Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system

Cisplatin (CDDP) plays a central role in chemotherapy for head and neck squamous cell carcinoma (HNSCC), but drug resistance in HNSCC chemotherapy remains a problem, and the mechanism of CDDP resistance is unclear. We investigated CDDP‐resistance mechanisms mediated by extracellular vesicles (EVs) and ATPase copper transporting beta (ATP7B) in HNSCC.


K E Y W O R D S
ATPase copper transporting beta, cisplatin resistance, extracellular vesicle, GW4869, head and neck squamous cell carcinoma

| INTRODUCTION
The standard treatment for head and neck squamous cell carcinoma (HNSCC) is surgical resection, with the aim of curative treatment.However, widespread damage by surgical resection for advanced cancer has significant adverse effects on the oral function and aesthetics of patients, resulting in a marked decline in quality of life.In light of these problems, HNSCC is also sometimes treated with less invasive modalities, such as chemotherapy and radiation therapy with anticancer agents.Combination chemotherapy for HNSCC is expected to improve the overall survival of patients with advanced cases.Nonetheless, despite the progress achieved with these treatment methods, the overall results of HNSCC treatment have hardly improved, a discouraging outcome that is partly attributable to the treatment resistance to anticancer drugs. 1,2is-diamminedichloro-platinum (CDDP) is commonly used in cancer chemotherapy and in many cases of HNSCC. 3 CDDP is a small molecule consisting of platinum bound to two amines and two chloride ions. 4DDP affects cells by altering the conformation of DNA and thereby altering how the DNA interacts with proteins.Within the DNA-binding protein family, for example, high mobility group (HMG) domain proteins protect DNA damaged by CDDP from other proteins involved in DNA replication, repair, and transcription. 5hese events accumulate to cause programmed cell death.Namely, CDDP exerts an anticancer effect by inducing apoptosis due to cells' DNA damage, impaired replication, transcriptional inhibition, and cell-cycle arrest. 6It has been reported that the mechanism of resistance to CDDP is multifactorial, including variables such as an extracellular efflux, decreased uptake of CDDP, 7,8 a cytoplasmic detoxification mechanism, 9 and a DNA repair mechanism. 10][14] A direct correlation has been demonstrated between changes in the expressions of ATP7A and ATP7B and resistance to platinum agents. 14Intracellular copper transport systems may modulate the intracellular concentrations of platinum drugs and thus affect the cancer chemotherapy response rate. 150][21][22][23] The elucidation of factors that regulate this copper transport system may thus provide a major step toward solving the difficult problem of drug resistance in chemotherapy for HNSCC.
5][26][27] EVs are lipid-membrane-surrounded vesicles released by many types of cells, 28,29 and they are present in virtually all bodily fluids, including serum, urine, saliva, and milk. 30,312][33] EVs (including exosomes) contain various proteins, nucleic acids, and lipids from their donor cells. 315][36] It has been suggested that (i) the mechanisms underlying the functions of EVs have the potential to provide a direct interaction with cells through receptor-mediated interactions, and (ii) EVs function by transferring various endogenous molecules such as proteins, membrane receptors, mRNAs, and microRNAs from their donor cells to recipient cells. 37,38umor-derived EVs are known to be involved in the process of chemotherapy resistance. 24,39,40One of our earlier studies clarified the mechanism by which HNSCC cells secrete epidermal growth factor receptor (EGFR)-containing EVs, which act to confer resistance to the anti-EGFR antibody cetuximab. 39In addition, the depletion of ATP-binding cassette transporter G1 (ABCG1), a cholesterol lipid efflux pump, promoted both the intracellular accumulation of EVs and tumor mass regression, indicating the potential for novel therapeutic strategies targeting tumorderived EVs. 40However, our knowledge of the role of EVs in the tumor control and progression of HNSCC is still quite limited, and more research is urgently needed. 31,41e conducted the present study to elucidate the mechanism of CDDP resistance via EVs and the copper transport pathway in HNSCC.To the best of our knowledge, there are no published reports linking the copper transport pathway and EVs.Our experiments revealed several drug resistance-associated molecules involved in EV dynamics, and suggest new therapeutic strategies to overcome CDDP resistance.
Cisplatin (Randa ® Injection) was obtained from Nippon Kayaku (Tokyo).GW4869, a selective inhibitor of sphingomyelinase, was obtained from Cayman Chemicals (Ann Arbor, MI) and suspended in DMSO solvent as an EV secretion inhibitor before use.

