| Research backgroundPrimary liver cancer (PLC) is one of the most common malignant tumors in human body, with the characteristics of aggressive, high-grade malignancy, short survival, and the lack of typical symptoms in the early stage. Most of the patients were diagnosed in the late-stage and were difficult to cure. The five-year survival rate of PLC is still dissatisfactory, which can not be solved only by clinical surgical treatment. Chemotherapy is an essential method for PLC, but the long-term use of traditional antitumor drugs would induce the drug resistance of tumor cells, which greatly affected the clinical application of chemotherapy drugs. The side-effects of chemotherapy could make the patients hard to tolerate, such as the suppression of bone marrow and immunization and the side-effects of gastrointestinal tract. Combined chemotherapy may reduce the side-effects and delay the onset of drug resistance.Leukemia, as one of the most common hematological malignancy, is malignant clone diseases of hematopoietic stem cells. Nowadays chemotherapy is still the main treatment for leukemia. It has better prognosis for Leukemia with the detailed understanding of pathogenesis, the improvement of combined treatment and the application of hematopoietic stem cell transplantation in recent years. However, the treatment of leukemia is significantly restricted by the following factors, such as the multi-drug resistance (MDR), the expensive support treatments after chemotherapy, the side-effects of chemotherapy drugs and the low success rate/higher cost of hematopoietic stem cells transplantation. Thus, it is focused on the effective, little side-effects, low cost and less resistant antitumor drugs nowadays.Artemisinin is a kind of colorless crystal with sesquiterpene lactone, extracted from the stems of compositae artemisia annua. Artesunate (Art) is a derivative of artemisinin, with good water-solubility and high antimalarial activity. The studies on the metabolism, pharmacokinetics and toxicology of Art are clear now. Art has been used as a malarial for the past two decades, with a solid foundation for the next studies on its clinical application.In recent years, the studies have showed that Art could kill tumor cells selectively, with less toxic effects on normal cells. Art can enhance the cytotoxic effects of some antitumor drugs, increase the sensibility of tumor cells to antitumor drugs and reduce the side effect of those drugs.Cisplatin (DDP) belongs to the cell cycle nonspecific antumor agents, with broad spectrum antitumor activity by its cytotoxicity and the ability to restrain the DNA replication of tumor cells. However, DDP also has many side-effects which restrict its applications in tumor chemotherapy. Art can inhibit the proliferation and induce the apoptosis of tumor cells, and has synergistic effect with some other antitumor drugs. The combination of these drugs can increase the sensitivity of tumor cells to antitumor drugs and reduce the side-effects of drugs. But there are no evidences if Art and DDP have synergistic effects on the inhibition of proliferation and the induction of apoptosis of hepatoma carcinoma HepG2cells or leukemia K562cells. On the other hand, the mechanism of reaction is unclear for the combination of drugs to induce apoptosis of hepatoma carcinoma cell and leukemia. Therefore, the MTT assay and CCK8assay were used to investigate the effect of Art or DDP on HepG2cells and K562cells in this study. Annexin V-PE/PI was applied to detect the early apoptotic of HepG2cells and K562cells. Enzyme-linked immunosorbent assay was applied to detect the telomerase activity of HepG2cells and K562cells.Ferrochelatase (FECH) is also called heme synthase and located on the mitochondrial membrane. It is one of the key enzymes in the synthesis of heme. and catalyze ferrous ion and protoporphyrin IX into heme. FECH is important to maintain the function of hemoglobin, myoglobin and cytochrome. It was reported that Art can block the hemoglobin biosynthesis and the mature of hematopoietic precursors in bone marrow. Art can also inhibit the proliferation and differentiation of bone marrow hematopoietic stem/progenitor cells in mice. So the effects of Art on heme biosynthesis may be associated with its antitumor activities. The aim of our study is to examine the synergetic effects and the mechanisms of Art and DDP on cell proliferation and apoptosis of hepatocellular carcinoma HepG2cells and leukemia K562cells. Thus, enzyme-linked immunosorbent assay was applied to investigate the serum concentration of ferrochelatase in KM mice after treated by Art and (or) DDP to discover the influence of ferrochelatase in tumor cells.Objectives1. To study the effect of combined Art and DDP on cell proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2and human leukemia cell line K562.2. To study the molecular mechanisms of the combined Art and DDP on the apoptosis induction of tumor cells.Methods1. MTT assay was used to investigate the effect of Art or DDP on HepG2cells in vitro.HepG2cells were divided into the control group with normal culture medium, and the different treatment groups. The treatment groups were cultured with different concentrations of Art (0.16,0.8,4,20,100μmol/L), DDP (0.16,0.8,4,20,100mg/L) or both Art (4μmol/L) and DDP (4mg/L), respectively. The MTT assay was used to identify the cell viability after the incubation for24h,48h and72h. 2. CCK8assay was applied to investigate the effect