Radiosensitizing effects of curcumin alone or combined with GLUT1 siRNA on laryngeal carcinoma cells through AMPK pathway‐induced autophagy

In this study, we investigated the ability of curcumin alone or in combination with GLUT1 siRNA to radiosensitize laryngeal carcinoma (LC) through the induction of autophagy. Protein levels in tumour tissues and LC cells were measured by immunohistochemistry and Western blotting. In vitro, cell proliferation, colony formation assays, cell death and autophagy were detected. A nude mouse xenograft model was established through the injection of Tu212 cells. We found that GLUT1 was highly expressed and negatively associated with autophagy‐related proteins in LC and that curcumin suppressed radiation‐mediated GLUT1 overexpression in Tu212 cells. Treatment with curcumin, GLUT1 siRNA, or the combination of the two promoted autophagy. Inhibition of autophagy using 6‐amino‐3‐methypourine (3‐MA) promoted apoptosis after irradiation or treatment of cells with curcumin and GLUT1 siRNA. 3‐MA inhibited curcumin and GLUT1 siRNA‐mediated non‐apoptotic programmed cell death. The combination of curcumin, GLUT1 siRNA and 3‐MA provided the strongest sensitization in vivo. We also found that autophagy induction after curcumin or GLUT1 siRNA treatment implicated in the AMP‐activated protein kinase‐mTOR‐serine/threonine‐protein kinase‐Beclin1 signalling pathway. Irradiation primarily caused apoptosis, and when combined with curcumin and GLUT1 siRNA treatment, the increased radiosensitivity of LC occurred through the concurrent induction of apoptosis and autophagy.

Curcumin is a flavouring agent extracted from the Curcuma longa rhizome, [6][7][8][9] which has attracted a great deal of attention due to its antioxidant, anti-inflammatory, anti-tumour and antifibrosis properties. 6 Curcumin can inhibit cell growth and enhance the radiosensitivity of many cancers, including LC, possibly through the regulation of autophagy. [7][8][9] Many studies have linked autophagy inhibitors with improvement in the radiosensitivity of some solid malignant tumours. [10][11][12][13] Previous studies have shown that the expression of autophagy-associated genes (ATGs) is reduced in the tissues of laryngeal squamous cell carcinoma and is related to prognosis, lymph node metastasis and T stage. [14][15][16] Increasing evidence indicates a relationship between autophagy and the radioresistance of LC, but the detailed mechanism remains unclear.
The PI3K/Akt pathway plays a key role in autophagy regulation, and blockage of PI3K/Akt may enhance the radiosensitivity of some malignant tumours, suggesting that PI3K/Akt affects radioresistance through the regulation of autophagy. [17][18][19] The inhibition of glycolysis increases the AMP/ATP content, 20 which then activates the AMP-activated protein kinase (AMPK) pathway, which is closely associated with autophagy. [21][22][23][24][25] The relationship between AMPK and autophagy may be associated with the serine/threonine-protein kinase (ULK1) complex, a key regulator of autophagy, as a bridge between the upstream energy receptors mTOR and AMPK and downstream autophagosome formation. AMPK/mTOR signalling regulates the activation of ULK1. AMPK also inactivates mTOR, 26 along with increases in GLUT1 expression and glucose uptake in thyroid PCCL3 cells 24 and blood-brain barrier pericytes. 27 Curcumin also activates the AMPK pathway. 25 Therefore, we speculate that GLUT1 siRNA and curcumin treatment would activate the AMPK pathway and induce activation of ULK1 to enhance autophagy. However, few studies have considered this or the involvement, if any, of autophagy in the radiosensitizing process and whether the PI3K/Akt and AMPK pathways regulate autophagy in this process.
Here, we investigated the expression of GLUT1 and Beclin-1 and LC3-II in human LC tissues using immunohistochemical methods, and analysed the relationships between these markers and LC. We further investigated whether combining curcumin with GLUT1 siRNA has a synergistic effect on the radiosensitivity of LC and whether the mechanism is through autophagy modulation by the PI3K/Akt or AMPK pathway.

| Patient samples
Forty-five fresh tissues from patients with LC admitted to our hospital between May 2013 and October 2016 were collected. Twelve fresh paracarcinoma tissue samples were obtained from the negative margin (approximately 0.5 cm from the edge) of patients who underwent partial or total laryngectomy. No patients underwent chemotherapy or radiotherapy preoperatively. All fresh samples were immediately frozen in liquid nitrogen and maintained for subsequent testing.

