LAGE3 promotes angiogenesis on hepatocellular carcinoma by stabilizing VEGFA mRNA.

RNA modification plays important roles in various physiological and pathological process. LAGE3 is a component of EKC/KEOPS complex, which is probably involved in the formation of a threonylcarbamoyl group on adenosine at position 37 (t(6)A37) in tRNAs, but its exact role in HCC is less studied. Our study reveals that LAGE3 exhibits upregulated expression in HCC compared with normal hepatocellular tissue. High expression of LAGE3 promotes hepatocellular cell proliferation and migration. Further investigations suggest that the increased expression of LAGE3 cloud lead to upregulated VEGFA secretion and angiogenesis in HCC. The mechanistic study reveals LAGE3 is required for the VEGFA mRNA stability. This research may open new avenues for diagnosis and targeted therapy in HCC.

SOX12 expression is associated with progression and poor prognosis in human breast cancer.

The sex-determining region Y-box 12 (SOX12) is implicated in several oncogenic signaling pathways of multiple types of cancer; however, the biological effects of SOX12 on breast cancer has yet to be elucidated. Here, we assessed SOX12 expression using real-time quantitative PCR in 142 pairs of breast cancer and adjacent normal tissues (ANTs) and immunohistochemistry in 524 breast cancer and 147 ANTs. The effects of SOX12 on breast cancer progression, clinicopathological variables, and prognostic value were then investigated. SOX12 expression was markedly elevated in breast cancer tissues relative to that in ANTs at both mRNA and protein levels. Positive SOX12 expression was correlated to tumor size (P = 0.005), estrogen receptor (ER) (P = 0.018) and human epidermal growth factor receptor (HER2) (P = 0.004) status, lymph node metastasis (P < 0.001), and the tumor-node-metastasis (TNM) stage (P < 0.001). Notably, the positive rate of SOX12 expression gradually increased with breast cancer progression. Multivariate analysis indicated that SOX12 was an independent prognostic factor for overall survival (OS, P = 0.023) and distant metastasis-free survival (DMFS, P = 0.012). Subgroup analysis revealed that luminal and HER2 patients with positive SOX12 expression had a shorter OS period than those with negative SOX12 expression. Moreover, SOX12 expression was associated with a high risk of distant metastasis in invasive carcinoma with the lymph node metastasis subgroup. In summary, SOX12 correlates with progression and poor prognosis in human breast cancer, suggesting that SOX12 is a potential target for breast cancer treatment and warrants further functional research.

The SNP of rs6854845 suppresses transcription via the DNA looping structure alteration of super-enhancer in colon cells.

Single-nucleotide polymorphisms (SNPs) identified by Genome-Wide Association Studies (GWASs) have been determined to closely connect with multiple diseases. Previous studies revealed one underlying mechanism that SNPs located within the regulatory elements could affect the encoding gene expression through long-range regulation. SNP rs6854845 was suggested to be a risk of colon cancer in human population. Nevertheless, the underlying molecular mechanism for colon carcinogenesis remains largely unknown. In present study, rs6854845 with G > T mutation in situ in FHC, HCT-116 and SW-480 cells were generated by Crispr/Cas9. The nearby chromatin organization was investigated by chromatin conformation capture (3C). And the expression of coding gene regulated by super-enhancer (SE) was detected by real-time PCR. We observed a significantly different pattern of the genome organization upon rs6854845 generation in colon epithelial cells but not in colon cancer cells. Moreover, we observed the shifted enrichment of H3K4me1 and H3K27ac at the SE (chr4:75.7M-76.0 M) where rs6854845 located. Furthermore, we observed that the transcription of the gene clusters regulated by SE were affected by rs6854845 in colon cells. Overall, our results demonstrated that SNP rs6854845 located in SE could destroy the long-range chromosomal interaction between SE and target gene clusters thereby affecting the transcription of these genes.

Long-Circulating Liposomal Delivery System Targeting at PDGFR-ß Enhances the Therapeutic Effect of IFN-α on Hepatic Fibrosis.

