F. Combination of NTP with cetuximab attenuates invasion via the NF-ƍB signaling pathway in SCCQLL1 cells
To identify the underlying mechanism of the effect of NTP in combination with cetuximab via the NF-ƈB pathway, we assessed whether overexpression of NF-ƈB expression modulated the effect of the combination of NTP with cetuximab. As demonstrated in Figure 8A, transfection of NF-ƈB cDNA upregulated NF-ƈB expression compared with control-transfected cells. As expected, NF-ƈB augmentation itself noticeably increased migration and invasion of SCCQLL1 cells, and the combination treatment showed no definite significant effect on migration (Figures 8B and C) and invasion (Figures 8D and E) of NF-ƈB upregulated SCCQLL1 cells, respectively.
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H[SRVXUHWReach condition (gas, 1 kV of NTP, 10 țg/ml of cetuximab, and combination of NTP and cetuximab). (A) Western blotting. p65 (1)ƈ% SURWHLQ H[SUHVVLRQ ZDV
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FHWX[LPDEGHFUHDVHGWKHOHYHORIS 1)ƈ% (B and C) Scratch-based migration assay using 1)ƈ%ėXSUHJXODWHG6&&4//FHOOV % $IWHUKRILQFXEDWLRQ, ZRXQGKHDOLQJZDV
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H[SHULPHQWVSHUIRUPHGLQWULSOLFDWHScale bar = 500 țm. (C) Percentage of closure of the denuded zone was analyzed. The data represent the means S.D. of three independent experiments. NS, not significant; 3 3 'DQG( ,QYDVLRQDVVD\XVLQJ
1)ƈ%ėXSUHJXODWHG6&&4//FHOOV ' $IWHUH[SRVXUHto gas (He plus O2 only), NTP (1 kV) or/and cetuximab (10 țg/ml), cells were incubated in the upper part of the Transwell chamber for 24 h. Attached cells in the lower section (invading cells) were stained with H&E.
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WULSOLFDWH(E) Stained cells in the lower chamber were counted using light microscopy (200u).
The data represent the means S.D. of three independent experiments. *P < 0.05 3
ୌ
IV. DISCUSSION
EGFR is commonly overexpressed or constitutively activated in HNC, including OSCC, and is known to contribute to their uncontrolled proliferation, poor prognosis, and survival(Chung, Ely, et al., 2006). Thus, blocking the EGFR pathway has been regarded as a promising molecular target for HNC(Boeckx et al., 2014). Unfortunately, only 10-20% of patients with HNC tumors display a favorable response to cetuximab monotherapy and even the combination of standard chemotherapies with cetuximab treatment prolongs overall survival by a few months because of resistance to EGFR pathway inhibition(Boeckx et al., 2014; Ratushny, Astsaturov, Burtness, Golemis, & Silverman, 2009). Therefore, new therapeutic approaches, including rational combination strategies, are needed to increase the long-term survival of OSCC patients.
Evidences from recent literatures suggested NTP as a promising anti-cancer therapeutic method by inducing growth arrest and cell death in various types of cancer cells(Chang, Kang, Shin, Kim, Seo, Yang, Lee, Moon, Baek, et al., 2014; Chang, Kang, Shin, Kim, Seo, Yang, Lee, Moon, Lee, et al., 2014; Guerrero-Preston et al., 2014; S. U. Kang et al., 2014; Kim, Bahn, et al., 2010; Ma et al., 2014). Although the mechanisms underlying the anticancer effects of NTP have not been fully elucidated, the biological effects of NTP are known to depend mainly on reactive oxygen/nitrogen species (ROS/RNS), which are generated when cells and fluid are brought into contact with NTP(S. U. Kang et al., 2014;
Ma et al., 2014). Previously, we demonstrated that NTP induced anticancer effects via ROS generation(S. Y. Lee et al., 2014). However, we also suggested a novel NTP anticancer mechanism other than ROS signaling(Chang, Kang, Shin, Kim, Seo, Yang, Lee, Moon, Baek, et al., 2014; Chang, Kang, Shin, Kim, Seo, Yang, Lee, Moon, Lee, et al., 2014). Especially in thyroid papillary cancer cells, NTP ameliorated the invasive characteristics of cancer cells via FAK inhibition, which is associated with both cytoskeleton modulation and inhibition of MMPs/uPA system activities(Chang, Kang, Shin, Kim, Seo, Yang, Lee, Moon, Lee, et al., 2014). The purpose of the current study was to explore the effect of combination treatment with NTP on migration and invasion, rather than cell death, of cetuximab-resistant OSCC
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intensity (1 kV NTP) that does not result in cell death even in combination. Moreover, in this study, each mono- or combination therapy alone had no significant effect on HaCaT normal oral epithelial cells.
