Formation Of Cytoplasmic Vacuoles Research Articles

Carvedilol induces pyroptosis through NLRP3-caspase1-ASC inflammasome by nuclear migration of NF-κB in prostate cancer models.

Pyroptosis is an inflammatory type of programmed cell death, and could overcome the drug-resistance induced by anti-apoptotic effect of cancers. Carvedilol (CVL), a β-adrenergic receptors antagonist, has shown anti-inflammatory response and anti-cancer effect. The aim of this study is to investigate whether pyroptosis can be activated by CVL in prostate cancer (PCa). Datasets were used to analyze the expressions of pyroptosis-related proteins. Intracellular morphological change, cell viability, LDH and Il-1β release by cells,, and Hoechst/PI staining were used to detect the occurrence of pyroptosis. Realtime-PCR, western blot, immunofluorescence, and immunohistochemistry (IHC) were used to investigate the expressions of pyroptosis-related proteins. Datasets analyze showed the expressions of NLRP3, Caspase 1, ASC and GSDMD were all decreased in PCa comparing with normal tissues, but without prognostic significance. CVL treatment weakened the viabilities of PCa cells. Cell morphology changing, cytoplasmic vacuole formation, membrane integrity loss, LDH and IL-1β release and PI positive cells increasing were observed. NLRP3, Caspase 1, ASC, GSDMD and N-GSDMD expressions were elevated after CVL treatment, accompanied by a tendency of NF-κB transferring into nucleus. In vivo, CVL inhibited the growth of subcutaneous transplanted tumor. IHC showed CVL increased the expressions of NLRP3, ASC, and GSDMD, and decreased the expression of Ki-67 in transplanted tumor tissues. This study demonstrated that CVL could induce pyroptosis in PCa cells through NLRP3-caspase1-ASC inflammasome by promoting nuclear translocation of NF-κB, which would lay a foundation for the application of adrenergic receptor antagonist in PCa.

Zearalenone Induces Blood-Testis Barrier Damage through Endoplasmic Reticulum Stress-Mediated Paraptosis of Sertoli Cells in Goats.

Zearalenone (ZEA) is present worldwide as a serious contaminant of food and feed and causes male reproductive toxicity. The implication of paraptosis, which is a nonclassical paradigm of cell death, is unclear in ZEA-induced male reproductive disorders. In this study, the toxic effects of ZEA on the blood-testis barrier (BTB) and the related mechanisms of paraptosis were detected in goats. ZEA exposure, in vivo, caused a significant decrease in spermatozoon quality, the destruction of seminiferous tubules, and damage to the BTB integrity. Furthermore, ZEA exposure to Sertoli cells (SCs) in vitro showed similar dysfunction in structure and barrier function. Importantly, the formation of massive cytoplasmic vacuoles in ZEA-treated SCs corresponded to the highly swollen and dilative endoplasmic reticulum (ER), and paraptosis inhibition significantly alleviated ZEA-induced SC death and vacuolization, which indicated the important contribution of paraptosis in ZEA-induced BTB damage. Meanwhile, the expression of ER stress marker proteins was increased after ZEA treatment but decreased under the inhibition of paraptosis. The vacuole formation and SC death, induced by ZEA, were remarkably blocked by ER stress inhibition. In conclusion, these results facilitate the exploration of the mechanisms of the SC paraptosis involved in ZEA-induced BTB damage in goats.

Ferritinophagy-mediated apoptosis and paraptosis induction involved MAPK and PI3K/AKT pathway in mechanism of an iron chelator