BACKGROUND: In this study, we developed a drug of IFN-α combined with pPB-SSLs, which specifically target at platelet-derived growth factor receptor-ß (PDGFR-ß). AIM: The aim of this study is to improve the limitations of IFN-α including insufficient drug concentration for the target cells and side-effects causing serious concerns in treatment of hepatic fibrosis. METHODS: We constructed the targeted stable liposomes (SSLs) that not only increase the half-life period of IFN- α, but also can deliver IFN-α to hepatic stellate cells (HSCs). Subsequently, the anti-hepatic-fibrosis effect of pPB-SSL-IFN-α was evaluated both in vitro and in vivo. Immunofluorescent assay showed that the pPB-SSL particles were able to be easily taken up by 3T3 cells. The cellular distribution experiment demonstrated that most of the pPB-SSL-IFN-α would accumulate around the fibroblast, and the cell would be invaded by pPB-SSLIFN- α. RESULTS: The pPB-SSL-IFN-α showed an entrapment efficiency of 39.73 ± 5.21% for IFN-α and the particles reached nanoscale level. It showed more significant alleviated performance for hepatic fibrosis than IFN-α. Both in vitro and in vivo, the pPB-SSL-IFN-α could contribute to reduction or inhibition in the expression of TGF-ß1 and α-SMA even cleavage of caspase-3. Moreover, it was found that the pPB-SSL-IFN-α induced the apoptosis of 3T3 cells by inhibiting the expression of TGF-ß1 as well as α-SMA. Under observation for fibrotic liver of mice treated with pPB-SSL-IFN-α, the semiquantitative score for collagen I, TGF-ß1 and α-SMA were all inferior to the control group and those treated with PEG-IFN-α, SSL-IFN-α or IFN-α. In addition, pPB-SSL-IFN-α has been detected to down-regulate the expression of TNF-α and IL-1ß in comparison with model group (P<0.01). And the phosphorylations of JAK1 and STAT1 were enhanced by pPB-SSL-IFN-αin comparison with model groups (P < 0.01). CONCLUSION: All results of our present research indicated that the pPB-SSL-IFN-α might be an alternative antiliver fibrotic drug and the synthetic method may offer a new access to the anti-hepatic fibrosis research and development.

Krüppel-like factor 2 promotes cell proliferation in hepatocellular carcinoma through up-regulation of c-myc.

Members of krüppel-like factor family have been shown to play critical roles in the development, metabolism and tumorigenesis. Our previous study demonstrated that up-regulationupregulation of KLF2 in livers from obese mice could promote hepatosteatosis. However, little is known about its effects in hepatocellular carcinogenesis. In the present study, we found that mRNA and protein expression of KLF2 was increased in primary HCC, when compared with that in adjacent normal liver. In vitro studies further showed that enforced overexpression of KLF2 expression enhanced, while its knockdown inhibited cell proliferation and invasion. At the molecular level, c-myc was a direct transcriptional target of KLF2 and a KLF2-binding site in the c-myc promoter bound specifically to KLF2 protein. Indeed, ablation of c-myc largely attenuated the proliferative roles of KLF2 in HCC cells. Therefore, our data highlight an important role for KLF2/c-myc pathway in HCC development and progression.

Overexpression of LIMK1 promotes tumor growth and metastasis in gastric cancer.

Gastric cancer is the second-leading cause of cancer death in Asia. Despite improvement of therapies, the outcome in patients remains extremely poor because of metastasis. In the present study, we found that LIMK1 is overexpressed in gastric cancer, and its expression level correlate with tumor size, lymph node metastasis and TNM stage. Knockdown of LIMK1 expression could inhibit cell proliferation, migration and invasion in vitro, as well as suppress the activation of FAK/paxillin pathway. Moreover, knockdown of LIMK1 expression retarded tumor growth and peritoneal ametastasis in vivo. This highlights that LIMK1 might be used as a potential target in the treatment of gastric cancer.

