A polymorphism in the 3'-untranslated region of the matrix metallopeptidase 9 gene is associated with susceptibility to idiopathic calcium nephrolithiasis in the Chinese population.

OBJECTIVE: To investigate whether single nucleotide polymorphisms (SNPs) in the 3' untranslated region (UTR) of the matrix metallopeptidase 9 gene (MMP9) are associated with susceptibility to calcium oxalate stones. METHODS: A total of 428 patients with kidney stone disease (KSD) and 450 control individuals were enrolled. Three MMP9 SNPs (rs20544, rs9509, and rs1056628) were genotyped, and MMP9 mRNA and protein expression was determined in patients and controls. The dual luciferase reporter gene assay was conducted by transfecting HEK293 cells with miR-491-5p mimics and plasmids containing MMP9 with rs1056628 AA/CC genotypes. RESULTS: The rs1056628 CC genotype was significantly increased in KSD patients compared with controls (CC vs AA: odds ratio [OR] = 2.279, 95% confidence interval [CI] = 1.048-4.956). The rs1056628 C allele frequency was higher in KSD patients than controls. The increased KSD risks associated with rs1056628 were more evident in individuals aged <30 years (OR = 3.504, 95% CI = 1.102-11.139) and men (OR = 2.522, 95% CI = 1.004-6.334). mRNA and protein levels of MMP9 were significantly higher in KSD patients with the CC genotype than in those with the AA genotype. CONCLUSION: This study demonstrates that MMP9 SNP rs1056628 is associated with a significant KSD risk in Chinese Han individuals.

A novel gene mutation of Runx2 in cleidocranial dysplasia.

Haploinsufficiency of the runt-related transcription factor 2 (Runx2) gene is widely known to be responsible for cleidocranial dysplasia (CCD). To date, more than 190 mutations in Runx2 gene have been reported to be related to CCD. In this study, a novel mutation of Runx2 gene was observed in a female with CCD. Genomic DNA was extracted from peripheral venous blood of the proband and eleven members of her family. Genetic testing on these twelve people identified a novel missense mutation (c.895 T>C, Y299H) in exon 5 of the RUNX2 gene in the proband. This mutation results in an amino acid change at codon 895 (P.Tyr 299 His.) from a tryptophan codon (TAT) to a histidine codon (CAT). Our finding may further extend the known mutation spectrum of the RUNX2 gene, and facilitate prenatal genetic diagnosis of CCD in the future.

[Effect of triptolide on sensitivity of K562/A02 cell line to adriamycin].

This study was aimed to evaluate the effect of triptolide (TPL) on the reversal of multidrug resistance in K562/A02 cell line. The sensitivity of K562 and K562/A02 to adriamycin (ADM) and reversal of drug resistance were determined with MTT method. The concentration of intracellular ADM and P-glycoprotein expression were detected by flow cytometry. Luciferase reporter gene assay was used to detect the transcriptional activity of MDR1 promoter. The results showed that TPL significantly decreased the resistance degree of K562/A02 cells, inhibited P-glycoprotein expression (mean fluorescent intensity decreased from 123 ± 13 to 39 ± 13) and increased the intracellular concentration of ADM (mean fluorescent intensity increased from 18 ± 5 to 34 ± 6) in K562/A02 cells. Luciferase reporter gene assay demonstrated that TPL inhibited the transcriptional activity of MDR1 promoter by 75%. It is concluded that TPL may effectively reverse the multidrug resistance in K562/A02 cells via modulating P-glycoprotein expression and increasing intracellular ADM accumulation.

[Effect of regulation of Y-box protein 1 by RNA interference on the doxorubicin induced mdr1 gene expression in K562 cells].

