Preclinical trial of targeting the Hic-5-mediated pathway to prevent the progression of hepatocellular carcinoma.

The poor prognosis of hepatocellular carcinoma (HCC) was ascribed to metastasis. Targeted therapy aiming at the molecules along the metastatic pathway is a promising therapeutic strategy. Among them, hydrogen peroxide inducible clone-5 (Hic-5) is highlighted. Hic-5, discovered as a reactive oxygen species (ROS)-inducible gene, was identified to be an adaptor protein in focal adhesion and a critical signaling mediator upregulated in various cancers including HCC. Moreover, Hic-5 may regulate epithelial-mesenchymal transition (EMT) transcription factor Snail and its downstream mesenchymal genes including fibronectin and matrix metalloproteinase-9 required for migration and invasion of HCC. However, the comprehensive Hic-5-mediated pathway was not established and whether Hic-5 can be a target for preventing HCC progression has not been validated in vivo. Using whole-transcriptome mRNA sequencing, we found reactive oxygen species modulator (ROMO) and ZNF395 were upregulated by Hic-5 in a patient-derived HCC cell line, HCC372. Whereas ROMO was involved in Hic-5-mediated ROS signaling, ZNF395 locates downstream of Snail for mesenchymal genes expression required for cell migration. Also, ZNF395 but not ROMO was upregulated by Hic-5 for migration in another patient-derived HCC cell line, HCC374. Further, by in vivo knock down of Hic-5 using the Stable Nucleic Acids Lipid nanoparticles (SNALP)-carried Hic-5 siRNA, progression of HCC372 and HCC374 in SCID mice was prevented, coupled with the decrease of the downstream mesenchymal genes. Our study provides the preclinical evidence that targeting Hic-5 is potentially able to prevent the progression of HCCs with Hic-5 overexpression.

Suppressing of Src-Hic-5-JNK-AKT Signaling Reduced GAPDH Expression for Preventing the Progression of HuCCT1 Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a malignant neoplasm of the bile ducts, being the second most common type of cancer in the liver, and most patients are diagnosed at a late stage with poor prognosis. Targeted therapy aiming at receptors tyrosine kinases (RTKs) such as c-Met or EGFR have been developed but with unsatisfactory outcomes. In our recent report, we found several oncogenic molecules downstream of RTKs, including hydrogen peroxide clone-5 (Hic-5), Src, AKT and JNK, were elevated in tissues of a significant portion of metastatic CCAs. By inhibitor studies and a knockdown approach, these molecules were found to be within the same signal cascade responsible for the migration of HuCCT1 cells, a conventionally used CCA cell line. Herein, we also found Src inhibitor dasatinib and Hic-5 siRNA corporately suppressed HuCCT1 cell invasion. Moreover, dasatinib inhibited the progression of the HuCCT1 tumor on SCID mice skin coupled with decreasing the expression of Hic-5 and EGFR and the activities of Src, AKT and JNK. In addition, we found a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and several cytoskeletal molecules such as tubulin and cofilin were dramatically decreased after a long-term treatment of the HuCCT1 tumor with a high dose of dasatinib. Specifically, GAPDH was shown to be a downstream effector of the Hic-5/Src/AKT cascade involved in HuCCT1 cell migration. On the other hand, TFK1, another CCA cell line without Hic-5 expression, exhibited very low motility, whereas an ectopic Hic-5 expression enhanced the activation of Src and AKT and marginally increased TFK1 migration. In the future, it is tempting to investigate whether cotargeting Src, Hic-5 and/or GAPDH is efficient for preventing CCA progression in future clinical trials.

Targeting Src-Hic-5 Signal Cascade for Preventing Migration of Cholangiocarcinoma Cell HuCCT1.

