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Previously, we reported that the secreted lysyl oxidase like 2 (LOXL2) from hepatocellular carcinoma (HCC) cells under higher stiffness stimulation contributed to the formation of lung premetastatic niche. To further clarify whether matrix stiffness also alters LOXL2 expression in other cells within tumor microenvironment, we developed a gel-based culture system combined with a model of macrophage polarization to evaluate the effects of matrix stiffness on the polarization of M2 macrophages and their LOXL2 expression. THP-1 cells cultured on 6KPa, 10KPa, and 16KPa stiffness substrates were first incubated with 100nM phorbol 12-myristate 13-acetate (PMA) for 24 hours and subsequently treated with 20nM interleukin-4 (IL-4) and 20nM interleukin-13 (IL-13) for 48 hours. The polarization states of M2 macrophages under different stiffness stimulation were comparatively analyzed, and their LOXL2 expressions as well as the underlying molecular mechanism were further explored. Our results demonstrated that increased matrix stiffness remarkably strengthened M2 macrophage polarization and promoted their LOXL2 expression. Activation of integrin β5-FAK-MEK1/2-ERK1/2 pathway participated in matrix stiffness-mediated HIF-1α upregulation, and HIF-1α upregulation resulted in a significant improvement in LOXL2 expression. Additionally, M2 macrophage polarization state and LOXL2 expression in HCC tissues with COL1High /LOXHigh were consistent with the results in vitro, further confirming the regulation roles of matrix stiffness in macrophage polarization and LOXL2 expression. The findings about LOXL2 upregulation in the polarized macrophages under higher stiffness stimulation will be helpful to better understand the underlying mechanism of matrix stiffness-induced premetastatic niche formation in HCC.

Background Metformin, a traditional first-line anti-hyperglycemic agent for diabetes, recently exhibits better antitumor effect in hepatocellular carcinoma (HCC). However, its resistance and tolerance mechanism in HCC remains largely unknown. Here, we investigated whether increased matrix stiffness attenuated the intervention effects of metformin on HCC invasion and metastasis, and explored its underlying molecular mechanism. Methods FN-coated gel substrates with 6, 10, and 16 kPa, which simulated the stiffness of normal, fibrotic, and cirrhotic liver tissues respectively, were established to evaluate matrix stiffness-mediated effects on HCC cells. Alterations in morphology, proliferation, motility, and invasive/metastatic-associated genes (PTEN, MMP2, MMP9) of HCC cells grown on different-stiffness substrates were comparatively analyzed before and after metformin intervention. Subsequently, the underlying molecular mechanism by which higher matrix stiffness attenuates antitumor effects of metformin in HCC was further elucidated. Results Metformin significantly inhibited proliferation, migration, and invasion of HCC cells. Compared with the controls on lower-stiffness substrate, HCC cells grown on higher-stiffness substrate exhibited an obvious resistance to intervention effects of metformin on proliferation, migration, invasion and metastasis. High stiffness stimulation significantly activated the miR-17-5p/PTEN/PI3K/Akt signaling pathway in HCC cells via integrin β1 and in turn resulted in MMP2 and MMP9 upregulation. Meanwhile, integrin β1 knockdown or PI3K inhibitor partially reversed the activation of the above signaling molecules. For HCC cells grown on the same-stiffness substrate, metformin remarkably upregulated PTEN expression and suppressed the activation of the PI3K/Akt/MMP pathway, but no effect on integrin β1 expression. Importantly, the increase in fold of PTEN expression and decrease in folds of Akt phosphorylation level and MMP2 and MMP9 expressions in the treated HCC cells with metformin on 16-kPa stiffness substrate were evidently weakened compared with those in the controls on the 6-kPa stiffness substrate. Conclusions Increased matrix stiffness significantly attenuates the inhibitory effect of metformin on HCC invasion and metastasis, and a common pathway of PTEN/PI3K/Akt/MMPs activated by mechanical stiffness signal and inactivated by metformin contributes to matrix stiffness-caused metformin resistance. To the best of our knowledge, this is the first report to clarify the mechanism of metformin intervention resistance from the perspective of tumor biophysical microenvironment.

Background: The mechanisms underlying the contribution of primary tumor to pre-metastatic niche formation remains largely unknown in HCC. We previously reported that the released Lysyl oxidase like 2 (LOXL2) from HCC cells under higher stiffness stimulation facilitated the formation of lung pre-metastatic niche. Here we further clarified the pathological role of LOXL2 in promoting lung pre-metastatic niche formation in HCC and its relevant molecular mechanism. Methods: Lung pre-metastatic niche nude mouse model and LOXL2-overexpressed xenograft HCC models were respectively developed to validate the significance of LOXL2 in pre-metastatic niche formation and pulmonary metastasis. Simultaneously, a gel-based cell culture system mirroring lung tissue stiffness in normal and pathological state was constructed to investigate the underlying mechanism of LOXL2-induced pre-metastatic niche formation.Results: LOXL2 by tail vein injection had obvious inducting effects on CD11b+/CD45+ BMDCs recruitment and fibronectin expression in lung tissue during the development of lung pre-metastatic niche. Tumor-secreted LOXL2 also promoted the occurrence of pulmonary metastasis and the number of metastasis lesions remarkably in LOXL2-overexpressed xenograft HCC models. Applying a gel-based cell culture system, we discovered that LOXL2 and LOXL2-caused matrix stiffening all upregulated the expressions of MMP9 and fibronectin in lung fibroblasts significantly, but little effect on MMP2 expression. Moreover, the activation of PI3K-AKT pathway participated in the regulation of LOXL2-upregulated fibronectin. Additionally, LOXL2 and LOXL2-caused matrix stiffening also increased the number of adherent HCC cells evidently, as well as the expression of chemokine CXCL2. Clinical data analysis demonstrated that HCC patients in High-LOXL2 group had higher ratio of microvascular invasion (OR=10.563, P=0.005) and tumor recurrence (OR=8.556, P=0.013) than HCC patients in Low-LOXL2 group, also revealing a significance of LOXL2 in HCC progression and unfavorable outcomeConclusion: Primary tumor-released LOXL2 contributes to the formation of lung pre-metastatic niche and the occurrence of lung metastasis. LOXL2 and LOXL2-caused matrix stiffening work together to induce pre-metastatic niche formation through influencing matrix remodeling and cell colonization. This study sheds light on the pathological significance of LOXL2 in lung pre-metastatic niche formation in HCC, also implicates a new intervention approach for HCC metastasis through reversal of lung pre-metastatic niche.

