IDF-11774 Induces Cell Cycle Arrest and Apoptosis by Inhibiting HIF-1α in Gastric Cancer

Abstract

Hypoxia-inducible factor-1 alpha (HIF-1α) is a regulatory factor of intracellular oxygen supersession. The expression or increased activity of HIF-1α is closely related to various human cancers. Previously, IDF-11774 was demonstrated to inhibit HSP70 chaperone activity and suppress the accumulation of HIF-1α. In this study, we aimed to determine the effects of IDF-11774 on gastric cancer cell lines. Treatment with IDF-11774 was found to markedly decrease the proliferation, migration, and invasion of the gastric cancer cell lines. Furthermore, the phosphorylation levels of extracellular signal-regulated kinase 1/2, p38, and Jun N-terminal kinase in the mitogen-activated protein kinase signaling pathways were markedly increased in a dose-dependent manner, ultimately promoting apoptosis via the induction of cell cycle arrest. Our findings indicate that HIF-1α inhibitors are potent drugs for the treatment of gastric cancer.

Keywords: HIF-1α; HIF-1α inhibitor; IDF-11774; apoptosis; cell cycle arrest; gastric cancer.

Figure 1

Baseline expression of hypoxia-inducible factor-1 alpha (HIF-1α) in gastric cancer cells and HIF-1α knockdown-induced suppression of gastric cancer cell proliferation. (A) Immunoblotting analysis of HIF-1α expression under normoxia in MKN74 and MKN45 cells. (B) Immunoblotting analysis of HIF-1α expression in MKN74 and MKN45 cells after dimethyloxalylglycine treatment in the dose and time-dependent manners. (C) Immunoblotting analysis of HIF-1α expression in MKN45 cells transfected with small interfering ribonucleic acids (siRNAs) for 48 h. (D) Effects of HIF-1α knockdown using siRNAs on the viability of MKN45 cells (n = 3). Cell viability was monitored for 3 days using a CellTiter-Glo luminescent assay (normalized to control wild-type cells on day 0; ** p < 0.01). (E) Effects of HIF-1α knockdown using siRNAs on colony formation in MKN45 cells (n = 3). MKN45 cells transfected with HIF-1α siRNAs were cultured for 48 h and stained with crystal violet to visualize and determine the number of colonies (** p < 0.01). (F) Kaplan–Meier representation of the overall survival between the two groups of patients with high (red line) and low (black line) HIF-1α expression.

Figure 2

Hypoxia-inducible factor-1 alpha (HIF-1α) knockdown suppresses the growth of gastric cancer cells via cell cycle arrest, apoptosis, and the mitogen-activated protein kinase (MAPK) pathway. (A) MKN45 cells were transfected with HIF-1α siRNA for 48 h and then lysed and immunoblotted with HIF-1α, cell cycle arrest-associated proteins (Cyclin B1, D1, and E1), and β-actin. (B) MKN45 cells were transfected with HIF-1α siRNA for 48 h and nocodazole for 16 h, and then released into G1 for up to 40 h; nocodazole was replaced with fresh media. Samples were lysed and immunoblotted with HIF-1α, cell cycle arrest-associated proteins (Cyclin B1, D1, E1, Cyclin-dependent kinase 4, p53, and p21), and β-actin. (C) MKN45 cells were transfected with HIF-1α siRNA for 48 h and then lysed and immunoblotted with HIF-1α, apoptosis-associated proteins (poly(ADP ribose) polymerase, cleaved caspase-3), and β-actin. (D) MKN45 cells were transfected with HIF-1α siRNAs for 48 h and then lysed and immunoblotted with HIF-1α, MAPK pathway proteins (phospho-extracellular signal-regulated protein kinases (ERK), ERK, phosphor-Jun N-terminal kinase (JNK), JNK, phosphor-p38, and p38), and β-actin.