Cholangiocarcinoma (CCA) is the second most common primary liver cancer with poor prognosis. The deregulation of a lot of oncogenic signaling molecules, such as receptor tyrosine kinases (RTKs), has been found to be associated with CCA progression. However, RTKs-based target therapy showed limited improvement suggesting a need to search for alternative targets for preventing CCA progression. To address this issue, we screened the oncogenic signal molecules upregulated in surgical tissues of CCAs. Interestingly, over-expression of hydrogen peroxide inducible clone-5 (Hic-5) coupled with over-activation of Src, AKT, JNK were observed in 50% of the cholangiocarcinoma with metastatic potential. To investigate whether these molecules may work together to trigger metastatic signaling, their up-and-down relationship was examined in a well-established cholangiocarcinoma cell line, HuCCT1. Src inhibitors PP1 (IC50, 13.4 µM) and dasatinib (IC50, 0.1 µM) significantly decreased both phosphorylated AKT (phosphor-AKT Thr450) and Hic-5 in HuCCT1. In addition, a knockdown of Hic-5 effectively suppressed activation of Src, JNK, and AKT. These implicated a positive cross-talk occurred between Hic-5 and Src for triggering AKT activation. Further, depletion of Hic-5 and inhibition of Src suppressed HuccT1 cell migration in a dose-dependent manner. Remarkably, prior transfection of Hic-5 siRNA for 24 h followed by treatment with PP1 or dasatinib for 24 h resulted in additive suppression of HuCCT1 migration. This suggested that a promising combinatory efficacy can be achieved by depletion of Hic-5 coupled with inhibition of Src. In the future, target therapy against CCA progression by co-targeting Hic-5 and Src may be successfully developed in vivo.

Regulation on tumor metastasis by Raf kinase inhibitory protein: New insight with reactive oxygen species signaling.

Targeted therapy aiming at the metastatic signal pathway, such as that triggered by receptor tyrosine kinase (RTK), for the prevention of tumor progression is promising. However, RTK-based targeted therapy frequently suffered from drug resistance due to the co-expression of multiple growth factor receptors that may raise compensatory secondary signaling and acquired mutations after treatment. One alternative strategy is to manipulate the common negative regulators of the RTK signaling. Among them, Raf kinase inhibitory protein (RKIP) is highlighted and focused on this review. RKIP can associate with Raf-1, thus suppressing the downstream mitogen-activated protein kinase (MAPK) cascade. RKIP also negatively regulates other metastatic signal molecules including NF-κB, STAT3, and NOTCH1. In general, RKIP achieves this task via associating and blocking the activity of the critical molecules on upstream of the aforementioned pathways. One novel RKIP-related signaling involves reactive oxygen species (ROS). In our recent report, we found that PKCδ-mediated ROS generation may interfere with the association of RKIP with heat shock protein 60 (HSP60)/MAPK complex via oxidation of HSP60 triggered by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. The departure of RKIP may impact the downstream MAPK in two aspects. One is to trigger the Mt→cytosol translocation of HSP60 coupled with MAPKs. The other is to change the conformation of HSP60, favoring more efficient activation of the associated MAPK by upstream kinases in cytosol. It is worthy of investigating whether various RTKs capable of generating ROS can drive metastatic signaling via affecting RKIP in the same manner.

Snail Upregulates Transcription of FN, LEF, COX2, and COL1A1 in Hepatocellular Carcinoma: A General Model Established for Snail to Transactivate Mesenchymal Genes.

SNA is one of the essential EMT transcriptional factors capable of suppressing epithelial maker while upregulating mesenchymal markers. However, the mechanisms for SNA to transactivate mesenchymal markers was not well elucidated. Recently, we demonstrated that SNA collaborates with EGR1 and SP1 to directly upregulate MMP9 and ZEB1. Remarkably, a SNA-binding motif (TCACA) upstream of EGR/SP1 overlapping region on promoters was identified. Herein, we examined whether four other mesenchymal markers, lymphoid enhancer-binding factor (LEF), fibronectin (FN), cyclooxygenase 2 (COX2), and collagen type alpha I (COL1A1) are upregulated by SNA in a similar fashion. Expectedly, SNA is essential for expression of these mesenchymal genes. By deletion mapping and site directed mutagenesis coupled with dual luciferase promoter assay, SNA-binding motif and EGR1/SP1 overlapping region are required for TPA-induced transcription of LEF, FN, COX2 and COL1A1. Consistently, TPA induced binding of SNA and EGR1/SP1 on relevant promoter regions of these mesenchymal genes using ChIP and EMSA. Thus far, we found six of the mesenchymal genes are transcriptionally upregulated by SNA in the same fashion. Moreover, comprehensive screening revealed similar sequence architectures on promoter regions of other SNA-upregulated mesenchymal markers, suggesting that a general model for SNA-upregulated mesenchymal genes can be established.

Dengue Virus Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent NETosis-Mediated Inflammation in Mice.