Background Sustained adrenergic signaling secondary to chronic stress promotes cancer progression; however, the underlying mechanisms for this phenomenon remain unclear. Hepatocellular carcinoma (HCC) frequently develops within fibrotic livers rich in activated hepatic stellate cells (HSCs). Here, we examined whether the stress hormone norepinephrine (NE) could accelerate HCC progression by modulating HSCs activities. Methods HCC cells were exposed to conditioned medium (CM) from NE-stimulated HSCs. The changes in cell migration and invasion, epithelial-mesenchymal transition, parameters of cell proliferation, and levels of cancer stem cell markers were analyzed. Moreover, the in vivo tumor progression of HCC cells inoculated with HSCs was studied in nude mice subjected to chronic restraint stress. Results CM from NE-treated HSCs significantly promoted cell migration and invasion, epithelial-mesenchymal transition (EMT), and expression of cell proliferation-related genes and cancer stem cell markers in HCC cells. These pro-tumoral effects were markedly reduced by depleting secreted frizzled related protein 1 (sFRP1) in CM. The pro-tumoral functions of sFRP1 were dependent on β-catenin activation, and sFRP1 augmented the binding of Wnt16B to its receptor FZD7, resulting in enhanced β-catenin activity. Additionally, sFRP1 enhanced Wnt16B expression, reinforcing an autocrine feedback loop of Wnt16B/β-catenin signaling. The expression of sFRP1 in HSCs promoted HCC progression in an in vivo model under chronic restraint stress, which was largely attenuated by sFRP1 knockdown. Conclusions We identify a new mechanism by which chronic stress promotes HCC progression. In this model, NE activates HSCs to secrete sFRP1, which cooperates with a Wnt16B/β-catenin positive feedback loop. Our findings have therapeutic implications for the treatment of chronic stress-promoted HCC progression.

Our previous study has validated that higher matrix stiffness obviously improves vascular endothelial growth factor (VEGF) expression in HCC cells, highlighting a linkage between matrix stiffness and HCC angiogenesis. However, the effects of matrix stiffness on vascular endothelial cells in HCC and its underlying mechanism remain largely uncharacterized. Here we further analyzed the expression of vascular endothelial growth factor receptor 2 (VEGFR2) in human umbilical vein endothelial cells (HUVECs) grown on different stiffness substrates and explored its regulatory mechanism for better understanding matrix stiffness-regulated angiogenesis in HCC. Our results revealed that increased matrix stiffness significantly upregulated the expression of VEGFR2 in HUVECs, and the expression level of VEGFR2 was positively correlated with the expression levels of COL1 and lysyl oxidase in human HCC tissues and rat HCC tissue, moreover VEGFR2 and CD34 were co-localized at blood vessel of HCC tissues, indicating an obvious regulation role of matrix stiffness in VEGFR2 expression. Simultaneously, increased matrix stiffness also elevated the phosphorylation level of Akt and the expressions of integrin αV/β5 and nuclear Sp1 in HUVECs. Inhibition of integrin αVβ5 remarkably reversed the expression of VEGFR2 and phosphorylation level of Akt in HUVECs grown on higher stiffness substrate. Except that, PI3K inhibitor also suppressed the phosphorylation level of Akt and the expressions of VEGFR2 and nuclear Sp1 evidently. Taken together, higher matrix stiffness increased VEGFR2 expression in HUVECs, and integrin αVβ5/Akt/Sp1 pathway participated in stiffness-mediated effects on VEGFR2 upregulation. This study combining with our previous report discloses a new paradigm in which higher matrix stiffness as an initiator drives HCC angiogenesis via upregulating both VEGFR2 expression in vascular endothelial cells and VEGF expression in HCC cells.

Background Accelerated malignant behaviors induced by insufficient thermal ablation have been increasingly reported, however, the exact mechanisms are still unclear. Here, we investigated the importance of the extracellular matrix (ECM) in modulating the progression of residual hepatocellular carcinoma (HCC) after heat treatment. Methods Heat-exposed residual HCC cells were cultured in different ECM gels. We used basement membrane gel (Matrigel) to simulate the normal microenvironment and collagen I to model the pathological stromal ECM. The alterations of morphology and parameters of proliferation, epithelial-mesenchymal transition (EMT) and stemness were analyzed in vitro and in vivo. Results Increased collagen I deposition was observed at the periablational zone after incomplete RFA of HCC in a xenograft model. The markers of cell proliferation, EMT, motility and progenitor-like traits of heat-exposed residual HCC cells were significantly induced by collagen I as compared to Matrigel (p values all

The aim of this study was to investigate whether treatment-damaged hepatocellular carcinoma (HCC) would accelerate liver cirrhosis through promoting the activities of hepatic stellate cells (HSCs). HCC cells were exposed to chemotherapeutic agent or hypoxia to mimic the transarterial chemoembolization (TACE)-like treatment. Growth differentiation factor 15 (GDF15) expression was increased in cisplatin- or hypoxia-treated HCC cells. Treatment-induced GDF15 increase in HCC cells was mediated by p38MAPK, JNK, ERK1/2 activation. GDF15 from treatment-damaged HCC cells enhanced the proliferation and collagen synthesis of HSCs through ERK1/2- and Smad3-dependent pathways. Metformin significantly reduced the GDF15 production from treatment-damaged HCC cells by targeting JNK. The use of metformin could attenuate the in vivo fibrotic activities of HSCs promoted by treatment-damaged HCC cells and inhibit GDF15 expression. In conclusion, treatment-damaged HCC accelerates fibrosis by promoting the activities of HSCs via GDF15 secretion, which could be reversed by metformin. This provides a potential therapeutic target for alleviating TACE-aggravated liver cirrhosis.

