Effect of Inhibition of GLUT1 Expression and Autophagy Modulation on the Growth and Migration of Laryngeal Carcinoma Stem Cells Under Hypoxic and Low-Glucose Conditions

doi:10.21203/rs.3.rs-38920/v1

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Research article

Effect of Inhibition of GLUT1 Expression and Autophagy Modulation on the Growth and Migration of Laryngeal Carcinoma Stem Cells Under Hypoxic and Low-Glucose Conditions

https://doi.org/10.21203/rs.3.rs-38920/v1

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posted 10 Aug, 2020

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Background: Enhanced glucose uptake and autophagy are means by which cells adapt to stressful microenvironments. We investigated the roles of glucose transporter-1 (GLUT-1) and autophagy in laryngeal carcinoma stem cells under hypoxic and low-glucose conditions.

Methods: CD133⁺ Tu212 laryngeal carcinoma stem cells were purified by magnetic-activated cell sorting and subjected to hypoxic and/or low-glucose conditions. Proliferation was evaluated using a cell-counting kit and a clone-formation assay, and migration was evaluated through a Transwell assay. Autophagy was assessed via transmission electron microscopy. GLUT-1 and beclin-1 expression were silenced using an shRNA and autophagy was manipulated using rapamycin, 3-MA, or chloroquine. Gene expression levels were evaluated by quantitative reverse transcription-polymerase chain reaction and protein concentrations were assessing via Western blotting.

Results: Compared to CD133^– stem cells, CD133⁺ cells showed increased proliferation and migration, and reduced apoptosis, under hypoxic or low-glucose conditions. They also showed increased expression of GLUT-1 and autophagy markers. Finally, GLUT-1 knockdown or autophagy inhibition reduced their proliferation and migration.

Conclusions: Enhanced glucose uptake and autophagy maintain the functions of CD133⁺ laryngeal carcinoma stem cells under hypoxic and low-glucose conditions.

Molecular Biology

General Cell Biology & Physiology

Laryngeal carcinoma

cancer stem cell

CD133+ cell

GLUT-1

autophagy

Laryngeal cancer is a common malignant tumor of the head and neck. The 5-year survival rate of laryngeal cancer has not improved in the past 40 years despite the near-continuous development of diagnostic and treatment methods [1]. The lack of understanding of the mechanisms underlying the functions of laryngeal cancer cells has hampered the development of effective therapeutic strategies.

Compared to ordinary tumor cells, cancer stem cells (CSCs) are typically resistant to apoptosis and therapeutic agents [2, 3]. CSCs are closely related to the occurrence, development, metastasis, recurrence, and chemoradiotherapy resistance of tumors [4]. Laryngeal CSCs reportedly express CD133 as a marker [5]. CD133⁺ CSCs have higher tumorigenic and invasive abilities than CD133⁻ cancer cells [5–7, 10]. However, whether CD133⁺ CSCs have specific metabolic mechanisms is unclear.

Autophagy provides energy for cells from the degradation of proteins and organelles. It can restrain the growth of tumor cells. However, it is also an adaptive response to stressful tumor microenvironments, preventing apoptosis of tumor cells [11, 12]. A basal level of autophagy may promote the survival of cancer cells [11, 12]. However, prolonged and excessive activation of autophagy may induce self-degradation and death of tumor cells [13].

