For patients who are treated with anti-cancer drugs,DNA repair is often a critical mechanism,which determines therapeutic efficacy and/or adverse biological consequences such as mutagenesis,carcinogenesis and teratogenesis. This brief review is focused onto DNA repair activities on damage resulting from exposure to methylating anticancer drugs. Although these drugs unselectively target DNA from cancer and normal cells,the cancer-specific killing effect is likely due to downregulation of specific DNA repair activities,thereby killing more cancer than normal cells. The monofunctional alkylating agents exhibit methylating properties (procarbazine,dacarbazine,streptozotocine, temozolomide) or they chloroethylate the DNA forming monoadducts and,in a second step,DNA interstrand crosslinks (lomustine,nimustine,carmustine,fotemustine). A major mechanism of defense of cancer cells to these drugs is direct reversal of the critical DNA damage,O⁶-methylguanine as well as O⁶-chloroethylguanine,by the suicide enzyme O⁶-methylguanine-DNA methyltransferase (MGMT),which represents an important drug resistance marker especially in high-grade malignant gliomas. Since MGMT has a significant impact on the outcome of anti-cancer therapy,it is a predictive marker of the effectiveness of methylating anticancer drugs,and clinical trials are underway analyzing the influence of MGMT inhibition on the therapeutic success. Other DNA repair factors involved in methylating drug resistance are mismatch repair,DNA double-strand break (DSB) repair by homologous recombination and DSB signaling. Base excision repair and alkB homologous proteins (such as ABH2) might also contribute to alkylating drug resistance notably in cells expressing a high amount of MGMT. Better understanding of these mechanisms will be helpful in designing more effective therapies that have less adverse outcomes.

OBJECTIVE: Melanoma usually has poor prognosis,thus the aims of this study were to find potential targets of melanoma in serum and to understand their roles in the progression of melanoma. METHODS: Serum samples from patients and healthy controls were collected. From the samples,IgG and albumin were removed,and proteins were digested and labeled with iTRAQ labeling reagent. Differentially expressed serum proteins were identified by LC-MS/MS and validated by ELISA. TGF-β1 overexpressed melanoma cells were constructed using PLVX plasmid and lentivirus transfection. Cell apoptosis were analyzed by flow cytomeric assay. RESULTS: 81 melanoma-associated proteins were identified,33 were up-regulated and 49 were down-regulated. TGF-β1 was found significantly down-regulated (3.17±0.76,P<0.05) in melanoma patients' serum and validated by ELISA. TGF-β1 overexpressed melanoma cells (A-375 and SK-MEL-1) were constructed,the apoptosis analysis showed that TGF-β1 could significantly induced apoptosis of melanoma cells. CONCLUSION: TGF-β1 was reduced in melanoma patients' serum and was involved with inhibition of tumor cell proliferation. Thus,the findings suggest that TGF-β1 could be an important potential target in melanoma.

OBJECTIVE: To investigate the differential expression level of Gadd45α in human gastric cancer with Helicobacter Pylori (HP) infection status and the clinical significance of the expression for improvedrclinical diagnosis and treatment for gastric cancer. METHODS: 70 paraffin-embeded samples of gastric cancerwith HP infection status and 56 normal tissues from surgically resected tissues were selected at random. The expressions of Gadd45α protein in the samples were detected by immuno-histochemical staining. The relationship between the positive expression of Gadd45α protein and the clinico-pathological parameters of gastric cancer was analyzed. Furthermore,expression levels of Gadd45α mRNA and protein in 8 fresh gastric cancer specimens were detected using Western blot and RT-PCR. RESULTS: Immuno-histochemical analyses showed that the positive expression rate of Gadd45α protein in HP⁺ gastric carcinoma tissues,HP^- gastric carcinoma tissues and HP⁺ adjacent non-neoplastic tissues were 77.5%,46.67% and 34.21%,respectively. Their differences were significant (P<0.05). The expression rate in gastric carcinoma tissues was 64.29% and it was correlated with tumor size. The expression of Gadd45α protein in gastric carcinoma was positively correlated with HP infection status (r_s=0.318,P<0.05) by a correlated statistical analysis of Spearman's Rho. Western blot and RT-PCR detection results showed that HP+gastric cancer tissues had a higher expression in protein and mRNA levels of Gadd45α in fresh specimens than in adjacent non-neoplastic tissues. CONCLUSION: Expression of Gadd45α protein was up-regulated,especially in HP⁺ gastric carcinomas. The expression level was closely related to the occurrence and development of gastric carcinoma (especially HP+). The detection of the expression of Gadd45α protein was helpful for early diagnosis and for prediction of prognosis of gastric carcinoma. The expression of Gadd45α protein and Helicobacter Pylori infection status in gastric carcinoma were positively correlated. This was probably correlated with the development of gastric carcinoma. The prognosis of HP+gastric carcinoma might be affected by anti HP treatment.

