Evaluation of the medicinal herb Graptopetalum paraguayense as a treatment for liver cancer.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third most common cause of cancer-related death worldwide. Sorafenib is the only drug for patients with advanced-stage hepatocellular carcinoma (HCC) that has been shown to confer a survival benefit to patients with HCC; however, it has many side effects. Thus, alternate therapeutic strategies with improved safety and therapeutic efficacy for the management of HCC should be developed. METHODS AND FINDINGS: We demonstrate that an extract of Graptopetalum paraguayense (GP) down-regulated the expression levels of several onco-proteins, including AURKA, AURKB, and FLJ10540, in HCC cells. To isolate the active components in the GP extracts, we prepared extracts fractions and assessed their effects on the expression of onco-proteins in HCC cells. The fraction designated HH-F3 was enriched in active ingredients, exhibited cytotoxic effects, and suppressed the expression of the onco-proteins in HCC cells. The structure of the main active compound in HH-F3 was found to be similar to that of the proanthocyanidin compounds derived from Rhodiola rosea. In addition, a distinct new compound rich in 3, 4, 5-trihydroxy benzylic moieties was identified in the HH-F3 preparations. Mechanistic studies indicated that HH-F3 induced apoptosis in HCC cells by promoting the loss of mitochondrial membrane potential and the production of reactive oxygen species. HH-F3 also enhanced PTEN expression and decreased AKT phosphorylation at Ser473 in a concentration-dependent manner in HCC cells. Moreover combination of GP or HH-F3 and sorafenib synergistically inhibits the proliferation of Huh7 cells. The treatment of a rat model with diethylnitrosamine (DEN)-induced liver cancer with extracts of GP and HH-F3 decreased hepatic collagen contents and inhibited tumor growth. CONCLUSIONS: These results indicate that GP extracts and HH-F3 can protect the liver by suppressing tumor growth; consequently, these compounds could be considered for the treatment of HCC.

Gluconeogenesis, lipogenesis, and HBV replication are commonly regulated by PGC-1α-dependent pathway.

PGC-1α, a major metabolic regulator of gluconeogenesis and lipogenesis, is strongly induced to coactivate Hepatitis B virus (HBV) gene expression in the liver of fasting mice. We found that 8-Br-cAMP and glucocorticoids synergistically induce PGC-1α and its downstream targets, including PEPCK and G6Pase. Also, HBV core promoter activity was synergistically enhanced by 8-Br-cAMP and glucocorticoids. Graptopetalum paraguayense (GP), a herbal medicine, is commonly used in Taiwan to treat liver disorders. Partially purified fraction of GP (named HH-F3) suppressed 8-Br-cAMP/glucocorticoid-induced G6Pase, PEPCK and PGC-1α expression and suppressed HBV core promoter activity. HH-F3 blocked HBV core promoter activity via inhibition of PGC-1α expression. Ectopically expressed PGC-1α rescued HH-F3-inhibited HBV surface antigen expression, HBV mRNA production, core protein levels, and HBV replication. HH-F3 also inhibited fatty acid synthase (FASN) expression and decreased lipid accumulation by down-regulating PGC-1α. Thus, HH-F3 can inhibit HBV replication, gluconeogenesis and lipogenesis by down-regulating PGC-1α. Our study indicates that targeting PGC-1α may be a therapeutic strategy for treatment of HBV infections. HH-F3 may have potential use for the treatment of chronic hepatitis B patients with associated metabolic syndrome.

Effects of Scutellaria baicalensis Georgi on macrophage-hepatocyte interaction through cytokines related to growth control of murine hepatocytes.