of Art or DDP on K562cells in vitro.K562cells were divided into the control group with normal culture medium, and the treatment groups were cultured with different concentrations of Art (0.16,0.8,4,20,100μmol/L), DDP (0.16,0.8,4,20,100mg/L) or both Art (4μmol/L) and DDP (4mg/L), respectively. The CCK8assay was used to identify the cell viability after the incubation for24h,48h and72h.3. Annexin V-PE/PI was applied to detect the early apoptotic of HepG2cells and K562cells.HepG2or K562cells were seeded in6-well plate and treated with normal culture medium. The treatment groups were cultured with Art (4μmol/L), DDP (4mg/L) or both Art (4μmol/L) and DDP (4mg/L), respectively. When the cells were grown up to70%-80%confluence, the cells were digested after48h, washed twice with pre-cold PBS and resuspended with lxbinding buffer. Then5μL Annexin V and10μL PI were added into1×105cells to stain cells for15min at room temperature. At last the cell apoptosis were analyzed by Flow Cytometry.4. Enzyme-linked immunosorbent assay was applied to detect the telomerase activity of HepG2cells and K562cells.HepG2or K562cells were seeded in6-well plate and treated with normal culture medium. The treatment groups were cultured with Art (4μmol/L), DDP (4mg/L) or both Art (4μmol/L) and DDP (4mg/L), respectively. When the cells were grown up to70%-80%confluence, the cells were digested at48h, repeated freeze-thaw cycles, extracted the cell contents. The telomerase activities of HepG2cells or K562cells in the cytoplasm were determined by human TE ELISA kits. The specific steps were according to the instructions of the ELISA kits.5. Enzyme-linked immunosorbent assay was applied to investigate the serum concentration of ferrochelatase in KM mice.After intraperitonealy injected with Art (30mg/kg/d), DDP (lmg/kg/d), or both Art and DDP (Art30mg/kg/d+DDP1mg/kg/d) for14d, the KM mice were decapitated and blood samples were collected. The serum concentrations of ferrochelatase in KM mice were determined by the mice FECH ELISA kits. The specific steps were according to the instructions of the ELISA kits.6. Statistical methodsThe assay data can be expressed as Mean+SD. The2-independent-sampIes T test was used to analyze the differences between the groups of treatments. The univariate analysis of variance was used to analyze multi-groups data. Multiple comparisons can be used on the premise that there are significant differences among groups. The difference (P<0.05) was considered with statistically significance. The software of SPSS13.0for windows was used to analyze all the data of the studies.Results1. Artesunate can effectively inhibit the cell proliferation and induce the apoptosis in HepG2cells.The survival rate of HepG2cells treated by Art and(or) DDP for24h were (116.09±18.40)%、(84.08±6.54)%、(53.85±2.18)%. he survival rate of HepG2cells treated by Art and(or) DDP for48h were (100.55±10.19)%,(70.48±8.84)%and (52.36±9.83)%repectively. he survival rate of HepG2cells treated by Art and (or) DDP for72h were (89.05±12.89)%,(37.69±3.87)%and (5.18±2.99)%. Compared with Art or DDP treatment alone, Art can significantly enhance the inhibitory effect of DDP on the cell proliferation of HepG2(P<0.05). The combined Art and DDP treatment can significantly increase the induction of apoptosis in HepG2cells compared with Art or DDP treatment alone (P<0.05).2. Artesunate can effectively inhibit the proliferation and induce the apoptosis of K562cells.he survival rate of K562cells treated by Art and(or) DDP for24h were (100.63±9.71)%,(80.92±4.45)%,(59.85±7.93)%. he survival rate of K562cells treated by Art and(or) DDP for48h were (92.13±10.59)%,(67.40±4.53)%,(61.14±6.11)%. The survival rate of K562cells treated by Art and(or) DDP for72h were (44.78±2.40)%,(25.16±1.29)%,(22.82±6.43)%. Compared with Art or DDP treatment alone, Art can significantly enhance the inhibitory effect of DDP on the cell proliferation of K562(P<0.05). The combined Art and DDP treatment can significantly increase the induction of apoptosis of K562cells compared with Art or DDP treatment alone (P<0.05).3. The telomerase activity of K562cells and HepG2cells treated by Art and (or) DDP were detected by ELISA assay.The telomerase activity of HepG2cells treated by Art and(or) DDP for48h were (9.114±0.004) U/L,(7.806±0.005) U/L,(13.317±0.004) U/L; The telomerase activity of HepG2cells treated by Art and (or) DDP for48h were (10.731±0.003) U/L,(7.806±0.008) U/L,(12.372±0.006)U/L. The telomerase activity of the combination group was significantly higher than Art or DDP group (P<0.05).4. the serum concentration of ferrochelatase in mice treated by Art and (or) DDP were detected by ELISA assay.After intraperitonealy injected with normal saline, Art, DDP, or both Art and DDP for14d, the serum concentration of ferrochelatase in mice were (343.00±47.50) pg/mL,(196.15±37.77) pg/mL,(235.04±41.12) pg/mL,(161.59±23.78) pg/mL. Compared with NS-treated or DDP-treated mice, the serum concentration of ferrochelatase in mice with combined Art and DDP treatment was reduced obviously (P<0.05).Conclusions1. Artesunate can effectively inhibit the proliferation and induce the apoptosis of HepG2cells and K562cells. Art can also enhance the cytotoxic effect of cis-platinum on proliferation and apoptosis of those two cell lines.2. The inhibition of ferrochelatase by Art might be the possible machanism of the synergy between Art and DDP on tumour cells. |
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