Transarterial chemoembolization (TACE) is the standard treatment for unresectable hepatocellular carcinoma (HCC). Hypoxia‐induced angiogenesis by TACE is linked to treatment failure; however, whether the chemotherapeutic damage of TACE to HCC could increase tumor angiogenesis has not been explored. The molecular effects of chemotherapy‐damaged HCC cells on the neo‐angiogenesis were investigated in vitro and in vivo. The expression of growth differentiation factor 15 (GDF15) was significantly upregulated in HCC cells exposed to chemotherapeutic agents. GDF15 from chemotherapy‐damaged HCC cells promoted the in vitro proliferation, migration, and tube formation of endothelial cells. The pro‐angiogenic effect of GDF15 was through the activation of Src and its downstream AKT, MAPK, and NF‐κB signaling, which was blocked by thalidomide. The use of thalidomide significantly attenuated the in vivo chemotherapy‐damaged HCC cells‐promoted angiogenesis in nude mice. In conclusion, the chemotherapeutic damage in TACE to HCC could promote tumor angiogenesis via the increased release of GDF15. Thalidomide could reverse these pro‐angiogenic effects.

Additional file 8: Table S2. Different phosphorylation signal proteins in expression pattern of “a”. Table S3. Different phosphorylation signal proteins in expression pattern of“b”. Table S4. Different phosphorylation signal proteins in expression pattern of “c”. Table S5. Different phosphorylation signal proteins in expression pattern of “d”.

Background: Microscopic residual tumor often occurs after thermal ablation for medium-large hepatocellular carcinoma (HCC), leading to early aggressive recurrence or late relapse during follow-up. The mechanism how microscopic residual HCC cells survive sublethal heat stress and develop rapid outgrowth remains poorly understood. Methods: HCC cells were exposed to sublethal heat treatment and co-cultured with conditioned media from activated HSCs (HSC-CM). Changes of cell proliferation, parameters of cell autophagy and activation of signaling pathways in heat-treated residual HCC cells were analyzed. An HCC orthotopic model was subjected to partial thermal ablation and antitumor effects of a combined treatment regimen were studied. Results: HCC cells survived sublethal heat stress via activation of autophagy. HSC-CM enhanced autophagic survival within 24 h and then promoted proliferation of heat-treated residual HCC cells through HGF/c-Met signaling. Inhibition of autophagy or c-Met increased apoptosis of heat-treated residual HCC cells and reversed the protective effect of HSC-CM. HGF modulated biological status in autophagic survival or proliferation of heat-treated residual HCC through HGF/c-Met/ERK signaling and downstream components of ATG5/Beclin1 or cyclinD1. In an animal model, inhibiting autophagy in combination with c-Met inhibitor significantly thwarted tumor progression of residual HCC after incomplete thermal ablation via the suppressed autophagy, the decreased proliferation and the increased apoptosis. Conclusions: Activated HSCs promote progression of residual HCC cells after sublethal heat treatment from autophagic survival to proliferation via HGF/c-Met signaling. A combined treatment regimen of inhibiting autophagy and c-Met signaling could be used to suppress tumor progression of residual HCC after incomplete thermal ablation.

Background Incomplete thermal ablation may induce invasiveness of hepatocellular carcinoma (HCC). Here, we investigated whether activated hepatic stellate cells (HSCs) would accelerate the progression of residual HCC after sublethal heat treatment, and thus sought to identify the potential targets. Methods Hepatocellular carcinoma cells were exposed to sublethal heat treatment and then cultured with the conditioned medium from activated HSCs (HSC-CM). The cell proliferation, migration, invasion and parameters of epithelial–mesenchymal transition (EMT) were analyzed. In vivo tumor progression of heat-treated residual HCC cells inoculated with activated HSCs was studied in nude mice. Results HSC-CM significantly enhanced the proliferation, motility, invasion, prominent EMT activation and decreased apoptosis of heat-exposed residual HCC cells. These increased malignant phenotypes were markedly attenuated by neutralizing periostin (POSTN) in HSC-CM. Furthermore, exogenous POSTN administration exerted the similar effects of HSC-CM on heat-treated residual HCC cells. POSTN induced the prominent activation of p52Shc and ERK1/2 via integrin β1 in heat-exposed residual HCC cells. Vitamin D analog calcipotriol blocked POSTN secretion from activated HSCs. Calcipotriol plus cisplatin significantly suppressed the activated HSCs-enhanced tumor progression of heat-treated residual HCC cells via the inhibited POSTN expression and the increased apoptosis. Conclusions Activated HSCs promote the tumor progression of heat-treated residual HCC through the release of POSTN, which could be inhibited by calcipotriol. Calcipotriol plus cisplatin could be used to thwart the accelerated progression of residual HCC after suboptimal heat treatment. Electronic supplementary material The online version of this article (10.1186/s12967-018-1676-3) contains supplementary material, which is available to authorized users.