Our current data indicate that NTP in combination with cetuximab had synergistic antitumor effects by inhibiting migration and invasion of cetuximab-resistant cells via suppression of NF-ț% VLJQDOLQJ HYHQ WKRXJK ZDs no significant effect on cancer cell viability. This is the first report to present an anticancer effect of NTP other than apoptosis in OSCC, along with the molecular mechanism, although Guerrero et al. suggested the preliminary possibility of non-apoptotic mechanisms without specific mechanistic explanation in KXPDQSDSLOORPDYLUXV-negative HNC with a minimal effect on the normal adjacent tissue(Guerrero-Preston et al., 2014). )XUWKHUPRUH WKLV LV WKH ILUVW UHSRUW RI D
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ORFDOO\ DGYDQFHPHQW RI WXPRU RU GLVWDQW PHWDVWDVLV In normal cells, quiescent NF-ț% LV
activated by inflammatory stimuli. In most cancers, including HNC, NF-ț%LVLQYROYHGLQ
tumorigenesis, tumor maintenance or progression, and resistance to cytotoxic chemotherapy (Tanaka et al., 2011). In addition, the abnormal constitutive activation of NF-ț%FRQWULEXWHV
to malignant progression and resistance to therapy (Xie et al., 2001). In our data, all cetuximab-resistant cell lines and tumor tissues showed significant NF-ț% SURWHLQ
overexpression. Moreover, cetuximab sensitive cells demonstrated few NF-ț%H[SUHVVLRQ
and down regulation of NF-ț%XVLQJ51$LQWHUIHUHQFHUHFRYHUHGFHWX[LPDEVHQVLWLYLW\RQ
cetuximab-resistance cells (Figure 9). Thus we postulated that constitutive NF-ț% VLJQDO
activation was related to cetuximab resistance.
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Previous studies indicated that NF-ƈB can induce EMT via various molecular pathways, which differ according to cell type, but resulting in tumor progression and metastasis (Julien et al., 2007; Li et al., 2012) and thus, the NF-ƈB signal is a potential target for anti-metastatic therapy (Julien et al., 2007; Wang et al., 2015). To verify the effect of the combination of NTP with cetuximab on invasive cellular phenotypes at the molecular level, we analyzed protein levels of Slug and Snail, which are transcription factors and master regulators of the epithelial-to-mesenchymal transition (EMT); as well as E-cadherin or vimentin, which are cellular machinery associated with the invasive phenotype of cancer cells and thus hallmarks of the EMT (Scanlon, Van Tubergen, Inglehart, & D'Silva, 2013;
Smith, Teknos, & Pan, 2013). Different from our previous report of anti-EMT effects of 2 and 4 kV of NTP (Chang, Kang, Shin, Kim, Seo, Yang, Lee, Moon, Lee, et al., 2014), 1 kV of NTP monotherapy did not decrease EMT marker expression and did not inhibit EMT phenotypes, likely because of the low intensity of NTP. However, the combination of NTP with cetuximab showed significant inhibition of both EMT marker expression and characteristics, indicating that the combination treatment exerted a synergistic effect. In addition, because the MMP/uPA system plays an important role in ECM degradation and facilitates tumor migration and invasion (Chang, Kang, Shin, Kim, Seo, Yang, Lee, Moon, Lee, et al., 2014; Smith et al., 2013), we assessed the effect of the combination of NTP with cetuximab on the MMP/uPA system. Consistent with the previous report of a relationship between NF-ƈB and the MMP/uPA system (Adhikary et al., 2010; Ji et al., 2015), we found decreased activities of MMP-2/-9 and uPA as well as decreased MMP-2/-9 protein levels following NTP combination induced NF-ƈB suppression.
Intriguingly, although a synergistic effect of NTP in combination with cetuximab on the MSKQLL1 and SCCQLL1 cell lines was noted, NF-ƈB suppression was noted only in SCCQLL1, not MSKQLL1, cells in the present study (among EGFR overexpressing cell lines, NTP combination with cetuixmab also attenuated NF-ƈB in HN6 cells whereas not in SCC25 cells, Figure 10). Because differences in gene expression profiles are related to
heterogeneous biological responses, such as sensitivity to chemotherapy and the malignant phenotype of cancer cells(T. L. Lee et al., 2007), we focused on the differential expression of p53 in the two cell lines. The tumor suppressor protein p53 regulates the cellular response to DNA damage by mediating cell cycle arrest, DNA repair, and cell death(Shieh, Taya, &
Prives, 1999). Inactivation of p53 is reported in most human cancers, with mutations in p53 occurring in about 50% of all tumors(T. L. Lee et al., 2007). In addition to ęloss of functionĚ mutants that lack the tumor-suppressive function, ęgain of functionĚ p53 mutant variants lose their sequence-specific DNA binding but exert complex DNA interactions instead, thereby modifying the set of target genes related to tumorigenesis and drug resistance(Muller, Beissert, & Kulms, 2015). For HNC, about 50% tumors harboring p53 dysfunction related to aberrant overexpression for p53 and inactivation of p53 is observed in most of the rest(T.
