Reduction of Intracellular-Reactive Oxygen Species and Diminished Mitogen-Activated Protein Kinases (MAPKs) Activation are Associated with Oral Squamous Cell Carcinoma Cell Aggressiveness
AbstractOral squamous cell carcinoma (OSCC) is a serious health problem in many countries. Several drugs have been used to treat head and neck and oral cavity cancers. However, the success rate has not been impressive because of the heterogeneity of cancerous cells, resulting in differential responsiveness to chemotherapy. Two distinct phenotypes of OSCC cells, the CLS-354/WT and CLS-354/DXcells, have been used as in vitro cell models for this study. CLS-354/DXcells were more aggressive than CLS-354/WTcells, supported by the observation that CLS-354/DXcells can undergo epithelial-mesenchymal transition (EMT), grow anchorage-independently, and increase invasiveness. We investigated the preliminary redox status of these 2 cell lines, including levels of reactive oxygen species (ROS) and cellular antioxidants, using flow cytometry analysis and ABTS+ free radical scavenging assay, respectively. A 7-fold decrease in ROS level was detected in CLS-354/DXcells, comparing with CLS-354/WTcells, while antioxidant capacity was not different from that of CLS-354/WT cells. Hydrogen peroxide, a ROS modulating agent, could induce ROS levels, and caused cell death in CLS-354/WT greater than that of CLS-354/DX cells. Of note, hydrogen peroxide-induced cytotoxicity could be rescued by N-acetyl cysteine, confirming ROS-mediated cytotoxicity in both cell lines. ROS-sensitive mitogen-activated protein kinases (MAPKs) were observed using immunoblot assay. The expressions of p-JNK1/2 and p-p38 MAPK in CLS-354/DX cells were absent, while these expressions were abundantly detected in CLS-354/WTcells. This suggests that lower ROS levels, with the concomitant reduction of JNK and p38 MAPK activation in CLS-354/DX cells, are associated with cancer cell aggressiveness. These findings provide significant evidence of the resistance to ROS-modulating agents in aggressive OSCC cells.
J Massano, FS Regateiro, G Januário and A Ferreira. Oral squamous cell carcinoma: Review of prognostic and predictive factors. Oral Surg Oral Med. Oral Pathol. Oral Radiol. Endod. 2006; 102, 67-76.
S Warnakulasuriya. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol. 2009; 45, 309-16.
X Zhang,Y Liu, MZ Gilcrease,XH Yuan, GL Clayman, K Adler-Storthz and Z Chen. A lymph node metastatic mouse model reveals alterations of metastasis-related gene expression in metastatic human oral carcinoma sublines selected from a poorly metastatic parental cell line. Cancer 2002; 95, 1663-72.
L Olasz, E Orsi, T Markó and J Szalma. Induction chemotherapy response and recurrence rates in correlation with N0 or N+ stage in oral squamous cell cancer (OSCC). Cancer Metastasis Rev. 2010; 29, 607-11.
JM Matés, JA Segura, FJ Alonso and J Márquez. Intracellular redox status and oxidative stress: Implications for cell proliferation, apoptosis, and carcinogenesis. Arch. Toxicol. 2008; 82, 273-99.
D Trachootham, W Lu, MA Ogasawara, NRD Valle and P Huang. Redox regulation of cell survival. Antioxid. Redox Signal 2008; 10, 1343-74.
T Ishimoto, O Nagano, T Yae, M Tamada, T Motohara, H Oshima, M Oshima, T Ikeda, R Asaba, H Yagi, T Masuko, T Shimizu, T Ishikawa, K Kai, E Takahashi, Y Imamura, Y Baba, M Ohmura, M Suematsu, H Baba and H Saya. CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby promotes tumor growth. Cancer Cell. 2011; 19, 387-400.
L Chaiswing, W Zhong, Y Liang, DP Jones and TD Oberley. Regulation of prostate cancer cell invasion by modulation of extra- and intracellular redox balance. Free Radic. Biol. Med. 2012; 52, 452-61.
L Chaiswing, JM Bourdeau-Heller, W Zhong and TD Oberley. Characterization of redox state of two human prostate carcinoma cell lines with different degrees of aggressiveness. Free Radic. Biol. Med. 2007; 43, 202-15.
CW Chang, YS Chen, SH Chou, CL Han, YJ Chen, CC Yang, CY Huang and JF Lo. Distinct subpopulations of head and neck cancer cells with different levels of intracellular reactive oxygen species exhibit diverse stemness, proliferation, and chemosensitivity. Cancer Res. 2014; 74, 6291-305.
