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.