Study on changes of proliferation, migration and invasion abilities of radiation-induced polyploid colon cancer SW1116 cells and their progeny and the related mechanisms / 肿瘤研究与临床

Objective:To investigate the characteristics related to proliferation, migration and invasion of radiation-induced polyploid colon cancer SW1116 cells and their progeny.Methods:Colon cancer SW1116 cells were conventionally cultured in Leibovitz's L-15 medium containing 10% fetal bovine serum. SW1116 cells at logarithmic growth stage were irradiated with 7 Gy X-ray, and the morphological changes of the cells were observed by inverted microscope on days 3, 5, 10 and 19 after radiation induction. According to the morphological changes of the cells, the cells at day 3 after radiation induction were labeled as polyploid giant cancer cell (PGCC) group, and the cells at day 19 were recorded as PGCC progeny group. SW1116 cells without radiation induction were used as control group. Flow cytometry was used to detect cell ploidy in the control, PGCC and PGCC progeny groups, CCK-8 assay was used to detect the proliferation ability of the three groups, cell migration and invasion abilities of the three groups were detected by cell scratch assay and Transwell assay, and Western blotting was used to detect the expressions of cell cycle and proliferation-related proteins and epithelial-mesenchymal transition (EMT) marker N-cadherin (N-cad) in the three groups.Results:The volume of SW1116 cells gradually became larger on days 3, 5 and 10 after radiation induction, and returned to normal on day 19. The proportions of polyploid (DNA content >4N) cell subsets in the control group, PGCC group and PGCC progeny group were (2.3±1.1)%, (23.1±8.1)% and (3.2±0.5)%, the difference was statistically significant ( F = 18.52, P < 0.05), and the proportion of polyploid cell subpopulations in the PGCC group was higher than that in the control group ( t = 5.38, P < 0.01), but the differences between the PGCC progeny group and the control group were not statistically significant ( t = 0.22, P > 0.05). After 72 h of culture, the cell proliferation rates of the control, PGCC and PGCC progeny groups were (100.0±4.1)%, (73.5±0.7)% and (123.9±3.5)%, and the difference was statistically significant ( F = 190.27, P < 0.001). After 48 h of cell scratching, the scratch healing rates in the control, PGCC and PGCC progeny groups were (38.0±2.7)%, (41.5±4.0)% and (63.7±4.2)%, and the difference was statistically significant ( F = 43.05, P < 0.001). After 24 h of culture, the number of invasive cells in the control, PGCC and PGCC progeny groups was 12.9±1.2, 3.4±0.6 and 23.7±1.5, and the difference was statistically significant ( F = 63.64, P < 0.001). The expression levels of cell cycle-related proteins P-cdc25c, cdc25c and cdc2 in the PGCC group were lower than those in the control group (all P < 0.05), and the expression levels of transcription factor-related proteins E2F-2, E2F-3 and EMT marker N-cad were downregulated compared with the control group (all P < 0.05); the expression levels of P-cdc25c, cdc25c, cdc2, E2F-2, E2F-3 and N-cad proteins in the PGCC progeny group were higher than those in the control group (all P < 0.05). Conclusions:Radiation can induce colon cancer SW1116 cells to produce polyploid, which may then generate daughter cells through asymmetric mitosis and gain new life, and then promote the recurrence and metastasis of colon cancer.

Effects of human umbilical cord-derived mesenchymal stem cells and their conditioned medium on proliferation, migration and apoptosis of non-small cell lung cancer polyploid A549 cells / 肿瘤研究与临床

