ERα/LKB1 complex upregulates E-cadherin expression and stimulates breast cancer growth and progression upon adiponectin exposure.

Adiponectin is the major adipocytes-secreted protein involved in obesity-related breast cancer growth and progression. We proved that adiponectin promotes proliferation in ERα-positive breast cancer cells, through ERα transactivation and the recruitment of LKB1 as ERα-coactivator. Here, we showed that adiponectin-mediated ERα transactivation enhances E-cadherin expression. Thus, we investigated the molecular mechanism through which ERα/LKB1 complex may modulate the expression of E-cadherin, influencing tumor growth, progression and distant metastasis. We demonstrated that adiponectin increases E-cadherin expression in ERα-positive 2D and higher extent in 3D cultures. This occurs through a direct activation of E-cadherin gene promoter by ERα/LKB1-complex. The impact of E-cadherin on ERα-positive breast cancer cell proliferation comes from the evidence that in the presence of E-cadherin siRNA the proliferative effects of adiponectin is no longer noticeable. Since E-cadherin connects cell polarity and growth, we investigated if the adiponectin-enhanced E-cadherin expression could influence the localization of proteins cooperating in cell polarity, such as LKB1 and Cdc42. Surprisingly, immunofluorescence showed that, in adiponectin-treated MCF-7 cells, LKB1 and Cdc42 mostly colocalize in the nucleus, impairing their cytosolic cooperation in maintaining cell polarity. The orthotopic implantation of MCF-7 cells revealed an enhanced E-cadherin-mediated breast cancer growth induced by adiponectin. Moreover, tail vein injection of MCF-7 cells showed a higher metastatic burden in the lungs of mice receiving adiponectin-treated cells compared to control. From these findings it emerges that adiponectin treatment enhances E-cadherin expression, alters cell polarity and stimulates ERα-positive breast cancer cell growth in vitro and in vivo, sustaining higher distant metastatic burden.

Valproic acid inhibits cell growth in both MCF-7 and MDA-MB231 cells by triggering different responses in a cell type-specific manner.

BACKGROUND: Breast cancer is the second leading cause of death among women after lung cancer. Despite the improvement in prevention and in therapy, breast cancer still remains a threat, both for pre- and postmenopausal women, due to the development of drug resistance. To counteract that, novel agents regulating gene expression have been studied in both hematologic and solid tumors. The Histone Deacetylase (HDAC) inhibitor Valproic Acid (VA), used for epilepsy and other neuropsychiatric diseases, has been demonstrated a strong antitumoral and cytostatic activity. In this study, we tested the effects of Valproic Acid on the signaling pathways involved in breast cancer cells viability, apoptosis and in Reactive Oxygen Species (ROS) production using ER-α positive MCF-7 and triple negative MDA-MB-231 cells. METHODS: Cell proliferation assay was performed by MTT Cell cycle, ROS levels and apoptosis were analyzed by flow cytometry, protein levels were detected by Western Blotting. RESULTS: Cell treatment with Valproic Acid reduced cell proliferation and induced G0/G1 cell cycle arrest in MCF-7 and G2/M block in MDA-MB-231 cells. In addition, in both cells the drug enhanced the generation of ROS by the mitochondria. In MCF-7 treated cells, it has been observed a reduction in mitochondrial membrane potential, a down regulation of the anti-apoptotic marker Bcl-2 and an increase of Bax and Bad, leading to release of cytochrome C and PARP cleavage. Less consistent effects are recorded in MDA-MB-231 cells, in which the greater production of ROS, compared to MCF-7cells, involves an inflammatory response (activation of p-STAT3, increased levels of COX2). CONCLUSIONS: Our results have demonstrated that in MCF-7 cells the Valproic Acid is a suitable drug to arrest cell growth, to address apoptosis and mitochondrial perturbations, all factors that are important in determining cell fate and health. In a triple negative MDA-MB 231 cells, valproate directs the cells towards the inflammatory response with a sustained expression of antioxidant enzymes. Overall, the not always unequivocal data between the two cellular phenotypes indicate that further studies are needed to better define the use of the drug, also in combination with other chemotherapy, in the treatment of breast tumors.

