Up-regulation Of Metallometrix Protease In Type 2 Diabetes Mellitus Promoting Pancreatic Cancer Progression

Part Ⅰ Type 2 diabetes mellitus induced up-regulation of matrix metalloproteinases expression in pancreatic cancerObjective This study aimed to compare the matrix metalloproteinases(MMPs)in pancreatic cancer subjects with and without type 2 diabetes(T2DM),and to investigate whether MMPs would be the underlying factors promoting malignant actions of pancreatic cancer under diabetic conditions.Materials and Methods Murine pancreatic cancer cells,panc02 cells,used in this study were labelled with reporter of firefly luciferase.The mouse models of pancreatic cancer with T2 DM were prepared by utilizing streptozotocin-induced(STZ,25 mg/kg,once a day for 5 days)and genetically modified diabetic(db/db)mice,and pancreatic cancer without T2 DM were conducted on wild type C57BL/6J mice(WT).T2-weighted MR imaging(T2WI)was performed to confirm cancer lesions and dynamically monitor tumor growth.In vivo bioluminescence imaging by IVIS Spectrum imaging system was carried out to detect the metastasis after D-luciferin(i.p.,150 mg/kg,Promega,USA)administration.The m RNA expression of MMPs family members(MMP-1,MMP-2,MMP-3,MMP-7,MMP-9,MMP-10,MMP-11,MMP-13,MMP-14)in tumors from WT,STZ and db/db mice were analysed by q RT-PCR.On 4,8 and 12 days after tumor transplantation,the protein expression levels of MMP-9 and MMP-2 in tumor tissues were detected by western blot and imminohistochemistric staining(IHC).In addition,paraffin-embedded tissue samples from pancreatic cancer patients with T2DM(n=22)and without T2DM(n=42)were obtained from the Department of Pathology at Zhongda Hospital affiliated to Southeast University.The MMP-2 and MMP-9 expression levels in patients’ tissues were detected by IHC and analysed the correlation with gender,age,size of tumor,regional lymph node metastasis(LNM),and pathologic TNM stage(p TNM).Results We found that T2 DM promoted the malignant behaviors of concurrent pancreatic cancer,as demonstrated by larger tumor size,especially in db/db group,and more metastasis(P<0.05).Among the tumor-associated MMPs,MMP-2 and MMP-9 m RNA expression were found to be significantly higher in pancreatic cancer of STZ and db/db mice compared with the WT group(P<0.05).Consistent tendency of protein levels of further elevation of MMP-2 and MMP-9 induced by diabetic conditions were observed in both patient’s tissues and mouse models(all P<0.05).In addition,higher lymph node metastasis rate was also observed in diabetic pancreatic cancer patients than nondiabetic pancreatic cancer.Conclusion T2 DM promoted the malignant behaviors of concurrent pancreatic cancer,which were parallel with further elevation of MMP-2 and MMP-9 promoted by diabetic conditions.It suggests that up-regulating MMP-2 and MMP-9 can potentially serve as targets for prognosis and treatment of pancreatic cancer with diabetes.Part Ⅱ Dynamic visualization and therapeutic evaluation of matrix metalloproteinases in vivo in diabetic pancreatic cancer by an activatable probeObjective An MMP activatable probe was synthesized utilizing a long wavelength near-infrared(NIR)fluorophore to realize the dynamic visualization and therapeutic evaluation of matrix metalloproteinases(MMPs)in orthotopic pancreatic cancer with and without type 2 diabetes(T2DM)in vivo.Methods MMP activatable probe,I780BP-PEG12,consisted of an NIR dye(IR780)-labelled GPLGVRGKGG peptide that acted as a side chain-protected MMP substrate.The peptide was linked to the quencher BHQ-3,and the molecular beacons were PEGylated via conjugation of PEG12.The CCK-8 assays were conducted to evaluate the cytotoxicity of I780BP-PEG12.The in vitro effects of I780BP-PEG12 were evaluated by incubating the probe with MMP-2,MMP-9,and MMP-2/9 inhibitor,SB-3CT.The mouse models of pancreatic cancer were prepared by utilizing wild type C57BL/6J mice(WT),streptozotocin-induced(STZ,25 mg/kg,once a day for 5 days)and genetically modified diabetic(db/db)mice.T2-weighted MR imaging(T2WI)were performed dynamically to confirm cancer lesions and detect tumor growth.To obtain optimal near infrared fluorescence(NIRF)images,NIRF imaging was performed at a series of time points from 15 min to 24 h after injecting with an IR780-labelled MMP-targeted probe(200 μL,8 n M)dissolved in PBS via the tail vein(excitation wavelength 745 nm,emission wavelength 815 nm;CRi,Woburn,MA,USA).To dynamically visualize MMP activity in vivo over time,NIRF imaging of pancreatic cancer in WT,STZ,and db/db groups were performed on day 4,8,and 12 after tumour transplantation