The Experimental Study Of Cisplatin Combined With Exosomes On Tumor Inhibition

ObjectiveCisplatin is one of the anti-cancer drug which is the most widely used both at home and abroad. High-dose cisplatin chemotherapy for cancer is fast and strong,but it cause major side effects to the body. Tumor vaccine is a new category of treatment.It relies on the body's own strength to kill the tumor cells. This destruction is limited and slow. In our study ,we use low-dose cisplatin combined with H22 cell-derived exosomes. To study the anti-tumor effects and mechanisms of cisplatin(DDP) combined with exosomes.Our data may provide a basis to clinical application of chemotherapy combined with tumor vaccine in the future.Methods1.Taking the murine hepatocarcinoma cell line H22 as our experimental model. We observed the H22 cell's proliferation inhibition treated with cisplatin and its effect on Fas expression on H22 cell:the inhibitory rate of cell proliferation was detected using MTT assay. The effect of DDP on mRNA and protein levels of Fas in H22 was analyzed using RT-PCR and Western blot.2. Hepato-carcinoma H22 cell-derived exosomes were isolated and purified from the culture supernatants by serial ultracentrifugation and sugar density ultracentrifugation. The mice were immunized by H22 cell-derived exosomes. The expression of FasL mRNA on spleen lymphocyte was determined by RT-PCR.3.In vitro,we observed the specific cytotoxic activity of mice spleen lymphocyte elicited by exosomes,and DDP enhanced cytotoxicity in combination with CTL by MTT assay. H22 cell apoptosis was analyzed by Hoechest33258 stain and electron microscopy.4. H22 xenograft tumor models were established in BABL/C.We observed the inhibitory activity of cisplatin combined with exosomes' tumor vaccine on the growth of mice bearing transplanted H22 hepatocarcinom in vivo from the following aspects:the xenograft tumor were excised and weighted to calculate the tumor growth inhibitory rate;the survival of the mice was observed;sections from BABL/C xenograft tumor was subjected to HE staining to reveal the changes of histomorphology;transmission electron microscopy was used to test the ultramicro-structure alternations of BALB/C xenograft tumor.5.Based on the same H22 xenograft tumor models,we surveyed the effects of cisplatin combined with exosomes' tumor vaccine on the immune function in BALB/C mice from the following aspects: The spleens and thymuses were weighed,and the effect of cisplatin combined with exosomes on spleens and thymus index was determined;The proliferation and cytotoxicity of splenocytes were detected by MTT assay; Serum level of IL-2 and INF-γwas detected by enzyme-linked immunoabsorbent assay(ELISA).Results1.MTT test showed the the inhibitory rate that DDP inhibit the murine hepatocarcinoma cell line H22 at all experimental dose. The inhibitory rate increased with the dose increasing. DDP inhibited H22 cell in a dose-dependant manner.We chosed the low concentrations inhibition dose: 2.5μg/ml and 5μg/ml DDP as the concentration of following experiments. RT-PCR and Western blot showed: Fas increased 3.1 times and 4.9 times on the the mRNA level and 3.3times and 5.1times on the protein level after administering 2.5μg/ml and 5μg/ml DDP on H22 cells for 24 hours.2.RT-PCR showed: spleen lymphocyte could amplify specific target band of 456 bp after the mice were immunized by H22 cell-derived exosomes.The control group spleen lymphocyte could not see any specific target band.3.The exosomes elicited CTL-specific cytotoxicity was enhanced when H22 cells pretreatment with low dose(2.5μg/ml)DDP for 24 hours.The killing rate was higher than CTL itself when the effector/target rate was 10∶1,20∶1,40∶1,80∶1 respectively. Apoptosis was seen under Hoechest33258 staining and electron microscopy.4. The inhibitory activity was most strong in cisplatin combined with exosomes group,with the inhibition rate reached 64.4%. HE staining showed a large number of infiltrating lymphocytes in H22 xenograft tumor tissues in combination group. The electron microscope demonstrated the existence of apoptotic cells and apoptotic bodies in H22 xenograft tumor tissues in combination group.5. The spleen indexes and thymus indexes of mice bearing tumor in combination group were 9.96±0.91 and 3.51±4.79. Compared with control group,cisplatin combined with exosomes could induce lymphocyte proliferation( P <0.01),and generate strong specific cytotoxic spleen lymphocyte response(P <0.01).The serum level of IL-2 and INF-γwas (421.51±39.17) pg/mL and (39.86±3.15) pg/mL in combination group,and the difference was significant compared with the control group(P <0.01).Conclusions1.Fas increased gradually after administering DDP on H22 cells. FasL on mice spleen lymphocyte increased after the mice were immunized by exosomes. The therapy of DDP combined with exosomes had significant synergistic effect against tumor. The mechanism of synergistic effect includes enhancement of CTL activity.2. Our data verified that cisplatin combined with exosomes markedly inhibit tumor growth in vivo,with the inhibition rate reached 64.4%. Low-dose cisplatin had not significantly affect on the immune system. Combination of cisplatin and exosomes exerts strong inhibitory activity on the growth of mice bearing transplanted H22 hepatocarcinom. The effect is correlated to the enhanced immune activity in mice.