Liver Cancer Xenograft Model Research Articles

Anti-cancer Activity of Novel TM4SF5-Targeting Antibodies through TM4SF5 Neutralization and Immune Cell-Mediated Cytotoxicity.

The transmembrane four L6 family member 5 (TM4SF5) protein is a novel molecular target for the prevention and treatment of hepatocellular carcinoma. TM4SF5 is highly expressed in liver, colon, esophageal, and pancreatic cancers and is implicated in tumor progression. Here, we screened monoclonal antibodies that specifically bound to the extracellular loop 2 (EC2) of TM4SF5 from a phage-displayed murine antibody (single-chain variable fragment; scFv) library. We constructed and characterized chimeric antibodies, Ab27 and Ab79, of scFv fused with Fc domain of human IgG1. The affinity (KD) of Ab27 and Ab79 for soluble EC2 was approximately 9.2 nM and 16.9 nM, respectively, as determined by surface plasmon resonance analysis. Ab27 and Ab79 efficiently bound to native TM4SF5 on the cell surface were internalized into the cancer cells, leading to a decrease in cell surface TM4SF5. Ab27 and Ab79 inhibited the proliferation and invasion of TM4SF5-positive liver and colon cancer cells and reduced FAK and c-Src phosphorylation. Ab27 and Ab79 also enhanced anoikis sensitivity and reduced survivin. Ab27 mediated antibody-dependent cell-mediated cytotoxicity in vitro. Ab27 and Ab79 efficiently inhibited tumor growth in a liver cancer xenograft model. These results strongly support the further development of Ab27 as a novel anti-cancer agent in the clinic.

Combination therapy with doxorubicin-loaded galactosylated poly(ethyleneglycol)-lithocholic acid to suppress the tumor growth in an orthotopic mouse model of liver cancer.

Despite advances in technology, neither conventional anti-cancer drugs nor current nanoparticle (NP) drugs have gained substantial success in cancer treatment. While conventional chemotherapy drugs have several limitations such as low potency, poor invivo stability and limited bioavailability, non-specific targeting of NP drugs diminishes their potency at actual target sites. In addition, the development of drug resistance to anti-cancer drugs is another challenging problem. To overcome these limitations, we aimed to develop a polymer-drug conjugate, which functions as an active NP drug and drug carrier both, to deliver a chemotherapeutic drug for combination therapy. Accordingly, we made targeting NP carrier of lithocholic acid-poly(ethylene glycol)-lactobionic acid (LPL) loading doxorubicin (Dox) to produce Dox/LPL NPs. The cellular uptake of Dox/LPL NPs was relatively higher in human liver cancer cell line (SK-HEP-1) due to galactose ligand-asialoglycoprotein receptor interaction. Consequently, the cellular uptake of Dox/LPL NPs led to massive cell death of SK-HEP-1cells by two different mechanisms, particularly apoptotic activity by LPL and mitotic catastrophe by Dox. Most importantly, Dox/LPL NPs, when administered to orthotopic xenograft model of liver cancer, greatly reduced proliferation, invasion, migration, and angiogenesis of liver tumor invivo. Thus, this study exemplifies the superiority of combination therapy over individual NP drug or conventional small molecule drug for cancer therapy. Overall, we present a promising approach of combinatorial therapy to inhibit the hepatic tumor growth and metastasis in the orthotopic xenograft model mice, thus representing an effective weapon for cancer treatment.

Melittin-MIL-2 fusion protein as a candidate for cancer immunotherapy.