OBJECTIVE: To investigate the interference of sal-like 2 (SALL2) gene on proliferation and apoptosis of the cervical carcinoma HeLa cells. METHODS: 3 small interference RNA (siRNA-1,2,3) which targeted SALL2 were structured,and a carrier group and a control group were set. Liposome was used to transfect SALL2-siRNA into HeLa cells. Expression of SALL2 mRNA and protein in the transfected cells were detected by the method of RT-PCR and Western blot by screening the siRNA fragment with the best silencing effect. After the most suitable siRNA fragment was transfected into HeLa cells,CCK-8 was used to detect the cell proliferation rate,and flow cytometry was used to test cell cycle and apoptosis rates. RESULTS: SALL2 was highly expressed in HeLa cells of the control group. Compared with the control group,SALL2 mRNA and protein expression in the carrier group and the siRNA-3 transfection group showed no statistically significant differences (P>0.05),while those in HeLa cells were significantly reduced after transfected with siRNA-1 and siRNA-2 for 48 h (P<0.05). Among them,siRNA-1 showed the best silencing effect,thus siRNA-1 was subsequently selected as the interference group. In contrast with the control group,cell proliferation rate increased significantly after the transfection with siRNA-1 for 48 and 72 h (P<0.05);the proportion of cells in the G0/G1 phase decreased dominantly and apoptosis rate reduced significantly in the siRNA-1 group after transfection for 48 h (P<0.05). CONCLUSION: SALL2 gene interference improved the proliferation rate and reduced the apoptosis rate in HeLa cells,suggesting that SALL2 gene served as a tumor suppressor gene in cervical carcinoma HeLa cells.

OBJECTIVE: To investigate the mechanisms and effect of ultra violet A (UVA) radiation on vitality and apoptosis of human keratinocytes (HaCaT). METHODS: After different doses of UVA irradiation to keratinocytes, MTT and flow cytometric analysis were used to detect cell survival and apoptosis;immunofluorescence and Western blot were used to detect the formation of intracellular γH2AX focus and its protein levels;and atomic force microscopy was used to detect DNA breakage. RESULTS: UVA irradiation inhibited survival of HaCaT cells in a dose-response manner at the range of 5-30 J/cm² (r=0.982,P=0.009). Early apoptotic cell fraction increased significantly at 20^th hour after UVA irradiation,and had an increasing trend at the range of 10-40 J/cm² (r=0.936,P=0.008) with the rising radiation doses. The 30 and 40 J/cm² doses of UVA induced γH2AX foci formation. After different doses of irradiation,γH2AX expression could be observed,moreover,the expression of γH2AX reached the highest level at 5 J/cm² and 10 J/cm² doses. DNA breaks increased significantly over the control group after irradiation,as detected by atomic force microscopy. CONCLUSION: UVA exposure inhibited HaCaT cell survival,promoted its apoptosis and induced DNA chain break,leading to cell death.

OBJECTIVE: The aim of this study was to investigate the association between IL-2 and IL-10 cytokine levels in plasma,and clinical pathologic factors in cervical cancer of Uighur women. METHODS: Peripheral blood specimens from 50 cervical cancer patients,20 patients with CIN Ⅲ precancerous lesions and 18 controls were used to detect IL-2 and IL-10 by ELISA. RESULTS: The results show that expressions of IL-2 in plasma from patients with cervical cancer or precancerous lesions was significantly reduced compared with those from the controls (P<0.05),while expressions of IL-10 was significantly increased (P<0.05). From the cervical cancer group,IL-10 expression was gradually increased with tumor pathological staging (P<0.05),while IL-2 expression level did not show obvious relationship with tumor pathological staging (P>0.05). Moreover,there was no significant difference in IL-2 expression (P>0.05) between normal Han and Uighur populations,but among those with cervical cancer,Uighur patients' IL-2 expression level was significantly lower than that in Han patients (P<0.05). CONCLUSION: Our results show that immunosuppression occurred despite having low cellular immune level among cervical cancer patients. The observation suggests that one of the mechanisms may be immune escape of tumor cells. In addition,IL-2 may have played an important role in cervical carcinogenesis among Uighur women.