Unraveling the Role of Adiponectin Receptors in Obesity-Related Breast Cancer.

Obesity has a noteworthy role in breast tumor initiation and progression. Among the mechanisms proposed, the most validated is the development of chronic low-grade inflammation, supported by immune cell infiltration along with dysfunction in adipose tissue biology, characterized by an imbalance in adipocytokines secretion and alteration of their receptors within the tumor microenvironment. Many of these receptors belong to the seven-transmembrane receptor family, which are involved in physiological features, such as immune responses and metabolism, as well as in the development and progression of several malignancies, including breast cancer. These receptors are classified as canonical (G protein-coupled receptors, GPCRs) and atypical receptors, which fail to interact and activate G proteins. Among the atypical receptors, adiponectin receptors (AdipoRs) mediate the effect of adiponectin, the most abundant adipocytes-derived hormone, on breast cancer cell proliferation, whose serum levels are reduced in obesity. The adiponectin/AdipoRs axis is becoming increasingly important regarding its role in breast tumorigenesis and as a therapeutic target for breast cancer treatment. The objectives of this review are as follows: to point out the structural and functional differences between GPCRs and AdipoRs, and to focus on the effect of AdipoRs activation in the development and progression of obesity-dependent breast cancer.

Poncirus trifoliata (L.) Raf. Seed Extract Induces Cell Cycle Arrest and Apoptosis in the Androgen Receptor Positive LNCaP Prostate Cancer Cells.

Prostate cancer (PCa) is the second most common male cancer. Its incidence derives from the interaction between modifiable and non-modifiable factors. The progression of prostate cancer into a more aggressive phenotype is associated with chronic inflammation and increased ROS production. For their biological properties, some phytochemicals from fruits and vegetable emerge as a promise strategy for cancer progression delay. These bioactive compounds are found in the highest amounts in peels and seeds. Poncirus trifoliata (L.) Raf. (PT) has been widely used in traditional medicine and retains anti-inflammatory, anti-bacterial, and anticancer effects. The seeds of P. trifoliata were exhaustively extracted by maceration with methanol as the solvent. The cell proliferation rate was performed by MTT and flow cytometry, while the apoptosis signals were analyzed by Western blotting and TUNEL assay. P. trifoliata seed extract reduced LNCaP and PC3 cell viability and induced cell cycle arrest at the G0/G1phase and apoptosis. In addition, a reduction in the AKT/mTOR pathway has been observed together with the up-regulation of stress-activated MAPK (p38 and c-Jun N-terminal kinase). Based on the study, the anti-growth effects of PT seed extract on prostate tumor cells give indications on the potential of the phytochemical drug for the treatment of this type of cancer. However, future in-depth studies are necessary to identify which components are mainly responsible for the anti-neoplastic response.

Cdk4 Regulates Glioblastoma Cell Invasion and Stemness and Is Target of a Notch Inhibitor Plus Resveratrol Combined Treatment.

Glioblastoma multiforme (GBM) is one of the most aggressive types of cancer characterized by poor patient outcomes. To date, it is believed that the major cause of its recurrence and chemoresistance is represented by the enrichment of GBM stem cells (GSCs) sustained by the abnormal activation of a number of signaling pathways. In this study, we found that in GBM cells, treatment with low toxicity doses of the γ-secretase inhibitor RO4929097 (GSI), blocking the Notch pathway activity, in combination with resveratrol (RSV) was able to reverse the basal mesenchymal phenotype to an epithelial-like phenotype, affecting invasion and stemness interplay. The mechanism was dependent on cyclin D1 and cyclin-dependent kinase (CDK4), leading to a reduction of paxillin (Pxn) phosphorylation. Consequently, we discovered the reduced interaction of Pxn with vinculin (Vcl), which, during cell migration, transmits the intracellular forces to the extracellular matrix. The exogenous expression of a constitutively active Cdk4 mutant prevented the RSV + GSI inhibitory effects in GBM cell motility/invasion and augmented the expression of stemness-specific markers, as well as the neurosphere sizes/forming abilities in untreated cells. In conclusion, we propose that Cdk4 is an important regulator of GBM stem-like phenotypes and invasive capacity, highlighting how the combined treatment of Notch inhibitors and RSV could be prospectively implemented in the novel therapeutic strategies to target Cdk4 for these aggressive brain tumors.

