| Part 1 Preparation and identification of SKOV3-derived ovarian cancer stem-like cells and tumor associated macrophageObjective To investigate the interaction between SKOV3-derived ovarian cancer stem-like cells(OCSLC)and THP-1-derived tumor associated macrophages(TAM)and the intervention mechanism of genistein,SKOV3-derived OCSLC and TAM cultured and proliferated in vitro.Methods Spheroids were obtain from established human ovarian cancer SKOV-3 cell line by suspension culture in stem cell-condition medium using ultra-low adhesion plate.Compared with the parental cells,the second generation spheroids were identified as OCSLC by sphere-forming assay,clone forming assay,the expression of cancer stem cell markers(CD133 and CD44)and tumorigenicity in vivo.Then,THP-1 monocyte cell line cells were activated by treatment of Phorbol Myristate Acetate(PMA;final concentration: 100ng/ml)for 48 h.Compared with THP-1 macrophages,TAM was identified as THP-1 derivedmacrophage by comparing the expression of TAM markers(CD68 and CD163)and cytokines(IL-8,IL-10,IL-12,IL-1β,VEGF and mm P-9)and the concentration of NO products.Results 1.Dispersive primary spheroids from SKOV3 cell line could generate the secondary spheroids,and then generate the third and the fourth generation spheroids.The secondary spheroids possessed highest sphere-forming efficiency(P<0.05).2.Compared with SKOV3 cells,the secondary spheroids possessed higher sphere forming ratio,clone forming ratio,and elevated expression of cancer stem cell markers such as CD133 and CD44(P<0.05).3.Compared with the parental cells,SKOV3-derived OCSLC had stronger tumorigenicity in nude mice(P<0.05).4.Compared with THP-1 macrophages,THP-1 macrophage co-cultured with SKOV3-derived OCSLC over-expressed CD163,p-STAT3 and NF-κBp65(P<0.05).However,there was no significant difference at the expression of CD68.5.Compared with SKOV3-derived OCSLC condition medium(OCSLC-CM)and THP-1 macrophage condition medium(THP-1-CM),the concentration of IL-8,IL-10,VEGF,mm P-9 and NO products was higher and the concentration of IL-12 was decreased(P<0.05)in SKOV3-derived OCSLC/THP-1 macrophage co-culture conditional medium(Co-CM).However,there was no significant difference at the concentration of IL-1β between THP-1-CM and Co-CM.6.Compared with SKOV3 cells treated with THP-1-CM,sphere formation,clone formation and expression of CD133 and CD44 of SKOV3 cells were induced by Co-CM(P<0.05).7.SKOV3-derived OCSLC/THP-1 macrophage co-injection promoted growth of SKOV3-derived OCSLC xenograft in nude mice.Conclusion 1.The secondary spheroids derived from SKOV3 cell line possessed OCSLC characteristics.2.THP-1 macrophage co-cultured with SKOV3-derived OCSLC had characteristics of TAM,and SKOV3-derived OCSLC/THP-1 macrophages co-injection promoted growth of SKOV3-derived OCSLC xenograft in nude mice.Part 2 Impaction of IL-8 on the OCSLC characteristics of SKOV3 cells induced by SKOV3-derived OCSLC/THP-1 macrophage co-cultureObjective To investigate whether high excretion of IL-8 from SKOV3-derived OCSLC/THP-1 macrophage co-culture was associated with promotion interaction between SKOV3-derived OCSLC and THP-1-derived TAM,and promotion of OCSLC characteristics of SKOV3 cells induced by SKOV3 derived OCSLC/THP-1macrophage co-culture.Methods The expression of TAM marker(CD163),cytokines(IL-10 and IL-12),the concentration of NO products and sphere forming ratio,clone forming ratio and the expression of cancer stem cell markers(CD133 and CD44)were detected respectively using western blot,ELISA,sphere forming assay and clone forming assay in THP-1 macrophage conditional medium or cancer stem cell conditional medium with or without IL-8.These experiments were aim to ensuring whether IL-8 take participate in SKOV3-derived OCSLC/THP-1 macrophage co-culture induced M2 phenotype of THP-1 macrophage and OCSLC characteristics of SKOV3 cells.Results: 1.THP-1 macrophage conditional medium added with IL-8 increased the expression of CD163 and the concentration of IL-8 in THP-1 macrophage(P<0.05).Simultaneously,it decrease the excretion of IL-12 and the generation of NO products(P<0.05).2.Stem cell conditional medium