Corrigendum: Dual-Targeting Nanoparticle-Mediated Gene Therapy Strategy for Hepatocellular Carcinoma by Delivering Small Interfering RNA.

[This corrects the article DOI: 10.3389/fbioe.2020.00512.].

Dual-Targeting Nanoparticle-Mediated Gene Therapy Strategy for Hepatocellular Carcinoma by Delivering Small Interfering RNA.

As a gene therapy strategy, RNA interference (RNAi) offers tremendous tumor therapy potential. However, its therapeutic efficacy is restricted by its inferior ability for targeted delivery and cellular uptake of small interfering RNA (siRNA). This study sought to develop a dual-ligand nanoparticle (NP) system loaded with siRNA to promote targeted delivery and therapeutic efficacy. We synthesized a dual receptor-targeted chitosan nanosystem (GCGA), whose target function was controlled by the ligands of galactose of lactobionic acid (LA) and glycyrrhetinic acid (GA). By loading siPAK1, an siRNA targeting P21-activated kinase 1 (PAK1), a molecular-targeted therapeutic dual-ligand NP (GCGA-siPAK1) was established. We investigated the synergistic effect of these two targeting units in hepatocellular carcinoma (HCC). In particular, GCGA-siPAK1 enhanced the NP targeting ability and promoted siPAK1 cell uptake. Subsequently, dramatic decreases in cell proliferation, invasion, and migration, with an apparent increase in cell apoptosis, were observed in treated cells. Furthermore, this dual-ligand NP gene delivery system demonstrated significant anti-tumor effects in tumor-bearing mice. Finally, we illuminated the molecular mechanism, whereby GCGA-siPAK1 promotes endogenous cell apoptosis through the PAK1/MEK/ERK pathway. Thus, the dual-target property effectively promotes the HCC therapeutic effect and provides a promising gene therapy strategy for clinical applications.

P-21-activated kinase 1 contributes to tumor angiogenesis upon photodynamic therapy via the HIF-1α/VEGF pathway.

Photodynamic therapy (PDT) is an effective oncotherapy and has been approved for clinical application. Unfortunately, its therapeutic efficacy is usually overshadowed by tumor angiogenesis. Thus, a detailed understanding of the tumor angiogenesis upon PDT is imperative. This study aimed to investigate the potential contribution and mechanism of P-21-activated kinase 1 (PAK1) in PDT-induced tumor angiogenesis. Firstly, we found that PAK1 was upregulated upon PDT and associated with tumor angiogenesis. Then, we elucidated the underlying molecular mechanism. Activation of PAK1 prevents hypoxia-inducible factor 1 alpha (HIF-1α) protein from ubiquitin-mediated degradation. Thereafter, HIF-1α accumulation results in the upregulation of vascular endothelial growth factor (VEGF), thus promoting tumor angiogenesis. More importantly, we determined that PAK1 knockdown effectually repressed tumor angiogenesis, which contributes to enhance the therapeutic effect of PDT. Together, PAK1 is a potential novel pharmaceutical target for inhibiting PDT-induced tumor angiogenesis, and PAK1 suppression in combination with PDT may be a potentially effective strategy for anti-tumor therapy.

Biodistribution and biocompatibility of glycyrrhetinic acid and galactose-modified chitosan nanoparticles as a novel targeting vehicle for hepatocellular carcinoma.

Aim: The dual-ligand glycyrrhetinic acid and galactose-modified chitosan nanoparticles were designed to further improve the targeting capability to hepatocellular carcinoma (HCC). Materials & methods: The dual-ligand glycyrrhetinic acid and galactose-modified chitosan nanoparticles were fabricated by using ionic gelation method and their characteristics have been measured. Furthermore, the biodistribution and biocompatibility of this targeting vehicle were investigated in vitro and in vivo, respectively. Results: The targeting vehicle was specifically internalized into hepatoma cells in vitro and accumulated into tumor tissue in vivo with high efficacy. Moreover, the vehicle did not induce inflammation reaction and affect morphologies and organ functions. Conclusion: The targeting accumulation in HCC tissue and great biocompatibility of the dual-ligand modified chitosan nanoparticles highlight the potential of delivering anticancer agents into HCC cells.

Higd1a Protects Cells from Lipotoxicity under High-Fat Exposure.