CD133⁺ CSCs show greater proliferation and a lower frequency of apoptosis compared to CD133⁻ cancer cells in multiple types of tumors, particularly under hypoxic or nutrient-deprived conditions [14, 15]. Interestingly, autophagy induction reportedly promotes the conversion of non-stem pancreatic cancer cells into CD133⁺ stem-like cells under intermittent hypoxia [16]. The expression of glucose transporter-1 (GLUT-1) is reportedly associated with autophagy activation in CSCs under hypoxic or nutrient-deprived conditions [17–20]. Autophagy may also affect cellular glucose uptake [21–23]. Beclin-1, an autophagy marker, plays an important role in the initiation of autophagy [24]. It promotes the localization of other autophagy-related proteins to autophagosomes, thus promoting the formation and maturation of autophagosomes. High expression of beclin-1 is typically accompanied by enhanced autophagy, increased GLUT-1 expression, and increased glucose uptake in certain types of tumors, such as non-small-cell lung carcinoma and breast cancer [25–27]. However, in one study, beclin-1 and GLUT-1 expression were negatively correlated in 29 cases of head-and-neck squamous cell carcinoma [28], indicating that the association between autophagy and glucose metabolism may be cancer-type related. In this work, we investigated the regulation by GLUT-1 and autophagy of the functions of laryngeal carcinoma stem cells under hypoxic or low-glucose conditions, as well as the underlying mechanisms.

Cell culture and treatment

Tu212 cells were purchased from the Cell Research Institute of the Chinese Academy of Sciences (Shanghai, China). Tu212 cells were cultured in Roswell Park Memorial Institute-1640 medium (Gibco-BRL, Gaithersburg, MD), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT), 100 U/mL penicillin, and 100 g/mL streptomycin at 37 °C in an atmosphere containing 5% CO₂.

Sorting and identification of Tu212 laryngeal carcinoma cells

Magnetic sorting. Briefly, 1 × 10⁷ Tu212 cells were resuspended in phosphate-buffered saline (PBS). The resuspended cells were added to 100 µL FcR Blocking Reagent and 100 µL CD133 MicroBeads, mixed, and incubated at 4 °C for 30 min. Next, 2 mL PBS was added, and the cells were centrifuged at 300 × g for 10 min; the supernatant was discarded. Subsequently, the cell pellet was resuspended in 500 µL PBS. The magnetic separation (MS) column was clipped to the magnetic separator and 500 µL PBS was added to moisten the column. Next, the cell suspension was added to the MS sorting column. The MS separation column was washed three times with 500 µL PBS to remove unbound cells. The column was removed from the magnetic separator, 1 mL PBS was added, and the cells were expelled from the column using a push rod. After centrifugation at 300 × g for 10 min, the supernatant was discarded, and the cells were resuspended in 1 mL PBS and enumerated.

Determination of the purity of Tu212 CD133 ⁺ cells. Cells (2 × 10⁵) were removed before and after separation, centrifuged, and the supernatant was discarded. Next, 80 µL PBS and 10 µL anti-human CD133-PE were added. The sample was gently mixed using a micropipette and incubated at 4 °C for 10 min. The cells were centrifuged at 1000 rpm for 5 min and the supernatant was discarded. Precooled PBS (1 mL) was added, and unbound excess antibody components were removed by two centrifugation and washing steps. After adding 4% paraformaldehyde and incubation at 4 °C for 20 min, the supernatant was centrifuged. The cells were transferred to a flow tube and stored at 4 °C protected from light. Flow cytometry was performed using the standard procedure (Beckman, Fullerton, CA).

Experimental groups

Relationship between the proliferation and migration of Tu212 CD133 + cells and the levels of GLUT-1 and autophagy under hypoxia and low-glucose conditions. Eight groups were used in this experiment: CD133⁺ (20% O₂, 25 mM Glu); CD133⁻ (20% O₂, 25 mM Glu); CD133⁺+hypoxia (1% O₂, 25 mM Glu); CD133⁻+hypoxia (1% O₂, 25 mM Glu); CD133⁺+low Glu (20% O₂, 2.5 mM Glu); CD133⁻+low Glu (20% O₂, 2.5 mM Glu); CD133⁺+hypoxia + low Glu (1% O₂, 2.5 mM Glu); and CD133⁻+hypoxia + low Glu (1% O₂, 2.5 mM Glu).

