Fatty acid binding protein 5 suppression attenuates obesity-induced hepatocellular carcinoma by promoting ferroptosis and intratumoral immune rewiring.

Due to the rise in overnutrition, the incidence of obesity-induced hepatocellular carcinoma (HCC) will continue to escalate; however, our understanding of the obesity to HCC developmental axis is limited. We constructed a single-cell atlas to interrogate the dynamic transcriptomic changes during hepatocarcinogenesis in mice. Here we identify fatty acid binding protein 5 (FABP5) as a driver of obesity-induced HCC. Analysis of transformed cells reveals that FABP5 inhibition and silencing predispose cancer cells to lipid peroxidation and ferroptosis-induced cell death. Pharmacological inhibition and genetic ablation of FABP5 ameliorates the HCC burden in male mice, corresponding to enhanced ferroptosis in the tumour. Moreover, FABP5 inhibition induces a pro-inflammatory tumour microenvironment characterized by tumour-associated macrophages with increased expression of the co-stimulatory molecules CD80 and CD86 and increased CD8+ T cell activation. Our work unravels the dual functional role of FABP5 in diet-induced HCC, inducing the transformation of hepatocytes and an immunosuppressive phenotype of tumour-associated macrophages and illustrates FABP5 inhibition as a potential therapeutic approach.

Heterogeneity of hepatocyte dynamics restores liver architecture after chemical, physical or viral damage.

Midlobular hepatocytes are proposed to be the most plastic hepatic cell, providing a reservoir for hepatocyte proliferation during homeostasis and regeneration. However, other mechanisms beyond hyperplasia have been little explored and the contribution of other hepatocyte subpopulations to regeneration has been controversial. Thus, re-examining hepatocyte dynamics during regeneration is critical for cell therapy and treatment of liver diseases. Using a mouse model of hepatocyte- and non-hepatocyte- multicolor lineage tracing, we demonstrate that midlobular hepatocytes also undergo hypertrophy in response to chemical, physical, and viral insults. Our study shows that this subpopulation also combats liver impairment after infection with coronavirus. Furthermore, we demonstrate that pericentral hepatocytes also expand in number and size during the repair process and Galectin-9-CD44 pathway may be critical for driving these processes. Notably, we also identified that transdifferentiation and cell fusion during regeneration after severe injury contribute to recover hepatic function.

Diabetic individuals with COVID-19 exhibit reduced efficacy of gliptins in inhibiting dipeptidyl peptidase 4 (DPP4). A suggested explanation for increased COVID-19 susceptibility in patients with type 2 diabetes mellitus (T2DM).

AIMS: Dipeptidyl peptidase 4 (DPP4) has been proposed as a coreceptor for SARS-CoV-2 cellular entry. Considering that type 2 diabetes mellitus (T2DM) has been identified as the most important risk factor for SARS-CoV-2, and that gliptins (DPP4 inhibitors) are a prescribed diabetic treatment, this study aims to unravel the impact of DPP4 in the intersection of T2DM/COVID-19. MATERIALS AND METHODS: We analyzed 189 serum human samples, divided into six clinical groups (controls, T2DM, T2DM + gliptins, COVID-19, COVID-19 + T2DM, and COVID-19 + T2DM + gliptins), measuring DPP4 protein concentration and activity by Western blot, ELISA, and commercial activity kits. The obtained results were verified in Huh-7 cellular models. KEY FINDINGS: Both DPP4 concentration and activity were decreased in COVID-19 patients, and as in T2DM patients, compared to controls. Despite these lower levels, the ratio of DPP4 activity/concentration in COVID-19 sera was the highest (0.782 ± 0.289 µU/ng vs. 0.547 ± 0.050 µU/ng in controls, p < 0.0001), suggesting a compensating mechanism in these patients. Supernatants of Huh-7 cells incubated with COVID-19 serum showed a consistent and significantly lower DPP4 concentration and activity. Furthermore, COVID-19 + T2DM + gliptins patients showed a higher serum DPP4 concentration and activity than T2DM + gliptin subjects (p < 0.05), indicating that sera from COVID-19 convalescents interfere with gliptins. SIGNIFICANCE: Either SARS-CoV-2 or some metabolites present in the sera of COVID-19-convalescent patients interact with soluble DPP4 or even gliptins themselves since the inhibitory effect of gliptins on DPP4 activity is being prevented. The interactions between DPP4, gliptins, and SARS-CoV-2 should be further elucidated to reveal the mechanism of action for these interesting observations.