// Yang You 1, * , Qiongdan Zheng 1, * , Yinying Dong 1 , Xiaoying Xie 1 , Yaohui Wang 2 , Sifan Wu 1 , Lan Zhang 1 , Yingcong Wang 1 , Tongchun Xue 1 , Zhiming Wang 3 , Rongxin Chen 1 , Yanhong Wang 1 , Jiefeng Cui 1 , Zhenggang Ren 1 1 Liver Cancer Institute, Zhongshan Hospital, Fudan University & Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai 200032, PR China 2 Department of Interventional Radiology, Shanghai Cancer Center, Fudan University, Shanghai 200032, PR China 3 Department of Oncology, Zhongshan Hospital Subdivision, Fudan University, Shanghai 200052, PR China * These authors have contributed equally to this work Correspondence to: Jiefeng Cui, e-mail: cui.jiefeng@zs-hospital.sh.cn Zhenggang Ren, e-mail: ren.zhenggang@zs-hospital.sh.cn Keywords: matrix stiffness, hepatocellular carcinoma, stemness, mTOR, integrin β1 Received: November 13, 2015 Accepted: February 23, 2016 Published: March 31, 2016 ABSTRACT Matrix stiffness as an important physical attribute of extracellular matrix exerts significant impacts on biological behaviors of cancer cells such as growth, proliferation, motility, metabolism and invasion. However, its influence on cancer stemness still remains elusive. Here, we explore whether matrix stiffness-mediated effects on stemness characteristics occur in HCC cells. As the substrate stiffness increased, HCC cells exhibited high proportion of cells with CD133(+)/EpCAM(+), high expression levels of CD133, EpCAM, Nanog and SOX2, greater self-renewing ability and oxaliplatin resistance. Simultaneously, their phosphorylation levels of Akt and mTOR, as well as p-4E-BP and SOX2 expressions were also obviously upregulated. Conversely, knockdown of integrin β1 partially attenuated higher stiffness-mediated stemness characteristics in HCC cells, and reversed the phosphorylation levels of Akt and mTOR, and expressions of p-4E-BP and SOX2, suggesting that integrin β1 may deliver higher stiffness signal into HCC cells and activate mTOR signaling pathway. Additionally, mTOR inhibitor suppressed the mTOR phosphorylation level and expression levels of p-4E-BP and SOX2 in HCC cells grown on higher stiffness substrate, as well as depressed their stemness properties significantly, favoring a regulating role of mTOR signaling pathway in matrix stiffness-mediated effects on stemness. In summary, matrix stiffness may be involved in the process of stemness regulation via activating integrin β1/Akt/mTOR/SOX2 signaling pathway. To the best of our knowledge, this study first reveals a novel regulating pathway to direct the stemness characteristics in HCC cells.

Background Higher matrix stiffness affects biological behavior of tumor cells, regulates tumor-associated gene/miRNA expression and stemness characteristic, and contributes to tumor invasion and metastasis. However, the linkage between higher matrix stiffness and pre-metastatic niche in hepatocellular carcinoma (HCC) is still largely unknown. Methods We comparatively analyzed the expressions of LOX family members in HCC cells grown on different stiffness substrates, and speculated that the secreted LOXL2 may mediate the linkage between higher matrix stiffness and pre-metastatic niche. Subsequently, we investigated the underlying molecular mechanism by which matrix stiffness induced LOXL2 expression in HCC cells, and explored the effects of LOXL2 on pre-metastatic niche formation, such as BMCs recruitment, fibronectin production, MMPs and CXCL12 expression, cell adhesion, etc. Results Higher matrix stiffness significantly upregulated LOXL2 expression in HCC cells, and activated JNK/c-JUN signaling pathway. Knockdown of integrin β1 and α5 suppressed LOXL2 expression and reversed the activation of above signaling pathway. Additionally, JNK inhibitor attenuated the expressions of p-JNK, p-c-JUN, c-JUN and LOXL2, and shRNA-c-JUN also decreased LOXL2 expression. CM-LV-LOXL2-OE and rhLOXL2 upregulated MMP9 expression and fibronectin production obviously in lung fibroblasts. Moreover, activation of Akt pathway contributed to LOXL2-induced fibronectin upregulation. LOXL2 in CM as chemoattractant increased motility and invasion of BMCs, implicating a significant role of LOXL2 in BMCs recruitment. Except that, CM-LV-LOXL2-OE as chemoattractant also increased the number of migrated HCC cells, and improved chemokine CXCL12 expression in lung fibroblasts. The number of HCC cells adhered to surface of lung fibroblasts treated with CM-LV-LOXL2-OE was remarkably higher than that of the control cells. These results indicated that the secreted LOXL2 facilitated the motility of HCC cells and strengthened CTCs settlement on the remodeled matrix “soil”. Conclusion Integrin β1/α5/JNK/c-JUN signaling pathway participates in higher matrix stiffness-induced LOXL2 upregulation in HCC cells. The secreted LOXL2 promotes fibronectin production, MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation. Electronic supplementary material The online version of this article (10.1186/s13046-018-0761-z) contains supplementary material, which is available to authorized users.

Sequence of human LOXL2 overexpression. (DOCX 16 kb)

The purpose of this work is to examine the serum proteomic profiles associated with the subsequent development of diabetic nephropathy (DN) in patients with type 2 diabetes and to develop and validate a decision tree based on the profiles to predict the risk of DN in advance by albuminuria. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to obtain the proteomic profiles from baseline serum samples of 84 patients with type 2 diabetes with normal albuminuria, including 42 case subjects who developed DN after 4 years and 42 control subjects who remained normoalbuminuric over the same 4 years. From signatures of protein mass, a decision tree was established for predicting DN. At baseline, urinary albumin/creatinine ratio was similar between the case and control groups. The intensities of 5 peaks detected by CM10 chips appeared up-regulated, whereas 18 peaks were down-regulated more than twofold in the case group than compared with the control group in the training set. An optimum discriminatory decision tree for case subjects created with four nodes using four distinct masses was challenged with testing set. The positive predictive value was 77.8 % (7/9), and the negative predictive value was 72.7 % (8/11). We developed and validated a decision tree to predict DN in patients with type 2 diabetes.

The cAMP-regulated phosphoprotein 19 (ARPP-19) plays a key role in cell mitotic G2/M transition. Expression of ARPP-19 was increased in human hepatocellular carcinoma (HCC) compared to adjacent non-tumorous liver tissues in 36 paired liver samples, and the level of ARPP-19 in HCC tissues was positively correlated with the tumor size. To determine the interrelationship between ARPP-19 expression and HCC, we silenced ARPP-19 expression in the human hepatocarcinoma HepG2 and SMMC-7721 cells using lentivirus encoding ARPP-19 siRNA. HepG2 and SMMC-7721 cells with ARPP-19 knockdown displayed lowered cell growth rate, retarded colony formation and increased arrest at the G2/M phase transition. Silencing ARPP-19 in HCC cells resulted in decreased protein levels of phospho-(Ser) CDKs substrates and increased levels of inactivated cyclin division cycle 2 (Cdc2). Therefore, ARPP-19 may play a role in HCC pathogenesis through regulating cell proliferation.

