Soloxolone methyl induces apoptosis and oxidative/ER stress in breast cancer cells and target cancer stem cell population.

One of the most prevalent malignancies in women and one of the leading causes of cancer-related death is breast cancer. There is a need for new treatment approaches and drugs for breast cancer. Many studies show the high potential of triterpene compounds and their semisynthetic derivatives as anticancer agents due to their ability to induce apoptosis and suppress tumorigenesis. The effects of soloxolone methyl (SM), a semisynthetic derivative of 18-H-glycyrrhetinic acid, on the cytotoxicity and apoptosis of human breast cancer cell line (T-47D) and cancer stem cell (CSCs) population (mammospheres; CD44+/CD24-antigen) derived from breast cancer cells, were examined in this work. The ATP assay was used to determine SM growth-inhibitory effects. Fluorescent staining, caspase-cleaved cytokeratin 18, and flow cytometry analysis were used to determine the mode of the cell death. In addition, cell death was investigated at protein and gene levels by Western Blotting and PCR, respectively. SM resulted in cytotoxicity in a time and dose dependent manner via ROS production and ER stress in T-47D cells in 2 models. The mode of cell death was apoptosis, evidenced by phosphatidylserine exposure, caspase activation, and bax overexpression. In mammospheres as 3D model, SM decreased stem cell properties and induced cell death. Taken together, SM may be a promising agent in the treatment of breast cancer, especially due to its antigrowth activity on CSCs.

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.

Novel Epoxides of Soloxolone Methyl: An Effect of the Formation of Oxirane Ring and Stereoisomerism on Cytotoxic Profile, Anti-Metastatic and Anti-Inflammatory Activities In Vitro and In Vivo.

It is known that epoxide-bearing compounds display pronounced pharmacological activities, and the epoxidation of natural metabolites can be a promising strategy to improve their bioactivity. Here, we report the design, synthesis and evaluation of biological properties of αO-SM and ßO-SM, novel epoxides of soloxolone methyl (SM), a cyanoenone-bearing derivative of 18ßH-glycyrrhetinic acid. We demonstrated that the replacement of a double-bound within the cyanoenone pharmacophore group of SM with α- and ß-epoxide moieties did not abrogate the high antitumor and anti-inflammatory potentials of the triterpenoid. It was found that novel SM epoxides induced the death of tumor cells at low micromolar concentrations (IC50(24h) = 0.7-4.1 µM) via the induction of mitochondrial-mediated apoptosis, reinforced intracellular accumulation of doxorubicin in B16 melanoma cells, probably by direct interaction with key drug efflux pumps (P-glycoprotein, MRP1, MXR1), and the suppressed pro-metastatic phenotype of B16 cells, effectively inhibiting their metastasis in a murine model. Moreover, αO-SM and ßO-SM hampered macrophage functionality in vitro (motility, NO production) and significantly suppressed carrageenan-induced peritonitis in vivo. Furthermore, the effect of the stereoisomerism of SM epoxides on the mentioned bioactivities and toxic profiles of these compounds in vivo were evaluated. Considering the comparable antitumor and anti-inflammatory effects of SM epoxides with SM and reference drugs (dacarbazine, dexamethasone), αO-SM and ßO-SM can be considered novel promising antitumor and anti-inflammatory drug candidates.

Novel Soloxolone Amides as Potent Anti-Glioblastoma Candidates: Design, Synthesis, In Silico Analysis and Biological Activities In Vitro and In Vivo.

The modification of natural or semisynthetic triterpenoids with amines can be explored as a promising strategy for improving their pharmacological properties. Here, we report the design and synthesis of 11 novel amide derivatives of soloxolone methyl (SM), a cyano enone-bearing derivative of 18ßH-glycyrrhetinic acid. Analysis of their bioactivities in vitro and in silico revealed their high toxicity against a panel of tumor cells (average IC50(24h) = 3.7 µM) and showed that the formation of amide moieties at the C-30 position of soloxolone did not enhance the cytotoxicity of derivatives toward tumor cells compared to SM, though it can impart an ability to pass across the blood-brain barrier. Further HPLC-MS/MS and mechanistic studies verified significant brain accumulation of hit compound 12 (soloxolone tryptamide) in a murine model and showed its high anti-glioblastoma potential. It was found that 12 induced ROS-dependent and autophagy-independent death of U87 and U118 glioblastoma cells via mitochondrial apoptosis and effectively blocked their clonogenicity, motility and capacity to form vessel-like structures. Further in vivo study demonstrated that intraperitoneal injection of 12 at a dosage of 20 mg/kg effectively inhibited the growth of U87 glioblastoma in a mouse xenograft model, reducing the proliferative potential of the tumor and leading to a depletion of collagen content and normalization of blood vessels in tumor tissue. The obtained results clearly demonstrate that 12 can be considered as a promising leading compound for drug development in glioblastoma treatment.

Soloxolone methyl, as a 18ßH-glycyrrhetinic acid derivate, may result in endoplasmic reticulum stress to induce apoptosis in breast cancer cells.

