Publications by authors named "Giampietro Viola"

134 Publications

Concise synthesis and biological evaluation of 2-Aryl-3-Anilinobenzo[b]thiophene derivatives as potent apoptosis-inducing agents.

Bioorg Chem 2021 Jul 20;112:104919. Epub 2021 Apr 20.

Department of Medical Sciences, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, 44121 Ferrara, Italy.

Many clinically used agents active in cancer chemotherapy exert their activity through the induction of cell death (apoptosis) by targeting microtubules, altering protein function or inhibiting DNA synthesis. The benzo[b]thiophene scaffold holds a pivotal place as a pharmacophore for the development of anticancer agents, and, in addition, this scaffold has many pharmacological activities. We have developed a flexible method for the construction of a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophenes as potent antiproliferative agents, giving access to a wide range of substitution patterns at the 2-position of the 6-methoxybenzo[b]thiophene common intermediate. In the present study, all the synthesized compounds retained the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety, and the structure-activity relationship was examined by modification of the aryl group at its 2-position with electron-withdrawing (F) or electron-releasing (alkyl and alkoxy) groups. We found that small substituents, such as fluorine or methyl, could be placed in the para-position of the 2-phenyl ring, and these modifications only slightly reduced antiproliferative activity relative to the unsubstituted 2-phenyl analogue. Compounds 3a and 3b, bearing the phenyl and para-fluorophenyl at the 2-position of the 6-methoxybenzo[b]thiophene nucleus, respectively, exhibited the greatest antiproliferative activity among the tested compounds. The treatment of both Caco2 (not metastatic) and HCT-116 (metastatic) colon carcinoma cells with 3a or 3b triggered a significant induction of apoptosis as demonstrated by the increased expression of cleaved-poly(ADP-ribose) polymerase (PARP), receptor-interacting protein (RIP) and caspase-3 proteins. The same effect was not observed with non-transformed colon 841 CoN cells. A potential additional effect during mitosis for 3a in metastatic cells and for 3b in non-metastatic cells was also observed.
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http://dx.doi.org/10.1016/j.bioorg.2021.104919DOI Listing
July 2021

Developing novel classes of protein kinase CK1δ inhibitors by fusing [1,2,4]triazole with different bicyclic heteroaromatic systems.

Eur J Med Chem 2021 Apr 2;216:113331. Epub 2021 Mar 2.

Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste, Via Licio Giorgieri 1, 34127, Trieste, Italy. Electronic address:

Protein kinase CK1δ expression and activity is involved in different pathological situations that include neuroinflammatory and neurodegenerative diseases. For this reason, protein kinase CK1δ has become a possible therapeutic target for these conditions. 5,6-fused bicyclic heteroaromatic systems that resemble adenine of ATP represent optimal scaffolds for the development of a new class of ATP competitive CK1δ inhibitors. In particular, a new series of [1,2,4]triazolo[1,5-c]pyrimidines and [1,2,4]triazolo[1,5-a][1,3,5]triazines was developed. Some crucial interactors have been identified, such as the presence of a free amino group able to interact with the residues of the hinge region at the 5- and 7- positions of the [1,2,4]triazolo[1,5-c]pyrimidine and [1,2,4]triazolo[1,5-a][1,3,5]triazine scaffolds, respectively; or the presence of a 3-hydroxyphenyl or 3,5-dihydroxyphenyl moiety at the 2- position of both nuclei. Molecular modeling studies identified the key interactions involved in the inhibitor-protein recognition process that appropriately fit with the outlined structure-activity relationship. Considering the fact that the CK1 protein kinase is involved in various pathologies in particular of the central nervous system, the interest in the development of new inhibitors permeable to the blood-brain barrier represents today an important goal in the pharmaceutical field. The best potent compound of the series is the 5-(7-amino-5-(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)benzen-1,3-diol (compound 51, IC = 0.18 μM) that was predicted to have an intermediate ability to cross the membrane in our in vitro assay and represents an optimal starting point to both studies the therapeutic value of protein kinase CK1δ inhibition and to develop new more potent derivatives.
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http://dx.doi.org/10.1016/j.ejmech.2021.113331DOI Listing
April 2021

A facile synthesis of diaryl pyrroles led to the discovery of potent colchicine site antimitotic agents.

Eur J Med Chem 2021 Mar 29;214:113229. Epub 2021 Jan 29.

Istituto di Ricerca Pediatrica (IRP), Corso Stati Uniti 4, 35128, Padova, Italy. Electronic address:

Three different series of cis-restricted analogues of combretastatin A-4 (CA-4), corresponding to thirty-nine molecules that contained a pyrrole nucleus interposed between the two aryl rings, were prepared by a palladium-mediated coupling approach and evaluated for their antiproliferative activity against six human cancer cell lines. In the two series of 1,2-diaryl pyrrole derivatives, results suggested that the presence of the 3',4',5'-trimethoxyphenyl moiety at the N-1 position of the pyrrole ring was more favorable for antiproliferative activity. In the series of 3,4-diarylpyrrole analogues, three compounds (11i-k) exhibited maximal antiproliferative activity, showing excellent antiproliferative activity against the CA-4 resistant HT-29 cells. Inhibition of tubulin polymerization of selected 1,2 pyrrole derivatives (9a, 9c, 9o and 10a) was similar to that observed with CA-4, while the isomeric 3,4-pyrrole analogues 11i-k were generally from 1.5- to 2-fold more active than CA-4. Compounds 11j and 11k were the only compounds that showed activity as inhibitors of colchicine binding comparable to that CA-4. Compound 11j had biological properties consistent with its intracellular target being tubulin. This compound was able to block the cell cycle in metaphase and to induce significant apoptosis at a concentration of 25 nM, following the mitochondrial pathway, with low toxicity for normal cells. More importantly, compound 11j exerted activity in vivo superior to that of CA-4P, being able to significantly reduce tumor growth in a syngeneic murine tumor model even at the lower dose tested (5.0 mg/kg).
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http://dx.doi.org/10.1016/j.ejmech.2021.113229DOI Listing
March 2021

Synthesis, DNA-binding and antiproliferative properties of diarylquinolizinium derivatives.

