Antitumor Effects And Mechanism Of SLP4,a Novel Naphthalene Polyamine Conjugate,on Hepatocellular Carcinoma In Vitro And In Vivo

Objective:Hepatocellular carcinoma?HCC?is one of the most common causes of cancer-related death in the worldwide.There are no obvious symptoms in early-stage HCC,and a majority of the patients are diagnosed when the disease is advanced which are no longer candidates for surgical curative therapy.Therefore,there are extremely limited choices of advanced liver cancer treatment.Moreover,due to the high rate of recurrence and metastasis,five-year survival rate of liver cancer patients is still not satisfactory.Hence,novel antitumor drugs were exploited has great significance for the treatment of liver cancer.The naphthalimide compounds,as DNA intercalators,have shown good antitumor activity.Natural polyamines or synthetic polyamine analogs are targeted vectors for efficient use of polyamine transporters on tumor cell membranes and have the ability to enhance cell selectivity and bioactivity of compounds.When conjugated to polyamine,the water solubility and antitumor activity of naphthalimide were obviously increased and allowed it to enter tumor cells selectivity.Therefore,the research and development of naphthalene lactam-polyamine conjugates with targeted transporter naphthalene into tumor cells is a new hotspot in the field of chemotherapy today.In this thesis,we evaluated the antitumor effect and molecular mechanism of SLP4,a novel naphthalene imide-polyamine conjugated,on hepatocellular carcinoma in vitro and in vivo.Methods:1 The effects of SLP4 on proliferation of SMMC-7721 and Hep G2 cells were evaluated in vitro and in vivo.The inhibitory effect of SLP4 on the proliferation of hepatoma cells?SMMC-7721,Hep G2?was determined by MTT assay,and the IC₅₀ values were calculated at the determined time?24 h and 48 h?.The effect of SLP4 on the cell cycle of SMMC-7721 and Hep G2 was detected using flow cytometry.SMMC-7721 and Hep G2 cells were stained by AO/EB,Rh123/Hoechst 33342,and AnnexinV/PI,and then the apoptosis was determined using HCS and flow cytometry.The expression of cell cycle and apoptosis-related protein influenced by SLP4 were detected by western blot.In addition,the effect of SLP4on the amount of polyamine in SMMC-7721 and Hep G2 cells was determined by high performance liquid chromatography?HPLC?.Based on H22 tumor mice model,the effect of SLP4 on the proliferation of solid tumors was evaluated in vivo.2 The effects of SLP4 on the migration and metastasis of HCC were investigated in vitro and in vivo.The migration of hepatoma cells was evaluated by cell scratches and Transwell chamber experiments.The effect of SLP4 on the expression of migration-related proteins was detected by western blot.The effect of SLP4 on lung metastasis and the prolongation of life of mice were evaluated with H22 tumor model in mice.Results:1 SLP4 inhibits the proliferation of HCC.The IC₅₀ values of SLP4 on the proliferation of SMMC-7721 and Hep G2 cells were?17.5±0.95??M and?19.4±1.35??M after co-incubation for 24 h,respectively.After treatment for 48 h,the IC₅₀ values were?8.56±0.86??M and?9.88±1.21??M,respectively.In addition,SLP4 could induce G₂/M phase arrest and apoptosis in SMMC-7721 and Hep G2 cells.Western blot analysis showed that SLP4 could up-regulate the protein expression of p53,p21,Cyclin B1,p-Cdc2,Bax,c-PARP1 and SSAT,while it down-regulated the expression of Cdc2,Bcl-2 and CyclinD1.The results of HPLC showed that SLP4 could decrease the amount of polyamines in SMMC-7721 and Hep G2 cells.Furthermore,SLP4 have significantly reduced solid tumor volume in the H22 tumor model in mice.2 SLP4 can inhibit the migration and metastasis of HCC.Cell scratch test and Transwell chamber assay showed that SLP4 could inhibit the migration of SMMC-7721 and Hep G2 cells.Western blot analysis showed that the expression of?-catenin,MMP7 and N-cadherin were down-regulated by SLP4,and the E-cadherin was up-regulated in both cells.The results showed that SLP4 could inhibit tumor metastasis and could prolong its survival time moderately in the H22tumor model in mice.Conclusions:SLP4 have induced G₂/M phase arrest and apoptosis in SMMC-7721 and Hep G2 cells via p53-mediated signaling pathway.On the other hand,SLP4 could decrease intracellular amount of polyamine via up-regulating the expression of SSAT,and then it exerted an important role in inhibiting tumor proliferation,migration and metastasis.