Relationship Of MEKK1 And Pancreatic Cancer Development

Pancreatic cancer is one of the gastrointestinal malignancies with extremely poor prognosis.Although the incidence rate remains relatively low,pancreatic cancer contributes the eighth leading cause of cancer death.To date,few therapeutic options are available for patients with this metastatic disease.The main problem of this cancer is the late diagnosis as its cancer-specific symptoms occur only at an advanced stage when the tumor size is too large(above 4 cm in diameter) with local spread or lymph node metastasis.Many factors have been identified to be closely related to the carcinogenesis and progression of pancreatic cancer.The factors include those involved in VEGF,EGF, Notch,IGF,MUC,K-Ras,PI3K-AKT,and sonic hedgehog signaling pathways. Importantly,the factors in the Ras-MAPK,PI3K-AKT-mTOR and Hedgehog pathways were reported to be the most important ones that contribute to invasion and metastasis of pancreatic cancer.Ras/Raf/MAPK pathway plays an important role in most of tumor invasion and metastasis.In pancreatic cancer,activating mutation of K-Ras is frequently found.Ras activation leads to activation of downstream signaling proteins including MEKK1 and MEK.MEKK1 is a Ser/Thr protein kinase,with 196 KD in mass,belonging to the MEKK/STE11 subgroup of the MAPKKK family,and is a hub of the MAPK signal pathway.MEKK1 functions as to interact with many proteins such as scaffold proteins, serine-threonine kinase,and a focal adhesion-associated kinase(FAK).MEKK1 is able to activate its cascade proteins including ERK1/2,JNK and P38 pathways,and it has also been shown to regulate both IKK-NFκB and JAK-STAT pathways.MEKK1 plays a critical role in cell growth,cell differentiation as well as invasion and metastasis.To date, MEKK1 has been reported to mediate tumor metastasis of gastric carcinomas,mammary and ovarian cancers.However,its role in pancreatic cancer has not been well documented.To observe whether the MEKK1 expression is related to pancreatic cancer in vitro,we examined the MEKK1 mRNA and protein levels in different pancreatic cancer cell lines (BxPC3,Capan2,SW1990 and PANC1).The data showed that MEKK1 mRNA level remained highly abundant in BxPC3 and Capan2 cell lines while it had weak expression in the SW1990 and PANC1 cell lines.A Western blot analysis and immunofluence assay further confirmed the MEKK1 protein expression in a similar pattern in the four observed pancreatic cell lines.To clarify whether the MEKK1 expression level is related to the malignancy of pancreatic cancer cells,softagar assay,woudhealing assay,adhesion assay and a tumor invasion assay were performed by use of the four human pancreatic cancer cell lines.The data revealed that BxPC3 cells had stronger abilities of adhesion,molity and invasion. These results suggest that MEKK1 expression is positively associated with metastatic potential of the human pancreatic cancer cells.To examine whether the highly expressed MEKK1 is the cause for the strong invasive ability,we depleted MEKK1 by using the RNA interference method in BxPC3 cells.We selected and synthesized three siRNAs against MEKK1.The efficiency of the selected siRNAs was examined by transfection in the BxPC3 cells.RT-PCR analysis revealed that siRNAl and siRNA3 have a significant inhibitory effect on the endogenous MEKK1 expression while the siRNA2 showed no effect.The effects of the selected siRNAs were further confirmed by a Western blot analysis and the results consistently showed that siRNA3 was the strongest one to inhibit MEKK1 expression.Based on these results we determined to use siRNA3 for the following experiments.To study the role of MEKK1 on the cell invasion,motility and adhesion,which are the major characteristics of the metastasis,we used siRNA3,the most powerful one we selected,to deplete the endogenous expression of MEKK1 in the BxPC3 cells.In an invasion assay,we calculated the number of cells that migrated to the bottom side of the membrane on a chamber where the cells were seeded.The data showed that wild type BxPC3 cells had more numbers of the migrated cells with or without transfection of a control siRNA.However,when siRNA3 targeting MEKK1 was transfected into the cells, the number of the migrated cells decreased dramatically.These results suggested that depletion of MEKK1 significantly suppressed the migration ability of BxPC3 cells.To examine whether depletion of MEKK1 has any effect on the motive ability of the cells, we performed a wound healing experiment using BxPC3 cells transfected with or without control-siRNA and siRNA3.The data showed that the wild type BxPC3 cells had no difference in the relative wound closure with or without transfection of the control siRNA,but a significant difference was observed for the cells transfected with or without siRNA3 targeting MEKK1 both for 15 and 24 hours.The highly expressed MEKK1 correlated to the pancreatic tumor metastasis in patients with the tumor reminded us of examining whether MEKK1 has an effect on cell adhesion to ECM(extracellular matrix).For this purpose,we performed a cell adhesive assay by using siRNA3 targeting MEKK1 in the BxPC3 cells.The results showed that the cells, when transfected with siRNA3,obtained low adhesion ability,compared with the wild type cells.The results were in consistence during different times as we observed in 30,60 and 120 min,respectively.These results indicated that MEKK1 might help the tumor cells adhere to the ECM.Taking together,our data