| Background:Acute leukemia is acquired clonal disease caused by variation of hematopoietic progenitor cells, characterized by a marked proliferation of blast cell and dysdifferentiation of myeloid cells in the bone marrow. The incidence of the acute leukemia is about 2.76/10 million, is still the most common hematological malignancy, and the incidence has increased yearly since the past few years. Survival outcomes have greatly improved with combination chemotherapy, radiation therapy, immunotherapy, stem cell transplantation and molecular targeted therapy;however,Disease-free Survival (diseases-Free Survival, DFS) in acute leukemia is only about 30-40%, and the the long-term prognosis is very poor except for children with acute lymphoblastic leukemia (acute lymphoblastic leukemia, ALL). which has prompted us to further explore the new drugs.Differentiation-inducing therapy has been suggested in recent years, Its theoretical basis is that the specific genes that inhibit the differentiation of cells are reversible under certain conditions.Tumor cells can be induced to normal cells by differentiation inducer. ATRA and AS2O3 have been used in the clinical treatment of APL as differentiation inducing agents and greatly improve the remission rate and long-term survival rate of patients with APL.It is the first successful model of differentiation and treatment of malignant diseases, and it is also the most successful and a typical target treatment model. ATRA has been widely used in treatment of APL, AS2O3 always is used in relapsed or refractory cases. However, it is found that both ATRA and AS2O3 have distinct individual differences on curative effect, drug resistance, recurrence rate and other side effects with the expansion of clinical application to cases.Other differentiation inducing agents such as vitamin D3, interferon, and cytokine class are prevented to be widespread used in the clinical treatment of tumors due to various limitations. Therefore, it is an urgent need to find a new inducing differentiation and apoptosis agents with less toxic or side effect.Marine creatures can produce certain unique bioactive substances that accumulate in them in the process of their growth and metabolism, Some of these bioactive substances have shown the anti-tumor effects in vitro study. Tegillarca granosa is a sea creature that has been used as a traditional Chinese medicine to treat cancer for more than a century. It is rich in peptide and vitamin B12, with anti tumor, anti-virus and bacteria, immune regulation, and other effects. Haishengsu (Hss) is a purified protein from Tegillarca granosa, and recent research has confirmed that Hss has a distinct effect in both inhibiting the proliferation of renal carcinoma cells and leukemic cells and inducing the apoptosis of cells.However, these studies are mostly in terms of solid tumors or inhibition of tumor proliferation respect, if Hss can induce tumor cell differentiation or not and the mechanism has not been reported yet.This study focuses on the impact of Hss on the proliferation and differentiation of HL-60 cells in the leukemic cell line by taking tretinoin and AS2O3 as a positive control and make a comparative analysis between the effect of Hss and tretinoin and AS2O3, which are currently the most commonly applied clinical differentiation inducers,in the hope to find a new differentiation and apoptosis inducing agent.Objectives:1. To investigate the effects of Hss on proliferation of HL-60 cells.2. To study impact of Hss on the differentiation and apoptosis of the HL-60 cells and make a comparative analysis between Hss, ATRA and AS2O3.3. To illustrate the mechanism which HSS exerts its effect on the differentiation of HL-60 cells.Methods:1. Effects of Hss on proliferation of HL-60 cells1.1 HL-60 cells were exposed to various dosages of Hss(0.1mg/l, 1mg/ L, 10mg/L, 100mg/L and 1000 mg/L) and cultured separately for 12h,24h,48h,72h.1.2 MTT assay was used to examine absorbance value of HL-60 cells with different concentration Hss.1.3 The inhibitory rates of proliferation were calculated according to the absorbance values.I.4 IC50 of Hss was calculated by Graphpad Prism 5 according to concentrations and the relative proliferation inhibition rates.2. To observe the impact of Hss on the proliferation and differentiation of HL-60 cells and make a comparative analysis between the effects of Hss,tretinoin and AS2O32.1 ATRA,AS203 and Hss were added separately into each experimental group. The final concentration was ATRA 5umol/L, AS2O35umol/L and Hss 100mg/L,cultured for 72 hours.2.2 The proliferation of HL-60 cells was evaluated after the culture via MTT method.2.3 Morphological observation under light microscope afer Giemasa stain, followed by observation with photos.2.4 Measure the expression of CD11b and CD15 by means of FCM2.5 Cell apoptosis was detected by flow cytometry using Annexin V FITC/PI double staining before and after treatment.2.6 The cell cycle distribution of HL-60 cells was detected by flow cytometry.3. The mechanism which HSS exerts its effect on the differentiation and proliferation of HL-60 cells3.1 The Hss100mg/L group, ATRA5umol/L group,5umol/L AS2O3 group and control group were set up respectively, and cultured for 72 hours.3.2 The expression levels of bcl-2, bax and mpo mRNA were examined by RT-PCR.3.3 The protein levels of bcl-2 and bax were examined by western blotting.3.4 Taking ATRA and AS2O3 as positive control and made comparative analysis between the effect of Hss,ATRA and AS2O3.Result:1. Effect of Hss on proliferation of HL-60 cellsHss significantly inhibited the proliferation of HL-60 cells which followed a concentration-depedent and time-dependent manner at 100mg/L,1000 mg/L.The difference of absorbance value was not statistically significant between 0 mg/L,0.1mg/L, 1mg/L, 10mg/L at 12h?24h?48h?72h of treatment (P>0.05). The difference of absorbance value was not statistically significant between 0 mg/L, 100mg/L, 1000mg/L at 12h of treatment (P>0.05),but it was statistically significant at 24h?48h?72h of