OBJECTIVE: To investigate the effect of phenylethanol glycosides from cistanchetubulosa(CPhGs) on cell proliferation and activation of TGF-β1-induced hepatic stellate cells (HSC-T6). METHODS: HSC-T6 cells were cultivated with CPhGs in different concentrations (25,50,75,100 μg/mL). After the cells were stimulated with TGF-β1,cell activity and drug cytotoxicity were determined by MTT and LDH,respectively. Smad2,Smad3,Smad7 mRNA and Smad2,Smad3,p-Smad2,p-Smad3,Smad7 protein expressions were assayed by qPCR and Western blot. RESULTS: CPhGs at 50-100 μg/mL concentrations effectively inhibited TGF-β1-mediated cell proliferation (P<0.05) and CPhGs at 25-100 μg/mL concentrations did not induce significant cytotoxicity. CPhGs at 25-100 μg/mL concentrations,inhibited Smad2 and Smad3 mRNA and protein levels,as well as the phosphorylation levels of Smad2 and Smad3 proteins,but increased Smad7 mRNA and protein expression significantly on HSC-T6 (P<0.05 or P<0.01). CONCLUSION: CPhGs demonstrated the protective effect against hepatic fibrosis. The mechanism of this process could involve interference with the TGF-β1/Smad signaling pathway,and inhibition of activation and proliferation of HSC-T6.

OBJECTIVE: To investigate the protective effects of salidroside on acute lung injury (ALI) in rats which were exposed to chlorine,and to explore the protective mechanism. METHODS: Fortymale Sprague-Dawley (SD) rats were randomly divided into four groups:control,chlorine,chlorine+salidroside and salidroside. Chlorine and chlorine+salidroside groups were exposed to chlorine of 1 200 mg/m³ for 5 minutes. Chlorine+salidroside and salidroside groups were treated with salidroside (300 mg/kg) at 30 minutes before chlorineexposure and at 15 minutes after chlorine exposure. At 3 hours after chlorine exposure,histopathological analysis was conducted to evaluate the degree of ALI and dihydroethidium (DHE) assay was used to determine the level of pulmonary reactive oxygen species (ROS). Lung permeation index was calculated by protein detecting. The malondialdehyde (MDA),superoxide dismutase (SOD),L-glutathione (GSH) and oxidized glutathione (GSSG) in BALF were measured by kits. The protein level of SOD1 and SOD2 were detected by western blotting. RESULTS: At 3 hours after chlorine exposure,there appeared marked histopathological changes in the lung,including macrophage and neutrophile granulocyte infiltration and edema emerging in bronchiolar and pulmonary alveolar. The ROS level in lung and the lung permeation index were higher. MDA, SOD,GSSG of BALF increased significantly (P<0.05) compared with control group. The GSH and GSH/GSSG ratio in BALF were lower than that in the control group (P<0.05). SOD activity in BALF,and SOD1 and SOD2 protein expression were significantly elevated in chlorine group. In the chlorine+salidroside group,the lung permeation index,ROS level in the lung,MDA and GSSG in BALF decreased significantly (P<0.05) compared with the chlorine group. The GSH/GSSG ratio in BALF were higher than that in the chlorine group (P<0.05). SOD1 and SOD2 protein expression were significantly reduced (P<0.05). CONCLUSION: Our data show that chlorine exposure induced ALI. Administration of salidroside reduce the chlorine-induced ALI via reduction of oxidative stress.

OBJECTIVE: To evaluate the mutagenicity and pollution indexes of mercury,arsenic, formaldehyde and their combinations in Vicia faba root tip cells. METHODS: Cells treated with distilled water were used as negative control group,and with potassium dichromate (50 mg/L) as positive control group. The micronucleus assay of Vicia faba root tip cells were used to determine genotoxicity after exposure to mercury (0.001-0.03 mg/L),arsenic (0.01-0.3 mg/L),formaldehyde (0.9-7.2 mg/L) and combined pollution (MIX I,MIX Ⅱ,MIX Ⅲ). Pollution indexes were calculated. Cells exposed to water or to potassium dichromate (50 mg/L) were used as negative and positive controls,respectively. RESULTS: When the concentration of mercury was ≥0.009 mg/L,arsenic ≥0.3 mg/L, formaldehyde ≥3.6 mg/L in the water,the micronucleus rate were significantly higher compared with the negative control group,respectively (P<0.05). There were four kinds of combination,included mercury-arsenic;mercury-formaldehyde; arsenic-formaldehyde;mercury-arsenic-formaldehyde. In group MIX I,the micronucleus rate of mercury-arsenic and mercury-formaldehyde were significantly higher compared with the negative control group,and also the mercury, arsenic,formaldehyde single exposure,respectively (P<0.05). In group MIX Ⅱ and Ⅲ,The micronucleus rate of all these four combinations were significantly higher compared with the negative control group,and the mercury, arsenic,formaldehyde single exposure,respectively (P<0.05). CONCLUSION: Mercury (≥0.009 mg/L), arsenic (≥0.3 mg/L),formaldehyde (≥3.6 mg/L) induced high frequency of micronucleiin Vicia faba root tip cells. Exposure to combined pollutants induced higher frequency of micronuclei than to single pollutants in varying degrees. There was a certain dose-response relationship between the exposure doses and micronucleus rates.

