scholarly journals Evaluation of Cocaine Effect on Endogenous Metabolites of HepG2 Cells Using Targeted Metabolomics

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4610
Author(s):  
Adamantios Krokos ◽  
Olga Deda ◽  
Christina Virgiliou ◽  
Helen Gika ◽  
Nikolaos Raikos ◽  
...  

Cocaine toxicity has been a subject of study because cocaine is one of the most common and potent drugs of abuse. In the current study the effect of cocaine on human liver cancer cell line (HepG2) was assessed. Cocaine toxicity (IC50) on HepG2 cells was experimentally calculated using an XTT assay at 2.428 mM. The metabolic profile of HepG2 cells was further evaluated to investigate the cytotoxic activity of cocaine at 2 mM at three different time points. Cell medium and intracellular material samples were analyzed with a validated HILIC-MS/MS method for targeted metabolomics on an ACQUITY Amide column in gradient mode with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring. About 106 hydrophilic metabolites from different metabolic pathways were monitored. Multivariate analysis clearly separated the studied groups (cocaine-treated and control samples) and revealed potential biomarkers in the extracellular and intracellular samples. A predominant effect of cocaine administration on alanine, aspartate, and glutamate metabolic pathway was observed. Moreover, taurine and hypotaurine metabolism were found to be affected in cocaine-treated cells. Targeted metabolomics managed to reveal metabolic changes upon cocaine administration, however deciphering the exact cocaine cytotoxic mechanism is still challenging.

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4593
Author(s):  
Deepthi Venkatachalapathy ◽  
Chandan Shivamallu ◽  
Shashanka Prasad ◽  
Gopenath Thangaraj Saradha ◽  
Parthiban Rudrapathy ◽  
...  

The edible parts of the plants Camellia sinensis, Vitis vinifera and Withania somnifera were extensively used in ancient practices such as Ayurveda, owing to their potent biomedical significance. They are very rich in secondary metabolites such as polyphenols, which are very good antioxidants and exhibit anti-carcinogenic properties. This study aims to evaluate the anti-cancerous properties of these plant crude extracts on human liver cancer HepG2 cells. The leaves of Camellia sinensis, Withania somnifera and the seeds of Vitis vinifera were collected and methanolic extracts were prepared. Then, these extracts were subjected to DPPH, α- amylase assays to determine the antioxidant properties. A MTT assay was performed to investigate the viability of the extracts of HepG2 cells, and the mode of cell death was detected by Ao/EtBr staining and flow cytometry with PI Annexin- V FITC dual staining. Then, the protein expression of BAX and BCl2 was studied using fluorescent dye to determine the regulation of the BAX and BCl2 genes. We observed that all the three extracts showed the presence of bioactive compounds such as polyphenols or phytochemicals. The W. somnifera bioactive compounds were found to have the highest anti-proliferative activity on human liver cancer cells.


2021 ◽  
Author(s):  
Jian-Bo Yang ◽  
Yun-Fei Song ◽  
Yue Liu ◽  
Hui-Yu Gao ◽  
Qi Wang ◽  
...  

