ObjectiveTo investigate the effect of expressions of nucleoside transporters subtype (hENT1 and hENT2) on 5-fluorouracil(5-FU) cytotoxicity in breast cancer cell lines(MDA-MB-231, MDA-MB-468, SK-BR-3, MCF-7). MethodsFour breast cancer cell lines were chosen to detect the mRNA expressions of hENT1 and hENT2 by RT-PCR. Cells were incubated in the medium with a serial concentrations of 5-FU from 1.28×104 ng/L to 2.00×108 ng/L for 48 h. Then the cell proliferation in each cell line was measured by MTT assay and the IC50 was evaluated. Results①The mRNA expressions of hENT1 and hENT2 in the MDA-MB-231, MDA-MB-468, or SK-BR-3 cells were significantly higher than thoes in the MCF-7 cells(P < 0.05). The mRNA expression of hENT2 was detected in the MDA-MB-231, MDA-MB-468, or SK-BR-3 cells, not detected in the MCF-7 cells. 2MTT showed that IC50 of 5-FU in the MDAMB-231, MDA-MB-468, or SK-BR-3 cells was significantly lower than that in the MCF-7 cells(P < 0.05). There was no statistical difference of IC50 among the three lines(MDA-MB-231, MDA-MB-468, and SK-BR-3)(P > 0.05).③The three lines(MDA-MB-231, MDA-MB-468, and SK-BR-3) with lower IC50 of 5-FU highly expressed hENTs, and MCF-7 cell with the higher IC50 of 5-FU expressed less hENTs. ConclusionsThe expressions of hENTs in breast cancer cell lines can significantly influence 5-FU cytotoxic effect. It is implicated that the hENTs expressions might be the clue to the choice of nucleoside anticancer drugs in clinic.
ObjectiveTo investigate the value of different minimally invasive surgical techniques, stent placement, laparoscopic surgery, and sustained-releasing 5-fluorouracil, in solving intestinal obstruction due to colorectal cancer. MethodsFrom May 2000 to May 2010, total 68 patients with obstructed colorectal cancers in three centers were treated in two ways in terms of the stage: The first, patients with resectable tumors underwent colorectal stent placement as a ‘bridge to surgery’ guided by enteroscope under X-ray. After clinical decompression and bowel preparation, laparoscopic radical resection was performed. The second, patients with unresectable tumors underwent rectal stent placement just for palliation. Sustained-releasing 5-fluorouracil was implanted into the local cancerous intestinal tract through stent walls. ResultsFifty-one of 52 patients underwent laparoscopic radical resection successfully following stent placement, while one failed and died during follow-up 93 d postoperatively. Forty patients with successful laparoscopic surgery were followed up in 3 to 36 months (with an average of 15 months) without tumor planting in the incision, postoperative local recurrence or anastomotic stricture. Fifteen unresectable patients and one high-risk, intolerable patient underwent rectal stent placement and implantation of sustained-releasing 5fluorouracil. During follow-up 3 to 24 months (with an average of 14 months), 11 died, who survived for (350±222) d (range 101-720 d), and 5 were still alive for 3 to 13 months (with an average of 9 months) without intestinal obstruction. ConclusionsLaparoscopic surgery combined with stent placement is an effective and safe procedure for resectable obstructed colorectal cancer. For unresectal obstructed rectal cancer, rectal stent placement combined with sustained-releasing 5-fluorouracil can prolong survival time avoiding colostomy.