Objective:To investigate the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) and their conditioned medium on proliferation, migration and apoptosis of human non-small cell lung cancer (NSCLC) polyploid A549 cells.Methods:A549 cells in logarithmic phase were selected. After induction treatment with 1 μmol/L docetaxel for 24 h, DMEM/F12 medium with 10% fetal bovine serum was used to culture the cells for 3 d, finally the polyploid A549 cells model was successfully established. After finishing the separation and culture of hUC-MSC, hUC-MSC conditioned medium was prepared. Normally cultured polyploid A549 cells were treated as the control group, conditioned medium cultured polyploid A549 cells were treated as the conditioned medium group. hUC-MSC was co-cultured with polyploid A549 cells, and the ratio of the total number of cells was 2:1 and 5:1, respectively, which were recorded as MSC 1 group and MSC 2 group. Cells in each group were continually cultured for 48 h or 72 h. Proliferation and apoptosis of polyploid A549 cells in each group were detected by using flow cytometry, cell migration ability was detected by using Transwell assay, and the expressions of migration and apoptosis-related proteins were detected by using Western blotting.Results:Polyploid A549 cells model was successfully established and hUC-MSC was cultured separately. The result of cell proliferation detected by flow cytometry showed that at 48 h, the mean fluorescence intensity of the control group, conditioned medium group, MSC 1 group and MSC 2 group was 1 695±305, 2 020±85, 1 259±35 and 1 356±33, respectively, and the difference was statistically significant ( F = 14.00, P < 0.05); at 72 h, the mean fluorescence intensity of the control group, conditioned medium group, MSC 1 group and MSC 2 group was 1 052±77, 1 309±24, 864±201 and 1 103±237, respectively, and the difference was statistically significant ( F = 3.90, P > 0.05). The result of Transwell assay showed that at 48 h, the number of cell migration in the control group, conditioned medium group, MSC 1 group and MSC 2 group was 52±9, 57±12, 68±8 and 75±11, respectively, and the difference was statistically significant( F = 32.16, P < 0.05); the number of cell migration in each experimental group was all higher than that in the control group (all P < 0.05). The percentage of apoptotic cells in the control group, conditioned medium group, MSC 1 group and MSC 2 group was (15.53±4.27)%, (13.77±1.75)%, (3.60±0.50)% and (2.33±0.06)%, respectively, and the difference was statistically significant ( F = 182.36, P < 0.05); there was no statistically significant difference between the control group and conditioned medium group ( P > 0.05); there were statistically significant differences between MSC 1 group and the control group, MSC 2 group and the control group (both P < 0.05). Western blotting results showed that compared with the control group, the expression of migration-related protein matrix metallopeptidase 9 (MMP-9) was increased, the expression of pro-apoptotic protein bax was reduced, the expression of anti-apoptotic protein bcl-xL was increased in conditioned medium group, MSC 1 group and MSC 2 group. Conclusions:hUC-MSC can improve the migration and anti-apoptotic ability of polyploid A549 cells, suggesting that hUC-MSC may affect the survival of tumor cells during the process of chemotherapy damage and repair.

Study on the biological characteristics of polyploid cervical cancer HeLa cells induced by radiation / 国际肿瘤学杂志

