An evaluation of the effects of medium pH on the viability of the HepG2 cell line

Year 2020, Volume: 31 Issue: 2, 107 - 114, 31.12.2020

Neşe Güvenalp , Dilek Güvenç

https://doi.org/10.35864/evmd.742482

Cited By: 1

Abstract

Cancer cells are characterized by increased glucose uptake and the production of lactate, which leads to acidification of the tumor microenvironment. This acidification facilitates the development of invasiveness and metastasis. In this study, we investigated the effects of medium pH manipulation on the proliferation and viability of human hepatocellular carcinoma (HepG2) cells in vitro. HepG2 cells were grown in media with pH ranging from 6.0 to 8.5 for 24 h. The cells were then subjected to WST-1 and trypan blue exclusion assays to evaluate viability and cell proliferation, respectively. At pH 6.8 and 6.6, HepG2 cell viability was not significantly different from the control group (pH 7.2) (p>0.05) but there was a significant decrease at pH 6.4, 6.2 and 6.0 (p < 0.05). Furthermore, there was a significant decrease in cell viability at pH’s 7.8, 8.0 and 8.5 (p<0.05). Cell numbers decreased at pH 6.8 and increased at pH 6.6, although not significantly (p>0.05), and decreased at pH 7.6 and 7.8 (significant at pH 7.8; p<0.05). In acidic environments, the cells were spindle-shaped and formed islands but they became more spherical and had reduced adhesion capacity in alkaline media. In this study, an alkaline environment reduced the proliferation and viability of the cell line, HepG2. Therefore, after further investigations, in addition to current treatments, systemic alkalization that appropriately increases the pH of the tumor microenvironment may suppress the activity of tumor cells and increase the efficacy of normal HCC treatment.

Keywords

Alkali medium, HepG2, microenvironment, proliferation, Warburg effect

Supporting Institution

Ondokuz Mayis University Research Foundation

Project Number

PYO.VET.1904.19.001

Thanks

The authors thank Dr. Gregory T. Sullivan for proofreading an earlier version of this manuscript.

HepG2 hücre hattının canlılığı üzerine medium pH'sının etkisinin incelenmesi

Abstract

Kanser hücreleri, tümör mikroçevresinin asitleşmesine neden olan artmış glikoz alımı ve laktat üretimi ile karakterizedir. Asidik mikro-çevre kanser hücrelerinin invazyon ve metastaz gelişimini kolaylaştırır. Bu çalışmada medium pH değişiminin, in vitro insan hepatoselüler karsinom (HepG2) hücrelerinin proliferasyon ve canlılığına etkisinin değerlendirilmesi amaçlanmıştır. HepG2 hücreleri pH değeri 6.0 ila 8.5 arasında değişen medium ortamlarında 24 saat boyunca inkübe edildi. Daha sonra hücre canlılığı ve proliferasyon değerlendirmeleri için sırasıyla WST-1 ve tripan mavisi hücre canlılık testleri uygulandı. HepG2 hücre canlılığında, medium pH’sı 6.6 ve 6.8 olan gruplar ile kontrol grubu (pH 7.2) karşılaştırıldığında önemli bir farkın bulunmadığı (p> 0.05), ancak pH 6.4, 6.2, 6.0'da önemli oranda azalmanın olduğu saptandı (p <0.05). Ayrıca alkali gruplardan pH 7.8, pH 8.0 ve pH 8.5'te hücre canlılık oranında belirgin bir azalma gözlemlendi (p<0.05). Hücre sayısında pH 6.8'de artış ve pH 6.6'da azalmanın olduğu ancak istatistiksel olarak önemli fark olmadığı (p>0.05), pH 7.6 ve 7.8'de azalmanın bulunduğu ve bu azalmanın pH 7.8’de önemli olduğu saptandı (p<0.05). Asidik ortamlarda, iğ şeklindeki hücrelerin adalar oluşturduğu, alkali ortamda ise yapışma kapasiteleri azalmış hücrelerin yuvarlak şekilde olduğu gözlendi. Bu çalışmada, alkali mikro-çevrenin HepG2 hücre hattının canlılığını ve proliferasyonunu azalttığı tespit edilmiştir. Bu nedenle, daha ileri araştırmalardan sonra mevcut tedavi yöntemlerine ek olarak tümör mikro-çevre pH'sını uygun bir şekilde yükselten sistemik alkalizasyonun, tümör hücrelerinin aktivitesini baskılayabileceği ve normal HCC tedavisinin etkinliğini artırabileceği sonucuna varıldı.

