Human Kidney Epithelial Cells Research Articles

Alterations in the proteomes of HepG2 and IHKE cells inflicted by six selected mycotoxins.

Toxic fungal secondary metabolites, referred to as mycotoxins, emerge in moldy food and feed and constitute a potent but often underestimated health threat for humans and animals. They are structurally diverse and can cause diseases after dietary intake even in low concentrations. To elucidate cellular responses and identify cellular targets of mycotoxins, a bottom-up proteomics approach was used. We investigated the effects of the mycotoxins aflatoxin B1, ochratoxin A, citrinin, deoxynivalenol, nivalenol and penitrem A on the human hepatoblastoma cell line HepG2 and of ochratoxin A and citrinin on the human kidney epithelial cell line IHKE. Incubations were carried out at sub-cytotoxic concentrations to monitor molecular effects before acute cell death mechanisms predominate. Through these experiments, we were able to detect specific cellular responses that point towards the mycotoxins' mode of action. Besides very well-described mechanisms like the ribotoxicity of the trichothecenes, we observed not yet described effects on different cellular mechanisms. For instance, trichothecenes lowered the apolipoprotein abundance and aflatoxin B1 affected proteins related to inflammation, ribogenesis and mitosis. Ochratoxin A and citrinin upregulated the minichromosomal maintenance complex and nucleotide synthesis in HepG2 and downregulated histones in IHKE. Penitrem A reduced enzyme levels of the sterol biosynthesis. These results will aid in the elucidation of the toxicodynamic properties of this highly relevant class of toxins.

A promising class of antiprotozoal agents, design and synthesis of novel Pyrimidine–Cinnamoyl hybrids

Malaria, caused by parasitic protozoans of the Plasmodium genus, continues to be one of the greatest global health crises, especially in Africa. The emergence of antimalarial drug resistance continues to be a health problem necessitating an urgent need for alternative and cost-effective antimalarials. Using a molecular hybridization approach, we report the design and synthesis of an efficacious novel class of antiprotozoal agents; (E)-1-(4-(4,6-diphenylpyrimidin-2-yl)piperazin-1-yl)-3-phenyl prop-2-en-1-one derivatives (8a-r). The in vitro inhibitory activity of the synthesized compounds was evaluated against the NF54 chloroquine-sensitive strain of Plasmodium falciparum. From the antiprotozoal screening, three compounds displayed propitious activity with IC50 values (0.18–0.21 μM), using quinine and chloroquine as standard antimalarials. Compounds 8o and 8l emerged as the most potent candidates with IC50 values of 0.18 ± 0.02 μM and 0.21 ± 0.001 μM with an associated good safety index of 18.59 and 16.75 to human kidney epithelial (HEK293) cells, respectively. The synthesized analogues present a new chemical architecture structurally unrelated to the current regime of antimalarial drugs, representing a valid strategy to combat resistance in P. falciparum species to current commercial drugs. We further investigated the binding affinities of the compounds against recombinant forms of two P. falciparum heat shock protein 70 homologues; PfHsp70-1 and PfHsp70-z, both of which are essential and promising druggable candidates. Compound 8l exhibited the highest binding affinity for PfHsp70-1 and PfHsp70-z. Furthermore, molecular docking revealed that compounds 8k, 8l, 8m, and 8o exhibited better fitness to PfHsp70-1, with compounds 8l and 8o showing the highest binding affinity of −10.5 kcal/mol and −10.1 kcal/mol, respectively. Therefore, it can be speculated that PfHsp70–1 may be a possible target of some of the inhibitors tested in this study. The presence of electron-donating groups on the phenyl ring of 4,6-pyrimidine moiety and cinnamoyl group demonstrated a positive correlation between the observed computational data and the biological activity. Taken together, this paper demonstrates the importance of using the molecular hybridization approach in the development of newer cinnamoyl clubbed with 4,6-diphenyl pyrimidine hybrids as potential antiprotozoal agents.

