HepG2 Tumor-bearing Nude Mice Research Articles

Uncovering the Mechanisms of Active Components from Toad Venom against Hepatocellular Carcinoma Using Untargeted Metabolomics.

Toad venom, a dried product of secretion from Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider, has had the therapeutic effects of hepatocellular carcinoma confirmed. Bufalin and cinobufagin were considered as the two most representative antitumor active components in toad venom. However, the underlying mechanisms of this antitumor effect have not been fully implemented, especially the changes in endogenous small molecules after treatment. Therefore, this study was designed to explore the intrinsic mechanism on hepatocellular carcinoma after the cotreatment of bufalin and cinobufagin based on untargeted tumor metabolomics. Ultraperformance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was performed to identify the absorbed components of toad venom in rat plasma. In vitro experiments were determined to evaluate the therapeutic effects of bufalin and cinobufagin and screen the optimal ratio between them. An in vivo HepG2 tumor-bearing nude mice model was established, and a series of pharmacodynamic indicators were determined, including the body weight of mice, tumor volume, tumor weight, and histopathological examination of tumor. Further, the entire metabolic alterations in tumor after treating with bufalin and cinobufagin were also profiled by UHPLC-MS/MS. Twenty-seven active components from toad venom were absorbed in rat plasma. We found that the cotreatment of bufalin and cinobufagin exerted significant antitumor effects both in vitro and in vivo, which were reflected in inhibiting proliferation and inducing apoptosis of HepG2 cells and thereby causing cell necrosis. After cotherapy of bufalin and cinobufagin for twenty days, compared with the normal group, fifty-six endogenous metabolites were obviously changed on HepG2 tumor-bearing nude mice. Meanwhile, the abundance of α-linolenic acid and phenethylamine after the bufalin and cinobufagin intervention was significantly upregulated, which involved phenylalanine metabolism and α-linolenic acid metabolism. Furthermore, we noticed that amino acid metabolites were also altered in HepG2 tumor after drug intervention, such as norvaline and Leu-Ala. Taken together, the cotreatment of bufalin and cinobufagin has significant antitumor effects on HepG2 tumor-bearing nude mice. Our work demonstrated that the in-depth mechanism of antitumor activity was mainly through the regulation of phenylalanine metabolism and α-Linolenic acid metabolism.

Design, Synthesis and Preclinical Assessment of 99mTc-iFAP for In Vivo Fibroblast Activation Protein (FAP) Imaging.

Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. 68Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl2 for labeling with 99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, 1H–NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The 99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the 99mTc-iFAP.

A carrier-free multifunctional nano photosensitizer based on self-assembly of lactose-conjugated BODIPY for enhanced anti-tumor efficacy of dual phototherapy

We reported a simple strategy for constructing carrier-free multifunctional NPSs based on one single amphiphilic small molecule of glycosylated BODIPY for tumor targeting and microenvironment responsive DPT with enhanced anti-tumor efficacy. • A multifunctional nano photosensitizer assembled from a glycosylated BODIPY. • Excellent biocompatibility, targetability, and GSH responsiveness in vitro. • Biosafety, accumulation at tumors and effective destruction of tumors in vivo. Carrier-free multifunctional nano photosensitizers (NPSs) based on amphiphilic small molecules with a single photoactivable motif that combine targeted delivery, tumor microenvironment (TME) responsive release, and dual phototherapy with a single irradiation hold great potential to enhance efficacy, safety, and accuracy of cancer treatment as well as facilitate clinical translation. However, the fabrication of such NPSs is challenging. Herein, we report the synthesis of an amphiphilic small glycosylated BODIPY (BSL) molecule with the ability of self-assembly into NPS (BSL NPS). BSL NPS shows excellent biocompatibility, targetability to HepG2 cells via overexpressed galactose receptors on the cell membrane, and responsiveness to high level of glutathione (GSH) in TME. BODIPY released from intracellular BSL NPS by GSH generated reactive oxygen species and hyperthermia upon irradiation of 685 nm NIR laser resulted in significant enhanced cytotoxicity through the synergy of photodynamic and photothermal therapies. More importantly, in vivo experiments with BSL NPS using HepG2 tumor-bearing nude mice model demonstrated good biosafety, enrichment at tumors, and effective destruction of tumors. This work provides a good example of constructing carrier-free multifunctional NPSs based on one single glycosylated amphiphilic small molecule for dual phototherapy against cancers, which may eventually contribute to bridging the translational gap to the clinic.

