The Nexus of Inflammation-Induced Epithelial-Mesenchymal Transition and Lung Cancer Progression: A Roadmap to Pentacyclic Triterpenoid-Based Therapies.

Lung cancer is the leading cause of cancer-related death worldwide. Its high mortality is partly due to chronic inflammation that accompanies the disease and stimulates cancer progression. In this review, we analyzed recent studies and highlighted the role of the epithelial-mesenchymal transition (EMT) as a link between inflammation and lung cancer. In the inflammatory tumor microenvironment (iTME), fibroblasts, macrophages, granulocytes, and lymphocytes produce inflammatory mediators, some of which can induce EMT. This leads to increased invasiveness of tumor cells and self-renewal of cancer stem cells (CSCs), which are associated with metastasis and tumor recurrence, respectively. Based on published data, we propose that inflammation-induced EMT may be a potential therapeutic target for the treatment of lung cancer. This prospect is partially realized in the development of EMT inhibitors based on pentacyclic triterpenoids (PTs), described in the second part of our study. PTs reduce the metastatic potential and stemness of tumor cells, making PTs promising candidates for lung cancer therapy. We emphasize that the high diversity of molecular mechanisms underlying inflammation-induced EMT far exceeds those that have been implicated in drug development. Therefore, analysis of information on the relationship between the iTME and EMT is of great interest and may provide ideas for novel treatment approaches for lung cancer.

Soloxolone Methyl Reduces the Stimulatory Effect of Leptin on the Aggressive Phenotype of Murine Neuro2a Neuroblastoma Cells via the MAPK/ERK1/2 Pathway.

Despite the proven tumorigenic effect of leptin on epithelial-derived cancers, its impact on the aggressiveness of neural crest-derived cancers, notably neuroblastoma, remains largely unexplored. In our study, for the first time, transcriptome analysis of neuroblastoma tissue demonstrated that the level of leptin is elevated in neuroblastoma patients along with the severity of the disease and is inversely correlated with patient survival. The treatment of murine Neuro2a neuroblastoma cells with leptin significantly stimulated their proliferation and motility and reduced cell adhesion, thus rendering the phenotype of neuroblastoma cells more aggressive. Given the proven efficacy of cyanoenone-bearing semisynthetic triterpenoids in inhibiting the growth of neuroblastoma and preventing obesity in vivo, the effect of soloxolone methyl (SM) on leptin-stimulated Neuro2a cells was further investigated. We found that SM effectively abolished leptin-induced proliferation of Neuro2a cells by inducing G1/S cell cycle arrest and restored their adhesiveness to extracellular matrix (ECM) proteins to near control levels through the upregulation of vimentin, zonula occludens protein 1 (ZO-1), cell adhesion molecule L1 (L1cam), and neural cell adhesion molecule 1 (Ncam1). Moreover, SM significantly suppressed the leptin-associated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and ribosomal protein S6 kinase A1 (p90RSK), which are key kinases that ensure the survival and proliferation of cancer cells. Further molecular modeling studies demonstrated that the inhibitory effect of SM on the mitogen-activated protein kinase (MAPK)/ERK1/2 signaling pathway can be mediated by its direct interaction with ERK2 and its upstream regulators, son of sevenless homolog 1 (SOS) and mitogen-activated protein kinase kinase 1 (MEK1). Taken together, our findings in murine Neuro2a cells provide novel evidence of the stimulatory effect of leptin on the aggressiveness of neuroblastoma, which requires further detailed studies in human neuroblastoma cells and relevant animal models. The obtained results indicate that SM can be considered a promising drug candidate capable of reducing the impact of adipokines on tumor progression.