Hypoxia-inducible gene domain family member 1A (Higd1a) has recently been reported to protect cells from hypoxia by helping to maintain normal mitochondrial function. The potential induction of Higd1a under high-fat exposure and whether it could protect cells from oxidative stress attracted our attention. Initially, 0.4 mM oleic acid and 0.2 mM palmitate were added to the growth media of HepG2 and LO2 cells for 72 hours. We discovered increased Higd1a expression, and knocking down Higd1a impaired mitochondrial transmembrane potential and induced cell apoptosis. We then identified that elevated reactive oxygen species (ROS) is responsible for increased Higd1a expression. Furthermore, we found that ROS promoted Higd1a expression by upregulating HIF-1a and PGC-1a expressions, and these two proteins could exert synergistic effects in inducing Higd1a expression. Taken together, these data suggest that Higd1a plays positive roles in protecting cells from oxidative stress, and ROS could induce Higd1a expression by upregulating PGC-1a and HIF-1a expressions.

Theranostic Nanodots with Aggregation-Induced Emission Characteristic for Targeted and Image-Guided Photodynamic Therapy of Hepatocellular Carcinoma.

Photosensitizer (PS) serves as the central element of photodynamic therapy (PDT). The use of common nanoparticles (NPs) for PDT has typically been rendered less effective by the undesirable aggregation-caused quenching (ACQ) effect, resulting in quenched fluorescence and reduced reactive oxygen species (ROS) generation that diminish the imaging quality and PDT efficacy. To overcome the ACQ effect and to enhance the overall efficacy of PDT, herein, integrin ανß3-targeted organic nanodots for image-guided PDT were designed and synthesized based on a red emissive aggregation-induced emission (AIE) PS. Methods: The TPETS nanodots were prepared by nano-precipitation method and further conjugated with thiolated cRGD (cRGD-SH) through a click reaction to yield the targeted TPETS nanodots (T-TPETS nanodots). Nanodots were characterized for encapsulation efficiency, conjugation rate, particle size, absorption and emission spectra and ROS production. The targeted fluorescence imaging and antitumor efficacy of T-TPETS nanodot were evaluated both in vitro and in vivo. The mechanism of cell apoptosis induced by T-TPETS nanodot mediated-PDT was explored. The biocompatibility and toxicity of the nanodots was examined using cytotoxicity test, hemolysis assay, blood biochemistry test and histological staining. Results: The obtained nanodots show bright red fluorescence and highly effective 1O2 generation in aggregate state. Both in vitro and in vivo experiments demonstrate that the nanodots exhibit excellent tumor-targeted imaging performance, which facilitates image-guided PDT for tumor ablation in a hepatocellular carcinoma model. Detailed analysis reveals that the nanodot-mediated PDT is able to induce time- and concentration-dependent cell death. The use of PDT at a high PDT intensity leads to direct cell necrosis, while cell apoptosis via the mitochondria-mediated pathway is achieved under low PDT intensity. Conclusion: Our results suggest that well-designed AIE nanodots are promising for image-guided PDT applications.

Effect of Huaier granule on recurrence after curative resection of HCC: a multicentre, randomised clinical trial.

OBJECTIVE: There is little evidence that adjuvant therapy after radical surgical resection of hepatocellular carcinoma (HCC) improves recurrence-free survival (RFS) or overall survival (OS). We conducted a multicentre, randomised, controlled, phase IV trial evaluating the benefit of an aqueous extract of Trametes robinophila Murr (Huaier granule) to address this unmet need. DESIGN AND RESULTS: A total of 1044 patients were randomised in 2:1 ratio to receive either Huaier or no further treatment (controls) for a maximum of 96 weeks. The primary endpoint was RFS. Secondary endpoints included OS and tumour extrahepatic recurrence rate (ERR). The Huaier (n=686) and control groups (n=316) had a mean RFS of 75.5 weeks and 68.5 weeks, respectively (HR 0.67; 95% CI 0.55 to 0.81). The difference in the RFS rate between Huaier and control groups was 62.39% and 49.05% (95% CI 6.74 to 19.94; p=0.0001); this led to an OS rate in the Huaier and control groups of 95.19% and 91.46%, respectively (95% CI 0.26 to 7.21; p=0.0207). The tumour ERR between Huaier and control groups was 8.60% and 13.61% (95% CI -12.59 to -2.50; p=0.0018), respectively. CONCLUSIONS: This is the first nationwide multicentre study, involving 39 centres and 1044 patients, to prove the effectiveness of Huaier granule as adjuvant therapy for HCC after curative liver resection. It demonstrated a significant prolongation of RFS and reduced extrahepatic recurrence in Huaier group. TRIAL REGISTRATION: NCT01770431; Post-results.