Org Biomol Chem 2021 01 7;19(4):878-890. Epub 2021 Jan 7.

Department of Women's and Child's health, Oncohematology laboratory, University of Padova, Via Giustiniani 2, I-35128 Padova, Italy.

A series of ten 2,7- and 2,8-diarylquinolizinium derivatives was synthesized and their DNA-binding and cytotoxic properties were investigated. Except for one nitro-substituted derivative all tested diarylquinolizinium ions bind to DNA with sufficient affinity (2 × 10 M-2 × 10 M). It was shown with photometric, fluorimetric and polarimetric titrations as well as with flow-LD analysis that the ligands bind mainly by intercalation to duplex DNA, however, depending on the ligand-DNA ratio, groove binding and backbone association were also observed with some derivatives. The biological activity was further investigated with tests of cytotoxicity and antiproliferative properties towards non-tumor cells and selected cancer cells, along with cell cycle analysis and an annexin-V assay. Notably, substrates that carry donor-functionalities in the 4-position of the phenyl substituents revealed a strong, and in some cases selective, antiproliferative activity as quantified by the growth inhibition, GI, at very low micromolar and even submicromolar level both in leukemia and solid tumors.
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http://dx.doi.org/10.1039/d0ob02298eDOI Listing
January 2021

Insight on [1,3]thiazolo[4,5-e]isoindoles as tubulin polymerization inhibitors.

Eur J Med Chem 2021 Feb 24;212:113122. Epub 2020 Dec 24.

Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Università degli Studi di Palermo, Via Archirafi 32, 90123, Palermo, Italy.

A series of [1,3]thiazolo[4,5-e]isoindoles has been synthesized through a versatile and high yielding multistep sequence. Evaluation of the antiproliferative activity of the new compounds on the full NCI human tumor cell line panel highlighted several compounds that are able to inhibit tumor cell proliferation at micromolar-submicromolar concentrations. The most active derivative 11g was found to cause cell cycle arrest at the G2/M phase and induce apoptosis in HeLa cells, following the mitochondrial pathway, making it a lead compound for the discovery of new antimitotic drugs.
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http://dx.doi.org/10.1016/j.ejmech.2020.113122DOI Listing
February 2021

Verteporfin induces apoptosis and reduces the stem cell-like properties in Neuroblastoma tumour-initiating cells through inhibition of the YAP/TAZ pathway.

Eur J Pharmacol 2021 Feb 18;893:173829. Epub 2020 Dec 18.

Fondazione Istituto di Ricerca Pediatrica Città Della Speranza (IRP) - Neuroblastoma Laboratory, Corso Stati Uniti 4, 35127, Padova, Italy.

Neuroblastoma is an embryonal malignancy of early childhood arising from the embryonic sympatho-adrenal lineage of the neural crest. About half of all cases are currently classified as high-risk of disease recurrence, with an overall survival rate of less than 40% at 5 years despite intensive therapy. Recent studies on matched primary tumours and at the relapse revealed downregulation of genes transcriptionally silenced by YAP as significant association with neuroblastoma relapse. Here, we evaluated the pharmacological targeting of YAP/TAZ with the YAP/TAZ-TEAD inhibitor Verteporfin (VP) in Tumour Initiating Cells (TICs) derived from High-Risk Neuroblastoma patients. VP treatment suppresses YAP/TAZ expression, induces apoptosis and causes the re-organization of the cytoskeleton reducing cells migration and clonogenic ability. Moreover, VP reduces the percentage of side population cells and ABC transporters involved in drug resistance, and the percentage of stem cell subpopulations CD133+ and CD44 of TICs. Finally, we demonstrated that VP sensitizes TICs to the standard drugs used for neuroblastoma therapy etoposide and cis-platin opening the way to use VP as drug repositioning candidate for recurrent neuroblastoma.
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http://dx.doi.org/10.1016/j.ejphar.2020.173829DOI Listing
February 2021

Pyrrolo[2',3':3,4]cyclohepta[1,2-][1,2]oxazoles, a New Class of Antimitotic Agents Active against Multiple Malignant Cell Types.

J Med Chem 2020 10 11;63(20):12023-12042. Epub 2020 Oct 11.

Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Via Archirafi 32, 90123 Palermo, Italy.

A new class of pyrrolo[2',3':3,4]cyclohepta[1,2-][1,2]oxazoles was synthesized for the treatment of hyperproliferative pathologies, including neoplasms. The new compounds were screened in the 60 human cancer cell lines of the NCI drug screen and showed potent activity with GI values reaching the nanomolar level, with mean graph midpoints of 0.08-0.41 μM. All compounds were further tested on six lymphoma cell lines, and eight showed potent growth inhibitory effects with IC values lower than 500 nM. Mechanism of action studies showed the ability of the new [1,2]oxazoles to arrest cells in the G2/M phase in a concentration dependent manner and to induce apoptosis through the mitochondrial pathway. The most active compounds inhibited tubulin polymerization, with IC values of 1.9-8.2 μM, and appeared to bind to the colchicine site. The G2/M arrest was accompanied by apoptosis, mitochondrial depolarization, generation of reactive oxygen species, and PARP cleavage.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901646PMC
October 2020

Synthesis, biological evaluation, in silico modeling and crystallization of novel small monocationic molecules with potent antiproliferative activity by dual mechanism.

Eur J Med Chem 2020 Dec 6;207:112797. Epub 2020 Sep 6.

Department of Pharmaceutical and Organic Chemistry, Faculty of Pharmacy, Campus Cartuja S/n. University of Granada, 18010, Granada, Spain. Electronic address:

Seeking for new anticancer drugs with strong antiproliferative activity and simple molecular structure, we designed a novel series of compounds based on our previous reported pharmacophore model composed of five moieties. Antiproliferative assays on four tumoral cell lines and evaluation of Human Choline Kinase CKα1 enzymatic activity was performed for these compounds. Among tested molecules, those ones with biphenyl spacer showed betters enzymatic and antiproliferative activities (n-v). Docking and crystallization studies validate the hypothesis and confirm the results. The most active compound (t) induces a significant arrest of the cell cycle in G0/G1 phase that ultimately lead to apoptosis, following the mitochondrial pathway, as demonstrated for other choline kinase inhibitors. However additional assays reveal that the inhibition of choline uptake could also be involved in the antiproliferative outcome of this class of compounds.
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http://dx.doi.org/10.1016/j.ejmech.2020.112797DOI Listing
December 2020

Autophagic flux inhibition enhances cytotoxicity of the receptor tyrosine kinase inhibitor ponatinib.

