BackgroundSTAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH+), or cell surface molecule CD44-positive (CD44+) but CD24-negative (CD24−) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24− subpopulations of breast cancer cells is unknown.Methods and ResultsWe examined STAT3 activation in ALDH+ and ALDH+/CD44+/CD24− subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH+) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH−) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH+ subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH+ subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH+ subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH+ breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH+ cells were further selected for the stem cell markers CD44+ and CD24−.ConclusionThese studies demonstrate an important role for STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24− subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.

Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated in human liver and colon cancer cells and is required for cancer cell viability, survival and migration. Therefore, inhibition of STAT3 signaling may be a viable therapeutic approach for these two cancers. We recently designed a non-peptide small molecule STAT3 inhibitor, LY5, using in silico site-directed Fragment-based drug design (FBDD). The inhibitory effect on STAT3 phosphorylation, cell viability, migration and colony forming ability by LY5 were examined in human liver and colon cancer cells. We demonstrated that LY5 inhibited constitutive Interleukin-6 (IL-6)-induced STAT3 phosphorylation, STAT3 nuclear translocation, decreased STAT3 downstream targeted gene expression and induced apoptosis in liver and colon cancer cells. LY5 had little effect on STAT1 phosphorylation mediated by IFN-γ. Inhibition of persistent STAT3 phosphorylation by LY5 also inhibited colony formation, cell migration, and decreased the viability of liver cancer and colon cancer cells. Furthermore, LY5 inhibited STAT3 phosphorylation and suppressed colon tumor growth in a mouse model in vivo. Our results suggest that LY5 is a potent STAT3 inhibitor and may be a potential drug candidate for liver and colon cancer therapy.

The interleukin (IL)‐6/glycoprotein (GP)130/signal transducer and activator of transcription (STAT)3 pathway is emerging as a target for the treatment of hepatocellular carcinoma. IL‐6 binds to IL‐6R, forming a binary complex, which further combines with GP130 to transduce extracellular signaling by activating STAT3. Therefore, blocking the interaction between IL‐6 and GP130 may inhibit the IL‐6/GP130/STAT3 signaling pathway and its biological effects. It has been reported that bazedoxifene acetate (BAZ), a selective estrogen receptor modulator approved by the US Food and Drug Administration, could inhibit IL‐6/GP130 protein‐protein interactions. Western blot, immunofluorescence staining, wound healing and colony formation assays were used to detect the effect of BAZ on liver cancer cells. Cell viability was evaluated by MTT assay. Apoptosis of cells was determined using the Annexin V‐FITC detection kit. Mouse xenograft tumor models were utilized to evaluate the effect of BAZ in vivo. Our data showed that BAZ inhibited STAT3 phosphorylation (P‐STAT3) and expression of STAT3 downstream genes, inducing apoptosis in liver cancer cells. BAZ inhibited P‐STAT3 induced by IL‐6, but not by leukemia inhibitory factor. BAZ inhibited P‐STAT1 and P‐STAT6 less significantly as elicited by interferon‐α, interferon‐γ and IL‐4. In addition, pretreatment of BAZ impeded the translocation of STAT3 to nuclei induced by IL‐6. BAZ inhibited cell viability, wound healing and colony formation in vitro. Furthermore, tumor growth in HEPG2 mouse xenografts were significantly inhibited by daily intragastric gavage of BAZ. Our results suggest that BAZ inhibited the growth of hepatocellular carcinoma in vitro and in vivo, indicating another potential strategy for HCC prevention and therapy.

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BackgroundInterleukin-6 (IL-6) is a multifunctional cytokine, which is involved in the regulation of differentiation and growth of certain types of tumor cells. Constitutive activation of Signal Transducer and Activator of Transcription 3 (STAT3) induced by IL-6 is frequently detected in liver cancer and has emerged as a viable molecular target for liver cancer treatment. However, few inhibitors targeting up-streams of STAT3 are available for the therapy of liver cancer. We reported the discovery of EVISTA (Raloxifene HCl) as novel inhibitor of IL-6/GP130 protein-protein interactions (PPIs) using multiple ligand simultaneous docking (MLSD) and drug repositioning. The possible effect of Raloxifene in STAT3 signaling or liver cancer cells is still unclear.ResultsRaloxifene inhibited the P-STAT3 stimulated by IL-6, but not the induction of STAT1 and STAT6 phosphorylation by IFN-γ, IFN-α, and IL-4. Raloxifene inhibited STAT3 phosphorylation and resulted in the induction apoptosis on human liver cancer cell-lines. Raloxifene inhibited the targets of STAT3, such as Bcl-2, Bcl-xl and survivin and cell viability, cell migration, and colony formation in liver cancer cells. Further, daily administration of Raloxifene suppressed the Hep-G2 tumor growth in mice in vivo.Materials and MethodsThe inhibitory effect on STAT3 phosphorylation and activity as well as cell viability, migration, and colony forming ability by Raloxifene was examined in human liver cancer cells. Tumor growth was detected via mouse xenograft tumor mode.ConclusionsOur results suggest that Raloxifene is a potent IL-6/GP130 inhibitor and may be a chemoprevention agent for liver cancer by targeting persistent STAT3 signaling.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are the major cause of in-stent restenosis (ISR). Intervention proliferation and migration of VSMCs is an important strategy for antirestenotic therapy. Roscovitine, a second-generation cyclin-dependent kinase inhibitor, can inhibit cell cycle of multiple cell types. We studied the effects of roscovitine on cell cycle distribution, proliferation and migration of VSMCs in vitro by flow cytometry, BrdU incorporation and wound healing assay, respectively. Our results showed that roscovitine increased the proportion of G0/G1 phase cells after 12 h (69.57±3.65 vs. 92.50±1.68, P=0.000), 24 h (80.87±2.24 vs. 90.25±0.79, P=0.000) and 48 h (88.08±3.86 vs. 88.87±2.43, P=0.427) as compared with control group. Roscovitine inhibited proliferation and migration of VSMCs in a concentration-dependent way. With the increase of concentration, roscovitine showed increased capacity for growth and migration inhibition. Roscovitine (30 μmol/L) led to an almost complete VSMCs growth and migration arrest. Combined with its low toxicity and selective inhibition to ISR-VSMCs, roscovitine may be a potential drug in the treatment of vascular stenosis diseases and particularly useful in the prevention and treatment of ISR.

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