Preclinical Trials for Prevention of Tumor Progression of Hepatocellular Carcinoma by LZ-8 Targeting c-Met Dependent and Independent Pathways

<div><p>Hepatocellular carcinoma (HCC) is among the most lethal cancers. Mounting studies highlighted the essential role of the HGF/c-MET axis in driving HCC tumor progression. Therefore, c-Met is a potential therapeutic target for HCC. However, several concerns remain unresolved in c-Met targeting. First, the status of active c-Met in HCC must be screened to determine patients suitable for therapy. Second, resistance and side effects have been observed frequently when using conventional c-Met inhibitors. Thus, a preclinical system for screening the status of c-Met signaling and identifying efficient and safe anti-HCC agents is urgently required. In this study, immunohistochemical staining of phosphorylated c-Met (Tyr1234) on tissue sections indicated that HCCs with positive c-Met signaling accounted for approximately 46% in 26 cases. Second, many patient-derived HCC cell lines were established and characterized according to motility and c-Met signaling status. Moreover, LZ8, a medicinal peptide purified from the herb Lingzhi, featuring immunomodulatory and anticancer properties, was capable of suppressing cell migration and slightly reducing the survival rate of both c-Met positive and negative HCCs, HCC372, and HCC329, respectively. LZ8 also suppressed the intrahepatic metastasis of HCC329 in SCID mice. On the molecular level, LZ8 suppressed the expression of c-Met and phosphorylation of c-Met, ERK and AKT in HCC372, and suppressed the phosphorylation of JNK, ERK, and AKT in HCC329. According to receptor array screening, the major receptor tyrosine kinase activated in HCC329 was found to be the epidermal growth factor receptor (EGFR). Moreover, tyrosine-phosphorylated EGFR (the active EGFR) was greatly suppressed in HCC329 by LZ8 treatment. In addition, LZ8 blocked HGF-induced cell migration and c-Met-dependent signaling in HepG2. In summary, we designed a preclinical trial using LZ8 to prevent the tumor progression of patient-derived HCCs with c-Met-positive or -negative signaling.</p></div

Comparison of cell morphologies and motilities among patients derived HCCs.

<p>The patient-derived HCC cells were established according to the description in Materials and Methods. Morphologies (A) and (B) motilities (by wound healing assay) of the indicated HCC cell lines were imaged using phase contrast microscopy (200X). (C) Quantitation for comparing the motility of the indicated HCCs. Relative motility of the HCCs was calculated; taking motility of HCC340 as 1.0. (**) and (*) represent statistical significance (p<0.005 and p<0.05, respectively, n = 4) for differences in motility between the indicated HCCs and the HCC340.</p

LZ8, not JNJ, prevents tumor metastasis of HCC329.

<p>HCC329 was inoculated onto the middle lobe of SCID mouse livers through orthotropic transplantation. Subsequently, the mice were injected intraperitoneally twice per week with DMEM medium, JNJ-38877605 (JNJ) 26.5 nmole/g per mouse, or LZ8 (4.0–20.0 μg/g per mouse). The mice were sacrificed 2 months after injection; intrahepatic metastasis was observed in the medium- and JNJ- but not LZ8-treated group. White arrow heads indicate the site of secondary tumors on the left and right liver lobes. Data were representative of two reproducible experiments.</p

Comparison of various signaling molecules in HCC cell lines and suppression of cell migration by JNK and MEK inhibitors.

<p>(A) Western blots of indicated signal molecules in the indicated HCC cell lines using GAPDH as a loading control. Data were representative of two reproducible experiments. (B) Cells were untreated or treated with SP (SP600125), JNK inhibitor, or PD (PD98059), and subjected to wound healing migration analysis for 24 h. Relative migration was calculated and taking the data of untreated HCC372 or HCC329 as 100%. (**) and (#) represent statistical significance (p<0.005 and p<0.05, respectively, n = 3) between the indicated inhibitor-treated sample and untreated HCC329 or HCC372 control group.</p

LZ8 prevented cell migration and reduced HCC survival.