Cell growth, migration, and levels of GLUT-1 and autophagy-related proteins. Fourteen groups were used in this experiment: CD133⁺+hypoxia + low Glu (1% O₂, 2.5 mM Glu); CD133⁻+hypoxia + low Glu (1% O₂, 2.5 mM Glu); CD133⁺+hypoxia + low Glu (1% O₂, 2.5 mM Glu) + NC shRNA; CD133⁻+hypoxia + low Glu (1% O₂, 2.5 mM Glu + NC shRNA); CD133⁺+hypoxia + low Glu (1% O₂, 2.5 mM Glu + GLUT-1 shRNA); CD133⁻+ hypoxia + low Glu (1% O₂, 2.5 mM Glu) + GLUT-1 shRNA); CD133⁺+hypoxia + low Glu (1% O₂, 2.5 mM Glu + beclin-1 shRNA); CD133⁻+hypoxia + low Glu (1% O₂, 2.5 mM Glu + beclin-1 shRNA); CD133⁺+hypoxia + low Glu (1% O₂, 2.5 mM Glu + 3-MA); CD133⁻+hypoxia + low Glu (1% O₂, 2.5 mM Glu + 3-MA); CD133⁺+hypoxia + low Glu (1% O₂, 2.5 mM Glu + CQ); CD133⁻+hypoxia + low Glu (1% O₂, 2.5 mM Glu + CQ); CD133⁺+hypoxia + low Glu (1% O₂, 2.5 mM Glu) + rapamycin); and CD133⁻+hypoxia + low Glu (1% O₂, 2.5 mM Glu) + rapamycin).

Clonogenic assay

The cell suspension was dispersed; the percentage of individual cells was greater than 95%. Next, cells were counted, and the cell density was adjusted to 250/mL by adding culture medium. The cell suspension was added to the wells of a six-well plate (2 mL per well), and the plate was gently shaken. The plate was placed in an incubator for 2 to 3 weeks, and the medium was replaced every 3 days. The culture was terminated when clones became visible. The medium was discarded, and the cells were gently washed twice with PBS, stained with 1% crystal violet at room temperature for 1 h, and photographed.

Cell-Counting Kit-8 assay

Cells were incubated at 37 °C in an atmosphere containing 5% CO₂ for 48 h. Next, 20 µL Cell Counting Kit-8 (CCK-8) solution was added, and the cells were incubated in the dark for 1 h. The absorption at 450 nm of the suspension was measured using a Spectra Plus Microplate Reader (Molecular Devices, Sunnyvale, CA).

Flow cytometry

Briefly, 10 × Binding Buffer was diluted 1:10 with deionized water. Cells were collected by centrifugation for 5 min, exposed to reagents, digested, and resuspended in 500 µL Annexin V binding buffer. Next, 5 µL fluorescein isothiocyanate and 10 µL propidium iodide (Sigma Aldrich Co., St. Louis, MO) were added for 10 min in darkness at RT. Finally, the proportions of non-apoptotic and apoptotic cells were determined in triplicate by flow cytometry with ModFit LT software (Becton Dickinson, Mountain View, CA)

Transwell assay

Cells were digested with trypsin and the culture medium was discarded. Next, the cells were washed once or twice with PBS and resuspended in serum-free medium (containing 0.2% bovine serum albumin) to a density of 1 × 10⁶/mL. Cell suspension (200 µL) was added to the upper Transwell chamber and 600 µL FBS was added to the lower chamber. The cells were incubated in a 5% CO₂ atmosphere at 37 °C for 24 h. The Transwell chamber was removed, and the culture medium was discarded. Then the chamber was washed twice with calcium-free PBS, fixed in formaldehyde for 30 min, air-dried, and the cells were stained with 0.1% crystal violet for 20 min. Finally, the upper layer of unmigrated cells was gently removed using cotton swabs and washed three times with PBS.