L. Lee et al., 2007). Moreover, both the mutation and loss of expression of p53 are implicated in reduced tumor cell apoptosis and resistance to chemotherapy(Hwang et al., 2012). In our study, similarly, cetuximab resistant cell lines showed aberrant overexpression for p53 or inactivation of p53 with little expression: SCCQLL1 cells showed depleted p53 expression, which was restored by treatment with NTP in combination with cetuximab. On the other hand, MSKQLL1 cells with aberrant overexpression of mutated p53 showed no significant changes in expression regardless of application of mono- or combination therapy. In contrast with SCCQLL1 cells, in MSKQLL1 cells, other mechanisms such as the HER-3 pathway, which is a salvage signal against EGFR inhibition(Yonesaka et al., 2015), appear to be linked to the effect of treatment with the combination of NTP with cetuximab (Figure 11).
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Recent studies demonstrated that besides the constitutive activation of NF-ƈB, inactivation of p53 is important for invasiveness of many types of cancer(Hastak et al., 2003;
Hwang et al., 2012; T. L. Lee et al., 2007). p53 has been implicated in malignant progression, metastasis, resistance to chemoradiotherapy, and poorer prognosis through modulating NF-ƈB activity in several studies(Bradford et al., 2003; Hwang et al., 2012; T. L. Lee et al., 2007). Bradford et al. demonstrated that wild-type p53 with low expression is related to cisplatin resistance in vitro(Bradford et al., 2003), and chemoradiation resistance in the clinical setting(Bradford et al., 1995). Tergaonkar et al. suggested that p53 stabilization is decreased upon NF-ƈB activation and that NF-ƈB is associated with acquisition of chemotherapy resistance(Tergaonkar, Pando, Vafa, Wahl, & Verma, 2002). Similarly, Gurova et al. reported that decreased expression of p53 can result from NF-ƈB activation(Gurova et al., 2005). Furthermore, Tin et al. found evidence for an inverse association between inactivation of p53 and NF-ƈB activation via bioinformatics analysis of genome-wide gene expression data in HNC(T. L. Lee et al., 2007). These observations support our hypothesis that differential molecular responses (NF-ƈB suppression or not) to NTP in a subset of OSCC cells (SCCQLL1 and MSKQLL1) is linked to p53 status. In the future, however, it will be necessary to evaluate whether p53 activation is the primary event regulating NF-ƈB inactivation and the link between p53 activation and NF-ƈB suppression in OSCC.
Our findings provide new insight into the mechanisms of not only resistance toward cetuximab in OSCC but also treatment with NTP in combination with cetuximab. Several p53-inducing anticancer drugs have been reported to induce p53 as well as NF-ƈB in various cell types(Hwang et al., 2012). Under this condition, despite p53-induced apoptosis, NF-ƈB activation aggravate invasiveness or promote resistance to apoptosis. Therefore, NTP, which has the ability to repress NF-ƈB signaling associated with cetuximab resistance while also
activating the p53 pathway, might possess greater anticancer efficacy. Thus, we can highlight the development of a rational therapeutic strategy comprising NTP in combination with conventional anticancer modalities.
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ୌ
V. CONCLUSION
We demonstrated that the combination of NTP with cetuximab inhibited invasion/migration of OSCC cells by simultaneously modulating the p53 and NF-ƈB signaling pathways. Although further investigation, including clinical trials, is needed to prove the usefulness of NTP in combination with cetuximab, it may be a useful and novel strategy for OSCC treatment.
୍
<PART TWO>
I. INTRODUCTION
Oral cavity squamous cell carcinoma (OSCC) is the most common head and neck cancer, accounting for ~3% of all newly diagnosed cancer cases (Argiris et al., 2008).
Despite recent advances in surgery, radiotherapy and chemotherapy treatment protocols, the long-term survival of patients with OSCC has remained almost unchanged over the past decade (Bolt et al., 2005b). Resection margin is a well-known prognostic indicator; it is important to obtain tumor-free resection margins in patients with oral cancer (Cavicchi et al., 2000). To achieve this, the surgeon usually removes the tumor with a margin of 10 mm of macroscopically normal tissue, owing to microscopic involvement, and, in more than half of patients with OSCC, the surgical defect is large enough that free flap reconstruction is necessary (Lam & Samman, 2013). Moreover, adjuvant chemoradiation to control the local disease itself may induce many morbidities that affect quality of life and prognosis (Lam &
Samman, 2013). Therefore, novel methods of preventing micro-involvement after surgical resection in OSCC are needed; non-thermal atmospheric pressure plasma (NTP) may be a promising tool with which to address tumor micro-involvement.
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In this study, we evaluated whether NTP induced apoptotic cell death in OSCC cell lines and investigated the molecular mechanism of NTP-induced apoptosis in terms of DNA damage, cell cycle arrest and the ataxia telangiectasia mutated (ATM)/p53 signaling pathway. To our knowledge, this is the first report of a possible mechanism of the anti-cancer effect of NTP related to the ATM/p53 pathway.