Inhibition of hepatocellular carcinoma by fulvestrant involves the estrogen receptor α and Wnt pathways in vitro and in patients.

The aim of the present study was to investigate the effect of anti-estrogen treatment (fulvestrant) on the biological activity of hepatocellular carcinoma (HCC), involving the estrogen receptor α (ERα) and Wnt pathways, and to evaluate whether ERα and Wnt inhibitory factor-1 (WIF1) could be biomarkers for anti-estrogen clinical therapy. H22 and HepG2 cells were treated with 0.04 to 625 nM fulvestrant and the WST-8 method was used to assess the inhibition rate after 72 h. Furthermore, prolactin (PRL) secretion by HepG2 cells was assessed at 24 h using an enzyme immunoassay. Quantitative polymerase chain reaction and western blot analysis were used to analyze the mRNA and protein expression levels of ERα, ß-catenin and WIF1, respectively, in HepG2 cells. For clinical patient analysis, the tumor volume was analyzed by magnetic resonance imaging methods, and PRL in the blood was detected by an enzyme immunoassay. In HepG2 cells, the mRNA and protein expression levels of ERα were downregulated (P<0.01), while ß-catenin expression remained unchanged and WIF1 expression was upregulated (P<0.01). Analysis of samples from clinical patients demonstrated that there was a positive correlation between PRL levels and tumor volume. In addition, as compared with non-cancerous tissues, the ERα mRNA levels in tumor tissue were upregulated (P<0.05), particularly in that of male patients, while WIF1 expression was significantly downregulated (P<0.01). In conclusion, fulvestrant inhibited the proliferation of HepG2 cells, involving the ERα and non-canonical Wnt pathways, and it may be a promising therapeutic for HCC.

HMGA2 induces transcription factor Slug expression to promote epithelial-to-mesenchymal transition and contributes to colon cancer progression.

Epithelial-to-mesenchymal transition (EMT) is considered to play an essential role in progression and metastasis. This study aims to investigate the expression and underlying molecular targets of high-mobility group AT-hook 2 (HMGA2) in the progression of colon cancer. The expression of HMGA2 is upregulated by both active extracellular signal-regulated kinase (ERK)1/2 and TGF-ß signaling in colon cancer cells through a series of lentiviral infection and pharmacological assays. HMGA2 knockdown by specific shRNAs attenuates proliferation, motility and invasion of colon cancer cells in vitro and in vivo. Besides, exogenous HMGA2 expression caused EMT in colon cancer cells, which was confirmed by the downregulation of the epithelial markers and the upregulation of the mesenchymal markers. Moreover, HMGA2 positively regulates the Slug expression by directly binding to the regulatory region in Slug promoter. Importantly, the knockdown of Slug could reverse the HMGA2-induced EMT and decrease the migration and invasion ability of colon cancer cells. Taken together, our results reveal a critical role for HMGA2 in promoting EMT, migration, invasion, and proliferation of colon cancer cells, suggesting HMGA2 as a potential molecular target to prevent colon cancer progression.

Cortactin expression confers a more malignant phenotype to gastric cancer SGC-7901 cells.