Quantitative real-time polymerase chain reaction

The cells were collected, washed three times with precooled PBS, centrifuged at 1500 rpm for 3 min, and lysed on ice in the presence of TRIzol. Total RNA was extracted from the cells according to the manufacturer’s instructions. Briefly, 1 µg RNA was reverse-transcribed using a First-Strand cDNA Synthesis Kit (K1622; Fermentas, Burlington, ON, Canada) and amplified by PCR using a SYBR Green qPCR Kit (Merck, Darmstadt, Germany). The PCR program was 37 °C for 60 min, 85 °C for 5 min, and 4 °C for 5 min. The amplification products were stored at − 20 °C. The primers for GLUT-1, Beclin-1, Atg7, Atg5, and LC3 were designed and synthesized by Sangon Biotech (Table 1). The 2^ΔΔCt method was used to calculate relative gene expression levels.

Table 1

The primers for GLUT-1, Beclin-1, Atg7, Atg5, and LC3
	forward	reverse
GLUT-1	5′-GTCAACACGGCCTTCACTG-3′	5′GGTCATGAGTATGGCACAACC-3′
Beclin-1	5′-CCATGCAGGTGAGCTTCGT-3′	5′-GAATCTGCGAGAGACACCATC-3′
Atg7	5′-CTGCCAGCTCGCTTAACATTG-3′	5′-CTTGTTGAGGAGTACAGGGTTTT-3′
Atg5	5′-TCAGCCACTGCAGAGGTGTTT-3′	5′-GGCTGCAGATGGACAGTTGCA-3′
LC3	5′-CATGAGCGAGTTGGTCAAGAT-3′	5′-TCGTCTTTCTCCTGCTCGTAG-3
GAPDH	5′-GAGCCCGCAGCCTCCCGCTT-3′	5′-CCCGCGGCCATCACGCCACAG-3′

Western blotting

Total proteins were extracted from cells and tumor tissues in radioimmunoprecipitation assay buffer. The cells were collected, washed three times with precooled PBS, and centrifuged at 1500 rpm for 3 min. An appropriate volume of cell lysate was added, and the cells were left on ice for 30 min. The supernatant was centrifuged at 1200 rpm at 4 °C for 30 min and stored at − 80 °C. After assaying the protein concentration, samples were added to 4⋅ sodium dodecyl sulfate loading buffer, boiled for 5 to 10 min, and centrifuged at 12,000 ⋅ g for 1 min. Proteins (30 µg) were subjected to SDS-polyacrylamide gel electrophoresis (SDSPAGE) and transferred to a polyvinylidene difluoride membrane (Millipore). Primary antibodies against GLUT-1 (Abcam), beclin-1, LC3 (Proteintech), Atg7, Atg5, and β-actin (Abcam) were added and incubated at 4 °C overnight; β-actin served as the internal control. After washing three times with Tris-buffered saline/Tween 20, the secondary antibodies were added for 1 h at room temperature. The signal was developed using an enhanced chemiluminescence assay kit (Beyotime Biotech) and analyzed semi-quantitatively using the ChemiDoc XRS + System (Bio-Rad).

Transmission electron microscopy

Cells were collected, washed in PBS, fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, and dehydrated in ethanol and acetone. After embedding in epoxy resin, sections were cut and stained with uranyl acetate and lead citrate. Autophagy was visualized by transmission electron microscopy (TEM; Thermo Fisher Scientific, Waltham, MA).

The proliferation, migration and apoptosis of CD133 ⁺ laryngeal carcinoma stem cells under oxygen- or glucose-deprived conditions

To investigate the role of GLUT-1 in LCSCs, we first sorted CD133⁺ cells from laryngeal carcinoma Tu212 cells by MACS. Cell purity was examined by flow cytometry. The results showed that the proportion of CD133⁺ Tu212 cells was only ~ 8% before sorting, and reached more than 90% after sorting. Moreover, the isolated Tu212 CD133⁺ cells showed good viability as demonstrated by SSC/FSC plots (Fig. 1a).

