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

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

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.


Reference

1. Poole-Wilson PA. Measurement of myocardial intracellular pH in pathological states. J Mol Cell Cardiol. 1978; 10:511–526.
Article
2. Reichert M, Steinbach JP, Supra P, Weller M. Modulation of growth and radiochemosensitivity of human malignant glioma cells by acidosis. Cancer. 2002; 95:1113–1119.
Article
3. Thews O, Gassner B, Kelleher DK, Schwerdt G, Gekle M. Impact of hypoxic and acidic extracellular conditions on cytotoxicity of chemotherapeutic drugs. Adv Exp Med Biol. 2007; 599:155–161.
Article
4. Wilting RH, Dannenberg JH. Epigenetic mechanisms in tumorigenesis, tumor cell heterogeneity and drug resistance. Drug Resist Updat. 2012; 15:21–38.
Article
5. Raghunand N, Gillies RJ. pH and drug resistance in tumors. Drug Resist Updat. 2000; 3:39–47.
Article
6. Fais S, De Milito A, You H, Qin W. Targeting vacuolar H+-ATPases as a new strategy against cancer. Cancer Res. 2007; 67:10627–10630.
7. Taylor S, Spugnini EP, Assaraf YG, Azzarito T, Rauch C, Fais S. Microenvironment acidity as a major determinant of tumor chemoresistance: Proton pump inhibitors (PPIs) as a novel therapeutic approach. Drug Resist Updat. 2015; 23:69–78.
Article
8. Bertuccio P, Chatenoud L, Levi F, Praud D, Ferlay J, Negri E, Malvezzi M, La Vecchia C. Recent patterns in gastric cancer: a global overview. Int J Cancer. 2009; 125:666–673.
Article
9. Peleteiro B, Severo M, La Vecchia C, Lunet N. Model-based patterns in stomach cancer mortality worldwide. Eur J Cancer Prev. 2014; 23:524–531.
Article
10. Deng K, Lin S, Zhou L, Li Y, Chen M, Wang Y, Li Y. High levels of aromatic amino acids in gastric juice during the early stages of gastric cancer progression. PLoS One. 2012; 7:e49434.
Article
11. Estrella V, Chen T, Lloyd M, Wojtkowiak J, Cornnell HH, Ibrahim-Hashim A, Bailey K, Balagurunathan Y, Rothberg JM, Sloane BF, Johnson J, Gatenby RA, Gillies RJ. Acidity generated by the tumor microenvironment drives local invasion. Cancer Res. 2013; 73:1524–1535.
Article
12. Wang S. The promise of cancer therapeutics targeting the TNF-related apoptosis-inducing ligand and TRAIL receptor pathway. Oncogene. 2008; 27:6207–6215.
Article
13. Gura T. How TRAIL kills cancer cells, but not normal cells. Science. 1997; 277:768.
14. Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med. 1999; 5:157–163.
Article
15. Kashii Y, Giorda R, Herberman RB, Whiteside TL, Vujanovic NL. Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells. J Immunol. 1999; 163:5358–5366.
16. Baker SJ, Reddy EP. Modulation of life and death by the TNF receptor superfamily. Oncogene. 1998; 17:3261–3270.
Article
17. Shin JN, Park SY, Cha JH, Park JY, Lee BR, Jung SA, Lee ST, Yun CW, Seol DW, Kim TH. Generation of a novel proform of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein that can be reactivated by matrix metalloproteinases. Exp Cell Res. 2006; 312:3892–3898.
Article
18. Lim SC, Choi JE, Kang HS, Han SI. Ursodeoxycholic acid switches oxaliplatin-induced necrosis to apoptosis by inhibiting reactive oxygen species production and activating p53-caspase 8 pathway in HepG2 hepatocellular carcinoma. Int J Cancer. 2010; 126:1582–1595.
Article
19. Luo S, Rubinsztein DC. Atg5 and Bcl-2 provide novel insights into the interplay between apoptosis and autophagy. Cell Death Differ. 2007; 14:1247–1250.
Article
20. Apel A, Herr I, Schwarz H, Rodemann HP, Mayer A. Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy. Cancer Res. 2008; 68:1485–1494.
Article
