Luigi Sapio

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.

cAMP Elevation Down-Regulates β3 Integrin and Focal Adhesion Kinase and Inhibits Leptin-Induced Migration of MDA-MB-231 Breast Cancer Cells.

Abstract Breast cancer is one of the most common malignancies and a major cause of cancer death among women worldwide. The high mortality rate associated with breast cancer is mainly due to a propensity of the tumor to metastasize, even if small or undetectable. Given the relevant role of leptin in breast cancer growth and metastasis, novel strategies to counteract biological effects of this obesity-linked cytokine are warranted. Recently, we demonstrated that in MDA-MB-231 breast cancer cells, intracellular cAMP elevation completely abrogates both ERK1/2 and STAT3 phosphorylation in response to leptin. Very surprisingly, this provided evidence that when cAMP levels are increased, leptin drives cells towards apoptosis associated with a marked decrease of Bcl2 protein levels and accompanied by down-regulation of protein kinase A (PKA). The aim of the current study was to investigate the role of cAMP in leptin-associated motility of breast cancer cells. Here we show that cAMP elevation completely prevents leptin-induced migration of MDA-MB-231 breast cancer cells. Interestingly, the inhibition by cAMP-elevating agents of leptin-mediated cell migration is accompanied by a strong decrease of β3 integrin subunit and focal adhesion kinase (FAK) protein levels. Analysis of the underlying cAMP-dependent molecular mechanisms revealed that PKA blockers partly counteract the inhibition of leptin-induced migration and completely prevent the antiproliferative action by cAMP elevation. Moreover, a cAMP analogue that specifically activates Epac and not PKA has an inhibitory effect on leptin-induced cell migration as well. The present study confirms initial evidence for the efficacy of cAMP elevation against oncogenic effects of leptin, identifies β3 integrin subunit and FAK as proteins strongly down-regulated by cAMP elevation, and suggests that both cAMP/PKA- and cAMP/Epac-dependent pathways are involved in inhibition of leptin-induced migration of MDA-MB-231 breast cancer cells. The potential clinical significance and therapeutic applications of our data are discussed.

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.

Lactic dehydrogenase and cancer: an overview.

Despite the intense scientific efforts made, there are still many tumors that are difficult to treat and the percentage of patient survival in the long-term is still too low. Thus, new approaches to the treatment of cancer are needed. Cancer is a highly heterogeneous and complex disease, whose development requires a reorganization of cell metabolism. Most tumor cells downregulate mitochondrial oxidative phosphorylation and increase the rate of glucose consumption and lactate release, independently of oxygen availability (Warburg effect). This metabolic rewiring is largely believed to favour tumor growth and survival, although the underlying molecular mechanisms are not completely understood. Importantly, the correlation between the aerobic glycolysis and cancer is widely regarded as a useful biochemical basis for the development of novel anticancer strategies. Among the enzymes involved in glycolysis, lactate dehydrogenase (LDH) is emerging as a very attractive target for possible pharmacological approaches in cancer therapy. This review addresses the state of the art and the perspectives concerning LDH both as a useful diagnostic marker and a relevant molecular target in cancer therapy and management.

Biological influence of Ca/P ratio on calcium phosphate coatings by sol-gel processing

The objective of this work has been to develop low temperature sol-gel glass coatings to modify the substrate surface and to evaluate their bioactivity and biocompatibility. Glasses, based on SiO2·CaO·P2O5, were synthesized by the sol-gel technique using tetraethyl orthosilicate, calcium nitrate tetrahydrate and triethyl phosphate as precursors of SiO2, CaO and P2O5, respectively. Those materials, still in the sol phase, have been used to coat substrates by means of the dip-coating technique. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) has been used for characterize coatings and a microstructural analysis has been obtained using scanning electron microscopy (SEM). The potential applications of the coatings in the biomedical field were evaluated by bioactivity and biocompatibility tests. The coated substrate was immersed in simulated body fluid (SBF) for 21days and the hydroxyapatite deposition on its surface was subsequently evaluated via SEM-EDXS analysis, as an index of bone-bonding capability. In order to study the cell behavior and response to our silica based materials, prepared via the sol-gel method, with various Ca/P ratio and coating substrate, we have used the human osteoblast-like U2OS cell line.

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.