Resistance to 2-Hydroxy-Flutamide in Prostate Cancer Cells Is Associated with the Downregulation of Phosphatidylcholine Biosynthesis and Epigenetic Modifications.

In this study, we examined the metabolic adaptations of a chemoresistant prostate cancer cell line in comparison to a sensitive cell line. We utilized prostate cancer LNCaP cells and subjected them to a stepwise increase in the antiandrogen 2-hydroxy-flutamide (FLU) concentration to generate a FLU-resistant cell line (LN-FLU). These LN-FLU cells displayed characteristics of cancer stem cells, exhibited drug resistance, and showed a significantly reduced expression of Cyclin D1, along with the overexpression of p16, pointing to a proliferation arrest. In comparing the cancer stem-like LN-FLU cells to the LNCaP cells, we observed a decrease in the expression of CTP-choline cytidylyl transferase α (CCTα), as well as a decline in choline kinase, suggesting altogether a downregulation of the phosphatidylcholine biosynthetic pathway. In addition, we found decreased levels of the protein methyl transferase PRMT2 and the upregulation of the histone deacetylase Sirtuin1 (Sirt1). Analysis of the human prostate cancer samples revealed similar results in a population with high expressions of the stem cell markers Oct4 and ABCB1A1. Our findings suggest that the adaptation of prostate cancer cells to antiandrogens could induce reprogramming into stem cells that survive in a low phosphocholine metabolism and cell cycle arrest and display drug resistance.

Alteration of the HIF-1α/VEGF Signaling Pathway and Disruption of the Cell Cycle by Second Generation Carbosilan Dendrimers.

Current therapies against prostate cancer (PCa) disease, such as surgery, radiotherapy, or in last term chemical castration by androgen deprivation, have led to significant reduction of the incidence of PCa throughout the world. Worse prognosis is found in those patients which exhibit castration resistance, relapsing into the disease with even greater aggressiveness. Hypoxia cancer cell adaption has been observed to be closely connected to fatal prognostic tumor features. Therefore, hypoxia adaptive mechanisms of cancer cells have attracted large interest as a relevant biological target for treatment-resistant patients. Dendrimers have been established as a promising nanotechnological tool owing to their beneficial physicochemical features such as multivalency and monodispersity. Herein, we have completed a thorough study to better understand the effect within the cell of the already published ruthenium(II)-N-heterocyclic carbene metallodendrimer (G2Ru) that was able to drastically reduce HIF-1α stabilization and exhibited antiproliferative capability against androgen-sensitive (LNCaP) and androgen-resistant prostate cancer cells (LNFLU) in vitro. G2Ru, as well as its cationic imidazolium precursor (G2P), displayed scavenging properties against intracellular and externally stimulated ROS levels, which would presumably hinder the stabilization of HIF-1α by prolyl hydroxylase (PHD) inhibition. Furthermore, these dendrimers have shown considerably beneficial properties against tumor progression capability in terms of apoptosis, cell cycle, CSCs expression, and epithelial phenotype promotion. Taken all together, in this study we could demonstrate the extraordinary anticancer properties of NHC-based carbosilane dendrimers against androgen-resistant prostate cancer cells in vitro.

Metabolic fingerprinting of chemotherapy-resistant prostate cancer stem cells. An untargeted metabolomic approach by liquid chromatography-mass spectrometry.

Chemoresistance is one of the most important challenges in cancer therapy. The presence of cancer stem cells within the tumor may contribute to chemotherapy resistance since these cells express high levels of extrusion pumps and xenobiotic metabolizing enzymes that inactivate the therapeutic drug. Despite the recent advances in cancer cell metabolism adaptations, little is known about the metabolic adaptations of the cancer stem cells resistant to chemotherapy. In this study, we have undertaken an untargeted metabolomic analysis by liquid chromatography-high-resolution spectrometry combined with cytotoxicity assay, western blot, quantitative real-time polymerase chain reaction (qPCR), and fatty acid oxidation in a prostate cancer cell line resistant to the antiandrogen 2-hydroxiflutamide with features of cancer stem cells, compared to its parental androgen-sensitive cell line. Metabolic fingerprinting revealed 106 out of the 850 metabolites in ESI+ and 67 out of 446 in ESI- with significant differences between the sensitive and the resistant cell lines. Pathway analysis performed with the unequivocally identified metabolites, revealed changes in pathways involved in energy metabolism as well as posttranscriptional regulation. Validation by enzyme expression analysis indicated that the chemotherapy-resistant prostate cancer stem cells were metabolically dormant with decreased fatty acid oxidation, methionine metabolism and ADP-ribosylation. Our results shed light on the pathways underlying the entry of cancer cells into dormancy that might contribute to the mechanisms of drug resistance.