Evaluation of the Antitumor Potential of Soloxolone Tryptamide against Glioblastoma Multiforme Using in silico, in vitro, and in vivo Approaches.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by uncontrollable diffusive growth, resistance to chemo- and radiotherapy, and a high recurrence rate leading to a low survival rate of patients with GBM. Due to a large number of signaling pathways regulating GBM pathogenesis, one of the promising directions is development of novel anti-glioblastoma compounds based on natural metabolites capable of affecting multiple targets. Here, we investigated the antitumor potential of the semisynthetic triterpenoid soloxolone tryptamide (STA) against human glioblastoma U87 cells. STA efficiently blocked the growth of U87 cells in 2D and 3D cultures, enhanced adhesiveness of tumor cells, and displayed synergistic cytotoxicity with temozolomide. In silico analysis suggested that the anti-glioblastoma activity of STA can be explained by its direct interaction with EGFR, ERBB2, and AKT1 which play an important role in the regulation of GBM malignancy. Along with direct effect on U87 cells, STA normalized tumor microenvironment in murine heterotopic U87 xenograft model by suppressing the development of immature blood vessels and elastin production in the tumor tissue. Taken together, our results clearly demonstrate that STA can be a novel promising antitumor candidate for GMB treatment.

Protective effect of soloxolone derivatives in carrageenan- and LPS-driven acute inflammation: Pharmacological profiling and their effects on key inflammation-related processes.

The anti-inflammatory potential of three cyanoenone-containing triterpenoids, including soloxolone methyl (SM), soloxolone (S) and its novel derivative bearing at the C-30 amidoxime moiety (SAO), was studied in murine models of acute inflammation. It was found that the compounds effectively suppressed the development of carrageenan-induced paw edema and peritonitis as well as lipopolysaccharide (LPS)-driven acute lung injury (ALI) with therapeutic outcomes comparable with that of the reference drugs indomethacin and dexamethasone. Non-immunogenic carrageenan-stimulated inflammation was more sensitive to the transformation of C-30 of SM compared with immunogenic LPS-induced inflammation: the anti-inflammatory properties of the studied compounds against carrageenan-induced paw edema and peritonitis decreased in the order of SAO > S > > SM, whereas the efficiency of these triterpenoids against LPS-driven ALI was similar (SAO ≈ S ≈ SM). Further studies demonstrated that soloxolone derivatives significantly inhibited a range of immune-related processes, including granulocyte influx and the expression of key pro-inflammatory cytokines and chemokines in the inflamed sites as well as the functional activity of macrophages. Moreover, SM was found to prevent inflammation-associated apoptosis of A549 pneumocytes and effectively inhibited the protease activity of thrombin (IC50 = 10.3 µM) tightly associated with rodent inflammatome. Taken together, our findings demonstrate that soloxolone derivatives can be considered as novel promising anti-inflammatory drug candidates with multi-targeted mechanism of action.

Uncovering the anti-angiogenic effect of semisynthetic triterpenoid CDDO-Im on HUVECs by an integrated network pharmacology approach.

AIM: To reveal the molecular mechanism of anti-angiogenic activity of semisynthetic triterpenoid CDDO-Im. MATERIALS AND METHODS: Using re-analysis of cDNA microarray data of CDDO-Im-treated human vascular endothelial cells (HUVECs) (GSE71622), functional annotation of revealed differentially expressed genes (DEGs) and analysis of their co-expression, the key processes induced by CDDO-Im in HUVECs were identified. Venn diagram analysis was further performed to reveal the common DEGs, i.e. genes both susceptible to CDDO-Im and involved in the regulation of angiogenesis. A list of probable protein targets of CDDO-Im was prepared based on Connectivity Map/cheminformatics analysis and chemical proteomics data, among which the proteins that were most associated with the angiogenesis-related regulome were identified. Finally, identified targets were validated by molecular docking and text mining approaches. KEY FINDINGS: The effect of CDDO-Im in HUVECs can be divided into two main phases: the short early phase (0.5-3 h) with an acute FOXD1/CEBPA/JUNB-regulated pro-angiogenic response induced by xenobiotic stress, and the second anti-angiogenic step (6-24 h) with massive suppression of various angiogenesis-related processes, accompanied by the activation of cytoprotective mechanisms. Our analysis showed that the anti-angiogenic activity of CDDO-Im is mediated by its inhibition of the expression of PLAT, ETS1, A2M, SPAG9, RASGRP3, FBXO32, GCNT1 and HDGFRP3 and its direct interactions with EGFR, mTOR, NOS2, HSP90AA1, MDM2, SYK, IRF3, ATR and KIF14. SIGNIFICANCE: Our findings provide valuable insights into the understanding of the molecular mechanisms of the anti-angiogenic activity of cyano enone-bearing triterpenoids and revealed a range of novel promising therapeutic targets to control pathological neovascularization.

