Molecular Mechanism Of The Specific Mechanism Of Aurora A And Aurora B Inhibitor APIO-EE-9 Suppressed Esophageal Cancer Growth

Esophageal cancer (EC) is one of the aggressive malignancies of the upper aerodigestive tract.In china, EC mortality and morbidity rates are 4th and 6th highest,respectively. The main type of the EC is esophageal squamous cell carcinoma (ESCC)and by the characterized with high invasiveness and poor prognosis. Despite remarkable advances in multimodal therapies combining surgery, chemotherapy,radiotherapy and chemoradiotherapy, the prognosis of ESCC patients remains poor,the five year survive rate only below 20%. Thus identifying novel molecular targets and developing therapeutic agents are critical.Aurora kinases are the member of serine/threonine Aurora kinase family and play a crucial role in mitosis and cell division, currently discussed as an attractive therapeutic target in cancer. Aurora kinases regulate spindle assembly, chromosome segregation and its overexpression can induce abnormal of mitosis. As reported,Aurora kinase overexpression associated with many types of malignant cancer.According to the literary, Aurora kinase are a family of conserved mitotic regulators consisting of Aurora kinase A, B and C in human, each kinase performs a distinct task.Aurora A is localized at the spindle poles and required for centrosome maturation,separation and spindle assembly. Aurora B kinase is a chromosome passenger protein required for phosphorylation of histone H3 correct alignment of chromosomes on the metaphase plate and cytokinases. Much less is known about the function of Aurora C.Expression of Aurora C is restricted to germ cells, where it is believed to regulate spermatogenesis. Aurora kinase has been shown to be overexpressed in many types of cancers, including pancreatic, ovarian, breast, colorectal, lung and bladder cancers.Aurora kinase as an attracted therapy target that many companies pay attention to Aurora inhibitor. Until now, many publication about Aurora inhibitors, but few inhibitors can inhibit both Aurora A and Aurora B activity but with toxicity. Thus,identifying small-molecule inhibitors of Aurora kinase is crucial for testing in translation studies and early-phase clinical trials to facilitate the development of potential drugs for cancer therapy.Herein, we reported that a newly synthesized Aurora inhibitor APIO-EE-9 suppresses esophageal cancer cell growth, meanwhile, also can induce cell apoptosis by inhibiting Aurora A or Aurora B activity and downstream phosphorylation of Histone H3 in vitro and in cells. Based on this information, we knocked down Aurora A or Aurora B expression to study the function of Aurora A and Aurora B kinase in esophageal cancer. Results showed that esophageal cancer cell growth was inhibited after knocking down Aurora A and Aurora B expression. Moreover, APIO-EE-9 strongly inhibited esophageal tumor growth in a patient-derived xenograft (PDX)mouse model.Chapter I APIO-EE-9 inhibited anchorage-independent growth and proliferation in esophageal cancer cellsMethods1. By using computational methods to design and synthesize small molecule inhibitor APIO-EE-9 against Aurora.2. The MTS assay was used to determine whether APIO-EE-9 exerted any cytotoxic XII effects against normal esophageal cells.3. The soft agar assay was performed to examine the effects of APIO-EE-9 on esophageal cancer cell lines.4. Detect the change of esophageal cell viability after APIO-EE-9 treatment by MTS.Results1. Designed and synthesized a potential Aurora A and Aurora B small molecular inhibitor APIO-EE-92. APIO-EE-9 had no cytotoxicity against on normal esophageal cellsThe MTS result showed that different concentration of APIO-EE-9 treated nonnal esophageal cell line Het-1A for 24 h or 48 h, the OD492 absorbance has no obviously decreasedcompared with control group. This results reveals that APIO-EE-9 had no cytotoxicity against Het-1 A cells.3. APIO-EE-9 inhibited the anchorage-independent cell growth in esophageal cancer cellsSoft Agar results showed that APIO-EE-9 inhibit the anchorage-independent cell growth in esophageal cancer cell KYSE450, KYSE510 and KYSE30 0.25μM APIO-EE-9 can had significantly inhibition on esophageal cancer cells (P<0.01)and inhibit 50% anchorage-independent cell growth with dose dependent. 