Chlormequat Chloride Inhibits TM3 Leydig Cell Growth via Ferroptosis-Initiated Inflammation.

Ferroptosis hallmarked by lipid peroxidation and iron homeostasis imbalance is involved in the occurrence and development of various diseases. The plant growth regulator chlormequat chloride (CCC) can contribute to the causality and exacerbation of reproductive disorders. However, the mechanism by which CCC may cause Leydig cell attenuation remains poorly understood. In this study, TM3 Leydig cells were used to investigate the inhibitory effect of CCC on cell growth and its possible mechanism. The results showed that CCC caused apoptosis, pyroptosis, ferroptosis and necroinflammation in TM3 cells. By comparing the effects of ferroptosis inhibitor Ferrostatin-1 (Fer-1) and pan-Caspase inhibitor Z-VAD-FMK (ZVF) on lipid peroxidation and Caspase-mediated regulated cell death (RCD), we found that Fer-1 was better at rescuing the growth of TM3 cells than ZVF. Although ZVF reduced mitochondrial ROS level and inhibited the activation of Caspase3 and Caspase1, it could not significantly ameliorate lipid peroxidation and the levels of IL-1ß and HMGB1 like Fer-1. Therefore, ferroptosis might be a key non apoptotic RCD mode responsible for CCC-driven inflammation, leading to weakened viability and proliferation of TM3 cells. In addition, overexpression of ferritin light chain (FTL) promoted the resistance of TM3 cells to CCC-induced ferroptosis-mediated inflammation and to some extent improved the inhibition of viability and proliferation. Altogether, ferroptosis-initiated inflammation might play a key role in CCC-impaired TM3 cell growth.

Granulocyte pro-myeloperoxidase is redundantly processed by proprotein convertase furin and PC7 in HL-60 cells.

Neutrophil myeloperoxidase/H2O2/chloride system is a key mechanism to control pathogen infection. This enzyme, myeloperoxidase, plays a pivotal role in the arsenal of azurophilic granules that are released through degranulation upon neutrophil activation, which trigger local hypochlorous acid production. Myeloperoxidase gene encodes a protein precursor named promyeloperoxidase that arbors a propeptide that gets cleaved later during secretory routing in post-endoplasmic reticulum compartments. Although evidence suggested that this processing event was performed by one or different enzymes from the proprotein convertases family, the identity of this enzyme was never investigated. In this work, the naturally producing myeloperoxidase promyelocytic cell line HL-60 was used to investigate promyeloperoxidase cleavage during granulocytic differentiation in response to proprotein convertase inhibitors decanoyl-RVKR-chloromethylketone and hexa-d-arginine. Stable PC knockdown of endogenously expressed proprotein convertases, furin and PC7, was achieved using lentiviral delivery of shRNAs. None of the knockdown cell line could reproduce the effect of the pan-proprotein convertases inhibitor decanoyl-RVKR-chloromethylketone that accumulated intracellular promyeloperoxidase stores in HL-60 cells, therefore illustrating that both furin and PC7 redundantly process this proprotein.

Effective Prevention of Arterial Thrombosis with Albumin-Thrombin Inhibitor Conjugates.

Thromboprophylaxis is indicated in patients at an elevated risk of developing thrombotic disorders, typically using direct oral anticoagulants or low-molecular-weight heparins. We postulated that transient thromboprophylaxis (days-weeks) could be provided by a single dose of an anticoagulant engineered for prolonged pharmacokinetics. In the present work, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (PPACK) was used as a model anticoagulant to test the hypothesis that conjugation of thrombin inhibitors to the surface of albumin would provide durable protection against thrombotic insults. Covalent conjugates were formed between albumin and PPACK using click chemistry, and they were tested in vitro using a thrombin activity assay and a clot formation assay. Thromboprophylactic efficacy was tested in mouse models of arterial thrombosis, both chemically induced (FeCl3) and following ischemia-reperfusion (transient middle cerebral artery occlusion; tMCAO). Albumin-PPACK conjugates were shown to have nanomolar potency in both in vitro assays, and following intravenous injection had prolonged circulation. Conjugates did not impact hemostasis (tail clipping) or systemic coagulation parameters in normal mice. Intravenous injection of conjugates prior to FeCl3-induced thrombosis provided significant protection against occlusion of the middle cerebral and common carotid arteries, and injection immediately following ischemia-reperfusion reduced stroke volume measured 3 days after injury by ∼40% in the tMCAO model. The data presented here provide support for the use of albumin-linked anticoagulants as an injectable, long-circulating, safe thromboprophylactic agent. In particular, albumin-PPACK provides significant protection against thrombosis induced by multiple mechanisms, without adversely affecting hemostasis.