OBJECTIVE: To investigate the effect of YB-1 on the transcription of induced mdr1 gene expression in K562 cells. METHODS: K562 cells were treated with doxorubicin (DOX) at different concentrations and times. Expression of mdr1 and YB-1 genes was examined by RT-PCR and P-glycoprotein (P-gp) by flow cytometry. Cyto/nuclear protein was extracted for YB-1 detection by Western blotting. The expression of YB-1 gene in K562 cells was inhibited by YB-1 gene specific RNA interference (RNAi), then the expression of mdr1 and P-gp in YB-1 gene silenced cells treated with DOX was detected. RESULTS: The mdr1 gene as well as its corresponding protein P-gp was highly expressed in DOX exposed K562 cells. DOX up-regulated the expression of YB-1 gene, and promoted YB-1 protein nuclear translocation. On YB-1 gene silenced, the expressions of mdr1 gene and P-gp were obviously down-regulated in DOX treated K562 cells. CONCLUSION: Doxorubicin can induce the expression of mdr1 gene in K562 cells, which may result from the transcription of mdr1 gene by activated YB-1.

[Influence of YB-1 protein on the biological behaviour in K562/A02 cells].

The aim of this study was to investigate whether the growth, apoptosis and sensitivity to anticancer agent could be altered after introduction of YB-1 shRNA eukaryotic expression vector into the K562/A02 cells, and its possible molecular mechanisms. The recombinant eukaryotic expression plasmids including YB-1 shRNA and the vector-random-sequence were introduced into K562/A02 cells by lipofectamine mediation, and the positive clones were screened by G418. RT-PCR and Western blot were employed to detect the expression of mRNA and protein of YB-1 in leukemia cells, respectively. The proliferative ability of the cells was determined by MTT assay and cell cycle analysis. Apoptosis of K562/A02 cells was assayed by AnnexinV-FITC/PI double labeled flow cytometry. The drug sensitivity to anticancer agent was determined by MTT assay. The expressions of MDR1 gene and P-gp were detected by RT-PCR and flow cytometry respectively. The results indicated that the levels of mRNA and protein of YB-1 decreased dramatically in three groups of positively transfected cells when compared with control cells. The inhibitory rates of 3 different shRNA sequences targeting YB-1 gene were (65.1 ± 2.1)%, (27.4 ± 1.3)% and (67.4 ± 1.6)% respectively. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased levels of the proliferative ability in K562/A02 cells, and displayed higher at G(1), lower at G(2) and S phase in cell cycle distribution in comparison with the control groups. AnnexinV/PI detection indicated higher AnnexinV(+) ratio in 3 groups of positively transfected cells after being treated with As(2)O(3) of 0.5 µmol/L for 24 hours. The IC(50) values of doxorubicin in 3 groups of positively transfected cells were significantly lower than that in control group. The level of MDR1 gene and P-gp decreased significantly in 3 groups of positively transfected cells. It is concluded that the transfection with YB-1 shRNA gene can inhibit the proliferation of leukemia cells and induce cell apoptosis. The expression of MDR1 mRNA and P-gp decrease after transfection of YB-1 shRNA into K562/A02 cells.

[Mechanisms of preventive effect of tetrandrine on acquired multidrug resistance in K562 cells].

This study was purposed to explore the mechanisms of preventive effect of tetrandrine (TTD) on doxorubicin (ADM)-induced multidrug resistance (MDR) in human leukemia cell line K562 from two aspects of the transcription control of MDR1 gene and cell apoptosis. The experiment was divided into 3 groups: group I-blank control; group II-ADM-induced drug-resistance; group III-ADM-induced drug-resistance after pretreatment with TTD. Reverse transcription-PCR (RT-PCR) was used to detect the mRNA expression levels of c-Jun, YB-1 and Survivin genes. Western blot was used to determine the nuclear protein expression levels of c-Jun and YB-1. Flow cytometry was used to assay the apoptosis of cells. The results showed that as compared with group I, the expression levels of c-Jun mRNA and nuclear protein decreased (p < 0.05), as well as the expression levels of YB-1 mRNA and nuclear protein increased in group II (p < 0.05). However, the expression of Survivin mRNA had no change (p > 0.05); the apoptosis rate of cells was 8.31%. As compared with group II, the expression levels of c-Jun mRNA and nuclear protein increased (p < 0.05), expression levels of YB-1 mRNA and nuclear protein as well as Survivin mRNA decreased in group III (p < 0.05). The apoptosis of cells was 97.2%. It is concluded that TTD can inhibit the expression of YB-1 and up-regulate the expression of c-Jun, thus inhibit the expression of MDR1 gene. TTD can also inhibit the expression of Survivin and increase the apoptosis of cells induced by ADM.

