FBX8 Acts as an Invasion and Metastasis Suppressor and Correlates with Poor Survival in Hepatocellular Carcinoma

<div><p>Background</p><p>F-box only protein 8 (FBX8), a novel component of F-box proteins, is lost in several cancers and has been associated with invasiveness of cancer cells. However, its expression pattern and role in the progression of hepatocellular carcinoma remain unclear. This study investigated the prognostic significance of FBX8 in hepatocellular carcinoma samples and analyzed FBX8 function in hepatocellular carcinoma cells by gene manipulation.</p><p>Methodology</p><p>The expression of FBX8 was detected in 120 cases of clinical paraffin-embedded hepatocellular carcinoma tissues, 20 matched pairs of fresh tissues and five hepatocellular carcinoma cell lines by immunohistochemistry with clinicopathological analyses, real-time RT-PCR or Western blot. The correlation of FBX8 expression with cell proliferation and invasion in five HCC cell lines was analyzed. Moreover, loss of function and gain of function assays were performed to evaluate the effect of FBX8 on cell proliferation, motility, invasion <i>in vitro</i> and metastasis <i>in vivo</i>.</p><p>Conclusions</p><p>We found that FBX8 was obviously down-regulated in HCC tissues and cell lines (P<0.05). The FBX8 down-regulation correlated significantly with poor prognosis, and FBX8 status was identified as an independent significant prognostic factor. Over-expression of FBX8 decreased proliferation, migration and invasion in HepG2 and 97H cells, while knock-down of FBX8 in 7721 cells showed the opposite effect. FBX8 negatively correlated with cell proliferation and invasion in 7701, M3, HepG2 and 97H cell lines. In vivo functional assays showed FBX8 suppressed tumor growth and pulmonary metastatic potential in mice. Our results indicate that down-regulation of FBX8 significantly correlates with invasion, metastasis and poor survival in hepatocellular carcinoma patients. It may be a useful biomarker for therapeutic strategy and control in hepatocellular carcinoma treatment.</p></div

FBX8 inhibits proliferation of HCC cells in vitro.

<p>(A) FBX8 expression in FBX8 overexpressing 97H and HepG2 cells by real-time RT-PCR. (B) FBX8 expression in FBX8 overexpressing 97H and HepG2 cells by Western blotting. (C) FBX8 expression in FBX8 depleting 7721 cells by real-time RT-PCR. (D) FBX8 expression in FBX8 depleting 7721 cells by Western blotting. (E) Effect of ectopic FBX8 on cell proliferation in vitro by MTT assay. (F) Effect of FBX8 knockdown on cell proliferation in vitro by MTT assay. (G) Effect of ectopic FBX8 on cell proliferation in vitro by colony formation assay. *P<0.05.</p

FBX8 inhibits tumor growth and metastasis of HCC cells in vivo.

<p>(A, B) Subcutaneous tumors of mice injected with 97H/mock and 97H/FBX8 cells. (C) Effects of FBX8 on subcutaneous tumor growth by MTT assay. (D) Pulmonary metastases of mice injected with 97H/mock and 97H/FBX8 cells. (E) Number of metastatic intestinal or hepatic nodules per mice. The number of metastatic nodules in individual mice was counted under the microscope. * P<0.05.</p

FBX8 is down-regulated in HCC cell lines and fresh HCC tissues.

<p>(A) Kaplan-Meier survival analysis of primary HCC patients with high and low FBX8 expressions. (B) Real-time RT-PCR analysis of FBX8 in six cell lines. The relative mRNA levels with the use of control LO2 were normalized to 1. (C) Western blotting analysis of FBX8 in six cell lines. (D) Real-time PCR analysis of FBX8 expression in 8 paired HCC tissues. (E) Western blotting analyses of FBX8 expression in the same 8 paired HCC tissues. N = normal mucosa and T = tumor.</p

FBX8 is down-regulated in clinical paraffin-embedded HCC tissues.

<p>(A) Strong positive expression of FBX8 in adjacent normal livers (×100, ×400). (B) Positive expression of FBX8 in the cirrhotic livers (×100, ×400). (C) Week expression of FBX8 in HCC tissues while strong positive expression in adjacent livers (×200, ×400). (D) Negative expression of FBX8 in HCC tissues compared with strong positive expression in adjacent livers (×200, ×400). (E) Negative expression of FBX8 in HCC tissues with low differentiation (×200, ×400). (F) Weak expression of FBX8 in HCC tissues with high differentiation (×200, ×400).</p

BTG3 is down-regulated in clinical paraffin-embedded HCC tissues and a prognostic factor for poor overall survival in HCC patients.