Identification of Kukoamine A, Zeaxanthin, and Clexane as New Furin Inhibitors.

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.

Cellular autophagy, an unbidden effect of caspase inhibition by zVAD-fmk.

zVAD-fmk is a widely used pan-caspase inhibitor that blocks apoptosis but has undesirable side effects, including autophagy. In this issue, Needs et al. propose that zVAD-fmk induces autophagy by inhibiting the N-glycanase NGLY1 rather than caspases. NGLY1 is essential for the ERAD response and patients with inactivating mutations in NGLY1 present with neurodevelopmental defects and organ dysfunction. The ability of NGLY1 to inhibit basal levels of autophagy may contribute to this pathology. This study demonstrates possible crosstalk between protein turnover and autophagy while also underscoring the importance of specificity when using chemical tools to interrogate these pathways. Comment on https://doi.org/10.1111/febs.16345.

Role of HMGB1 in TNF-α Combined with Z-VAD-fmk-Induced L929 Cells Necroptosis.

The present study established a necroptosis model in vitro and investigated the role of HMGB1 in cell necroptosis. A combination of tumor necrosis factor-α and z-VAD-fmk was used to induce necroptosis in L929 cells with necroptosis inhibitor necrostatin-1 applied as an intervention. Flow cytometry and transmission electron microscopy (TEM) were used to measure cell necroptosis. Western blotting assay was applied to detect the expression of receptor-interacting serine/threonine-protein kinase 3 (RIPK3), mixed lineage kinase domain-like pseudokinase (MLKL) and HMGB1. Co-immunoprecipitation (Co-IP) assay was used to confirm the interaction between HMGB1 and RIPK3. Our study demonstrated that HMGB1 migrated from the nucleus to the cytoplasm at the onset of necroptosis and was subsequently released passively to the extracellular matrix. Further experiments determined that the binding of HMGB1 with RIPK3 in the cytoplasm was loose during necroptosis. By contrast, when necroptosis was inhibited, the interaction in the cytoplasm was tight suggesting that this association between HMGB1 and RIPK3 might affect its occurrence. In conclusion, the transfer of HMGB1 from nucleus to cytoplasm, and its interaction with RIPK3 might be potentially involved in necroptosis.

Inhibition of FOSL2 aggravates the apoptosis of ovarian cancer cells by promoting the formation of inflammasomes.

BACKGROUND: Ovarian cancer is a common gynecological malignancy among female patients and poses a serious threat to women's health. Although it has been established that Fos-like antigen 2 (FOSL2) is linked to ovarian cancer (OC), its exact role in the development of OC remains unknown. OBJECTIVE: This article aims to investigate the role of FOSL2 in ovarian cancer development. METHODS: FOSL2 expression in ovarian carcinoma and adjacent tissues was assessed using real-time fluorescent quantitative PCR and western blot. We constructed OE/sh-FOSL2 plasmids and Caspase-1 specific inhibitors (Yvad-CMK) and transfected A 2780 cells with them to identify the relevant cell functions. Furthermore, we used western blot assay to determine the changes in expression of apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartate-specific proteasezymogen procaspase 1 (pro-caspase-1), cysteinyl aspartate-specific proteinase-1 (caspase-1), interleukin-1ß precursor (pro-IL-1ß), interleukin-1ß (IL-1ß), interleukin-18 precursor (pro-IL-18), and interleukin-18 (IL-18). In addition, we measured the concentration of IL-1ß and IL-18 using an enzyme-linked immunosorbent assay (ELISA). Moreover, Tthe level of lactate dehydrogenase (LDH) in the cell supernatant was measured by LDH release assay kit. RESULTS: The expression of FOSL2 was significantly higher compared with the surrounding tissues. The proliferation, migration, and invasion of A2780 cells were enhanced after transfection with OE-FOSL2 plasmids; however, the cell apoptosis was significantly decreased. When FOSL2 was overexpressed, the inflammasome-associated proteins such as ASC, caspase-1, IL-1ß, and IL-18 were downregulated. Furthermore, FOSL2 induced apoptosis and activated the production of inflammasomes in A2780 cells. Co-therapy with Yvad-CMK and substantially inhibited apoptosis and activation of inflammasomes. CONCLUSIONS: Inhibition of FOSL2 promotes the apoptosis of OC cells by mediating the formation of an inflammasome.