Objective:To investigate the biological characteristics of proliferation, apoptosis, migration and invasion of radiation-induced polyploid cervical cancer HeLa cells, and to analyze the potential facilitation of polyploid HeLa cells in cervical cancer recurrence after radiotherapy.Methods:HeLa cells were irradiated by 6 MV-X ray with 7 Gy and 14 Gy, the cells were cultured until the third day, and then they were recorded as 7 Gy group and 14 Gy group respectively. The unirradiated HeLa cells were recognized as the control group. The cell morphology was checked under optical microscope. Flow cytometry was used to determine cell ploidy. MTT assay was applied to detect cell proliferation. Flow cytometry by AnnexinⅤ-FITC/PI double labeling was used to detect apoptosis. The ability of migration and invasion was detected by Transwell assay. The expression levels of STAT3 signal pathway-related proteins were analyzed by Western blotting.Results:Compared with the control group, the volume of HeLa cells in 7 Gy group and 14 Gy group increased significantly. The percentages of polyploid HeLa cell subsets in the control group, 7 Gy group and 14 Gy group were (6.33±1.26) %, (21.13±0.50) % and (46.07±1.68) % respectively, with a statistically significant difference ( F=780.47, P<0.001) . The absorbance values in the control group, 7 Gy group and 14 Gy group of polyploidy HeLa cells were 0.21±0.01, 0.23±0.02, 0.16±0.01 at 24 h, 0.37±0.03, 0.38±0.06, 0.21±0.00 at 48 h, 0.66±0.02, 0.55±0.01, 0.28±0.01 at 72 h, and there were statistically significant differences ( F=31.62, P=0.001; F=20.10, P=0.002; F=708.52, P<0.001) . Further pairwise comparison showed that the proliferation abilities of polyploidy HeLa cells of the 14 Gy group at 24, 48 and 72 h were significantly lower than those of the control group and the 7 Gy group (all P<0.05) . The proportions of apoptotic cell subset in the control group, 7 Gy group and 14 Gy group were (3.67±1.16) %, (3.07±0.81) %, (3.83±0.91) %, the proportions of early apoptotic subset were (2.33±0.35) %, (2.13±0.61) %, (2.23±0.32) %, and the proportions of late apoptotic subset were (1.33±0.81) %, (0.93±0.31) %, (1.60±0.60) % respectively. There were no statistically significant differences ( F=0.52, P=0.620; F=0.15, P=0.864; F=0.92, P=0.450) . The migrated numbers of cells in the control group, 7 Gy group and 14 Gy group were 297.40±26.53, 121.33±15.16, 18.40±4.79, and the invaded numbers were 195.67±20.26, 63.60±6.91, 9.47±3.23 respectively, with statistically significant differences ( F=647.28, P<0.001; F=213.94, P<0.001) . Compared with the control group, the migration and invasion abilities of polyploid HeLa cells in the 7 Gy and the 14 Gy groups were significantly decreased, and the migration and invasion abilities of polyploid HeLa cells in the 14 Gy group were significantly lower than those in the 7 Gy group (all P<0.001) . The expression levels of P-STAT3 (Tyr 705) and Bcl-2 in radiation-induced polyploidy HeLa cells were higher than those in the control group, and the expression levels were further increased with the increase of radiation dose. Compared with the control group, the expression levels of Survivin and Mcl-1 in polyploid HeLa cells in the 14 Gy group were up-regulated (both P<0.05) . There was no significant difference in Bcl-xL expression among the three groups ( F=0.52, P=0.618) . Conclusion:The proliferation, migration and invasion abilities of polyploid HeLa cells are reduced by radiation, and the proportion of apoptotic subset is not significantly changed, but the activation of STAT3 signaling pathway is accompanied by up-regulation of downstream anti-apoptotic related proteins, which is favorable for the polyploid tumor cells to be the potential risk factor of recurrent cervical cancer after radiotherapy.

Study on the proliferation and apoptosis characteristics of polyploid non-small cell lung cancer A549 cells induced by docetaxel / 肿瘤研究与临床

