Post-Transcriptional Regulation of Rab7a in Lysosomal Positioning and Drug Resistance in Nutrient-Limited Cancer Cells.

Limited nutrient availability in the tumor microenvironment can cause the rewiring of signaling and metabolic networks to confer cancer cells with survival advantages. We show here that the limitation of glucose, glutamine and serum from the culture medium resulted in the survival of a population of cancer cells with high viability and capacity to form tumors in vivo. These cells also displayed a remarkable increase in the abundance and size of lysosomes. Moreover, lysosomes were located mainly in the perinuclear region in nutrient-limited cells; this translocation was mediated by a rapid post-transcriptional increase in the key endolysosomal trafficking protein Rab7a. The acidic lysosomes in nutrient-limited cells could trap weakly basic drugs such as doxorubicin, mediating resistance of the cells to the drug, which could be partially reversed with the lysosomal inhibitor bafilomycin A1. An in vivo chorioallantoic membrane (CAM) assay indicated a remarkable decrease in microtumor volume when nutrient-limited cells were treated with 5-Fluorouracil (5-FU) and bafilomycin A1 compared to cells treated with either agent alone. Overall, our data indicate the activation of complementary pathways with nutrient limitation that can enable cancer cells to survive, proliferate and acquire drug resistance.

Glutamine withdrawal leads to the preferential activation of lipid metabolism in metastatic colorectal cancer.

INTRODUCTION: Glutamine is a non-essential amino acid that is critical for cell growth. However, the differential metabolism of l-glutamine in metastatic versus primary colorectal cancer (CRC) has not been evaluated adequately. MATERIALS AND METHODS: Differential expression of glutamine-related genes was determined in primary versus metastatic CRC. Univariate Cox regression and hierarchical clustering were used to generate a gene signature for prognostication. Untargeted metabolomics and 18O based fluxomics were used to identify differential metabolite levels and energy turnover in the paired primary (SW480) and metastatic (SW620) CRC cells. Western blot and qRT-PCR were used to validate differential gene expression. Subcellular localization of E-cadherin was determined by immunocytochemistry. Lipid droplets were visualized with Nile Red. RESULTS: The GO term "Glutamine metabolism" was significantly enriched in metastatic versus primary tumors. Supporting this, SW620 cells showed decreased membrane localization of E-cadherin and increased motility upon l-Glutamine withdrawal. A glutamine related signature associated with worse prognosis was identified and validated in multiple datasets. A fluxomics assay revealed a slower TCA cycle in SW480 and SW620 cells upon l-Glutamine withdrawal. SW620 cells, however, could maintain high ATP levels. Untargeted metabolomics indicated the preferential metabolism of fatty acids in SW620 but not SW480 cells. Lipids were mainly obtained from the environment rather than by de novo synthesis. CONCLUSIONS: Metastatic CRC cells can display aberrant glutamine metabolism. We show for the first time that upon l-glutamine withdrawal, SW620 (but not SW480) cells were metabolically plastic and could metabolize lipids for survival and cellular motility.

The Chorioallantoic Membrane (CAM) Assay for the Analysis of Starvation-Induced Autophagy.

During avian development, the chorioallantoic membrane (CAM) is generated around 4 days after fertilization following the fusion of the allantois and the chorion. The CAM develops rapidly over the next several days and gets heavily vascularized and therefore has been explored widely as a tool for the study of angiogenesis. Additionally, being immunodeficient, the CAM can be used for tumor growth of human origin and its metastasis. Of note, the CAM assay is minimally invasive for the chicken embryo and lacks innervation, which gives this in vivo model a low ethical burden. Here, we describe the protocol for the generation of microtumors from human colorectal cancer cell lines on the CAM, incubated in a nutrient-deficient medium for the activation of autophagy. We show that pre-inoculation markers of autophagy induced through nutrient deficiency are retained in the microtumors generated on the CAM.

A partial epithelial-mesenchymal transition signature for highly aggressive colorectal cancer cells that survive under nutrient restriction.