Abstract Background: The raw and processed roots of Polygonum multiflorum Thunb (PM) are commonly used in clinical practice to treat diverse diseases; however, the reports of hepatotoxicity induced by Polygoni Multiflori Radix (PMR) and Polygoni Multiflori Radix Praeparata (PMRP) have emerged worldwide. Thus, it is necessary for researcher to explore the methods to improve its quality standards and further ensure its quality and treatment effect.Methods: In the present study, an ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ- MS/MS) method has been optimized and validated for the determination of dianthrones in PMR and PMRP, using bianthronyl as the internal standard. Chromatographic separation with a gradient mobile phase (A: acetonitrile and B: water containing 0.1% formic acid (v/v)) at a flow rate of 0.25 mL/min was achieved on a Waters Acquilty UPLC BEH b) C18 column (2.1 mm × 50 mm, 1.7 µm). A triple quadrupole mass spectrometer (TQMS) was operated in negative ionization mode with multiple reaction monitoring for the quantitative analysis of six dianthrones. Meanwhile, compounds 5 and 6 were further evaluated for cytotoxicity of HepaRG cells by CCK8 assay.Results: The UHPLC-QQQ-MS/MS method was first developed to simultaneous determination of six dianthrones in PMR and PMRP, namely polygonumnolides C1–C4 (1–4), trans-emodin dianthrones (5), and cis-emodin dianthrones (6). The contents of 1~6 in 90 batches of PMR were in the range of 0.027-19.04, 0.022-13.86, 0.073 -15.53, 0.034 -23.35, 0.38-83.67 and 0.29 -67.00 µg/g, respectively. The contents of 1~6 in 86 batches of commercial PMRP were in the range of 0.020-13.03, 0.051-8.94, 0.022-7.23, 0.030 -12.75, 0.098-28.54 and 0.14-27.79 µg/g, respectively. The six dianthrones were almost completely gone after reasonable processing for 24 h. Meanwhile, compounds 5 and 6 showed the inhibitory activity against HepaRG cells with the IC50 values of 10.98 and 15.45 μM, respectively. Furthermore, a systematic five-step strategy to realize the standardization of TCMs with endogenous toxicity is proposed for the first time, involving the establishment of determination methods, determination of the toxic markers, the standardization of processing method, the development of limit standards and benefit-risk assessment.Conclusion: The results of cytotoxicity evaluation of dianthrone indicated that trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be selected as the toxic markers of PMRP. Taking PMR and PMRP for example, we hope this study provided insight into the standardization and internationalization of endogenous toxic TCMs, with the main purpose of improving public health by scientifically using TCMs to treat diverse complex diseases in future.


