Biological Effects Of Itraconazole Plus Anthracyclines On Leukemia Stem-like Cell KG1α Cell Lines And Its Mechanism

BackgroundAcute myeloid leukemia (AML) originated in hematopoietic stem and progenitor cells is a kind of malignant tumor of myeloid hematopoietic system. With the development of new drugs, and the application of hematopoietic stem cell transplantation(HSCT), the treatment effect of AML has made breakthrough progress, the current main method of treatment of AML is combined chemotherapy and hematopoietic stem cell transplantation after the remission. Early high-dose chemotherapy can make many patients achieved complete remission (CR), but is accompanied by severe side effects, elderly patients can’t tolerate and easy to relapse. With the application of high doses of chemotherapy,immunosuppressants and hematopoietic stem cell transplantation,invasive fungal infection(IFI) increased year by year,become one of the causes of death in patients with AML.Accumulating evidences support the notion that AML is organized in a hierarchical system, originating from a special proportion of leukemia stem cells (LSCs). Similar to their normal counterpart hematopoietic stem cells (HSC), possess self-renewal capacity and are responsible for the continued growth and proliferation of the bulk of leukemia cells in the blood and bone marrow. It is believed that LSCs are also the root cause for the treatment failure and relapse of AML because LSCs are often resistant to chemotherapy. Therefore, in order to improve the traditional radiotherapy and chemotherapy, it can through small dose of the drugs combination, to improve the sensitivity of LSCs for drugs and achieve the purpose of high efficiency and low toxicity for teatment.It can provide more and more evidences of the evidence-based medicine for treatment reseach in the blood diseases.Hedgehog (Hh) signaling pathway is one of the most conservative signaling pathways of biological species. Hedgehog has three kinds of homologous genes: SonicHedgehog (SHH), Indian Hedgehog (IHH) and Desert Hedgehog (DHH), coding Shh, Ihh and Dhh protein respectively. Hh signaling pathway contains Patched (Ptc), Smoothened (Smo) and pathways downstream of Costal2 (Cos2), Fused (Fu), Suppressor of Fused (Su (Fu)), Cubitus interruptus (Ci) and other proteins. In normal circumstances (no Hh), Ptc inhibits the activity of Smo protein, while the full length of Ci is phosphorylated by PKA, GSK3 and CKI, and degrades to fragmented Ci75, enters the cell nucleus, inhibit the transcription of downstream target genes. In the case of Hh existence, Hh combined with Ptc can remove the inhibition of Smo, Smo is phosphorylated by the PKA, CKI and accumulates on the membrane, while the Smo molecular’s C-terminal conformation is changed form the closed inactive state transforms into the open active state, activating downstream pathways. It regulates the key events during the developmental processes i.e growth and patterning of multicellar embryos.It can influences stem cell fate,differentiation,proliferation,and apoptosis in responsive tissues.In adult organisms,Hh signaling pathway activity is required for aspects of tissue maintenance and regeneration. Mainly due to Glil transcriptional activator in the Hh pathway downstream can induce G1/S cell cycle regulatory protein expression and improve the proliferation of cells; it can directly induce expression of anti-apoptotic factor Bcl-2 to inhibit apoptosis; it also can directly activate the transcription of the factor that can promote epithelial tissue to mesenchymal transformation to enhance tumor invasiveness.As a result,the disorder of Hh signaling pathway is associated with the formation,growth and maintenance of malignant tumor such as melanoma, prostate cancer, pancreatic cancer, colon cancer, breast cancer and glioblastoma. Some hematological malignancies such as acute leukemia (AMLand ALL), chronic myelogenous leukemia (CML), chronic lymphatic leukemia (CLL), lymphoma, multiple myeloma (MM) are dependent on Hedgehog signaling pathway.Recent evidences suggest that Hh signaling pathway is critical for the maintenance and expansion