Partial epithelial-mesenchymal transition (p-EMT) has recently been identified as a hybrid state consisting of cells with both epithelial and mesenchymal characteristics and is associated with the migration, metastasis, and chemoresistance of cancer cells. Here, we describe the induction of p-EMT in starved colorectal cancer (CRC) cells and identify a p-EMT gene signature that can predict prognosis. Functional characterisation of starvation-induced p-EMT in HCT116, DLD1, and HT29 cells showed changes in proliferation, morphology, and drug sensitivity, supported by in vivo studies using the chorioallantoic membrane model. An EMT-specific quantitative polymerase chain reaction (qPCR) array was used to screen for deregulated genes, leading to the establishment of an in silico gene signature that was correlated with poor disease-free survival in CRC patients along with the CRC consensus molecular subtype CMS4. Among the significantly deregulated p-EMT genes, a triple-gene signature consisting of SERPINE1, SOX10, and epidermal growth factor receptor (EGFR) was identified. Starvation-induced p-EMT was characterised by increased migratory potential and chemoresistance, as well as E-cadherin processing and internalisation. Both gene signature and E-cadherin alterations could be reversed by the proteasomal inhibitor MG132. Spatially resolving EGFR expression with high-resolution immunofluorescence imaging identified a proliferation stop in starved CRC cells caused by EGFR internalisation. In conclusion, we have gained insight into a previously undiscovered EMT mechanism that may become relevant when tumour cells are under nutrient stress, as seen in early stages of metastasis. Targeting this process of tumour cell dissemination might help to prevent EMT and overcome drug resistance. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Lysosomal alkalinization in nutrient restricted cancer cells activates cytoskeletal rearrangement to enhance partial epithelial to mesenchymal transition.

INTRODUCTION: Nutrient restriction in cancer cells can activate a number of stress response pathways for cell survival. We aimed to determine mechanistically how nutrient depletion in colorectal cancer (CRC) cells leads to cellular adaptation. MATERIALS AND METHODS: Cell survival under nutrient depletion (ND) was evaluated by colony formation and in vivo tumor formation assays. Lysosomes are activated with ND; therefore, we incubated the ND cells with the V-ATPase inhibitor Bafilomycin A1 (ND+Baf). The expression of epithelial and mesenchymal markers with ND+Baf was determined by RNA sequencing and RT-qPCR while motility was determined with an in vivo Chorioallantoic membrane (CAM) assay. Reorganization of cytoskeletal network and lysosomal positioning was determined by immunocytochemistry. RESULTS: 4 different colorectal cancer (CRC) cell lines under ND showed high viability, tumor forming ability and increased expression of one or more epithelial and mesenchymal markers, suggesting the activation of partial (p)-EMT. We observed a further increase in p-EMT markers, numerous membrane protrusions, decreased cell-cell adhesion in 3D, and increased motility in ND+Baf cells. The protrusions in the ND+Baf cells were primarily mediated by microtubules and enabled the relocalization of lysosomes from the perinuclear region to the periphery. CONCLUSIONS: ND activated p-EMT in CRC cells, which was exacerbated by lysosomal alkalinization. The ND+Baf cells also showed numerous protrusions containing lysosomes, which may lead to lysosomal exocytosis and enhanced motility.

The indispensability of macrophage adaptor proteins in chronic inflammatory diseases.

Adaptor proteins represent key signalling molecules involved in regulating immune responses. The host's innate immune system recognizes pathogens via various surface and intracellular receptors. Adaptor molecules are centrally involved in different receptor-mediated signalling pathways, acting as bridges between the receptors and other molecules. The presence of adaptors in major signalling pathways involved in the pathogenesis of various chronic inflammatory diseases has drawn attention toward the role of these proteins in such diseases. In this review, we summarize the importance and roles of different adaptor molecules in macrophage-mediated signalling in various chronic disease states. We highlight the mechanistic roles of adaptors and how they are involved in protein-protein interactions (PPI) via different domains to carry out signalling. Hence, we also provide insights into how targeting these adaptor proteins can be a good therapeutic strategy against various chronic inflammatory diseases.

ATF2 loss promotes tumor invasion in colorectal cancer cells via upregulation of cancer driver TROP2.