AIM: To study the effects of cortactin on the tumor biology of SGC-7901 cells and identify the mechanism involved in the process. METHODS: Cell lines in which cortactin was stably overexpressed or knocked down as well as the respective control cell lines were established by standard molecular methods. The effects of cortactin on the proliferation, migration and invasion capacity of SGC-7901 cells were assessed by the MTT assay, colony formation, flow cytometry, transwell migration and matrigel invasion. Nude mouse models were also used to assess the role of cortactin in the growth and metastasis of SGC-7901 cells in vivo. Western blotting analysis was performed to detect the expression of epidermal growth factor receptor (EGFR) and downstream molecules. RESULTS: Cell lines in which cortactin was stably overexpressed or knocked down as well as control cell lines were successfully established and designated as LV5-cortactin-SGC, LV5-SGC, LV3-shRNA-SGC and LV3-SGC. Cortactin overexpression promoted SGC-7901 cell migration (340.7 ±12.6 vs 229.1 ± 23.2, P < 0.01) and invasion (71.6 ± 5.2 vs 48.4 ± 3.6, P < 0.01). Cortactin downregulation impaired SGC-7901 cell migration (136.2 ± 19.8 vs 225 ± 17) and invasion (29.2 ± 5.2 vs 49.6 ± 3.8, P < 0.01). The results from the MTT and colony formation assays results indicated increased LV5-cortactin-SGC cell proliferation and decreased LV3-shRNA-SGC cell proliferation compared to the control cells. Flow cytometry analysis demonstrated that cortactin overexpression promoted the proliferation index of SGC-7901 cells, and the results were reversed when cortactin was downregulated. Mouse tumor models confirmed that cortactin expression increased SGC-7901 cell proliferation and metastasis in vivo. Western blotting analysis revealed that cortactin elevated EGFR expression and activated the downstream molecules. CONCLUSION: Cortactin expression promoted the migration, invasion and proliferation of SGC-7901 cells both in vivo and in vitro. The EGFR signaling pathway is mechanistically involved.

Prognostic significance of cyclin D1 expression in colorectal cancer: a meta-analysis of observational studies.

OBJECTIVE: Cyclin D1 plays a vital role in cancer cell cycle progression and is overexpressed in many human cancers, including colorectal cancer (CRC). However, the prognostic value of cyclin D1 overexpression in colorectal cancer is conflicting and heterogeneous. We conducted a meta-analysis to more precisely evaluate its prognostic significance. METHODS: A comprehensive literature search for relevant studies published up to January 2014 was performed using PubMed, EMBASE, and ISI Web of Science. The pooled hazard ratio (HR) with 95% confidence intervals (CI) was used to estimate the effects. RESULTS: 22 studies with 4150 CRC patients were selected to evaluate the association between cyclin D1 and overall survival (OS), disease-free survival (DFS) and clinicopathological parameters. In a random-effects model, the results showed that cyclin D1 overexpression in CRC was significantly associated with both poor OS (HR = 0.73, 95% CI: 0.63-0.85, P<0.001) and DFS (HR = 0.60, 95% CI: 0.44-0.82, P = 0.001). Additionally, cyclin D1 overexpression was significantly associated with more relative older patients (≥ 60 years) (OR 0.62, 95% CI 0.44-0.89, P = 0.009), T3,4 tumor invasion (OR 0.70, 95% CI 0.57-0.85, P<0.001), N positive (OR 0.75, 95% CI 0.60-0.95, P = 0.016) and distant metastasis (OR 0.60, 95% CI 0.36-0.99, P = 0.047) of CRC. CONCLUSION: The meta-analysis results indicated that cyclin D1 is an unfavorable prognostic factor for CRC. Cyclin D1 overexpression might be associated with poor clinical outcome and some clinicopathological factors such as age, T category, N category and distant metastasis in CRC patients.

Increased SSeCKS expression in rat hepatic stellate cells upon activation in vitro and in vivo.

Recent reports suggest that src suppressed c kinase substrates (SSeCKS) are early inflammatory response protein. However, there is only scarce knowledge on the functional role of SSeCKS in liver under conditions of acute inflammation. In the present study, we investigated SSeCKS expression in liver after administration of carbon tetrachloride (CCl4) in rats and in isolated primary hepatic stellate cells (HSCs) upon activation on a plastic dish. We found that SSeCKS mRNA was hardly detectable in healthy liver tissue and further increased in carbon tetrachloride-mediated acute liver failure. SSeCKS protein expression was mainly found in hepatic stellate cells. In vitro, SSeCKS expression in activated rat HSCs was dramatically increased. The upregulation of SSeCKS protein expression in rat HSCs during activation in vitro and in vivo suggested the possibility of SSeCKS, an important part of function of the activated HSCs, perhaps through modulation of liver regeneration or formation of liver fibrosis after various injuries.

