Chitosan-enclosed SLN improved SV-induced hepatocellular cell carcinoma death by modulation of IQGAP gene expression, JNK, and HDAC activities.

BACKGROUND: Hepatocellular carcinoma (HCC) is a global life-threatening problem and therapeutic interventions are still encountered. IQGAP genes are involved in HCC oncogenesis. The modulatory effect of statins on the expression of IQGAP genes is still unclear. This study aims to study the effect of free SV and chitosan (CS) decorated simvastatin (SV) loaded solid lipid nanoparticles (C-SV-SLNs) on HCC mortality. METHODS AND RESULTS: Plain, SV-SLN, and C-SV- SLN were prepared and characterized in terms of particle size (PS), zeta potential (ZP), and polydispersity index (PDI). The biosafety of different SLN was investigated using fresh erythrocytes, moreover, cytotoxicity was investigated using HepG2 cell lines. The effect of SLNs on IQGAPs gene expression as well as JNK, HDAC6, and HDAC8 activity was investigated using PCR and MOE-docking. The current results displayed that SV-SLNs have nanosized, negative ZP and are homogenous, CS decoration shifts the ZP of SLN into cationic ZP. Furthermore, all SLNs exhibited desirable biosafety in terms of no deleterious effect on erythrocyte integrity. SV solution and SV-SLN significantly increase the mortality of HepG2 compared to undertreated cells, however, the effect of SV-SLN is more pronounced compared to free SV. Remarkably, C-SV-SLN elicits high HepG2 cell mortality compared to free SV and SV-SLN. The treatment of HepG2 cells with SV solution, SV-SLN, or C-SV-SLN significantly upregulates the IQGAP2 gene with repression of IQGAP1 and IQGAP3 genes. MOE-docking studies revealed both SV and tenivastatin exhibit interactions with the active sites of JNK, HDAC6, and HDAC8. Moreover, tenivastatin exhibited greater interactions with magnesium and zinc compared to SV. CONCLUSIONS: This research provides novel insights into the therapeutic potential of SV, SV-SLN and C-SV-SLNs in HCC treatment, modulating critical signaling cascades involving IQGAPs, JNK, and HDAC. The development of C-SV-SLNs presents a promising strategy for effective HCC therapy.

α-Fetoprotein fragment synergizes with sorafenib to inhibit hepatoma cell growth and migration and promote the apoptosis.

Alpha fetoprotein (AFP) is associated with hepatocellular carcinoma (HCC) by stimulating the proliferation, metastasis and drug resistance. The application of AFP fragments to inhibit the malignant behaviours induced by AFP is a new strategy for the treatment of HCC. In an effort to design, screen and discover drugs, we attempted to express different human AFP fragments (AFP220-609 , AFP390-609 and AFP460-609 ) in a Bac-to-Bac system. We found that the AFP390-609 fragment was highly expressed in the system. Then, we assessed the bioactivity of the fragment in the human liver cancer cell line Bel7402, and the results indicated that the AFP fragment synergized with sorafenib to inhibit the hepatoma cell growth and migration and promote the apoptosis. This study provides a method to produce significant AFP fragments to screen AFP inhibitors for use in HCC therapy.

Role of MRI with hepatospecific contrast agent in the identification and characterization of focal liver lesions: pathological correlation in explanted livers.

