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Objective To explore the effect of oxaliplatin ( OXA) on enhancing radiosensitivity in human hepatocellular carcinoma cell line HepG2 . Methods 50% inhibition concentration ( IC50 ) of HepG2 cells treated with OXA was measured by using MTT method at 6, 12, 24, 48 hours. Then clone formation assay was applied to obtain sensitizing enhancement ratio ( SER) of OXA combing IR, according to the survival fraction of three groups 10?14 days after treatments:placebo?treated group ( C) ,radiation group ( IR, single dose of 1 Gy,2 Gy,4 Gy,6 Gy,8 Gy,10 Gy) and IR synchronizing OXA group ( IR+3 mg/L OXA) . The proportions of cell apoptosis were analyzed using flow cytometry at 24 hours after treatment. At last, we semi?quantitative tested the expression of extracellular regulated protein kinase 1/2 ( ERK 1/2 ) and DNA damage repair protein Ku?70 of the C,IR and IR+OXA groups. Statistical analysis was performed by T test. Results The IC50 of OXA on HepG2 cells is 54?4 mg/L at 6 hours,29?1 mg/L at 12 hours,17?8 mg/L at 24 hours and 10?5 mg/L at 48 hours.3 mg/L was selected in clone formation assay at which 80?90% HepG2 cells survived at 24 hours. The SER ( SF2 ) is calculated as 1?59. Flow cytometry showed the proportion of survival cells in IR+OXA group is significantly lower than those of IR group ( P=0?005) ,OXA group ( P=0?008) and C group ( P=0?001) . The expressions of ERK 1/2 were inhibited in IR and IR+OXA groups compared by that of control group. But the expression of ERK 1/2 in IR group showed increasing after 48 hours which was higher than that of IR+OXA group. For Ku?70,the changes of expression were similar with that of ERK 1/2. Conclusion Oxaliplatin presented enhancing radiosensitivity in human hepatocellular carcinoma cell line HepG2 in vitro.
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Objective To analyze the genomic evolution characteristics of pathogenicity islands (PAIs)in Deng strain of enteropathogenic Escherichia coli (E.coli,EPEC Deng).Methods EPEC Deng was isolated from infant stool specimen,serotypes were identified and antimicrobial susceptibility testing was performed;whole-genome se-quencing was performed by Illumina 2000 system,the locations of prophages(PPs)in the chromosome were detected using PHAST software,collinearity analysis was performed by MUMmer software,phylogenetic trees of homolo-gous gene were constructed in order to understand the evolutional rule of homology gene.PAIs prediction was per-formed using PAI finder software,the homologous evolutionary rule of PAIs core region(LEE)and core genes were clarified,genetic polymorphism was analyzed.Results The serotype of EPEC Deng strain was O119:H6,the strain was resistant to ciprofloxacin,levofloxacin,and ampicillin,but sensitive to other antimicrobial agents.The complete circular chromosome contained 5 025 482 bp with a GC content of 50.52 %,and the plasmid contained 207 564 bp with a GC content of 49.50%.A total of 17 PPs in the chromosomal genome were discovered,phyloge-netic trees analysis suggested that EPEC Deng strain was highly homologous with O26:H11 and O111 :H strains;PAIs and core genes were highly homologous with RDEC-1 and O26:H413/89-1 strains;genetic diversity analysis showed that the intimin (eae)and its receptor tir had high polymorphism,with the pi (π)value>0.10,the genes in type III secretion system was relatively stable.Conclusion The study clarified the genomic evolution characteris-tics of EPEC Deng genome and it’s PAIs,and is helpful for understanding genetic characteristics of native EPEC.
