Michela Illiano

Targeting protein kinase A in cancer therapy: an update.

Protein Kinase A (PKA) is a well known member of the serine-threonin protein kinase superfamily. PKA, also known as cAMP-dependent protein kinase, is a multi-unit protein kinase that mediates signal transduction of G-protein coupled receptors through its activation upon cAMP binding. The widespread expression of PKA subunit genes, and the myriad of mechanisms by which cAMP is regulated within a cell suggest that PKA signaling is one of extreme importance to cellular function. It is involved in the control of a wide variety of cellular processes from metabolism to ion channel activation, cell growth and differentiation, gene expression and apoptosis. Importantly, since it has been implicated in the initiation and progression of many tumors, PKA has been proposed as a novel biomarker for cancer detection, and as a potential molecular target for cancer therapy. Here, we highlight some features of cAMP/PKA signaling that are relevant to cancer biology and present an update on targeting PKA in cancer therapy.

The Natural cAMP Elevating Compound Forskolin in Cancer Therapy: Is It Time?

Cancer is a major public health problem and the second leading cause of mortality around the world. Although continuous advances in the science of oncology and cancer research are now leading to improved outcomes for many cancer patients, novel cancer treatment options are strongly demanded. Naturally occurring compounds from a variety of vegetables, fruits, and medicinal plants have been shown to exhibit various anticancer properties in a number of in vitro and in vivo studies and represent an attractive research area for the development of new therapeutic strategies to fight cancer. Forskolin is a diterpene produced by the roots of the Indian plant Coleus forskohlii. The natural compound forskolin has been used for centuries in traditional medicine and its safety has also been documented in conventional modern medicine. Forskolin directly activates the adenylate cyclase enzyme, that generates cAMP from ATP, thus, raising intracellular cAMP levels. Notably, cAMP signaling, through the PKA‐dependent and/or ‐independent pathways, is very relevant to cancer and its targeting has shown a number of antitumor effects, including the induction of mesenchymal‐to‐epithelial transition, inhibition of cell growth and migration and enhancement of sensitivity to conventional antitumor drugs in cancer cells. Here, we describe some features of cAMP signaling that are relevant to cancer biology and address the state of the art concerning the natural cAMP elevating compound forskolin and its perspectives as an effective anticancer agent. J. Cell. Physiol. 232: 922–927, 2017.

Inorganic Phosphate Prevents Erk1/2 and Stat3 Activation and Improves Sensitivity to Doxorubicin of MDA-MB-231 Breast Cancer Cells.

Due to its expression profile, triple-negative breast cancer (TNBC) is refractory to the most effective targeted therapies available for breast cancer treatment. Thus, cytotoxic chemotherapy represents the mainstay of treatment for early and metastatic TNBC. Therefore, it would be greatly beneficial to develop therapeutic approaches that cause TNBC cells to increase their sensitivity to cytotoxic drugs. Inorganic phosphate (Pi) is emerging as an important signaling molecule in many cell types. Interestingly, it has been shown that Pi greatly enhances the sensitivity of human osteosarcoma cell line (U2OS) to doxorubicin. We investigated the effects of Pi on the sensitivity of TNBC cells to doxorubicin and the underlying molecular mechanisms, carrying out flow cytometry-based assays of cell-cycle progression and cell death, MTT assays, direct cell number counting and immunoblotting experiments. We report that Pi inhibits the proliferation of triple-negative MDA-MB-231 breast cancer cells mainly by slowing down cell cycle progression. Interestingly, we found that Pi strongly increases doxorubicin-induced cytotoxicity in MDA-MB-231 cells by apoptosis induction, as revealed by a marked increase of sub-G1 population, Bcl-2 downregulation, caspase-3 activation and PARP cleavage. Remarkably, Pi/doxorubicin combination-induced cytotoxicity was dynamically accompanied by profound changes in Erk1/2 and Stat3 protein and phosphorylation levels. Altogether, our data enforce the evidence of Pi acting as a signaling molecule in MDA-MB-231 cells, capable of inhibiting Erk and Stat3 pathways and inducing sensitization to doxorubicin of TNBC cells, and suggest that targeting Pi levels at local sites might represent the rationale for developing effective and inexpensive strategies for improving triple-negative breast cancer therapy.

Oxidative Stress and Cellular Response to Doxorubicin: A Common Factor in the Complex Milieu of Anthracycline Cardiotoxicity

The production of reactive species is a core of the redox cycling profile of anthracyclines. However, these molecular characteristics can be viewed as a double-edged sword acting not only on neoplastic cells but also on multiple cellular targets throughout the body. This phenomenon translates into anthracycline cardiotoxicity that is a serious problem in the growing population of paediatric and adult cancer survivors. Therefore, better understanding of cellular processes that operate within but also go beyond cardiomyocytes is a necessary step to develop more effective tools for the prevention and treatment of progressive and often severe cardiomyopathy experienced by otherwise successfully treated oncologic patients. In this review, we focus on oxidative stress-triggered cellular events such as DNA damage, senescence, and cell death implicated in anthracycline cardiovascular toxicity. The involvement of progenitor cells of cardiac and extracardiac origin as well as different cardiac cell types is discussed, pointing to molecular signals that impact on cell longevity and functional competence.

