(-)-Guaiol triggers immunogenic cell death and inhibits tumor growth in non-small cell lung cancer

Abstract

(-)-Guaiol is a sesquiterpenoid found in many traditional Chinese medicines with potent antitumor activity. However, its therapeutic effect and mechanism in non-small cell lung cancer (NSCLC) have not been fully elucidated. In this study, (-)-Guaiol was found to induce immunogenic cell death (ICD) in NSCLC in vitro. Using (-)-Guaiol in vivo, we found that (-)-Guaiol could suppress tumor growth, increase dendritic cell activation, and enhance T-cell infiltration. Vaccination experiments suggest that cellular immunoprophylaxis after (-)-Guaiol intervention can suppress tumor growth. Previous studies have found that (-)-Guaiol induces apoptosis and autophagy in NSCLC. Apoptosis and autophagy are closely related to ICD. To explore whether autophagy and apoptosis are involved in (-)-Guaiol-induced ICD, we used inhibitors of apoptosis and autophagy. The results showed that the release of damage-associated molecular patterns (DAMPs) was partly reversed after inhibition of apoptosis and autophagy. In conclusion, these results suggested that the (-)-Guaiol triggers immunogenic cell death and inhibits tumor growth in NSCLC.

Keywords: (−)-Guaiol; Apoptosis; Autophagy; DAMPs; ICD; NSCLC.

Fig. 1

DAMP release after (–)-Guaiol treatment in vitro. A The CRT level in three separate experiments detected by flow cytometry. B Representative image of CRT location and expression of three confocal images. Green, CRT; Red, β-catenin indicates the cell membrane; Blue, DAPI indicates the nucleus. C ATP content in the supernatant detected by an ATP assay kit. D Content of HMGB1 in the cell supernatant detected by enzyme-linked immunosorbent assay (ELISA). E Content of HSP70/90 in the cell supernatant detected by ELISAs. **p < 0.01, ***p < 0.001 vs. the control group

Fig. 2

Effect of (–)-Guaiol on tumor growth and immune infiltration in vivo. A Tumor volume in the 28 day experiment (n = 5). B Tumor weight at the end of the experiment (n = 5). C Representative image of MHC II, CD11c, CD86, CD4+ T, CD8+ T, CD69+ T of three immunohistochemical (IHC) stains. (D) Representative images of three HE stains in the heart, liver, spleen, lung, and kidney. *p < 0.05, ***p < 0.001 vs. the control group

Fig. 3

The vaccine effect of (–)-Guaiol in vivo. A Tumor volume and body weight during the 22 days of experiment (Control n = 3, LLC, n = 4, (–)-Guaiol + LLC, n = 4). B Tumor weight in the end of the experiment. Ns, not significant vs Control group; **p < 0.01, ***p < 0.001 vs. Control group; ##p < 0.01 vs. (–)-Guaiol + LLC Group

Fig. 4

Effect of apoptosis inhibitor on (–)-Guaiol induced DAMP release. A Apoptosis detected by flow cytometry. B The CRT level detected by flow cytometry. C ATP content in the supernatant detected by an ATP assay kit. D Content of HMGB1 in the cell supernatant detected by enzyme-linked immunosorbent assays (ELISAs). E Content of HSP70/90 in the cell supernatant detected by ELISAs. **p < 0.01, ***p < 0.001 vs. the control group; #p < 0.01, ##p < 0.01, ###p < 0.001 vs. the (–)-Guaiol group

Fig. 5

Effect of autophagy inhibitor on (–)-Guaiol induced DAMP release A LC3 I and LC3 II detected by WB. B The CRT level detected by flow cytometry. C ATP content in the supernatant detected by an ATP assay kit. D Content of HMGB1 in the cell supernatant detected by enzyme-linked immunosorbent assays (ELISAs). E Content of HSP70/90 in the cell supernatant detected by ELISAs. ***p < 0.001 vs. the control group; #p < 0.01, ##p < 0.01, ###p < 0.001 vs. the (–)-Guaiol group