The anticarcinogenic effect of eugenol on lung cancer induced by diethylnitrosamine/2-acetylaminofluorene in Wistar rats: insight on the mechanisms of action.

This study was designed to assess the ameliorative effects of eugenol and to propose the possible mechanisms of action of eugenol in diethylnitrosamine (DENA)/acetylaminofluorene (AAF)-caused lung cancer in Wistar rats. To induce lung cancer, DENA at a dose of 150 mg/kg body weight (b.wt) for 2 weeks were intraperitoneally injected once each week and AAF was administered orally at a dose of 20 mg/kg b.wt. four times each week for the next 3 weeks. DENA/AAF-administered rats were orally supplemented with eugenol at a dose of 20 mg/kg b.wt administered once a day until 17 weeks starting from the 1st week of DENA administration. Lung histological lesions, including sheets of tumor cells, micropapillary adenocarcinoma, and apoptotic cells, resulting from the DENA/AAF dosage, were ameliorated by eugenol treatment. However, a significant drop in the levels of LPO in the lungs and a remarkable rise in GSH content and GPx and SOD activities were observed in DENA/AAF-administered rats treated with eugenol compared with those in DENA/AAF-administered controls. Moreover, in DENA/AAF-administered rats, eugenol supplementation significantly reduced TNF-α and IL-1ß levels and mRNA expression levels of NF-κB, NF-κB p65, and MCP-1 but significantly elevated the level of Nrf2. Furthermore, the DENA/AAF-administered rats treated with eugenol exhibited a significant downregulation of Bcl-2 expression levels in addition to a significant upregulation in P53 and Bax expression levels. Otherwise, the administration of DENA/AAF elevated the protein expression level of Ki-67, and this elevation was reversed by eugenol treatment. In conclusion, eugenol has effective antioxidant, anti-inflammatory, proapoptotic, and antiproliferative properties against lung cancer.

Chronic Administration of Diethylnitrosamine and 2-Acetylaminofluorene Induces Hepatocellular Carcinoma in Wistar Rats.

This study aimed to analyze the biochemical, histological, and gene expression alterations produced in a hepatocarcinogenesis model induced by the chronic administration of diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) in Wistar rats. Thirteen rats weighing 180 to 200 g were divided into two groups: control and treated. Rats in the treated group were administered an intraperitoneal (i.p.) injection of DEN (50 mg/kg/week) and an intragastric (i.g.) dose of 2-AAF (25 mg/kg/week) for 18 weeks. The treated group had significant increases in their total cholesterol, HDL-C, AST, ALT, ALKP, and GGT levels. Furthermore, a histological analysis showed the loss of normal liver architecture with nuclear pleomorphism in the hepatocytes, atypical mitosis, and fibrous septa that were distributed between the portal triads and collagen fibers through the hepatic sinusoids. The gene expressions of 24 genes related to fibrosis, inflammation, apoptosis, cell growth, angiogenesis, lipid metabolism, and alpha-fetoprotein (AFP) were analyzed; only TGFß, COL1α1, CYP2E1, CAT, SOD, IL6, TNF-α, and ALB showed significant differences when both groups were compared. Additionally, lung histopathological alterations were found in the treated group, suggesting metastasis. In this model, the chronic administration of DEN+2-AAF induces characteristic alterations of hepatocellular carcinoma in Wistar rats without AFP gene expression changes, highlighting different signatures in hepatocellular carcinoma heterogeneity.

In silico and in vivo hepatoprotective activity of the synthesized 5-benzylidene-2-thiohydantoin against diethylnitrosamine-induced liver injury in a rat model.

