Fu-Nian Li

Ganoderic acids suppress growth and angiogenesis by modulating the NF-κB signaling pathway in breast cancer cells.

It has been demonstrated that ganoderma acids suppress growth, angiogenesis and invasiveness of highly invasive and metastatic breast cancer cells in vitro and vivo. However, the mechanism of action of ganoderma acids in breast cancer remains unknown. In the present study, we looked into the effect of ganoderic acid Me (GA-Me) on cellular phenotypes and tumor growth in the MDA-MB-231 breast cancer cell line. The results indicated the GA-Me inhibited nuclear factor kappaB (NF-κB) activity at 24 h in MDA-MB-231 cells. When MDAMB- 231 cells were stimulated with tumor necrosis factor-alpha (TNF-α), the inhibitory effects of GA-Me were still maintained. We demonstrated that GA-Me inhibited proliferation and invasion and induced apoptosis in MDA-MB-231 cells via suppressing the NF-κB activity. However, GA-Me did not inhibit the phosphorylation and degradation of IkappaB-α (IkB-α). GA-Me down-regulated the expression of various NF-κB-regulated genes including genes involved in cell proliferation (c-Myc and cyclin D1), anti-apoptosis (Bcl-2), invasion (MMP-9) and angiogenesis (VEGF, interleukin (IL)-6 and -8). I.P. administration of GA-Me inhibited tumor growth of MDA-MB-231 cells in vivo. Our results demonstrated that GA-Me inhibited proliferation, angiogenesis, invasion and induced apoptosis in MDA-MB-231 cells via suppressing NF-κB activity and the expression profile of its downstream genes. These findings provide evidence for a novel role of GA-Me in the prevention and treatment of breast cancer by its ability to modulate the NF-κB signaling pathway.

Wortmannin Reduces Metastasis and Angiogenesis of Human Breast Cancer Cells via Nuclear Factor-κB-Dependent Matrix Metalloproteinase-9 and Interleukin-8 Pathways

OBJECTIVE: To investigate whether inhibition of Akt phosphorylation by the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, reduces metastasis and angiogenesis in a human breast cancer cell line via nuclear factor (NF)-κB-dependent matrix metalloproteinase (MMP)-9 and interleukin (IL)-8 pathways. METHODS: MDA-MB-231 cells were treated with wortmannin 0 – 200 nM for 4 h. Restoration of Akt activity was evaluated by transfection of cells with constitutively active myristoylated Akt (myr-Akt). NF-κB, MMP-9 and IL-8 proteins were detected by electrophoretic mobility shift assay, Western blot or enzyme-linked immunosorbent assay. The chicken embryo chorio-allantoic membrane assay, cell motility and migration assays were used to evaluate angiogenesis and invasion in vitro. A mouse pseudo metastatic breast cancer model was used to assess the effects of wortmannin on metastasis and angiogenesis in vivo. RESULTS: Wortmannin inhibited the phosphorylation of Akt, upregulation of NF-κB, MMP-9, IL-8, and in vitro cell invasion and angiogenesis, in a dose-dependent manner. Transfection of myr-Akt reversed the cellular and biochemical effects of wortmannin in vitro. Wortmannin also significantly inhibited tumour metastasis and angiogenesis in vivo. CONCLUSION: The findings of the present study suggest that wortmannin inhibits metastasis and angiogenesis in breast cancer cells via PI3K/Akt/NF-κB-mediated MMP-9 and IL-8 signalling pathways.

Mesenchymal stem cells expressing interleukin-18 suppress breast cancer cells in vitro

Breast cancer is the most common malignancy among females throughout the world. Current treatments have unsatisfactory outcomes due to the dispersed nature of certain types of the disease. The development of a more effective therapy for breast cancer has long been one of the most elusive goals of cancer gene therapy. In the present study, human mesenchymal stem cells derived from umbilical cord (hUMSCs) genetically modified with interleukin 18 (IL-18) gene were used to study the effect of hUMSCs/IL-18 on the growth, migration and invasion of MCF-7 and HCC1937 cells in vitro. The hUMSCs could be efficiently modified by lentiviral systems and stably expressed IL-18 protein. hUMSCs/IL-18, but not hUMSCs without the IL-18 gene transduction, significantly suppressed the proliferation, migration and invasion of the MCF-7 and HCC1937 cells. The mechanism of this proliferation suppression may have involved the induction of G1- to S-phase arrest of the breast cancer cells by the hUMSCs/IL-18. In conclusion, hUMSCs/IL-18 can suppress the proliferation, migration and invasion of breast cancer cells in vitro and may provide an approach for a novel antitumor therapy in breast cancer.