Clinical application of carbon nanoparticles in curative resection for colorectal carcinoma.

PURPOSE: To explore the potential of carbon nanoparticles (CNs) for the intraoperative detection of positive and negative lymph nodes in the treatment of colorectal cancer. PATIENTS AND METHODS: The clinical data of 470 patients undergoing surgical procedures for colorectal cancer from June 2010 to February 2013 were analyzed retrospectively. The patients were divided into the CN group (183 males and 161 females; mean age, 58.6±12.4 years), who were given a CN suspension, and the control group (78 males and 48 females; mean age, 59.1±12.2 years), who were not given a CN suspension. The operative time, blood loss, number of lymph nodes detected/positive lymph nodes, and prevalence of postoperative complications were compared between the two groups. Three years after surgery, 444 cases (327 cases in the CN group and 117 cases in the control group) were interviewed, with the remaining 26 cases lost to follow-up. With regard to tumor, node, metastasis staging, the survival and prevalence of recurrence in each group at 3 years were analyzed. RESULTS: The number of positive lymph nodes was higher and the prevalence of blood loss was lower in the CN group than in the control group (p<0.05). There were no significant differences in the operative time, number of lymph nodes detected, or the prevalence of postoperative complications, survival, metastasis, or recurrence between the two groups at 3 years (p>0.05). CONCLUSION: The application of CNs is convenient for the detection of lymph nodes to reduce blood loss and increase the probability of detecting positive lymph nodes accurately and rapidly.

One-Step Formulation of Targeted Aggregation-Induced Emission Dots for Image-Guided Photodynamic Therapy of Cholangiocarcinoma.

Photodynamic therapy (PDT) is a palliative technique that can improve median survival with minimal invasion for cholangiocarcinoma (CC) patients. An ideal photosensitizer (PS) is critical to guarantee the efficacy of PDT. However, conventional PSs have some obvious drawbacks, such as lack of specificity and easy aggregation in aqueous media that limit their further application in the clinic. We herein fully take advantage of a red emissive aggregation-induced emission (AIE) PS to fabricate integrin ανß3 targeted organic AIE dots for image-guided PDT via a simple and straightforward one-step strategy. The obtained AIE dots exhibit high specificity to CC as well as excellent antitumor effect both in vitro and in vivo. Different from conventional PSs and previously reported PS-loaded nanostructures, the AIE dots do not suffer from aggregation-caused fluorescence quenching and reduction in reactive oxygen species production when the AIE PS molecules are in an aggregated state. The significant antitumor effect, as well as good biocompatibility and negligible toxicity, makes the AIE dots promising for future translational research in CC diagnosis and therapy.

Mechanism of dynamic near-infrared fluorescence cholangiography of extrahepatic bile ducts and applications in detecting bile duct injuries using indocyanine green in animal models.

Fluorescence intraoperative cholangiography (IOC) is a potential alternative for identifying anatomical variation and preventing iatrogenic bile duct injuries by using the near-infrared probe indocyanine green (ICG). However, the dynamic process and mechanism of fluorescence IOC have not been elucidated in previous publications. Herein, the optical properties of the complex of ICG and bile, dynamic fluorescence cholangiography and iatrogenic bile duct injuries were investigated. The emission spectrum of ICG in bile peaked at 844 nm and ICG had higher tissue penetration. Extrahepatic bile ducts could fluoresce 2 min after intravenous injection, and the fluorescence intensity reached a peak at 8 min. In addition, biliary dynamics were observed owing to ICG excretion from the bile ducts into the duodenum. Quantitative analysis indicated that ICG-guided fluorescence IOC possessed a high signal to noise ratio compared to the surrounding peripheral tissue and the portal vein. Fluorescence IOC was based on rapid uptake of circulating ICG in plasma by hepatic cells, excretion of ICG into the bile and then its interaction with protein molecules in the bile. Moreover, fluorescence IOC was sensitive to detect bile duct ligation and acute bile duct perforation using ICG in rat models. All of the results indicated that fluorescence IOC using ICG is a valid alternative for the cholangiography of extrahepatic bile ducts and has potential for measurement of biliary dynamics.

Initial 12-h operative fluid volume is an independent risk factor for pleural effusion after hepatectomy.