Goat lactoferrin–pterostilbene complexes as novel edible functional proteins with enhanced ultraviolet stabilization and antioxidant properties

The use of polyphenols for enhancing protein function in the food industry is limited by their low bioavailability and stability. In this study, we developed complexes of the polyphenol pterostilbene (PTE) and goat lactoferrin (GLF) with the aim of developing a nutritionally enhanced food base with improved functional properties. Specifically, we investigated the binding affinity, structure, and functional modifications of PTE encapsulated with GLF. Fluorescence spectroscopy revealed notable changes in GLF, characterized by reduced fluorescence intensity and a blue shift in the emission peak, following interaction with varying concentrations of PTE. Fourier transformer infrared spectroscopy demonstrated a decrease in β-sheet content and an increase in β-turn structure with increasing PTE level. These structural alterations were furthered confirmed through molecular docking and molecular dynamics simulations. Furthermore, GLF–PTE complexes exhibited improved surface hydrophobicity, foaming capability, and emulsifying activity properties. Importantly, GLF maintained the antioxidant capacity of PTE under UV light irradiation and improved the bioaccessibility of PTE according to in-vitro intestinal digestion simulation assays. Furthermore, the GLF–PTE complexes showed no significant cytotoxicity to mouse fibroblast-like, goat mammary epithelial, or human kidney epithelial cells. This study elucidates the interaction mechanisms between GLF and PTE and supports the potential application of GLF–PTE complexes in food systems owing to their enhanced functional properties and bioactivity.

Molecular Basis for Surface-Initiated Non-Thrombin-Generated Clot Formation Following Viral Infection.

In this paper, we propose a model that connects two standard inflammatory responses to viral infection, namely, elevation of fibrinogen and the lipid drop shower, to the initiation of non-thrombin-generated clot formation. In order to understand the molecular basis for the formation of non-thrombin-generated clots following viral infection, human epithelial and Madin-Darby Canine Kidney (MDCK, epithelial) cells were infected with H1N1, OC43, and adenovirus, and conditioned media was collected, which was later used to treat human umbilical vein endothelial cells and human lung microvascular endothelial cells. After direct infection or after exposure to conditioned media from infected cells, tissue surfaces of both epithelial and endothelial cells, exposed to 8 mg/mL fibrinogen, were observed to initiate fibrillogenesis in the absence of thrombin. No fibers were observed after direct viral exposure of the endothelium or when the epithelium cells were exposed to SARS-CoV-2 isolated spike proteins. Heating the conditioned media to 60 °C had no effect on fibrillogenesis, indicating that the effect was not enzymatic but rather associated with relatively thermally stable inflammatory factors released soon after viral infection. Spontaneous fibrillogenesis had previously been reported and interpreted as being due to the release of the alpha C domains due to strong interactions of the interior of the fibrinogen molecule in contact with hydrophobic material surfaces rather than cleavage of the fibrinopeptides. Contact angle goniometry and immunohistochemistry were used to demonstrate that the lipids produced within the epithelium and released in the conditioned media, probably after the death of infected epithelial cells, formed a hydrophobic residue responsible for fibrillogenesis. Hence, the standard inflammatory response constitutes the ideal conditions for surface-initiated clot formation.

Enhanced stability and dissolution of curcumin nanocrystals stabilized by octenyl succinic anhydride modified starch

Curcumin (CUR) has known to have poor oral bioavailability due to its poor aqueous solubility and stability in gastrointestinal fluid condition as well as its limited permeability which greatly limits its applications in food and medical fields. The present study reported the development of CUR nanocrystal suspension (CUR-NS) for enhanced bioavailability, stabilized with octenyl succinic anhydride modified starch (OSA-S), a naturally renewable and biocompatible biomolecule. This preparation was achieved via an antisolvent precipitation combined with ultrasonic dispersion method, following which the suspension was freeze-dried to form powder CUR nanocrystals (CUR-NCs). Physicochemical properties of the prepared CUR-NS and CUR-NCs powders were extensively characterized. The particle size of the nanocrystals was found to be around 322.8 ± 15.7 nm with a small polydispersity index (PDI) of about 0.230 ± 0.039. Rod like crystal shape was observed by the SEM. Results from powder X-ray diffraction (pXRD) and differential scanning calorimetry (DSC) indicated that the crystal form of CUR in CUR-NCs was a monoclinic polymorph Form 2. Stability results indicated that CUR-NCs possessed an excellent storage-, thermal-, and photo-stabilities. CUR-NCs had a significantly higher dissolution rate, a 68-fold increase in its solubility than that of raw CUR. The cytotoxicity assay demonstrated that CUR-NCs exhibited lower toxicity towards normal human kidney epithelial cells compared to raw CURs. Parallel artificial membrane permeability assay (PAMPA) characterization confirmed that the flux of CUR was significantly enhanced after forming nanocrystals with OSA-S, probably resulting from its increased solubility and dissolution rate. The study showed that OSA-S could effectively stabilize CUR-NCs, and CUR-NCs developed in the current work exhibited significant enhancement in the stability and dissolution rate leading to an improved bioavailability which might be helpful for further development of CUR based products.