added with IL-8 induced sphere forming,clone forming and the expression of CD133 and CD44 in SKOV3 cells.3.THP-1 macrophage conditional medium added with IL-8 up-regulated the protein expression of p-STAT3 and NF-κBp65 in THP-1 macrophages.4.SKOV3-derived OCSLC/THP-1 macrophage co-culture conditional medium with i mmunodepletion of IL-8 decreased the expression of CD163 and the concentration of IL-10 in THP-1 macrophage(P<0.05).Simultaneously,it promoted the excretion of IL-12 and generation of NO products in THP-1 macrophage(P<0.05).5.SKOV3-derived OCSLC/THP-1 macrophage co-culture condition medium with i mmunodepletion of IL-8 inhibited sphere-forming,clone forming and the expression of CD133 and CD44 in induced SKOV3 cells.6.SKOV3 derived OCSLC/THP-1 macrophage co-culture condition medium with i mmunodepletion of IL-8 suppressed the expression of p-STAT3 and down-regulated NF-κBp65 expression in THP-1 macrophages(P<0.05).Conclusion M2 phenotype of THP-1 macrophages and OCSLC characteristics of SKOV3 cells induced by SKOV3-derived OCSLC/THP-1 macrophage co-culture was depended on high excretion of IL-8.Part 3 Effects of genistein on OCSLC characteristics of SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-cultureObjective To investigate the inhibitory effects of genistein on M2 phenotype of THP-1 macrophages and OCSLC characteristics of SKOV3 cells induced by SKOV3-derived OCSLC/THP-1 macrophage co-cultureMethods The effects of genistein of indicated concentration(10.0,20.0,and 40.0 μM)on M2 phenotype of TAM induced by SKOV3-derived OCSLC/THP-1 macrophage co-culture and OCSLC characteristics of SKOV3 cell and the expression of p-STAT3 and NF-κBp65 were detected using western blot,ELISA measure,sphere forming assay and clone forming assay.Results 1.Genistein of indicated concentration(10.0,20.0 and 40.0 μM)decreased the expression of CD163 and the concentration of IL-10 in THP-1 macrophages co-cultured with SKOV3-derived OCSLC,in a dose-dependent manner(P<0.05).Simultaneously,it promoted the excretion of IL-12 and generation of NO products(P<0.05).2.Genistein(10.0,20.0 and 40.0 μM)inhibited sphere forming,clone forming and the expression of CD133 and CD44 in SKOV3 cells induced by SKOV3-derived OCSLC/THP-1 macrophage co-culture,in a dose-dependent manner(P<0.05).3.Genistein(10.0,20.0,and 40.0 μM)inhibited the protein expression of p-STAT3 and NF-κBp65 in THP-1 macrophages co-cultured with SKOV3 derived OCSLC(P<0.05).Conclusion Genistein could inhibit M2 phenotype of THP-1 macrophage and OCSLC characteristics of SKOV3 cells induced by SKOV3-derived OCSLC/THP-1 macrophage.Part 4 Mechanism that genistein may inhibit TAM-derived ovarian cancer stem-like cells and tumor associated macrophage via suppressing IL-8/STAT3 signal axisObjective To reveal the mechanism that genistein inhibits M2 phenotype of THP-1 macrophages and OCSLC characteristics of SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture via suppressing IL-8/STAT3 signal axis.Methods In order to explore the effects of genistein on M2 phenotype of THP-1 macrophage and the protein expression of p-STAT3 and NF-κBp65 and OCSLC characteristics of SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture,genistein was combined with addition of IL-8 or i mmunodepletion of IL-8.To explore whether genistein inhibits M2 phenotype of THP-1 macrophage and OCSLC characteristics of SKOV3 cells induced by SKOV3 derived OCSLC co-culture via suppressing STAT3 in THP-1 macrophage,STAT3 gene was silenced or over-expressed in THP-1 macrophage using adenovirus gene delivery system.Moreover,to evaluate effectiveness in vivo of genistein and STAT3 sh RNA expressed adenovirus on therapy of transplantation tumor in nude mouse induced by the co-injection of SKOV3 derived OCSLC/THP-1 macrophages.Results 1.Addition of 10 ng/ml IL-8 could antagonize effectively the effects of genistein(10 μM)on CD163 expression and IL-10 excretion(P<0.05)and could promote IL-12 excretion and NO products generation in THP-1 macrophages in THP-1 macrophage induced by SKOV3-derived OCSLC co-culture(P<0.05).2.2 Addition of 10 ng/ml IL-8 could effectively antagonize the inhibitory effects