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Objective To determine the optimized fractionated radiation schedule by comparing the dose-response relationship between different fractionated radiation schedules with a total dose of 40 Gy or 60 Gy in subclinical breast tumor.Methods Balb/c nude mice bearing subclinical human breast cancer (injected subcutaneously into the hind legs with 1.5 × 105 or 3.1 × 105 exponentially growing MCF-7 cells) were assigned randomly to blank control group (without radiation),conventionally fractionated radiation group (200 cGy,once daily,10 times/week),hyperfractionated radiation group (160 cGy,twice daily with an interval of 6 h,5 times/week),first hypofractionated radiation group (300 cGy,once daily,5 times/ week),and second hypofractionated radiation group (400 cGy,once every other day,3 times/week) ;the total dose was 40 Gy or 60 Gy.The measurement indices were tumor formation rate,short-term tumor control rate,long-term tumor control rate,the time of tumor recurrence,and the maximum diameter of the bottom of tumor.The observation lasted 24 weeks.Data were compared between these groups by chi-square test.Results With a total dose of 40 Gy (the number of injected cells was 1.5 × 105,the tumor formation rate of the blank control group was 2/8),hyperfractionated radiation was the optimized schedule.With a total dose of 60 Gy (the number of injected cells was 3.1 × 105,the tumor formation rate of the blank control group was 11/11),the first hypofractionated radiation (300 cGy,once daily,5 times/week) was the optimized schedule (P =0.001);the short-term and long-term tumor control rates of the conventionally fractionated radiation group,hyperfractionated radiation group,second hypofractionated radiation group,and first hypofractionated radiation group were 0/0 (tumor formation rates:8/8 and 8/8),50％/25％ (tumor formation rates:4/8 and 6/8),25 ％/25 ％ (tumor formation rates:6/8 and 6/8)),and 67 ％/67 ％ (tumor formation rates:4/12 and 4/12),respectively.Conclusions The optimized fractionated radiation schedule for subclinical breast cancer and its total dose vary with the number of injected tumor cells.When the tumor formation rate is 100％,hypofractionated radiation (300 cGy,once daily,5 times/week) is the optimized schedule in terms of long-term tumor control.
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Objective To study dose-response relationship and screen the optimized fractionated irradiation schedules in subclinical tumors of malignant glioma.Methods Balb/c-nude mice bearing human malignant glioma xenograft were assigned randomly into control group,fractionated irradiation schedules group and nimotuzumab-conventional fraction group.The fractionated schedules were 200 cGy x 5f/w,300 cGy ×5f/w,160 cGy ×2f/d x5 d and 400 cGy ×3f/w with total dose of 40 Gy and 60 Gy,respectively.Measurement indexes were tumor-forming rate,average recurrence time and maximum diameter of the tumor bottom.The observation lasted 24 weeks.Results With the total dose of 40 Gy,none of the significant long-term tumor regression were detected in any fractionated irradiation schedules; 400 cGy x 3f/w with complete tumor response at the end of treatment showed a better short-term curative effect.With the total dose of 60 Gy,long-term control rate of each fractionated irradiation schedule group was improved with prolonged average recurrence time of varable degrees,except 200 cGy x 5f/w fractionated schedule (tumor formation rate was 100％ at the end of treatment and average recurrence time was the poorest of 108 d).160 cGy × 2f/d × 5 d fractionated schedule showed the best curative effect with no tumor formation in 2 of 8 mice and longest recurrence time of 143 d.300 cGy x 5f/w fractionated schedule ranked second with no tumor formation in 1 of 8 mice and average recurrence time was 137 d.400 cGy x 3f/w fractionated schedule produced the poorest outcome with no case cured.There were no significant changes in the tumor-forming rate or average recurrence time when nimotuzumab was concurrently used for subclinical tumors of malignant glioma with total dose of 60 Gy.Conclusions Conventional fractionated irradiation is not the best option to control the sustained growth.160 cGy ×2f/d ×5 d and 300 cGy × 5f/w might be the optimized fractionated irradiation schedules for subclinical tumors of malignant glioma.