Abnormal immune responses and cytokine storm are involved in the development of severe dengue, a life-threatening disease with high mortality. Dengue virus-induced neutrophil NETosis response is associated with cytokine storm; while the role of viral factors on the elicitation of excessive inflammation mains unclear. Here we found that treatments of dengue virus envelope protein domain III (EIII), cellular binding moiety of virion, is sufficient to induce neutrophil NETosis processes in vitro and in vivo. Challenges of EIII in inflammasome Nlrp3-/- and Casp1-/- mutant mice resulted in less inflammation and NETosis responses, as compared to the wild type controls. Blockages of EIII-neutrophil interaction using cell-binding competitive inhibitor or selective Nlrp3 inflammasome inhibitors OLT1177 and Z-WHED-FMK can suppress EIII-induced NETosis response. These results collectively suggest that Nlrp3 inflammsome is a molecular target for treating dengue-elicited inflammatory pathogenesis.

PKCδ mediates mitochondrial ROS generation and oxidation of HSP60 to relieve RKIP inhibition on MAPK pathway for HCC progression.

Both protein kinase C (PKC) and reactive oxygen species (ROS) are well-known signaling messengers cross-talking with each other to activate mitogen-activated protein kinases (MAPKs) for progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms are not well elucidated. Especially, whether mitochondrial ROS (mtROS) is involved and how it triggers MAPK signaling are intriguing. In this study, we found mtROS generation and phosphorylation of MAPKs were mediated by PKCδ in HCCs treated with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Heat shock protein 60 (HSP60), one of the chaperones in mitochondria was the major protein oxidized in TPA-treated HCCs. Moreover, depletion of HSP60 or expression of HSP60 cysteine mutant prevented TPA-induced phosphorylation of MAPKs. To delineate how HSP60 mediated MAPK activation, the role of Raf kinase inhibitor protein (RKIP), a negative regulator of MAPK, was investigated. TPA dissociated RKIP from HSP60 in both mitochondria and cytosol, concurrently with translocation of HSP60 and MAPK from mitochondria to cytosol, which was associated with robust phosphorylation of MAPKs in the cytosol. Moreover, TPA induced opposite phenotypical changes of HCCs, G1 cell cycle arrest, and cell migration, which were prevented by mtROS scavengers and depletion of PKCδ and HSP60. Consistently, TPA increased the migration-related genes, hydrogen peroxide inducible clone5, matrix metalloproteinase-1/3, lamininγ2, and suppressed the cell cycle regulator cyclin E1 (CCNE1) via PKCδ/mtROS/HSP60/MAPK-axis. Finally, c-jun and c-fos were required for TPA-induced expression of the migration-related genes and a novel microRNA, miR-6134, was responsible for TPA-induced suppression of CCNE1. In conclusion, PKCδ cross-talked with mtROS to trigger HSP60 oxidation for release of RKIP to activate MAPK, regulating gene expression for migration, and G1 cell cycle arrest in HCC. Targeted therapy aiming at key players like PKCδ, RKIP, and HSP60 is promising for preventing HCC progression.

The role of hydrogen peroxide-inducible clone-5 in tumor progression.

The poor prognosis of cancers such as hepatocellular carcinoma is due to high recurrence rate mainly caused by metastasis. Target therapy aiming at critical signal molecules within these pathways is one of the promising strategies for the prevention of metastasis. Hydrogen peroxide-inducible clone-5 (Hic-5), which belongs to the paxillin superfamily, is emerging as a potential target along the metastatic signaling pathway. Hic-5 and paxillin share similar structural features; however, there are a lot of different biochemical properties between them, including tissue-specific distribution, regulation of gene expression, critical signal cascade, and the impacts on cellular phenotypes. This review focus on the recent studies of Hic-5 related to its impacts on signal transduction and transcription responsible for tumor progression. Hic-5 may regulate mitogen-activated protein kinase cascade for cell migration and invasion in various systems. Hic-5 can mediate transforming growth factor-ß1-induced epithelial-mesenchymal transition (EMT) via RhoA- and Src-dependent signaling. Moreover, Hic-5 plays a central role in a positive feedback Hic-5-NADPH oxidase-ROS-JNK signal cascade. This sustained signaling is required for regulating EMT-related genes including E-cadherin, Snail, MMP9, and Zeb-1. In addition, Hic-5 can be a transcription coregulatory factor for a lot of nuclear receptors. Owing to the critical role of Hic-5 in signal transduction and transcription responsible for tumor progression, it can be a potential therapeutic target for the prevention of tumor metastasis.