of genistein(10 μM)on the capability of sphere forming,clone forming and the expression of CD133 and CD44 in SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture(P<0.05).3.Addition of 10ng/ml IL-8 could effectively antagonize the inhibitory effects of genistein(10 μM)on the protein expression of p-STAT3 and NF-κBp65 in THP-1 macrophages co-cultured by SKOV3 derived OCSLC/THP-1 macrophage condition medium.4.I mmunodepletion of IL-8 and genistein(10 μM)could cooperatively inhibited CD163 expression and IL-10 excretion and could promoted IL-12 excretion and NO products generation in in THP-1 macrophage induced by SKOV3 derived OCSCLCs co-culture.5.I mmunodepletion of IL-8 and genistein(10 μM)could cooperatively inhibited the capability of sphere forming,clone forming and the expression of CD133 and CD44 in SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture(P<0.05).6.I mmunodepletion of IL-8 and genistein(10 μM)could cooperatively inhibited the expression of p-STAT3 and NF-κ Bp65 in THP-1 macrophage co-cultured by SKOV3 derived OCSLC/THP-1 macrophages(P<0.05).7.STAT3 sh RNA and genistein(10 μM)could cooperatively inhibited CD163 expression and IL-10 excretion and could promoted IL-12 excretion and NO products generation in THP-1 macrophage induced by SKOV3 derived OCSCLCs co-culture.8.STAT3 sh RNA and genistein(10.0 μM)could cooperatively inhibited the capability of sphere forming,clone forming and the expression of CD133 and CD44 in SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture(P<0.05).9.STAT3 sh RNA and genistein(10 μM)could cooperatively inhibited the expression of p-STAT3 and NF-κ Bp65 in THP-1 macrophage co-cultured by SKOV3 derived OCSLC/THP-1 macrophages(P<0.05).10.STAT3 gene transduction effectively antagonize the effects of genistein(10 μM)on CD163 expression and IL-10 excretion(P<0.05)and promote IL-12 excretion and NO products generation in THP-1 macrophage induced by SKOV3 derived OCSLC co-culture(P<0.05).11.STAT3 gene transduction effectively antagonize the inhibitory effects of genistein(10 μM)on the capability of sphere forming,clone forming and the expression of CD133 and CD44 in SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture(P<0.05).12.STAT3 gene transductions effectively antagonize the inhibitory effects of genistein(10 μM)on the protein expression of p-STAT3 and NF-κBp65 in THP-1 macrophages co-cultured by SKOV3 derived OCSLC/THP-1 macrophage.13.STAT3 gene transduction antagonize the inhibitory effects of the combination of STAT3 sh RNA and genistein(10 μM)on CD163 expression and IL-10 excretion(P<0.05)and the promotion effects on IL-12 excretion and NO products generation in THP-1 macrophage induced by SKOV3 derived OCSCLC co-culture(P<0.05).14.STAT3 gene transduction could antagonize the inhibitory effects of the combination of STAT3 sh RNA and genistein(10 μM)on the capability of sphere forming,clone forming and the expression of CD133 and CD44 in SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture(P<0.05).15.STAT3 gene transduction antagonize the inhibitory effects of the combination of STAT3 sh RNA and genistein(10 μM)on the expression of p-STAT3 and NF-κ Bp65 in THP-1 macrophage co-cultured by SKOV3 derived OCSLC/THP-1 macrophage(P<0.05).16.Genistein and STAT3 sh RNA cooperatively inhibited the growth of xenograft in nude mouse of SKOV3 derived OCSLC/THP-1 macrophage co-injection(P<0.05).Conclusion Genistein inhibited M2 phenotype of THP-1 macrophage and OCSLC characteristics of SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture via blocking IL-8/STAT3 signal axis.Conclusion of the Fulltext 1.Activation of IL-8/STAT3 signal axis promoted M2 phenotype of THP-1 macrophage and OCSLC characteristics of SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture.2.Genistein could inhibit M2 phenotype of THP-1 macrophage and OCSLC characteristics of SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture.3.Genistein inhibited M2 phenotype of THP-1 macrophage and OCSLC characteristics of SKOV3 cells induced by SKOV3 derived OCSLC/THP-1 macrophage co-culture via blocking IL-8/STAT3 signal axis in THP-1 macrophages. |
PDF Full Text Request