Zinc finger protein 91 mediates necroptosis by initiating RIPK1-RIPK3-MLKL signal transduction in response to TNF receptor 1 ligation.

Necroptosis is a form of regulated programmed cell death that is mediated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting serine/threonine protein kinase-3 (RIPK3), and mixed lineage kinase domain-like protein (MLKL); however, it is not known whether zinc finger protein 91 (ZFP91) is involved in this process. Here, we investigated ZFP91 as a potential mediator of necroptosis. Our mechanistic study demonstrates that ZFP91 promotes RIPK1-RIPK3 interaction, thereby stabilizing the RIPK1 and RIPK3 proteins and facilitating necroptosis. ZFP91 stabilized RIPK1 to promote cell death by inducing RIPK1 de-ubiquitination. ZFP91 also significantly increased production of mitochondrial reactive oxygen species (ROS). Accumulation of ROS promoted RIPK3-independent necroptosis triggered by tumor necrosis factor (TNF). in vivo, ZFP91 knockdown alleviated TNFα-induced systemic inflammatory response syndrome (SIRS). These results provide direct evidence that ZFP91 plays an important role in the initiation of RIPK1/RIPK3-dependent necroptosis in vitro and in vivo. We discussed the potential of ZFP91 as a novel therapeutic target for necroptosis-associated diseases.

Coronavirus Infection-Associated Cell Death Signaling and Potential Therapeutic Targets.

COVID-19 is the name of the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that occurred in 2019. The virus-host-specific interactions, molecular targets on host cell deaths, and the involved signaling are crucial issues, which become potential targets for treatment. Spike protein, angiotensin-converting enzyme 2 (ACE2), cathepsin L-cysteine peptidase, transmembrane protease serine 2 (TMPRSS2), nonstructural protein 1 (Nsp1), open reading frame 7a (ORF7a), viral main protease (3C-like protease (3CLpro) or Mpro), RNA dependent RNA polymerase (RdRp) (Nsp12), non-structural protein 13 (Nsp13) helicase, and papain-like proteinase (PLpro) are molecules associated with SARS-CoV infection and propagation. SARS-CoV-2 can induce host cell death via five kinds of regulated cell death, i.e., apoptosis, necroptosis, pyroptosis, autophagy, and PANoptosis. The mechanisms of these cell deaths are well established and can be disrupted by synthetic small molecules or natural products. There are a variety of compounds proven to play roles in the cell death inhibition, such as pan-caspase inhibitor (z-VAD-fmk) for apoptosis, necrostatin-1 for necroptosis, MCC950, a potent and specific inhibitor of the NLRP3 inflammasome in pyroptosis, and chloroquine/hydroxychloroquine, which can mitigate the corresponding cell death pathways. However, NF-κB signaling is another critical anti-apoptotic or survival route mediated by SARS-CoV-2. Such signaling promotes viral survival, proliferation, and inflammation by inducing the expression of apoptosis inhibitors such as Bcl-2 and XIAP, as well as cytokines, e.g., TNF. As a result, tiny natural compounds functioning as proteasome inhibitors such as celastrol and curcumin can be used to modify NF-κB signaling, providing a responsible method for treating SARS-CoV-2-infected patients. The natural constituents that aid in inhibiting viral infection, progression, and amplification of coronaviruses are also emphasized, which are in the groups of alkaloids, flavonoids, terpenoids, diarylheptanoids, and anthraquinones. Natural constituents derived from medicinal herbs have anti-inflammatory and antiviral properties, as well as inhibitory effects, on the viral life cycle, including viral entry, replication, assembly, and release of COVID-19 virions. The phytochemicals contain a high potential for COVID-19 treatment. As a result, SARS-CoV-2-infected cell death processes and signaling might be of high efficacy for therapeutic targeting effects and yielding encouraging outcomes.