BackgroundCytokine fusion protein that modulates the immune response holds great potential for cancer immunotherapy. IL-2 is an effective treatment against advanced cancers. However, the therapeutic efficacy of IL-2 is limited by severe systemic toxicity. Several mutants recombinant IL-2 can increase antitumor activity and minimize systemic toxicity. Melittin is an attractive anticancer candidate because of its wide-spectrum lytic properties. We previously generated a bifunctional fusion protein melittin-MIL-2, composed of melittin and a mutant IL-2. The melittin-MIL-2 inhibited the growth of human ovarian cancer SKOV3 cells in vitro and in vivo tumor growth. However, whether this antitumor effect could also be used in cancer immunotherapy was unknown. To assess its cancer immunotherapy potential, we further investigated its more effective antitumor immune response and antitumor effect against cancers of different tissue origins in vitro and in vivo.MethodsThe specific IL-2 activity of the melittin-MIL-2 fusion protein was tested on the cytokine growth dependent cell line CTLL-2. The cytolytic activity was detected by standard 4-h 51Cr-release assays. PBMC stimulation in response to the melittin-MIL-2 was determined by IFN-γ release assay. We observed the cancer cell proliferation of different tissue origins by MTT assay. The ability of melittin-MIL-2 to inhibit tumor growth in vivo was evaluated by using human liver (SMMC-7721 cancer cells), lung (A549 cancer cells) and ovarian (SKOV3 cancer cells) cancer xenograft models. To assess the immunity within the tumor microenvironment, the level of some cytokines including IFN-γ, TNF-α, IL-12 and IL-4 was analyzed by ELISA. We injected the MDA-MB-231 cells and the melittin-MIL-2 into mice, and the anti-metastatic effect was examined by counting nodules in the lung.ResultsThe melittin-MIL-2 was more effective in inducing T cell and NK-cell cytotoxicity. The fusion protein significantly increased IFN-γ production in PBMCs. In vitro, the melittin-MIL-2 mediated immune cells killing or directly killed the cancer cell lines of different tissue origins. In vivo, the fusion protein exhibited stronger inhibition on the growth of transplanted human tumors compared to rIL-2. The melittin-MIL-2 treatment promoted the IFN-γ secretion in tumor tissues and decreased the immunosuppressive cells in vivo. Furthermore, the fusion protein reduced lung metastasis of breast cancer.ConclusionsThis study provides the evidence that the melittin-MIL-2 can produce stronger immune stimulation and antitumor effects, and the fusion protein is a potent candidate for cancer immunotherapy.

Efficient and Targeted Transduction of Nonhuman Primate Liver With Systemically Delivered Optimized AAV3B Vectors

Recombinant adeno-associated virus serotype 3B (rAAV3B) can transduce cultured human liver cancer cells and primary human hepatocytes efficiently. Serine (S)- and threonine (T)-directed capsid modifications further augment its transduction efficiency. Systemically delivered capsid-optimized rAAV3B vectors can specifically target cancer cells in a human liver cancer xenograft model, suggesting their potential use for human liver-directed gene therapy. Here, we compared transduction efficiencies of AAV3B and AAV8 vectors in cultured primary human hepatocytes and cancer cells as well as in human and mouse hepatocytes in a human liver xenograft NSG-PiZ mouse model. We also examined the safety and transduction efficacy of wild-type (WT) and capsid-optimized rAAV3B in the livers of nonhuman primates (NHPs). Intravenously delivered S663V+T492V (ST)-modified self-complementary (sc) AAV3B-EGFP vectors led to liver-targeted robust enhanced green fluorescence protein (EGFP) expression in NHPs without apparent hepatotoxicity. Intravenous injections of both WT and ST-modified rAAV3B.ST-rhCG vectors also generated stable super-physiological levels of rhesus chorionic gonadotropin (rhCG) in NHPs. The vector genome predominantly targeted the liver. Clinical chemistry and histopathology examinations showed no apparent vector-related toxicity. Our studies should be important and informative for clinical development of optimized AAV3B vectors for human liver-directed gene therapy.

Chloroquine inhibits hepatocellular carcinoma cell growth in vitro and in vivo.

Recently, chloroquine (CQ) has been widely used to improve the efficacy of different chemotherapy drugs to treat tumors. However, the effects of single treatment of CQ on liver cancer have not been investigated. In the present study, we examined the effects of CQ on the growth and viability of liver cancer cells in vitro and in vivo, and revealed that CQ treatment triggered G0/G1 cell cycle arrest, induced DNA damage and apoptosis in a dose- and time-dependent manner in liver cancer cells. Moreover, administration of CQ to tumor-bearing mice suppressed the tumor growth in an orthotopic xenograft model of liver cancer. These findings extend our understanding and suggest that CQ could be repositioned as a treatment option for liver cancer as a single treatment or in combination.

Targeted expression of BikDD combined with metronomic doxorubicin induces synergistic antitumor effect through Bax activation in hepatocellular carcinoma.

