Fayun Zhang

Naringenin Decreases Invasiveness and Metastasis by Inhibiting TGF-β-Induced Epithelial to Mesenchymal Transition in Pancreatic Cancer Cells

Epithelial to mesenchymal transition (EMT) promotes cellular motility, invasiveness and metastasis during embryonic development and tumorigenesis. Transforming growth factor-β (TGF-β) signaling pathway is a key regulator of EMT. A lot of evidences suggest that this process is Smad3-dependent. Herein we showed that exposure of aspc-1 and panc-1 pancreatic cancer cells to TGF-β1 resulted in characteristic morphological alterations of EMT, and enhancement of cell motility and gemcitabine (Gem) resistance along with an up-regulation of EMT markers genes such as vimentin, N-cadherin, MMP2 and MMP9. Naringenin (Nar) down-regulated EMT markers expression in both mRNA and protein levels by inhibiting TGF-β1/Smad3 signal pathway in the pancreatic cancer cells. Consequently, Nar suppressed the cells migration and invasion and reversed their resistance to Gem.

Naringenin reduces lung metastasis in a breast cancer resection model

Metastasis is the main cause of death in cancer patients. To improve the outcomes of patients undergoing a surgery, new adjuvant therapies that can effectively inhibit metastases have to be developed. Studies have shown that flavonoid naringenin, a natural product that is mainly present in grapes and citrus, may contribute to cancer prevention. It has many advantages compared to traditional chemotherapeutic drugs, such as low toxicity. To determine whether naringenin can also inhibit metastases, a breast cancer resection model that mimics clinical situations was established. We found that orally administered naringenin significantly decreased the number of metastatic tumor cells in the lung and extended the life span of tumor resected mice. Flow cytometry analysis revealed that T cells displayed enhanced antitumor activity in naringenin treated mice, with an increased proportion of IFN-γ and IL-2 expressing T cells. In vitro studies further demonstrated that relief of immunosuppression caused by regulatory T cells might be the fundamental mechanism of metastasis inhibition by naringenin. These results indicate that orally administered naringenin can inhibit the outgrowth of metastases after surgery via regulating host immunity. Thus, naringenin can be an ideal surgical adjuvant therapy for breast cancer patients.

Polymeric micelles for enhanced lymphatic drug delivery to treat metastatic tumors

Polymeric micelles have been proven to be a promising nano-sized system for drug delivery. Understanding its in vivo behaviors at the whole body, tissue and cellular levels is critical for translating this drug delivery system into clinical practice. In this study, the 14.5 nm micelles made of polyethylene glycol-phosphatidylethanolamine (PEG-PE) for delivery of doxorubicin and vinorelbine were investigated. Using confocal and two-photon microscopy imaging of live mice or tissue sections, we observed that after systemic administration, the fluorescently labeled PEG-PE micelles encapsulating doxorubicin migrated through blood vessels in entirety into the interstitial tissue, collected by lymphatic vessels, and accumulated in lymph nodes. Importantly, encapsulated drugs such as vinorelbine (Nanovin), preferentially accumulate in lymph nodes when compared to the free drugs. Moreover, the in vivo bioluminescent imaging showed that Nanovin significantly reduced lymph node metastasis rate (P<0.05) in 4 T1-luc2 murine breast tumor bearing mice. Finally, we observed that Nanovin enhanced antitumor activity against primary tumors and lung metastases while having low toxicity in various 4 T1 tumor models. This study suggests that PEG-PE micelle is a promising drug delivery system for the treatment of lymphatic metastases, and may also have important applications in other lymphatic system-related diseases.