Melanoma cells were more resistant to ferroptosis with still poor therapy outcomes. Sensitizing melanoma cell to the ferroptosis inducer was a crucial strategy for treatment of melanoma. In the present study, 2,2′-di-pyridylketone hydrazone dithiocarbamate s-butyric acid (DpdtbA) displayed superior inhibitory activity than ferroptosis inducer Erastin in melanoma cells, which prompt us to explore the underlying mechanism. The analyses from flow cytometry and Western blot showed that the growth inhibition of DpdtbA against SK-MEL-28 and A375 cells involved apoptosis induction and G1 phase arrest. Surprisingly, the cytoplasmic vacuoles were found upon the treatment; transmission electron microscopy and endoplasmic reticulum (ER) staining revealed that the cytoplasmic vacuoles were in ER; while down-regulation of alix and requirement of protein synthesis suggested there was an occurrence of paraptosis. However, both NAC and 3-MA could significantly attenuate the cytoplasmic vacuolization and growth inhibition, hinting that both ROS and autophagy involved the paraptosis induction. The additional evidence revealed that there was an occurrence of continuous ferritinophagy, which was responsible for the ROS production. Downregulation of NCOA4 clearly attenuated the apoptosis and paraptosis induction. In addition, activation of MAPK involved regulation of paraptosis, but only ERK and JNK had role in the formation of cytoplasmic vacuoles and growth inhibition. Furthermore, a ROS dependent regulation of PI3K/AKT pathway was also involved. Taken together, our result firstly demonstrated that a continuous ferritinophagy contributed to the apoptosis and paraptosis induction, highlighting that the lysosomal labile iron pool had a crucial role in control of melanoma cell fate.

Species diversity of grasshoppers in Kamrup district of Assam and their management by aqueous extracts of <i>Azadirachta indica</i> A. Juss and <i>Aegle marmelos</i> L.

Grasshoppers pose a significant threat to agricultural productivity worldwide, causing substantial damage to various crops such as cereals, legumes, orchards, vegetables, grasslands, and forest plantations. This research aimed to study the species diversity of grasshoppers in five selected paddy field locations of Kamrup district, Assam, and assess the efficacy of aqueous extracts from the leaves of Azadirachta indica A. Juss (neem) and Aegle marmelos L. (bael) for controlling the most common grasshopper species, Acrida exaltata, found in the study locations. A total of 36 species of grasshoppers were collected and identified, out of which Acrididae was the most predominant grasshopper family, constituting 77.78% of the total collected species. The experimental results demonstrated abnormal changes in behaviour, including slower movement and increased inactivity among the aqueous extracts treated insects. Both the treated groups showed morphological and behavioural change with the more prominent effects observed from the neem-treated group. Haemolymph analysis revealed the presence of severe deformities in both treated groups, such as the spindle-shaped formation of cells, nuclear membrane disintegration, cell fusion and stacking, cytoplasmic vacuole formation, and cytoplasm degeneration. These findings demonstrate the pesticidal potential of neem and bael extracts against the Acrida exaltata grasshopper species, following its toxic impact on haematological, physiological, and morphological behaviour, suggesting the need for further research on their use as effective and environmentally friendly control measures against grasshopper pest infestations in densely vegetated and paddy fields in the region.

Osimertinib induces paraptosis and TRIP13 confers resistance in glioblastoma cells

The efficacy of osimertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor, has been evaluated in glioblastoma (GBM) through preclinical and clinical trials. However, the underlying mechanism of osimertinib-induced GBM cell death and the underlying resistance mechanism to osimertinib remains unclear. Here, we demonstrate that Osimertinib induces paraptosis in GBM cells, as evidenced by the formation of cytoplasmic vacuoles, accumulation of ubiquitinated proteins, and upregulation of endoplasmic reticulum (ER) stress markers like CHOP. Additionally, neither apoptosis nor autophagy was involved in the osimertinib-induced cell death. RNAseq analysis revealed ER stress was the most significantly downregulated pathway upon exposure to osimertinib. Consistently, pharmacologically targeting the PERK-eIF2α axis impaired osimertinib-induced paraptosis. Notably, we show that the expression of thyroid receptor-interacting protein 13 (TRIP13), an AAA+ATPase, alleviated osimertinib-triggered paraptosis, thus conferring resistance. Intriguingly, MK-2206, an AKT inhibitor, downregulated TRIP13 levels and synergized with Osimertinib to suppress TRIP13-induced high GBM cell growth in vitro and in vivo. Together, our findings reveal a novel mechanism of action associated with the anti-GBM effects of osimertinib involving ER stress-regulated paraptosis. Furthermore, we identify a TRIP13-driven resistance mechanism against Osimertinib in GBM and offer a combination strategy using MK-2206 to overcome such resistance.