21. Han W, Sun J, Feng L, Wang K, Li D, Pan Q, Chen Y, Jin W, Wang X, Pan H, Jin H. Autophagy inhibition enhances daunorubic-ininduced apoptosis in K562 cells. PLoS One. 2011; 6:e28491.
Article
22. Buzzard KA, Giaccia AJ, Killender M, Anderson RL. Heat shock protein 72 modulates pathways of stress-induced apoptosis. J Biol Chem. 1998; 273:17147–17153.
Article
23. Su C, Su B, Tang L, Zhao Y, Zhou C. Effects of collagen iv on cisplatin-induced apoptosis of non-small cell lung cancer cells. Cancer Invest. 2007; 25:542–549.
Article
24. Januchowski R, Świerczewska M, Sterzyńska K, Wojtowicz K, Nowicki M, Zabel M. Increased expression of several collagen genes is associated with drug resistance in ovarian cancer cell lines. J Cancer. 2016; 7:1295–1310.
Article
25. Vukovic V, Tannock IF. Influence of low pH on cytotoxicity of paclitaxel, mitoxantrone and topotecan. Br J Cancer. 1997; 75:1167–1172.
Article
26. Wachsberger PR, Landry J, Storck C, Davis K, O'Hara MD, Owen CS, Leeper DB, Coss RA. Mammalian cells adapted to growth at pH 6.7 have elevated HSP27 levels and are resistant to cisplatin. Int J Hyperthermia. 1997; 13:251–255. discussion 257-9.
Article
27. Kudo-Saito C, Shirako H, Takeuchi T, Kawakami Y. Cancer metastasis is accelerated through immunosuppression during Snail-induced EMT of cancer cells. Cancer Cell. 2009; 15:195–206.
Article
28. Tan X, Banerjee P, Liu X, Yu J, Gibbons DL, Wu P, Scott KL, Diao L, Zheng X, Wang J, Jalali A, Suraokar M, Fujimoto J, Behrens C, Liu X, Liu CG, Creighton CJ, Wistuba II, Kurie JM. The epithelial-to-mesenchymal transition activator ZEB1 initiates a prometastatic competing endogenous RNA network. J Clin Invest. 2018; 128:1267–1282.
Article
29. Hardy J, Jones A, Gore ME, Viner C, Selby P, Wiltshaw E. Treatment of advanced ovarian cancer with intraperitoneal tumour necrosis factor. Eur J Cancer. 1990; 26:771.
Article
30. Barnhart BC, Legembre P, Pietras E, Bubici C, Franzoso G, Peter ME. CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells. EMBO J. 2004; 23:3175–3185.
Article
31. Srivastava RK. TRAIL/Apo-2L: mechanisms and clinical applications in cancer. Neoplasia. 2001; 3:535–546.
Article
32. Lemke J, von Karstedt S, Zinngrebe J, Walczak H. Getting TRAIL back on track for cancer therapy. Cell Death Differ. 2014; 21:1350–1364.
Article
33. Lim SC, Parajuli KR, Han SI. The alkyllysophospholipid edelfosine enhances TRAIL-mediated apoptosis in gastric cancer cells through death receptor 5 and the mitochondrial pathway. Tumour Biol. 2016; 37:6205–6216.
Article
34. Jin CY, Park C, Cheong J, Choi BT, Lee TH, Lee JD, Lee WH, Kim GY, Ryu CH, Choi YH. Genistein sensitizes TRAIL-resistant human gastric adenocarcinoma AGS cells through activation of caspase-3. Cancer Lett. 2007; 257:56–64.
Article
35. Jung EM, Park JW, Choi KS, Park JW, Lee HI, Lee KS, Kwon TK. Curcumin sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through CHOP-independent DR5 upregulation. Carcinogenesis. 2006; 27:2008–2017.
Article
36. Yoon MJ, Kang YJ, Kim IY, Kim EH, Lee JA, Lim JH, Kwon TK, Choi KS. Monensin, a polyether ionophore antibiotic, overcomes TRAIL resistance in glioma cells via endoplasmic reticulum stress, DR5 upregulation and c-FLIP downregulation. Carcinogenesis. 2013; 34:1918–1928.
Article
37. Huang Y, Yang X, Xu T, Kong Q, Zhang Y, Shen Y, Wei Y, Wang G, Chang KJ. Overcoming resistance to TRAIL-induced apoptosis in solid tumor cells by simultaneously targeting death receptors, c-FLIP and IAPs. Int J Oncol. 2016; 49:153–163.
Article
38. Lee YJ, Song JJ, Kim JH, Kim HR, Song YK. Low extracellular pH augments TRAIL-induced apoptotic death through the mitochondria-mediated caspase signal transduction pathway. Exp Cell Res. 2004; 293:129–143.
Article
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