Heterocoumarins Are Selective Carbonic Anhydrase IX and XII Inhibitors with Cytotoxic Effects against Cancer Cells Lines.
ABSTRACT: We have synthesized a new series of coumarin-based compounds demonstrating high selectivity and potent effects with low nanomolar affinity against the tumor associated carbonic anhydrase (CA, EC 22.214.171.124) isoforms hCA IX and XII. A number of these compounds were evaluated ex vivo against human prostate (PC3) and breast (MDA-MB-231) cancer cell lines. Compounds 4b and 15 revealed effective cytotoxic effects after 48 h of incubation in both normoxic and hypoxic conditions with PC3 cancer cell line. However, compound 3 showed selective cytotoxic effects against MDA-MB-231 in hypoxic condition. These results may be of particular importance for the choice of future drug candidates targeting hypoxic tumors and metastases, considering the fact that a selective carbonic anhydrase CA IX inhibitor (SLC-0111) is presently in phase II clinical trials.
Project description:A series of benzenesulfonamides bearing selenourea moieties was obtained considering the ureido-sulfonamide SLC-0111, in Phase I clinical trials as antitumor agent, as a lead molecule. All compounds showed interesting inhibition potencies against the physiologically relevant human (h) carbonic anhydrase (hCAs, EC 126.96.36.199) isoforms I, II, IV, and IX. The most flexible analogues in the series 14-19 showed low nanomolar inhibition constants against hCA I, II, and IX. We assessed selected compounds on the in vitro antioxidant properties and binding modes and evaluated ex vivo human prostate (PC3), breast (MDA-MB-231), and colon-rectal (HT-29) cancer cell lines both in normoxic and hypoxic conditions.
Project description:Human carbonic anhydrase (CA) IX has emerged as a promising anticancer target and a diagnostic biomarker for solid hypoxic tumors. Novel fluorinated CA IX inhibitors exhibited up to 50 pM affinity towards the recombinant human CA IX, selectivity over other CAs, and direct binding to Zn(II) in the active site of CA IX inducing novel conformational changes as determined by X-ray crystallography. Mass spectrometric gas-analysis confirmed the CA IX-based mechanism of the inhibitors in a CRISPR/Cas9-mediated CA IX knockout in HeLa cells. Hypoxia-induced extracellular acidification was significantly reduced in HeLa, H460, MDA-MB-231, and A549 cells exposed to the compounds, with the IC50 values up to 1.29 nM. A decreased clonogenic survival was observed when hypoxic H460 3D spheroids were incubated with our lead compound. These novel compounds are therefore promising agents for CA IX-specific therapy.
Project description:Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC.
Project description:Herein, fifteen new compounds containing coumarin, 1,2,3-triazole and benzoyl- substituted arylamine moieties were designed, synthesized and tested in vitro for their anticancer activity. The results showed that all tested compounds had moderate antiproliferative activity against MDA-MB-231, a human breast cancer cell line, under both normoxic and hypoxic conditions. Furthermore, the 4-substituted coumarin linked with benzoyl 3,4-dimethoxyaniline through 1,2,3-triazole (compound 5e) displayed the most prominent antiproliferative activities with an IC50 value of 0.03 ?M, about 5000 times stronger than 4-hydroxycoumarin (IC50 > 100 ?M) and 20 times stronger than doxorubicin (IC50 = 0.60 ?M). Meanwhile, almost all compounds revealed general enhancement of proliferation-inhibiting activity under hypoxia, contrasted with normoxia. A docking analysis showed that compound 5e had potential to inhibit carbonic anhydrase IX (CA IX).
Project description:Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs.We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia- and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared.MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1alpha) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1alpha target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts.CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo.
Project description:Carbonic anhydrase IX (CAIX) is a tumor-specific protein that is upregulated during hypoxic conditions where it is involved in maintaining the pH balance. CAIX causes extracellular acidification, thereby limiting the uptake of weak basic chemotherapeutic agents, such as doxorubicin, and decreasing its efficacy. The aim of this study was to determine if doxorubicin efficacy can be increased when combined with the selective sulfamate CAIX inhibitor S4. The effect of S4 on doxorubicin efficacy was tested in vitro using cell viability assays with MDA-MB-231, FaDu, HT29 -CAIX high and HT29 -CAIX low cell lines. In addition, the efficacy of this combination therapy was investigated in tumor xenografts of the same cell lines. The addition of S4 in vitro increased the efficacy of doxorubicin in the MDA-MB-231 during hypoxic exposure (IC50 is 0.25 versus 0.14 µM, p = 0.0003). Similar results were observed for HT29-CAIX high with S4 during normoxia (IC50 is 0.20 versus 0.08 µM, p<0.0001) and in the HT29 -CAIX low cells (IC50 is 0.09 µM, p<0.0001). In vivo doxorubicin treatment was only effective in the MDA-MB-231 xenografts, but the efficacy of doxorubicin was decreased when combined with S4. In conclusion, the efficacy of doxorubicin treatment can be increased when combined with the selective sulfamate CAIX inhibitor S4 in vitro in certain cell lines. Nevertheless, in xenografts S4 did not enhance doxorubicin efficacy in the FaDu and HT29 tumor models and decreased doxorubicin efficacy in the MDA-MB-231 tumor model. These results stress the importance of better understanding the role of CAIX inhibitors in intratumoral pH regulation before combining them with standard treatment modalities, such as doxorubicin.
