| Part one:Synergistic anti-pancreatic cancer activities by suppressing EGFR,PI3K/Akt/mTOR,STAT3 and ERK signalingObjectiveSTAT3 is the primary therapeutic target of CuB in pancreatic cancer.Interestingly,accumulated data have demonstrated that CuB can selectively inhibit other signaling pathways,which is dependent on cancer cell contexts.For example,CuB suppressed the expression and activity of HER2 as well as EGFR in HER2-overexpressed breast cancer cells.It is reported that epidermal growth factor receptor（EGFR）plays a key role in the development of pancreatic cancer and is highly expressed in 30%-89%of patients.Therefore,EGFR may be a potential therapeutic target for pancreatic cancer.But due to EGFR-independent constitutive activation of downstream effectors or complex crosstalk among EGFR downstream signaling pathways,EGFR inhibitors are insufficient in effectively treating human pancreatic cancer.Therefore,blocking EGFR and its downstream signaling targets might be a rational strategy for pancreatic cancer therapy.In this study,we investigated whether CuB inhibited pancreatic cancer cell growth by modulating EGFR and its downstream signaling targets and tested whether CuB in combination with downstream EGFR signaling inhibitor could enhance its therapeutic efficacy.Methods（1）Effect of Cu B on cell cycle.Bxpc-3 and HPAC cells were treated respectively with different concentrations of CuB for 24 h,respectively.The staining was carried out by PI（5 mg/ml）after the fixed cell.The cell cycle was analyzed by flow cytometry.Bxpc-3 and HPAC cells were treated respectively with different concentrations of CuB for 24 h,respectively.Western blot was used to analyze the expression of CDK1、pCDK1 and Cyclin B1.（2）Effect of Cu B on cell death.Bxpc-3 and HPAC cells were treated respectively with different concentrations of CuB for 24 h,respectively.Trypan blue staining,LDH assay and flow cytometer were used to detect the cell death caused by Cu B.（3）CuB inhibits the cell proliferation by suppressing the expression of EGFR.Bxpc-3 and HPAC cells were treated with different concentrations of CuB respectively.RNA was extracted from cells and the expression level of EGFR was detected by RT-PCR.After Bxpc-3 and HPAC cells were transfected with sh-NTC or sh-EGFR,the cells were treated with different concentrations of CuB for 24 h and MTT assay was used to analyze the availabe cells.（4）Effect of Cu B on the activity of EGFR,STAT3,Akt and S6.Bx PC-3 and HPAC were treated with different concentrations of CuB for 24 h,respectively.Western blot was used to detect the expression levels of EGFR,pEGFR,STAT3,pSTAT3,Akt,pAkt（T308）,pAkt（S473）and pS6 proteins.（5）Effect of Cu B on ERK activity.Bx PC-3 and HPAC were treated with different concentrations of CuB,respectively.The expression levels of ERK,pERK,AMPK and pAMPK protein were detected by western blot.After that,BxPC-3 and HPAC were treated with 0.3μM CuB for 6 h,12h and 24 h,respectively.The expression levels of ERK,pERK,AMPK and pAMPK protein were detected by western blot.（6）Combination of CuB and ERK inhibitor（SCH772984）.Bx PC-3,ASPC-1,MiaPaCa-2 and HPAC were treated with different doses of CuB,SCH772984 or combination.IC500 of CuB in the absence or presence of SCH772984 was detected by MTT assay.The standard equivalent diagram was used to explain the joint effect.And the CI（combination index）was calculated by CalcuSyn software.（7）Effects of CuB and SCH772984 on cell death.Bx PC-3 and HPAC cells were treated with 0.3μM CuB or 2μM SCH772984,separately or jointly,the cell death was detected by flow cytometry.（8）Effects of CuB and SCH772984 on related proteins.Bx PC-3 and HPAC cells were treated with 0.3μM CuB or 2μM SCH772984,separately or jointly,the related proteins were detected by western blot.