Differential proteomic analysis of cancer stem cell properties in hepatocellular carcinomas by isobaric tag labeling and mass spectrometry.

Malignant tumors are relatively resistant to treatment due to their heterogeneous nature, drug resistance, and tendency for metastasis. Recent studies suggest that a subpopulation of cancer cells is responsible for the malignant outcomes. These cells are considered as cancer stem cells (CSC). Although a number of molecules have been identified in different cancer cells as markers for cancer stem cells, no promising markers are currently available for hepatocellular carcinoma cells. In this study, two clones of Hep3B cell lines were functionally characterized as control or CSC-like cells, based on properties including spheroid formation, drug resistance, and tumor initiation. Furthermore, their protein expression profiles were investigated by isobaric tags for relative and absolute quantitation (iTRAQ), and a total of 1,127 proteins were identified and quantified from the combined fractions; 50 proteins exhibited at least 2-fold differences between these two clones. These 50 proteins were analyzed by GeneGo and were found to be associated with liver neoplasms, hepatocellular carcinoma (HCC), and liver diseases. They were also components of metabolic pathways, immune responses, and cytoskeleton remodeling. Among these proteins, the expressions of S100P, S100A14, and vimentin were verified in several HCC cell lines, and their expressions were correlated with tumorigenicity in HCC cell lines. The functional significance of vimentin and S100A14 were also investigated and verified.

Chlorella sorokiniana-Induced Activation and Maturation of Human Monocyte-Derived Dendritic Cells through NF-κB and PI3K/MAPK Pathways.

Chlorella sorokiniana (CS) is a unicellular green alga. The extracts of Chlorella have been used as treatments for relieving hypertension and modulating immune response. The detailed mechanisms are not clear yet. In this study, we sought to study the molecular mechanisms for the polysaccharide fraction of CS-induced immune response. We pulsed dendritic cells (DCs) with CS and found that CS could maturate DCs. CS-maturated DC could activate naïve T cells and stimulate T-cell proliferation and IFN-γ secretion. Furthermore, CS activated PI3K and MAPKs signaling pathways in DCs by interacting with TLR4 receptor. These CS-activated signaling pathways could further activate NF-κB and induce IL-12 production in DCs. This study provides molecular mechanisms for CS-induced DCs activation and immune response.

Astragalus Polysaccharide (PG2) Suppresses Macrophage Migration Inhibitory Factor and Aggressiveness of Lung Adenocarcinoma Cells.

Astragalus membranaceus is the most popular traditional Chinese medicine for managing vital energy deficiency. Its injectable polysaccharide PG2 has been used for relieving cancer-related fatigue, and PG2 has immune-modulatory and anti-inflammatory effects. In this study, we explored the effects of PG2 in lung adenocarcinoma A549 and CL1-2 cells and investigated its anticancer activity, and the results were validated in severe combined immunodeficiency (SCID) mice. Although PG2 did not inhibit the growth of these cells, it dose-dependently suppressed their migration and invasion, accompanied by reduced vimentin and AXL and induced epithelial cadherin (E-cadherin) expression. Regarding the underlying molecular mechanism, PG2 treatment reduced the macrophage migration inhibitory factor (MIF), an inflammatory cytokine that promotes the epithelial-mesenchymal transition and aggressiveness of cancer cells. Consistent with the previous finding that MIF regulates matrix metalloproteinase-13 (MMP-13) and AMP-activated protein kinase (AMPK), treatment with PG2 reduced MMP-13 and activated AMPK in A549 and CL1-2 cells in this study. In SCID mice injected with A549 cells through the tail vein, intraperitoneal injection with PG2 reduced lung and abdominal metastases in parallel with decreased immunohistochemical staining of AXL, vimentin, MMP-13, and MIF in the tumor. Collectively, data revealed a potential application of PG2 in integrative cancer treatment through the suppression of MIF in cancer cells and their aggressiveness.

Karnofsky Performance Status as A Predictive Factor for Cancer-Related Fatigue Treatment with Astragalus Polysaccharides (PG2) Injection-A Double Blind, Multi-Center, Randomized Phase IV Study.

Fatigue is a common and debilitating symptom in patients with advanced cancer, resulting in poor quality of life and reduced treatment efficacy. Phytotherapeutic agents have shown potential effects to relieve cancer-related fatigue in these patients. The aim of this study was to evaluate the efficacy and safety of Astragalus Polysaccharides injection and identify predictive factors associated with this treatment. Patients with advanced cancer receiving palliative care with moderate to severe cancer-related fatigue were enrolled in this study for two treatment cycles. Fatigue improvement response rates were analyzed as the primary endpoint at the end of the first cycle to determine treatment efficacy. The drug safety profile was evaluated by the reporting of adverse events. Three hundred and ten patients were enrolled in this study and 214 patients were included ITT population. Improvement in fatigue scores by at least 10% was observed in greater than 65% of subjects after one treatment cycle compared to scores at baseline. Patients with higher Karnofsky Performance Status (KPS) responded better to the Astragalus Polysaccharides injection. Drug-related adverse event rates were less than 9%. This study identified KPS as a promising predictive factor for the therapeutic efficacy of Astragalus Polysaccharides injection.

