Lenvatinib Synergistically Promotes Radiation Therapy in Hepatocellular Carcinoma by Inhibiting Src/STAT3/NF-κB-Mediated Epithelial-Mesenchymal Transition and Metastasis.

PURPOSE: This study suggested that lenvatinib may incapacitate hepatocellular carcinoma (HCC) to radiation treatment by abrogating radiation-induced Src/signal transducer and the activator of transcription 3 signaling (STAT3)/nuclear factor-κB (NF-κB) to escalate radiation-induced extrinsic and intrinsic apoptosis. These findings uncover the role of targeting Src and its arbitrating epithelial-mesenchymal transition (EMT), which could increase the anti-HCC efficacy of radiation therapy (RT). Lenvatinib and sorafenib are multikinase inhibitors used to treat HCC. Lenvatinib is noninferior to sorafenib in the therapeutic response in HCC. However, whether lenvatinib intensifies the anti-HCC efficacy of RT is ambiguous. Several oncogenic kinases and transcription factors, such as Src, STAT3, and NF-κB, enhance the radiosensitivity of cancers. Therefore, we aimed to investigate the roles of the Src/STAT3/NF-κB axis in HCC after RT treatment and assessed whether targeting Src by lenvatinib may enhance the effectiveness of RT. METHODS AND MATERIALS: Hep3B, Huh7, HepG2, and SK-Hep1 HCC cells and 2 types of animal models were used to identify the efficacy of RT combined with lenvatinib. Cellular toxicity, apoptosis, DNA damage, EMT/metastasis regulation, and treatment efficacy were validated by colony formation, flow cytometry, Western blotting, and in vivo experiments, respectively. Knockdown of Src by siRNA was also used to validate the role of Src in RT treatment. RESULTS: Silencing Src reduced STAT3/NF-κB signaling and sensitized HCC to radiation. Lenvatinib reversed radiation-elicited Src/STAT3/NF-κB signaling while enhancing the anti-HCC efficacy of radiation. Both lenvatinib and siSrc promoted the radiation effect of cell proliferation on suppression, inhibition of the invasion ability, and induction of apoptosis in HCC. Lenvatinib also alleviated radiation-triggered oncogenic and EMT-related protein expression. CONCLUSIONS: Our findings uncovered the role of the Src/STAT3/NF-κB regulatory axis in response to radiation-induced toxicity and confirmed Src as the key regulatory molecule for radiosensitization of HCC evoked by lenvatinib.

DNA damage and NF-κB inactivation implicate glycyrrhizic acid-induced G1 phase arrest in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the most frequently occurring liver malignancy in Asia. Glycyrrhizic acid is known to reduce the risk of HCC formation in patients with chronic hepatitis C. To identify whether glycyrrhizic acid may play a role in anti-HCC therapy as an adjuvant is important. However, the inhibitory effect of glycyrrhizic acid on cell cycle progression in HCC cells and the mechanism of such have not been fully elucidated. This study used the comet assay, cell cycle analysis, immunofluorescence staining, the TUNEL assay, and Western blotting to identify the anti-HCC role of glycyrrhizic acid. Glycyrrhizic acid may induce DNA damage, apoptosis, activation of ATM, and expression of p21, and p27 in HCC cells. In addition, glycyrrhizic acid may also induce G1 phase arrest and suppress NF-κB-mediated Cyclin D1 expression. DNA damage and NF-κB inactivation may be associated with glycyrrhizic acid-induced G1 phase arrest in HCC cells.

Glycyrrhizic Acid Modulates Apoptosis through Extrinsic/Intrinsic Pathways and Inhibits Protein Kinase B- and Extracellular Signal-Regulated Kinase-Mediated Metastatic Potential in Hepatocellular Carcinoma In Vitro and In Vivo.

A previous study presented that glycyrrhizic acid as the hepatoprotective agent inhibits total parenteral nutrition-associated acute liver injury in rats. However, the anticancer effect and mechanism of glycyrrhizic acid in human hepatocellular carcinoma (HCC) is ambiguous. The purpose of the present study was to investigate the effect of glycyrrhizic acid on apoptosis dysregulation and metastatic potential in HCC in vitro and in vivo. Both SK-Hep1 and Hep3B cells were treated with different concentrations of glycyrrhizic acid for 24 or 48h. SK-Hep1/luc2 tumor-bearing mice were treated with vehicle or glycyrrhizic acid (50mg/kg/day by intraperitoneal injection) for 7 days. Tumor cells growth, apoptotic, and metastatic signaling transduction were evaluated by using MTT assay, digital caliper, bioluminescence imaging (BLI), flow cytometry, western blotting assay, and immunohistochemistry (IHC) staining. The results demonstrated glycyrrhizic acid significantly inhibits tumor cell growth, cell invasion, and expression of AKT (Ser473), extracellular-signal-regulated kinase (ERK), epidermal growth factor receptor (EGFR) phosphorylation, anti-apoptotic and metastatic proteins in HCC in vitro and in vivo. Glycyrrhizic acid also significantly triggered apoptosis and extrinsic/intrinsic apoptotic signaling transduction. In addition, PD98059 (ERK inhibitor) and LY294002 (AKT inhibitor) obviously reduced cell invasion and expression of metastasis-associated proteins. Taken together, these results indicated that glycyrrhizic acid induces apoptosis through extrinsic/intrinsic apoptotic signaling pathways and diminishes EGFR/AKT/ERK-modulated metastatic potential in HCC in vitro and in vivo.

