Evaluation of the Medicinal Herb <i>Graptopetalum paraguayense</i> as a Treatment for Liver Cancer

<div><p>Background</p><p>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.</p><p>Methods and Findings</p><p>We demonstrate that an extract of <i>Graptopetalum paraguayense</i> (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 <i>Rhodiola rosea</i>. 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.</p><p>Conclusions</p><p>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.</p></div

GP extracts and their fractions regulate oncogenic protein expression in HCC cell lines.

<p>Huh7 and Mahlavu cells were treated with GP extracts prepared in water, butanol, acetone, methanol, 100% ethanol, 70% ethanol, 50% ethanol, 100% DMSO, or 30% DMSO at concentrations of 100, 150, 250, 500, 750, 1000 and 1500 μg/ml for 72 hrs. The viability of the treated cells was determined using MTT assays. The 30% DMSO GP extracts was associated with the greatest inhibition of growth of Huh7 and Mahlavu cells after 72 hrs. (B) Huh7 and Mahlavu cells were treated with concentrations of 0, 250, 500, 750 and 1000 μg/ml of the 30% DMSO GP extracts for 24, 48, and 72 hrs, and MTT assays were conducted. The IC₅₀ concentrations of the 30% DMSO GP extracts for growth inhibition of the Huh7 and Mahlavu cells were approximately 500 and 250 μg/ml, respectively, after 72 hrs of treatment. (C) HepG2 and Huh7 cells were treated with the GP extracts prepared in 30% DMSO, water or butanol (BuOH) for 48 hrs. The levels of AURKA, AURKB and FLJ10540 were measured by Western blot analysis. The expression levels of AURKA, AURKB and FLJ10540 in HepG2 and Huh7 cells were suppressed by the 30% DMSO GP extracts but not by the fractions prepared in water or butanol. (D) HepG2 cells were treated with 75 ng/ml of the synchronizing agent nocodazole (NOC) for 18 hrs; the cells were then treated with 750 μg/ml of the 30% DMSO GP extracts or vehicle control (30% DMSO) for another 3 hrs. Western blotting was performed using anti-FLJ10540, anti-AURKA and anti-AURKB antibodies. (E) HepG2 cells were treated with the 30% DMSO GP extracts and its fractions (HH-F1, HH-F2, HH-F3 and HH-F4) for 3 hrs. AURKA and AURKB expression levels in HepG2 cells were suppressed by the HH-F3 fraction but not by the other fractions. (F) Huh7, Mahlavu and PLC5 cells were treated with various concentrations of HH-F3 for 48 hrs. The expression of AURKA and FLJ10540 proteins was assessed by immunoblot analysis. HH-F3 reduced AURKA and FLJ10540 protein levels in a concentration-dependent manner.</p

The HH-F3 fraction inhibits the growth of HCC cells.

<p>(A) Human hepatocyte were treated with various concentrations of 30% DMSO GP extracts/HH-F3 for 72 hrs. Data are expressed as the mean ± standard deviation (SD) of three independent experiments. Huh7, Mahlavu and PLC5 cells were treated with 5, 25, 50 and 75 μg/ml of the HH-F3 fraction at for 24, 48 and 72 hrs then subjected to the MTT assay (B) and trypan blue assay (C). The IC₅₀ concentrations of HH-F3 for the growth inhibition of Huh7, Mahlavu and PLC5 cells were approximately 50, 37.5 and 75 μg/ml, respectively, after 72 hrs of treatment.</p

HH-F3 induces apoptosis in HCC cells.

<p>Huh7 and Mahlavu cells were treated with 5, 25 and 50 μg/ml of HH-F3 for 48 hrs. The cells were stained with propidium iodide (PI) and the DNA content of the cells was analyzed by flow cytometry to identify the distribution of sub-G1 cells in the cell cycle (*P < 0.05, **P < 0.005). (B) The cell lysates treated with the HH-F3 fraction were subjected to immunoblot analysis for anti-cleaved caspase9, anti-cleaved caspase-3 and cleaved PARP. The expression levels of cleaved caspase-3, caspase-9 and PARP were increased, indicating that the cells had undergone apoptosis. HH-F3 treatment also resulted in the down-regulation of the expression of BCL2 and BCL-XL. (C) The expression of p-AKT-Ser⁴⁷³ and p-p70S6K was down-regulated and the expression of PTEN was up-regulated by HH-F3 in a concentration-dependent manner, as shown by the results of the immunoblot analysis with anti-p70S6K, anti-p-p70S6K, anti-p-AKT-Ser⁴⁷³, anti-AKT and anti-PTEN antibodies.</p

Disruption of mitochondrial membrane potential and increase ROS generation in HCC cells treated with 30% DMSO GP extracts and HH-F3.