Toward Multitasking Pharmacological COX-Targeting Agents: Non-Steroidal Anti-Inflammatory Prodrugs with Antiproliferative Effects.

The antitumor activity of certain anti-inflammatory drugs is often attributed to an indirect effect based on the inhibition of COX enzymes. In the case of anti-inflammatory prodrugs, this property could be attributed to the parent molecules with mechanism other than COX inhibition, particularly through formulations capable of slowing down their metabolic conversion. In this work, a pilot docking study aimed at comparing the interaction of two prodrugs, nabumetone (NB) and its tricyclic analog 7-methoxy-2,3-dihydro-1H-cyclopenta[b]naphthalen-1-one (MC), and their common active metabolite 6-methoxy-2-naphthylacetic acid (MNA) with the COX binding site, was carried out. Cytotoxicity, cytofluorimetry, and protein expression assays on prodrugs were also performed to assess their potential as antiproliferative agents that could help hypothesize an effective use as anticancer therapeutics. Encouraging results suggest that the studied compounds could act not only as precursors of the anti-inflammatory metabolite, but also as direct antiproliferative agents.

Uncoupling effects of estrogen receptor α on LKB1/AMPK interaction upon adiponectin exposure in breast cancer.

Adipose tissue is a metabolic and endocrine organ that secretes bioactive molecules called adipocytokines. Among these, adiponectin has a crucial role in obesity-associated breast cancer. The key molecule of adiponectin signaling is AMPK, which is mainly activated by liver kinase B1 (LKB1). Here, we demonstrated that estrogen receptor-α (ERα)/LKB1 interaction may negatively interfere with the LKB1 capability to phosphorylate AMPK and inhibit its downstream signaling TSC2/mTOR/p70S6k. In adiponectin-treated MCF-7 cells, AMPK signaling was not working, resulting in its downstream target acetyl-CoA carboxylase (ACC) being still active. In contrast, in MDA-MB-231 cells, AMPK and ACC phosphorylation was enhanced by adiponectin, inhibiting lipogenesis and cell growth. Upon adiponectin, ERα signaling switched the energy balance of breast cancer cells toward a lipogenic phenotype. Therefore, adiponectin played an inhibitory role on ERα-negative cell growth and progression in vitro and in vivo. In contrast, low adiponectin levels, similar to those circulating in obese patients, acted on ERα-positive cells as a growth factor, stimulating proliferation. The latter effect was blunted in vivo by high adiponectin concentration. All this may have translational relevance, addressing how the handling of adiponectin, as a therapeutic tool in breast cancer treatment, needs to be carefully considered in ERα-positive obese patients, where circulating levels of this adipocytokine are relatively low. In other words, in ERα-positive breast cancer obese patients, higher adiponectin doses should be administered with respect to ERα-negative breast cancer, also opportunely combined with antiestrogen therapy. -Mauro, L., Naimo, G. D., Gelsomino, L., Malivindi, R., Bruno, L., Pellegrino, M., Tarallo, R., Memoli, D., Weisz, A., Panno, M. L., Andò, S. Uncoupling effects of estrogen receptor α on LKB1/AMPK interaction upon adiponectin exposure in breast cancer.

Estrogen receptor-α drives adiponectin effects on cyclin D1 expression in breast cancer cells.