A Highly Sensitive Immunoassay for Determination of Immune Response to SARS-CoV-2 in Capillary Blood Samples.

Throughout the pandemic, serological assays have been revealed as crucial for detecting previous exposures to the virus and determining the timing of antibody maintenance after vaccination or natural infection. This study aimed to develop an optimized enzyme-linked immunosorbent assay (ELISA)-based serology, which could be used in case of reagent shortages, such as that occurred in the beginning of this health emergency. As a result, we present a high-sensitive immunoassay for the determination of IgG levels in venous serum samples, using 2 µg/mL antigen (receptor-binding domain of the spike protein S1) for coating the plate and utilizing human samples at a dilution 1:1000. This method showed non-inferiority features versus a commercial kit, is less expensive, and has a higher spectrophotometric range that allows for a better quantification of the antibody titers. The optical density values before and after heating venous serum samples at 56 °C during 30 min was quite similar, showing that heat inactivation can be used to reduce the biohazardous risks while handling samples. Furthermore, we show that finger-stick capillary blood samples can also serve as a suitable source for IgG detection, bypassing the need for serum isolation and being suitable for point-of-care application (Pearson's coefficient correlation with capillary serum was 0.95, being statistically significant).

Role of Dipeptidyl Peptidase-4 (DPP4) on COVID-19 Physiopathology.

DPP4/CD26 is a single-pass transmembrane protein with multiple functions on glycemic control, cell migration and proliferation, and the immune system, among others. It has recently acquired an especial relevance due to the possibility to act as a receptor or co-receptor for SARS-CoV-2, as it has been already demonstrated for other coronaviruses. In this review, we analyze the evidence for the role of DPP4 on COVID-19 risk and clinical outcome, and its contribution to COVID-19 physiopathology. Due to the pathogenetic links between COVID-19 and diabetes mellitus and the hyperinflammatory response, with the hallmark cytokine storm developed very often during the disease, we dive deep into the functions of DPP4 on carbohydrate metabolism and immune system regulation. We show that the broad spectrum of functions regulated by DPP4 is performed both as a protease enzyme, as well as an interacting partner of other molecules on the cell surface. In addition, we provide an update of the DPP4 inhibitors approved by the EMA and/or the FDA, together with the newfangled approval of generic drugs (in 2021 and 2022). This review will also cover the effects of DPP4 inhibitors (i.e., gliptins) on the progression of SARS-CoV-2 infection, showing the role of DPP4 in this disturbing disease.

The Natural Chemotherapeutic Capsaicin Activates AMPK through LKB1 Kinase and TRPV1 Receptors in Prostate Cancer Cells.

The natural bioactive compound capsaicin has been reported to have anticancer activity, although the underlying mechanism of action has not been completely clarified. Herein, we investigated the mechanism whereby capsaicin exerts antitumor effects on prostate cancer cells. We found that capsaicin activated AMP-activated kinase (AMPK) and promoted cell death in the LKB1-expressing prostate cancer cell lines LNCaP and PC3, but not in the liver kinase B1 (LKB1)-null cell line DU-145. Capsaicin treatment stimulated LKB1 phosphorylation and activated AMPK in LKB1-expressing cells. In addition, LKB1 silencing in LNCaP and PC3 cells abrogated capsaicin-induced AMPK activation, while the overexpression of LKB1 by lentiviral infection in DU-145 cells induced capsaicin-triggered AMPK phosphorylation. Moreover, the calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) inhibitor STO-609 did not modify the activation of AMPK induced by capsaicin, suggesting a CaMKK2-independent mechanism. Capsaicin-induced LKB1 phosphorylation was dependent on the transient receptor potential cation channel subfamily V member 1 (TRPV1), since TRPV1 knocked down by shRNA abolished LKB1 and AMPK phosphorylation in LKB1-expressing cells. Altogether, our results showed that capsaicin affected AMPK activity in an LKB1- and TRPV1-dependent fashion, linking TRPV1 with cell fate. These data also suggest that capsaicin may be a rational chemotherapeutic option for prostate tumors.

