Heated garlic mustard oil macerate: A novel formulation with the capacity to reduce various pro-inflammatory cytokine and Cox2 expression in lipopolysaccharide-induced inflammation in THP-1 cell line

Nosocomial infections caused by antibiotic-resistant Gram-negative pathogens are of grave concern today. Polymyxins are considered as the last resorts of therapy to treat these multi-drug resistant (MDR) bacteria. But their associated nephrotoxicity and neurotoxicity calls for the development of safer polymyxin therapy until novel and less toxic antibiotics are discovered. No other polymyxin molecule except polymyxin B and E (colistin) is explored thoroughly in literature to demonstrate its clinical relevance. In the present study, we have isolated two antimicrobial compounds named P1 and P2 from the soil isolate Paenibacillus dendritiformis strain PV3-16, which we later identified as polymyxin A2 and A1 respectively. We tested their minimum inhibitory concentrations (MICs) against MDR clinical isolates, performed membrane permeabilization assays and determined their interaction with lipopolysaccharide (LPS). Finally, we studied their toxicity against human Leukemic monocyte cell line (THP-1) and embryonic kidney cell line (HEK 293). Both compounds displayed equal efficacy when compared with standard polymyxins. P1 was 2–4 fold more active in most of the clinical strains tested. Moreover, P1 showed higher affinity toward LPS. In cytotoxicity studies, P1 had IC50 value (>1000 μg/ml) similar to colistin against HEK cells but immune cells, i.e., THP-1 cell lines were more sensitive to polymyxins. P1 showed less toxicity in THP-1 cell line than all other polymyxins checked. To sum up, P1 (polymyxin A2) possessed better efficacy than polymyxin B and E and had least toxicity to immune cells. Since polymyxin A was not investigated thoroughly, we performed the comprehensive in vitro assessment of this molecule. Moreover, this is the first report of isolation and characterization of polymyxin A from P. dendritiformis. This compound should be further investigated for its in vivo efficacy and toxicity to develop it as a drug candidate.

Mitogen-activated protein kinases, originally known as microtubule-associated protein (MAP) kinases, are activated in response to a variety of stimuli. Here we report that microtubule-depolymerizing agents such as colchicine or nocodazole induced strong activation of MAP kinases including JNK, ERK, and p38. This effect was markedly attenuated by parkin, whose mutations are linked to Parkinson disease (PD). Our previous study has shown that parkin stabilizes microtubules through strong interactions mediated by three independent domains. We found that each of the three microtubule-binding domains of parkin was sufficient to reduce MAP kinase activation induced by microtubule depolymerization. The ability to attenuate microtubule depolymerization and the ensuing MAP kinase activation was abrogated in B-lymphocytes and fibroblasts derived from PD patients with parkin mutations such as exon 4 deletion. Such mutations produced truncated parkin proteins lacking any microtubule binding domain and prevented parkin from protecting midbrain dopaminergic neurons against microtubule-depolymerizing toxins such as rotenone or colchicine. Consistent with these, blocking MAP kinase activation in midbrain dopaminergic neurons by knocking down MAP kinase kinases (MKK) significantly reduced the selective toxicity of rotenone or colchicine. Conversely, overexpression of MAP kinases caused marked toxicities that were significantly attenuated by parkin. Thus, the results suggest that parkin protects midbrain dopaminergic neurons against microtubule-depolymerizing PD toxins such as rotenone by stabilizing microtubules to attenuate MAP kinase activation.