NOD mouse dorsal root ganglia display morphological and gene expression defects before and during autoimmune diabetes development.

During the development of Autoimmune Diabetes (AD) an autoimmune attack against the Peripheral Nervous System occurs. To gain insight into this topic, analyses of Dorsal Root Ganglia (DRG) from Non-Obese Diabetic (NOD) mice were carried out. Histopathological analysis by electron and optical microscopy in DRG samples, and mRNA expression analyzes by the microarray technique in DRG and blood leukocyte samples from NOD and C57BL/6 mice were performed. The results showed the formation of cytoplasmic vacuoles in DRG cells early in life that could be related to a neurodegenerative process. In view of these results, mRNA expression analyses were conducted to determine the cause and/or the molecules involved in this suspected disorder. The results showed that DRG cells from NOD mice have alterations in the transcription of a wide range of genes, which explain the previously observed alterations. In addition, differences in the transcription genes in white blood cells were also detected. Taken together, these results indicate that functional defects are not only seen in beta cells but also in DRG in NOD mice. These results also indicate that these defects are not a consequence of the autoimmune process that takes place in NOD mice and suggest that they may be involved as triggers for its development.

Disulfiram oxy-derivatives induce entosis or paraptosis-like death in breast cancer MCF-7 cells depending on the duration of treatment

BackgroundDithiocarbamates and derivatives (including disulfiram, DSF) are currently investigated as antineoplastic agents. We have revealed earlier the ability of hydroxocobalamin (vitamin В12b) combined with diethyldithiocarbamate (DDC) to catalyze the formation of highly cytotoxic oxidized derivatives of DSF (DSFoxy, sulfones and sulfoxides). MethodsElectron and fluorescent confocal microscopy, molecular biology and conventional biochemical techniques were used to study the morphological and functional responses of MCF-7 human breast cancer cells to treatment with DDC and B12b alone or in combination. ResultsDDC induces unfolded protein response in MCF-7 cells. The combined use of DDC and B12b causes MCF-7 cell death. Electron microscopy revealed the separation of ER and nuclear membranes, leading to the formation of both cytoplasmic and perinuclear vacuoles, with many fibers inside. The process of vacuolization coincided with the appearance of ER stress markers, a marked damage to mitochondria, a significant inhibition of 20S proteasome, and actin depolimerization at later stages. Specific inhibitors of apoptosis, necroptosis, autophagy, and ferroptosis did not prevent cell death. A short- time (6-h) exposure to DSFoxy caused a significant increase in the number of entotic cells. ConclusionsThese observations indicate that MCF-7 cells treated with a mixture of DDC and B12b die by the mechanism of paraptosis. A short- time exposure to DSFoxy caused, along with paraptosis, a significant activation of the entosis and its final stage, lysosomal cell death. General significanceThe results obtained open up opportunities for the development of new approaches to induce non-apoptotic death of cancer cells by dithiocarbamates.

Structure-based discovery of receptor tyrosine kinase AXL degraders with excellent anti-tumor activity by selectively degrading AXL and inducing methuosis

The receptor tyrosine kinase (RTK) anexelekto (AXL) is mutated and/or overexpressed in various malignancies, and plays a central role in tumor development and acquired drug resistance. Although highly selective inhibitors have been developed in recent years, direct inhibition of AXL may block its ubiquitination, eventually leading to surface accumulation of the protein. Herein, we designed and synthesized a series of AXL degraders with high selectivity and without compensatory increase of AXL. In particular, compounds 20 and 22 showed significant AXL degradation capacity, which inhibited the proliferation and migration of cancer cells in vitro. In addition, these compounds induced the formation of cytoplasmic vacuoles and triggered methuosis, a new type of non-apoptotic cell death, by stimulating excessive production of macropinosomes. Vacuole formation was mediated via H-Ras activation, and was attenuated upon inhibition of its downstream regulatory factor Rac1. Furthermore, compound 20 inhibited the growth of tumor cell xenografts in vivo, and prolonged the survival of the tumor-bearing mice.

