Palladium (II) compounds containing oximes as promising antitumor agents for the treatment of osteosarcoma: An in vitro and in vivo comparative study with cisplatin.

Drug resistance, evasion of cell death and metastasis are factors that contribute to the low cure rate and disease-free survival in osteosarcomas (OS). In this study, we demonstrated that a new class of oxime-containing organometallic complexes called Pd-BPO (O3) and Pd-BMO (O4) are more cytotoxic than cisplatin (CDDP) for SaOS-2 and U2OS cells using the MTT assay. Annexin-FITC/7-AAD staining demonstrated a greater potential for palladium-oxime complexes to induce death in SaOS-2 cells than CDDP, an event confirmed using the pan-caspase inhibitor Z-VAD-FMK. Compared to CDDP, only palladium-oxime complexes eradicated the clonogenicity of SaOS-2 cells after 7 days of treatment. The involvement of the lysosome-mitochondria axis in the cell death-inducing properties of the complexes was also evaluated. Using LysoTracker Red to label the acidic organelles of SaOS-2 cells treated with the O3 and O4 complexes, a decrease in the fluorescence intensity of this probe was observed in relation to CDDP and the control. Lysosomal membrane permeabilization (LMP) was also induced by the O3 and O4 complexes in an assay using acridine orange (A/O). The greater efficiency of the complexes in depolarizing the mitochondrial membrane compared to SaOS-2 cells treated with CDDP was also observed using TMRE (tetramethyl rhodamine, ethyl ester). For in vivo studies, C. elegans was used and demonstrated that both complexes reduce body bends and pharyngeal pumping after 24 h of treatment to the same extent as CDDP. We conclude that both palladium-oxime complexes are more effective than CDDP in inducing tumor cell death. The toxicity of these complexes to C. elegans was like that induced by CDDP. These results encourage preclinical studies aimed at developing more effective drugs for the treatment of osteosarcoma (OS). Furthermore, we propose palladium-oxime complexes as a new class of antineoplastic agents.

Two-pore channel-2 and inositol trisphosphate receptors coordinate Ca2+ signals between lysosomes and the endoplasmic reticulum.

Lysosomes and the endoplasmic reticulum (ER) are Ca2+ stores mobilized by the second messengers NAADP and IP3, respectively. Here, we establish Ca2+ signals between the two sources as fundamental building blocks that couple local release to global changes in Ca2+. Cell-wide Ca2+ signals evoked by activation of endogenous NAADP-sensitive channels on lysosomes comprise both local and global components and exhibit a major dependence on ER Ca2+ despite their lysosomal origin. Knockout of ER IP3 receptor channels delays these signals, whereas expression of lysosomal TPC2 channels accelerates them. High-resolution Ca2+ imaging reveals elementary events upon TPC2 opening and signals coupled to IP3 receptors. Biasing TPC2 activation to a Ca2+-permeable state sensitizes local Ca2+ signals to IP3. This increases the potency of a physiological agonist to evoke global Ca2+ signals and activate a downstream target. Our data provide a conceptual framework to understand how Ca2+ release from physically separated stores is coordinated.

Artemisia vulgaris Induces Tumor-Selective Ferroptosis and Necroptosis via Lysosomal Ca2+ Signaling.

OBJECTIVE: To evaluate the chemical composition and effects of Artemisia vulgaris (AV) hydroalcoholic extract (HEAV) on breast cancer cells (MCF-7 and SKBR-3), chronic myeloid leukemia (K562) and NIH/3T3 fibroblasts. METHODS: Phytochemical analysis of HEAV was done by high-performance liquid chromatography-mass (HPLC) spectrometry. Viability and cell death studies were performed using trypan blue and Annexin/FITC-7AAD, respectively. Ferrostatin-1 (Fer-1) and necrostatin-1 (Nec-1) were used to assess the mode of HEAV-induced cell death and acetoxymethylester (BAPTA-AM) was used to verify the involvement of cytosolic calcium in this event. Cytosolic calcium measurements were made using Fura-2-AM. RESULTS: HEAV decreased the viability of MCF-7, SKBR-3 and K562 cells (P<0.05). The viability of HEAV-treated K562 cells was reduced compared to HEAV-exposed fibroblasts (P<0.05). Treatment of K562 cells with HEAV induced cell death primarily by late apoptosis and necrosis in assays using annexin V-FITC/7-AAD (P<0.05). The use of Nec-1 and Fer-1 increased the viability of K562 cells treated with HEAV relative to cells exposed to HEAV alone (P<0.01). HEAV-induced Ca2+ release mainly from lysosomes in K562 cells (P<0.01). Furthermore, BAPTA-AM, an intracellular Ca2+ chelator, decreased the number of non-viable cells treated with HEAV (P<0.05). CONCLUSIONS: HEAV is cytotoxic and activates several modalities of cell death, which are partially dependent on lysosomal release of Ca2+. These effects may be related to artemisinin and caffeoylquinic acids, the main compounds identified in HEAV.

