Lorlatinib-mediated ALK pathway inhibition results in apoptosis and senescence induction, and the senolytic agent ABT-263 enhance the efficacy of lorlatinib against HCC cells
(A) HepG2/C3A and Huh7 cells were treated with lorlatinib (10 μM) or DMSO for 96 h and then analyzed by fluorescence-activated cell sorting (FACS)-based annexin V-PE staining to analyze the apoptotic rates. For HepG2/C3A, p = 0.0275 (DMSO versus lorlatinib (n = 3 each)), t = 3.391837, df = 4. For Huh7, p = 0.0186 (DMSO versus lorlatinib (n = 3 each)), t = 3.830, df = 4. p values were calculated using a two-tailed, unpaired Student’s t test.
(B) HepG2/C3A and Huh7 cells were treated with lorlatinib (10 μM) or DMSO for 96 h and then analyzed by fluorescence-activated cell sorting (FACS)-based cleaved caspase-3 staining. For HepG2/C3A, p < 0.0001 (DMSO versus lorlatinib (n = 3 each)), t = 39.89, df = 4. For Huh7, p < 0.0001 (DMSO versus lorlatinib (n = 3 each)), t = 24.36, df = 4. p values were calculated using a two-tailed, unpaired Student’s t test.
(C) HepG2/C3A and Huh7 cells were treated with lorlatinib or DMSO for 96 h and then analyzed for cleaved-caspase3 and cleaved PARP. ACTINB was used as a loading control.
(D) HepG2/C3A and Huh7 cells were treated with lorlatinib or DMSO for 96 h and then analyzed for senescence-associated β-galactosidase assay (SA β-gal). Representative images for HepG2/C3A and Huh7 cells treated with either lorlatinib or DMSO. Scale bar, 100 μm.
(E) HepG2/C3A, Huh7 cells treated with lorlatinib (10 μM) or DMSO and IL-8 gene expression was analyzed using RT-qPCR. ACTINB mRNA expression was used as an internal normalization control. Relative IL-8 mRNA expression is plotted. For HepG2/C3A, p < 0.0001 (DMSO versus lorlatinib (n = 3 each)), t = 974.0, df = 4. For Huh7, p < 0.0001 (DMSO versus lorlatinib (n = 3 each)), t = 67.71, df = 4. p values were calculated using a two-tailed, unpaired Student’s t test.
(F) HepG2/C3A, Huh7cells treated with lorlatinib (10 μM) or DMSO and IL-8 protein expression was analyzed using immunoblotting. ACTINB was used as a loading control.
(G) HepG2/C3A and Huh7 cells were treated with DMSO, lorlatinib, ABT-263, or both lorlatinib and ABT-263, and clonogenic assay was performed. Representative wells for the HepG2/C3A and Huh7 cells under the indicated treatment conditions are shown. All quantitative data represent the mean ± SEM.