Mouse Liver Cancer Cells Research Articles

Effects of Phellinus ribis Water Extracts(PRWE) on Apoptosis‐Induction in Liver Cancer Cell(Hepa1c1c7 cell)

The Phellinus ribis has been known to exhibit potent biological activities, such as anti‐viral, anti‐cancer and anti‐diabetes activities. This study investigated the effects of apoptosis induction in Hepa1c1c7 mouse liver cancer cells by treatment of PRWE. There were no cytotoxicity of PRWE evaluated by MTT assay with NCTC1469 normal cells and Hepa1c1c7 mouse cells. The changes of mRNA and proteins pivotally related to apoptosis were investigated using RT‐PCR and Western blots. The mRNA and protein expressions of TNF‐α, Caspase‐8 and Fas associated with death ligand/receptor‐dependent apoptosis pathway were increased in PRWE treatment group. The treatment of PRWE regulated mitochondria‐dependent apoptosis pathway by inducing the increase of Caspase‐9, Caspase‐3 and the decrease of Bcl‐2 in Hepa1c1c7 cells. In addition, the expression of p53 was increased in PRWE treatment group, indicating a direct damage on the cells. These results showed that PRWE effectively induced death of liver cancer cell by activating apoptosis.

Fluorescent quantum dot-labeled aptamer bioprobes specifically targeting mouse liver cancer cells

Fluorophore-labeled bioprobes are the key for fluorescent-labeled imaging technology. In the present work, mouse liver hepatoma cell line BNL 1ME A.7R.1 (MEAR)-specific ssDNA aptamer TLS9a was used to fabricate quantum dot-labeled aptamer bioprobe (QD-Apt), which was obtained by conjugating streptavidin-modified quantum dots (SA-QDs) with biotin-derived aptamer via the interaction between biotin and streptavidin. The QD-Apt was of monodispersity and excellent fluorescence properties. When the optimum ratio of SA-QDs to aptamer, which is 1:16, was used in the preparation of the QD-Apt, the resultant QD-Apt was of satisfactory bioactivity. They could specifically recognize MEAR cells and could not recognize BNL cells and Hela cells. Particularly, the growth and viability of QD-Apt bound MEAR cells were not affected by QD-Apt within 84 h compared to control cells, indicating that the probe was biocompatible and suitable for live cell imaging.

Abnormal blood vessels formed by human liver cavernous hemangioma endothelial cells in nude mice are suitable for drug evaluation

Cavernous hemangioma is vascular malformation with developmental aberrations. It was assumed that the abnormality of endothelial cells contributed greatly to the occurrence of cavernous hemangioma. In our previous study, we have found distinct characteristics of endothelial cells derived from human liver cavernous hemangioma (HCHEC). Here, we reported the abnormal vascular vessels formed by primary HCHEC in nude mice and that the drug podophyllotoxin can destroy HCHEC in vitro and in vivo. HCHEC was isolated from a human liver cavernous hemangioma specimen, and the HCHEC generated a red hemangioma-like mass 7 days after subcutaneously co-inoculating HCHEC and human liver cancer cells (Bel-7402) in nude mice. Lentiviral expression of GFP and immunohistochemistry for human CD31 was used to confirm that the HCHEC formed the blood vessels in nude mice. And the pathological features of vascular vessels formed by HCHEC were very similar to clinical cavernous hemangioma. In addition, by MTT assay, the drug podophyllotoxin was found inhibiting HCHEC viability, and by TUNEL and DNA ladder assays, podophyllotoxin was found inducing apoptosis of HCHEC. Moreover, podophyllotoxin was also effective for destroying the abnormal vascular vessels in the hemangioma-like mass in nude mice. In summary, the HCHEC can form abnormal blood vessels in nude mice, and we can evaluate drugs for cavernous hemangioma by using HCHEC in vitro and in vivo.

Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor by Hypoxia-Inducible Factor 1 Is Crucial for Invasion of Pancreatic and Liver Cancer

Angioinvasion is critical for metastasis with urokinase-type plasminogen activator receptor (uPAR) and tumor hypoxia-activated hypoxia-inducible factor 1 (HIF-1) as key players. Transcriptional control of uPAR expression by HIF has never been reported. The aim of the present study, therefore, was to test whether tumor hypoxia-induced HIF expression may be linked to transcriptional activation of uPAR and dependent angioinvasion. We used human pancreatic cancer cells and a model of parental and derived HIF-1β-deficient mouse liver cancer cell lines and performed Northern blot analysis, nuclear runoff assays, electrophoretic mobility shift assay, polymerase chain reaction-generated deletion mutants, luciferase assays, Matrigel invasion assays, and in vivo angioinvasion assays in the chorioallantoic membrane of fertilized chicken eggs. Urokinase-type plasminogen activator receptor promoter analysis resulted in four putative HIF binding sites. Hypoxia strongly induced de novo transcription of uPAR mRNA. With sequential deletion mutants of the uPAR promoter, it was possible to identify one HIF binding site causing a nearly 200-fold increase in luciferase activity. Hypoxia enhanced the number of invading tumor cells in vitro and in vivo. In contrast, HIF-1β-deficient cells failed to upregulate uPAR expression, to activate luciferase activity, and to invade on hypoxia. Taken together, we show for the first time that uPAR is under transcriptional control of HIF and that this is important for hypoxia-induced metastasis.

Validation of tissue-specific promoter-driven tumor-targeting trans-splicing ribozyme system as a multifunctional cancer gene therapy device in vivo

A trans-splicing ribozyme that can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA was previously suggested as a useful tool for tumor-targeted gene therapy. In this study, we applied transcriptional targeting with the RNA replacement approach to target liver cancer cells by combining a liver-selective promoter with an hTERT-mediated cancer-specific ribozyme. To validate effects of this system in vivo, we constructed an adenovirus encoding for the hTERT-targeting trans-splicing ribozyme under the control of a liver-selective phosphoenolpyruvate carboxykinase promoter. We observed that intratumoral injection of this virus produced selective and efficient regression of tumors that had been subcutaneously inoculated with hTERT-positive liver cancer cells in mice. Importantly, the trans-splicing reaction worked equally well in a nude mouse model of hepatocarcinoma-derived peritoneal carcinomatosis, inducing the highly specific expression of a transgene, and moreover, the efficient regression of the hTERT-positive liver tumors with minimal liver toxicity when systemically delivered with the adenovirus. In addition to the observed hTERT-dependent therapeutic gene induction, significant reductions in the levels of hTERT RNA (approximately 75%) were also observed. In conclusion, this study demonstrates that a cancer-specific RNA replacement approach using trans-splicing ribozyme with a tissue-selective promoter represents a promising strategy for cancer treatment.

Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3

To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22. Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry. MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G(1) phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.

Metabolism and Anticancer Activity of the Curcumin Analogue, Dimethoxycurcumin

The plant-derived compound curcumin has shown promising abilities as a cancer chemoprevention and chemotherapy agent in vitro and in vivo but exhibits poor bioavailability. Therefore, there is a need to investigate modified curcumin congeners for improved anticancer activity and pharmacokinetic properties. The synthetic curcumin analogue dimethoxycurcumin was compared with curcumin for ability to inhibit proliferation and apoptosis of human HCT116 colon cancer cells in vitro by estimating the GI(50) and LC(50) values and detecting the extent of apoptosis by flow cytometry analysis of the cell cycle. Metabolic stability and/or identification of metabolites were evaluated by recently developed mass spectrometric approaches after incubation with mouse and human liver microsomes and cancer cells in vitro. Additionally, circulating levels of dimethoxycurcumin and curcumin were determined in mice following i.p. administration. Dimethoxycurcumin is significantly more potent than curcumin in inhibiting proliferation and inducing apoptosis in HCT116 cells treated for 48 h. Nearly 100% of curcumin but <30% of dimethoxycurcumin was degraded in cells treated for 48 h, and incubation with liver microsomes confirmed the limited metabolism of dimethoxycurcumin. Both compounds were rapidly degraded in vivo but dimethoxycurcumin was more stable. Compared with curcumin, dimethoxycurcumin is (a) more stable in cultured cells, (b) more potent in the ability to kill cancer cells by apoptosis, (c) less extensively metabolized in microsomal systems, and (d) more stable in vivo. It is likely that the differential extent of apoptosis induced by curcumin and dimethoxycurcumin in vitro is associated with the metabolite profiling and/or the extent of stability.

