Curcuminoid EF24 enhances the anti-tumour activity of Akt inhibitor MK-2206 through ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction in gastric cancer

Abstract

Background and purpose: Gastric cancer is one of the leading causes of morbidity and mortality worldwide. Akt is an anti-apoptotic kinase that plays a dynamic role in cell survival and is implicated in the pathogenesis of gastric cancer. MK-2206, the first allosteric inhibitor of Akt, is in clinical trials for a number of cancers. Although preclinical studies showed promise, clinical trials reported it had no effect when given alone at tolerated doses. The aim of our study was to delineate the effects of MK-2206 on gastric cancer cells and explore the ability of combination treatments to enhance the anti-tumour activity of MK-2206.

Experimental approach: SGC-7901, BGC-823 cells and immunodeficient mice were chosen as a model to study the treatment effects. Changes in cell viability, apoptosis and ROS, endoplasmic reticulum stress and mitochondrial dysfunction in the cells were analysed by MTT assays, ROS imaging and FACSCalibur, electron microscopy, JC-1 staining and western blotting.

Key results: MK-2206 induced apoptotic cell death through the generation of ROS. We utilized ROS production to target gastric cancer cells by combining MK-2206 and an ROS inducer EF24. Our in vitro and in vivo xenograft studies showed that combined treatment with MK-2206 and EF24 synergistically induced apoptosis in gastric cancer cells and caused cell cycle arrest. These activities were mediated through ROS generation and the induction of endoplasmic reticulum stress and mitochondrial dysfunction.

Conclusion and implications: Targeting ROS generation by using a combination of an Akt inhibitor and EF24 could have potential as a therapy for gastric cancer.

Figure 1

MK‐2206 reduces gastric cancer cell viability and generates ROS. (A) Effect of MK‐2206 treatment on gastric cancer cell lines showing effective inhibition of Akt phosphorylation. (B–C) Cells were treated with MK‐2206 for 24 h, and cell viability was measured. Figure showing reduced viability of human gastric cancer cells (B) and normal cells (C) upon MK‐2206 exposure. (D) Gastric cancer cells were treated with MK‐2206 for 16 h, and cleaved‐PARP levels were determined by western blot. (E) Induction of apoptosis in human gastric cancer cells was determined by Annexin V/PI staining following treatment with MK‐2206 for 24 h (CON = control; MK‐10, −20 and −40 = 10, 20 and 40 μM MK‐2206). (F) Intracellular ROS generation by MK‐2206 was measured in SGC‐7901 and BGC‐823 cells by redox‐sensitive dye DCFH‐DA. (G) Quantification of DCF data from (F). All representative images are from five independent experiments. Data are reported as mean ± SEM and analysed by Student's t‐test; n = 5 independent experiments; *P < 0.05 compared with DMSO control.

Figure 2

EF24 potentiates ROS production by MK‐2206. (A) Quantification of DCF flow cytometry data of intracellular ROS levels in SGC‐7901 and BGC‐823 cells treated with different concentrations of MK‐2206 (5, 10 and 20 μM) with or without 2 μM EF24 (Median = mean fluorescence intensity). (B) Quantification of DCF flow cytometry data showing the effect of NAC pretreatment for 2 h on ROS levels. Data are reported as mean ± SEM and analysed by Student's t‐test; n = 6 independent experiments; *P < 0.05. (C) Representative DCF fluorescence images of cells exposed to MK‐2206 and EF24 with or without NAC pretreatment. All images are representative of six independent experiments.

Figure 3

The combination treatment of MK‐2206 and EF24 induces apoptosis in human gastric cancer cells. (A) The effects of combined MK‐2206 and EF24 treatment on the viability of human gastric cancer cells. Cells were treated with increasing concentrations of MK‐2206 and 2 μM EF24 for 24 h. (B) The induction of apoptosis as assessed by Annexin V/PI staining following combined treatment with MK‐2206 and EF24 for 24 h. (C) Western blot analysis of apoptosis‐related proteins in cells treated with MK‐2206 and EF24 for 16 h. Figure showing levels of Bcl‐2, Bax, cleaved‐PARP and cleaved‐caspase 3. (D) Effect of combined treatment on colony formation. Cells were treated for 24 h and stained with crystal violet on day 8. (E) Gastric cancer cells were pretreated with 5 mM NAC for 2 h before exposure to MK‐2206 and EF24 for 24 h. Apoptosis was detected by Annexin V/PI staining. (F) Quantification of data presented in panel (E). Data are reported as mean ± SEM and analysed by Student's t‐test; n = 5 independent experiments; *P < 0.05. (G) Analysis of cleaved‐PARP, Bcl‐2 and Bax levels in SGC‐7901 cells pretreated with NAC. All representative images are from five independent experiments.

