| ObjectiveLeukemia is a kind of malignancy of hematopoietic system. Now, theprimary method to treat leukemia is high-dose combination chemotherapywhich has serious damage on immune/hematopoietic system and tissues/organs of the body. Patients usually die from systemic failure induced bychemotherapy. It will bring us a new idea to treat leukemia and othertumors if we can find a natural senescence or apoptosis inducers ofleukemia cells.Angelica sinensis polysaccharide(ASP) is the effective component ofAngelica which can supply and promote blood. Experiments showed thatASP has some Pharmacological effects, such as resisting to radiationdamage, promoting hematopoietic, confronting tumor and regulatingimmune, and so on. Our recent studies showed that ASP can delayhematopoietic stem/progenitor cells senescence definitely. Chinesemedicine theory holds that: many herbal medicine or its effectivecomponents have the effects of "two-way adjustment" or "strengthen body resistance and eliminating evil". It has not been reported whether ASP canpromote leukemia cells senescence. It will bring us a new strategy and wayto treat leukemia, if this scientific problem can be elucidated.Firstly, acute myeloid leukemia KG1α cell lines were used to researchthe senescent effect of ASP and cytarabine(Ara-C) on human leukemiacells in vitro. Sencondly, we established the NOD/SCID leukemia mousemodel. At the base of that, we researched the senescent effect of ASP andAra-C on human transplanted leukemia cells in vivo, we also discussed thestructure and function of the liver and spleen. Through the two parts ofexperiments via vitro and vivo, we elucidated the senescent effect and therelated mechanism of ASP and Ara-C on leukemia cells, the protectioneffect and mechanism of them on important viscera. We hope to providetheoretical and experimental foundation to the therapeutic method oftumors that regulating the malignant tumor cells senescence induced by theeffective components of natual medicine combined with anti-cancer drugs,to provide new ideas and methods to treat leukemia and other tumors.Methods1Leukemia KG1α cell lines in logarithmic growth phase were dividedinto4groups: ASP, Ara-C, ASP+Ara-C, and control groups.The cells weretreated with different concentration of ASP, Ara-C and ASP+Ara-C fordifferent time. The inhibited proliferation of KG1α cells was detected byEnzyme-linked immunosorbent assay Via CCK-8. Distribution of cell cycles in KG1α cells were examined by flow cytometry(FCM).Morphological alterations of senescent KG1α cells were observed byWright ’ s staining. The senescent cells were counted by SA-β-Gal staining.The senescence related proteins of P16and Rb were detected by Westernblotting. The senescent effect and the related mechanism of combinedadministration of ASP and Ara-C on KG1α cells were then observed.2K562cells were transplanted into the tail vein of mice to establishthe transplanted human leukemia NOD/SCID mouse model. Then weobserved the disease process of leukemia mice. we collected the eyeballblood to detect the amount and classification of white blood cells(WBC);took the femurs to count the bone marrow mononuclear cells (BMNCs).Morphological alterations of bone marrow cells of leukemia mice wereobserved by wright’s staining.The expression of DQα in bone marrow ofleukemia mice was detected by PCR and the source of the leukemia cellswas identified.3After the establishment of the transplanted human leukemiaNOD/SCID mouse model, leukemia mice were randomly divided intocontrol, ASP, Ara-C and ASP+Ara-C groups. The mice wereintraperitoneally injected with ASP, Ara-C and ASP+Ara-C. The eyeballblood was collected to detect the amount and classification of WBC. Thefemurs were taken to count BMNCs. The proliferation of BMNCs wasdetected by CCK-8; the distribution of cell cycles was analyzed by FCM; the capability of colony forming was examined by CFU-Mix cultivation;the senescent BMNCs were counted by SA-β-Gal staining; the senescencerelated proteins of P16、Rb、CDK4and CyclinD1were detected by Westernblotting. The senescent effect and possible mechanism of ASP and Ara-Cthat inducing BMNCs in the transplanted human leukemia mouse modelwere exploreted.4After the treatment, the same as step3, the eyeball blood wascollected to detect the liver function, such as alanineaminotransferase(ALT), aspartate aminotransferase(AST), albumin(Alb)and total bilirubin(TBiL). The liver and splenic index were calculated, thefresh liver and spleen frozen sections were perpareed and stained with HEand TUNEL to observe pathological structure and cell apoptosis of liverand spleen. By preparation and collection of the supernatants of liver andspleen tissue homogenates，the levels of superoxide dismutase (SOD),antioxidant(GSH-Px), glutathione(GSH), malonaldehyde(MDA) andpro-inflammatory cytokines of IL-1β and IL-6were assayed by chemicalcolorimetric analysis and ELISA.Results1Both the ASP or Ara-C used alone and in combination can inhibitthe proliferation of KG1α cells significantly in vitro, and arrested the cellsin G0/G1stages. Cells showed the aging morphological feature. Thesenescent cells stained by SA-β-Gal was significantly increased. The expression of the senescence related proteins of P16and RB wassignificantly up-regulated. The group of ASP+Ara-C was more obvious inthe above results.2After the establishment of the transplanted human leukemiaNOD/SCID mouse model, the amount of the peripheral blood WBC, thepercentage of neutrophiles, the amount of bone marrow myeloblasts ofleukemia mice increased significantly，DQα in bone marrow of leukemiamice expressed positively. It suggests that transplanted K562cells into thetail vein of the NOD/SCID mice can successfully establish leukemia mice.3Compared with the control group, both the ASP or Ara-C injectedalone and in combination can reduce the amount of the peripheral bloodWBC, the percentage of neutrophiles, the number of femur BMNCsobviously; effectively inhibit the proliferation of the BMNCs，the CFU-Mixforming, the ratio of S stages; markedly raise the percentage oflymphocytes, the ratio of G1stages, the percentage of SA-β-Gal positivecells; down-regulate the expression of the senescence related proteins ofCDK4and cyclinD1; up-regulate the expression of P16and Rb proteins.The effects of ASP+Ara-C group were much better than other groups.4Compared with the model group, both the ASP or Ara-C injectedalone and in combination can obviously reduce the liver and spleen index,liver function damage was alleviated: ALT, AST and TBiL weresignificantly reduced, while Alb was notably increased; the leukemia cells infiltrated into the liver and spleen were reduced and apoptotic positivecells were increased remarkably; SOD, GSH-Px were significantlyincreased and GSH,MDA were raised; the inflammatory cytokines ofIL-1β and IL-6were decreased dramatically. The effects of combinedinjection were much better than other groups.Conclusions1ASP and Ara-C can induce KG1α cells senescence that may attributeto the senescence related proteins.2Leukemia mice can be established successfully by transplantingK562cells into the tail vein of the NOD/SCID mice.3ASP and Ara-C can induce BMNCs in the transplanted humanleukemia mice senescence, and it may attribute to the senescence relatedproteins of P16、Rb、CDK4and CyclinD1.4ASP combined with Ara-C can reduce the accumulation of leukemiacells within the liver and spleen，protect the structure and function of liverand spleen. |
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