AIM: To assess the diagnostic performance of magnetic resonance imaging (MRI) with gadoxetic acid in the identification of hepatocellular carcinoma (HCC) nodules by comparison with histological findings. METHODS: In a cohort of patients suffering from cirrhosis of various etiologies (chronic hepatitis C virus (HCV) or hepatitis B virus (HBV), alcohol abuse, cryptogenic forms), we selected 17 patients affected by HCC who were eligible for liver transplantation on the basis of a computed-tomography (CT) total-body examination. Such patients also underwent an MRI examination under basal conditions, and with four dynamic phases, as well as a hepatobiliary phase acquired after at least 20 min and recognized by the excretion of contrast agent into the bile duct, following intravenous administration of 0.05 mol/kg of gadoxetic acid (gadoxetate disodium, Primovist(®); Bayer, Osaka, Japan). The MRI images were then evaluated in a double-blinded experimental setup by two radiologists experienced in imaging of the liver. The diagnosis of HCC was made in the presence of nodular lesions that showed typical or atypical enhancement patterns. The liver was subsequently explanted (on average 47.4 days after MRI evaluation), dissected into 1-cm samples, and histologically evaluated according to the classification of Edmondson-Steiner. RESULTS: At the histopathological examination, 46 nodules were identified, on average 2.7 nodules for each patient. Of these, 37 were hepatocellular carcinomas, 3 were characterized by histologically unrecognizable complete necrosis, and 6 showed high-grade dysplasia. MRI with hepatospecific contrast medium showed inter-observer average values of sensitivity, specificity, and diagnostic accuracy of 94.6, 90, and 93.6 %, respectively. In one case, a nodule was not identified by MRI with gadoxetic acid, even in the hepatospecific phase (false negative (FN)). This result could be implicated to the long time interval between the analysis and the explant (88 days). In another case, there was an overdiagnosis of a HCC with a typical nodular pattern (false positive (FP)), but which most likely should have been attributed to a previous echinococcus cyst. MRI analysis, in combination with the study of the hepatobiliary phase, also showed a greater sensitivity, the same specificity, and a greater diagnostic accuracy compared to MRI evaluated only in the dynamic phases (with an average percentage between the two operators, respectively, of 75.7, 90, and 78 %). CONCLUSIONS: MRI with gadoxetic acid shows a diagnostic accuracy superior to contrast-enhanced MRI, allowing for the diagnosis of additional lesions, and it could be considered as an imaging method to carry out a more appropriate management of waiting lists for liver transplants.

Repolarization of macrophages to improve sorafenib sensitivity for combination cancer therapy.

Sorafenib is the first line drug for hepatocellular carcinoma (HCC) therapy. However, HCC patients usually acquire resistance to sorafenib treatment within 6 months. Recent evidences have shown that anticancer drugs with antiangiogenesis effect (e.g., sorafenib) can aggravate the hypoxia microenvironment and promote the infiltration of more tumor-associated macrophages (TAMs) into the tumor tissues. Therefore, repolarization of TAMs phenotype could be expected to not only eliminate the influence of TAMs on sorafenib lethality to HCC cells, but also provide an additional anticancer effect to achieve combination therapy. However, immune side effects remain a great challenge due to the non-specific macrophage repolarization in normal tissues. We herein employed a tumor microenvironment (TME) pH-responsive nanoplatform to concurrently transport sorafenib and modified resiquimod (R848-C16). This nanoparticle (NP) platform is made with a TME pH-responsive methoxyl-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) copolymer. After intravenous administration, the co-delivery NPs could highly accumulate in the tumor tissues and then respond to the TME pH to detach their surface PEG chains. With this PEG detachment to enhance uptake by TAMs and HCC cells, the co-delivery NPs could combinatorially inhibit HCC tumor growth via sorafenib-mediated lethality to HCC cells and R848-mediated repolarization of TAMs into tumoricidal M1-like macrophages. STATEMENT OF SIGNIFICANCE: Anticancer drugs with antiangiogenesis effect (e.g., sorafenib) can aggravate the hypoxia microenvironment and promote the infiltration of more tumor-associated macrophages (TAMs) into the tumor tissues to restrict the anticancer effect. In this work, we designed and developed a tumor microenvironment (TME) pH-responsive nanoplatform for systemic co-delivery of sorafenib and resiquimod in hepatocellular carcinoma (HCC) therapy. These co-delivery NPs show high tumor accumulation and could respond to the TME pH to enhance uptake by TAMs and HCC cells. With the sorafenib-mediated lethality to HCC cells and R848-mediated repolarization of TAMs, the co-delivery NPs show a combinational inhibition of HCC tumor growth in both xenograft and orthotopic tumor models.