Next, we investigated whether the growth, proliferation, apoptosis, and migration of laryngeal carcinoma Tu212 CD133⁺ cells under hypoxia and low glucose were significantly higher than those in CD133⁻cells or CD133⁺ under normal condition. Clonal formation test showed that the clonal numbers of Tu212 CD133⁺cells were significantly higher than that of Tu212 CD133⁻cells under hypoxia (1%O₂), low glucose (2.5 mM glucose) and hypoxia + low glucose conditions. However, the clonal-forming ability was similar between Tu212 CD133⁺cells and Tu212 CD133⁻cells under normal culture condition (Fig. 1b). CCK-8 assay also revealed that compared with CD133⁻ cells, the proliferation of Tu212 CD133⁺cells was significantly increased under hypoxia, low glucose and hypoxia + low glucose conditions (Fig. 1c). In addition, flow cytometry showed that the apoptotic rate of Tu212 CD133⁺ cells was significantly decreased compared to Tu212 CD133⁻ cells under hypoxia, low glucose and hypoxia + low glucose conditions (Fig. 1d). Transwell assay demonstrated that the migratory ability of Tu212 CD133⁺ cells was also higher than that of Tu212 CD133⁻ cells under the above three stress conditions (Fig. 1e). In contrast, the proliferation and apoptosis were comparable between Tu212 CD133⁺ cells and Tu212 CD133⁻cells under normal culture condition, with CD133⁺ cells showing only a marginal increase in the migratory ability. Hence, CD133⁺ laryngeal carcinoma stem cells showed increased proliferative and migratory capacity than CD133⁻ cells under stressful conditions.

The expression of GLUT-1 and cell autophagy in CD133 + laryngeal carcinoma stem cells under oxygen- or glucose-deprived conditions

Previous studies showed that GLUT-1 overexpression promoted cancer cell proliferation, invasion and metastasis, especially in hypoxia and starvation conditions [6, 7]. Besides, activated autophagy promoted the functions of CD133⁺ cells under hypoxia [15, 16], probably by enhancing cell glucose intake [21–23]. Thus, we explored whether the increased proliferation and migration of laryngeal carcinoma CD133⁺ Tu212 cells were associated with high level of GLUT-1 expression and cell autophagy. To this end, we first investigated the expression of GLUT-1 and autophagy in Tu212 cells under hypoxia and low glucose conditions. qRT-PCR results showed that the expression of GLUT-1 mRNA in Tu212 CD133⁺ cells was significantly higher than that in Tu212 CD133⁻ cells under normal, hypoxia, low glucose, and hypoxia + low glucose conditions. The expression of GLUT-1 mRNA of Tu212 CD133⁺ cells under hypoxia, low glucose, and hypoxia and low glucose was higher than that of Tu212 CD133⁺cells under normal culture condition (Fig. 2a). Western blotting also showed that the protein level of GLUT-1 in Tu212 CD133⁺ was significantly higher than that in Tu212 CD133⁻ cells under the above four conditions (Fig. 2b, S1).

In terms of cell autophagy, we found that hypoxia or low glucose increased the expression of Beclin-1, Atg7, Atg5, and LC3 in Tu212 CD133⁺ cells, but to a lesser extent in Tu212 CD133⁻ cells. Compared with Tu212 CD133⁻ cells, the levels of these autophagy markers were significantly higher in Tu212 CD133⁺ cells under hypoxia, low glucose, and hypoxia + low glucose conditions (Fig. 2a, b, S1). Transmission Electron Microscopy (TEM) imaging also showed that the number of autophagosomes in Tu212 CD133⁺ cells was significantly higher compared with that in Tu212 CD133⁻ cells under the above three conditions, indicating the enhanced autophagy in stressed-laryngeal carcinoma stem cells (Fig. 2c). Thus, the increased survival and migration of laryngeal carcinoma stem cells under hypoxia and low glucose maybe associated with high GLUT-1 expression and cell autophagy.

