SP-8356, a (1S)-(-)-Verbenone Derivative, Inhibits the Growth and Motility of Liver Cancer Cells by Regulating NF-κB and ERK Signaling.

Liver cancer is a common tumor and currently the second leading cause of cancer-related mortality globally. Liver cancer is highly related to inflammation as more than 90% of liver cancer arises in the context of hepatic inflammation, such as hepatitis B virus and hepatitis C virus infection. Despite significant improvements in the therapeutic modalities for liver cancer, patient prognosis is not satisfactory due to the limited efficacy of current drug therapies in anti-metastatic activity. Therefore, developing new effective anti-cancer agents with anti-metastatic activity is important for the treatment of liver cancer. In this study, SP-8356, a verbenone derivative with anti-inflammatory activity, was investigated for its effect on the growth and migration of liver cancer cells. Our findings demonstrated that SP-8356 inhibits the proliferation of liver cancer cells by inducing apoptosis and suppressing the mobility and invasion ability of liver cancer cells. Functional studies revealed that SP-8356 inhibits the mitogen-activated protein kinase and nuclear factor-kappa B signaling pathways, which are related to cell proliferation and metastasis, resulting in the downregulation of metastasis-related genes. Moreover, using an orthotopic liver cancer model, tumor growth was significantly decreased following treatment with SP-8356. Thus, this study suggests that SP-8356 may be a potential agent for the treatment of liver cancer with multimodal regulation.

CXCR7: a ß-arrestin-biased receptor that potentiates cell migration and recruits ß-arrestin2 exclusively through Gßγ subunits and GRK2.

BACKGROUND: Some chemokine receptors referred to as atypical chemokine receptors (ACKRs) are thought to non-signaling decoys because of their inability to activate typical G-protein signaling pathways. CXCR7, also known as ACKR3, binds to only two chemokines, SDF-1α and I-TAC, and recruits ß-arrestins. SDF-1α also binds to its own conventional receptor, CXCR4, involving in homeostatic modulation such as development and immune surveillance as well as pathological conditions such as inflammation, ischemia, and cancers. Recently, CXCR7 is suggested as a key therapeutic target together with CXCR4 in such conditions. However, the molecular mechanisms underlying cellular responses and functional relation with CXCR7 and CXCR4 have not been elucidated, despite massive studies. Therefore, we aimed to reveal the molecular networks of CXCR7 and CXCR4 and compare their effects on cell migration. METHODS: Base on structural complementation assay using NanoBiT technology, we characterized the distinct mechanisms underlying ß-arrestin2 recruitment by both CXCR4 and CXCR7. Crosslinking and immunoprecipitation were conducted to analyze complex formation of the receptors. Gene deletion using CRISPR and reconstitution of the receptors were applied to analysis of ligand-dependent ERK phosphorylation and cell migration. All experiments were performed in triplicate and repeated more than three times. Unpaired Student's t-tests or ANOVA using PRISM5 software were employed for statistical analyses. RESULTS: Ligand binding to CXCR7 does not result in activation of typical signaling pathways via Gα subunits but activation of GRK2 via ßγ subunits and receptor phosphorylation with subsequent ß-arrestin2 recruitment. In contrast, CXCR4 induced Gαi activation and recruited ß-arrestin2 through C-terminal phosphorylation by both GRK2 and GRK5. SDF-1α-stimulated ERK phosphorylation was facilitated by CXCR4, but not CXCR7. Heterodimerization of CXCR4 and CXCR7 was not confirmed in this study, while homodimerization of them was verified by crosslinking experiment and NanoBiT assay. Regarding chemotaxis, SDF-1α-stimulated cell migration was mediated by both CXCR4 and CXCR7. CONCLUSION: This study demonstrates that SDF-1α-stimulated CXCR7 mediates ß-arrestin2 recruitment via different molecular networking from that of CXCR4. CXCR7 may be neither a simple scavenger nor auxiliary receptor but plays an essential role in cell migration through cooperation with CXCR4.

Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels.

