The Role Of Notch Signaling In Drug Resistance Of Osteosarcoma In Hypoxia And Involved Mechanism

Research Background:Osteosarcoma(OS)is the most common primary malignant bone tumor that seriously affects the physical and mental health of young adults.Chemoresistance(especially multi-drug resistance)is the main factor leading to the failure of osteosarcoma treatment.Therefore,to explore the method of reversal of drug resistance is of great significance.Studies have shown that tumor microenvironment and a variety of signaling pathways involved in tumor drug resistance.Hypoxic microenvironment is an important factor affecting tumor development.Hypoxia inducible factor-1(HIF-1)is a key nuclear transcription factor that regulates gene expression under hypoxia.HIF-1αis the active functional subunit of HIF-1,which is closely related to the stability of HIF-1.Local hypoxia in tumor tissue can stimulate the expression of HIF-la protein significantly,which regulates transcription of downstream target genes and produces corresponding tumor biological effects.Studies have shown that hypoxia can induce a variety of tumor chemoresistance.Notch signaling pathway is involved in the regulation of the normal cell survival,proliferation,differentiation,apoptosis and some other physiological processes.Notch signaling pathway consists of Notch receptors,Notch ligands and cellular effectors.In a variety of tumors,Notch was significantly abnormal expression and closely related to the development and progression of tumors.Notch is involved in the proliferation,apoptosis and drug resistance of many kinds of tumors.Recent studies have found that interaction between hypoxia and Notch involved in the development of a variety of tumors,and is a key factor affecting tumor growth and chemoresistance.Section ⅠExpression of HIF-1α,Notchl,MDRl/P-gp and MRP1 in human osteosarcoma and correlational researchObjective:The expression of HIF-1α,Notchl,MDR1/P-gp and MRP1 in human osteosarcoma pathological specimens and the classic cell line MG-63 were detected and their correlations were analyzed.It can provide the theoretical basis for the follow-up study on the interaction among HIF-1α,Notch1,MDR1/P-gp and MRP1 in the pathogenesis and progression of osteosarcoma.Materials and Methods:1.Immunohistochemistry was used to detect the expression of HIF-1α,Notchl,MDR1/P-gp and MRP 1 protein in osteosarcoma and osteochondroma clinical specimens.2.Statistical analysis of the correlation among expression of HIF-1α,Notchl,MDR1/P-gp and MRP1 protein in clinical specimens.3.The mRNA expression of HIF-1α,Notchl,MDR1/P-gp and MRP1 in human osteosarcoma cell line MG-63 was detected by RT-PCR.Results:1.Immunohistochemical staining results showed that the positive rate of HIF-la in osteosarcoma was 83.3%,osteochondroma control group was 25%,the difference was significant(P<0.01);The positive rate of Notchl was 77.8%in osteosarcoma group and 21.4%in control group,the difference was significant(P<0.01).The positive rate of MDR1/P-gp protein was 44.4%in osteosarcoma group and 14.3%in control group,the difference between the two groups was statistically significant(P<0.05).The positive rate of MRP 1 was 69.4%in osteosarcoma group and 28.6%in control group,the difference was significant(P<0.01).The results showed that the positive expression rates of HIF-1α,Notchl,MDR1/P-gp and MRP1 in osteosarcoma tissue were higher than those in osteochondroma tissue.2.Correlation analysis of the expression of HIF-1α,Notchl,MDR1/P-gp and MRP1 protein in osteosarcoma tissue was performed by spearman rank correlation statistical analysis.The results showed that there was a significant positive correlation between the expression of HIF-1α and Notchl,the expression of Notch1 and MRP1,as well as the expression of HIF-la and MRP 1.However,no significant correlation was found between the expression of MDRl/P-gp and HIF-1α,Notch1 or MRP1.3.The mRNA expression of HIF-la,Notchl,MDR1/P-gp and MRP1 in human osteosarcoma cell line MG-63 was detected.The results showed that in MG-63,Notchl was highly expressed,MRP1 was low,while HIF-la and MDR1/P-gp were moderately expressed.Conclusion:1.The expression of HIF-1α,Notch1,MDR1/P-gp and MRP1 in osteosarcoma and osteochondroma were statistical different.The positive rates of four proteins in osteosarcoma tissue were higher than those of osteochondroma.2.There was a significant positive correlation between HIF-la