Korean J Physiol Pharmacol.  2017 Jul;21(4):397-405. 10.4196/kjpp.2017.21.4.397.

MDL-12330A potentiates TRAIL-induced apoptosis in gastric cancer cells through CHOP-mediated DR5 upregulation

Affiliations
  • 1Department of Pathology, College of Medicine, Chosun University, Gwangju 61501, Korea.
  • 2Division of Premedical Science, College of Medicine, Chosun University, Gwangju 61501, Korea. sihan@chosun.ac.kr

Abstract

MDL-12330A is a widely used adenylyl cyclase (AC) inhibitor that blocks AC/cAMP signaling. In this study, we demonstrated a novel antitumor activity of this drug in gastric carcinoma (GC) cell lines. In these GC cells, MDL-12330A reduced cell viability and induced cell death in a concentration-dependent manner. At a moderate concentration (~20 µM), MDL-12330A mainly induced apoptotic death whereas at concentrations greater than 20 µM, it increased non-apoptotic cell death. The induction of apoptosis was at least partially regulated by CHOP-mediated DR5 upregulation, as detected by immunoblotting and gene interference assays. More importantly, low concentrations of MDL-12330A effectively enhanced recombinant human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (rhTRAIL)-induced apoptosis and clonogenicity in these gastric cancer cells. This study demonstrates a possible role of MDL-12330A as a potential sensitizer to TRAIL, and suggests a novel therapeutic strategy targeting gastric cancer cells.

Keyword

Apoptosis; CHOP; DR5; Gastric cancer; MDL-12330A; TRAIL

MeSH Terms

Adenylyl Cyclases
Apoptosis*
Cell Death
Cell Line
Cell Survival
Humans
Immunoblotting
Stomach Neoplasms*
Tumor Necrosis Factor-alpha
Up-Regulation*
Adenylyl Cyclases
Tumor Necrosis Factor-alpha

Figure

  • Fig. 1 MDL-12330A induces an antitumor effect in human GC cells. (A, B) Cells were treated with the indicated concentrations of MDL-12330A (MDL) for 24 h and subjected to the EZ-cytox assay for measurement of cell viability (A), or stained with Hoechst 33342/PI to assess the number of cells undergoing apoptotic or non-apoptotic cell death (B) as described in the Methods section. *p<0.05, **p<0.01 vs. control.

  • Fig. 2 Apoptosis triggered by the combined action of MDL-12330A and rhTRAIL involves activation of caspase-3, caspase-8, and caspase-4, but not caspase-9. SNU601 and AGS cells were treated with 20 µM MDL-12330A in the absence or presence of z-DEVD, z-IETD, z-LEHD, z-LEVD, or z-VAD for 24 h. The treated cells were stained with Hoechst33342, and apoptotic body was counted under a fluorescence microscope. The number of apoptotic cells was expressed as a percentage of the total number of cells counted. *p<0.05 vs. control; #p<0.05 vs. MDL-12330A (MDL) treated cells.

  • Fig. 3 DR5 plays an important role in MDL-12330A-mediated apoptosis. (A) SNU601 cells were exposed to 20 µM MDL-12330A, and the mRNA level of DR5 was determined using real-time PCR. (B) SNU601, SNU638, and AGS cells were treated with various concentrations of MDL-12330A for 24 h, and then analyzed by immunoblotting with antibodies against DR5, DR4, or α-tubulin. (C) SNU601 cells transfected with scrambled small interfering RNA (CTL RNAi), DR5 RNAi or DR4 RNAi were treated with 20 µM MDL-12330A for 24 h (left graph), or 10 µM MDL-12330A for 24 h (right graph). Treated cells were then stained using Hoechst 33342, and apoptotic body was counted under a fluorescence microscope. The number of apoptotic cells was expressed as a percentage of the total number of cells counted. *p<0.05 vs. control; #p<0.05 vs. MDL-12330A treated and CTL RNAi cells.

  • Fig. 4 MDL-12330A-mediated DR5 expression is regulated by CHOP. (A) SNU601 or SNU638 cells were transfected with a scrambled small interfering RNA (CTL RNAi), p65 RNAi, CHOP RNAi, and C-EBPβ RNAi, and then treated with 20 µM MDL-12330A (MDL) for 24 h. Cell lysates were prepared and analyzed by immunoblotting to assess DR5 expression. Silencing effect of each siRNA was confirmed by immunoblotting in vehicle treated control samples. (B, C) SNU601 or SNU638 cells were exposed to the indicated concentrations of MDL-12330A for 16 h and cell lysates were analyzed by immunoblotting with an antibody to CHOP (B), and to BiP and p-PERK (C). Alpha-tubulin was used as a loading control.

  • Fig. 5 Antitumor effect of MDL-12330A is not linked to inhibition of adenylate cyclase activity. (A) SNU601 and SNU638 cells were exposed to other type of adenylate cyclase inhibitors NKY80 and NB001 at indicated concentrations for 24 h, and cell viability was assessed by the EZ-cytox assay. (B) SNU601 and SNU638 cells were incubated with NKY80 or NB001 at 0, 10, 30 and 50 µM for 24 h, and cell lysates were prepared and analyzed by immunoblotting with an antibody to DR5 and BiP.

  • Fig. 6 MDL-12330A enhances rhTRAIL-induced apoptosis and clonogenicity. (A) Cells were pre-treated with 5 or 10 µM MDL-12330A for 24 h and then further exposed to rhTRAIL for 6 h. Apoptosis was detected by staining cells with Hoechst 33342 and assessing the ratio of apoptotic nuclei to normal nuclei under a fluorescence microscope. (B) Cells were incubated in the absence or presence of 5 µM MDL-12330A for 24 h and then exposed to 0, 5, or 10 ng/ml rhTRAIL for the last 2 h. Following this, 2×103 cells were re-plated on 60mm dishes. Colonies were stained with crystal violet and counted at 2 weeks post-incubation. #p<0.05 vs. rhTRAIL treated alone.


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