Tetrastigma hemsleyanum flavones exert antihepatic carcinoma property both in vitro and in vivo

Abstract Tetrastigma hemsleyanum (T. hemsleyanum) has been regarded as an anticancer food in China. However, its corresponding mechanisms remains unclear. Thus, in this study, the antitumor activity of flavones-rich fraction of root of T. hemsleyanum (FRTH) was investigated in vitro and in vivo. The results indicated that FRTH could inhibit the proliferation and migration of human hepatoellular carcinomas (HepG2) cells in vitro by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. FRTH could increase the level of reactive oxygen species and change the mitochondrial membrane potential in HepG2 cells. In addition, FRTH treatment (300 mg/kg and 600 mg/kg, body weight) significantly suppressed tumor growth on HepG2 tumor-bearing nude mice. Besides, immunohistochemistry assays and western blotting revealed that FRTH enhanced the expression level of Bcl-2 associated X protein/B-cell lymphoma-2 (Bax/Bcl-2), cytochrome C, caspase-3, caspase-9, and cleaved-caspase-3, and downregulated the expression level of platelet endothelial cell adhesion molecule-1 (CD31), ki67, and vascular endothelial growth factor (VEGF) factor in HepG2 tumor-bearing mice. Our study suggests T. hemsleyanum as a kind of promising candidate medicine for liver cancer treatment.

Investigating the Antitumor Activities of Curcumae Rhizoma by Building HepG2 Tumor-bearing Nude Mice Models and Exploring its Anticancer Mechanism by Observing Glycoprotein Expression of Tumor Tissues Using Lectin Microarray Technology

Background: Curcumae Rhizoma (CR) comes from Curcuma genus, functional breaking blood stasis, detumescence and acesodyne for treatment of Zhengjia accumulation, amenorrhea, traumatic injury and bruising pain. Modern pharmacological studies have shown that the main monomer compositions of Curcumae Rhizoma, such as curcumol, β-elemene, curcumin, have good anti-tumor effects. However, the mechanisms are not clear yet. Previous studies have revealed that the associated glycoprotein showed significant differences with normal people after the development of hepatocellular carcinoma (HCC). Methods: In this study, the anticancer activities of CR extract were investigated by constructing HepG2 tumor bearing nude mice models. Furthermore, glycan profiles of tumor tissues were thoroughly characterized by lectin microarrays, a high-throughput technique, in an effort to explore the anticancer mechanism of Curcumae Rhizoma. Results: It is indicated that CR extract might inhibit the tumor proliferation in tumor-bearing model and the potential mechanisms might be CR treatment altered protein glycosylation of tumor cell, which plays an important role in the pathogenesis and progression of HCC. In detail, fucosylation (identified by PSA and UEA-I), bisecting GlcNAc or multianternnary (identified by PHA-E+L) and terminal GalNAc (identified by BPL) decreased, while sialylation (identified by WGA and SNA), high-mannose (identified by ConA) and T-antigen/TN-antigen/sialyl-T antigen (identified by ACA) increased in CR treatment group compared to model group. Similar phenomenon also occurred in two positive groups, western medicine cyclophosphamide (CTX) and Chinese medicine monomer β-elemene administration groups, especially in β-elemene administration one. The liver contains various receptors on sinusoidal and hepatocyte surfaces, and many proteins that bind to these receptors reply on carbohydrate moieties during the development of HCC. Conclusion: In this point of view, a search for the biological significance of glycosylation expression and its function after Chinese Medicine administration in HCC may open a new direction in glycobiology.

ROS triggered cleavage of thioketal moiety to dissociate prodrug nanoparticles for chemotherapy

With the utilization of high concentration reactive oxygen species (ROS) in tumor microenvironment, PEG-doxorubicin (PEG-DOX) prodrug was synthesized via a thioketal moiety as the linker, which was ROS cleavable to trigger DOX release from the self-assembled prodrug nanoparticles. The in vitro ROS sensitivity of prodrug nanoparticles (NPs) was investigated in Fenton agent and H2O2, and the disassembly of NPs was more sensitive to Fenton reagent. After internalized in HepG2 cells via endocytosis, the cellular ROS consuming test revealed intracellular DOX release. The pharmacokinetics and biodistribution study demonstrated that the in vivo elimination of NPs was significantly improved and the NPs were passively targeted to tumor tissues via EPR effect. The ROS-responsive prodrug NPs exhibited excellent antitumor activity in HepG2 tumor-bearing nude mice, remarkably induced tumor cells apoptosis and reduced the systemic toxicity of DOX. Our study revealed the ROS responsive prodrug nanoparticle is an effective strategy to fabricate nanomedicine for cancer chemotherapy.

