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Sabatino M.A.,Laboratory of Molecular Pharmacology
Epigenetics : official journal of the DNA Methylation Society | Year: 2013

Brostallicin is a DNA minor groove binder that shows enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. Prostate cancer cells present, almost invariably, methylation of the GSTP1 gene promoter and, as a consequence, low levels of GST-pi expression and activity. In these cells, brostallicin shows very little activity. We tested whether pretreatment of heavily GST-methylated prostate cancer cells with demethylating agents could enhance the activity of brostallicin. Human prostate cancer cells LNCaP and DU145 were used for these studies both in vitro and in vivo. The demethylating agent zebularine was used in combination with brostallicin. Methylation specific PCR and pyrosequencing were used to determine the level of GST methylation. Pretreatment with demethylating agents enhanced the in vitro activity of brostallicin in LNCaP cells. Zebularine, in particular, induced an enhancement of activity in vivo comparable to that obtained by transfecting the human GSTP1 gene in LNCaP cells in vitro. Molecular analysis performed on tumor xenografts in mice pretreated with zebularine failed to detect re-expression of GST-pi and demethylation of GSTP1. However, we found demethylation in the GSTM1 gene, with consequent re-expression of GST-mu at the mRNA level. These results indicate that zebularine, both in vitro and in vivo, enhances the activity of brostallicin and that this enhancement correlates with re-expression of GST-pi and GST-mu. These findings highlight the potential therapeutic value of combining demethylating agents and brostallicin in tumors with GST methylation that poorly respond to brostallicin.

Mazzoletti M.,Laboratory of Molecular Pharmacology | Broggini M.,Laboratory of Molecular Pharmacology
Current Medicinal Chemistry | Year: 2010

Phisiological activation of PI3K pathway is necessary for cells to regulate many different physiological processes such as transcription, protein synthesis, metabolic responses and membrane trafficking. Abnormal activation of the PI3K pathway leads to an increased activity resulting in tumor onset, maintenance, progression and invasion. Both genetic and epigenetic alterations could affect the normal pathway's activation. Ovarian cancer is the leading cause of death from gynaecological malignancies in the western world. PI3K pathway has been recorded as one of the most deregulated signalling pathway in many tumors, including ovarian ones. So it could be considered an attractive target to be investigated with the various classes of chemical compounds already present or in development. In this rewiew we'll try to discuss the published data of the inhibitors targeting members of the PI3K/akt/mTOR pathway in the ovarian cancer setting from a preclinical and clinical point of view, with particular emphasis on drugs combination and strategies of administration. Relevant issues and limitations to the use of particular compounds will be also addressed. © 2010 Bentham Science Publishers Ltd.

Carrassa L.,Laboratory of Molecular Pharmacology | Chila R.,Laboratory of Molecular Pharmacology | Lupi M.,Laboratory of Cancer Pharmacology | Ricci F.,Laboratory of Molecular Pharmacology | And 4 more authors.
Cell Cycle | Year: 2012

Targeting Chk1 protein kinase can enhance the antitumor effects of radio- and chemotherapy. Recent evidence disclosed a role of Chk1 in unperturbed cell proliferation and survival, implying that Chk1 inhibitors could also be effective as single agents in tumors with a specific genetic background. To identify genes in synthetic lethality with Chk1, we did a high-throughput screening using a siRNA library directed against 719 human protein kinases in the human ovarian cancer cell line OVCAR-5, resistant to Chk1 inhibitors. Wee1 tyrosine kinase was the most significant gene in synthetic lethality with Chk1. Treatment with non-toxic concentrations of a Chk1 inhibitor (PF-00477736) and a Wee1 inhibitor (MK-1775) confirmed the marked synergistic effect in various human cancer cell lines (breast, ovarian, colon, prostate), independently of the p53 status. Detailed molecular analysis showed that the combination caused cancer cells to undergo premature mitosis before the end of DNA replication, with damaged DNA leading to cell death partly by apoptosis. In vivo treatment of mice bearing OVCAR-5 xenografts with the combination of Chk1 and Wee1 inhibitors led to greater tumor growth inhibition than with the inhibitors used as single agents with no toxicity. These data provide a strong rationale for the clinical investigation of the combination of a Chk1 and a Wee1 inhibitor. © 2012 Landes Bioscience.

Chila R.,Laboratory of Molecular Pharmacology | Celenza C.,Laboratory of Molecular Pharmacology | Lupi M.,Laboratory of Cancer Pharmacology | Damia G.,Laboratory of Molecular Pharmacology | Carrassa L.,Laboratory of Molecular Pharmacology
Cell Cycle | Year: 2013

Chk1 is implicated in several checkpoints of the cell cycle acting as a key player in the signal transduction pathway activated in response to DNA damage and crucial for the maintenance of genomic stability. Chk1 also plays a role in the mitotic spindle checkpoint, which ensures thefidelity of mitotic segregation during mitosis, preventing chromosomal instability and aneuploidy. Mad2 is one of the main mitotic checkpoint components and also exerts a role in the cellular response to DNA damage. To investigate a possible crosslink existing between Chk1 and Mad2, we studied Mad2 protein levels after Chk1 inhibition either by specific siRNAs or by a specific and selective Chk1 inhibitor (PF-00477736), and we found that after Chk1 inhibition, Mad2 protein levels decrease only in tumor cells sensitive to Chk1 depletion. We then mapped six Chk1's phosphorylatable sites on Mad2 protein, and found that Chk1 is able to phosphorylate Mad2 in vitro on more than one site, while it is incapable of phoshorylating the Mad2 form mutated on all six phosphorylatable sites. Moreover our studies demonstrate that Chk1 co-localizes and physically associates with Mad2 in cells both under unstressed conditions and after DNA damage, thus providing new and interesting evidence on Chk1 and Mad2 crosstalk in the DNA damage checkpoint and in the mitotic spindle checkpoint. Copyright © 2013 Landes Bioscience.

Rusconi P.,Laboratory of Molecular Pharmacology | Caiola E.,Laboratory of Molecular Pharmacology | Broggini M.,Laboratory of Molecular Pharmacology
Current Medicinal Chemistry | Year: 2012

The RAS/RAF/MEK signaling pathway plays a central role in mediating both proliferation and survival of cancer cells. These proteins are a group of serine/threonine kinases activated in response to a variety of extracellular stimuli and mediate signal transduction from the cell surface towards both nuclear and cytosolic targets. In combination with several other signaling pathways, they can differentially alter phosphorylation status of the transcription factors. A controlled regulation of these cascades is involved in cell proliferation and differentiation, whereas an unregulated activation of these kinases can result in oncogenesis. Dysregulation of the RAS/RAF/MEK pathway has been detected in more than 30% of human tumors, however mutations in the MEK1 and MEK2 genes are seldom, so that hyperactivation of MEK1/2 usually results from gain-of-function mutations in RAS and/or B-RAF. In addition, alteration of the pathways is often associated with drug resistance in the clinic, such as the case of K-RAS mutant expressing tumors. Since RAS protein is a difficult target, alternative ways altering post-translational modifications using farnesyl transferase inhibitors have been adopted. Drug discovery programs have therefore largely focused on B-RAF and MEK. In this review we will discuss the most promising strategies developed to target these kinases and the most recent inhibitors facing the preclinical and clinical setting, also considering their structure-activity relationship (SAR). © 2012 Bentham Science Publishers.

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