Involvement of PPARy in the antitumoral action of cannabinoids on hepatocellular carcinoma

Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In\ud this study, we examined whether the PPARc-activated pathway contributed to the antitumor effect of two cannabinoids,\ud D9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity\ud and intracellular level of PPARc mRNA and protein, which was abolished by the PPARc inhibitor GW9662. Moreover, genetic\ud ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARc decreased the cannabinoid-induced\ud cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC\ud tumors in mice. In addition, PPARc knockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62,\ud suggesting that PPARc is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the\ud endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARc expression and induced p62\ud accumulation, which was counteracted by overexpression of PPARc in TRIB3-knocked down cells. Taken together, we\ud demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo,\ud are modulated by upregulation of PPARc-dependent pathways

Involvement of PPARy in the antitumoral action of cannabinoids on hepatocellular carcinoma

Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In this study, we examined whether the PPARc-activated pathway contributed to the antitumor effect of two cannabinoids, D9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity and intracellular level of PPARc mRNA and protein, which was abolished by the PPARc inhibitor GW9662. Moreover, genetic ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARc decreased the cannabinoid-induced cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC tumors in mice. In addition, PPARc knockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62, suggesting that PPARc is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARc expression and induced p62 accumulation, which was counteracted by overexpression of PPARc in TRIB3-knocked down cells. Taken together, we demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo, are modulated by upregulation of PPARc-dependent pathways

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 exerts synergistic antitumor effect with sorafenib inhepatocellular 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 the antiandrogen flutamide on the expression of protein kinase C isoenzymes in LNCaP and PC3 human prostate cancer cells

Flutamide is a nonsteroidal antiandrogen that is frequently used for total androgen blockage in the treatment of advanced prostate cancer. We investigated the effect of this antiandrogen on the expression of protein kinase C (PKC) isoenzymes (a, b1, e, f) that are involved in cell growth, apoptosis and neoplastic transformation. Androgen-dependent (LNCaP) and independent (PC3) human prostate cancer cells were cultured in a medium that contained fetal bovine serum (FBS) or charcoal-stripped serum (CSS) and treated with 10 lM flutamide. The expression of PKC isoenzymes and the androgen receptor (AR) were analyzed by Western blot and RT-PCR, respectively. Serum steroids differentially regulate the expression of PKC isoenzymes in LNCaP and PC3 cells. Flutamide up-regulated the expression of a, b1 and f, but not e, PKC isoenzymes in CSS-LNCaP cells. These results were not homogeneously reproduced in the presence of androgens. We observed an opposite effect of flutamide, compared to CSS, on PKCb1 isoform expression in CSS-LNCaP suggesting that this antiandrogen exerts an agonistic effect. In PC3 cells flutamide potentiated the expression of the four PKC isoenzymes in almost all conditions tested (FBS- and CSScultured cells). Such effect of flutamide in PC3 cells is independent of AR since no expression of AR was detected. These results provide new evidence on antagonistic/agonistic responses of prostate cancer cells to antiandrogen drugs that are widely used in therapy and show that flutamide can elicit responses in prostate cancer cells that do not express AR

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 cannabinoid WIN 55,212-2 prevents neuroendocrine differentiation of LNCaP prostate cancer cells

BACKGROUND: Neuroendocrine (NE) differentiation represents a common feature of prostate cancer and is associated with accelerated disease progression and poor clinical outcome. Nowadays, there is no treatment for this aggressive form of prostate cancer. The aim of this study was to determine the influence of the cannabinoid WIN 55,212-2 (WIN, a non-selective cannabinoid CB1 and CB2 receptor agonist) on the NE differentiation of prostate cancer cells. METHODS: NE differentiation of prostate cancer LNCaP cells was induced by serum deprivation or by incubation with interleukin-6, for 6 days. Levels of NE markers and signaling proteins were determined by western blotting. Levels of cannabinoid receptors were determined by quantitative PCR. The involvement of signaling cascades was investigated by pharmacological inhibition and small interfering RNA. RESULTS: The differentiated LNCaP cells exhibited neurite outgrowth, and increased the expression of the typical NE markers neuron-specific enolase and ?III tubulin (?III Tub). Treatment with 3??M WIN inhibited NK differentiation of LNCaP cells. The cannabinoid WIN downregulated the PI3K/Akt/mTOR signaling pathway, resulting in NE differentiation inhibition. In addition, an activation of AMP-activated protein kinase (AMPK) was observed in WIN-treated cells, which correlated with a decrease in the NE markers expression. Our results also show that during NE differentiation the expression of cannabinoid receptors CB1 and CB2 dramatically decreases. CONCLUSIONS: Taken together, we demonstrate that PI3K/Akt/AMPK might be an important axis modulating NE differentiation of prostate cancer that is blocked by the cannabinoid WIN, pointing to a therapeutic potential of cannabinoids against NE prostate cancer.Ministerio de Economía y CompetitividadJunta de Comunidades de Castilla-La ManchaFundación Tatiana Pérez de Guzmán el BuenoFondo Europeo de Desarrollo RegionalComunidad de Madri

