ISL Attenuates The Invasive Capacity Of Breast Cancer Cells Via Up-regulating The Tumor Suppressor RECK

Breast cancer is the primary cause of cancer-related deaths in women worldwide, mainly due to tumor cell metastasis. Abnormal degradation of extracellular martrix (ECM) induced by matrix metalloproteases (MMPs) played a key role in tumor cell invasion composed of multiple steps. As proved in vivo and vitro, the tumor suppressor RECK showed anti-tumor effects via the repressive effects on MMP2 and MMP9. And the down-regulation or loss of RECK is frequently observed during tumor progression. Thus, restoration of RECK in tumor cells may play an important role in inhibition of tumor cell invasion. Isoliquiritigenin (ISL), a dietary compound extracted from licorice, has been well studied for its anti-oxidant and anti-inflammatory activity, however whether the involvement of induction of RECK in its anti-tumor invasive capacity remain largely unknown.Objective:In the present study, we used triple negative breast cancer cell line Hs-578T and MDA-MB-231 to explore the mechanism whereby ISL exerted repressive effects on breast cancer cell invasion and the role of RECK protein in it. As a result, our data may provide more evidence for tumor dietary intervention in the future.Methods:With MTT assay, we could determine the cell viability of breast cancer cells treated by ISL at different concentrations. Application of transwell with pre-coated martrigel, we could observe the alteration of the invasive capacity of breast cancer cells after individual treatment. With gelatin zymography assay, we could measure whether the activity of MMP2 and MMP9 changed in the conditioned medium collected from culture dish. Use of RT-PCR and real time PCR assay, we could determine the mRNA level of certain genes in breast cancer cells after individual treatment. With real time PCR assay, we could measure the expression level of miR-21 in breast cancer cells. With ChIP assay, we could observe the enrichment of transcriptional factor stat3 at the promoter of miR-21 in breast cancer cells. We could artificially up-or down-regulate the expression of certain genes in breast cancer cells by application of cell transient transfection assay.Results:(1) ISL could attenuate the invasive capacity of breast cancer Hs-578T and MDA-MB-231 cells:Firstly, due to the null influence on cell viability observed in MTT assay, we selected 20.0 and 10.0μM of ISL as the highest concentrations for treatment of 24 and 48h, respectively. With transwell assay, the invasive capacity of breast cancer cells was inhibited by ISL, without affecting the cell viability. The results of RT-PCR showed that ISL could inhibit the transcription and activity of MMP9 in dose- and time-dependent manner in breast cancer cells. However, neither the expression nor the activity of MMP2 changed due to the exposure of ISL.(2) Induction of RECK expression is involved in the repressive effects of ISL on MMP9:Mechanistical evidence demonstrated that the RECK protein not the mRNA was up-regulated in breast cancer cells exposed to ISL. Moreover, data showed that knockdown of RECK expression with small interfering RNA reversed the inhibitory activity of ISL on MMP9 and tumor cell invasive capacity.(3) ISL up-regulates the RECK protein via decreasing the miR-21 expression: The results of real time PCR showed that ISL could inhibit the expression of miR-21 in dose- and time-dependent manner in breast cancer cells. Knockdown of miR-21 by specific antagonist caused increase of the RECK protein not the mRNA, indicating translation of RECK mRNA triggered by miR-21. Reversion of miR-21 expression by miR-21 mimics attenuated the inductive and inhibitory effects of ISL on RECK protein and the invasive capacity of breast cancer cells, respectively.(4) ISL decreases the enrichment of stat3 at the promoter of miR-21:The data of ChIP assay and immunoblotting assay showed the inhibitory effects of ISL on the enrichment of stat3 at the promoter of miR-21 and this modification occured at post-phosphorylation level.(5) Modulation of PIAS3 is involved in the down-regulation of the enrichment of stat3 at the promoter of miR-21 caused by ISL treatment:Immunoblotting assay confirmed the induction of PIAS3, a specific endogenous inhibitor of activated stat3, in breast cancer cells treated with ISL. Furthermore, knockdown of PIAS3 expression with small interfering RNA reversed the modulation of enrichment of stat3 at the promoter of miR-21 and subsequent miR-21 expression by ISL.Conclusion:In human triple negative breast cancer Hs-578T and MDA-MB-231 cells, ISL decreased the miR-21 expression through reduced enrichment of stat3 at the promoter of miR-21 by inducing PIAS3 expression. Subsequently, the protein level of RECK increased without no alteration of the mRNA. Finally, the expression and enzymic activity of MMP9 decreased, leading to the repression of the invasive capacity of breast cancer cells.