Objective:To investigate the proliferation and apoptosis characteristics of polyploid non-small cell lung cancer (NSCLC) cell model induced by docetaxel (Doc), and to analyze the potential role of polyploid tumor cells in chemotherapy resistance and tumor recurrence.Methods:NSCLC A549 cells were treated with dimethyl sulfoxide (DMSO) or 1 μmol/L Doc for 24 h. After drug removal, the cells were cultured in complete medium until the third day or the 5th day, and then they were recorded as the control group, Doc 24 h group, Doc 24 h+ 3 d group, Doc 24 h + 5 d group, respectively. The cell morphology was detected by using immunofluorescence staining. Flow cytometry was used to determine cell ploidy and cell cycle. Dil labeling and CFSE labeling were applied to detect cell proliferation. Flow cytometry by Annexin-V/PI double labeling was used to detect apoptosis. The changes of cyclin and apoptotic protein were analyzed by using Western blot.Results:Immunofluorescence staining results showed that compared with the control group, the volume of a small number of surviving cells in Doc 24 h group was increased slightly and the cells showed multinuclear status; while the cell volume in Doc 24 h+ 3 d group and Doc 24 h+ 5 d group continued to increase, and the nucleus remained multinuclear. The results of cell ploidy analysis also showed that the percentage of polyploid cell subsets was (3.40±0.95)%, (20.80±2.87)% in Doc 24 h group, (55.67±3.85)% in Doc 24 h+3 d group and (76.20±2.51)% in Doc 24 h+5 d group. With the prolongation of withdrawal time, the percentage of polyploid cell subsets was increased, and the difference was statistically significant ( F= 478.054, P < 0.05). The percentage of G 1 and S phase cell subsets in Doc 24 h group was lower than that in the control group, and the percentage of G 2/M phase cell subsets was higher than that in the control group, and the difference was statistically significant (both P < 0.05). The protein expression level of cdc2, P-cdc2 (Thr14), P-cdc2 (Tyr15), P-cyclin B1 (Ser128), P-cyclin B1 (Ser147) in the cells of the control group, Doc 24 h group, Doc 24 h+ 3 d group and Doc 24 h+ 5 d group was down-regulated in sequence, while the expression level of cyclin B1 was up-regulated, and cdc25c was down-regulated in Doc 24 h + 3 d group and Doc 24 h+ 5 d group. Dil staining results showed that the fluorescence of cell-labeled Dil in Doc 24 h group, Doc 24 h+ 3 d group and Doc 24 h + 5 d group did not decrease significantly. CFSE staining showed that the fluorescence intensity of CFSE labeled by polyploid A549 cells did not change significantly with the prolonged withdrawal time. Annexin-V/PI double staining showed that the percentage of apoptotic cell subsets in Doc 24 h group was higher than that in the control group ( P < 0.05), but the percentage of apoptotic cell subsets in Doc 24 h + 3 d group and Doc 24 h + 5 d group was lower than that in Doc 24 h group, while there was no statistically significant difference when compared with the control group ( P > 0.05). Western blot results showed that the expression of bcl-xl and mcl-1 in the control group, Doc 24 h group, Doc 24 h + 3 d group and Doc 24 h + 5 d group was up-regulated in sequence, while the expression of bax and bak in Doc 24 h + 3 d group and Doc 24 h + 5 d group was up-regulated, but down-regulated in Doc 24 h+5 d group. Conclusions:Doc can induce polyploidy of A549 cells in vitro. The cell cycle is blocked in G 2/M phase. After doc treatment, the proliferation of A549 cells is significantly decreased, and the apoptosis of A549 cells is promoted. However, with the prolongation of withdrawal time, apoptosis resistance occurs, and the expression levels of corresponding pro-apoptosis and anti-apoptosis proteins show significant changes. This may be helpful for polyploid tumor cells to produce drug resistance and tumor recurrence after chemotherapy intervention.

Role of docetaxel induced polyploid tumor cells in tumor recurrence / 国际肿瘤学杂志

Objective:To study the migration of polyploid tumor cells induced by docetaxel, the characteristics of epithelial-mesenchymal transition, and the killing effect of immune cells on them, in order to explore the potential role of polyploid tumor cells in tumor recurrence.Methods:The human non-small cell lung cancer A549 cells were treated with 1 μmol/L docetaxel for 24 h, and the cells were collected as Doc 1 d group. After drug removal, the cells were cultured in fresh and complete medium for 3 or 5 days, then the cells were collected as Doc 3 d group or Doc 5 d group respectively. The A549 cells were treated with DMSO for 24 h as control group. Immunofluorescence staining was used to detect cell morphology, flow cytometry was used to analyze cell ploidy, scratch test was used to detect cell migration, Western blotting was used to detect the expression of epithelial-mesenchymal transition related proteins, and lactate dehydrogenase release method was used to evaluate the killing activity of cytokine-induced killer (CIK) cells.Results:Compared with the control group, most of the cells in the Doc 1 d group, Doc 3 d group and Doc 5 d group were apoptotic, a few of the surviving cells were significantly larger, and the nucleus was polynuclear. The proportions of polyploid cell subset (DNA content > 4N) in the control group, Doc 1 d group, Doc 3 d group and Doc 5 d group were (1.93±0.55)%, (22.97±2.37)%, (51.30±12.51)% and (67.87±8.31)% respectively, and the difference among the four groups was statistically significant ( F=26.521, P<0.001). The proportion of polyploid cell subset in Doc 1 d group, Doc 3 d group and Doc 5 d group was significantly higher than that in the control group (all P<0.001). With the prolongation of withdrawal time, the proportion of polyploid cell subset in Doc 3 d group and Doc 5 d group was significantly higher than that in Doc 1 d group ( P=0.009; P=0.004). After 24 h and 48 h culture, the wound healing rates of the control group were both 100%, and the wound healing rates of the Doc 3 d group were (39.10±2.12)% and (46.13±5.32)% respectively, with no significant difference ( t=2.126, P=0.051). Compared with the control group at 24 h and 48 h, the cell migration abilities of Doc 3 d group were significantly lower ( t=49.756, P<0.001; t=30.825, P<0.001). Compared with the control group, the expression of E-cadherin protein decreased gradually in the Doc 1 d group, Doc 3 d group and Doc 5 d group, the expression of Vimentin protein increased gradually, and the expressions of Snail protein and N-cadherin protein did not change significantly. The killing efficiencies of CIK cells against the cells of the control group, Doc 3 d group and Doc 5 d group were (27.27±1.91)%, (17.87±2.35)%, (9.47±0.51)% respectively, and the difference was statistically significant ( F=11.294, P<0.001). The killing efficiency of Doc 3 d group and Doc 5 d group was significantly lower than that of the control group ( P=0.004; P<0.001). The killing efficiency of Doc 5 d group was significantly lower than that of Doc 3 d group ( P=0.003). Conclusion:The migration ability of polyploid tumor cells induced by docetaxel is weakened, but epithelial-mesenchymal transition is likely to occur, and the killing effect of immune cells on them is reduced.