[VEGF shRNA enhances the sensitivity of multidrug-resistant leukemia cells to anticancer agent].

This study was aimed to explore the effect of vascular endothelial growth factor (VEGF) on sensitivity of leukemia cell line K562/A02 to doxorubicin by using RNA interference, and to investigate its mechanism. The 3 shRNA targeting human vegf gene were synthesized, then transfected into K562/A02 cells by lipofectamine 2000 reagent. RT-PCR was used to detect the expression of vegf and mrp1 at the mRNA level;Western blot was used to analyze the expression of VEGF, MRP1, AKT, P-AKT at the protein level; MTT was used to determine the IC(50) value of transfected cells to doxorubicin (DOX); flow cytometry was used to detect cell apoptosis and intracellular Rho123 retention. The results showed that after vegf shRNA were transfected into K562/A02 cells, the expression of vegf at the mRNA level decreased, and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was statistically significant (p < 0.05), the greatest decrease was observed in the cells transfected with vegf shRNA3; and the protein level of VEGF was also down-regulated. The IC(50) value of positively transfected group was lower than that of control groups, and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was significant (p < 0.05). The retention of intracellular Rho123 was enhanced in three positively transfected groups (p < 0.05). Cell apoptosis increased in positively transfected groups, and there was statistically difference between vegf shRNA2 group or vegf shRNA3 group and HK group (p < 0.05). The expression of mrp1 at the mRNA level were decreased, and there were statistical difference between vegf shRNA3 group and HK group (p < 0.05), and the protein level of mrp1 was also down-regulated; the expression of P-AKT at protein level decreased in positively transfected groups, and the greatest decrease was seen in vegf shRNA3 group. It is concluded that the transfection with exogenous vegf shRNA can inhibit the expression of vegf at both mRNA and protein levels, and enhance the sensitivity of K562/A02 cell to doxorubicin, the mechanism of which may be the inhibition of apoptosis and down-regulation of MRP1 by inactivating PI3K/AKT signaling pathway.

Preventing chemoresistance of human breast cancer cell line, MCF-7 with celecoxib.

PURPOSE: To investigate the preventive effect of celecoxib, a specific cyclooxygenase-2 (Cox-2) inhibitor, on the development of chemoresistance in breast cancer cell line, MCF-7, and explore the mechanism of the action. METHODS: Chemoresistance phenotype was established by treating MCF-7 cells with 0.05 µg/ml doxorubicin for 7 days, and then the effect of preventive chemoresistance was investigated by the combination of same dose of doxorubicin with 10 µM celecoxib. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay was used to assess cytostatic efficacy of doxorubicin and 50% inhibiting concentration (IC(50)) of MCF-7 cells. RT-PCR was performed to examine mRNA expression of multidrug resistance gene 1 (MDR1) and its transcription factors c-Jun and NF-κB. Western blotting analysis was performed to detect the expression of protein. Flow cytometry (FCM) was applied to analyze the expression and function of P-glycoprotein (P-gp). Electrophoretic gel mobility shift assay (EMSA) was performed to determine the DNA-binding activity of nuclear transcription factors AP-1 and NF-κB. RESULTS: Compared with sensitive MCF-7 cells, MDR1 and its transcription factors c-Jun and NF-κB were up-regulated at both mRNA level (P < 0.01) and protein level (P < 0.01) by treatment with 0.05 µg/ml doxorubicin for 7 days. After co-incubation with both the same dose of doxorubicin and 10 µM celecoxib for 7 days, both mRNA level and protein level of MDR1, c-Jun and NF-κB up-regulated by doxorubicin were partly reversed (P < 0.01); DNA-binding activity of nuclear transcription factors AP-1 and NF-κB were inhibited; and the function of P-gp was decreased (P < 0.01). When MCF-7 cells were treated with increasing doses of doxorubicin in the presence of 10 µM celecoxib, IC50 value of doxorubicin and doxorubicin plus 10 µM celecoxib was 0.67 ± 0.03 and 0.38 ± 0.04 µg/ml, respectively (P < 0.01). CONCLUSION: Celecoxib effectively prevents the development of chemoresistance in breast cancer cell line MCF-7 induced by doxorubicin, which was partly involved in inhibiting the expression and DNA-binding activity of nuclear transcription factors AP-1 and NF-κB and downstream expression and function of P-gp. Furthermore, cytostatic efficacy of celecoxib and doxorubicin on MCF-7 cell was synergistic.