Obesity is a risk factor for breast cancer, largely due to altered expression of various adipocytokines. As it concerns adiponectin, there are not univocal results regarding its role in breast cancer occurrence and progression. Here, we demonstrate that in animals injected with human estrogen receptor (ER)-α-negative MDA-MB-231 cells pretreated with adiponectin (1 and 5 µg/ml), a significant reduction (60 and 40%, respectively) in tumor volume is observed, whereas an increased tumor growth (54 and 109%, respectively) is evidenced in the animals receiving human ER-α-positive MCF-7 cells. Moreover, cyclin D1 (CD1) mRNA and protein levels are decreased in MDA-MB-231 cells, whereas they are up-regulated in ER-α-positive cells by adiponectin. These findings fit with the opposite effects of adiponectin on CD1 promoter: 0.44- and 0.34-fold decrease in MDA-MB-231 cells and 0.63- and 0.95-fold increase in MCF-7 cells, treated with 1 and 5 µg/ml, respectively. Functional studies indicate that these effects are mediated by the specific protein 1 motif located in the CD1 promoter. In the absence of ER-α, the adiponectin-mediated down-regulation of CD1 involves the recruitment of corepressors. In the presence of ER-α, the adiponectin-induced expression of CD1 requires the involvement of an activator complex. In conclusion, we propose that a possible mechanism through which adiponectin differently affects breast cancer growth is the opposite modulation of CD1 levels accordingly to ER-α expression.

A novel functional interplay between Progesterone Receptor-B and PTEN, via AKT, modulates autophagy in breast cancer cells.

The tumour suppressor activity of the phosphatase and tensin homologue on chromosome 10 (PTEN) is subject of intense investigative efforts, although limited information on its regulation in breast cancer is available. Herein, we report that, in breast cancer cells, progesterone (OHPg), through its cognate receptor PR-B, positively modulates PTEN expression by inducing its mRNA and protein levels, and increasing PTEN-promoter activity. The OHPg-dependent up-regulation of PTEN gene activity requires binding of the PR-B to an Sp1-rich region within the PTEN gene promoter. Indeed, ChIP and EMSA analyses showed that OHPg treatment induced the occupancy of PTEN promoter by PR and Sp1 together with transcriptional coactivators such as SRC1 and CBP. PR-B isoform knockdown abolished the complex formation indicating its specific involvement. The OHPg/PR-B dependent induction of PTEN causes the down-regulation of PI3K/AKT signal, switching on the autophagy process through an enhanced expression of UVRAG and leading to a reduced cell survival. Altogether these findings highlight a novel functional connection between OHPg/PR-B and tumour suppressor pathways in breast cancer.

Epigallocatechin gallate inhibits growth and epithelial-to-mesenchymal transition in human thyroid carcinoma cell lines.

Well-differentiated papillary and follicular thyroid carcinoma are the most frequent types of thyroid cancer and the prognosis is generally favorable however, a number of patients develops recurrences. Epigallocatechin-3-gallate (EGCG), a major catechin in green tea, was shown to possess remarkable therapeutic potential against various types of human cancers, although data on thyroid cancer cells are still lacking. The aim of this study was to investigate the effect of EGCG on the proliferation and motility of human thyroid papillary (FB-2) and follicular (WRO) carcinoma cell lines. Our results demonstrate that EGCG (10, 40, 60 µM) treatment inhibited the growth of FB-2 and WRO cells in a dose-dependent manner. These changes were associated with reduced cyclin D1, increased p21 and p53 expression. Furthermore, EGCG suppressed phosphorylation of AKT and ERK1/2. In addition EGCG treatment results in reduction of cell motility and migration. Changes in motility and migration in FB-2 were associated with modulation in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. After 24 h EGCG caused an increase of the E-cadherin expression and a concomitant decrease of SNAIL, ZEB and the basic helix-loop-helix transcription factor TWIST. Besides expression of Vimentin, N-cadherin and α5-integrin was down-regulated. These data well correlate with a reduction of MMP9 activity as evidenced by gelatin zymography. Our findings support the inhibitory role of EGCG on thyroid cancer cell proliferation and motility with concomitant loss of epithelial-to-mesenchymal cell transition markers.