Keywords

Alkali ortam, HepG2, mikro-çevre, proliferasyon, Warburg etkisi

Project Number

PYO.VET.1904.19.001

References

• Alfarouk KO, Stock CM, Taylor S, Walsh M, Muddathir AK, Verduzco D, Bashir AH, Mohammed OY, Elhassan GO, Harguindey S, Reshkin SJ, Ibrahim ME, Rauchet C. (2015). Resistance to cancer chemotherapy: failure in drug response from ADME to P-gp. Cancer Cell Int. 15,71 DOI: 10.1186/s12935-015-0221-1.
• Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68, 394-424 DOI: 10.3322/caac.21492. Chen M, Chen C,Shen Z, Zhang X, Chen Y, Lin F, Ma X, Zhuang C, Mao Y, Gan H, Chen P, Zong X, Wu R. (2017). Extracellular pH is a biomarker enabling detection of breast cancer and liver cancer using CEST MRI. Oncotarget. 8, 45759- 45767 DOI: 10.18632/oncotarget.17404.
• Chen Y, Chen CH, Tung PY, Huang SH, Wang SM. (2009). An acidic extracellular pH disrupts adherens junctions in HepG2 cells by Src kinases-dependent modification of E-cadherin. J Cell Biochem,851-859
• DeBerardinis RJ, Sayed N, Ditsworth D, Thompson CB. (2008). Brick by brick: metabolism and tumor cell growth. Curr Opin Genet Dev. 18, 54-61 DOI: 10.1016/j.gde.2008.02.003
• 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 et al. (2013). Acidity generated by the tumor microenvironment drives local invasion. Cancer Res. 73, 1524-1535 DOI: 10.1016/j.gde.2008.02.003
• Feng Q, Shen Y, Fu Y, Muroski ME, Zhang P, Wang Q, Xu C, Lesniak MS, Li G, Cheng Y.(2017). Self-assembly of gold nanoparticles shows microenvironment-mediated dynamic switching and enhanced brain tumor targeting. Theranostics, 1875-1889 DOI: 10.7150/thno.18985.
• Fukamachi T, Ikeda S, Wang X, Saito H, Tagawa M, Kobayashi H. (2013). Gene expressions for signal transduction under acidic conditions. Genes , 65-85
• Gatenby RA, Gillies RJ. (2008). A microenvironmental model of carcinogenesis. Nat Rev Cancer, 56-61
• Gatenby RA, Gillies RJ.( 2004). Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4, 891-899
• Gatenby RA, Gawlinski ET, Gmitro AF, Kaylor B, Gillies RJ.( 2006). Acid-mediated tumor invasion: a multidisciplinary study. Cancer Res, 5216-5223
• Hamaguchi R, Okamoto T, Sato M, Hasegawa M, Wada H. (2017). Effects of an alkaline diet on EGFR-TKI therapy in EGFR mutation-positive NSCLC. Anticancer Res, 37, 5141-5145
• Hao G , Xu ZP, Li L. (2018). Manipulating extracellular tumour pH: an effective target for cancer therapy. RSC Adv. 8, 22182-22192
• Hsu PP, Sabatini DM. (2018). Cancer cell metabolism: Warburg and beyond. Cell 134, 703-707
• Ibrahim-Hashim A, Cornell HH, Abrahams D, Lloyd M, Bui M, Gillies RJ, Gatenby RA. (2012). Systemic buffers inhibit carcinogenesis in TRAMP mice. J Urol, 188, 624-631
• Izumi H, Torigoe T, Ishiguchi H, Uramoto H, Yoshida Y, Tanabe M, Ise T, Murakami T, Yoshida T, Nomoto M, Kohno K. (2003). Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy. Cancer Treat Rev, 541-549
• Kato Y,Ozawa S, Miyamoto C, Maehata Y, Suzuki A, Maeda T, Baba Y. (2013). Acidic extracellular microenvironment and cancer. Cancer Cell Int, 13, 89
• Lacroix R, Rozeman EA, Kreutz M, Renner K, Blank CU. (2018). Targeting tumor-associated acidity in cancer immunotherapy. Cancer Immunol Immunother. 67, 1331-1348
• Lodish H, , Berk A, Kaiser CA, Paul M. (2016) Molecular Cell Biology. W. H. Freeman, 5th edition, New York
• Moellering RE, Black KC, Krishnamurty C, Baggett BK, Stafford P, Rain M, Gatenby RA, Gillies RJ. (2008). Acid treatment of melanoma cells selects for invasive phenotypes. Clin Exp Metastasis, 411-425
• Neri D, Supuran CT. (2011). Interfering with pH regulation in tumours as a therapeutic strategy. Nat Rev Drug Discov. 10, 767-777
• Park H, Lyons JC, Ohtsubo T, Song CW. (2000). Cell cycle progression and apoptosis after irradiation in an acidic environment. Cell Death Differ, 7, 729-738
• Park HJ, Lee SH, Chung H, Rhee YH, Lim BU, Ha SW, Griffin RJ, Lee HS, Song CW, Choiet EK. (2003). Influence of environmental pH on G2-phase arrest caused by ionizing radiation. Radiat Res. 159, 86-93 DOI: 10.1667/0033-7587(2003)159[0086:ioepog]2.0.co;2.
