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M cell cycle arrest in pancreatic cancer cells through JNK pathway
Abstracts / Pancreatology 16 (2016) S1eS63
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repressed growth and metastasis of several kinds of cancer cells, including pancreatic cancer and breast cancer. In the present study, we investigated the regulation on MMP2 expression by PP2A inhibitors. Methods: mRNA levels were determined by using real-time PCR and microarray. Gene transcription was evaluated by luciferase reporter gene assay. mRNA degradation was tested by using RNA stability assay. Small interfering RNA (siRNA) was used to knockdown target gene. Results: By using real-time PCR, we found that PP2A 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. By using RNA stability assay, we confirmed the accelerated degradation of MMP2 mRNA. By using microarray assay, we identified that multiple genes participating in RNA degradation pathway were up-regulated after treatment with PP2A inhibitors. Among these genes, up-regulation of members of deadenylation pathway was most remarkable. Blocking deadenylation pathway by using siRNA targeting CNOT7, RHAU or PARN could respectively attenuate the repression on MMP2 expression by PP2A inhibitors. Conclusions: PP2A inhibitors repressed MMP2 expression through accelerated MMP2 mRNA degradation by activated deadenylation mechanism.
PP2A (protein phosphatase 2A). In the present study, we tried to investigate whether the cell cycle arrest could be due to PP2A inhibition. Methods: Gene expression was evaluated by real-time PCR and western blot. Cell viability was determined by MTT assay. PP2A catalytic C subunit (PP2Ac) targeting siRNA was used for a specific inhibition on PP2A. Expression profiles were determined by using microarray and analyzed by spearman's rank correlation analysis. Cell cycle was analyzed by flow cytometry. Cell viability was determined by MTT assay. Results: The gene expression profile of cantharidin treated group correlated with the one of PP2Ac siRNA group. By cross comparing the profiles of these groups, we identified that expression of CDK1 could be repressed by both cantharidin and PP2Ac siRNA. The repression on CDK1 by its inhibitor RO-3306 resulted in G2/M cell cycle arrest and growth inhibition in pancreatic cancer cells. Conclusions: The regulation of cantharidin on gene expressions was mainly through inhibition on PP2A. The inhibition on PP2A could result in down-regulation CDK1, leading to G2/M cell cycle arrest and repression on cell growth. Keywords: Cantharidin; PP2A; CDK1; Pancreatic cancer
Keywords: Pancreatic cancer; PP2A; MMP2; mRNA degradation 15475. PP2A inhibitors repress growth of pancreatic cancer cells through JNK/SP1/CDK1 pathway 15473. PP2A inhibitors induce G2/M cell cycle arrest in pancreatic cancer cells through JNK pathway Abstract Objective: In our previous studies, we demonstrated that PP2A inhibitors repressed pancreatic cancer cell growth through JNK pathway dependent manner and arrest cell cycle in G2/M phase. In the present study, we tried to investigate whether this cell cycle arrest was also executed through JNK pathway dependent manner. Methods: Cell cycle was analyzed by flow cytometry. As flow cytometry analysis based on PI staining cannot distinguish between G2 and M cell cycle arrest, because cells at both these phases are diploid; flow cytometry analysis based on mithramycin A staining was performed. Regulation on expressions of cell cycle-related genes was evaluated by microarray. Results: PP2A inhibitors induced cell accumulation at G2/M phase in a dose-dependent manner, with concurrent decreases in cell populations at G0/G1 and S phases, after 24 h treatment. The level of G2/M aggregation was reduced by pretreatment with SP600125, suggesting that PP2A inhibitors induced G2/M arrest through a JNK dependent pathway. Flow cytometry analysis based on mithramycin A staining suggested that PP2A inhibitors arrested cell cycle in G2 phase. To investigate which genes were involved in the cell cycle alteration triggered by PP2A inhibitors, we performed microarray analyses to determine the mRNA expressions of 72 genes participating in cell cycle regulation. Among these genes, expression changes of 21 genes consist with the decreases in cell populations at G0/G1 and S phases, as well as arrested G2/M transition in PP2A inhibitor treated groups, suggesting multiple genes were involved in the cell cycle regulation by PP2A inhibitors. Conclusions: PP2A inhibitors arrested G2/M cell cycle transition through JNK pathway dependent manner. Keywords: PP2A; JNK; G2/M cell cycle arrest; Pancreatic cancer