J Exp Clin Cancer Res 2020 Sep 22;39(1):195. Epub 2020 Sep 22.

Neuroblastoma Laboratory, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Corso Stati Uniti 4, 35121, Padova, Italy.

Background: Despite reported advances, acquired resistance to tyrosine kinase inhibitors still represents a serious problem in successful cancer treatment. Among this class of drugs, ponatinib (PON) has been shown to have notable long-term efficacy, although its cytotoxicity might be hampered by autophagy. In this study, we examined the likelihood of PON resistance evolution in neuroblastoma and assessed the extent to which autophagy might provide survival advantages to tumor cells.

Methods: The effects of PON in inducing autophagy were determined both in vitro, using SK-N-BE(2), SH-SY5Y, and IMR-32 human neuroblastoma cell lines, and in vivo, using zebrafish and mouse models. Single and combined treatments with chloroquine (CQ)-a blocking agent of lysosomal metabolism and autophagic flux-and PON were conducted, and the effects on cell viability were determined using metabolic and immunohistochemical assays. The activation of the autophagic flux was analyzed through immunoblot and protein arrays, immunofluorescence, and transmission electron microscopy. Combination therapy with PON and CQ was tested in a clinically relevant neuroblastoma mouse model.

Results: Our results confirm that, in neuroblastoma cells and wild-type zebrafish embryos, PON induces the accumulation of autophagy vesicles-a sign of autophagy activation. Inhibition of autophagic flux by CQ restores the cytotoxic potential of PON, thus attributing to autophagy a cytoprotective nature. In mice, the use of CQ as adjuvant therapy significantly improves the anti-tumor effects obtained by PON, leading to ulterior reduction of tumor masses.

Conclusions: Together, these findings support the importance of autophagy monitoring in the treatment protocols that foresee PON administration, as this may predict drug resistance acquisition. The findings also establish the potential for combined use of CQ and PON, paving the way for their consideration in upcoming treatment protocols against neuroblastoma.
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http://dx.doi.org/10.1186/s13046-020-01692-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507635PMC
September 2020

Ecdysteroid Derivatives that Reverse P-Glycoprotein-Mediated Drug Resistance.

J Nat Prod 2020 08 13;83(8):2434-2446. Epub 2020 Aug 13.

Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Università degli Studi di Padova, Via Giustiniani 2, Padova, 35128, Italy.

The expression of multidrug resistance P-glycoprotein (P-gp) by cancer cells represents one of the major drawbacks to successful cancer therapy. Accordingly, the development of drugs that inhibit the activity of this transporter remains a major challenge in cancer drug discovery. In this context, several new ecdysteroid derivatives have been synthesized and evaluated as P-gp inhibitors. Two of them (compounds and ) were able to resensitize CEM and LoVo resistant cell lines to vinblastine and doxorubicin, respectively. Indeed, both compounds and increased the cellular accumulation of rhodamine 123 in cells expressing P-gp and stimulated basal P-glycoprotein-ATPase activity at a 1 μM concentration, demonstrating their interference with the transport of other substrates in a competitive mode. Moreover, in a medulloblastoma cell line (DAOY), compounds and reduced the side population representing cancer stem cells, which are characterized by a high expression of ABC drug transporters. Further, in DAOY cells, the same two compounds synergized with cisplatin and vincristine, two drugs used commonly in the therapy of medulloblastoma. Molecular docking studies on the homology-modeled structure of the human P-glycoprotein provided a rationale for the biological results, validating the binding mode within the receptor site, in accordance with lipophilicity data and observed structure-activity relationship information. Altogether, the present results endorse these derivatives as promising P-gp inhibitors, and they may serve as candidates to reverse drug resistance in cancer cells.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009596PMC
August 2020

Design, synthesis, in vitro and in vivo biological evaluation of 2-amino-3-aroylbenzo[b]furan derivatives as highly potent tubulin polymerization inhibitors.

Eur J Med Chem 2020 Aug 12;200:112448. Epub 2020 May 12.

Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Università di Padova, 35131, Padova, Italy. Electronic address:

A new class of inhibitors of tubulin polymerization based on the 2-amino-3-(3',4',5'-trimethoxybenzoyl)benzo[b]furan molecular scaffold was synthesized and evaluated for in vivo and in vitro biological activity. These derivatives were synthesized with different electron-releasing or electron-withdrawing substituents at one of the C-4 through C-7 positions. Methoxy substitution and location on the benzene part of the benzo[b]furan ring played an important role in affecting antiproliferative activity, with the greatest activity occurring with the methoxy group at the C-6 position, the least with the substituent at C-4. The same effect was also observed with ethoxy, methyl or bromine at the C-6 position of the benzo[b]furan skeleton, with the 6-ethoxy-2-amino-3-(3',4',5'-trimethoxybenzoyl)benzo[b]furan derivative 4f as the most promising compound of the series. This compound showed remarkable antiproliferative activity (IC: 5 pM) against the Daoy medulloblastoma cell line, and 4f was nearly devoid of toxicity on healthy human lymphocytes and astrocytes. The potent antiproliferative activity of 4f was derived from its inhibition of tubulin polymerization by binding to the colchicine site. The compound was also examined for in vivo activity, showing higher potency at 15 mg/kg compared with the reference compound combretastatin A-4 phosphate at 30 mg/kg against a syngeneic murine mammary tumor.
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http://dx.doi.org/10.1016/j.ejmech.2020.112448DOI Listing
August 2020

Design, synthesis and biological evaluation of 2-alkoxycarbonyl-3-anilinoindoles as a new class of potent inhibitors of tubulin polymerization.

Bioorg Chem 2020 04 18;97:103665. Epub 2020 Feb 18.

Rega Institute for Medical Research, KU Leuven, Laboratory of Virology and Chemotherapy, Leuven, Belgium.