Synthesis and anticancer evaluation of copper(II)- and manganese(II)- theophylline mixed ligand complexes

Metals complexed to organic ligands have attracted much attention as potential treatments for cancer. In this paper we present the synthesis and anticancer activity of four metal complexes [Cu(theo)2(H2O)3]·2H2O (1), Cu(theo)2bpy(H2O)·4H2O (2), Cu(theo)2phen(H2O)·5H2O (3), and [Mn(theo)2(H2O)4] (4) (Theo = theophylline; Bpy = 1,2-bipyridine; Phen = 1,10-phenanthroline). The compounds were characterized by Single Crystal X-ray diffraction (SC-XRD), UV–Vis diffuse reflectance spectroscopy (UV–Vis-DRS), Fourier-transform infrared spectroscopy (FT-IR) and Thermogravimetry-Differential Scanning Calorimetry (TGA-DSC). Based on crystal data, copper(II) and manganese(II) complexes exhibited square pyramidal and octahedral configurations, respectively. The complexes were assessed for anticancer activity against a panel of cell lines, representing breast-, lung-, colorectal-, and pancreatic cancer, as well glioblastoma and neuroblastoma; cancers of the central nervous system. Cu(theo)2phen(H2O)·5H2O (complex 3) showed superior and broad spectrum antitumor activity compared to complexes 1, 2 and 4. The IC50 values of complex 3 ranged between 1.5 and 5 µM making it a promising lead compound. Complex 3 showed similar or better activity to doxorubicin on the panel of cell lines evaluated. An evaluation of the ligands, 1,2-theophylline and 1,10-phenanthroline showed that theophylline did not contribute to the cytotoxic effect of the complexes. In contrast, 1,10-phenanthroline showed poor cytotoxic activity at a concentration of 5 µM but caused significant cytotoxicity at 50 µM. Preliminary analysis of the effect of the complexes on cell morphology indicates the absence of necrotic cell death. Formation of cytoplasmic vacuoles, indicative of paraptosis was commonly observed. Complex 3 decreased the binding of Hoechst 33342 to some cells and changed the appearance of the nuclei. Molecular modelling of the complex supports the notion that the complex is able to bind to DNA. The formation of apoptotic nuclei was observed for all cell lines, whilst overt apoptosis was evident in the glioblastoma cell line. The data presented here indicates that complex 3 is worthy of further evaluation as a potential anticancer agent.

Cytotoxicities and wound healing effects of contact lens multipurpose solution on human corneal epithelial cell

ABSTRACT Clinical Relevance Contact lens multipurpose solutions (MPSs) contain several components that have the potential to cause corneal epithelial cell toxicity. Evaluating the components and the toxic effect of MPS should be considered for effective eye care. Background The cytotoxic and wound healing effects of five commercially available MPSs on human corneal epithelial cells (HCECs) are is investigated. Methods The following commercially available MPSs were used: Queen’s PLURISOL®, Frenz®, Boston SIMPLUS®, DL+PLUS EYE® (DL), and NEW YORK DEFINE® (NY). The proliferation of HCECs exposed to each MPS for 1, 6, and 24 h and the cytotoxicity of these solutions were analyzed using methyl thiazolyl tetrazolium-based colorimetric and lactate dehydrogenase leakage assays, respectively. The cellular morphology was evaluated by inverted phase-contrast and electron microscopy. A scratch-wound assay was performed to measure wound widths 24 h after confluent HCEC monolayers were scratch-wounded. Results The tested MPS had a time-dependent inhibitory effect on HCEC proliferation and cytotoxicity, significantly at 24 h after exposure (p< 0.05 in all MPSs). HCECs exposed to MPS detached from the bottom of the culture dishes, showed degenerative changes such as loss of microvilli, cytoplasmic vacuole formation and nuclear condensation, and decreased wound healing, compared to the controls (p< 0.001 in Boston, DL and NY). Among the tested MPS, DL and NY were more cytotoxic and showed less wound healing. Conclusion MPS has a toxic effect on HCECs, which is dependent on the concentration of the disinfecting component. Since the components that constitute the MPS are absorbed and retained in the lens, cautious scrutiny of the concentration and attention to lens cleaning are warranted to mitigate the related cytotoxicity.