The association between the expression of GLUT-1 and autophagy markers in CD133 + laryngeal carcinoma stem cells

To further study the association between the levels of GLUT-1 and autophagy marker genes. We adopted the following strategies: (1) silenced GLUT-1 expression by shRNA. (2) silenced Beclin-1 expression by shRNA. (3) inhibited autophagy by 3-MA and chloroquine. (4) activate autophagy by RAPA in Tu212 CD133⁺ cells, then cell functions were evaluated. qRT-PCR and Western blotting results showed that the levels of GLUT-1, Beclin-1, Atg7, Atg5, and LC3II/LC3I ratio in Tu212 CD133⁺ cells were higher than that in Tu212 CD133⁻ cells under hypoxia + low glucose condition. After transfection with GLUT-1 shRNA, the levels of Beclin-1, Atg7, Atg5 mRNA and LC3II/LC3I ratio in Tu212 CD133⁺ cells were significantly descended. After transfection with Beclin-1 shRNA, the LC3II/LC3I ratio in Tu212 CD133⁺ cells were significantly descended, whereas the expression of GLUT-1, ATG7 and ATG5 did not obviously changed upon Beclin-1 silencing. In terms of autophagy inhibitors, 3-MA treatment significantly decreased the levels of Beclin-1, Atg7, Atg5 and LC3II/LC3I ratio in Tu212 CD133⁺ cells, whereas GLUT-1 expression did not markedly change. In contrast, CQ treatment failed to obviously change the expression of GLUT-1, Beclin-1, Atg7, Atg5 and, LC3II/LC3I ratio in Tu212 CD133⁺. On the other hand, RAPA treatment significantly increased the expression of Beclin-1 and LC3II/LC3I ratio in Tu212 CD133⁺ cells (Fig. 3a, b, S2). The above results suggested that the expression of GLUT-1 and autophagy markers is closely associated in stressed laryngeal carcinoma stem cells. In addition, TEM imaging showed that silencing of GLUT-1 and Beclin-1, or inhibiton of autophagy significantly decreased the number of autophagosomes in Tu212 CD133 + cells under hypoxia + low glucose condition. In contrast, activation of autophagy by RAPA treatment significantly enhanced the number of autophagosomes in Tu212 CD133⁺ cells (Fig. 3c).

GLUT-1 knockdown or autophagy inhibition reduced the proliferation and migration of CD133 + laryngeal carcinoma stem cells

In the functional analysis, we found that silencing of GLUT-1 markedly decreased the clonal-forming capacity, cell proliferation, and migration of Tu212 CD133⁺ cells under oxygen- and glucose-deprived condition (Fig. 4a-c). In contrast, the apoptotic rate of Tu212 CD133⁺ cells was significantly increased by the silencing of GLUT-1 (Fig. 4d). Similarly, Beclin-1 silencing or autophagy inhibitor (3-MA, CQ) treatment also significantly decreased the above malignant behaviors of CD133⁺ cells. Importantly, upon GLUT-1 silence or autophagy inhibition, the survival and migratory advantages of CD133⁺ cells over CD133⁻ counterparts were greatly compromised. To our surprise, autophagy activator Rapamycin also reduced the malignant behaviors of CD133⁺ cells, suggesting that the excessive autophagy may be harmful for stem cells as well (Fig. 4a-d). Taken together, the enhanced glucose uptake and autophagy are responsible for maintaining the growth and migration of the stressed laryngeal carcinoma stem cells.

Glucose is the main energy source for tumor cells and GLUT-1 is a key transporter of extracellular glucose [6, 7, 29]. GLUT-1 is overexpressed in many cancers, including laryngeal carcinoma, and is associated with their metastasis, drug resistance, and prognosis [6, 7, 30–36]. We investigated the role of GLUT-1 in Tu212 CD133⁺ laryngeal carcinoma cells under hypoxic and low-glucose conditions.

