寡霉素 A 促进 HT29 结直肠癌细胞的放射抗性及其机制。
Oligomycin A promotes radioresistance in HT29 colorectal cancer cells and its mechanisms.
机构信息
Department of Oncology, Third Xiangya Hospital, Central South University, Changsha 410013, China.
出版信息
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2021 Feb 28;46(2):113-120. doi: 10.11817/j.issn.1672-7347.2021.200063.
OBJECTIVES
Radiotherapy is one of the main therapies for colorectal cancer, but radioresistance often leads to radiotherapy failure. To improve the radioresistance, we explore the effect of oligomycin A, the H-ATP synthase inhibitor, on the sensitivity of HT29 colorectal cancer cells to irradiation and its underlying mechanisms.
METHODS
The effects of different concentrations of oligomycin A on the survival rate and glycolysis of HT29 colorectal cancer cells at different time points were investigated via MTT and glycolysis assay. siRNA-PFK1 was synthesized in vitro and transfected into HT29 cells. The effects of oligomycin A on radiosensitivity of HT29 colorectal cancer cells were measured via MTT and colony formation assay. Western blotting was used to detect the effect of oligomycin A on the expression of glycolytic enzyme PFK1. We compared difference between the effects of siRNA-PFK1 group and oligomycin A combined with siRNA-PFK1 group on cell survival and glycolysis. After 4 Gy X-ray irradiation, the effects of cell survival and glycolysis between the siRNA-PFK1 group and the oligomycin A combined with siRNA-PFK1 group were compared.
RESULTS
Compared with the 0 μmol/L oligomycin A group, the cell survival rate of HT29 cells treated with 4 μmol/L oligomycin A was significantly increased (<0.05), and the glucose uptake, the lactic acid, and the ATP production were also significantly increased (all <0.01). After X-ray irradiation at different doses (0, 2, 4, 6, and 8 Gy), the colony formation rate and cell survival rate of the 4 μmol/L oligomycin A treated group were significantly higher than those in the 0 μmol/L oligomycin A group (both <0.01). The sensitization enhancement ratio of oligomycin A on HT29 colorectal cancer cells was 0.4886. The expression of PFK1 in the 4 μmol/L oligomycin A group was significantly higher than that in the 0 μmol/L oligomycin A group (<0.001). The glycolysis level, colony formation rate, and cell survival rate of the siRNA-PFK1 HT29 cells group were significantly lower than those in the 0 μmol/L oligomycin A group (all <0.05), while the results in the 4 μmol/L oligomycin A combined with siRNA-PFK1 group were significantly higher than those in the siRNA-PFK1 group (all <0.001). After 4 Gy X-ray irradiation, the colony formation rate and cell survival rate in the siRNA-PFK1 group were decreased compared with those in the irradiation group (<0.01 or <0.001), while the results of the 4 μmol/L oligomycin A combined with siRNA-PFK1 group were significantly higher than those in the siRNA-PFK1 group (both <0.001).
CONCLUSIONS
Oligomycin A can promote the radioresistance of HT29 colorectal cancer cells, which may be related to up-regulation of the PFK1 expression and increase of cell glycolysis.
目的
放疗是结直肠癌的主要治疗方法之一，但放射抵抗常常导致放疗失败。为了提高放射抵抗性，我们研究了寡霉素 A（H+-ATP 合酶抑制剂）对 HT29 结直肠癌细胞对辐射敏感性的影响及其潜在机制。
方法
通过 MTT 和糖酵解试验，研究不同浓度的寡霉素 A 在不同时间点对 HT29 结直肠癌细胞存活率和糖酵解的影响。体外合成 siRNA-PFK1 并转染 HT29 细胞。通过 MTT 和集落形成试验测量寡霉素 A 对 HT29 结直肠癌细胞放射敏感性的影响。Western blot 用于检测寡霉素 A 对糖酵解酶 PFK1 表达的影响。我们比较了 siRNA-PFK1 组和寡霉素 A 联合 siRNA-PFK1 组对细胞存活和糖酵解的影响差异。在 4 Gy X 射线照射后，比较 siRNA-PFK1 组和寡霉素 A 联合 siRNA-PFK1 组之间细胞存活和糖酵解的差异。
结果
与 0 μmol/L 寡霉素 A 组相比，用 4 μmol/L 寡霉素 A 处理的 HT29 细胞的细胞存活率明显增加（<0.05），葡萄糖摄取、乳酸和 ATP 生成也明显增加（均<0.01）。在不同剂量（0、2、4、6 和 8 Gy）的 X 射线照射后，用 4 μmol/L 寡霉素 A 处理的细胞集落形成率和细胞存活率明显高于 0 μmol/L 寡霉素 A 组（均<0.01）。寡霉素 A 对 HT29 结直肠癌细胞的增敏增强比为 0.4886。寡霉素 A 组的 PFK1 表达明显高于 0 μmol/L 寡霉素 A 组（<0.001）。siRNA-PFK1 HT29 细胞组的糖酵解水平、集落形成率和细胞存活率明显低于 0 μmol/L 寡霉素 A 组（均<0.05），而寡霉素 A 联合 siRNA-PFK1 组的结果明显高于 siRNA-PFK1 组（均<0.001）。在 4 Gy X 射线照射后，siRNA-PFK1 组的集落形成率和细胞存活率较照射组下降（均<0.01 或<0.001），而寡霉素 A 联合 siRNA-PFK1 组的结果明显高于 siRNA-PFK1 组（均<0.001）。
结论
寡霉素 A 可促进 HT29 结直肠癌细胞的放射抵抗性，这可能与 PFK1 表达上调和细胞糖酵解增加有关。
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