DNA binding, potential anticancer, antioxidant and molecular docking simulations of some isonicotinohydrazide metal complexes with the impact of high energy γ-rays irradiation doses: Synthesis and structural characterization

This work focuses on the preparation and characterization of some VO(II), Co(II), Cu(II), Zn(II) and Hg(II) complexes (1–5) of N'-(2-aminobenzoyl)isonicotinohydrazide ligand (HL). The compounds were characterized using various analytical and spectral tools. The resulting powder samples of the as prepared complexes (3,5) were exposed to two gamma ray doses up to 150 and 200 kGy from highly-energetic 60Co γ-irradiation (hereafter referred to as (3F,5F) at dose value of 150 kGy and (3F*,5F*) at dose value of 200 kGy). The impact of γ-irradiation on the possible structural changes of these samples after gamma irradiation was studied. The ligand and as prepared Cu(II) (3), Hg(II) (5) and their γ-irradiated (3F,5F) and (3F*,5F*) samples were tested for free radical scavenging activity using the DPPH assay. The DNA cleavage activity using supercoiled plasmid DNA (calf thymus ct-DNA) was explored. The tested ligand (HL) and complexes (3,5) showed excellent DNA binding. Additionally, the in vitro antitumor activity of (HL) and its Cu(II) (3) and Hg(II) (5) complexes was scrutinized against the human breast carcinoma (MCF-7), human hepatocarcinoma (Hep-G2) cell lines and kidney epithelial normal cells extracted from African green monkey kidney (Vero) to investigate the potential safety towards the normal cells. Also, docking simulation was done on the active site of 4FM9 and 1H7K protein for liver cancer and breast cancer cell line, respectively and showed excellent results.

Comprehensive assessment of per and polyfluoroalkyl substances (PFAS) contamination in groundwater of Kamrup, Assam, India: occurrence, health risks, and metabolomic insights.

Per-and polyfluoroalkyl substances (PFAS) are synthetic chemicals that are known for their environmental persistence and adverse health effects. This study comprehensively assessed PFAS contamination in the Kamrup region of Assam, India, focusing on its presence in groundwater and associated health risks. The analysis detected 12 PFAS in groundwater samples from both the Kamrup Metro and Rural regions. In Kamrup Rural, Perfluorohexanoic acid (PFHxA), perfluorononanoic acid (PFNA), and perfluorooctanesulfonic acid (PFOS) were prevalent, whereas in Kamrup Metro, PFNA and PFOS were dominant, based on detection frequencies. These findings are noteworthy, as they demonstrate the widespread presence of PFAS in groundwater, a vital source of drinking water in the region. The assessment of PFAS health risks in India involved hazard quotient calculations for different age groups. Perfluorobutanesulfonic acid (PFBS) posed the highest risk, ranking children > boys > men > girls > women. Overall, ∑PFAS had low hazard (HQ: 0.27-0.41). Further, this study assessed PFBS and PFOS toxicity in human kidney epithelial cell lines (HEK293T) cells, revealing that PFBS was more cytotoxic than PFOS. The study examined the metabolomics of HEK293T cells after PFBS exposure, revealing significant alterations in lipid metabolism, particularly glycerophospholipids, potentially affecting cellular function and health. These findings underscore the importance of monitoring PFAS contamination in drinking water sources, especially in regions such as Kamrup, where groundwater is a primary source. Our metabolomics results show significant health effects at the cellular level, raising concerns about the impact of PFAS exposure on human health. This study highlights PFAS contamination in Kamrup, Assam's groundwater and its health risks, providing valuable insights for policymakers and public health management.

The Apoptotic Property of Nymphaea Caerulea Flower Extract on Acute Myeloid Leukaemia Cell Line, THP-1.