Gender disparity is well known in hepatocellular carcinoma (HCC). SRY is a critical sex-determination gene involved in embryonic development. The potential relevance of SRY to HCC progression was evaluated. SRY expression in HCC cell lines and tissues was evaluated. Invasion and wound healing assays were used to evaluate the role of SRY in HCC cell migration. The prognostic value of SRY for HCC patient survival was evaluated. SRY was highly expressed in HCC cell lines and tumor tissues. Downregulation of SRY expression decreased migration and invasion potential of HCC cells. High SRY levels correlated with poor HCC patient survival. Additionally, neither spatial position nor expression intensity of SRY was correlated with HCC gender disparity. High levels of SRY expression correlated with cancer progression and poor HCC patient survival. However, high SRY levels are not significantly correlated with HCC sex bias.

This study investigated the effects of extremely low frequency electromagnetic fields (ELF-EMFs) on human fetal scleral fibroblasts (HFSFs). HFSFs were subjected to 50 Hz artificial ELF-EMFs generated by Helmholtz coils with 0.1, 0.2, 0.5, and 1.0 mT field intensities for 6 to 48 h. The viability and factors involved in scleral structuring of HFSFs were determined. The growth rate of HFSFs significantly decreased after only 24 h of exposure to ELF-EMFs (0.2 mT). The messenger RNA (mRNA) expression of collagen type I (COL1A1) decreased and expression of matrix metalloproteinase-2 (MMP-2) increased significantly. There was a decrease in tissue inhibitor of MMP-2 mRNA levels between treated and control cells only at the 1.0 mT intensity level. Transforming growth factor beta-2 mRNA increased in exposed cells, and, simultaneously, fibroblast growth factor-2 mRNA levels decreased. The protein expressions of COL1A1 and MMP-2 were also significantly altered subsequent to exposure ( p < 0.05). This study shows that ELF-EMFs had biological effects on HFSFs and could cause abnormality in scleral collagen.

Matrix stiffness as a novel regulation factor involves in modulating the pathogenesis of hepatocellular carcinoma (HCC) invasion or metastasis. However, the mechanism by which matrix stiffness modulates HCC angiogenesis remains unknown. Here, using buffalo rat HCC models with different liver matrix stiffness backgrounds and an in vitro cell culture system of mechanically tunable Collagen1 (COL1)-coated polyacrylamide gel, we investigated the effects of different matrix stiffness levels on vascular endothelial growth factor (VEGF) expression in HCC cells and explored its regulatory mechanism for controlling HCC angiogenesis. Tissue microarray analysis showed that the expression levels of VEGF and CD31 were gradually upregulated in tumor tissues with increasing COL1 and lysyl oxidase (LOX) expression, indicating a positive correlation between tumor angiogenesis and matrix rigidity. The expression of VEGF and the phosphorylation levels of PI3K and Akt were all upregulated in HCC cells on high-stiffness gel than on low-stiffness gel. Meanwhile, alteration of intergrin β1 expression was found to be the most distinctive, implying that it might mediate the response of HCC cells to matrix stiffness simulation. After integrin β1 was blocked in HCC cells using specific monoclonal antibody, the expression of VEGF and the phosphorylation levels of PI3K and Akt at different culture times were accordingly suppressed and downregulated in the treatment group as compared with those in the control group. All data suggested that the extracellular matrix stiffness stimulation signal was transduced into HCC cells via integrin β1, and this signal activated the PI3K/Akt pathway and upregulated VEGF expression. This study unveils a new paradigm in which matrix stiffness as initiators to modulate HCC angiogenesis.

Some evidences show that residual tumor after thermal ablation will progress rapidly. However, its mechanisms remain unclear. Here, we assessed whether activated HSCs could regulate stem cell-like property of residual tumor after incomplete thermal ablation to promote tumor progression. Human HCC cell lines were exposed to sublethal heat treatment to simulate the peripheral zone of thermal ablation. After residual HCC cells were cultured with conditional medium (CM) from activated HSCs, parameters of the stem cell-like phenotypes were analyzed. Nude mice bearing heat-exposed residual HCC cells and HSCs were subjected to metformin treatment to thwarter tumor progression. CM from activated primary HSCs or LX-2 cells significantly induced the stem cell-like phenotypes of residual HCC cells after heat treatment. These effects were significantly abrogated by neutralizing periostin (POSTN) in the CM. POSTN regulated the stemness of heat-exposed residual HCC cells via activation of integrin β1/AKT/GSK-3β/β-catenin/TCF4/Nanog signaling pathway. Metformin significantly inhibited in vivo progression of heat-exposed residual HCC via suppressing POSTN secretion and decreasing cancer stem cell marker expression. Our data propose a new mechanism of activated HSCs promoting the stemness traits of residual HCC cells after incomplete thermal ablation and suggest metformin as a potential drug to reverse this process.

Background It is well established that some irradiated liver non-parenchymal cells secrete pro-inflammatory cytokines to facilitate the development of radiation-induced liver disease. However, little is known on whether the irradiated hepatoma cells-mediated non-irradiated hepatocyte injury occurs in HCC patients. Here, we elucidated the roles of the irradiated hepatoma cells in driving non-irradiated hepatocyte injury and its underlying mechanism. Methods SMMC7721 cells were cultured and divided into irradiated (4-Gy X-ray, R) and non-irradiated (NR) groups. At 24th hour after irradiation, conditioned medium (CM) from these cultures was mixed with normal culture medium in specific proportions, and termed as 7721-R-CM and 7721-NR-CM. Following incubation with these CM compound, the biological characteristics of L02 cells related to liver cell injury including viability, apoptosis and liver dysfunction indices were comparatively analyzed. Simultaneously, the levels of proliferation- and apoptosis-related cytokines in irradiated and non-irradiated SMMC7721 cells were also measured. FasL as a cytokine with significantly differential expression, was selected to clarify its effects on L02 apoptosis. Subsequently, FasL expression following irradiation was examined in SMMC7721 and other HCC cells with varying malignant potentials, as well as in HCC tissues, the related mechanism of higher expression of FasL in irradiated HCC cells was further investigated. Results Apoptosis and liver dysfunction indices were all significantly enhanced in L02 cells treated with 7721-R-CM, whereas viability was suppressed, compared to those with 7721-NR-CM stimulation. FasL was identified as a leading differential cytokine in the irradiated SMMC7721 cells. Higher proportion of apoptosis was also found in L02 cells following FasL incubation. A recombinant Fas-Fc protein, which blocks Fas-FasL interaction, ameliorated 7721-R-CM-induced apoptosis in L02 cells. FasL was highly expressed in a dose-dependent manner, and peaked at the 24th hour post-irradiation in different HCC cells and their culture supernatant. Meanwhile, phosphorylation levels of JNK, ERK, Akt, and p38 were all upregulated significantly in irradiated HCC cells. But, only JNK inhibition was validated to block radiation-induced FasL expression in HCC cells. c-Jun, the target transcription factor of JNK, was also activated. Conclusion In HCC cells, the JNK-c-Jun pathway plays an important role in mediating irradiation- induced FasL expression, which may be critical in determining non-irradiated hepatocyte injury. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0394-z) contains supplementary material, which is available to authorized users.

