Chlorogenic Acid Enhances Doxorubicin-Mediated Cytotoxic Effect in Osteosarcoma Cells.

Despite the recurring outbreak of resistance mechanisms and adverse reactions, doxorubicin (Doxo) still remains the standard-of-care for several cancers, including osteosarcoma (OS). As an appealing source of phytochemical compounds, naturally occurring molecules have extensively been reported to overcome Doxo limitations in preclinical models. Unlike other dietary polyphenols, only few studies recognize chlorogenic acid (CGA) as a potential partner in combination therapy, while, conversely, its anticancer evidence is steadily growing, ultimately in OS. On this basis, herein we examine the cooperating effects between CGA and Doxo in U2OS and MG-63 human OS cells. With respect to Doxo alone, the concomitant administration of CGA further decreased cell viability and growth, promoting cell death potentially via apoptosis induction. Furthermore, a longer-lasting reduction in clonogenic potential deeply supported the CGA ability to improve Doxo efficacy in those cells. Remarkably, CGA treatment ameliorated Doxo-induced cytotoxicity in H9c2 rat cardiomyocyte cells instead. Although inactivation of p44/42 MAPK was detected in response to CGA plus Doxo, PD98059-mediated p44/42 MAPK impairment enhanced the combination outcome in OS cells. These findings firstly propose CGA as a promising chemosensitizer and cardioprotective agent in OS therapy, suggesting the p44/42 MAPK pathway as relevantly involved in CGA-mediated Doxo susceptibility.

Chlorogenic acid activates ERK1/2 and inhibits proliferation of osteosarcoma cells.

Osteosarcoma (OS) is a very aggressive metastatic pediatric and adolescent tumor. Due to its recurrent development of chemotherapy resistance, clinical outcome for OS patients remains poor. Therefore, discovering more effective anticancer agents is needed. Chlorogenic acid (CGA) is a phenolic compound contained in plant-related products that modulates many cellular functions and inhibits cell proliferation in several cancer types. However, few evidence is available in OS. Here, we investigate the effects of CGA in U2OS, Saos-2, and MG-63 OS cells. By multiple approaches, we demonstrate that CGA acts as anticancer molecule affecting the cell cycle and provoking cell growth inhibition mainly by apoptosis induction. We also provide evidence that CGA strongly activates extracellular-signal-regulated kinase1/2 (ERK1/2). Strikingly, ERK1/2 inhibitor PD98059 sensitizes the cells to CGA. Altogether, our data enforce the evidence of the anticancer activity mediated by CGA and provide the rationale for the development of innovative therapeutic strategies in OS cure.

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. © 2016 Wiley Periodicals, Inc.

Adiponectin down-regulates CREB and inhibits proliferation of A549 lung cancer cells.

INTRODUCTION: Adipokines are known to play a relevant role in a number of cancer related molecular pathways. Adiponectin is a major adipokine with anti-inflammatory and beneficial metabolic actions. Furthermore, it has been shown to exert anti-carcinogenic effects in various tumor models and some clinical studies suggested an inverse relationship between circulating levels of adiponectin and an increased risk for development of malignancies. On the other hand, the cyclic AMP response element binding (CREB) transcription factor has been clearly linked to lung cancer. METHODS: we analyzed cell proliferation, cell cycle of A549 cells treated with adiponectin as well as CREB activation status in human lung adenocarcinoma A549 cells and in non-small cell lung cancer (NSCLC) samples. RESULTS: adiponectin treatment, at concentrations ranging between 5 and 50 µg/ml mimicking human serum levels, has a significant effect on reducing tumor cell proliferation of A549 cells, mainly by altering cell cycle progression. Importantly, we provide evidence that adiponectin clearly inhibits in a dose- and time-dependent manner CREB phosphorylation (activation) and, at least in part, also the level of CREB protein itself, preceding and accompanying the anti-proliferative effects in response to adiponectin. Moreover, in agreement with previous studies demonstrating that CREB over-expression occurs in many tumors, we also show by western-blotting from lung specimen that CREB is significantly up-regulated in NSCLC samples compared to adjacent normal tissues from six patients. CONCLUSIONS: Overall, our results represent the first evidence of CREB inhibition by adiponectin and may provide new insight into therapeutic strategies for lung cancer.

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.

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.

Inorganic phosphate enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin via a p53-dependent pathway.

Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. The clinical outcome for osteosarcoma remains discouraging despite aggressive surgery and intensive radiotherapy and chemotherapy regimens. Thus, novel therapeutic approaches are needed. Previously, we have shown that inorganic phosphate (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. In this study, we investigated whether Pi could affect chemosensitivity of osteosarcoma cells and the underlying molecular mechanisms. Here, we report that Pi inhibits proliferation of p53-wild type U2OS cells (and not of p53-null Saos and p53-mutant MG63 cells) by slowing-down cell cycle progression, without apoptosis occurrence. Interestingly, we found that Pi strongly enhances doxorubicin-induced cytotoxicity in U2OS, and not in Saos and MG63 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 accompanied by an increase of p53 protein levels and of p53 target genes mdm2, p21 and Bax, and was significantly reduced by the p53 inhibitor pifithrine-alpha. Moreover, the doxorubicin-induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Altogether, our data enforce the evidence of Pi as a novel signaling molecule capable of inhibiting ERK pathway and inducing sensitization to doxorubicin of osteosarcoma cells by p53-dependent apoptosis, implying that targeting Pi levels might represent a rational strategy for improving osteosarcoma therapy.

Forskolin affects proliferation, migration and Paclitaxel-mediated cytotoxicity in non-small-cell lung cancer cell lines via adenylyl cyclase/cAMP axis.

Non-Small-Cell Lung Cancer (NSCLC) is considered one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths worldwide. Despite the undoubted therapeutic advances that have occurred in clinical practice over time, due to its high degree in both heterogeneity and resistance, NSCLC remains largely incurable. As a natural cAMP elevating agent, Forskolin has shown anti-cancer properties in different tumor types, thus supposing its possible usage in treating malignancies. In this study, we investigated the Forskolin outcome in H1299 and A549 NSCLC cell lines, either alone or in combination with Paclitaxel. We proved that Forskolin impairs cell growth and migration ability of these cells, concurrently. Albeit with a different extent between H1299 and A549, changes in cell-cycle progression and epithelial-mesenchymal markers were observed in response to Forskolin administration. Interestingly, comparable cell growth impairment was also obtained with the cAMP phosphodiesterase inhibitor IBMX, while the employment of adenylyl cyclase inhibitor SQ22536 counteracted, at least in part, the Forskolin-mediated anticancer effects. Besides as a single agent, we also demonstrated that Forskolin strongly enhances Paclitaxel-induced cytotoxicity, affecting cell death mainly via apoptosis induction. Notably, H89-mediated protein kinase A (PKA) inhibition further deteriorated the combination outcome. Altogether, our data designate Forskolin as a possible anticancer molecule in NSCLC, and recognize the adenylyl cyclase/cAMP axis as one of the pathways involved in. Although achieved at preclinical stage, our findings encourage the design of future studies aimed at further exploring the Forskolin employment in NSCLC treatment.

AdipoRon and Pancreatic Ductal Adenocarcinoma: a future perspective in overcoming chemotherapy-induced resistance?

The latest scientific knowledge has provided additional insights accountable for the worst prognosis for pancreatic ductal adenocarcinoma (PDAC). Among the causative factors, the aptitude to develop resistance towards approved medications denotes the master key for understanding the lack of improvement in PDAC survival over the years. Even though several compounds have achieved encouraging results at preclinical stage, no new adjuvant agents have reached the bedside of PDAC patients lately. The adiponectin receptor agonist AdipoRon is emerging as a promising anticancer drug in different cancer models, particularly in PDAC. Building on the existing findings, we recently reinforced its candidacy in PDAC cells, proposing AdipoRon either as a suitable partner in gemcitabine-based treatment or as an effective drug in resistant cells. Crossing the current state-of-the-art, herein we provide a critical perspective on AdipoRon to figure out whether this receptor agonist can potentially be considered a future therapeutic choice in overcoming chemotherapy-induced resistance, expressly in PDAC.

Integrating Gemcitabine-Based Therapy With AdipoRon Enhances Growth Inhibition in Human PDAC Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of all pancreatic cancers. Albeit its incidence does not score among the highest in cancer, PDAC prognosis is tremendously fatal. As a result of either aggressiveness or metastatic stage at diagnosis, chemotherapy constitutes the only marginally effective therapeutic approach. As gemcitabine (Gem) is still the cornerstone for PDAC management, the low response rate and the onset of resistant mechanisms claim for additional therapeutic strategies. The first synthetic orally active adiponectin receptor agonist AdipoRon (AdipoR) has recently been proposed as an anticancer agent in several tumors, including PDAC. To further address the AdipoR therapeutic potential, herein we investigated its pharmacodynamic interaction with Gem in human PDAC cell lines. Surprisingly, their simultaneous administration revealed a more effective action in contrasting PDAC cell growth and limiting clonogenic potential than single ones. Moreover, the combination AdipoR plus Gem persisted in being effective even in Gem-resistant MIA PaCa-2 cells. While a different ability in braking cell cycle progression between AdipoR and Gem supported their cooperating features in PDAC, mechanistically, PD98059-mediated p44/42 MAPK ablation hindered combination effectiveness. Taken together, our findings propose AdipoR as a suitable partner in Gem-based therapy and recognize the p44/42 MAPK pathway as potentially involved in combination outcomes.