Hydrogen peroxide inducible clone-5 sustains NADPH oxidase-dependent reactive oxygen species-c-jun N-terminal kinase signaling in hepatocellular carcinoma.

Target therapy aiming at critical molecules within the metastatic signal pathways is essential for prevention of hepatocellular carcinoma (HCC) progression. Hic-5 (hydrogen peroxide inducible clone-5) which belongs to the paxillin superfamily, can be stimulated by a lot of metastatic factors, such as transforming growth factor (TGF-ß), hepatocyte growth factor (HGF), and reactive oxygen species (ROS). Previous studies implicated Hic-5 cross-talks with the ROS-c-jun N-terminal kinase (JNK) signal cascade in a positive feedback manner. In this report, we addressed this issue in a comprehensive manner. By RNA interference and ectopic Hic-5 expression, we demonstrated Hic-5 was essential for activation of NADPH oxidase and ROS generation leading to activation of downstream JNK and c-jun transcription factor. This was initiated by interaction of Hic-5 with the regulator and adaptor of NADPH oxidase, Rac1 and Traf4, respectively, which may further phosphorylate the nonreceptor tyrosine kinase Pyk2 at Tyr881. On the other hand, promoter activity assay coupled with deletion mapping and site directed mutagenesis strategies demonstrated the distal c-jun and AP4 putative binding regions (943-1126 bp upstream of translational start site) were required for transcriptional activation of Hic-5. Thus Hic-5 was both downstream and upstream of NADPH oxidase-ROS-JNK-c-jun cascade. This signal circuit was essential for regulating the expression of epithelial mesenchymal transition (EMT) factors, such as Snail, Zeb1, E-cadherin, and matrix metalloproteinase 9, involved in HCC cell migration and metastasis. Due to the limited expression of Hic-5 in normal tissue, it can be a promising therapeutic target for preventing HCC metastasis.

Involvement of N-glycan in Multiple Receptor Tyrosine Kinases Targeted by Ling-Zhi-8 for Suppressing HCC413 Tumor Progression.

The poor prognosis of hepatocellular carcinoma (HCC) is resulted from tumor metastasis. Signaling pathways triggered by deregulated receptor tyrosine kinases (RTKs) were the promising therapeutic targets for prevention of HCC progression. However, RTK-based target therapy using conventional kinase-based inhibitors was often hampered by resistances due to compensatory RTKs signaling. Herein, we report that Ling-Zhi-8 (LZ-8), a medicinal peptide from Ganoderma lucidium, was effective in suppressing cell migration of HCC413, by decreasing the amount and activity of various RTKs. These led to the suppression of downstream signaling including phosphorylated JNK, ERK involved in HCC progression. The capability of LZ-8 in targeting multiple RTKs was ascribed to its simultaneous binding to these RTKs. LZ-8 may bind on the N-linked glycan motif of RTKs that is required for their maturation and function. Notably, pretreatment of the N-glycan trimming enzyme PNGase or inhibitors of the mannosidase (N-glycosylation processing enzyme), kifunensine (KIF) and swainsonine (SWN), prevented LZ-8 binding on the aforementioned RTKs and rescued the downstream signaling and cell migration suppressed by LZ-8. Moreover, pretreatment of KIF prevented LZ-8 triggered suppression of tumor growth of HCC413. Our study suggested that a specific type of N-glycan is the potential target for LZ-8 to bind on multiple RTKs for suppressing HCC progression.

Snail collaborates with EGR-1 and SP-1 to directly activate transcription of MMP 9 and ZEB1.

