Impaired death-associated protein kinase-mediated survival signals in 5-fluorouracil-resistant human endometrial adenocarcinoma cells

Abstract

Abstract.A recent study showed that both 5-fluorouracil (5FU)-stimulated apoptosis and Fas-mediated apoptosis in human endometrial adenocarcinoma cells are enhanced by targeted knockdown of endogenous death-associated protein kinase (DAPK) with DAPK small-interfering RNAs. Therefore, we investigated the DAPK survival signals in three 5FU-resistant subclones. DAPK knockdown did not enhance 5FU-stimulated or Fas-mediated apoptosis in any of the three 5FU-resistant subclones, but the subclones acquired resistance to VP16-stimulated cell death that was DAPK-independent. Semiquantitative flow cytometric analyses showed that there was no differential expression in nine cell surface antigens, including Fas, and six intracellular molecules, including DAPK, that may regulate cell death or survival between the parent cells and 5FU-resistant cells. DAPK mRNA and protein were expressed in the 5FU-resistant subclones at similar levels to the parent cells. These results indicate that acquisition of 5FU-resistance may be accompanied by impairment of common apoptotic signals regulating both DAPK-dependent and DAPK-independent pathways. IntroductionThe death-associated protein kinase (DAPK) cDNA was isolated from human cervical carcinoma cells as a positive mediator of apoptosis triggered by interferon-γ (1). Recent investigations have revealed that DAPK functions as a Ca 2+ /calmodulin-dependent serine/threonine kinase to regulate cell death or survival (1-16). However, the physiological functions of DAPK have not been fully clarified. Loss of DAPK expression has been implicated in tumorigenesis and metastasis (3,4,13), suggesting a crucial role for DAPK in the apoptotic process under pathological conditions. On the other hand, inhibition of DAPK expression in HeLa cells, 3T3 fibroblasts, primary human vascular smooth muscle cells and various human uterine cancer cells using an antisense DAPK or small-interfering RNAs (siRNAs) for DAPK was found to increase apoptosis (6,12,15,16).Specific stimulation of Fas antigen can easily induce apoptotic cell death in differentiated human endometrial adenocarcinoma HHUA cells (17), and Fas-mediated apoptosis can be enhanced in a dose-dependent manner by targeted knockdown of endogenous DAPK protein expression by treatment with specific DAPK siRNAs (DAPK-KD) (18). Since DAPK-KD induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HHUA cells (15), it is highly possible that DAPK-mediated signals constitutively suppress the tumor necrosis factor receptor-mediated cell death signaling common to the Fas-mediated and TRAIL-mediated pathways. Although typical apoptosis with DNA fragmentation can easily be induced in HHUA cells by treatment with 5-fluorouracil (5FU) (19), DAPK-KD increases apoptotic susceptibility to 5FU in HHUA cells (20). However, DAPK-KD has no effect on etoposide (VP16)-sensitivity in HHUA cells (20). Therefore, there are two apoptotic signals in HHUA cells, comprising DAPK-regulated and DAPK-independent death signals.In the human cervi...

Novel Functions of Death-Associated Protein Kinases through Mitogen-Activated Protein Kinase-Related Signals

“…Moreover, it was shown that DAPK1 was capable of suppressing oncogenic transformation caused by c-Myc and E2F, which blocks the tumor metastasis [ 13 , 14 ]. DAPK1 is also mediated in anti-cancer drug resistance to 5-fluorouracil in endometrial adenocarcinoma cells [ 56 ], anti-epidermal growth factor receptor antibodies in lung cancer cells [ 57 ], gemcitabine in pancreatic cancer cells [ 58 ], and cisplatin in cervical squamous cancer cells [ 59 ].…”

Targeting Death-Associated Protein Kinases for Treatment of Human Diseases: Recent Advances and Future Directions

“…Notably, DAPK1 can modulate the resistance to some antitumor drugs. For instance, in human endometrial adenocarcinoma cells, the knockdown of DAPK1 enhances 5-fluorouracil-stimulated apoptosis and Fas-mediated apoptosis, thus enhancing the drug treatment effect . Additionally, in drug-resistant NSCLC cells, hypermethylation of DAPK1 in derivatives of epidermal growth factor receptor (EGFR) therapeutics resensitizes cells to erlotinib and cetuximab.…”

“…For instance, in human endometrial adenocarcinoma cells, the knockdown of DAPK1 enhances 5-fluorouracil-stimulated apoptosis and Fas-mediated apoptosis, thus enhancing the drug treatment effect. 117 Additionally, in drug-resistant NSCLC cells, hypermethylation of DAPK1 in derivatives of epidermal growth factor receptor (EGFR) therapeutics resensitizes cells to erlotinib and cetuximab. Consequently, in order to increase therapeutic effectiveness and overcome anti-EGFR medication resistance, DAPK1 may therefore be a novel target.…”

MicroRNAs mediate therapeutic and preventive effects of natural agents in breast cancer

“…In breast cancer MDA-MB-231 and MDA-MB-468 cells with stably expressed miR-510h, resistance to paclitaxel is significantly increased when compared to the cells transfected with vehicle vectors. After searching the global putative targets of miR-520h and sorting those associated to apoptosis, DAPK2 is picked based on its documented role as a pro-apoptotic gene [15] . MiR-520h targeting DAPK2 is verified by using Western blotting and luciferase reporter assays, respectively.…”