S-Adenosylmethionine Affects ERK1/2 and Stat3 Pathways and Induces Apotosis in Osteosarcoma Cells.

Osteosarcoma is a very aggressive bone tumor. Its clinical outcome remains discouraging despite intensive surgery, radiotherapy, and chemotherapy. Thus, novel therapeutic approaches are demanded. S‐Adenosylmethionine (AdoMet) is a naturally occurring molecule that is synthesized in our body by methionine adenosyltransferase isoenzymes and is also available as a nutritional supplement. AdoMet is the principal methyl donor in numerous methylation reactions and is involved in many biological functions. Interestingly, AdoMet has been shown to exert antiproliferative action in various cancer cells. However, the underlying molecular mechanisms are just starting to be studied. Here, we investigated the effects of AdoMet on the proliferation of osteosarcoma U2OS cells and the underlying mechanisms. We carried out direct cell number counting, MTT and flow cytometry‐based assays, and immunoblotting experiments in response to AdoMet treatment. We found that AdoMet strongly inhibits proliferation of U2OS cells by slowing‐down cell cycle progression and by inducing apoptosis. We also report that AdoMet consistently causes an increase of p53 and p21 cell‐cycle inhibitor, a decrease of cyclin A and cyclin E protein levels, and a marked increase of pro‐apoptotic Bax/Bcl‐2 ratio, with caspase‐3 activation and PARP cleavage. Moreover, the AdoMet‐induced antiproliferative effects were dynamically accompanied by profound changes in ERK1/2 and STAT3 protein and phosphorylation levels. Altogether, our data enforce the evidence of AdoMet acting as a biomolecule with antiproliferative action in osteosarcoma cells, capable of down‐regulating ERK1/2 and STAT3 pathways leading to cell cycle inhibition and apoptosis, and provide a rationale for the possible use of AdoMet in osteosarcoma therapy. J. Cell. Physiol. 231: 428–435, 2016.

The Influence of the Polymer Amount on the Biological Properties of PCL/ZrO2 Hybrid Materials Synthesized via Sol-Gel Technique

Organic/inorganic hybrid materials are attracting considerable attention in the biomedical area. The sol-gel process provides a convenient way to produce many bioactive organic–inorganic hybrids. Among those, poly(e-caprolactone)/zirconia (PCL/ZrO2) hybrids have proved to be bioactive with no toxic materials. The aim of this study was to investigate the effects of these materials on the cellular response as a function of the PCL content, in order to evaluate their potential use in the biomedical field. For this purpose, PCL/ZrO2 hybrids containing 6, 12, 24, and 50 wt % of PCL were synthesized by the sol-gel method. The effects of their presence on the NIH-3T3 fibroblast cell line carrying out direct cell number counting, MTT, cell damage assays, flow cytometry-based analysis of cell-cycle progression, and immunoblotting experiments. The results confirm and extend the findings that PCL/ZrO2 hybrids are free from toxicity. The hybrids containing 12 and 24 wt % PCL, (more than 6 and 50 wt % ones) enhance cell proliferation when compared to pure ZrO2 by affecting cell cycle progression. The finding that the content of PCL in PCL/ZrO2 hybrids differently supports cell proliferation suggests that PCL/ZrO2 hybrids could be useful tools with different potential clinical applications.

Forskolin improves sensitivity to doxorubicin of triple negative breast cancer cells via Protein Kinase A-mediated ERK1/2 inhibition

Graphical abstract Figure. No Caption available. ABSTRACT Triple negative breast cancer (TNBC) is an invasive, metastatic, highly aggressive tumor. Cytotoxic chemotherapy represents the current treatment for TNBC. However, relapse and chemo‐resistance are very frequent. Therefore, new therapeutic approaches that are able to increase the sensitivity to cytotoxic drugs are needed. Forskolin, a natural cAMP elevating agent, has been used for several centuries in medicine and its safeness has also been demonstrated in modern studies. Recently, forskolin is emerging as a possible novel molecule for cancer therapy. Here, we investigate the effects of forskolin on the sensitivity of MDA‐MB‐231 and MDA‐MB‐468 TNBC cells to doxorubicin through MTT assay, flow cytometry‐based assays (cell‐cycle progression and cell death), cell number counting and immunoblotting experiments. We demonstrate that forskolin strongly enhances doxorubicin‐induced antiproliferative effects by cell death induction. Similar effects are observed with IBMX and isoproterenol cAMP elevating agents and 8‐Br‐cAMP analog, but not by using 8‐pCPT‐2′‐O‐Me‐cAMP Epac activator. It is important to note that the forskolin‐induced potentiation of sensitivity to doxorubicin is accompanied by a strong inhibition of ERK1/2 phosphorylation, is mimicked by ERK inhibitor PD98059 and is prevented by pre‐treatment with Protein Kinase A (PKA) and adenylate cyclase inhibitors. Altogether, our data indicate that forskolin sensitizes TNBC cells to doxorubicin via a mechanism depending on the cAMP/PKA‐mediated ERK inhibition. Our findings sustain the evidence of anticancer activity mediated by forskolin and encourage the design of future in‐vivo/clinical studies in order to explore forskolin as a doxorubicin sensitizer for possible use in TNBC patients.