PKCdelta clustering at the leading edge and mediating growth factor-enhanced, but not ecm-initiated, dermal fibroblast migration.

We have previously shown that the immobilized extracellular matrices (ECMs) initiate cell migration and soluble growth factors (GFs) further enhance ECM-initiated cell migration. GFs alone cannot initiate cell migration. To further investigate the specificity of the two signaling mechanisms, we focused on the protein kinase C (PKC) family genes in primary human dermal fibroblasts (DFs). We here show that platelet-derived growth factor-BB (PDGF-BB) strongly stimulates membrane translocation and leading edge clustering of protein kinase Cdelta (PKCdelta). In contrast, attachment to collagen matrix alone does not cause the translocation. Although the kinase function of PKCdelta is dispensable for initial membrane translocation, it is critical for its sustained presence at the cells's leading edge. Blockade of endogenous PKCdelta signaling with dominant-negative kinase-defective PKC (PKCdelta-KD) or PKCdelta-small interfering RNA (siRNA) completely inhibited PDGF-BB-stimulated DF migration. In contrast, neither PKCdelta-KD nor PKCdelta-siRNA affected collagen-induced initiation of DF migration. Overexpression of a constitutively activated PKCdelta (PKCdelta-R144/145A) partially mimics the effect of PDGF-BB. However, PKCdelta-KD, PKCdelta-siRNA, or PKCdelta-R144/145A does not affect PDGF-BB-stimulated activation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase1/2, or c-Jun N-terminal kinase. Instead, inhibition of PKCdelta blocks PDGF-BB-stimulated activation of signal transducer and activator of transcription 3 (Stat3). This study unveiled the specificity of PKCdelta in the control of DF migration.

High-purity separation of cancer cells by optically induced dielectrophoresis.

Detecting and concentrating cancer cells in peripheral blood is of great importance for cancer diagnosis and prognosis. Optically induced dielectrophoresis (ODEP) can achieve high resolution and low optical intensities, and the electrode pattern can be dynamically changed by varied light patterns. By changing the projected light pattern, it is demonstrated to separate high-purity gastric cancer cell lines. Traditionally, the purity of cancer cell isolation by negative selection is 0.9% to 10%; by positive selection it is 50% to 62%. An ODEP technology is proposed to enhance the purity of cancer cell isolation to about 77%.

Extracellular heat shock protein 90 signals through subdomain II and the NPVY motif of LRP-1 receptor to Akt1 and Akt2: a circuit essential for promoting skin cell migration in vitro and wound healing in vivo.

Normal cells secrete heat shock protein 90 alpha (Hsp90α) in response to tissue injury. Tumor cells have managed to constitutively secrete Hsp90α during invasion and metastasis. The sole function of extracellular Hsp90α (eHsp90α) is to promote cell motility, a critical event for both wound healing and tumor progression. The mechanism of promotility action by eHsp90α, however, has remained elusive. A key issue is whether eHsp90α still acts as a chaperone outside the cells or is a new and bona fide signaling molecule. Here, we have provided evidence that eHsp90α utilizes a unique transmembrane signaling mechanism to promote cell motility and wound healing. First, subdomain II in the extracellular part of low-density lipoprotein receptor-related protein 1 (LRP-1) receives the eHsp90α signal. Then, the NPVY but not the NPTY motif in the cytoplasmic tail of LRP-1 connects eHsp90α signaling to serine 473 but not threonine 308 phosphorylation in Akt kinases. Individual knockdown of Akt1, Akt2, or Akt3 revealed the importance of Akt1 and Akt2 in eHsp90α-induced cell motility. Akt gene rescue experiments suggest that Akt1 and Akt2 work in concert, rather than independently, to mediate eHsp90α promotility signaling. Finally, Akt1 and Akt2 knockout mice showed impaired wound healing that cannot be corrected by topical application with the eHsp90α protein.

Antitumor Effect of Periplocin in TRAIL-Resistant Human Hepatocellular Carcinoma Cells through Downregulation of IAPs.