and Notchl,MRP1 and Notchl,MRP1 and HIF-1α protein expression in osteosarcoma tissue.However there was no correlation between MDR1/P-gp and the other three proteins.The results suggest that there are interactions between HIF-la,Notchl,MRP1 proteins in the pathogenesis and progression of osteosarcoma.3.HIF-1α,Notch1,MDR1/P-gp and MRP 1 are expressed in human osteosarcoma cell line MG-63.Section ⅡThe role of Notch signaling pathway in drug resistance of hypoxic osteosarcoma cellsObjective:Investigate the effect of hypoxic environment on the proliferation,cycle and drug resistance of osteosarcoma cells;To study the expression of HIF-la,Notchl,MDR1/P-gp and MRP1 in hypoxic osteosarcoma;Clarify the mechanism of hypoxia and Notch pathway in osteosarcoma drug resistance by downregulating Notch1.Materials and Methods:1.MTT method was used to detect the effect of hypoxia on proliferation of osteosarcoma cell line MG-63:Osteosarcoma cell line MG-63 was exponentially growing,seeded in 96-well cell culture plate and placed respectively in normoxia and hypoxic environment(1%O2,94%N2 and 5%CO2)cultured 12 h,24 h,48 h,72 h.MTT assay was used to detect the effect of hypoxia on osteosarcoma cell proliferation.2.The effect of hypoxia on the cell cycle of osteosarcoma cell line MG-63 by flow cytometry:MG-63 cells were cultured in hypoxia for 48 h,and the cell cycle was detected by flow cytometry after PI stained.3.The effect of hypoxia on chemosensitivity of osteosarcoma MG-63 cells by MTT assay:MG-63 cells were cultured under normoxia and hypoxia for 24 h,then treated with different concentrations of cisplatin(0-80 μm),Doxorubicin(0-5μm),methotrexate(0-5 μm)or ifosfamide(0-12 μg/ml)for 24 h under normoxia and hypoxia.MTT assay was used to detect cell proliferation.Then we conducted the analysis of chemotherapy sensitivity.4.MG-63 cells were cultured in hypoxia and normoxia,total cellular protein was extracted and Western blot was used to detect the expression of HIF-1α,Notch1,MDR1/P-gp and MRP1.5.The effect of siRNA-mediated downregulation of Notchl on the proliferation of osteosarcoma cells in hypoxia by MTT assay:In hypoxia,Notch1 was downregulated by siRNA transfected into osteosarcoma cells.MTT assay was used to detect the expression of 12 h,24 h,48 h and 72 h osteosarcoma cell proliferation.6.Flow cytometry was used to detect the effect of down-regulating Notch1 on the cell cycle and apoptosis of osteosarcoma cells in hypoxia.In hypoxia,osteosarcoma cells down-regulated Notch1 by siRNA transfection,PI staining and flow cytometry were used to detect cell cycle changes and apoptosis.7.MTT assay detects the effect of Notch on the drug sensitivity of osteosarcoma cells in hypoxia:Notch1 was downregulated by siRNA transfected into MG-63 cells cultured in hypoxia for 24 h.Then MG-63 cells were treated with hypoxia for 24 h.After being treated with different concentrations of cisplatin(0-80 μM),doxorubicin(0-5 μM),methotrexate(0-5 μM)or ifosfamide(0-12 μg/ml),MG-63 cells were treated in hypoxia for another 24 h.The cells were then assayed by MTT assay.8.Western blot was used to detect the effect of down-regulating Notch1 on the expression of MRP1 in osteosarcoma MG-63 cells in hypoxia:Notchl siRNA was used to block the expression of Notch1 in hypoxic MG-63 cells.Then the cells were cultured in hypoxia for 48 h.Western blot assay was used to detect the protein expression.Results:1.MTT results showed that hypoxia promoted the proliferation of MG-63 cells,and a statistically difference was found between the two groups(P<0.05,P<0.01,P<0.05)at 24 h,48 h,72 h respectively.Among them,compared with normoxia control group,the effect of hypoxia for 48 h on cell proliferation was significantly different(P<0.01).The results showed that hypoxia 48 h had a strong effect on promoting proliferation of MG-63 cells.2.Flow cytometry was used to detect the cell cycle distribution of MG-63 cells cultured in hypoxia for 48 h.The results showed that the proportion of cells in G0/G1 phase of hypoxia group(41.79%± 3.25%)and normoxia group(53.87%±2.31%).A statistically significant difference was found between the two group(P<0.01).Hypoxia can promote cell cycle progression,suggesting that hypoxia can promote the proliferation of osteosarcoma MG-63 cells.3.MG-63 cells were cultured in normoxia and hypoxia for 24 h,then treated with cisplatin(0-80 μM),doxorubicin(0-5 μM),methotrexate 0-5 μM)or ifosfamide(0-12 μg/ml)for 