Objective To investigate the effects and mechanism of 3-methyladenine （3-MA， an autophagy inhib-itor） on the apoptosis of hepatocellular carcinoma cell line SMMC7721 induced by 5-fluorouracil （5-FU）. Methods The autophagy was observed using fluorescent microscope by monodansyicadaverin （MDC） staining. The viability of SMMC-7721 cell induced by 5-FU was measured using CCK8 assay before and after autophagy inhibited by 3-MA， meanwhile the apoptosis of SMMC7721 cell was determined via AnnexinⅤ/PI assay. The light chain 3 protein （LC3， the autophagy specific protein） and caspase-3， PARP protein were detected by Western blot. Results The autophagy of SMMC7721 cell could be induced by 5-FU after treatment for 48 h， the cell survival rate was （60.73±2.65）%， and the apoptosis rate was （40.42±2.34）%. Compared with the group of 5-FU treatment， the survival rate of SMMC7721 cell in the combination of 5-FU and 3-MA after treatment for 48 h decreased to （42.31±1.32）% （P＜0.01）， and the apoptosis rate increased to （60.92±2.99）% （P＜0.01）， meanwhile the expressions of LC3-Ⅱ， activation fragment of caspase-3， and cleavage frag-ment of PARP significantly increased （P＜0.01）. Conclusions Autophagy is a protective phenomenon during the SMMC7721 cell line apoptosis induced by 5-FU， and autophagy inhibition may enhance the sensitivity of SMMC7721 cell line to 5-FU treatment， which is probably associated with the activation of caspase-3 and cleavage of PARP. Therefore， autophagy inhibition could be a promising strategy for adjuvant chemotherapy in hepatocellular carcinoma.
Objective To explore the postoperative influence of intra-abdominal implantation of sustained-releasing 5fluorouracil on the hepato-renal function, immune function, nutritional state and complications in patients with gastric and colorectal cancer. Methods Sixty-five patients with gastric or colorectal cancer were included into this study from January to June 2009. The patients (35 cases of gastric cancer, 18 cases of colon cancer and 12 cases of rectal cancer) were randomly divided into experimental group (n=25) and control group (n=40). In experimental group, 400 mg sustained-releasing 5-fluorouracil was used. Blood samples were gained before operation, the second day and the seventh day after operation to examine the indexes of hepato-renal function, immune function and nutritional state. Complications， venting time and length of stay after operation were also recorded. Results There was no statistical significance for distribution of tumor stages and patients’ gender between experimental group and control group (Pgt;0.05). Preoperative indexes of hepato-renal function, immune function and nutritional state were also not reached statistical significance between two groups (Pgt;0.05). Compared with control group, the indexes of total protein and transferrin were decreased and urea nitrogen and IgM were increased in the second day after operation in experimental group (Plt;0.05). The number of lymphocyte was increased, while CD4, Alb, total protein and IgA were decreased in the seventh day after operation in experimental group, respectively. The time of passage of gas of experimental group was longer than that of control group (Plt;0.05).Conclusion Intra-abdominal implantation of sustained-releasing 5-fluorouracil is safe and feasible, which does not increase the complications and the time of length stay after operation. However, there is a little influence on immune function and gastrointestinal function after operation for intra-abdominal implantation of sustained-releasing 5-fluorouracil.
Objective To investigate the effect of imbedding chemotherapy of sustained release of 5-fluorouracil on the healing of colonic stoma in dog. Methods Twenty-eight adult hybrid dogs were randomly divided into chemotherapy group （n=22) and control group (n=6). The canine sigmoid colon were firstly detached and then anastomosed via median abdominal incision, 200 mg sustained release of 5-fluorouracil was imbedded in the mesentery 1.0-1.5 cm away from colonic stoma in chemotherapy group, whereas the control substance was injected into the dogs in control group. Tissue samples were collected from mesentery and stomas on 3, 5, 7, 10 and 15 days after operation, respectively, in order to observe the healing of stoma. The drug concentrations in the stoma and in the tissues that were 0, 1, 3, 5, 7, 10 and 15 cm away from the imbedding point were also measured by high performance liquid chromatographymethod at different phases. Results The tissues from colonic stoma only showed inflammatory reaction at early stage, with no necrosis and cellular degeneration. It was observed that the stoma healed basically on the tenth day after operation. The drug concentrations in the tissues gradually decreased at the range of 0-15 cm over time, but all of which were higher than the anti-tumor effective concentration （0.10 μg/g）. Conclusion The imbedding chemotherapy of sustained release of 5-fluorouracil in mesentery has little effect on the healing of stoma, and it could remain an effective anti-tumor concentration in a period of time.