The analysis of alcohol consumption during the severe acute respiratory syndrome Coronavirus 2 Italian lockdown.

BACKGROUND: The SARS-CoV-2 lockdown resulted in deep changes of lifestyles, promoting in many people the onset of psychological symptoms generally associated with drug and alcohol abuse. The aim of this study was to assess the variation of alcohol drinking habits in a sample of Italian citizens during lockdown and to identify the psychosocial factors surrounding it. METHODS: An online anonymous questionnaire was created and submitted from April 9 to April 28, 2020. Questions were related to personal psychosocial details and alcohol drinking habits during the lockdown, including Alcohol Use Disorders Identification Test (AUDIT C) questions. RESULTS: On a total of 1234 surveys the increase of both anxiety and fear was largely detected (63% and 61% respectively). The 18% increased alcohol consumption during the lockdown and it showed a significant correlation with anxiety and fear experienced (both P<0.001). The relative risk for 7 to 9 and more than 10 drinks per day consumption were directly linked to these symptoms (P<0.001). The most involved categories of participants showed this harmful association were self-employed workers and participants who live alone, subject aged 30-50 with high level of instruction or students, and not occupied people in the age range 18-19 (all P<0.001). Additionally, the subset of the study population that showed low alcohol consumption before the lockdown has demonstrated the worsening of alcohol assumption during the quarantine (P<0.0001). CONCLUSIONS: Several psychosocial factors are involved in determining the increase of alcohol consumption during lockdown and need the healthcare support to avoid awful impact on human life.

Alcoholic Consumption of Young Italians During the SARS-CoV-2 Pandemic.

BACKGROUND: The international health emergency caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which, at the end of 2019, hit the world, forced the governments of all countries to adopt stringent restrictive measures to contain the spread of the virus. Several studies have revealed worsening levels of anxiety, depression and perceived stress related to these restrictions and the resulting lifestyle changes. Some studies have also confirmed the presence of a relationship between SARS-CoV-2-related emotional distress and drinking behavior. Indeed, is a wellknown fact that alcohol consumption is one of the behavioral strategies used to reduce negative emotional states. However, it was documented that young people developed different responses to alcohol use during the pandemic than adults. OBJECTIVE: The aim of this work was to investigate the consumption habits of young Italians and how the consumption and purchase of alcoholic beverages have changed following the pandemic. New ways of drinking alcohol were also interesting to observe, such as online. METHODS: Young people between 18 and 35 years old were subjected to an anonymous questionnaire of 22 questions on the adoption of forms of behavior at risk through alcohol consumption, the quantity and occasions of preferential consumption, and on the methods and quantities of alcoholic beverage purchase, before and during the SARS-CoV-2 pandemic. The subjects who declared themselves "non-drinkers" were not included in the statistical survey. RESULTS: About 33% of the enrolled "drinkers" (268/823), adopted risky forms of alcoholic behavior. Males reported a higher average habit of drinking wine or alcohol (M = 1.9953 ± 1.39743, F = 1.7373 ± 1.36688, p <0.005); an increased frequency of drinking (M = 2.3025 ± 0.80610 F = 2.0494 ± 0.75043 p <0.001); a higher average number of drinks consumed (M = 1.5182 ± 0.85646, F = 1.2618 ± 0.53292, p <0.001) and binge drinking to the greatest extent (M = 1.1933 ± 0.96522 F = 0.8176 ± 0.85446 p <0.001). Education and employment were significantly correlated with the frequency of alcohol consumption (r = 0.107 p <0.005 and r = 0.120 p = 0.001 respectively). Subjects reported buying alcoholic beverages during the pandemic with a frequency of "less than once a month" (N = 291, 35.36%) and mainly in shops (N = 556, 67.56%), while before the pandemic they mainly bought alcohol once a week (N = 431, 52.37%) and predominantly in bars / clubs (N = 619, 75.21%). New ways of drinking alcohol such as online drinking, have not been significantly identified. CONCLUSION: A change in alcohol consumed and alcohol purchased before and during the SARSCoV- 2 pandemic was revealed.

The Lesson from the First Italian Lockdown: Impacts on Anxiety and Depressive Symptoms and Sleep Quality in Patients with Remission of Inflammatory Bowel Disease.