Author(s):  
Do Huu Nghi ◽  
Vo Thi Ngoc Hao ◽  
Nguyen Thi Hong Nhung

This study discusses the results of the experimental application of high-content screening (HCS) techniques in evaluating the induction of cell-cycle arrest and apoptosis on human liver cancer cell line, Hep-G2. Accordingly, the bisbenzimide-stained cells (Hoechst 33342; 350 to 500 nM) were analyzed by using an Olympus scanˆR HCS-system to determine the cell-cycle phases (G1, S, and G2/M) and apoptosis as well. As a result, the cell-cycle arrest could be indicated by an increase in G2/M population of Hep-G2 cells after 24h exposure to zerumbone (Zer4; 9 µg/mL) and a similar observation could be made for paclitaxel (Pac; 4 µg/mL) as a reference substance. Keywords Apoptosis, cell-cycle arrest, high-content screening, human liver cancer cell line Hep-G2. References [1] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646–674.[2] M. Malumbres, M. Barbacid, Cell cycle, CDKs and cancer: a changing paradigm, Nat. Rev. Cancer 9 (2009) 153–166.[3] S. Diermeier-Daucher, et al., Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry, Cytometry A 75 (2009) 535-546.[4] J. Essers, et al., Nuclear dynamics of PCNA in DNA replication and repair, Mol. Cell Biol 25 (2005) 9350- 9359. [5] V. Roukos, et al., Dynamic recruitment of licensing factor Cdt1 to sites of DNA damage. J. Cell Sci. 124 (2011) 422-434.[6] M. Hesse, et al., Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle, Nat. Commun 3 (2012) 1076. doi: 10.1038/ncomms2089.[7] P. Cappella, F. Gasparri, M. Pulici, J. Moll, A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining, Cytometry A 73 (2008) 626–636. [8] S. Diermeier-Daucher, et al., Cell type specific applicability of 5-ethynyl-2’-deoxyundine (EdU) for dynamic proliferation assessment in flow cytometry, Cytometry A 75 (2009) 535–546.[9] T. Yokochi, D.M. Gilbert, Replication labeling with halogenated thymidine analogs, Curr. Protoc. Cell Biol, 35 (2007) 22.10.1–22.10.14. [10] T.J. McGarry, M.W. Kirschner, Geminin, an inhibitor of DNA replication, is degraded during mitosis, Cell 93 (1998) 1043–1053. [11] H. Nishitani, S. Taraviras, Z. Lygerou, T. Nishimoto, The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase. J. Biol. Chem 276 (2001) 44905–44911.[12] J. Pines, T. Hunter, Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B, Nature 346 (1990) 760–763. [13] A. Stathopoulou, et al., Cdt1 is differentially targeted for degradation by anticancer chemotherapeutic drugs. PLoS ONE 7, e34621 (2012). [14] M. Hesse, A. Raulf, G.A. Pilz, C. Haberlandt, A.M. Klein, R. Jabs, H. Zaehres, C.J. Fügemann, K. Zimmermann, J. Trebicka, A. Welz, A. Pfeifer, W. Röll, M.I. Kotlikoff, C. Steinhäuser, M. Götz, H.R. Schöler, B.K. Fleischmann, Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle, Nat. Commun 3 (2012): 1076.[15] D.A. Ridenour, M.C. McKinney, C.M. Bailey, P.M. Kulesa, CycleTrak: a novel system for the semiautomated analysis of cell cycle dynamics. Dev. Biol 365 (2012) 189–195. [16] A. Roukos, et al., Cell cycle staging of individual cells by fluorescence microscopy, Nat. Protoc 10 (2015) 334-348.[17] E. Harlow, D. Lane, Fixing attached cells in paraformaldehyde, CSH Protoc 3 (2006) doi: 10.1101/pdb.prot4294.[18] G. Mazzini, M. Danova, Fluorochromes for DNA staining and quantitation, Method. Mol. Biol 1560 (2017) 239-259.[19] A. Gottfried, E. Weinhold, Sequence-specific covalent labelling of DNA, Biochem. Soc. Trans 39 (2011) 623-628.[20] J. Bucevičius, G. Lukinavičius, R. Gerasimaitė, The use of Hoechst dyes for DNA staining and beyond, Chemosensor 6 (2018) 1-18.[21] V. Kumar, A.K. Abbas, J.C. Aster, Robbins and Cotran Pathologic Basis of Disease, Ninth ed., Elsevier/Saunders, Philadelphia (2015).[22] N.A. Jensen et al., Establishment of a high content assay for the identification and characterisation of bioactivities in crude bacterial extracts that interfere with the eukaryotic cell cycle, J. Biotechnol 140 (2009) 124-134.[23] H.S. Rahman, et al., Zerumbone induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in Jurkat cell line, Nat. Prod. Commun 9 (2014) 1237-1242.[24] S.I. Abdelwahab, et al., Zerumbone inhibits interleukin-6 and induces apoptosis and cell cycle arrest in ovarian and cervical cancer cells, Intern. Immunopharm 12 (2012) 594-602.[25] M. Xian, et al., Zerumbone, A bioactive sesquiterpene, induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas- and mitochondria-mediated pathway, Cancer Sci 98 (2007) 118-126.[26] A. Sehrawat, et al., Zerumbone causes Bax-and Bak-mediated apoptosis in human breast cancer cells and inhibits orthotopic xenograft growth in vivo, Breast Cancer Res. Treat. 136 (2012) 429-441.[27] Y.Z. Zhou, et al., Zerumbone induces G1 cell cycle arrest and apoptosis in cervical carcinoma cells, Int. J. Clin. Exp. Med. 10 (2017) 6640-6647.