Increase in Ischemia-Modified Albumin and Pregnancy-Associated Plasma Protein-A in COVID-19 Patients.

This study was undertaken due to the urgent need to explore reliable biomarkers for early SARS-CoV-2 infection. We performed a retrospective study analyzing the serum levels of the cardiovascular biomarkers IL-6, TNF-α, N-terminal pro-B natriuretic peptide, cardiac troponin T (cTnT), ischemia-modified albumin (IMA) and pregnancy-associated plasma protein-A (PAPP-A) in 84 patients with COVID-19.Patients were divided into three groups according to their RT-qPCR and IgG values: acute infection (n = 35), early infection (n = 25) or control subjects (n = 24). Levels of biomarkers were analyzed in patient serum samples using commercially available ELISA kits. Results showed a significant increase in IMA and PAPP-A levels in the early infected patients. Moreover, multivariate analysis and receiver operating characteristic (ROC) curve showed that IMA and PAPP-A had excellent discrimination value for the early stage of COVID-19. For IMA, the area under the ROC curve (AUC) had a value of 0.94 (95% confidence interval (CI): 0.881-0.999). Likewise, the serum level of PAPP-A was significantly higher in patients with early infection than in the control subjects (AUC = 0.801 (95% CI: 0.673-0.929)). The combined use of IMA and PAPP-A enhanced the sensitivity for total SARS-CoV-2-infected patients to 93%. These results suggest that the increased levels of PAPP-A and IMA shed light on underlying mechanisms of COVID-19 physiopathology and might be used as efficient biomarkers with high sensitivity and specificity for the early stage of COVID-19. Importantly, when monitoring pregnancy and cardiovascular diseases using PAPP-A or IMA levels, a SARS-CoV-2 infection should be discarded for proper interpretation of the results.

Androgen Deprivation Induces Reprogramming of Prostate Cancer Cells to Stem-Like Cells.

In the past few years, cell plasticity has emerged as a mode of targeted therapy evasion in prostate adenocarcinoma. When exposed to anticancer therapies, tumor cells may switch into a different histological subtype, such as the neuroendocrine phenotype which is associated with treatment failure and a poor prognosis. In this study, we demonstrated that long-term androgen signal depletion of prostate LNCaP cells induced a neuroendocrine phenotype followed by re-differentiation towards a "stem-like" state. LNCaP cells incubated for 30 days in charcoal-stripped medium or with the androgen receptor antagonist 2-hydroxyflutamide developed neuroendocrine morphology and increased the expression of the neuroendocrine markers ßIII-tubulin and neuron specific enolase (NSE). When cells were incubated for 90 days in androgen-depleted medium, they grew as floating spheres and had enhanced expression of the stem cell markers CD133, ALDH1A1, and the transporter ABCB1A. Additionally, the pluripotent transcription factors Nanog and Oct4 and the angiogenic factor VEGF were up-regulated while the expression of E-cadherin was inhibited. Cell viability revealed that those cells were resistant to docetaxel and 2-hidroxyflutamide. Mechanistically, androgen depletion induced the decrease in AMP-activated kinase (AMPK) expression and activation and stabilization of the hypoxia-inducible factor HIF-1α. Overexpression of AMPK in the stem-like cells decreased the expression of stem markers as well as that of HIF-1α and VEGF while it restored the levels of E-cadherin and PGC-1α. Most importantly, docetaxel sensitivity was restored in stem-like AMPK-transfected cells. Our model provides a new regulatory mechanism of prostate cancer plasticity through AMPK that is worth exploring.

Dysregulated lipid metabolism in hepatocellular carcinoma cancer stem cells.