5μM APIO-EE-9 can inhibit esophageal cancer cells colony formation completely.4. APIO-EE-9 inhibited cell proliferation in esophageal cancer cellsMTS results showed thatAPIO-EE-9 markedly suppresses cell proliferation of cell line KYSE450, KYSE510 and KYSE30 in dose dependent manner. The OD492 absorbance was dramatically decreased after treated 5μM APIO-EE-9 for 72 h(P<0.01).Chapter Ⅱ APIO-EE-9 induced Esophageal Cancer CellsapoptosisMethods1. To detect the effect of apoptosis with APIO-EE-9 treatmentin esophageal cancer cells or normal esophageal cells by Flow cytometry.2. To detect the effect of apoptosis with APIO-EE-9 treatmentin esophageal cancer cells by WBResults1. APIO-EE-9 induced apoptosis in esophageal cancer cell lines, but has no effect on normal esophageal cellsFlow cytometry results showed that different concentration of APIO-EE-9 treated normal esophageal cell line Het-1A for 72h, compared with control group, 5μM APIO-EE-9 can induce 8.5% apoptosis. However, 5μM APIO-EE-9 treated KYSE450 cell for 72h, can induce 83.6% apoptosis and had a significant difference compared with control group (P<0.01).2. APIO-EE-9 increaseed expression of pro-apoptosis markers and decrease expression of anti-apoptotic markersWestern Blot results showed that KYSE450, KYSE510 and KYSE30 treated with different concentration of APIO-EE-9 for 72h, it can increase expression of pro-apoptosis markers, including cleaved caspase 3, cleaved PARP and Bax and decreased expression of anti-apoptotic Bcl-2.Chapter III Aurora A and Aurora B were highly expressed in esophageal cancer cell lines and tissuesMethods1. Western Blot to detect Aurora A and Aurora B expression in esophageal cell lines KYSE450, KYSE510 and KYSE30 compare with normal esophageal cell Het-1A2. Immunohistochemical to detect Aurora A and Aurora B expression in esophageal cancer tissues.3. Western Blot to detect phosphorylation of Histone H3 after treated APIO-EE-9 for 24h.4. Immunofluorescence to detect multi-nucleation and multi-centrosome after treated with 5μM APIO-EE-9.Results1. Esophageal cancer cells highly expression Aurora A and Aurora B kinaseWestern Blot results showed that compared with normal esophageal cell Het-IA,Aurora A or Aurora B is highly expressed in esophageal cancer lines.2. Esophageal cancer tissues highly expression Aurora A and Aurora B kinaseImmunohistochemical results showed that Aurora A and Aurora B is highly expressed in esophageal cancer tissues compared with normal adjacent tissues.3. APIO-EE-9 inhibited Histone H3 phosphorylation in dose dependent mannerWestern Blot analysis to determine the level of phosphorylated histone H3 (Ser10)after treatment with various concentration of APIO-EE-9. Results demonstrated that APIO-EE-9 inhibits histone H3 (Ser10) phosphorylation in a dose-dependent manner.4. APIO-EE-9 did indeed induce multi-nucleation and multi-centrosome formation in KYSE450 cellsAfter treatment with 5μM APIO-EE-9 for 2 hours, immunofluorescence results revealed that APIO-EE-9 can induce multi-nucleation and multi-centrosoime formation in KYSE450 cells, but no this phenotype in control group.Chapter IV APIO-EE-9 binded and inhibited both Aurora A and Aurora B activities in vitro and ex vivo XVMethods1. To better understand the mechanisms and the activity of APIO-EE-9, we conducted an in silico docking study and an energy minimization and molecular dynamics (MD) simulation.2. Aurora A or Aurora B in vitro kinase assays were conducted to determine the inhibiting efficiency of APIO-EE-9.3. To further validate these simulations, we conducted ATP competition assays with APIO-EE-9-conjugated SepharoseTM 4B beads.4. Experiment of ex vivo binding assay between APIO-EE-9-conjuated SepharoseTM 4B beads and cell lysate overexpressing Aurora A or Aurora B.Results1. APIO-EE-9 binded Aurora A or Aurora B at the ATP-binding pocketResults of the final computational model docking results showed that APIO-EE-9 formed hydrogen bonds with Aurora A and Aurora B at their respective ATP binding pocket.2. APIO-EE-9 inhibited both Aurora A and Aurora B activities in vitroThe in vitro kinase assay showed APIO-EE-9 strongly inhibited both Aurora A and Aurora B kinase activity in vitro.3. APIO-EE-9 binded Aurora A or Aurora B at the ATP-binding pocket,compete with ATPAn ATP competition assay further showed that either Aurora A or Aurora B