Objective To study the effect of 5-fluorouracil (5-FU) induced apoptosis of the rectal carcinoma cell line HR8348 in vitro and the relationship between apoptosis induced by 5-FU and the expression of bcl-2，bcl-xl，bax and p53,and to investigate the possible mechanism of apoptosis of rectal carcinoma cell line HR8348 induced by 5-FU.Methods After treatment with 5-FU for 24 h,the apoptotic index was detected by methyl green and pyronine Y staining and by terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick end labeling (TUNEL).The bcl-2,bcl-xl,bax and p53 gene expression of HR8348 cells were examined by immunohistochemical method.Results After treatment with 5-FU,the apoptotic index of experiment group was significantly increased,there was significant difference as compared with the control.Exposed to 5-FU for 12 h,24 h and 36 h,the expression of bcl-2 of HR8348 cell line remained unchanged,but the expression of bcl-xl slightly diminished,while the expression of bax was remarkly increased,the expression of p53 was not detected in both experiment and control groups.Conclusion This results indicate that 5-FU may induce apoptosis of rectal carcinoma cell line HR8348 and the possible mechanism of apoptosis induction is through upregulation of bax expression and the change of bax to bcl-xl ratio.
ObjectiveTo investigate the effects of dipyridamole (DP), one of the human equilibrative nucleoside transporters (hENTs) blockers, on 5-fluorouracil (5-FU) induced cell apoptosis and cell cycle changes of the pancreatic cancer Panc-1 cell line. MethodsPancreatic cancer cell line Panc-1 was divided into hENTs blocked group and hENTs unblocked group. The hENTs blocked group was further divided into two subgroups according to the DP concentration, 5 μmol/L DP group and 10 μmol/L DP group. Each group was incubated in culture medium with or without 1.5×106 ng/L 5-FU for 24 h. Cell apoptosis and cell cycle were detected by flow cytometry. Results①The apoptosis results of each group： Incubated in culture medium with 1.5×106 ng/L 5-FU for 24 h, the apoptosis rates of 5 μmol/L DP group and 10 μmol/L DP group were higer than those of hENTs unblocked group （Plt;0.05）, and which of 10 μmol/L DP group was higer than that of 5 μmol/L DP group (Plt;0.05). Incubated in culture medium without 5-FU for 24 h, there were no significant differences of the apoptosis rates among three groups (Pgt;0.05). ②The cell cycle results of each group: Incubated in culture medium with 1.5×106 ng/L 5-FU for 24 h, the percentages of S phase cells in the 5 μmol/L DP group and 10 μmol/L DP group were less than those of hENTs unblocked group (Plt;0.05). The percentage of S phase cell of 5 μmol/L DP group reduced to 87.09% and that of 10 μmol/L DP group reduced to 74.06% as compared with hENTs unblocked group. 5-FU had little influence on G2 phase (Pgt;0.05) except for the percentage of G2 phase cells in the 5 μmol/L DP group increased significantly (Plt;0.05) as compared with the hENTs unblocked group. Incubated in culture medium without 5-FU for 24 h, there were no significant differences of the cell cycles among three groups (Pgt;0.05). ConclusionsIn pancreatic cancer Panc-1 cell, DP could enhance the cytotoxicity of 5-FU by blocking hENTs. The enhanced cytotoxicity is related to elevation of 5-FU concentration in cells, and unrelated to DP itself.
