| Objective:To explore whether the protein kinase Ca (PKCa) is activated in the TF-VIIa-PAR2signal transduction axis as well as its roles in the proliferation, migration and survival of colon cancer cell lines SW620, and to further analyze upstream and downstream signaling molecules of PKCa and its regulation of effector molecules in the process to clarify the underlying molecular mechanism that TF-VIIa-PAR2-PKCa promotes SW620cell proliferation, migration, and survival.Methods:(1) SW620cells were treated with factor Ⅶa (10nM), protease activated receptor-2agonists (PAR2-AP,100μM), phorbol-12-myristate-13-acetate (PMA,100nM) and medium for different times, and the expression of PKC a and p-PKC a was detected by Western blot, and meanwhile the distribution of PKC a was observed by immunofluorescence assay, respectively.(2) With inhibition antibody (a-TF, a-PAR2), isotype control antibody (mopc-21), PKC a inhibitor (safingol,10μM) and ERK1/2inhibitor (U0126,10μM), inhibition experiments were carried out and western blot was used to detect the expression of PKCa and p-PKCa, ERK1/2and p-ERK1/2as well as NF-κB and p-NF-KB, so as to explore upstream and downstream signaling molecules of PKCa.(3) After SW620cells were treated with different combinations of PMA (100nM), Ⅶa (10nM) and PKC a inhibitor(safingol,10μM), the cell proliferation ability was detected by MTT assay, the cell migratory potential was tested by transwell experiment and the cell survival ability was assessed by flow cytometry.(4) After SW620cells were treated with different combinations of VIIa(10nM), PKC α inhibitor(safingol,10μM) and NF-κB inhibitor (PDTC,5μM), real-time fluorescence quantitative PCR and western blot were used to detect the expression of MMP-9, caspase-3, TF, and Bcl-2/Bax, and TF activity kits was used to detect TF activity.Results:(1) Compared with the medium group,Ⅶa (10nM), PAR2-AP (100μM) and PMA (100nM, positive control) could obviously induced phosphorylation of PKCα (p-PKCα) but did not affect the expression of total PKCα. Immunofluorescence results showed that PKCα was distributed mostly in the cytoplasm with irregularity but not detected in the nucleus in untreated cells; while in the Ⅶa or PAR2-AP or PMA treated cells, PKCα was mainly localized in the perinuclear region with slight distribution in the nucleus.(2) Compared with the medium group, α-TF or α-PAR2could dramatically attenuate phosphorylation of PKCα (p-PKCα) induced by Ⅶa (p<0.05, compared to Ⅶa treatment alone), which was not found in pretreatment with isotype control antibody (Mopc-21). Compared with the Ⅶa treatment alone group, PKCα inhibitor (safingol,10μM) significantly inhibited Ⅶa-induced downstream signaling molecules ERK1/2and NF-κB phosphorylation (p<0.05).(3) The cell proliferation ability, migratory potential and survival ability were significantly enhanced by stimulation with Vila or PMA (p<0.05, compared to medium control). However, the enhanced effects by Vila or PMA on cell proliferation, migration and survival were blocked by safingol (p<0.05, compared to Vila or PMA treatment alone).(4) Compared with the medium group, obvious downregulation of caspase-3and Bax expression and significant upregulation of MMP-9, TF and Bcl-2expression as well as TF activity were elicited by Vila (p<0.05). However, the induced effects by Vila were dramatically neutralized by safingol or PDTC treatment (p<0.05, compared to Vila treatment alone).Conclusions:(1) Activation of PKCa can be caused by PAR2-AP and Vila in SW620cells.(2) Ⅶa-induced PKCa activation is dependent on TF and PAR2, and consequently activates downstream of ERK1/2and NF-κB.(3) PKCa activation is necessary for the proliferation, migration and survival of SW620cells stimulated by Ⅶa.(4) PKCa activation is involved in the expression of MMP-9, caspase-3, TF, and Bcl-2/Bax induced by Vila. |
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