Acute Myeloid Leukaemia (AML) is considered to be an extremely heterogeneous malignancy of bone marrow and blood. The first line of therapy for AML is prolonged chemotherapy. Due to the presence of molecular heterogeneity in AML as confirmed by next-generation sequencing, researchers are planning to develop newer strategies of therapy. In the present study we have explored the anti-cancer potentiality of the hydro-ethanolic extract (50% and 70%) of the whole flower of Nymphaea caerulea against the Acute Myeloid Leukaemia cell line, THP-1 with control of normal human kidney epithelial cell line (HEK 293). The present study is a novel contribution to the existing scientific knowledge as at present no study as an anti-leukaemic agent is available on N. caerulea (blue lotus) extract and exploring its action mechanism on in-vitro cell line model. Some targeted cytokine and apoptotic genes genes to deduce the anti-cancer mechanism of action of the crude extract (hydro-ethanolic extract (50% and 70%) of the whole flower) were selected as Interferon (IFN) γ, Interleukins - IL-6, IL-8, IL- 10, IL-1β, Transforming Growth Factor (TGF β1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9 (CAS 9), CD95 (Fas), Tumor Necrosis Factor Receptor 1 (TNFRSF1A) to observe relative fold changes of the expression using Real-Time PCR with housekeeping gene β-actin. Cellular cytopathic effect (CPE), cell viability assay by methylene blue assay, and cell cytotoxicity of the crude extract against the THP-1 cell line were also studied along with it's bio-active compositional analysis of the extract was explored using ultra-performance liquid chromatography followed by mass spectra. The N. caerulea flower extract is capable of inducing apoptosis in AML and it can balance cytokine alterations in such diseases. Nymphaea caerulea flower extract appears to be a good anti-leukemia agent.

Epigallocatechin-3-gallate attenuates arsenic-induced fibrogenic changes in human kidney epithelial cells through reversal of epigenetic aberrations and antioxidant activities.

Renal fibrosis is a pathogenic intermediate stage of chronic kidney disease (CKD). Nephrotoxicants including arsenic can cause kidney fibrosis through induction of oxidative stress and epigenetic aberrations. Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, is known to have antioxidant and epigenetic modulation properties. Whether EGCG, through its antioxidant and epigenetic modulating activities, can attenuate fibrogenesis is not known. Therefore, the objective of this study was to determine whether EGCG can attenuate arsenic-induced acute injury and long-term exposure associated fibrogenicity in kidney epithelial cells. To address this question, two human kidney epithelial cell lines Caki-1 and HK-2 exposed to arsenic for both acute and long-term durations were treated with EGCG. The protective effect of EGCG on arsenic-induced cytotoxicity and fibrogenicity were evaluated by measuring the cell growth, reactive oxygen species (ROS) production, genes expression, and epigenetic changes in histone marks. Results revealed that EGCG has a protective effect in arsenic-induced acute cytotoxicity in these cells. EGCG scavenges the increased levels of ROS in arsenic exposed cells. Aberrant expression of fibrogenic genes in arsenic exposed cells were restored by EGCG. Abrogation of arsenic-induced fibrogenic changes was also associated with EGCG-mediated restoration of arsenic-induced aberrant expression of epigenetic regulatory proteins and histone marks. Novel findings of this study suggest that EGCG, through its antioxidant and epigenetic modulation capacities, has protective effects against arsenic-induced cytotoxicity and fibrogenic changes in kidney epithelial cells.

Abstract B104: U18666A-induced cholesterol accumulation decreases tumor-derived exosomes load and modulates malignant transformation