To evaluate the effect of 5-fluorouracil (5-FU) appl ied topically on preventing adhesion andpromoting functional recovery after tendon repair. Methods From August 2003 to June 2007, 48 patients with flexor tendonrupture of the fingers by sharp instrument were treated and randomly divided into two groups. In 5-FU group, 39 fingers of 26 patients included 17 males and 9 females, aged (29.3 ± 9.8) years; the locations were zone I in 19 fingers and zone II in 20 fingers; single finger was involved in 12 cases and more than 2 fingers were involved in 14 cases; and the time from injury to operation was (2.4 ± 1.6) hours. In control group, 36 fingers of 22 patients included 14 males and 8 females; aged (26.1 ± 8.7) years; the locations were zone I in 16 fingers and zone II in 20 fingers; single finger was involved in 10 cases and more than 2 fingers were involved in 12 cases; and the time from injury to operation was (2.1 ± 1.8) hours. No statistically significant difference was found in constituent ratio of age, gender, injured fingers and their zones, between two groups (P gt; 0.05). The repair site in 5-FU group was given 5-FU at a concentration of 25 mg/mL with a soaked sponge, and the synovial sheath of the repaired site was wrapped with the 5-FU-soaked sponge for 1 minute for 4 times after the tendons were repaired; normal sal ine was used in the control group. Results Wound healed by first intention and no infection and tendon rupture occurred in two groups. The patients were followed up for 3-8 months (mean 4.1 months) and 3-8 months (mean 3.9 months) in 5-FU group and in control group respectively. The functional recovery degrees of the fingers were evaluated with total active movement (TAM) evaluation system. In 5-FU group, the results were excellent in 22 fingers, good in 13 fingers, fair in 3 fingers and poor in 1 finger; the excellentand good rate was 89.7%. In control group, the results were excellent in 11 fingers, good in 15 fingers, fair in 9 fingers andpoor in 1 finger; the excellent and good rate was 72.2%. There was statistically significant difference in the functional recovery degrees of fingers between two groups (P lt; 0.05). The 2 fingers which had a poor result in 5-FU group and control group were served with tenolysis was performed in 2 cases having poor results after 6 months of operation and had an excellent result at last. Conclusion 5-FU appl ied topically can reduce tendon adhesions after the ruptured tendon repair.
Objective To assess the effect of topical appl ication of 5-fluorouracil (5-FU) on intimal hyperplasia in rabbit vein graft. Methods Sixty-four male New Zealand white rabbits, aged 5 months and weighing 2.8-3.0 kg, were randomly divided into group A, B, C, and D (n=16 rabbits per group). Artery defect model was establ ished by cutting about 1 cm artery from the middle part of the dissociated left common carotid artery. A section about 3 cm was cut from the right external jugular vein, and the harvested vein was inverted and end-to-end anastomosed to the artery defect with 9-0 non-traumatic suture. After anastomosis, the extima of the grafted veins in group A, B, and C was completely wrapped with cotton sheet (12 mm × 30 mm × 1 mm in size) immersed by 5-FU at a concentration of 50.0, 25.0, and 12.5 mg/mL, respectively, and eachvein was treated 5 times (1 minute at a time). In group D, the extima of the graft veins was treated with normal sal ine instead of 5-FU. The grafted veins were obtained 1, 2, 4, and 6 weeks after operation, HE staining and Masson staining were preformed for histological changes of grafted vein wall, prol iferating cell nuclear antigen (PCNA) immunohistochemistry staining and TUNEL label ing staining were conducted for prol iferation and apoptosis of smooth muscle cell of the grafted vein, and transmission electron microscope observation was performed for cellular ultrastructure. Results The HE staining, Masson staining, and PCNA immunohistochemistry staining showed that the thickness of intima in group A and B was obviously less than that in group C and D at 1, 2, 4, and 6 weeks after operation, and the prol iferation cells in group A and B were less than that in group C and D at 1, 2, and 4 weeks after operation. The thickness of the intima, the degree of intima hyperplasia, the degree of vessel lumen stenosis of four groups at different time points were as follows: at 1 week after operation, group A [(12.69 ± 1.68) μm, 0.73 ± 0.05, 0.025 ± 0.003], group B [(17.52 ± 2.01) μm, 0.86 ± 0.06, 0.027 ± 0.004], group C [(21.92 ± 1.85) μm, 1.06 ± 0.09, 0.036 ± 0.006] and group D [(26.45 ± 3.86) μm, 1.18 ± 0.08, 0.041 ± 0.005]; at 2 weeks after operation, group A [(24.61 ± 2.91) μm, 0.86 ± 0.06, 0.047 ± 0.003], group B [(37.28 ± 2.78) μm, 1.17 ± 0.09, 0.060 ± 0.004], group C [(46.52 ± 2.25) μm, 1.44 ± 0.08, 0.073 ± 0.003], and group D [(52.07 ± 3.29) μm, 1.45 ± 0.05, 0.081 ± 0.006]; at 4 weeks after operation, group A [(61.09 ± 6.84) μm, 1.38 ± 0.08, 0.106 ± 0.007], group B [(63.61 ± 8.25) μm, 1.40 ± 0.07, 0.107 ± 0.010], group C [(80.04 ± 7.65) μm, 1.64 ± 0.07, 0.129 ± 0.011], and group D [(84.45 ± 9.39) μm, 1.68 ± 0.10, 0.139 ± 0.014]; at 6 weeks after operation, group A [(65.27 ± 5.25) μm, 1.46 ± 0.07, 0.113 ± 0.005], group B [(65.82 ± 7.12) μm, 1.45 ± 0.05, 0.112 ± 0.011], group C [(84.45 ± 9.39) μm, 1.69 ± 0.09, 0.135 ± 0.007], and group D [(87.27 ± 8.96) μm, 1.76 ± 0.05, 0.140 ± 0.012]. Group A and B were inferior to group C and D in terms of the above three parameters and cell prol iferation index 1, 2 and 4 weeks after operation (P lt; 0.05). Group A and B were superior to group C and D in terms of cell apoptosis index of intima and media 1 and 2 weeks after operation (P lt; 0.05). Transmission electron microscope observation showed that the synthetic cell organelles such as rough endoplasmic reticulum, golgi apparatus, and ribosome in group A and B were obviously less than those in group C and D (P lt; 0.05). Conclusion Topicalappl ication of 5-FU can effectively inhibit intima hyperplasia of the vein grafts.