The NO-dependent caspase signaling pathway is a target of deoxynivalenol in growth inhibition in vitro.

DON is commonly found in foods and feeds; it presents health risks, especially an increase of growth inhibition in humans, particularly infants and young children. However, there are relatively few research studies devoted to the mechanism of DON-mediated growth retardation. Interestingly, our results showed that DON does not cause any significant production of ROS but results in a persistent and significant release of NO with iNOS increasing activity, mitochondrial ultrastructural changes and decreasing ΔΨm. Moreover, the significant decreases in GH production and secretion induced by DON were dose-dependent, accompanied by an increase of caspase 3, 8 and 9, IL-11, IL-lß and GHRH. NO scavenging agent (haemoglobin) and free radical scavenging agent (N-acetylcysteine) partially reversed mitochondrial damage, and Z-VAD-FMK increased the levels of GH and decreased the levels of caspase 3, 8 and 9, while haemoglobin decreased the levels of caspase 3, 8 and 9, indicating that NO is the primary target of DON-mediated inhibition. Present research study firstly demonstrated that NO is a key mediator of DON-induced growth inhibition and plays critical roles in the interference of GH transcription and synthesis. The current research is conducive to future research on the molecular mechanisms of DON-induced growth inhibition in humans, especially children.

Caspase-1-Inhibitor AC-YVAD-CMK Inhibits Pyroptosis and Ameliorates Acute Kidney Injury in a Model of Sepsis.

OBJECTIVE: To observe the protective effect of AC-YVAD-CMK on sepsis-induced acute kidney injury in mice and to explore its possible mechanisms primarily. METHODS: Eighteen male C57BL/6 mice were randomly divided into sham-operated group (Control), cecal ligation and puncture group (CLP), and CLP model treated with AC-YVAD-CMK group (AC-YVAD-CMK) (n = 6 in each group). Mice were sacrificed at 24 h after operation, and blood and kidney tissue samples were collected for analyses. Histologic changes were determined microscopically following HE staining. The expression of Ly-6B and CD68 was investigated using immunohistochemistry. Serum concentrations of creatinine (sCR) and blood urea nitrogen (BUN) were measured. Serum levels of interleukin-1ß (IL-1ß), interleukin-18 (IL-18), TNF-α, and interleukin-6 (IL-6) were determined by ELISA. The expressions of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues were investigated using Western blot. Immunofluorescence staining was used to detect the expression of GSDMD protein in renal tissues. RESULTS: AC-YVAD-CMK treatment significantly alleviates sepsis-induced acute kidney injury, with decreased histological injury in renal tissues, suppresses the accumulation of neutrophils and macrophages in renal tissues, and decreased sCR and BUN level (P < 0.05). Attenuation of sepsis-induced acute kidney injury was due to the prohibited production of inflammatory cytokines and decrease expression of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues. In addition, AC-YVAD-CMK treatment significantly reduced the expression of GSDMD in renal tissues compared to those observed in controls (P < 0.05). CONCLUSIONS: We demonstrated a marked renoprotective effect of caspase-1-inhibitor AC-YVAD-CMK in a rat model of sepsis by inhibition of pyroptosis.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease.

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

FURIN and placental syncytialisation: a cautionary tale.

FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.

The antiandrogenic effect of neferine, liensinine, and isoliensinine by inhibiting 5-α-reductase and androgen receptor expression via PI3K/AKT signaling pathway in prostate cancer.