Blocking Autophagy by the Two-Pore Channels Antagonist Tetrandrine Improves Sorafenib-Induced Death of Hepatocellular Carcinoma Cells.

Sorafenib, an oral multi-kinase inhibitor, used to treat hepatocellular carcinoma (HCC). However, drug resistance is still common in several HCC patients. This complex mechanism is not yet fully elucidated, driving the search for new therapeutic targets to potentiate the antitumoral effect of sorafenib. Recent findings have linked the expression of Two-Pore Channels (TPCs) receptors with the development and progression of cancer. TPCs receptors are stimulated by NAADP, a Ca2+ messenger, and inhibited by their antagonists Ned-19 and tetrandrine. Here, we investigate the participation of TPCs inhibition in cell death and autophagy in sorafenib-treated HCC cells. Here, we show that the association of sorafenib with tetrandrine increased sorafenib-induced cell death accompanied by increased lysotracker fluorescence intensity. In contrast, these effects were not observed after treating these cells with Ned-19. The pharmacological TPC antagonists by Ned-19 and tetrandrine or siRNA-mediated TPC1/2 inhibition decreased sorafenib-induced Ca2+ release, reinforcing the participation of TPCs in sorafenib HCC responses. Furthermore, the association tetrandrine and sorafenib blocked autophagy through ERK1/2 pathway inhibition, which represents a putative target for potentiating HCC cell death. Therefore, our study proposes the use of tetrandrine analogs with the aim of improving sorafenib therapy. Also, our data also allow us to suggest that TPCs may be a new target in anticancer therapies.

Cannabidiol Recovers Dopaminergic Neuronal Damage Induced by Reserpine or α-synuclein in Caenorhabditis elegans.

Progressive neurodegenerative disorders such as Parkinson Disease (PD) lack curative or long-term treatments. At the same time, the increase of the worldwide elderly population and, consequently, the extension in the prevalence of age-related diseases have promoted research interest in neurodegenerative disorders. Caenorhabditis elegans is a free-living nematode widely used as an animal model in studies of human diseases. Here we evaluated cannabidiol (CBD) as a possible neuroprotective compound in PD using the C. elegans models exposed to reserpine. Our results demonstrated that CBD reversed the reserpine-induced locomotor alterations and this response was independent of the NPR-19 receptors, an orthologous receptor for central cannabinoid receptor type 1. Morphological alterations of cephalic sensilla (CEP) dopaminergic neurons indicated that CBD also protects neurons from reserpine-induced degeneration. That is, CBD attenuates the reserpine-induced increase of worms with shrunken soma and dendrites loss, increasing the number of worms with intact CEP neurons. Finally, we found that CBD also reduced ROS formation and α-syn protein accumulation in mutant worms. Our findings collectively provide new evidence that CBD acts as neuroprotector in dopaminergic neurons, reducing neurotoxicity and α-syn accumulation highlighting its potential in the treatment of PD.

Voluntary physical activity mitigates alveolar bone loss in mice with ligature-induced experimental periodontitis.

