[Molecular mechanism of HL-60 cell apoptosis induced by baicalin].

This study was aimed to investigate the effect of baicalin on proliferation and apoptosis of HL-60 cells and its mechanism. Cell proliferation was assayed by using Cell Counting Kit-8. The morphological changes of HL-60 cells were examined by light microscopy and nucleolus morphological changes were observed by fluorescent microscopy after Hoechst 33342 staining. The early cell apoptosis was detected by using flow cytometry with Annexin V-FITC/PI double staining. The expression of caspase-3, caspase-9, Bcl-2 and Bax mRNA was detected by RT-PCR and Western blot assay was carried out to examine Bax, Bcl-2, caspase-8 and cleaved caspase-3 expression. The results showed that Baicalin inhibited the proliferation of HL-60 cells in a time- and concentration-dependent manner. HL-60 cells exhibited typical morphological features (for example, cell shrinkage, membrane blebbing and formation of apoptotic bodies). Cell apoptosis in early stage could be detected, the expression of caspase-3, caspase-9 and Bax mRNA was obviously up-regulated, while the Bcl-2 expression down-regulated, and accordingly Bcl-2/Bax ratio decreased. Such results were consistent with the expression of these proteins. In addition, the expression of cleaved caspase-8 protein was induced significantly after treated with baicalin. It is concluded that baicalin can significantly inhibit the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells, which may occur through decreasing Bcl-2/Bax ratio by intrinsic pathway and through extrinsic pathway. It suggests that baicalin may be a promising drug for the therapy of acute myeloid leukemia.

Inhibition of C35 gene expression by small interfering RNA induces apoptosis of breast cancer cells.

C35 was reported to be a new biomarker and therapeutic target for breast cancer. To explore the functional importance of C35, we constructed small interfering RNA (siRNA) targeting C35 and investigated the effects of the siRNAs on C35 expression and apoptosis of T47D cells. C35 siRNAs were constructed and named psiRNA-C35-1 and psiRNA-C35-2. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blots were used to detect the effects of the siRNAs on mRNA and protein expression of C35 in T47D cells. The effects of the two siRNAs on apoptosis of T47D cells were detected by flow cytometry and terminal dUTP nicked-end labelling assays. Also, the apoptosis related molecule caspase-3 was detected using Western blots. The psiRNA-C35-1 and psiRNA-C35-2 siRNAs were verified by both EcoR I/Hind III digestion analysis and automated DNA sequencing. RT-PCRs and Western blots showed that C35 mRNA and protein expression in T47D cells were obviously inhibited after psiRNA-C35-1 and psiRNA-C35-2 transfection. Flow cytometry and terminal dUTP nicked-end labelling assays showed that apoptosis of T47D cells was significantly induced after transfection with psiRNA-C35-1 and psiRNA-C35-2 (p < 0.05). Also, caspase-3 expression in the psiRNA-C35-1 and psiRNA-C35-2 transfected cells was obviously higher than that of the Lipofectamine and pTZU6+1 transfected cells. This study showed that apoptosis of T47D cells can be significantly induced by inhibiting C35 expression using siRNAs, which may be caused by activating caspase-3. C35 might play an important role in apoptosis of breast cancer cells, and therapeutic strategies targeting C35 may be useful for breast cancer treatment.

[Molecular mechanism underlying differentiation of HL-60 cells induced by hexamethylene bisacetamide].

The objective of this study was to investigate the effect of hexamethylene bisacetamide (HMBA) on differentiation of HL-60 cells and its possible molecular mechanism. HL-60 cells were co-cultured with different concentrations of HMBA (0.5, 1, 2 mmol/L) for 4 days, then the proliferation was assayed by MTT at different time points. Wright-Giemsa staining was used to observe the change in morphology. Cell differentiation antigen CD11b expression and the altered distribution of cell cycle in HL-60 induced by HMBA were analyzed by flow cytometry. The expressions of c-myc, mad1, p21, p27, hTERT and HDAC1 mRNA were detected by RT-PCR. The results indicated that the proliferation of HL-60 cells was inhibited by HMBA in a time-and-dose-dependent manner. Upon 2 mmol/L HMBA treatment, the HL-60 cells arrested at G(0)/G(1) phase and differentiated into granular line in morphology, with the up-regulation of CD11b expression. The expression of c-myc and hTERT mRNA obviously down-regulated, the expression of p21, p27 and mad1 mRNA up-regulated, while there was no change of the expression of hTERT mRNA. It is concluded that effect of HMBA on the differentiation of HL-60 cells may partly contribute to switch from c-myc to mad1 expression, to up-regulate expressions of p21 and p27 mRNA, and down-regulate hTERT mRNA expression, while there is no relation with the expression of HDAC1 mRNA.