The effect of entinostat on NK killing effect on non-small cell lung cancer in vitro / 中国肿瘤临床

Objective: To investigate the Effects of entinostat on the expression of NKG2D ligands in the non-small cell lung cancer (NSCLC) cell lines, A549 and HCC-827, and to detect the effect of entinostat-mediated NK cell killing of A549 and HCC-827 cells. Meth-ods: The effect of entinostat on A549 and HCC-827 cell proliferation was measured by MTT assay. Flow cytometry was used to detect the expression of NKG2D ligands. mRNA levels of the ligands were detected by RT-PCR . The level of soluble MICA in cell culture super-natant was evaluated by ELISA. The cytotoxicity of NK cells against A549 and HCC-827 cell lines (treated with entinostat) was assessed using lactate dehydrogenase release assay. Results: Entinostat showed a time-and dose-dependent inhibition effect on the prolifera-tion of A549 and HCC-827 cell lines. The expression of NKG2D ligands and mRNA transcription levels of MICA and MICB were en-hanced after treatment with 0.5, 1μmol/L entinostat for 48 h. The soluble MICA level in A549 cell culture supernatant was increased by 1μmol/L entinostat. The sensitivity of HCC-827 cells to NK cells was enhanced upon treatment with 0.5, 1μmol/L entinostat. Con-clusions: entinostat enhanced the killing effect of NK cells on non-small cell lung cancer cells by up-regulating the expression of NKG2D ligands. This provides a new method and theory for the treatment of NSCLC.

Application effect of comprehensive intervention on perioperative period of patients with limb salvage surgery for lower extremity malignant tumor / 中国基层医药

Objective To analyze the application effect of comprehensive intervention in perioperative period of patients with limb salvage surgery for malignant tumors of lower extremity.Methods According to the order of admission,77 patients received lower extremity malignant tumor limb salvage surgery were divided into control group (n =34)and observation group(n =35).The control group was given routine nursing intervention,the observation group underwent perioperative comprehensive intervention.The nursing effect of different clinical nursing intervention was compared between the two groups.Results The incidence rate of complications of the observation group was 5.71%,which was lower than 23.53% of the control group,the difference was statistically significant(χ2 =4.417, P <0.05).The satisfaction rate of patients in the observation group was 97.14%,which was significantly higher than 76.47% in the control group(χ2 =6.498,P <0.05).The nursing,anxiety depression score of the two groups were significantly decreased,which of the observation group decreased more significantly compared with the control group(t =25.200,28.660,all P =0.000).Conclusion The effect of comprehensive nursing intervention in the perioperative period of patients with lower extremity malignant tumor limb salvage surgery is obvious.

