Zhi-Dong Lv

Mesothelial cells differentiate into fibroblast-like cells under the scirrhous gastric cancer microenvironment and promote peritoneal carcinomatosis in vitro and in vivo

Peritoneal metastases are one reason for the poor prognosis of scirrhous gastric cancer (SGC), and myofibroblast provides a favorable environment for the peritoneal dissemination of gastric cancer. The aim of this study was to determine whether myofibroblast originates from peritoneal mesothelial cells under the influence of the tumor microenvironment. Immunohistochemical studies of peritoneal biopsy specimens from patients with peritoneal lavage cytological (+) status demonstrate the expression of the epithelial markers cytokeratin in fibroblast-like cells entrapped in the stroma, suggesting that these cells stemmed from local conversion of mesothelial cells. To confirm this hypothesis in vitro, we co-incubated mesothelial cells with SGC or non-SGC to investigate morphology and function changes. As we expected, mesothelial cells undergo a transition from an epithelial phenotype to a mesenchymal phenotype with loss of epithelial morphology and decrease in the expression of cytokeratin and E-cadherin when exposed to conditioned medium from HSC-39, and the induction of mesothelial cells can be abolished using a neutralizing antibody to transforming growth factor-beta1 (TGF-β1) as well as by pre-treatment with SB431542. Moreover, we found that these mesothelial cells-derived cells exhibit functional properties of myofibroblasts, including the ability to increase adhesion and invasion of SGC. In summary, our current data demonstrated that mesothelial cells are a source of myofibroblasts under the SGC microenvironment which provide a favorable environment for the dissemination of gastric cancer; TGF-β1 produced by autocrine/paracrine in peritoneal cavity may play a central role in this pathogenesis.

Astragalus Extract Attenuates Allergic Airway Inflammation and Inhibits Nuclear Factor κB Expression in Asthmatic Mice

Background:Astragalus membranaceus from traditional Chinese herbal medicines previously showed that it possesses a strong anti-inflammatory activity. The purpose of this study was to elucidate the effect of astragalus on allergen-induced airway inflammation and airway hyperresponsiveness and investigate its possible molecular mechanisms. Methods:Female BALB/c mice sensitized and challenged with ovalbumin (OVA) developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts and cytokine and chemokine levels. In vivo airway responsiveness to increasing concentrations of methacholine was measured 24 hours after the last OVA challenge using whole-body plethysmography. The expression of inhibitory &kgr;B-&agr; and p65 in lung tissues was measured by Western blotting. Results:Astragalus extract attenuated lung inflammation, goblet cell hyperplasia and airway hyperresponsiveness in OVA-induced asthma and decreased eosinophils and lymphocytes in bronchoalveolar lavage fluid. In addition, astragalus extract treatment reduced expression of the key initiators of allergic TH2-associated cytokines (interleukin 4, interleukin 5) (P < 0.05). Furthermore, astragalus extract could inhibit nuclear factor &kgr;B (NF-&kgr;B) expression and suppress NF-&kgr;B translocation from the cytoplasm to the nucleus in lung tissue samples. Conclusions:Taken together, our current study demonstrated a potential therapeutic value of astragalus extract in the treatment of asthma and it may act by inhibiting the expression of the NF-&kgr;B pathway.

miR-655 suppresses epithelial-to-mesenchymal transition by targeting Prrx1 in triple-negative breast cancer.

Triple‐negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial‐to‐mesenchymal transition (EMT) is a key contributor in the metastatic process. In this study, we found that miR‐655 was down‐regulated in TNBC, and its expression levels were associated with molecular‐based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR‐655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR‐655 not only induced the up‐regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal‐like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR‐655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR‐655 significantly suppressed Prrx1, as demonstrated by Prrx1 3′‐untranslated region luciferase report assay. Our study demonstrated that miR‐655 inhibits the acquisition of the EMT phenotype in TNBC by down‐regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression.

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.