| Cell culture
The HNSCC cell lines SAS, HSC-3, and HSC-4 were obtained from the JCRB Cell Bank (Osaka, Japan) and cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) in a humidified incubator at 37 C and 5% CO 2 as described previously. 42

| Tissue microarray analysis
The expression of ATP7B was analyzed in head and neck cancer tissue and in a normal tissue microarray (#OR601c, #HN811; US Biomax, Rockville, MD).The antigen was activated by cooking in a citric acid solution.For the immunohistochemical analysis, the specimens were incubated with anti-ATP7B antibody (1:250) overnight at 4 C.The slides were then treated with a streptavidin-biotin complex (EnVision System Labeled Polymer, HRP; Dako; Agilent Technologies, Santa Clara, CA) for 60 min at a dilution of 1:100.The immunoreaction was visualized with the use of a DAB substrate-chromogen solution (Dako Cytomation Liquid DAB Substrate Chromogen System; Dako, Glostrup, Denmark).The cells were counted using a light microscope and evaluated.

| CDDP-resistance model
We developed the CDDP-resistance model by growing the HNSCC cell lines SAS and HSC-3 in increasing sub-lethal concentrations of CDDP in the growth medium.The starting dose of CDDP was approx. 1 μg/mL for 72 h.The medium was then replaced to let the cells recover for a further 72 h.Thereafter, CDDP concentrations were augmented by 0.5 μg/mL every 72 h.The medium was changed every 3 days and maintained at the established CDDP concentration.The final CDDP concentration was set at a maximum of 3 μg/mL.This development phase was conducted for approx.6 months, after which the halfmaximal inhibitory concentration (IC50) was reassessed.The resistant cell lines obtained (SAS-R and HSC-3-R) were cultivated under the same conditions as the parental cell line.

| shRNA transfection
SAS and HSC-3 cells were transfected with 5.0 μg of control short hairpin (sh)RNA plasmid (sc-108 060; Santa Cruz Biotechnology) or ATP7B shRNA plasmid (sc-44 491-SH; Santa Cruz Biotechnology) with the use of 4D-Nucleofector™ (Lonza Group, Walkersville, MD).Two days later, the cells were cultured in DMEM +10% FBS for 5 days in the presence of 1.6 μg/mL puromycin dihydrochloride for the selection of cells that stably expressed the shRNAs.

| EV fractions
EV fractions were prepared from the serum-free culture supernatants at 48 h after medium replacement using a modified polymer-based precipitation method as described previously. 43,44Whole cell lysates were prepared at the same time as described below.Briefly, the cell culture supernatant was centrifuged at 2000g for 30 min at 4 C and then centrifuged at 10 000g for 30 min at 4 C.The supernatants were filtered with a 0.2-μm pore filter in a few experiments as described in the Supplementary Materials.The pass-through was concentrated using an ultrafiltration device for molecular weight 100 K to separate the EV fraction and soluble EV-free fraction.
The concentrate was applied to polymers of Total Exosome Isolation (Thermo Fisher Scientific, Carlsbad, CA).The EV fractions were suspended in 100-200 μL phosphate-buffered saline (PBS) without calcium and magnesium.For the protein assay, 10 μL of 10Â RIPA buffer and 10 μL of 100Â protease inhibitor cocktail (Sigma, St. Louis, MO) were added to 100 μL of the EV fraction and incubated on ice for 15 min.Protein concentrations were analyzed with a micro-BCA protein assay (Thermo Fisher Scientific).

| Western blotting
The whole cell lysate (WCL) was prepared as described previously. 43,45Briefly, cells cultured in a 10-cm dish were lysed in 200 μL/dish of RIPA buffer (1% NP-40, 0.1% SDS, 0.5% deoxycholate, and EDTA-free protease inhibitor cocktail in PBS) and collected by a cell scraper.The cells were further lysed using a 25-gauge syringe for 10 strokes and then incubated for 30 min on ice.Spheroids were treated with ultrasonic crushing.The protein concentrations were analyzed using a BCA protein assay (Thermo Fisher Scientific).
Equal amounts of the WCL were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane using a semi-dry method.Membranes were blocked in 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 for 60 min, incubated with primary antibodies, and then incubated with HRP-conjugated secondary antibodies.Blots were visualized using a Clarity ECL substrate and a ChemiDoc MP system (Bio-Rad, Hercules, CA).