Neferine, liensinine, and isoliensinine are bisbenzylisoquinoline alkaloids extracted from seed-embryos of Nelumbo nucifera Gaertn. In this study, we evaluated the anticancer activities and mechanism of action of these natural products in prostate cancer cells by MTT, wound healing, ELISA and Western blotting. Neferine, liensinine, and isoliensinine showed growth inhibition and displayed a significant anti-migration activity in prostate cancer cells. They induced apoptosis and autophagy by activating cleaved caspase-9, cleaved PAPR, Bax, LC3B-II, but decreased Bcl-2 and PARP protein expression in LNCaP cells 24 h after treatments. The apoptotic and cytotoxic effects of neferine, liensinine, and isoliensinine were significantly attenuated in the presence of the caspase inhibitor, Z-VAD-FMK. However, the effects were enhanced in the presence of Akt inhibitor (MK2206) and PI3K inhibitor (LY294002). Moreover, neferine, liensinine, and isoliensinine also downregulated the protein expression of androgen receptor, prostate-specific antigen, and type II 5-α-reductase. These results demonstrated that these bisbenzylisoquinoline alkaloids have the potential as promising therapeutics agents. They induced apoptosis via inactivation with the PI3K/AKT signal pathway.

Caspase-1 inhibition ameliorates murine acute graft versus host disease by modulating the Th1/Th17/Treg balance.

Our previous studies have implicated Caspase-1 signaling in driving the proinflammatory state of acute graft versus host disease (aGVHD). Therefore, we aimed to elucidate the mechanism of Caspase-1 in in murine models of aGVHD through specific inhibition of its activity with the decoy peptide Ac-YVAD-CMK. We transplanted bone marrow from donor C57BL/6 (H-2b) mice into recipient BALB/c (H-2Kd) mice and randomized the recipients into the following treatment cohorts: (1) allogeneic hematopoietic stem cell transplantation and splenic cell infusion control (PBS group); (2) low dose Ac-YVAD-CMK (AC low group); (3) and high dose Ac-YVAD-CMK (AC high group). Indeed, we observed that Caspase-1 inhibition by Ac-YVAD-CMK ameliorated pathological damage and inflammation in the liver, lungs, and colon elicited by aGVHD. This was associated with reduced mortality secondary to aGVHD. Mechanistically, we found that Caspase-1 inhibition modulated donor T cell expansion, restored the balance of Th1/Th17/Treg subsets, and markedly decreased serum levels and aGVHD target organ mRNA expression of IL-1ß, IL-18, and HMGB1. Thus, we demonstrate that inhibition of Caspase-1 by Ac-YVAD-CMK mitigates murine aGVHD by regulating Th1/Th17/Treg balance and attenuating its characteristic proinflammatory state.

Caspase-1/IL-1ß represses membrane transport of GluA1 by inhibiting the interaction between Stargazin and GluA1 in Alzheimer's disease.

BACKGROUND: Alzheimer's disease is a neurodegenerative disease. Previous study has reported that caspase-1/IL-1ß is closely associated with Alzheimer's disease. However, the biological role of caspase-1/IL-1ß in Alzheimer's disease has not been fully elucidated. This study aimed to explore the mechanism of action of caspase-1/IL-1ß in Alzheimer's disease. METHODS: Mouse hippocampal neurones were treated with Aß1-42 to induce Alzheimer's disease cell model. APP/PS1 mice and Aß1-42-induced hippocampal neurones were treated with AC-YVAD-CMK (caspase-1 inhibitor). Spatial learning and memory ability of mice were detected by morris water maze. Flow cytometry, TUNEL staining, Thioflavin S staining and immunohistochemistry were performed to examine apoptosis and senile plaque deposition. Enzyme linked immunosorbent assay and western blot were performed to assess the levels of protein or cytokines. Co-Immunoprecipitation was performed to verify the interaction between Stargazin and GluA1. RESULTS: AC-YVAD-CMK treatment improved spatial learning and memory ability and reduced senile plaque deposition of APP/PS1 mice. Moreover, AC-YVAD-CMK promoted membrane transport of GluA1 in APP/PS1 mice. In vitro, Aß1-42-induced hippocampal neurones exhibited an increase in apoptosis and a decrease in the membrane transport of GluA1, which was abolished by AC-YVAD-CMK treatment. In addition, Stargazin interacted with GluA1, which was repressed by caspase-1. Caspase-1/IL-1ß inhibited membrane transport of GluA1 by inhibiting the interaction between Stargazin and GluA1. CONCLUSIONS: Our data demonstrate that caspase-1/IL-1ß represses membrane transport of GluA1 by inhibiting the interaction between Stargazin in Alzheimer's disease. Thus, caspase-1/IL-1ß may be a target for Alzheimer's disease treatment.

