Experimental Study On Stromal Remodeling In Colorectal Cancer Mediated By Rosiglitazone And Asiatic Acid And Its Role In Improving The Efficacy Of Pegylated Liposomal Doxorubicin

Objective:To observe the effect of rosiglitazone and asiatic acid on the remodeling of stromal mediated by colorectal cancer associated fibroblast,and its role in regulation the antitumor effect of pegylated liposomal doxorubicin(PLD)and the intratumoral accumulation.Methods:①The primary colorectal-cancer associated fibroblats(pCAF)was isolated,cultured and identified from patients diagnosed with colorectal cancer.And the co-culture models of pCAF and cancer cell lines were established and utilized as the in vitro research model.The maximal safety concentrations of both rosiglitazone and asiatic acid for pCAF,SW620,HT-29,and Caco-2 were tested by MTT assay.And the rosiglitazone-induced the gene encoded collagens expression changes were determined by gene array analysis.②The most sensitive cell line to doxorubicin treatment were measured by MTT assay.And the cell line was used to build the colorectal cancer xenograft models.The synergetic effects of rosiglitazone or asiatic acid with pegylated liposomal doxorubicin were observed.Histomorphometric analysis and stromal fibrosis were achieved by Hematoxylin-Eosin staining and sirius red/fast green double staining.The intratumoral fluorescence intensity of doxorubicin was evaluated by whole-body fluorescence imaging.③The in vitro synergetic effects of rosiglitazone and doxorubicin was measured by MTT assay.The role of rosiglitazone in epithelial-mesenchymal-transition of SW620 cell line was examined by the changes of biomarkes determined by the western blot test.And the rosiglitazone-induced the genome expression changes were determined by gene array analysis.Results:①The pCAF was presented as e-cadherin negative,vimentin and α-smooth muscle actin(α-SMA)dual positive cells.The inhibiting effect of rosiglitazone and asiatic acid on SW620,HT-29,Caco-2,and pCAFs cells was similar(all IC10 values were 10 μM approx.).There are ninety-nine collagen-related gene probes,seventy percent of which changed under the induction of rosiglitazone.Among them,COL1A1 and COL3A1 genes were down-regulated more than 3 folds.The COL4A3,and COL6A6 genes were down-regulated more than 22 folds and 31 folds respectively.②The 72 h IC50 values of doxorubicin against SW620,HT-29,and Caco-2 cell lines were 0.746 pM,3.827 μM,and 4.046 μM respectively.As a result,SW620 cells were used to establish the xenograft models.Rosiglitazone and asiatic acid have no observed adverse effect on the mice body weight and behavior.The in vivo inhibitory effect of PLD(4 mg/kg)was 48.5%,while rosiglitazone showed no significant antitumor effect in the same experiment(p=0.794).Interestingly,high,mid and low dosage of rosiglitazone combined with PLD exhibited great antitumor effect,with an inhibition rate of 67.40%(10mg/kg),66.67%(4mg/kg)and 60.46%(1 mg/kg)respectively.The weight of the dissected tumor mass in combination group is significantly less than the PLD alone(p=0.031).The inhibition rates of high,mid and low dosage of asiatic acid combined with PLD were 59.06%(10mg/kg),71.07%(4mg/kg)and 59.52%(1mg/kg),which were much higher than asiatic acid alone(inhibition rate=19.47%).The tumor weight in combination group(asiatic acid 4 mg/kg+PLD)was significantly less than PLD alone(p=0.043).An obvious intratumoral stroma sediments was seen by H&E stain in the saline control group,indicating the undisturbed collagen,and the boundary between stromal cells and cancer cells remained clear.In rosiglitazone and asiatic acid alone group,the microscopy field was filled with packed tumor cells but no observable collagen.Meanwhile,dramatic necrosis was seen in PLD group,the stromal cells and collagen was much more than either rosiglitazone or asiatic acid alone.The percentage of collagen within tumor tissue was 0.942±0.111、0.742±0.119 and 0.800±0.17 respectively in saline control,rosiglitazone and asiatic acid group.Rosiglitazone and asiatic acid treatment was able to dramatically increase the tumor accumulation of doxorubicin,through the analysis of tumor fluorescence intensity.③MTT results showed that rosiglitazone failed to synergistically increased the anti-proliferative ability of doxorubicin,the IC50 values were 3.84 μM(10μM),3.67μM(4μM)and 9.18μM(10μM)in high,mid and low dose rosiglitazone group.The expression of E-cadherin and Vimentin was unchanged after cells were challenged with 4 μM of rosiglitazone,suggesting that rosiglitazone would not influence the EMT in SW620 cells.The microarray resulted showed that 485 genes were up-regulated at least three fold and 448 genes were down-regulated at least three fold after rosiglitazone treatment.Both TGF-β and Smad signaling pathways were irrelevant to rosiglitazone induced collagen depletion.Conclusion:Either rosiglitazone or asiatic acid could increase the intratumoral accumulation of PLD via the depletion of tumor stromal collagen,thus improving the antitumor efficacy of chemotherapy.