Conventional chemotherapy is commonly used to treat advanced non-resectable hepatocellular carcinoma (HCC) but this treatment modality has not demonstrated convincing survival benefit in HCC patients. Our previous studies indicated that targeted expression of therapeutic BikDD driven by a liver cancer-specific α-fetoprotein promoter/enhancer (eAFP) in the VISA backbone (eAFP-VISA-BikDD) significantly and specifically kills HCC cells in multiple orthotopic animal models. To enhance its therapeutic efficacy, we combined eAFP-VISA-BikDD with chemotherapeutic agents and found that eAFP-VISA-BikDD plus doxorubicin (Dox) or 5-fluorouracil (5-FU) demonstrated synergistic cytotoxicity in HCC cells. Specifically, the combination of eAFP-VISA-BikDD plus Dox markedly induced apoptosis via increased Bax mitochondrial translocation and cytoplasmic cytochrome c release. Compared with either agent alone, a low dose of Dox combined with eAFP-VISA-BikDD induced better antitumor effect and prolonged longer survival of mice in two orthotopic liver cancer xenograft models. Our findings provide strong preclinical support for evaluating the combined therapy of eAFP-VISA-BikDD and Dox in a clinical setting as a treatment option for HCC.

Selective in vivo targeting of human liver tumors by optimized AAV3 vectors in a murine xenograft model.

Current challenges for recombinant adeno-associated virus (rAAV) vector-based cancer treatment include the low efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to liver damage. Our research provides a novel means to achieve not only targeted delivery but also the potential for gene therapy of human liver cancer.

Tetrandrine induces G1/S cell cycle arrest through the ROS/Akt pathway in EOMA cells and inhibits angiogenesis in vivo.

Tetrandrine, a bisbenzylisoquinoline alkaloid, is known to inhibit tumor cell proliferation and induce apoptosis in cancer models invitro and invivo. In the present study, tetrandrine significantly inhibited the proliferation of mouse endothelial cells (EOMA cell) and induced G1/S arrest in EOMA cells, in which the expressions of cyclinD and cyclinE and CDKs were downregulated. Tetrandrine treatment also caused intracellular accumulation of reactive oxygen species (ROS). Pretreatment with NAC, which is a ROS inhibitor, blocked G1/S cell arrest and cyclin regulation induced by tetrandrine, implying that ROS generation plays an important role in tetrandrine-induced cell cycle arrest. Furthermore, a decreased phospho-Akt protein level after tetrandrine treatment was reversible with the removal of the intracellular ROS by NAC. Notably, overexpression of Akt decreased tetrandrine-induced G1/S arrest. Finally, we verified the antiangiogenic effects of tetrandrine invivo in a liver cancer xenograft model in nude mice. In conclusion, tetrandrine inhibits EOMA cell growth through the ROS/Akt pathway, and it could be a promising compound for cancer therapy as an inhibitor of tumor vascular growth.

The isothiocyanate erucin abrogates telomerase in hepatocellular carcinoma cells in vitro and in an orthotopic xenograft tumour model of HCC.

In contrast to cancer cells, most normal human cells have no or low telomerase levels which makes it an attractive target for anti-cancer drugs. The small molecule sulforaphane from broccoli is known for its cancer therapeutic potential in vitro and in vivo. In animals and humans it was found to be quickly metabolized into 4-methylthiobutyl isothiocyanate (MTBITC, erucin) which we recently identified as strong selective apoptosis inducer in hepatocellular carcinoma (HCC) cells. Here, we investigated the relevance of telomerase abrogation for cytotoxic efficacy of MTBITC against HCC. The drug was effective against telomerase, independent from TP53 and MTBITC also blocked telomerase in chemoresistant subpopulations. By using an orthotopic human liver cancer xenograft model, we give first evidence that MTBITC at 50 mg/KG b.w./d significantly decreased telomerase activity in vivo without affecting enzyme activity of adjacent normal tissue. Upon drug exposure, telomerase decrease was consistent with a dose-dependent switch to anti-survival, cell arrest and apoptosis in our in vitro HCC models. Blocking telomerase by the specific inhibitor TMPyP4 further sensitized cancer cells to MTBITC-mediated cytotoxicity. Overexpression of hTERT, but not enzyme activity deficient DNhTERT, protected against apoptosis; neither DNA damage nor cytostasis induction by MTBITC was prevented by hTERT overexpression. These findings imply that telomerase enzyme activity does not protect against MTBITC-induced DNA damage but impacts signalling processes upstream of apoptosis execution level.

