HIF-1α inhibition by MO-2097, a novel chiral-free benzofuran targeting hnRNPA2B1.

INTRODUCTION: Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator mediating adaptive responses to hypoxia. It is up-regulated in the tumor microenvironment and recognized as an effective anticancer drug target. Previously, we discovered that the natural compound moracin-O and its synthetic derivative MO-460 inhibited HIF-1α via hnRNPA2B1. OBJECTIVES: This study aimed to develop novel HIF-1 inhibitors for cancer chemotherapy by harnessing the potential of the natural products moracins-O and P. METHODS: In an ongoing search for novel HIF-1 inhibitors, a series of nature-inspired benzofurans with modifications on the chiral rings of moracins-O and P were synthesized. They showed improved chemical tractability and were evaluated for their inhibitory activity on HIF-1α accumulation under hypoxic conditions in HeLa CCL2 cells. The most potent derivative's chemical-based toxicities, binding affinities, and in vivo anti-tumorigenic effects were evaluated. Further, we examined whether our compound, MO-2097, exhibited anticancer effects in three-dimensional cultured organoids. RESULTS: Herein, we identified a novel synthetic chiral-free compound, MO-2097, with reduced structural complexity and increased efficiency. MO-2097 exhibited inhibitory effects on hypoxia-induced HIF-1α accumulation in HeLa CCL2 cells via inhibition of hnRNPA2B1 protein, whose binding affinities were confirmed by isothermal titration calorimetry analysis. In addition, MO-2097 demonstrated in vivo efficacy and biocompatibility in a BALB/c mice xenograft model. The immunohistochemistry staining of MO-2097-treated tissues showed decreased expression of HIF-1α and increased levels of apoptosis marker cleaved caspase 3, confirming in vivo efficacy. Furthermore, we confirmed that MO-2097 works effectively in cancer patient-based organoid models. CONCLUSION: MO-2097 represents a promising new generation of chemotherapeutic agents targeting HIF-1α inhibition via hnRNPA2B1, requiring further investigation.

Amphipathic Small Molecule AZT Compound Displays Potent Inhibitory Effects in Cancer Cell Proliferation.

Cancer has been identified as a leading cause of death worldwide, and the increasing number of cancer cases threatens to shorten the average life expectancy of people. Recently, we reported a 3-azido-3-deoxythymidine (AZT)-based amphipathic small molecule, ADG-2e that revealed a notable potency against tumor metastasis. To evaluate the anticancer potential of ADG-2e, we assessed its anticancer potency in vitro and in vivo. Anticancer screening of ADG-2e against cervical cancer cells, HeLa CCL2, and BT549 mammary gland ductal carcinoma showed significant inhibition of cancer cell proliferation. Furthermore, mechanistic investigations revealed that cancer cell death presumably proceeded through an oncosis mechanistic pathway because ADG-2e treated cells showed severe damage on the plasma membrane, a loss of membrane integrity, and leakage of α-tubulin and ß-actin. Finally, evaluation of the antitumorigenic potential of ADG-2e in mouse xenograft models revealed that this compound potentially inhibits cancer cell proliferation. Collectively, these findings suggest that ADG-2e can evolve as an anticancer agent, which may represent a model for nucleoside-based small molecule anticancer drug discovery.

CPPF, A Novel Microtubule Targeting Anticancer Agent, Inhibits the Growth of a Wide Variety of Cancers.

In the past, several microtubule targeting agents (MTAs) have been developed into successful anticancer drugs. However, the usage of these drugs has been limited by the acquisition of drug resistance in many cancers. Therefore, there is a constant demand for the development of new therapeutic drugs. Here we report the discovery of 5-5 (3-cchlorophenyl)-N-(3-pyridinyl)-2-furamide (CPPF), a novel microtubule targeting anticancer agent. Using both 2D and 3D culture systems, we showed that CPPF was able to suppress the proliferation of diverse cancer cell lines. In addition, CPPF was able to inhibit the growth of multidrug-resistant cell lines that are resistant to other MTAs, such as paclitaxel and colchicine. Our results showed that CPPF inhibited growth by depolymerizing microtubules leading to mitotic arrest and apoptosis. We also confirmed CPPF anticancer effects in vivo using both a mouse xenograft and a two-step skin cancer mouse model. Using established zebrafish models, we showed that CPPF has low toxicity in vivo. Overall, our study proves that CPPF has the potential to become a successful anticancer chemotherapeutic drug.