OBJECTIVE: To investigate the effect of voluntary physical activity (VPA) on inflammatory profile and the progression of experimental periodontal disease (PD) in mice. METHODS: Male C57BL/6 mice were randomly distributed into Control; VPA; PD and PD/VPA groups. We registered VPA (total volume of revolutions) and average speed (revolutions/minute) in a free running wheel for 30 days. On the 15th day, animals from the PD and PD/VPA groups received ligatures on the upper second molars bilaterally. On the 30th day animals were euthanized, and PD progression was assessed by measuring alveolar bone loss (ABL - the linear distance between the cemento-enamel junction and the alveolar bone crest on the teeth buccal surface). Gene expression of RANKL (kappa nuclear factor B receptor) OPG (osteoprotegerin), IL-1ß (interleukin 1 beta), IL-6 (interleukin 6) and TNF-α (tumor necrosis factor alpha) were evaluated by real-time PCR (quantitative Polymerase Chain Reaction - relative gene expression). RESULTS: The total volume of physical activity and the activity speed decreased along the seven days after ligature-placement (p < 0.05), returning to a similar pattern in relation to VPA group. Ligature placement produced significant bone resorption, and increased RANKL, IL-1ß, IL-6 and TNF-α expression. VPA reduced ABL (p < 0,05) and the expression of TNF-α and IL-1ß, whereas increased OPG expression. CONCLUSION: Animals induced to PD with access to the VPA wheel presented both lower gingival inflammation and less alveolar bone resorption in comparison to animals without access to the wheel.

Rapamycin Improves the Response of Effector and Memory CD8+ T Cells Induced by Immunization With ASP2 of Trypanosoma cruzi.

Deficiency in memory formation and increased immunosenescence are pivotal features of Trypanosoma cruzi infection proposed to play a role in parasite persistence and disease development. The vaccination protocol that consists in a prime with plasmid DNA followed by the boost with a deficient recombinant human adenovirus type 5, both carrying the ASP2 gene of T. cruzi, is a powerful strategy to elicit effector memory CD8+ T-cells against this parasite. In virus infections, the inhibition of mTOR, a kinase involved in several biological processes, improves the response of memory CD8+ T-cells. Therefore, our aim was to assess the role of rapamycin, the pharmacological inhibitor of mTOR, in CD8+ T response against T. cruzi induced by heterologous prime-boost vaccine. For this purpose, C57BL/6 or A/Sn mice were immunized and daily treated with rapamycin for 34 days. CD8+ T-cells response was evaluated by immunophenotyping, intracellular staining, ELISpot assay and in vivo cytotoxicity. In comparison with vehicle-injection, rapamycin administration during immunization enhanced the frequency of ASP2-specific CD8+ T-cells and the percentage of the polyfunctional population, which degranulated (CD107a+) and secreted both interferon gamma (IFNγ) and tumor necrosis factor (TNF). The beneficial effects were long-lasting and could be detected 95 days after priming. Moreover, the effects were detected in mice immunized with ten-fold lower doses of plasmid/adenovirus. Additionally, the highly susceptible to T. cruzi infection A/Sn mice, when immunized with low vaccine doses, treated with rapamycin, and challenged with trypomastigote forms of the Y strain showed a survival rate of 100%, compared with 42% in vehicle-injected group. Trying to shed light on the biological mechanisms involved in these beneficial effects on CD8+ T-cells by mTOR inhibition after immunization, we showed that in vivo proliferation was higher after rapamycin treatment compared with vehicle-injected group. Taken together, our data provide a new approach to vaccine development against intracellular parasites, placing the mTOR inhibitor rapamycin as an adjuvant to improve effective CD8+ T-cell response.

Angiotensin II modulates the murine hematopoietic stem cell and progenitors cocultured with stromal S17 cells.

Although the existence of the renin-angiotensin system (RAS) in the bone marrow is clear, the exact role of this system in hematopoiesis has not yet been fully characterized. Here the direct role of angiotensin II (AngII) in hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs), and megakaryocytes/erythroid progenitors (MEPs), using a system of coculture with stromal S17 cells. Flow cytometry analysis showed that AngII increases the percentage of HSC and GMP, while reducing CMP with no effect on MEP. According to these data, AngII increased the total number of mature Gr-1+ /Mac-1+ cells without changes in Terr119+ cells. AngII does not induce cell death in the population of LSK cells. In these populations, treatment with AngII decreases the expression of Ki67+ protein with no changes in the Notch1 expression, suggesting a role for AngII on the quiescence of immature cells. In addition, exposure to AngII from murine bone marrow cells increased the number of CFU-GM and BFU-E in a clonogenic assay. In conclusion, our data showed that AngII is involved in the regulation of hematopoiesis with a special role in HSC, suggesting that AngII should be evaluated in coculture systems, especially in cases that require the expansion of these cells in vitro, still a significant challenge for therapeutic applications in humans.