Isolation of Bovine leukemia virus from cows with persistent lymphocytosis in Iraq

This is the first study to report on the isolation of bovine leukemia virus (BLV) from peripheral blood mononuclear cells of two cross bred cows in Iraq. The cattle were seropositive by ELISA when selected while being surveyed for the detection of BLV. Among six cows, two were cases of persistent lymphocytosis (PL). Cytopathology was characterized by the formation of multinucleated giant cells (syncytia) and cytoplasmic vacuoles. Moreover, the viruses produced clear plaques on the monolayer of the primary fetal calf kidney (FCK) cells. Inhibition of plaque formation by BLV-antisera suggested a diagnosis of BLV, which was further confirmed by PCR. Cells infected with the isolates were positive to a monoclonal antibody against the viral gp51 trans-membrane glycoprotein by immunocytochemistry. Both isolates replicated and induced cytopathic effects in bovine and human cell line cultures. Phylogenetic analysis based on partial gp51 env gene sequences revealed that Iraqi strain highly homogenous with Turkey strain (100%) and had 1% distance value with other world strains. In conclusion, this present study found that BLV-infected cattle with PL can be a source for viral isolation, and the cytopathological features of the virus infection are arranged and differ depending on the cell type. This is the first study to report on the isolation of the EBL virus in Iraq, and it provides the basis for further studies about a BLV Iraqi strain that can help control this disease.

1, 8-cineole attenuates cardiac hypertrophy in heart failure by inhibiting the miR-206-3p/SERP1 pathway

Background1,8-Cineole (1,8-CIN) is a monoterpene found in diverse dietary and medicinal herbs that has been reported to be effective against cardiovascular diseases. PurposeThe present research was designed to elucidate the treatment effects and the underlying mechanism of 1,8-CIN on heart failure (HF). MethodAn in vitro cardiac hypertrophy model and an in vivo heart failure (HF) model induced by isoprenaline (ISO) were established and treated with or without 1,8-CIN. In vitro miR-206-3p mimic or inhibitors were created. MiR-206-3p, SERP1 and related mRNAs or proteins were detected using qPCR or western blotting. Cell viability was tested by MTT assay, and apoptosis was measured using TUNEL assay, AO/EB assay and flow cytometry. Actin was stained with FITC-phalloidin. MiR-206-3p and related mRNAs or proteins in cardiac muscle tissues were measured using qPCR or western blotting, HE staining, Masson staining. ResultsISO subcutaneous injection increased cardiac hypertrophy, cytoplasmic vacuole formation, myofiber loss and fibrosis and decreased cardiomyocyte viability. 1,8-CIN treatment improved cardiomyocyte viability and reduced cardiac hypertrophy, cytoplasmic vacuole formation, myofibre loss and fibrosis. We found that 1,8-CIN attenuated apoptosis. We observed that expression of miR-206-3p was dramatically increased in ISO-exposed cardiomyocytes or ISO-treated rat hearts. MiR-206-3p was identified to target the 3’UTR of SERP1, resulting in the accumulation of un- or misfolded proteins, leading to endoplasmic reticulum (ER) stress. ConclusionThese results suggest that 1,8-CIN reduces the apoptosis induced by ER stress through inhibiting miR-206-3p, which inhibits the expression of SERP1.