<p>(A) Strong positive expression of BTG3 in adjacent normal livers (×200, ×400). (B) Positive expression of BTG3 in the cirrhotic livers (×100, ×400). (C) Negative expression of BTG3 in HCC tissues compared with strong positive expression in adjacent livers (×200, ×400). (D) Week expression of BTG3 in HCC tissues while strong positive expression in adjacent livers (×200, ×400). (E) Negative expression of BTG3 in HCC tissues with low differentiation (×200, ×400). (F) Weak expression of BTG3 in HCC tissues with high differentiation (×200, ×400). (G) Kaplan-Meier survival analysis of primary HCC patients with high and low BTG3 expressions.</p

BTG3 knockdown promotes proliferation, invasion and G1/S phase transition of HCC cells in vitro.

<p>(A) BTG3 expression in BTG3 depleting 97H and 7701 cells by Western blot. (B) Effect of BTG3 knockdown on cell proliferation in vitro by MTT assay. (C) Effect of BTG3 knockdown on cell invasion in vitro by using Boyden chambers. (D) Morphological comparison of BTG3-depleting cells and control cells penetrating the artificial basement membrane was shown. (E) Effect of BTG3 kncokdown on cell cycle transition in vitro by flow cytometry. (F) Expression levels of Cyclin D1 and p27 in BTG3-overexpressing and depleting cells by Western blot. Tubulin was shown as a control.</p

BTG3 inhibits proliferation, invasion and induces G1/S cycle arrest of HCC cells in vitro.

<p>(A) BTG3 expression in FBX8 over-expressing 7721 and HepG2 cells by Western blot. Tubulin was shown as a control. (B) Effect of ectopic BTG3 on cell proliferation in vitro by MTT assay. (C) Effect of ectopic BTG3 on cell invasion in vitro by using Boyden chambers. (D) Morphological comparison of BTG3-overexpressing cells and control cells penetrating the artificial basement membrane was shown. (E) Effect of ectopic BTG3 on cell cycle transition in vitro by flow cytometry.</p

Promoter hypermethylation contributes to the transcriptional silencing of BTG3 in HCC.

<p>(A) Real-time PCR analysis of BTG3 expression after 5Aza-C treatment in HepG2 and 7721 cells. The relative mRNA levels with the use of control cells without 5Aza-C treatment were normalized to 1. (B) Western blotting analysis of BTG3 expression in HepG2 and 7721 cells after 5Aza-C treatment. Tubulin was shown as a control. (C) DNA sequencing results of methylation sites in LO2 cells. (D) DNA sequencing results of methylation sites in HepG2 and 97H cells. (E) Ratio of methylation sites of HepG2 or 97H cells was compared with that of LO2 cells. </p

Design, Synthesis, X‑ray Crystallographic Analysis, and Biological Evaluation of Thiazole Derivatives as Potent and Selective Inhibitors of Human Dihydroorotate Dehydrogenase

Human dihydroorotate dehydrogenase (<i>Hs</i>DHODH) is a flavin-dependent mitochondrial enzyme that has been certified as a potential therapeutic target for the treatment of rheumatoid arthritis and other autoimmune diseases. On the basis of lead compound <b>4</b>, which was previously identified as potential <i>Hs</i>DHODH inhibitor, a novel series of thiazole derivatives were designed and synthesized. The X-ray complex structures of the promising analogues <b>12</b> and <b>33</b> confirmed that these inhibitors bind at the putative ubiquinone binding tunnel and guided us to explore more potent inhibitors, such as compounds <b>44</b>, <b>46</b>, and <b>47</b> which showed double digit nanomolar activities of 26, 18, and 29 nM, respectively. Moreover, <b>44</b> presented considerable anti-inflammation effect in vivo and significantly alleviated foot swelling in a dose-dependent manner, which disclosed that thiazole-scaffold analogues can be developed into the drug candidates for the treatment of rheumatoid arthritis by suppressing the bioactivity of <i>Hs</i>DHODH