Inhibition of osteoclasts differentiation by CDC2-induced NFATc1 phosphorylation.

Bone homeostasis is regulated by a balance of bone formation and bone resorption; dysregulation of bone homeostasis may cause bone-related diseases (e.g., osteoporosis, osteopetrosis, bone fracture). Members of the nuclear factor of activated T cells (NFAT) family of transcription factors play crucial roles in the regulation of immune system, inflammatory responses, cardiac formation, skeletal muscle development, and bone homeostasis. Of these, NFATc1 is a key transcription factor mediating osteoclast differentiation, which is regulated by phosphorylation by distinct NFAT kinases including casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), and dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs). In this study, we report that cell division control protein 2 homolog (cdc2) is a novel NFAT protein kinase that inhibits NFATc1 activation by direct phosphorylation of the NFATc1 S263 residue. Cdc2 inhibitors such as Roscovitine and BMI-1026 induce reduction of phosphorylation of NFATc1, and this process leads to the inhibition of NFATc1 translocation from the nucleus to the cytoplasm, consequently increasing the nuclear pool of NFATc1. Additionally, the inhibition of cdc2-mediated NFATc1 phosphorylation causes an elevation of osteoclast differentiation or TRAP-positive staining in zebrafish scales. Our results suggest that cdc2 is a novel NFAT protein kinase that negatively regulates osteoclast differentiation.

Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability.

The initiation of centrosome duplication is regulated by the Plk4/STIL/hsSAS-6 axis; however, the involvement of other centrosomal proteins in this process remains unclear. In this study, we demonstrate that Cep131 physically interacts with Plk4 following phosphorylation of residues S21 and T205. Localizing at the centriole, phosphorylated Cep131 has an increased capability to interact with STIL, leading to further activation and stabilization of Plk4 for initiating centrosome duplication. Moreover, we found that Cep131 overexpression resulted in centrosome amplification by excessive recruitment of STIL to the centriole and subsequent stabilization of Plk4, contributing to centrosome amplification. The xenograft mouse model also showed that both centrosome amplification and colon cancer growth were significantly increased by Cep131 overexpression. These findings demonstrate that Cep131 is a novel substrate of Plk4, and that phosphorylation or dysregulated Cep131 overexpression promotes Plk4 stabilization and therefore centrosome amplification, establishing a perspective in understanding a relationship between centrosome amplification and cancer development.

Enhanced anticancer effects of a methylation inhibitor by inhibiting a novel DNMT1 target, CEP 131, in cervical cancer.

Methylation is a primary epigenetic mechanism regulating gene expression. 5-aza-2'-deoxycytidine is an FDA-approved drug prescribed for treatment of cancer by inhibiting DNA-Methyl-Transferase 1 (DNMT1). Results of this study suggest that prolonged treatment with 5-aza-2'-deoxycytidine could induce centrosome abnormalities in cancer cells and that CEP131, a centrosome protein, is regulated by DNMT1. Interestingly, cancer cell growth was attenuated in vitro and in vivo by inhibiting the expression of Cep131. Finally, Cep131-deficient cells were more sensitive to treatment with DNMT1 inhibitors. These findings suggest that Cep131 is a potential novel anti-cancer target. Agents that can inhibit this protein may be useful alone or in combination with DNMT1 inhibitors to treat cancer. [BMB Reports 2019; 52(5): 342-347].

Mechanism of the natural product moracin-O derived MO-460 and its targeting protein hnRNPA2B1 on HIF-1α inhibition.

