Inhibition of autophagy enhanced chemosensitivity in cisplatin resistant hypopharyngeal squamous carcinoma cells

Hypopharyngeal carcinoma is characterized by high degree of malignancy. The most common pathological type is squamous cell carcinoma (HSCC). It has been conrmed that high autophagy level promotes the development of hypopharyngeal cancer in recent years. Clinical researches have reported that high autophagy level often caused insensitivity to chemotherapy, a common phenomenon that greatly reduces therapeutic effect in cisplatin-resistant tumor cell lines. Therefore, exploring internal mechanisms of autophagy on cisplatin resistant HSCC is necessary for founding theoretical basis for synergistic antitumor drugs by interfering with autophagy. Observed signicant reduction in the number of autophagy vesicles conrms the decline in autophagy levels. These data indicated that inhibition of autophagy can lead to increasement of cell death rate of cisplatin-resistant cells at the same concentration of cisplatin, which means chemotherapy sensitivity was recovered. Furthermore, by analyzing the expression of downstream factors lc3-1, lc3-2, atg12-atg5 and P62, results suggest that Beclin 1 plays an important role in the development of autophagy in cisplatin resistant FaDu cells. 3-MA, as an autophagy inhibitor, may achieve its function partially by inhibiting Beclin 1. Taken together, we suggest that in cisplatin induced chemotherapy resistance, autophagy inhibit is a very effective method to regain chemosensitivity. It should be used combined with chemotherapy drugs for increasing the treatment ecacy. Beclin1 might be a potential lead research target that regulates autophagy and chemotherapy resistance.


Background
Squamous cell carcinoma, the main pathological types of hypopharyngeal carcinoma, representing approximately 97% of such cases. [1] . It has been reported that the 5-year survival rate for this disease is estimated between 30% − 40% [2] . There are many treatment methods, among them, Currently, chemotherapy is the main treatment for hypopharyngeal squamous cell carcinoma(HSCC), especially for the late stage and elder patients. The major challenge for HSCC chemotherapy is drug resistance, which is easy to occur in the course of chemotherapy and greatly affects the prognosis [3] . Therefore, intervention of drug resistance and improved sensitivity to chemotherapy is an existing research gap for in-depth studies on hypopharyngeal cancer [4] .
In the mechanism of tumor drug resistance, apoptosis-resistance has been proved to be a key reason of chemotherapy insensitivity [5] . Several studies have shown that apoptosis-resistance is linked to autophagy [6] . As a process of material catabolism in cells, autophagy [7] is a form of programmed cell death. Changes in autophagy level is related to the occurrence and development of human tumor, this affects tumor cell apoptosis, angiogenesis and anti-tumor treatment [8] .
Beclin1 is an autophagic activator. It has been reported that Beclin1 interacts with anti-apoptotic Bcl-2 family members through a BH3 domain [9] ,which hinders Beclin 1's participation in the assembly of preautophage and the binding of Bcl-2 with apoptotic protein, resulting in the reduction of autophagy level and the promotion of apoptosis [10,11] . Furthermore, chemotherapy has been proved to induce autophagy in several cancer cells [12] . The sensitivity of tumor to chemotherapy drugs is reduced by the activation of protective autophagy phenomenon, which leads to the emergence of drug resistance in tumor cells [13] .
To sum up, we raised three questions in our research: Is there a high autophagy level in drug resistant hypopharyngeal squamous cell carcinoma?
Can the use of autophagy inhibitors and the reduction of the expression of Beclin 1 reverse the drug resistance of hypopharyngeal squamous cell carcinoma cells?
Do autophagy inhibitors work through Beclin1 signaling pathway?
In this study, we constructed a stable cisplatin-resistant FaDu cell models(Human hypopharyngeal squamous cell carcinoma cells)using cisplatin. Observed autophagic bodies and detected the expression of Beclin1 gene to determine the autophagic level. Based on the above experimental data, plasmids transfected (siRNA) method was used to construct Beclin1 knocked down group, and 3-MA was used to intervene cisplatin-resistant cell lines to construct inhibitor group. Cell cycle, apoptosis and cell survival rate were analyzed to determine the effect of intervention on cisplatin-resistant cells. We further detected the expressions of various autophagic proteins to clarify the internal mechanism. Taken together, our research indicates that inhibition of autophagy in drug resistance of hypopharyngeal cancer might be a crucial means to reverse chemotherapy insensitivity, Beclin 1 plays an important role in the induction of autophagy in cisplatin-resistant cells and can be a therapeutic target that cannot be ignored.