Clinicopathological Significance of Mesothelin Expression in Invasive Breast Cancer

OBJECTIVES: This study evaluated the expression profile of the mesothelin (MSLN) gene and its prognostic significance in breast cancer. METHODS: To evaluate the diagnostic and prognostic significance of mesothelin, immunohistochemistry was used to assess the level of mesothelin protein in surgically resected, formalin-fixed, paraffin-embedded invasive breast carcinoma specimens. Associations between mesothelin and other biomarkers, including oestrogen receptor (OR), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2/neu), were also evaluated. RESULTS: A total of 182 breast carcinoma specimens were included. Mesothelin protein was present in the membrane of malignant cells. There was correlation between the presence of mesothelin in tumour cells and tumour infiltration of the lymph node. There was no correlation between the presence of mesothelin and HER2/neu protein, OR and PR in tumour cells. Mesothelin levels were significantly associated with decreased overall survival. CONCLUSIONS: Lymph node status, tumour size, HER2/neu and mesothelin protein levels in breast cancer cells were independent prognostic factors. Mesothelin could be useful as a prognostic marker of overall survival in invasive breast cancer.

Silencing of Prrx1b suppresses cellular proliferation, migration, invasion and epithelial–mesenchymal transition in triple-negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive tumour subtype associated with poor prognosis. The mechanisms involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumour subtype. Paired‐related homeobox 1b (Prrx1b), one of major isoforms of Prrx1, has been identified as a new epithelial–mesenchymal transition (EMT) inducer. However, the function of Prrx1b in TNBC has not been elucidated. In this study, we found that Prrx1b was significantly up‐regulated in TNBC and associated with tumour size and vascular invasion of breast cancer. Silencing of Prrx1b suppressed the proliferation, migration and invasion of basal‐like cancer cells. Moreover, silencing of Prrx1b prevented Wnt/β‐catenin signaling pathway and induced the mesenchymal‐epithelial transition (MET). Taken together, our data indicated that Prrx1b may be an important regulator of EMT in TNBC cells and a new therapeutic target for interventions against TNBC invasion and metastasis.

MiR-212-5p Suppresses the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer by Targeting Prrx2.

Background/Aims: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Our study investigated the functional role of miR-212-5p in TNBC. Methods: Realtime PCR was used to quantify miR-212-5p expression levels in 30 paired TNBC samples and adjacent normal tissues. Wound healing and Transwell assays were used to evaluate the effects of miR-212-5p expression on the invasiveness of TNBC cells. Luciferase reporter and Western blot assays were used to verify whether the mRNA encoding Prrx2 is a major target of miR-212-5p. Results: MiR-212-5p was downregulated in TNBC, and its expression levels were related to tumor size, lymph node status and vascular invasion in breast cancer. We also observed that the miR-212-5p expression level was significantly correlated with a better prognosis in TNBC. Ectopic expression of miR-212-5p induced upregulation of E-cadherin expression and downregulation of vimentin expression. The expression of miR212-5p also suppressed the migration and invasion capacity of mesenchymal-like cancer cells accompanied by a morphological shift towards the epithelial phenotype. Moreover, our study observed that miR-212-5p overexpression significantly suppressed Prrx2 by targeting its 3’-untranslated region (3’-UTR) region, and Prrx2 overexpression partially abrogated miR-212-5p-mediated suppression. Conclusions: Our study demonstrated that miR-212-5p inhibits TNBC from acquiring the EMT phenotype by downregulating Prrx2, thereby inhibiting cell migration and invasion during cancer progression.