Circadian clock gene expression regulates cancer cell growth through glutaminase

Glutamine is an essential amino acid for malignant tumor cells. Glutaminase that metabolizes glutamine reaches a maximum expression in tumors immediately before the maximum proliferation rate. Tumor cells grow at different rates during the day. We postulated that the activity of glutaminase in tumor cells is subject to the regulation of circadian clock gene. We measured glutaminase by western blot analysis and circadian clock gene expression by real-time polymerase chain reaction in the liver and tumor cells at six equispaced time points of the day in individual mice of a 12/12 h light/dark schedule. The results showed that the tumor-bearing mice, under normal diurnal conditions, are circadianly entrained, as reflected by the normal host locomotor activity rhythms and rhythmic liver clock gene expression. The tumors within these mice are also circadianly organized, as reflected by circadian clock gene (Bmal1) expression. What is most remarkable is that kidney-type glutaminase also showed circadian rhythms in the same pattern with tumor circadian clock gene expression in liver cancer xenograft model, indicating that conditionally inhibiting glutaminase activity may provide a new target for cancer therapy.

Targeting GRP75 Improves HSP90 Inhibitor Efficacy by Enhancing p53-Mediated Apoptosis in Hepatocellular Carcinoma

Heat shock protein 90 (HSP90) inhibitors are potential drugs for cancer therapy. The inhibition of HSP90 on cancer cell growth largely through degrading client proteins, like Akt and p53, therefore, triggering cancer cell apoptosis. Here, we show that the HSP90 inhibitor 17-AAG can induce the expression of GRP75, a member of heat shock protein 70 (HSP70) family, which, in turn, attenuates the anti-growth effect of HSP90 inhibition on cancer cells. Additionally, 17-AAG enhanced binding of GRP75 and p53, resulting in the retention of p53 in the cytoplasm. Blocking GRP75 with its inhibitor MKT-077 potentiated the anti-tumor effects of 17-AAG by disrupting the formation of GRP75-p53 complexes, thereby facilitating translocation of p53 into the nuclei and leading to the induction of apoptosis-related genes. Finally, dual inhibition of HSP90 and GRP75 was found to significantly inhibit tumor growth in a liver cancer xenograft model. In conclusion, the GRP75 inhibitor MKT-077 enhances 17-AAG-induced apoptosis in HCCs and increases p53-mediated inhibition of tumor growth in vivo. Dual targeting of GRP75 and HSP90 may be a useful strategy for the treatment of HCCs.

Thermosensitive hydrogels based on polypeptides for localized and sustained delivery of anticancer drugs

Thermosensitive hydrogels based on poly(γ-ethyl-l-glutamate)-poly(ethylene glycol)-poly(γ-ethyl-l-glutamate) triblock copolymers (PELG-PEG-PELG) were prepared for localized and sustained delivery of anticancer drugs. The polypeptide-based hydrogels showed much lower critical gelation concentration than the traditional polyester-based hydrogels. In vivo biocompatibility studies revealed that the in situ formed gels in the subcutaneous layer last for ∼21 days, and H&E staining study suggested acceptable biocompatibility of our materials in vivo. Then the hydrogels were tried as injectable implants to encapsulate antitumor drug, paclitaxel (PTX), to assess the in situ anti-tumoral activity using liver cancer xenograft model. The results demonstrated that the PTX-incorporated hydrogels could efficiently suppress the tumor growth, and did not result in obvious damage to normal organs. Therefore, the polypeptide-based thermosensitive hydrogels designed in the present study have great potential to serve as an effective platform for localized anti-cancer drug delivery.

Abstract 5086: The side effects of sorafenib in treating hepatocellular carcinoma.