Abstract Background: Tumor-derived exosomes (TDEs) play an important role in the horizontal transfer of oncogenic information from the tumor to other sites, where they regulate pre-metastatic niche formation, organotropism, migration, invasion and survival. Cellular cholesterol homeostasis emerges as an important factor for exosomal biogenesis and cellular release. In this study, we utilized U18666A to trigger cholesterol accumulation within late endosomes of MDA-MB231 cells and evaluated the ability of exosomes derived from both U18666A-treated and untreated cells to initiate malignant transformation in recipient human epithelial kidney (HEK293) cells. Methods: Exosomes were isolated and characterized from MDA-MB231 (untreated and U18666A-treated) and HEK293 cells. Cholesterol content of the cells and their associated exosomes were measured to confirm its accumulation following U18666A treatment. The effects of exosomes derived from untreated MDA-MB231 (UCE) and U18666A-treated MDA-MB231 (UTCE) cells on HEK293 cells were examined to determine their transforming potential. Results: Exosomes derived from MDA-MB231 cells induced proliferation, migration, malignant transformation and epithelial-mesenchymal transition (EMT) process in non-cancerous recipients’ cells. U18666A treatment led to accumulation of cholesterol within late endosomes and significantly reversed the EMT process in MDA-MB231 cells. It also reduced TAEs load from the cancer cells and rendered them less oncogenic which was evident from their inability to induce malignant transformation in the recipient HEK293 cells. Conclusion: Modulation of cholesterol homeostasis and disruption of cholesterol supply to aggressive cancer cells can be an attractive strategy for limiting TAEs release as well as their subsequent role in cancer progression and metastasis. Citation Format: Syed Sultan Beevi, Vinod Kumar Verma. U18666A-induced cholesterol accumulation decreases tumor-derived exosomes load and modulates malignant transformation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B104.

Enhanced fatty acid oxidation through metformin and baicalin as therapy for COVID-19 and associated inflammatory states in lung and kidney

Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. It is the final outcome of the acute respiratory distress syndrome (ARDS), characterized by an initial exacerbated inflammatory response, metabolic derangement and ultimate tissue scarring. A positive balance of cellular energy may result crucial for the recovery of clinical COVID-19. Hence, we asked if two key pathways involved in cellular energy generation, AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling and fatty acid oxidation (FAO) could be beneficial. We tested the drugs metformin (AMPK activator) and baicalin (CPT1A activator) in different experimental models mimicking COVID-19 associated inflammation in lung and kidney. We also studied two different cohorts of COVID-19 patients that had been previously treated with metformin. These drugs ameliorated lung damage in an ARDS animal model, while activation of AMPK/ACC signaling increased mitochondrial function and decreased TGF-β-induced fibrosis, apoptosis and inflammation markers in lung epithelial cells. Similar results were observed with two indole derivatives, IND6 and IND8 with AMPK activating capacity. Consistently, a reduced time of hospitalization and need of intensive care was observed in COVID-19 patients previously exposed to metformin. Baicalin also mitigated the activation of pro-inflammatory bone marrow-derived macrophages (BMDMs) and reduced kidney fibrosis in two animal models of kidney injury, another key target of COVID-19. In human epithelial lung and kidney cells, both drugs improved mitochondrial function and prevented TGF-β-induced renal epithelial cell dedifferentiation. Our results support that favoring cellular energy production through enhanced FAO may prove useful in the prevention of COVID-19-induced lung and renal damage.

Activation of immune evasion machinery is a part of the process of malignant transformation of human cells

Malignant transformation of human cells is associated with their re-programming which results in uncontrolled proliferation and in the same time biochemical activation of immunosuppressive pathways which form cancer immune evasion machinery. However, there is no conceptual understanding of whether immune evasion machinery pathways and expression of immune checkpoint proteins form a part of the process of malignant transformation or if they are triggered by T lymphocytes and natural killers (NK) attempting to attack cells which are undergoing or already underwent malignant transformation. To address this fundamental question, we performed experimental malignant transformation of BEAS-2B human bronchial epithelium cells and RC-124 non-malignant human kidney epithelial cells using bracken extracts containing carcinogenic alkaloid called ptaquiloside. This transformation led to a significant upregulation of cell proliferation velocity and in the same time led to a significant upregulation in expression of key immune checkpoint proteins – galectin-9, programmed death ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO1). Their increased expression levels were in line with upregulation of the levels and activities of HIF-1 transcription complex and transforming growth factor beta type 1 (TGF-β)-Smad3 signalling pathway. When co-cultured with T cells, transformed epithelial cells displayed much higher and more efficient immune evasion activity compared to original non-transformed cells. Therefore, this work resolved a very important scientific and clinical question and suggested that cancer immune evasion machinery is activated during malignant transformation of human cells regardless the presence of immune cells in microenvironment.