In cancer, the activating transcription factor 2 (ATF2) has pleiotropic functions in cellular responses to growth stimuli, damage, or inflammation. Due to only limited studies, the significance of ATF2 in colorectal cancer (CRC) is not well understood. We report that low ATF2 levels correlated with worse prognosis and tumor aggressiveness in CRC patients. NanoString gene expression and ChIP analysis confirmed trophoblast cell surface antigen 2 (TROP2) as a novel inhibitory ATF2 target gene. This inverse correlation was further observed in primary human tumor tissues. Immunostainings revealed that high intratumoral heterogeneity for ATF2 and TROP2 expression was sustained also in liver metastasis. Mechanistically, our in vitro data of CRISPR/Cas9-generated ATF2 knockout (KO) clones revealed that high TROP2 levels were critical for cell de-adhesion and increased cell migration without triggering EMT. TROP2 was enriched in filopodia and displaced Paxillin from adherens junctions. In vivo imaging, micro-computer tomography, and immunostainings verified that an ATF2KO/TROP2high status triggered tumor invasiveness in in vivo mouse and chicken xenograft models. In silico analysis provided direct support that ATF2low/TROP2high expression status defined high-risk CRC patients. Finally, our data demonstrate that ATF2 acts as a tumor suppressor by inhibiting the cancer driver TROP2. Therapeutic TROP2 targeting might prevent particularly the first steps in metastasis, i.e., the de-adhesion and invasion of colon cancer cells.

Structural and functional evaluation mammalian and plant lipoxygenases upon association with nanodics as membrane mimetics.

Lipoxygenases (LOX) are a family lipid oxygenating enzymes that can generate bioactive lipids of clinical relevance from polyunsaturated fatty acids. Most LOXs display a Ca2+-dependent association with membranes for their activity. Nanodiscs (ND) are stable self-assembled discoidal fragments of lipid bilayers that can mimic the plasma membrane. In this study, we evaluated the association of mammalian 15-LOXs (ALOX15 and ALOX15B) and soybean LOX-1 with NDs (LOX-ND), their enzymatic activities and inhibition. Mammalian LOXs associated with NDs showed better retention of enzymatic function compared to soybean LOX-1. Treatment of both LOX-NDs and free enzymes with the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA) showed an approximately 5-fold more effective inhibition of the enzymes associated with NDs compared to the free form. NDs are easy to generate membrane mimics that can be used as an effective tool to determine enzymatic function and inhibition of membrane associated proteins.

Development of ACE2 loaded decoy liposomes and their effect on SARS-CoV-2 for Covid-19 treatment.

SARS-CoV-2 is a novel coronavirus with a positively oriented single-stranded RNA that first appeared in December 2019. In this study, Angiotensin Converting Enzyme 2 (ACE2) loaded decoy liposomes were developed and characterized. ACE2 protein was loaded onto a liposomal carrier system and its toxicity and effectiveness were evaluated in cell culture and in vitro virus neutralization studies. Liposomes were prepared with the film hydration method and adjusted for size with the dialysis membrane method or the ultrasonic homogenization method. All formulations showed high entrapment efficiency between 99.98-79.6%. Liposomes with two different particle sizes above 2 µm and below 500 nm were obtained with the dialysis membrane method and homogenization method. Two optimum formulations, M6-S90 with a PDI value of 0.383 ± 0.053 and particle size of 397.7 ± 28.25 nm which was produced by ultrasonic homogenization and M6-4 with a PDI 0.769 ± 0.205 and particle size of 2606 ± 1396.00 were chosen as optimum formulations for further studies. M6-S90 was stable and showed low toxicity on Calu3 lung epithelial cells. Pre-incubation of M6-S90 with with 3.1 × 105 PFU/mL of SARS-CoV-2 followed by incubation with Vero E6 cells resulted in a 4 log fold change reduction in cell death compared to virus alone. This suggests that MS6-S90 had good neutralization activity on SARS-CoV-2 whilst maintaining viability of the host cell. The novel ACE-2 loaded decoy liposomes described in this study can be further evaluated for the treatment of COVID-19.