In the present study, the hepatoprotective effect of 5-benzylidine-2-thiohydantoin (5B2T), a unique derivative of the thiohydantoin group, on liver injury induced by diethylnitrosamine (DEN) in male rats was investigated. The experimental animals were divided into three groups, each with 14 rats. Rats in group I were considered to be controls and received only 10% Tween 80. Rats in group II were injected with 200 mg/kg DEN intraperitoneally. Rats in group III were injected with a single dose of DEN 200 mg/kg intraperitoneally and received the treatment orally (50 mg/kg, 5B2T) for two durations, 3 and 6 weeks. At the end of the experiment, blood was collected for the analysis of liver function and pro-inflammatory cytokine IL-6 and tumor necrosis factor α (TNF-α) levels. Additionally, liver specimens were used for histopathological examination and immunohistochemistry. The single intraperitoneal injection of 200 mg/kg DEN into rats resulted in significant elevation of serum enzyme levels of AST, ALT and ALP, which are indicators of hepatocellular damage, along with elevation in TNF-α and IL-6 in the DEN group. The results of both LFTs and ELISA in the treatment group showed improvements and a decline in the levels of the markers. Histopathological examination showed fibrosis, necrosis and infiltration of inflammatory cells in the DEN group, with lower intensity in the treatment group. The results of immunohistochemical staining revealed strong positive staining of both HSA and Ki-67 antibodies in the DEN group, with much lower intensity in the treatment group. The results of the docking study indicated that 5B2T has a remarkable interaction with TNF-α (PDB ID: 1TNF) and human IL-6 (PDB ID: 1IL6) with binding site energies of - 7.1 and - 6.1 (kcal/mol), respectively. The correct absorption and binding between the drug and the receptor was evaluated through computerized molecular docking by using the AutoDock program. The conclusion of the results from the current study reflected the interesting hepatoprotective abilities of 5B2T against DEN-induced hepatocellular damage and cancer in experimental rats.

Experimental VX2 Rabbit Liver Tumor Model in Carbon Tetrachloride-Induced Cirrhosis of the Liver.

Liver cirrhosis is a major underlying factor in the development of hepatocellular carcinoma. Currently, there is an unmet need for midsize experimental vertebrate models that would offer reproducible implantable liver tumors in a cirrhotic liver background. This study establishes a protocol for a syngeneic rabbit model of VX2 liver cancer with underlying liver cirrhosis induced using carbon tetrachloride (CCl4). Male New Zealand white rabbits (n = 3) received CCl4 by intragastric administration once weekly. Concentrations started at 5% v/v CCl4 dissolved in olive oil. CCl4 dosing was progressively increased every week by 2.5% v/v increments for the duration of treatment (16 weeks total). VX2 tumors were then orthotopically implanted into the left hepatic lobe and allowed to grow for 3 weeks. Cross-sectional imaging confirmed the presence of hepatic tumors. Gross and histopathological evaluations showed reproducible tumor growth in the presence of liver cirrhosis in all animals.

Modulation of EphA7 and pEphA7 Protein Expression: Potential Biomarkers for Early Detection of Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading drivers of cancer-related mortality in the world. As a result, researchers are constantly looking for ways to optimize the screening and diagnosis of the said malignancy. OBJECTIVE: To establish the mice model of hepatocellular carcinoma with the administration of a suitable dose of diethylnitrosamine (DEN) and examine the utility of EphA7 and pEphA7 as ideal diagnostic markers in HCC. METHODS: Swiss Albino (BALB/c) mice of around 10-12 weeks old were exposed to a known hepatocarcinogen-diethylnitrosamine at a dose of 20 mg/kg body weight at weekly intervals for a period of 4, 8, 12, & 16 weeks. Blood was collected from mice of different experimental groups, and age-matched control and serum were separated from whole blood samples. The liver homogenate was prepared after completion of treatment, and the resulting supernatant was used for enzyme assays. A range of liver biomarker enzyme assays such as Gamma-glutamyl transpeptidase (GGT), Acetylcholine esterase (AChE), GPx activity and GSH level, Heme oxygenase-1 (HO-1), GPC3 and alpha-fetoprotein (AFP) level along with the expression of Caspase-3, EphA7 and pEphA7 were evaluated. RESULTS: An elevation in body weight and relative liver weight across the treatment period (4, 8, 12, 16 weeks) was observed in DEN-treated mice. Significant differences in GGT levels between control and DEN treated mice were noted in the present study (P < 0.005). In the 16th week of the treatment period, a significant difference in AchE level was noted between the treated and control group (P < 0.001). However, there was no statistically significant difference in the levels of SGOT and SGPT levels between the control and DEN treated groups (P > 0.001). Lower GSH and GPx levels were demonstrated in the treated mice as compared to control over all the treatment period. Loss of Caspase-3 expression and significant differences in expression of HO-1 activity in treated vs. non-treated group of mice were observed. Significant differences in EphA7 and pEphA7 protein expression levels were noted in the DEN-treated vs. control groups across all the treatment periods (4 weeks: P < 0.05; 8 weeks: P < 0.05; 12 weeks:  P < 0.005; 16 weeks: P < 0.05). CONCLUSION: The present study indicated that EphA7 and phosphoEphA7 over-expression might contribute to the malignancy transition, invasion development, and metastasis of HCC. As a result, along with the known markers such as AFP and others, EphA7 and pEphA7 could be a very putative biomarkers of HCC, particularly at a very early stage of cancer development.

