Inhibition of autophagy by chloroquine makes chemotherapy in nasopharyngeal carcinoma more efficient

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Inhibition of autophagy by chloroquine makes chemotherapy in nasopharyngeal carcinoma more efficient Tomomi Aga a, Kazuhira Endo a, Akira Tsuji b, Mitsuharu Aga a, Makiko Moriyama-Kita a, Takayoshi Ueno a, Yosuke Nakanishi a, Miyako Hatano a, Satoru Kondo a, Hisashi Sugimoto a, Naohiro Wakisaka a, Tomokazu Yoshizaki a,* a b

Division of Otolaryngology — Head and Neck Surgery, Graduate School of Medicine, Kanazawa University, Kanazawa, Japan Division of Otolaryngology, Toyama City Hospital, Toyama, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 June 2018 Accepted 10 October 2018 Available online xxx

Objectives: A combination of platinum-based chemotherapy and radiotherapy is the standard treatment for nasopharyngeal carcinoma (NPC). However, the efficacy of chemotherapy has reached a plateau. Many autophagy studies suggest that autophagy can either promote or suppress to cancer progression. Thus, a role of autophagy in the acquisition of chemoradioresistance has recently been a notable event. Therefore, we examined the relationship between autophagy and chemotherapy in NPC. Methods: The expression of Beclin 1 and microtubule-associated protein light chain 3 (LC3), a marker of autophagy, was determined by immunohistochemistry in the biopsy samples of patients with NPC before and after the first course of chemotherapy. Additionally, to investigate in the effect of autophagy suppression in chemotherapy, NPC cell line C666-1 cells were treated with cisplatin and/or chloroquine, an inhibitor of autophagy. Results: The expression of Beclin 1 increased after chemotherapy in all patients. In NPC cell line C666-1, compared to cisplatin alone, combination therapy (cisplatin and chloroquine) reduced cell viability, and promoted cell apoptosis. Conclusions: These results suggest that autophagy, represented by Beclin 1, is upregulated after chemotherapy in both in vitro and in vivo NPC studies. Inhibition of autophagy could therefore be new strategy for NPC treatment. © 2018 Elsevier B.V. All rights reserved.

Keywords: Autophagy Beclin 1 Chloroquine LC3 Nasopharyngeal carcinoma

1. Introduction Platinum-based chemotherapy and radiotherapy are the current standard treatments for locally advanced nasopharyngeal carcinoma (NPC), which is highly metastatic and chemoradiosensitive [1,2]. The prognosis remains poor in * Corresponding author at: Division of Otolaryngology — Head and Neck Surgery, Graduate School of Medicine, Kanazawa University, Takara-machi 13-1, Kanazawa, Ishikawa 920-8640, Japan. E-mail address: [email protected] (T. Yoshizaki).

NPC patients with recurrence or distant metastasis. An intervention of surgical approach and re-irradiation has little effect for those patients, and the therapeutic role of chemotherapy is quite important. Thus, in recurrent and metastatic carcinoma, chemoresistance is one of the most serious problems [3,4] and it is very important to find a way to improve chemosensitivity. Intracellular proteins are degraded by two main cellular systems: apoptosis and autophagy. Autophagy is a survival mechanism under conditions of stress and is a fundamental cellular homeostatic mechanism whereby damaged proteins,

https://doi.org/10.1016/j.anl.2018.10.013 0385-8146/© 2018 Elsevier B.V. All rights reserved.