Nutritional and immunization status of under-five children of India and Bangladesh.

BACKGROUND: The nutritional and immunization status of children can play an important role in determining their future health status of a particular country. The aim of the present study is to investigate the nutritional and immunization status of under-five children in India and Bangladesh, and to find the difference in the status between these two countries. METHODS: We have used the National Family Health Survey data, 2015-2016 of India and Bangladesh Demographic Health Survey, 2017-2018 datasets. The sample sizes are 222,418, among them 8759 and 8759 children for India and Bangladesh respectively. The nutritional status of under-five children is measured by standard anthropometric indicators of height-for-age (HAZ) and weight-for-age (WAZ). Regarding child immunization status, only BCG, DPT, polio and measles vaccinations are considered for the present study. Multiple binary logistic model has been used for analysing the data. RESULTS: This study reveals that the prevalence of stunting and underweight of under-five children in India are higher than Bangladeshi children. Secondary and higher educated mothers are more likely of having normal HAZ and WAZ children than up to primary educated mothers for both countries. Chances of having normal HAZ and WAZ are higher among non-poor category for both countries. The present study also shows that immunization status of Bangladeshi children is better than Indian children except measles. Religion of mother also shows influence on immunization status of children in India whereas Bangladesh shows no significant results regarding religion. Mother's educational attainment and wealth index show influence on immunization status among children for both countries. CONCLUSIONS: The study concludes that a remarkable number of under-five children are suffering from under nutrition for both countries, however Bangladeshi children have better nutritional and immunization status compared to Indian children. Higher wealth index, better educational attainment and lower unemployment of Bangladeshi mothers may be the causes for better nutritional and immunization status of children. Mother's socio-economic factors have significant impact on determining the child's health status. Our findings can help to government of Indian and Bangladesh for taking health policy to improve under-five children nutritional and immunization status.

A pan-cancer transcriptomic study showing tumor specific alterations in central metabolism.

Recently, there has been a resurgence of interest in metabolic rewiring of tumors to identify clinically relevant genes. However, most of these studies have had either focused on individual tumors, or are too general, providing a broad outlook on overall changes. In this study, we have first curated an extensive list of genes encoding metabolic enzymes and metabolite transporters relevant to carbohydrate, fatty acid and amino acid oxidation and biosynthesis. Next, we have used publicly available transcriptomic data for 20 different tumor types from The Cancer Genome Atlas Network (TCGA) and focused on differential expression of these genes between tumor and adjacent normal tissue. Our study revealed major transcriptional alterations in genes that are involved in central metabolism. Most tumors exhibit upregulation in carbohydrate and amino acid transporters, increased glycolysis and pentose phosphate pathway, and decreased fatty acid and amino acid oxidation. On the other hand, the expression of genes of the tricarboxylic acid cycle, anaplerotic reactions and electron transport chain differed between tumors. Although most transcriptomic alterations were conserved across many tumor types suggesting the initiation of common regulatory programs, expression changes unique to specific tumors were also identified, which can provide gene expression fingerprints as potential biomarkers or drug targets. Our study also emphasizes the value of transcriptomic data in the deeper understanding of metabolic changes in diseases.

A Novel Therapeutic Peptide Blocks SARS-CoV-2 Spike Protein Binding with Host Cell ACE2 Receptor.

BACKGROUND AND OBJECTIVE: Coronavirus disease 2019 is a novel disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 virus. It was first detected in December 2019 and has since been declared a pandemic causing millions of deaths worldwide. Therefore, there is an urgent need to develop effective therapeutics against coronavirus disease 2019. A critical step in the crosstalk between the virus and the host cell is the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to the peptidase domain of the angiotensin-converting enzyme 2 (ACE2) receptor present on the surface of host cells. METHODS: An in silico approach was employed to design a 13-amino acid peptide inhibitor (13AApi) against the RBD of the SARS-CoV-2 spike protein. Its binding specificity for RBD was confirmed by molecular docking using pyDockWEB, ClusPro 2.0, and HDOCK web servers. The stability of 13AApi and the SARS-CoV-2 spike protein complex was determined by molecular dynamics simulation using the GROMACS program while the physicochemical and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of 13AApi were determined using the ExPASy tool and pkCSM server. Finally, in vitro validation of the inhibitory activity of 13AApi against the spike protein was performed by an enzyme-linked immunosorbent assay. RESULTS: In silico analyses indicated that the 13AApi could bind to the RBD of the SARS-CoV-2 spike protein at the ACE2 binding site with high affinity. In vitro experiments validated the in silico findings, showing that 13AApi could significantly block the RBD of the SARS-CoV-2 spike protein. CONCLUSIONS: Blockage of binding of the SARS-CoV-2 spike protein with ACE2 in the presence of the 13AApi may prevent virus entry into host cells. Therefore, the 13AApi can be utilized as a promising therapeutic agent to combat coronavirus disease 2019.