Chemical analysis and anti-proliferative activity of Campania Thymus Vulgaris essential oil

Abstract In this study, the potential antitumor activity of an essential oil (EO) obtained from Thymus vulgaris L. plants cultivated in Campania (Southern Italy) was evaluated. The gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy were used to investigate the chemical composition of EO. The monoterpenes thymol, p-cymene and carvacrol are the more abundant compounds detected. The anti-proliferative activity of the EO as a function of dose and time of exposure was evaluated by MTT assay on human osteosarcoma U2OS and pancreatic cancer PANC-1 cell lines. EO inhibits proliferation of both cell lines, but the effect on PANC-1 cells was more extensive. Flow cytometry and Western blot analyses revealed that the anti-proliferative effect is due to a decrease of cells in the S phase and their accumulation in the G1 phase, associated with the decrease of cyclin A and the increase of cell cycle inhibitor p27 protein levels, respectively.

Silica/Polyethylene Glycol Hybrid Materials Prepared by a Sol-Gel Method and Containing Chlorogenic Acid

Chlorogenic acid (CGA) is a very common dietary polyphenolic compound. CGA is becoming very attractive due to its potential use as preventive and therapeutic agent in many diseases, including cancer. Inorganic/organic hybrid materials are gaining considerable attention in the biomedical field. The sol-gel process provides a useful way to obtain functional organic/inorganic hybrids. The aim of this study was to synthesize silica/polyethylene glycol (PEG) hybrids with different percentages of CGA by sol-gel technique and to investigate their impact on the cancer cell proliferation. Synthesized materials have been chemically characterized through the FTIR spectroscopy and their bioactivity evaluated looking by SEM at their ability to produce a hydroxyapatite layer on their surface upon incubation with simulated body fluid (SBF). Finally, their effects on cell proliferation were studied in cell lines by direct cell number counting, MTT, flow cytometry-based cell-cycle and cell death assays, and immunoblotting experiments. Notably, we found that SiO2/PEG/CGA hybrids exhibit clear antiproliferative effects in different tumor, including breast cancer and osteosarcoma, cell lines in a CGA dependent manner, but not in normal cells. Overall, our results increase the evidence of CGA as a possible anticancer agent and illustrate the potential for clinical applications of sol-gel synthesized SiO2/PEG/CGA materials.

Forskolin sensitizes human acute myeloid leukemia cells to H3K27me2/3 demethylases GSKJ4 inhibitor via Protein Kinase A

Acute myeloid leukemia (AML) is an aggressive hematological malignancy occurring very often in older adults, with poor prognosis depending on both rapid disease progression and drug resistance occurrence. Therefore, new therapeutic approaches are demanded. Epigenetic marks play a relevant role in AML. GSKJ4 is a novel inhibitor of the histone demethylases JMJD3 and UTX. To note GSKJ4 has been recently shown to act as a potent small molecule inhibitor of the proliferation in many cancer cell types. On the other hand, forskolin, a natural cAMP raising compound, used for a long time in traditional medicine and considered safe also in recent studies, is emerging as a very interesting molecule for possible use in cancer therapy. Here, we investigate the effects of forskolin on the sensitivity of human leukemia U937 cells to GSKJ4 through flow cytometry-based assays (cell-cycle progression and cell death), cell number counting, and immunoblotting experiments. We provide evidence that forskolin markedly potentiates GSKJ4-induced antiproliferative effects by apoptotic cell death induction, accompanied by a dramatic BCL2 protein down-regulation as well as caspase 3 activation and PARP protein cleavage. Comparable effects are observed with the phosphodiesterase inhibitor IBMX and 8-Br-cAMP analogous, but not by using 8-pCPT-2′-O-Me-cAMP Epac activator. Moreover, the forskolin-induced enhancement of sensitivity to GSKJ4 is counteracted by pre-treatment with Protein Kinase A (PKA) inhibitors. Altogether, our data strongly suggest that forskolin sensitizes U937 cells to GSKJ4 inhibitor via a cAMP/PKA-mediated mechanism. Our findings provide initial evidence of anticancer activity induced by forskolin/GSKJ4 combination in leukemia cells and underline the potential for use of forskolin and GSKJ4 in the development of innovative and effective therapeutic approaches for AML treatment.