Figure 3

IDF-11774 promotes hypoxia-inducible factor-1 alpha (HIF-1α) degradation in gastric cancer cells. (A) MKN74 and MKN45 cells were treated with 15 μM and 30 μM IDF-11774 for 4 h and then lysed and immunoblotted with HIF-1α and β-actin. (B) After induction of hypoxia with dimethyloxalylglycine for 4 h, MKN74 and MKN45 cells were treated with 15 μM and 30 μM IDF-11774 for 4 h, and then lysed and immunoblotted with HIF-1α and β-actin. (C) MKN74 and MKN45 cells were treated with DMSO or 30 μM IDF-11774 for 4 h. Thereafter, de novo protein synthesis was inhibited using the cycloheximide treatment. Lysis and immunoblotting with HIF-1α and β-actin were performed 0, 5, 10, 20, and 30 min after cycloheximide treatment. (D) MKN74 and MKN45 cells were treated with DMSO, 15 μM or 30 μM IDF-11774 for 4 h, followed by inhibition of the proteasome using MG132. Lysis and immunoblotting with HIF-1α were subsequently performed. (E) Fractionation assay with total cell lysate, nuclear and cytoplasmic HIF-1α in MKN74 and MKN45 cells after treatment with 30 μM IDF-11774 for 4 h.

Figure 4

IDF-11774 inhibits the proliferation, migration, and invasion of gastric cancer cells. (A) Effect of IDF-11774 (15 and 30 μM) on the viability of MKN45 and MKN74 cells (n = 3). Cell viability was monitored for 3 days using a CellTiter-Glo luminescent assay (normalized to control wild-type cells at day 0; ** p < 0.01). (B) Effects of IDF-11774 (15 and 30 μM) on colony formation in MKN45 and MKN74 cells (n = 3). MKN45 and MKN74 cells transfected with hypoxia-inducible factor-1 alpha (HIF-1α) short interfering ribonucleic acids were cultured for 48 h and stained with crystal violet to visualize and determine the number of colonies (*** p < 0.001). (C) Effects of IDF-11774 (15 and 30 μM) on the migration of MKN45 and MKN74 cells. Cell migration was estimated using a Transwell migration assay, and data are presented as the mean ± standard deviation of the number of migrated cells (n = 5, (** p < 0.01). (D) Effects of IDF-11774 (15 and 30 μM) on the invasion of MKN45 and MKN74 cells. Cell invasion was estimated using a Transwell migration assay, and the data are presented as the mean ± standard deviation of the number of migrated cells (n = 5, (** p < 0.01).

Figure 5

IDF-11774 induces cell cycle arrest and apoptosis by downregulating hypoxia-inducible factor-1 alpha (HIF-1α) in gastric cancer cells. (A) MKN45 and MKN74 cells were treated with 30 μM IDF-11774 for 4 h and then lysed and immunoblotted with HIF-1α, cell cycle arrest-associated proteins (Cyclin B1, D1, and E1), and β-actin. (B) MKN45 and MKN74 cells were treated with 30 μM IDF-11774 for 4 h and nocodazole for 16 h. Thereafter, the cells were released into G1 for up to 40 h; nocodazole was replaced with fresh media. Samples were lysed and immunoblotted with HIF-1α, cell cycle arrest-associated proteins (Cyclin B1, D1, E1, cyclin-dependent kinase 4, p53, and p21), and β-actin. (C) MKN45 and MKN74 cells were treated with 30 μM IDF-11774 for 4 h and then lysed and immunoblotted with HIF-1α, apoptosis-associated proteins (poly(ADP ribose) polymerase, cleaved caspase-3), and β-actin. (D) MKN45 and MKN74 cells were treated with 30 μM IDF-11774 for 4 h and then lysed and immunoblotted with HIF-1α, mitogen-activated protein kinase pathway proteins (phospho-extracellular signal-regulated protein kinases (ERK), ERK, phosphor-Jun N-terminal kinase (JNK), JNK, phosphor-p38, and p38), and β-actin.

Figure 6

Graphical overview of the effect of IDF-11774 on the gastric cancer cell lines, MKN45 and MKN74. IDF-11774 mediates the degradation of hypoxia-inducible factor-1 alpha, arresting cell cycle progression and increasing mitogen-activated protein kinase activation, ultimately causing apoptosis.