Cannabidiol induces autophagy via ERK1/2 activation in neural cells.

Autophagy is a lysosomal catabolic process essential to cell homeostasis and is related to the neuroprotection of the central nervous system. Cannabidiol (CBD) is a non-psychotropic phytocannabinoid present in Cannabis sativa. Many therapeutic actions have been linked to this compound, including autophagy activation. However, the precise underlying molecular mechanisms remain unclear, and the downstream functional significance of these actions has yet to be determined. Here, we investigated CBD-evoked effects on autophagy in human neuroblastoma SH-SY5Y and murine astrocyte cell lines. We found that CBD-induced autophagy was substantially reduced in the presence of CB1, CB2 and TRPV1 receptor antagonists, AM 251, AM 630 and capsazepine, respectively. This result strongly indicates that the activation of these receptors mediates the autophagic flux. Additionally, we demonstrated that CBD activates autophagy through ERK1/2 activation and AKT suppression. Interestingly, CBD-mediated autophagy activation is dependent on the autophagy initiator ULK1, but mTORC1 independent. Thus, it is plausible that a non-canonical pathway is involved. Our findings collectively provide evidence that CBD stimulates autophagy signal transduction via crosstalk between the ERK1/2 and AKT kinases, which represent putative regulators of cell proliferation and survival. Furthermore, our study sheds light on potential therapeutic cannabinoid targets that could be developed for treating neurodegenerative disorders.

α-Synuclein Overexpression Induces Lysosomal Dysfunction and Autophagy Impairment in Human Neuroblastoma SH-SY5Y.

Although the etiology of Parkinson's disease (PD) is multifactorial, it has been linked to abnormal accumulation of α-synuclein (α-syn) in dopaminergic neurons, which could lead to dysfunctions on intracellular organelles, with potential neurodegeneration. Patients with familial early-onset PD frequently present mutation in the α-syn gene (SNCA), which encodes mutant α-syn forms, such as A30P and A53T, which potentially regulate Ca2+ unbalance. Here we investigated the effects of overexpression of wild-type α-syn (WT) and the mutant forms A30P and A53T, on modulation of lysosomal Ca2+ stores and further autophagy activation. We found that in α-syn-overexpressing cells, there was a decrease in Ca2+ released from endoplasmic reticulum (ER) which is related to the increase in lysosomal Ca2+ release, coupled to lysosomal pH alkalization. Interestingly, α-syn-overexpressing cells showed lower LAMP1 levels, and a disruption of lysosomal morphology and distribution, affecting autophagy. Interestingly, all these effects were more evident with A53T mutant isoform when compared to A30P and WT α-syn types, indicating that the pathogenic phenotype for PD is potentially related to impairment of α-syn degradation. Taken together, these events directly impact PD-related dysfunctions, being considered possible molecular targets for neuroprotection.

Effective Synergy of Sorafenib and Nutrient Shortage in Inducing Melanoma Cell Death through Energy Stress.

Skin melanoma is one of the most aggressive and difficult-to-treat human malignancies, characterized by poor survival rates, thus requiring urgent novel therapeutic approaches. Although metabolic reprogramming has represented so far, a cancer hallmark, accumulating data indicate a high plasticity of cancer cells in modulating cellular metabolism to adapt to a heterogeneous and continuously changing microenvironment, suggesting a novel therapeutic approach for dietary manipulation in cancer therapy. To this aim, we exposed melanoma cells to combined nutrient-restriction/sorafenib. Results indicate that cell death was efficiently induced, with apoptosis representing the prominent feature. In contrast, autophagy was blocked in the final stage by this treatment, similarly to chloroquine, which also enhanced melanoma cell sensitization to combined treatment. Energy stress was evidenced by associated treatment with mitochondrial dysfunction and glycolysis impairment, suggesting metabolic stress determining melanoma cell death. A reduction of tumor growth after cycles of intermittent fasting together with sorafenib treatment was also observed in vivo, reinforcing that the nutrient shortage can potentiate anti-melanoma therapy. Our findings showed that the restriction of nutrients by intermittent fasting potentiates the effects of sorafenib due to the modulation of cellular metabolism, suggesting that it is possible to harness the energy of cancer cells for the treatment of melanoma.