CAR-T Therapy Targets Extra Domain B of Fibronectin Positive Solid Tumor Cells

ABSTRACT Background CAR-T cell immunotherapy has achieved remarkable success in malignant B-cell malignancies, but progress in solid tumors is slow, and one of the key reasons is the lack of ideal targets. Cancer-specific extra domain B of fibronectin (EDB-FN) is widely upregulated in solid tumors and expressed at low levels in normal tissues. Many imaging and targeted cancer therapies based on EDB-FN targets have been developed and tested in clinical trials, making EDB-FN an ideal target for immunotherapy. Methods We constructed two EDB-FN-targeted CAR-Ts based on the peptide APT0 and the single-chain antibody CGS2 in a lentiviral infection manner for the first time. Luciferase cytotoxicity assay to assess CAR-T killing of tumor cells. An enzyme-linked immunosorbent assay was used to detect the release of the cytokine IFN-γ. Fluorescence imaging to evaluate the dynamics of CAR-T cell and tumor cell coculture. Knockdown assays were used to validate the target specificity of CAR-T cells. Results In this research, two CAR-Ts targeting EDB-FN, APT0 CAR-T, and CGS2 CAR-T, were constructed. In vitro, both CAR-T cells produced broad-spectrum killing of multiple EDB-FN-positive solid tumor cell lines and were accompanied by cytokine IFN-γ release. Regarding safety, the two CAR-T cells did not affect T cells’ normal growth and proliferation and were not toxic to HEK-293T human embryonic kidney epithelial cells. Conclusion APT0 CAR-T and CGS2 CAR-T cells are two new CAR-Ts targeting EDB-FN. Both CAR-T cells can successfully identify and specifically kill various EDB-FN-positive solid tumor cells with potential clinical applications.

Combination Organelle Mitochondrial Endoplasmic Reticulum Therapy (COMET) for Multidrug Resistant Breast Cancer

It is time for the story of mitochondria and intracellular communication in multidrug resistant cancer to be rewritten. Herein we characterize the extent and cellular advantages of mitochondrial network fusion in multidrug resistant (MDR) breast cancer and have designed a novel nanomedicine that disrupts mitochondrial network fusion and systematically manipulates organelle fusion and function. Combination Organelle Mitochondrial Endoplasmic reticulum Therapy (COMET) is an innovative translational nanomedicine for treating MDR triple negative breast cancer (TNBC) that has superior safety and equivalent efficacy to the current standard of care (paclitaxel). Our study has demonstrated that the increased mitochondrial networks in MDR TNBC contribute to apoptotic resistance and network fusion is mediated by mitofusin2 (MFN2) on the outer mitochondrial membrane. COMET consists of three components; Mitochondrial Network Disrupting (MiND) nanoparticles (NPs) that are loaded with an anti-MFN2 peptide, tunicamycin, and Bam7. The therapeutic rationale of COMET is to reduce the apoptotic threshold in MDR cells with MiND NPs, followed by inducing the endoplasmic reticulum mediated unfolded protein response (UPR) by stressing MDR cells with tunicamycin, and finally, directly inducing mitochondrial apoptosis with Bam7 which is a specific bcl-2 Bax activator. MiND NPs are PEGylated liposomes with the 21 amino acid (2577.98 MW) anti-MFN2 peptide compartmentalized in the aqueous core. Hypoxia (0.5% oxygen) was used to create MDR derivatives of MDA-MB-231 cells and BT-549 cells. Mitochondrial networks were quantified using 3D analysis of 60× live cell images acquired with a Keyence BZ-X710 microscope and MiND NPs effectively fragmented mitochondrial networks in drug sensitive and MDR TNBC cells. The IC50 values, combination index, and dose reduction index derived from dose response studies demonstrate that MiND NPs decrease the apoptotic threshold of both drug sensitive and MDR TNBC cells and COMET is a synergistic drug combination. Complex V (ATP synthase) extracted from bovine cardiac mitochondria was used to assess the effect of MiND NPs on OXPHOS; both MiND NPs and anti-MFN2 peptide solution significantly decrease the activity of mitochondrial complex V and decrease the capacity of OXPHOS. A BacMam viral vector based fluorescent biosensor was used to quantify the unfolded protein response (UPR) at the level of the endoplasmic reticulum and tunicamycin specifically induces the UPR in drug sensitive and MDR TNBC cells. A caspase 3 colorimetric assay demonstrated that the synergistic triple drug combination of COMET increases the ability of Bam7 to specifically induce apoptosis. Dose limiting toxicity and off target effects are a significant challenge for current chemotherapy regimens including paclitaxel. COMET has significantly lower cytotoxicity than paclitaxel in human embryonic kidney epithelial cells and has the potential to fulfill the clinical need for safer cancer therapeutics. COMET is a promising early stage translational nanomedicine for treating MDR TNBC. Manipulating intracellular communication and organelle fusion is a novel approach to treating MDR cancer. The data from this study has rewritten the story of mitochondria, organelle fusion, and intracellular communication and by targeting this intersection, COMET is an exciting new chapter in cancer therapeutics that could transform the clinical outcome of MDR TNBC.