JY Fang and BC Richardson. The MAPK signaling pathways and colorectal cancer. Lancet Oncol. 2005; 6, 322-7.
X Sui, N Kong, L Ye, W Han, J Zhou, Q Zhang, C He and H Pan. p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents. Cancer Lett. 2014; 344, 174-9.
I Dolado, A Swat, N Ajenjo, G De Vita, A Cuadrado and AR Nebreda. p38α MAP kinase as a sensor of reactive oxygen species in tumorigenesis. Cancer Cell. 2007; 11, 191-205.
P Bragado, A Armesilla, A Silva and A Porras. Apoptosis by cisplatin requires p53 mediated p38α MAPK activation through ROS generation. Apoptosis 2007; 12, 1733-42.
L Pereira, A Igea, B Canovas, I Dolado and AR Nebreda. Inhibition of p38 MAPK sensitizes tumour cells to cisplatin-induced apoptosis mediated by reactive oxygen species and JNK. EMBO Mol. Med. 2013; 5, 1759-74.
T Utaipan, A Athipornchai, A Suksamrarn, S Chunsrivirot and W Chunglok. Isomahanine induces endoplasmic reticulum stress and simultaneously triggers p38 MAPK-mediated apoptosis and autophagy in multidrug-resistant human oral squamous cell carcinoma cells. Oncol. Rep. 2017; 37, 1243-52.
W Chunglok, W Ittarat, P Tomakidi, R Schmidt, W Stremmel and W Chamulitrat. Human gingival mucosal keratinocytes exhibiting anchorage-independent growth express increased inducible nitric oxide synthase: regulation by MAP kinases. Nitric Oxide. 2004; 11, 237-46.
R Kalluri and RA Weinberg. The basics of epithelial-mesenchymal transition. J. Clin. Invest. 2009; 119, 1420-8.
C Morata-Tarifa, G Jiménez, MA García, JM Entrena, C Griñán-Lisón, M Aguilera, M Picon-Ruiz and JA Marchal. Low adherent cancer cell subpopulations are enriched in tumorigenic and metastatic epithelial-to-mesenchymal transition-induced cancer stem-like cells. Sci. Rep. 2016; 6, 18772.
A Singh and J Settleman. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 2010; 29, 4741-51.
SI Shin, VH Freedman, R Risser and R Pollack. Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc. Natl. Acad. Sci. USA 1975; 72, 4435-9.
W Chamulitrat. Role of gp91phox homolog Nox1 in induction of premalignant spindle phenotypes of HPV 16 E6/E7-immortalized human keratinocytes. Sci. World J. 2010; 10, 1435-49.
J Yang, E Lam, H Hammad,T Oberley and L Oberley. Antioxidant enzyme levels in oral squamous cell carcinoma and normal human oral epithelium. J. Oral Pathol. Med. 2002; 31, 71-7.
F Hecht, JM Cazarin, CE Lima, CC Faria, AAdC Leitão, ACF Ferreira, DP Carvalho and RS Fortunato. Redox homeostasis of breast cancer lineages contributes to differential cell death response to exogenous hydrogen peroxide. Life Sci. 2016; 158, 7-13.
AM Faten, AA Abo-Aziza and AA Zaki. Confluence-associated proliferation and osteogenic differentiation of bone marrow mesenchymal stem cell (BMMSCs). Int. Res. J. Biological Sci. 2016; 5, 44-56.
Q Zhang, N Yu and C Lee. Mysteries of TGF-β paradox in benign and malignant cells. Front Oncol. 2014; 4, 94.
ER Butch and KL Guan. Characterization of ERK1 activation site mutants and the effect on recognition by MEK1 and MEK2. J. Biol. Chem. 1996; 271, 4230-5.
YC Hseu, MS Lee, CR Wu, HJ Cho, KY Lin, GH Lai, SY Wang, YH Kuo, KJ Kumar and HL Yang. The chalcone flavokawain B induces G2/M cell-cycle arrest and apoptosis in human oral carcinoma HSC-3 cells through the intracellular ROS generation and downregulation of the Akt/p38 MAPK signaling pathway. J. Agric. Food Chem. 2012; 60, 2385-97.
L Si, L Zheng, L Xu, L Yin, X Han, Y Qi, Y Xu, C Wang and J Peng. Dioscin suppresses human laryngeal cancer cells growth via induction of cell-cycle arrest and MAPK-mediated mitochondrial-derived apoptosis and inhibition of tumor invasion. Eur. J. Pharmacol. 2016; 774, 105-17.