Korean J Physiol Pharmacol.  2018 Sep;22(5):513-523. 10.4196/kjpp.2018.22.5.513.

Extracellular acidity enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis via DR5 in gastric cancer cells

• Hong R
• Han SI

• KMID: 2419001
• DOI: http://doi.org/10.4196/kjpp.2018.22.5.513

Abstract

The tumor microenvironment greatly influences cancer cell characteristics, and acidic extracellular pH has been implicated as an essential factor in tumor malignancy and the induction of drug resistance. Here, we examined the characteristics of gastric carcinoma (GC) cells under conditions of extracellular acidity and attempted to identify a means of enhancing treatment efficacy. Acidic conditions caused several changes in GC cells adversely affecting chemotherapeutic treatment. Extracellular acidity did inhibit GC cell growth by inducing cell cycle arrest, but did not induce cell death at pH values down to 6.2, which was consistent with down-regulated cyclin D1 and up-regulated p21 mRNA expression. Additionally, an acidic environment altered the expression of atg5, HSPA1B, collagen XIII, collagen XXAI, slug, snail, and zeb1 genes which are related to regulation of cell resistance to cytotoxicity and malignancy, and as expected, resulted in increased resistance of cells to multiple chemotherapeutic drugs including etoposide, doxorubicin, daunorubicin, cisplatin, oxaliplatin and 5-FU. Interestingly, however, acidic environment dramatically sensitized GC cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Consistently, the acidity at pH 6.5 increased mRNA levels of DR4 and DR5 genes, and also elevated protein expression of both death receptors as detected by immunoblotting. Gene silencing analysis showed that of these two receptors, the major role in this effect was played by DR5. Therefore, these results suggest that extracellular acidity can sensitize TRAIL-mediated apoptosis at least partially via DR5 in GCs while it confers resistance to various type of chemotherapeutic drugs.

Keyword

Acidity; Gastric cancer; Resistance; TRAIL

MeSH Terms

Apoptosis*
Cell Cycle Checkpoints
Cell Death
Cisplatin
Collagen
Cyclin D1
Daunorubicin
Doxorubicin
Drug Resistance
Etoposide
Fluorouracil
Gastropoda
Gene Silencing
Hydrogen-Ion Concentration
Immunoblotting
Necrosis*
Receptors, Death Domain
RNA, Messenger
Snails
Stomach Neoplasms*
Treatment Outcome
Tumor Microenvironment
Cisplatin
Collagen
Cyclin D1
Daunorubicin
Doxorubicin
Etoposide
Fluorouracil
RNA, Messenger
Receptors, Death Domain

Figure

• 

  Fig. 1 GC cell growth was inhibited in acidic culture medium. (A, B) AGS and SNU-601 cells were cultured in growth medium adjusted to the indicated pH for 24, 48, or 72 h, and counted at each time point. (C–E) Cells were incubated for 48 h in normal growth medium (pH 7.4) or acidic medium adjusted to pH 6.8, 6.5, or 6.2. Then cells are collected and stained with Hoechst 33342 to assess apoptotic cell death (C) or cells are fixed and stained with propidium iodide and analyzed by fluorescence-activated cell sorting analysis (E). Cell-free culture supernatants were subjected to an LDH release assay (D). *p<0.05 vs. pH 7.4.

• 

  Fig. 2 Extracellular acidity altered the expression of various genes. AGS cells were incubated for 48 h in growth medium adjusted to pH 6.5, and mRNA expression of the genes encoding cyclin D1 (A), p21 (B), ATG5 (C), HSP70 (D), collagen XIII (E), collagen XXA1 (F), SLUG (G), SNAIL (H), ZEB1 (I), FAS (J), DR4 (K), and DR5 (L) was analyzed by real-time PCR. *p<0.05, **p<0.01 vs. pH 7.4.

• 

  Fig. 3 Extracellular acidic conditions decreased GC cell sensitivity to various chemotherapeutic drugs. SNU-601 cells were cultured for 24 h in growth medium adjusted to pH 7.4 or 6.5, and subsequently exposed for 48 h to the indicated concentration of doxorubicin, daunorubicin, oxaliplatin, cisplatin, etoposide, or 5-fluorouracil in each pH-adjusted medium. The cells were then subjected to an EZ-cytox assay for measurement of cell viability (A–F), or immunoblotting of total protein extracts (for caspase-3 and α-tubulin) or cytosolic protein extracts (for released cytochrome c) (G–L).

• 

  Fig. 4 Expression of TRAIL-Rs in GC cells was upregulated in acidic culture conditions. AGS and SNU-601 cells were exposed for 48 h to growth medium adjusted to pH 7.4, 6.8, 6.5, or 6.2. The treated cells were collected and mRNA expression of the genes FAS, DR4, and DR5 was analyzed by real-time PCR (A–F) or total protein extracts were prepared by cell lysis and analyzed by immunoblotting with antibodies against the corresponding proteins and α-tubulin as a loading control (G, H). *p<0.05, **p<0.01 vs. pH 7.4.

• 

  Fig. 5 Acidic culture conditions sensitized GC cells to rhTRAIL-induced apoptosis. AGS (A, B) and SNU-601 (C, D) cells were exposed to normal growth medium (pH 7.4) or acidic medium (pH 6.5) for 48 h, before addition of 2 or 4 ng rhTRAIL and incubation for a further 6 h. The cells were then stained with Hoechst 33342, and images were captured under a fluorescence microscope (A, C) or apoptotic bodies were counted (B, D). The number of apoptotic cells is expressed as a percentage of the total number of cells counted. #p<0.05 vs. pH 7.4.

• 

  Fig. 6 TRAIL-R2/DR5 plays an essential role in acidity-promoted rhTRAIL-induced apoptosis. (A, B) SNU-601 cells transfected with scrambled small interfering RNA (CTL RNAi) were exposed to normal growth medium (pH 7.4) and those transfected with CTL RNAi, DR5 RNAi, or DR4 RNAi were exposed to acidic culture medium (pH 6.5) for 42 h, before addition of 2 ng rhTRAIL and incubation for a further 6 h. The treated cells were then stained with Hoechst 33342, and apoptotic bodies were counted under a fluorescence microscope (A). The silencing effect of each small interfering RNA was confirmed by immunoblotting analysis of cells not administered rhTRAIL (B). (C, D) SNU-601 cells transfected with CTL RNAi, DR5 RNAi, or DR4 RNAi were exposed to growth medium at pH 6.5 for 42 h, before addition of 2 ng rhTRAIL and incubation for a further 4 h. The treated cells were subjected to a caspase-8 assay (C) and -3 activity assay (D). #p<0.05 vs. CTL RNAi- and rhTRAIL-treated cells.

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