Effects of two different immunotherapies on triple negative breast cancer in animal model

The ability of immune system to react specifically against tumors inspirited the study of triple negative breast cancer (TNBC) immunotherapies. Sixty spontaneous breast cancer TA2 mice were randomly divided into three groups: GM-CSF group, with therapy of granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with breast cancer stem cells associated antigens and cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs); DC-CIK group, with infusions of dendritic cells/cytokine-induced killer (DC/CIK) cells; and PBS group as controls. After therapy, the cellular immunity of mice in GM-CSF group and DC-CIK group was obviously increased, especially for GM-CSF group (P<0.05), tumor regression was obviously observed in GM-CSF group. The survival rate of mice in GM-CSF group was significantly higher compared to DC-CIK group and PBS group. These results indicated that tumor immunotherapy manifested strong killing activity against TNBC. The therapeutic effect of GM-CSF combined with antigens and CpG was better than DC-CIK cells.

Mesenchymal stem cells expressing interleukin-18 inhibit breast cancer in a mouse model

Development of an improved breast cancer therapy has been an elusive goal of cancer gene therapy for a long period of time. Human mesenchymal stem cells derived from umbilical cord (hUMSCs) genetically modified with the interleukin (IL)-18 gene (hUMSCs/IL-18) were previously demonstrated to be able to suppress the proliferation, migration and invasion of breast cancer cells in vitro. In the present study, the effect of hUMSCs/IL-18 on breast cancer in a mouse model was investigated. A total of 128 mice were divided into 2 studies (the early-effect study and the late-effect study), with 4 groups in each, including the PBS-, hUMSC-, hUMSC/vector- and hUMSC/IL-18-treated groups. All treatments were injected along with 200 µl PBS. Following therapy, the tumor size, histological examination, and expression of lymphocytes, Ki-67, cluster of differentiation 31 and cytokines [interleukin (IL)-18, IL-12, interferon (IFN)-γ and TNF-α] in each group were analyzed. Proliferation of cells (assessed by measuring tumor size and Ki-67 expression) and metastasis, (by determining pulmonary and hepatic metastasis) of breast cancer cells in the hUMSC/IL-18 group were significantly decreased compared with all other groups. hUMSCs/IL-18 suppressed tumor cell proliferation by activating immunocytes and immune cytokines, decreasing the proliferation index of proliferation marker protein Ki-67 of tumor cells and inhibiting tumor angiogenesis. Furthermore, hUMSCs/IL-18 were able to induce a more marked and improved therapeutic effect in the tumor sites, particularly in early tumors. The results of the present study indicate that hUMSCs/IL-18 were able to inhibit the proliferation and metastasis of breast cancer cells in vivo, possibly leading to an approach for a novel antitumor therapy in breast cancer.

Silencing of Prrx2 Inhibits the Invasion and Metastasis of Breast Cancer both In Vitro and In Vivo by Reversing Epithelial-Mesenchymal Transition

Background/Aims: Epithelial-mesenchymal transition (EMT) is recognized as a crucial mechanism in breast cancer progression and metastasis. Paired-related homeobox 2 (Prrx2) has been identified as a new EMT inducer in cancer, but the underlying mechanisms are still poorly understood. Methods: The expression of Prrx2 was assessed by immunohistochemistry in breast cancer tissues to evaluate the clinicopathological significance of Prrx2, as well as the correlation between Prrx2 and EMT. Short hairpin RNA knockdown of Prrx2 was used to examine cellular effects of Prrx2, detecte the expression of Wnt/β-catenin signaling and EMT-associated proteins, and observe cell proliferation, invasion and migration abilities in vitro and in vivo. Results: Clinical association studies showed that Prrx2 expression was related to tumor size, lymph node metastasis, tumor node metastasis stages, EMT and poor survival. Results also showed that knockdown of Prrx2 could alter cell morphology, suppressed the abilities of cell proliferation, invasion and migration in breast cancer. Moreover, silencing of Prrx2 induced the mesenchymal-epithelial transition and prevented nuclear translocation of β-catenin, inhibited wnt/β-catenin signaling pathway. Conclusion: Our study indicated that Prrx2 may be an important activator of EMT in human breast cancer and it can serve as a molecular target of therapeutic interventions for breast cancer.