This study was approved by the Institutional Review Board of The
First Affiliated Hospital, College of Medicine, Zhejiang University (number: 2019-566-1). Written informed consent was obtained from all patients before sample collection.

| Immunohistochemistry
Briefly, samples were fixed, embedded and cut into sections 5µm thick. After deparaffinization and hydration, sections underwent antigen retrieval. Endogenous peroxidase activity was blocked by H 2 O 2 , and slides were incubated with primary antibodies against GLUT1, Beclin1 and LC3-II diluted in working solutions (1:50) overnight. Sections were incubated with secondary antibodies labelled with streptavidin-horseradish peroxidase. Then, the slides were stained using a 3,3'-diaminobenzidine (DAB) staining kit and subjected to haematoxylin and eosin staining. The slides were photographed under a microscope (Olympus BX41; Olympus Corp., Tokyo, Japan); cells labelled with brownish-yellow granules were identified as positive cells. Five high-magnification fields were randomly selected, and 100cells were counted in each field.
Immunohistochemical expression was assessed by the total score according to the following formula: the score of the rate of positive cells + the score of dye depth. Total scores of 0-1, 2, 3-4 and 5-6 were considered negative (-), weak-positive (+), positive (++) and strong-positive (+++), respectively.

| Cell culture and treatments
Tu212 cells were purchased from the Cell Research Institute of the Chinese Academy of Sciences (Shanghai, China). Tu212 cells were maintained in RPMI-1640 (Gibco-BRL, Gaithersburg, MD, USA), supplemented with 10% heat-inactivated foetal bovine serum albumin (BSA; Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 g/mL streptomycin at 37°C in a 5%CO 2 atmosphere. After digesting using trypsin and harvesting after growing to 80%-90% confluence, the cells were seeded in six-well plates and transfected with GLUT1-siRNA (GLUT1- or a combination of these treatments for an additional 24 hours. siRNA for GLUT1 was synthesized by GenePharma (Shanghai, China). X-ray radiation was performed on a linear accelerator (Clinac 23EX, Varian Medical Systems, Orlando, USA); the plate distance was 100 cm, the radiation field area was 35 cm × 35 cm, the X-ray energy was 6 MV, and the dose rate was 500 MU/min.
All assays were carried out in triplicate.

| Clonogenic assay
Briefly, the well surface of a 6 cm plate was covered with a mixture of 1.2% agarose and Dulbecco's modified eagle's medium(DMEM) medium and was allowed to cool for solidification. After transfection with GLUT1-siRNA or control siRNA, cells were exposed to 10 Gy irradiation, curcumin treatment or the combination for 24 hours. The cell suspension was mixed with 0.7% agarose and medium and seeded in a 6 cm plate coated with 1.2% agarose. After solidification of agarose-containing cells, all plates were incubated at 37°C in a 5%CO 2 atmosphere for 10 days. Images of the colonies were acquired after crystal violet staining.

| Flow cytometry
After treatment, cells were harvested and resuspended. Next, Each experiment was performed in triplicate.

| CCK8 assay
Cells transfected with GLUT1-siRNA were subjected to 10 Gy irradiation, curcumin treatment or the combination. All cell groups were exposed to phosphate-buffered saline (PBS) or z-VAD-fmk for 48 hours. Subsequently, 10 µL cell counting solution was added. The absorption at 450 nm was measured using a Spectra Plus microplate reader (Molecular Devices Co., Sunnyvale, CA, USA). The relative cell inhibition rate (%) was determined using the following formula: 100 -(sample absorption/control mean absorption) × 100%.

| Immunofluorescence
After incubation for 48 hours, cells were washed three times with PBS and fixed with 4% paraformaldehyde. Subsequently, cells were permeabilized using 0.2% Triton X-100 and blocked using 5% BSA.

| Western blotting
Total proteins from cells and tumour tissues were extracted using radioimmunoprecipitation assay lysis buffer. Protein samples (30 μg) were subjected to sodium dodecyl sulphate-polyacrylamide gel
Approximately 0.2 mL (2 × 10 7 /mL) Tu212 cells transfected with GLUT1-siRNA or control siRNA were inoculated subcutaneously into the right flank of mice. Mice were randomly divided into 11 groups and treated with 10 Gy X-ray irradiation, curcumin, 3-MA or their combination (n = 6). The groups were as follows:Tu212, Tu212 + 10 Gy, Tu212 + 3-MA, Tu212 + 3-MA + 10 Gy,and Tu212 + 3-MA + GLUT1-siRNA + Curcumin+10 Gy. X-ray radiation was performed on a linear accelerator (Clinac 23EX, Varian Medical Systems). The source-skin distance was 100 cm, the radiation field area was 35 cm × 35 cm, the X-ray energy was 6 MV, and the dose rate was 500 MU/min. Tumour For the X-ray irradiation group, each mouse was subjected to concurrent 10 Gy X-ray irradiation after the first drug treatment. Two weeks later, mice were euthanized under general anaesthesia using pentobarbital, and the tumours were excised. After weighing, the tumour tissues were stored at −80°C until further analysis.