24 h in the respective environment.In hypoxia,osteosarcoma MG-63 cells significantly increased resistance to different chemotherapeutic drugs.The median inhibitory concentrations(IC50)of hypoxic osteosarcoma MG-63 cells to cisplatin,doxorubicin,methotrexate or ifosfamide were 42.55±1.62 μM,3.13 ± 0.08 μM,3.05 ± 0.07 μM,8.18±0.04 μg/ml.The IC50 of cisplatin,doxorubicin,methotrexate or ifosfamide in normoxia osteosarcoma MG-63 cells were 59.63 ± 1.49 μM,3.97 ± 0.03 μM,3.98 v 0.03 μM,9.97 ± 0.07μg/ml.Compared with the IC50 of cisplatin,doxorubicin,methotrexate or ifosfamide in hypoxia and normoxia MG-63 cells,there was a statistical difference(P<0.015 P<0.05,P<0.01,P<0.01).4.The expression of HIF-1α,Notch 1-ICN and MRP1 protein in hypoxia group was detected by Western blot.The results showed that the expression of HIF-la,Notchl-ICN and MRP1 in hypoxic group increased.However,the expression of MDRl/P-gp protein was not significantly different.The results show that hypoxia activates Notch signaling pathway and up-regulates MRP1 in MG-63 cell.5.The results of MTT assay showed that the cell proliferation was decreased in hypoxia-interference group,and the cell proliferation decreased significantly after 48h(P<0.01).However,there was no statistical difference in the group of hypoxia or hypoxia-interference control group.Hypoxia may promote osteosarcoma cell proliferation by upregulating Notchl expression.6.Flow cytometry was used to detect the effect of down-regulating Notch1 on the cell cycle of MG-63 cells in hypoxia.The results showed that downregulation of Notchl could lead to an increase in the proportion of G0/G1 phase in Hypoxia-interference group compared with hypoxia group and the hypoxia-interference control group.Compared with the control group,the hypoxia-interference group showed 17.53%apoptosis.Downregulation of Notchl led to an increase in the proportion of G0/G1 phase in hypoxia-interference group compared with hypoxia group and the hypoxia-interference control group.7.To clarify the effect of Notch on the chemoresistance of osteosarcoma cells in hypoxia,MG-63 cells were treated in hypoxia for 24 h,and then transfected by Notchl siRNA.Next day,MG-63 cells were treated with different concentrations of four drugs after another 24 h hypoxia culture for MTT test.The results showed that siRNA-mediated downregulation of Notchl can lead to enhanced drug sensitivity of osteosarcoma cells to cisplatin,doxorubicin,methotrexate or ifosfamide in hypoxia.Downregulation of Notchl can reverse the hypoxic osteosarcoma cells resistant.Meanwhile,the median inhibitory concentrations(IC50)of hypoxia-interference group in cisplatin,doxorubicin,methotrexate or ifosfamide were 33.39 ± 1.62 μM,2.72 ± 0.02 μM,2.01 ± 0.10 μm,5.79 ± 0.15±g/ml respectively.The IC50 values of half-cell inhibitory concentration of cisplatin,doxorubicin,methotrexate or ifosfamide in osteosarcoma MG-63 cells in hypoxia-interference control group were 58,08 ± 3.49 μM and 4.18 ± 0.07μM,3.94 ± 0.12 μM,9.61 ± 0.98 μg/ml.Compared with IC50 of cisplatin,doxorubicin,methotrexate or ifosfamide,the IC50 of MG-63 cells in hypoxia-interference group and hypoxia-interference control group were statistically different(P<0.01,P<0.01,P<0.01,P<0.05).8.In order to clarify whether MRP1 protein is regulated by Notch pathway in hypoxia,MG-63 cells with downregulation of Notchl were cultured in hypoxic for 48h.The results of Western blot showed that the expression of Notchl-ICN and MRP1 protein in the cells of hypoxia-interference group decreased after Notchl knockdown(P<0.01).There was no significant difference in the expression of MDR1/P-gp between hypoxia-interference group and hypoxia-interference control group.Conclusion:1.Hypoxia can promote osteosarcoma cell proliferation by regulating cell cycle.Down-regulation of Notch1 expression can increase osteosarcoma cells in G0/G1 phase and decrease osteosarcoma cell proliferation.2.Hypoxia induces drug resistance in osteosarcoma cells.Down-regulation of Notch1 can promote the apoptosis of osteosarcoma cells in hypoxia and reverse the hypoxia-mediated drug resistance of osteosarcoma cells.3.Expression of MRP1 protein can increase in hypoxic osteosarcoma cells.Down-regulation of Notchl can reverse hypoxia-induced multidrug resistance in osteosarcoma cells by decreasing MRP1 protein expression.4.Notchl is an effective molecular target for reversing drug resistance in osteosarcoma.