Quantitative Secretome Analysis Reveals Clinical Values of Carbonic Anhydrase II in Hepatocellular Carcinoma.

Early detection and intervention are key strategies to reduce mortality, increase long-term survival, and improve the therapeutic effects of hepatocellular carcinoma (HCC) patients. Herein, the isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic strategy was used to study the secretomes in conditioned media from HCC cancerous tissues, surrounding noncancerous tissues, and distal noncancerous tissues to identify diagnostic and prognostic biomarkers for HCC. In total, 22 and 49 dysregulated secretory proteins were identified in the cancerous and surrounding noncancerous tissues, respectively, compared with the distal noncancerous tissues. Among these proteins, carbonic anhydrase II (CA2) was identified to be significantly upregulated in the secretome of cancerous tissues; correspondingly, the serum concentrations of CA2 were remarkably increased in HCC patients compared with that in normal populations. Interestingly, a significant increase of serum CA2 in recurrent HCC patients after radical resection was also confirmed compared with HCC patients without recurrence, and the serum level of CA2 could act as an independent prognostic factor for time to recurrence and overall survival. Regarding the mechanism, the secreted CA2 enhances the migration and invasion of HCC cells by activating the epithelial mesenchymal transition pathway. Taken together, this study identified a novel biomarker for HCC diagnosis and prognosis, and provided a valuable resource of HCC secretome for investigating serological biomarkers.

A near-infrared turn-on fluorescence probe for glutathione detection based on nanocomposites of semiconducting polymer dots and MnO2 nanosheets.

Fluorescence biosensors that enable highly sensitive detection of glutathione (GSH) are in great demand for various biological investigations and early disease diagnoses. Here, we report a turn-on fluorescence nanoplatform based on fluorescent semiconducting polymer nanoparticle@MnO2 nanosheets (P-dot@MnO2) nanocomposites for rapid homogeneous determination of GSH. The near-infrared (NIR) fluorescent P-dots were prepared by doping NIR dyes into polymer matrix and then encompassed by MnO2, which resulted in a remarkable fluorescence quenching. Owing to the selective decomposition of MnO2 by GSH, the fluorescence recovery was achieved in the presence of GSH. On the basis of the target-induced turn-on fluorescence response, the developed nanoplatform can readily detect GSH with a high sensitivity up to 0.26 µM, as well as a superior specificity. Furthermore, it was successfully applied in monitoring the intracellular GSH in living cells, revealing its great potential in biomedical applications. Graphical abstract.

4E-BP1Thr46 Phosphorylation Association with Poor Prognosis in Quantitative Phosphoproteomics of Portal Vein Tumor Thrombus Revealed that 4E-BP1Thr46 Phosphorylation is Associated with Poor Prognosis in HCC.

PURPOSE: Early formation of portal vein tumor thrombosis (PVTT) is a key characteristic of hepatocellular carcinoma (HCC) metastasis, but to date, the aetiology of PVTT in HCC metastasis is largely unknown. We aim to find highly sensitive and specific biomarkers for the prediction of HCC prognosis. PATIENTS AND METHODS: We used isobaric tags for relative and absolute quantitation (iTRAQ) based quantitative phosphoproteomics approach to investigate the molecular signatures of the HCC with PVTT in primary HCC tissues, surrounding non-cancerous tissues and PVTT tissues. The different proteome profiles in three groups were investigated and might reveal different underlying molecular mechanisms. RESULTS: In total, we identified 1745 phosphoproteins with 2724 phosphopeptides and 4594 phosphorylation sites in three groups. Among these phosphoproteins, 80 phosphoproteins were dysregulated in PVTT/Pan group, 51 phosphoproteins were dysregulated in HCC/Pan group, and 10 phosphoproteins were dysregulated in PVTT/HCC group. Furthermore, the phosphorylation of 4E-BP1 was elevated in HCC tissues and PVTT tissues in comparison with surrounding non-cancerous tissues,  and the elevated fold change of phosphorylation level was higher than that in expression level of 4E-BP1. The further IHC analysis in acohort of 20 HCC tissues showed that the phosphorylation of 4E-BP1 on Thr46 might be closely related to HCC prognosis. CONCLUSION: The high phosphorylation level of 4E-BP1Thr46 might serve as a biomarker for the diagnosis of early recurrence and metastasis of HCC.

Ku80 negatively regulates the expression of OCT4 via competitive binding to SALL4 and promoting lysosomal degradation of OCT4.