was pulled down by APIO-EE-9-conjugated SepharoseTM 4B beads but not SepharoseTM 4B beads alone.4. APIO-EE-9 binded Aurora A or Aurora B in esophageal cancer cellsWe observed ex vivo binding between APIO-EE-9 and Aurora A or Aurora B in KYSE450 cells. APIO-EE-9 could strongly bind with Aurora A or Aurora B in esophageal cancer cells.Chapter V Knocking down Aurora A increases apoptosis in esophageal cancer cellsMethods1. Small hairpin RNA (shRNA) constructs against Aurora A were used to packageparticles, shMock was used as a negative control. Western blotting to detect the knocking down efficiency of Aurora A after KYSE450 or KYSE30 cells infected with shAurora A particles, using β-actin as control.2. After knocking down Aurora A expression, soft agar assay was performed to examine the effects on esophageal cancer cell lines colony formation.3. MTS assay to detect esophageal cancer cells proliferation after knocking down Aurora A expression.4. Flow cytometry analysis to detect esophageal cancer cells apoptosis after knockdown Aurora A expression.5. Western Blot to detect histone H3 phosphorylation level and apoptosis markers after knockdown Aurora A expression.Results1. ShAurora A#1 and shAurora A#2 particles successfully knocking down Aurora A expression in KYSE450 and KYSE510 cellsThe knocking down efficiency was assessed by Western blotting, and the results indicated that the Aurora A expression level was suppressed in KYSE450 and KYSE510 cells expressing shAurora A#1 or shAurora A#2, compared with control KYSE450 or KYSE510 cells that expressed shmock.2. Knocking down Aurora A expression inhibits colony formation in esophageal cancer cellsSoft agar assay results showed that after knocking down Aurora A expression, it can dramatically inhibit colony formation compared with the shMock group.3. Knocking down Aurora A expression suppresses cell proliferation in esophageal cancer cellsMTS assay results showed that KYSE450 and KYSE510 cell growth were strongly inhibited after knocking down Aurora A expression.4. Apoptosis was increased in KYSE450 and KYSE510 esophageal cancer cells after Aurora A knocking downFlow cytometry results showed that shAurora A#1 or shAurora A#2 group can induce 18% and 10% apoptosis compared with 1% of shMock group in KYSE450 cellsafter knocking down Aurora A expression, respectively (p<0.01).shAurora A#1 or shAurora A#2 group can induce 9% and 12% apoptosis compared with 2%of shMock group in KYSE510 cells, respectively (p<0.01).5. Knocking down Aurora A expression, it increases apoptosis markers including cleaved PARP, cleaved caspase3 and Bax, decreases Histone H3(Ser10) phosphorylation levelWestern Blot results showed that compared with shMock group,shAurora A#1 or shAurora A#2 group can increase the expression of pro-apoptosis markers including cleaved PARP, cleaved caspase3 and BAX. Meanwhile, knocking down Aurora A expression also can decrease the expression level of Histone H3 (Ser10)phosphorylation.Chapter VI Knocking down Aurora B increases apoptosis in esophageal cancer cellsMethods1. Small hairpin RNA (shRNA) constructs against Aurora B were used to packageparticles, shMock was used as a negative control. Western blotting to detect theknockdown efficiency of Aurora B after KYSE450 or KYSE30 cells infected with shAurora B particles, using β-actin as control.2. After knocking down Aurora B expression, soft agar assay was performed to examine the effects on esophageal cancer cell lines colony formation.3. MTS assay to detect esophageal cancer cells proliferation after knocking down Aurora B expression.4. Flow cytometry analysis to detect esophageal cancer cells apoptosis after knocking down Aurora B expression.5. Western Blot to detect histone H3 phosphorylation level and apoptosis markers after knocking down Aurora B expression.Results1. ShAurora B#1 and shAurora B#2 particles successfully knocking down Aurora B expression in KYSE450 and KYSE510 cellsThe knocking down efficiency was assessed by Western blotting, and the results indicated that the Aurora B expression level was suppressed in KYSE450 and KYSE510 cells expressingshAurora B#1 or shAurora B#2,compared with control KYSE450 or KYSE510 cells that expressed shmock.2. Knocking down Aurora B expression can inhibit colony formation in esophageal cancer cellsSoft agar assay results showed that after