Synergistic cytotoxic effects of inorganic phosphate and chemotherapeutic drugs on human osteosarcoma cells

Novel therapeutic approaches are required for the treatment of osteosarcoma. Combination chemotherapy is receiving increased attention in order to identify compounds that may increase the therapeutic index of clinical anticancer drugs. In this regard, naturally occurring molecules with antitumor activity and with limited toxicity to normal tissues have been suggested as possible candidates for investigation of their synergistic efficacy in combination with antineoplastic drugs. Inorganic phosphate (Pi) is an essential nutrient for living organisms. Relevantly, Pi has emerged as an important signaling molecule capable of modulating multiple cellular functions by altering signal transduction pathways, gene expression and protein abundance in many cell types. Previously, we showed that Pi inhibits proliferation and aggressiveness of U2OS human osteosarcoma cells and that Pi is capable of inducing sensitization of osteosarcoma cells to doxorubicin in a p53-dependent manner. In this study, we extended the role of Pi in the chemosensitivity of osteosarcoma cells to other anticancer drugs. Specifically, we report and compare the antiproliferative effects of a combination between Pi and doxorubicin, Taxol® and 5-fluorouracil (5-FU) treatments. We found that Pi increases the antiproliferative response to both Taxol and doxorubicin to a similar extent. On the other hand, Pi did not potentiate the anticancer effects induced by 5-FU. These effects were paralleled by apoptosis induction and were cell cycle-dependent. The clinical significance of our data and their potential therapeutic applications for improving osteosarcoma treatment are discussed.

Inorganic Phosphate as a Novel Signaling Molecule with Antiproliferative Action in MDA-MB-231 Breast Cancer Cells

Abstract Inorganic phosphate (Pi) is an essential nutrient for living organisms. It plays a key role in diverse physiological functions, including osteoblast differentiation and skeletal mineralization. Relevantly, Pi is emerging as an important signaling molecule capable of modulating multiple cellular functions by altering signal transduction pathways, gene expression, and protein abundance in many cell types. To our knowledge, the consequences of elevated Pi on behavior of breast cancer cells have been poorly addressed. In this study we investigate the effects of Pi on proliferation of MDA-MB-231 breast cancer cells. We report that Pi inhibits proliferation of MDA-MB-231 cells by slowing cell cycle progression, without apoptosis occurrence. We found that Pi causes cells to accumulate in G1 phase in a time-dependent manner. Accordingly, G1 accumulation was associated with a decrease of cyclin A and cyclin E and an increase of cell cycle inhibitors p21 and p27 protein levels, respectively. Moreover, the Pi-induced antiproliferative effect was dynamically accompanied by profound changes in ERK1/2 and STAT3 protein and phosphorylation levels in response to Pi. Altogether, our data represent the first evidence of Pi acting as a novel signaling molecule in MDA-MB-231 breast cancer cells, capable of eliciting a strong antiproliferative action and suggest that targeting Pi levels at local sites might represent the rationale for developing novel strategies for therapeutic intervention in triple-negative breast cancer.

Inorganic phosphate as a signaling molecule: a potential strategy in osteosarcoma treatment.

Inorganic phosphate (Pi) is an essential nutrient to living organisms. It plays a key role in diverse biological processes, including osteoblast differentiation and skeletal mineralization. Maintenance of proper Pi homeostasis is a critical event, as any deviation from that state can lead to several acute and chronic disease states and influence the ageing process and lifespan. Serum Pi level is maintained within a narrow range through a complex interplay between intestinal absorption, exchange with intracellular and bone storage pools, renal tubular reabsorption and depends mainly on the activity of Na/Pi cotransporters. Pi is abundant in the diet and intestinal absorption of Pi is efficient and minimally regulated. The kidney is a major regulator of Pi homeostasis and can increase or decrease its Pi reabsorptive capacity to accommodate Pi need. Relevantly, Pi is emerging as an important signalling molecule capable of modulating multiple cellular functions by altering signal transduction pathways, gene expression and protein abundance in many cell types. However, little is known about the initial events involving the detection of changes in serum or local Pi concentrations and the subsequent downstream regulation cascade. Previously, we provided evidence that Pi inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. More recently, we demonstrated that Pi is capable also of inducing sensitization of osteosarcoma cells to doxorubicin in a p53-dependent manner and through a mechanism involving ERK1/2 down-regulation. This review summarizes the current knowledge regarding inorganic phosphate as a novel specific signaling molecule in bone and other cell types in mammals and discuss how targeting Pi levels at local sites might represent a potential strategy for improving osteosarcoma 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.