Identification of EHMT2 As a New Target in T-Cell Acute Lymphoblastic Leukemia

▪Acute lymphoblastic leukemia (ALL) constitutes a family of genetically heterogeneous lymphoid neoplasms and is the most common cancer in childhood. T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive subtype of ALL and accounts for approximately 15-20% of all cases. Despite remarkable improvement in the prognosis of childhood ALL over the past few decades, many patients will require additional therapy for relapsed/refractory disease, and ALL remains the second leading cause of cancer-related death in children. Genomic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in ALL, suggesting emerging opportunities for novel therapeutic interventions.To identify new potential targets in T-ALL we tested a collection of inhibitors of epigenetic modifiers, including inhibitors of the histone acetyltransferase (HAT) p300 (CTPB and C646) and inhibitors of protein methyltransferases (PMTs), including targeting EZH2 (GSK126), DOT1L (SGC0946), and G9a (EHMT2)/G9a-like (GLP) (EHMT1) (UNC0642 and BIX01294). We demonstrated that the G9a/GLP inhibitors significantly impaired cell viability compared to other molecules as measured by the reduction of cellular ATP content.G9a/EHMT2 and GLP/EHMT1 are conserved protein lysine methyltransferases that contain a Su(Var), Enhancer of Zeste, Trithorax (SET) domain. G9a localizes in euchromatin regions and regulates gene expression and chromosome structure through de novo mono- and di- methylation of histone H3 lysine 9 (H3K9me1/2). In particular, di- and trimethylation of H3K9 in gene promoters have been associated with transcriptional repression. Despite recent evidence that G9a is involved in different tumor types, the role of the G9a/GLP complex has not been systematically studied in T-ALL.Because cancer driver genes and response to targeted therapy are frequently correlated with activating somatic mutations and/or expression levels we analyzed several transcriptional databases, including the Differentiation Map (DMAP), Cancer Cell Line Encyclopedia, and primary T-ALL datasets. Collectively, the results revealed that EHMT2 is a preferential marker of lymphoid differentiation and is highly expressed in T-ALL compared to other cancer subtypes or normal bone marrow cells. We next validated the effects of UNC0642 and extended testing to BRD4770, another selective G9a/GLP inhibitor. Competitive inhibition of G9a with both molecules impaired cell growth and triggered starvation-induced autophagic activity as shown by the detection of LC3-II by immunoblotting and formation of cytoplasmic autophagic vacuoles. Moreover, di-methylated histone H3 lysine 9 (H3K9me2) levels were decreased as measured by western blotting after histone extraction, supporting the notion that phenotypic changes upon small molecule treatment may be due to H3K9me2 loss and epigenetic reprogramming.To genetically validate G9a as a potential new target in T-ALL we intersected our chemical screening results with a previously published short hairpin RNA (shRNA) screen targeting nearly 350 chromatin regulator genes (Knoechel B. et al., Cancer Cell, 2014). EHMT2 scored among the top 20 hits that impaired the viability of T-ALL cells, further supporting our initial observation of the dependency of T-ALL on EHMT2. Because shRNA-based screens are limited by the occurrence of off-target effects and potentially the incomplete repression of target genes, we took a CRISPR/Cas9 approach and designed sgRNA guides targeting the SET domain of EHMT2. Consistently, genetic knockout of EHMT2 in T-ALL cell lines impaired cell growth and induced apoptosis measured by Annexin V/ Propidium iodide staining and quantified by flow cytometry.In conclusion, the intersection of multiple chemical, genetic, and genomic approaches led to the identification of G9a/EHMT2 as a druggable candidate in T-ALL. Our preliminary data strongly support the further investigation of EHMT2 in this aggressive subset of leukemia. DisclosuresStegmaier:Novartis: Consultancy, Research Funding.

Licarin-B Exhibits Activity Against the Toxoplasma gondii RH Strain by Damaging Mitochondria and Activating Autophagy.

Toxoplasma gondii is an obligate intracellular pathogen that infects warm-blooded animals and humans. However, side effects limit toxoplasmosis treatment, and new drugs with high efficiency and low toxicity need to be developed. Natural products found in plants have become a useful source of drugs for toxoplasmosis. In this study, twenty natural compounds were screened for anti-T. gondii activity by Giemsa staining or real-time fluorescence quantitative polymerase chain reaction (qPCR) in vitro. Among these, licarin-B from nutmeg exhibited excellent anti-T. gondii activity, inhibiting T. gondii invasion and proliferation in a dose-dependent manner, with an EC50 of 14.05 ± 3.96 μg/mL. In the in vivo, licarin-B treatment significantly reduced the parasite burden in tissues compared to no treatment, protected the 90% infected mice from to death at 50 mg/kg.bw. Flow cytometry analysis suggested a significant reduction in T. gondii survival after licarin-B treatment. Ultrastructural changes in T. gondii were observed by transmission electron microscopy (TEM), as licarin-B induced mitochondrial swelling and formation of cytoplasmic vacuoles, an autophagosome-like double-membrane structure and extensive clefts around the T. gondii nucleus. Furthermore, MitoTracker Red CMXRos, MDC, and DAPI staining showed that licarin-B promoted mitochondrial damage, autophagosome formation, and nuclear disintegration, which were consistent with the TEM observations. Together, these findings indicate that licarin-B is a promising anti-T. gondii agent that potentially functions by damaging mitochondria and activating autophagy, leading to T. gondii death.