Autophagy: Dual Response in the Development of Hepatocellular Carcinoma.

Autophagy is an evolutionary conserved intracellular mechanism which helps eukaryotic cells in maintaining their metabolic state to afford high-efficiency energy requirements. In the physiology of a normal liver and the pathogenesis of liver diseases, autophagy plays a crucial role. Autophagy has been found to be both upregulated and downregulated in different cancers providing the evidence that autophagy plays a dual role in suppressing and promoting cell survival. Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the major leading cause of cancer mortality worldwide. In light of its high complexity and poor prognosis, it is essential to improve our understanding of autophagy's role in HCC. In this review, we summarize the dual mechanism of autophagy in the development of HCC and elucidate the currently used therapeutic strategies for anti-HCC therapy.

Co-delivery of sorafenib and VEGF-siRNA via pH-sensitive liposomes for the synergistic treatment of hepatocellular carcinoma.

Non-viral nanocarrier affords a platform for drug and siRNA combination, the focus of which is to load drug and siRNA into a single carrier, allowing for co-delivery and a synergistic effect at tumour site. In our previous study, pH-sensitive carboxymethyl chitosan-modified liposomes (CMCS-SiSf-CL) were assembled for sorafenib (Sf) and Cy3-siRNA co-loaded. The present study evaluated in vitro and in vivo co-delivery of the co-loaded liposomes. Further, in vitro inhibiting hepatocellular carcinoma of the pH-sensitive sorafenib (Sf) and VEGF-siRNA co-loaded liposomes was discussed. The experimental results demonstrated co-delivery and penetration into 2-dimensional (2D) cultured HepG2 cells, 3-dimensional (3D) cultured HepG2 tumour spheroids and tumour regions of H22 tumour-bearing mice. Compared with free siRNA and single loaded carrier, co-delivery liposomes exhibited enhanced VEGF downregulating effect, inducing cell early apoptosis. Therefore, the CMCS-SiSf-CL delivery system can lay the foundation for the co-delivery systems development and provide new area for HCC therapy.

MicroRNA-203 impacts on the growth, aggressiveness and prognosis of hepatocellular carcinoma by targeting MAT2A and MAT2B genes.

Hepatocellular carcinoma (HCC) is characterized by the down-regulation of the liver-specific methyladenosyltransferase 1A (MAT1A) gene, encoding the S-adenosylmethionine synthesizing isozymes MATI/III, and the up-regulation of the widely expressed methyladenosyltransferase 2A (MAT2A), encoding MATII isozyme, and methyladenosyltransferase 2B (MAT2B), encoding a ß-subunit without catalytic action that regulates MATII enzymatic activity. Different observations showed hepatocarcinogenesis inhibition by miR-203. We found that miR-203 expression in HCCs is inversely correlated with HCC proliferation and aggressiveness markers, and with MAT2A and MAT2B levels. MiR-203 transfection in HepG2 and Huh7 liver cancer cells targeted the 3'-UTR of MAT2A and MAT2B, inhibiting MAT2A and MAT2B mRNA levels and MATα2 and MATß2 protein expression. These molecular events were paralleled by an increase in SAM content and were associated with growth restraint and apoptosis, inhibition of cell migration and invasiveness, and suppression of the expression of CD133 and LIN28B stemness markers. In contrast, MAT2B transfection in the same cell lines led to a rise of both MATß2 and MATα2 expression, associated with increases in cell growth, migration, invasion and overexpression of stemness markers and p-AKT. Altogether, our results indicate that the miR-203 oncosuppressor activity may at least partially depend on its inhibition of MAT2A and MAT2B and show, for the first time, an oncogenic activity of MAT2B linked to AKT activation.

Sphk2 RNAi nanoparticles suppress tumor growth via downregulating cancer cell derived exosomal microRNA.

