br Overexpression of CPA promoted the
3.4. Overexpression of CPA4 promoted the proliferation and tumorigenesis of CRC cells
CRC cell lines SW620 and RKO stably overexpressing CPA4 were established and the overexpression of CPA4 in these cells was confirmed by western blot (Fig. 3A, B). In comparison with control cells, the growth of SW620 and RKO cells was promoted by CPA4 overexpression, as de-termined by CCK-8 and colony formation assays (Fig. 3C–F). CPA4 over-expression also enhanced the migration and invasion of SW620 and RKO cells (Fig. 3G, H). Furthermore, to confirm the growth-promoting effects in vivo, tumorigenesis assays by subcutaneous injection were performed in nude mice. Overexpression of CPA4 in the RKO cells mark-edly promoted tumor growth in vivo (Fig. 3I, J). In addition, the IHC anal-ysis revealed that expression of CPA4, STAT3 and ERK was increased in CPA4 overexpression group compared with control group (Fig. 3K).
Fig. 4. CPA4 Knockdown inhibits G1/S transition and apoptosis of colorectal cancer (CRC) cells. (A) and (B) Representative images of the T16Ainh A01 assays in HCT116 and LS123 cells after transfection with shNC or shCPA4. Cells were stained with propidium iodide and analyzed by flow cytometry. (C) and (D) Apoptosis of HCT116 and LS123 cells was determined by flow cytometry. Cells stained with annexin-V-APC were considered apoptotic.
3.5. CPA4 knockdown results in G1 arrest in CRC
Changes in the cell cycle profile following CPA4 knockdown were analyzed by flow cytometry. The percentage of cells in the G1 phase was significantly increased for shCPA4-transfected HCT116 and LS123 cells compared with that of shNC-transfected cells (P b 0.05) (Fig. 4A, B). However, no significant difference was found in the percentage of cells in the S or G2/M phases after CPA4 knockdown. Together, the data show that CPA4 knockdown attenuates CRC cell proliferation by inhibiting the G1/S transition.
3.6. CPA4 inhibits apoptosis in CRC cells
The percentages of CRC cells that underwent apoptosis upon shCPA4-mediated knockdown were determined for both HCT116
and LS123 cell lines. For HCT116 cells, the percentages of cells that underwent apoptosis were 4.29% and 23.07% (p b 0.01) for shNC and shCPA4 cells, respectively. For LS123 cells, the percentages of cells that underwent apoptosis were 4.57% and 13.23% (p b 0.01) for shNC and shCPA4 cells, respectively (Fig. 4C, D). The levels of caspases, a family of central regulators of apoptosis, were assessed by western blot. Cleavage of caspase-9 and caspase-3 was increased in CPA4 knockdown cells, while levels of cleaved caspases de-creased in CPA4-overexpressing cells compared with those in con-trol cells (Fig. 5A).
3.7. CPA4 activates STAT3 and ERK pathways in CRC cells
To explore the regulatory mechanism of CPA4 in CRC, we examined changes in intracellular signaling molecules upon CPA4 knockdown in
Fig. 5. CPA4 activated STAT3 and ERK signaling pathways in colorectal cancer (CRC) cells. (A–B) The phosphorylation of intracellular signaling in HCT-116 and LS-123 with CPA4 knockdown was examined by intracellular protein assays. (C) The levels of phosphorylated ERK, total ERK, phosphorylated STAT3, total STAT3, and cleavage of caspase-9 and caspase-3 were detected in CPA4-knockdown and -overexpressing cells by western blot analysis. GAPDH was used as the loading control. (D–G) The relative grey value of phosphorylated STAT3 and phosphorylated ERK were measured by Image J software.
HCT116 and LS123 cells. A significant decrease in phosphorylation of STAT3-Tyr705 and ERK1/2-Tyr202/Tyr204 was detected (Fig. 5A, B). To further validate these findings, we measured the phosphorylation and total protein levels of STAT3 and ERK in CPA4 knockdown and over-expression cell lines using western blot. Relative to levels in control cells, levels of phosphorylated STAT3 and ERK were decreased in HCT116 and LS123 CPA4 knockdown cells, whereas phosphorylation of STAT3 and ERK was increased in SW620 and RKO cells
overexpressing CPA4 (Fig. 5C). However, the total protein levels of STAT3 and ERK did not change in any cell line.
Then the CPA4 overexpressing RKO cells were treated with an STAT3 inhibitor (cryptotanshinone) or ERK inhibitor (U0126). The result of Western blots showed that STAT3 and ERK phosphorylation was subse-quently inhibited in the CPA4 overexpressing RKO cells (Fig. 6A). In ad-dition, CCK-8, colony formation assays and transwell assays revealed that the cell proliferation and growth, as well as migration and invasion
Fig. 6. The RKO cells with CPA4 overexpression were treated with the STAT3 inhibitor cryptotanshinone, the ERK inhibitor U01226. (A) The expression levels of the indicated proteins were measured by western blot. (B–D) The abilities of cell proliferation, growth, migration and invasion of RKO cells with CPA4 overexpression was determined by CCK-8 (B), colony formation assay (C), and transwell assays (D) after treatment with cryptotanshinone, U0126 or DMSO. ( p b 0.001).