Mental health disorders(MHD) in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) have been widely studied. However, the underlying role of inflammatory cytokines and their associated signaling pathways have not been investigated. Here, we report the potential role of cytokines and associated signaling pathways in CP/CPPS patients with MHD and in a CP/CPPS animal model. CP/CPPS patients (n = 810) and control subjects (n = 992) were enrolled in this case-control multicenter study, and serum cytokine levels were measured. Male Sprague-Dawley rats received multiple intracutaneous injections of an immuno-agent along with a pertussis-diphtheria-tetanus triple vaccine for autoimmune CP/CPPS development. The results revealed that, in CP/CPPS patients with significant MHD, elevated IL-1α, IL-1β, IL-4, IL-13, and TNF-α serum levels were observed. The above five cytokines in CP/CPPS rats were significantly elevated in prostate tissue (p p

The extracellular matrix metalloproteases MMP-9 and MMP-2 are critical for the invasive potential of tumors. However, it is not clear which of the two plays the predominant role in tumor invasion and progression. In the present study, we compared the clinical efficacy of MMP-9 and MMP-2 overexpression for predicting tumor recurrence and survival after surgical resection in HCC patients.MMP-9 and MMP-2 expression in HCC cell lines and in vitro HCC invasion model were detected by quantitative RT-PCR and immunofluorescence. The expression levels of MMP-9 and MMP-2 were assessed by immunohistochemistry in HCC tissue microarrays from HCC patients (study set) who underwent curative resection. The clinicopathological data were retrospectively analyzed. The results were further verified in an independent cohort of 92 HCC patients (validation set).Univariate analysis demonstrated that high expression of MMP-9 was associated with both time to recurrence (TTR, P = .015) and overall survival (OS, P = .024), whereas high expression of MMP-2 was only correlated with TTR (P = .041). Multivariate analysis confirmed that MMP-9 expression was an independent predictor of TTR and OS. The coindex of MMP-9 and preoperative serum AFP levels was significantly correlated with TTR and OS (P = .036 and P = .040), but the coindex of MMP-2 and AFP did not show prognostic significance for either TTR or OS (P = .067 and P = .053). The prognostic value of MMP-9 overexpression was validated in the independent data set.MMP-9 is superior to MMP-2 for the prediction of tumor recurrence and survival in HCC patients after surgical resection.

We established an in vitro 3-D model of metastatic hepatocellular carcinoma (HCC) by culturing MHCC97H cells on molecular scaffolds within a rotating wall vessel bioreactor. Morphological and biochemical analyses revealed that the 3-D HCC model mirrored many clinical pathological features of HCC in vivo, including cancer cell morphology, tissue ultrastructure, protein production and secretion, glucose metabolism, tissue-specific gene expression, and apoptosis. Xenografts into livers of nude mice resulted in tumorigenesis and distant metastasis. This 3-D HCC spheroid is a promising model for HCC tumor biology, anticancer drug screening, and for the establishment of HCC animal models.

Considerable interests have recently been focused on mechanism of human hepatocellular carcinoma (HCC) metastasis-the most fundamental characteristics of HCC and the ultimate cause of most HCC mortality, so screening more potential early prognostic marker and therapeutic target is urgent. In this study, we screened genome of three HCC cell lines with consistently increased metastatic potentials and sharing same genetic background, through DNA microarray and found consecutively up-regulated expression of PKCbeta in these cell lines compared to others PKCs, which was reconfirmed by real time RT-PCR and western blot analysis. Moreover, it was found, after efficient silence of PKCbeta by RNAi assay or inhibition of PKCbeta activity by a specific inhibitor LY317615, migration and invasion of HCC cells significantly decreased. In addition, depletion of PKCbeta protein significantly reversed the enhancement of PMA-stimulated HCC migration and invasion ability in vitro. All the data suggest a key role of PKCbeta in HCC motility and PKCbeta may be a potential therapeutic target.

Proteomic profiling of serum is an emerging technique to identify new biomarkers indicative of disease severity and progression. Our study was to assess the use of surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify multiple serum protein biomarkers for early detection of laryngeal squamous cell carcinoma (LSCC), establish predictive model, and accurately distinguish LSCC patients with or without lymph node metastasis. A cohort of 252 serum samples with LSCC (n = 142) and normal control (n = 110) were consented into this study. These serum samples were randomly divided into training set (including 89 LSCC patients at stages I–II and 65 normal controls, 30 LSCC patients with lymph node metastasis) and blind testing set (including 53 LSCC patients at stages III–IV and 45 normal controls). Serum protein profiles on weak cationic exchange (WCX2) were performed by SELDI-TOF MS and then analyzed by Biomarker Wizard software. The Decision Tree classification algorithm and blind validation were determined by Biomarker Pattern Software (BPS). A panel of 18 biomarkers ranging 2–30 kDa was selected based on their collective contribution to the optimal separation between stages I–II LSCC patients and healthy controls. Among them, one candidate protein peak with an m/z value of 4,176 Da was selected to establish predictive model by BPS with sensitivity of 86.52% and specificity of 84.62%. The ability to detect LSCC patients was evaluated using blinding test data in stages III and IV cancer patients. A sensitivity of 84.91% and specificity of 82.22% were validated in blind testing set. Meanwhile 14 potential biomarkers could differentiate LSCC patients with or without lymph node metastasis (P