Exosomes secreted from cancer cells promote tumor progression through the transfection of containing microRNA (miRNA), mRNAs and proteins. Yet, little of this knowledge has translated into the therapeutic application. Herein, we propose a tumor therapeutic strategy via decreasing exosomal miRNA secretion. The study designed small interfering RNA (siRNA) loaded nanoparticles to downregulate sphingosine kinase 2 (Sphk2) and investigate their potential in decreasing exosomal oncogenic miRNA content and inhibiting tumor growth. The synthesized lipid (2E)-4-(dioleostearin)-amino-4­carbonyl-2-butenoic (DC) and chitosan were utilized to produce siRNA loaded nanoparticles (DC/CS-siRNA NPs), with optimal siRNA complexation and high transfection efficacy. We demonstrated that Sphk2 gene silencing induced by nanoparticles in hepatocellular carcinoma (HCC) cells could reduce miRNA-21 sorting into exosomes, contributing to the inhibition of tumor cell migration and tumorigenic function of exosomes to normal liver cells. Furthermore, in xenograft mouse model, Sphk2 siRNA loaded DC/CS NPs could significantly block tumor progression of malignancy HCC. These results suggest a new therapeutic approach for tumor treatment by ablating oncogenic miRNA in malicious exosomes.

When and how should we perform a biopsy for HCC in patients with liver cirrhosis in 2018? A review.

The role of liver biopsy in the diagnosis of hepatocellular carcinoma (HCC) has changed over time. The diagnostic algorithm for this tumor is nowadays mainly based on radiological imaging, relegating histology to controversial cases, in which imaging techniques cannot establish a clear-cut diagnosis. This most commonly happens in small lesions, where biopsies frequently become mandatory, or in larger hypovascularized lesions. In this case however, the histological examination may not be reliable enough to grade the lesion, as different cell clones, deriving from sequential mutations, can originate heterogeneous cell populations. The risk of complications of liver biopsy, such as tumor seeding and intra-abdominal bleeding, needs to be reconsidered in light of new scientific evidence and of the technical improvements that have been introduced. Furthermore, increasing knowledge of the immunohistochemical and molecular characteristics of hepatocellular carcinoma opens a new scenario in which biopsy may play a decisive role in defining prognosis, and even treatment, by identifying the patient populations who could most benefit from target-driven hepatocellular carcinoma treatments, and therefore improving the success rate of experimental therapies. All the above reasons suggest that, overall, the role of liver biopsy in the management of HCC needs a reappraisal.

Effectiveness of localized ultrasound-targeted microbubble destruction with doxorubicin liposomes in H22 mouse hepatocellular carcinoma model.

OBJECTIVE: In order to increase local drug concentration and reduce systemic side effects of liver cancer chemotherapy, it is desirable to develop novel non-invasive technologies for drug targeting, such as ultrasound-targeted microbubble destruction (UTMD). METHODS: H22 hepatocellular carcinoma (HCC) xenograft transplantation model was generated in UTMD study. BALB/c mice were randomly divided into six groups: doxorubicin HCl liposomal injection (DOX), DOX + US, UTMD, DOX + UTMD, H22 liver tumor control (CH control) and blank control group. The therapeutic schedule started on day 4 after tumor inoculation. RESULTS: Average survival time of the animal model was approximately 18 d. The UTMD therapy parameters were optimized in the H22 mouse model to be: microbubble (MB) diameter, 2.30 ± 0.25 µm; MB density, 4.0 × 10(9) bubbles/ml; treatment dose, 0.2 ml per 20 g mouse body weight; sonication frequency, 1.3 MHz; and sonication power, 2.06 W/cm(2). Mice treated with DOX + UTMD had the smallest tumor volume and weight (p < 0.001), and the highest tumor inhibition rate (p < 0.01), intratumoral DOX concentration (p < 0.001) and survival rate among all tumor-burden groups (p < 0.001). Cell viability in different treatment groups was also assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. CONCLUSION: An improved antitumor effect was observed with the combination therapy of DOX and UTMD, as compared with treatment with DOX, DOX + US or UTMD, which implicates a novel approach for HCC treatment.