According to the stem cell theory for cancer, hepatocellular carcinomas are sustained by a group of cancer stem cells (CSCs) which are responsible for resistance to chemotherapy. In the present study we aimed to examine lipid metabolism in cancer stem cells induced by long-term treatment with sorafenib and its relationship with acquisition of a CSC-like phenotype. Two cell lines (HepG2SF1 and Huh7SF1) were generated by incubation with a step-wise increase of sorafenib concentrations for 10 months. These cell lines displayed stem-like characteristics like increase in the expression of ABCB1A, Nanog and Oct4 as well as an E-cadherin/N-cadherin switch. HepG2SF1 and Huh7SF1 cells showed intracellular accumulation of neutral lipids, assessed by flow cytometry and confocal microscopy. The exam of lipid metabolism revealed that HepG2SF1 and Huh7SF1 cells increased the expression of the enzymes involved in de novo lipid synthesis ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) and that of the fatty acid transporter CD36. In addition, these CSC-like cells had enhanced expression of the lipogenic transcription factor SREBP1c. Analysis of the key metabolic sensor AMP-activated kinase (AMPK) demonstrated that both AMPK phosphorylation and levels were decreased in the CSC-like cells compared to their parental cells. Interestingly, transfection of HepG2SF1 and Huh7SF1 cells with AMPK, restored the levels of the lipogenic enzymes and SREBP1c and decreased the intracellular lipid accumulation. Furthermore, AMPK transfection decreased the stemness markers and inhibited the E-cadherin/N-cadherin switch. Targeting AMPK and lipid metabolism of hepatocellular cancer stem cells is a promising strategy to face stemness and chemotherapy resistance.

Targeting AMP-activated kinase impacts hepatocellular cancer stem cells induced by long-term treatment with sorafenib.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. HCC treatment is hindered by the frequent emergence of chemoresistance to the multikinase inhibitor sorafenib, which has been related to the presence of cancer stem cells (CSCs) that self-renew and often escape therapy. The key metabolic sensor AMP-activated kinase (AMPK) has recently been recognized as a tumour growth regulator. In this study, we aimed to elucidate the role of AMPK in the development of a stem cell phenotype in HCC cells. To this end, we enriched the CSC population in HCC cell lines that showed increased expression of drug resistance (ALDH1A1, ABCB1A) and stem cell (CD133, Nanog, Oct4, alpha fetoprotein) markers and demonstrated their stemness phenotype. These cells were refractory to sorafenib-induced cell death. We report that sorafenib-resistant cells had lower levels of total and phosphorylated AMPK as well as its downstream substrate, ACC, compared with the parental cells. Interestingly, AMPK knockdown with siRNA or inhibition with dorsomorphin increased the expression of stem cell markers in parental cells and blocked sorafenib-induced cell death. Conversely, the upregulation of AMPK, either by transfection or by pharmacological activation with A-769662, decreased the expression of ALDH1A1, ABCB1A, CD133, Nanog, Oct4, and alpha fetoprotein, and restored sensitivity to sorafenib. Analysis of the underlying mechanism points to hypoxia-inducible factor HIF-1α as a regulator of stemness. In vivo studies in a xenograft mouse model demonstrated that stem-like cells have greater tumourigenic capacity. AMPK activation reduced xenograft tumour growth and decreased the expression of stem cell markers. Taken together, these results indicate that AMPK may serve as a novel target to overcome chemoresistance in HCC.

Capsaicin Targets Lipogenesis in HepG2 Cells Through AMPK Activation, AKT Inhibition and PPARs Regulation.

Obesity, a major risk factor for chronic diseases such as type 2 diabetes (T2D), represents a serious primary health problem worldwide. Dietary habits are of special interest to prevent and counteract the obesity and its associated metabolic disorders, including lipid steatosis. Capsaicin, a pungent compound of chili peppers, has been found to ameliorate diet-induced obesity in rodents and humans. The purpose of this study was to examine the effect of capsaicin on hepatic lipogenesis and to delineate the underlying signaling pathways involved, using HepG2 cells as an experimental model. Cellular neutral lipids, stained with BODIPY493/503, were quantified by flow cytometry, and the protein expression and activity were determined by immunoblotting. Capsaicin reduced basal neutral lipid content in HepG2 cells, as well that induced by troglitazone or by oleic acid. This effect of capsaicin was prevented by dorsomorphin and GW9662, pharmacological inhibitors of AMPK and PPARγ, respectively. In addition, capsaicin activated AMPK and inhibited the AKT/mTOR pathway, major regulators of hepatic lipogenesis. Furthermore, capsaicin blocked autophagy and increased PGC-1α protein. These results suggest that capsaicin behaves as an anti-lipogenic compound in HepG2 cells.

Combination of the natural product capsaicin and docetaxel synergistically kills human prostate cancer cells through the metabolic regulator AMP-activated kinase.