| Cell viability assay
The resistance of HNSCC cells to CDDP was evaluated at IC50.The cell viability used to determine the IC50 value was measured by an MTT assay kit (Cayman Chemicals).In brief, all three lines of HNSCC cells were further cultured and then treated with 10 μL of MTT.After incubation for 3 h, the medium was removed and 100 μL of DMSO was added.The optical density at 570 nm was measured, and the cell survival rate in each group was calculated.The IC50 of the HNSCC cells to CDDP was then obtained using relative survival curves.
The cell viability assay was performed as described previously. 42Briefly, CDDP was added to the HNSCC cells at IC50 24 h after cell seeding.PBS was added to the control group as a substitute for cisplatin.Cells were detached or disassembled using Trypsin/EDTA at 24 h after the addition of CDDP or PBS, and the number of cells was counted using a Countess ® Automated Cell Counter (Thermo Fisher Scientific).Cell viability was determined by converting the ratio of the number of cells in the cisplatin-added group to that in the PBS-added group.

| Cisplatin concentration assay
The CDDP concentrations in the cell suspensions were measured by a cisplatin assay kit (MicroMolar Cisplatin Assay Kit; ProFoldin, Hudson, MA).The samples, buffer, and chelate color solution were mixed and incubated for 60 min at 65 C. The absorbance was then read at a wavelength of 535 nm using a microplate reader (SH-1000; Hitachi, Tokyo).

| Transmission electron microscopy
Transmission electron microscopy (TEM) and a particle diameter distribution analysis were carried out as described previously. 43,44Briefly, a 400-mesh copper grid coated with formvar/carbon films was treated hydrophilically.The EV suspension (5-10 μL) was placed on Parafilm ® , and the grid was floated on the EV liquid and left for 15 min.The sample was negatively stained with 2% uranyl acetate solution for 2 min.Extracellular vesicles on the grid were visualized with 20 000Â magnification with an H-7650 transmission electron microscope (Hitachi).

| Particle size distribution
A part of the EV fraction was diluted with PBS (À) to a volume up to 40 μL and then analyzed using a Zetasizer Nano ZSP (Malvern Panalytical, Malvern, UK) in a range of diameters from 0.3 to 10 000 nm, as described previously. 43,44

| Statistical analysis
Statistical significance was calculated using Microsoft Excel and EZR software (http://jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedEN.html).Differences between the two data sets were examined with Welch's t-test, and more than three sets of data were examined with the Kruskal-Wallis test.Probability ( p)-values <0.05 were considered significant.The data are expressed as the mean ± standard deviation (SD) unless otherwise specified.

| High ATP7B expression correlates with HNSCC malignancy
Our earlier investigation demonstrated that the ATP7B expression was higher in tumor tissues than in normal tissues in the head and neck region, 16 and to further investigate the relationship between ATP7B expression and HNSCC stage, we classified HNSCC tissues in the head and neck region in the present study according to their grade and then performed tissue microarrays (Figure 1A,B) (Table 1).The results revealed that ATP7B was more highly expressed in the advanced-stage cases compared to the early-stage cases (Figure 1A,B).This result suggested that ATP7B may play an important role in HNSCC, especially in advanced cases.
We performed in vitro experiments with the three HNSCC cell lines to verify the significance of ATP7B expression in HNSCC tissues.First, we performed a quantitative comparison of ATP7B protein expression using the HNSCC cell lines (Figure 1C).The results showed significantly higher expression of ATP7B in the poorly differentiated HNSCC cell lines SAS and HSC-3, whereas only slight expression of ATP7B was observed in the well differentiated HNSCC cell line HSC-4 (Figures 1C and S1). 42Next, to investigate whether CDDP affects the secretion of EVs from HNSCC, we prepared EVs from culture supernatants of the three HNSCC cell lines and analyzed the morphological differences among the cells' EVs under TEM and by a particle size measurement (Figure 1D,E).Extracellular vesicles with a cup-shaped morphology were observed in EV fractions of all three HNSCC cell lines, and their peak particle size values were around 150-200 nm (Figure 1D,E).
We further examined the changes in EV secretion upon the addition of CDDP, and the results demonstrated that the addition of CDDP markedly increased the secretion of EVs (Figure S2).For the subsequent experiments, we used SAS cells and HSC-3 cells to test the correlation between ATP7B and EV secretion.

