium nitrite enhanced cell autophagy flux. Under the transmission electron microscopy (TEM), more autolysosomes formed after sodium nitrite treatment and NAC could prevent autophagosome degradation. RT-PCR results indicated that the expression levels of COX I and COXIV mRNA were decreased significantly after sodium nitrite treatment. Meanwhile, laser scanning confocal microscopy showed that sodium nitrite significantly reduced mitochondrial mass detected by Mito-Tracker Green staining. The expression levels of HIF-1α, Beclin-1 and Bnip3 (mitophagy marker molecular) increased remarkably after sodium nitrite treatment, which were reversed by NAC. Our results demonstrated that sodium nitrite (16 mg x L(-1)) increased the potentials of invasion and migration of hepatic cancer SMMC-7721 cells through induction of ROS and HIF-1α mediated mitophagy.
Acetylcysteine ; pharmacology ; Autophagy ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Movement ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Liver Neoplasms ; pathology ; Mitochondrial Degradation ; Neoplasm Invasiveness ; Nitrites ; metabolism ; Reactive Oxygen Species ; metabolism ; Sodium Nitrite ; pharmacology

This study was aimed to compare K562 cell proliferation, chemo-sensitivity and alteration of MDR1 before and after adhesive culture with MSC, so as to evaluate the relationship between chemodrug-resistance of leukemia cells and hemopoietic microenvironment. K562 cell cultivated in suspension and adhesively cultivated with MSC were collected respectively and cell proliferation curves were drawn; the cell cycle was determined by flow cytometry; the effect of chemotherapy on cellular viability and apoptosis of K562 cell was investigated, the MDR1 gene expression was determined by RT-PCR. The results showed that K562 cells adhesively cultivated with MSC were inhibited and cells in G0/G1 increased (P < 0.05), cells in S phase decreased (P < 0.05) and those in G0/G1 increased (P < 0.01), compared with that cultivated in suspension. In process of daunomycin-inducing apoptosis, K562 cell apoptosis in the adhesive culture with MSC was inhibited (P < 0.05). MDR1 gene expression in K562 cells was not induced or altered by adhesive co-cultivation. It is concluded that by co-culture of cell-cell contact with MSC, growth suppression and induction of chemo-resistance of K562 cells take place. The mechanism, however, seems not relevant with MDR1.
ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Apoptosis ; physiology ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Daunorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; K562 Cells ; Mesenchymal Stromal Cells ; cytology

Objective To investigate the clinical significance of intraportal vein anticoagulation for the prevention of portal vein thrombosis after portaazygous devascularization and splenorenal shunt. Methods In this study 67 patients of portal hypertension undergoing surgery were randomly divided into two groups,receiving respectively intraportal vein heparin injection by 100 U?kg~(-1)?d~(-1)?7 d in group A (32 patients)and placebo in group B(35 patients).Portal vein thrombosis,the recurrent bleeding after operation and portal hypertensive gastropathy were compared between the two groups.Results The occurrence of portal vein thrombosis after operation in group A(0)was significantly lower than that in group B(20%,X~2=5.169,P

BACKGROUNDThe hematopoietic microenvironment (HM) plays a critical role in malignant cell growth, patient survival, and response to chemotherapy in hematologic malignancies. However, mechanisms associated with this environmental influence remain unclear. In this study, we investigated the role of bone marrow derived mesenchymal stem cells (MSCs) in U937 cell line, to find out the relations between leukemia drug resistance and the MSCs.

METHODSU937 cells were cultured in suspension or grew adherently with MSCs. The cell growth curve was drawn and the cell cycle was measured by flow cytometry. Apoptosis and sensitivity of U937 to daunoblastina (DNR) were quantified by DNA ladder detection and trypan blue exclusion assays, respectively. The gene expression profile chip technology was used to determine and analyze the changes in apoptosis-related gene expression after adherent culture and the expression of MDR1 mRNA was assessed by reverse transcriptional polymerase chain reaction (RT-PCR) at the same time.