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lipids, and organelles in the cytoplasm are autodigested for removal or turnover [5,6]. Impaired autophagy occurs in the pathogenesis of various diseases, such as myopathy, microbial infection, neuronal degeneration, inflammatory bowel disease aging and cancer [7–12]. Autophagy is now regarded to have a context-dependent role in cancer [7]. Beclin 1 is one of the most important autophagyrelated genes, which was discovered as a Bcl-2-interacting protein. When Beclin 1 is bound to Bcl-2, autophagy levels remain within a homeostatic range [13–15]. Beclin 1 is encoded by the BECN1 gene located on chromosome 17q21. Monoallelic loss of BECN1 on chromosome 17q21 occurs in 40%– 75% of human breast, ovarian, and prostate tumors [12,14,16– 18]. These studies suggest that Beclin 1 has an important role in several kinds of cancer. Under- or overexpression of Beclin 1 is associated with poor prognosis, which is dependent on the type of cancer [19,20]. These studies suggest that Beclin 1 has a biologically as well as clinically important role in such cancers. Microtubule-associated protein light chain 3 (LC3) is also used as markers of autophagy. LC3 comprises three isoforms LC3A, B and C [21]. LC3 is incorporated into the inner and outer membrane of autophagosomes. LC3-I is cytosolic, whereas LC3-II is membrane bound. Conversion of LC3-I to LC3-II is enhanced by culturing cells under starvation conditions. The amount of LC3-II is related with the number of autophagosomes [22,23]. Therefore, LC3 is widely used to monitor autophagy status. In normal cells, autophagy suppresses tumorigenesis by removing damaged organelles and proteins and by limiting genomic instability [24–26]. This helps to maintain cellular homeostasis. Thus, autophagy defects can promote and accelerate tumorigenesis. Paradoxically, in established tumors, autophagy promotes the growth of the tumors under stressed conditions [27]. Thus, autophagy is also reported to be activated by chemotherapy, and promotes resistance to chemotherapy in laryngeal cancer [28]. On the other hand, there are contrasting findings suggesting that autophagy facilitates chemotherapeutic cytotoxicity in apoptosis-resistant tumor cells through the activation of autophagy-associated cell-death pathways [7]. Therefore, it is unclear whether it acts as an enhancer or an inhibitor of chemotherapeutic agents in NPC tumors. The main purpose of this study is to clarify the role of autophagy in NPC and to explore the possibility of a new therapeutic strategy to target autophagy. To ascertain this, we used NPC tissues and NPC cell lines to examine the relationship between autophagy and chemotherapy. We also studied whether the inhibition of autophagy increases the efficacy of chemotherapy in NPC cell lines. 2. Materials and methods

study was approved by the Institutional Review Board of Kanazawa University (application number: 1270) and we received consent from each participating patient. All specimens were fixed in 10% neutral formalin and embedded in paraffin. After a review of all H&E-stained slides of the specimens, they were classified histopathologically according to the 2005 WHO histological classification [31]. The clinical staging of NPC was evaluated on the basis of the tumor-node metastasis classification of the Union Internationale Contre le Cancer [32]. 2.2. Reagents and materials Cisplatin (CDDP) was used as a chemotherapeutic agent. Chloroquine (CQ) was used as an inhibitor of autophagy. CDDP was purchased from Nippon Kayaku (Chiyoda, Tokyo, Japan). CQ was purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-Beclin 1 was purchased from Novus Biologicals (Littleton, CO, USA), and anti-b-actin was purchased from Gene Tex (Irvine, CA, USA). Anti-LC3 was obtained from Medical & Biological Laboratories Co., LTD (Nagoya, Aichi, Japan). 2.3. Immunohistochemical analysis Consecutive 3-mm sections were cut from each block. Deparaffinized sections were treated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase activity. The sections were incubated with protein blocker (Dako, Glostrup, Denmark) for 20 min and incubated with primary antibodies Beclin 1 and LC3 (diluted 1:500) at 4  C overnight. After washing three times with phosphate-buffered saline (PBS), sections were incubated with secondary antibodies conjugated to a peroxidaselabeled polymer (EnVisionTM + system, DAKO) for 30 min at room temperature. After washing three times with PBS, the sections were color-developed with 3,3-diaminobenzidine tetrahydrochloride solution. The sections were counterstained with hematoxylin and dehydrated. The stained sections were independently examined by two of authors. The portion of Beclin 1 or LC3 positive cells was scored in each section using 400 magnification as follows: negative (score 0), 0%–25% (score 1), 26%–50% (score 2), 51%–75% (score 3) and 76%– 100% (score 4) [33]. The average of the score was calculated without knowledge of the clinical feature. 2.4. Cell lines and cell culture Nasopharyngeal carcinoma cell line C666-1 is an EBVpositive NPC cell line [34]. C666-1 cells were cultured in RPMI medium 1640 (Gibco BRL, Grand Island, NY, USA) with contained 10% fetal bovine serum (FBS) (Gibco BRL), 1% penicillin, and streptomycin. C666-1 cells were incubated in a humidified incubator containing 5% CO2 atmosphere at 37  C.