| CDDP-resistant HNSCC cell lines have an enhanced expression of ATP7B
To investigate the association between CDDP resistance and ATP7B expression, we attempted to establish a CDDP-resistant subline of HNSCC cells (Figure 2).After prolonged culture in maintenance medium containing a low concentrations of CDDP, both of the HNSCC cell lines SAS and HSC-3 acquired CDDP resistance.In both cell lines, the IC50 to CDDP was enhanced by two-to threefold in the resistant subline compared to the parental cell line (Figure 2A).More interestingly, the intracellular CDDP accumulation tended to be significantly lower in the resistant HNSCC cells than in the parental cells (Figure 2B).The weakening of the cell-killing effect of CDDP on resistant HNSCC cells was also indicated by the decreased expression of the apoptotic product, cleaved caspase 3 (Figure 2C).
We therefore conducted western blotting and immunocytochemistry analyses to evaluate the relationship between the CDDP resistance capacity and the expression of copper transporter protein.The results showed that the ATP7B expression was significantly enhanced in both HNSCC cell lines compared to the parental cell line in the CDDP-resistant subline (Figure 2D,E).Conversely, the expression of CTR1 tended to be downregulated in the CDDP-resistant subline (Figure 2D).Incidentally, the CDDP-resistant sublines, especially HSC-3, showed many cells with irregular cell morphology, such as brittle intercellular bridges, flattened and protruding morphology, marked enlargement of nucleus and cytoplasm, and multiple nucleoli compared to the parental cells (Figure 2E).These cell morphological changes are consistent with events previously attributed to long-term exposure to CDDP and acquisition of drug resistance. 46,47These results suggested that an enhancement of ATP7B is involved in CDDP resistance in HNSCCs.

| ATP7B knockdown enhanced the CDDP sensitivity and suppressed EV secretion
Next, we performed ATP7B knockdown by shRNA transfection in HNSCC cells to determine whether ATP7B truly affects CDDP sensitivity (Figure 3A,B).HNSCC cells transfected with shATP7B showed significantly reduced cell viability in response to CDDP (Figure 3C).Furthermore, the EV fractions recovered from HNSCC cells transfected with shATP7B showed no obvious change in EV morphology or size (Figure 3D,E), but did show a decrease in EV content at the protein level (Figure 3F).In addition, the EV marker molecules CD9, EpCAM, HSP90 and GAPDH in the EV fractions recovered from each group were attenuated in the shATP7B-transfected group (Figure 3G).In other words, interestingly, the transfection of shATP7B not only enhanced the CDDP sensitivity of the HNSCC cells; it also had a tendency to decrease EV secretion (Figure 3F,G).These results suggested that the expression of ATP7B in HNSCC cells is also involved in EV-secretion mechanisms.

| GW4869 enhanced the cell-killing effect of CDDP by suppressing EV secretion and ATP7B expression in HNSCC cells
GW4869 is a selective neutral sphingomyelinase (nSMase) inhibitor that is known to inhibit the production of exosomes and other EVs. 48,49When we treated HNSCC cells with GW4869, the EV secretion in the culture supernatant tended to decrease at the protein level (Figure 4A).GW4869 also did not induce morphological changes, toxicity, or reduced proliferation or viability on HNSCC cells (Figure 4B).We thus investigated how GW4869 affects the ATP7B expression in HNSCC cells, and the results demonstrated that GW4869 caused a clear down-regulation of ATP7B expression in both HNSCC cell lines (Figure 4C,D).
Intrigued by the effects of GW4869, we next examined whether GW4869 augments the cell-killing effect of CDDP (Figure 5).As mentioned earlier, no cytotoxicity to or decreased viability of HNSCC cells was observed when GW4869 was used alone (Figure 5A,B).However, CDDP caused a clear decrease in the number of viable HNSCC cells (Figure 5A,B).It is important to note that the combination of CDDP + GW4869 resulted Abbreviations: F, female; M, male; mon., month; SCC, squamous cell carcinoma; SD, standard deviation.
in a significant decrease in the number of viable cells compared to the CDDP-alone group (Figure 5A,B).The CDDP-induced enhancement of cleaved caspase 3 expression was also more pronounced in the CDDP + GW4869 combination group compared to the CDDPalone group (Figure 5C).Moreover, the inhibitory effect of GW4869 on ATP7B expression was still observed even when GW4869 was used in combination with CDDP (Figure 5D).The results of these experiments with GW4869 suggest that the suppression of EV secretion and the expression of ATP7B may potentiate the therapeutic effect of CDDP.