Rapid solid-liquid dynamic extraction of Cachrys pungens Jan ex Guss. aerial parts: influence on the photobiological and antioxidant properties.

Cachrys pungens Jan ex Guss. (Apiaceae) is a perennial plant native to Italy and Northwestern Africa. This species is known for its content in furanocoumarins, and the methanolic extract obtained with maceration previously demonstrated significant phototoxicity on 375 melanoma cells. Here, in order to better explain the biological effects, the apoptotic responses on melanoma cancer cell line were verified. The aerial parts were extracted with methanol through an innovative solid-liquid extraction technology, the Naviglio extractor®, and the raw extract was tested for its photobiological properties on human melanoma C32 cells irradiated with UVA light. The in vitro antioxidant potential was assessed as well. The sample exerted a concentration-dependent photocytotoxic activity (IC50 value = 3.00 ± 0.16 µg/mL). In line with these evidences, in C32-treated cells subjected to UV irradiation, further data have reported an up-regulation of p53 and PARP, both proteins involved in apoptotic response and DNA repair.

Phytochemical Profile and In Vitro Antioxidant and Photobiological Properties of Different Extracts from Prangos ferulacea Lindl.

Interesting photobiological properties have been demonstrated for some Cachrys species, including C. libanotis L., C. sicula L., and C. pungens Jan. The present study was designed to assess the photocytotoxic activity of Prangos ferulacea Lindl. (synonym of C. ferulacea (L.) Calest.). This plant was previously considered a Cachrys species but, at present, it is part of the Prangos genus. P. ferulacea is an orophilous plant present in the eastern Mediterranean and in western Asia. Three different extraction techniques were utilized. Obtained extracts were compared both for their phytochemical content and for their photobiological properties on human melanoma cells irradiated with UVA light. The apoptotic responses, together with the antioxidant activity, were also assessed. P. ferulacea extracts were able to affect cell viability in a concentration-dependent manner, with the sample obtained through supercritical CO2 extraction showing the highest activity (IC50 = 4.91 µg/mL). This research points out the interesting content in the photoactive compounds of this species, namely furanocoumarins, and could provide a starting point for further studies aimed at finding new photosensitizing agents useful in cancer photochemotherapy.

The G protein-coupled receptor 30 is up-regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) in breast cancer cells and cardiomyocytes.

GPR30, also known as GPER, has been suggested to mediate rapid effects induced by estrogens in diverse normal and cancer tissues. Hypoxia is a common feature of solid tumors involved in apoptosis, cell survival, and proliferation. The response to low oxygen environment is mainly mediated by the hypoxia-inducible factor named HIF-1α, which activates signaling pathways leading to adaptive mechanisms in tumor cells. Here, we demonstrate that the hypoxia induces HIF-1α expression, which in turn mediates the up-regulation of GPER and its downstream target CTGF in estrogen receptor-negative SkBr3 breast cancer cells and in HL-1 cardiomyocytes. Moreover, we show that HIF-1α-responsive elements located within the promoter region of GPER are involved in hypoxia-dependent transcription of GPER, which requires the ROS-induced activation of EGFR/ERK signaling in both SkBr3 and HL-1 and cells. Interestingly, the apoptotic response to hypoxia was prevented by estrogens through GPER in SkBr3 cells. Taken together, our data suggest that the hypoxia-induced expression of GPER may be included among the mechanisms involved in the anti-apoptotic effects elicited by estrogens, particularly in a low oxygen microenvironment.

Resveratrol, through NF-Y/p53/Sin3/HDAC1 complex phosphorylation, inhibits estrogen receptor alpha gene expression via p38MAPK/CK2 signaling in human breast cancer cells.