knocking down Aurora B expression, it can dramatically inhibit colony formation compared with the shMock group.3. Knocking down Aurora B expression suppresses cell proliferation in esophageal cancer cellsMTS assay results showed that KYSE450 and KYSE510 cell growth were strongly inhibited after knocking down Aurora A expression.4. Apoptosis was increased in KYSE450 and KYSE510 esophageal cancer cells after Aurora B protein expressionFlow cytometry results showed that shAurora B#1 or shAurora B#2 group can induce 88% and 85% apoptosis compared with 16% of shMock group in KYSE450 cellsafter knocking down Aurora B expression, respectively(p<0.01).shAurora B#1 or shAurora B#2 group can induce 22% and 15%apoptosis compared with 6% of shMock group in KYSE510 cells,respectively(p<0.01).5. Knocking down Aurora B expression, it increases apoptosis markers including cleaved PARP and cleaved caspase3, decreases Histone H3(Ser10)phosphorylation levelWestern Blot results showed that compared with shMock group, shAurora B#1 or shAurora B#2 group can increase the expression of pro-apoptosis markers including cleaved PARP and cleaved caspase3. Meanwhile, knocking down Aurora B expression also can decrease the expression level of Histone H3 (Ser10)phosphorylation and the anti-apoptosis marker Bcl-2.ChapterVII Knocking down Aurora A or Aurora B expression suppresses tumor growth of esophageal cancer cells in a xenograft mouse modelMethods1 . To determine whether knocking down Aurora A or Aurora B expression has effect on athymic nude xenograft mouse model of KYSE450 cells.2. Immunohistochemical analysis the expression level of Histone H3(Ser10)phosphorylation and apoptosis markers after knocking down Aurora A or Aurora B expression in athymic nude xenograft mouse model of KYSE450 cells.Results1. Knocking down Aurora A or Aurora B expression dramatically suppresses tumor growth in KYSE450 xenograft mouse modelXenograft mouse model results showed that after knocking down Aurora A or Aurora B expression, it can suppressed KYSE450 xenograft tumor volume compared with shMock group (p<0.01).2. Knocking down Aurora A or Aurora B expression decreased the phosphorylation level of Histone H3 (Ser 10)and Ki-67, increase the expression level of apoptosis markersImmunohistochemical results showed that compared with shMock group, after knocking down Aurora A or Aurora B expression, the expression level of Ki-67 was dramatically decreased (p<0.01); phosphorylation level of Histone H3 (Ser10) was decreased; Meanwhile, the expression level of pro-apoptosis marker was increased(p<0.01). All the results illustrated that Aurora A and Aurora B play a crucial role in tumor genesis, so target Aurora kinase can as a therapy for esophageal cancer.ChapterVIII APIO-EE-9 suppressed tumor growth in a esophageal PDX modelMethods 1. To detect the chemotherapeutic effect of APIO-EE-9 in a esophageal Patient-derived Xenograft (PDX) model.2. Immunohistochemical analysis of APIO-EE-9 treated PDX tumors was conducted to evaluate the related-protein expression levels.Results1. APIO-EE-9 inhibited the growth of tumor weight and volume in PDX modelPDX results showed that APIO-EE-9 (40 or 200 mg/kg) effectively inhibited PDX tumor growth compared with the vehicle-treated group with no significant loss in body weight, suggesting minimal toxicity (p<0.01).2. APIO-EE-9 inhibited the expression level of Ki-67 and phosphorylation level of Histone H3 (Ser10)Immunohistochemical results showed that Ki-67 and phosphorylation of Histone H3 (Ser10) were significantly suppressed and cleaved caspase3 increased in the APIO-EE-9 treated-groups compared with the vehicle group.Conclusions1. APIO-EE-9 was synthesized by our lab and several different approaches,supercomputer simulation, in vitro, ex vivo and in vivo, all confirmed that APIO-EE-9 binds and inhibits Aurora A and Aurora B activities by competing ATP in Aurora A or Aurora B binding pocket, and attenuated primary downstream pathways of Aurora signaling and prevent the tumor genesis.2. APIO-EE-9 markedly inhibites EC tumor growth of both in vivo xenograft study and PDX model.APIO-EE-9 exerts a substantial chemotherapeutic effect against EC without affecting body weight of mice, all these data indicated that APIO-EE-9 might be a very promising novel drug candidate tobe evaluated in clinical trials.