The aim of this study is to elucidate the effects of Scutellaria baicalensis Georgi (SbG) extract and its constituents on macrophage-hepatocyte interaction in primary cultures. By using trans-well primary Kupffer cell culture or conditioned medium (CM) from murine macrophage RAW264.7 cell line (RAW cells), effects of SbG on hepatocyte growth were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and trypan blue exclusion assay. Cytokine production, antibody-neutralization studies, and molecular mechanisms of transforming growth factor (TGF)-beta1 gene expression were elucidated on SbG-treated RAW264.7 cells. In addition, recombinant human TGF-beta1 (r-human TGF-beta1) was added to elucidate the mechanisms of SbG effects on cultured hepatocytes. Immunohistochemistry using anti-NF-kappaB antibody was used to determine the possible signal transduction pathways in primary hepatocyte culture. The results showed that SbG stimulated the proliferation of cultured hepatocytes, possibly through NF-kappaB, but not of Toll-like receptor 4 activation; whereas SbG-RAW-CM and SbG in trans-well significantly suppressed the proliferation of hepatocytes. Antibody-neutralization studies revealed that TGF-beta1 was the main antimitotic cytokine in SbG-treated RAW cells CM. The growth stimulation effect of SbG on cultured hepatocytes was inhibited by exogenous administration of r-human TGF-beta1. Furthermore, SbG induced NF-kB translocation into the nuclei of cultured cells. In the RAW264.7 line, SbG and baicalin stimulated TGF-beta1 gene expression via NF-kappaB and protein kinase C activation. We conclude that SbG stimulates hepatocyte growth via activation of the NF-kappaB pathway and induces TGF-beta1 gene expression through the Kupffer cell-hepatocyte interaction, which subsequently results in the inhibition of SbG-stimulated hepatocyte growth.

Gene expression profiling predicts liver responses to a herbal remedy after partial hepatectomy in mice.

The aim of this study was to demonstrate that regenerating liver responses to a herbal remedy could be presented by gene expression profiling. Compositions of the ingredients in the remedy containing Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Wild (S/B remedy) were analyzed and quantified by high performance liquid chromatography. By using a 70% partial hepatectomy in BALB/c mice as an in vivo model, the effects of high dose (50 mg/kg) and low dose (1 mg/kg) S/B remedy were evaluated by cDNA microarray, followed by RT-PCR and real-time PCR confirmation. Factors affecting proliferative activities of mouse hepatocytes were measured by DNA flow cytometry, BrdU incorporation assay and serum interleukin-6 (IL-6) level. Based on global gene expression profiles, the results showed that the low dose S/B remedy down-regulated expression of immediate early genes and cell cycle-related genes, whereas the high dose had opposite effects. The gene expression was further verified by real-time RT-PCR. Proliferative activities, in terms of synthetic phase fractions and G2/M phase fractions, in vehicle, low dose, and high dose groups were 18.45+/-2.56%, 14.65+/-1.06%; 9.27+/-0.85%, 7.80+/-0.11%; and 18.90+/-2.17%, 22.95+/-0.25%, respectively. The serum IL-6 level was also dose-dependent in both low and high dose S/B remedy-treated mice. We conclude that in vivo gene expression profiling correlates with liver responses to a herbal remedy, which provides a new direction for pharmaceutical studies on human diseases.

Selenite-induced survival of HuH7 hepatoma cells involves activation of focal adhesion kinase-phosphatidylinositol 3-kinase-Akt pathway and Rac1.

Selenium has been shown to sustain the growth of selected human hepatocellular carcinoma cell lines under serum-free conditions, but the detailed mechanism remained undetermined. In the present study, the molecular mechanism(s) involving sodium selenite (Na2SO3, Se) as a survival agent were determined. Selenite not only protects HuH7 cells from serum deprivation-induced apoptosis, it also supports its long-term growth in sodium selenite (10(-7)m) supplemented serum-free medium. The anti-apoptotic effect correlates with activation of focal adhesion kinase and the phosphatidylinositol 3-kinase (PI3K)-Akt kinase pathway. Using HuH7 cells stably transfected with a constitutively active Akt kinase and PI3K inhibitor LY294002, selenite-induced cell survival was shown to be PI3K-Akt-dependent. Parallel changes included a significant reduction in the intracellular reactive oxygen species content, the reversal of DNA fragmentation, and the suppression of caspase and apoptosis signal-regulating kinase 1 activities. HuH7 cells stably expressing a Rac1 mutant N17 (Rac1N17-HuH7) are refractory to selenite treatment. In these cells selenite supplement neither triggers Akt activation nor supports cell proliferation. Participation of Rac1 activation in this event is supported by the fact that selenite treatment drastically enhanced activation of Rac1. The exact link between selenite treatment, Rac1 activation, and activation of the focal adhesion kinase-PI 3-kinase, however, remains to be characterized. The mitogenic signaling mediated by selenite may involve unconventional growth stimuli including higher glutathione peroxidase 1 activity and higher transcription levels of selenoprotein P. The selenium-HuH7 system we have established thus provides a unique tool that will allow the biological role of selenite in growth regulation of hepatocytes to be studied in detail.