Enhancing Anti-Tumor Activity of Sorafenib Mesoporous Silica Nanomatrix in Metastatic Breast Tumor and Hepatocellular Carcinoma via the Co-Administration with Flufenamic Acid.

IntroductionBecause tumor-associated inflammation is a hallmark of cancer treatment, in the present study, sorafenib mesoporous silica nanomatrix (MSNM@SFN) co-administrated with flufenamic acid (FFA, a non-steroidal anti–inflammatory drug (NSAID)) was investigated to enhance the anti-tumor activity of MSNM@SFN.MethodsMetastatic breast tumor 4T1/luc cells and hepatocellular carcinoma HepG2 cells were selected as cell models. The effects of FFA in vitro on cell migration, PGE2 secretion, and AKR1C1 and AKR1C3 levels in 4T1/luc and HepG2 cells were investigated. The in vivo anti-tumor activity of MSNM@SFN co-administrating with FFA (MSNM@SFN+FFA) was evaluated in a 4T1/luc metastatic tumor model, HepG2 tumor-bearing nude mice model, and HepG2 orthotopic tumor-bearing nude mice model, respectively.ResultsThe results indicated that FFA could markedly decrease cell migration, PGE2 secretion, and AKR1C1 and AKR1C3 levels in both 4T1/luc and HepG2 cells. The enhanced anti-tumor activity of MSNM@SFN+FFA compared with that of MSNM@SFN was confirmed in the 4T1/luc metastatic tumor model, HepG2 tumor-bearing nude mice model, and HepG2 orthotopic tumor-bearing nude mice model in vivo, respectively.DiscussionMSNM@SFN co-administrating with FFA (MSNM@SFN+FFA) developed in this study is an alternative strategy for improving the therapeutic efficacy of MSNM@SFN via co-administration with NSAIDs.

Improved Stability and Enhanced Anti-Tumor Activity of Hyaluronic Acid Modified ES2-AF Nanoparticle-Like Conjugate.

The growth, migration and spread of malignant tumors requires a large number of new blood vessels to supply nutrients. In our previous study, it was found that the hyaluronic acid (HA) modified novel antiangiogenic peptide ES2-AF has better solubility, longer half-life, stronger targeting than non-modified ES2-AF, and it significantly inhibited the formation of new blood vessels. In the current work, we studied the stability of ES2-AF after HA modification, including heat stability, longterm stability, and pH stability. After treatment with HA-ES2-AF, cell apoptosis and the cell cycle were analyzed using flow cytometry. The inhibitory effect of HA-ES2-AF on tumors was investigated in vivo in HepG2 tumor-bearing nude mice. All the above results showed that HA-ES2-AF significantly improved stability, effectively induced apoptosis of endothelial cells, caused G1 phase arrest of endothelial cells, and decreased the percentage of cells in G2 and S phases. In vivo, HA-ES2-AF exhibited an inhibitory effect on tumors growth. In addition, the results provide a reliable basis for studying the inhibition of neovascularization and anti-tumor drugs in the future.

Synthesis of theranostic epithelial cell adhesion molecule targeted mesoporous silica nanoparticle with gold gatekeeper for hepatocellular carcinoma.

In this study, we report the fabrication of epithelial cell adhesion molecule targeted 5-fluorouracil (5-FU) encapsulated PEGylated mesoporous silica nanoparticles (NPs) hybridized with gold NPs (PEG-Au@Si-5-FU) as gatekeeper for theranostic applications. The prepared targeted and nontargeted formulations were evaluated in vitro in terms of their cellular internalization and toxicity. The prepared theranostic hybrid system was also implemented for computed tomography of HepG2 tumor-bearing nude mice in vivo. Fluorescence microscopy and MTT assay demonstrated that the developed epithelial cell adhesion molecule-PEG-Au@Si-5-FU had higher cytotoxicity than nontargeted PEG-Au@Si-5-FU in 2D and 3D HepG2 cell cultures. Moreover, the targeted hybrid system was preferentially accumulated in HepG2 tumor cells in vitro and in vivo. This work introduces a novel strategy for developing multimodal NPs via nanoparticulate hybrid materials.

Investigation of the anti-cancer effect of quercetin on HepG2 cells in vivo.