Dietary folate drives methionine metabolism to promote cancer development by stabilizing MAT IIA.

Folic acid, served as dietary supplement, is closely linked to one-carbon metabolism and methionine metabolism. Previous clinical evidence indicated that folic acid supplementation displays dual effect on cancer development, promoting or suppressing tumor formation and progression. However, the underlying mechanism remains to be uncovered. Here, we report that high-folate diet significantly promotes cancer development in mice with hepatocellular carcinoma (HCC) induced by DEN/high-fat diet (HFD), simultaneously with increased expression of methionine adenosyltransferase 2A (gene name, MAT2A; protein name, MATIIα), the key enzyme in methionine metabolism, and acceleration of methionine cycle in cancer tissues. In contrast, folate-free diet reduces MATIIα expression and impedes HFD-induced HCC development. Notably, methionine metabolism is dynamically reprogrammed with valosin-containing protein p97/p47 complex-interacting protein (VCIP135) which functions as a deubiquitylating enzyme to bind and stabilize MATIIα in response to folic acid signal. Consistently, upregulation of MATIIα expression is positively correlated with increased VCIP135 protein level in human HCC tissues compared to adjacent tissues. Furthermore, liver-specific knockout of Mat2a remarkably abolishes the advocating effect of folic acid on HFD-induced HCC, demonstrating that the effect of high or free folate-diet on HFD-induced HCC relies on Mat2a. Moreover, folate and multiple intermediate metabolites in one-carbon metabolism are significantly decreased in vivo and in vitro upon Mat2a deletion. Together, folate promotes the integration of methionine and one-carbon metabolism, contributing to HCC development via hijacking MATIIα metabolic pathway. This study provides insight into folate-promoted cancer development, strongly recommending the tailor-made folate supplement guideline for both sub-healthy populations and patients with cancer expressing high level of MATIIα expression.

hnRNPC induces isoform shifts in miR-21-5p leading to cancer development.

MicroRNA (miRNA) processing is a critical step in mature miRNA production. Its dysregulation leads to an increase in miRNA isoforms with heterogenous 5'-ends (isomiRs), which can recognize distinct target sites because of their shifted seed sequence. Although some miRNA genes display productive expression of their 5'-isomiRs in cancers, how their production is controlled and how 5'-isomiRs affect tumor progression have yet to be explored. In this study, based on integrative analyses of high-throughput sequencing data produced by our group and publicly available data, we demonstrate that primary miR-21 (pri-miR-21) is processed into the cancer-specific isomiR isomiR-21-5p | ±1, which suppresses growth hormone receptor (GHR) in liver cancer. Treatment with antagomirs against isomiR-21-5p | ±1 inhibited the in vitro tumorigenesis of liver cancer cells and allowed the recovery of GHR, whereas the introduction of isomiR-21-5p | ±1 mimics attenuated these effects. These effects were validated in a mouse model of spontaneous liver cancer. Heterogeneous nuclear ribonucleoprotein C and U2 small nuclear RNA auxiliary factor 2 were predicted to bind upstream of pre-miR-21 via a poly-(U) motif and influence Drosha processing to induce the production of isomiR-21-5p | ±1. Our findings suggest an oncogenic function for the non-canonical isomiR-21-5p | ±1 in liver cancer, and its production was shown to be regulated by hnRNPC.