Integra BioFis 5.0. A collaborative, participatory and interdisciplinary experience for undergraduates in nursing

The pandemic has forced us to reinvent ourselves and to consider new strategies in education. Motivation, fundamental to student performance, has been seriously compromised. In this sense, the type of motivation we are interested in "fostering" is intrinsic motivation, closely linked to the concept of learning-centered goals and objectives. The action implemented is committed to the approach to challenge-based learning-gamification in the Degree in Nursing (UAH), in order to develop an integrative training with an interdisciplinary focus. Biochemistry and Physiology came together in Integra BioFis 5.0 and through participatory and collaborative techniques we pursued meaningful learning. All the students of the Biochemistry and Physiology subjects (n = 120) took part in the learning experience, organized in 12 teams. The action was carried out online through the virtual platform: - An initial session, in which the objectives, methodology, timetable and evaluation criteria were clarified. Topics that aroused the students? interest were randomly assigned (https://bit.ly/3tfJaCi). The assigned tutors guided the students in overcoming the challenges of each stage. - The development of the action consisted of a series of phases: i) documentation and literature search; ii) integration of objectives and choice of presentation format; iii) elaboration of the graphical document; iv) peer review of presentations and voting for the best contribution. The students' papers, as well as the rubrics with comments and suggestions from each of the instructors, were returned to the teams immediately. - A final session, in which they reflected on the activity they had carried out, highlighting the positive aspects of the training for the development of competences and skills: i) search for information from quality sources; ii) synthesis of contents; iii) work as a team; iv) elaboration of an original and own work. Voting was then shown for the papers presented, revealing the names of the three teams with the most votes, finalists and winners. Learning assessment was conducted by taking into account the influence of learning on motivation and the student's self-esteem and competences, with indicators such as progress, content, sources, graphical document production, teamwork and responsibility, among others. As an important element that makes gamification work, a reward for participation (awarding of a participation diploma) and for the best Integra BioFis 5.0 graphic document presented (awarding of diplomas and prizes-gifts to the members of the winning team) was considered as an important element that makes gamification work. From this educational strategy, it can be concluded that gamification is a constructive experience, taking advantage of all the benefits of implementing the overcoming of challenges in the educational environment. This has had an impact on teaching practice, to the extent that what has been "reflected" and "worked" in Integra BioFis 5.0 has contributed to improving the quality of virtual teaching, "fostering" intrinsic motivation in students.Universidad de Alcal

Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells

Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and ?III tubulin (?III tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1?M AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer

Designing a biochemical escape room for undergraduate students

Gamification is increasingly used in Higher Education to foster intrinsic motivation of undergraduate students. In this sense, we designed the Biochemical Escape Room. The students, organized in teams and through the overcoming of challenges, tackled the contents reviewed in the face-to-face classes. In addition, the tests helped in the training of different soft skills such as creative thinking, deductive thinking, collaborative work, teamwork, manual dexterity, communication and time management. The activity was designed in several phases: i) setting the learning objectives; ii) adapting the physical spaces to the teams involved in the "game"; iii) acquiring the material; iv) preparing, placing the tests and the clues and general rehearsal before D-day; and v) D-day. The results obtained after the design of the Biochemical Escape Room and its implementation show that one of the objectives of the activity had been achieved: it provided an "injection" of motivation for teachers and students. And although there are certain aspects that need to be improved, designing the escape room for the biochemistry laboratories has meant a significant change both for the instructors, in the way we teach, and for the students, in the manner they learn.Universidad de Alcalá. Vicerrectorado de Innovación Docente y Transformación Digita