Aldolase A promotes cervical cancer cell radioresistance by regulating the glycolysis and DNA damage after irradiation

Abstract

Radioresistance is the major obstacle that affects the efficacy of radiotherapy which is an important treatment for cervical cancer. By analyzing the databases, we found that aldolase A (ALDOA), which is a key enzyme in metabolic reprogramming, has a higher expression in cervical cancer patients and is associated with poor prognosis. We detected the expression of ALDOA in the constructed cervical cancer radioresistance (RR) cells by repetitive irradiation and found that it was upregulated compared to the control cells. Functional assays were conducted and the results showed that the knockdown of ALDOA in cervical cancer RR cells inhibited the proliferation, migration, and clonogenic abilities by regulating the cell glycolysis. In addition, downregulation of ALDOA enhanced radiation-induced apoptosis and DNA damage by causing G2/M phase arrest and further promoted radiosensitivity of cervical cancer cells. The functions of ALDOA in regulating tumor radiosensitivity were also verified by the mouse tumor transplantation model in vivo. Therefore, our study provides new insights into the functions of ALDOA in regulating the efficacy of radiotherapy and indicates that ALDOA might be a promising target for enhancing radiosensitivity in treating cervical cancer patients.

Keywords: ALDOA; DNA damage; cervical cancer; glycolysis; radioresistance; radiotherapy.

Figure 1.

ALDOA is overexpressed in CC RR cell lines and correlated with prognosis. (a) By analyzing the UCSC XENA database (https://xenabrowser.Net/datapages/), ALDOA expression was significantly increased in CC tissues compared with normal cervical tissues (***P < .001). (b) According to the TCGA database (https://portal.Gdc.cancer.gov/), CC patients with higher ALDOA expression have a lower overall survival rate (*P < .05). (c) According to the AUC curve of the UCSC XENA database (https://xenabrowser.Net/datapages/), ALDOA can be used as a better diagnostic standard for CC (AUC = 86.20% with 95% CI = 74.1–98.2%). (d) Colony formation assay showed that the CC RR cells (SiHa-RR and C33A-RR) had stronger resistance to RT. (e) The expression of ALDOA in CC cell lines SiHa, SiHa-RR, C33A and C33A-RR was measured by qRT-PCR. (f) The protein expression level of ALDOA in SiHa, SiHa-RR, C33A and C33A-RR was detected by western blot. *P < .05, **P < .01, ***P < .001.

Figure 2.

Knockdown ALDOA inhibits cell clonal formation, proliferation, and migration. (a) The mRNA expression of ALDOA in transfected SiHa-RR or C33A-RR cells was measured by qRT-PCR. (b) The protein expression level of ALDOA in transfected SiHa-RR, C33A-RR cells was measured by western blot. (c) The clonal formation experiment showed that the cloning ability of SiHa-RR and C33A-RR decreased after ALDOA knockdown. (d) Cell proliferation of transfected SiHa-RR or C33A-RR cells was analyzed by CCK-8 assays. OD 450 values at indicated time points were displayed to indicate cell proliferation. (e-f) The transfected cells were used for migration measurement, stained with crystal violet, and counted under the light microscope. *P < .05, **P < .01, ***P < .001.

Figure 3.

Targeting ALDOA promotes CC cell apoptosis, G2/M phase arrest, and DNA damage induced by irradiation. (a-b) Apoptosis was assessed by flow cytometry in the control group, ALDOA knockdown group, irradiation group and combined treatment group. (c-d) Cell cycle of the control group, ALDOA knockdown group, irradiation group and combined treatment group was measured by flow cytometry. (e-f) Immunofluorescence was used to detect the number of γ-H2AX foci. *P < .05, **P < .01.

Figure 4.

Knockdown ALDOA in CC RR cells combined with irradiation inhibits the growth of CC xenograft in vivo. (a) Representative images of xenograft tumors in mice. The animal study was repeated three times (n = 5). (b) Tumor weight of mice in different groups. (c) Changes in tumor volume in different groups. (d-e) Representative images and statistical analysis of immunohistochemical staining of tumor tissues. *P < .05, **P < .01, ***P < .001.

Figure 5.

Knockdown ALDOA in CC RR cells inhibits the glycolysis pathway. (a) Seahorse Bioscience XFp analyzer added with the glucose (10 mM), oligomycin (1.0 μM), and 2-deoxy-d-glucose (2-DG, 50 mM) was used to detect the cellular acidification rate of transfected cells. (b) Determination of glucose consumption in SiHa-RR and C33A-RR by stably knockdown ALDOA. (c) Determination of lactic acid production in SiHa-RR and C33A-RR cells by stably knockdown ALDOA. (d-e) The expression levels of glycolytic proteins LDHA, PKM2, and HK1 in transfected SiHa-RR, C33A-RR cells were measured by western blot. *P < .05, **P < .01, ***P < .001.