A new class of inhibitors of tubulin polymerization based on the 2-alkoxycarbonyl-3-(3',4',5'-trimethoxyanilino)indole molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization and cell cycle effects. The results presented show that the methoxy substitution and location on the indole nucleus plays an important role in inhibition of cell growth, and the most favorable position for the substituent was at C-6. In addition, a small-size ester function (methoxy/ethoxycarbonyl) at the 2-position of the indole core was desirable. Also, analogues that were alkylated with methyl, ethyl or n-propyl groups or had a benzyl moiety on the N-1 indolic nitrogen retained activity equivalent to those observed in the parent N-1H analogues. The most promising compounds of the series were 2-methoxycarbonyl-3-(3',4'.5'-trimethoxyanilino)-5-methoxyindole 3f and 1-methyl-2-methoxycarbonyl-3-(3',4'.5'-trimethoxyanilino)-6-methoxy-indole 3w, both of which target tubulin at the colchicine site with antitubulin activities comparable to that of the reference compound combretastatin A-4.
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http://dx.doi.org/10.1016/j.bioorg.2020.103665DOI Listing
April 2020

A BAG's life: Every connection matters in cancer.

Pharmacol Ther 2020 05 27;209:107498. Epub 2020 Jan 27.

Neuroblastoma Laboratory, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Corso Stati Uniti 4, 35128 Padova, Italy.

The members of the BCL-2 associated athanogene (BAG) family participate in the regulation of a variety of interrelated physiological processes, such as autophagy, apoptosis, and protein homeostasis. Under normal circumstances, the six BAG members described in mammals (BAG1-6) principally assist the 70 kDa heat-shock protein (HSP70) in protein folding; however, their role as oncogenes is becoming increasingly evident. Deregulation of the BAG multigene family has been associated with cell transformation, tumor recurrence, and drug resistance. In addition to BAG overexpression, BAG members are also involved in many oncogenic protein-protein interactions (PPIs). As such, either the inhibition of overloading BAGs or of specific BAG-client protein interactions could have paramount therapeutic value. In this review, we will examine the role of each BAG family member in different malignancies, focusing on their modular structure, which enables interaction with a variety of proteins to exert their pro-tumorigenic role. Lastly, critical remarks on the unmet needs for proposing effective BAG inhibitors will be pointed out.
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http://dx.doi.org/10.1016/j.pharmthera.2020.107498DOI Listing
May 2020

Human Medulloblastoma Cell Lines: Investigating on Cancer Stem Cell-Like Phenotype.

Cancers (Basel) 2020 Jan 17;12(1). Epub 2020 Jan 17.

Laboratory of Biomedical Technologies, ENEA CR-Casaccia Via Anguillarese 301, 00123 Rome, Italy.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite the progress of new treatments, the risk of recurrence, morbidity, and death remains significant and the long-term adverse effects in survivors are substantial. The fraction of cancer stem-like cells (CSCs) because of their self-renewal ability and multi-lineage differentiation potential is critical for tumor initiation, growth, and resistance to therapies. For the development of new CSC-targeted therapies, further in-depth studies are needed using enriched and stable MB-CSCs populations. This work, aimed at identifying the amount of CSCs in three available human cell lines (DAOY, D341, and D283), describes different approaches based on the expression of stemness markers. First, we explored potential differences in gene and protein expression patterns of specific stem cell markers. Then, in order to identify and discriminate undifferentiated from differentiated cells, MB cells were characterized using a physical characterization method based on a high-frequency dielectrophoresis approach. Finally, we compared their tumorigenic potential in vivo, through engrafting in nude mice. Concordantly, our findings identified the D283 human cell line as an ideal model of CSCs, providing important evidence on the use of a commercial human MB cell line for the development of new strategic CSC-targeting therapies.
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http://dx.doi.org/10.3390/cancers12010226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016648PMC
January 2020

Design, synthesis and biological evaluation of novel vicinal diaryl-substituted 1H-Pyrazole analogues of combretastatin A-4 as highly potent tubulin polymerization inhibitors.

Eur J Med Chem 2019 Nov 1;181:111577. Epub 2019 Aug 1.

Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Università di Padova, 35131, Padova, Italy; Istituto di Ricerca Pediatrica (IRP), Corso Stati Uniti 4, 35128, Padova, Italy. Electronic address:

A series of 3-(3',4',5'-trimethoxyphenyl)-4-substituted 1H-pyrazole and their related 3-aryl-4-(3',4',5'-trimethoxyphenyl)-1-H-pyrazole regioisomeric derivatives, prepared as cis-rigidified combretastatin A-4 (CA-4) analogues, were synthesized and evaluated for their in vitro antiproliferative against six different cancer cell lines and, for selected highly active compounds, inhibitory effects on tubulin polymerization, cell cycle effects and in vivo potency. We retained the 3',4',5'-trimethoxyphenyl moiety as ring A throughout the present investigation, and a structure-activity relationship (SAR) information was obtained by adding electron-withdrawing (OCF, CF) or electron-releasing (alkyl and alkoxy) groups on the second aryl ring, corresponding to the B-ring of CA-4, either at the 3- or 4-position of the pyrazole nucleus. In addition, the B-ring was replaced with a benzo[b]thien-2-yl moiety. For many of the compounds, their activity was greater than, or comparable with, that of CA-4. Maximal activity was observed with the two regioisomeric derivatives characterized by the presence of a 4-ethoxyphenyl and a 3',4',5'-trimethoxyphenyl group at the C-3 and C-4 positions, and vice versa, of the 1H-pyrazole ring. The data showed that the 3',4',5'-trimethoxyphenyl moiety can be moved from the 3- to the 4-position of the 1H-pyrazole ring without significantly affecting antiproliferative activity. The most active derivatives bound to the colchicine site of tubulin and inhibited tubulin polymerization at submicromolar concentrations. In vivo experiments, on an orthotopic murine mammary tumor, revealed that 4c inhibited tumor growth even at low concentrations (5 mg/kg) compared to CA-4P (30 mg/kg).
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http://dx.doi.org/10.1016/j.ejmech.2019.111577DOI Listing
November 2019

Evaluating the effects of fluorine on biological properties and metabolic stability of some antitubulin 3-substituted 7-phenyl-pyrroloquinolinones.

