Chrysin-induced ERK1/2 Phosphorylation Enhances the Sensitivity of Human Hepatocellular Carcinoma Cells to Sorafenib.

BACKGROUND/AIM: Sorafenib is now standard treatment for advanced hepatocellular carcinoma (HCC). However, therapeutic efficacy is not as good as was predicted. Many efforts are being made to improve HCC sensitivity to sorafenib. Our previous study demonstrated that co-treatment with chrysin enhanced sorafenib sensitivity through inhibition of ATP-binding cassette super-family G member 2 (ABCG2). Whether there is another mechanism other than inhibition of ABCG2 underlying chrysin-mediated synergistic effect is still not completely elucidated. MATERIALS AND METHODS: Phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) was examined by western blot. Cell viability was examined by crystal violet staining. The importance of ERK1/2 phosphorylation was assessed by overexpression and blockage of mitogen-activated protein kinase kinase 1 (MEK1). RESULTS: Chrysin induced sustained ERK1/2 phosphorylation of HCC cells in both time- and dose-dependent manners. Overexpression of MEK1 enhanced, whereas blockage of MEK1 led to loss of chrysin-synergized sorafenib effect, through modulating ERK1/2 phosphorylation level. CONCLUSION: These results identify another novel mechanism underlying chrysin-mediated synergistic effect on sorafenib activity in HCC cells.

c-Met inhibition is required for the celecoxib-attenuated stemness property of human colorectal cancer cells.

Cyclooxygenase-2 (COX-2) is frequently overexpressed and enhances colorectal cancer (CRC) tumorigenesis, including cancer stem cell (CSC) regulation. Accordingly, nonsteroidal anti-inflammatory drugs (NSAIDs), inhibiting COX-1/2 activity, are viewed as potential drugs for CRC treatment. Accumulated evidence indicates that celecoxib has the most potency for antitumor growth among NSAIDs and the underlying mechanism is only partly dependent on COX-2 inhibition. However, the potency of these NSAIDs on CSC inhibition is still not known. In this study, we found that among these NSAIDs, celecoxib has the most potency for CSC inhibition of CRC cells, largely correlating to inhibition of c-Met, not COX-2. Further analysis reveals that c-Met activity was required for basal CSC property. Silence of c-Met blocked whereas overexpression of c-Met enhanced the celecoxib-inhibited CSC property. Collectively, these results not only first elucidate the mechanism underlying celecoxib-inhibited CSC but also indicate c-Met as a critical factor for the CSC property of CRC cells.

Chrysin Attenuates Cell Viability of Human Colorectal Cancer Cells through Autophagy Induction Unlike 5-Fluorouracil/Oxaliplatin.

Chemotherapeutic 5-fluorouracil (5-FU) combined with oxaliplatin is often used as the standard treatment for colorectal cancer (CRC). The disturbing side effects and drug resistance commonly observed in chemotherapy motivate us to develop alternative optimal therapeutic options for CRC treatment. Chrysin, a natural and biologically active flavonoid abundant in propolis, is reported to have antitumor effects on a few CRCs. However, whether and how chrysin achieves similar effectiveness to the 5-FU combination is not clear. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), western blotting, fluorescence microscopy, and reactive oxygen species (ROS) production were assayed. We found that chrysin exhibited similar inhibition of cell viability as the 5-FU combination in a panel of human CRC cells. Furthermore, the results showed that chrysin significantly increased the levels of LC3-II, an autophagy-related marker, in CRC cells, which was not observed with the 5-FU combination. More importantly, blockage of autophagy induction restored chrysin-attenuated CRC cell viability. Further mechanistic analysis revealed that chrysin, not the 5-FU combination, induced ROS generation, and in turn, inhibited the phosphorylation of protein kinase B (Akt) and mammalian target of rapamycin (mTOR). Collectively, these results imply that chrysin may be a potential replacement for the 5-FU and oxaliplatin combination to achieve antitumor activity through autophagy for CRC treatment in the future.

GPR56/ADGRG1 Activation Promotes Melanoma Cell Migration via NTF Dissociation and CTF-Mediated Gα12/13/RhoA Signaling.

GPR56/ADGRG1 is a versatile adhesion G protein-coupled receptor with diverse biological functions. GPR56 expression is variably detected in human melanoma cell lines and correlates inversely with the metastatic potential of melanoma lesions. GPR56 associates with the tetraspanins CD9 and CD81 on the melanoma cell surface. GPR56 activation by immobilized CG4 monoclonal antibody facilitates N-terminal fragment dissociation in a CD9/CD81-dependent manner specifically inducing IL-6 production, which promotes cell migration and invasion. Interestingly, expression of GPR56-C-terminal fragment alone recapitulates the antibody-induced receptor function, implicating a major role for the C-terminal fragment in GPR56 activation and signaling. Analysis of site-directed mutant receptors attests the importance of the conserved N-terminal residues of the C-terminal fragment for its self-activation. Finally, we show that the GPR56-induced signaling in melanoma cells is mediated by the Gα12/13/RhoA pathway. In summary, the expression and activation of GPR56 may modulate melanoma progression in part by inducing IL-6 production after N-terminal fragment dissociation and C-terminal fragment self-activation.