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ObjectiveTo evaluate the effect on lung injury of gefitinib or/and radiation.Methods Totally 160 mice were divided into five groups:control (C) ;gefitinib (G) ;radiation (R) ;gefitinib followed by irradiation ( G + R) ;and R + G.12 Gy irradiation was delivered.Geiitinib fed by 200 mg/kg once daily for 3 weeks.Mice were sacrificed on 1,2,4 or 6 months after radiation.Macrophage count of lung lavage fluid and hydroxyproline assessed,lung fibrosis scored.and plasma TGF-β1 concentration assayed.One-way ANOVA was used to test the significance. Results The lung lavage macrophage cell number were significantly higher in group R,R + G and G + R than group C ( q =2.95 - 8.61,all P ＜ 0.05 ) on 4 and 6months,yet no significant difference between the three groups ( q =0.37 -3.49,all P ＜ 0.05 ) ; The macrophage was significantly lower in month 1,4 and 6 in group G than R,R + G and G + R ( q =3.37- 6.25,all P ＜ 0.05 ).The hydroxyproline content and the fibrosis score of G,R,R + G and G + R were significantly higher than C ( q =3.14 - 4.76,all P ＜ 0.05 ),but no significant difference between the four groups ( q =0.70 - 4.19,all P ＞ 0.05 ).The TGF-β1 concentration of R,G + R,R + G at all time points and TGF-β1 concentration of G at 1 st and 2nd months were significantly higher than C ( q =3.76 -8.09,all P ＜ 0.05).ConclusionsGefitinib could cause lung fibrosis in vivo in BalB/C mouse.The combination of gefitinib and radiation did not significantly exacerbate lung injury caused byeither alone.The mechanism of lung fibrosis caused by gefitinib might be different from that by radiation which needs further research.
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ObjectiveTo evaluate whether oral administration of Chinese tradiational medicine,Qing-Xue granula,can prevent mouse lung injury caused by thoracic radiation.Methods128 BalB/C mice were divided into 4 groups:control (C) group; radiation (R) group; radiation plus high dose Qing-Xue granula (H) group and radiation plus median dose Qing-Xue granula ( M ) group.The H and M groups were fed 0.64 g and 0.32 g of Qing-Xue granula dissolved in 0.5 nl anline once daily for two months,which were 4 and 2 times of human dosage,respectively.Whole thorax radiation of 12 Gy was delivered with a single ventral-dorsal field with 6 MV X-ray.Group C and group R received 21 days of 0.5 ml saline feeding.Mice were sacrificed at 1,2,4 or 6 months after radiation. Macrophage cell count of lung lavage fluid and hydroxyproline content of left lung were assayed,and the lung fibrosis was scorred according to the Ashcroft's criteria.The plasma interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) concentration were assayed with ELISA method.The One-way ANOVA was used to test the significance of any differences between groups at each time point. Results The macrophage cell number of lung lavage fluid was significantly lower in the 1st month in group M than in group R (2∶4,q =3.92,P ＜ 0.05 ),but had no significant difference between group M and C ( 1 ∶ 4,q =2.13,P＞0.05 ).The hydroxyproline content of group H was significantly lower than group R in the 1st and 6th months (q =3.62,3.54,all P ＜ 0.05 ),but still higher than group C ( q =4.09,3.72,all P ＜ 0.05 ).The fibrosis score of group H was significantly lower than group R in the 2nd,4th and 6th months (q=3.38 -4.16,all P＜0.05).The IL-6 concentration of group H was significantly lower than group R in the 1st month ( q=3.53,P＜0.05 ),but not significantly higher than group C (q =1.41,P＞0.05).The VEGF concentration was significantly higher in group R than group C since the 2nd month ( q =3.12 - 3.78,P ＜ 0.05 ).The VEGF concentration was significantly higher in group H and M than group R in the 2nd and 6th months ( q =3.08 - 3.92,all P ＜ 0.0 5 ).Conclusions Oral Chinese traditional medicine,Qing-Xue granula,could prevent radiation induced lung fibrosis in mice,especially at high dosage.The degree of elevation of VEGF in plasma was not parallel with that of lung fibrosis.
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Objective To indentify the gene expression on different fractionated radiation regimens with the same total radiation dose in xenografts with human lung adenocareinoma. Methods Forty-eight BALB/c-nu mice, implanted with human lung adenocarcinoma (Anip973), were randomized into 4 groups: normal control greup,60 Gy in 30 fractions conventional radiation group (2 Gy group) ,60 Gy in 10 fractions hypofractionated radiation group (6 Gy group) ,60 Gy in 6 fractions hypofractionaed radiation group (10 Gy group). Gene alterations were investigated with the microchip analytical procedures covering the entire genome. Genes with significantly different expression were further validated by the quantitative real-time polymerase chain reaction (RT-PCR). Results Compared to the 2 Gy group, the expression of the genes related with the cell growth inhibition and apoptesis was increased, while the genes related with the cell proliferation, anti-apoptosis and DNA damage repair were decreased in the 6 Gy and 10 Gy groups. Confirmed by RT-PCR, c-myc gene was distinctly suppressed in the 6 Gy group (2. 9%) comparing with 2 Gy (5.6%) group and 10 Gy (4.8%) group (P=0. 000,P=0. 002) , and was slightly suppressed in the 10 Gy group comparing with 2 Gy group (P = 0. 069). Conclusions In the BALB/c-nu mice implanted with human lung adenocarcinoma, the hypofractionated radiation regimens clearly inhibit the tumor growth more than conventional fractionation group, though with the same total dose. The 6 Gy group seem to be more effective than 10 Gy group in the inhibition of tumor growth.