Being one of the leading causes of cancer death among women, various chemotherapeutic agents isolated from natural compounds are used in breast cancer treatment and consequently studies to develop new drugs still continue. There are several studies on 18ßH-glycyrrhetinic acid, a secondary metabolite which is found in Glycyrrhiza glabra (liquorice roots), as a potential anticancer agent. In this study, the cytotoxic and apoptotic effects of Soloxolone methyl compound, a semisynthetic derivative of 18ßH-glycyrrhetinic acid were investigated on breast cancer cells (MCF-7, MDA-MBA-231). Soloxolone methyl is found to be cytotoxic on both MCF-7 and MDA-MBA-231 breast cancer cells by inducing apoptosis. Especially in MDA-MB-231 cells apoptosis is detected to be triggered by ER stress. The antigrowth effects of Soloxolone methyl were determined using MTT and ATP assays. To identify the mode of cell death (apoptosis/necrosis), fluorescent staining (Hoechst 33342 and Propidium iodide) and caspase-cleaved cytokeratin 18 (M30-antigen) analyses were used. In addition, apoptosis was investigated on gene and protein levels by PCR and Western Blotting. Soloxolone methyl decreased cell viability on cells in a dose and time-dependent manner and induced apoptosis markers. An increase on apoptotic proteins related to endoplasmic reticulum stress (IRE1-α, Bip, CHOP) was also determined in MDA-MB-231 cells. Moreover, an increase of apoptotic gene expressions was determined in both cells treated with Soloxolone methyl. Advance analyses should be performed to elucidate the potential of Soloxolone methyl as an anticancer agent in breast cancer treatment.

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.

Trioxolone Methyl, a Novel Cyano Enone-Bearing 18ßH-Glycyrrhetinic Acid Derivative, Ameliorates Dextran Sulphate Sodium-Induced Colitis in Mice.

Semi-synthetic triterpenoids, bearing cyano enone functionality in ring A, are considered to be novel promising therapeutic agents with complex inhibitory effects on tissue damage, inflammation and tumor growth. Previously, we showed that the cyano enone-containing 18ßH-glycyrrhetinic acid derivative soloxolone methyl (SM) effectively suppressed the inflammatory response of macrophages in vitro and the development of influenza A-induced pneumonia and phlogogen-stimulated paw edema in vivo. In this work, we reported the synthesis of a novel 18ßH-glycyrrhetinic acid derivative trioxolone methyl (TM), bearing a 2-cyano-3-oxo-1(2)-en moiety in ring A and a 12,19-dioxo-9(11),13(18)-dien moiety in rings C, D, and E. TM exhibited a high inhibitory effect on nitric oxide (II) production by lipopolysaccharide-stimulated J774 macrophages in vitro and dextran sulfate sodium (DSS)-induced colitis in mice, displaying higher anti-inflammatory activity in comparison with SM. TM effectively suppressed the DSS-induced epithelial damage and inflammatory infiltration of colon tissue, the hyperproduction of colonic neutral mucin and TNFα and increased glutathione synthesis. Our in silico analysis showed that Akt1, STAT3 and dopamine receptor D2 can be considered as mediators of the anti-colitic activity of TM. Our findings provided valuable information for a better understanding of the anti-inflammatory activity of cyano enone-bearing triterpenoids and revealed TM as a promising anti-inflammatory candidate.

Deep insights into the response of human cervical carcinoma cells to a new cyano enone-bearing triterpenoid soloxolone methyl: a transcriptome analysis.

Semisynthetic triterpenoids, bearing cyano enone functionality in ring A, are considered now as novel promising anti-tumor agents. However, despite the large-scale studies, their effects on cervical carcinoma cells and, moreover, mechanisms underlying cell death activation by such compounds in this cell type have not been fully elucidated. In this work, we attempted to reconstitute the key pathways and master regulators involved in the response of human cervical carcinoma KB-3-1 cells to the novel glycyrrhetinic acid derivative soloxolone methyl (SM) by a transcriptomic approach. Functional annotation of differentially expressed genes, analysis of their cis- regulatory sequences and protein-protein interaction network clearly indicated that stress of endoplasmic reticulum (ER) is the central event triggered by SM in the cells. A range of key ER stress sensors and transcription factor AP-1 were identified as upstream transcriptional regulators, controlling the response of the cells to SM. Additionally, by using Gene Expression Omnibus data, we showed the ability of SM to modulate the expression of key genes involved in regulation of the high proliferative rate of cervical carcinoma cells. Further Connectivity Map analysis revealed similarity of SM's effects with known ER stress inducers thapsigargin and geldanamycin, targeting SERCA and Grp94, respectively. According to the molecular docking study, SM could snugly fit into the active sites of these proteins in the positions very close to that of both inhibitors. Taken together, our findings provide a basis for the better understanding of the intracellular processes in tumor cells switched on in response to cyano enone-bearing triterpenoids.

[Novel Glycyrrhetinic Acid Derivative Soloxolone Methyl Inhibits the Inflammatory Response and Tumor Growth in vivo].

Due to wide spreading of inflammatory disease and imperfection of available anti-inflammatory drugs, mainly associated with their serious side effects, searching for new anti-inflammatory agents is a pressing problem. Natural triterpenoids and their synthetic analogs are a promising source of new drugs. In this study, we have investigated the anti-inflammatory and antitumor effects in vivo of the glycyrrhetinic acid derivative soloxolone methyl (SM), or methyl 2-cyano-3,12-dioxo-18ßH-olean-9(ll),l(2)-dien-30-oate. SM was shown to efficiently suppress the development of edema in a mouse model of carrageenan- or histamine-induced acute inflammation. SM also inhibited the tumor growth and reduced the tumor cell count in the ascitic fluid in mice bearing Krebs-2 carcinoma, the development of which is accompanied by an inflammatory process in the surrounding tissues.