Differentiation Mechanism Of Leukemia Cells Induced By As₂O₃-created Warm Microenvironment

In recent years,tumor has became the first killer in many kinds of fatal diseases,which seriously affects the health of human beings.Conventional methods for tumor treatment are radiotherapy and chemotherapy,which have many disadvantages such as bad side effects,poor prognosis,and drug resistance.It is necessary to find new drugs with high efficiency and low toxicity for tumor treatment in the field of cancer research.The tumor differentiation therapy is a novel way for tumor treatment which prevents tumor growth by inducing tumor cell differentiation with differentiation agents,it has broken the traditional view that"once the cancer cells,cancer cells will always be.".The clinical study found that tumor differentiation therapy can not only overcome many disadvantages of radiotherapy and chemotherapy,such as liver and kidney toxicity,poor tolerance and immune suppression,but also can effectively improve the life quality and prolong the life span in cancer patients.The cell surrounding temperature is an important factor for tumor cell differentiation.Warm temperature can promote blood circulation,enhance immunity and induce tumor cell apoptosis.In the 70s of last century,As₂O₃ was found to have a significant effect on acute myeloid leukemia,and its effective rate was over 90%.It was found that As₂O₃ could not only promote tumor cell differentiation,but also reverse the multidrug resistance of tumor cells to other differentiation agents.It is generally believed that the differentiation mechanism of As₂O₃ may be related to the degradation of PML-RAR alpha fusion protein,the recovery of retinoic acid signaling pathway and the inhibition of JAK-STAT signaling pathway,ignoring the fact that As₂O₃ is a hot drug in herbal medicine.Warm herbal medicine can stimulate cell energy metabolism and create a"Warm Yang"environment.The aim of this study is to demonstrate the differentiation mechanism of leukemic cells induced by As₂O₃-created warm microenvironment based on the warm herbal property.This mechanism was supplementary verified by leukemia cell differentiation induced by Chuanwu Decoction-created or the nature-created warm environment.These findings can provide a new idea for tumor differentiation therapy.The subject include in vitro study,in vivo study and action mechanism.Firstly,in vitro,magnetic bead separator coated by CD34⁺/CD38^-antibody was used to sort leukemia cells into the non leukemia stem cells?K562?and leukemia stem cells?K562s?,and flow cytometry was used to detect the expression levels of the stem cell markers Nanog and Oct_4.Self renewal was detected by soft agar cloning.After two sorted cells were treated with As₂O_3,in vitro,Brdu-incorporation was used to detect cell proliferation,ATP kit and oxygen electrode were used to detect cell energy metabolism,temperature probe was used to detect the temperature change in culture medium,cell self-renewal ability was detected by cell ball formation under methylcellulose condition,benzidine staining was used to detect erythroid differentiation,Western blot was used to detect the expression levels of r-globin and CD235a?two erythroid differentiation markers?.In order to further prove the differentiation effect of warm microenvironment on leukemia cells,we also evaluated the leukemia cell differentiation induced by Chuanwu Decoction-created or nature-created warm microenvironment,ATP kit and oxygen electrode were used to detect cell energy metabolism,cell proliferation was detected by Brdu-incorporation,cell self-renewal ability was detected by cell ball formation under methylcellulose condition,benzidine staining was used to detect leukemia cell differentiation,Western blot was used to detect stem cell markers Oct₄ and Nanog and erythroid differentiation markers r-globin and CD235a.Then,subcutaneous transplantation tumor model of K562 and K562s was established to evaluate effects of As₂O₃ on the tumor differentiation in nude mice.Tumor-bearing mice survival rate was recorded,body temperature were measured to determine As₂O₃-created warm microenvironment,tumor volumes were measured to evaluate tumor proliferation,mice survival status were evaluated by autonomic activity.Elisa assay was used to detect plasma coagulation factor,plasma viscosity were detected by Hemorheology.Red or white blood cell count was detected by Blood cell analyzer,erythroid differentiation markers r-globin was detected by Western blot,erythroid differentiation markers CD235a was detected by Immunohistochemistry.In order to further prove the differentiation effect of drug-created warm microenvironment on leukemia cells,we treated mice with Chuanwu Decoction and then evaluated whether Chuanwu Decoction had a similar effect to As₂O₃ by detecting survival rate,body temperature,tumor size,autonomic activity,red or white blood cell count,coagulation factor,plasma viscosity and subcutaneous transplantation tumor differentiation in mouse model.Finally,we explore action mechanism of As₂O₃-created warm microenvironment on leukemia cell differentiation.:In vitro,after two sorted cells were treated with As₂O_3,AO staining was used to detect autophagy,immunofluorescence was used to detect the expression levels of LC3-B and p62?two autophagy related markers?,and immunohistochemistry was used to detect the expression level of P16INK4a?a senescence marker?.Simultaneously,we also evaluated the leukemia cell differentiation induced by Chuanwu Decoction-created or nature-created warm microenvironment in vitro,AO staining was used to detect autophagy,SA-beta-gal staining was used to detect senescence,immunohistochemistry was used to detect the expression level of P16INK4a.In vivo,after leukemia subcutaneous tumor-bearing mice were treated with As₂O₃,immunohistochemistry were used to detect the expression levels of P62?a autophagy related marker?and P16INK4a?a senescence marker?.In order to further prove the differentiation effect of drug-created warm microenvironment on mouse model,we also treated mice with Chuanwu Decoction,AO staining was used to detect autophagy,SA-beta-gal staining was used to detect cell senescence.Flow cytometry assay showed that the stem cell markers Nanog and Oct₄ in K562s cells were significantly higher than those in K562,and soft agar cloning assay showed that K562s cell had stronger self-renewal ability than K562 cell.In vitro,As₂O₃ at dose of 0.5u M which has no effect on cell proliferation within three days could promote cell heat production,slightly raise cell temperature and create a warm microenvironment,reduce the tumor sphere formation,increase expressions of?-globin and CD235a in K562s cells,but not in K562 cells.Leukemia cell differentiation induced by Chuanwu Decoction-created or nature-created warm microenvironment had a similar effect to As₂O₃ in vitro.In vivo,As₂O₃ at dose of 2mg/kg without side effect could improve survival rate,inhibit tumor growth,slightly raise body temperature,decrease coagulation factor content in plasma,decrease plasma viscosity,increase red blood cells and decrease white blood cells in blood.Chuanwu Decoction had a similar effect to As₂O₃of 2mg/kg in vivo.Mechanism research showed that As₂O₃ of 0.5 uM suppressed autophagy to result in cell differentiation senescence in K562s cells but promoted autophagy to directly induce senescence in K562cells in vitro.In vivo,As₂O₃ of 2mg/kg can not also significantly promote K562s subcutaneous transplanted tumor differentiation senescence but also directly induce K562 subcutaneous transplanted tumor senescence.The mechanism of As₂O₃-created warm microenvironment on leukemia cell differentiation was confirmed by Chuanwu Decoction-created warm microenvironment in vitro and in vivo..In summary,As₂O₃-created warm microenvironment can induce leukemic cell differentiation.The mechanism is that As₂O₃-created warm microenvironment is not directly kill leukemic cells but can change cell autophagic state to promote K562s cell differentiation and to directly induce K562 cell senescence,which ultimately prevents tumor growth.