Methods
Construction of drug resistance FaDu cell model FaDu cell line from human hypopharyngeal squamous cell carcinoma was obtained from Shanghai Branch of Chinese Academy of Sciences. Cells were cultured in DMEM medium composed of 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in 37℃ and 5% CO 2 incubator.
FaDu cell line of cisplatin resistant hypopharyngeal squamous cell carcinoma was induced and cultured by gradually increasing the concentration and intermittent action [14] . The initial cisplatin intervention concentration was 0.1 µg mL,the concentration was doubled after every two weeks in a gradient of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 µg mL concentration. FaDu cell line of hypopharyngeal squamous cell carcinoma resistant to cisplatin was obtained at the intervention concentration of 1.0 µg mL. Drug Resistance Validation CCK-8 kit was used to determine cell drug resistance condition. FaDu cells in in logarithmic growth phase were prepared into cell suspension with a density of 3 × 104/ml, and blank control group was established.
Cells in logarithmic growth phase were prepared into cell suspension with a density of 3 × 104/ml and blank control group was established. After overnight culture at 37℃, conventional FaDu cell lines and cisplatin-resistant groups were treated with 0/2.5/5/10 ummol cisplatin, respectively. 450 nm wavelength absorbance was measured and recorded by a microplate reader at 0 h/24 h/48 h/72 h timepoint.

Observation Of Autophagosomes By Mdc Staining
Cells from each group were seeded on 24-well plates with a density of 3 × 10 4 /ml. After 1-day culturing, the autophagic vacuoles were subjected to MDC staining and observed under electron microscopy.
Autophagic vesicles that emitted green uorescence were noted.

Cell Cycle Detection
Cell cycle was detected by ow cytometry. Cells were digested with trypsin, centrifuged(1000 RPM for 15 minutes), resuspended. Thereafter, xation was done with absolute ethanol, stained with PI (Propidium Iodide) after careful removal of RNA with RNase A solution. Red uorescence was observed at 488 nm excitation wavelength by ow cytometry within 24 hours and light scattering was detected. Cell cycle analysis was performed using FLOWJO software.

Cell Apoptosis
Apoptosis was also detected using ow cytometry method. Cells were seeded in 6-well plates, cells were seeded in 6-well plates and collected after 24 h grouping treatment. After PBS washing, the cells were digested with trypsin and counted. 50,000-100,000 resuspended cells were taken for centrifuged, discarded the supernatant then resuspended with 195 L Annexin V-FITC binding solution, 5 L Annexin V-FITC was added, incubated at 4º C in dark for 15 min. PI staining was performed and tube without AnnexinV-FITC and PI was used as a negative control. Annexin V-FITC was observed as green uorescence and propidium iodide (PI) was red uorescence.

Cell Survival
Cell survival was monitored using CCK-8 kit. Grouped cells were grown in 96-well plates and cell survival was measured after 24 hours following the manufacturer's protocol. CCK-8 and serum-free medium were mixed at a volume ratio of 1:10 and incubated in a 5% CO 2 at 37 ºC for 1 hour. Absorbance at 450 nm wavelength (OD value) was measured by a microplate reader.

Western Blotting Assay
Total proteins were extracted from FaDu cells using One Step Animal Tissue/Cell Active Protein Extraction buffer (RIPA; Thermo Fisher Scienti c). The cells were seeded in 6-well plates and incubated overnight in 37℃ and 5% CO2 incubator, thereafter, siRNAmethod was used to establish the knockdown group. All groups were starved for 48 hours in FBS-free medium to achieve synchronization. Cells cultured in drug-free medium represented the control group. Samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a Bio-Rad Electrophoresis System. The proteins were transferred to a nitrocellulose membrane gel electrophoresis separation. After blocking with 5% skim milk for 1 hour, Tris-buffered saline with Tween 20 (TBST) was used to wash the membranes.
Overnight incubation of the membranes with a 1:1000 dilution of rabbit primary antibodies: Beclin1, lc3i, lc3ii, atg-5, p62 and GAPDH (Abcam) was done at 4 °C. This followed another incubation with a 1:10000 dilution of Sheep anti-rabbit HRP labeled secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 hour. A Bio-Rad ChemiDoc MP was used to expose the membranes, and analysis was performed using Image Lab software (Bio-Rad).