treatment.The IC50 value at 72h's treatment was 40mg/L.2. The impact of Hss on the proliferation and differentiation of HL-60 cells and a comparative analysis between the effect of Hss,tretinoin and AS2O32.1 Cell proliferation assay:Hss, ATRA and AS2O3 all inhibited the proliferation of HL-60 cells which followed a time effect manner. With the passage of time, their inhibition rate gradually increased. Each time point had statistical significance.The inhibition rate of Hss at 24h?48h? 72h of treatment were 25.23±5.13%, 39.13±3.14%?70.45±8.20%, tretinoin group were 56±3.31% 23.20±4.32%?44.22±6.26%, AS2O3 group were 4.25±3.82%, 36.80±2.45%?73.62±7.28%.Inhibition rate of Hss was clearly superior to that of tretinoin and AS2O3 at 24h of treatment.Inhibition rate of Hss at 48h?72h of treatment was statistically higher than ATRA (P<0.05), there was no significant difference compared with the AS2O3 (P>0.05).2.2 Morphological observation:after Giemsa stain was applied,control group was found that HL-60 cells were relatively large,the cell nucleus was big and round,and chromosomes were dispersed.The cell contained 2-4 nucleoli,the cytoplasm was basophilic and hyperchromatic,and the nucleo-cytoplasmic ratio was large. On the tretinoin side,after 72 hours'culturing, the cell morphology was significantly changed.The cell differentiated into a mature granulocyte,the cell body shrank,the cell nucleus was depressed,and the chromatin became dense and thick.The cell nucleus decreased and disappeared,the nucleo-cytoplasmic ratio was reduced,and some cells began to die.On the AS2O3 side,after 72 hours'culturing,most of the cells began to become apoptotic,the cell body shrank,karyopyknosis occurred,and the acidophilia of chromatin was enhanced.Part of the cell nucleus broke into several round granules of different sizes.Apoptotic bodys were observed in some cells.On the Hss side,after 72 hours'culturing,both differentiation and apoptosis could be observed.The cell body shrank,the cell nucleus was depressed,and the chromatin became dense and thick.The cell nucleus decreased and disappeared, the nucleo-cytoplasmic ratio was reduced,and also karyopyknosis and apoptotic body appeared.2.3 The change of differentiation antigen on cell surface:after 72 hours' culturing of Hss,ATRA and AS203,the expression of CD11b significantly increased.There was a statistical difference in comparison with the control group. Difference was observed between Hss and AS2O3. No difference was observed between Hss and ATRA. CD 15 showed a high expression in both the control group and the experimental group, without statistical difference.2.4 Cell apoptosis:after 72 hours'culturing with Hss,ATRA,AS2O3 in HL-60 cells,the apoptosis rate was significant increased compared with the control group. Apoptosis rate of Hss group was not significantly stronger than that of 5 ?m ATRA and 5?m AS2O3.2.5 The change of cell cycle:HL-60 cells in the exponential phase were mainly located in s phase and G2/M phase. After 72 hours culturing of Hss, ATRA and AS2O3, most of the cells remained in the G0/G1 phases. The number of cells in S and G2/M phases significantly decreased. There was a statistical difference in comparison with the control group. Differences were also observed between Hss and tretinoin, but not observed between Hss and AS2O3.3. The mechanism which HSS exerts its effect on the differentiation and proliferation of HL-60 cells.3.1 After 72 hours'culturing of 100mg/L Hss, the expression of bcl-2 in HSS group was markedly decreased from 0.89+0.12 to 0.68+0.06, compard with the control group,and the expression of bcl-2 in AS2O3 group and ATRA group was also decreased,while there was no statistical difference in comparison with the control group. No difference was observed between Hss and ATRA,or Hss and AS2O3. The expression of Bax in Hss group,AS2O3 group and ATRA group were 0.89±0.15,0.50±0.05,0.89±0.06, the control group was 0.18±0.07.The expression of the Bax gene was higher in experimental group than that in control group. There was no statistical difference between Hss group and ATRA group. The expression of the Bax gene was higher in Hss group than that in AS2O3 group.3.2 The expression of MPO in Hss group, AS2O3 group and ATRA group were 0.44±0.14?0.86±0.08?0.83±0.15, the control group was 0.95±0.17.The expression of the MPO gene was weaker in experimental group than that in control group.Expression of the MPO gene was mostly weakened in Hss group, and then the ATRA group. Differences were observed both between Hss and tretinoin and between Hss and AS2O3.3.3 Western blotting showed that expression of bcl-2 protein in Hss group, AS2O3 group and ATRA group were 0.45±0.09,0.36±0.11? 0.39±0.15, the control group was 0.76±0.08,decreased expression levels of bcl-2 protein were observed (P<0.05)? Expression of bax protein in Hss group, AS2O3 group and ATRA group were 0.70±0.12?0.74±0.10?0.69±0.09, the control group was 0.35±0.10,increased expression levels of bax protein were observed (P<0.05)?No significant chang was observed in expression of bcl-2 and bax protein between experimental group.Conclusion:1. Hss significantly inhibited the proliferation of HL-60 cells which followed a concentration-depedent and time-dependent manner,low concentration Hss couldn't inhibit the HL-60 cell proliferation, high concentration Hss could significantly inhibit the HL-60 cell proliferation. IC50 was 40mg/L.2. Hss had a significant effect in both inhibiting the proliferation and inducing the differentiation of HL-60 cells. Its ability to inhibite HL-60 cell proliferation and induce cell differentiation was close to 5umol/L ATRA and 5umol/L AS2O3,ever superior to them.3. HSS could down-regulate the expression of MPO gene and bcl-2 gene,and up-regulate the expression of Bax gene,which may affect the apoptosis of the cells via the bcl-2/bax pathway.4. There was a strong possibility that Hss may be a new kind of differentiation inducer.Of course,it was still requires further study,especially in vivo. |
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