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Objective To investigate the mechanism of an antifibrotic drug, pirfenidone, in preventing radiation-induced pulmonary fibrosis. Methods Male BALB/C mice were randomized into 4 groups:control group (C), radiation alone group (R), pirfenidone alone group (P), and pirfenidone plus radiation group (P + R). Irradiation was administrated to the whole pulmonary with a single fraction of 12 Gy. The pirfenidone was given 0. 3 ml/kg/d from 3 days prior to irradiation to 12 weeks after.Bronchoalveolar lavage fluid (BALF) from right lung was collected for macrophages counting every monthmonthly until 6 months after irradiation, and left lungs were collected and fixed for. The pulmonary fibrosis was assessed by Masson trichrome staining. The plasma transforming growth factor β(TGF-β) was measured by ELISA. The lung hydroxyproline was evaluated by alkaline solution. Results Compared to group R, the counts of macrophages in BALF in group P + R were reduced by 76% and 62%, and hydroxyproline levels were reduced by 21% and 24% at the 4th and 5th months, respectively. The plasma TGF-β decreased from the 3rd month to 5th month. Pirfenidone markedly ameliorated the severity of lung fibrosis at the 4 - 6th month after radiation. Conclusions Pirfenidone can prevent radiation-induced pulmonary fibrosis, the mechanism of which may be the reduced of inflammation and collagen deposition by decreasing macrophages and hydroxyproline.
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Objective To evaluate the radiobiological effect of different irradiation fractionated regimens in human glioma cells ( BT 325 cell line). Methods The xenografts in Balb/c-nude mice were irradiated with different single and fractionated regimens. The single fraction dose was 10, 20, 30, 40 and 60 Gy, respectively. The fractionated regimens were 2 Gy × 5 fractions ( irradiated every day), and 3 Gy ×3 fractions (irradiated every other day), 3 Gy × 5 fractions (irradiated every day) and 4 Gy × 3 fractions (irradiated every other day), with total doses of 125 Gy, 114 Gy, 126 Gy and 112 Gy, respectively. The growth curve was used to evaluate the tumor doubling time. clonogenic assays was performed to draw the cell survival curve and analyze the radiobiological parameters with doses of 1, 2, 4, 6, 8 and 10 Gy. T1/2 was measured by comet assay. Results Tumor regression were not observed by single fraction irradiation, 2 Gy × 5 fractons and 3 Gy × 3 fractions irradiation regimens. The tumor regress was more significant with the increas of fraction dose. The 4 Gy × 3 fractionrs inhibited tumor more though not curing tumor. The cell doubling time of the BT 325 cell was 30. 16 h and the tumor doubling time of the xenograft was 43 days.When fitted with L-Q model ,α was 0. 36 Gy -1 and β was 0. 057 Gy -2. When fitted with the single-hit multitarget model, D0 was 1. 394 Gy, Dq was 2. 127 Gy and SF2 was 0. 714, respectively. The T1/2 was 9. 999min. Conclusions Glioma is a radioresistant tumor. Increase of the fraction dose improves recent effect.Further study is needed to control the tumor stem cells.