Quercetin, a natural polyphenolic flavonoid compound, can inhibit the growth of several malignant cancers. However, the mechanism still remains unclear. Our previous findings have suggested that quercetin can significantly inhibit HepG2 cell proliferation and induce cell apoptosis in vitro. It can also affect cell cycle distribution and significantly decrease cyclin D1 expression. In this study, we investigated the anti-cancer effect of quercetin on HepG2 tumor-bearing nude mice and its effect on cyclin D1 expression in the tumor tissue. First, the nude murine tumor model was established by subcutaneous inoculation of HepG2 cells, then quercetin was administered intraperitoneally, and the mice injected with saline solution were used as controls. The daily behavior of the tumor-bearing mice was observed and differences in tumor growth and survival rate were monitored. The expression of cyclin D1 in isolated tumor sections was evaluated by immunohistochemistry. We found that HepG2 tumor became palpable in the mice one-week post-inoculation. Tumors in the control group grew rapidly and the daily behavior of the mice changed significantly, including listlessness, poor feeding and ataxia. The mice in quercetin-treated group showed delayed tumor growth, no significant changes in daily behavior, and the survival rate was significantly improved. Finally, we observed increased tumor necrosis and a lighter cyclin D1 staining with reduced staining areas. Our findings thus suggest that quercetin can significantly inhibit HepG2 cell proliferation, and this effect may be achieved through the regulation of cyclin D1 expression.

A tenascin C targeted nanoliposome with navitoclax for specifically eradicating of cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) play a vitally important role during tumor progression. Navitoclax (Nav) can specifically induce apoptosis in CAFs. The present study aims to develop a novel CAF-targeted nanoliposome for cancer therapy. Nav-loaded nanoliposomes modified with peptide FH (FH-SSL-Nav), which specifically binds to tenascin C, a protein mainly expressed by CAFs, were formulated and characterized. Several experiments were performed to evaluate CAFs selective apoptosis, targeting and eradicating. Compared with SSL-Nav, FH-SSL-Nav achieved higher cellular uptake, and exhibited stronger cytotoxicity in vitro. The in vivo tumor stroma targeting effect was further confirmed by near infrared imaging. Accordingly, FH-SSL-Nav displayed improved tumor growth inhibition by eradicating CAFs in Hep G2 tumor-bearing nude mice model. In conclusion, FH-SSL-Nav could achieve targeting delivery of Nav to CAFs in vitro and in vivo, and may offer a potential strategy for cancer therapy based on tumor stroma. From the Clinical EditorThe progression of cancer cells often depends on the underlying tumor microenvironment, in which cancer-associated fibroblasts play an important role. In this article, the authors developed targeted therapy against CAFs using liposomes as carriers. This new modality was shown to be more effective in tumor killing both in vitro and in vivo. The finding may open a new era in cancer therapy.

Core-shell nanoparticles based on pullulan and poly(β-amino) ester for hepatoma-targeted codelivery of gene and chemotherapy agent.

This study designs a novel nanoparticle system with core-shell structure based on pullulan and poly(β-amino) ester (PBAE) for the hepatoma-targeted codelivery of gene and chemotherapy agent. Plasmid DNA expressing green fluorescent protein (pEGFP), as a model gene, was fully condensed with cationic PBAE to form the inner core of PBAE/pEGFP polycomplex. Methotrexate (MTX), as a model chemotherapy agent, was conjugated to pullulan by ester bond to synthesize polymeric prodrug of MTX-PL. MTX-PL was then adsorbed on the surface of PBAE/pEGFP polycomplex to form MTX-PL/PBAE/pEGFP nanoparticles with a classic core-shell structure. MTX-PL was also used as a hepatoma targeting moiety, because of its specific binding affinity for asialoglycoprotein receptor (ASGPR) overexpressed by human hepatoma HepG2 cells. MTX-PL/PBAE/pEGFP nanoparticles realized the efficient transfection of pEGFP in HepG2 cells and exhibited significant inhibitory effect on the cell proliferation. In HepG2 tumor-bearing nude mice, MTX-PL/PBAE/pEGFP nanoparticles were mainly distributed in the tumor after 24 h postintravenous injection. Altogether, this novel codelivery system with a strong hepatoma-targeting property achieved simultaneous delivery of gene and chemotherapy agent into tumor at both cellular and animal levels.