BACKGROUND: Current chemotherapy for castration-resistant prostate cancer is established on taxane-based compounds like docetaxel. However, eventually, the development of toxic side effects and resistance limits the therapeutic benefit being the major concern in the treatment of prostate cancer. Combination therapies in many cases, enhance drug efficacy and delay the appearance of undesired effects, representing an important option for the treatment of castration-resistant prostate cancer. In this study, we tested the efficacy of the combination of docetaxel and capsaicin, the pungent ingredient of hot chili peppers, on prostate cancer cells proliferation. METHODS: Prostate cancer LNCaP and PC3 cell lines were used in this study. Levels of total and phosphorylated forms of Akt, mTOR, S6, LKB1, AMPK and ACC were determined by Western blot. AMPK, LKB1 and Akt knock down was performed by siRNA. PTEN was overexpressed by transient transfection with plasmids. Xenograft prostate tumors were induced in nude mice and treatments (docetaxel and capsaicin) were administered intraperitoneally. Statistical analyses were performed with GraphPad software. Combination index was calculated with Compusyn software. RESULTS: Docetaxel and capsaicin synergistically inhibited the growth of LNCaP and PC3 cells, with a combination index lower than 1 for most of the combinations tested. Co-treatment with docetaxel and capsaicin notably decreased Akt and its downstream targets mTOR and S6 phosphorylation. Overexpression of PTEN phosphatase abrogated the synergistic antiproliferative effect of docetaxel and capsaicin. The combined treatment also increased the phosphorylation of AMP-activated kinase (AMPK) and the phosphorylation of its substrate ACC. In addition, pharmacological inhibition of AMPK with dorsomorphin (compound C) as well as knock down by siRNA of AMPK or its upstream kinase LKB1, abolished the synergy of docetaxel and capsaicin. Mechanistically, we showed that the synergistic anti-proliferative effect may be attributed to two independent effects: Inhibition of the PI3K/Akt/mTOR signaling pathway by one side, and AMPK activation by the other. In vivo experiments confirmed the synergistic effects of docetaxel and capsaicin in reducing the tumor growth of PC3 cells. CONCLUSION: Combination of docetaxel and capsaicin represents a therapeutically relevant approach for the treatment of Prostate Cancer.

The red pepper's spicy ingredient capsaicin activates AMPK in HepG2 cells through CaMKKß.

Capsaicin is a natural compound present in chili and red peppers and the responsible of their spicy flavor. It has recently provoked interest because of its antitumoral effects in many cell types although its action mechanism is not clearly understood. As metabolic dysregulation is one of the hallmarks of cancer cells and the key metabolic sensor in the AMP-activated kinase (AMPK), in this study we explored the ability of capsaicin to modulate AMPK activity. We found that capsaicin activated AMPK in HepG2 cells by increasing AMPK phosphorylation and its downstream target ACC. Mechanistically, we determined that capsaicin activated AMPK through the calcium/calmodulin-dependent protein kinase kinase ß, CaMKKß as either the CaMKK inhibitor STO-609 or CaMKK knock down with siRNA abrogated the activation of AMPK. Moreover, capsaicin decreased cell viability, inhibited Akt/mTOR pathway and increased reactive oxygen species (ROS) in HepG2 cells. AMPK activation was involved in the underpinning mechanism of capsaicin-induced cell death.

Hierarchical Self-Assembly of BODIPY Dyes as a Tool to Improve the Antitumor Activity of Capsaicin in Prostate Cancer.

Capsaicin (CAP) has been long known for its analgesic properties and more recently for its antitumor activity in various cell types. However, its pungency and the high doses needed to achieve a significant activity have precluded its application in cancer therapy. Herein, we propose a straightforward novel strategy to improve the antitumor effect of CAP based on the enhancement of its aggregation propensity in aqueous media by covalent attachment of a BODIPY (BDP) dye. The target CAP-BDP 1 self-assembles in aqueous solutions into weakly fluorescent globular assemblies that become highly emissive upon cell uptake-induced disassembly. Remarkably, due to the improved delivery to the tumour tissue upon aggregation, we have succeeded in reducing the doses of CAP-based drugs in vivo in prostate cancer by two orders of magnitude while maintaining a substantial antitumor activity.

Identification of a novel 2-oxindole fluorinated derivative as in vivo antitumor agent for prostate cancer acting via AMPK activation.