90 Complement activation during hypoxia-reoxygenation in human kidney epithelial cells

90 Complement activation during hypoxia-reoxygenation in human kidney epithelial cells

RhMYDGF Alleviates I/R-induced Kidney Injury by Inhibiting Inflammation and Apoptosis via the Akt Pathway.

Renal ischemia/reperfusion (I/R) injury is one of the crucial factors affecting the outcome of renal transplantation. In recent years, myeloid-derived growth factor (MYDGF) has received a lot of attention for its extensive beneficial effects on cardiac repair and protection of cardiomyocytes from cell death. Therefore, we hypothesized that the recombinant human MYDGF (rhMYDGF) protein might play an essential role in safeguarding renal I/R injury. In vivo experiments were conducted using a mouse unilateral I/R model. Mice were pretreated with rhMYDGF by intraperitoneal injection to study the potential mechanism of renal protection. In vitro, we established hypoxia/reoxygenation and H 2 O 2 treatment models to pretreat cells with rhMYDGF. The expression levels of oxidative stress, inflammation, and apoptosis-related factors in tissues and cells were detected. Finally, we explored the role of the protein kinase B (Akt) pathway in the renal protective mechanism of rhMYDGF. In this study, we found that intraperitoneal injection of 1.25 μg rhMYDGF could significantly improve renal function of I/R mice, and reduce oxidative stress, inflammation, and apoptosis. For the human proximal tubular epithelial cell line and human kidney cell line, pretreatment with 0.3 μg/mL rhMYDGF for 24 h significantly downregulated oxidative stress, inflammation, and apoptosis via the phosphorylation of Akt, which could be ameliorated by LY294002. rhMYDGF protects kidney from I/R injury by attenuating oxidative stress, inflammation, and apoptosis through the activation of the Akt pathway.

OCT4 induces long-lived dedifferentiated kidney progenitors poised to redifferentiate in 3D kidney spheroids

Upscaling of kidney epithelial cells is crucial for renal regenerative medicine. Nonetheless, the adult kidney lacks a distinct stem cell hierarchy, limiting the ability to long-term propagate clonal populations of primary cells that retain renal identity. Toward this goal, we tested the paradigm of shifting the balance between differentiation and stemness in the kidney by introducing a single pluripotency factor, OCT4. Here we show that ectopic expression of OCT4 in human adult kidney epithelial cells (hKEpC) induces the cells to dedifferentiate, stably proliferate, and clonally emerge over many generations. Control hKEpC dedifferentiate, assume fibroblastic morphology, and completely lose clonogenic capacity. Analysis of gene expression and histone methylation patterns revealed that OCT4 represses the HNF1B gene module, which is critical for kidney epithelial differentiation, and concomitantly activates stemness-related pathways. OCT4-hKEpC can be long-term expanded in the dedifferentiated state that is primed for renal differentiation. Thus, when expanded OCT4-hKEpC are grown as kidney spheroids (OCT4-kSPH), they reactivate the HNF1B gene signature, redifferentiate, and efficiently generate renal structures invivo. Hence, changes occurring in the cellular state of hKEpC following OCT4 induction, long-term propagation, and 3D aggregation afford rapid scale-up technology of primary renal tissue-forming cells.