Eur J Med Chem 2019 Sep 3;178:297-314. Epub 2019 Jun 3.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131, Padova, Italy. Electronic address:

A small number of fluorinated 7-phenyl-pyrroloquinolinone (7-PPyQ) derivatives was synthesized in an attempt to improve the metabolic stability of 3N-ethyl-7-PPyQ and 3N-benzoyl-7-PPyQ. The possible impacts of the fluorine-hydrogen isosterism on both biological activity and metabolic stability were evaluated. Introduction of a fluorine atom in the 2 or 3 position of the 7-phenyl ring yielded the 7-PPyQ derivatives 12, 13 and 15, which showed potent cytotoxicity (low micromolar and sub-nanomolar GIs) both in human leukemic and solid tumor cell lines. None of them induced significant cell death in quiescent and proliferating human lymphocytes. Moreover, 12, 13 and 15 exhibited remarkable cytotoxic activity in the multidrug-resistant cell line CEM, suggesting that they are not substrates for P-glycoprotein. All compounds inhibited tubulin assembly and the binding of [H]colchicine to tubulin, with the best activity occurring with compound 15. Mechanistic studies carried out on compound 12 indicated that it caused (a) a strong G2/M arrest; (b) apoptosis in a time- and concentration-dependent manner; (c) a significant production of ROS (in good agreement with the observed mitochondrial depolarization); (d) caspase-3 and poly (ADP-ribose) polymerase activation; and (e) a decrease in the expression of anti-apoptotic proteins. In vivo experiments in a murine syngeneic tumor model demonstrated that compounds 12 and 15 significantly reduced tumor mass at doses four times lower than that required for the reference compound combretastatin A-4 phosphate. Neither monofluorination of the 7-phenyl ring of 3N-ethyl-7-PPyQ nor replacement of the benzoyl function of 3N-benzoyl-7-PPyQ with a 2-fluorobenzoyl moiety led to any improvement in the metabolic stability.
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http://dx.doi.org/10.1016/j.ejmech.2019.05.092DOI Listing
September 2019

Identification of novel indole derivatives acting as inhibitors of the Keap1-Nrf2 interaction.

J Enzyme Inhib Med Chem 2019 Dec;34(1):1152-1157

c Dipartimento di Salute della Donna e del Bambino , Università di Padova , Padua , Italy.

Nine indole derivatives () were tested as potential inhibitors of the Keap1-Nrf2 interaction. This class of compounds increases the intracellular levels of the transcription factor Nrf2 and the consequent expression of enzymes encoded by genes containing the antioxidant response element (ARE). In the ARE-luciferase reporter assay only - revealed to be remarkably more active than -butylhydroxyquinone (-BHQ), with standing out as the best performing compound. While and are weak acids, is an ampholyte prevailing as a zwitterion in neutral aqueous solutions. The ability of to significantly increase levels of Nrf2, NADPH:quinone oxidoreductase 1, and transketolase (TKT) gave further support to the hypothesis that these compounds act as inhibitors of the Keap1-Nrf2 interaction. Docking simulations allowed us to elucidate the nature of the putative interactions between and Keap1.
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http://dx.doi.org/10.1080/14756366.2019.1623209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567006PMC
December 2019

Synthesis, in vitro and in vivo biological evaluation of substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones as new potent anticancer agents.

Eur J Med Chem 2019 Mar 26;166:514-530. Epub 2019 Jan 26.

Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Università di Padova, 35128, Padova, Italy. Electronic address:

A small library of 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones has been synthesized and screened according to protocols available at the National Cancer Institute (NCI). Some derivatives were potent antiproliferative agents, showing GI values in the nanomolar range. Remarkably, when most active compounds against leukemia cells were tested in human peripheral blood lymphocytes from healthy donors, were 100-200 times less cytotoxic. Some compounds, selected by the Biological Evaluation Committee of NCI, were examined to determine tubulin assembly inhibition. Furthermore, flow cytometric studies performed on HeLa, HT-29, and A549 cells, showed that compounds 14 and 25 caused a block in the G2/M phase. Interestingly, these derivatives induced apoptosis through the mitochondrial death pathway, causing in parallel significant activation of both caspase-3 and -9, PARP cleavage and down-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1. Finally, compound 25 was also tested in vivo in the murine BL6-B16 melanoma and E0771 breast cancer cells, causing in both cases a significant reduction in tumor volume.
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http://dx.doi.org/10.1016/j.ejmech.2019.01.049DOI Listing
March 2019

Results of a multicenter universal newborn screening program for sickle cell disease in Italy: A call to action.

Pediatr Blood Cancer 2019 05 5;66(5):e27657. Epub 2019 Feb 5.

Clinic of Pediatric Hematology Oncology, Department of Woman's and Child Health, Azienda Ospedaliera-Università di Padova, Padova, Italy.

Background: Sickle cell disease (SCD) is a chronic multisystem disorder requiring comprehensive care that includes newborn screening (NBS) as the first step of care. Italy still lacks a national SCD NBS program and policy on blood disorders. Pilot single-center screening programs and a regional targeted screening have been implemented so far, but more evidence is needed in order to impact health policies.

Population And Methods: NBS was offered to parents of newborns in gynecology clinics in Padova and Monza, tertiary care university hospitals in northern Italy. High-performance liquid chromatography (HPLC) was performed as the first test on samples collected on Guthrie cards. Molecular analysis of the beta-globin gene was performed on positive samples.

Results: A total of 5466 newborns were enrolled; for 5439, informed consents were obtained. A similar family origin was seen in the two centers (65% Italians, 9% mixed couples, 26% immigrants). Compared with SCD NBS programs in the United States and Europe, our results show a similar incidence of SCD patients and carriers. All SCD patients had a Sub-Saharan family background; HbS carriers were 15% Caucasians (Italian, Albanians) and 10% from other areas (North Africa-India-South America); carriers of other hemoglobin variants were mainly (47%) from other areas.

