A novel method to detect intracellular metabolite alterations in MCF-7 cells by doxorubicin induced cell death

Kumar, Ajay; Patel, Sheetal; Bhatkar, Devyani; Sharma, Nilesh Kumar

doi:10.1101/812255

Cited by 3 publications

(1 citation statement)

References 45 publications

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“…One major limitation is on the lack of data that show physiological abundance of lactyl-CoA in cancer cells using comprehensive metabolomics approaches (17)(18)(19)(20)(21)(22)(23)(24)(25). In our lab, we have not detected lactyl-CoA in cancer cells such as breast and colorectal cancer (34).…”

Section: Limitationsmentioning

confidence: 91%

Molecular Docking and Simulation Studies Predict Lactyl-CoA as the Substrate for P300 Directed Lactylation

Patel¹,

Kumar²,

Lokhande³

et al. 2020

Preprint

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Background <div>According to Warburg effects, cancer cells are known to produce lactate as an end product instead of pyruvate and accumulation of lactate is linked to metabolic reprogramming. Hiowever, substantial exist in terms of the non-metabolic role of lactate including modification of histones during epigenetic regulation, in particular. </div><div>Methods</div><div> This study employs in silico molecular docking and molecular dynamics study to determine the potential mechanisms of lactylation on histone proteins that achieve epigenetic changes in cancer and non-cancer cells. Here, we tested three potential substrate sources for lactylation, namely lactate (CHEBI ID:24996), lactyl-CoA (CHEBI ID:15529) and (R)-Slactoylglutathione (PubChem ID-440018). A histone acetyltransferase p300 (HAT p300) enzyme (PDB ID: 6GYR) was considered as a potential candidate for the lactylation process. </div><div>Results </div><div>Among the studied substrates for the lactylation process, molecular docking reveals a highly efficient binding affinity (docking energy -8.6 Kcal/Mol) of lacyl-CoA with p300 enzyme. On the other hand, lactate and (R)-S-Lactoylglutathione did not shown any significant and specific binding to HAT p300 enzyme. Furthermore, molecular dynamics simulation study suggestsa stable binding of lactyl-CoA at the substrate-binding site of p300 with amino acid residues ASP-1399, HIS1402, ARG-1410, THR1411, TYR1414, TRP1436, ASP1454 and LYS1456. </div><div>Conclusion</div><div> In conclusion, our data support that lactyl-CoA is a potential substrate for lactylation carried out by the HAT p300 enzyme. However, lactyl-CoA is not detected at the physiological level in cancer and non-cancer supporting cells such as macrophage. Based on our data and existing views on lactylation, the authors propose an involvement of pro-tumor bacteria in this that converts lactate to lactyl-CoA and lactyl-CoA is shuttled to the macrophage within the tumor microenvironment. Due to lactyl-CoA entry into macrophages (anti-tumorigenic)s, lactylation process allow the transcriptional changes and achieve the M1 to M2 macrophage polarization (pro-tumor) and in turn, promotes the tumor growth and survival.</div>

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Section: Limitationsmentioning

confidence: 91%

Molecular Docking and Simulation Studies Predict Lactyl-CoA as the Substrate for P300 Directed Lactylation

Patel¹,

Kumar²,

Lokhande³

et al. 2020

Preprint

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Novel antiproliferative tripeptides block AP-1 transcriptional complex by in silico approach