BF175 inhibits endometrial carcinoma through SREBP-regulated metabolic pathways in vitro.

Elevated lipogenesis is an important metabolic hallmark of rapidly proliferating tumor such as endometrial carcinoma (EC). The sterol regulatory element-binding protein 1 (SREBP1) is a master regulator of lipogenesis and involved in EC proliferation. BF175 is a novel chemical inhibitor of SREBP pathway, and has shown potent anti-lipogenic effects. However, the effect of BF175 on EC cells are yet to be determined. In the present study, we found that BF175 decreased cell viability, colony formation and migratory capacity, inducing autophagy and mitochondrial related apoptosis in EC cell line AN3CA. Z-VAD-FMK partially attenuated the effect of BF175 on AN3CA. In addition, BF175 significantly downregulated SREBPs and their downstream genes. The levels of free fatty acids and total cholesterol were also inhibited. Microarray analysis suggested BF175 treatment obviously affected lipid metabolic pathways in EC. Taken together, we validated BF175 exhibited anti-tumor activity by targeting SREBP-dependent lipogenesis and inducing apoptosis which mitochondrial pathway involved in, suggesting that it's potential as a novel therapeutic reagent for EC.

Effect of simvastatin on cell proliferation and Ras activation in canine tumour cells.

Statins are inhibitors of the mevalonate cascade that is responsible for cholesterol biosynthesis and the formation of intermediate metabolites, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) used in the prenylation of proteins. Although statins are widely used in the treatment of hypercholesterolemia, recent studies suggest that they also inhibit proliferation of tumour cells by reducing prenylation of small GTP-binding proteins, such as, Ras. This study aimed to evaluate the effect of simvastatin on cell proliferation and Ras activation in various canine tumour cell lines, including hemangiosarcoma (HSA), melanoma, and lymphoma cell lines. Simvastatin inhibited cell proliferation of all cell lines tested in a concentration- and time-dependent manner, but the susceptibilities were different amongst the cell lines. Simvastatin induced apoptotic cell death via activation of caspase-3 and cell cycle arrest. The cytotoxic effects of simvastatin were attenuated by GGPP and FPP. Simvastatin decreased the amount of prenylated Ras and GTP-bound Ras in HSA and melanoma cell lines, but not in lymphoma cell lines. These results indicate that simvastatin induces cytotoxic effects through the depletion of GGPP and FPP in a variety of canine tumour cells, whereas multiple mechanisms are involved in the effects. Further study is required to elucidate the underlying mechanisms of simvastatin-induced cytotoxic effects in a variety of canine tumour cells.

Identification of two subtilisin-like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana.

The tobacco variant Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti-HIV-1 antibodies 2F5 and PG9 as well as the protease inhibitor α1 -antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin-like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals. DATABASES: NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005.

Proprotein convertases blockage up-regulates specifically metallothioneins coding genes in human colon cancer stem cells.

Despite continuous exertion made, colon cancer still represents a major health problem and its incidence continues being high worldwide. There is growing evidence in support of the cancer stem cells (CSCs) being central in the initiation of this cancer, and CSCs have been the focus of various studies for the identification of new ways of treatment. Lately, the proprotein convertases (PCs) were reported to regulate the maturation and expression of various molecules involved in the malignant phenotype of colon cancer cells, however, the identity of the molecules regulated by these serine proteases in CSCs is unknown. In this study, we used the general PCs inhibitor, the Decanoyl-RVKR-chloromethylketone (Decanoyl-RVKR-CMK) that inhibits all the PCs found in the secretory pathway, and analyzed its effect on CSCs using RNA-seq analysis. Remarkably, from the only 9 up-regulated genes in the human SW620-derived sphere-forming cells, we identified 7 of the 11 human metallothioneins, all of them localized on chromosome 16, and zinc related proteins as downstream effectors of the PCs. The importance of these molecules in the regulation of cell proliferation, differentiation and chemoresistance, and their reported potential tumor suppressor role and loss in colon cancer patients associated with worse prognosis, suggests that targeting PCs in the control of the malignant phenotype of CSCs is a new potential therapeutic strategy in colon cancer.