The Snail transcription factor plays as a master regulator of epithelial mesenchymal transition (EMT), one of the steps of tumor metastasis. Snail enhances expressions of a lot of mesenchymal genes including the matrix degradation enzyme matrix metalloproteinases 9 (MMP9) and the EMT transcription factor zinc finger E-box binding homeobox 1 (ZEB1), however, the underlying mechanisms are not clarified. Herein, we investigated how Snail upregulated transcription of ZEB1 and MMP9 induced by the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) in hepatoma cell HepG2. According to deletion mapping and site directed mutagenesis analysis, the TPA-responsive elements on both MMP9 and ZEB1 promoters locate on a putative EGR1 and SP1 overlapping region coupled with an upstream proposed Snail binding motif TCACA. Consistently, chromatin immunoprecipitation (ChIP) assay showed TPA triggered binding of Snail, EGR1 and SP1 on MMP9 and ZEB1 promoters. Double ChIP further indicated TPA induced association of Snail with EGR1 and SP1 on both promoters. Also, electrophoresis mobility shift assay revealed TPA enhanced binding of Snail with a MMP9 promoter fragment. According to shRNA techniques, Snail was essential for gene expression of both ZEB1 and MMP9. In conclusion, Snail transactivates genes involved in tumor progression via direct binding to a specific promoter region.

High mobility group B1 up-regulates angiogenic and fibrogenic factors in human retinal pigment epithelial ARPE-19 cells.

Hypoxia-induced retinal neovascularization plays a central role in the pathogenesis of diabetic retinopathy. This study aimed to investigate whether hypoxia leads to the release of nuclear high mobility group box 1 (HMGB1) peptides from cultured retinal pigment epithelial ARPE-19 cells, to determine the effect of HMGB1 on angiogenic cytokine production and elucidate the involved signaling pathways. A chemical hypoxia mimetic agent, cobalt chloride, induced SIRT1 downregulation, HMGB1 nucleocytoplasmic relocation and extracellular release from ARPE-19 cells, implicating its autocrine function. Resveratrol treatment significantly reduced secretion of HMGB1 from ARPE-19 cells exposed to hypoxia. Cell proliferation and cell cycle analyses demonstrated that exogenous HMGB1 caused significant growth suppression and G1 cell cycle arrest in ARPE-19 cells. Morphological observations showed that HMGB1 enhanced adhesion, but suppressed migration of ARPE-19 cells. More intriguingly, HMGB1 up-regulated expression of angiofibrogenic factors in ARPE-19 cells, including VEGF, bFGF, TGF-ß2, and CTGF. Signal profiling characterization indicated that HMGB1 triggered hyperphosphorylation of Akt, p38 MAPK, and NF-κB, but not that of ERK, JNK, and Smad2, whereas inhibition of PI3K, MAPK, or NF-κB significantly attenuated the HMGB1-driven cytokine overproduction in ARPE-19 cells. Functional neutralization with anti-TLR4 and -RAGE antibodies confirmed that both receptors were involved in the cytokine overproduction. In conclusion, chemically-mimicked hypoxia induced nucleocytoplasmic relocation and release of HMGB1 peptides, which in turn up-regulated the production of angiofibrogenic factors in RPE cells, thereby contributing to the pathogenesis of hypoxia-associated diabetic retinopathies. Conversely, blockades of intraocular HMGB1 bioavailability or signal activation may prevent angiofibrogenesis in development of diabetic retinopathy.

The Therapeutic Targeting of HGF/c-Met Signaling in Hepatocellular Carcinoma: Alternative Approaches.

The poor prognosis of hepatocellular carcinoma (HCC), one of the most devastating cancers worldwide, is due to frequent recurrence and metastasis. Among the metastatic factors in the tumor microenvironment, hepatocyte growth factor (HGF) has been well known to play critical roles in tumor progression, including HCC. Therefore, c-Met is now regarded as the most promising therapeutic target for the treatment of HCC. However, there are still concerns about resistance and the side effects of using conventional inhibitors of c-Met, such as tyrosine kinase inhibitors. Recently, many alternative strategies of c-Met targeting have been emerging. These include targeting the downstream effectors of c-Met, such as hydrogen peroxide-inducible clone 5 (Hic-5), to block the reactive oxygen species (ROS)-mediated signaling for HCC progression. Also, inhibition of endosomal regulators, such as PKCε and GGA3, may perturb the c-Met endosomal signaling for HCC cell migration. On the other hand, many herbal antagonists of c-Met-dependent signaling, such as saponin, resveratrol, and LZ-8, were identified. Taken together, it can be anticipated that more effective and safer c-Met targeting strategies for preventing HCC progression can be established in the future.

Synthesis and evaluation of cholesterol-grafted PEGylated peptides with pH-triggered property as novel drug carriers for cancer chemotherapy.

