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  • br AKT phosphorylation of HK

    2020-07-06


    3.6. AKT2 phosphorylation of HK2 can reduce apoptosis induced by serum starvation
    Following the treatment of HCT-116 and HT-29 Okadaic acid with serum starvation for 48 h, we found that the rate of OV AKT2 cell apoptosis was significantly lower than that of normal control cells (Fig. 3E and F; p < .01). Knocking out the hk2 gene in OV AKT2 cells increased the rate of apoptosis induced by serum starvation (p < .05); however, the level of apoptosis was still lower than that of the control group (p < .05). This finding indicates that knocking out the hk2 gene in OV AKT2 cells did not completely reduce the ability of these cells to resist apoptosis induced by AKT2 overexpression. The stable rescue of HK2 overexpression in OV AKT2 hk2 cells significantly increased the re-
    sistance to apoptosis induced by serum starvation (p < .01). However, the rescue of overexpressed HK2T473A mutants in OV AKT2 hk2 cells
    did not alter the ability of cells to resist apoptosis (p > .05). These results suggest that AKT2 phosphorylation of HK2 at T473 can increase cell resistance to apoptosis induced by serum starvation.
    3.7. AKT2 mediates HK2 to increase invasion ability of colon cancer cells
    It has been well-established that AKT2 plays a very important role in tumor cell invasion and metastasis [27]. However, there is currently no study on whether AKT2 can mediate HK2 to increase cellular invasion. Using a transwell experiment as shown in Fig. 4A - D, in HCT-116 and HT-29 cells, AKT2 overexpression significantly increased cellular in-vasiveness compared to the control group (p < .01). Knocking out the hk2 gene in OV AKT2 cells can reduce a certain level of cellular invasion (p < .05). However, the ability of AKT2 overexpression to promote cellular invasion was not completely reduced. It is suggested that AKT2 may also mediate cell invasion through other downstream molecules. The rescued overexpression of HK2 in OV AKT2 hk2 cells significantly
    increased the invasive ability of the cells (p < .05); however, the rescue of overexpressed HK2T473A mutants in OV AKT2 hk2 cells did
    not alter the invasive ability of the cells (p > .05). These results in-dicate that AKT2 phosphorylation of HK2 at T473 can increase the invasive capacity of these cells.
    3.8. AKT2-stimulated tumorigenesis is dependent on HK2
    We next explored the biological significance of AKT2-induced HK2 activation in colon cancer HT-29 cells. As shown in Fig. 4E and F, AKT2 overexpression significantly increased xenograft tumor growth in BABL/c nude mice compared with the control group (p < .01). Knocking out the hk2 gene in OV AKT2 cells reduced the growth of xenograft tumors at a certain level (p < .01); however, the tumor weight was significantly higher than that of the control group (p < .01), indicating that knocking out the hk2 gene did not com-pletely inhibit the growth of the xenograft tumor induced by AKT2 overexpression. This outcome may be related to AKT2 through other downstream molecules that mediate colon cancer xenograft tumor growth. Stably rescuing the overexpression of HK2 in OV AKT2 hk2
    cells significantly increased the growth of the xenograft tumor (p < .01), rather than the HK2T473A mutant cells (p > .05). These
    results indicate that AKT2 phosphorylation of HK2 at T473 is required for colon cancer xenograft tumor growth. In these groups of xenograft tumor tissues, we also detected hexokinase activity and lactic acid
    Fig. 3. AKT2 phosphorylates HK2 at T473 can increase hexokinase activity and production of lactic acid and reduce apoptosis induced by serum starvation in colon cancer cells.
    production similar to those obtained from these cellular experiments (Fig. 4G and H).
    3.9. HK2 activity is required for AKT2-stimulated tumor metastasis
    As shown in Fig. 4I and J, we found that AKT2 overexpression significantly increased the number of nodules in lung metastases (p < .01). A knockout of the hk2 gene after the overexpression of AKT2 significantly reduced the number of lung metastasis nodules in nude mice (p < .05); however, the number of metastatic nodules was sig-nificantly higher than that of the control group (p < .05), indicating that knocking out the hk2 gene did not completely reduce the lung 
    metastasis increased due to AKT2 overexpression. Rescuing the over-expression of HK2 in OV AKT2 hk2 cells significantly increased the
    number of lung metastasis nodules (p < .01). However, rescuing the overexpressed HK2T473A mutants in OV AKT2 hk2 cells did not alter
    the number of lung metastases (p > .05). These findings indicate that AKT2 phosphorylates HK2 to increase the capacity for lung metastases by colon cancer cells.
    3.10. Upstream molecules regulate the AKT2/HK2-mediated progression of colon cancer