Nrf2 activation contributes to hepatic tumor-augmenting effects of developmental arsenic exposure.

Developmental arsenic exposure increases cancer risk in later life with the mechanism elusive. Oxidative stress is a dominant determinant in arsenic toxicity. However, the role of Nrf2, a key regulator in antioxidative response, in tumor-augmenting effects by developmental arsenic exposure is unclear. In the present study, wild-type C57BL/6J and Nrf2-konckout (Nrf2-KO) were developmentally exposed to inorganic arsenic via drinking water. For hepatic tumorigenesis analysis, mice were intraperitoneally injected with diethylnitrosamine (DEN) at two weeks of age. Developmental arsenic exposure aggravated tumor multiplicity and burden, and expression of PCNA and AFP in hepatic tumors induced by DEN. Nrf2 activation as indicated by over-expression of Nrf2 and its downstream genes, including Gss, Gsr, p62, Gclc and Gclm, was found in liver tumors, as well as in the livers in developmentally arsenic-exposed pups at weaning. Notably, Nrf2 deficiency attenuated tumor-augmenting effects and over-expression of Nrf2 downstream genes due to developmental arsenic exposure. Furthermore, the levels of urinary DEN metabolite (acetaldehyde) and hepatic DNA damage markers (O6-ethyl-2-deoxyguanosine adducts and γ-histone H2AX) after DEN treatment were elevated by Nrf2 agonist, 2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide. Collectively, our data suggest that augmentation of DEN-induced hepatic tumorigenesis by developmental arsenic exposure is dependent on Nrf2 activation, which may be related to the role of Nrf2 in DEN metabolic activation. Our findings reveal, at least in part, the mechanism underlying increased susceptibility to developing cancer due to developmental arsenic exposure.

The mouse Anxa6/miR-9-5p/Anxa2 axis modulates TGF-ß1-induced mouse hepatic stellate cell (mHSC) activation and CCl4-caused liver fibrosis.

Chronic liver disease such as hepatic fibrosis is a major cause of morbidity and mortality and has been related to high individual risk of hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) activation is a central event of hepatic fibrosis progression. In this study, the up-regulation of lncRNA ANXA2P2 (mouse Anxa6) was found in liver fibrosis. Within CCl4-caused liver fibrosis murine model, Anxa6 knockdown partially ameliorated CCl4-induced hepatic fibrosis and blocked the PI3K/Akt signaling activation. In TGF-ß1-stimulated HSCs, Anxa6 knockdown partially inhibited TGF-ß1-induced HSC activation and blocked the PI3K/Akt signaling activation. Mouse Anxa6 downstream mmu-miR-9-5p directly targeted Anxa2; Anxa6 negatively regulated mmu-miR-9-5p, and mmu-miR-9-5p negatively regulated mouse Anxa2. In TGF-ß1-stimulated HSCs, miR-9-5p inhibitor promoted TGF-ß1-induced HSC activation and PI3K/Akt signaling activation, whereas Anxa2 knockdown exerted opposite effects; Anxa2 knockdown significantly attenuated miR-9-5p inhibitor effects upon TGF-ß1-stimulated HSCs. In conclusion, lncRNA ANXA2P2 (mouse Anxa6) expression is up-regulated in hepatic fibrosis and exerts pro-fibrotic effects on CCl4-caused liver fibrosis model mice and TGF-ß1-stimulated HSCs. The mouse Anxa6/miR-9-5p/Anxa2 axis and the PI3K/Akt pathway might participate in the functions of lncRNA ANXA2P2 (mouse Anxa6) on hepatic fibrosis.

Senescence Connects Autophagy Deficiency to Inflammation and Tumor Progression in the Liver.