of malignant stem cells. Zhao e.1 believed that the Smo’s activation of murine and human is necessary to maintain the number of CML stem cells, Smo can increase the number of stem cells in CML and make the deterioration, whereas the lack of the Smo could make stem cells depletion. It suggests that the Smo inhibitors may effectively reduce leukemia stem cells, which become a kind of effective treatment.Itraconazole (ITC) is a kind of antifungal drugs,as the prevention and treatment of malignancies IFI line drugs and a potent antagonist of the Hh signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. The study found that in serum which from rat medulloblastoma homograft tumor and antifungal treatment of patients, itraconazole can significantly inhibit the activity of the Hedgehog signaling pathway, to prevent the growth of tumor. ITC combinated with arsenic trioxide can synergistically inhibit Hh signaling pathway.It is reported that itraconazole inhibit non-small cell lung cancer blood vessels and the growth of tumor cells.And clinical studies have shown that Itraconazole can be used in the treatment of recurrent prostate cancer, and achieved good results. Thus, for Hh signaling pathway dependent tumors, itraconazole alone or in combination with other chemotherapy drugs for anti-tumor therapy will have a good prospect.Anthracyclines is a commonly used chemotherapy drugs in clinical. It can inhibit the synthesis of RNA and DNA, the most strongest inhibitory effect is for RNA synthesis.Anthracyclines has a broad antineoplastic spectrum and has an effect on a wide variety of tumor.It is non-specific drug to cell cycle,can kill tumor cells of various growth cycle. It is mainly used for acute leukemia. However, anthracyclines has serious side effects such as cardiac toxicity, and now the leukemia cells resistant to anthracyclines.At present, there is not yet anthracyclines plus itraconazole applied to acute leukemia. In this experiment, KG1α cells is as experimental subjects, KG1α cell lines derived from male patients with AML,which can not differentiate and have self-renewal potential and has Chemotherapy resistance (resistance to anthracyclines).So we can be considered KGla cells as "leukemia stem cell-like cells". In order to investigate whether the Hedgehog signaling pathway as a new target used in the treatment of leukemia, and combined with the inhibition of Hh pathway and RNA, DNA synthesis whether has a synergistic effect for killing the leukemia cells and its mechanism. The study will take morphological, molecular and cell biology methods to studied the molecular mechanism of chemotherapy drug adriamycin plus Hh inhibitor itraconazole on acute myeloid cell lines KGla Cells in vitro, which is expected that provide a scientific basis for the new method which is low toxicity and high efficient and targeting leukemia stem cells applies to leukemia prevention.Part 1 Biological effects of itraconazole plus anthracyclines on leukemia stem-like cell KGla cell linesObjectiveTo explore the effects of itraconazole (ITC) plus anthracyclines on proliferation and apoptosis of leukemia stem-like cell KG1α cell lines in vitro.MethodFlow cytometry was used to detect the expression of CD34 and CD38 antigens what were in the surface of KGla cells.The growth inhibition effects of different concentrations of ITC, ADM,DNR or ITC (2,6,15μmol/L) plus ADM (0.30,0.75 μmol/L) or DNR (0.6、1.5μmol/L) were detected by CCK8 assay in KG1α cells. Different concentrations of ITC for 7,14 and 21 days were applied for observing the effect on colony formation. After 48 h treatments with 6μmol/L ITC,0.75μmol/L ADM/1.5μmol/L DNR or ITC (6μmol/L) plus ADM (0.75μmol/L)/DNR (1.5μmol/L), the morphological changes of cells were observed by Wright staining. Flow cytometry was used to detect cell apoptotic rate, mitochondrial membrane potential and cell cycle arrest..Statistical analysisThe analysis was performed using SPSS 17.0 software package.All experiments were repeated three times, the experimental data was expresses as mean ±standard deviation(x±s).Comparisons of means among groups were performed using factorial design analysis