Role of DNA damage response in cyclophosphamide-induced premature ovarian failure in mice.

AIM: To investigate the DNA damage response (DDR) in a cyclophosphamide (CTX)-induced mouse model of premature ovarian failure (POF). METHODS: The POF model was established by injecting mice with CTX. The body, ovarian weights, the estrus cycle, and pathological changes of the ovaries were recorded. The serum levels of 17 ß-estradiol (E2) and follicle-stimulating hormone (FSH) were measured. The expression of Ki67, ß-galactosidase (ß-gal), p21, p53, γH2AX, and pATM in ovarian tissues was detected by immunohistochemistry. The expression of ß-gal, γH2AX, and pATM was analyzed by immunofluorescence staining of primary cultured granulosa cells (GCs). RESULTS: The body and ovarian weights decreased, the estrus cycles were erratic, and the FSH level increased, whereas the E2 level decreased in POF mice compared to controls. The pathological consequences of POF revealed an increase in atretic follicles, corpus luteum, and primordial follicles and a decrease in the number of primary, secondary, and tertiary follicles. Ki67 expression was reduced, ß-gal, p21, p53, γH2AX, and pATM expression were elevated in the ovaries of POF mice. The expression of ß-gal, γH2AX, and pATM increased in GCs with the concentration in a time-dependent manner. CONCLUSION: In total, CTX induced POF in mice, which was mediated by the DDR pathway of ATM-P53-P21.

Induction of H2AX phosphorylation in tumor cells by gossypol acetic acid is mediated by phosphatidylinositol 3-kinase (PI3K) family.

BACKGROUND: H2AX is phosphorylated (γH2AX) by members of the phosphatidylinositol 3-kinase (PI3K) family, including Ataxia telangiectasia-mutated (ATM), ATM- and Rad3-related (ATR) and DNA-PK in response to DNA damage. Our study shows that gossypol acetic acid (GAA) alone can induce γH2AX in Human mucoepidermoid carcinoma cell line (MEC-1) in vitro. Thus, we further examined the possible mechanisms of GAA to induce γH2AX in tumor cells. MATERIALS AND METHODS: The PI3K inhibitors caffeine and wortmannin were used in an effort to identify the kinase(s) responsible for GAA -induced γH2AX in MEC-1 cells. DNA dependent protein kinase (DNA-PK) - proficient and -deficient cells, human glioma cell lines M059K and M059J, were also used to evaluate the kinases responsible for GAA induced H2AX phosphorylation. γH2AX expression was detected by immunofluorescent microscopy. Flow cytometry assay was used to assay γH2AX and cell cycle. RESULTS: GAA induced H2AX phosphorylation in a cell cycle-dependent manner and a significant G0/G1 phase arrest in MEC-1 cells was shown. Caffeine and wortmannin significantly inhibited GAA-induced H2AX phosphorylation in MEC-1 cells. GAA induced H2AX phosphorylation in M059K, but not in M059J. Taken together, these data suggested that GAA treatment alone could induce H2AX phosphorylation in a cell cycle dependent manner in MEC-1 and M059K, but not in M059J cells. A significant G0/G1 phase arrest was shown in MEC-1. CONCLUSIONS: The member of PI3K family, DNA-PK, ATM and ATR are involved in the H2AX phosphorylation of MEC-1 cells.

[Gossypol acetic acid induces DNA double-strand breaks in human mucoepidermoid carcinoma cell MEC-1].

The present study was conducted to investigate the effects of gossypol acetic acid (GAA) on the proliferation of human mucoepidermoid carcinoma cell line MEC-1 in vitro and its possible molecular mechanisms of DNA double-strand breaks (DSB). MTT assay was performed to test the inhibition of proliferation of MEC-1 cells by GAA. DSB and γH2AX foci formation induced by GAA were detected by neutral comet assay and immunostaining. GAA (5-40 µmol/L) inhibited the growth of MEC-1 cells in a dose- and time-dependent manner. One of the indexes of comet assay, percentage of head DNA was decreased, however other indexes, including tail length, percentage of tail DNA, tail moment (TM) and Olive tail moment (OTM) were increased when treated with 2.5- 40 µmol/L GAA for 24 h or 20 µmol/L GAA for 3-48 h, compared with those in control. The percentage of γH2AX-positive cells was also increased when MEC-1 was treated with 2.5-20 µmol/L GAA for 24 h or 20 µmol/L GAA for 3-48 h, compared with that in control. All these results show that GAA inhibits the proliferation of MEC-1, and DSB maybe one of the mechanisms of inhibitory effect of GAA on the growth of tumor cells.

[Effects of matrine on airway inflammation and early airway remodeling in asthmatic mice].