Potent saccharinate-containing palladium(II) complexes for sensitization to cancer therapy

Palladium(II) (PdII) complexes are among the most promising anticancer compounds. Both 2`-benzoylpyridine thiosemicarbazone (BpT) and saccharinate (Sac) are efficient metal chelators with potent anticancer activity. To explore a more effective new anticancer drug, we synthesized a series of Sac and BpT-containing PdII complexes coordinated with thiosemicarbazone (TSC)-derived ligands, and characterized them through nuclear magnetic resonance (NMR), Fourier transformed infrared spectroscopy (FT-IR), elemental analysis, ultraviolet-visible spectroscopy (UV–Vis) and thermogravimetric analysis (TGA). Each target complex was composed of PdII, BpT, and one or two Sac molecules. Both the in vitro and in vivo anti-growth effects of those ligands and the obtained PdII complexes were investigated in the human lung adenocarcinoma cell lines A549 and Spc-A1. The coordination of PdII with the TSC-derivatives and Sac resulted in clearly greater anticancer activity than single ligands. These compounds were demonstrated to be safe for 293 T normal human kidney epithelial cells. The introduction of Sac into the TSC-derived PdII complex significantly enhanced anti-growth effects, and induced apoptosis of human lung cancer cells in vitro and in vivo in a dose dependent manner. Moreover, the PdII complex containing two Sac molecules showed the most promising therapeutic effects, thereby confirming that Sac increases the cancer therapeutic efficacy of PdII complexes and providing a new strategy for exploring anticancer drugs for potential clinical treatment.

Identification of an unrecognized circRNA associated with development of renal fibrosis.

Backgroud: Renal fibrosis is the common characteristic of chronic kidney disease. Circular RNA plays an essential role in the occurrence and development of Renal fibrosis, but its regulative mechanism remains elusive. Methods: The animal and cell model of Renal fibrosis was established, and RNA-sequencing and real-time polymerase chain reaction (qRT-PCR) experiments were implemented. Subsequently, experiments for detecting apoptosis and proliferation of cell, were carried out, and the isobaric tags for relative and absolute quantification proteomics analyses were performed accordingly. Results: It was found that a newly discovered Circular RNA (circRNA_0002158), is highly expressed in kidneys or cells with fibrosis, implying that this Circular RNA might be associated with the occurrence and development of Renal fibrosis. Subsequently, the overexpression and knockdown of circRNA_0002158 were conducted in the human kidney epithelial cell line (HK-2) cells, and the results indicated that the circRNA_0002158 could inhibit apoptosis, and promote proliferation of cells. The kidney injury-related factors, including Fibronectin and plasminogen activator inhibitor-1 (PAI-1), were decreased in HK-2 cells with overexpression of circRNA_0002158, while the results were reversed in cells with knockdown of circRNA_0002158. Finally, to explore the regulative mechanism of circRNA_0002158, the iTRAQ proteomics analyses were implemented for the cell samples with OE of circRNA_0002158 and its control, it showed that multiple genes and functional pathways were associated with the occurrence and development of Renal fibrosis. Conclusion: CircRNA_0002158 is associated with regulating Renal fibrosis, and may contribute to ameliorating the progression of Renal fibrosis in the future.

Overexpressed c-Myc Sensitizes Cells to TH1579, a Mitotic Arrest and Oxidative DNA Damage Inducer.

Previously, we reported that MTH1 inhibitors TH588 and TH1579 selectively induce oxidative damage and kill Ras-expressing or -transforming cancer cells, as compared to non-transforming immortalized or primary cells. While this explains the impressive anti-cancer properties of the compounds, the molecular mechanism remains elusive. Several oncogenes induce replication stress, resulting in under replicated DNA and replication continuing into mitosis, where TH588 and TH1579 treatment causes toxicity and incorporation of oxidative damage. Hence, we hypothesized that oncogene-induced replication stress explains the cancer selectivity. To test this, we overexpressed c-Myc in human epithelial kidney cells (HA1EB), resulting in increased proliferation, polyploidy and replication stress. TH588 and TH1579 selectively kill c-Myc overexpressing clones, enforcing the cancer cell selective killing of these compounds. Moreover, the toxicity of TH588 and TH1579 in c-Myc overexpressing cells is rescued by transcription, proteasome or CDK1 inhibitors, but not by nucleoside supplementation. We conclude that the molecular toxicological mechanisms of how TH588 and TH1579 kill c-Myc overexpressing cells have several components and involve MTH1-independent proteasomal degradation of c-Myc itself, c-Myc-driven transcription and CDK activation.