Stereoselective glucuronidation of ornidazole in humans: predominant contribution of UDP-glucuronosyltransferases 1A9 and 2B7.

Ornidazole [R,S-1-chloro-3-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ol] is a chiral 5-nitroimidazole class antimicrobial agent. This study aimed to investigate the principal metabolic pathway of ornidazole in humans and identify the major enzymes involved. A total of 19 metabolites were identified in human urine collected from patients with hepatobiliary diseases after an intravenous drip infusion of 500 mg of racemic ornidazole. Stereoselective glucuronidation, followed by renal excretion, was the principal metabolic pathway of ornidazole in humans, accounting for 37.3% of the administered dose. Screening assays with 12 available human recombinant UDP-glucuronosyltransferases (UGTs) demonstrated that UGT1A9 was the predominant UGT isoform involved in R-ornidazole glucuronidation, whereas S-ornidazole glucuronidation was almost exclusively catalyzed by UGT2B7. Chemical inhibition study with niflumic acid and flurbiprofen supported these findings. Enzyme kinetic parameters were then determined in human liver microsomes (HLMs), human kidney microsomes (HKMs), UGT1A9, and 2B7. The K(m) values for UGT1A9 (15.6 ± 1.6 mM for R-ornidazole) and 2B7 (3.8 ± 0.9 mM for S-ornidazole) were quite similar to those determined in HLMs and HKMs (20.1 ± 1.4 and 17.7 ± 4.0 mM for R-ornidazole; 6.6 ± 1.3 and 3.2 ± 0.4 mM for S-ornidazole). The in vitro intrinsic clearance (CL(int)) ratios of S- to R-ornidazole were approximately 4.3 in HLMs and 6.5 in HKMs, respectively. The hepatic and renal clearances were estimated based on the well-stirred model. Overall, stereoselective glucuronidation was the principal metabolic pathway of ornidazole in humans. Furthermore, UGT1A9 and 2B7 were the predominant UGT isoforms responsible for R- and S-ornidazole glucuronidation in humans, respectively.

Pancreaticobiliary maljunction is associated with common bile duct carcinoma: a meta-analysis.

OBJECTIVE: Pancreaticobiliary maljunction (PBM) has been reported to be associated with an increased risk of gallbladder carcinoma. However, the relationship between PBM and common bile duct carcinoma (CBDC) remains unclear. We aimed to conduct a meta-analysis to determine the available evidence on the association between PBM and CBDC. METHODS: The pooled odds ratio (OR) and standard mean differences (SMD) with 95% confidence interval (95% CI) were used to estimate the effects. RESULTS: A total of eight case-control studies and two cohort studies were identified. The incidence of PBM was higher in CBDC patients than in controls (OR = 1.45; 95% CI, 1.19-1.76). Compared with patients without PBM, CBDC patients with PBM were younger at the diagnosis age (SMD = -0.46; 95% CI, -0.64 to -0.28). A difference in the incidence of associated CDC was found between CBDC patients with or without PBM (OR = 2.14; 95% CI, 1.60-2.87). CONCLUSIONS: Compared with benign biliary tract diseases, the incidence of PBM was higher in CBDC patients, especially in relatively young patients. We also found that the incidence of CDC was higher in CBDC patients with PBM. These findings showed positive association between PBM and CBDC, which may help in identifying high-risk individuals.

Mechanistic studies on the absorption and disposition of scutellarin in humans: selective OATP2B1-mediated hepatic uptake is a likely key determinant for its unique pharmacokinetic characteristics.