• Pillai SR, Damaghi M, Marunaka Y, Spugnini EP, Fais S, Gillies RJ. (2019). Causes, consequences, and therapy of tumors acidosis. Cancer Metastasis Rev. 38, 205-222 DOI: 10.1007/s10555-019-09792-7
• Raghunand N, Gillies RJ.( 2000). pH and drug resistance in tumors. Drug Resist Upda,t 3, 39-47
• Robey IF, Baggett BK, Kirkpatrick ND, Roe DJ, Dosescu J, Sloane BF, Ibrahim Hashim A, Morse DL, Raghunand N, Gatenby RA, Gillies RJ. (2009). Bicarbonate increases tumor pH and inhibits spontaneous metastases. Cancer Res. 69, 2260-2268 DOI: 10.1158/0008-5472.CAN-07-5575.
• Rodríguez-Hernández MA, González R, de la Rosa ÁJ, Mauriz JL, Padillo FJ, Muntane J. (2018). Molecular characterization of autophagic and apoptotic signaling induced by Sorafenib in liver cancer cells: In vitro and in vivo studies. J Hepatol 68, 670-671 DOI:https://doi.org/10.1016/S0168-8278(18)31599-X
• Rofstad EK, Mathiesen B, Kindem K, Galappathi K. (2006). Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice. Cancer Res. 66, 6699-6707 DOI: 10.1158/0008-5472.CAN-06-0983.
• Ruddon RW. (2007). Characteristics of Human Cancer. In: Cancer Biology, 4th Edition. Oxford University Press Inc, New York. p. 3-16
• Schwartz L, Seyfried T, Alfarouk KO, Moreira JV, Fais S. ( 2017). Out of Warburg effect: an effective cancer treatment targeting the tumor specific metabolism and dysregulated pH. Semin Cancer Biol, 43, 134-138 DOI: 10.1016/j.semcancer.2017.01.005.
• Silva AS, Yunes JA, Gillies RJ, Gatenby RA. (2009). The potential role of systemic buffers in reducing intratumoral extracellular pH and acid-mediated invasion. Cancer Res. 69, 2677-2684 DOI: 10.1158/0008-5472.CAN-08-2394
• Stern R, Shuster S, Neudecker B, Formby B. (2002). Lactate stimulates fibroblast expression of hyaluronan and CD44: the Warburg effect revisited. Exp Cell Res, 24-31.
• Swietach P, Vaughan-Jones RD, Harris AL. (2007). Regulation of tumor pH and the role of carbonic anhydrase 9. Cancer Metastasis Rev. 26, 299-310
• Tian XP, Wang CY, Jin XH, Li M,Wang FW, Huang WJ, Yun JP,Xu RH, Cai QQ,Xie D. (2019) Acidic Microenvironment Up-Regulates Exosomal miR-21 and miR-10b in Early-Stage Hepatocellular Carcinoma to Promote Cancer Cell Proliferation and Metastasis. Theranostics. 9, 1965-1979
• Torre LA, Siegel RL, Ward EM, Jemal A. ( 2016). Global cancer incidence and mortality rates and trends—an update. Cancer Epidemiol Biomarkers Prev 25, 16-27 DOI: 10.1158/1055-9965.EPI-15-0578.
• Trivedi A, Ahmad R, Misra A.( 2018). Effect of alkaline pH on cytotoxicity profile of neem (Azadirachta indica) ethanolic extract against human breast cancer cell line MDA-MB-231. Eur J Integr Med, 1-7
• Tuccitto A. ( 2018). pH regulatory molecules in the tumour microenvironment: modulators of aggressiveness and immune profile of human hepatocellular carcinoma. The Open University, Faculty of Science Technology Engineering and Mathematics (STEM), PhD thesis, Milton Keynes (UK)
• Xia F, Hou W, Zhang C, Zhi X, Cheng J, Fuente JM, Song J, Cui D. (2018). pH-responsive gold nanoclusters-based nanoprobes for lung cancer targeted near-infrared fluorescence imaging and chemo-photodynamic therapy. Acta Biomater, 308-319 DOI: 10.1016/j.actbio.2017.12.034.
• Yin P, Yang L, Xue Q, Yu M, Yao F, Sun L, Liu Y. (2018). Identification and inhibitory activities of ellagic acid- and kaempferol-derivatives from Mongolian oak cups against α-glucosidase, α-amylase and protein glycation linked to type II diabetes and its complications and their influence on HepG2 cells’ viability. Arab J Chem, 11, 1247-1259
• Zhang X, Lin Y, Gillies RJ.( 2010). Tumor pH and its measurement. J Nucl Med. 51, 1167-1170, DOI: 10.2967/jnumed.109.068981.
• Zhang Y, Zhang T, Wu C, Xia Q, Xu D. (2017). ASIC1a mediates the drug resistance of human hepatocellular carcinoma via the Ca 2+/PI3-kinase/AKT signaling pathway. Lab Invest. 97, 53-69 DOI: 10.1038/labinvest.2016.127