Identification of DNA-PKcs as a primary resistance factor of TIC10 in hepatocellular carcinoma cells

Abstract

The current study tested the anti-hepatocellular carcinoma (HCC) cell activity of TIC10, a first-in-class small-molecule tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) inducer. TIC10 exerted potent anti-proliferative and pro-apoptotic actions in primary and established human HCC cells. TIC10 blocked Akt-Erk activation, leading to Foxo3a nuclear translocation, as well as TRAIL and death receptor-5 (DR5) transcription in HCC cells. We propose that DNA-PKcs is a major resistance factor of TIC10 possibly via inhibiting Foxo3a nuclear translocation. DNA-PKcs inhibition, knockdown or mutation facilitated TIC10-induced Foxo3a nuclear translocation, TRAIL/DR5 expression and cell apoptosis. Reversely, exogenous DNA-PKcs over-expression inhibited above actions by TIC10. In vivo, oral administration of TIC10 significantly inhibited HepG2 tumor growth in nude mice, which was further potentiated with Nu7026 co-administration. Thus, TIC10 shows promising anti-HCC activity, alone or together with DNA-PKcs inhibitors.

Keywords: DNA-PKcs; TIC10; TRAIL and chemosensitization; hepatocellular carcinoma (HCC).

Figure 1. TIC10 inhibits HCC cell proliferation in vitro

Established HCC cell lines, HepG2 (A–C) and Huh-7 (D), primary human HCC cells (D, “Pri_1/Pri _2”), as well as HL-7702 human hepatocytes (D) and primary human adult hepatocytes (“Hepatocytes”, D), were either left untreated (“C”, same for all figures), or treated with applied concentration of TIC10 (0.1–30 μM), cells were then cultured in conditional medium for applied time; Cell proliferation was tested by MTT assay (A and D), clonogenicity assay (B) and [H³] Thymidine incorporation assay (C). Experiments in this figure were repeated for five times, with similar results obtained. n = 5 for each repeat. Bars stand for mean ± SD. *p < 0.05 vs. group “C”.

Figure 2. TIC10 induces TRAIL and DR5 expression, provokes apoptosis in HCC cells

HepG2 cells (A–F) Huh-7 cells (G and H), primary human HCC cells (G and H, “Pri_1/Pri _2”), as well as HL-7702 human hepatocytes (G and H) and primary human adult hepatocytes (“Hepatocytes”, G and H), were treated with applied concentration of TIC10 (0.1–30 μM), cells were then cultured in conditional medium for applied time; TRAIL/DR5 mRNA expression (A and G) and capase-3/−8 (“Cas-3/−8”) activation (B) were tested; Apoptosis was tested by listed assays (C, D and H). HepG2 cells, pretreated for 1 hour with 50 μM of z-IETD-fmk (“IETD”), z-DEVD-fmk (“DEVD”) or z-VAD-fmk (“VAD”), were treated with TIC10 (10 μM) for applied time; Cell apoptosis (E, TUNEL assay) and proliferation (F, MTT assay) were tested. Experiments in this figure were repeated for four times, with similar results obtained. n=5 for each repeat. Bars stand for mean ± SD *p < 0.05 vs. group “C”. ^#p < 0.05 vs. TIC10 only (E and F).

Figure 3. DNA-PKcs could be a primary resistance factor of TIC10 in HCC cells

HepG2 cells, expressing dominant negative DNA-PKcs (“dnDNA-PKcs”, Flag-tagged), DNA-PKcs shRNA (“shDNA-PKcs”), wild-type DNA-PKcs (“wtDNA-PKcs”, Flag-tagged), or the parental control HepG2 cells (“Ctrl”), were treated with applied concentration of TIC10, or together with the DNA-PKcs inhibitor Nu7026 (10 μM), cells were further cultured in the conditional medium for applied time; Cell proliferation was tested by MTT assay (A and D); Cell apoptosis was tested by the Histone DNA ELISA assay (B and E) or TUNEL staining assay (C); Expression of DNA-PKcs (both wild-type and mutant) and Tubulin (loading control) were shown (A and D, upper panels). The primary human HCC cells (“Pri_1”), transfected with DNA-PKcs siRNAs (“-1/−2”) or scramble siRNA (“si-scr”), were treated with TIC10 (10 μM) or plus Nu7026 (10 μM) for indicated time; Cell proliferation was tested by MTT assay (F); Expressions of DNA-PKcs and Tubulin (loading control) were shown (F, upper panel). Experiments in this figure were repeated for three times, with similar results obtained. n = 5 for each repeat. Bars stand for mean ± SD *p < 0.05 vs. group “C”. ^#p < 0.05 vs. “Ctrl” (A–E) or “si-scr” (F).

Figure 4. DNA-PKcs counteracts TIC10-induced Foxo3p nuclear translocation

HepG2 cells were treated with applied concentration of TIC10 (0.1-30 μM), cells were then cultured in conditional medium for applied time; Expressions of listed proteins in cytosol (A) or nuclear (B) fraction lysates were tested by Western blotting assay, and five sets of repeated blots were quantified (A-B). HepG2 cells, expressing dominant negative DNA-PKcs (“dnDNA-PKcs”), DNA-PKcs shRNA (“shDNA-PKcs”), wild-type DNA-PKcs (“wtDNA-PKcs”), or the parental control cells (“Ctrl”), were treated with TIC10 (10 μM), or together with Nu7026 (10 μM), cells were further cultured in the conditional medium for applied time; Expressions of listed proteins in cytosol (C) or nuclear (D) fraction lysates were tested by Western blotting assay, and five sets of repeated blots were quantified (C-D); TRAIL (E) and DR5 (F) mRNA expression was also tested. *p < 0.05 vs. group “C”. ^#p < 0.05 vs. “Ctrl” cells.

Figure 5. Nu7026 facilitates TIC10-induced anti-HepG2 tumor activity in vivo

HepG2 bearing nude mice (n = 10 for each group) were administrated daily with TIC10 (30 mg/kg, oral administration) and/or Nu7026 (50 mg/kg, intraperitoneal injection, i.p.) for a total of 21 days; Weekly tumor volumes (in mm³) (A) and mice body weights (in gram) (D) were shown; Tumor daily growth was also calculated (B). At the end of the experiments (“W7”), tumors were isolated and weighted (C). Bars stand for mean ± SD. “W” stands for Week. *p < 0.05 vs. group “Vehicle”. ^#p < 0.05 vs. TIC10 treatment only (A-C).