Conclusions: Our results demonstrate the feasibility of a multicentric NBS program for SCD, give information on HbS epidemiology in two Northern Italian Areas, and support previous European recommendation for a universal NBS program for SCD in Italy: a high incidence of patients and carriers has been detected, with a high percentage of Caucasian carriers, impossible to identify in a targeted NBS.
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http://dx.doi.org/10.1002/pbc.27657DOI Listing
May 2019

Design, Synthesis, and Biological Evaluation of 6-Substituted Thieno[3,2- d]pyrimidine Analogues as Dual Epidermal Growth Factor Receptor Kinase and Microtubule Inhibitors.

J Med Chem 2019 02 18;62(3):1274-1290. Epub 2019 Jan 18.

Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia , Università di Padova , 35131 Padova , Italy.

The clinical evidence for the success of tyrosine kinase inhibitors in combination with microtubule-targeting agents prompted us to design and develop single agents that possess both epidermal growth factor receptor (EGFR) kinase and tubulin polymerization inhibitory properties. A series of 6-aryl/heteroaryl-4-(3',4',5'-trimethoxyanilino)thieno[3,2- d]pyrimidine derivatives were discovered as novel dual tubulin polymerization and EGFR kinase inhibitors. The 4-(3',4',5'-trimethoxyanilino)-6-( p-tolyl)thieno[3,2- d]pyrimidine derivative 6g was the most potent compound of the series as an antiproliferative agent, with half-maximal inhibitory concentration (IC) values in the single- or double-digit nanomolar range. Compound 6g bound to tubulin in the colchicine site and inhibited tubulin assembly with an IC value of 0.71 μM, and 6g inhibited EGFR activity with an IC value of 30 nM. Our data suggested that the excellent in vitro and in vivo profile of 6g may be derived from its dual inhibition of tubulin polymerization and EGFR kinase.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01391DOI Listing
February 2019

Evaluation of Technical Issues in a Pilot Multicenter Newborn Screening Program for Sickle Cell Disease.

Int J Neonatal Screen 2019 Mar 21;5(1). Epub 2018 Dec 21.

Dipartimento di Salute della Donna e del Bambino, Università di Padova, 35128 Padova, Italy.

A multicenter pilot program for universal newborn screening of Sickle cell disease (SCD) was conducted in two centres of Northern Italy (Padova and Monza). High Performance Liquid Chromatography (HPLC) was performed as the first test on samples collected on Guthrie cards and molecular analysis of the β-globin gene () was the confirmatory test performed on the HPLC-positive or indeterminate samples. 5466 samples of newborns were evaluated. Of these, 5439/5466 were submitted to HPLC analysis and the molecular analysis always confirmed in all the alteration detected in HPLC (62/5439 newborns); 4/5439 (0.07%) were SCD affected, 37/5439 (0.68%) were HbAS carriers and 21/5439 (0.40%) showed other hemoglobinopathies. Stored dried blood spots were adequate for HPLC and β-globin gene molecular analysis. Samples were suitable for analysis until sixteen months old. A cut-off of A percentage, in order to avoid false negative or unnecessary confirmation tests, was identified. Our experience showed that several technical issues need to be addressed and resolved while developing a multicenter NBS program for SCD in a country where there is no national neonatal screening (NBS) program for SCD and NBS programs occur on a regional basis.
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http://dx.doi.org/10.3390/ijns5010002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510190PMC
March 2019

Phytosterol and γ-Oryzanol Conjugates: Synthesis and Evaluation of their Antioxidant, Antiproliferative, and Anticholesterol Activities.

J Nat Prod 2018 10 17;81(10):2212-2221. Epub 2018 Oct 17.

Dipartimento di Chimica , Università degli Studi di Milano , Via Golgi 19 , 20133 , Milano , Italy.

Fifteen new multifunctional conjugates were designed and synthesized by chemically linking the steroidal framework of natural occurring γ-oryzanol and γ-oryzanol-derived phytosterols to a wide range of bioactive natural compounds (fatty acids, phenolic acids, amino acids, lipoic acid, retinoic acid, curcumin, and resveratrol). Starting from γ-oryzanol, which is the main component of rice bran oil, this study was aimed at assessing if the conjugation strategy might enhance some γ-oryzanol bioactivities. The antioxidant activity was evaluated through three different mechanisms, namely, DPPH-scavenging activity, metal-chelating activity, and β-carotene-bleaching inhibition. Measurement of the in vitro cell growth inhibitory effects on three different human cancer cellular lines was also carried out, and the potential hypocholesterolemic effect was studied. Compounds 10 and 15 displayed an improved antioxidant activity, with respect to that of γ-oryzanol. Compounds 2, 6, and 12 exerted an antiproliferative activity in the low micromolar range against HeLa and DAOY cells (GI < 10 μM). As for the claimed hypocholesterolemic effect of γ-oryzanol, none of the synthesized compounds inhibited the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis.
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http://dx.doi.org/10.1021/acs.jnatprod.8b00465DOI Listing
October 2018

Choline Kinase Alpha Inhibition by EB-3D Triggers Cellular Senescence, Reduces Tumor Growth and Metastatic Dissemination in Breast Cancer.

Cancers (Basel) 2018 Oct 22;10(10). Epub 2018 Oct 22.

Pediatric Hematooncology Laboratory, Department of Women's and Children's health, University of Padova, 35128 Padova, Italy.

Choline kinase (ChoK) is the first enzyme of the Kennedy pathway leading to the biosynthesis of phosphatidylcholine (PtdCho), the most abundant phospholipid in eukaryotic cell membranes. EB-3D is a novel choline kinase α1 (ChoKα1) inhibitor with potent antiproliferative activity against a panel of several cancer cell lines. ChoKα1 is particularly overexpressed and hyperactivated in aggressive breast cancer. By NMR analysis, we demonstrated that EB-3D is able to reduce the synthesis of phosphocholine, and using flow cytometry, immunoblotting, and q-RT-PCR as well as proliferation and invasion assays, we proved that EB-3D strongly impairs breast cancer cell proliferation, migration, and invasion. EB-3D induces senescence in breast cancer cell lines through the activation of the metabolic sensor AMPK and the subsequent dephosphorylation of mTORC1 downstream targets, such as p70S6K, S6 ribosomal protein, and 4E-BP1. Moreover, EB-3D strongly synergizes with drugs commonly used for breast cancer treatment. The antitumorigenic potential of EB-3D was evaluated in vivo in the syngeneic orthotopic E0771 mouse model of breast cancer, where it induces a significant reduction of the tumor mass at low doses. In addition, EB-3D showed an antimetastatic effect in experimental and spontaneous metastasis models. Altogether, our results indicate that EB-3D could be a promising new anticancer agent to improve aggressive breast cancer treatment protocols.
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http://dx.doi.org/10.3390/cancers10100391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6209942PMC
October 2018

Improvement and extension of anti-EGFR targeting in breast cancer therapy by integration with the Avidin-Nucleic-Acid-Nano-Assemblies.