Statistical analysis
All experiments were repeated in triplicate and the results were presented as the mean ± standard deviation (SD). All statistical analyses were performed using SPSS version 21.0 (IBM, Armonk, NY, USA) and GraphPad Prism 7 (La Jolla, CA, USA). Statistically signi cant differences were determined at P < 0.05.

Active Autophagy In Drug Resistant Fadu Cells
To determine whether the level of autophagy is increased in cisplatin-resistant cells, we detected the expression of autophagy-speci c factor Beclin 1 using Western-blot method. Beclin1 expression was signi cantly higher in cisplatin resistant FaDu cells as compared to conventional FaDu cells. This implied that high autophagic activity existed in cisplatin resistant FaDu cells. Further, Autophagic vesicles between the two groups was observed aby MDC uorescence staining. The number of autophagic vesicles in cisplatin-resistant cells increased signi cantly and the uorescence staining was more obvious.
Effect of 3-MA and Beclin 1 siRNA transfection on autophagy In this experiment, autophagy inhibitor 3-Ma was used for intervention and Beclin 1 siRNA gene transfection was used to knock down Beclin1 expression with the aim of inhibiting autophagy. To verify a successful Beclin1 knocked down, expression of Beclin1 mRNA in cells was detected by RT-PCR, whereas expression level of Beclin1 protein was detected by Western blot. Acquired data con rmed that the knockdown e ciency of Beclin1 was about 84%.
To verify the speci c effects of these two interventions on autophagy, MDC staining was used to compare autophagy in each group. The autophagic vacuoles stained by uorescence were more signi cant in the control group compared to 3-MA group and the Beclin1-siRNA group. Fewer autophagic vacuoles were observed in the Beclin1-siRNA group than in the 3-MA group. These results indicate that 3-MA intervention and siRNA gene transfection effectively inhibited the high autophagy level of cisplatinresistant FaDu cells.

Inhibition of autophagy decreases survival of cisplatinresistant cells
The CCK-8 assay was used to detect cell survival after intervention. Compared with each group at 24 h, the survival rate of cisplatin-resistant cells in Beclin1-siRNA group and 3-MA group decreased signi cantly compared with the control group, and the survival rate of Beclin1-siRNA group was even lower compared with the 3-MA group. These results indicate that inhibition of autophagy can effectively reduce cell resistance and increase the mortality of tumor cells.

Inhibition of autophagy causes cell cycle arrest and increased apoptosis
To explore the mechanism of increased cell death in drug resistant FaDu cells, we adopted ow cytometry to study cell cycle changes and apoptosis in each group of cells. After 24 hours of intervention, the proportion of Beclin1-siRNA group and 3-MA group in G0/G1 phase increased while the number of cells in S phase and G2/M phase decreased compared with control group. This was more pronounced in the Beclin1-siRNA group. These data indicated that autophagy inhibition caused G0/G1 arrest of cell cycle.
To further determine apoptosis condition, the early and late stage apoptosis of cells were increased after the intervention of Beclin1-siRNA and 3-MA, which was more signi cant in the Beclin1-siRNA group. Similar results were obtained using ow cytometric analysis of necrotic cells. The above data con rmed that inhibiting autophagy increased the sensitivity of drug resistant FaDu cells, by arresting the cell cycle, promoting cell apoptosis and necrosis.

3-ma Inhibits Autophagy Partly Through Beclin1
Western blot method was used to investigate the protein expression of each group to help elucidate the autophagy of cisplatin-resistant group, study the intrinsic working mechanism of beclin1and further explore the drug mechanism of 3-MA.Results showed that the expression of LC3-1, LC3-2, ATG12-ATG5 decreased signi cantly, however in the expression increased in P62 in the absence of Beclin1 when compared with the control group. This phenomenon may be caused by the accumulation of P62 as a result of blocking the initial stage and oe of autophagy. By using 3-MA, protein expression of Beclin1 and above was also signi cantly inhibited. Western blot results indicate that Beclin1 might be a key factor in regulating autophagy in drug resistant FaDu cells, 3-MA could inhibit Beclin1 expression and play a role in inhibiting autophagy.

