Radiosensitizing Effect And Mechanism Of Action By Antihelminthic Niclosamide On Triple-negative Breast Cancer Cells

Breast cancer is one kind of malignant tumor with the highest incidence in women. The combination of surgery and radio-chemotherapy is the best treatment for breast cancer nowadays. However, due to high heterogeneity of breast cancers, some molecular subtypes of breast cancer, especially the triple negative breast cancer (TNBC), do not respond well to traditional therapy and has relatively poor prognosis. It has been the focus in the diagnosis and treatment of breast cancer, while radioresistance is still the important problem in treatment of TNBC. Many studies indicate that the Wnt/β-catenin and AKT signaling pathway, which are particularly over-activated in breast cancer, especially in TNBC, and are associated with the radioresistance and poor prognosis of breast cancer. Furthermore, the activation of Wnt/β-catenin signaling can also increase the repair capacity of DNA duble strand breaks (DSBs) induced by radiation in breast cancer cells. Therefore, for the first time, we choose niclosamde, the Wnt/β-catenin signaling target inhibitor, to demonstrate its radiosensitivity effect in breast cancer from cell molecular level. The inhibiting effect of Wnt/p-catenin and AKT signaling activation, and the inbibiting effect of non-homologous end joining (NHEJ) repair of DNA duble strand breaks (DSBs) induced by radiation are the important mechanisms of its radiosensitization. In this study, we expected to explore a new tageted radiosensitizer of TNBC, besides to provide experimental basis for the combined application of niclosamide and radiotherapy in improving therapeutic effect of TNBC.Part Ⅰ Radiosensitivity effect of niclosamide on triple negative breast cancer cells and the potential mechanisms of its inhibition effect related to Wnt/β-catenin signaling pathway and AKT activity.Objective To investigate the radiosensitivity effect of niclosamide which is recommended by the world health organization (WHO) to kill tapeworm on triple negative breast cancer cells and the potential mechanisms of its inhibition effect related to Wnt/p-catenin signaling pathway and AKT activity.Methods Four methyl thiazolyl tetrazolium (MTT) assay was used to measure the dose dependent growth inhibition effect of niclosamide on triple negative breast cancer MDA-MB-231/Hs578T cells and non-triple negative breast cancer T-47D/MCF-7 cells and the growth inhibition effect of different concentration of LiCl on MDA-MB-231 cells.50% inhibitory concentration (IC50) value of the four kinds of breast cancer was calculated. Cells were divided into 4 groups:untreated group, niclosamide or LiCl treated group, radiation group and niclosamide or LiCl combined radiation treated group. The cells were pretreated or unpretreated with a concentration of niclosamide or LiCl lower than 20% IC50, then were irradiated with 137Cs γ-rays at a dose range of 0,2,4 and 6 Gy. Effect of niclosamide on the radiosensitivity of these four human breast cancer cells was assayed with the colony formation method. The location of β-catenin within the nuclear induced by radiation was analyzed with immunofluorescence. Fold change of phospho-β-catenin and β-catenin, phospho-AKT and AKT, Cyclin D1, cleaved-caspase-3、caspase-3、Bax. Bcl-2 and LC-3β protein vs control group were measured with Western blot. Cell cycle progression and apoptosis were assayed with flow cytometry.Results Respective SF6 of MDA-MB-231、Hs578T、T-47D、MCF-7 cells were 0.17±0.05,0.12±0.02、0.06±0.01 and 0.03±0.01. It indicated that triple negative breast cancer cells showed much more radioresisitant than non-triple negative breast cancer cells. Niclosamde obviously inhibited the viability of MDA-MB-231、 Hs578T、T-47D、MCF-7 cells in a dose-dependent manner with a IC50 value of 13.63 ±0.43、25.32±0.54、3.87±0.11、3.68±0.13μM respectively. LiCl inhibited the viability of MDA-MB-231 cells in a dose-dependent manner with a IC50 value of 154.11±2.86mM. Pretreated with 1 and 1.5μM niclosamide evidently enhanced the radiosensitivity of MDA-MB-231 cells to γ-rays, and the SER were 1.40±0.06 and 1.63±0.07. Pretreated with 1 and 1.2μM niclosamide evidently enhanced the radiosensitivity of Hs578T cells to y-rays, and the SER were 1.54±0.06 and 1.63± 0.06. Pretreaed with 0.4 and 0.8μM niclosamide evidently enhanced the radiosensitivity of T-47D and MCF-7 cells to y-rays, and the SER were 0.96± 0.04,0.99±0.03,1.12±0.06, and 1.18±0.10. After pretreatment with 10mM LiCl and 1.5μM niclosamide, the SER of MDA-MB-231 cells was 1.24±0.09. Pretreatment of niclosamide significantly inhibited radiation-induced expression of p-β-catenin (S675)、β-catenin、Cyclin D1、p-AKT(S473) and p-AKT (T308) proteins in MDA-MB-231 and Hs578T cells, reduced radiation-induced β-catenin nuclear location (P<0.05), increased radiation-induced cleaved-caspase-3 and LC-3p expression, diminished the radiation-induced G2/M arrest (P<0.05), increased apoptosis (P<0.05). LiCl eliminated the decreased expression of p-β-catenin (S675)、 β-catenin、Cyclin D1 after treatment with niclosamide, thus eliminated G0/G1 arrest effect of niclosamide.Conclusions Niclosamide enhance the sensitivity of breast cancer cells to y-ray irradiation selectively. To triple negative MDA-MB-231-Hs578T cells, its radiosensitization effect is very significant, while to estrogen and progestogen receptor-positive T-47D、MCF-7 cells, the effect is not obvious, which reminders that radiosensitization effect of niclosamide may put up cell specificity. Niclosamide can selectively enhance the radiosensitization through inhibiting Wnt/β-catenin signaling pathway and AKT activity, which results in the reduction of radiation-induced G2/M arrest and increasing of the apoptosis mediated by caspase and autophagy. Wnt/p-catenin signaling pathway may serve as an important signal for radiosensitization of triple-negative breast cancer.Part II The potential mechanisms related to the radiosensitization effect and the inhibition of DNA double strand break repair of niclosamide on triple negative breast cancer cells.Objective To investigate the relationship between DNA double strand break repair and radiosensitization effect of niclosamide on MDA-MB-231 cells.Methods Cells were divided into 4 groups:untreated group, niclosamide treated group, radiation group and niclosamide combined radiation group. Before irradiated with 137Cs y-rays at a dose of 6Gy, the cells were pretreated or unpretreated with 1.5μM niclosamide. yH2AX、p-DNA-PKcs (T2609)、Rad51 foci induced by radiation were analyzed with immunofluorescence, and the fold change of 53BP1 protein expressions was measured with Western blot.Results Pretreatment with 1.5μM niclosamide evidently enhanced radiation-induced yH2AX foci formation (P<0.05) in MDA-MB-231 cells, diminished the radiation-induced 53BP1 protein expression (P<0.01) and phospho-DNA-PKcs (T2609) foci formation (P<0.05), but had no significant effect on Rad51 foci formation.Conclusions Niclosamide can enhance radiosensitivity of MDA-MB-231 cells by aggravating the γ-ray irradiation-induced DSBs and inhibiting non-homologous end-joining, but have no effect on homologous recombination repair pathway.