Apoptosis induction and ERK/NF-κB inactivation are associated with magnolol-inhibited tumor progression in hepatocellular carcinoma in vivo.

Although hepatitis B and/or hepatitis C virus were recognized as major risk factor for the development of hepatocellular carcinoma (HCC), certain occupational, environmental, and lifestyle factors also play key roles in HCC tumorigenesis. Moreover, in molecular signaling route, extracellular signal-regulated kinase (ERK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was found to be overexpressed and linked to poor prognosis in HCC. Thus, to identify possible nature compound that can suppress ERK/NF-κB may be benefit to HCC patient. Magnolol, a natural compound derived from herbal plant Magnolia officinalis, has been recognized as a liver protection and antitumor reagent. However, whether magnolol-inhibited HCC progression correlates with disruption of ERK/NF-κB signaling is remained unclear. In this studies, we performed SK-Hep1/luc2 HCC bearing animal model to investigate the anticancer efficacy and mechanism of magnolol on tumor progression. Tumor size and tumor growth rate were dramatically suppressed after treatment of magnolol. In addition, expression of phospho-ERK (p-ERK), NF-κB p65 (Ser536), and tumor progression-associated proteins, such as matrix metallopeptidase 9 (MMP-9), vascular endothelial growth factor (VEGF), X-linked inhibitor of apoptosis protein (XIAP), and CyclinD1 were all significantly decreased by magnolol. Most important, major extrinsic and intrinsic apoptosis signaling factors, including active caspase-8 and caspase-9 were both enhanced by magnolol. This study indicated that apoptosis induction through extrinsic/intrinsic pathways and blockage of ERK/NF-κB activation were associated with magnolol-inhibited tumor progression in HCC in vivo.

Amentoflavone Enhances the Therapeutic Efficacy of Sorafenib by Inhibiting Anti-apoptotic Potential and Potentiating Apoptosis in Hepatocellular Carcinoma In Vivo.

BACKGROUND/AIM: In a previous study, we showed that amentoflavone promotes sorafenib-induced apoptosis in hepatocellular carcinoma (HCC) cells in vitro. However, whether amentoflavone augments anticancer efficacy of sorafenib in HCC in vivo is unknown. The aim of the present study was to verify the anticancer effect of amentoflavone combined with sorafenib in HCC in vivo. MATERIALS AND METHODS: HCC SK-Hep1 tumor-bearing mice were treated with vehicle, sorafenib, amentoflavone, or combination for 14 days, respectively. Effect of sorafenib, amentoflavone, or their combination on tumor growth, anti-apoptotic potential, apoptotic signaling and general toxicity were evaluated with digital caliper, immunohistochemistry staining and body weight. RESULTS: Our results demonstrated that amentoflavone significantly enhanced sorafenib-inhibited tumor growth and expression of ERK/AKT phosphorylation and anti-apoptotic proteins compared to single-agent treatment. Additionally, amentoflavone also triggered sorafenib-induced apoptosis through extrinsic and intrinsic apoptotic pathways. CONCLUSION: Amentoflavone boosts therapeutic efficacy of sorafenib through blockage of anti-apoptotic potential and induction of apoptosis in HCC in vivo.

Glycyrrhizin represses total parenteral nutrition-associated acute liver injury in rats by suppressing endoplasmic reticulum stress.

Total parenteral nutrition (TPN) is an artificial way to support daily nutritional requirements by bypassing the digestive system, but long-term TPN administration may cause severe liver dysfunction. Glycyrrhizin is an active component of licorice root that has been widely used to treat chronic hepatitis. The aim of this study is to investigate the hepatoprotective effect of glycyrrhizin on TPN-associated acute liver injury in vivo. Liver dysfunction was induced by intravenous infusion of TPN at a flow rate of 20 mL/kg/h for three h in Sprague Dawley rats. The rats were pretreated with Glycyrrhizin (1, 3 and 10 mg/kg intravenously). After receiving TPN or saline (control group) for three h, the rats were sacrificed, blood samples were collected for biochemical analyses and liver tissue was removed for histopathological and immunohistochemical examination. We found that aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB) and triglyceride (TG) levels were significantly increased in the TPN group without glycyrrhizin pretreatment and decreased in the glycyrrhizin-pretreated TPN group in a dose-dependent manner. The stained liver sections showed that glycyrrhizin relieved acute liver injury. The upregulation of serum protein biomarkers of reactive nitrogen species, including nitrotyrosine and inducible NO synthase (iNOS), were attenuated by glycyrrhizin pretreatment. Levels of endoplasmic reticulum (ER) stress factors, such as phosphorylation of JNK1/2, p38 MAPK and CHOP, were decreased by glycyrrhizin pretreatment. In summary, our results suggest that glycyrrhizin decreases TPN-associated acute liver injury factors by suppressing endoplasmic reticulum stress and reactive nitrogen stress.