using an in vivo optical imaging system.In addition,the response to selective MMP-2/9 inhibitory therapy in three groups are monitored non-invasively by MMPs molecular imaging,followed by histological validation.Results In vitro I780BP-PEG12 presented very low cytotoxicity,high quenching efficiency,and high sensitivity with the capacity to detect nanomolar concentrations of enzymes.In vivo NIRF imaging showed that the tumour regions presented ultrafast signal enhancement as early as 15 min,which was sustained for as long as 24 h,presenting the peak intensity at 6 h post-injection.Immunofluorescence staining confirmed that MMP-2 and MMP-9 were well co-localized with the IR780 fluorescence of I780BP-PEG12.Furthermore,much higher levels of MMPs activity were detected in pancreatic cancer under diabetic conditions,including both STZ and db/db groups.After SB-3CT treatment,NIRF imaging showed that in contrast to the strong fluorescence signal enhancement observed in vehicle-treated pancreatic cancer,the signal intensity was significantly decreased in STZ and db/db mice after SB-3CT treatment,as determined by quantified TBR both in vivo and ex vivo.However,the signal alteration in the WT group after SB-3CT treatment did not reach statistical significance.Conclusion A probe,I780BP-PEG12,targeting MMPs can facilitate dynamic monitoring of MMPs and inhibitory treatment in vivo,which showed the significantly higher MMP levels in pancreatic cancer in diabetic condition than in non-diabetic pancreatic cancer.It would be beneficial for personal therapeutic strategy planning and optimization of pancreatic cancer management,especially with diabetic conditions.Part Ⅲ Evaluation the potential therapeutic effects of MMP-2/9 inhibitor on pancreatic cancer with and without type 2 diabetesObjective To investigate the potential effects of MMP-2/9 inhibitor on pancreatic cancer with and without type 2 diabetes(T2DM)and further to investigate whether MMP-2/9 inhibitor would enhance anti-cancer effect of the gemcitabine.Methods The mouse models of pancreatic cancer were prepared by utilizing C57BL/6J mice(WT),streptozotocin-induced(STZ,25 mg/kg,once a day for 5 days)and genetically modified diabetic(db/db)mice.T2-weighted imaging was performed dynamically to monitor tumor growth.Bioluminescence imaging by IVIS Spectrum imaging system was carried out to detect the tumor metastasis after D-luciferin(i.p.,150 mg/kg,Promega,USA)administration.Pancreatic cancer in WT,STZ,and db/db mice were respectively divided into four groups and treated for 20 days: i)PBS group was administered with an equal volume of PBS as the control group;ii)SB-3CT group received an intraperitoneal injection(i.p.)of the selective MMP-2/9 inhibitor SB-3CT(Selleck,USA)diluted in 10% DMSO at the dosage of 25 mg/kg once daily;iii)gemcitabine group was administered with gemcitabine(25 mg/kg,i.p.)every three days for 20 days.iv)Combination group was administered with SB-3CT(25 mg/kg,i.p.)daily for 20 days,during this period,gemcitabine(25 mg/kg,i.p.)was injected every three days.After treatments,IHC was conducted to evaluate tumour microvessel density(marked by CD31)and cell proliferation(marked by Ki67).Results After treatment with gemcitabine,diabetic STZ mice(non-obesity)and db/db mice(obesity)presented much more metastasis than WT group.Selective MMP-2/9 inhibitor effectively reversed the malignancy-promoting actions in diabetic STZ mice bearing pancreatic cancer,inclulding tumor growth and metastasis(all P<0.05).In diabetic STZ mice bearing pancreatic cancer,the antitumour effect of the combination therapy was significantly better than that in gemcitabine treated group,presenting smaller tumour volume,lower metastatic number(all P<0.05).Besides,in diabetic db/db mice with obesity,metastatic number after the combination therapy was significantly less than that in gemcitabine treated group.IHC detection showed that,the combination therapy presented significantly lower cell proliferation level and less microvessel density in diabetic STZ and diabetic db/db mice than that with either gemcitabine or MMP-2/9 inhibitor treated group(all P<0.05).Conclusion This study demonstrated that the combination of gemcitabine and MMP-2/9 inhibitors remarkably augmented the therapeutic effect of gemcitabine in pancreatic cancer with diabetes but not in nondiabetic pancreatic cancer,especially for reducing metastasis,which suggests that MMP-2/9 can potentially serve as targets for treatment for personal therapeutic strategy planning and optimization of pancreatic cancer management,especially for diabetic individuals.