Kumar

Kothari

Sinchana

et al. 2020

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BACKGROUNDThe complexity and heterogeneity at genetic, epigenetic and microenvironment levels are key attributes of tumors. Genetic heterogeneity encompasses one of key factors at transcriptional gene regulation that promote abnormal proliferation, invasiveness and metastasis. Among various key pro-tumor transcriptional complexes, activating protein-1 (AP-1) transcriptional complex controls the transcriptional expression of key oncogenes in cancer cells. Therefore, an avenue to search for a chemical inhibition approach of the AP-1 transcriptional complex is warranted in cancer therapeutics.METHODSTo achieve chemical inhibition of AP-1 transcriptional complex, we report novel tripeptides identified from the goat urine DMSO fraction as potential agents that bind to AP-1 responsive TPA element and heterodimer c-Jun:c-Fos. Novel tripeptides enriched GUDF were tested against DNA substrates to assess DNA metabolizing activity. Further, Novel tripeptides enriched GUDF were treated upon HCT-116 cells to estimate the nature of tripeptides entered into the intracellular compartment of HCT-116 cells. Here, we report on a novel methodology that employ VTGE assisted intracellular metabolite purification and is analyzed with the help of LC-HRMS technique. Post purification of intracellular metabolites that included tripeptides of GUDF, these tripeptides from DMSO and GUDF treated HCT-116 cells were subjected to molecular docking and ligand-DNA:AP-1 (PDB ID: 1FOS) interaction study by using bioinformatics tools AutoDock Vina and PyMol.RESULTSGUDF enriched with tripeptides and other metabolites show appreciable instability of DNA substrates plasmid and genomic DNA to an extent of 90%. Interestingly, LC-HRMS analysis of intracellular metabolite profiling of GUDF treated HCT-116 cells reveal the appreciable abundance of tripeptides Glu-Glu-Arg, Gly-Arg-Pro, Gln-Lys-Arg, Glu-Glu-Lys, Trp-Trp-Val. On the other hand, DMSO treated HCT-116 cells show the presence of Ser-Trp-Lys, Glu-Glu-Gln, Glu-Glu-Lys, Ser-Leu-Ser. Interestingly, GUDF treated HCT-116 cells show inhibition of proliferation by more than 70%. Among the identified intracellular tripeptides, Glu-Glu-Arg (9.1 Kcal/Mol), Gly-Arg-Pro (8.8 Kcal/Mol), and Gln-Lys-Arg (6.8) show a precise and strong binding to heptameric TPA response element 5’ TGAGTCA 3’ and key amino acid residue within the AP-1 transcriptional complex.CONCLUSIONIn summary, this study suggests the potential of novel tripeptides, those are reported from GUDF intracellularly in HCT-116 cells to destabilize the AP-1 transcriptional complex. Data indicate that cellular arrest in HCT-116 cells treated by GUDF is well supported by the molecular docking observations that destabilization of AP-1 complex is linked to reduced growth and proliferation.

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Doxorubicin-Conjugated Iron Oxide Nanoparticles Synthesized by Laser Pyrolysis: In Vitro Study on Human Breast Cancer Cells

et al. 2020

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Even today, breast cancer remains a global public problem, with a high mortality rate among women. Nanoparticle (NP) based systems are developed to enhance drug delivery, reducing the toxic effect of medicine molecules. By using iron oxide nanoparticles for cancer treatment, several advantages were highlighted: the ability to target specific locations derived from their magnetic properties and reduced side effects. The aim of this study was to examine on breast cancer cell line the anticancer potential of γ-Fe2O3 NPs loaded with doxorubicin (DOX) and stabilized with carboxymethylcellulose sodium (CMCNa). The γ-Fe2O3 NPs were synthesized by laser pyrolysis technique and their nanometric size and crystallinity were confirmed by X-ray diffraction and transmission electron microscopy. The loading efficiency was estimated by using absorption and fluorescence spectroscopy. The DOX conjugated//CMCNa coated γ-Fe2O3 NPs proved through the biological studies to have a good anticancer effect through the inhibition of tumoral cell proliferation, disruption of the cellular membrane, induction of cell death and reduced effects on normal breast cells. Our data showed that DOX cytotoxicity increases significantly when conjugated with ɣ-Fe2O3 and ɣ-Fe2O3_CMCNa, a 50% reduction of cancer cell viability was obtained with a concentration around 0.1 µg/mL.

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A novel method to detect intracellular metabolite alterations in MCF-7 cells by doxorubicin induced cell death

Cited by 3 publications

References 45 publications

Molecular Docking and Simulation Studies Predict Lactyl-CoA as the Substrate for P300 Directed Lactylation

Molecular Docking and Simulation Studies Predict Lactyl-CoA as the Substrate for P300 Directed Lactylation

Novel antiproliferative tripeptides block AP-1 transcriptional complex by in silico approach

Doxorubicin-Conjugated Iron Oxide Nanoparticles Synthesized by Laser Pyrolysis: In Vitro Study on Human Breast Cancer Cells

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