Combination of Ad-sTRAIL with the chemotherapeutic drug cisplatin synergistically enhances their pro-apoptotic ability in human breast cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand (TRAIL or Apo2L) is a member of the tumor necrosis factor superfamily that induces apoptosis in various cancer cell types but not in most normal cells. However, it is clear that not all cancer cells are sensitive to the killing effects of TRAIL, including breast cancer. Previous studies have demonstrated that chemotherapeutic drugs sensitize tumor cells to apoptosis induced by TRAIL in several types of malignancies. In the present study, we studied the effects of TRAIL combined with cisplatin on two breast cancer cell lines MDA-MB-468 and HCC-1937 in vitro. MTT assay, crystal violet staining assay, DAPI staining assay and flow cytometric analysis were undertaken to evaluate the enhancement of breast cancer cell death using Ad-sTRAIL and/or cisplatin. The levels of apoptotic molecules in signal transduction pathways were analyzed by real-time RT-PCR and western blotting. We found that co-treatment with Ad-sTRAIL and cisplatin exhibited stronger cytotoxicity and induced more significant apoptosis in breast cancer cells compared with Ad-sTRAIL or cisplatin alone. Pretreatment with cisplatin significantly enhanced the expression of DR5. Moreover, the induction of apoptosis by TRAIL plus cisplatin was accompanied by the downregulation of cFLIP and BCL2L1, and simultaneously robust enzymatic activation of caspase-8, culminating in decreased cancer cell survival. The present study revealed that TRAIL conjugated with cisplatin exhibited a markedly increased cytotoxic and apoptosis-inducing effect on breast cancer cells.

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.

The cytotoxic effect of TGF-β1 on mesothelial cells via apoptosis in early peritoneal carcinomatosis.

Peritoneal dissemination is one of the main causes of death in gastric cancer patients. We have previously reported that gastric cancer cells can induce peritoneal apoptosis, lead to damage of peritoneum integrity, and therefore promote peritoneal metastasis. However, the soluble factors secreted by cancer cells to trigger the damaging cascade remain unclear. TGF-β1, a cytokine known for its capacity to induce proliferative and transformative changes of cells is found in significantly higher quantities correlated with peritoneal metastasis and TNM stages of gastric cancer. High levels of TGF-β1 in the subperitoneal milieu may affect the morphology and function of mesothelial cells, so that the resulting environment becomes favorable for peritoneal metastases. We observed apoptosis induced by TGF-β1 in mesothelial cells in peritoneal carcinomatosis. Knockdown of the smad2 gene by siRNA silencing can partially inhibit these effects. TGF-β1 could upregulate the expressions of Bax and suppress Bcl-2 in mesothelial cells. We conclude that TGF-β1 could induce apoptosis of mesothelial cells, which involves the smad2 signaling pathway in peritoneal carcinomatosis. Bcl-2 and Bax may contribute to this phenomenon.

Blocking TGF-β1 by P17 peptides attenuates gastric cancer cell induced peritoneal fibrosis and prevents peritoneal dissemination in vitro and in vivo

Our previous study demonstrated that the peritoneal stroma environment favors proliferation of tumor cells by serving as a rich source of growth factors and chemokines known to be involved in tumor metastasis. In this study, we investigated the interaction between gastric cancer cells and peritoneal mesothelial cells, and determined the effects of TGF-β1 in this processing. Human peritoneal tissues and peritoneal wash fluid were obtained, which examined by hematoxylin and eosin staining or ELISA for measurements of TGF-β1 levels. The peritoneal mesothelial cells were co-incubated with the supernatants of gastric cancer, the expression of TGF-β1, collagen and fibronectin was observed by ELISA and western blot. We then investigated the effects of serum-free conditioned media from HSC-39 gastric cancer cells on the peritoneum of nude mice, and the effects of peritoneal fibrosis on the development of peritoneal metastasis in vivo. The peritoneum from gastric patients were thickened and contained extensive fibrosis. After co-culture both gastric tumor cells and mesothelial cells, we found that TGF-β1 expression was greatly increased in the co-culture system compared to individual culture condition. Serum-free Conditioned Media from HSC-39 was able to induce extracellular matrix expression in vitro and in vivo, and tumorigenicity in mice with peritoneal fibrosis was greater than in mice with normal peritoneum, while blocking TGF-β1 by peptide P17 can partially inhibit these effects. In conclusion, these results indicated that the interaction of gastric cancer with peritoneal fibrosis and determined that TGF-β1 plays a key role in induction of peritoneal fibrosis, which in turn affected dissemination of gastric 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.