Abstract 3569: Photodynamic activity of the glucoconjugated silicon(IV) phthalocyanine SiPcGlu

Abstract Photodynamic therapy involves the use of a non-toxic photosensitizer which exhibits killing effect upon activation by light. It has been used successfully in treating age-related macular degeneration, actinic keratosis and various cancers like lung, bladder and esophageal cancers. The success of photodynamic therapy depends on the properties of the photosensitizer and there is a continuous search for better photosensitizers with desirable photophysical and biochemical effects. In the last few years, we have synthesized a series of silicon(IV) phthalocyanines with different axial substituents and demonstrated their photocytotoxic effects in the in vitro heptocellular carcinoma HepG2 cells. Among them the glucoconjugated silicon(IV) phthalocyanine SiPcGlu is most promising with IC50 value down to nanomolar range in the cell culture system. In the present investigation, the in vivo effect of SiPcGlu was studied in the HepG2 tumor-bearing nude mice model. The biodistribution of SiPcGlu was followed to evaluate its tumor-selectivity and the tumor volume was measured after photodynamic therapy. The level of intrinsic toxicity induced was also investigated by examining the level of plasma enzyme markers. The distribution and clearance results showed that the concentration of SiPcGlu in the tumor was relatively higher at 24 hr post-injection when compared with all the other tissues examined. One percent of initial dose per gram tissue could be found in the tumor while the corresponding values for the other tissues ranged from 0.8% (lung) to 2.5% (skin). Thus, a drug-light interval of 24 hr was selected in the photodynamic therapy experiment. A laser light dose of 30 J/cm2 (bandwidth 675 ± 3 nm) was spotted onto the tumor 24 hr after intravenous injection of the drug (one μmol per kg body weight). Significant tumor regression was observed for the experimental group of mice. Fifteen days after photodynamic treatment, the volume of the tumor was less than 20% of the initial volume while mice in the two control groups, viz. dark control in which the mice received drug but no irradiation and the light control group in which the mice received irradiation but no drug, did not show any tumor regression. The photodynamic treatment did not cause any apparent toxicity to the animals as the plasma levels of the three enzyme markers, alanine transaminase, aspartate transaminase and creatine kinase, were similar in all the three groups of mice. To conclude, the present results suggest that SiPcGlu is a good candidate for further evaluation on its anti-tumor potential. (This project is supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, Project No. CUHK 4569/05M.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3569.

The Retrovirus-mediated Antisense Human Telomerase RNA (hTR) Gene Limits the Growth of Hepatocellular Carcinoma Growth in Cell Culture and Animals

To investigate the inhibitory effect of retrovirus-mediated antisense human telomerase RNA (hTR) gene therapy on hepatocelluar carcinoma. We first constructed the sense and antisense hTR vectors and then transfected these into HepG2 cells. Telomerase activity, cell growth curves, proliferating cell nuclear antigen expression (PCNA), cell cycle distribution, and cell apoptosis were detected by the means of telemere repeat amplification protocol (TRAP), MTT assay, immunofluorescence, flow cytometric analysis, and transferase-mediated nick end labeling (TUNEL), respectively. In order to further confirm the therapeutic effect of this gene therapy, we developed an experimental line of HepG2 tumor-bearing nude mice by and directly injected these with retrovirus expressing the antisense hTR gene. Tumor growth was determined by tumor volume, and cell apoptosis was analyzed by TUNEL. The antisense hTR gene was shown to be successfully integrated into the target cells' genome. HepG2 cells transfected with the antisense hTR gene showed down-regulated telomerase activity, inhibited cell growth, decreased PCNA expression, and increased apoptotic rate. Moreover, flow cytometry revealed a decrease of cells in the S phase with cell cycle arrest at the G2/M phase. In the antisense hTR-treated group, tumor growth was significantly reduced and showed an increase of apoptotic cells. The results indicate that the specific inhibitor of the hTR template is likely to be a very efficient tool for hepatocellular carcinoma research and may possess potential therapeutic significance in the future clinical practice.

Norcantharidin inhibits growth of human HepG2 cell-transplanted tumor in nude mice and prolongs host survival.

In this study, norcantharidin was compared with adriamycin and mitomycin C for its inhibitory action in the growth of cultured human hepatocellular carcinoma HepG2 cells. The IC50 of adriamycin and mitomycin C on HepG2 cells was 7.3 microM and 27 microM, respectively, whereas the IC50 of norcantharidin for inhibiting the growth of HepG2 cells was as high as 1900 microM. After HepG2 tumor-bearing nude mice were treated with 12 daily intraperitoneal injections of norcantharidin (2 mg/kg), the increase in tumor size was significantly slower than that of untreated controls. The mean survival time of untreated tumor-bearing nude mice was 129 days, whereas in the tumor-bearing nude mice treated with norcantharidin, the mean survival time was significantly prolonged to 194 days (P < 0.0001). It is concluded that norcantharidin may have a potential role in the treatment of human hepatocellular carcinoma.