（9）Antitumor efficacy of CuB and SCH772984 in vivo.We used a mouse HPAC xenograft model to evaluate the effects of CuB and SCH772984 on pancreatic tumor growth.When the xenografts reached a volume of106.9±13.4 mm3,mice were randomized into 4 groups（5 animals per group,with mean tumor volumes of 104.1±7.7,105.7±7.3,104.7±8.3 and 115.2±9.0 mm3 for the vehicle control,CuB,SCH772984,and combination group,respectively）and treated with（i）vehicle control,（ii）0.5 mg/kg CuB three times per week by intraperitoneal injection,（iii）25 mg/kg SCH772984 daily by intraperitoneal injection,or（iv）0.5 mg/kg CuB three times a week by intraperitoneal injection and 25 mg/kg SCH772984 daily by intraperitoneal injection for 4 weeks.Tumor diameters were measured with a caliper daily.Results（1）Effect of Cu B on cell cycle.Bxpc-3 and HPAC cells were treated with different concentrations of CuB and the distribution of the cell cycle was studied by flow cytometry after PI staining.CuB induced cell cycle arrest in a dose-dependent manner in G2/M and decreased with G0/G1 phase.After that,we used western blot to investigate the expression of related proteins in G2/M phase.The results showed that CuB could lead to decreasing phosphorylation of CDK1（Tyr15）.In HPAC cells,Cyclin B1 protein expression was reduced,but no change in Bxpc-3 cells.（2）Effect of Cu B on cell death.Bxpc-3 and HPAC cells were treated with different concentrations of CuB for 24h.After PI staining,flow cytometry,trypan blue staining and LDH assay were used to detect cell death.The results showed that the cell death rate was about 15%after high concentration of CuB.These indicated that the ability of CuB to induce cell death was limited.（3）CuB inhibits the cell proliferation by suppressing the expression of EGFR.In the knockdown of EGFR cells,different concentrations of CuB were treated respectively for 24 h,and the proliferation of cells was detected by MTT assay.When the EGFR was knocked down,the growth ability of the cells was reduced compared to the control group.The growth inhibition rate was significantly higher than that in the control group.（4）Effect of Cu B on the activity of EGFR,STAT3,Akt and S6.After Bxpc-3 and HPAC cells were treated with different concentrations of CuB for 24 h,the related proteins were detected by western blot.The results showed that CuB inhibited the activity of EGFR,STAT3,Akt and S6.（5）Effect of Cu B on ERK activity.The phosphorylation of ERK and AMPK were induced by using western blot,which were dependent on time and dose.The use of AMPK inhibitors or AMPK knockout to destroy AMPK could abolish the ERK phosphorylation caused by CuB.（6）Combination of CuB and ERK inhibitor（SCH772984）.By MTT and western blot,CuB and SCH772984 had a good synergistic effect in pancreatic cancer cells and SCH772984 was able to enhance the effect of CuB on pancreatic cancer cells.（7）Effects of CuB and SCH772984 on cell death.Annexin V/PI double staining and flow cytometry analyses showed that combination treatment significantly increased cell apoptosis relative to individual treatment in BxPC-3 and HPAC cells.（8）Effects of CuB and SCH772984 on related proteins.SCH772984 at 2μM abrogated both ERK protein levels and ERK phosphorylation.And CuB-activated ERK phosphorylation was markedly suppressed by combination treatment with SCH772984 and CuB.In addition,EGFR,pEGFR,pAkt（T308）,pAkt（S473）,pS6 and pSTAT3 levels were lower after the combined treatment than after vehicle control treatment in BxPC-3 and HPAC cells.We also found Mcl-1 protein levels were much lower after the combined treatment than after CuB or SCH772984treatment alone.The combined treatment also resulted in an increase in Bim levels and a decrease in Bcl-2,Bcl-xl and survivin compared to vehicle control.