15474. Cantharidin induces G2/M cell cycle arrest through inhibition on PP2A Abstract Objective: Cantharidin is the active extractive of canthari, a traditional Chinese medicine. In our previous studies, we demonstrated that cantharidin repressed pancreatic cancer cell growth and arrest cell cycle in G2/M phase. It has been proved that cantharidin is a selective inhibitor of
Abstract Objective: In our previous studies, we demonstrated that PP2A (protein phosphatase 2A) inhibitors repressed growth of pancreatic cancer cells through JNK panthway dependent manner. PP2A inhibitors arrested cell cycle at G2/M phase and down-regulated expression of CDK1, the key regulator of G2/M cell cycle transition. SP1 is the critical downstream of JNK pathway. In the present study, we tried to investigate whether the anti-cancer effect of PP2A inhibitors was executed through JNK/SP1/CDK1 pathway dependent manner. Methods: Gene expression was evaluated by real-time PCR and western blot. Cell viability was determined by MTT assay. Transcriptional activity was measured by luciferase reporter gene assay. For cell signaling transduction investigation, JNK inhibitor SP600125, CDK1 inhibitor RO3306, and SP1 targeting siRNA were used. Results: PP2A inhibitors induced transcriptional activation of SP1. SP600125 attenuated the activation of SP1 by PP2A inhibitors. Both SP600125 and SP1 siRNA could impair the repression on CDK1 expression by PP2A inhibitors. Both RO-3306 and SP1 siRNA could attenuate the repression on cell growth by PP2A inhibitors. Conclusions: PP2A inhibitors repressed pancreatic cancer cell growth through JNK/SP1/CDK1 pathway dependent manner. Keywords: PP2A; JNK; SP1; CDK1; Pancreatic cancer
15476. PP2A inhibitors repress transcription of CDK1 through JNK/SP1 pathway Abstract Objective: In our previous studies, we demonstrated that PP2A (protein phosphatase 2A) inhibitors repressed growth of pancreatic cancer cells through JNK panthway dependent manner. PP2A inhibitors arrested cell cycle at G2/M phase and down-regulated expression of CDK1, the key regulator of G2/M cell cycle transition. SP1 is the critical downstream of JNK pathway. In the present study, we tried to explore the mechanism involved in the JNK/SP1 pathway dependent regulation on CDK1 transcription. Methods: Gene expression was evaluated by real-time PCR and western blot. Cell viability was determined by MTT assay. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified the element in the CDK1 promoter that responded to the JNK/ Sp1 pathway after stimulation with PP2A inhibitors.
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repressed growth and metastasis of several kinds of cancer cells, including pancreatic cancer and breast cancer. In the present study, we investigated the regulation on MMP2 expression by PP2A inhibitors. Methods: mRNA levels were determined by using real-time PCR and microarray. Gene transcription was evaluated by luciferase reporter gene assay. mRNA degradation was tested by using RNA stability assay. Small interfering RNA (siRNA) was used to knockdown target gene. Results: By using real-time PCR, we found that PP2A 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. By using RNA stability assay, we confirmed the accelerated degradation of MMP2 mRNA. By using microarray assay, we identified that multiple genes participating in RNA degradation pathway were up-regulated after treatment with PP2A inhibitors. Among these genes, up-regulation of members of deadenylation pathway was most remarkable. Blocking deadenylation pathway by using siRNA targeting CNOT7, RHAU or PARN could respectively attenuate the repression on MMP2 expression by PP2A inhibitors. Conclusions: PP2A inhibitors repressed MMP2 expression through accelerated MMP2 mRNA degradation by activated deadenylation mechanism.