Cyano Enone-Bearing Triterpenoid Soloxolone Methyl Inhibits Epithelial-Mesenchymal Transition of Human Lung Adenocarcinoma Cells In Vitro and Metastasis of Murine Melanoma In Vivo.

Introduction of α-cyano α,ß-unsaturated carbonyl moiety into natural cyclic compounds markedly improves their bioactivities, including inhibitory potential against tumor growth and metastasis. Previously, we showed that cyano enone-bearing derivatives of 18ßH-glycyrrhetinic (GA) and deoxycholic acids displayed marked cytotoxicity in different tumor cell lines. Moreover, GA derivative soloxolone methyl (SM) was found to induce ER stress and apoptosis in tumor cells in vitro and inhibit growth of carcinoma Krebs-2 in vivo. In this work, we studied the effects of these compounds used in non-toxic dosage on the processes associated with metastatic potential of tumor cells. Performed screening revealed SM as a hit compound, which inhibits motility of murine melanoma B16 and human lung adenocarcinoma A549 cells and significantly suppresses colony formation of A549 cells. Further study showed that SM effectively blocked transforming growth factor ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of A549 cells: namely, inhibited TGF-ß-stimulated motility and invasion of tumor cells as well as loss of their epithelial characteristics, such as, an acquisition of spindle-like phenotype, up- and down-regulation of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin, zona occludens-1 (ZO-1)) markers, respectively. Network pharmacology analysis with subsequent verification by molecular modeling revealed that matrix metalloproteinases MMP-2/-9 and c-Jun N-terminal protein kinase 1 (JNK1) can be considered as hypothetical primary targets of SM, mediating its marked anti-EMT activity. The inhibitory effect of SM on EMT revealed in vitro was further confirmed in a metastatic model of murine B16 melanoma: SM was found to effectively block metastatic dissemination of melanoma B16 cells in vivo, increase expression of E-cadherin and suppress expression of MMP-9 in lung metastatic foci. Altogether, our data provided valuable information for a better understanding of the antitumor activity of cyano enone-bearing semisynthetic compounds and revealed SM as a promising anti-metastatic drug candidate.

Dual Effect of Soloxolone Methyl on LPS-Induced Inflammation In Vitro and In Vivo.

Plant-extracted triterpenoids belong to a class of bioactive compounds with pleotropic functions, including antioxidant, anti-cancer, and anti-inflammatory effects. In this work, we investigated the anti-inflammatory and anti-oxidative activities of a semisynthetic derivative of 18ßH-glycyrrhetinic acid (18ßH-GA), soloxolone methyl (methyl 2-cyano-3,12-dioxo-18ßH-olean-9(11),1(2)-dien-30-oate, or SM) in vitro on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and in vivo in models of acute inflammation: LPS-induced endotoxemia and carrageenan-induced peritonitis. SM used at non-cytotoxic concentrations was found to attenuate the production of reactive oxygen species and nitric oxide (II) and increase the level of reduced glutathione production by LPS-stimulated RAW264.7 cells. Moreover, SM strongly suppressed the phagocytic and migration activity of activated macrophages. These effects were found to be associated with the stimulation of heme oxigenase-1 (HO-1) expression, as well as with the inhibition of nuclear factor-κB (NF-κB) and Akt phosphorylation. Surprisingly, it was found that SM significantly enhanced LPS-induced expression of the pro-inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) in RAW264.7 cells via activation of the c-Jun/Toll-like receptor 4 (TLR4) signaling axis. In vivo pre-exposure treatment with SM effectively inhibited the development of carrageenan-induced acute inflammation in the peritoneal cavity, but it did not improve LPS-induced inflammation in the endotoxemia model.