RESULTSIn the adherent culture, the proliferation of the U937 cells was inhibited, the G0/G1 phase cells increased (F = 64.9726, P < 0.0001), G2/M phase cells were decreased (F = 98.1361, P < 0.0001) and the natural apoptosis rate was decreased (F = 24.0866, P < 0.0001) compared with those in the suspended culture. U937 cell viability was enhanced and cell apoptosis was blocked during DNR treatment in adherent culture with MSCs. Thirty-nine differently expressed genes were screened from the 487 apoptosis related genes in the adherent culture U937 cells. Among the 37 upregulated genes, Bcl-XL was upregulated most significantly. Two genes were downregulated. Adherent culture did not induce MDR1 mRNA expression in U937 cells.

CONCLUSIONSMSCs play a role in modulating the proliferation of U937 cells and response of U937 cells to DNR, and Bcl-XL apoptosis-inhibiting gene may be most important in determining the sensitivity of leukemic cells to treatment, which is not related to MDR1.

Apoptosis ; drug effects ; Bone Marrow Cells ; physiology ; Cell Proliferation ; Daunorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Genes, MDR ; Humans ; Immunophenotyping ; Mesenchymal Stromal Cells ; physiology ; U937 Cells ; drug effects

OBJECTIVETo explore the role of reversal multidrug resistance (MDR) using short hairpin RNA (shRNA) expression vectors in multidrug resistance human leukemia cell line K562/ADM.

METHODSThe oligonucleotides with 19-mer hairpin structure were synthesized. The shRNA expression vectors were constructed and introduced into K562/ADM cells. Expression of mdr1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western blot. The apoptosis and sensitivity of the K562/ADM cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscope (LCSM).

RESULTSIn positive clones of K562/ADM cells stably transfected with pSilencer 3.1-HI neo mdr1-A and mdr1-B shRNA expression vectors, RT-PCR showed that mdr1 mRNA expression was significantly reduced to 35.9% (P < 0.05), 27.5% (P < 0.01), respectively. Western blot showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 79-fold to 38-fold (P < 0.05), 30-fold (P < 0.01) respectively. Furthermore, the fluorescence intensity of K562/ADM cells was increased significantly compared with the control. shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The percent of the apoptosis cell was significantly enhanced to 18.1% (P < 0.05) , 54.4% (P < 0.01) respectively.

CONCLUSIONSshRNA expression vectors can effectively reverse MDR, and restore the sensitivity of drug-resistance K562/ADM cells to conventional chemotherapeutic agents.

ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Apoptosis ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Genetic Vectors ; Humans ; K562 Cells ; RNA Interference ; RNA, Messenger ; genetics ; Transfection

OBJECTIVETo investigate the relationship between the gene mutations of mannose binding protein(MBP) and the progression of hepatitis B.

METHODSThe MBP gene mutations in 52 patients with chronic hepatitis B and 62 patients with severe hepatitis B and 64 HBsAg-negative healthy controls were investigated. The mutations in MBP gene were analyzed by polymerase chain reaction (PCR) and direct DNA sequencing.

RESULTSA mutation of MBP gene codon 54 was found. The mutation frequency in the group of severe hepatitis B (35.5%, 22/62) was higher than those in the chronic hepatitis B group (15.4%, 8/52) and the HBsAg-negative healthy controls(14.1%, 9/64), respectively, and their difference was significant (chi-square was 7.79, P< 0.01; chi-square was 5.89,lzP<0.05). The difference between the chronic hepatitis B group and the HBsAg-negative healthy control group was not significant (P > 0.05).

CONCLUSIONThere is only mutation in codon 54 of the MBP gene in patients with hepatitis B infection in the area analyzed. Codon 54 mutation of MBP gene is not related to the persistence of hepatitis B, but it was associated with the progression of hepatitis B infection.

