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(−)-Oleocanthal inhibits growth and metastasis by blocking activation of STAT3 in human hepatocellular carcinoma

Abstract

We explored the anti-cancer capacity of (−)-oleocanthal in human hepatocellular carcinoma (HCC). (−)-Oleocanthal inhibited proliferation and cell cycle progression and induced apoptosis in HCC cells in vitroand suppressed tumor growth in an orthotopic HCC model. (−)-Oleocanthal also inhibited HCC cell migration and invasion in vitro and impeded HCC metastasis in an in vivo lung metastasis model. ( )-Oleocanthal acted by inhibiting epithelial-mesenchymal transition (EMT) through downregulation Twist, which is a direct target of STAT3. (−)-Oleocanthal also reduced STAT3 nuclear translocation and DNA binding activity, ultimately downregulating its downstream effectors, including the cell cycle protein Cyclin D1, the anti-apoptotic proteins Bcl-2 and survivin, and the invasion-related protein MMP 2. Overexpression of constitutively active STAT3 partly reversed the anti cancer effects of (−)-oleocanthal, which inhibited STAT3 activation by decreasing the activities of JAK1 and JAK2 and increasing the activity of SHP-1. These data suggest that (−)-oleocanthal may be a promising candidate for HCC treatment.

Results

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Effects of anti-proliferation and pro-apoptosis by (−)-oleocanthal in an orthotopic tumor model of HCC in vivo.

(A) Representative images of mice from bioluminescent imaging at the first, third and fifth week after (−)-oleocanthal treatment, respectively. The mice were sacrificed at the end of treatment and representative images of gross specimen were shown. (B) Quantification of the tumor growth based on the luciferase intensity. (C) The tumor volumes were measured with vernier calipers. (D) Immunohistochemical analysis of Ki-67 for cell proliferation in tumor tissues. Ki-67-positive cells were counted to evaluate the proliferation index. Scale bars = 200 μm. (E) TUNEL analysis was used to detect the apoptosis in tumor tissues. TUNEL-positive cells were counted to calculate the apoptosis index. Scale bars = 200 μm. Data was presented as the means ± SD of three independent experiments. * compared with control, P < 0.05. ** compared with control, P < 0.01. *** compared with control, P < 0.001.

(−)-Oleocanthal inhibits HCC migration and invasion in vitro and in vivo

To explore the effect of (−)-oleocanthal on cell motility, Huh-7 and HepG2 cells were treated with 10 or 15 μM (−)-oleocanthal; these doses did not affect cell viability. (−)-Oleocanthal decreased Huh-7 and HepG2 cell migration ability in a wound-healing assay (Figure  (Figure4A).4A). (−)-Oleocanthal also suppressed Huh-7 and HepG2 cell invasion ability in a matrigel-coated transwell assay (Figure  (Figure4B).4B). To further investigate the effects of (−)-oleocanthal on HCC in vivo, we injected luciferase-expressing HCCLM3 cells into the tail veins of nude mice and monitored tumor metastasis using bioluminescence imaging. Illumination signals were stronger in control group than in the (−)-oleocanthal-treated group (Figure  (Figure4C4C and  and4E).4E). At the end of treatment, the mice were sacrificed and lungs were excised to perform hematoxylin and eosin staining. The (−)-oleocanthal-treated group had fewer and smaller lung metastases compared to the control group (Figure  (Figure4D4D and  and4F4F).

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(−)-Oleocanthal inhibits migration and invasion abilities of HCC in vitro and in vivo

(A) Representative images of cell migration for Huh-7 and HepG2 cells using wound-healing assay after the treatment with 10 μM of (−)-oleocanthal (left panel). The wound closure was quantified at 24 h and 48 h post-wound by measuring the migrated area (right panel). Scale bar = 50 μm. (B) Representative images of invasion assay for Huh-7 and HepG2 cells after the pre-treatment with increasing doses of (−)-oleocanthal for 24 h (top panel). The number of invaded cells was counted (bottom panel). Scale bar = 100 μm. (C) Representative images of mice from bioluminescent imaging at the sixth and eighth week, respectively. (D) The mice were sacrificed and lungs were excised at the end of treatment. Representative images of gross specimen were shown in the top and middle panel. Hematoxylin and eosin staining of lung tissue samples from the different experimental groups were shown in the bottom panel. Black scale bar = 100 μm. Red scale bar = 0.5 cm. (E) Quantification of the tumor growth based on the luciferase intensity. (F) Number of metastatic lung foci was detected in each group. Data was presented as the means ± SD of three independent experiments. ** compared with control, P < 0.01. *** compared with control, P < 0.001.

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Authors: Tiemin Pei,#1 Qinghui Meng,#1 Jihua Han,#1 Haobo Sun,1 Long Li,1 Ruipeng Song,1 Boshi Sun,1 Shangha Pan,1Desen Liang,1 and Lianxin Liu
Source: NCBI