Objective To investigate the effect of irradiation combined with ligustrazine on the malignant biological behaviors of hepatocellular carcinoma cells.

Methods Hepatocellular carcinoma cells (HepG2, Huh-7, SK-Hep1, SMMC-7721, and Hep-3B) were irradiated with 2 Gy of γ-rays and cultured for different durations (12, 24, and 48 h). Cell counting kit-8 (CCK-8) proliferation assay was used to detect cell viability to screen for the irradiation-insensitive cells. Irradiation-insensitive cells were taken as the research object and divided into 0, 2, 4, and 6 Gy groups in accordance with irradiation doses. The 0 Gy group was subdivided into control and ligustrazine groups (final drug concentration of 4.4 mmol/L), whereas the 2, 4, and 6 Gy groups were each subdivided into irradiation and irradiation+ligustrazine groups (final drug concentration of 4.4 mmol/L). CCK-8 proliferation assay was used to detect cell viability to screen the irradiation dose for subsequent experiments. Subsequently, control, ligustrazine (final drug concentration of 4.4 mmol/L), irradiation, and irradiation+ligustrazine groups (final drug concentration of 4.4 mmol/L) were established, and wound healing and Transwell invasion assays were conducted to detect the effects of ligustrazine and irradiation on cell viability and migration and invasion abilities. The effects of ligustrazine and irradiation on the mRNA and protein relative expression levels of X-ray repair cross-complementing protein 6 (also known as KU70) and cyclin-dependent kinase 12 (CDK12) were examined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence assays. Independent samples t-test was used to compare measurement data between two groups.

Results The results of the CCK-8 proliferation assay showed that compared with 12 h after irradiation, there was no statistically significant difference in the viability of Huh-7 cells at 48 h after irradiation (t=1.28, P>0.05). Therefore, the Huh-7 cells were the most insensitive to irradiation. Compared with Huh-7 cells in the 2, 4, and 6 Gy irradiation groups, the viability of Huh-7 cells in the 2, 4, and 6 Gy irradiation+ligustrazine groups were significantly decreased (t=2.42, 2.68, 3.12; all P<0.05). At 12 h after wound healing, the wound healing ability of Huh-7 cells in the irradiation+ligustrazine group was also significantly decreased and the percentage of remaining scrath area was significantly increased compared with that in the irradiation group and the ligustrazine group (t=3.05, 3.72; both P<0.05). In the Transwell invasion assay, the A₄₅₀ of the Huh-7 cells in the ligustrazine (0.66±0.05) and irradiation+ligustrazine groups (0.48±0.04) had significantly decreased compared with those in the irradiation group (0.82±0.06) (t=2.89, 3.41; both P<0.05). In addition, qRT-PCR results showed that compared with those in the irradiation group, the relative expression levels of KU70 and CDK12 mRNA and proteins in the cells in the ligustrazine group and the irradiation+ligustrazine group had all decreased (t=2.81, 3.57, 2.95, 3.62; all P<0.05). Immunofluorescence assays further demonstrated that compared with the relatively strong fluorescence signals in the control group and the irradiation group, the fluorescence signals of KU70 and CDK12 proteins in the cells of the ligustrazine group were weakened, and the decrease in fluorescence signal intensity was most pronounced in the irradiation+ligustrazine group, confirming that their protein relative expression levels were significantly down-regulated.

Conclusion Ligustrazine may enhance the sensitivity of Huh-7 cells to irradiation by inhibiting the expression of the deoxyribonucleic acid double-strand break repair related proteins KU70 and CDK12, thereby inhibiting the malignant biological behaviors, such as proliferation, migration, and invasion, of hepatocellular carcinoma cells.