2.1. Tissue samples 2.5. Western blot analysis The biopsy specimens were obtained from patients who had been diagnosed with NPC and had been treated with alternating chemoradiotherapy at Kanazawa University Hospital from 2008 to 2017 [29,30]. We had 42 cases of NPC in the period. Of them, the pair biopsy samples were examined in 25 cases. This

Western blotting was performed as previously described [35]. 100 mg protein of each sample was solubilized. Then, proteins were separated by 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes

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(Bio-Rad, CA, USA). The membrane was incubated with the primary antibodies Beclin 1 (dilution 1:3000), LC3 (dilution 1:1000) and b-actin (dilution 1:1000) at 4  C overnight. Further, the membrane was incubated by goat anti-rabbit IgGHRP conjugated secondary antibody (dilution 1:2000) for 1 h at room temperature. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia, Buckinghamshire, UK). b-actin was used as the loading control. The protein expression levels were measured by densitometer in the 5 sets of cultured C666-1 cells. 2.6. Measurement of cell viability 1

To measure the viability of C666-1 cells, CellTiter 96 AQueous One Solution Cell Proliferation Assay was carried out. Cells were seeded in 96-well flat-bottom microtiter plates at a density of 5  103 cells in a volume of 100 ml per well. After 24 h, culture medium was changed with fresh medium, which graded concentrations of CDDP, CQ or a combination of CDDP, and CQ. After 24 h, 20 mM CellTiter 961 AQueous One Solution Reagent was added per well, and plates were incubated for 2 h at 37  C. Cell viability curves were then prepared according to the OD values at the wave length of 490 nm.

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Table 1 Clinicopathological parameters of patients. Parameter

Total

Age

16–79 (median 54.2)

Gender Male Female

23 2

Tumor state T1–T2 T3–T4

13 12

Lymph node N0–N1 N2–N3

11 14

Distant metastasis M0 M1

22 3

Clinical stage Ⅱ Ⅲ Ⅳ

4 14 7

Histopathological classification (WHO) Ⅱ Ⅲ

13 12

2.7. Measurement of cell apoptosis To measure the apoptosis of C666-1 cells, annexin V/ propidium iodide apoptosis assay was carried out. Apoptosis was detected by the translocation of phosphatidylserine (PS) to the cell surface using an Annexin V-fluorescein isothiocyanate (FITC) antibody. C666-1 cells were treated with CDDP and/or CQ for 24 h. After trypsinization, 2  105 cells were washed once with medium next with PBS. The cell pellet was resuspended in 85 ml of binding buffer at a density of 2  105 cells per ml and incubated with 10 ml of annexin V-FITC and 5 ml of propidium iodide (PI) for 15 min at room temperature in the dark. Apoptotic cells were detected using visualization with a fluorescent microscope.

carcinoma and were EBER positive (data was not shown). To assess the baseline status of autophagy in the NPC tumor tissues, the expression of Beclin 1 or LC3 was investigated. In the NPC biopsy tissues before chemotherapy, the high expression of Beclin 1 and LC3 were detected in 7 NPC and 15 NPC specimens, respectively. However, there was no statistically significant association with age, tumor size, lymph node metastasis, and/or distant metastasis (Tables 2 and 3). The score before and after the treatment of CDDP in the same case is stated in Table 4. 3.2. Treatment of CDDP increases autophagy

3. Results

We examined the effect of cisplatin administration on autophagy through immunohistochemical study of Beclin 1 and LC3 in the NPC tissues obtained before and after the first course of chemotherapy (Figs. 1 and 2). The expression of Beclin 1 in NPC biopsies after the first course of chemotherapy was higher than before the treatment (Table 5; P < 0.05). There was no statistically significant association with the expression of LC3 and chemotherapy (P = 0.217). However, the expression of LC3 tends to increase after chemotherapy. The effect of CDDP on autophagy in the NPC cell line was then examined using western blot (Fig. 3A). The expression of Beclin 1 and LC3-II/LC3-I in the CDDP group was higher than that in the control group (Fig. 3A, B). These results suggest that CDDP upregulates autophagic response represented by Beclin 1 and LC3 in NPC.

