Study On The Activity Of Chloroquine Enhancing 20(S)-PPD Induced Cytotoxicity In Human Hepatoma HepG2 Cells

Liver cancer is a common malignancy and is hard to detect in its early stages.As a result of the high morbidity and mortality,this disease has become a great threat to public health.At present,the most widely used treatment of liver cancer is surgery.However,even tumors have been removed,patients are still bearing high risk for recurrence.In this case,the adjuvant medication is needed.Moreover,for patients who cannot get surgical treatment,systematic treatment including chemotherapy and radiotherapy is the main choice of treatments.However,the present clinical outcomes suggest that the prognosis of such treatment is not satisfactory.Therefore,new anti-cancer medicines need to be developed.Ginseng is a famous Chinese herbal medicine.Ginsenoside is the main active ingredient in ginseng.A variety of types of ginseng saponins have been found efficient and effective on suppressing tumor growth.The capabilities of these ginsenosides include killing tumor cells directly,inhibiting tumor angiogenesis,invasion and metastasis,and strengthening the body’s immune system.20(S)-PPD was found as a small molecule compound with good anti-proliferation activity on tumor cells in former research.Chloroquine has long been used to prevent and to treat malaria.But recent studies have shown that chloroquine also has good anti-tumor potential because of its ability of suppressing autophagy and activating the immune system.More importantly,chloroquine may have the ability to sensitize cancer cells to radiation and chemotherapy.Since liver cancer is prone to relapse and is susceptible to drug resistance,it would be nice if we can find two drugs that can synergize and inhibit liver cancer by multiple targets.Based on the anticancer properties of 20(S)-PPD and chloroquine,it is suspected that these two drugs may synergize against cancer.Then next,we carried out study as follows.In order to find out if the 20(S)-PPD and chloroquine could synergistically inhibit the growth of human liver cancer cell,we draw the agent-efficiency curves of the two drugs used alone and in combination on HepG2 cells and calculated the combination index of these two drugs.It turns out that the combination treatment of 20(S)-PPD and chloroquine could inhibit the growth of HepG2 cells.Next,DAPI staining was used to observe morphological changes of HepG2 cells.It shows that as time went on,increasing HepG2 cells upon combination treatment of 20(S)-PPD and chloroquine start to shrink,condense and blister,the chromatin start to condense and fracture,and finally,the apoptosis body appeared.It is hypothesized that the synergistic effect of 20(S)-PPD and chloroquine could promote the apoptosis of HepG2 cells.To further verify the result,we conducted western blot assay on HepG2 cells treated with 20(S)-PPD and chloroquine in combination.The results show that PARP,the caspase-3 substrate,was cleaved.The enzyme activity of caspases was detected,proving that caspase-3,and-9 were activated significantly.The flow cytometry was carried out.Comparing to which of cells treated by compound alone,the apoptosis rate of HepG2 cells under the synergistic effect of 20(S)-PPD and chloroquine markedly increased.Next,we tried to explore by which pathway,the apoptosis induced by combination treatment of 20(S)-PPD and chloroquine was activated.Western blot data show that the abundance of cleaved-caspase-9 is far higher than that of cleaved-caspase-8 at the same time.Therefore,we detected the mitochondria membrane potential changes in HepG2 cells,and the western blot was used to detect the inversion of Bax/Bak and the release of the Cyt c and Smac.All these results prove that the 20(S)-PPD and chloroquine could synergize with each other to activate the cell apoptosis by endogenous mitochondrial pathway.In addition,western blot assay of LC3-II showed that 20(S)-PPD and chloroquine synergistically inhibited the autophagy flux in HepG2 cells and released apoptosis from the antagonism produced by autophagy.There are good reasons to believe that the adverse effect on autophagy could be one of mechanisms for the synergistic effect of 20(S)-PPD and chloroquine.