Cardiotoxin III suppresses hepatocyte growth factor–stimulated migration and invasion of MDA‐MB‐231 cells

The hepatocyte growth factor (HGF)/c‐Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA‐MB‐231 cells. When cells were treated with non‐toxic doses of CTX III, CTX III inhibited the HGF‐promoted cell migration and invasion. CTX III significantly suppressed the HGF‐induced c‐Met phosphorylation and downstream activation of phosphatidylinositol 3‐kinase (PI3k)/Akt and extracellular signal‐regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF‐κB) as well as a reduction of matrix metalloproteinase‐9 (MMP‐9) activity. Furthermore, the c‐Met inhibitor PHA665752 inhibited HGF‐induced MMP‐9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c‐Met activation. Taken together, these findings suggest that CTX III inhibits the HGF‐induced invasion and migration of MDA‐MB‐231 cells via HGF/c‐Met‐dependent PI3K/Akt, ERK1/2 and NF‐κB signalling pathways, leading to the downregulation of MMP‐9 expression. Copyright © 2014 John Wiley & Sons, Ltd.     

Interleukin‐6 contributes to chemoresistance in MDA‐MB‐231 cells via targeting HIF‐1α

Chemoresistance is a critical challenge in the clinical treatment of triple‐negative breast cancer (TNBC). It has been well documented that inflammatory mediators from tumor microenvironment are involved in the pathogenesis of TNBC and might be related to chemoresistance of cancer cells. In this study, the contribution of interleukin‐6 (IL‐6), one of the principal oncogenic molecules, in chemoresistance of a TNBC cell line MDA‐MB‐231 was first investigated. The results showed that IL‐6 treatment could induce upregulation of HIF‐1α via the activation of STAT3 in MDA‐MB‐231 cells, which consequently contributed to its effect against chemotherapeutic drug‐induced cytotoxicity and cell apoptosis. However, knockdown of HIF‐1α attenuated such effect via affecting the expressions of apoptosis‐related molecules as Bax and Bcl‐2 and drug transporters as P‐gp and MRP1. This study indicated that targeting at IL‐6/HIF‐1α signaling pathway might be an effective strategy to overcome chemoresistance in TNBC therapy.     

A sea urchin in vivo model to evaluate Epithelial‐Mesenchymal Transition

Epithelial‐mesenchymal transition (EMT) is an evolutionarily conserved cellular program, which is a prerequisite for the metastatic cascade in carcinoma progression. Here, we evaluate the EMT process using the sea urchin Paracentrotus lividus embryo. In sea urchin embryos, the earliest EMT event is related to the acquisition of a mesenchymal phenotype by the spiculogenetic primary mesenchyme cells (PMCs) and their migration into the blastocoel. We investigated the effect of inhibiting the epidermal growth factor (EGF) signaling pathway on this process, and we observed that mesenchyme cell differentiation was blocked. In order to extend and validate our studies, we investigated the migratory capability and the level of potential epidermal growth factor receptor (EGFr) targets in a breast cancer cell line after EGF modulation. Altogether, our data highlight the sensitivity of the sea urchin embryo to anti‐EMT drugs and pinpoint the sea urchin embryo as a valuable in vivo model system for studying EMT and the screening of anti‐EMT candidates.     

Selective Activation of Phospholipase Cγ1 and Distinct Protein Kinase C Subspecies in Intracellular Signaling by Hepatocyte Growth Factor/Scatter Factor in Primary Cultured Rat Neocortical Cells

Abstract: Hepatocyte growth factor/scatter factor (HGF) was recently reported to function as a neurotrophic factor in the CNS. To investigate the intracellular signal pathways after activation of the HGF receptor c-Met in primary cultured rat neocortical cells, in vitro kinase assays were performed. HGF stimulation enhances the phosphorylation of endogenous 80- and 45-kDa substrates. Studies with protein kinase inhibitors and phorbol 12-myristate 13-acetate showed that protein kinase C (PKC) is activated intracellularly. The 80-kDa protein was identified to be the major PKC substrate MARCKS. Although four PKC subspecies, PKCα, PKCε, PKCγ, and PKCλ, were expressed in the cells, only PKCα, PKCε, and PKCγ were selectively translocated in the plasma membrane after HGF stimulation. As expected from these three PKC subspecies, phosphorylation of phospholipase Cγ1 (PLCγ1) but not phosphatidylinositol 3-kinase was enhanced, although the stimulation of brain-derived neurotrophic factor induced phosphorylation of phosphatidylinositol 3-kinase. In contrast to the neocortical cells, HGF did not enhance phosphorylation of PLCγ1 in primary astrocytes. We also found that activated PKC(s) served as a major mitogen-activated protein kinase activator in this pathway. These findings suggest that HGF exerts neurotrophic effects through selective phosphorylation of PLCγ1 and activation of distinct PKC subspecies in neocortical cells, most likely neurons.     