Pleural effusion after hepatectomy is associated with significant morbidity and prolonged hospital stays. Several studies have addressed the risk factors for postoperative pleural effusion. However, there are no researches concerning the role of the initial 12-h operative fluid volume. The aim of this study was to evaluate whether the initial 12-h operative fluid volume during liver resection is an independent risk factor for pleural effusion after hepatectomy. In this study, we retrospectively analyzed clinical data of 470 patients consecutively undergoing elective hepatectomy between January 2011 and December 2012. We prospectively collected and retrospectively analyzed baseline and clinical data, including preoperative, intraoperative, and postoperative variables. Univariate and multivariate analyses were carried out to identify whether the initial 12-h operative fluid volume was an independent risk factor for pleural effusion after hepatectomy. The multivariate analysis identified 2 independent risk factors for pleural effusion: operative time [odds ratio (OR)=10.2] and initial 12-h operative fluid volume (OR=1.0003). Threshold effect analyses revealed that the initial 12 h operative fluid volume was positively correlated with the incidence of pleural effusion when the initial 12-h operative fluid volume exceeded 4636 mL. We conclude that the initial 12-h operative fluid volume during liver resection and operative time are independent risk factors for pleural effusion after hepatectomy. Perioperative intravenous fluids should be restricted properly.

Ligand-based targeted therapy: a novel strategy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with high morbidity and mortality worldwide. Chemotherapy is recommended to patients with intermediate or advanced stage cancer. However, the conventional chemotherapy yields low desired response rates due to multidrug resistance, fast clearance rate, nonspecific delivery, severe side effects, low drug concentration in cancer cells, and so on. Nanoparticle-mediated targeted drug delivery system can surmount the aforementioned obstacles through enhanced permeability and retention effect and active targeting as a novel approach of therapeutics for HCC in recent years. The active targeting is triggered by ligands on the delivery system, which recognize with and internalize into hepatoma cells with high specificity and efficiency. This review focuses on the latest targeted delivery systems for HCC and summarizes the ligands that can enhance the capacity of active targeting, to provide some insight into future research in nanomedicine for HCC.

A new alkaloid from Nodulisporium sp.

The genus Nodulisporium, is known to produce secondary metabolites with structural diversity. A new alkaloid, 2-hy- droxy-1,1-dimethyl-1,2,3,9-tetrahydro-4H-carbazol-4-one(1), was isolated from the extract of a fungal strain Nodulisporium sp. fermented with rice, together with three known phenols, tyrosol(2), hydroxytyrosol(3), and hydroxytyrosol acetate(4). Their structures were identified by detailed spectroscopic analyses.

Nodulisporipyrones A-D, new bioactive α-pyrone derivatives from Nodulisporium sp.

Four new α-pyrone derivatives, nodulisporipyrones A-D (1-4), were isolated from the extract of an endolichenic fungal strain Nodulisporium sp. (65-12-7-1) that was fermented with rice. The structures of 1-4 were elucidated by extensive spectroscopic analysis, and the absolute configurations were determined by modified Mosher's method and electronic circular dichroism experiments. Their antimicrobial activities against Staphylococcus aureus 209P, Escherichia coli ATCC0111, Aspergillus niger R330, and Candida albicans FIM709 were evaluated using a paper disk diffusion method. Nodulisporipyrones A-D (1-4) are the first α-pyrone derivatives from Nodulisporium fungi.

Emerging role of Toll-like receptor 4 in hepatocellular carcinoma.

Toll-like receptor (TLR) signaling has been implicated in inflammatory-related cancers. The upregulation of TLR signaling in hepatocellular carcinoma (HCC) suggests that it may play an essential role in the prognosis of chronic and inflammatory diseases that ultimately culminate in HCC. Here, we provide evidence about the involvement of the TLR pathway in the initiation, progression, and metastasis of HCC. The differential expression of TLR in epithelial cells has also been discussed. In particular, we emphasize the physiological role of TLR4 in the development and pathogenesis of HCC and propose novel and promising approaches for HCC therapeutics with the aid of TLR ligands.

Transarterial (chemo)embolization for curative resection of hepatocellular carcinoma: a systematic review and meta-analyses.