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Objective To evaluate the radiosensitization effect of gemcitabine in human cholangiocarcinoma cells(QBC939)in vitro. Methods The IC10,IC50 and IC90 of gemcitabine in QBC939 cells were determined by clonogenic assay,which were adopted in the following experiments.The cells were divided into five groups:control group,chemotherapy group,radiation group,radiation after chemotherapy(C+R)group and radiation before chemotherapy(R+C)group.The radiation dose ranged from 1 Gy to 10 Gy.The cell survival Curves were fit using two-component equation and the sensitive enhancement ratio(SER)was calculated.Results The IC10,IC50 and IC90 of gemcitabine in QBC939 cells were 0.1 nmol/L(low concentration),11.0 nmol/L(moderate concentration)and 21.5 nmol/L(high concertration),resDectively.Gemcitabine in low concentration radiosensitized the cells at low irradiation dose(≤2 Gy)in R +C group(SERDq=1.52).Gemcitabine in moderate concentration had radiosensitization effect at all irradiation doses in C+R group(SERD0=1.27,SERDq=116.93),and at low irradiation dose(≤2 Gy)in R+C group.Gemcitabine in high concentration had radiosensitization effect at all irradiation doses in both R+C and C+R groups,which was most markedly at low irradiation dose(≤2 Gy)in R+C group(SERDq =323.30). Conclusions Gemeitabine has radiosensitization effect in QBC939 cells,which is based on the optimal scheduling and concentration.
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Objective To impmve the method of "modified comet assay" in predicting the radiosensitivitv of solid tumor. Methods A single cell suspension from biopsy sample was lmdlated on ice with a dose of 5 Gy.The microscope slide was spread with agarose,lysed for 50 minutes,rinsed 3 times rinse solution,and given electrophoresis for 20 minutes. After being stained with PI,cell images were collected through the microscope and analyzed with Lucia G software(Version 4.6).In order to check system/ background errors,every sample was made into control slide and irradiation slide.The end-points were cell DNA contents and tail moment. Results The factors influencing the results included:(1)Sample was iaulty tor the biopsv taken from mucosa and no tumor cells were contained. (2)The slides with a high backgmund ( induced by necrosis) disturbed the measurement of comet assay. (3) Setting lymphocytes as control to check svstem errors was very important. (4)To separately collect images of the normal tissue cells and tumor cells from the biopsy sample improved the conformity between the clinical obscrvation and the lab result. Conclusions To increase the correlation between comet assay and clinical response,it is very helpful to set double control for checking system/background errors and to collect images of the normal tissue cells and tumor cells through the microscope,respectively.
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Objective To evaluate the relationship between single nucleotide polymorphism(SNP) of candidate genes and radiation-induced esophagitis (RIE) in patients with lung cancer. Methods Between Jan. 2004 and Aug. 2006,170 patients with pathologically diagnosed lung cancer were enrolled in this study. The total target dose was 45-70 Gy( median 60 Gy). One hundred and thirty-two patients were treated with three-dimensional conformal radiotherapy(3DCRT) and 38 with two-dimensional radiotherapy(2DRT).Forty-one patients received radiotherapy alone, 78 received sequential chemoradiotherapy and 51 received concurrent chemoradiotherapy. Thirty-seven SNPs in 20 DNA repair genes were analyzed by using PCR-based restrieted fragment length polymorphism(RFLP). These genes were apoptosis and inflammatory cytoking genes including ATM, ERCC1, XRCC3, XRCC1, XPD, XPC, XPG, NBS1, STK15, ZNF350, ADPRT,TP53, FAS, FASL, CYP2D6 * 4, CASPASE8, COX2,TGF-β, CD14 and ACE. The endpoint was grade ≥2 R I E. Results Forty of the 170 patients developed grade ≥2 R I E, including 36 in grade 2 and 4 in grade 3. Univariate analysis revealed that radiation technique and concurrent chemoradiotherapy were statistically significant relatives to the incidence of R I E (P = 0. 032,0.049) , and both of them had the trend associating with the esophagitis( P = 0.072,0. 094 ). An increased incidence of esophagitis was observed associating with the TGF-β1-509T and XPD 751 Lys/Lys genotypes ( χ2 = 5.65, P = 0.017 ;χ2 = 3.84, P = 0. 048 )in multivariate analysis. Conclusions Genetic polymorphisms in TGF-β1 gene and XPD gene have a significant association with radiation-induced esophagitis.