3.1. Patient characteristics and expression of Beclin 1 or LC3 in NPC tissues

3.3. Inhibition of autophagy modulates the cytotoxicity of CDDP

The clinicopathological parameters of 25 patients are summarized in Table 1. All cases were non-keratinizing

We investigated whether the inhibition of autophagy influences the cytotoxicity of CDDP in NPC cells using

2.8. Statistical analysis Statistical analysis was performed using SPSS for Windows software version 11.0.1. The expression of Beclin 1 in relation to the clinicopathological parameters or chemotherapy was analyzed with Fisher’s exact test. The statistical comparison of protein expression level in western blot was analyzed with oneway analysis of variance (ANOVA); where indicated, individual comparisons between the control group and each group were performed using Student’s t-test. Cell viability were determined using Student’s t-test. A P value of <0.05 was considered statistically significant.

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Table 2 The association between the expression of Beclin 1 and the clinicopathological parameters. Before chemotherapy

After chemotherapy

Score22

Score33

P-value

Score22

Score33

P-value

Age 250 years >50 years

5 13

3 4

0.634

1 8

7 9

0.182

Tumor stage T1–2 T3–4

8 10

5 2

0.378

4 5

9 7

0.688

Lymph nodes N0–1 N2–3

7 11

4 3

0.656

2 7

9 7

0.208

Metastasis M0 M1

16 2

6 1

1

8 1

14 2

1

The expression of Beclin 1 in relation to the clinicopathological parameters were analyzed using Fisher’s exact test.

Table 3 The association between the expression of LC3 and the clinicopathological parameters. Before chemotherapy

After chemotherapy

Score22

Score33

P-value

Score22

Score33

P-value

Age 250 years >50 years

4 6

4 11

0.667

3 2

5 15

0.283

Tumor stage T1–2 T3–4

3 7

10 5

0.111

2 3

11 9

0.645

Lymph nodes N0–1 N2–3

6 4

5 10

0.241

3 2

8 12

0.623

Metastasis M0 M1

9 1

13 2

1

5 0

17 3

1

The expression of LC3 in relation to the clinicopathological parameters were analyzed using Fisher’s exact test.

western blot and MTS assay. First, cell viability was examined with a graded CDDP concentration. CDDP treatment reduced the cell viability in a dose-dependent manner (data not shown). However, low-dose cisplatin, 50 mM or less, did not affect cell viability. The expression of Beclin 1 was significantly enhanced in the CDDP group and CQ 5 mM groups compared with the control. In the CDDP + CQ group, the Beclin 1 expression levels were lower than the CDDP group. (Fig. 3B). In the 5 mM CQ group cell viability was 104%, and almost the same as that in the control group. However, when 50 mM CQ was added, cell viability was only 51%. Low-dose CQ (5 mM) itself did not affect cell viability (Fig. 3C). In the 50 mM CDDP + 5 mM CQ group, cell viability was 50% and was significantly lower than that in the 50 mM CDDP group. The combination of CDDP and autophagy inhibition was more effective than CDDP alone in the NPC cells. These results suggest that inhibition of autophagy improves response to CDDP treatment.

3.4. Inhibition of autophagy enhances the apoptotic effect of cisplatin We examined if the inhibition of autophagy can induce apoptosis. Three types of micrographs, bright field, Annexin V (green), and PI (red), were taken (Fig. 4A). Cells were classified into the following three groups: living cells (Annexin V /PI ), early apoptotic cells (Annexin V+/PI ), and late apoptotic cells (Annexin V+/PI+). Annexin V positive cells were regarded as apoptotic cells, and the number of apoptotic cells per 100 in three fields was counted and the average was taken. The portion of apoptotic cells in the control, CDDP, CQ, and CDDP + CQ groups was 4.3, 6.7, 14.3 and 27.7%, respectively. There was no obvious difference between the control and CDDP groups. However, the portion of apoptotic cells in the CDDP + CQ group was significantly higher than in the control group or in the CDDP group (Fig. 4B). These results suggest that CQ enhances cisplatin mediated induction of apoptosis.