Scutellarin [scutellarein-7-O-glucuronide (S-7-G)] displayed a unique pharmacokinetic profile in humans after oral administration: the original compound was hardly detected, whereas its isomeric metabolite isoscutellarin [scutellarein-6-O-glucuronide (S-6-G)] had a markedly high exposure. Previous rat study revealed that S-7-G and S-6-G in the blood mainly originated from their aglycone in enterocytes, and that the S-7-G/S-6-G ratio declined dramatically because of a higher hepatic elimination of S-7-G. In the present study, metabolite profiling in human excreta demonstrated that the major metabolic pathway for S-6-G and S-7-G was through further glucuronidation. To further understand the cause for the exposure difference between S-7-G and S-6-G in humans, studies were conducted to uncover mechanisms underlying their formation and elimination. In vitro metabolism study suggested that S-7-G was formed more easily but metabolized more slowly in human intestinal and hepatic microsomes. Efflux transporter study showed that S-6-G and S-7-G were good substrates of breast cancer resistance protein and multidrug resistance-associated protein (MRP) 2 and possible substrates of MRP3; however, there was no preference great enough to alter the S-7-G/S-6-G ratio in the blood. Among the major hepatic anion uptake transporters, organic anion-transporting polypeptide (OATP) 2B1 played a predominant role in the hepatic uptake of S-6-G and S-7-G and showed greater preference for S-7-G with higher affinity than S-6-G (K(m) values were 1.77 and 43.9 µM, respectively). Considering the low intrinsic permeability of S-6-G and S-7-G and the role of OATP2B1 in the hepatic clearance of such compounds, the selective hepatic uptake of S-7-G mediated by OATP2B1 is likely a key determinant for the much lower systemic exposure of S-7-G than S-6-G in humans.

Interferon-α enhances antitumor activities of oncolytic adenovirus-mediated IL-24 expression in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) has a dismal 5-year-survival rate of 10%, so novel strategies are warranted. IL-24 mediates anti-tumor activity reducing STAT3 expression, which suggests that interferon (IFN) alpha may augment tumor cell lysis and reduce angiogenesis. We investigated the antitumor activity of treatment with IFN-α, with the oncolytic adenovirus SG600-IL-24, or the combination of both in HCC in vitro and in vivo. RESULTS: RT-PCR, ELISA assay and Western-blot confirmed that the exogenous IL-24 gene was highly expressed in HCC cells infected with SG600-IL-24. Treatment with combined IFN-α and SG600-IL-24 suppressed growth and promoted apoptosis of the HepG2, MHCC97L, and HCCLM3 cell lines compared with the normal cell line L02. The combined therapy increased STAT1 and SOCS1 and apoptosis, but decreased the expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF, which are regulated by STAT3 in HCC cells in vitro. To assess the effects in vivo, the HCC cell line HCCLM3 was transplanted subcutaneously into the right flanks of nude mice. Mice in the IFN-α group, the SG600-IL-24 group, or the combined therapy group had significantly suppressed growth of the HCC xenografted tumors compared to the PBS control group of mice. Among the mice treated with the combination of IFN-α and SG600-IL-24, three of those eight mice had long-term survival and no evidence of a tumor. These mice also had decreased expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF. CONCLUSIONS: The present study demonstrated for the first time the potential antitumor activity of IFN-α combined with the oncolytic adenovirus SG600-IL-24 in HCC both in vitro and in vivo, and suggests its further development as a potential candidate for HCC cancer gene therapy.

ShRNA-mediated gene silencing of heat shock protein 70 inhibits human colon cancer growth.

Heat shock protein 70 (Hsp70), a chaperone involved in tumor progression, is overexpressed in various human tumors. However, its role in colon cancer progression is not completely understood. In the present study, two shRNA plasmid vectors against Hsp70 were constructed and stably transfected into the colon cancer cell line HT29 to determine the effect of Hsp70 on cell proliferation, cell cycle distribution and cell apoptosis in HT29 cells in vitro, and its effect on xenograft tumor growth and apoptosis in vivo. Cell proliferation was determined using MTT assay. The results revealed that Hsp70 silencing efficiently inhibited the growth of HT29 cells in culture, induced cell cycle arrest at the G1 phase, and significantly increased apoptosis. Moreover, stable clones from the Hsp70 shRNA-2 vector suppressed xenograft tumor growth and enhanced apoptosis in vivo compared with a mock and vector control group. In conclusion, specific Hsp70 shRNA silencing may inhibit colon cancer growth, indicating that Hsp70 silencing is a potential therapeutic strategy for the treatment of colon cancer.