| DISCUSSION
We conducted this study to identify proteins associated with drug resistance in tumor cells and to elucidate the underlying mechanisms.The results showed that the copper transporter molecule ATP7B is highly expressed in CDDP-resistant HNSCC cells and is associated with EV-mediated drug resistance mechanisms.It has been reported that EVs facilitate the transport of several drugs (doxorubicin, CDDP, paclitaxel, and others) out of the cell. 50,51In addition, tumor cells can secrete more EVs under drug stimulation (Figure S2). 52EVs may therefore play an important role in tumor-cell drug resistance.
Much of the research on drug resistance has focused on the multidrug resistance-associated protein family, which includes the ATP-binding cassette (ABC) transporters. 53These transporter have been implicated in an EV-mediated induction of drug resistance, especially in EV-secreting cells. 54,55For example, ABCG2 is one of the key transporters, and studies of ABCG2-rich EVs have shown that drugs such as riboflavin, topotecan, and methotrexate are exported out of cells through EVs. 56We have also shown that the depletion of ABCG1 results in an intracellular accumulation of EVs and tumor regression. 40On the other hand, our report is the first study to demonstrate that ATP7B, a copper transporter but not an ABC transporter, is associated with EVs.More interestingly, we had proposed a novel drug-resistance mechanism that protects HNSCC cells that had previously been attacked with cetuximab via EV secretion. 39These previous reports led us to the idea of elucidating the molecular mechanism by which the copper transporter responsible for platinum efflux plays a role in EV secretion and CDDP resistance in cancer cells.A clarification of the links among drug resistance, transporters, and EVs remains an active area of research, and these links deserve attention for their potential involvement in a novel drug-resistance mechanism.Our initial study confirmed that ATP7B is more highly expressed in advanced cases of HNSCC compared to early cases (Figure 1A,B).Patients with HNSCC usually respond well to CDDP-based chemotherapy, but more advanced cases are resistant to CDDP.Our results suggest that ATP7B expression is involved in CDDP resistance in advanced HNSCC (Figure 1A,B).The efficacy of CDDP and other platinum-based drugs against malignant tumors depends on the drug resistance of cancer cells, but the exact mechanism is still unknown.ATP7A and ATP7B are known to induce drug resistance to CDDP. 16,57Both ATP7A and ATP7B are membranebound molecular pumps belonging to P-type ATPases and are involved in copper homeostasis.It has also been suggested that CDDP is transported by ATP7B via lysosomal exocytosis, 58 but similar studies of head and neck cancers have not been conducted.In the present experiments, we detected the expression of ATP7B in HNSCC cells (Figure 1C), and we observed that ATP7B was expressed to a significantly greater degree in CDDPresistant HNSCC cells compared to the parental cells (Figure 2), further suggesting that suppression of ATP7B expression may be involved in EV regulation and resistance mechanisms (Figure 3).
Dimethyl amiloride (DMA), GW4869, and a glucosyl ceramide synthase inhibitor have been reported to inhibit EV secretion in several cell lines. 59Among them, we selected GW4869, a selective neutral sphingomyelinase (N-SMase) inhibitor.Our validation indeed showed that the action of GW4869 on HNSCC cells clearly suppressed EV secretion at the protein level (Figure 4A).Several research groups have shown that EV and exosome secretion can be inhibited by GW4869. 48,49Interestingly, in our validation, the treatment of HNSCC cells with this known inhibitor of EV biosynthesis did not significantly affect cell viability or cell morphology, but it enhanced the antitumor effect of CDDP, which in turn led to a clear decrease in ATP7B expression (Figures 4 and 5).Rimmer et al. reported that the antitumor effect of gemcitabine on pancreatic cancer cells was enhanced under a GW4869 combination. 60Others have reported that the combination of GW4869 and doxorubicin against acute myeloid leukemia similarly enhanced sensitivity to chemotherapy. 51Abad et al. revealed a new mechanism of acquired chemotherapy resistance in triple negative breast cancer (TNBC) cells, in which EVs transport mitochondria to susceptible cancer cells and enhance their resistance to chemotherapy. 61They also reported that the inhibition of mitochondrial transport by EV inhibitors, including GW4869, reduced the chemotherapy resistance of acquired TNBC cells. 61Our present findings suggest that GW4869 may be useful in addressing CDDP chemotherapy resistance because it potentiates the cell-killing effect of cisplatin in vitro (Figure 5).The results of our validation indicate that GW4869 should be further explored as an option for combination therapy with chemotherapeutic agents for the better management of CDDP-resistant HNSCC.
ATP7B is abundant in the Golgi body and has been shown to function in the luminal uptake to late endosomes and in the extracellular elimination of CDDP bound to the copper chaperone Atox1. 62It has also been reported that the localization of ATP7B itself induces its redistribution in late endosomes upon the administration of copper or CDDP. 63We hypothesized that the attenuation of ATP7B by GW4869 occurred as a result of its action on this late endosomal-localized ATP7B (Figure 6).The inhibition of CDDP uptake into the endosomal lumen by ATP7B may suppress the uptake of CDDP into EVs and a CDDP efflux, resulting in an enhanced effect of CDDP.In other words, GW4869 is expected to inhibit the drug efflux pathway by late endosome-localized ATP7B in addition to inhibiting EV formation itself (Figure 6).