Nat Commun 2018 10 4;9(1):4070. Epub 2018 Oct 4.

Dipartimento di Scienze del Farmaco, Università di Padova, Via Marzolo, 5, 35131, Padova, Italy.

Nowadays, personalized cancer therapy relies on small molecules, monoclonal antibodies, or antibody-drug conjugates (ADC). Many nanoparticle (NP)-based drug delivery systems are also actively investigated, but their advantage over ADCs has not been demonstrated yet. Here, using the Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS), a class of polyavidins multifuctionalizable with stoichiometric control, we compare quantitatively anti-EGFR antibody(cetuximab)-targeted NPs to the corresponding ADC. We show that ANANAS tethering of cetuximab promotes a more efficient EGFR-dependent vesicle-mediated internalization. Cetuximab-guided ANANAS carrying doxorubicin are more cytotoxic in vitro and much more potent in vivo than the corresponding ADC, leading to 43% tumor reduction at low drug dosage (0.56 mg/kg). Advantage of cetuximab-guided ANANAS with respect to the ADC goes beyond the increase in drug-to-antibody ratio. Even if further studies are needed, we propose that NP tethering could expand application of the anti-EGFR antibody to a wider number of cancer patients including the KRAS-mutated ones, currently suffering from poor prognosis.
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http://dx.doi.org/10.1038/s41467-018-06602-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172284PMC
October 2018

Lead optimization-hit expansion of new asymmetrical pyridinium/quinolinium compounds as choline kinase α1 inhibitors.

Future Med Chem 2018 08 25;10(15):1769-1786. Epub 2018 Jul 25.

Department of Pharmaceutical & Organic Chemistry, Faculty of Pharmacy, University of Granada, 18010 Granada, Spain.

Aim: Choline kinase α inhibitors represent one of the newest classes of cytotoxic drugs for cancer treatment, since aberrant choline metabolism is a characteristic shared by many human cancers.

Results: Here, we present a new class of asymmetrical pyridinium/quinolinium derivatives developed and designed based on drug optimization.

Conclusion: Among all compounds described here, compound 8, bearing a 7-chloro-4N-methyl-p-chloroaniline quinolinium moiety, exhibited the greatest inhibitory activity at the enzyme (IC = 0.29 μM) and antiproliferative activity in cellular assays (GI = 0.29-0.92 μM). Specifically, compound 8 strongly induces a cell-cycle arrest in G1 phase, but it does not significantly induce apoptosis while causing senescence in the MDA-MB-231 cell line.
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http://dx.doi.org/10.4155/fmc-2018-0059DOI Listing
August 2018

EB-3D a novel choline kinase inhibitor induces deregulation of the AMPK-mTOR pathway and apoptosis in leukemia T-cells.

Biochem Pharmacol 2018 09 11;155:213-223. Epub 2018 Jul 11.

Department of Woman's and Child's Health, Oncohematology Laboratory, University of Padova, 35128 Padova, Italy. Electronic address:

Choline kinase alpha 1 (ChoKα1) has recently become an interesting therapeutic target since its overexpression has been associated to tumorigenesis in many cancers. Nevertheless, little is known regarding hematological malignancies. In this manuscript, we investigated the effect of a novel and selective ChoKα inhibitor EB-3D in T acute lymphoblastic leukemia (T-ALL). The effect of EB-3D was evaluated in a panel of T-leukemia cell lines and ex-vivo primary cultures derived from pediatric T-ALL patients. We also evaluated in detail, using Reverse Phase Protein Array (RPPA), protein phosphorylation level changes in T-ALL cells upon treatment. The drug exhibits a potent antiproliferative activity in a panel of T-leukemia cell lines and primary cultures of pediatric patients. Moreover, the drug strongly induces apoptosis and more importantly it enhanced T-leukemia cell sensitivity to chemotherapeutic agents, such as dexamethasone and l-asparaginase. In addition, the compound induces an early activation of AMPK, the main regulator of cellular energy homeostasis, by its phosphorylation at residue T712 of catalytic subunit α, and thus repressing mTORC1 pathway, as shown by mTOR S2448 dephosphorylation. The inhibition of mTOR in turn affects the activity of several known downstream targets, such as 4E-BP1, p70S6K, S6 Ribosomal Protein and GSK3 that ultimately may lead to a reduction of protein synthesis and cell death. Taken together, our findings suggest that targeting ChoKα may be an interesting option for treating T-ALL and that EB-3D could represent a valuable therapeutic tool.
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http://dx.doi.org/10.1016/j.bcp.2018.07.004DOI Listing
September 2018

3-Aryl/Heteroaryl-5-amino-1-(3',4',5'-trimethoxybenzoyl)-1,2,4-triazoles as antimicrotubule agents. Design, synthesis, antiproliferative activity and inhibition of tubulin polymerization.

Bioorg Chem 2018 10 30;80:361-374. Epub 2018 Jun 30.

Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Università di Padova, 35131 Padova, Italy. Electronic address:

Many natural and synthetic substances are known to interfere with the dynamic assembly of tubulin, preventing the formation of microtubules. In our search for potent and selective antitumor agents, a novel series of 1-(3',4',5'-trimethoxybenzoyl)-5-amino-1,2,4-triazoles were synthesized. The compounds had different heterocycles, including thiophene, furan or the three isomeric pyridines, and they possessed a phenyl ring bearing electron-releasing or electron-withdrawing substituents at the 3-position of the 5-amino-1,2,4-triazole system. Most of the twenty-two tested compounds showed moderate to potent antiproliferative activities against a panel of solid tumor and leukemic cell lines, with four (5j, 5k, 5o and 5p) showing strong antiproliferative activity (IC < 1 μM) against selected cancer cells. Among them, several molecules preferentially inhibited the proliferation of leukemic cell lines, showing IC values 2-100-fold lower for Jurkat and RS4;11 cells than those for the three lines derived from solid tumors (HeLa, HT-29 and MCF-7 cells). Compound 5k strongly inhibited tubulin assembly, with an IC value of 0.66 μM, half that obtained in simultaneous experiments with CA-4 (IC = 1.3 μM).
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http://dx.doi.org/10.1016/j.bioorg.2018.06.037DOI Listing
October 2018

BMP9 counteracts the tumorigenic and pro-angiogenic potential of glioblastoma.