BACKGROUND & AIMS: Cellular senescence frequently is present in injured livers. The induction mechanism and the pathologic role are not always clear. We aimed to understand the dynamics of senescence induction and progression, and the mechanism responsible for the pathology using a mouse model that disables the essential process of autophagy. METHODS: Mice deficient in key autophagy genes Atg7 or Atg5 in the liver were used. Senescence was measured using established cellular and molecular signatures. The mechanistic roles of nuclear factor erythroid 2 (NRF2), forkhead box K1, and C-C motif chemokine receptor 2 (CCR2) were assessed using mouse genetic models. Liver functions, pathology, and tumor development were measured using biochemical and histologic approaches. RESULTS: Inducible deletion of Atg7 rapidly up-regulated cyclin-dependent kinase inhibitors independently of injury and induced senescence-associated ß-galactosidase activities and senescence-associated secretory phenotype (SASP). Sustained activation of NRF2 was the major factor causing senescence by mediating oxidative DNA damage and up-regulating C-C motif chemokine ligand 2, a key component of autophagy-related SASP, via the NRF2-forkhead box K1 axis. Senescence was responsible for hepatic inflammation through CCR2-mediated recruitment of CD11b+ monocytes and CD3+ T cells. The CCR2-mediated process in turn enhanced senescence and SASP by up-regulating cyclin-dependent kinase inhibitors and chemokines. Thus, senescence and inflammation can mutually augment each other, forming an amplification loop for both events. The CCR2-mediated process also modulated liver injury and tumor progression at the later stage of autophagy deficiency-related pathology. CONCLUSIONS: These results provide the insight that hepatic senescence can occur early in the disease process, triggers inflammation and is enhanced by inflammation, and has long-term effects on liver injury and tumor progression.

Farnesoid X receptor (FXR) agonists induce hepatocellular apoptosis and impair hepatic functions via FXR/SHP pathway.

Farnesoid X receptor (FXR) plays an indispensable role in liver homeostasis and has been a promising drug target for hepatic diseases. However, the concerns of undesired biological actions limit the clinical applications of FXR agonists. To reveal the intrinsic mechanism of FXR agonist-induce hepatotoxicity, two typical FXR agonists with different structures (obeticholic acid (OCA) and Px-102) were investigated in the present study. By detecting MMP, ROS, and ATP and analyzing the fate of cells, we found that both OCA and Px-102 reduced the mitochondrial function of hepatocytes and promoted cell apoptosis. Gene ablation or inhibition of FXR or SHP ameliorated the cytotoxicities of OCA and Px-102, which indicated the adverse actions of FXR/SHP activation including down-regulation of phosphorylation of PI3K/AKT and functional hepatic genes. The dose-related injurious effects of OCA (10 mg/kg and 30 mg/kg) and Px-102 (5 mg/kg and 15 mg/kg) on the liver were confirmed on a high-fat diet mouse model. The decrease of hepatocyte-specific genes and augmenter of liver regeneration in the liver caused by OCA or Px-102 suggested an imbalance of liver regeneration and a disruption of hepatic functions. Exploration of intestinally biased FXR agonists or combination of FXR agonist with apoptosis inhibitor may be more beneficial strategies for liver diseases.

6-Amino-2,4,5-trimethylpyridin-3-ol and 2-amino-4,6-dimethylpyrimidin-5-ol derivatives as selective fibroblast growth factor receptor 4 inhibitors: design, synthesis, molecular docking, and anti-hepatocellular carcinoma efficacy evaluation.

A novel series of aminotrimethylpyridinol and aminodimethylpyrimidinol derivatives were designed and synthesised for FGFR4 inhibitors. Structure-activity relationship on the FGFR4 inhibitory activity of the new compounds was clearly elucidated by an intensive molecular docking study. Anti-cancer activity of the compounds was evaluated using hepatocellular carcinoma (HCC) cell lines and a chick chorioallantoic membrane (CAM) tumour model. Compound 6O showed FGFR4 inhibitory activity over FGFR1 - 3. Compared to the positive control BLU9931, compound 6O exhibited at least 8 times higher FGFR4 selectivity. Strong anti-proliferative activity of compound 6O was observed against Hep3B, an HCC cell line which was a much more sensitive cell line to BLU9931. In vivo anti-tumour activity of compound 6O against Hep3B-xenografted CAM tumour model was almost similar to BLU9931. Overall, compound 6O, a novel derivative of aminodimethylpyrimidinol, was a selective FGFR4 kinase inhibitor blocking HCC tumour growth.