Risk factors leading to pulmonary embolism in patients of lower extremity deep vein thrombosis with iliac vein compression / 中华普通外科杂志

Objective To evaluate risks of symptomatic pulmonary embolism (PE) in deep vein thrombosis (DVT) patients complicating iliac vein compression syndrome (IVCS).Methods Between January 2010 and January 2015,patients diagnosed with lower extremity DVT and IVCS at our institution were included.The odds of symptomatic PE versus lower extremity DVT complicated with IVCS were assessed using Logistic regression models.Its association with age,gender,thrombophilic risk factors,degree of stenosis,filter implantation and symptomatic PE were assessed.Results Of 759 patients,410 patients had inferior vena cava (IVC) filters implanted and 349 had not.In filter placement group,3 patients suffered from symptomatic PE among 32 with thrombi intercepted in IVC filters.In the group with no IVC filters placement,39 cases suffered from symptomatic PE.Symptomatic PE was in positive correlation to respiratory disease and right lower extremity DVT,and negative correlation to IVC filter implantation.Conclusions In lower extremity DVT patients complicated with IVCS,the right lower extrenity DVT and respiratory disease are risk factors of symptomatic PE.IVC filter implantation prevents symptomatic PE from happening in most patients.

Comparison of effect of formulas clearing away heat and promoting blood circulation on prevention and treatment of liver fibrosis in CCl4 mice / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the effect of traditional Chinese medicine formulas clearing away heat and promoting blood circulation-Biejiayinzi (BJYZ), Gexiazhuyu Tang (GXZYT) and Fugan Wan (FGW) on liver fibrosis in CCl4 mice by screening and analyzing formula-syndrome database of kidney and liver fibrosis based on the principle of formula-syndrome, compared with pivot-harmonizing decoction.</p><p><b>METHOD</b>Ten-week-old male C57BL/6 mice, with the weight of (20 +/- 3) g, were randomly divided into 6 groups: the normal group, the model group, the BJYZ group, the GXZY group, the FGW group and the XST group. Except the normal group, other groups were abdominally injected with 10% CCl4 olive oil solution a dose of 2 mL x kg(-1) body weight for four weeks, three times each week. Meanwhile, the latter four groups were administered with extracts of BJYZ, GXZYT, FGW and XST, respectively, once every day, concomitantly continued CCl4 administration. The normal and the model groups were given the same volume of deionized water. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (gamma-GT), Alb and TBil were detected by chemiluminescence. The hydroxyproline (HYP) content was detected by acid hydrolysis method. The hepatic collagen deposition was evaluated with Sirius red staining.</p><p><b>RESULT</b>Compared with the normal group, the model group recorded notable decrease in weight and increase in the ratio of liver weight and body weight and the ratio of spleen weight and body weight, with obvious fatty degeneration and inflammatory necrosis in liver cells. Collagen fiber deposition was so notable to form fibrous septums and pseudolobules. The levels of serum ALT, AST, TBil, gamma-GT, the HYP content in liver tissue and the deposition of hepatic collagen were significantly increased in the model group. Compared with model group, Serum AST were significantly decreased in BJYZ group as gamma-GT decreased in the GXZYT group, without notable decrease in degeneration and inflammatory necrosis in liver cells and collagen deposition. The GXZYT group showed significant decrease in gamma-GT, with slight improvement in degeneration and inflammatory necrosis in liver cells and reduction in collagen deposition. The ratio of liver weight and body weight, AST, gamma-GT and HYP content were significantly decreased in the FGW group, with slight improvement in degeneration and inflammatory necrosis in liver cells and reduction in collagen deposition. The XST group showed decrease in the ratio of liver weight and body weight, with no obvious change in inflammation and fibrosis of hepatic tissue.</p><p><b>CONCLUSION</b>FGW shows the best effect of prevention and treatment of liver fibrosis in CCl4 mice.</p>

THE EFFECTS OF BASIC FIBROBLAST GROWTH FACTOR ON PROTEIN KINASE B ACTIVITY / 解剖学报

Objective To study the effects of basic fibroblast growth factor(bFGF) on protein kinase B(PKB) activity. Methods The cytosol and membrane activity of PKB at the indicated time periods was determined by [?-()~(32)P]ATP incorporation assay. Results Both membrane and cytosol activity of PKB in HEK293 cell treated with bFGF(75??g/L) reached to the peak at 10?min.Wortmannin efficiently inhibited the activity of PKB in HEK293 cell.Conclusion bFGF rapidly stimulats both membrane and cytosol activity of PKB via P13K in HEK293 cell.