Multifunctional core/shell micelles were self-assembled from triblock copolymers poly(ethylene glycol) methyl ether-b-peptide-g-cholesterol (mPEG-b-P-g-Chol) and used as the doxorubicin delivery carriers for cancer chemotherapy. The copolymers were designed and synthesized successfully based on peptides containing histidine residues (pH-trigger) with different topological structures. The peptides were modified by mPEG (hydrophilic) and cholesterol motifs (hydrophobic) on the terminus, resulting in pH-sensitive amphiphilic copolymers. The critical micelle concentrations (CMCs) of the micelles were determined as 4.79, 2.50 and 1.86mg/L for the linear, Y-shape and fork-shape copolymers, respectively, demonstrating the formation of micelle even at low concentration. The pKb values of three copolymers were found to be around 6.1-6.3 by potentiometric titration test, showing the satisfied pH-sensitivity. The average diameter and zeta potential of blank micelles were 170nm and +20mV at pH 7.4, and increased to 250nm and +35mV at pH 5.0. DOX was loaded into the core of polymeric micelles by dialysis method, and the drug loading capacity slightly increased when the copolymer topological structure changed from linear to Y- and fork-shape. The drug release rate from the system was obviously influencing by the pH values according to the results of in vitro DOX release experiment. Moreover, to investigate the structure-property relationship, the drug release mechanism was preliminarily explored by the semi-empirical equations. Toxicity test showed that three copolymers had bare toxicity whereas the DOX-loaded micelles remained high cytotoxicity for tumor cells. The results indicate the synthesized copolymers might be a potential hydrophobic drug delivery carrier for cancer targeting therapy with controlled drug release.

Oxidation of heat shock protein 60 and protein disulfide isomerase activates ERK and migration of human hepatocellular carcinoma HepG2.

Hepatocyte growth factor (HGF) and its receptor c-Met were frequently deregulated in hepatocellular carcinoma (HCC). Signaling pathways activated by HGF-c-Met are promising targets for preventing HCC progression. HGF can induce the reactive oxygen species (ROS) signaling for cell adhesion, migration and invasion of tumors including HCC. On the other hand, extracellular signal-regulated kinases (ERK), member of mitogen activated kinase, can be activated by ROS for a lot of cellular processes. As expected, HGF-induced phosphorylation of ERK and progression of HCC cell HepG2 were suppressed by ROS scavengers. By N-(biotinoyl)-N'-(iodoacetyl)-ethylenediamine (BIAM) labeling method, a lot of cysteine (-SH)-containing proteins with M.W. 50-75 kD were decreased in HepG2 treated with HGF or two other ROS generators, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phenazine methosulfate. These redox sensitive proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among them, two chaperones, heat shock protein 60 (HSP60) and protein disulfide isomerase (PDI), were found to be the most common redox sensitive proteins in responding to all three agonists. Affinity blot of BIAM-labeled, immunoprecipitated HSP60 and PDI verified that HGF can decrease the cysteine (-SH) containing HSP60 and PDI. On the other hand, HGF and TPA increased cysteinyl glutathione-containing HSP60, consistent with the decrease of cysteine (-SH)-containing HSP60. Moreover, depletion of HSP60 and PDI or expression of dominant negative mutant of HSP60 with alteration of Cys, effectively prevented HGF-induced ERK phosphorylation and HepG2 migration.In conclusion, the redox sensitive HSP60 and PDI are required for HGF-induced ROS signaling and potential targets for preventing HCC progressions.

Hydrogen peroxide inducible clone-5 mediates reactive oxygen species signaling for hepatocellular carcinoma progression.