OBJECTIVE: To investigate the metabolic activities of rat liver S9 after using 7 different kinds of inducting agents and cryopreservation methods. METHODS: The S9 fraction was prepared from male Sprague-Dawley rats with phenobarbital,β-naphthoflavone or PCB induction,using different routes of administration,dosage and time by the way of combination or single inducing method. The S9 fractions were stored at 0,-20 and -40℃. Three indirect mutagens were selected and used to test for mutagenicity:2-amino-fluorene(2AF),acridine orange(AO) and cyclophosphamide (CP). RESULTS: Each group of liver homogenates showed positive results when 2AF,AO and CP were used as indirect mutagens. The activity of S9 from the combined induction method was higher. S9 could be preserved with glycerinum and stored at -20 or -40℃ without obvious loss of activity. CONCLUSION: Our data showed the following levels of S9 metabolic activities:PCB induced group=combination induced group >single induced group >non-induced group. The different routes of administration,dosage and time had little influence on the activity of S9. The S9 fraction retained good activities after storage in -20 and -40℃.

OBJECTIVE: Fox is a seasonal single-estrus animal and its spermatogenesis is stage-specific. The objective of our study was to ascertain the impact of seasonal variations on silver fox spermatogenesis. METHODS: Silver fox testicles were taken in September,November and January for measurement of testicular volume,weight and other basic morphological parameters. Testosterone in the testes was measured by enzyme-linked assay. Tissue slice seminiferous tubules from all kinds of raw composition and proportion of sperm cells were analyzed. RESULTS: In September,November,January,the average testicular weights were (0.896±0.002) g,(3.365±0.081) g and (5.241 ±0.100) g;volume were (1.215±0.036) cm³,(5.558±0.794) cm³ and (14.112±2.854) cm³ and testosterone contents were (0.211±0.060) μg/g,(2.040±0.202) μg/g,(3.240±0.432) μg/g,respectively. Testicular volume, weight and testosterone concentrations in the three months were significantly different (P<0.05). The content of testosterone and the weight changes were positively correlated (r=0.991). In the different seasons,the composition and number of the spermatogenic cell in seminiferous tubules changed with the seasons significantly (P<0.05). CONCLUSION: Our results indicate that spermatogenesis in silver fox showed typical seasonal patterns. The information can be used to determine the best time of semen collection and the approach to enhance artificial fertilization rate.

OBJECTIVE: To compare the mutagenicity among four components of aristolochic acid (AA-I,AA-Ⅱ,AA-Ⅲ and AA-IV) using the Ames fluctuation test,and to evaluate the relationship between toxicity mechanisms and molecular structures of aristolochic acid. METHODS: Two Salmonella typhimurium tester strains (TA98 and TA100) were employed to perform the Ames fluctuation test in the absence and presence of the S9 exogenous metabolic activation system. RESULTS: The mean number of revertant wells of AA-I and AA-Ⅱ were significantly increased in tester strains TA98 and/or TA100,with and without S9 (P<0.05). AA-Ⅱ showed a clear dose-response increase and the fold inductions in mean number of revertant wells over the baseline of AA-Ⅱ and more than AA-I. Both AA-Ⅲ and AA-IV did not induce significant increase with the two test strains,but AA-IV showed a trend of dose-response increase. CONCLUSION: AA-I and AA-Ⅱ were mutagenic with or without metabolic activation,and the mutagenicity of AA-Ⅱ was stronger than that of AA-I. AA-IV demonstrated mutagenicity with metabolic activation. AA-Ⅲ did not show mutagenicity in the two tester strains. The four aristolochic acids exhibited different levels of mutagenicity which might be due to their different structural and metabolic characteristics.

OBJECTIVE: To investigate the acute and genetic toxicity of aqueous extract of Eriobotrya japonica leaves. METHODS: Acute oral toxicity,bacterial reverse mutation (Ames test),mammalian erythocyte micronucleus and in vitro mammalian cells chromosome aberration assays were used. RESULTS: The median lethal dose (LD₅₀) of aqueous extract of Eriobotrya japonica leaves was more than 20.00 g/kg. Using 8-5 000 μg/plate of Eriobotrya japonica leaves in the Ames test,the number of spontaneous revertants did not reach levels 2 times higher than the spontaneous mutation rate,and there was no dose-effect relationship. At the doses of 20.0,10.0 and 5.00 g/kg,the micronucleus rate was not significantly different from the negative control group (P>0.05). At the doses of 625-5 000 μg/mL,the chromosome aberration rate was not significant different from the negative control group(P>0.05). CONCLUSION: Under our test conditions,aqueous extract of Eriobotrya japonica leaves did not show acute or genetic toxicity in a variety of tests.