PURPOSE: To assess the beneficial and harmful effects of transarterial embolization (TAE) or transarterial chemoembolization (TACE) for curative resection of hepatocellular carcinoma (HCC). METHODS: The authors conducted an extensive search of studies on this strategy. All randomized controlled trials comparing TACE or TAE plus operation versus operation only were considered for inclusion, regardless of blinding, language, or publication status. Results were performed with disease-free survival (DFS) and overall survival (OS) as the primary endpoint. Tumor response and adverse events were secondary endpoints. RESULTS: A total of 10 studies involving 909 HCC participants finally fulfilled the predefined inclusion criteria. Four trials assessed preoperative TACE versus control and six trials assessed postoperative TACE versus control. There were significant improvements for DFS [HR 0.62 (95 % CI 0.49-0.79)] and OS [HR 0.60 (0.46-0.79)] in the postoperative TACE compared with the control when the mean tumor size was bigger than 5 cm. However, preoperative TACE did not improve DFS [HR 0.92 (0.71-1.20)] and OS [HR 1.07 (0.78-1.46)] for curative resection of HCC. Substantial differences in criteria for assessing tumor response did not allow quantitative analyses. Fever (26.7-85.9 %), abdominal pain (19.3-71.2 %), and nausea/vomiting (27.4-66.3 %) were common adverse events. Relatively rare but more serious complications were also reported. CONCLUSIONS: Postoperative TACE offers potential benefits for curative resection of HCC when the mean tumor size is bigger than 5 cm.

Mouse A6-positive hepatic oval cells derived from embryonic stem cells.

Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were used to induce differentiation of mouse embryonic stem (ES) cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells' differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1+/CD34+ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1+/CD34+ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1+/CD34+ cells on the day 8 were A6 positive. Highly purified A6+/Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group (up to 4.46%) was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells' membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.

Chaetoglobosin Y, a new cytochalasan from Chaetomium globosum.

Chaetoglobosin Y (1), was isolated from the endolichenic fungal strain Chaetomium globosum (No. 64-5-8-2), along with related six known cytochalasans, chaetoglobosin Fex (2), chaetoglobosin E (3), isochaetoglobosin D (4), chaetoglobosin G (5), cytoglobosin B (6), and cytoglobosin C (7). Their structures were determined by detailed spectroscopic analyses and comparison with those of the closely related compounds previously reported. The cytotoxicity to HCT-116 cell line of 2-7 was evaluated in vitro with doxorubicin as positive control.

Antiviral therapy after curative treatment of hepatitis B/C virus-related hepatocellular carcinoma: A systematic review of randomized trials.

AIM: Available published work on the benefit of adjuvant antiviral therapy after curative treatment of hepatocellular carcinoma (HCC) reports controversial results. The objective of this systematic review was to evaluate the effect of adjuvant antiviral therapy on recurrence and survival after curative treatment of HCC. METHODS: We conducted an extensive search strategy. All randomized controlled trials comparing adjuvant antiviral therapy versus placebo or no treatment were considered for this review. Results were expressed as hazard ratio for time-to-event outcomes with 95% confidence intervals using RevMan 5. RESULTS: We included nine trials (three of low risk of bias and six of unclear risk of bias) with 954 patients. All the included studies used conventional interferon (IFN) as adjuvant antiviral therapy; none of them used pegylated IFN or nucleoside analogs. There were significant improvements for recurrence-free survival and overall survival in the adjuvant IFN group compared with the control group. Subgroup analysis also showed a significant difference favoring IFN therapy in hepatitis C virus (HCV)-related HCC patients, but for hepatitis B virus (HBV)-related patients, the difference failed to reach statistical significance. A dose reduction was needed in 28.3% of patients and discontinuation of IFN therapy happened in 8.2% of patients due to moderate to severe side-effects. CONCLUSION: Our study suggested potential benefits of adjuvant IFN therapy following curative treatment of HCC, especially for HCV-related HCC. Further high-quality randomized controlled trials of more effective adjuvant antiviral regimens, either used alone or in combination, for virus-related HCC, especially HBV-related HCC, are needed.

Nodulisporisteriods A and B, the first 3,4-seco-4-methyl-progesteroids from Nodulisporium sp.

Two new 4-methyl-progesteroids, nodulisporisteriod A (1) and nodulisporisteriod B (2), were isolated from the extract of an endolichenic fungal strain Nodulisporium sp. (No. 65-17-2-1), along with two related metabolites, demethoxyviridin (3) and inoterpene B (4). Their structures were determined by detailed spectroscopic analyses, X-ray crystallographic analysis and comparison of the NMR data with those of the closely related compounds previously reported. Nodulisporisteriod A (1) and nodulisporisteriod B (2) possess new carbon skeletons, which are the first cases of fission at C-3,4 in 4-methyl-progesteroids. A hypothetical biosynthetic pathway for 1 and 2 was proposed. Moreover, the Aß42 aggregation inhibitory activities of 1-4 were evaluated using standard thioflavin T (ThT) fluorescence assay with epigallocatechin gallate (EGCG) as positive control. Demethoxyviridin (3) displayed anti-Aß42 aggregation activity with IC50 value of 13.4µM.