[Relationship between susceptibility of formaldehyde metabolism and genetic polymorphisms of ALDH2 and cytochrome P4502E1].

OBJECTIVE: To study the relationship between occupational hazard susceptibility of formaldehyde and genetic polymorphisms of ALDH2 and CYP2E1. METHODS: Genotypes of ALDH2 and CYP2E1 (Rsa I/Pst I site) of 107 subjects exposed to formaldehyde were determined with PCR-RFLP through testing peripheral blood lymphocytes, and the concentration of air formaldehyde in workplace and urine formic acid of the subjects were measured with HPLC. The relationship between genotypes and the urine formic acid increment was analyzed with nonparametric rank sum testing. RESULTS: The concentration of urine formic acid increment was related with ALDH2 genotypes (chi2 = 9.241, P < 0.05), and the means of urinary formic acid of subjects with GG, GA, AA genotype were (15.84 +/- 6.86), (12.06 +/- 7.94) and (7.31 +/- 5.37) mg/g creatinine, respectively. Mann-Whitney U test showed the formic acid increment between allele G homozygotes and allele A homozygotes was significantly different (U=26, P= 0.033). Our data indicated that the formaldehyde metabolism of ALDH2 GG homozygotic genotype was more active than ALDH2 AA homozygotic genotype(the difference of the two mean rank was 13.30). But the polymorphism of Rsa I / Pst I site of CYP2E1 5'-franking region was not correlated with the concentration of urine formic acid (chi2 = 4.285, P=0.117), and the urinary formic acid means of subjects with C1/C1, C1/C2, C2/C2 genotype were (11.14 +/- 7.91), (12.13 +/- 8.16) and (16.51 -/+ 3.78) mg/g creatinine, respectively. By Stepwise Multiple Regression Analysis, it showed that the urinary formic acid increment might be influenced by FA exposure concentration and ALDH2 genotype, and the model's R2 was 0.196. CONCLUSION: The metabolism of formaldehyde in human body was related with the genotypes of ALDH2, but not with the CYP2E1 (Rsa I/Pst I) polymorphisms.

Down-regulation of CD44 contributes to the differentiation of HL-60 cells induced by ATRA or HMBA.

CD44 is highly expressed in human acute myeloid leukemia (AML) cells. Some experiments had shown that it was possible to reverse differentiation blockage in AML cells by CD44 ligation with specific antibodies, indicating that CD44 was closely related to the differentiation of leukemia cells. The differentiation of acute promyelocytic leukemia cell line HL-60 cells could be induced by all trans-retinoic acid (ATRA) and hexamethylene bisacetamide (HMBA), but so far the mechanism was not demonstrated clearly. In the present study, we investigated whether ATRA or HMBA induced the growth arrest of HL-60 cells by down-regulating the expression of CD44. The results indicated that the proliferation of HL-60 cells was obviously inhibited and the differentiation was induced by both ATRA and HMBA. The decreased expression of CD44 and cyclin E mRNA, and the increased expression of p27 and p21 at mRNA levels were observed. Furthermore, there was a negative correlation between the expression of CD44 and p27. It was concluded that ATRA and HMBA played a role in the differentiation induction of HL-60 cells, which was mediated by the down-regulation of CD44, accompanied by down-regulation of cyclin E, and up-regulation of p27 and p21 mRNA.

[A study on K469E polymorphism of ICAM1 gene and ICAM1 plasma level in patients with coronary heart disease].

OBJECTIVE: To study the linkage between K469E polymorphism of intercellular adhesion molecule 1(ICAM1) gene with ICAM1 plasma level and coronary heart disease (CHD) in Han population of China. METHODS: One hundred and sixty-four controls without CHD and 160 patients with CHD were enrolled in our study. By nested PCR with allele-specific oligonucleotide primers, all patients and controls were genotyped for the ICAM1 polymorphism. And the ICAM1 plasma level was measured by ELISA. RESULTS: In the patients with CHD, both K allele frequency and the plasma level of ICAM1 were higher than those in control (P<0.05). The individual with K allele had higher plasma level of ICAM1 than that without K allele (344.34+/-128.59 microg/L vs 303.54+/-108.74 microg/L, P=0.008). K allele enhanced the risk of CHD (P<0.01, OR=2.158, 95%CI: 1.250-3.727). There was the K allele cooperation with smoking in influencing the risk of CHD. CONCLUSION: There is the polymorphism of ICAM1 K469E gene in Han population of China, and the K allele may be a genetic factor influencing the risk of CHD.

[Plasma activated coagulation factor VII and Msp I polymorphism in elderly patients with coronary heart disease].