（9）Antitumor efficacy of CuB and SCH772984 in vivo.Individual and combined drug treatments were well tolerated,as indicated by the lack of a significant loss of body weight.Compared to vehicle control treatment,successive 4-week treatment with CuB or SCH772984 alone significantly reduced tumor growth,resulting in lower mean tumor volumes（63.8%and 54.7%on day 28,respectively,）.In particular,the combined drug treatment resulted in significant delay of tumor growth during the treatment period compared to single drug treatment,with85.0%tumor growth inhibition on day 28.Individual drug treatment resulted in increased tumor necrosis,which was further increased following combination treatment as indicated in arrows in HE staining.Proliferation was substantially lower in the combination group compared to the single groups,as indicated by lower PCNA staining.The combined drug treatment resulted in increased cell apoptosis,as measured by the TUNEL assay.Conclusion1 CuB mainly inhibits cell proliferation.2 CuB inhibits the activity of EGFR and downstream signaling（STAT3 and PI3K/AKT）.3 CuB induces phosphorylation of AMPK,leading to activation of ERK signalingpathway.4 SCH772984 synergizes with CuB to induce growth inhibition and apoptosis of PCcells in vivo and in vitro.Part two: Cucurbitacin B inhibits the proliferation of pancreatic cancer cells through destroying AFAP1-AS1/mi RNA-146b-5p/EGFR axisObjectivePancreatic cancer（PC）is an oncogene-driven tumor with multiple genetic and epigenetic alterations that may contribute to its aggressive nature and confer resistance to conventional and targeted agents.Although previous studies have identified numerous susceptibility loci for PC,the mechanism underlying transcriptome regulation is not well elucidated.Inspiringly,recent studies have demonstrated that lnc RNAs are a critical factor in the pathogenesis of PC.Among these lnc RNAs,the overexpression of lnc RNA-AFAP1-AS1 was observed in most PC patients and was associated with poor survival.Therefore,AFAP1-AS1 has potential value as a prognostic biomarker and therapeutic target in PC.Cucurbitacins are natural tetracyclic triterpene compounds derived from the Cucurbitaceae plant family and show a wide spectrum of pharmacological activities,such as anticancer,anti-inflammatory and hepatoprotective effects.In recent years,many studies have addressed the pharmacological activities of cucurbitacin B（Cu B）,especially its anticancer activities.To data,it is not clear whether Cu B can affect the expression of AFAP1-AS1 in PC.Growing evidences have suggested that the lnc RNA acts as a mi RNA sponge or as competing endogenous RNA（ce RNA）,reducing the availability of mi RNAs for m RNA target binding.However,Since AFAP1-AS1 research is still at an early stage,AFAP1-AS1–related mi RNAs or m RNAs have not been found,to date.Therefore,in this part,we study the effect of Cu B on the proliferation of PC cells by destroying AFAP1-AS1/mi RNA/m RNA network.Methods（1）The relation between the sensitivity of pancreatic cancer cells and the expression of the AFAP1-AS1.In this study,we chose Bx PC-3,HPAC,Mia Pa Ca-2,ASPC-1 and human normal pancreatic duct epithelial cells: HPDE6-C7.RNA was extracted from the cells,and the expression level of AFAP1-AS1 was detected by RT-PCR and HPDE6-C7 was used as the quantitative reference.After that,different concentrations of Cu B were used to treat Bx PC-3,HPAC,Mia Pa Ca-2,ASPC-1 and HPDE6-C7 cells respectively.The availabe cells were analyzed by MTT assay.The IC50 value was calculated using the Graph Pad Prism 5.0 software.（2）Cu B inhibits the cell proliferation by suppressing the expression of AFAP1-AS1.Bxpc-3 and HPAC cells were treated with different concentrations of Cu B respectively.RNA was extracted from cells,and the expression level of AFAP1-AS1 was detected by RT-PCR.After Bxpc-3 and HPAC cells were transfected with si-NTC or si-AFAP1-AS1 for 72 h,the cells were treated with different concentrations of Cu B for 24 h,and MTT assay was used to analyze the availabe cells.The other cells were stained with PI after the cells were fixed.The cell cycle was analyzed by flow cytometry.