Death-associated protein kinase 3 controls the tumor progression of A549 cells through ERK MAPK/c-Myc signaling

Abstract

Death-associated protein kinases (DAPKs) are members of the serine/threonine protein kinase family, which regulate cell death. Although DAPK3 has been implicated as a tumor suppressor, a recent study revealed an oncogenic role of DAPK3. However, the role of DAPK3 in non-small cell lung cancer (NSCLC) remains unclear. Therefore, we examined whether DAPK3 controls the progression of NSCLC using the NSCLC cell line, A549. We generated A549 cells stably expressing small hairpin RNA (shRNA) targeting DAPK3. In the A549 cells, the protein level of DAPK3 was decreased and the cell proliferation was inhibited. DAPK3 knockdown caused G1/G0 cell cycle arrest as assessed by flow cytometric assay and reduced cyclin D1 expression in A549 cells. Phosphorylation of ERK and c-Myc, but not Akt and JNK, was inhibited by DAPK3 knockdown. Cell migration and invasion were also inhibited by DAPK3 knockdown as determined by a Boyden chamber assay and an invasion assay, respectively. Moreover, DAPK3 knockdown inhibited anchorage-independent cell growth as determined by soft-agar colony formation assay. In a mouse xenograft model, tumors derived from DAPK3-knockdown cells exhibited reduced tumor growth. The present results demonstrated for the first time that DAPK3 controls proliferation, migration, invasion, soft‑agar colony formation and tumor growth through activation of ERK/c-Myc signaling in A549 cells. These findings indicate that DAPK3 may be a novel target for the treatment of NSCLC.

Establishment of a dog primary prostate cancer organoid using the urine cancer stem cells

“…Dog mammary tumor cells, CIP‐p and CIP‐m, and dog osteosarcoma cells, C‐HOS, were cultured in RPMI‐1640 supplemented with 10% FBS (Thermo Fisher Scientific) as described previously …”

“…Cells were incubated with propidum iodide before flow cytometric analysis to exclude dead cells. The samples were analyzed using BD Accuri C6 (BD Biosciences) as described previously . The cell population data were analyzed using BD Accuri software (BD Bioscience) and the positive cells were determined based on each isotype control.…”

“…Dog mammary tumor cells, CIP-p and CIP-m, and dog osteosarcoma cells, C-HOS, were cultured in RPMI-1640 supplemented with 10% FBS (Thermo Fisher Scientific) as described previously. (16) H&E staining of organoids. After the organoids were fixed with 4% paraformaldehyde (PFA) at 4°C overnight, they were embedded in paraffin.…”

“…The samples were analyzed using BD Accuri C6 (BD Biosciences) as described previously. (16) The cell population data were analyzed using BD Accuri software (BD Bioscience) and the positive cells were determined based on each isotype control. Cell sorting was performed using Sony Cell Sorter (SH800, Sony, Tokyo, Japan) after staining the cells using the method as described above.…”

“…Western blotting was performed as described previously. (16,19) Protein lysates were obtained by homogenizing the cells with Triton-based lysis buffer (50 mM Tris-HCl [pH 8.0], 5 mM EDTA, 5 mM EGTA, 1% Triton X100, 1 mM Na 3 VO 4 , 20 mM sodium pyrophosphate and Roche Complete protease inhibitor mixture). Loading proteins (10-20 lg) were separated by SDS-PAGE (10%) and transferred to a nitrocellulose membrane (Wako, Osaka, Japan).…”