Inhibitory Effects And Mechanisms Of Resveratrol On Breast Cancer Stem-like Cells

Breast cancer is the most common malignancy in women worldwide, accounting for29per cent of all new attacked female cancer cases, with approximately70,000new casesdiagnosed each year. Despite advances in the detection and treatment of breast cancer,mortality from this disease remains high, ranking only second to lung cancer, accounting for14per cent of that from all female cancer. The National Cancer Institute has recognized thatprevention is a critical component in minimizing the number of individuals that are afflictedwith breast cancer.The cancer stem cell hypothesis suggested that cancers be driven by a smallsubpopulation of stem cells. The CSCs possess such capacities as self-renewal and tumorgeneration. The concept of CSCs has profound clinical implications for cancer preventionand therapeutic strategies. With the identification of cancer-initiating cells and the assay ofnonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse xenotransplants,this hypothesis is well established. The initial discovery of breast CSCs revealed a cellularsubpopulation from human breast cancer tumors characterized by the cell-surface markersESA+/CD44+/CD24/low. This subpopulation of cells in tumors was highly tumorigenic. Asfew as200ESA+/CD44+/CD24/lowcells or1000CD44+/CD24/lowcells, could producetumors when xenotransplanted into NOD/SCID mice. While more than50,000unsortedcells were required to generate tumors. Subsequent studies found that the aldehydedehydrogenase (ALDH1) activity could also be used to enrich for breast CSCs and normalmammary stem cells. The convenient isolation of CSCs has allowed the investigation of themolecular mechanisms involved in their origin, self-renewal, differentiation into cancercells, resistance to radiation therapy and chemotherapy, and invasiveness and metastaticability. Clinical analyses of CSCs in breast tumors have found a correlation between theproportion of CSCs and poor prognosis. Therefore, chemoprevention and therapy strategiesthat specifically target CSCs are an urgent need. Because a diet rich in plant foods reduced the risks of cancer, there were more andmore interest in isolating and characterizing the nutritive and non-nutritive components infruits and vegetables for potential chemo-preventive agents. Resveratrol(3,4′,5-trihydroxy-trans-stilbene) is a polyphenol compound which is abundantly found inplant foods including grapes, red wine, berries, peanuts，Polygonum cuspidatum andmulberry. Studies revealed a wide spectrum of pharmacological bioactivities of resveratrol,such as antioxidant, anti-inflammatory, anti-tumor and anti-atherosclerotic properties. Theanti-cancer effect of resveratrol has been shown to modulate various steps of carcinogenesisand development such as initiation, progression, and metastasis. However, the underlyingmolecular mechanism of anti-cancer effect has not been clearly defined.Given the important role of CSCs in breast tumorigenesis and development, it is worthinvestigating the effects of resveratrol on breast CSCs and exploring the potentialmechanisms. The purpose of this work was to develop an understanding of the effects ofresveratrol on breast CSCs to determine its therapeutic value in preventing or treating thisdisease.The article is an attempt to investigate the effects and related molecular mechanism ofresveratrol on breast cancer stem cells. Firstly, after breast cancer cells MCF-7andSUM159marked with ALDH1and7AAD, ALDH1positive breast cancer stem cells wereobtained with flow activated cell sorting. Sencondly, the follow experiments wereevaluated the effect of resveratrol on breast cancer stem cells in vitro. The cytotoxic effectsof resveratrol treatment (0,10,20and40μM) for24h on breast epithelial cells MCF10Aand breast cancer cells SUM159, MCF-7were detected by CCK-8assay. The cytotoxiceffects of resveratrol treatment for24h on BCSCs isolated from SUM159and MCF-7cellswere also detected by CCK-8assay.After treatment with resveratrol for24h, the percentageof ALDH1-positive population in SUM159cells or MCF-7cells were detected with flowcytometry. The effect of resveratrol treatment for7days on the formation of mammospheresin BCSCs was evaluated. Thirdly, the assay in vivo was evaluated for resveratrol inhibitesbreast cancer stem cells with primary NOD/SCID mouse model and secondary NOD/SCIDmouse model. SUM159cells (5×106) were injected to the mammary fat pads of5-week-oldfemale NOD/SCID mice. Two weeks after the cell injection, mice were randomly separatedinto two groups: one group was injected (i.p.) with control (0.9%NaCl solution) and the other group was injected with100mg/kg resveratrol (dissolved in0.9%NaCl solution)daily for2weeks. The volumes of tumors were measured every two days. Mice werehumanely euthanized and tumors were harvested. Tumor tissues were dissociatedmechanically and enzymatically to obtain a single-cell suspension. Living cells from thedissociated tumors were sorted out by fluorescence-activated cell sorting. Mice wereimplanted with tumor cells separately. Each secondary NOD/SCID mouse was inoculatedwith5×104cells from control mouse tumors in one side of inguinal mammary fat pad andanother5×104cells from resveratrol-treated tumors in the contralateral mammary fat pad.Lastly，the molecular mechanism of resveratrol inhibited breast cancer stem cells wereexplored by plasmid transfections, confocal microscopy, TEM analysis, western boltting.The results and conclusions are summarized as follows.(1) Resveratrol significantly decreased the cells viability of SUM159and MCF-7,while showed no apparent cytotoxicity on MCF10A cells. Resveratrol treatmentsignificantly decreased the percentage of ALDH1-positive population in SUM159cells andMCF-7. After the treatment of resveratrol on the formation of mammospheres in BCSCs,the number of spheres declined and the size of the spheres was reduced.(2) Using a xenograft model of SUM159cells in NOD/SCID mice，resveratroldisplayed no apparent toxicity as determined by body weight, and the tumor volume inresveratrol-treated mice was significantly smaller than that in the control. The analysisrevealed that resveratrol significantly reduced the ALDH1-positive populations in tumorcells compared with those in the control mice. Furthermore, we examined the ability ofresidual cancer cells to initiate tumors in NOD/SCID mice inoculated with primary tumorcells obtained from the primary xenografts. After inoculation for30d, all6controlinoculations produced tumors, while tumor cells derived from resveratrol-treated mice onlycaused1tumor of6inoculations.(3) TEM analysis showed that resveratrol treatment increased the number ofautophagosomes in BCSCs from SUM159and MCF-7. GFP-LC3-II puncta formation assayconfirmed that resveratrol-treated breast CSCs displayed a significant increase in thepercentage of cells with autophagosomes (GFP-LC3-II dots) compared with the control.Then we performed western blot analysis because LC3-II, Beclin1and Atg7are indicatorsof autophagy and they are required for autophagosome formation. LC3-II, Beclin1and Atg 7were significantly up-regulated by resveratrol in breast CSCs in a dose-dependent manner.In all, these findings indicated that resveratrol induces autophagy in breast CSCs.(4) Resveratrol treatment decreased the expression of β-catenin and cyclin D1in breastCSCs in vitro. Accordingly, the immunohistochemistry detection indicated that resveratroladministration could significantly reduce the expression of β-catenin and cyclin D1inxenograft breast tumors in vivo. Inhibition of autophagy or over-expression of β-cateninattenuates the effects of resveratrol on BCSCs, CQ co-treatment significantly compromisesresveratrol-induced reduction of ALDH1-positive populations in breast cancer cells. CQco-treatment blocked autophagic induction of resveratrol in BCSCs, and simultaneouslyattenuated the anti-proliferation effect of resveratrol on BCSCs. These data addressed theimportant role of autophagy in the anticancer mechanism of resveratrol on BCSCs.Transient transfection of the plasmid pcDNA3-S33Y β-catenin led to substantial productionof β-catenin in BCSCs and abrogated the inhibitory effect of resveratrol on Wnt/β-cateninsignal. In transfected cells, over-expressed β-catenin markedly reduced resveratrol-inducedcytotoxicity and autophagy, indicating that resveratrol inhibits breast CSCs and inducesautophagy, at least partially, via suppressing Wnt/β-catenin signaling pathway.ConclusionIn summary, resveratrol could effectively inhibit breast cancer stem like cells.Resveratrol induces autophagy via suppressing Wnt/β-catenin signaling pathway, whichcould give rise to the autophagic cell death of breast cancer stem cells. The results wouldfurther support the prevention and treatment of resveratrol on breast cancer.