BACKGROUND: During the COVID-19 pandemic in Italy, decisions were taken to adopt restrictive legislative measures, such as the first half of the 2020 lockdown. In those months, patients with inflammatory bowel disease experienced social isolation and reduced access to health care. OBJECTIVE: We aimed to evaluate, in this condition, the presence of remission subgroups that were most impacted by the lockdown. METHODS: During the first Italian lockdown, we recruited patients with remission of inflammatory bowel disease by administering an online questionnaire including patient demographics, the Beck Anxiety Questionnaire Inventory, the Beck Depression Inventory questionnaire, and the Pittsburg Sleep Quality Index, all validated standardized questionnaires for anxiety symptom levels, depression, and sleep quality. RESULTS: Our results showed how female patients (p<0.0001) with Crohn's disease (p<0.001) experienced worse levels of anxiety symptoms. Female patients (p<0.0001) between 50 and 60 years of age (p=0.013) with Crohn's disease (p=0.047) experienced worse levels of depressive symptoms. Females also experienced significantly worse sleep levels (p<0.001). We found a correlation between the number of sleeping hours (p<0.001) and the time taken to fall asleep (p<0.001) and the Beck Anxiety Questionnaire Inventory,which showed a linear worsening of the number of minutes it took to fall asleep, and the Beck Depression Inventory questionnaire. CONCLUSION: Among patients with remission of inflammatory bowel disease, female patients, patients with Crohn's disease, and people aged between 50 and 70 years should be considered for screening for anxiety and depression disorders and an assessment of sleep quality.

Protein Kinase A Detection in Human Urine Samples.

Actively involved in tumor maintenance, cAMP-dependent protein kinase A (PKA) has been proposed as a putative biomarker in cancer. Recently, an active PKA form has been identified in human sera and PKA autoantibodies have been detected in cancer patients. However, their serum functions, as well as diagnostic significance, remain largely unknown. Although several PKA detection assays have been developed, none refer to a laboratory diagnostic procedure. Among these, ELISA and Western blotting (WB) assays have been employed in PKA detection. Since, to the best of our knowledge, there are no data showing its presence in human urine samples, herein, we explore the possibility of PKA's existence in this biological specimen. Interestingly, among the 30 screened urines by quantitative sandwich ELISA, we recognized detectable PKA levels in 5 different samples, and of those two exhibited a considerable high concentration. To corroborate these results, we also evaluated PKA's presence in both positive and negative ELISA urines by WB. Remarkably, immunoblotting analysis confirmed PKA's existence in certain, but not in all, human urine specimens. Despite being quite preliminary, these findings firstly identify PKA in urine samples and provide evidence for its potential clinic usage as a diagnostic analyte in laboratory medicine.

Leptin potentiates antiproliferative action of cAMP elevation via protein kinase A down-regulation in breast cancer cells.

Previously, we have shown that leptin potentiates the antiproliferative action of cAMP elevating agents in breast cancer cells and that the protein kinase A (PKA) inhibitor KT-5720 prevented the antiproliferative effects induced by the leptin plus cAMP elevation. The present experiments were designed to gain a better understanding about the PKA role in the antitumor interaction between leptin and cAMP elevating agents and on the underlying signaling pathways. Here we show that exposure of MDA-MB-231 breast cancer cells to leptin resulted in a strong phosphorylation of both ERK1/2 and STAT3. Interestingly, intracellular cAMP elevation upon forskolin pretreatment completely abrogated both ERK1/2 and STAT3 phosphorylation in response to leptin and was accompanied by a consistent CREB phosphorylation. Notably, leptin plus forskolin cotreatments resulted in a strong decrease of both PKA regulatory RIα and catalytic subunits protein levels. Importantly, pretreatment with the PKA inhibitor KT-5720 blocked the forskolin-induced CREB phosphorylation and prevented both the inhibition by forskolin of leptin-induced ERK1/2 and STAT3 phosphorylation and the PKA subunits down-regulation induced by the combination of leptin and forskolin. Altogether, our results indicate that leptin-dependent signaling pathways are influenced by cAMP elevation and identify PKA as relevantly involved in the pharmacological antitumor interaction between leptin and cAMP elevating drugs in MDA-MB-231 cells. We propose a molecular model by which PKA confers its effects. Potential therapeutic applications by our data will be discussed.

Targeting CREB in Cancer Therapy: A Key Candidate or One of Many? An Update.