SALL4 and OCT4, along with other pluripotency-associated transcription factors, play critical roles in maintaining embryonic stem cell pluripotency and self-renewal. Ku80 is a component of the protein complex called DNA-dependent protein kinase, which mainly involved in DNA double-strand break repair. In this study, we show evidence that Ku80 physically interacted with SALL4. The interaction competitively disrupts the SALL4-OCT4 complex and result in OCT4 lysosomal degradation. Finally, Ku80 inhibits self-renewal and metastasis of hepatocellular carcinoma cells through breaking the SALL4-OCT4 interactions and down-regulating the expression of OCT4. Our study reveal novel function of Ku80 in stemness maintaining of cancer stem cells via its interaction with SALL4 and highlight the double-sidedness of Ku80 as an anti-cancer target.

UHRF1 downmodulation enhances antitumor effects of histone deacetylase inhibitors in retinoblastoma by augmenting oxidative stress-mediated apoptosis.

Identification of new genetic pathways or molecular targets that sensitize cancer cells to chemotherapeutic drugs may improve the efficacy of current chemotherapy. Here, we report that downmodulation of UHRF1 (ubiquitin-like with PHD and RING finger domains 1) in retinoblastoma (RB) cells increases the sensitivity to histone deacetylase (HDAC) inhibitors, augmenting apoptotic cell death. We found that UHRF1 depletion downregulates two redox-responsive genes GSTA4 (glutathione S-transferase α4) and TXN2 (thioredoxin-2) in RB cells, and increases the basal level of intracellular oxidative stress. Antioxidant treatment significantly reduced both basal and HDAC inhibitor-induced DNA damage and apoptosis in UHRF1-depleted cells. Knockdown of GSTA4 or TXN2 sensitized RB cells to HDAC inhibitors, demonstrating that GSTA4 and TXN2 play key roles in redox homeostasis in RB cells and the susceptibility to HDAC inhibitor treatment upon UHRF1 depletion. In human primary RB, GSTA4 and TXN2 proteins were found to be mostly elevated along with high UHRF1 expression. In addition to augmentation of apoptosis in UHRF1-depleted RB cells, we also show that UHRF1 downmodulation derepresses the expression of photoreceptor-specific genes in RB cells in cooperation with a HDAC inhibitor MS-275 and promotes neuron-like differentiation. However, further investigation revealed that the enhanced growth-inhibitory effects of MS-275 in UHRF1-depleted cells were still mainly due to robust apoptosis induction rather than differentiation-mediated growth arrest. Consistent with our findings, UHRF1 depletion in RB cells increased the therapeutic efficacy of MS-275 in murine orthotopic xenografts. These results provide a novel basis for potential benefits of UHRF1 targeting for RB treatment.

MT1G serves as a tumor suppressor in hepatocellular carcinoma by interacting with p53.

Poor prognosis of hepatocellular carcinoma (HCC) patients is frequently associated with rapid tumor growth, recurrence and drug resistance. MT1G is a low-molecular weight protein with high affinity for zinc ions. In the present study, we investigated the expression of MT1G, analyzed clinical significance of MT1G, and we observed the effects of MT1G overexpression on proliferation and apoptosis of HCC cell lines in vitro and in vivo. Our results revealed that MT1G was significantly downregulated in tumor tissues, and could inhibit the proliferation as well as enhance the apoptosis of HCC cells. The mechanism study suggested that MT1G increased the stability of p53 by inhibiting the expression of its ubiquitination factor, MDM2. Furthermore, MT1G also could enhance the transcriptional activity of p53 through direct interacting with p53 and providing appropriate zinc ions to p53. The modulation of MT1G on p53 resulted in upregulation of p21 and Bax, which leads cell cycle arrest and apoptosis, respectively. Our in vivo assay further confirmed that MT1G could suppress HCC tumor growth in nude mice. Overall, this is the first report on the interaction between MT1G and p53, and adequately uncover a new HCC suppressor which might have therapeutic values by diminishing the aggressiveness of HCC cells.

Inflammatory Micro-environment Contributes to Stemness Properties and Metastatic Potential of HCC via the NF-κB/miR-497/SALL4 Axis.

Increasing evidence has demonstrated the essential role of inflammatory micro-environment in tumorigenesis and tumor progression. Some cancer cells in tumor maintain typical stemness properties and, with the capacity of self-renewal, are thought to be crucial for the initiation and maintenance of tumors as well as their metastasis. Although both inflammatory micro-environment and stemness properties played crucial roles in tumor initiation and development, currently it is still unclear whether and how the inflammatory micro-environment promotes cancer stemness properties. Here, we show the first evidence that the inflammatory micro-environment promotes the stemness properties and metastatic potential of hepatocellular carcinoma (HCC) via the NF-κB/miR-497/SALL4 axis. We discover that miR-497 directly targets SALL4, negatively regulates its expression, and further inhibits the self-renewal and metastasis of HCC; more importantly, inflammatory factor TNF-α inhibits the expression of miR-497 via NF-kB-mediated negative transcriptional regulation and simultaneously upregulates the expression of SALL4 and promotes the self-renewal and metastasis phenotypes of HCC cells. Moreover, lower expression of miR-497 is significantly associated with poor prognosis in HCC patients. Taken together, our findings not only revealed a novel signaling pathway (NF-κB/miR-497/SALL4 axis) to connect inflammation with stemness properties, and clarified the molecular mechanisms underlying the inflammation-mediated self-renewal and metastasis phenotypes, but also provided novel molecular targets for developing new anticancer strategies.