Adult ; Aged ; Codon ; genetics ; Female ; Hepatitis B ; genetics ; Humans ; Male ; Mannose-Binding Lectin ; genetics ; Middle Aged ; Mutation ; genetics ; Polymerase Chain Reaction ; Young Adult

OBJECTIVETo search novel SNPs in exons and regulatory regions of CDKN2A and two novel putative tumor suppressor genes NGX6 and UBAP1, which all reside on chromosome 9p21-22.

METHODSThe exons and regulatory regions of those genes were amplified and sequenced in 96 subjects.

RESULTSTwo novel SNPs were found, one resides on the sixth exon of UBAP1 gene and the other on the fourth exon of CDKN2A gene. Two novel SNPs were submitted to the dbSNP database, and their access ID are rs3135929 and rs3088440. The polymorphic information contents of them are 0.102 and 0.213 respectively. There is linkage equilibrium between them, and the polymorphic information content of their haplotype is 0.302, higher than any of them individually.

CONCLUSIONThe polymorphic information content can be improved by using haplotype analysis of several SNPs.

Asian Continental Ancestry Group ; genetics ; Base Sequence ; Carrier Proteins ; genetics ; China ; ethnology ; Chromosomes, Human, Pair 9 ; genetics ; Genes, p16 ; physiology ; Genetic Predisposition to Disease ; Humans ; Membrane Proteins ; genetics ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA ; Tumor Suppressor Proteins ; genetics

AIM:To investigate the neuroprotective effect of progesterone against adenosine triphosphate (ATP)-injured human neuroblastoma SH-SY5Y cells.METHODS:The SH-SY5Y cells in the logarithmic phase were divided into different groups according to the progesterone and ATP concentrations.The cell viability was measured by CCK-8 assay.The membrane permeability was detected using fluorescent dye YO-PRO-1.Cytosolic Ca2+ concentration was measured with fluorescent dye Fluo-3/AM.The expression of purinergic P2X7 receptor was assessed by Western blot.RESULTS:The viability of the SH-SY5Y cells was significantly decreased (P ＜ 0.05) and YO-PRO-1 uptake was obviously increased (P ＜ 0.05) in a concentration-dependent manner compared with control group when SH-SY5Y cells were treated with ATP at 1,3,5 and 7 mmol/L for 2 h.The viability reduction of the SH-SY5Y cells induced by ATP was obviously counteracted by treatment with progesterone at 3,10 and 30 nmol/L for 30 min (P ＜ 0.05) as compared with ATP group.YO-PRO-1 fluorescence enhancement induced by ATP in SH-SY5Y cells was significantly reduced (P ＜ 0.05) by progesterone (30 nmol/L) or P2X7 receptor antagonist KN-62 (500 nmol/L) pretreatment for 30 min,and no obvious difference between treatments with progesterone and KN-62 was observed.Cytosolic Ca2+ fluorescence intensity in normal group was a little,but that in ATP group was increased (P ＜ 0.05).Progesterone or KN-62 pretreatment significantly decreased the cytosolic fluorescence intensity of Ca2+ induced by ATP (P ＜ 0.05).However,no obvious difference between treatments with progesterone and KN-62 was found.The expression of P2X7 receptor in ATP group was significantly higher than that in control group (P ＜ 0.05),and progesterone inhibited ATP-induced P2X7 receptor expression (P ＜ 0.05).CONCLUSION:Progesterone inhibits P2X7 receptor expression,membrane pore formation,intracellular Ca2+ increase and cell death induced by ATP,so progesterone may protect SH-SY5Y cells against ATP-induced injuries.

OBJECTIVETo investigate the safety and effectiveness of one-stage posterior correction of scoliosis associated with little symptomatic syringomyelia.