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ANL-2526; No. of Pages 8 T. Aga et al. / Auris Nasus Larynx xxx (2018) xxx–xxx Table 4 The score of Beclin 1 and LC3 before and after chemotherapy in each case. Case number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Before chemotherapy

After chemotherapy

Beclin 1

LC3

Beclin 1

LC3

1.0 1.5 2.0 3.0 1.0 1.5 1.0 2.5 1.0 2.0 2.0 3.0 2.0 3.0 3.0 1.5 2.5 3.0 1.0 2.0 1.0 1.0 1.5 3.0 4.0

1.0 1.0 2.5 3.0 4.0 1.0 1.0 2.0 2.0 2.0 2.5 2.0 3.0 3.5 3.0 4.0 4.0 4.0 3.0 3.0 4.0 4.0 4.0 4.0 4.0

3.0 1.5 1.0 4.0 2.5 2.0 3.0 2.0 3.0 3.0 2.0 4.0 3.5 3.0 4.0 2.0 3.0 3.0 3.0 3.5 2.0 3.0 2.0 4.0 4.0

1.0 2.0 2.0 2.5 2.0 3.0 3.0 3.0 3.5 3.0 3.0 3.5 3.0 3.0 3.0 3.0 3.5 3.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0

4. Discussion Autophagy may reduce or accelerate tumor development depending on the tumor type, microenvironment, and the timepoint of malignant progression road [5,36]. Impaired autophagy, such as in Beclin 1 knockdown, leads to an oncogenic stimulus and accelerates carcinogenesis [37]. It is prevalent that lower amount of Beclin 1 is expressed in the tumor cells than the normal cells in many cancers [38–41]. In hypopharyngeal squamous cell carcinoma, both Beclin 1 and LC3 are significantly downregulated [42]. Moreover, the expression of Beclin 1 is associated with clinical factors and survival of the patients with various cancers. On the other hand, the expression levels of Beclin 1 in colorectal cancer are higher than those in normal tissues [43].

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In our immunohistochemical analysis, the expression of Beclin 1 in NPC was higher than that in the surrounding normal tissues. Through cancer development time-course, tumor tissues are exposed to metabolic stress, which make cancer cells promote autophagy to gain nutrients and overcome starvation-related cell death. This may be related to Beclin 1 overexpression in NPC. There was no obvious correlation of Beclin 1 expression with clinicopathological parameters. Similar to Beclin 1, LC3II expression is receiving mixed review. Lower LC3-II is associated with advanced T stage, lymph node metastasis and poor prognosis in hypopharyngeal squamous cell carcinoma [44]. On the other hand, in gastrointestinal cancers, overexpression of LC3 predicts poor prognosis [45]. Thus, it is unrevealed that LC3 inhibits or promotes tumorigenesis. A study with a larger sample size may reveal the role of Beclin 1 or LC3 in NPC more precisely. The important finding in this study is that the administration of CDDP upregulates the expression of Beclin 1 and promotes Beclin 1-mediated autophagy in both the NPC tumor tissue and in the NPC cell lines. This suggests that tumor cells promote Beclin 1-mediated autophagy to acquire resistance to anticancer agents-mediated cell death. The upregulated expression of LC3 also appeared to be associated with chemotherapy treatment, but this trend did not achieve statistical significant. Although LC3-II sensitively reflects autophagy, the antibody we used in immunohistochemistry can detect both LC3-I and LC3-II, which, we speculated, was the reason why there was no significant difference in LC3 in immunohistochemical analysis. Therefore, baseline activity of Beclin1-associated autophagic pathway is slightly up-regulated in NPC, and chemotherapeutic stress enhances its activity to survive against apoptosis. Although our study did not induce conclusive result for the relationship with the expression of Beclin1 or LC3 and clinicopathological features in NPC, the result suggests that upregulation of Beclin 1-included autophagic pathway is a common and an early event in NPC development. CQ inhibits the acidification of lysosomes and prevents the fusion of autophagosomes to lysosomes, which leads to autophagy disruption [46]. In hepatocellular carcinoma, breast cancer and human salivary gland adenoid cystic carcinoma, the inhibition of autophagy enhances the cytotoxicity of CDDP in vitro [47–49]. Furthermore, CQ enhances CDDP-induced