Identification of amiodarone metabolites in human bile by ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Amiodarone is recognized as an effective drug in the treatment of arrhythmias. Previous experiments demonstrated that mono-N-desethylamiodarone (MDEA) was the major circulating metabolite in humans. In addition, dealkylation, hydroxylation, and deamination were minor metabolic pathways. The purpose of this study was to identify the metabolites of amiodarone in the bile obtained from patients with T-tube drainage after oral drug administration. Amiodarone metabolism in vitro was also investigated using human liver microsomes (HLMs) and S9 fraction. Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) revealed 33 metabolites in human bile, including 22 phase I and 11 phase II metabolites. The major metabolites were MDEA (M7) and ω-carboxylate amiodarone (M12). Metabolite M12 was isolated from human bile, and the chemical structure was confirmed using UPLC-Q/TOF MS and ¹H NMR. Moreover, the authentic standards of two hydroxylated metabolites, 2-hydroxylamiodarone and 3'-hydroxylamiodarone, were obtained through microbial transformation. Several novel metabolic pathways of amiodarone in human were proposed, including ω-carboxylation, deiodination, and glucuronidation. The in vitro study demonstrated that incubation of HLMs with amiodarone did not give rise to any carboxyl metabolites. In contrast, M12 and its metabolites were detected in human liver S9 incubation samples, and the production of these metabolites were inhibited almost completely by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, suggesting the involvement of alcohol dehydrogenase in the ω-carboxylation of amiodarone. Overall, UPLC-Q/TOF MS analysis leads to the discovery of several novel amiodarone metabolites in human bile and underscores the importance of bile as an excretion pathway.

Transfection of IL-2 and/or IL-12 genes into spleen in treatment of rat liver cancer.

AIM: To test the efficacy of gene therapy in rat liver tumor. METHODS: A retroviral vector GCIL12EIL2PN encoding human IL-2 (hIL-2) and mouse IL-12 (mIL-12) fused gene and its packaging cell were constructed. The packaging cell lines contained of IL-2 and/or IL-12 genes were injected intrasplenically to transfect splenocyte at different time. The therapeutic effect, immune function and toxic effect were evaluated. RESULTS: The average survival times of the 4 groups using IL genes at days 1, 3, 5 and 7 after tumor implantation were 53.3+/-3.7, 49.3+/-4.2, 31.0+/-2.1 and 24.3+/-1.4 d respectively in IL-2/IL-12 fused gene group, 25.0+/-2.5, 23.5+/-2.0, 18.3+/-2.4 and 12.0+/-1.8 d respectively in IL-2 gene treatment group, and 39.0+/-4.8, 32.0+/-3.9, 23.0+/-2.5 and 19.4+/-2.1 d respectively in IL-12 gene treatment group (P<0.01, n=10). In the IL-12/IL-2 fused gene treatment group, 30% of rats treated at days 1 and 3 survived more than 60 d and serum mIL-12 and hIL-2 levels were still high at day 3 after treatment. Compared with IL alone, NK cell activity was strongly stimulated by IL-2/IL-12 gene. Microscopy showed that livers were infiltrated by a number of lymphocytes. CONCLUSION: IL-2 and/or IL-12 genes injected directly into spleen increase serum IL-2 and IL-12 levels and enhance the NK cell activity, which may inhibit the liver tumor growth. The therapy of fused gene IL-2/IL-12 is of low toxicity and relatively high NK cell activity. Our data suggest that IL-2/IL-12 fused gene may be a safe and efficient gene therapy for liver tumor. The gene therapy should be administrated as early as possible.

[Treatment of hepatocellular carcinoma by transfecting interleukin-12 and interleukin-2 fusion gene intrasplenically, an experimental study].