Synergistic Effects of Flavonoids on Cell Proliferation in Hepa‐1c1c7 and LNCaP Cancer Cell Lines

ABSTRACT: Fruits and vegetables contain a variety of phytochemicals, including flavonoids, which have antioxidant and anticancer properties. The purpose of this study was to evaluate the antiproliferative effects and synergistic interactions of a variety of flavonoids in Hepa‐1c1c7, a mouse liver cancer cell line, and LNCaP, a human prostate cancer cell line. Aglycone flavonoids, such as quercetin, kaempferol, and naringenin (at 12.5 to 50 μM), inhibited cancer cell proliferation in both cell lines in a dose dependent manner without cytotoxicity. In contrast, glycone flavonoids (rutin, quercetrin, and naringin) did not inhibit cell growth. Significant synergistic antiproliferative effects were demonstrated in both cancer cell lines when flavonoids were provided in combination treatments. These results suggest that combinations of flavonoids, which are naturally present in whole fruits and vegetables, are more effective in cancer cell growth inhibition than the individual flavonoids.

Structures, biogenetic relationships, and cytotoxicity of pimarane-derived diterpenes from Petalostigma pubescens

Extraction of Petalostigma pubescens heartwood followed by chromatographic purifications and crystallizations afforded five tricyclic diterpenes: 5,9-syn-rosanes petalostigmones A and B ( 1 and 2), the erythroxylane petalostigmone C ( 3), the norditerpene lactone pubescenone ( 4), and the known ent-cleistanthane diterpene (−)-sonderianol ( 5). The structures and relative stereochemistry were elucidated by means of spectroscopic methods, chemical correlations, and, in the cases of 1 and 4, by X-ray crystallographic analyses. The new isolates 1– 4 are assumed to belong to the same absolute configurational family (9αCH 3) of ent-pimarane-derived diterpenes as the known co-occurring (−)- 5 (10αCH 3). Biogenetic schemes originating from a common ent-copalyl diphosphate intermediate are presented to rationalize the structures of these natural products. A novel ring contraction–ring expansion mechanism is suggested to account for the 7-membered B ring of pubescenone. Compounds 1– 5 were evaluated for their cytotoxicity; sonderianol ( 5) showed the highest activity against mouse leukemia cell lines L1210, P388 and mouse liver cancer cells HEPA1c1c7.

Effective separation of potent antiproliferation and antiadhesion components from wild blueberry (Vaccinium angustifolium Ait.) fruits.

Extracts from wild blueberry (Vaccinium angustifolium Ait.) were separated into proanthocyanidin-rich fractions using liquid vacuum and open column chromatography on Toyopearl and Sephadex LH-20, respectively. Fractions were characterized using analytical tools including mass spectrometry and NMR spectroscopy; fraction composition was correlated with bioactivity using antiproliferation and antiadhesion in vitro assays. There was a significant positive correlation between proanthocyanidin content of different fractions and biological activity in both the antiproliferation and antiadhesion assays. Two fractions containing primarily 4-->8-linked oligomeric proanthocyanidins with average degrees of polymerization (DPn) of 3.25 and 5.65 inhibited adhesion of Escherichia coli responsible for urinary tract infections. Only the fraction with a DPn of 5.65 had significant antiproliferation activity against human prostate and mouse liver cancer cell lines. These findings suggest both antiadhesion and antiproliferation activity are associated with high molecular weight proanthocyanidin oligomers found in wild blueberry fruits.

8-chloroadenosine induces apoptosis in human MOLT-4 cell line

THE 8-chloroadenosine (8CA) is a novel analog of adenosine synthesized in the National Research Laboratories of Natural and Biomimetic Drugs. Fang et al. reported that 8CA is capable of inhibiting the cell growth in vitro of mouse liver cancer cells and human HL-60