Agents to counteract acquired resistance to hormonal therapy for breast cancer would substantially enhance the long-term benefits of hormonal therapy. In the present study, we demonstrate how resveratrol (Res) inhibits human breast cancer cell proliferation, including MCF-7 tamoxifen-resistant cells (IC(50) values for viability were in the 30-45 µM range). We show that Res, through p38(MAPK) phosphorylation, causes induction of p53, which recruits at the estrogen receptor α (ERα) proximal promoter, leading to an inhibition of ERα expression in terms of mRNA and protein content. These events appear specifically p53 dependent, since they are drastically abrogated with p53-targeting siRNA. Coimmunoprecipitation assay showed specific interaction between p53, the Sin3A corepressor, and histone deacetylase 1 (HDAC1), which was phosphorylated. The enhancement of the tripartite complex p53/Sin3A/HDAC1, together with NF-Y on Res treatment, was confirmed by chromatin immunoprecipitation analyses, with a concomitant release of Sp1 and RNA polymerase II, thereby inhibiting the cell transcriptional machinery. The persistence of such effects in MCF-7 tamoxifen-resistant cells at a higher extent than parental MCF-7 cells addresses how Res may be considered a useful pharmacological tool to be exploited in the adjuvant settings for treatment of breast cancer developing hormonal resistance.

Ischemic Preconditioning Modulates the Peripheral Innate Immune System to Promote Anti-Inflammatory and Protective Responses in Mice Subjected to Focal Cerebral Ischemia.

The development of tolerance triggered by a sublethal ischemic episode (preconditioning, PC) involves a complex crosstalk between neurons, astrocytes and microglia, although the role of the peripheral immune system in this context is largely unexplored. Here, we report that severe cerebral ischemia caused by transient middle cerebral artery occlusion (MCAo) in adult male mice elevates blood counts of inflammatory neutrophils and monocytes, and plasma levels of miRNA-329-5p. These inflammatory responses are prevented by ischemic PC induced by 15 min MCAo, 72h before the severe insult (1h MCAo). As compared with sham-operated animals, mice subjected to either ischemic PC, MCAo or a combination of both (PC+MCAo) display spleen contraction. However, protein levels of Ym1 (a marker of polarization of myeloid cells towards M2/N2 protective phenotypes) are elevated only in spleen from the experimental groups PC and PC+MCAo, but not MCAo. Conversely, Ym1 protein levels only increase in circulating leukocytes from mice subjected to 1h MCAo, but not in preconditioned animals, which is coincident with a dramatic elevation of Ym1 expression in the ipsilateral cortex. By immunofluorescence analysis, we observe that expression of Ym1 occurs in amoeboid-shaped myeloid cells, mainly representing inflammatory monocytes/macrophages and neutrophils. As a result of its immune-regulatory functions, ischemic PC prevents elevation of mRNA levels of the pro-inflammatory cytokine interleukin (IL)-1ß in the ipsilateral cortex, while not affecting IL-10 mRNA increase induced by MCAo. Overall, the elevated anti-inflammatory/pro-inflammatory ratio observed in the brain of mice pre-exposed to PC is associated with reduced brain infarct volume and ischemic edema, and with amelioration of functional outcome. These findings reaffirm the crucial and dualistic role of the innate immune system in ischemic stroke pathobiology, extending these concepts to the context of ischemic tolerance and underscoring their relevance for the identification of novel therapeutic targets for effective stroke treatment.

Farnesoid X receptor, through the binding with steroidogenic factor 1-responsive element, inhibits aromatase expression in tumor Leydig cells.