Tumor metastasis might be associated with the expression levels of cellular glycoproteins and the alteration of their glycan parts. In order to screen the aberrantly alpha1,6-fucosylated glycoproteins related to hepatocellular carcinoma (HCC) metastasis, a high-throughput glycomic approach which consisted of 2-DE, electronic transfer of proteins, lectin affinity blot and precipitation, and MALDI-TOF-MS/MS, was established. Lens culinaris agglutinin (LCA) affinity glycoprotein profiles of higher and lower metastatic HCC cell lines were compared and analyzed. Seven out of 34 identified glycoproteins were differentially displayed; they were cytokeratin 8 (CK8), annexin I, annexin II, heterogeneous nuclear ribonucleoprotein A/B, PDZ and LIM domain 1, RNA-binding motif protein 4, and poly(rC)-binding protein 1. On comparison with Hep3B, CK8 showed a higher affinity to Ricinus communis agglutinin 1 (RCA-I) and LCA, and annexin I presented a higher affinity to LCA and Con A by the lectin-binding assay. Furthermore, the up-regulation of CK8, annexin I, and annexin II were found by Western blot and immunofluorescence analysis in higher metastatic HCC cell lines. This implied that the alteration of CK8, annexin I, and annexin II both in their expression levels and their glycan parts might be related to metastatic ability, and play a critical role in the process of HCC metastasis.

Purpose: A comparative proteomic approach was used to identify and analyze proteins related to metastasis of hepatocellular carcinoma (HCC). Methods: Proteins extracted from 12 HCC tissue specimens (six with metastases and six without) were separated by two-dimensional gel electrophoresis (2-DE). The protein spots exhibiting statistical alternations between the two groups through computerized image analysis were then identified by mass spectrometry. In addition immunohistochemistry (IHC), Western blotting and RT-PCR were performed to verify the expression of certain candidate proteins. Results: 16 proteins including HSP27, S100A11, CK18 were annotated by mass spectrometry, relevant to chaperone function, cell mobility, cytoskeletal architecture, respectively. Most were previously unconnected with metastasis of HCC. Of these HSP27 was found overexpressed consistently in 2-DE patterns of all metastatic HCC tissues compared with nonmetastatic ones. IHC and Western blotting of HCC tissues confirmed this difference while RT-PCR did not. Conclusion: There are various proteins joined together in HCC metastasis. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets unique to the metastatic phenotype of HCC.

Acetylation is a single labeling process to label peptides in control and experimental samples universally, and is independent of amino acid composition or post-translational modification. Here, we propose a new strategy especially useful to quantify either hydrophobic or extremely acidic and basic proteins involved in acetylation of tryptic peptides after sodium dodecyl sulfate polyarcylamide gel electrophoresis (SDS-PAGE) separation. We studied some essential parameters of acetylation labeling reactions in either in-solution tryptic peptides or in-gel digested extracts systematically. We have found that the acetylation efficiency varies markedly on account of different reactive systems, and demonstrated that stable isotope labeling can be steadily obtained with in-gel digested peptides under optimized conditions. We use this protocol to quantify some proteins of two kinds of hepatocellular carcinoma cell line, non-metastatic hepatocellular carcinoma cells, Hep3B, and metastatic hepatocellular carcinoma cells, MHCC97-H. The experimental results provide positive evidence for the potential application of an acetylation labeling strategy in quantitative proteomics, and an efficient way for global proteome quantification.

Previous research showed that microRNA-612 (miR-612) has inhibitory effects on cell proliferation, migration, invasion, and metastasis of hepatocellular carcinoma (HCC). AKT2 was confirmed to be a direct target of miR-612, through which the epithelial-mesenchymal transition (EMT) and metastasis of HCC were inhibited. Our present findings reveal that miR-612 is able to suppress the stemness of HCC by reducing the number and size of tumorspheres as well as clone formation in soft agar, and to relieve drug resistance to cisplatin and 5-fluorouracil. In addition, miR-612 hampered the capacity of tumorigenesis in NOD/SCID mice and redistributed the tumor invasive frontier of miR-612-modulating cells. Finally, our findings suggest that Wnt/β-catenin signaling is required in the regulation of EMT-associated stem cell-like traits by miR-612.

Purpose: Previously, we have established a tissue-like HCC spheroid which better mirrors the biological features of tumorigenesis and metastasis. This study was to find out metastasis-associated genes between two 3D HCC spheroids with different metastasis potential using comparative PCR arrays. Materials and Methods: Two HCC spheroids derived from high-metastatic MHCC97H cells and low-metastatic Hep3B cells were formed respectively in a rotating wall vessel bioreactor after 3D culture for 15 days. The candidate metastasis-associated genes related to cell adhesion, matrix secretion and invasion in HCC spheroids were screened by RT² profiler PCR arrays. The expression patterns of several differentially-expressed genes were further confirmed by real-time RT-PCR. Results: Total of 123 differential expression genes (fold-change >2) were found between two HCC spheroids, including 70 up-regulated genes (VCAM-1, IL-1β, CD44, tenascin C, SPP1, fibronectin, MMP-2, MMP-7, etc) and 53 down-regulated genes (E-cadherin, CTNND2, etc) in the high-metastatic spheroid. Function classification showed that the number of up-regulated genes related to adhesion molecules mediating cell-matrix interactions and matrix secretion was significantly higher in high-metastatic spheroid than that in low-metastatic spheroid. In contrast, the expressions of adhesion molecules maintaining homotypic tumor cell adhesion were decreased in metastatic spheroid as compared with that in low-metastatic spheroid. In addition, the expression pattern of seven selected genes associated with tumor metastasis measured by real-time RT-PCR were consistent with results of PCR arrays. Conclusions: Obvious differences between two HCC spheroids in gene expression patterns of adhesion molecules, matrix secretion, invasion and other molecules may determine the different metastatic characteristics and malignant phenotype of HCC spheroid.