| CONCLUSION
The results of this study indicate that (i) the release of EVs via ATP7B is a major obstacle to CDDP treatment of head and neck squamous cell carcinoma, and (ii) ATP7B is a potential new therapeutic target.Combinations of EV inhibitors may be a novel adjunctive treatment option in CDDP treatment for HNSCC.We will continue to search for ATP7B potentiators in EVs and to develop novel biomarkers.

AUTHOR CONTRIBUTIONS
KiO, TOk, and SI conceptualized and designed the study.KiO, SR, NY, TOk, HN, and SI prepared resources.KiO, SR, HoK, KyO, TOk, and KuO devised the methodologies.TOg, KiO, TN, KU, KY, and HiK carried out the experiments.HiK, HoK, and KyO performed the formal analyses.KiO and KyO interpreted the data.KiO wrote the manuscript.TOg, KiO, TN, and KuO revised and edited the manuscript.All authors reviewed the manuscript.

1
Expression of ATP7B and secretion of EVs by HNSCC cells.(A) Representative immunohistochemistry images of ATP7B expression in normal tissues and tumor tissues in the head and neck region.The upper row shows the low-magnification image and the lower row shows the high-magnification image.The box indicated by a dotted line shows the coincident area in each image.Scale bar: 200 μm.(B) Box and whisker plot of ATP7B-positive cells (%) in normal tissue (n = 5), tumor tissue from early-stage cases (stage I/II, n = 12) and tumor tissue from advanced cases (stage III/IV, n = 15) in the head and neck region.*p < 0.05, Kruskal-Wallis test.(C) Western blotting (WB) showing ATP7B protein expression in HNSCC cell lines.β-Actin was used as a loading control.(D) Representative TEM images of EVs derived from HNSCC cell lines.Scale bar: 100 nm.(E) Representative particle diameter distribution of EVs derived from HNSCC cell lines.Peak values were 100-200 nm.[Color figure can be viewed at wileyonlinelibrary.com]

4
The effect of GW4869 on EV secretion and ATP7B expression.(A) Amount of EV protein per WCL protein (%) of HNSCC cells treated with or without GW4869 for 24 h (n = 6).*p < 0.05, Kruskal-Wallis test.(B) Representative images of HNSCC cells treated with or without GW4869 for 24 h.Scale bar: 100 μm.(C) ICC showing ATP7B protein expression in HNSCC cells treated with or without GW4869 for 24 h.Scale bar: 100 μm.(D) WB showing ATP7B protein expression in HNSCC cells treated with or without GW4869 for 24 h.β-Actin = loading control.[Color figure can be viewed at wileyonlinelibrary.com]

5
Additive effect of GW4869 on the cell-killing of CDDP.(A) Representative images of HNSCC cells treated with GW4869 and/or CDDP for 24 h.Scale bar: 200 μm.(B) Number of surviving HNSCC cells treated with GW4869 and/or CDDP for 24 h (n = 9).*p < 0.05, Kruskal-Wallis test.(C) WB showing cleaved caspase 3 and caspase 3 protein expressions in HNSCC cells treated with GW4869 and/or CDDP for 24 h.β-Actin = loading control.(D) ICC showing ATP7B protein expression in HNSCC cells treated with GW4869 and/or CDDP for 24 h.Scale bar: 200 μm.[Color figure can be viewed at wileyonlinelibrary.com] Details of each tissue used for the head and neck cancer tissue and normal tissue microarray.
T A B L E 1