Cortex periplocae is the dried root bark of Periploca sepium Bge., a traditional Chinese herb medicine. It contains high amounts of cardiac glycosides. Several cardiac glycosides have been reported to inhibit tumor growth or induce tumor cell apoptosis. We extracted and purified cortex periplocae and identified periplocin as the active ingredient that inhibited the growth of TNF-related apoptosis-inducing ligand-(TRAIL-) resistant hepatocellular carcinoma cells. The antitumor activity of periplocin was further increased by TRAIL cotreatment. Periplocin sensitized TRAIL-resistant HCC through the following two mechanisms. First, periplocin induced the expression of DR4 and FADD. Second, the cotreatment of TRAIL and periplocin suppressed several inhibitors of apoptosis (IAPs). Both mechanisms resulted in the activation of caspase 3, 8, and 9 and led to cell apoptosis. In addition, intraperitoneal injection (IP) of periplocin repressed the growth of hepatocellular carcinoma (HCC) in xenograft tumor model in mice. In summary, periplocin sensitized TRAIL-resistant HCC cells to TRAIL treatment and resulted in tumor cell apoptosis and the repression of tumor growth in vivo.

A potentially common peptide target in secreted heat shock protein-90α for hypoxia-inducible factor-1α-positive tumors.

Deregulated accumulation of hypoxia-inducible factor-1α (HIF-1α) is a hallmark of many solid tumors. Directly targeting HIF-1α for therapeutics is challenging. Our finding that HIF-1α regulates secretion of heat shock protein-90α (Hsp90α) for cell migration raises the exciting possibility that targeting the secreted Hsp90α from HIF-1α-positive tumors has a better clinical outlook. Using the HIF-1α-positive and metastatic breast cancer cells MDA-MB-231, we show that down-regulation of the deregulated HIF-1α blocks Hsp90α secretion and invasion of the cells. Reintroducing an active, but not an inactive, HIF-1α into endogenous HIF-1α-depleted cells rescues both Hsp90α secretion and invasion. Inhibition of Hsp90α secretion, neutralization of secreted Hsp90α action, or removal of the cell surface LRP-1 receptor for secreted Hsp90α reduces the tumor cell invasion in vitro and lung colonization and tumor formation in nude mice. Furthermore, we localized the tumor-promoting effect to a 115-amino acid region in secreted Hsp90α called F-5. Supplementation with F-5 is sufficient to bypass the blockade of HIF-1α depletion and resumes invasion by the tumor cells under serum-free conditions. Because normal cells do not secrete Hsp90α in the absence of stress, drugs that target F-5 should be more effective and less toxic in treatment of HIF-1α-positive tumors in humans.

A fragment of secreted Hsp90α carries properties that enable it to accelerate effectively both acute and diabetic wound healing in mice.

Wounds that fail to heal in a timely manner, for example, diabetic foot ulcers, pose a health, economic, and social problem worldwide. For decades, conventional wisdom has pointed to growth factors as the main driving force of wound healing; thus, growth factors have become the center of therapeutic developments. To date, becaplermin (recombinant human PDGF-BB) is the only US FDA-approved growth factor therapy, and it shows modest efficacy, is costly, and has the potential to cause cancer in patients. Other molecules that drive wound healing have therefore been sought. In this context, it has been noticed that wounds do not heal without the participation of secreted Hsp90α. Here, we report that a 115-aa fragment of secreted Hsp90α (F-5) acts as an unconventional wound healing agent in mice. Topical application of F-5 peptide promoted acute and diabetic wound closure in mice far more effectively than did PDGF-BB. The stronger effect of F-5 was due to 3 properties not held by conventional growth factors: its ability to recruit both epidermal and dermal cells; the fact that its ability to promote dermal cell migration was not inhibited by TGF-ß; and its ability to override the inhibitory effects of hyperglycemia on cell migration in diabetes. The discovery of F-5 challenges the long-standing paradigm of wound healing factors and reveals a potentially more effective and safer agent for healing acute and diabetic wounds.

Participation of the lipoprotein receptor LRP1 in hypoxia-HSP90alpha autocrine signaling to promote keratinocyte migration.

Hypoxia is a microenvironmental stress in many pathological conditions, including wound healing and tumor invasion. Under hypoxia, the cells are forced to adapt alternative and self-supporting mechanisms. Understanding these mechanisms may lead to new insights into human disorders. We report here a novel autocrine signaling mechanism by which hypoxia promotes human keratinocyte (HK) migration. First, hypoxia triggers HKs to secrete heat shock protein 90-alpha (HSP90alpha) via a HIF1-dependent pathway. The secreted HSP90alpha in turn promotes migration, but not proliferation, of the cells. Disruption of the secretion or extracellular function of HSP90alpha blocked hypoxia-stimulated HK migration. The ubiquitously expressed surface receptor, LRP1 (LDL-receptor-related protein 1), mediates the HSP90alpha signaling. Inhibition of LRP1 binding to extracellular HSP90alpha by neutralizing antibodies or genetic silencing of the LRP1 receptor by RNAi completely nullified hypoxia-driven HK migration. Finally, re-introducing a RNAi-resistant LRP1 cDNA into LRP1-downregulated HKs rescued the motogenic response of the cells to hypoxia. We propose that the hypoxia-HSP90alpha-LRP1 autocrine loop provides previously unrecognized therapeutic targets for human disorders such as chronic wounds and cancer invasion.