One of the signaling components involved in hepatocellular carcinoma (HCC) progression is the focal adhesion adaptor paxillin. Hydrogen peroxide inducible clone-5 (Hic-5), one of the paralogs of paxillin, exhibits many biological functions distinct from paxillin, but may cooperate with paxillin to trigger tumor progression. Screening of Hic-5 in 145 surgical HCCs demonstrated overexpression of Hic-5 correlated well with intra- and extra-hepatic metastasis. Hic-5 highly expressed in the patient derived HCCs with high motility such as HCC329 and HCC353 but not in the HCCs with low motility such as HCC340. Blockade of Hic-5 expression prevented constitutive migration of HCC329 and HCC353 and HGF-induced cell migration of HCC340. HCC329Hic-5(-), HCC353Hic-5(-), HCC372Hic-5(-), the HCCs stably depleted of Hic-5, exhibited reduced motility compared with each HCC expressing Scramble shRNA. Moreover, intra/extrahepatic metastasis of HCC329Hic-5(-) in SCID mice greatly decreased compared with HCC329Scramble. On the other hand, ectopic Hic-5 expression in HCC340 promoted its progression. Constitutive and HGF-induced Hic-5 expression in HCCs were suppressed by the reactive oxygen species (ROS) scavengers catalase and dithiotheritol and c-Jun N-terminal kinase (JNK) inhibitor SP600125. On the contrary, depletion of Hic-5 blocked constitutive and HGF-induced ROS generation and JNK phosphorylation in HCCs. Also, ectopic expression of Hic-5 enhanced ROS generation and JNK phosphorylation. These highlighted that Hic-5 plays a central role in the positive feedback ROS-JNK signal cascade. Finally, the Chinese herbal derived anti-HCC peptide LZ-8 suppressed constitutive Hic-5 expression and JNK phosphorylation. In conclusion, Hic-5 mediates ROS-JNK signaling and may serve as a therapeutic target for prevention of HCC progression.

PKCε-mediated c-Met endosomal processing directs fluctuant c-Met-JNK-paxillin signaling for tumor progression of HepG2.

Hepatocyte growth factor (HGF) induced c-Met signaling play critical roles in the progression of hepatocellular carcinoma (HCC). However, c-Met targeting approaches suffered resistance and side effect, thus identification of more suitable downstream targets is needed. Recently, we demonstrated HGF-induced fluctuant ERK/paxillin signaling within 24h. We further examined the underlying mechanisms for fluctuant c-Met/JNK/paxillin signal cascade within 12h. HGF-induced phosphorylation of c-Met, JNK, and paxillin (Ser178) shared a common fluctuation pattern characterized by an initial peak at 0.5h, a middle drop at 4h, and a later peak at 10h. Dynasore, the inhibitor of dynamin, suppressed HGF-induced c-Met internalization and phosphorylation of JNK and paxillin (Ser178) at 0.5h, indicating that endosome formation is required for initial signal enhancement. Further, depletion of PKCε not only enhanced HGF-induced phosphorylation of JNK and paxillin (Ser178) but also prevented c-Met degradation at 0.5h, suggesting that PKCε mediated c-Met degradation for signal declination. On the other hand, HGF induced colocalizations of both phosphorylated JNK and paxillin with the endosomal recycling protein GGA3 at 10h and depletion of GGA3 abolished membrane recycling of c-Met and phosphorylation of JNK/paxillin at the same time point. Interestingly, HGF induced GGA3 phosphorylation in a PKCε-dependent manner during 0.5-4h, which is associated with c-Met degradation in the same period. Finally, HGF-induced cell migration, invasion and intrahepatic metastasis of HepG2 were prevented by the inhibitors of endocytosis. Our results suggest that critical endosomal components are promising therapeutic targets for preventing HGF-induced progression of HCC.

Preclinical trials for prevention of tumor progression of hepatocellular carcinoma by LZ-8 targeting c-Met dependent and independent pathways.

Hepatocellular carcinoma (HCC) is among the most lethal cancers. Mounting studies highlighted the essential role of the HGF/c-MET axis in driving HCC tumor progression. Therefore, c-Met is a potential therapeutic target for HCC. However, several concerns remain unresolved in c-Met targeting. First, the status of active c-Met in HCC must be screened to determine patients suitable for therapy. Second, resistance and side effects have been observed frequently when using conventional c-Met inhibitors. Thus, a preclinical system for screening the status of c-Met signaling and identifying efficient and safe anti-HCC agents is urgently required. In this study, immunohistochemical staining of phosphorylated c-Met (Tyr1234) on tissue sections indicated that HCCs with positive c-Met signaling accounted for approximately 46% in 26 cases. Second, many patient-derived HCC cell lines were established and characterized according to motility and c-Met signaling status. Moreover, LZ8, a medicinal peptide purified from the herb Lingzhi, featuring immunomodulatory and anticancer properties, was capable of suppressing cell migration and slightly reducing the survival rate of both c-Met positive and negative HCCs, HCC372, and HCC329, respectively. LZ8 also suppressed the intrahepatic metastasis of HCC329 in SCID mice. On the molecular level, LZ8 suppressed the expression of c-Met and phosphorylation of c-Met, ERK and AKT in HCC372, and suppressed the phosphorylation of JNK, ERK, and AKT in HCC329. According to receptor array screening, the major receptor tyrosine kinase activated in HCC329 was found to be the epidermal growth factor receptor (EGFR). Moreover, tyrosine-phosphorylated EGFR (the active EGFR) was greatly suppressed in HCC329 by LZ8 treatment. In addition, LZ8 blocked HGF-induced cell migration and c-Met-dependent signaling in HepG2. In summary, we designed a preclinical trial using LZ8 to prevent the tumor progression of patient-derived HCCs with c-Met-positive or -negative signaling.