<p>HCC329 and HCC372 cells were untreated (control) or treated with LZ8 (2.0 μg/mL) for 72 h (A and B), and LZ8 (2.0 μg/mL) or JNJ (26.5 nM) for 48 h (C). HepG2 cells were untreated (control), treated with 25 nM HGF with or without indicated inhibitors for 48 h (E). Doubling time determination (A) and cell cycle analysis (B) at 72 h, and wound healing migration analysis (C) and (E) of the HCCs were performed. In (A), relative doubling time was evaluated taking the data of untreated HCC372 or HCC329 as 100%. In (B), the cell counts of each cell cycle phase are depicted in the left panel (PI: propidium iodide), whereas the percentage of each phase is shown in the right panel. Data were the average of three experiments; CV = 5.8%. (D) and (F) are quantitative figures for (C) and (E), respectively. In (A) and (D), (**) and (*) (#) represent statistical significance (p<0.005 and p<0.05, respectively, n = 3) between the indicated inhibitor-treated samples and the untreated HCC329 or untreated HCC372 group. In (F), (**) represent statistical significance (p<0.005, n = 3) between the indicated HGF/inhibitor samples and the HGF-only group.</p

LZ8 block signal transduction in c-Met positive and negative HCCs and HGF-induced HepG2.

<p>HCC372 and HCC329 were treated with 2.0 μg/mL LZ8 for 4 h and 24 h, respectively (A), and HepG2 was untreated (control), treated with HGF only, or treated with HGF coupled with 2.0 μg/mL of LZ8 (C). Western blotting of indicated signal molecules was performed using GAPDH (A) and ERK (C) as loading controls. (B) and (D) are quantitative figures for (A) and (C), respectively. Relative band intensity of each molecule was calculated; taking the data of untreated HCC372 and HCC329 as 100% (B) and untreated HepG2 as 1.0 (D). In (B), (**) (^##) and (*) (^#) represent statistical significance (p<0.005 and p<0.05, respectively, n = 3) between the indicated inhibitor-treated sample and untreated HCC329 or HCC372 control group. In (D), (**) represent statistical significance (p<0.005, n = 3) between the indicated HGF/LZ8 cotreated and the HGF-only group.</p

Receptor array analysis for detecting RTK signaling in HCC329.

<p>Total cell lysate (300 μg/mL) of untreated HCC329 was used for analyzing the relative amounts of 49 phosphorylated RTKs by receptor array analysis, as described in Materials and Methods. The rectangles depicted by solid and dashed lines indicate the duplicated positions of p-EGFR and p-c-Met, respectively. The elliptic circles indicate the position of two positive controls.</p

Additional file 1: of Characterization of the hepatitis B virus DNA detected in urine of chronic hepatitis B patients

Urine DNA analysis by qPCR assays targeting multiple locations in the HBV genome. Raw data for Fig. 2. (XLSX 18 kb

Methylation status of the sense (S) and antisense (AS) strands of the promoter and the first exon region of the <i>GSTP1 gene</i> (Genebank accession #M24485, nt. 999–1387) of the DNA isolated from normal adult livers, fetal liver, and normal nonliver tissues.

<p>The open boxes indicate unmethylated CpG sites; the filled boxes indicate methylation detected.</p

Scatter plot distribution of serum AFP levels (x-axis) and the amount of methylated 5′-end of the <i>GSTP1</i> DNA (<i>mGTSP1</i>) (y-axis) for 115 HCC samples.

<p>Each circle represents the value for an individual HCC case. A vertical reference line intersects at an AFP value of 20 ng/ml. A horizontal reference line intersects right above the MSP value of 0 as the reference for undetectable (ND), which is less than 10 copies per assay. The number of HCC cases and the percent of the total HCC in each of four areas are indicated.</p