Novel Combination Therapy With Doxorubicin,Bioengineered MiRNA-34a Prodrug And Sorafenib Suppresses Osteosarcoma And Pulmonary Metastases

Part ? tRNA/ncRNAProdrugs:Biosynthesis,Purification and Application in Osteosarcoma TreatmentObjective To develop a novel method for tRNA scaffold-based biosynthesis and purification of large-scale,high-purity recombinant RNA molecules,providing a cost-effective and reliable source for basic research and clinical application of functional non-coding RBNA(ncRNA).To select optimal tRAN/ncRNA prodrug through screening anti-proliferative activity of osteosarcoma cell in a series of tRNA/ncRNAs.To examine the efficacy and mechanism of selected bioengineered tRNA/ncRNA in treatment of orthotopic osteosarcoma in xenograft mouse model.Methods A series of ncRNA sequences,including miR-34a,miR-124a,miR-144,miR-126,simiR-21 were cloned and inserted into fused tRNA-pre-miRNA-34a(pre-miR-34a without mature miR-34a sequence)scaffolds and formed chimeric tRNA/ncRNAs.The expression plasmid was constructed and transfected into Escherichia coli(HST08 strain).Anion exchange fast protein liquid chromategraphy(FPLC)was employed to isolate and purify the desired tRNA/ncRNAs.The yield and purity of recombinant ncRNAs were qualitatively analyzed by denatured urea polyacrylamide gel electrophoresis(urea PAGE).The purity of tRNA/ncRNAs was quantitatively estimated by high performance liquid chromatography(HPLC).The CleanAll DNA/RNA Clean-up kit was used to remove the residue of endotoxin.The concentration of endotoxin was determined by Pyrogent-5000 dynamic endotoxin turbidimetric assay.The concentrateion of purified tRNA/ncRNAs was determined by Nanodrop 2000 spectrophotometer and the yield was calculated.Human osteosarcoma 143B cells were transfected with pCCLc-Luc-EGFP lentiviral vector to select luciferase and eGFP fluorescent protein positive cell lines for the following in vitro and in vivo studies.A series of biosynthetic tRNA/ncRNAs were transfected into 143B cells and MG-63 cells.72 hours later,cell viability was determined by Celltiter-Glo luminescence assay.Then,143B cells were treated with gradient concentration of selected tRNA/ncRNA(tRNA/miR-34a)prodrug.The cell viability was measured.The pharmacokinetic parameters were calculated.The level of miR-34a and its targets,c-MET and SIRT1,in prodrug-treated 143B cells were analyzed by qRT-PCR and western blotting,respectively.The orthotopic osteosarcoma mouse model was established by subperiosteal injection of 143B cell into right tibia.The tRNA/miR-34a prodrug was systemically administrated via tail vein injection.The tumor signal intensity was measured by the small animal bioluminescence imaging system and the tumor size was monitored by caliper measurement.All control groups were performed with vehicle or tRNA/MSA.GraphPad Prism software was used to perform statistical analysis.Difference was considered as statistically significant at the level ofp<0.05.Results Bioengineered tRNA/ncRNAs accumulated to quite high level in E.coli HST08 strain.FPLC quickly separated the target recombinant ncRNAs.After purification,the yield of tRNA/ncRNA molecules accounted for about 20?30%of total RNA extraction.Urea PAGE analysis showed that chimeric tRNA/ncRNAs presented high homogeneity and purity.HPLC analysis showed that the purities of tRNA/ncRNA molecules were above 98%.Pyrogent-5000 dynamic endotoxin turbidimetric assay showed that the concentrations of endotoxin were<3.0EU per l?g purified tRNA/ncRNAs;Nanodrop 2000 detection indicated that about 10 to 20 mg of tRNA/ncRNA chimeras can be easily obtained from 1 L bacterial culture.The anti-proliferative activity screening of tRNA/ncRNAs,including tRNA/miR-34a,tRNA/miR-124a,tRNA/miR-144,tRNA/miR-126,tRNA/simiR-21,showed that all tRNA/ncRNA prodrugs significantly inhibited the proliferation of 143B cells and MG-63 cells in comparinsion with vehicle and tRNA/MSA.The inhibitory rate of