The homeobox gene, goosecoid (GSC), is a transcription factor that participates in cell migration during embryonic development. Because cell migration during development has characteristics similar to cell invasion during metastasis, we evaluated the potential role of GSC in the metastasis of hepatocellular carcinoma (HCC). GSC expression in HCC cell lines and tissues was evaluated, and its effects on the migration potential of HCC cells were determined by GSC knock-down and overexpression methods. In addition, the prognostic role of GSC expression in the metastasis of cancer cells in HCC patients was determined. Our data showed that GSC was highly expressed in several HCC cell lines, particularly in a highly metastatic HCC cell line. Overexpression of GSC promoted cell migration and invasion of HCC cells in vitro. Gain-of-function induced the epithelial-mesenchymal transition but not collective cell migration, whereas loss-of-function induced the reverse change. High-level expression of GSC correlated closely with poor survival and lung metastasis in HCC patients; lung metastases showed more upregulated GSC expression than the primary tumor. We conclude that GSC promotes metastasis of HCC potentially through initiating the epithelial-mesenchymal transition. GSC is also a prognostic factor for poor survival and metastasis of HCC, which suggests its potential as a therapeutic target for metastatic HCC.

Background Tumor cell invasion into the surrounding matrix has been well documented as an early event of metastasis occurrence. However, the dynamic expression patterns of proteins during early invasion of hepatocellular carcinoma (HCC) are largely unknown. Using a three-dimensional HCC invasion culture model established previously, we investigated the dynamic expression patterns of identified proteins during early invasion of HCC. Materials and Methods Highly metastatic MHCC97H cells and a liver tissue fragment were long-term co-cultured in a rotating wall vessel (RWV) bioreactor to simulate different pathological states of HCC invasion. The established spherical co-cultures were collected on days 0, 5, 10, and 15 for dynamic expression pattern analysis. Significantly different proteins among spheroids at different time points were screened and identified using quantitative proteomics of iTRAQ labeling coupled with LC–MS/MS. Dynamic expression patterns of differential proteins were further categorized by K-means clustering. The expression modes of several differentially expressed proteins were confirmed by Western blot and qRT–PCR. Results Time course analysis of invasion/metastasis gene expressions (MMP2, MMP7, MMP9, CD44, SPP1, CXCR4, CXCL12, and CDH1) showed remarkable, dynamic alterations during the invasion process of HCC. A total of 1,028 proteins were identified in spherical co-cultures collected at different time points by quantitative proteomics. Among these proteins, 529 common differential proteins related to HCC invasion were clustered into 25 types of expression patterns. Some proteins displayed significant dynamic alterations during the early invasion process of HCC, such as upregulation at the early invasion stage and downregulation at the late invasion stage (e.g., MAPRE1, PHB2, cathepsin D, etc.) or continuous upregulation during the entire invasion process (e.g., vitronectin, Met, clusterin, ICAM1, GSN, etc.). Conclusions Dynamic expression patterns of candidate proteins during the early invasion process of HCC facilitate the discovery of new molecular targets for early intervention to prevent HCC invasion and metastasis.

Purpose To investigate the expression differences of type I collagen (COL1A1) and its underlying mechanisms in human fetal scleral fibroblasts (HFSFs) that were treated with conditioned medium from retinal pigment epithelial (RPE) cells under extremely low-frequency electromagnetic fields (ELF-EMFs). Methods The ELF-EMFs used in this study were established by slidac and artificial coils. Growth of the treated HFSFs was evaluated by a cell-counting kit-8 assay. The expression of COL1A1 and matrix metalloproteinases-2 (MMP-2) in the treated HFSFs was detected by reverse transcription PCR (RT-PCR) and western blot, and the expression of transforming growth factor-β2 (TGF-β2) and basic fibroblast growth factor-2 (FGF-2) in RPE cells exposed to EMFs was detected by RT-PCR. The expression of COL1A1 and MMP-2 in HFSFs was further confirmed by immunofluorescence staining. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2 also called p44/p42 mitogen-activated protein kinases [MAPK]) and p38 in HFSFs was measured by western blot. Results We found that exposure to ELF-EMFs resulted in a decreased proliferation rate of HFSFs and that addition of RPE supernatant medium could enhance this effect. Compared with that of the control cells, a significant decrease in collagen synthesis was detected in HFSFs under ELF-EMFs. However, the expression of MMP-2 was upregulated, which could be further enhanced via an RPE supernatant additive. The activities of ERK1/2 and p38 were significantly increased in HFSFs exposed to ELF-EMFs, and this effect could be enhanced by RPE supernatant medium additive. Conclusions Our results suggested that ELF-EMFs can inhibit the expression of type I collagen in HFSFs and contribute to the remodeling of the sclera.

To explore the molecular basis of inducible differentiation of embryonic stem cells into hepatocyte-like cells, a proteomic strategy was utilized to examine the global protein expression alterations after early-stage differentiation of a mouse D3 embryonic stem (ES) cell line along hepatic lineage. The undifferentiated D3 cells were treated stepwise with combinations of defined chemicals and growth factors. The differentiated cells were identified by hepatocyte-like morphology, expressed liver-specific markers as well as the evidence of glycogen storage. The subsequent proteomic separation and identification were performed with 2-DE followed by MALDI-TOF-MS/MS analysis. Of the 119 differentially displayed protein spots analyzed, 90 spots presenting 64 distinct proteins were finally identified. The interested protein expressions were validated by Western blotting such as albumin and cytokeratin-8. Bioinformatic annotations indicated that this set of proteins was enriched with transcription, translation regulation and protein processing, energy/metabolism and chaperone functions. A part of them had been found to be involved in the differentiation of mouse ES cells. Interestingly, approximately 40% of these proteins had been previously reported as being dysregulated in hepatocellular carcinoma. It suggested that these changed proteins may be candidate regulators of ES cell differentiation, some of them may be specific to hepatic differentiation.