Discussion
HSCC accounts for 95% of the pathological classi cation of hypopharyngeal cancer. It is the sixth most common malignant tumor worldwide [15] . HSCC lesions occur in in concealed areas for example, piriform fossa and posterior sections of pharyngeal wall. In addition, the rich lymphatic network is a predisposing factor to the occurrence of lymphatic metastasis in its early stage. Poor prognosis has been observed in most patients diagnosed in the middle and late stage at the time of initial diagnosis. [16] . At present, hypopharyngectomy, radiotherapy and chemotherapy are the main clinical treatment methods, among which, chemotherapy plays a pivotal role in delaying the recurrence of tumors and prolonging the survival of patients [17] .
Among various HSCC chemotherapy regimens including induction chemotherapy and combined chemotherapy, chemoresistance is regarded as the most critical factor that causes unsuccessful treatment. Current studies suggest that series of factors for example, apoptosis, cell cycle arrest, drug transport, DNA damage and autophagy promotes intrinsic and acquired resistance [18] of HSCC. Tumor resistance to chemotherapeutics, like tumorigenesis, is a multifactorial, multistep process. Among these factors, autophagy is considered by most researchers to play a key role in regulating drug resistance in multiple tumors [10,19,20] . So, is there a link between autophagy and HSCC resistance as well?
In order to verify this interpretation, We constructed a stable cisplatin-resistant FaDu cell model,inhibiting autophagy by reducing the expression of autophagy factor Beclin1 and using inhibitor 3-MA. In this way, we found that the level of autophagy was signi cantly increased in drug resistant FaDu cells, inhibition of autophagy led to G0/G1 phase arrest, promoted cell apoptosis.
Autophagy is an evolutionarily conserved catabolic process which is widespread within eukaryotic cells [21] . The role of autophagy is very complex [21] , In the study of lymphocytic leukemia [22,23] and multiple myeloma [24,25] , arsenic trioxide has been adopted to up-regulate the expression of Beclin1, a key initiating molecule of autophagy, which induce autophagic cell death (type II programmed death), thereby alleviating the disease [26] . However, more studies have shown that the increased autophagy under this condition is regarded as a protective mechanism of tumor cells against foreign interventions, thus, resulting in drug resistance [27] .
A report by Yorimitsu [28] et al. revealed that the intrinsic mechanism is endoplasmic reticulum stress induced by chemotherapy can lead to increased autophagy, so that the misfolded protein accumulated in the endoplasmic reticulum cavity can be removed. Furthermore, interfering with the expression of autophagy-related genes accelerates the death of chemo-resistant tumor cells [29] . By using some autophagy inhibitors such as chloroquine [30] 3-methyladenine [31] will effectively induces apoptosis in cisplatin-resistant tumor cells. To sum up, in anti-tumor therapy, if we can inhibit the high level of autophagy caused by chemotherapy in some way,initiate apoptosis-related signaling pathways, we can improve the treatment e ciency by promoting the death and chemo sensitization of cisplatin-resistant tumor cells.
Consistent with the above conclusions, our experiments showed that knocking down Beclin1 and using 3-MA to inhibit autophagy effectively lead to cell cycle G0/G1 arrest, promoted cell apoptosis of all phase in cisplatin-resistant FaDu cells. Demonstrated that increased autophagy in HSCC leads to the occurrence of cisplatin resistance, inhibition of autophagy and its initiating factor Beclin1 effectively promote the death of cisplatin-resistant tumor cells, render them more sensitive to cisplatin chemotherapy.
This research however, present several limitations. Even though apoptosis and autophagic cell death are both programmed cell death modes, great differences exist between them. Some studies suggests that during cellular stress response, several abnormal proteins are formed within the cells, this is accompanied by mitochondrial swelling and release of apoptotic factors such as cytochrome C [32] . At this stage, cells activate autophagy to remove the damaged components, so as to avoid the release of apoptosis factors into the cytoplasm. In the process, there are typical node molecules for example, Bcl-2 and JNK-1, which needs further study to establish their role. Also, in order to clarify the mechanism of 3-MA, there is need to carry out in-depth study on the role of inherent cell signaling pathway in inhibiting autophagy. Researchers, therefore, needs to further explore the mechanism of autophagy in drug resistant FaDu cells and develop targeted drugs based on autophagy genes. This will be signi cant in developing multi drug combination therapy and the improving the therapeutic potential of HSCC.