OBJECTIVE: To observe the influence of matrine on airway inflammation and early airway remodeling in asthmatic mice. METHODS: Fifty BALB/c mice were randomly divided into 5 groups: a normal control group (A), an asthmatic group (B), a dexamethasone (DXM) group (C, 2 mg/kg), a high-dose matrine group (D, 50 mg/kg) and a low-dose matrine group (E, 25 mg/kg). The mice model of asthma in the B, C, D, and E groups was established by ovalbumin (OVA) intraperitoneal injections and aerosolization. Intra-gastric administration of different medications in C, D, E groups and 0.9% sodium chloride in B group were carried out 1 hour before provocation. 0.9% sodium chloride was used for intraperitoneal injection, aerosolization and intra-gastric administration in group A. The lung tissue slices were stained, and then the grade of inflammation around the wall of bronchi, mucous secretion, and the percentage of goblet-cells were counted. The areas of bronchial smooth muscle and of collagen deposition in airway wall were analyzed. The transcriptions and protein expressions of transforming growth factor-beta(1) (TGF-beta(1)) and connective tissue growth factor (CTGF) were measured respectively by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: In the A, B, C, D, E groups, the grades of inflammation were 1.5 (1, 2), 4 (4, 5), 2 (1, 3), 2 (2, 3), 3 (2, 3.3), respectively; the degrees of mucous secretions were 1.5 (1, 2), 5 (4, 6), 2 (1, 3), 2 (2.5, 4), 3 (3, 4), respectively. These airway inflammatory parameters in group B were significantly higher than in group A (chi(2) = 21.3, 22.6, P all < 0.01), while they were remarkably decreased in group C compared to group B (chi(2) = 13.3, 15.0, P all < 0.01). These parameters in group D and group E were also lower than those in group B (chi(2) = 9.1, 10.9; 9.8, 9.7; P all < 0.05). The percentage of goblet cells in airway epithelium was (1.7 +/- 0.5)%, (54.7 +/- 15.5)%, (20.4 +/- 5.9)%, (31.7 +/- 7.6)% and (36.2 +/- 10.8)%, respectively; it was significantly higher in group B than in group A (t = 12.0, P < 0.01), and remarkably lower in groups C and D than in group B (t = 7.7, 5.1, P all < 0.01), and lower in group E than in B group (t = 4.2, P < 0.05). In these 5 groups, the area of bronchial smooth muscle was (11.5 +/- 2.1) microm(2)/microm, (30.0 +/- 3.3) microm(2)/microm, (15.2 +/- 3.1) microm(2)/microm, (22.2 +/- 4.8) microm(2)/microm and (26.5 +/- 3.4) microm(2)/microm, respectively; it was significantly higher in group B than in group A (t = 11.4, P < 0.01), and remarkably lower in groups C, D and E than in group B (t = 9.1, 4.7, 2.2, P all < 0.01). The area of collagen deposition was (3.9 +/- 1.8) microm(2)/microm, (24.4 +/- 6.1) microm(2)/microm, (15.4 +/- 3.5) microm(2)/microm, (16.6 +/- 6.0) microm(2)/microm and (17.5 +/- 4.4) microm(2)/microm, respectively; it was also significantly higher in group B than in group A (t = 9.3, P < 0.01), and remarkably lower in groups C and D than in group B (t = 4.1, 3.5, P all < 0.01), and lower in group E than in B group (t = 3.2, P < 0.05). The mRNA levels of TGF-beta(1) were 160 +/- 25, 247 +/- 37, 174 +/- 23, 195 +/- 25 and 207 +/- 42, respectively, and those of CTGF were 86 +/- 8, 160 +/- 24, 94 +/- 10, 93 +/- 14 and 104 +/- 10, respectively in the 5 groups. The levels were remarkably increased in group B, as compared to group A (t = 6.1, 11.6, P all < 0.01), and the levels in groups C, D and E were remarkably decreased, as compared to group B, the difference being significant (t = 3.7, 2.7, 5.1; 10.6, 8.6, 10.3; P all < 0.01). The protein level of TGF-beta(1) in lung tissues was 21 +/- 5, 36 +/- 8, 26 +/- 5, 26 +/- 5 and 26 +/- 5, respectively, and that of CTGF was 15 +/- 4, 27 +/- 5, 21 +/- 4, 22 +/- 3 and 23 +/- 4, respectively in the 5 groups. The levels in B group were significantly increased, as compared to group A (t = 5.7, 6.4, P all < 0.01), and those in groups C and D were significantly decreased (t = 3.9, 3.9; 3.2, 2.8, P all < 0.01), and that in group E was also lower (t = 3.8, 2.5, P all < 0.05), as compared to group B. In all the groups, the protein levels of TGF-beta(1) and CTGF were positively correlated with the area of bronchial smooth muscle and with the area of collagen deposition (r = 0.435, 0.583, 0.522, 0.590, P all < 0.01). CONCLUSIONS: Matrine inhibited airway inflammation and early airway remodeling in asthmatic mice. The signal transduction of TGF-beta(1) and CTGF maybe involved.