RasGRP1 induces autophagy and transformation-associated changes in primary human keratinocytes

Ras mutations are present in only a subset of sporadic human cutaneous squamous cell carcinomas (cSCC) even though Ras is activated in most. This suggests that other mechanisms of Ras activation play a role in the disease. The aberrant expression of RasGRP1, a guanyl nucleotide exchange factor for Ras, is critical for mouse cSCC development through its ability to increase Ras activity. However, the role of RasGRP1 in human keratinocyte carcinogenesis remains unknown. Here we report that RasGRP1 is significantly elevated in human cSCC and that high RasGRP1 expression in human primary keratinocytes triggered activation of endogenous Ras and significant morphological changes including cytoplasmic vacuole formation and growth arrest. Moreover, RasGRP1-expressing cells were autophagic as indicated by LC3-II increase and the formation of LC3 punctae. In an in vitro organotypic skin model, wild type keratinocytes generated a well-stratified epithelium, while RasGRP1-expressing cells failed to do so. Finally, RasGRP1 induced transformation-like changes in skin cells from Li-Fraumeni patients with inactivating p53 mutations, demonstrating the oncogenic potential of this protein. These results support a role for RasGRP1 in human epidermal keratinocyte carcinogenesis and might serve as an important new therapeutic target.

LZ-106, a potent lysosomotropic agent, causing TFEB-dependent cytoplasmic vacuolization.

Cytoplasmic vacuolization usually occurs in cells treated with different agents and substances. We found that LZ-106, an analog of enoxacin, is a potent lysosomotropic agent, contributing to the formation of cytoplasmic vacuoles in cells. Studies of LZ-106-induced vacuolization in H460 cells showed acid environment inside these vacuoles. Further study demonstrated that markers in the late endosomes and lysosomes, like LAMP1 and RAB7, on the surface of the vacuoles, implying that these vacuoles might derive from endosomes and/or lysosomes. By studying the fluorescence intensity of LZ-106, we discovered that LZ-106 tended to locate in acid organelles, and Bafilomycin A1, a V-ATPase inhibitor, was able to suppress its acid organelles localization. Also, we noticed that LZ-106 could induce lysosome stress, involving pH increment and lysosomal membrane damage. Moreover, the expression levels of some lysosome-related proteins, like LAMP1, EEA1, and Cathepsin B, were also altered upon LZ-106 treatment. At last, we confirmed LZ-106 can activate TFEB, a key regulator of lysosomes. Knockdown of TFEB could also reverse LZ-106's effect on vacuolization in H460 cells. Taken together, due to LZ-106's lysosomotropic properties, it is able to accumulate in the acid organelles and induce lysosomal dysfunction in H460 cells, leading to TFEB activation and the following cytoplasmic vacuolization.

Abemaciclib induces atypical cell death in cancer cells characterized by formation of cytoplasmic vacuoles derived from lysosomes.

In the cell cycle, the G1/S transition is controlled by the cyclin‐dependent kinase (CDK) 4/6‐cyclin D complex. Constitutive activation of CDK4/6 dysregulates G1/S transition, leading to oncogenic transformation. We found that 3 CDK4/6 inhibitors, abemaciclib, ribociclib, and palbociclib, exerted a cytocidal effect as well as a cytostatic effect at the G1 phase in cancer cell lines, including A549 human non–small cell lung cancer cells. Among these inhibitors, abemaciclib exhibited the most potent cytotoxic effect. The cell‐death phenotype induced by abemaciclib, which entailed formation of multiple cytoplasmic vacuoles, was not consistent with apoptosis or necroptosis. Abemaciclib blocked autophagic flux, resulting in accumulation of autophagosomes, however vacuole formation and cell death induced by abemaciclib were independent of autophagy. In addition, methuosis, a cell‐death phenotype characterized by vacuole formation induced by excessive macropinocytosis, was excluded because the vacuoles did not incorporate fluorescent dextran. Of note, both formation of vacuoles and induction of cell death in response to abemaciclib were inhibited by vacuolar‐type ATPase (V‐ATPase) inhibitors such as bafilomycin A1 and concanamycin A. Live‐cell imaging revealed that the abemaciclib‐induced vacuoles were derived from lysosomes that expanded following acidification. Transmission electron microscopy revealed that these vacuoles contained undigested debris and remnants of organelles. Cycloheximide chase assay revealed that lysosomal turnover was blocked by abemaciclib. Furthermore, mTORC1 inhibition along with partial lysosomal membrane permeabilization occurred after abemaciclib treatment. Together, these results indicate that, in cancer cells, abemaciclib induces a unique form of cell death accompanied by swollen and dysfunctional lysosomes.