| Transmission electron microscopy
After 48 hours of treatment, the tumour tissues were fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, and gradually dehydrated in ethanol and acetone. After embedding into epoxy resin, tumours were cut into sections and stained with uranyl acetate and lead citrate. Autophagy levels were determined by transmission electron microscopy (Thermo Fisher Scientific, Waltham, MA, USA).

| TUNEL assay
Tumour sections were stained with the in situ Cell Death Detection Kit-POD (Roche, Shanghai, China). In brief, tissue sections were fixed using the fixation solution and incubated with the blocking buffer.
The sections were incubated for 60 minutes at 37°C and in the dark with a TUNEL reaction mixture and subsequently with 100 µL termination solution at room temperature for 10 minutes. Then, they were incubated with 50 µL streptavidin-HRP conjugate, stained with 200 µL DAB solution, mounted and observed under a fluorescence microscope.

| Statistical analyses
Data are expressed as the mean ± SEM and were analysed using the SPSS 25.0software (IBM Corp., Armonk, NY, USA). The variance was similar between the groups that are being statistically compared.
Statistical significance was determined using one-way ANOVA; P <.05 was considered statistically significant.

| GLUT1 expression is negatively associated with Beclin-1 and LC3-II in LC
Immunohistochemical analyses showed that GLUT1 expression that occurred in a diffuse pattern localized in the cell membrane was robustly enhanced in LC compared to paracarcinoma tissue ( Figure   S1A). In all, 42 of 45 LC patients (93.3%) presented with GLUT1positive expression, while 50% (6/12) were positive in paracarcinoma samples (Table S1; P = 0.0015). However, Beclin-1 and LC3-II were predominantly expressed in the cytoplasm and showed low levels in tumour tissue compared to paracarcinoma in LC ( Figure   S1B). The positive rate of Beclin-1, LC3-II expression was 71.1% (32/45), 73.3% in LCs, respectively. The positive rate of Beclin-1, LC3-II expression was 75% (9/12), 100% in paracarcinoma samples, respectively. There was a significant negative correlation between GLUT1 expression status and Beclin-1 or LC3-II ( Figure S1C). Thus, there may be a negative association between ATGs and GLUT1 status in LC.

| Combination of curcumin with GLUT1 siRNA enhances the radiosensitivity of Tu212 cells
Exposure to radiation robustly reduced clone formation This suggests that the combination of curcumin with GLUT1siRNA enhances the radiosensitivity of LC, partly through modulation of GLUT1 expression.

| Combination of curcumin and GLUT1 siRNA induces both apoptotic and non-apoptotic cell death
As shown in Figure 3 Figures S7-S9). Taken together, these findings suggest that curcumin and GLUT1siRNA may induce apoptotic and non-apoptotic cell death, whereas irradiation preferentially promotes apoptosis and has no effect on non-apoptotic cell death.

| Curcumin and GLUT1 induce autophagy of LC cells
Immunoblotting showed that the levels of Beclin-1, LC3II/I and p62 were unchanged in cells transfected with NC siRNA or exposed to irradiation ( Figure 4A; Figure S10), whereas treatment with curcumin and GLUT1 siRNA alone or in combination significantly increased Beclin-1 expression and the LC3II/I ratio, as well as reducing the level of p62 ( Figure 4A; Figure S10). However, with irradiation, GLUT1 siRNA and curcumin alone and in combination induced changes in ATGs ( Figure 4A; Figure S10). Immunofluorescence analysis also indicated that the accumulation of LC3 was increased in the absence of GLUT1 and in the presence of curcumin, and greater accumulation was observed in cells exposed to a combination of curcumin and GLUT1siRNA ( Figure 4B). When administered along with irradiation, the accumulation of LC3 was not influenced by GLUT1 siRNA or curcumin alone or in combination. These results indicate that curcumin and GLUT1 siRNA cause autophagic events independent of irradiation in LC cells.