Figure 4

EF24 enhances MK‐2206‐mediated cell cycle arrest. (A) SGC‐7901 and BGC‐823 cells (SGC and BGC, respectively) were treated with a combination of MK‐2206 and EF24 for 18 h. The number of cells in the G2/M phase was determined by PI staining. (B) Representative histogram from cell cycle analysis presented in panel (A). (C) Expression of G2/M phase‐related proteins MDM‐2, cyclin B1 and Cdc2 in cells exposed to MK‐2206 and EF24 for 15 h. (D) Effect of NAC pretreatment on cell cycle distribution as determined by PI staining. Cells were pretreated with 5 mM NAC for 2 h before exposure to MK‐2206 and EF24 for 18 h. (E) Histogram generated from PI flow cytometry data presented in panel (D). (F) Total lysates from cells exposed to MK‐2206 and EF24, with or without NAC pretreatment, were subjected to analysis of cell cycle‐related proteins. All images are representative of five independent experiments.

Figure 5

ER stress and mitochondrial dysfunction induced by MK‐2206 and EF24 treatment. (A) Effect of combined treatment with MK‐2206 and EF24 on ER in SGC‐7901 cells as assessed by electron microscopy. Exposure of cells to 10 μM MK‐2206 in combination with 2 μM EF24 for 6 h induced ER alterations (arrows indicate swollen ER). However, the ER appears normal (indicated by arrows) in the DMSO control group, NAC‐treated cells and cells treated with NAC prior to MK‐2206 and EF24. Lower panel of (A) shows higher magnification. (B) Western blot analysis of ER stress pathway markers in cells treated with MK‐2206 and EF 24 for 3 h (ATF‐4 and p‐EIF2α) or 12 h (CHOP). (C) ER stress pathway proteins in cells exposed to MK‐2206 and EF24 as in panel (B) but with NAC pretreatment of 2 h. For panels (B) and (C), GAPDH and eIF2α served as internal controls. (D) Mitochondrial membrane potential (Δψm) was detected by JC‐1 dye. SGC‐7901 and BGC‐823 cells were exposed to 10 μM MK‐2206 in combination with 2 μM EF24 for 12 h, with or without 2 h pretreatment with NAC (scale bar = 20 μM). (E) Effect of MK‐2206 in combination with EF24 on the morphology of mitochondria in SGC‐7901 cells. Exposure of cells to 10 μM MK‐2206 in combination with 2 μM EF24 for 12 h induced mitochondrial dysfunction as illustrated by swollen mitochondria (arrows). These changes were not seen in cells treated with DMSO (control), NAC or MK and EF24 following NAC pretreatment (arrows indicate normal mitochondria). Lower panel of (E) shows higher magnification. (F) Western blot analysis of JNK phosphorylation in SGC‐7901 cells exposed to MK‐2206 in combination with EF24 for 6 h. (G) p‐JNK levels in cells cultured as in (F) but with NAC pretreatment. All images are representative of five independent experiments.

Figure 6

Anti‐tumour activity is enhanced by combining EF24 and MK‐2206 in gastric cancer xenograft model. (A–C) Tumour volume changes, harvested tumour specimens and tumour weight from mice treated with 10 mg·kg⁻¹ MK‐2206 (MK‐10) or 10 mg·kg⁻¹ MK‐2206 in combination with 3 mg·kg⁻¹ EF24. (D) Levels of oxidative stress marker MDA in the tumour tissues. (E) Immunohistochemical staining of tumour specimens for cell proliferation marker Ki‐67, apoptosis marker cleaved caspase 3 and phospho‐Akt (scale bar =50 μm). (F) Western blot analysis of cleaved‐PARP cleavage, cleaved‐caspase 3 and p‐EIF2α using tumour tissue lysates. Data are reported as mean ± SEM and analysed by Student's t‐test; n = 8 mice per group; *P < 0.05 and ns = not significant. All images are representative of eight mice per group.