Abstract Sorafenib, a tyrosine kinase inhibitor of VEGFR and B-Raf, is a standard treatment for advanced hepatocellular carcinoma (HCC). However, there are several clinical observations showing discontinuous sorafenib treatment associated with more metastasis or rapid relapse. We tested effect of sorafenib on tumor growth and metastasis in different dosing and schedules in liver cancer allograft or xenograft models. The results showed a lower dose sorafenib treatment will induce more intrahepatic or lung metastasis while the primary tumor growth is still inhibited in HCC with a higher expression of HTATIP2 but not in HCC with a lower expression of HTATIP2. Further studies showed sorafenib decreased HTATIP2 expression only in HCC with a higher expression of HTATIP2. The clinical data also supported the patients with lower HTATIP2 expression in tumor benefit from sorafenib treatment while patients with a higher HTATIP2 expression in tumor did not. We also observed the effect of sorafenib on host (mouse) immunity after we found pre-treatment by sorafenib before implantation of tumor resulted in more lung metastasis. We found interleukin 12 (IL12) was significantly inhibited by sorafenib, which is responsible for more lung metastasis. In conclusion, our study showed the side effect of sorafenib may promote tumor progression if sorafenib treatment is stopped or not given in a full dose. These findings may help to improve the efficacy of sorafenib by overcoming its side effects. Citation Format: Hui-Chuan Sun, Wei Zhang, Xiao-Dong Zhu, Lu Lu, Qiang-Bo Zhang, Zhao-You Tang. The side effects of sorafenib in treating hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5086. doi:10.1158/1538-7445.AM2013-5086 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

AL3810, a multi‐tyrosine kinase inhibitor, exhibits potent anti‐angiogenic and anti‐tumour activity via targeting VEGFR, FGFR and PDGFR

Angiogenesis plays an important role in neoplastic transformation and progression as well as in the metastasis process of most human cancers. Herein, we identified AL3810 as a novel and orally bioavailable small molecular inhibitor with potent inhibitory activity against multiple tyrosine kinases involved in the process of angiogenesis. We found that AL3810 substantially inhibited the autophosphorylation of VEGFR2, PDGFRβ and FGFR1 in endothelial cells. Moreover, AL3810 exhibited potent anti-angiogenesis activity, manifested by significant inhibition of microvessel outgrowth of rat arterial ring and chickallantochorion membrane (CAM) in ex vivo angiogenesis models. Daily dosing of AL3810 has shown broad-spectrum anti-tumour activity in human kidney, pancreas, liver cancer xenograft models. Importantly, immunohistochemistry results demonstrated that the anti-tumour activity of AL3810 was closely correlated with its anti-angiogenesis activity, as demonstrated by a decreased microvessel area and reduced microvessel numbers in tumour tissues. The overall pharmacological profiles of AL3810 are superior to sorafenib. The clinical trials of AL3810 will soon be launched in China.

Multiple Target-Specific Molecular Imaging Agents Detect Liver Cancer in a Preclinical Model

Liver cancer is the fifth most common cause of cancer deaths worldwide. Noninvasive diagnosis is difficult and the disease heterogeneity reduces the accuracy of pathological assays. Improvement in diagnostic imaging of specific molecular disease markers has provided hope for accurate and early noninvasive detection of liver cancer. However, all current imaging technologies, including ultrasonography, computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging, are not specific targets for detection of liver cancer. The aim of this study was to test the feasibility of injecting a cocktail of specific molecular imaging agents to noninvasively image liver cancer. The target-specific cocktail contained agents for imaging the neovasculature (RGD peptide), matrix metalloproteinase (MMP), and glucose transport (18F-fluorodeoxyglucose [18F-FDG]). Imaging studies were performed in liver cancer cells and xenograft models. The distribution of MMP at the intracellular level was imaged by confocal microscopy. RGD, MMP, and 18F-FDG were imaged on tumor-bearing mice using PET, CT, X-ray, and multi-wavelength optical imaging modalities. Image data demonstrated that each agent bound to a specific disease target component. The same liver cancer xenograft contained multiple disease markers. Those disease markers were heterogenetically distributed in the same tumor nodule. The molecular imaging agents had different distributions in the whole body and inside the tumor nodule. All target-specific agents yielded high tumor-to-background ratios after injection. In conclusion, target-specific molecular imaging agents can be used to study liver cancer in vitro and in vivo. Noninvasive multimodal/multi-target-specific molecular imaging agents could provide tools to simultaneously study multiple liver cancer components.