Fig. 1. Immunohistochemical analysis of Beclin1 expression in NPC biopsy tissues (400). (A) Low expressions of Beclin1 were observed before chemotherapy. (B) High expressions of Beclin1 were observed after chemotherapy.

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Fig. 2. Immunohistochemical analysis of LC3 expression in NPC biopsy tissues (400). (A) Low expressions of LC3 were observed before chemotherapy. (B) High expressions of LC3 were observed after chemotherapy.

Table 5 The association between the expression of Beclin 1 and chemotherapy.

Before chemotherapy After chemotherapy

Score <3

Score 33

P-value

18 9

7 16

0.013*

High expression of Beclin1 was significantly correlated with chemotherapy. Statistical analysis was performed by Fisher’s exact test (*<0.05).

apoptosis in hypopharyngeal cancer in xenograft mouse model [50]. In contrast, CQ does not increase CDDP-induced apoptosis in small-cell lung cancers [51]. Our data show that the inhibition of autophagy accelerated the cytotoxicity of CDDP in NPC. When CDDP alone was added, the viability of NPC cells did not decrease. However, combined CDDP with CQ, the viability of C666-1 cells decreased, and the number of apoptotic cells increased. It is also revealed that CQ induces

Fig. 3. Expression of autophagy-related protein and cell viability in NPC cells. (A) Expression of Beclin1 and LC3 in NPC cells by Western blot analysis. (B) Quantitation of Beclin1 protein level. (C) CellTiter 961 AQueous One Solution Cell Proliferation Assay of cell viability. Effects of autophagy inhibited by chloroquine in C666-1 cells. *P < 0.05.

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Fig. 4. Annexin V-FITC/PI double staining assay in C666-1. (A) Three types of micrographs were taken. (B) The number of apoptotic cells per 100 in C666-1 cells. Apoptotic cells increased in the combination of CDDP and CQ group. *P < 0.05, **P < 0.01.

tumor vessel normalization, and helps antimetastatic and chemotherapy-enhancing actions [52]. Thus, CQ has therapeutic potential and recent clinical trials have been carried out to evaluate the safety and efficacy of CQ in glioblastoma multiform [53], patients with brain metastases [54], small cell lung carcinoma (NCT00969306) and pancreatic cancer (NCT01777477). To date, a clinical trial of CQ therapy in head and neck cancer has not yet been carried out. Our data suggest that the inhibition of autophagy lead to increased chemosensitivity. However, autophagy plays a key role in the maintenance of intracellular homeostasis in normal tissues. Whole-body inhibition of autophagy may lead to treatment-related de novo oncogenesis or other disorders [55]. We should be careful about a situation where the usage of CQ is justified to improve the survival or quality of life of NPC patients. These findings suggest its benefit for the patients with multiple metastases. 5. Conclusion Beclin 1-mediated autophagy is increased after chemotherapy in NPC. Inhibition of autophagy enhances the effect of chemotherapy in NPC cells. Control of autophagy has a potential to be new strategies for NPC treatment, especially recurrent and/or metastatic diseases. Acknowledgements This study was partially supported by Grants-in-Aid for Scientific Research(C) (16K11227) and Challenging Exploratory Research (25670714) from the Japanese Ministry of Education, Culture, Sports, Science and Technology. References [1] Yoshizaki T, Ito M, Murono S, Wakisaka N, Kondo S, Endo K. Current understanding and management of nasopharyngeal carcinoma. Auris Nasus Larynx 2012;39:137–44.

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Please cite this article in press as: Aga T, et al. Inhibition of autophagy by chloroquine makes chemotherapy in nasopharyngeal carcinoma more efficient. Auris Nasus Larynx (2018), https://doi.org/10.1016/j.anl.2018.10.013