Department of Oncology, Third Xiangya Hospital, Central South University, Changsha 410013, China.
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.
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.
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.
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).
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.
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。
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。
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）。
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。