Sensitive fluorometric determination of glutathione using fluorescent polymer dots and the dopamine-melanin nanosystem.

A bioinspired fluorometric method has been developed for the detection of glutathione (GSH) in biological fluids. It is based on the use of near-infrared fluorescent semiconducting polymer dots (P-dots) and of the dopamine (DA)-melanin nanosystem. The P-dots were prepared from poly(styrene-co-maleic anhydride), the semiconducting polymer poly[(9,9'-dioctyl-2,7-divinylenefluorenylene)-alt-2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene] and the fluorescent dye tetraphenylporphyrin. They have excitation/emission maxima at 458/656 nm, and this enables measurement to be performed with low autofluorescence and scattering background. DA can self-polymerize on the surface of the P-dots to yield a poly-DA coating. This coating, at weak alkaline pH values, causes the quenching of the fluorescence of the P-dots. However, the polymerization of DA is inhibited by GSH. Hence, quenching of fluorescence is prevented. This effect was used to design a fluorometric assay for GSH that has good selectivity and sensitivity. Under optimal conditions, the method has a linear response in the 0.2 to 20 µM GSH concentration range and a 60 nM detection limit. It was successfully applied to the determination of GSH in HepG2 cells and in spiked human serum. Graphical abstract Schematic representation of using a NIR fluorescent P-dots and dopamine (DA)-melanin nanohybrid as a probe for glutathione (GSH) detection. The P-dots were prepared from poly(styrene-co-maleic anhydride) (PSMA), the semiconducting polymer poly[(9,9'-dioctyl-2,7-divinylenefluorenylene)-alt-2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene] (PEPV) and the fluorescent dye tetraphenylporphyrin (TPP).The GSH can inhibit the dopamine self-polymerization and prevented the formation of PDA and fluorescence quenching of P-dots.

An Optogenetic Controllable T Cell System for Hepatocellular Carcinoma Immunotherapy.

Rationale: T-cell based immunotherapy increasingly shows broad application prospects in cancer treatment, but its performance in solid tumors is far from our expectation, partly due to the re-inhibition of infiltrated T cells by immunosuppressive tumor microenvironment. Here we presented an artificial synthetic optogenetic circuit to control the immune responses of engineered T cells on demand for promoting and enhancing the therapeutic efficiency of cancer immunotherapy. Methods: We designed and synthesized blue-light inducible artificial immune signaling circuit and transgene expression system. The blue light triggered transgene expression was investigated by luciferase activity assay, qPCR and ELISA. The in vitro cytotoxicity and proliferation assays were carried out on engineered T cells. The in vivo anti-tumor activity of engineered T cells was investigated on xenograft model of human hepatocellular carcinoma. Results: Blue light stimulation could spatiotemporally control gene expression of specific cytokines (IL2, IL15, and TNF-α) in both engineered 293T cells and human primary T cells. This optogenetic engineering strategy significantly enhanced the expansion ability and cytolytic activity of primary T cells upon light irradiation, and the light activated T cells showed high-efficiency of elimination against xenograft of hepatocellular carcinoma cells. Conclusions: The current study represented an engineered remotely control T cell system for solid tumor treatment, and provided a potential strategy to partially overcome the intrinsic shortages of current immune cell therapy.

Highly efficient redox reaction between potassium permanganate and 3,3',5,5'-tetramethylbenzidine for application in hydrogen peroxide based colorimetric assays.

Potassium permanganate (KMnO4) is one of the most important oxidants, which plays important roles in many fields. Here, we found that KMnO4 could directly induce the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate an oxidized product with a color change. This redox reaction is highly efficient, and 1 µM KMnO4 is enough to cause detectable changes in the absorbance signal. Meanwhile, this reaction is very fast and the generated blue product can stabilize for a relatively long period, which has great advantages in practical applications. Since hydrogen peroxide (H2O2) is able to react with KMnO4 under acidic conditions, the KMnO4-TMB system can be used for the detection of H2O2; the absorbance signal induced by 5 µM H2O2 can be easily detected in this method. Meanwhile, the KMnO4-TMB system can also be used for the detection of glucose by monitoring the generation of H2O2, which is the main product of glucose oxidation; this method permits detection of concentrations as low as 10 µM glucose, and the sensitivity is comparable to or higher than most peroxidase mimetic based methods, but avoiding the preparation and storage of the nanomaterials. Furthermore, the KMnO4-TMB system can even be used for analyzing glucose in serum samples, which can also be expected to be used in immunoassays.