With the discovery of STIM1 and Orai1 and gating of both TRPC and Orai1 channels by STIM1, a central question is the role of each of the channels in the native store-operated Ca(2+) influx (SOCs). Here, we used a strategy of knockdown of Orai1 and of TRPC1 alone and in combination and rescue by small interfering RNA-protected mutants (sm) of smOrai1 and smTRPC1 to demonstrate that in human embryonic kidney (HEK) cells, rescue of SOCs required co-transfection of low levels of both smOrai1 and smTRPC1. The pore mutant Orai1(E106Q) failed to rescue the SOCs in the presence or absence of TRPC1 and, surprisingly, the pore mutant TRPC1(F562A) failed to rescue the SOCs in the presence or absence of Orai1. TRPC1 is gated by electrostatic interaction between TRPC1(D639D,D640D) with STIM1(K684K, K685K). Strikingly, the channel-dead TRPC1(D639K,D640K) that can be rescued only by the STIM1(K684E,K685E) mutant could restore SOCs only when expressed with Orai1 and STIM1(K684E,K685E). Accordingly, we found a mutual requirement of Orai1 and TRPC1 for their interaction with the native STIM1 in HEK cells. By contrast, SOC and the CRAC current in Jurkat cells were inhibited by knockdown of Orai1 but were not influenced by knockdown on TRPC1 or TRPC3. These findings define the molecular makeup of the native SOCs in HEK cells and the role of a STIM1-Orai1-TRPC1 complex in SOC activity.

Present study evaluates the effects of photodynamic therapy (PDT) with aminolevulinic acid (5-ALA) as photo sensitizer using Human embryonic kidney (HEK293T) cell line as an experimental model. Porphyrins derivatives are used as active cytotoxic antitumor agents in PDT. Above mentioned cell line were irradiated with red light (a diode laser, λ = 635 nm) at different doses (0–160 J/cm2) of light. The influence/effectiveness of incubation time, various concentrations of aminolevulinic acid (5-ALA) and light doses on the cellular viability was studied. HEK293T cells were deliberated by exposing the ALA-PpIX (0–1000 μg/ml) of concentrations. The optimal uptakes of photosensitizer (PS) in cell lines were investigated by means of spectro photo metric measurements. Cells viability was determined by means of neutral red assay (NRA). It was observed that alone, neither photosensitizer nor light dose have significant effect on cells viability, but optimal concentration of PS along with suitable dose of light exhibit effective impact on the viability of cell. Our results showed that light doses of 40 J/cm2 demonstrates effective PDT outcome for HEK293T cell line when incubated with 400 μg/ml, with wrapping up view that HEK293T cell line is very sensitive to ALA-mediated PDT as compared to cell line published in our data. At the end results has been verified by using reactive oxygen species (ROS) measure test.

Endogenous [ 3H]flunitrazepam binding in human embryonic kidney cell line 293

Microcystin-LR (MC-LR) is a potent hepatotoxin, and increasing evidence suggests that it might also induce kidney injury. The aim of the present work was to evaluate the cytotoxicity and possible apoptotic effects of MC-LR on a human embryonic kidney cell line (HEK-293) and human kidney adenocarcinoma cell line (ACHN). Cells were exposed for 24 h to pure MC-LR (1.0-200 µM) and the cytotoxic effects were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulphorhodamine B (SRB) cell viability assays. Cell viability in both cell lines was significantly decreased after treatment with MC-LR at 50 µM for 24 h (P<0.001). Moreover, MC-LR-treated ACHN and HEK-293 cells exhibited a marked dose-dependent loss of confluence as judged by phase-contrast microscopy. Similarly, fluorescence microscopic observations following acridine orange-ethidium bromide (AO/EB) staining confirmed that both cell types were undergoing apoptosis after treatment with MC-LR for 24 h. Expression of three apoptosis-related genes, Bax, Survivin and p53, was analysed by quantitative reverse transcriptase PCR analysis. Both Bax and p53 functioned as promoters of MC-LR-mediated apoptosis in ACHN and HEK-293 cells. The Survivin gene acted as a suppressor of apoptosis at lower MC-LR concentration (1 µM) and the gene was upregulated at higher MC-LR concentration (10 µM) (P<0.001). Significant increases of caspase 3 (P<0.0001) and caspase 9 (P<0.0001) activity were detected in both cell lines after exposure to MC-LR for 24 h, indicating the MC-LR induces cytotoxicity and a marked apoptosis in both ACHN and HEK-293 kidney cell lines.

Combined Raloxifene and Letrozole for Breast Cancer Patients

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.

