摘要
目的:探讨三氧化二砷(Arsenic trioxide, As_2O_3)及As_2O_3与放射线联合对食管癌Eca109细胞株的生长抑制作用;分析两者联合对Eca109细胞周期和凋亡的影响。为As_2O_3的临床应用提供理论依据。
方法:(1)As_2O_3对Eca109细胞增殖的抑制作用:采用MTT法检测不同浓度As_2O_3(0.05μmol/L、0.10μmol/L、0.15μmol/L、0.20μmol/L、0.25μmol/L、0.30μmol/L、0.35μmol/L、0.40μmol/L、0.45μmol/L、0.50μmol/L),在不同时间段(12 h、24 h、48 h、72h)对Eca109细胞增殖的抑制率;(2)放射线对Eca109细胞增殖的抑制作用:采用MTT法检测不同剂量的放射线(0.5Gy、1Gy、2Gy、4Gy、6Gy、8Gy),在不同时间段(12h、24h、48h、72h)对Eca109细胞增殖的抑制率;(3)As_2O_3联合放射线对Eca109细胞增殖的抑制作用:采用MTT法检测两种浓度的As_2O_3(0.20μmol/L、0.25μmol/L)联合(2Gy、4Gy)的放射线在不同的时间段(12h、24h、48h、72h)对Eca109细胞增殖的抑制率;(4)As_2O_3联合放射线对Eca109细胞周期及凋亡的影响:采用流式细胞技术检测0.20μmol/L的As_2O_3联合2Gy的放射线在不同时间(48h、72h)点对Eca109细胞周期及凋亡的影响;(5)采用免疫印迹技术检测0.20μmol/L的As_2O_3联合2Gy的放射线处理48h后,Eca109细胞中细胞色素(ccyt-c)和锰超氧化物歧化酶(Manganese superoxide dismutase, MnSOD)的表达变化情况。
结果:(1)As_2O_3在0.05～0.50μmol/L浓度范围内均能一定程度的抑制Eca109细胞的增殖,且随着浓度增加和作用时间延长,对细胞增殖的抑制作用增强。浓度在0.05～0.20μmol/L时,抑制率低,当浓度大于0.25μmol/L后,抑制率迅速升高。0.20～0.25μmol/L是抑制率由低到高的缓冲区。(2)放射线在0.5～8Gy范围内均对Eca109细胞增殖有一定程度的抑制作用。其中在0.5～2Gy之间,抑制率低,当剂量大于4Gy后,抑制作用明显增强。2～4Gy是抑制率由低到高的缓冲区。(3)0.20μmol/L、0.25μmol/L浓度的As_2O_3分别联合2Gy、4Gy的放射线在各时间段(12h、24h、48h、72h)对Eca109细胞的抑制率较单纯药物组和单纯照射组均高,并且抑制率大于单药组和照射组的抑制率之和。随着时间的延长,各个联合组抑制率随之升高。其中0.25μmol/L+4Gy联合组在72h的细胞抑制率最大,为83.56%,与其它联合组在对应时间段相比较,有显著性差异(P=0.000～0.001)。而0.25μmol/L+2Gy与0.20μmol/L+4Gy两组相比,在各对应时间段其抑制率无明显差异(P=0.057～0.298)。(4)流式细胞技术检测结果显示:0.20μmol/L+2Gy联合组在72h的凋亡率最高,为12.26%,与单药组和单纯照射组比较,有显著性差异(P=0.008～0.046)。联合组在72h细胞周期改变也最为明显,细胞被阻滞于G2/M期,该期所占比例为27.5%,与非联合组比较有显著性差异(P=0.000)。(5)Western blotting分析结果显示,cyt-c在各组未发生明显变化(P=0.27～0.86),而MnSOD在各处理组中均有所降低,且0.20μmol/L+2Gy联合组降低最为明显,两两比较均有显著性差异(P=0.000～0.028)。
结论:(1)As_2O_3能抑制食管癌Eca109细胞的增殖,具有明显的量效和时效关系。(2)As_2O_3能提高放射线对Eca109细胞增殖的抑制作用,有增敏作用。(3)As_2O_3可通过影响Eca109细胞周期,诱导细胞凋亡,来增强放射线对细胞的杀伤作用。(4)放射线可降低Eca109细胞中MnSOD蛋白的表达,与As_2O_3联合可使这一作用明显增强,二者单独或联合均对cyt-c的表达无影响。
Objective: To explore the effect of arsenic trioxide (Arsenic trioxide, As_2O_3) and As_2O_3 combined with radiation for the growth inhibition of esophageal cancer cell line -Eca109 and to analysis the impact of As_2O_3 joined with radiation for the cell cycle and apoptosis of Eca109 in order to provide a theoretical basis for As_2O_3 applied the treatment of esophageal cancer.