Photo-responsive hollow silica nanoparticles for light-triggered genetic and photodynamic synergistic therapy.

The development of multifunctional carriers incorporating genetic and photodynamic therapy (PDT) for synergistic antitumor treatment has attracted intensive interests very recently. However, most of the currently reported systems employ passive gene release strategies depending on tumor microenvironment, which are negatively affected by the heterogeneity of cancer cells, thus resulting in limited controllability in therapeutic progress. Herein, a novel photo-responsive hollow silica nanoparticle (HNP)-based gene and photosensitizer (PS) co-delivery nanovehicle is designed for dual-wavelength light-triggered synergistic gene and PDT therapy. The resultant HNP conjugated with PDMAEMA polycation through a 405-nm light-cleavable Cou-linker, namely, HNP-Cou-PD, exhibits excellent gene condensation capacity, good biocompatibility, outstanding PS loading ability, and light-triggered gene release properties. HNP-Cou-PD with Chlorin e6 (Ce6) loaded inside the silica cavity and a plasmid encoding caspase-8 gene (CSP8) attached to the PDMAEMA outside layer (Ce6-HNP-Cou-PD/CSP8) has been proven to possess better antitumor effects under the irradiation of pre-405-nm and post-670-nm light both in vitro and in vivo because of the light-triggered intracellular gene release and reactive oxygen species (ROS) generation. Therefore, HNP-Cou-PD designed as a gene and PS co-delivery carrier might have promising applications in the future to precisely treat various types of cancers. STATEMENT OF SIGNIFICANCE: Multifunctional carriers incorporating genetic and photodynamic therapy (PDT) have drawn intense attention very recently, ascribing to their enhanced anticancer effects. However, in the present gene and PDT synergistic system, gene release strategies passively relying on tumor microenvironment often result in no or poor controllability compared with PDT (a spatial and temporal therapeutic modal), which may hinder their synergistic efficacy, especially in an on-demand manner. To resolve this problem, we designed a hollow silica nanoparticle-based dual-wavelength light-responsive gene and photosensitizer (PS) co-delivery platform to achieve photo-triggered gene and PDT synergistic therapy. We believe that our work may have extensive application prospects in precise treatment of various cancers and be of interest to the readership.

Photoresponsive Nanovehicle for Two Independent Wavelength Light-Triggered Sequential Release of P-gp shRNA and Doxorubicin To Optimize and Enhance Synergistic Therapy of Multidrug-Resistant Cancer.

Prerelease of RNA molecules than chemotherapeutic drugs with a sufficient interval is a vital prerequisite for RNA/drug co-delivery strategy to overcome multidrug resistance (MDR) of cancer cells, but how to precisely control their release at different time points is still a grand challenge up to now. This study aims to on-demand remotely manipulate RNA and drug release in real time through single delivery system to sequentially play their respective roles for optimizing and enhancing their synergistic antitumor effects. To this end, a photoresponsive mesoporous silica nanoparticle (PMSN) is fabricated as a co-delivery vehicle of P-glycoprotein (P-gp) short-hairpin RNA (shRNA) and photocaged prodrug of doxorubicin (DOX), by which the orthogonal and sequential release of shRNA and DOX can be achieved using an external light. In our design, the cationic poly[2-( N, N-dimethylaminoethyl)methacrylate] is introduced onto the PMSN surface through a light-sensitive coumarin ester derivative linker to adsorb P-gp shRNA, whereas the photocleavable o-nitrobenzyl ester derivative-caged DOX is loaded into the inner pores of the PMSN. The PMSN is found to be effectively internalized by MDR cancer cells, and the release of the shRNA and DOX is demonstrated to be independently regulated by 405 and 365 nm light irradiations due to selectively cleaved coumarin and o-nitrobenzyl ester, resulting in enhanced drug retention, and finally bring out optimized and significantly improved chemotherapeutic effects both in vitro and in vivo for MDR cancer treatment, which might hold extensive application prospects in MDR cancer treatment in future.

A cancer cell specific targeting nanocomplex for combination of mRNA-responsive photodynamic and chemo-therapy.

We have developed a cancer cell specific targeting nanocomplex which combines photodynamic therapy with chemotherapy through precisely responding to the intracellular tumor-related mRNA. The combined treatment of these two modalities showed significantly enhanced therapeutic effects on cancer cells.