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that regulates bile acid homeostasis. It is expressed in the liver and the gastrointestinal tract, but also in several non-enterohepatic tissues including testis. Recently, FXR was identified as a negative modulator of the androgen-estrogen-converting aromatase enzyme in human breast cancer cells. In the present study we detected the expression of FXR in Leydig normal and tumor cell lines and in rat testes tissue. We found, in rat Leydig tumor cells, R2C, that FXR activation by the primary bile acid chenodeoxycholic acid (CDCA) or a synthetic agonist GW4064, through a SHP-independent mechanism, down-regulates aromatase expression in terms of mRNA, protein levels, and its enzymatic activity. Transient transfection experiments, using vector containing rat aromatase promoter PII, evidenced that CDCA reduces basal aromatase promoter activity. Mutagenesis studies, electrophoretic mobility shift, and chromatin immunoprecipitation analysis reveal that FXR is able to compete with steroidogenic factor 1 in binding to a common sequence present in the aromatase promoter region interfering negatively with its activity. Finally, the FXR-mediated anti-proliferative effects exerted by CDCA on tumor Leydig cells are at least in part due to an inhibition of estrogen-dependent cell growth. In conclusion our findings identify for the first time the activators of FXR as negative modulators of the aromatase enzyme in Leydig tumor cell lines.

A Notch inhibitor plus Resveratrol induced blockade of autophagy drives glioblastoma cell death by promoting a switch to apoptosis.

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain tumors and the hardest type of cancer to treat. Therapies targeting developmental pathways, such as Notch, eliminate neoplastic glioma cells, but their efficacy can be limited by various mechanisms. Combination regimens may represent a good opportunity for effective therapies with durable effects. We used low doses of the γ-secretase inhibitor RO4929097 (GSI), to block the Notch pathway activity, in combination with Resveratrol (RSV) and we evidenced the mechanisms of autophagy/apoptosis transition in GBM cells. Resveratrol and GSI combination results in the synergistic induction of cell death together with the block of the autophagic flux evidenced by a sustained increase of LC3-II and p62 protein content, due to the dramatic reduction of CDK4, an important regulator of lysosomal function. The ectopic overexpression of the constitutive active CDK4 mutant, greatly counteracted the RSV+GSI induced block of the autophagy. Triggering autophagy in RSV+GSI-treated cells, which have impaired lysosomal function, caused the collapse of the system and a following apoptosis. For instance, by combining the CDK4 mutant as well as the early stage autophagy inhibitor, 3-methyladenina, abolished the RSV+GSI induced caspases activation. The initiator caspases (caspases-8 and -9), effector caspase (caspase-3) and its downstream substrate PARP were induced after RSV+GSI exposure as well as the percentage of the TUNEL positive cells. Moreover, the pro-apoptotic signaling MAPK p38 was activated while the pro-survival MAPK p42/p44 signaling was inhibited. In short, we establish the role of CDK4 in the regulation of autophagy/apoptosis transition induced by RSV and GSI in GBM cells. This new synergistic therapeutic combination, increasing the accumulation of autophagosomes, may have therapeutic value for GBM patients.

Assessment of Photo-Induced Cytotoxic Activity of Cachrys sicula and Cachrys libanotis Enriched-Coumarin Extracts against Human Melanoma Cells.

Photochemotherapy is one of the most interesting current therapeutic approaches for the treatment of melanoma. Different classes of naturally occurring phytochemicals demonstrated interesting photoactive properties. The aim of this study was to evaluate the photocytotoxic potential of two Cachrys species from Southern Italy: C. sicula and C. libanotis (Apiaceae). The enriched-coumarin extracts were obtained from aerial parts through both traditional maceration and pressurized cyclic solid-liquid (PCSL) extraction using Naviglio extractor®. Qualitative and quantitative analyses of furanocoumarins were performed with GC-MS. The photocytotoxic effects were verified on C32 melanoma cells irradiated at a dose of 1.08 J/cm2. The apoptotic responses were also assessed. Moreover, phenolic content and the in vitro antioxidant potential were estimated. Xanthotoxin, bergapten, and isopimpinellin were identified. All the samples induced concentration-dependent photocytotoxic effects (IC50 ranging from 3.16 to 18.18 µg/mL). The C. libanotis sample obtained with Naviglio extractor® was the most effective one (IC50 = 3.16 ± 0.21 µg/mL), followed by C. sicula sample obtained with the same technique (IC50 = 8.83 ± 0.20 µg/mL). Both Cachrys samples obtained through PCSL induced up-regulation of apoptotic signals such as BAX (Bcl2-associated X protein) and PARP (poly ADP-ribose polymerase) cleavage. Moreover, these samples proved to be more photoactive, giving a greater upregulation of p21 protein in the presence of UVA radiation. Obtained results suggest that investigated species could be promising candidates for further investigations aimed to find new potential drugs for the photochemotherapy of skin cancer.