[CD44 variant increases the invasive ability of human breast cancer cell line MCF-7 cells].

OBJECTIVE: To evaluate the impact of a new CD44 variant on invasion of human breast cancer cell line MCF-7, and its possible mechanisms. METHODS: The full length cDNA encoding CD44v17 was obtained from the total RNA isolated from the MCF-7/ADR cells by reverse transcript-polymerase chain reaction (RT-PCR) and subcloned into pMD19-T vector. The CD44v17 gene sequence and reading frame were confirmed by two restriction enzymes and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44v17 was transfected into MCF-7 cells by Lipofectamine. The changes of MMP-2 and MMP-9 expression at gene and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells through the artificial matrix membrane in every group was counted to compare the change of the invasive ability regulated by CD44 variant. The ERK and p-ERK were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by CD44 variant. RESULTS: The new gene sequence was successfully cloned into recombinant vector pcDNA3.1 and identified by the two restriction enzymes. It was confirmed that the reconstructed plasmid contained the sequence of CD44 gene variant which was composed of 1 to 4 exons, 16 to 17 exons, and 1 to 205 bases of 18 exons. The new gene sequence was sent to NCBI for publication and obtained the registered number FJ216964. The up-regulated levels of the CD44 gene mRNA and protein were respectively detected by RT-PCR and flow cytometry in MCF-7 cells transfected with pcDNA3.1-CD44v17. The invasiveness of the cells and the activity of MMP-2 and MMP-9 were clearly activated by hyaluronic acid (HA) treatment and blocked by CD44 neutralizing antibody. Pretreated MCF-7/CD44v17 cells with the neutralizing antibody against CD44 and the inhibitor of MAPKs signaling pathway strongly block the expression of p-ERK. CONCLUSION: A new CD44 gene variant has been found in adriamycin-resistant human breast cancer MCF-7/ADR cells. The expression vector pcDNA3.1-CD44v17 has been cloned and constructed successfully. HA can be integrated with CD44 variant and then regulates the expression of MMP-2 and MMP-9, which increases the invasion ability of MCF-7 cells through the Ras/MAPK signaling pathway.

[Effect of YB-1 gene knockdown on human leukemia cell line K562/A02].

OBJECTIVE: To investigate the potential effects of YB-1 gene knockdown on gene expression profile, cell growth and apoptosis in leukemia cell line K562/A02. METHODS: The recombinant eukaryotic expression plasmid containing YB-1 short hairpin RNA (shRNA) or random-sequence (HK) were transfected into K562/A02 cells by lipofectamine mediation. cDNA microarray was performed to explore the alteration of gene expression profile when YB-1 gene expression was decreased. Expression of CARD8 and RHOC genes were verified by semi-quantitative reverse transcription-PCR (RT-PCR). The proliferative ability of the cells was determined by methyl thiazolyltetrazolium (MTT) assay and cell cycle analysis. Cell apoptosis was assayed by Annexin V-FITC/PI double labeled flow cytometry. RESULTS: The levels of YB-1 mRNA and protein decreased dramatically in three positively transfected cells when compared with untransfected K562/A02 cells or K562/A02-HK thansfected cells. Gene expression profile was altered by transfection of YB-1 shRNA into K562/A02 cells. Among 47,000 genes on the microarray, 252 genes were detected to have changes, with 143 down-regulated and 109 up-regulated. They were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, etc. An increase in CARD8 gene expression and a decrease in RHOC gene expression have been confirmed by RT-PCR in K562/A02-YBX13 cells. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased proliferation, higher G1, lower G2 and S ratio in cell cycle distribution in comparison with the control groups. Annexin V/PI detection indicated higher Annexin V+ ratio in the three positively transfected cells 24 hours after cells were treated with 0.5 micromol/L of As2O3. CONCLUSION: Down-regulation of YB-1 gene by shRNA-YB-1 can alter the gene expression profile in K562/A02 cells, leading to change of cell proliferation and apoptosis.