The Nedd8-Activating Enzyme Inhibitor MLN4924 Induces Autophagy and Apoptosis to Suppress Liver Cancer Cell Growth

Posttranslational neddylation of cullins in the Cullin-Ring E3 ligase (CRL) complexes is needed for proteolytic degradation of CRL substrates, whose accumulation induces cell-cycle arrest, apoptosis, and senescence. The Nedd8-activating enzyme (NAE) is critical for neddylation of CRL complexes and their growth-promoting function. Recently, the anticancer small molecule MLN4924 currently in phase I trials was determined to be an inhibitor of NAE that blocks cullin neddylation and inactivates CRL, triggering an accumulation of CRL substrates that trigger cell-cycle arrest, apoptosis, and senescence in cancer cells. Here, we report that MLN4924 also triggers autophagy in response to CRL inactivation and that this effect is important for the ability of MLN4924 to suppress the outgrowth of liver cancer cells in vitro and in vivo. MLN4924-induced autophagy was attributed partially to inhibition of mTOR activity, due to accumulation of the mTOR inhibitory protein Deptor, as well as to induction of reactive oxygen species stress. Inhibiting autophagy enhanced MLN4924-induced apoptosis, suggesting that autophagy is a survival signal triggered in response to CRL inactivation. In a xenograft model of human liver cancer, MLN4924 was well-tolerated and displayed a significant antitumor effect characterized by CRL inactivation and induction of autophagy and apoptosis in liver cancer cells. Together, our findings support the clinical investigation of MLN4924 for liver cancer treatment and provide a preclinical proof-of-concept for combination therapy with an autophagy inhibitor to enhance therapeutic efficacy.

FP3: A Novel VEGF Blocker with Anti-Angiogenic and Anti-Tumor Effects

Vascular endothelial growth fac-tor (VEGF) serves as a logical target for antiangiogenic cancer therapy. This study is to investigate the inhibitory effects of FP3, a novel VEGF blocker, on angiogenesis in vitro and in vivo as well as anti-tumor effects ona liver cancer xenograft model in vivo. The inhibitory effects of FP3 on angiogenesis were assessed by using human umbilical vein endothelial cells(HUVECs) in vitro and the chick embryo chorioallantoialc membrane (CAM) in viva. The inhibitory effect of FP3 on tumor growth in viva were evaluated in a human liver cancer cell line Hep-3B xenograft model in nude mice with the methods of tumor growth regression as-say. In experiments with HUVECs, FP3 inhibit-ed cell survival and tube formation. In CAM assay, FP3 suppressed MCF-7 human breast cancer cell-induced angiogenesis. In tumor growth regression assay, FP3 significantly blocked the growth of Hep-3B tumor cellin subcutaneous tumor xenograft model in nude mice. FP3 has excellent inhibitory effects on angiogenesis both in vitro and in viva and antitumor effect on liver cancer xenograft model; therefore, it might be used as an effective antiangiogenic agent in treatment of liver cancer.

Abstract 2535: PrevOnco® exhibits anti-tumor activity in in vitro and in vivo human tumor models: Combination therapy (PrevOnco® / Doxorubicin) demonstrates a better efficacy profile with enhanced survival rate in a liver cancer model