Fisetin induces apoptosis in breast cancer MDA‐MB‐453 cells through degradation of HER2/neu and via the PI3K/Akt pathway

Overexpression of human epidermal growth factor receptor 2 (HER2) is observed in breast cancer. The major snag faced by the human population is the development of chemoresistance to HER2 inhibitors by advanced stage breast cancer cells. Moreover, recent researchers focussed on fisetin as an antiproliferative and chemotherapeutic agent. Therefore, this study was intended to analyze the effects of fisetin on HER2/neu‐overexpressing breast cancer cell lines. Our results depicted that fisetin induced apoptosis of these cells by various mechanisms, such as inactivation of the receptor, induction of proteasomal degradation, decreasing its half‐life, decreasing enolase phosphorylation, and alteration of phosphatidylinositol 3‐kinase/Akt signaling.     

Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial‐to‐mesenchymal transition and immune modulation

Mesenchymal stem cells (MSCs) have been shown to improve the outcome of acute renal injury models; but whether MSCs can delay renal failure in chronic kidney disease (CKD) remains unclear. In the present study, the were cultured in media containing various concentrations of basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2‐phosphate to investigate whether hepatocyte growth factor (HGF) secretion could be increased by the stimulation of these growth factors. Then, TGF‐β1‐treated renal interstitial fibroblast (NRK‐49F), renal proximal tubular cells (NRK‐52E) and podocytes were co‐cultured with conditioned MSCs in the absence or presence of ascorbic acid 2‐phosphate to quantify the protective effects of conditioned MSCs on renal cells. Moreover, male Sprague‐Dawley rats were treated with 1 × 10⁶ conditioned MSCs immediately after 5/6 nephrectomy and every other week through the tail vein for 14 weeks. It was found that basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2‐phosphate promoted HGF secretion in MSCs. Besides, conditioned MSCs were found to be protective against TGF‐β1 induced epithelial‐to‐mesenchymal transition of NRK‐52E and activation of NRK‐49F cells. Furthermore, conditioned MSCs protected podocytes from TGF‐β1‐induced loss of synaptopodin, fibronectin induction, cell death and apoptosis. Rats transplanted with conditioned human MSCs had a significantly increase in creatinine clearance rate, decrease in glomerulosclerosis, interstitial fibrosis and increase in CD4⁺CD25⁺Foxp3⁺ regulatory T cells counts in splenocytes. Together, our studies indicated that conditioned MSCs preserve renal function by their anti‐fibrotic and anti‐inflammatory effects. Transplantation of conditioned MSCs may be useful in treating CKD.     

Growth cone neuropilin‐1 mediates collapsin‐1/sema III facilitation of antero‐ and retrograde axoplasmic transport

Collapsin‐1/SemaIII, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Cellular and molecular mechanisms by which collapsin‐1 exerts its action are not fully understood. Collapsin‐1 induces growth cone collapse via a pathway which may include neuropilin‐1, a cell‐surface collapsin‐1 binding protein, as well as intracellular CRMP‐62 and heterotrimeric G proteins. We previously identified a second action of collapsin‐1, the facilitation of antero‐ and retrograde axoplasmic transport. This response occurs via a mechanism distinct from that causing growth cone collapse. To investigate the possible involvement of neuropilin‐1 in the action of collapsin‐1 on axoplasmic transport, we produced a soluble neuropilin‐1 (sNP‐1) lacking the transmembrane and intracellular region. sNP‐1 progressively displaced the dose–response curve for collapsin‐1 to induce growth cone collapse to higher concentrations. sNP‐1 also inhibited collapsin‐1‐induced augmentation of both antero‐ and retrograde axoplasmic transport. Furthermore, an anti‐neuropilin‐1 antibody blocked the collapsin‐induced axoplasmic transport. These results together indicate that neuropilin‐1 mediates collapsin‐1 action on axoplasmic transport. To visualize collapsin‐1 binding to endogenous neuropilin‐1, we used a truncated collapsin‐1–alkaline phosphatase fusion protein (CAP‐4). CAP‐4 stains the growth cone, neurite, and cell body. However, local application of collapsin‐1 to growth cone but to neither neurite nor cell body promotes axoplasmic transport. Thus, growth cone NP‐1 mediates the facilitatory action of collapsin‐1 on antero‐ and retrograde axoplasmic transport. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 579–589, 1999