Sensitivity of MCF-7 mammosphere CSCs to neutron radiation

Aim:Cancer stem cells(CSCs)are highly resistant to chemotherapy andγ-irradiation.Neutrons have a high linear energy transfer,which can lead to extensive damage to the DNA of tumor cells and CSCs.The aim of this work was to compare the sensitivity of MCF-7 human breast adenocarcinoma cells and CSCs toγ-andγ,n-irradiation.Methods:To increase the number of CSCs,MCF-7 cells were cultured as mammospheres.γ-irradiation was carried out in a GUT-200M device(^(60)Co source)in the dose range of 1-8 Gy at a dose rate of 0.75 Gy/min.γ,n-irradiation was carried out in an IR-8 reactor in the dose range of 0.05-2 Gy at a dose rate of 0.06 Gy/min.DNA DSB formation was assessed by the level ofγH2AX foci using fluorescence microscopy and flow cytometry.CSCs were identified by flow cytometry as CD44^(+)/CD24^(-/low) cells.Results:We showed thatγ,n-irradiation induced the formation ofγH2AX foci of a larger size than didγ-irradiation and led to more severe DNA damage per 1 Gy.Moreover,γ,n-radiation was found to have a high relative biological effectiveness(RBE)as assessed by the cell survival rate,the number of CSCs in culture,and the ability of CSCs to repopulate.The highest RBE of neutron radiation was observed at low doses,when cell survival rate decreased by only 5%-10%.With an increase in the radiation dose,the RBE value decreased for all studied parameters,but it remained as high as 5.Conclusion:γ,n-radiation is highly effective against CSCs.Our results explain the efficacy of neutron therapy for resistant forms of breast cancer.

Twist促进乳腺癌细胞BT-549乳腺细胞小球形成及其迁移

目的探讨Twist基因功能缺失对乳腺肿瘤干细胞样细胞富集及其迁移的影响。方法采用shRNA干扰技术构建Twist基因敲减的BT-549乳腺癌细胞株(BT-549-sh Twist细胞株)和阴性对照细胞株BT-549-sh Vec;采用实时荧光定量PCR和Western blot法验证干扰效率;长程无血清悬浮培养方法富集具有干细胞特性的乳腺癌细胞小球(mammosphere);TranswellTM法检测乳腺癌细胞小球的迁移能力。结果采用shRNA干扰技术成功构建Twist基因敲减的BT-549乳腺癌细胞株BT-549-sh Twist细胞株;BT-549-sh Twist细胞株的mammosphere富集能力和迁移能力减弱。结论 Twist促进BT-549乳腺癌乳腺细胞小球形成及其迁移。

Aminoflavone upregulates putative tumor suppressor miR-125b-2-3p to inhibit luminal A breast cancer stem cell-like properties

Metastatic breast cancer is incurable and often due to breast cancer stem cell(CSC)-mediated self-renewal.We previously determined that the aryl hydrocarbon receptor(AhR)agonist aminoflavone(AF)inhibits the expression of the CSC biomarkerα6-integrin(ITGA6)to disrupt the formation of luminal(hormone receptor-positive)mammospheres(3D breast cancer spheroids).In this study,we performed miRNA-sequencing analysis of luminal A MCF-7 mammospheres treated with AF to gain further insight into the mechanism of AF-mediated anti-cancer and anti-breast CSC activity.AF significantly induced the expression of>70 microRNAs(miRNAs)including miR125b-2–3p,a predicted stemness gene regulator.AF-mediated miR125b-2–3p induction was validated in MCF-7 mammospheres and cells.miR125b-2–3p levels were low in breast cancer tissues irrespective of subtype compared to normal breast tissues.While miR125b-2–3p levels were low in MCF-7 cells,they were much lower in AHR100 cells(MCF-7 cells made unresponsive to AhR agonists).The miR125b-2–3p mimic decreased,while the antagomiR125b-2–3p increased the expression of stemness genes ITGA6 and SOX2 in MCF-7 cells.In MCF-7 mammospheres,the miR125b-2–3p mimic decreased only ITGA6 expression although the antagomiR125b-2–3p increased ITGA6,SOX2 and MYC expression.AntagomiR125b-2–3p reversed AF-mediated suppression of ITGA6.The miR125b-2–3p mimic decreased proliferation,migration,and mammosphere formation while the antagomiR125b-2–3p increased proliferation and mammosphere formation in MCF-7 cells.The miR125b-2–3p mimic also inhibited proliferation,mammosphere formation,and migration in AHR100 cells.AF induced AhR-and miR125b2-3p-dependent anti-proliferation,anti-migration,and mammosphere disruption in MCF-7 cells.Our findings suggest that miR125b-2–3p is a tumor suppressor and AF upregulates miR125b-2–3p to disrupt mammospheres via mechanisms that rely at least partially on AhR in luminal A breast cancer cells.