Dual regulating of mitochondrial fusion and Timp-3 by leflunomide and diallyl disulfide combination suppresses diethylnitrosamine-induced hepatocellular tumorigenesis in rats.

AIMS: Hepatocellular carcinoma (HCC) is considered one of the main causes of cancer-related death globally. Combination therapy targeting different pathways can improve the efficacy of HCC management. Mitofusin 2 (Mfn2), a mitochondrial fusion protein, and a tissue inhibitor of matrix metalloproteinase 3 (Timp-3) were found to be downregulated in various cancers, including HCC. Our study aimed to evaluate the possible antineoplastic effect of a novel combination in the treatment of HCC through targeting mitochondrial fusion and metastatic proteins. MAIN METHODS: HCC induction was performed using a single intraperitoneal dose of diethylnitrosamine (200 mg/kg), followed by adding phenobarbital sodium (0.05%) to the drinking water for successive 18 weeks. Then, leflunomide (LF, 10 mg/kg) was administered orally for 28 days. Diallyl disulfide (DADS, 50 mg/kg) was also given orally for 28 days, either alone or in combination with LF. KEY FINDINGS: Treatment with LF or DADS could alleviate the HCC- induced histological and biochemical variations, including liver enzyme activities (ALT, AST), alpha-fetoprotein, Bax, cyclin D1, Ki67, malondialdehyde, and reduced glutathione. They could shift the mitochondrial dynamics toward mitochondrial fusion through upregulating the expression of Mfn2 and also exhibited antimetastatic activity through upregulating the expression of Timp-3 and decreasing hepatic MMP9 content. SIGNIFICANCE: the treatment with a combination of LF and DADS displayed a more potent effect than the treatment with each drug alone. Our results suggest that the combined use of LF and a naturally occurring DADS can be used as a promising novel combination in managing HCC.

Preclinical mouse models of hepatocellular carcinoma: An overview and update.

Hepatocellular carcinoma (HCC) is by far the most common histological subtype of primary liver cancer. HCC often originates from chronic liver injuries and inflammation, subsequently leading to fibrosis and cirrhosis. Preclinical animal models, especially mice, are viewed as valuable and reliable tools for investigating the molecular processes involved in hepatocarcinogenesis and facilitating the evaluations of the efficacy of novel therapies for HCC. A wide range of mouse models of HCC has been established using various approaches including chemotoxic agents, genetic modifications, special diet administration, and tumor cells transplantation. Choosing a suitable model to represent certain genetic and physiological features of human HCC seems to be crucial. Here, we review the current preclinical mouse models that are frequently used to study HCC.

Preparation and structural properties of selenium modified heteropolysaccharide from the fruits of Akebia quinata and in vitro and in vivo antitumor activity.

Cancer is a complex disease, and blocking tumor angiogenesis has become one of the most promising approaches in cancer therapy. Here, an exopoly heteropolysaccharide (AQP70-2B) was firstly isolated from Akebia quinata. Monosaccharide composition indicated that the AQP70-2B was composed of rhamnose, glucose, galactose, and arabinose. The backbone of AQP70-2B consisted of →1)-l-Araf, →3)-l-Araf-(1→, →5)-l-Araf-(1→, →3,5)-l-Araf-(1→, →2,5)-l-Araf-(1→, →4)-d-Glcp-(1→, →6)-d-Galp-(1→, and →1)-d-Rhap residues. Based on the close relationship between selenium and anti-tumor activity, AQP70-2B was modified with selenium to obtain selenized polysaccharide Se-AQP70-2B. Then, a series of methods for analysis and characterization, especially scanning electron microscopy coupled with energy dispersive spectrometry (SEM-EDS), indicated that Se-AQP70-2B was successfully synthesized. Furthermore, zebrafish xenografts and anti-angiogenesis experiments indicated that selenization could improve the antitumor activity by inhibiting tumor cell proliferation and migration and blocking angiogenesis.

Mimic Lipoproteins Responsive to Intratumoral pH and Allosteric Enzyme for Efficient Tumor Therapy.