METHODSA total of 19 cases diagnosed as scoliosis with little symptomatic syringomyelia between January 2003 and November 2010 were included in this study (study group), the patients underwent one-stage posterior correction and instrumentation without neurosurgery for the syringomyelia. At the same time, 9 cases with severe symptomatic syringomyelia were included as the control group, the patients underwent neurosurgery before scoliosis correction, including suboccipital decompression and syrinx shunting. All patients underwent posterior pedicle screw or screw-hook hybrid instrumentation. The preoperative, postoperative and the last follow-up of the Cobb angle of the coronal main curve and thoracic kyphosis were measured. Also, the preoperative and postoperative of the apical vertebra translation, apical vertebra rotation and trunk shift were measured by the same person. The perioperative and the last follow-up complications of neurological injury were recorded. The surgical outcome and postoperative complications between the 2 groups were compared with the t student and chi-square statistics methods.

RESULTSThere were no significant differences in gender, age, the location, length and diameter of the syringomyelia of the 2 groups (P > 0.05). The follow-up period ranged from 6 to 45 months, with a mean of 28.6 months. The average preoperative Cobb angles of coronal main curves of the 2 groups were 71° ± 23° and 68° ± 19°, the postoperative Cobb angles were 27° ± 20° and 25° ± 16°, and the last follow-up Cobb angles were 29° ± 17° and 32° ± 20°. The coronal correction rate was 66% ± 19% in the study group and 65% ± 21% in the control group (t = 0.136, P = 0.893). There was no significant difference at the last follow-up(t = 0.210, P = 0.837). The average preoperative Cobb angles of thoracic kyphosis of the 2 groups were 35° ± 18° and 32° ± 19°, the postoperative Cobb angles were 25° ± 10° and 23° ± 9°, and the last follow-up Cobb angles were 24° ± 4° and 28° ± 8°. The mean sagittal correction rate of the 2 groups were 50% ± 58% and 57% ± 53% (t = -0.303, P = 0.764). There was also no significant difference at the last follow-up time (t = 0.769, P = 0.490). There were no significant difference, in terms of the postoperative of the apical vertebra translation, apical vertebra rotation and trunk shift between the 2 groups (P > 0.05). One case in the study group complicated with a pedicle screw breaking the anterior cortex of the vertebra and one in the control group complicated with a hook loosening, postoperatively. At the last follow-up time, the neurological symptoms of the 2 groups got no aggravating.

CONCLUSIONOne-stage posterior correction of scoliosis associated with little symptomatic syringomyelia may be effective and safe.

Adolescent ; Adult ; Child ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Scoliosis ; surgery ; Spinal Fusion ; methods ; Syringomyelia ; surgery ; Treatment Outcome ; Young Adult

OBJECTIVETo compare the effect of posterior correction and fusion between segmental pedicle screw instrumentation with hybrid constructs in adolescent idiopathic scoliosis (AIS).

METHODSStudy the clinical data of 40 AIS patients retrospectively. They were underwent posterior fusion and be distributed into two group, group A was hybrid instrumentation (20 cases) and group B was pedicle screw instrumentation (20 cases). Compared therapy effect, operative time, intraoperative blood loss.

RESULTSThe average major curve correction was 82.4% in the screw group and 71.8% in the hybrid group (P = 0.004). After one to three years follow-up, major curve correction was 77.0% and 62.5% respectively (P = 0.001). Average apical vertebral derotation showed 63% correction in the screw group and 32% in the hybrid group (P = 0.001). There was no statistical significance between two group in thoracic sagittal correction, the lowest instrumented vertebra below the lower end vertebra, trunk shift, operative time, and average estimated blood loss. There were no neurologic complications related to hybrid or pedicle screw instrumentation.

CONCLUSIONPedicle screw instrumentation was significantly better than hybrid constructs.

Adolescent ; Adult ; Bone Nails ; Bone Screws ; Child ; Female ; Follow-Up Studies ; Humans ; Male ; Retrospective Studies ; Scoliosis ; surgery ; Spinal Fusion ; instrumentation ; methods ; Treatment Outcome