Intratumor heterogeneity (ITH) is considered the major disorienting factor in cancer treatment. As a result of stochastic genetic and epigenetic alterations, the appearance of a branched evolutionary shape confers tumor plasticity, causing relapse and unfavorable clinical prognosis. The growing evidence in cancer discovery presents to us "the great paradox" consisting of countless potential targets constantly discovered and a small number of candidates being effective in human patients. Among these, cyclic-AMP response element-binding protein (CREB) has been proposed as proto-oncogene supporting tumor initiation, progression and metastasis. Overexpression and hyperactivation of CREB are frequently observed in cancer, whereas genetic and pharmacological CREB downregulation affects proliferation and apoptosis. Notably, the present review is designed to investigate the feasibility of targeting CREB in cancer therapy. In particular, starting with the latest CREB evidence in cancer pathophysiology, we evaluate the advancement state of CREB inhibitor design, including the histone lysine demethylases JMJD3/UTX inhibitor GSKJ4 that we newly identified as a promising CREB modulator in leukemia cells. Moreover, an accurate analysis of strengths and weaknesses is also conducted to figure out whether CREB can actually represent a therapeutic candidate or just one of the innumerable preclinical cancer targets.

The KDM Inhibitor GSKJ4 Triggers CREB Downregulation via a Protein Kinase A and Proteasome-Dependent Mechanism in Human Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a progressive hematopoietic-derived cancer arising from stepwise genetic mutations of the myeloid lineage. cAMP response element-binding protein (CREB) is a nuclear transcription factor, which plays a key role in the multistep process of leukemogenesis, thus emerging as an attractive potential drug target for AML treatment. Since epigenetic dysregulations, such as DNA methylation, histone modifications, as well as chromatin remodeling, are a frequent occurrence in AML, an increasing and selective number of epi-drugs are emerging as encouraging therapeutic agents. Here, we demonstrate that the histone lysine demethylases (KDMs) JMJD3/UTX inhibitor GSKJ4 results in both proliferation decrease and CREB protein downregulation in AML cells. We found that GSKJ4 clearly decreases CREB protein, but not CREB mRNA levels. By cycloheximide assay, we provide evidence that GSKJ4 reduces CREB protein stability; moreover, proteasome inhibition largely counteracts the GSKJ4-induced CREB downregulation. Very interestingly, a rapid CREB phosphorylation at the Ser133 residue precedes CREB protein decrease in response to GSKJ4 treatment. In addition, protein kinase A (PKA) inhibition, but not extracellular signal-regulated kinase (ERK)1/2 inhibition, almost completely prevents both GSKJ4-induced p-Ser133-CREB phosphorylation and CREB protein downregulation. Overall, our study enforces the evidence regarding CREB as a potential druggable target, identifies the small epigenetic molecule GSKJ4 as an "inhibitor" of CREB, and encourages the design of future GSKJ4-based studies for the development of innovative approaches for AML therapy.

AdipoRon Affects Cell Cycle Progression and Inhibits Proliferation in Human Osteosarcoma Cells.

AdipoRon (AdipoR) is the first synthetic molecule acting as a selective and potent adiponectin receptor agonist. Recently, the possible pharmacological use of AdipoR in different pathological conditions has been addressed. Interestingly, initial evidence suggests that AdipoR may have anticancer properties in different preclinical models, such as pancreatic and ovarian cancer. To our knowledge, so far no research has been directed at determining the impact of AdipoR on osteosarcoma, the most aggressive and metastatic bone malignancy occurring in childhood and adolescence age. Here, we investigate the possible antitumor effects of AdipoR in osteosarcoma cell lines. MTT and cell growth curve assays clearly indicate that AdipoR inhibits, at different extents, proliferation in both U2OS and Saos-2 osteosarcoma cell lines, the latter being more sensitive. Moreover, flow cytometry-based assays point out a significant G0/G1 phase accumulation and a contemporary S phase decrease in response to AdipoR. Consistent with the different sensitivity, a strong subG1 appearance in Saos-2 after 48 and 72 hours of treatment is also observed. The investigation of the molecular mechanisms highlights a common and initial ERK1/2 activation in response to AdipoR in both Saos-2 and U2OS cells. Interestingly, a simultaneous and dramatic downregulation of p70S6K phosphorylation, one of the main targets of mTORC1 pathway, has also been observed in AdipoR-treated Saos-2, but not in U2OS cells. Importantly, a strengthening of AdipoR-induced effects was reported upon everolimus-mediated mTORC1 perturbation in U2OS cells. In conclusion, our findings provide initial evidence of AdipoR as an anticancer molecule differently affecting various signaling pathways involved in cell cycle and cell death in osteosarcoma cells and encourage the design of future studies to further understand its pattern of activities.

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

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.

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.