A stressful extracellular microenvironment, such as hypoxia or low glucose, may upregulate GLUT-1 expression [37]. Indeed, hypoxia promotes the proliferation, migration, and chemoresistance of CSCs [4–7, 14, 15]. In this study, the growth and migration of CD133⁺ Tu212 cells were greater than those of CD133⁻ Tu212 cells under hypoxic and low-glucose conditions; this was associated with increased mRNA and protein levels of GLUT-1 in CD133⁺ Tu212 cells. These findings are consistent with previous reports that high GLUT-1 expression facilitates glucose uptake to meet the energy demand of cancer cells. This adaptive response enables cancer cells to overcome external stresses, such as hypoxia or nutrient deprivation [6, 38], thereby suppressing apoptosis.

The levels of autophagy markers in Tu212 CD133⁺ cells were increased by hypoxia and low glucose, whereas GLUT-1 silencing reduced the levels of these proteins. These results suggest mutual regulation of glucose uptake and autophagy in stressed laryngeal carcinoma stem cells. Autophagy modulates various metabolic pathways, including that centered on glucose [23]. Hypoxia and glucose deprivation may induce autophagy, promoting glucose uptake by upregulating GLUT-1 expression to increase the glycolytic flux and maintain nutrient uptake under stress conditions [21–23, 40, 41]. In airway progenitor cells, a lack of GLUT-1 impacts its recycling but not its expression, facilitating glucose uptake [40]. In mouse embryonic fibroblasts, however, autophagy enhances glucose uptake by increasing GLUT-1 expression and promoting GLUT-1 trafficking [21]. In this study, silencing of GLUT-1 decreased the levels of the autophagy markers beclin-1, Atg7, and Atg5, as well as the LC3II/LC3I ratio. However, inhibition or activation of autophagy by the beclin-1 shRNA/3-MA/CQ or rapamycin did not affect GLUT-1 expression. By contrast, rapamycin-induced autophagy activation increased the frequency of apoptosis of laryngeal CSCs, consistent with reports that excessive autophagy induces cell death.

This study had some limitations. First, we did not assay glucose metabolism, instead using GLUT-1 as a surrogate for glucose uptake. Second, we did not explore the in vivo implications of our findings using animal models. Third, the signaling mechanisms responsible for the enhanced GLUT-1 expression and autophagy in laryngeal carcinoma stem cells under hypoxic and low-glucose conditions warrant further investigation.

In summary, hypoxia and low glucose increased the growth and migration of CD133⁺ laryngeal carcinoma stem cells by enhancing the expression of GLUT-1 and activating autophagy.

3-MA: 3-Methyladenine; GLUT-1: glucose transporter-1; CSCs: cancer stem cells; HNSCC: head and neck squamous cell carcinoma; ATG: autophagy-related proteins

Ethics approval and consent to participate

This study was conducted under the guidelines and with the approval of Second Hospital of Jiaxin City, Jiaxin City, Zhejiang Province, 314000, China(No.jxey-201003).

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by Basic public welfare research plan of Zhejiang Province, China (No. LGF18H130001), Science and technology plan project of Medical and health of Zhejiang province, China (No. 2018KY801), and National Natural Science Foundation of China (No. 81372903). The funding LGF18H130001 has the role in the design of the study, the collection, analysis, and interpretation of data and in writing the manuscript. The funding 2018KY801 and 81372903 has the role in design of the study.

Authors’ contributions

XH C designed the study and wrote the manuscript. JL, JT Z and SH Z reviewed the literatures and analyzed results.SH Z designed the study and revised the manuscript. J F conducted the experiments. All authors reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgemets

Not applicable.

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posted 10 Aug, 2020

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Effect of Inhibition of GLUT1 Expression and Autophagy Modulation on the Growth and Migration of Laryngeal Carcinoma Stem Cells Under Hypoxic and Low-Glucose Conditions

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Abstract

Figures

Background

Methods

Cell culture and treatment

Sorting and identification of Tu212 laryngeal carcinoma cells

Experimental groups

Clonogenic assay

Cell-Counting Kit-8 assay

Flow cytometry

Transwell assay

Quantitative real-time polymerase chain reaction

Western blotting

Transmission electron microscopy

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

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Version 1