Conclusion
The present study aims at to explore whether autophagy is active in drug resistant HSCC and, more speci cally, examined whether inhibition of autophagy can reverse drug resistance and increase chemosensitivity. In order to achieve this, we constructed cisplatin resistant FaDu cells using a concentration gradient intervention method. Found that the expression of Beclin1 and the number of autophagy vesicles increased signi cantly in the resistant FaDu cells, which veri ed the previous hypothesis. Furthermore, we knocked down Beclin1 and adopted 3-MA, an autophagy inhibitor, to act on cisplatin-resistant FaDu cells. Results showed that after autophagy inhibition, the survival rate of cisplatin-resistant cells decreased, cell cycle G0 / G1 phase arrested and apoptosis rate increased.
Observed signi cant reduction in the number of autophagy vesicles con rms the decline in autophagy levels. These data indicated that inhibition of autophagy can lead to increasement of cell death rate of cisplatin-resistant cells at the same concentration of cisplatin, which means chemotherapy sensitivity was recovered. Furthermore, by analyzing the expression of downstream factors lc3-1, lc3-2, atg12-atg5 and P62, results suggest that Beclin 1 plays an important role in the development of autophagy in cisplatin resistant FaDu cells. 3-MA, as an autophagy inhibitor, may achieve its function partially by inhibiting Beclin 1. Taken together, we suggest that in cisplatin induced chemotherapy resistance, autophagy inhibit is a very effective method to regain chemosensitivity. It should be used combined with chemotherapy drugs for increasing the treatment e cacy. Beclin1 might be a potential lead research target that regulates autophagy and chemotherapy resistance.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.   Autophagy inhibition decreases cell viability by promoting apoptosis and necrosis. A A-1: Analysis of cell cycle by ow cytometry. A-2: Statistical analysis of cell cycle. Using 3-mA and Beclin 1 knockdown to inhibit autophagy resulted in G0 / G1 phase arrest and the proportion increased signi cantly. +, p < 0.05, ++, p < 0.01, compared with control group, *, p < 0.05, **, p < 0.01, compared with cisplatin-resistance group. B B-1: Analysis of cell apoptosis and necrosis by ow cytometry. A-2: Statistical analysis of cell apoptosis. Using 3-mA and Beclin 1 knockdown to inhibit autophagy resulted in both early and late stage apoptosis increased. +, p < 0.05, ++, p < 0.01, compared with control group, *, p < 0.05, **, p < 0.01, compared with cisplatin-resistance group. C: Statistical analysis of living cell percentage. Consist with apoptotic rates, autophagy inhibition decreases living cell percentages. D: Compartment of cell survival rate of each group. Autophagy inhibition decreases cell survival. Beclin1 Knockdown group showed signi cant lower survival rate. *, p < 0.05, **, p < 0.01, compared with cisplatin-resistance group. E Analysis of IC50 showed that after intervention, cisplatin resistance was reversed, this tendency is more obvious in Beclin 1 knockdown group. *, p < 0.05, **, p < 0.01, compared with cisplatin-resistance group. Data represents the results of three independent experiments, ± SE. p < 0.05 was considered signi cant.

Figure 4
The number of autophagic vesicles decreased after using 3-MA and Beclin 1 knockdown. A: Autophagic vesicles in each group stained with MDC. Using 3-MA and Beclin 1 knockdown signi cantly reduced the number of autophagic vesicles increased due to cisplatin resistance. B: Statistical analysis of autophagic vesicles number. p < 0.05, ++, p < 0.01, compared with control group, *, p < 0.05, **, p < 0.01, compared with cisplatin-resistance group. Data represents the results of three independent experiments, ± SE. p < 0.05 was considered signi cant.

Figure 5
A Western blot analysis of protein expression of autophagy related factors. GAPDH was served as the internal reference. B: Statistical analysis and comparison of protein expression in each group. Beclin1, lc3i, lc3ii, atg-5 expression analysis showed that 3-MA and Beclin 1 knockdown e ciently decreased protein expression induced by cisplatin resistance. C: While the expression of P62 increased compared to control group. p < 0.05, ++, p < 0.01, compared with control group, *, p < 0.05, **, p < 0.