Complete deficiency in activity-dependent neuroprotective protein (ADNP), a heterochromatin 1-binding protein, results in dramatic changes in gene expression, neural tube closure defects, and death at gestation day 9 in mice. To further understand the cellular roles played by ADNP, the HEK293 human embryonic kidney cell line that allows efficient transfection with recombinant DNA was used as a model for the identification of ADNP-interacting proteins. Recombinant green fluorescent protein (GFP)-ADNP was localized to cell nuclei. When nuclear extracts were subjected to immunoprecipitation with specific GFP antibodies followed by polyacrylamide gel electrophoresis, several minor protein bands were observed in addition to GFP-ADNP. In-gel protein digests followed by mass spectrometry identified BRG1, BAF250a, and BAF170, all components of the SWI/SNF (mating type switching/sucrose nonfermenting) chromatin remodeling complex, as proteins that co-immunoprecipitate with ADNP. These results were verified utilizing BRG1 antibodies. ADNP short hairpin RNA down-regulation resulted in microtubule reorganization and changes in cell morphology including reduction in cell process formation and cell number. These morphological changes are closely associated with the SWI/SNF complex multifunctionality. Taken together, the current study uncovers a molecular basis for the essential function of the ADNP gene and protein.

Objective To identify the role of miR153 in cell proliferation and apoptosis in Human Embryonic Kidney 293 (HEK293) cells. Methods miR153 was selected as a specific target miR by the technique of microRNA array and bioinformatics. Real-time PCR was used to detect the expression of miR153 in HEK293 cell lines. Anti-miR153 transfected by liposome was used to knockdown the expression of miR153 in HEK293 cell line. The quantity and the morphologic changes of the cells were recorded under optical microscope. The growth curve of HEK293 cells after antimiR153 transfection was determined with MTT assay. Flow cytometer was used to detect the cell death and apoptosis in order to analyze the change of cell cycle after antimiR153 transfection. Results miR153 expression was significantly higher in HEK293 cells than that in normal cells. Expression of miR153 in the antimiR group was significantly lower than that in the negative control group, with the knockdown rate in 80. 5-84. 3%. Cells in the anti-miR group grew more slowly than that in negative control group, which started from 24h after transfection and reached the significant time point at 48h. In the anti-miR group, cells became scattering and round, with the absence of cell synapses. Utilizing FCM, cells in the anti-miR153 group had higher apoptosis and necrotic rate (9. 39% ), which was 0. 7% higher than that in the negative control group. Cycle histogram showed that the peak of G2/M phase disappeared in anti-miR153 group, while more cells stayed in G0/G1 phase. Conclusions Cell functions of HEK293 cells can be affected by anti-miR targeting miR153, including induction of cell necrosis or apoptosis and inhibition of cell growth. Anti-miR153 application may be a new tool for treatment of tumors, while further investigation is still needed. Key words: Cell cycle; Cell proliferation; Apoptosis

Disclosure: A. Matveeva: None. T. Du Toit: None. A.V. Pandey: None.Introduction: P450 oxidoreductase (POR) is a flavoprotein that facilitates electron transfer from NADPH to microsomal cytochrome P450 proteins (CYPs) for metabolism of steroids, including sex hormones biosynthesis in adrenal glands and gonads. POR deficiency can lead to metabolic disorders in humans including disrupted sexual development, skeletal abnormalities, and congenital adrenal hyperplasia. To date, over 200 mutations in POR that cause loss of function have been discovered, with a few of them being characterized using purified protein systems. To gain a deeper understanding of POR activity within the living cell context, further studies using human cell lines are essential. Methods: The genomes of the NCI - H295R (adrenal carcinoma cells) and HEK 293T (human embryonic kidney cells) were edited using CRISPR/Cas9 system with sgRNAs targeting 4th exon of the POR gene. Resulting cells were subcloned to obtain monogenic cultures representing unified genotype. To evaluate the status of POR, we employed Sanger sequencing of POR gene, Western blot analysis, and the assays on POR function. For HEK 293T cells, the CYP17A1 and CYP21A2 activity were measured using thin layer chromatography. For NCI - H295R cells, the full steroid profile was obtained using LC-MS/MS. Results: POR gene knockout cell models were developed using human adrenal carcinoma and embryonic kidney cell lines. Adrenal - derived POR knockout cells were characterized by the significant inhibition of aldosterone, cortisol, and testosterone pathways indicating reduced activities of CYP21A2 and CYP17A1 - two key metabolic partners of POR in steroid biosynthesis. The Progesterone levels were significantly higher in POR knockout adrenal cells and 17OH-Progesterone levels were comparable to controls. In HEK293T cells, loss of POR showed 3.6 % of wild-type activity towards CYP21A2 and 0% activity towards CYP17A1. Conclusion: The generated cell lines resemble the steroid abnormalities found in patients with POR deficiency and have the advantage of lacking endogenous POR activity, which removes background interference. It allows us to study the patient-specific POR mutations, supporting the creation of personalized disease models. By reflecting individual genetic profiles, these cell models offer valuable insights into the complex metabolic processes in the adrenal glands while also serving as a screening platform to characterize the impact of the POR mutations on steroidogenesis and drug response. These models can greatly improve both the diagnosis and treatment of POR deficiency. This study was funded by the Swiss National Science Foundation grant number 310030M_204518 to Amit V Pandey (www.pandeylab.org)Presentation: Saturday, July 12, 2025