| Non-apoptotic cell death induced by combined treatment with curcumin and GLUT1 siRNA depends on autophagy
We observed that inhibition of autophagy using 3-MA did not affect the numbers of apoptotic or non-apoptotic cells, while 3-MA enhanced irradiation cytotoxicity. 3-MA alone or in combination with irradiation had no impact on non-apoptosis, suggesting that autophagy inhibitionmediated radiosensitivity with irradiation was not associated with non-apoptotic cell death ( Figure 5A). Compared to cells exposed to the combination of curcumin and GLUT1 siRNA after irradiation, the addition of 3-MA increased the apoptotic cell population, while also suppressing non-apoptotic cell death induced by the combination of curcumin and GLUT1 siRNA after irradiation( Figure 5A). In addition, Beclin-1 expression and the LC3II/I ratio were reduced, while p62 expression was upregulated by the addition of 3-MA ( Figure 5C; Figure S10). Irradiation did not affect 3-MA-mediated changes in Beclin-1, LC3II/I ratio and p62 expression ( Figure 5B; Figure S10). Moreover, the sharp increase in autophagy due to the combination of curcumin and GLUT1siRNA after irradiation was robustly blocked by 3-MA ( Figure 5B; Figure S11). These results indicated that the non-apoptotic cell death induced by curcumin and GLUT1 siRNA is a form of autophagy with or without irradiation (ie autophagy-associated cell death).

| Curcumin and GLUT1 siRNA increase the radiosensitivity of LC by promoting autophagy in vivo
Both tumour volume and weight were remarkably reduced when mice were subjected to irradiation or the intratumoural injection of curcumin or GLUT1siRNA, compared to the control group ( Figure 6A,B). Curcumin and GLUT1siRNA alone or in combination along with irradiation further inhibited tumour growth compared to a single-drug arm ( Figure 6A,B). Although 3-MA alone did not affect tumour growth, it limited the tumour size and weight when combined with irradiation ( Figure 6A,B). Combination of 3-MA, curcumin and GLUT1siRNA after irradiation exhibited the most potent inhibitory effect on tumour growth ( Figure 6A,B). Moreover, irradiation,

| The AMPK-mTOR-ULK1 pathway is required for curcumin and GLUT1 siRNA-mediated autophagy
Next, we determined that the PI3K/Akt pathway in Tu212 cells and Autophagy signalling, including increases in Beclin-1 expression and the LC3II/I ratio and a decrease in p62 expression, was activated by curcumin and GLUT1siRNA alone or in combination compared to control cells; this occurred along with increases in phosphorylated AMPK and ULK1 ser555 and decreases in phosphorylated mTOR and ULK1 ser757 ( Figure 7C) Thus, the AMPK-mTOR-ULK1 pathway was activated after treatment with curcumin and GLUT1siRNA with activation of autophagy ( Figure 7C; Figure S13).
In contrast, irradiation alone did not alter the autophagy activity or the AMPK-mTOR-ULK1 pathway compared to the control groups transfected with or without AMPK siRNA ( Figure 7C; Figure   S13C). There were also no effects of irradiation on the activation of AMPK-mTOR-ULK1 mediated by curcumin and GLUT1siRNA and subsequent autophagy events in the presence of NC siRNA or AMPK siRNA ( Figure 7C; Figure S13C). These results indicate that autophagy was evoked by the AMPK-mTOR-ULK1 signalling pathway after curcumin and GLUT1siRNA alone or in combination.