Exploring Modes of Photokilling by Hypericin.

Effects of photodynamic therapy (PDT) using the anthraquinone hypericin were explored with OVCAR-5 cells in vitro. Irradiation resulted in ER>lysosomal photodamage. Paraptosis was identified as a primary death pathway resulting from ER perturbation. This is characterized by an extensive pattern of cytoplasmic vacuole formation. As the PDT dose increased, apoptotic death was also detected. The cytoprotective effect of autophagy, observed when certain other subcellular sites are PDT targets, appears to be absent. These results, together with prior evidence that paraptosis can be lethal to cells with an impaired apoptotic pathway, suggest a role for agents with this targeting profile in photodynamic therapy. A limitation to be overcome for hypericin is a suboptimal absorbance profile.

DCZ5248, a novel dual inhibitor of Hsp90 and autophagy, exerts antitumor activity against colon cancer.

Hsp90 is a potential therapeutic target for tumor, as it maintains the stability of a variety of proteins related to tumor development and progression. Autophagy is a self-degradation process to maintain cellular homeostasis and autophagy inhibitors can suppress tumor growth. In this study, we identified DCZ5248, a triazine derivative, was a dual inhibitor of both Hsp90 and late-autophagy with potent antitumor activity against colon cancer cells in vitro and in vivo. We showed that DCZ5248 (0.1-10 μM) induced dose-dependent degradation of Hsp90 client proteins (AKT, CDK4, CDK6 and RAF-1) in HCT 116 colon cancer cells through a proteasome-dependent pathway. Meanwhile, DCZ5248 (0.3 μM) induced cytoplasmic vacuole formation, LC3 II conversion, p62 protein upregulation, and inhibited autophagy at the late stage in the colon cancer cell lines tested. We further revealed that the inhibition of autophagy was achieved by impairing lysosomal functions through induction of lysosomal acidification and attenuation of lysosomal cathepsin activity. The modulation of autophagy by DCZ5248 was independent of Hsp90 inhibition as the autophagy inhibition was not blocked by Hsp90 knockdown. Importantly, inhibition of both Hsp90 function and autophagy by DCZ5248 induced G1-phase cell cycle arrest, apoptosis, and exerted potent antitumor activity against colon cancer cells both in vitro and in vivo. These findings demonstrate that DCZ5248 is a novel dual inhibitor of Hsp90 and autophagy with potential for colon cancer therapy.

Synthesis and Photodynamic Activity of Vitamin-Chlorin Conjugates at Nanomolar Concentrations against Prostate Cancer Cells.

Phototoxicity response of synthesized vitamin–chlorin conjugates and their zinc and indium complexes was determined in the human PC-3 prostate cancer cell line, which was previously demonstrated to overexpress vitamin receptors on the cell surface. Pantothenic acid (Vit B5) and lipoic acid (or thioctic acid) were covalently linked to methyl pheophorbide (a chlorophyll derivative) and subsequently metallated with zinc and indium. Cell survival assay indicated that the vitamin–chlorin conjugates have better photodynamic activity against the PC-3 prostate cancer line at the nanomolar concentration range than the commercially available starting precursor methyl pheophorbide. Fluorescence and transmission electron microscopy studies indicated some formation of apoptotic cells and cytoplasmic vacuoles of photosensitized prostatic cells. Targeting vitamin receptors in prostatic cancer cells can be utilized to enhance specificity of photosensitizers for photodynamic therapy applications.