<p>(A-B) The mitochondrial membrane potential (ΔΨ) in Huh7 and Mahlavu cells was analyzed using the JC-1 mitochondrial membrane potential assay. The ΔΨ of the cells was lower in the HCC cells treated with the 30% DMSO GP extracts and HH-F3 than in the control HCC cells. The treatment effect as a function of time (0.25, 0.5, 1, 3, 6, 12, and 24 hrs) is shown (n = 3). (C-D) Intracellular peroxide levels, as measured by DCFH fluorescence, were increased after treatment with different concentrations of 30% DMSO GP extracts and HH-F3. The treatment effect as a function of time (0.25, 0.5, 1, 3, 6, 12, and 24 hrs) is shown (n = 3).</p

GP extracts and HH-F3 alleviate DEN-induced liver fibrosis and cancer in rats.

<p>The animals were divided into four groups and provided with tap water only (normal group) or with DEN-containing water (liver disease group). (A) Decreased bile flow in cirrhotic animals. The bile flow rate was recorded to measure liver function. *P < 0.05; ANOVA. (B) The spleen weight-to-body weight (BW) ratio of the animals in the high-dose GP treatment group was significantly lower than that of the animals in the DEN group (P < 0.05, ANOVA). (C) Treatment with GP extracts and HH-F3 significantly decreased liver hydroxyproline levels. (D) The percentages of α-SMA (+) areas were determined with a digital camera system using the 10 fields with the densest staining. *P < 0.05; **P < 0.005, ANOVA. (E) Tumor burdens were expressed as the sum of the volume of all tumor nodules. **P < 0.005, ***P < 0.001 relative to the DEN group.</p

Flux balance analysis predicts Warburg-like effects of mouse hepatocyte deficient in miR-122a

<div><p>The liver is a vital organ involving in various major metabolic functions in human body. MicroRNA-122 (miR-122) plays an important role in the regulation of liver metabolism, but its intrinsic physiological functions require further clarification. This study integrated the genome-scale metabolic model of hepatocytes and mouse experimental data with germline deletion of <i>Mir122a</i> (<i>Mir122a</i>^{<i>–/–</i>}) to infer Warburg-like effects. Elevated expression of <i>MiR-122a</i> target genes in <i>Mir122a</i>^{<i>–/–</i>}mice, especially those encoding for metabolic enzymes, was applied to analyze the flux distributions of the genome-scale metabolic model in normal and deficient states. By definition of the similarity ratio, we compared the flux fold change of the genome-scale metabolic model computational results and metabolomic profiling data measured through a liquid-chromatography with mass spectrometer, respectively, for hepatocytes of 2-month-old mice in normal and deficient states. The <i>Ddc</i> gene demonstrated the highest similarity ratio of 95% to the biological hypothesis of the Warburg effect, and similarity of 75% to the experimental observation. We also used 2, 6, and 11 months of mir-122 knockout mice liver cell to examined the expression pattern of DDC in the knockout mice livers to show upregulated profiles of DDC from the data. Furthermore, through a bioinformatics (LINCS program) prediction, BTK inhibitors and withaferin A could downregulate DDC expression, suggesting that such drugs could potentially alter the early events of metabolomics of liver cancer cells.</p></div

Metabolic reprogramming triggered by overexpression of <i>Ddc</i>.

<p>(A) Based on Recon2-hepatocyte model, three enzymatic reactions (red lines) are catalyzed by overexpression of Ddc. As a result of metabolic reprogramming, the fold change (FC) of the concentration of glutamine and glutamate are increased by 4.54 and 2.53, respectively (shown in blue). (B) Elevated expression of Ddc shift liver metabolism toward glycolysis and lactate synthesis in agreement with the hypothesis of the Warburg effect. The (FC) of metabolites in glycolysis, TCA cycle, and glutamine metabolism pathways are indicated in green (decrease) and red (increase).</p

Downregulation of DDC expression by treatment with ibrutinib, LFM-A13, and withaferin-A.

<p>(A) Schematic illustration of similar gene expression signatures between <i>DDC</i> shRNA and chemical compounds. (B) We queried the <i>DDC</i> shRNA gene signature via LINCS database and found BTK inhibitor (LFM-A13), withaferin A, and several compounds shared similar gene expression profiles with DDC shRNA gene signature. Score_best4 and score_best6 are the mean connectivity scores across the four and six cell lines, respectively, in which the perturbagen connected most strongly to the query (<i>DDC</i> shRNA). (C, D, E) DDC, P-BTK, and BTK were upregulated in liver tissues from mir-122 knockout mice. (F) Huh7 cells were treated with various concentrations of ibrutinib, LFM-A13, and withaferin-A for 24–72 hours, respectively. Huh7 cells were treated with (G) 10 and 20 μM ibrutinib or 5 μM sorafenib, (H) 5–20 μM LFM-A13, and (I) 1 and 2 μM withaferin A or 5 μM sorafenib for 24 hours. Cell lysates were subjected to western blot analysis. DDC was downregulated by the treatment of 20 μM ibrutinib and 2 μM withaferin A, but not the treatment of sorafenib. Both DDC and BTK were downregulated by the treatment of LFM-A13 in Huh7 cells.</p

The fold changes of the 35 intracellular metabolites from the LC/MS analysis and 16 miR122a target enzyme computational predictions.

<p>The fold changes of the 35 intracellular metabolites from the LC/MS analysis and 16 miR122a target enzyme computational predictions.</p