An Anti-PSMA Bivalent Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Imaging and Therapy

Prostate specific membrane antigen (PSMA) is overexpressed on prostate tumor cells and the neovascular endothelia various solid tumors. A bivalent immunotoxin generated by fusing a fold-back single-chain diabody derived from the Fv fragments of an anti-PSMA monoclonal antibody with a truncated diphtheria toxin (DT) containing the activity and translocation domains [A-dmDT390-scfbDb(PSMA)] might be suitable for targeted therapy of tumors that overexpress PSMA. In this study, a PSMA-positive and a PSMA-negative prostate cancer cell lines were treated with immunotoxin A-dmDT390-scfbDb(PSMA) in order to study the tumor targeting specificity and therapeutic potential of the immunotoxin. The cellular uptake and selective toxicity of the immunotoxin were evident in monolayer cultures of PSMA-positive LNCaP prostate cancer cells but not in cultures of PSMA-negative PC-3 prostate cancer cells. Cellular accumulation of A-dmDT390-scfbDb(PSMA) increased with increasing incubation times and concentrations in LNCaP cells. The proportion of apoptotic LNCaP cells increased upon incubation with increasing doses of the fold-back immunotoxin. Optical imaging and MRI with the Alexa Fluor 680-labeled A-dmDT390-scfbDb(PSMA) confirmed the specific targeting and therapeutic efficacy of this immunotoxin towards PSMA-positive LNCaP solid tumor xenografts in athymic nude mice.

Polymeric micelles as a drug delivery system enhance cytotoxicity of vinorelbine through more intercellular accumulation.

Polymeric micelles had been used as an efficacious carrier system for anti-cancer drug delivery. However, it is not clear whether the molecular mechanism of drug encapsulated in micelles is same as free drug. In this study, the mechanism of vinorelbine loaded in glycol-phosphatidylethanolamine (PEG-PE) micelles (M-Vino) on tumor cells was investigated. Compared with free vinorelbine (Free Vino), M-Vino was more effective in inhibiting the growth of tumor cells in vitro, inducing G2/M phase arrest and apoptosis of tumor cells. M-Vino showed a faster entry and higher accumulation in 4T1 cells than free vinorelbine. Therefore, M-Vino destabilized microtubules, induced cell death, and enhanced its cytotoxicity through more intercellular accumulation of vinorelbine.

Chrysin promotes osteogenic differentiation via ERK/MAPK activation

The effect of the anti-inflammatory flavonoid chrysin on osteogenesis was determined in preosteoblast MC3T3-E1 cells. Results demonstrated that chrysin could induce osteogenic differentiation in the absence of other osteogenic agents. Chrysin treatment promoted the expression of transcription factors (Runx2 and Osx) and bone formation marker genes (Col1A1, OCN, and OPN) as well as enhanced the formation of mineralized nodules. During osteogenic differentiation, chrysin preferentially activated ERK1/2, but not JNK nor the p38 MAPKs. Further experiments with inhibitors revealed the co-treatment of U0126, PD98059, or ICI182780 (a general ER antagonist) with chrysin effectively abrogated the chrysin-induced osteogenesis and ERK1/2 activation. Thus, the effect of chrysin on osteogenesis is ERK1/2-dependent and involves ER. Therefore, chrysin has the significant potential to enhance osteogenesis for osteoporosis prevention and treatment.

Naringenin prevents TGF-β1 secretion from breast cancer and suppresses pulmonary metastasis by inhibiting PKC activation