（3）Effect of Cu B on expression of AFAP1-AS1,EGFR and mi RNA-146b-5p in vivo and in vitro.First,the Bxpc-3 and HPAC cells were treated with different concentrations of Cu B and the expression of AFAP1-AS1,EGFR and mi RNA-146b-5p were detected by RT-PCR.And then we used a mouse HPAC xenograft model to evaluate the effects of Cu B on expression of AFAP1-AS1,EGFR and mi RNA-146b-5p.（4）The relation between AFAP1-AS1,EGFR and mi RNA-146b-5p.The relationship between the three genes were detected using si RNA,mi RNA minics and dual luciferase reporter gene system.Results（1）The relation between the sensitivity of pancreatic cancer cells and the expression of the AFAP1-AS1.We first examined the expression levels of AFAP1-AS1 in the five cells of Bx PC-3,HPAC,Mia Pa Ca-2,ASPC-1 and human normal pancreatic ductal epithelial cells: HPDE6-C7,which was used as a quantitative reference and an internal reference.We found that the expression of AFAP1-AS1 in Mia Pa Ca-2 cells was lowest than the remaining cells.After that,we examined the sensitivity of Cu B by MTT assay in Bx PC-3,HPAC,Mia Pa Ca-2,ASPC-1 and human normal pancreatic duct epithelial cells.The results showed that the IC50 range was between 0.02 and 0.28 μM after 72 h of treatment.4 cells showed different sensitivity to Cu B,and Mia Pa Ca-2 cells,which the IC50 was greater than 0.20 μM,were the most insensitive than other 3 PC cells.Coincidentally,in Mia Pa Ca-2 cells,AFAP1-AS1 gene expression level was relatively low,so there might be a correlation between the sensitivity of pancreatic cancer cells and the expression of the AFAP1-AS1.（2）Cu B inhibits the cell proliferation by suppressing the expression of AFAP1-AS1.In the knockdown of AFAP1-AS1 cells,different concentrations of Cu B were treated respectively for 24 h and the proliferation of cells was detected by MTT assay.When the AFAP1-AS1 was knocked down,the growth ability of the cells was reduced compared to the control group.The growth inhibition rate was significantly higher than that in the control group.These indicated that the down-regulation of Cu B-induced AFAP1-AS1 was the main cause of growth inhibition.After the AFAP1-AS1 gene knockdown,we found that the cell cycle was arrested in the G2/M phase.These results indicated that Cu B mainly inhibited the expression of AFAP1-AS1,thus inducing the cell cycle arrest of pancreatic cancer in G2/M period.（3）Effect of Cu B on expression of AFAP1-AS1,EGFR and mi RNA-146b-5p in vivo and in vitro.The Bxpc-3 and HPAC cells were treated with different concentrations of Cu B and the expressions of AFAP1-AS1,EGFR and mi RNA-146b-5p were detected by RT-PCR.We found that Cu B inhibited expression of AFAP1-AS1 and EGFR genes,and induced the expression of mi RNA-146b-5p.Finally,AFAP1-AS1 and EGFR were significantly down-regulated in Cu B-treated mice,compared to controls,while mi R-146b-5p was significantly up-regulated in Cu B-treated mice.（4）The relation between AFAP1-AS1,EGFR and mi RNA-146b-5p.Dual negative regulation between AFAP1-AS1 and mi R-146b-5p in PC cell lines was observed: after AFAP1-AS1 knockdown,mi R-146b-5p was overexpressed and AFAP1-AS1 expression could be down-regulated by mi R-146b-5p overexpression.Bioinformatics analysis confirmed the existence of mi R-146b-5p binding sites on EGFR and AFAP1-AS1 sequences and the results of luciferase experiments also showed that mi R-146b-5p could effectively interfere with AFAP1-AS1 and EGFR expression.Conclusion1.Cu B causes cell cycle arrest in G2/M by inhibiting the expression of AFAP1-AS1.2.Both in vivo and in vitro,Cu B inhibits the proliferation of pancreatic cancer cells through destroying the AFAP1-AS1 / mi RNA-146b-5p / EGFR axis. |
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