Aldo Keto Reductases AKR1B1 and AKR1B10 in Cancer: Molecular Mechanisms and Signaling Networks.

Deregulation of metabolic pathways has increasingly been appreciated as a major driver of cancer in recent years. The principal cancer-associated alterations in metabolism include abnormal uptake of glucose and amino acids and the preferential use of metabolic pathways for the production of biomass and nicotinamide adenine dinucleotide phosphate (NADPH). Aldo-keto reductases (AKRs) are NADPH dependent cytosolic enzymes that can catalyze the reduction of carbonyl groups to primary and secondary alcohols using electrons from NADPH. Aldose reductase, also known as AKR1B1, catalyzes the conversion of excess glucose to sorbitol and has been studied extensively for its role in a number of diabetic pathologies. In recent years, however, high expression of the AKR1B and AKR1C family of enzymes has been strongly associated with worse outcomes in different cancer types. This review provides an overview of the catalysis-dependent and independent data emerging on the molecular mechanisms of the functions of AKRBs in different tumor models with an emphasis of the role of these enzymes in chemoresistance, inflammation, oxidative stress and epithelial-to-mesenchymal transition.

Evaluation of an aldo-keto reductase gene signature with prognostic significance in colon cancer via activation of epithelial to mesenchymal transition and the p70S6K pathway.

AKR1B1 and AKR1B10, members of the aldo-keto reductase family of enzymes that participate in the polyol pathway of aldehyde metabolism, are aberrantly expressed in colon cancer. We previously showed that high expression of AKR1B1 (AKR1B1HIGH) was associated with enhanced motility, inflammation and poor clinical outcome in colon cancer patients. Using publicly available datasets and ex vivo gene expression analysis (n = 51, Ankara cohort), we have validated our previous in silico finding that AKR1B1HIGH was associated with worse overall survival (OS) compared with patients with low expression of AKR1B1 (AKR1B1LOW) samples. A combined signature of AKR1B1HIGH and AKR1B10LOW was significantly associated with worse recurrence-free survival (RFS) in microsatellite stable (MSS) patients and in patients with distal colon tumors as well as a higher mesenchymal signature when compared with AKR1B1LOW/AKR1B10HIGH tumors. When the patients were stratified according to consensus molecular subtypes (CMS), AKR1B1HIGH/AKR1B10LOW samples were primarily classified as CMS4 with predominantly mesenchymal characteristics while AKR1B1LOW/AKR1B10HIGH samples were primarily classified as CMS3 which is associated with metabolic deregulation. Reverse Phase Protein Array carried out using protein samples from the Ankara cohort indicated that AKR1B1HIGH/AKR1B10LOW tumors showed aberrant activation of metabolic pathways. Western blot analysis of AKR1B1HIGH/AKR1B10LOW colon cancer cell lines also suggested aberrant activation of nutrient-sensing pathways. Collectively, our data suggest that the AKR1B1HIGH/AKR1B10LOW signature may be predictive of poor prognosis, aberrant activation of metabolic pathways, and can be considered as a novel biomarker for colon cancer prognostication.

Tumor-derived exosomes in the regulation of macrophage polarization.