OBJECTIVE: To study the inhibitory effect of retroviral packaging cells injected intrasplenically encoding mouse interleukin-12 (mIL-12) and human interleukin-2 (hIL-2) fusion gene on the growth of hepatocellular carcinoma. METHODS: The retroviral vectors encoding mIL-12 gene, hIL-2 gene, and mIL-12 and hIL-2 genes, GCIL12EXPN, GCXEIL2PN, and GCIL12EIL2PN were constructed and then transfected into the retroviral packaging cells PA317 to construct cells PA317-GCIL12EXPN, PA317-GCXEIL2PN, and PA317-GCIL12EIL2PN. Rat hepatocellular carcinoma cells CBRH3 were implanted into the livers of Wistar rats to establish hepatoma animal model. Then the rats were divided into 5 groups to be injected intrasplenically with normal saline one day after the implantation (0.8 ml/rat, group I, n = 10), blank vector PA317-GCXEXPN one day after the implantation (10(7) cells/rat, group II, n = 10), PA317-GCIL12EXPN containing IL-2 gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group III, n = 40), PA317-GCXEIL2PN containing mIL-12 gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group IV, n = 40), and PA317-GCIL12EIL2PN containing IL-12-IL-2 fusion gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group V, n = 40) respectively. The rats surviving longer than 2 months were re-injected with hepatocellular carcinoma cells. The therapeutic effect, immune function and toxic effect were evaluated. CT was conducted on the liver before and after the experiment. Laparotomy was performed 3 and 7 days after treatment to resect some of the carcinoma tissues to undergo pathological examination and OX8 immunohistostaining. Serum mIL-12 and hIL-2 were detected one day before and 3, 7, 30, and 60 days after treatment. RESULTS: The average survival times of the rats treated with IL-12-IL-2 fusion gene at the first, third, fifth and seventh day after tumor implantation were 53.3 +/- 3.7 days, 49.3 +/- 4.2 days, 31.0 +/- 2.1 days, and 24.3 +/- 1.4 days respectively, longer than those treated with IL-2 gene (25.0 +/- 2.5 days, 23.5 +/- 2.0 days, 18.3 +/- 2.4 days, and 12.0 +/- 1.8 days respectively, P < 0.001), and those treated with IL-12 gene (39.0 +/- 4.8 days, 32.0 +/- 3.9 days, 23.0 +/- 2.5 days, and 19.4 +/- 2.1 days respectively, P < 0.001). Long survival (>or= 60 days) rate in the rats treated with IL-12-IL-2 fusion gene on the first and third day was 30%. The serum mIL-12 and hIL-2 levels in these rats remained high on the 60th day after treatment. The pathological study showed that the number of infiltrating lymphocytes in liver tumor tissues was increased in the IL-12-IL-2 fusion gene treatment group. CONCLUSION: The retroviral packaging cell line injected intrasplenically encoding mIL-12 and hIL-2 fusion gene inhibits the growth of hepatocellular carcinoma significantly in rats. The therapeutical efficacy of early administration is superior to that of late one.

Relationship between the imaging features and pathologic alteration in hepatoma of rats.

AIM: The imaging features of MRI and DSA, using the models of implanted and induced hepatoma, were investigated in rats. METHODS: CBRH3 cancer cells were implanted for different liver site of rat liver and the diethylnitrosoamine was given orally to rats in order to induce liver cancer. Both experimental groups were detected by magnetic resonance imaging (MRI), digital subtraction angiography (DSA) and morphologic assay. RESULTS: Hypointensity on T1WI and homogenous high signal intensity on T2WI in MRI, and ring-like abnormal stain on DSA were found in implanted cancer. Induced cancers appeared as homogeneous or heterogeneous hypointensity on T1WI (10 cases), and equal or slight high intensity on T2WI (8 cases), but some as hypointensity on T2WI (2 cases). CONCLUSION: The imaging features of implanted cancers were similar to that of human liver metastases. Therefore, it could serve as an experimental model of human liver metastatic tumor. The imaging feature of induced cancers, whereas, were similar to that of human primary liver cancer. It could be use as an experimental model of human primary liver cancer.