Abstract Background: The investigational anti-neoplastic agent PrevOnco® (know as Prevacid® /Lansoprazole®) is entering Phase II clinical trials in the US for hepatocellular carcinoma. Objectives: This study examined the efficacy of PrevOnco® in both in vitro and in vivo human tumor models. The optimal oral formulation, pharmacokinetic parameters and its combination therapy with doxorubicin were evaluated. Methods: Cytotoxicity was assessed in vitro by MTT analysis. In vivo efficacy was assessed in nude mice in 2 survival models of the pediatric kidney-derived G401 and SR leukemia, and one ectotopic HepG2 model. Results: In MTT assays, PrevOnco® demonstrated a concentration-dependent cytotoxic effect against a number of cancer cell lines (MC116, HOP-62, MOLT4, HCT-15, JMI, RPMI6666, RS 1184, STA 486, SF539, U251, SF295, UACC-62). Synergistic activity was observed with doxorubicin but not with cisplatin. PrevOnco® was significantly more effective than Omeprezole or SCH-28080. Pharmacokinetic analysis in nude mice showed 10% PEG400 to be a better vehicle than carboxy-methyl cellulose. A mean maximum concentration (Cmax) of 20,242 ng/mL was detected at 0.5 hours (Tmax) with the mean dose adjusted area under the curve (AUC) determined to be 846.5 nM*hr/mg. The half life (T1/2) of the compound was 0.9 hours. PrevOnco® at 100 mg/kg given orally significantly prolonged the survival rate in the G401 kidney xenograft by 60% at day 70 whereas all mice in vehicle treaded group died. At study end (day 83) 26% of the mice remained alive in the treated group. SR leukemia xenograft treated with PrevOnco® survived through the end of the study with >80% of the mice remaining alive. PrevOnco® induced a dose-dependent reduction in tumor growth in hepG2 model. A synergestic antitumoral effect was obtained with the combination treatment of PrevOnco® at 30 mg/kg and doxorubicin at10 mg/kg. PrevOnco® levels in hepG2 tumors following 100 mg/Kg BID treatment for 3 consecutive days indicated accumulation of the drug into tumor tissue. Conclusion: PrevOnco® exhibited anti-neoplastic activity in both in vitro and in vivo, enhancing survival and inhibiting tumor growth. Synergistic activity was obtained with doxorubicin in xenograft model of liver cancer, suggesting a potential new combination therapy for the treatment of liver cancer with less toxicological liability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2535. doi:10.1158/1538-7445.AM2011-2535

Establishment of in vivo Skin, Liver and Lung Cancer Xenograft Models using NCI-H640 and HepG2 Cell Lines in Athymic Nude Mice

Numerous murine models are available to study human cancer. These models are used to investigate the factors involved in malignant transformation, invasion and metastasis, as well as to examine response to therapy. One of the most widely used models is the human tumor xenograft. In this model, human tumor cells are transplanted, either under the skin or into the organ type in which the tumor originated, in immunocompromised mice that do not reject human cells. For example, the xenograft will be readily accepted by athymic nude mice, severely compromised immunodeficient (SCID) mice, or other immunocompromised mice. Depending upon the number of cells injected, or the size of the tumor transplanted, the tumor will develop over 1–8 weeks (or in some instances 1–4 months, or longer), and the response to appropriate therapeutic regimes can be studied in vivo. With this approach, using nude mice, we have established a subcutaneous xenograft and intrathoracic orthotopic models with human non-small cell lung cancer cell line (NCI-H460). These tumors grow to an appreciable volume within 10 days with 100% frequency subcutaneously and in the thorax. To test the validity of these models, we treated mice with known anticancer chemotherapeutic agent (Topotecan). This group showed significant reduction in the tumor volume compared to the untreated group. In addition, we have established a liver xenograft using HepG2 liver cell line. Compared to NCI-H460, HepG2 showed 50% frequency to tumors production. In conclusion, these models can be used to assess the anticancer property of different natural products in vivo.

RecQL1 DNA repair helicase: A potential tumor marker and therapeutic target against hepatocellular carcinoma

RecQL1 in the human RecQ DNA helicase family participates in DNA repair and recombination pathways in cell cycle replication. Immunohistochemical analysis of human hepatocellular carcinoma (HCC) tissues showed that RecQL1 expression is strongly correlated with histological grade and MIB-1 indices of HCC, and that the expression was greater in simple HCCs inducing extranodular growth or portal vein invasion than in HCCs not inducing extranodular growth or portal vein invasion. These histological data reveal the potential of RecQL1 as a biological marker predicting the malignancy and progression of liver cancer. High expression profiles were also produced by various HCC cells, including HCC cell lines established by us. When RecQL1 expression was silenced by siRNA in vitro, most HCC cells died of mitotic catastrophe. In a mouse orthotopic xenograft model of liver cancer with transplanted human HCC, RecQL1-siRNA mixed with cationic liposomes exhibited a strong anticancer effect that prevented the growth of the cancer. RecQL1-siRNA inhibited the growth of human HCC in the mouse liver, confirming that RecQL1 is an excellent molecular agent against liver cancer and suggests that RecQL1-siRNA formulated with liver-prone liposomes has excellent potential as a therapeutic drug against liver cancers.