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  • br increased the invasive ability of the cells p however

    2022-05-10


    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). Clozapine N-oxide 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 Clozapine N-oxide 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
    AKT can be activated by upstream kinases. In particular, it has been
    Fig. 4. HK2 activity is required for AKT2-stimulated tumor invasion, tumorigenesis and metastasis.
    A and B, HCT-116 cell migration assay; C and D, HT-29 cell migration assay; E, HK2 activity is required for AKT2-stimulated xenograft tumor growth. Tumors were collected and examined six weeks after inoculation of HT-29 cells, respectively. Pictures of isolated tumors were taken. F, Tumor weights were determined. G and H, Tumors shown in E were measured for HK activity and lactate production. I, Lung metastatic burden in BALB/c nude mice 45 days after tail vein injection of HT-29 cells, as determined by counting the number of micrometastases per section (arrows point to metastatic areas). J, The lung metastatic nodule numbers in BALB/c nude mice 45 days after tail vein injection of HT-29 cells. K, Representative HE staining of metastatic foci in the lung tissues. L, Similar changes in the lung metastatic nodule numbers were also detected in the ratio of metastatic tumor area/total lung area.