BackgroundTargeting the TGF-β1 pathway for breast cancer metastasis therapy has become an attractive strategy. We have previously demonstrated that naringenin significantly reduced TGF-β1 levels in bleomycin-induced lung fibrosis and effectively prevented pulmonary metastases of tumors. This raised the question of whether naringenin can block TGF-β1 secretion from breast cancer cells and inhibit their pulmonary metastasis.MethodsWe transduced a lentiviral vector encoding the mouse Tgf-β1 gene into mouse breast carcinoma (4T1-Luc2) cells and inoculated the transformant cells (4T1/TGF-β1) into the fourth primary fat pat of Balb/c mice. Pulmonary metastases derived from the primary tumors were monitored using bioluminescent imaging. Spleens, lungs and serum (n = 18–20 per treatment group) were analyzed for immune cell activity and TGF-β1 level. The mechanism whereby naringenin decreases TGF-β1 secretion from breast cancer cells was investigated at different levels, including Tgf-β1 transcription, mRNA stability, translation, and extracellular release.ResultsIn contrast to the null-vector control (4T1/RFP) tumors, extensive pulmonary metastases derived from 4T1/TGF-β1 tumors were observed. Administration of the TGF-β1 blocking antibody 1D11 or naringenin showed an inhibition of pulmonary metastasis for both 4T1/TGF-β1 tumors and 4T1/RFP tumors, resulting in increased survival of the mice. Compared with 4T1/RFP bearing mice, systemic immunosuppression in 4T1/TGF-β1 bearing mice was observed, represented by a higher proportion of regulatory T cells and myeloid-derived suppressor cells and a lower proportion of activated T cells and INFγ expression in CD8+ T cells. These metrics were improved by administration of 1D11 or naringenin. However, compared with 1D11, which neutralized secreted TGF-β1 but did not affect intracellular TGF-β1 levels, naringenin reduced the secretion of TGF-β1 from the cells, leading to an accumulation of intracellular TGF-β1. Further experiments revealed that naringenin had no effect on Tgf-β1 transcription, mRNA decay or protein translation, but prevented TGF-β1 transport from the trans-Golgi network by inhibiting PKC activity.ConclusionsNaringenin blocks TGF-β1 trafficking from the trans-Golgi network by suppressing PKC activity, resulting in a reduction of TGF-β1 secretion from breast cancer cells. This finding suggests that naringenin may be an attractive therapeutic candidate for TGF-β1 related diseases.

Vitamin C induces periodontal ligament progenitor cell differentiation via activation of ERK pathway mediated by PELP1

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our finding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.

A dual character of flavonoids in influenza A virus replication and spread through modulating cell-autonomous immunity by MAPK signaling pathways

Flavonoids are well known as a large class of polyphenolic compounds, which have a variety of physiological activities, including anti-influenza virus activity. The influenza A/WSN/33 infected A549 cells have been used to screen anti-influenza virus drugs from natural flavonoid compounds library. Unexpectedly, some flavonoid compounds significantly inhibited virus replication, while the others dramatically promoted virus replication. In this study, we attempted to understand these differences between flavonoid compounds in their antivirus mechanisms. Hesperidin and kaempferol were chosen as representatives of both sides, each of which exhibited the opposite effects on influenza virus replication. Our investigation revealed that the opposite effects produced by hesperidin and kaempferol on influenza virus were due to inducing the opposite cell-autonomous immune responses by selectively modulating MAP kinase pathways: hesperidin up-regulated P38 and JNK expression and activation, thus resulting in the enhanced cell-autonomous immunity; while kaempferol dramatically down-regulated p38 and JNK expression and activation, thereby suppressing cell-autonomous immunity. In addition, hesperidin restricted RNPs export from nucleus by down-regulating ERK activation, but kaempferol promoted RNPs export by up-regulating ERK activation. Our findings demonstrate that a new generation of anti-influenza virus drugs could be developed based on selective modulation of MAP kinase pathways to stimulate cell-autonomous immunity.

Naringenin Ameliorates Acute Inflammation by Regulating Intracellular Cytokine Degradation

Ungoverned activation of innate and adaptive immunity results in acute inflammatory disease, such as bacteria-induced endotoxemia and fulminant hepatitis by virus infection. Thus, therapeutic control of inflammation is crucial for clinical management of many human diseases. In murine models of LPS- and Con A–induced liver injury, we found that naringenin, a natural predominant flavanone, is capable of protecting against lethality induced by LPS and preventing inflammation-induced organ injury. The protective effect of naringenin is mediated by reducing the levels of several inflammatory cytokines. Unexpectedly, naringenin inhibits TNF-α and IL-6 secretion in macrophages and T cells without interfering with the TLR signaling cascade, cytokine mRNA stability, or protein translation. These results indicate the existence of a posttranslational control mechanism. Further studies show that naringenin enhances intracellular cytokine degradation through lysosome- and TFEB-dependent mechanisms. This study provides evidence that naringenin has the capacity to dampen cytokine production by regulating lysosome function. Thus, naringenin may represent a potential therapeutic agent for controlling inflammation-related diseases.