tRNA/miR-34a prodrug on 143B cells was up to 50%.tRNA/miR-34a prodrug inhibited 143 B cell growth in a dose-dependent manner.The tRNA/miR-34a prodrug inhibited 143B cell growth up to 100%,whereas maximum inhibition rate of tRNA/MSA was only 34%.The level of miR-34a in tRNA/miR-34a prodrug-treated 143B cells was about 200 times higher than that of tRNA/MSA or vehicle group.The tRNA/miR-34a significa-ntly reduced the intracellular miR-34a targets,c-MET and SIRT1.tRNA/miR-34a prodrug significantly suppressed the growth of orthotopic osteosarcoma on basis of both tumor signal intensity and tumor volume.Conclusions Bioengineering of tRNA scaffold-based recombinant RNA molecules is a cost-effective and high-yield method to provide highly homogeneous biological tRNA/ncRNA molecules.Bioengineered tRNA/miR-34a chimera molecule acts as a"prodrug" in the cell to be processed into mature miR-34a form to effectively control osteosarcoma progress.Bioengineered RNA molecules are valuable for development of new macromolecule drugs.Part II Combination Therapy with Doxorubicin,tRNA/miR-34a prodrug and Sorafenib Suppresses Osteosarcoma and Pulmanory MetastasesObjective To establish multiple highly clinical-relevant animal models to authentically and comprehensively evaluate the feasibility and efficacy of new antineoplastic drugs or regimens.Driven by multiple factors,osteosarcoma proliferation-invasion-metastasis cascade may not be succumbed by a single hit.Based on "saturation attack" strategy,doxorubicin,tRNA/miR-34a prodrug,and sorafenib are used to rationally combine and co-target critical DNA,RNA,protein kinase in osteosarcoma cellular process.To investigate the combination effects of these drugs and the mechanism of their interactions.To define the effectiveness and safety profile of triple-drug therapy in control of osteosarcoma growth and metastases and improvement of survival in both orthotopic osteosarcoma spontaneous metastases and experimental extensive lung metastases mouse models.Methods The level of ?H2A.X was analyzed by immunofluorescence assay under a confocal microscopy.The expressions of c-MET and p-Erkl/2 were determined by western blotting analysis.The gradient concentrations of doxorubicin,tRNA/miR-34a prodrug and sorafenib treated 143B cell alone or in combination.72 hours later,Celltiter-Glo luminescence assay was used to evaluate the cell viabilities.The dose-response relationships were assessed.The pharmacokinetic parameters of drugs(including ED50 and Hill slope)were calculated with Graphpad prism software.The Chou-Talalay method was employed to evaluate the interactions between the drugs.After treatment,the 143B cell invasion was examined by Transwell chamber with 8.0 mm PET film coated with matrix gel.The orthotopic osteosarcoma spontaneous metastases mouse model was established by intra-tibial injection of 143B cell into right tibia.Doxorubicin,tRNA/miR-34a prodrug and sorafenib were either intravenously or intragastrically administrated alone or in combination.The tumor signal intensity was measured by the small animal bioluminescence imaging system and the tumor size was monitored by caliper measurement.The serum samples were collected to perform blood biochemistry analysis.Orthotopic tumors and lung tissues were dissected and subject to weighting and histological analysis with H&E staining.The number of metastases and the sum of the longest diameter of metastases were measured independently.The experimental extensive lung metastases mouse model was established by tail vein injection of high dose 143B cells.Doxorubicin+sorafenib,tRNA/miR-34a prodrug were administrated alone or in combination,the mortalities and death time of SCID mice were recorded for survival analysis.The necropsy of all lung tissues was performed to confirm the presence of extensive lung metastases.GraphPad Prism software was used to perform statistical analysis.Difference was considered as statistically significant