Frontline Science: Autophagy is a cell autonomous effector mechanism mediated by NLRP3 to control Trypanosoma cruzi infection.

Autophagy and inflammasome activation are cell-autonomous and cross-regulated processes involved in host resistance against infections. Our group previously described that NLRP3 inflammasome is required for the control of Trypanosoma cruzi, the causative agent of Chagas disease. However, the involvement of autophagy in this process was unclear. Here, we demonstrated that T. cruzi was able to induce an increase in LC3-II expression as well as autophagosome and autolysosome formation in peritoneal macrophages (PMs) from C57BL/6 wild-type mice. Moreover, the pharmacologic inhibition of autophagic machinery impaired the ability of PMs to control T. cruzi replication. Importantly, NLRP3 was required for the induction of a regular autophagic flux in response to T. cruzi, an effect mediated by its participation in the autolysosomes formation. Together, these results indicate autophagy as an effector mechanism mediated by NLRP3 to control T. cruzi infection.

Medicinal properties of Angelica archangelica root extract: Cytotoxicity in breast cancer cells and its protective effects against in vivo tumor development.

OBJECTIVE: Although Angelica archangelica is a medicinal and aromatic plant with a long history of use for both medicinal and food purposes, there are no studies regarding the antineoplastic activity of its root. This study aimed to evaluate the cytotoxicity and antitumor effects of the crude extract of A. archangelica root (CEAA) on breast cancer. METHODS: The cytotoxicity of CEAA against breast adenocarcinoma cells (4T1 and MCF-7) was evaluated by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Morphological and biochemical changes were detected by Hoechst 33342/propidium iodide (PI) and annexin V/PI staining. Cytosolic calcium mobilization was evaluated in cells staining with FURA-4NW. Immunoblotting was used to determine the effect of CEAA on anti- and pro-apoptotic proteins (Bcl-2 and Bax, respectively). The 4T1 cell-challenged mice were used for in vivo assay. RESULTS: Using ultra-high-performance liquid chromatography-mass spectrometry analysis, angelicin, a constituent of the roots and leaves of A. archangelica, was found to be the major constituent of the CEAA evaluated in this study (73 µg/mL). The CEAA was cytotoxic for both breast cancer cell lines studied but not for human fibroblasts. Treatment of 4T1 cells with the CEAA increased Bax protein levels accompanied by decreased Bcl-2 expression, in the presence of cleaved caspase-3 and cytosolic calcium mobilization, suggesting mitochondrial involvement in breast cancer cell death induced by the CEAA in this cell line. No changes on the Bcl-2/Bax ratio were observed in CEAA-treated MCF7 cells. Gavage administration of the CEAA (500 mg/kg) to 4T1 cell-challenged mice significantly decreased tumor growth when compared with untreated animals. CONCLUSION: Altogether, our data show the antitumor potential of the CEAA against breast cancer cells in vitro and in vivo. Further research is necessary to better elucidate the pharmacological application of the CEAA in breast cancer therapy.

Estrogen receptors localization and signaling pathways in DU-145 human prostate cancer cells.