Discoid-reconstituted high-density lipoprotein (d-rHDL) is advantageous for tumor-targeted drug delivery due to its small size, long circulation, and efficient internalization into cancer cells. Nevertheless, an allosteric reaction catalyzed by serum lecithin-cholesterol acyltransferase (LCAT) may cause drug leakage from d-rHDL and reduce its targeting efficiency. Conversely, similar "structural weakening" catalyzed by acyl-coenzyme A-cholesterol acyltransferase (ACAT) inside tumor cells can stimulate precise intracellular drug release. Therefore, we synthesized and characterized a pH-sensitive n-butyraldehyde bi-cholesterol (BCC) to substitute for cholesterol in the d-rHDL particle, and bovine serum albumin (BSA) was used as the targeting agent. This dual pH- and ACAT-sensitive d-rHDL (d-d-rHDL) was small with a disk-like appearance. Morphological transformation observation, in vitro release assays, and differences in internalization upon LCAT treatment confirmed that BCC effectively inhibited the remodeling behavior and enhanced the tumor-targeting efficiency. The accumulation of d-d-rHDL in HepG2 cells was significantly higher than that in LO2 cells, and accumulation was inhibited by free BSA. The pH sensitivity was verified, and d-d-rHDL achieved efficient drug release in vitro and inside tumor cells after exposure to acidic conditions and ACAT. Confocal laser scanning microscopy demonstrated that d-d-rHDL escaped from lysosomes and became distributed evenly throughout cells. Moreover, in vivo imaging assays in a tumor-bearing mouse model demonstrated tumor-targeting properties of d-d-rHDL, and paclitaxel-loaded d-d-rHDL showed strong anticancer activity in these mice. This dual-sensitive d-d-rHDL thus combines structural stability in plasma and an intracellular pH/ACAT-triggered drug release to facilitate inhibition of tumor growth.

Kangxianruangan granule­containing serum mediated inhibition of hepatic oval cell differentiation into hepatocellular carcinoma cells via the Wnt­1/ß­catenin signaling pathway.

Hepatocellular carcinoma is a malignancy with poor clinical prognosis. Hepatic oval cells (HOCs) tend to differentiate into cancerous hepatocellular carcinoma cells (HCCs) in the tumor microenvironment. The purpose of the present study was to explore the role of kangxianruangan granule (KXRG)­containing serum in inhibiting the differentiation of HOCs into HCCs via the Wnt­1/ß­catenin signaling pathway. N­methyl­N'­nitro­N­nitrosoguanidine (MNNG) was applied to induce the transformation of the rat HOC cell line WB­F344 into HCCs. The overexpression plasmid, Wnt­1­up, was utilized to increase Wnt­1 expression. Subsequently, high, medium and low concentrations of KXRG were applied to MNNG­treated WB­F344 cells to assess the inhibitory effect of KXRG on cell differentiation. Flow cytometry was conducted to detect the cell cycle distribution, apoptotic rate and expression of cytokeratin­19 (CK­19) protein in cells. An immunofluorescence double staining protocol was used to detect the expression of Wnt­1 and ß­catenin. ELISAs were performed to detect α fetoprotein in the cell supernatants. Reverse transcription­quantitative PCR and western blotting were conducted to detect the mRNA and protein expression levels of Wnt­1, ß­catenin, Cyclin D1, C­myc, matrix metalloproteinase­7 (MMP­7), Axin2 and epithelial cell adhesion molecule (EpCAM) in cells. Compared with the normal group, the apoptotic rate, proportion of S phase cells, concentration of AFP in the cell supernatant, level of CK­19 protein, and mRNA and protein expression levels of Wnt­1, ß­catenin, Cyclin D1, C­myc, MMP­7, Axin2 and EpCAM were all significantly increased in the model group. Addition of KXRG significantly reduced the aforementioned indicators compared with the model group. Moreover, Wnt­1 overexpression further increased the aforementioned indicators compared with the model group, whereas KXRG significantly inhibited these effects. The results indicated that KXRG inhibited the differentiation of HOCs into HCCs via the Wnt­1/ß­catenin signaling pathway, which suggested the potential clinical application of KXRG for the prevention of hepatocellular carcinoma.

A branched small molecule-drug conjugate nanomedicine strategy for the targeted HCC chemotherapy.