of variance.Comparisons of means between teo groups were performed using T-text, P<0.05 were considered to be statistically significant.ResultKGlα cells have approximately 96.23% CD34+CD38- cells. ITC, ADM and DNR inhibited the proliferation of KGla cells and ITC reduced the colony-formation ability of KGla cells both in dose-dependent manners. Compared with control or single drug group, the changes of cell morphology were more apparent in combined group. Weeb coefficient test revealed a synergistic effect (D<70%C) of 0.75μmol/L ADM or 1.5μmol/L DNR plus 6μmol/L ITC. When KGla cells were treated with 6 μmol/L ITC plus 0.75μmol/L ADM, the apoptotic rate was 31.72%±1.58%. And it was significantly higher than that in control, ITC and ADM groups (4.17%±0.74%, 4.33%±1.12%,9.53%±1.15%,P<0.01). When KGla cells were treated with 6 μmol/L ITC plus 1.5μmol/L DNR, the apoptotic rate was 32.13%±4.11%. And it was significantly higher than that in control, ITC and DNR groups (3.61%±1.94%、 3.52%±1.92%、10.70%±2.15%, P<0.01). Detection of mitochondrial membrane potential showed that low red-fluorescent cells (P3) of combined groups were obviously higher than that in control, ADM and ITC single drug groups (18.82%±3.96% combined with 5.02%±2.11%、9.7%±1.56%、7.1%±2.82%, P<0.01); the P3 area of DNR+ITC groups were also obviously higher than that in control, DNR and ITC single drug groups (30.84% ± 4.01%combined with 10.51%±1.97%、9.53%±3.12%、7.0%±1.33%, P<0.01). KG1α cells were obviously arrested in G2 phase in combined group and it showed no statistical significance compared with control or single drug groups (P<0.05)Conclusion1. The most of KGla cells have stem cell properties,can be used as the experimental model of LSCs;2. ITC,ADM,DNR can inhibited cell proliferation in a time- and dose-dependent manner within limits;ITC can inhibit the different stages of KGla cells colony formation in a dose-dependent;3. ITC combined with ADM or DNR significantly inhibited cell proliferation in a time- and dose-dependent manner;4. ITC plus ADM or DNR can obviously increase KGla cells apoptotic rate, mitochondrial damage and G2 phase retardationPart 2 The mechanismas of apoptosis in leukemia stem-like cell KGla cell lines induced by itraconazole plus daunorubicinObjectiveTo explore the effects of itraconazole (ITC) plus Daunorubicin (DNR) on proliferation and apoptosis of leukemia stem-like cell KGla cell lines in vitro,then explore its possible mechanisms.MethodThe expression of apoptosis related proteins in difference drug groups were detected by protein chips.Western blot was used to detect the expression levels of Sonic Hedgehog (Shh) signaling pathway-related proteins Shh and Glil,and apoptosis-related proteins Bcl-2、XIAP、SMAC. The apoptotic rates of cells with DNR plus ITC that were treated with ABT-199 were detected by flow cytometry,and the expression levels of Bcl-2 were detected by western blot.Statistical analysisThe analysis was performed using SPSS 17.0 software package.All experiments were repeated three times, the experimental data was expresses as mean ±standard deviation(x±s). Comparisons of means among groups were performed using factorial design analysis of variance.Comparisons of means between teo groups were performed using T-text, P<0.05 were considered to be statistically significant.ResultWe can see from the results of protein chips,the expression levels of Survivin,HSP60,SMAC,HTRA,Bcl-2,p21 and Fas in the combined groups compared with the single drug groups and the control groups have varying degrees of increase;and the expression of XIAP、CD40L、CD40 and bax in combined groups compared with the single drug groups and the control groups have different degrees of reduction. Western blot showed that the expression levels of Shh、Gli1、Bcl-2、 XIAP and SMAC decreased with low concentration of ITC plus DNR.Conclusion1. ITC plus DNR can significantly inhibit Shh signaling pathway, as well as change the expression levels of apoptotic related proteins.2. Bcl-2 inhibitor ABT-199 can induce apotpsis of cells that were treated with DNR plus ITC,and obviously downregulate the expression of Bcl-2 of survival cells.