Interleukin-6 expression contributes to lapatinib resistance through maintenance of stemness property in HER2-positive breast cancer cells.

Lapatinib is an inhibitor of human epidermal growth factor receptor 2 (HER2), which is overexpressed in 20-25% of breast cancers. Clinically, lapatinib has shown promising benefits for HER2-positive breast cancer patients; however, patients eventually acquire resistance, limiting its long-term use. In a previous study, we found that interleukin-6 (IL-6) production was increased in acquired lapatinib-resistant HER2-positive breast cancer cells. In the present study, we confirmed that lapatinib-resistant cells had elevated IL-6 expression and also maintained both stemness population and property. The increase in IL-6 was required for stemness property maintenance, which was mediated primarily through the activation of signal transducer and activator of transcription 3 (STAT3). Blocking IL-6 activity reduced spheroid formation, cell viability and subsequently overcame lapatinib resistance, whereas stimulation of IL-6 rendered parental cells more resistant to lapatinib-induced cytotoxicity. These results point to a novel mechanism underlying lapatinib resistance and provide a potential strategy to overcome resistance via IL-6 inhibition.

miRNA-7/21/107 contribute to HBx-induced hepatocellular carcinoma progression through suppression of maspin.

Maspin suppresses tumor progression by promoting cell adhesion and apoptosis and by inhibiting cell motility. However, its role in tumorigenesis of hepatocellular carcinoma (HCC) remains unclear. The gene regulation of maspin and its relationship with HCC patient prognosis were investigated in this study. Maspin expression was specifically reduced in HBV-associated patients and correlated with their poor prognosis. Maspin downregulation in HCC cells was induced by HBx to promote their motility and resistance to anoikis and chemotherapy. HBx-dependent induction of microRNA-7, -107, and -21 was further demonstrated to directly target maspin mRNA, leading to its protein downregulation. Higher expressions of these microRNAs also correlated with maspin downregulation in HBV-associated patients, and were associated with their poor overall survival. These data not only provided new insights into the molecular mechanisms of maspin deficiency by HBx, but also indicated that downregulation of maspin by microRNAs confers HBx-mediated aggressiveness and chemoresistance in HCC.

Expression and immunoaffinity purification of recombinant soluble human GPR56 protein for the analysis of GPR56 receptor shedding by ELISA.

GPR56 is a multi-functional adhesion-class G protein-coupled receptor involved in biological systems as diverse as brain development, male gonad development, myoblast fusion, hematopoietic stem cell maintenance, tumor growth and metastasis, and immune-regulation. Ectodomain shedding of human GPR56 receptor has been demonstrated previously, however the quantitative detection of GPR56 receptor shedding has not been investigated fully due to the lack of appropriate assays. Herein, an efficient system of expression and immune-affinity purification of the recombinant soluble extracellular domain of human GPR56 (sGPR56) protein from a stably transduced human melanoma cell line was established. The identity and functionality of the recombinant human sGPR56 protein were verified by Western blotting and mass spectrometry, and ligand-binding assays, respectively. Combined with the use of two recently generated anti-GPR56 monoclonal antibodies, a sensitive sandwich ELISA assay was successfully developed for the quantitative detection of human sGPR56 molecule. We found that GPR56 receptor shedding occurred constitutively and was further increased in activated human melanoma cells expressing endogenous GPR56. In conclusion, we report herein an efficient system for the production and purification of human sGPR56 protein for the establishment of a quantitative ELISA analysis of GPR56 receptor shedding.

Hepatitis B virus X upregulates HuR protein level to stabilize HER2 expression in hepatocellular carcinoma cells.

Hepatitis B virus- (HBV-) associated hepatocellular carcinoma (HCC) is the most common type of liver cancer. However, the underlying mechanism of HCC tumorigenesis is very complicated and HBV-encoded X protein (HBx) has been reported to play the most important role in this process. Activation of downstream signal pathways of epidermal growth factor receptor (EGFR) family is known to mediate HBx-dependent HCC tumor progression. Interestingly, HER2 (also known as ErbB2/Neu/EGFR2) is frequently overexpressed in HBx-expressing HCC patients and is associated with their poor prognosis. However, it remains unclear whether and how HBx regulates HER2 expression. In this study, our data showed that HBx expression increased HER2 protein level via enhancing its mRNA stability. The induction of RNA-binding protein HuR expression by HBx mediated the HER2 mRNA stabilization. Finally, the upregulated HER2 expression promoted the migration ability of HBx-expressing HCC cells. These findings deciphered the molecular mechanism of HBx-mediated HER2 upregulation in HBV-associated HCC.