Pharmacological implications from the adhesion-induced signaling profiles in cultured human retinal pigment epithelial cells.

Extracellular matrix (ECM) plays an active and complex role in regulating cellular behaviors, including proliferation and adhesion. This study aimed at delineating the adhesion-induced signaling profiles in cultured human retinal pigment epithelium (RPE) cells and investigating the antiadhesion effect of antiproliferative drugs in this context. RPE R-50 cells grown on various ECM molecules, such as type I and IV collagens, fibronectin, and laminin, were used for adhesion assay and for examining the phosphorylation profiles of signaling mediators including Akt, extracellular signal-regulated kinase (ERK) 1/2, and integrin-linked kinase (ILK) using Western blotting. The cells receiving antiproliferative drug treatment at subtoxic doses were used to evaluate their antiadhesive and suppressive effects on kinase activities. ECM coating enhanced adhesion and spreading of RPE cells significantly. The cellular attachment onto ECM-coated surfaces differentially induced Akt, ERK1/2, and ILK phosphorylation, and concomitantly increased p53 phosphorylation and cyclin D1 expression, but decreased Bcl-2/Bax ratios. Treatment with antiproliferative agents, including 5-fluorouracil, mitomycin C, and daunomycin, at subtoxic doses suppressed the ability of RPE cells to adhere to ECM substratum significantly. This suppression was in part mediated through reduction of integrin ß1 and ß3 expressions and interfering Akt-ILK signaling activity. Mechanistically, blockade of PI3K/Akt signaling resulted in the suppressed adhesion of RPE cells to ECM. These findings support the hypothesis that, in addition to their antimitogenic effect, antiproliferative agents also exhibit suppressive effect on the adhesiveness of cultured RPE cells. Moreover, inhibitors of the PI3K/Akt signaling mediator can potentially be used as therapeutic agents for proliferative vitreoretinopathy.

Dissipative particle dynamics studies of doxorubicin-loaded micelles assembled from four-arm star triblock polymers 4AS-PCL-b-PDEAEMA-b-PPEGMA and their pH-release mechanism.

Dissipative particle dynamics (DPD) simulation was applied to investigate the microstructures of the micelles self-assembled from pH-sensitive four-arm star triblock poly(ε-caprolactone)-b-poly(2-(diethylamino)ethyl methacrylate)-b-poly(poly(ethylene glycol) methyl ether methacrylate) (4AS-PCL-b-PDEAEMA-b-PPEGMA). In the optimized system, the micelles have a core-mesosphere-shell three-layer structure. The drug-loading process and its distribution at different formulations in the micelles were studied. The results show that DOX molecules distributed in the core and the interface between the core and the mesosphere, suggesting the potential encapsulation capacity of DOX molecules. More drugs were loaded in the micelles with the increase in DOX, and the size of micelles became larger. However, some openings start to generate on the PEG shell when the DOX reaches a certain concentration. By changing the pH values of the system, different morphologies of the micelles were acquired after the pH-sensitive blocks PDEAEMA were protonated, the mechanism of which was also analyzed through correlating functions. The results indicated that the sudden increase in solubility parameter of the pH-sensitive blocks and the swelling of the micelles were the key factors on the change of morphologies. Furthermore, with the decrease in pH value, the number and size of the cracks on the surface of the micelles were larger, which may have a direct effect on the drug release. In conclusion, 4AS-PCL-b-PDEAEMA-b-PPEGMA has great promising applications in delivering hydrophobic anticancer drugs for improved cancer therapy.