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<p><b>BACKGROUND</b>Cells derived from lung cancer are biological heterogeneous and have different intrinsic radiosensitivity, it is a key question for us to investigate radiosensitive parameters for an individualized radiotherapy plan. The comet assay is a sensitive and facilitated method to detect single-cell DNA damage and repair, and the results from it have been proven to be so highly coincident with those from clonogenic assay by cell-line investigations that it has been considered as a promising method in predicting radiosensitivity. The study is designed to evaluate preliminarily the correlation between initial DNA damage detected by alkaline comet assay and the clinical endpoints.</p><p><b>METHODS</b>Biopsy samples from 31 lung cancer patients by fibrous bronchial endoscopy were detected by alkaline comet assay from April, 2002 to November, 2002. The adjusted tail moment (R TM) was measured and thoracic local-region lesions were measured by computer tomography scan. Response rate (RR) and time to progression (TTP) for the local-region lesions were as clinical endpoints. SPSS 10.0 software was used to compare median R TM of different RR and TTP groups by Mann-Whitney U and Kruskal-Wallis H rank test, the correlations between R TM with RR and TTP were estimated by Spearman's rank test.</p><p><b>RESULTS</b>There were no statistic differences of median R TM among different pathological types with a median R TM of 0.98, 1.27 and 1.05 in squamous cell carcinoma group, adenocarcinoma group and small cell lung cancer group, respectively (Chi-square=0.347, P=0.84). Through a median follow-up of 10 months, a median R TM of 1.08 and 1.21 for squamous cell carcinoma group and small cell lung cancer group in RR≥50% group was greater than 0.88 and 0.91 in RR < 50% group; median R TM in TTP > 9-monthgroups stratified according to pathological type was greater than that in TTP≤9-month groups (1.26, 1.38 and 1.39 versus 0.71, 0.48 and 1.03 for squamous cell carcinoma group, adenocarcinoma group and small cell lung cancer group respectively), but the differences of R TM classified by RR or TTP were not statistically significant (U=63.5, P=0.58; U=71, P=0.057); the Spearman's coefficients of R TM with RR and TTP were -0.105 (P=0.57) and 0.38 (P=0.035). The coefficients of R TM with TTP was 0.47 for non-small cell lung cancer indicating a modest correlation (P=0.048) and 0.043 for small cell lung cancer (P=0.89).</p><p><b>CONCLUSIONS</b>Although the results are confounded due to sampling and the greater background tail moments, Spearman's coefficient of R TM with TTP for non-small cell lung cancer indicates a modest positive correlation. The comet assay might be a promising method in predicting intrinsic radiosensitivity of lung cancer cells and techniques for sampling and assaying need to be further improved.</p>
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Objective:To evaluate the inhibitory effect of intraperitoneal sustained-release chemotherapy with 5-FU on the growth of H22 ascitic tumor in mice.Methods: Mouse H22 ascitic tumor model was established by intraperitoneal injection of(0.2 ml) H22 ascitic cells(4?10~(6)cells) and the animals were subsequently divided into 4 groups randomly, namely,the saline control group(received saline),peritoneal chemotherapy group(received common 5-FU),sustained-release chemotherapy group(received sustained-release 5-FU),and negative control group(received control sustained-release agent).The survival times of the mice were recorded in all groups.The apoptosis rates of H22 ascitic cells were analyzed with flow cytometry 9 and 12 days after injection of H22 cells and the proliferation index was calculated.Electron microscope was used to observe H22 cells 12 days after peritoneal injection.Results: The average survival time of peritoneal chemotherapy group([13.7?1.7] d) was significantly shorter than that of sustained-release chemotherapy group([15.3?2.0]d)(P
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Objective To define the correlation between the proliferating cell nuclear antigen (PCNA) and metastasis-related gene nm-23-H 1, and to correlate their espressions with clinical features,radiosensitivity and prognostic variables in exophageal squamous cell carcinoma (ESCC), to establish some biological parameters obtained prior to therapy though which we can predict radiosensitivity and outcome.Methods PCNA and nm-23-H 1 expression protein were determined by immunohistochemical technique with formalin-fixed paraffin-embedded specimens from 59 patients with ESCC who had received definitive radical radiation and had been followed up for more than 3 years. The values were assessed by distributions of patients , disease factors, including age,sex,lesion site,legth ,histological grade and prognosis.Results The findings showed that the mean labelling indices of PCNA and nm-23-H 1 were significantly higher in ESCC than in the normal esophageal tissue (P0.05).Conclusions PCNA and nm-23-H 1 indices can be taken as biological endpoints to predicting therapeutic response, local and systemic control of disease.