AG-1024 Sensitizes Sorafenib-Resistant Hepatocellular Carcinoma Cells to Sorafenib via Enhancing G1/S Arrest

Abstract

Purpose: The frequency in resistance to sorafenib accounts for the grim prognosis of advanced hepatocellular carcinoma (HCC). In the present study, we explore the anti-cancer efficacy of co-administration of sub-toxic AG-1024 with sorafenib in HCC cells to enhance the sensitivity of these cells to sorafenib.

Materials and methods: Two acquired sorafenib-resistant HCC cells, SNU-sora-5 and SK-sora-5, were established and verified. The MTT assay, colony formation assay, cell morphology detection and flow cytometric analysis were then used to determine the anti-tumor effects of the co-administration of sub-toxic AG-1024 and sorafenib. Finally, the potential molecular mechanism was preliminarily examined.

Results: Compared to parental cell lines, the acquired sorafenib-resistant cell lines, SNU-sora-5 and SK-sora-5, were more resistant to sorafenib. Sub-toxic AG-1024 markedly enhanced sorafenib-mediated cell inhibition in acquired sorafenib-resistant HCC strains, with a reversal index (RI) of 4.64 in SNU-sora-5 and 4.58 in SK-sora-5 cell lines. Moreover, co-administration of sub-toxic AG-1024 and sorafenib exerted dramatic cytotoxicity compared with sorafenib alone in the intrinsic sorafenib-resistant HCC-LM3 cells. In contrast to high-dose sorafenib, sub-toxic AG-1024 combined with sorafenib had less impact on apoptosis while significantly enhancing G1/S arrest via activation of the mTOR/p21 signaling pathway. The more, pharmacological inhibition of mTOR activity by inhibitor Palomid 529 significantly antagonized the synergistic anti-cancer effects of AG-1024 and sorafenib in HCC cells.

Conclusion: The current findings indicate that sub-toxic AG-1024 may be a promising therapeutic agent in enhancing the sensitivity in HCC cells to sorafenib, bringing hope to HCC patients refractory to sorafenib treatment.

Keywords: AG-1024; G1/S arrest; hepatocellular carcinoma; mTOR; sorafenib resistance.

Figure 1

Establishment and verification of acquired sorafenib-resistant HCC cells. (A and B) The IC₅₀ values of sorafenib were determined in SNU-sora-5, SK-sora-5, SNU-449 and SK-hep-1 after 72h treatment. (C and D) Colony formation of SNU-sora-5, SK-sora-5, SNU-449 and SK-hep-1 with various treatments of sorafenib (0 and 5μM). (E–H) Cell cycle analysis of SNU-sora-5, SK-sora-5, SNU-449 and SK-hep-1 after different treatments for 72h and the G1/S phase histogram. Data are shown as mean ± SD. Student’s t-test was used for two-group comparisons. **P <0.01.

Figure 2

Sub-toxic AG-1024 and sorafenib exerts synergistic anti-cancer effects in acquired sorafenib-resistant HCC cells. (A) SNU-sora-5 and SK-sora-5 were incubated with increasing doses of AG-1024 alone for 72h. (B and C) SNU-sora-5 and SK-sora-5 were exposed to a series of concentrations of sorafenib with or without 5μM AG-1024 for 72h. The IC₅₀ values of sorafenib were determined and the RI of 5μM AG-1024 combined with 2μM sorafenib was also calculated compared with 2μM sorafenib. (D–G) Colony formation of SNU-sora-5 and Sk-sora-5 with various treatments and the number of clones were counted. Data are shown as mean ± SD. Student’s t-test was used for two-group comparisons. **P <0.01.

Figure 3

Sub-toxic AG-1024 and sorafenib exerts synergistic anti-cancer effects in intrinsic sorafenib-resistant HCC cells. (A) The IC₅₀ values of sorafenib of different cells were calculated after being incubated with increasing doses of sorafenib alone for 72h. (B and C) HCC-LM3 was exposed to a series of concentrations of sorafenib with or without 5μM AG-1024 for 72h. The IC₅₀ values of sorafenib were calculated and the RI of 5μM AG-1024 combined with 2μM sorafenib was also calculated compared with 2μM sorafenib. (D and E) Colony formation of HCC-LM3 with various treatments and the number of clones were counted. Data are shown as mean ± SD. Student’s t-test was used for two-group comparisons. **P <0.01.

Figure 4

Sub-toxic AG-1024 combined with sorafenib has less impact on apoptosis. (A) Morphological images of SNU-sora-5 taken with an inverted microscope (x100) after various treatments for 72h. (B and C) Apoptosis assay of SNU-sora-5 after different treatments for 72h. (D and E) Western blot analysis of apoptosis-related proteins, PARP, Cle-PARP, Bax, Bcl-2 and Bcl-xl in SNU-sora-5. Data are shown as mean ± SD. Student’s t-test was used for two-group comparisons. **P <0.01.

Figure 5

Sub-toxic AG-1024 combined with sorafenib significantly enhance G1/S arrest. (A–C) Cell cycle analysis of SNU-sora-5 after different treatments for 72h and the G1/S phase histogram. (D) Western blot analysis of cell cycle-related proteins, CDK4, CDK6, Cyclin D, CDK2, Cyclin E and p-Rb in SNU-sora-5. Data are shown as mean ± SD. Student’s t-test was used for two-group comparisons. **P <0.01.

Figure 6

Sub-toxic AG-1024 combined with sorafenib enhance G1/S arrest via activating the mTOR/p21 signaling pathway. (A and B) Western blot analysis of IGF-1R, p-IGF-1R, AKT, p-AKT, mTOR, p-mTOR, p53 and p21 in SNU-sora-5. (C and D) Colony formation of SNU-sora-5 with various treatments and the number of clones were counted. SNU-sora-5 was exposed to co-administration treatment with or without Palomid 529. Data are shown as mean ± SD. Student’s t-test was used for two-group comparisons. **P <0.01.