Hypoxia-inducible factor-1α (HIF-1α) mediates tumor cell adaptation to hypoxic conditions and is a potentially important anticancer therapeutic target. We previously developed a method for synthesizing a benzofuran-based natural product, (R)-(-)-moracin-O, and obtained a novel potent analog, MO-460 that suppresses the accumulation of HIF-1α in Hep3B cells. However, the molecular target and underlying mechanism of action of MO-460 remained unclear. In the current study, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a molecular target of MO-460. MO-460 inhibits the initiation of HIF-1α translation by binding to the C-terminal glycine-rich domain of hnRNPA2B1 and inhibiting its subsequent binding to the 3'-untranslated region of HIF-1α mRNA. Moreover, MO-460 suppresses HIF-1α protein synthesis under hypoxic conditions and induces the accumulation of stress granules. The data provided here suggest that hnRNPA2B1 serves as a crucial molecular target in hypoxia-induced tumor survival and thus offer an avenue for the development of novel anticancer therapies.

A novel tubulin inhibitor STK899704 induces tumor regression in DMBA/TPA-induced skin carcinogenesis model.

Skin cancer is the most common type of cancer. The incidence rate of skin cancer has continuously increased over the past decades. In an effort to discover novel anticancer agents, we identified a novel tubulin inhibitor STK899704, which is structurally distinct from other microtubule-binding agents such as colchicine, vinca alkaloids and taxanes. STK899704 inhibited microtubule polymerization leading to mitotic arrest and suppressed the proliferation of various cancer cell lines as well as multidrug resistance cancer cell lines. In this study, our investigation is further extended into animal model to evaluate the effect of STK899704 on skin carcinogenesis in vivo. Surprisingly, almost 80% of the tumors treated with STK899704 were regressed with a one-fifth reduction in tumor volume. Furthermore, the efficacy of STK899704 was nearly 2 times higher than that of 5-fluorouracil, a widely used skin cancer therapeutic. Overall, our results suggest that STK899704 is a promising anticancer chemotherapeutic that may replace existing therapies, particularly for skin cancer.

Ginsenoside Re Promotes Osteoblast Differentiation in Mouse Osteoblast Precursor MC3T3-E1 Cells and a Zebrafish Model.

Bone homeostasis is tightly regulated to balance bone formation and bone resorption. Many anabolic drugs are used as bone-targeted therapeutic agents for the promotion of osteoblast-mediated bone formation or inhibition of osteoclast-mediated bone resorption. Previous studies showed that ginsenoside Re has the effect of the suppression of osteoclast differentiation in mouse bone-marrow derived macrophages and zebrafish. Herein, we investigated whether ginsenoside Re affects osteoblast differentiation and mineralization in in vitro and in vivo models. Mouse osteoblast precursor MC3T3-E1 cells were used to investigate cell viability, alkaline phosphatase (ALP) activity, and mineralization. In addition, we examined osteoblastic signaling pathways. Ginsenoside Re affected ALP activity without cytotoxicity, and we also observed the stimulation of osteoblast differentiation through the activation of osteoblast markers including runt-related transcription factor 2, type 1 collagen, ALP, and osteocalcin in MC3T3-E1 cells. Moreover, Alizarin red S staining indicated that ginsenoside Re increased osteoblast mineralization in MC3T3-E1 cells and zebrafish scales compared to controls. These results suggest that ginsenoside Re promotes osteoblast differentiation as well as inhibits osteoclast differentiation, and it could be a potential therapeutic agent for bone diseases.

Ginsenoside Re Inhibits Osteoclast Differentiation in Mouse Bone Marrow-Derived Macrophages and Zebrafish Scale Model.

Ginsenosides, which are the active materials of ginseng, have biological functions that include anti-osteoporotic effects. Aqueous ginseng extract inhibits osteoclast differentiation induced by receptor activator of NF-κB ligand (RANKL). Aqueous ginseng extract produces chromatography peaks characteristic of ginsenosides. Among these peaks, ginsenoside Re is a major component. However, the preventive effects of ginsenoside Re against osteoclast differentiation are not known. We studied the effect of ginsenoside Re on osteoclast differentiation, RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity, and formation of multinucleated osteoclasts in vitro. Ginsenoside Re hampered osteoclast differentiation in a dose-dependent manner. In an in vivo zebrafish model, aqueous ginseng extract and ginsenoside Re had anti-osteoclastogenesis effects. These findings suggest that both aqueous ginseng extract and ginsenoside Re prevent bone resorption by inhibiting osteoclast differentiation. Ginsenoside Re could be important for promoting bone health.