Off-target toxicity is one of the main challenges faced by anticancer chemotherapeutics. For tumor targeted and precision chemotherapy, we take the advantages of the ligand directed tumor active targeting of small molecule drug conjugates (SMDCs) and the passive tumor targeting of nanoparticles via the enhanced penetration and retention (EPR) effects, put forward a branched small molecule drug conjugate (BSMDC) nanomedicine design concept. In a proof of concept, we used pentaerythritol as the branched moiety, galactosamine (GalN) as the hepatocellular carcinoma (HCC) directing ligands, PTX as a payload, and a stearoyl moiety as the amphiphilic property adjusting group, designed and synthesized BSMDC 1 and prepared its NPs. In cellular level, the BSMDC 1 NPs targeted asialoglycoprotein receptor (ASGPR)-overexpressing HepG2 cells, were effectively taken up in the cells and released in tumor microenvironments, inhibited the HepG2 cell proliferation, arrested HepG2 cell in G2/M phase and induced tumor cell apoptosis. In HepG2 xenograft nude mice, the BSMDC 1 NPs were high specific to target the tumor and demonstrated a higher antitumor efficiency than BSMDC 1, having no apparent influences on mice body weights and major organs, supporting our BSMDC nanomedicine design concept. Therefore, this new strategy may find applications for cancer targeted and precision chemotherapy.

pH-Sensitive nanoparticles based on amphiphilic imidazole/cholesterol modified hydroxyethyl starch for tumor chemotherapy.

pH-Responsive nanoparticles (NPs) have emerged as an effective antitumor drug delivery system, promoting the drugs accumulation in the tumor and selectively releasing drugs in tumoral acidic microenvironment. Herein, we developed a new amphiphilic modified hydroxyethyl starch (HES) based pH-sensitive nanocarrier of antitumor drug delivery. HES was first modified by hydrophilic imidazole and hydrophobic cholesterol to obtain an amphiphilic polymer (IHC). Then IHC can self-assemble to encapsulate doxorubicin (DOX) and form doxorubicin-loaded nanoparticles (DOX/IHC NPs), which displayed good stability for one week storage and acidic sensitive long-term sustained release of DOX. As a result, cancer cell endocytosed DOX/IHC NPs could continuously release doxorubicin into cytoplasm and nucleus to effectively kill cancer cells. Additionally, DOX/IHC NPs could be effectively enriched in the tumor tissue, showing enhanced tumor growth inhibition effect compared to free doxorubicin. Overall, our amphiphilic modified HES-based NPs possess a great potential as drug delivery system for cancer chemotherapy.

Improvement in phenotype homeostasis of macrophages by chitosan nanoparticles and subsequent impacts on liver injury and tumor treatment.

When organic polymer-based drug nanocarriers become concentrated in macrophages, their influence on macrophage polarization has been rarely reported. This study prepared chitosan-based nanoparticles (CNs, 181.5 nm, +14.83 mV) and detected their impacts on macrophage reprogram. RT-PCR results showed in M1-like RAW264.7 cells (Mφ1), CNs decreased CD86 and iNOS expressions by 53.8% and 57.1%, and increased Arg-1 and IL-10 by 642.9% and 102.1%; in M2-like cells (Mφ2), CNs reduced Arg-1 and MR expressions by 70.7% and 93.0%, but increased CD86, iNOS and TNF-α by 290.4%, 86.2% and 728.6%; these results, consistent with cytokine secretions and surface CD86/CD206 expressions, showed CNs polarized Mφ1 and Mφ2 toward opposite type so as to improve the macrophage polarization homeostasis. In CCl4-induced mouse liver injury model, CNs reduced the hepatic Mφ1/Mφ2 ratio from 1.1 (model group) to 0.3, and then reduced the serum AST and ALT level by 42.3% and 39.0%; in mouse model of hepatocellular carcinoma, CNs decreased the number of CD163-positive cells and increased CD86-positive ones in tumor, and subsequently inhibited the tumor growth and metastasis. This study suggests CNs can improve the phenotype homeostasis of macrophages and subsequently promote the treatment of certain diseases such as liver injury and tumor.