BACKGROUND: This review focuses on exosomes derived from various cancer cells. The review discusses the possibility of differentiating macrophages in alternatively activated anti-inflammatory pro-tumorigenic M2 macrophage phenotypes and classically activated pro-inflammatory, anti-tumorigenic M1 macrophage phenotypes in the tumor microenvironment (TME). The review is divided into two main parts, as follows: (1) role of exosomes in alternatively activating M2-like macrophages-breast cancer-derived exosomes, hepatocellular carcinoma (HCC) cell-derived exosomes, lung cancer-derived exosomes, prostate cancer-derived exosomes, Oral squamous cell carcinoma (OSCC)-derived exosomes, epithelial ovarian cancer (EOC)-derived exosomes, Glioblastoma (GBM) cell-derived exosomes, and colorectal cancer-derived exosomes, (2) role of exosomes in classically activating M1-like macrophages, oral squamous cell carcinoma-derived exosomes, breast cancer-derived exosomes, Pancreatic-cancer derived modified exosomes, and colorectal cancer-derived exosomes, and (3) exosomes and antibody-dependent cellular cytotoxicity (ADCC). This review addresses the following subjects: (1) crosstalk between cancer-derived exosomes and recipient macrophages, (2) the role of cancer-derived exosome payload(s) in modulating macrophage fate of differentiation, and (3) intracellular signaling mechanisms in macrophages regarding the exosome's payload(s) upon its uptake and regulation of the TME. EVIDENCE: Under the electron microscope, nanoscale exosomes appear as specialized membranous vesicles that emerge from the endocytic cellular compartments. Exosomes harbor proteins, growth factors, cytokines, lipids, miRNA, mRNA, and DNAs. Exosomes are released by many cell types, including reticulocytes, dendritic cells, B-lymphocytes, platelets, mast cells, and tumor cells. It is becoming clear that exosomes can impinge upon signal transduction pathways, serve as a mediator of signaling crosstalk, thereby regulating cell-to-cell wireless communications. CONCLUSION: Based on the vesicular cargo, the molecular constituents, the exosomes have the potential to change the fate of macrophage phenotypes, either M1, classically activated macrophages, or M2, alternatively activated macrophages. In this review, we discuss and describe the ability of tumor-derived exosomes in the mechanism of macrophage activation and polarization.

Low-dose curcumin reduced TNBS-associated mucin depleted foci in mice by scavenging superoxide anion and lipid peroxides, rebalancing matrix NO synthase and aconitase activities, and recoupling mitochondria.

BACKGROUND: The role of mitochondrial dysfunction in the pathogenesis of inflammatory bowel diseases (IBD) is still being investigated. This study evaluated the therapeutic effect of curcumin (Cur), a polyphenolic electrophile in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis and mitochondrial dysfunction, in mice. METHODS: Colitis was induced by rectal instillation to mice of 30 mg kg-1 TNBS, alone or followed by daily intraperitoneal injections of Cur 25 mg kg-1. Animals were euthanized at days 3, 7, and 14, post TNBS challenge. Colon mitochondria of control mice were treated with 5 µM Cur, and TNBS (50, 100 µM)-toxicity was evaluated by measuring swelling, respiration, and aconitase and fumarase activities. Redox status was evaluated in colon mucosa and in mitochondria. RESULTS: In vitro, a short-term Cur treatment controlled the dose and time dependent mitochondrial toxicity induced by TNBS, by collapsing the generation of superoxide anion and hydroperoxy lipids, rebalancing nitric oxide synthase and aconitase activities, and recoupling mitochondria. In vivo, a daily low-dose Cur abolished mice mortality which reached 27% in model group. Cur improved in a time dependent manner mucosal redox homeostasis, cell apoptosis, mucin depleted crypts and crypt abscesses by controlling prooxidant activity of myeloperoxidase and NO synthase associated to phagocytes influx, quenching hydroperoxy lipids, and reboosting GSH levels. CONCLUSION: Cur, by quenching intra and extra mitochondrial ROS generation, rebalancing aconitase/fumarase and MDA/GSH ratios, and recoupling mitochondria, may support mithormesis priming and remitting in IBD.

15-LOX-1 has diverse roles in the resensitization of resistant cancer cell lines to doxorubicin.