The aim of the present study was to investigate the subcellular localization of estrogen receptors ERα and ERß in androgen-independent prostate cancer cell line DU-145, and the possible role of exportin CRM1 on ERs distribution. In addition, we evaluated the ERs contribution to activation of ERK1/2 and AKT. Immunostaining of ERα and ERß was predominantly found in the extranuclear regions of DU-145 cells. CRM1 inhibitor Leptomycin B reduced drastically the presence of ERα and ERß in the extranuclear regions and increased in the nuclei, indicating the possible involvement of CRM1 on ERs nuclear-cytoplasmic shuttling. 17ß-estradiol (E2), ERα-selective agonist PPT and ERß-selective agonist DPN induced a rapid increase on ERK1/2 phosphorylation. E2-induced ERK1/2 activation was partially inhibited when cells were pretreated with ERα- or ERß-selective antagonists, and blocked by simultaneous pretreatment with both antagonists, suggesting ERα/ß heterodimers formation. Furthermore, E2 treatment did not activate AKT pathway. Therefore, we highlighted a possible crosstalk between extranuclear and nuclear ERs and their upstream and downstream signaling molecules as an important mechanism to control ER function as a potential therapeutic target in prostate cancer cells.

Lithium, a classic drug in psychiatry, improves nilotinib-mediated antileukemic effects.

Although Tyrosine kinase inhibitors (TKIs) that target Bcr-Abl play a key role in Chronic Myeloid Leukemia (CML) therapy, they do not eradicate CML-initiating cells, which lead to the emergence of drug resistance. Here we used the lithium, a GSK-3 inhibitor, to attempt to potentiate the effects of nilotinib against leukemia cells. For this purpose, a K562 leukemia cell line and bone marrow cells from untreated Chronic Myeloid Leukemia (CML) patients, prior to any exposure to TKIs, were used as a model. Our results demonstrated that the combination of lithium + nilotinib (L + N) induced K562-cell death and cleaved caspase-3 when compared to lithium or nilotinib alone, accompanied by GSK-3ß phosphorylation and Bcr-Abl oncoprotein levels reduction. Interestingly, these events were related to autophagy induction, expressed by increased LC3II protein levels in the group treated with L + N. Furthermore, the clonogenic capacity of progenitor cells from CML patients was drastically reduced by L + N, as well as lithium and nilotinib when used separately. The number of cell aggregates (clusters), were increased by all treatments (L + N, lithium, and nilotinib). This pioneering research has demonstrated that lithium might be of therapeutic value when targeting Bcr-Abl cells with nilotinib because it triggers cell death in addition to exerting classical antiproliferative effects, opening new perspectives for novel target and therapeutic approaches to eradicate CML.

Cafestol, a diterpene molecule found in coffee, induces leukemia cell death.

To evaluate the antitumor properties of Cafestol four leukemia cell lines were used (NB4, K562, HL60 and KG1). Cafestol exhibited the highest cytotoxicity against HL60 and KG1 cells, as evidenced by the accumulation of cells in the sub-G1 fraction, mitochondrial membrane potential reduction, accumulation of cleaved caspase-3 and phosphatidylserine externalization. An increase in CD11b and CD15 differentiation markers with attenuated ROS generation was also observed in Cafestol-treated HL60 cells. These results were similar to those obtained following exposure of the same cell line to cytarabine (Ara-C), an antileukemic drug. Cafestol and Ara-C reduced the clonogenic potential of HL60 cells by 100%, but Cafestol spared murine colony forming unit- granulocyte/macrophage (CFU-GM), which retained their clonogenicity. The co-treatment of Cafestol and Ara-C reduced HL60 cell viability compared with both drugs administered alone. In conclusion, despite the distinct molecular mechanisms involved in the activity of Cafestol and Ara-C, a similar cytotoxicity towards leukemia cells was observed, which suggests a need for prophylactic-therapeutic pre-clinical studies regarding the anticancer properties of Cafestol.

Glutamate induces autophagy via the two-pore channels in neural cells.