Cateslytin abrogates lipopolysaccharide-induced cardiomyocyte injury by reducing inflammation and oxidative stress through toll like receptor 4 interaction.

Global public health is threatened by new pathogens, antimicrobial resistant microorganisms and a rapid decline of conventional antimicrobials efficacy. Thus, numerous medical procedures become life-threating. Sepsis can lead to tissue damage such as myocardium inflammation, associated with reduction of contractility and diastolic dysfunction, which may cause death. In this perspective, growing interest and attention are paid on host defence peptides considered as new potential antimicrobials. In the present study, we investigated the physiological and biochemical properties of Cateslytin (Ctl), an endogenous antimicrobial chromogranin A-derived peptide, in H9c2 cardiomyocytes exposed to lipopolysaccharide (LPS) infection. We showed that both Ctl (L and D) enantiomers, but not their scrambled counterparts, significantly increased cardiomyocytes viability following LPS, even if L-Ctl was effective at lower concentration (1 nM) compared to D-Ctl (10 nM). L-Ctl mitigated LPS-induced LDH release and oxidative stress, as visible by a reduction of MDA and protein carbonyl groups content, and by an increase of SOD activity. Molecular docking simulations strongly suggested that L-Ctl modulates TLR4 through a direct binding to the partner protein MD-2. Molecular analyses indicated that the protection mediated by L-Ctl against LPS-evoked sepsis targeted the TLR4/ERK/JNK/p38-MAPK pathway, regulating NFkB p65, NFkB p52 and COX2 expression and repressing the mRNA expression levels of the LPS-induced proinflammatory factors IL-1ß, IL-6, TNF-α and NOS2. These findings indicate that Ctl could be considered as a possible candidate for the development of new antimicrobials strategies in the treatment of myocarditis. Interestingly, L-enantiomeric Ctl showed remarkable properties in strengthening the anti-inflammatory and anti-oxidant effects on cardiomyocytes.

Combined low doses of PPARgamma and RXR ligands trigger an intrinsic apoptotic pathway in human breast cancer cells.

Ligand activation of peroxisome proliferator-activated receptor (PPAR)gamma and retinoid X receptor (RXR) induces antitumor effects in cancer. We evaluated the ability of combined treatment with nanomolar levels of the PPARgamma ligand rosiglitazone (BRL) and the RXR ligand 9-cis-retinoic acid (9RA) to promote antiproliferative effects in breast cancer cells. BRL and 9RA in combination strongly inhibit of cell viability in MCF-7, MCF-7TR1, SKBR-3, and T-47D breast cancer cells, whereas MCF-10 normal breast epithelial cells are unaffected. In MCF-7 cells, combined treatment with BRL and 9RA up-regulated mRNA and protein levels of both the tumor suppressor p53 and its effector p21(WAF1/Cip1). Functional experiments indicate that the nuclear factor-kappaB site in the p53 promoter is required for the transcriptional response to BRL plus 9RA. We observed that the intrinsic apoptotic pathway in MCF-7 cells displays an ordinated sequence of events, including disruption of mitochondrial membrane potential, release of cytochrome c, strong caspase 9 activation, and, finally, DNA fragmentation. An expression vector for p53 antisense abrogated the biological effect of both ligands, which implicates involvement of p53 in PPARgamma/RXR-dependent activity in all of the human breast malignant cell lines tested. Taken together, our results suggest that multidrug regimens including a combination of PPARgamma and RXR ligands may provide a therapeutic advantage in breast cancer treatment.