Cancer Res Treat. 2019;51(2):451-463.

Pan-Pim Kinase Inhibitor AZD1208 Suppresses Tumor Growth and Synergistically Interacts with Akt Inhibition in Gastric Cancer Cells.

, , , , , , , , , , , , , , ,

제1저자 이미소 · 교신저자 임석아

PMID 29879757 PMC6473293 DOI 10.4143/crt.2017.341 IF 4.2 혈액종양내과

소속 (3)

Cancer Research Institute, Seoul National University, Seoul, Korea.
Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.

Abstract

PURPOSE Pim kinases are highly conserved serine/threonine kinases, and different expression patterns of each isoform (Pim-1, Pim-2, and Pim-3) have been observed in various types of human cancers, including gastric cancer. AZD1208 is a potent and selective inhibitor that affects all three isoforms of Pim. We investigated the effects of AZD1208 as a single agent and in combination with an Akt inhibitor in gastric cancer cells.

MATERIALS AND METHODS The antitumor activity of AZD1208 with/without an Akt inhibitor was evaluated in a large panel of gastric cancer cell lines through growth inhibition assays. The underlying mechanism was also examined by western blotting, immunofluorescence assay, and cell cycle analysis.

RESULTS AZD1208 treatment decreased gastric cancer cell proliferation rates and induced autophagy only in long-term culture systems. Light chain 3B (LC3B), a marker of autophagy, was increased in sensitive cells in a dose-dependent manner with AZD1208 treatment, which suggested that the growth inhibition effect of AZD1208 was achieved through autophagy, not apoptosis. Moreover, we found that cells damaged by Pim inhibition were repaired by activation of the DNA damage repair pathway, which promoted cell survival and led the cells to become resistant to AZD1208. We also confirmed that the combination of an Akt inhibitor with AZD1208 produced a highly synergistic effect in gastric cancer cell lines.

CONCLUSION Treatment with AZD1208 alone induced considerable cell death through autophagy in gastric cancer cells. Moreover, the combination of AZD1208 with an Akt inhibitor showed synergistic antitumor effects through regulation of the DNA damage repair pathway.

Keywords

AZD1208AktAutophagyPim kinase inhibitorStomach neoplasms

Figures

**Fig. 1.** Effects of AZD1208 treatment on the inhibition of human gastric cancer cell growth. (A) Chemical structure of AZD1208. (B) The growth inhibition effects of AZD1208 were evaluated with a colony formation assay. The percentage of surviving cells was calculated by counting the number of colonies and are shown as a graph with standard error bars (n=3).

**Fig. 2.** Basal levels of Pim kinase protein expression in gastric cancer cell lines. Basal levels of Pim kinase proteins in human gastric cancer cell lines were confirmed by western blot analysis. BT-549 sample and α-tubulin were used as loading controls.

**Fig. 3.** Effects of AZD1208 treatment on the substrates of Pim kinase. Cells were treated with increasing doses of AZD1208 for 120 hours. Western blot analysis was performed with the indicated antibodies. α-Tubulin was used as a loading control.

**Fig. 4.** Induction of cell death by AZD1208 through stimulation of autophagy. (A) Cells were treated with dimethyl sulfoxide (DMSO; control) or 1 μM AZD1208 for 36 or 120 hours. The expression levels of light chain 3B (LC3B) and Beclin-1 were measured by western blot analysis. α-Tubulin was used as a loading control. (B) SNU-601 and SNU-638 cells transfected with GFP-LC3B were treated with 1 μM AZD1208 for 5 days. Confocal microscopy was used to observe the signals corresponding to LC3B expression (green fluorescence). DNA was counterstained with DAPI (blue). The merged images represent overlapping signals of the two channels. (C) SNU-638 cells were pre-treated with the autophagy inhibitor 3-methyladenine (3-MA; 10 μM) or caspase-3 inhibitor z-VAD-fmk (10 μM) for 24 hours. Next, the cells were treated with 1 μM AZD1208 every 3 days for 14 days. The percentages of surviving cells were calculated by counting the number of colonies and are presented in a bar graph with standard error bars (n=3). ^a)p=0.008.

**Fig. 5.** Association of the DNA damage repair pathway with AZD1208 resistance. Cells were treated with dimethyl sulfoxide (control) and 1 or 5 μM AZD1208 for 120 hours. The expression levels of ATM and Chk2 were measured by western blot analysis. α-Tubulin was used as a loading control.

**Fig. 6.** Enhanced antitumor effects of the combination of AZD1208 and an Akt inhibitor in gastric cancer cells. (A) Cells were seeded and cultured with increasing concentrations of AZD5363 and 1 μM AZD1208 every 3 days. The cells were cultured for 14 days until colonies formed and were then stained. The percentages of surviving cells were calculated by counting the number of colonies and are presented in a bar graph with standard error bars (n=3). ^a)p < 0.005. (B) Cells were treated for 120 hours with 1 μM AZD1208 and/or 100 nM AZD5363. The expression levels of the indicated proteins were determined by western blot analysis. α-Tubulin was used as a loading control. (C) Cells were treated with AZD1208 and AZD5363 alone or in combination for 5 days, and immunofluorescence analysis was subsequently performed. Confocal microscopy was used to observe the signals corresponding to p-Chk2 (red) and γ-H2AX (green). DAPI (blue) was used as a nuclear counterstain. CI, combination index. Scale bars=5 μm.

그림 출처: PubMed Central 공개 논문 (PMC6473293)

Conflict of interest statement

Yung-Jue Bang has advised or consulted for and received academic research funding from Bayer, Novartis, Boehringer-Ingelheim, Roche/Genentech, AstraZeneca, Merck Serano, MSD, BristolMyers Squibb, Eli Lilly, Pfizer, ONO, Taiho, and GreenCross. SeockAh Im is a recipient of academic research funds from AstraZeneca, Inc., and has served as a consultant and on the advisory board for AstraZeneca, Novartis, and Roche without compensation. Do-Youn Oh is a recipient of academic research funds from AstraZeneca, Inc. None of the other authors declare any potential conflicts of interest.

그 밖의 정보