Cytosolic Ca2+ levels ([Ca2+]c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca2+]c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca2+]c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca2+]c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca2+-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca2+]c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca2+]c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca2+]c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca2+]c in single cells and animal models.

SP-8356, a (1S)-(-)-verbenone derivative, exerts in vitro and in vivo anti-breast cancer effects by inhibiting NF-κB signaling.

Breast cancer exhibits high lethality in women because it is frequently detected at an advanced stage and aggressive forms such as triple-negative breast cancer (TNBC), which are often characterized by metastasis through colonization of secondary tumors. Thus, developing therapeutic agents that target the metastatic process is crucial to successfully treat aggressive breast cancer. We evaluated SP-8356, an anti-inflammatory synthetic verbenone derivative, with respect to its regulation of breast cancer cell behavior and cancer progression. Treatment of SP-8356 arrested cell cycle and reduced growth in various types of breast cancer cells with mild cytotoxicity. Particularly, SP-8356 significantly reduced the motility and invasiveness of TNBC cells. Assays using an in vivo xenograft mouse model confirmed the cell-specific anti-proliferative and anti-metastatic activity of SP-8356. Functional studies revealed that SP-8356 suppressed serum response element-dependent reporter gene expression and NF-κB-related signaling, resulting in downregulation of many genes related to cancer invasion. We conclude that SP-8356 suppresses breast cancer progression through multimodal functions, including inhibition of NF-κB signaling and growth-related signaling pathways.

Nafamostat mesilate negatively regulates the metastasis of triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) lacking of oestrogen receptor, progesterone receptor, and epidermal growth factor receptor type 2 is a highly malignant disease which results in a poor prognosis and rare treatment options. Despite the use of conventional chemotherapy for TNBC tumours, resistance and short duration responses limit the treatment efficacy. Therefore, a need exists to develop a new chemotherapy for TNBC. The aim of this study was to examine the anti-cancer effects of nafamostat mesilate (NM), a previously known serine protease inhibitor and highly safe drug on breast cancer cells. Here, we showed that NM significantly inhibits proliferation, migration, and invasion in MDA-MB231 cells, induces G2/M phase cell-cycle arrest, and inhibits the expression of cyclin-dependent kinase 1 (CDK1). Exposure of MDA-MB231 cells to NM also resulted in decreased transcription factor activities accompanied by the regulated phosphorylation of signalling molecules and a decrease in metalloproteinases, the principal modulators of the extracellular environment during cancer progression. Especially, inhibition of TGFß-stimulated Smad2 phosphorylation and subsequent metastasis-related gene expression, and downregulation of ERK activity may be pivotal mechanisms underlying inhibitory effects of NM on NM inhibits lung metastasis of breast cancer cells and growth of colonized tumours in mice. Taken together, our data revealed that NM inhibits cell growth and metastasis of TNBC cells and indicated that NM is a multi-targeted drug that could be an adjunct therapy for TNBC treatment.

Von Hippel-Lindau regulates interleukin-32ß stability in ovarian cancer cells.

Hypoxia-induced interleukin-32ß (IL-32ß) shifts the metabolic program to the enhanced glycolytic pathway. In the present study, the underlying mechanism by which hypoxia-induced IL-32ß stability is regulated was investigated in ovarian cancer cells. IL-32ß expression increased under hypoxic conditions in ovarian cancer cells as it did in breast cancer cells. The amount of IL-32ß was regulated by post-translational control rather than by transcriptional activation. Under normoxic conditions, IL-32ß was continuously eliminated through ubiquitin-dependent degradation by the von-Hippel Lindau (VHL) E3 ligase complex. Oxygen deficiency or reactive oxygen species (ROS) disrupted the interaction between IL-32ß and VHL, leading to the accumulation of the cytokine. The fact that IL-32ß is regulated by the energy-consuming ubiquitination system implies that it plays an important role in oxidative stress. We found that IL-32ß reduced protein kinase Cδ (PKCδ)-induced apoptosis under oxidative stress. This implies that the hypoxia- and ROS-stabilized IL-32ß contributes to sustain survival against PKCδ-induced apoptosis.