OBJECTIVE: To investigate the association of activated coagulation factor VII(F7a) and its gene Msp I polymorphism with coronary heart disease in elderly patients. METHODS: This was a case-control study, and the method of candidate gene was adopted. F7 genotypes were identified with polymerase chain reaction amplified genomic deoxyribonulieic acid (DNA) and Msp I restriction fragment length polymorphism analysis, and the level of plasma F7a was detected with recombinant tissue factor method for 108 elderly patients with coronary heart disease and 120 sex- and age-matched healthy control subjects. RESULTS: (1) Plasma F7a levels was significantly higher in elderly patients with coronary heart disease than in healthy control subjects (2.88 +/- 0.62 vs 2.58 +/- 0.60 microg/L, P < 0.05), and was significantly higher in old myocardial infarction than in stable angina pectoris (3.12 +/- 0.62 vs 2.76 +/- 0.60, P < 0.05). F7a was shown to be a risk factor for coronary heart disease in elderly patients by Logistic regression analysis (OR=1.21 P < 0.05). (2) The allelic frequencies were in accordance with Hardy-Weinberg equilibrium. The results suggested that the distribution of genotype and allelic frequencies in the groups displayed no significant difference, and there was no difference between the subgroups of coronary heart disease in elderly patients, either (P > 0.05). (3) F7a level was significantly higher in RR genotype than in Q allele carriers (2.72 +/- 0.60 vs 1.98 +/- 0.59 microg/L, P < 0.05) and was associated with F7 gene polymorphism. CONCLUSION: Plasma F7a level may be an independent risk factor of coronary heart disease in elderly patients, and it may be influenced by the Msp I polymorphism of F7 gene.

[The impact of gender factor on the candidate gene study of essential hypertension].

OBJECTIVE: To study the impact of gender factor on the candidate gene study of essential hypertension (EH). METHODS: The polymerase chain reaction (PCR) was performed to analyze the ACE gene I/D polymorphism of hypertensive patients (50 men and 50 women) and normal controls (50 men and 50 women). The investigation was further focused on possible influence of sex proportion on the conclusion of this kind of research. RESULTS: The frequency of DD genotype in male hypertensive patients is significantly higher than that in male normal controls (chi(2) = 6.98, P = 0.004). The frequency of D allele in male EH group is significantly higher than that of male normal controls (chi(2) = 6.87, P = 0.009), while no significant difference was observed for II and ID genotype between male EH group and control group (P > 0.05). For female EH group and normal controls, there were no significant differences in frequency of genotype and allele (P > 0.05), the distribution ratio of DD genotype in male EH group is significantly different from that of female EH group (chi(2) = 4.06, P = 0.044). Furthermore, males with DD genotype in EH group had higher SBP and PP than that of males with II and ID genotype (P < 0.05). However, there was no significant difference in DBP in all three genotypes (P > 0.05). At the same time, there was no difference in SBP, DBP and PP (P > 0.05) between II and ID genotype in male EH group. In female hypertensive patients, there was no significant difference in SBP, DBP and PP between all three genotypes (P > 0.05). CONCLUSIONS: The relationship between DD genotype in male and EH (especially SBP and PP) is closer than any other genotype-EH relationships in both male and female. The gender factor, as a probable confounding factor, can affect many candidate gene studies of essential hypertension including ACE gene I/D polymorphism, and thus biases the conclusion.

[Relationship between fibrinogen B beta gene FGB -455G/A polymorphism and atherosclerotic cerebral infarction].

OBJECTIVE: To explore the distribution of fibrinogen (FGB) B beta polymorphism in Chinese Han population and the association of the polymorphisms with the occurrence of atherosclerotic cerebral infarction (ACI). METHODS: The B beta gene FGB -455G/A polymorphism was identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) in 132 patients with ACI and 148 healthy controls matching on age and sex. Turbidimetric assays were performed to measure the plasma fibrinogen levels of all cases. RESULTS: The plasma fibrinogen level in ACI group (3.42+/-0.52 g/L), was significantly higher than that in the controls (2.96+/-0.42g/L), P<0.001. The A allele was associated with the elevated plasma fibrinogen levels in both patients and controls. Among the A allele carriers, smokers had significantly higher plasma fibrinogen levels than did the non-smokers (P<0.05). The distribution of B beta gene FGB -455G/A polymorphism was in accordance with the Hardy-Weinberg equilibrium (P>0.05). The A allelic frequency in ACI group (0.258) was significantly higher than that in the control group (0.152) (P<0.05). Logistic regression analysis showed that the cases carrying A allele (GA+AA genotype) had 1.653 times the risk of ACI. CONCLUSION: The study demonstrates that A allele of the B beta gene FGB -455G/A polymorphism may be a susceptible predictor of the occurrence of ACI, particularly in smokers.