Cell Death Differ 2018 11 5;25(10):1808-1822. Epub 2018 Jul 5.

Istituto di Ricerca Pediatrica - Città della Speranza - IRP, Padova, Italy.

Glioblastoma multiforme (GBM) is a highly vascularized and aggressive brain tumor, with a strong ability to disseminate and invade the surrounding parenchyma. In addition, a subpopulation of GBM stem cells has been reported to possess the ability to transdifferentiate into tumor-derived endothelial cells (TDECs), supporting the resistance to anti-angiogenic treatments of newly formed blood vessels. Bone Morphogenetic Protein 9 (BMP9) is critically involved in the processes of cancer cell differentiation, invasion and metastasis, representing a potential tool in order to impair the intrinsic GBM aggressiveness. Here we demonstrate that BMP9 is able to trigger the activation of SMADs in patient-derived GBM cells, and to strongly inhibit proliferation and invasion by reducing the activation of PI3K/AKT/MAPK and RhoA/Cofilin pathways, respectively. Intriguingly, BMP9 treatment is sufficient to induce a strong differentiation of GBM stem-like cells and to significantly counteract the already reported process of GBM cell transdifferentiation into TDECs not only in in vitro mimicked TDEC models, but also in vivo in orthotopic xenografts in mice. Additionally, we describe a strong BMP9-mediated inhibition of the whole angiogenic process engaged during GBM tumor formation. Based on these results, we believe that BMP9, by acting at multiple levels against GBM cell aggressiveness, can be considered a promising candidate, to be further developed, for the future therapeutic management of GBM.
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http://dx.doi.org/10.1038/s41418-018-0149-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180121PMC
November 2018

AKR1C enzymes sustain therapy resistance in paediatric T-ALL.

Br J Cancer 2018 04 8;118(7):985-994. Epub 2018 Mar 8.

Istituto di Ricerca paediatrica Città della Speranza-IRP, corso Stati Uniti 4, Padova, 35127, Italy.

Background: Despite chemotherapy intensification, a subgroup of high-risk paediatric T-cell acute lymphoblastic leukemia (T-ALL) patients still experience treatment failure. In this context, we hypothesised that therapy resistance in T-ALL might involve aldo-keto reductase 1C (AKR1C) enzymes as previously reported for solid tumors.

Methods: Expression of NRF2-AKR1C signaling components has been analysed in paediatric T-ALL samples endowed with different treatment outcomes as well as in patient-derived xenografts of T-ALL. The effects of AKR1C enzyme modulation has been investigated in T-ALL cell lines and primary cultures by combining AKR1C inhibition, overexpression, and gene silencing approaches.

Results: We show that T-ALL cells overexpress AKR1C1-3 enzymes in therapy-resistant patients. We report that AKR1C1-3 enzymes play a role in the response to vincristine (VCR) treatment, also ex vivo in patient-derived xenografts. Moreover, we demonstrate that the modulation of AKR1C1-3 levels is sufficient to sensitise T-ALL cells to VCR. Finally, we show that T-ALL chemotherapeutics induce overactivation of AKR1C enzymes independent of therapy resistance, thus establishing a potential resistance loop during T-ALL combination treatment.

Conclusions: Here, we demonstrate that expression and activity of AKR1C enzymes correlate with response to chemotherapeutics in T-ALL, posing AKR1C1-3 as potential targets for combination treatments during T-ALL therapy.
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http://dx.doi.org/10.1038/s41416-018-0014-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931104PMC
April 2018

Ribociclib, a Cdk4/Cdk6 kinase inhibitor, enhances glucocorticoid sensitivity in B-acute lymphoblastic leukemia (B-All).

Biochem Pharmacol 2018 07 3;153:230-241. Epub 2018 Feb 3.

Department of Woman's and Child's Heath, Oncohematology Lab, University of Padova, Italy. Electronic address:

Dysregulation of the cyclin D1-CDK4/CDK6 complex is frequently observed in almost all human cancer and contributes to aberrant cell proliferation and consequent tumorigenesis. Although many reports described the importance of CDK4/CDK6 in different set of human tumors, only few studies have been performed on leukemia. By gene expression analysis performed in a cohort of childhood patients affected by B-acute lymphoblastic leukemia (B-ALL) we found that both CDK4 and CDK6 are highly expressed. Moreover, reverse phase protein array (RPPA) analysis showed that cyclin D1 levels are higher in patients undergoing relapse. Starting from these considerations, we evaluated the effect of dual inhibition of CDK4/CDK6 in B-ALL and if this inhibition could enhance cytotoxic killing of leukemia cells after combination treatment with dexamethasone. We treated B-ALL cell lines with ribociclib, a highly specific CDK4/6 inhibitor. As expected, treatment with ribociclib induced growth inhibition of B-ALL cell lines, accompanied by strong cell cycle arrest in G1 phase, along with a dose-dependent decrease in phosphorylated retinoblastoma protein. Ribociclib exposure strongly synergizes with dexamethasone in SEM and RCH-ACV, two dexamethasone-resistant cell lines, along with a strong decrease in proliferation and a significant increase in apoptotic cell death. These results were also confirmed on primary cultures derived from bone marrow of pediatric patients affected by B-ALL. Immunoblot analysis showed a significant increase in glucocorticoid receptor (GR) along with some of its target genes, after combined treatment with ribociclib and dexamethasone. Altogether our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanism of glucocorticoid resistance.
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http://dx.doi.org/10.1016/j.bcp.2018.01.050DOI Listing
July 2018
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