Panoply™ Human CD24 Over-expressing Stable Cell Line

CD24 is able to regulate angiogenesis, drug sensitivity, and progression of colorectal cancer (CRC). This study investigated the functional role of altered CD24 expression in autophagy and apoptosis in HCT116 and HT29 human CRC cells. Results showed that CD24 expression levels were low in HCT116 cells, but high in HT29 cells. Induced overexpression or knockdown of CD24 did not affect the viability or spontaneous apoptosis of HCT116 and HT29 cells, respectively. Induced overexpression of CD24 significantly reduced the relative expression levels of Beclin-1, autophagy-related (Atg)3, and Atg5, and the number of microtubule-associated protein 1 light chain 3 (LC3)-positive dots, but increased the expression of p62 in HCT116 cells. In contrast, CD24 silencing increased the expression of Beclin-1, Atg3, and Atg5, and the number of LC3-positive dots, but reduced the expression of p62 in HT29 cells. Treatment with 3-methyladenine or knockdown of Atg5 by specific small interfering RNA to attenuate autophagy significantly increased the viability of CD24-overexpressing HCT116 cells, but decreased the viability of CD24-silenced HT29 cells, relative to the control group. Thus, attenuation of autophagy significantly reduced the frequency of apoptosis in CD24-overexpressing HCT116 cells, but increased the percentage of apoptosis in CD24-silenced HT29 cells. Overexpression of CD24 promoted the activation of nuclear factor (NF)-κBp65, while CD24 silencing attenuated this activity in CRC cells. In CRC cells, inhibition of NF-κB activation enhanced the reduction in autophagy caused by CD24 overexpression, but attenuated the increase in autophagy caused by CD24 silencing. Thus, CD24 inhibited autophagy in CRC cells, and the combination of targeting CD24 and inhibiting autophagy promoted apoptosis in CRC cells.

Here, the researchers evaluated the effects of altered CD24 expression on autophagy in CRC cells. The results showed that inducing CD24 overexpression reduced the relative expression levels of Beclin-1, Atg3, Atg5, LC3A, and LC3B in HCT116 cells, while silencing CD24 increased the relative expression levels of Beclin-1, Atg3, Atg5, LC3A, and LC3B in HT29 cells (Figures 1A and B). Compared with the control group, the expression level of p62 was increased in CD24-overexpressing HCT116 cells (CD24OE), while the expression level of p62 was decreased in CD24-knockdown HT29 cells (CD24KD) from 24 to 72 hours after culture (Figure 1C). At the same time, p-EGFP-LC3B plasmid was transfected into different groups of cells to monitor the autophagy process. In CD24-overexpressing HCT116 cells, the intermittent expression of LC3 was significantly reduced, whereas in CD24-knockdown HT29 cells, the intermittent expression of LC3 was increased (Figure 1D and E). These data suggest that CD24 inhibits autophagy and autophagic flux in CRC cells.

Figure 1. Altered expression of CD24 modulates autophagy in CRC cells. (Zhuo J, Wang X., 2019)