Project description:UNLABELLED: The aim of this study was to characterize the in vitro action of 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) on non-tumorigenic MCF-12A, tumorigenic MCF-7 and metastatic MDA-MB-231 breast cancer cells. ESE-16 is able to inhibit the activity of a carbonic anhydrase II and a mimic of carbonic anhydrase IX in the nanomolar range. Gene and protein expression studies using various techniques including gene and antibody microarrays and various flow cytometry assays yielded valuable information about the mechanism of action of ESE-16. The JNK pathway was identified as an important pathway mediating the effects of ESE-16 while the p38 stress-induced pathway is more important in MDA-MB-231 cells exposed to ESE-16. Lysosomal rupture and iron metabolism was identified as important mediators of mitochondrial membrane depolarization. Abrogation of Bcl-2 phosphorylation status as a result of ESE-16 also plays a role in inducing mitochondrial membrane depolarization. The study provides a basis for future research projects to develop the newly synthesized compound into a clinically usable anticancer agent either alone or in combination with other agents. KEYWORDS: Antimitotic, anticarbonic anhydrase IX, apoptosis, autophagy, cell cycle arrest, Bcl-2, JNK, p38, mitochondrial membrane depolarization, flow cytometry, gene expression and protein microarray, anticancer.
Project description:Cell plasticity is the ability that cells have to modify their phenotype, adapting to the environment. Cancer progression is under the strict control of the the tumor microenvironment that strongly determines its success by regulating the behavioral changes of tumor cells. The cross-talk between cancer and stromal cells and the interactions with the extracellular matrix, hypoxia and acidosis contribute to trigger a new tumor cell identity and to enhance tumor heterogeneity and metastatic spread. In highly aggressive triple-negative breast cancer, tumor cells show a significant capability to change their phenotype under the pressure of the hypoxic microenvironment. In this study, we investigated whether targeting the hypoxia-induced protein carbonic anhydrase IX (CA IX) could reduce triple-negative breast cancer (TNBC) cell phenotypic switching involved in processes associated with poor prognosis such as vascular mimicry (VM) and cancer stem cells (CSCs). The treatment of two TNBC cell lines (BT-549 and MDA-MB-231) with a specific CA IX siRNA or with a novel inhibitor of carbonic anhydrases (RC44) severely impaired their ability to form a vascular-like network and mammospheres and reduced their metastatic potential. In addition, the RC44 inhibitor was able to hamper the signal pathways involved in triggering VM and CSC formation. These results demonstrate that targeting hypoxia-induced cell plasticity through CA IX inhibition could be a new opportunity to selectively reduce VM and CSCs, thus improving the efficiency of existing therapies in TNBC.
Project description:Carbonic anhydrase (CA, EC 188.8.131.52) isoforms IX and XII are overexpressed in many hypoxic tumors as a consequence of the hypoxia inducible factor (HIF) activation cascade, being present in limited amounts in normal tissues. These enzymes together with many others are involved in the pH regulation and metabolism of hypoxic cancer cells, and were validated as antitumor targets recently. A multitude of targeting strategies against these enzymes have been proposed and are reviewed in this article. The small molecule inhibitors, small molecule drug conjugates (SMDCs), antibody-drug conjugates (ADACs) or cytokine-drug conjugates but not the monoclonal antibodies against CA IX/XII will be discussed. Relevant synthetic chemistry efforts, coupled with a multitude of preclinical studies, demonstrated that CA IX/XII inhibition leads to the inhibition of growth of primary tumors and metastases and depletes cancer stem cell populations, all factors highly relevant in clinical settings. One small molecule inhibitor, sulfonamide SLC-0111, is the most advanced candidate, having completed Phase I and being now in Phase Ib/II clinical trials for the treatment of advanced hypoxic solid tumors.
Project description:Antimitotic compounds are still one of the most widely used chemotherapeutic anticancer drugs in the clinic today. Given their effectiveness against cancer it is beneficial to continue enhancing these drugs. One way is to improve the bioavailability and efficacy by synthesizing derivatives that reversibly bind to carbonic anhydrase II (CAII) in red blood cells followed by a slow release into the blood circulation system. In the present study we describe the in vitro biological activity of a reduced derivative of 2-ethyl-3-O-sulphamoyl-estradiol (2EE), 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol). ESE-15-ol is capable of inhibiting carbonic anhydrase activity in the nanomolar range and is selective towards a mimic of carbonic anhydrase IX when compared to the CAII isoform. Docking studies using Autodock Vina suggest that the dehydration of the D-ring plays a role towards the selectivity of ESE-15-ol to CAIX and that the binding mode of ESE-15-ol is substantially different when compared to 2EE. ESE-15-ol is able to reduce cell growth to 50% after 48 h at 50-75 nM in MCF-7, MDA-MB-231, and MCF-12A cells. The compound is the least potent against the non-tumorigenic MCF-12A cells. In vitro mechanistic studies demonstrate that the newly synthesized compound induces mitochondrial membrane depolarization, abrogates the phosphorylation status of Bcl-2 and affects gene expression of genes associated with cell death and mitosis.