NAADP (nicotinic acid adenine dinucleotide phosphate) has been proposed as a second messenger for glutamate in neuronal and glial cells via the activation of the lysosomal Ca2+ channels TPC1 and TPC2. However, the activities of glutamate that are mediated by NAADP remain unclear. In this study, we evaluated the effect of glutamate on autophagy in astrocytes at physiological, non-toxic concentration. We found that glutamate induces autophagy at similar extent as NAADP. By contrast, the NAADP antagonist NED-19 or SiRNA-mediated inhibition of TPC1/2 decreases autophagy induced by glutamate, confirming a role for NAADP in this pathway. The involvement of TPC1/2 in glutamate-induced autophagy was also confirmed in SHSY5Y neuroblastoma cells. Finally, we show that glutamate leads to a NAADP-dependent activation of AMPK, which is required for autophagy induction, while mTOR activity is not affected by this treatment. Taken together, our results indicate that glutamate stimulates autophagy via NAADP/TPC/AMPK axis, providing new insights of how Ca2+ signalling glutamate-mediated can control the cell metabolism in the central nervous system.

Autophagy regulates Selumetinib (AZD6244) induced-apoptosis in colorectal cancer cells.

OBJECTIVE: As Selumetinib is a MEK1/2 inhibitor that has gained interest as an anti-tumor agent, the present study was designed to investigate autophagy involvement on Selumetinib-induced apoptosis in colorectal cancer (CRC) cells. METHODS: CRC cells death and cycle studies were assessed by AnnexinV-FITC and PI staining, respectively. Autophagy flux was analysed by Western Blot (LC3II and p62 protein levels) and retroviral infection of SW480 cells for siBecn1 RNA interference experiments. Confocal microscopy was used to determine mCherry-EGFP-LC3 distribution. KEY FINDINGS: The Selumetinib effects were concentration-dependent in SW480 cell line. Whereas 1 µM exerted an arrest in the cell cycle (G1 phase), higher concentrations (10 µM) induced cell death, which was accompanied by autophagy blockage in its last stages. Autophagy induction by Rapamycin (RAPA) increased cell survival, whereas pharmacology autophagy inhibition by Bafilomycin A1 (BAF), Chloroquine (CQ) or 3-Methyladenine (3-MA) increased Selumetinib-induced CRC cells death. CONCLUSIONS: Altogether, these results suggest that autophagy plays a fundamental role in CRC cells response to Selumetinib. In addition, the combination of Selumetinib with autophagy inhibitors may be a useful therapeutic strategy to enhance its activity against colorectal tumours.

The biphosphinic paladacycle complex induces melanoma cell death through lysosomal-mitochondrial axis modulation and impaired autophagy.

Recently, palladium complexes have been extensively studied as cyclization of these complexes by cyclometallation reactions increased their stability making them promising antitumor compounds. In this study, we have investigated apoptosis induced by the Biphosphinic Paladacycle Complex (BPC11) and possible cross talk between apoptosis and autophagy in cell line models of metastatic (Tm5) and non-metastatic (4C11-) melanoma. The BPC11-induced cell death in melanoma involved the lysosomal-mitochondrial axis, which is characterized by LMP, CatB activation and increased Bax protein levels following its translocation to mitochondria. Mitochondrial hyperpolarization, followed by membrane potential dissipation and cleavage of caspase-3, also resulted in cell death after 24 h of incubation. We also found that BPC11-mediated LC3II formation and increased p62 protein levels, suggesting blocked autophagy, probably due to LMP. Interestingly, the treatment of Tm5 and 4C11(-) cells with 3-methyladenine (3-MA), an inhibitor of the initial stage of autophagy, potentiated the effects of BPC11. We conclude that BPC11 is an anti-melanoma agent and that autophagy may be acting as a mechanism of melanoma cells resistance. Also, these data highlight the importance of studies involving autophagy and apoptosis during pre-clinical studies of new drugs with anticancer properties.

Bothropoides pauloensis venom effects on isolated perfused kidney and cultured renal tubular epithelial cells.

Snake envenomation (Bothrops genus) is common in tropical countries and acute kidney injury is one of the complications observed in Bothrops snakebite with relevant morbidity and mortality. Here, we showed that Bothropoides pauloensis venom (BpV) decreased cell viability (IC50 of 7.5 µg/mL). Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by apoptosis and late apoptosis, through caspases 3 and 7 activation, mitochondrial membrane potential collapse and ROS overproduction. BpV reduced perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, percentage of sodium, chloride or potassium tubular transportation. These findings demonstrated that BpV cytotoxicity on renal epithelial cells might be responsible for the nephrotoxicity observed in isolated kidney.