Objective Neuron purification is essential to procedure of various nerve cell experimental research, however, at present there is few reports on the effect of various factors on neural axons during purification. To find out a simple method of neuron purification, and to investigate the influence factors of corresponding purification culture in dorsal root gangl ion (DRG) tissue culture on β3-tubul in positive axon. Methods The DRGs were obtained from the 3 days neonatal SD rat microscopically and were made into cell suspension. Then, the amount of attached DRG neurons and non neuronal cells in poly-D-lysine (PDL) group, PDL/Laminin (PDL/LN) group and collagen-I (Col I) group was observed from 10 to 100 minutes. Then, the extension and arborization of β3-tubul in positive axons were observed after 72 hours completely randomised DRG tissue culture for the research of the influences among culture substrates (PDL, PDL/LN, and Col I), FBS (0, 5%, and 10%), 5 fluorouracil (5-Fu, 0, 20, and 40 μmol/L), and cytrarabine (Ara-C, 0, 10, and 20 μmol/L). Results Adherent cells were observed instantly after inoculation by inverted phase contrast microscope and inverted fluoresence microscope; after cell suspension was removed, adherent growth of DRGn cells and non-DRGn cells were still seen. In PDL group, the amount of NSE negative cells was significantly higher than that of NSE positive cells at 10 and 30 minutes (P lt; 0.05); the amount of NSE positive cells was significantly higher than that of NSE negative cells at 80, 90 and 100 minutes (P lt; 0.05). In PDL/LN gruop, there was no significant difference (P gt; 0.05) in the amount of NSE negative cells and NSE positive cells at 10, 20, 30, 40, and 50 minutes; the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 60, 70, 80, 90, 100 minutes. In Col I group, the amount of NSE negative cells was higher than that of NSE positive cells at 10-40 minutes, but showing no significant difference (P gt; 0.05); the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 70-100 minutes. At 72 hours after DRG tissue culture, the best result of β3-tubul in positive axon extension and arborization was obtained when the substrate level was PDL/LN, and the average length of PDL/LN level was significantly larger than that of other two substrates (P lt; 0.05). The highest number of β3-tubul in positive axon distal end was obtained at 5% concentration level of FBS (P lt; 0.05), but showing no significant differences in β3-tubul in positive axon length among three levels (P gt; 0.05). Both the most of β3-tubul in positive axon distal ends and the longest β3-tubul in positive axon average length were obtained at 0 μmol/L concentration level of 5-Fu, showing significant differences between 0 μmol/L level and 20, 40 μmol/L levels (P lt; 0.05). A similar result of β3-tubul in positive axon distal end was got at the 0 μmol/L level and 10 μmol/L level of Ara-C, which was significantly higher than that of 20 μmol/L level (Plt; 0.05). Conclusion? A purified DRG neuron suspension for neuron culture could be obtained via PDL differential attachment for 30 minutes. When DRG neuron culture, neuron special medium, PDL/LN substrate and 10 μmol/L Ara-C are recommended in β3-tubul in positive axon research.