| D ISCUSS I ON
The present study demonstrated an association between GLUT1 expression and the T stage of LC. GLUT1 expression was significantly increased after irradiation compared to control cells. These results are similar to those of our previous studies [3][4][5]28 and suggest a correlation between elevated GLUT1 expression and radioresistance of LC. 5,28,29 As a well-known antioxidant, curcumin has both radiosensitizing and radioprotective properties. We first observed that the combination of GLUT1siRNA and curcumin significantly increased the radiosensitivity of LC in vitro and in vivo, possibly by modulating GLUT1 expression.
As a type of programmed cell death, a higher proportion of apoptotic cells plays an important role in radiosensitivity. [30][31][32][33] Curcumin, GLUT1siRNA and the curcumin/GLUT1siRNA combi- As a type of non-apoptotic cell death, autophagy-associated cell death has a potent anti-cancer effect. 34,36,37 Here, 3-MA significantly reduced non-apoptotic cell death induced by curcumin/ GLUT1siRNA after irradiation. Meanwhile, it also changed the expression of ATGs. However, it did not change the apoptotic events in LCs. These findings suggest that non-apoptotic cell death induced by curcumin combined with GLUT1siRNA is dependent on autophagy.
The role of autophagy in radioresistance is paradoxical. 13,[38][39][40][41] In this study, the induction of autophagy by curcumin and GLUT1siRNA increased non-apoptotic cell death. Thus, induction of autophagy by curcumin and GLUT1siRNA may promote cytotoxicity and radiosensitivity in Tu212 cells. It was confirmed that irradiation can induce autophagy, thereby enhancing radiosensitivity. 42,43 Although Beclin-1 and LC3-II expression were low in LC, 44 we found that irradiation did not change the levels of autophagy-related proteins, but markedly increased the expression of apoptosis-related proteins and apoptosis rate. These results suggest that irradiation inhibited the proliferation of Tu212 cells through apoptotic rather than autophagy-associated cell death.
Theoretically, inhibition of autophagy induced by curcumin and GLUT1siRNA should decrease the radiosensitivity of LCs.
Interestingly, 3-MA combined with curcumin and GLUT1siRNA improved the radiosensitivity of Tu212 cells. We suggest that irradiation can increase apoptosis of Tu212 cells while inhibiting autophagy.
However, in the present study, the increase in apoptotic cell death was greater than the degree of inhibition of autophagy-associated cell death in the combined curcumin and GLUT1siRNA group after 3-MA treatment. Thus, the greatest inhibition was observed with 3-MA combined with curcumin and GLUT1siRNA after irradiation.
The possible mechanism underlying the elevation of radiosensitivity by GLUT1siRNA, curcumin and their combination may involve autophagy-associated cell death when the cytoprotective effect is excessive. Some studies have shown that low levels of autophagy may suppress radiosensitivity. 13,40,[45][46][47][48] Thus, the reasons for the differences in correlation between autophagy and radiosensitivity among studies may mainly be related to the double-edged effect of autophagy. An 'autophagic switch' has been proposed in which the term 'cytoprotective autophagy' is further distinguished as being 'cytotoxic' or 'cytostatic'. 49 The PI3K/Akt pathway plays an important role in the radioresistance of head and neck squamous cell carcinomas, possibly by modifying autophagy. 50,51 The pathway regulates GLUT1 expression, leading to the radioresistance of LC, and its inhibition (along with GLUT1 inhibition) improves the radiosensitivity of LCs. In this study, we found that curcumin, GLUT1siRNA and their combination did not affect the PI3K/Akt pathway in vitro or in vivo, implying that curcumin and GLUT1siRNA-induced autophagy are independent of this pathway. Given that the PI3K/Akt pathway is mainly responsible for cell proliferation, the addition of 3-MA may contribute to radiosensitivity by inhibiting proliferation via inactivation of the PI3K/ Akt pathway, with the exception of autophagy inhibition-mediated apoptosis induced by 3-MA.
Autophagy is a complicated process that can be activated by important role in autophagy. 23 AMPK activation also significantly up-regulates the GLUT1 content in thyroid PCCL3 cells. 24 Curcumin promotes autophagy by activating the AMPK pathway in lung adenocarcinoma cells. 25 However, the crosstalk among GLUT1, curcumin and the AMPK pathway in LC remains vague. In the present study, both GLUT1siRNA and curcumin alone or in combination increased AMPK activity, thereby inhibiting mTOR-dependent ULK1 inactivation, leading to the activation of autophagy. AMPK depletion robustly limited their effects in promoting the mTOR-ULK1-Beclin-1 pathway and autophagic events. Therefore, GLUT1siRNA and curcumin-mediated autophagy depends on the AMPK-mTOR-ULK1-Beclin-1 signalling cascade. However, irradiation had no effect on this pathway.
In conclusion, treatment with GLUT1siRNA alone or in combination with curcumin resulted in profound improvement of the radiosensitivity of LC cells after irradiation. Curcumin and GULT1siRNA alone or in combination not only promoted apoptosis of LC cells, but also induced autophagy-associated cell death through activation of AMPK/mTOR/ULK1 signalling-mediated autophagy with or without irradiation treatment ( Figure S14). This sensitization may have been due to increases in apoptotic and non-apoptotic cell death. In addition, non-apoptotic cell death is dependent on autophagy status, which is induced by activation of the AMPK-ULK1-mTOR-Beclin-1 pathway.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. Visualization (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.