Profiling motility signal-specific genes in primary human keratinocytes.

Regulation of human keratinocyte (HK) migration is critical for skin wound healing. Profiling HK migration-specific genes could help us gain a comprehensive understanding of the process. The main challenge is to separate genes that are unrelated to migration, but simultaneously induced by the same growth factor. In this study, we took advantage of a unique response of HKs to transforming growth factor-beta (TGF-beta), which inhibits proliferation but not migration of HKs, to suppress selectively the proliferation-related genes. Furthermore we stimulated HKs independently with TGF-alpha or insulin and identified the common genes and eliminated TGF-alpha- or insulin-specific genes. Under these conditions, we obtained profiles of the immediate-early genes (IEGs, at 30 minutes), early genes (EGs, at 60 minutes), and delayed-early genes (DEGs, at 120 minutes) by microarray analyses, followed by quantitative real-time reverse transcription-PCR (QRT-PCR) validation and functional characterization by RNA interference (RNAi). Our results revealed the following: (1) 25 upregulated and 1 downregulated IEGs; (2) 58 upregulated and 15 downregulated EGs, and (3) 13 upregulated and 3 downregulated DEGs in both TGF-alpha- and insulin-stimulated HKs. Three genes, all encoding secreted molecules, were investigated in HK migration. These cell motility-specific gene profiles may prove useful to skin wound healing.

Transforming growth factor alpha (TGFalpha)-stimulated secretion of HSP90alpha: using the receptor LRP-1/CD91 to promote human skin cell migration against a TGFbeta-rich environment during wound healing.

Jump-starting and subsequently maintaining epidermal and dermal cell migration are essential processes for skin wound healing. These events are often disrupted in nonhealing wounds, causing patient morbidity and even fatality. Currently available treatments are unsatisfactory. To identify novel wound-healing targets, we investigated secreted molecules from transforming growth factor alpha (TGFalpha)-stimulated human keratinoytes, which contained strong motogenic, but not mitogenic, activity. Protein purification allowed us to identify the heat shock protein 90alpha (hsp90alpha) as the factor fully responsible for the motogenic activity in keratinocyte secretion. TGFalpha causes rapid membrane translocation and subsequent secretion of hsp90alpha via the unconventional exosome pathway in the cells. Secreted hsp90alpha promotes both epidermal and dermal cell migration through the surface receptor LRP-1 (LDL receptor-related protein 1)/CD91. The promotility activity resides in the middle domain plus the charged sequence of hsp90alpha but is independent of the ATPase activity. Neutralizing the extracellular function of hsp90alpha blocks TGFalpha-induced keratinicyte migration. Most intriguingly, unlike the effects of canonical growth factors, the hsp90alpha signaling overrides the inhibition of TGFbeta, an abundant inhibitor of dermal cell migration in skin wounds. This finding provides a long-sought answer to the question of how dermal cells migrate into the wound environment to build new connective tissues and blood vessels. Thus, secreted hsp90alpha is potentially a new agent for wound healing.

Extracellular heat shock protein-90alpha: linking hypoxia to skin cell motility and wound healing.

Hypoxia is a microenvironmental stress in wounded skin, where it supports wound healing by promoting cell motility. The mechanism of the hypoxia action remained speculative. Here, we provide evidence that hypoxia promotes human dermal fibroblast (HDF) migration by inducing secretion of heat shock protein-90alpha (hsp90alpha) into the extracellular environment through hypoxia-inducible factor-1alpha (HIF-1alpha). The secreted hsp90alpha in turn executes hypoxia's pro-motility effect. Expression of an activated HIF-1alpha mimicked, whereas expression of an inactive HIF-1alpha or suppression of endogenous HIF-1alpha blocked, hypoxia-induced hsp90alpha secretion and HDF migration. Interestingly, the hypoxia-HIF-1 pathway-induced hsp90alpha secretion required neither changes in the steady-state mRNA level nor in the promoter activity of hsp90alpha. Recombinant hsp90alpha fully duplicated the hypoxia effect on HDFs. Inhibition of extracellular hsp90alpha function completely blocked the hypoxia-HIF-1 pathway-stimulated HDF migration. More intriguingly, topical application of hsp90alpha accelerated wound healing in mice. This study has demonstrated a novel mechanism of hypoxia>HIF-1>hsp90alpha secretion>skin cell migration>wound healing, and identified extracellular hsp90alpha as a potential therapeutic agent for skin wounds.