[Effect of tetrandrine on the doxorubicin-induced expression of mdr1 gene in K562 cells].

OBJECTIVE: To investigate the effect of tetrandrine (TTD) on doxorubicin-induced mdr1 gene expression and its mechanism. METHODS: MTT assay was used to detect the cytotoxicity of TTD to K562 cells. K562 cells were treated with doxorubicin alone or 0.6 microg/ml doxorubicin combined with various concentrations of TTD. RT-PCR was used to detect the mRNA expression of mdr1 and NF-kappa B. Flow cytometry was used to assay the expression of P-glycoprotein (P-gp). Intracellular rhodamine 123 (Rho123) retention assay was applied to test the P-gp function. RESULTS: After treatment with 0.6 microg/ml doxorubicin for 24 hours, the expressions of mdr1 mRNA, NF-kappa B mRNA and P-gp in K562 cells were increased from 0.171 +/- 0.012, 0.783 +/- 0.090, 7.85 +/- 0.15 to 0.428 +/- 0.012, 1.075 +/- 0.047 and 73.68 +/- 1.84, respectively. The intracellular Rho123 retention was decreased from 711.9 +/- 63.6 to 347.8 +/- 60.6, indicating up-regulation of P-gp function (P<0.05). Pretreatment of K562 cells with 2.0 microg/ml TTD for 24 hours and then incubated for another 24 h with doxorubicin, the expressions of mdr1 mRNA, NF-kappa B mRNA, P-gp and up-regulation of P-gp function induced by doxorubicin were prevented in K562 cells (0.148 +/- 0.006, 0.627 +/- 0.098, 7.18 +/- 0.38 and 799.7 +/- 45.8, respectively P<0.05). But 0.5 microg/ml and 1.0 microg/ml TTD had little effect. CONCLUSIONS: TTD inhibits the expression of mdr1 mRNA, P-gp and up-regulated P-gp function induced by doxorubicin in a dose dependent manner. The mechanism of this effect may be down-regulation of NF-kappa B by TTD.

Different roles of pummelo furanocoumarin and cytochrome P450 3A5*3 polymorphism in the fate and action of felodipine.

OBJECTIVE: Herein we aim to test if pummelo furanocoumarins can inhibit cytochrome P450 (CYP) 3A both in vitro and in vivo, and to explore the influence of CYP3A5*3 (GenBank AC005020: A22893-->G) polymorphism in the pharmacokinetics and pharmacological response to felodipine. METHOD: Fruit juices of pummelo grapefruit (Citrus paradisi Macf., G), 'Guanximiyou' (C. grandis Osbeck vs. Guanxi, P) and 'Changshanhuyou' (C. changshanhuyou Y.B. Chang, H) were selected by screening Citrus fruit juices for their furanocoumarin contents and their inhibition of testosterone 6beta-hydroxylation in human liver microsomes. Twelve healthy male Chinese were administered 250 mL G, P, H or water (W) alternatively with 26-mumol (10-mg) plain tablet felodipine, and were observed for 12 h. RESULTS: G had more furanocoumarins and at higher levels than P while H had none, and their potencies for in vitro CYP3A inhibition were in the order as G > P > H. The geometric mean and 90% confidence intervals of pharmacokinetic parameters for human oral felodipine with G, P, H and W were respectively as follows: peak plasma concentration (nmol.L(-1)), 37 (32-44), 25 (21-29), 19 (16-22) and 18 (15-21); area under the plasma concentration-time curve (nmol.h.L(-1)), 118 (103-136), 84 (73-97), 64 (56-74) and 59 (51-68). Subjects showed higher heart rates with G than with H or W. CYP3A5*3 polymorphism showed no significant effect on felodipine pharmacokinetics and related hemodynamic changes. CONCLUSIONS: This work supports the hypothesis that CYP3A inhibition by furanocoumarins caused pummelo fruit juice-drug interaction; while the role of CYP3A5 in the population pharmacokinetics of felodipine and blood pressure response appear to be limited.