Lipoxygenases (LOXs) are a family of enzymes that can oxygenate polyunsaturated fatty acids. As a member of the family, 15-lipoxygenase-1 (15-LOX-1) specifically metabolizes arachidonic acid and linoleic acid. 15-LOX-1 can affect physiological and pathophysiological events via regulation of the protein-lipid interactome, alterations in intracellular redox state and production of lipid metabolites that are involved in the induction and resolution of inflammation. Although several studies have shown that 15-LOX-1 has an antitumorigenic role in many different cancer models, including breast cancer, the role of the protein in cancer drug resistance has not been established yet. In this study, we, for the first time, aimed to show the potential role of 15-LOX-1 in acquired doxorubicin (DOX) resistance in MCF7 and HeLa cancer cell lines. Our results show that ALOX15 was transcriptionally downregulated in DOX-resistant cells compared with their drug-sensitive counterparts. Moreover, overexpression of ALOX15 in the drug-resistant cells resulted in resensitization of those cells to DOX in a cell-dependent manner. 15-LOX-1 expression could induce apoptosis by activating PPARγ and enhance the accumulation of DOX in drug-resistant MCF7 cells by altering cellular motility properties, and membrane dynamics. However, HeLa DOX cells did not show any of these effects but were susceptible to cell death when treated with 13(S)-HODE. These results underline the role and importance of 15-LOX-1 in cancer drug resistance, and points to novel mechanisms as a therapeutic approach to overcome cancer drug resistance.

Enhanced expression of HNF4α during intestinal epithelial differentiation is involved in the activation of ER stress.

Intestinal epithelial cells are derived from stem cells at the crypts that undergo differentiation into transit-amplifying cells, which in turn form terminally differentiated enterocytes as these cells reach the villus. Extensive alterations in both transcriptional and translational programs occur during differentiation, which can induce the activation of cellular stress responses such as ER stress-related unfolded protein response (UPR) and autophagy, particularly in the cells that are already committed to becoming absorptive cells. Using an epithelial cell model of enterocyte differentiation, we report a mechanistic study connecting enterocyte differentiation to UPR and autophagy. We report that differentiated colon epithelial cells showed increased cytosolic Ca2+ levels and activation of all three pathways of UPR: inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase, and activating transcription factor 6 (ATF6) compared to the undifferentiated cells. Enhanced UPR in the differentiated cells was accompanied by the induction of autophagy as evidenced by increased ratio of light chain 3 II/I, upregulation of Beclin-1, and downregulation of p62. We show for the first time that mechanistically, the upregulation of hepatocyte nuclear factor 4α (HNF4α) during differentiation led to increased promoter binding and transcriptional upregulation of two major proteins of UPR: X-box binding protein-1 and ATF6, implicating HNF4α as a key regulator of UPR response during differentiation. Integrating wet-lab with in silico analyses, the present study links differentiation to cellular stress responses, and highlights the importance of transcription factor signaling and cross-talk between the cellular events in the regulation of intestinal cell differentiation.

Synthesis, Characterization and Evaluation of Cytotoxic Activities of Novel Aziridinyl Phosphonic Acid Derivatives.

New aziridine 2-phosphonic acids were prepared by monohydrolysis of the aziridine 2-phosphonates that were obtained by the modified Gabriel-Cromwell reaction of vinyl phosphonate or α-tosylvinyl phosphonate with a primary amine or a chiral amine. The cellular cytotoxicity of these compounds was tested against the HCT-116 colorectal cancer cell lines and the CCD-18Co normal colon fibroblast lines using the MTT assay. Three of the synthesized phosphonic acid derivatives 2e (ethyl hydrogen {(2S)-1-[(1S)-1-(naphthalen-2-yl)ethyl]aziridin-2-yl}phosphonate), 2h (ethyl hydrogen (1-benzylaziridin-2-yl)phosphonate), and 2i (ethyl hydrogen (1-cyclohexylaziridin-2-yl)phosphonate) showed higher cytotoxicity than the reference cancer treatment agent etoposide. Cell death was through a robust induction of apoptosis even more effectively than etoposide, a well-known apoptosis inducing agent.