at the level ofp<0.05.Results yH2A.X immunofluorescence analysis showed that,?H2A.X signal intensity was quite high in doxorubicin treated cells.Co-administration of sorafenib and tRNA/miR-34a,alone or together,with doxorubicin sharply increased the levels of yH2A.X.Western blotting analysis demonstrated that compared to vehicle treatment,tRNA/miR-34a led to a 40%suppression of c-MET protein levels.Either doxorubicin or sorafenib alone or in combination led to an upregulation of c-MET.However,co-administration of miR-34a abrogated the increase of c-MET expression caused by doxorubicin and sorafenib.Likewise,sorafenib induced an 80%reduction in phospho-rylation of target Erkl/2.While doxorubicin and tRNA/miR-34a alone caused about 20%inhibition of p-Erkl/2 levels,triple-drug combination reduced p-Erkl/2 to a comparable level as sorafenib alone.Doxorubicin,sorafenib and miR-34a prodrug alone reduced the proliferation of 143B cells in a dose-dependent manner,and a dual-or triple-drug combination was more effective than individual agents.The anti-proliferation effects of sorafenib and tRNA/miR-34a combination were mainly additive or even antagonistic at lower concentrations.Co-administration of doxorubi-cin with sorafenib or tRNA/miR-34a or sorafenib plus miR-34a produced synergistic effects in suppressing the proliferation of 143B cells,while the synergism of triple-drug combination persisted at all concentrations.Combination therapy could offer remarkable degrees of dose reduction,especially for doxorubicin and miR-34a prodrug.Invasion assay demonstrated that tRNA/miR-34a alone suppressed the invasion capability of 143B cells by about 50%,and doxorubicin plus sorafenib reduced cell invasion by 74%,as compared to the vehicle control.Triple-drug combination with doxorubicin,sorafenib and tRNA/miR-34a led to over 90%suppression of cell invasiveness.In orthotopic osteosarcoma spontaneous metastases SCID mouse model,doxorubicin,sorafenib and tRNA/miR-34a prodrug,triple-drug therapy was more effective to inhibit osteosarcoma growth than that of mono-or dual-drug therapy on basis of the bioluminescence signal intensity and distribution or the volume and weight of orthotopic osteosarcoma.Triple-drug therapy also showed the greatest degree of inhibition of lung metastasis on basis of GFP signaling intensity and distribution.From histology of lung tissues,triple drug therapy consistently showed minimal lung metastases(number of metastases,2-4,mean 3.0;the sum of longest diameters of metastases,15.0-287.5 mm,mean 116.3 mm),significantly less than vehicle group(9-29,mean 18.75;475.0-1212.5 mm,mean 829.9 mm).At the end of study,all SCID mice showed 5-10%body weight loss.ALT,AST,total bilirubin,BUN and creatinine levels were changed,but within the normal range.cTnI levels were below 0.156ng/ml.One SC:ID mice in vehicle group showed severe weight loss and abnormal BUN and creatinine levels in the last week of study.In experimental extensive lung metastases SCID mouse model,survival analysis indicated that doxorubicin plus sorafenib therapy or tRNA/miR-34a prodrug alone significantly increased the overall survival and improved the median survival from 38 days to 54 days,triple-drug therapy improved the median survival to 60.5 days.In contrast to the death of all mice treated with vehicle control by day 56,50%of the mice under triple-drug therapy were still alive on day 56 and 25%of mice were alive at the termination of the study.Conclusions In orthotopic osteosarcoma SCID mouse model,combination therapy with doxorubicin,tRNA/miR-34a prodrug and sorafenib against osteosarcoma and metastasis is very effective and safe.Combination therapy with doxorubicin,tRNA/miR-34a prodrug and sorafenib can prolong the survival of advanced osteosarcoma and improve its prognosis.The new strategy,co-targeting DNA,RNA and protein molecules in the cells,provides a framework for developing new remedies for lethal tumor metastasis.