Methods: (1) As_2O_3 on the inhibition of proliferation: The inhibitory effect on proliferation was measured by MTT assay when Eca109 was treated by 0.05μmol/L, 0.10μmol/L, 0.15μmol/L, 0.20μmol/L, 0.25μmol/L, 0.30μmol/L, 0.35μmol/L, 0.40μmol/L, 0.45μmol/L, 0.50μmol/L As_2O_3 for four different treatment times (12h, 24h, 48h, 72h); (2) Radiation on the inhibition of proliferation: The inhibitory effect on proliferation Eca109 was measured by MTT assay when Eca109 was treated by 0.5Gy, 1Gy, 2Gy, 4Gy, 6Gy, 8Gy radiation for four different treatment times (12h, 24h, 48h, 72h); (3) As_2O_3 combined with radiation on the inhibition of proliferation: The inhibitory effect on proliferation was measured by MTT assay when Eca109 was treated by two concentrations of As_2O_3 (0.20μmol/L, 0.25μmol/L) combined with radiation (2Gy, 4Gy) for four different treatment times (12h, 24h, 48h, 72h); (4) As_2O_3 combined with radiation on the cell cycle and apoptosis of Eca109 cell: The effect on the cell cycle and apoptosis was detected by flow cytometry when Eca109 was treated by 0.20μmol/L As_2O_3 combined with 2Gy radiation for two different treatment times (48h, 72h); (5) The description changes of cyt-c and MnSOD were detected by Western blotting when Eca109 was treated by 0.20μmol/L As_2O_3 combined with 2Gy radiation for 48h.
Results: (1) As_2O_3 whose concentration range between 0.05 and 0.50μmol / L was able to a certain degree to inhibit the proliferation of esophageal cancer cells-Eca109. With the concentration increasing and the time prolonging, inhibition of cell proliferation enhanced. When the concentration between 0.05 and 0.20μmol/L, inhibition rate increased more moderate, but when exceeds 0.25μmol/L, it increased rapidly. 0.20～0.25μmol/L was a buffer area for inhibition rate from low to high. (2) Radiation whose dose range between 0.5 and 8Gy was able to a certain degree to inhibit the proliferation of esophageal cancer cells-Eca109. When the dose between 0.5 and 2Gy, inhibition rate either decreased or had no change, but when the dose exceeded 4Gy, inhibition rate increased rapidly. 2～4Gy was a buffer area for inhibition rate from low to high. (3) Compared with drug group or irradiation group, the inhibition rate of joined group was higher in the corresponding period times. What was more, it exceeded to the sum of inhibition rate of drug group and irradiation group. With the time prolonging, inhibition rate of every joint group increased. The largest of cell inhibitory rate was 83.56%, which occurred in the joint group after 72 hours. It had significant difference when compared with other joined group in the corresponding period(P=0.000～0.001). However, the comparison of the two joint group which was As_2O_3 (0.25μmol/L) combined with radiation (2Gy) and As_2O_3 (0.20μmol/L) combined with radiation (4Gy) had no significant difference in the corresponding period times ( P=0.057～0.298). (4) Flow cytometry results showed that the highest apoptosis rate occurred at 72 hours in the joint group which was 12.26%. Compared with single drug group or single radiation group, it had significant difference (P=0.008～0.046). The most obvious change of cell cycle happened in the joint Group after 72 hours, where G2/M phase arrest occurred, and the proportion of this period was 27.5% (P=0.000). (5) Western blotting analysis revealed that cyt-c in each group did not change significantly (P=0.27～0.86), while MnSOD expression in the treatment group had reduced, and the joint group which reduced the most obviously was As_2O_3 (0.20μmol/L) combined with radiation (2Gy). The comparison of any two group had significant difference (P=0.000～0.028).
Conclusion: (1) With a clear dose and time effect, As_2O_3 could inhibit the proliferation of esophageal cell line-Eca109. (2) As_2O_3 had a synergistic effect to improve radiation’s inhibition on Eca109 cells. (3) Via influencing cell cycle and inducing apoptosis, As_2O_3 could enhance radiation’s killing effect on Eca109 cells; (4) Radiation could reduce the expression of MnSOD protein, and this effect could be significantly enhanced when combined with As_2O_3. Neither individual nor joints had effect on the expression of cyt-c.
引文
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