Effects of anlotinib hydrochloride on the expression of immunogenic cell death-related molecules in Cal27 tongue cancer cells.

OBJECTIVE

To investigate the effects of anlotinib hydrochloride (AL3818) on the expression of Immunogenic Cell Death (ICD)-related molecules in tongue cancer cells.

METHODS

(1) The human Tongue Squamous Cell Carcinoma (TSCC) cell line Cal27 was cultured, and the half-maximal inhibitory concentration (IC50) of AL3818 and paclitaxel (PTX) on Cal27 cells was determined using the CCK-8 assay. The cells were divided into four groups: control group, AL3818 group, PTX group, and AL3818 + PTX group; (2) The apoptosis rate of each group was measured by flow cytometry; (3) The levels of calreticulin (CRT) and heat shock protein 70 (HSP70) were detected by immunofluorescence and flow cytometry; (4) The concentration of High Mobility Group Box 1 (HMGB1) was measured by Enzyme-Linked Immunosorbent Assay (ELISA); (5) Adenosine Triphosphate (ATP) levels were assessed using an ATP luminescence assay kit; and (6) The cells were divided into the control group, AL3818 group, AL3818 + CCT020312 (perk activator) treatment group, and AL3818 + ISRIB (perk inhibitor) treatment group, and detect the relative expression levels of total CRT, membrane-bound CRT, p-perk, and p-eIF2a in each group using the western blot method.

RESULTS

(1) The IC50 values of AL3818 and PTX at 24 h were 6.254 μmol/L and 1.718 μmol/L, respectively (P < 0.01); (2) AL3818 and PTX induced apoptosis in Cal27 cells (P < 0.05), with the highest apoptosis rate observed in the AL3818 + PTX group compared to the other three groups (P < 0.05); (3) AL3818 and PTX increased the expression of ICD-related molecules, including CRT, HMGB1, ATP, and HSP70, with the AL3818 + PTX group demonstrating the most significant effect (P < 0.05); and (4) AL3818 can induce early exposure of CRT on the membrane in the Cal27 cell line. Compared with the control group, the AL3818 + CCT020312 positive control group, and the AL3818 + ISRIB negative control group, the differences are statistically significant, while the total CRT remains roughly unchanged. The experimental results also indicate that after AL3818 acts on the Cal27 cell line for 24 h, there is an upregulation of p-perk and p-eIF2a expression along with the synchronous expression of CRT on the membrane.

CONCLUSION

AL3818 has the potential to induce ICD in the Cal27 TSCC cell line by modulating the levels of ICD-related molecules such as ATP, CRT, HSP70, and HMGB1. Moreover, the combination of AL3818 and PTX is more effective in inducing ICD in Cal27 cells compared to either agent alone. The process by which CRT is transferred from within the cell to the membrane during the induction of ICD in the Cal27 cell line by AL3818 may be related to the phosphorylation and activation of the PERK/elF2a signaling pathway.