The key metabolic sensor adenosine monophosphate-dependent kinase (AMPK) has emerged as a promising therapeutic target for cancer prevention and treatment. Besides its role in energy homeostasis, AMPK blocks cell cycle, regulates autophagy and suppresses the anabolic processes required for rapid cell growth. AMPK is especially relevant in prostate cancer in which activation of lipogenic pathways correlate with tumor progression and aggressiveness. This study reports the discovery of a new series of 2-oxindole derivatives whose AMPK modulatory ability, as well as the antitumoral profile in prostate cancer cells, was evaluated. One of the assayed compounds, compound 8c, notably activated AMPK in cultured PC-3, DU145 and LNCaP prostate cancer cells. Likewise, compound 8c caused PC-3, DU145 and LNCaP cells viability inhibition. Selective knocking down of α1 or α2 isoforms as well as in vitro assays using human recombinant α1ß1γ1 or α2ß1γ1 AMPK isoforms revealed that compound 8c exhibit preference for AMPKα1. Consistent with efficacy at the cellular level, compound 8c was potent in suppressing the growth of PC-3 xenograft tumors. In conclusion, our results show that a new 2-oxindole fluorinated derivative exerts potent in vivo antitumor actions against prostate cancer cells, indicating a promising clinical therapeutic strategy for the treatment of androgen-independent prostate cancer.

Capsaicin exerts synergistic antitumor effect with sorafenib in hepatocellular carcinoma cells through AMPK activation.

In this study, we investigated the antitumoral effects of combined treatment using sorafenib and capsaicin in hepatocellular carcinoma (HCC) cells. Here we showed that the combination of the two drugs had a much stronger inhibitory effect on both HepG2 and Huh-7 human HCC cells growth than either drug alone. The isobolograms demonstrated that the combinations investigated in this study produced a synergistic interaction. In the combination treatment using capsaicin and sorafenib, increased apoptosis, followed by the activation of caspase-9 and PARP, was observed. In addition, the present study demonstrated that sorafenib treatment induces activation of Akt, probably as a mechanism of resistance, whereas capsaicin inhibits Akt providing a possible pathway whereby capsaicin sensitizes to sorafenib in HCC cells. Moreover, capsaicin singly and the combination of capsaicin and sorafenib induce AMPK activation and Acetyl CoA carboxylase phosphorylation in HCC cells. Knocking down of AMPK by selective siRNA abrogates capsaicin-induced Akt inhibition, suggesting the involvement of AMPK in the antiproliferative effect. In vivo experiments further showed that that the anti-tumor effect of sorafenib was enhanced by its combination with 2.5 mg/Kg of capsaicin. Overall, these results show that combined treatment with capsaicin and sorafenib might improve sorafenib sensitivity and therefore it represents a promising and attractive strategy for the treatment of HCC.

Effects of JWH015 in cytokine secretion in primary human keratinocytes and fibroblasts and its suitability for topical/transdermal delivery.

Background JWH015 is a cannabinoid (CB) receptor type 2 agonist that produces immunomodulatory effects. Since skin cells play a key role in inflammatory conditions and tissue repair, we investigated the ability of JWH015 to promote an anti-inflammatory and pro-wound healing phenotype in human primary skin cells. Methods Human primary keratinocytes and fibroblasts were stimulated with lipopolysaccharide. The mRNA expression of cannabinoid receptors was determined using RT-PCR. The effects of JWH015 (0.05, 0.1, 0.5, and 1 µM) in pro- and anti-inflammatory factors were tested in lipopolysaccharide-stimulated cells. A scratch assay, using a co-culture of keratinocytes and fibroblasts, was used to test the effects of JWH015 in wound healing. In addition, the topical and transdermal penetration of JWH015 was studied in Franz diffusion cells using porcine skin and LC-MS. Results The expression of CB1 and CB2 receptors (mRNA) and the production of pro- and anti-inflammatory factors enhanced in keratinocytes and fibroblasts following lipopolysaccharide stimulation. JWH015 reduced the concentration of major pro-inflammatory factors (IL-6 and MCP-1) and increased the concentration of a major anti-inflammatory factor (TGF-ß) in lipopolysaccharide-stimulated cells. JWH015 induced a faster scratch gap closure. These JWH015'seffects were mainly modulated through both CB1 and CB2 receptors. Topically administered JWH015 was mostly retained in the skin and displayed a sustained and low level of transdermal permeation. Conclusions Our findings suggest that targeting keratinocytes and fibroblasts with cannabinoid drugs could represent a therapeutic strategy to resolve peripheral inflammation and promote tissue repair.