suggest that MEKK1 exerts its effects on metastasis by promoting invasion,migration and adhesion in BxPC3 cells.Matrix-metalloproteinases(MMPs) have been reported to play pivotal roles in tumor invasion through degradation of basement membrane and ECM.To reveal the mechanisms of the inhibition role of siRNA3 on pancreatic cell metastasis,the activity of MMP2 protein was investigated by a Gelatin zymography assay.The data showed that the activity of MMP2 can be observed in the gelatin zymography assay in HT1080 cells, a canonical cell line for the MMP2 activity measurement.When wild type BxPC3 cells were used,we observed high activities of MMP2 in the wild type and mock cells but a significant low activity of MMP2 in the cells transfected with siRNA3 targeting MEKK1. We did not observed the activity of MMP9 in BxPC3 cells although we examined that in HT1080 cells.This result indicated that MEEK1 contributed to the activation of MMP2 in the BxPC3 cells.MEKK1 functions as a hub in three signal pathways including ERK1/2,P38 and JNK.To identify which downstream pathways are critical in the metastasis of pancreatic tumor cells,we investigated the phosphorylation of ERK1/2,P38 and JNK in the MEKK-depleted BxPC3 cells.The results showed that the phosphorylation levels of ERK1/2 were significantly inhibited,while the levels of phosphorylated p38 and JNK were weakly affected by siRNA3 targeting MEKK1.These data suggest that ERK1/2 play a major role in the activation of the cascade of MEKK1 in the pancreatic tumor cells.To identify the detailed roles of MEKK1 in the regulation of malignant characteristics of human pancreatic adenocarcinoma in vivo.In this study,we analyzed MEKK1 expression in patient's samples by immunohistochemistry assay.The results demonstrated that MEKK1 positive staining(Figure 24) was noted in 32 samples among the 41 pancreatic adenocarcinomas(78.1%).All these results suggested that the MEKK1 expression was significantly associated with differentiation,TNM staning and lymph node metastasis in the observed pancreatic cancer patients(Table 8;P<0.05).The MEKK1 staining did not show significantly different among the patients of different gender,age,tumor location and tumor size(table 8;P>0.05).In addition,there is a mainly founding that MEKK1 cell location verified with cancer differentiation grade.The amounts of nuclear staining is 100%in poorly differentiated cancers,while the amount of nuclear staining in moderate and well differentiated cancers were 72.2%and 22.2%,respectively.In conclusion,this study demonstrates that MEKK1 plays a major role in the metastasis of pancreatic cancer,possibly through activating specifically ERK to regulate expression of MMP2.Our findings suggest that MEKK1 may be a potential molecule target for pancreatic cancer therapy. Icogenin is one of steroidal saponins extracted from plant named Dracaena Draco. Icogenin has been reported to have certain cytotoxicity in HL60 cells.It has been found that Icogenin prefers inhibiting the cancer cell growth of PANC1 and A431 among eight cancer cell lines by MTT assay.At present,it is still the best way to treat pancreatic cancer combining radical surgical resection with chemotherapy drugs.Chemotherapy is even more important for patients with unresected and advanced pancreatic cancer. Cytotoxic chemotherapy drugs lie in the dominated position.Therefore,it is of great significance to explore the activity of anti-pancreatic cancer and its mechanisms by Icogenin.In this study,we choose a pancreatic cancer cell line BxPC3 as cell model in vitro. Icogenin inhibited the abilities of cell proliferation and colony formation of BxPC3 cells, the IC₅₀ is 0.84±0.02μmol/L.Cell apoptosis was found at 6 hour by 5μmol/L Icogenin. At the same time,the results by flow cytometry,AO/EB staining and other methods have confirmed the existence of apoptosis.The DNA ladder was also found.BxPC3 cell cycle was arrested at G2/M phase.Western blot analysis results showed that BxPC3 cell apoptosis mainly related to inhibite the mitochondrial pathway bc1-2 family(Bax,Bak, bc1-2 and bcl-x1 were changed).The P53 level was not varied.The phosphorylation ERK and P38 were decreased and the phosphorylated JNK was increased in MAPK signal pathway by apoptosic dose Icogenin.We also found that Icogenin has the role against invasion and metastasis in pancreatic cancer at lower doses(1μmol/L is less than IC₁₀ at 48h point).To investigate the activities of Icogenin on anti-cancer and anti-metastasis and their mechanisms in pancreatic cancer cell line BxPC3 in vitro,using transwell assay,the effects of Icogenin on the invasion of BxPC3 cells were measured.The abilities of cell motility and adhesion in BxPC3 cells were detected by adhesion assay and wound healing assay respectively. The MAPK signal pathway proteins were analysed by Western blotting.At the same time, the activity of MMP2 was observed by zymography assay.Icogenin inhibited the abilities of motility,adhesion and invasion of pancreatic cancer BxPC3 cells in vitro,in dose-depended manner,and inhibited the secretion of MMP2 and phosphorylation of ERK.PD98059 and U0126 which were ERK inhibitors could suppress the abilities of invasion and metastasis of pancreatic cancer cells BxPC3(P<0.05). In summary,mechanism of Icogenin against pancreatic cancer may relate to inhibite the phosphoration of ERK and P38.On the other side,Icogenin induces pancreatic cancer cell apoptosis by activing the JNK proteins which induced Bcl-2 dependent apoptosis in mitochondria.