PP2A (protein phosphatase 2A). In the present study, we tried to investigate whether the cell cycle arrest could be due to PP2A inhibition. Methods: Gene expression was evaluated by real-time PCR and western blot. Cell viability was determined by MTT assay. PP2A catalytic C subunit (PP2Ac) targeting siRNA was used for a specific inhibition on PP2A. Expression profiles were determined by using microarray and analyzed by spearman's rank correlation analysis. Cell cycle was analyzed by flow cytometry. Cell viability was determined by MTT assay. Results: The gene expression profile of cantharidin treated group correlated with the one of PP2Ac siRNA group. By cross comparing the profiles of these groups, we identified that expression of CDK1 could be repressed by both cantharidin and PP2Ac siRNA. The repression on CDK1 by its inhibitor RO-3306 resulted in G2/M cell cycle arrest and growth inhibition in pancreatic cancer cells. Conclusions: The regulation of cantharidin on gene expressions was mainly through inhibition on PP2A. The inhibition on PP2A could result in down-regulation CDK1, leading to G2/M cell cycle arrest and repression on cell growth. Keywords: Cantharidin; PP2A; CDK1; Pancreatic cancer
Keywords: Pancreatic cancer; PP2A; MMP2; mRNA degradation 15475. PP2A inhibitors repress growth of pancreatic cancer cells through JNK/SP1/CDK1 pathway 15473. PP2A inhibitors induce G2/M cell cycle arrest in pancreatic cancer cells through JNK pathway Abstract Objective: In our previous studies, we demonstrated that PP2A inhibitors repressed pancreatic cancer cell growth through JNK pathway dependent manner and arrest cell cycle in G2/M phase. In the present study, we tried to investigate whether this cell cycle arrest was also executed through JNK pathway dependent manner. Methods: Cell cycle was analyzed by flow cytometry. As flow cytometry analysis based on PI staining cannot distinguish between G2 and M cell cycle arrest, because cells at both these phases are diploid; flow cytometry analysis based on mithramycin A staining was performed. Regulation on expressions of cell cycle-related genes was evaluated by microarray. Results: PP2A inhibitors induced cell accumulation at G2/M phase in a dose-dependent manner, with concurrent decreases in cell populations at G0/G1 and S phases, after 24 h treatment. The level of G2/M aggregation was reduced by pretreatment with SP600125, suggesting that PP2A inhibitors induced G2/M arrest through a JNK dependent pathway. Flow cytometry analysis based on mithramycin A staining suggested that PP2A inhibitors arrested cell cycle in G2 phase. To investigate which genes were involved in the cell cycle alteration triggered by PP2A inhibitors, we performed microarray analyses to determine the mRNA expressions of 72 genes participating in cell cycle regulation. Among these genes, expression changes of 21 genes consist with the decreases in cell populations at G0/G1 and S phases, as well as arrested G2/M transition in PP2A inhibitor treated groups, suggesting multiple genes were involved in the cell cycle regulation by PP2A inhibitors. Conclusions: PP2A inhibitors arrested G2/M cell cycle transition through JNK pathway dependent manner. Keywords: PP2A; JNK; G2/M cell cycle arrest; Pancreatic cancer
15474. Cantharidin induces G2/M cell cycle arrest through inhibition on PP2A Abstract Objective: Cantharidin is the active extractive of canthari, a traditional Chinese medicine. In our previous studies, we demonstrated that cantharidin repressed pancreatic cancer cell growth and arrest cell cycle in G2/M phase. It has been proved that cantharidin is a selective inhibitor of
Abstract Objective: In our previous studies, we demonstrated that PP2A (protein phosphatase 2A) inhibitors repressed growth of pancreatic cancer cells through JNK panthway dependent manner. PP2A inhibitors arrested cell cycle at G2/M phase and down-regulated expression of CDK1, the key regulator of G2/M cell cycle transition. SP1 is the critical downstream of JNK pathway. In the present study, we tried to investigate whether the anti-cancer effect of PP2A inhibitors was executed through JNK/SP1/CDK1 pathway dependent manner. Methods: Gene expression was evaluated by real-time PCR and western blot. Cell viability was determined by MTT assay. Transcriptional activity was measured by luciferase reporter gene assay. For cell signaling transduction investigation, JNK inhibitor SP600125, CDK1 inhibitor RO3306, and SP1 targeting siRNA were used. Results: PP2A inhibitors induced transcriptional activation of SP1. SP600125 attenuated the activation of SP1 by PP2A inhibitors. Both SP600125 and SP1 siRNA could impair the repression on CDK1 expression by PP2A inhibitors. Both RO-3306 and SP1 siRNA could attenuate the repression on cell growth by PP2A inhibitors. Conclusions: PP2A inhibitors repressed pancreatic cancer cell growth through JNK/SP1/CDK1 pathway dependent manner. Keywords: PP2A; JNK; SP1; CDK1; Pancreatic cancer
15476. PP2A inhibitors repress transcription of CDK1 through JNK/SP1 pathway Abstract Objective: In our previous studies, we demonstrated that PP2A (protein phosphatase 2A) inhibitors repressed growth of pancreatic cancer cells through JNK panthway dependent manner. PP2A inhibitors arrested cell cycle at G2/M phase and down-regulated expression of CDK1, the key regulator of G2/M cell cycle transition. SP1 is the critical downstream of JNK pathway. In the present study, we tried to explore the mechanism involved in the JNK/SP1 pathway dependent regulation on CDK1 transcription. Methods: Gene expression was evaluated by real-time PCR and western blot. Cell viability was determined by MTT assay. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified the element in the CDK1 promoter that responded to the JNK/ Sp1 pathway after stimulation with PP2A inhibitors.