Cantharidin Represses Invasion Of Pancreatic Cancer Cells Through Accelerated Degradation Of MMP2 MRNA

Objective:Pancreatic cancer cells have high invasive ability. Cantharidin, which targets protein phophatase 2A(PP2A), is the active extractive of Cantharis. Studies have proved that protein phophatase 2A(PP2A) inhibitors repressed growth and metastasis of several kinds of cancer cells, including pancreatic cancer. We previously demonstrated that repression on PP2 A by cantharidin led to activation of several kinase pathways, including ERK, JNK, NF-κB and PKC. Matrix metalloproteinase 2(MMP2) plays a central role in cancer cell invasion. In the present study, we investigated the effect on cell invasion by cantharidin and the mechanism involved, providing a basis for further development of cantharidin as a promising therapeutic agent for the treatment of pancreatic cancer.Methods:1. Invasion ability of pancreatic cancer cells was tested by transwell assays.2. Gene expression at the mRNA levels and the protein level were determined respectively by using real-time PCR and western blot assays.3. For cell signaling transduction investigation, ERK pathway inhibitor PD98059, JNK inhibitor SP600125, NF-κB pathway inhibitor Bay11-7082, PKC inhibitor GF109203 X and β-catenin pathway inhibitor FH535 were used.4. Small interfering RNA(siRNA) and short hairpin RNA(sh RNA) were used to knockdown target gene.5. Gene transcription of MMP2 was evaluated by luciferase reporter gene assay.6. mRNA degradation was tested by using RNA stability assay.7. Microarray analyses was used to test the expression of target genes.Results:1. Cantharidin repressed invasion of pancreatic cancer cell and inhibited expression of matrix metalloproteinase 2(MMP2).2. PP2 A catalytic C subunit(PP2Ac) targeting siRNA also repress MMP2 expression, suggesting cantharidin could donw-regulate MMP2 through PP2 A dependent manner.FH535 significantly inhibited migration of pancreatic cancer cells.3. PD98059, SP600125, Bay11-7082, GF109203 X and FH535 could attenuate the repression on MMP2 by cantharidin.4. By using real-time PCR, we found that PP2 A inhibitors repressed expression of MMP2. However, transcription of MMP2 was found to be up-regulated by using luciferase reporter gene assay, suggesting the repression on MMP2 expression was executed through post-transcriptional mechanism.5. By using RNA stability assay, we confirmed the accelerated degradation of MMP2 mRNA.6. By using microarray assay, we identified that multiple genes participating in RNA degradation pathway were up-regulated after treatment with PP2 A inhibitors. Among these genes, up-regulation of members of deadenylation pathway was most remarkable.7. Blocking deadenylation pathway by using shRNA targeting CNOT7, RHAU or PARN could respectively attenuate the repression on MMP2 expression by PP2 A inhibitors.Conclusion:Our present results indicated for the first time that cantharidin repressed MMP2 expression and pancreatic cancer cell invasion through PP2 A inhibition-induced activation of ERK, JNK, NF-κB, PKC and β-catenin pathways. PP2 A inhibitors repressed MMP2 expression through accelerated MMP2 mRNA degradation by activated deadenylation mechanism, shedding new lights on the therapy of pancreatic cancer.