Blue Light-induced Proliferation Inhibition And Apoptosis Of Acute Myeloid Leukemia Cells

Phototherapy is a very important method for tumor treatment,including photodynamics,photothermotherapy and photoimmunotherapy,which mainly use infrared light or ultraviolet light as the main therapeutic light source.Blue light(BL)is an important part of natural light.Its wavelength is between 400~495 nm and has relatively high energy as a cold light source.With the application of light emitting diodes(LEDs)which has long-life,low-power,high-brightness,it has been widely used in various fields and provides a very good illuminant for biomedical applications.Currently,blue light has been widely used in the clinical treatment of jaundice hepatitis and acne.In biological basic research,the study of blue light on proliferation inhibition,differentiation and related mechanisms of a variety of tumor cell types provides the basis for the research of blue light on the biological response of tumor cells in vitro and the possibility of blue light for clinical treatment of tumors.Acute myeloid leukemia(AML)is a subtype of common leukemia,and it is a highly heterogeneous and diversified malignant clonal blood disease,at present,the treatment of AML is mainly based on combination chemotherapy.Chinese scientists have used all-trans retinoic acid(ATRA),arsenic and other methods for the treatment of acute promyelocytic leukemia differentiation and have achieved very important clinical effect.Although the efficacy of combined chemotherapy is significant,with the increase in the number of chemotherapy,the patient eventually suffers from problems such as increased drug resistance,cytotoxicity,myelosuppression,and increased risk of infection.This type of induction chemotherapy cannot be continued for patients.Therefore,exploring a new type of treatment or improving current chemotherapy is an important frontier in this field.Blue light irradiation is an important physical therapy for leukemia.Currently,blue light has shown significant inhibition of proliferation in leukemic cells such as B cell lymphoma(A20)cells.In this thesis,blue light LED was used as the light source.AML-M3(HL60),AML-M5(U937)and AML-M2(Kasumi-1)AML cells models were selected to investigate the proliferation inhibition and apoptosis of three representative leukemia cells induced by blue light.Meanwhile the blue light was combined with other chemotherapy drugs,nano drugs to treat leukemia cells and provide research foundation for the clinical treatment of leukemia.The main research contents and results are as follows:1.HL60 cells are representative cell lines of AML-M3 leukemia.Blue light is combined with biocompatible nanodiamonds(NDs)and ATRA drugs(ATRA-BL-ND)to study proliferation inhibition and apoptosis of HL60 in vitro.The results showed that(1)blue light irradiation significantly enhanced the proliferation inhibition and apoptosis of HL60 cells.The apoptosis was caused by the increase of reactive oxygen species(ROS)content,decrease in the expression of anti-apoptotic gene B-cell leukemia/lymphoma 2(Bcl-2)mRNA,and mediated by the activity of apoptosis protein caspase-3.(2)ATRA-BL-ND synergistically promoted the proliferation inhibition of HL60 cells with a high inhibition rate of 75%,which was 4.4 times that of ATRA drug group.The inhibition of cell proliferation induced by ATRA-BL-ND could be attributed to safe and non-toxic blue light and high biocompatibility and drug-loaded NDs.2 The HL60 xenograft tumor model in nude mice was established.The inhibition of tumor under blue light irradiation and ATRA-BL-ND synergism was investigated for the first time.(1)The tumor inhibition rate of blue light and ATRA-BL-ND was more than 50% and 95% on the 7th day.(2)The tumor inhibition was achieved through mitochondrial apoptotic pathway.(3)From the perspective of toxicology,there was no toxicity of blue light irradiation on HL60 nude mice.From the above vitro and vivo experiments,blue light irradiation and ATRA-BL-ND provide a basis for development a high efficiency and low toxicity leukaemia treatment methods.3.AML-M5 leukemia is characterized by diffuse histiocytic lymphoma,and its representative cell line is U937 cells.The study on the inhibition and apoptosis caused by blue light on U937 cells showed that blue light could specifically inhibit the proliferation of U937 cells and promote apoptosis.The mechanism is as follows: blue light will cause the production of ROS and the decrease of mitochondrial membrane potential(??)regulated by the Bcl-2 apoptotic proteins family.The increase of mitochondrial membrane permeability will cause the release of apoptotic factors,which could lead to the activation of apoptosis-related proteins caspase-9 and caspase-3.We applied the homoharringtonine(HHT)and blue light synergistically on U937 cells.blue light induced cell proliferation inhibition was 2.2 times than that of HHT;The synergistic effect of HHT-BL has a better proliferation inhibition than that of blue light or HHT alone.Through studies on the expression of ROS,??,and Bcl-2 apoptosis-related genes and caspase-related proteins,it was determined that HHT-BL synergistically promotes apoptosis through mitochondrial apoptosis pathway.The above results provide the basis for the study of the use of blue light and drug in the treatment of clinical leukemia.4.Kasumi-1 cells are representative cell lines of AML-M2 leukemia.It was found that only blue light significantly inhibited cell proliferation(40%).According to apoptosis,quantification of gene expression,protein activity,and Western blot assays,it was preliminarily found that blue light promoted apoptosis of Kasumi-1 cells,ROS play an important role in the apoptosis of cells,and activation of caspase-3 and PARP which indicated that apoptosis induced by blue light was through the caspase protein-dependent apoptosis pathway.Blue light down-regulated the expression of gene and protein of AML1-ETO pathogenic fusion.After adding caspase inhibitor,it could reduce the degradation of ALM1-ETO expression of fusion protein induced by blue light,which proved that the degradation of fusion protein AML1-ETO depends on activation of caspase-3 protein induce by blue light.These results provide a basis for the application of blue light in the treatment of AML-M2 leukemia.This article explored the inhibition of proliferation and apoptosis of HL60,U937,Kasumi-1 leukemia cells by molecular biology and other experimental methods.It achieved a high proliferation inhibition rate and apoptosis in vitro cell experiments and achieved a higher tumor inhibition effect in vivo tumor inhibition experiments.On the other hand,the combination of blue light and drug/nano-drug complexes could provide a novel synergistic method that reduces drug toxicity for clinical treatment of leukemia.The above results provide new ideas and research basis and possibility for achieving the application of blue light in the clinical treatment of leukemia.