In the present study, natural phaeosphaeride A (PPA) derivatives are synthesized. Anti-tumor studies are carried out on the PC3, K562, HCT-116, THP-1, MCF-7, A549, NCI-H929, Jurkat, and RPMI8226 tumor cell lines, and on the human embryonic kidney (HEK293) cell line. All the compounds synthesized turned out to have better efficacy than PPA towards the tumor cell lines listed. Among them, three compounds exhibited an ability to overcome the drug resistance of tumor cells associated with the overexpression of the P-glycoprotein by modulating the work of this transporter. Luminex xMAP technology was used to assess the effect of five synthesized compounds on the activation of intracellular kinase cascades in A431 cells. MILLIPLEX MAP Multi-Pathway Magnetic Bead 9-Plex was used, which allowed for the simultaneous detection of the following nine phosphorylated protein markers of the main intracellular signaling pathways: a universal transcription factor that controls the expression of immune-response genes, apoptosis and cell cycle NFκB (pS536); cAMP-dependent transcription factor (CREB (pS133); mitogen-activated kinase p38 (pT180/pY182); stress-activated protein kinase JNK (pT183/pY185); ribosomal SK; transcription factors STAT3 (pS727) and STAT5A/B (pY694/699); protein kinase B (Akt) (pS473); and kinase regulated by extracellular signals ERK1/2 (pT185/pY187). The effect of various concentrations of PPA derivatives on the cell culture was studied using xCelligence RTCA equipment. The compounds were found to modulate JNK, ERK1/2, and p38 signaling pathways. The set of activated kinase cascades suggests that oxidative stress is the main probable mechanism of the toxic action of PPA derivatives.

Human embryonic kidney (HEK-293) cell line: An alternative for rabies virus diagnosis and research

Sting-Mediated Interferon Signaling Exerts Potent Antileukemic Effects in High-Risk Myeloid and Monocytic Malignancies

Objective To elucidate the effects of human Salvador 1 (hSav1 ) on cell proliferation of human embryonic kidney cell line HEK293. Methods The plasmid CFP-N1-hSav1 was constructed and transfected into HEK293 cells with lipofectamine 2000. The transfection efficiency was detected by fluorescent microscopy. The effects of hSav1 on cell proliferation were measured by MTT and BrdU incorporation. Results The transfection efficiency was about 70% -80%. The MTT results showed that the inhibition rate of cell proliferation in transfected group at the 12th, 24th, 36th, and 48th h was 2% , 5% , 15% , and 23% , respectively;while that in the control group was 2% , 3% , 2% , and 2% respectively. The BrdU incorporation revealed that the BrdU incorporation rate in transfected group (12. 9 ±5. 3)% was significantly lower than in control group (27.3±3.8)% (P<0.05). Conclusion hSav1 is a newly identified protein that can cause cell proliferation inhibition in HEK293 cells. Key words: hSav1; Cell proliferation; Human embryonic kidney cell