Experimental Study On Anti-tumor Effect Of Doxorubicin Prodrug

Malignant tumor is a severe disease. Nowadays, the number of patients who wasdiagnosed of cancer increases every year due to environmental factors. It is a maincause of disease leading death and threatens to human health. Traditional treatmentsof cancer include surgery, radiotherapy and chemotherapy, of which chemotherapy isstill an optimal therapy among all. However, the effect of chemotherapy isunsatisfactory due to high recurrence and death rate, causing injury and suppression tobody systems. Thus it is in a great need to develop new chemicals to treat tumors butwith a minimum harm to normal tissues and organs simultaneously. The developmentof targeting drug has solved this difficulty by targeting the drug to tumor cells directly,killing tumors without injuring normal tissues thus improving the efficiency of thetreatment. Doxorubicin is an antitumor drug with a broad spectrum, however, the drugresistance and cardiotoxicity caused by long-term application has restricted its futureuse in clinic. Multiple types of DOX has been developed to reduce the side effectcaused by the application of DOX and to improve the anti-tumor efficiency.Nowadays, it is a hot research focus to synthesize tumor targeting prodrug by bondingDOX to tumor targeting transporter or molecules which can be specifically identifiedby tumor tissue. The DOX prodrug in our research was synthesized by bonding an Xgroup to DOX and manufactured by SiTaoLi TianJin Co., Ltd.In this work, the biological activity of DOX was analyzed. The results are asfollows.1. The inhibitory effect of DOX-P to human HepG2cellsMTT method was used to examine the anti-tumor activity of DOX-P on humancancer cell lines of HepG2. The result showed that DOX-P could inhibit the growthand proliferation of human HepG2cells. The HepG2cells without treating DOX-Pwere seeded at1×104cells/ml in96-well plates, and incubated with differentconcentrations of DOX-P ranging from2μmol/L to64μmol/L for24,48,72h,respectively. The HepG2cells incubated with8μmol/L concentration of DOX was set as positive control group. The survival rate of the cells was decreased with theincrease of DOX-P concentration, and concentration of8μmol/L DOX-P has highertoxicity to HepG2cells than that of DOX. The cell survival rate decreased along withthe time extension.2. The inhibitory effect of DOX-P to human SKOV3cellsMTT method was used to examine the anti-cancer activity of DOX-P on humancancer cell lines of SKOV3. The result showed that DOX-P could inhibit the growthand proliferation of human SKOV3cells. The SKOV3cells without treating DOX-Pwere seeded at1×104cells/ml in96-well plates, and incubated with differentconcentrations of DOX-P ranging from2μmol/L to64μmol/L for24,48,72h,respectively. The SKOV3cells incubated with8μmol/L concentration of DOX wasset as positive control group. The survival rate of the cells was decreased with theincrease of DOX-P concentration, and concentration of8μmol/L DOX-P has highertoxicity to SKOV3cells than that of DOX. The cell survival rate decreased along withthe time extension.3. The intracellular distribution of DOX-PDue to the significant fluorescence feature of DOX, the intracellular distributionof drug was studied using fluorescence microscope. The SKOV3cells incubated with8μmol/L concentration of DOX was set as positive control group. The SKOV3cellsincubated with8μmol/L、16μmol/L and32μmol/L three different concentrations ofDOX-P were tested. The result of experiment on fluorescence microscope indicatedthat there were only a few of DOX could enter the cells after incubation, however,intracellular distribution of all three different concentrations of DOX-P were higherthan free DOX. The fluorescence signals get stronger with the increase of theconcentration of DOX-P, indicating the targeting capability of the prodrug to tumorcells.4. The uptake efficiency of DOX-P by tumorsThe SKOV3cells incubated with8μmol/L concentration of DOX was set aspositive control group. The SKOV3cells incubated with8μmol/L、16μmol/L and32μmol/L three different concentrations of DOX-P were tested. The uptake efficiencyof DOX and its prodrug by tumor cells was analyzed by flow cytometry. The uptakeexperiment indicated that prodrugs could increase the uptake efficiency. The uptakeefficiency by tumors gets more efficient with the increase of the concentration of DOX-P, which indicate that the targeting capability of the prodrug is stronger thanthat of free DOX.5. The anti-tumor activity of DOX-P in vivoThe HepG2cells cultured in logarithmic growth phase were inoculated toathymic mice under armpit to establish tumor-bearing models. Animals were treatedwith different concentrations of DOX-P according to the results of former experiments.The weight change, tumor growth of the athymic mice and the tumor volume werecalculated and recorded to evaluate the antitumor activity of DOX-P in vivo. Theresults indicated that the tumor-bearing athymic mice model was set up successfully,and the prodrug could inhibit the growth of tumor possessing a time-dependentmanner. The inhibiting rate of DOX was21.5±4.32%, while the maximum inhibitingrate of DOX-P was42.1±4.12%. In addition, data testified that the half-life of athymicmice treated with DOX-P was prolonged and their survival number was increased.We have proved that compared with DOX, DOX-P could prolong half-life andimprove bioavailability with lower toxicity. Our work presented that DOX-P could bea potent and efficient application on tumor therapy.