2021 ◽  
Vol 22 ◽  
Author(s):  
Jian Le ◽  
Yuehua Liao ◽  
Shengni Li ◽  
Xiujuan Chen ◽  
Zhanying Hong

Background: Pantoprazole and atorvastatin are often used jointly in the clinic. The drug-drug interaction of pantoprazole and atorvastatin is worthy of being investigated. Objective: A highly rapid, sensitive, and selective LC-MS/MS method was developed for simultaneous quantification of pantoprazole and atorvastatin in rat plasma. Methods: Omeprazole and atorvastatin-d5 were used as the internal standards (ISs) of pantoprazole and atorvastatin, respectively. Simple protein precipitation was used to extract analytes from 50.0 μL plasma samples. Results: The chromatographic separation was achieved on a C18 column and the total chromatographic run time was 3.2 min. Acquisition of mass spectrometric data was performed on a triple-quadrupole mass spectrometer in multiple- reaction-monitoring (MRM) mode with an ESI source using the transition m/z 384→ 200 for pantoprazole and m/z 559.4→ 440.2 for atorvastatin, respectively. The method was validated over the concentration range of 20.0 ∼ 5000 ng/mL for pantoprazole and 1.00 ∼ 250 ng/mL for atorvastatin. All the validation results, including linearity, specificity, precision, accuracy, extraction recovery, matrix effect, and stability, met the acceptance criteria as per FDA guidelines. Conclusion: This method was successfully applied to a pharmacokinetic interaction study in Wistar rats. The results revealed significant evidence for the drug-drug interaction between pantoprazole and atorvastatin.


2019 ◽  
Vol 14 (7) ◽  
pp. 1934578X1986031
Author(s):  
Xiao Yang ◽  
Ruixue Deng ◽  
Pu Liu ◽  
Jiangxia Hu ◽  
Wentian Niu ◽  
...  

As the main by-product during the oil production of peony seeds, the episperm is traditionally used as a lead component in folk herbal formulas for the cancer treatment in China. However, the investigation of its phytochemical foundation underlying anticancer effects remains an ongoing challenge. The work therefore determined growth inhibition activities of 8 solvent extracts of peony episperms in the human liver cancer cell line. This activity was then mapped onto the secondary metabolite profile of extracts by principal components analysis (PCA). The top 3 principal components of High Performance Liquid Chromatograph (HPLC)-PCA map discriminated extract activities mainly based on the differential content of 5 stilbene compounds, which were then tested individually. The trans-ε-viniferin, gnetin-H, suffruticosol A, suffruticosol B, and suffruticosol C were thus determined as growth inhibitors and apoptosis inducers of human liver cancer cells with activities comparable to that of the antineoplastic cisplatin. A partial least squares regression-HPLC model was also constructed for the prediction of inhibitory effects of peony episperm extracts. These results expand the fundamental understanding of the peony episperms and support its current medicinal uses in China. Moreover, PCA-mediated secondary metabolite mapping was proved to be an efficient approach to qualify biomedical products required for pharmaceutical and medicinal uses.


2016 ◽  
Vol 12 (2) ◽  
pp. 995-1000 ◽  
Author(s):  
Le-Wen Shao ◽  
Li-Hua Huang ◽  
Sheng Yan ◽  
Jian-Di Jin ◽  
Shao-Yan Ren

Medicine ◽  
2020 ◽  
Vol 99 (20) ◽  
pp. e20290
Author(s):  
Jing Li ◽  
Zhi-ye Liu ◽  
Hai-bo Yu ◽  
Qing Xue ◽  
Xiu-sheng Qu

Molecules ◽  
2019 ◽  
Vol 24 (11) ◽  
pp. 2031 ◽  
Author(s):  
Bing Zhang ◽  
Dongli Qi ◽  
Xiuping Deng ◽  
Zhe Ma ◽  
Yumei Wu ◽  
...  

A UHPLC-QQQ-MS/MS method was developed to quantify the significant constituents in Wen-Dan Decoction (WDD), a traditional Chinese medicine. Analysis of 19 compounds was conducted on an ACQUITY UPLC® BEH C18 Column (2.1 × 50 mm, 1.7 μm) using elution with a gradient elution of acetonitrile and 0.05% (v/v) formic acid in water. A triple quadrupole mass spectrometer was operated in negative ionization mode and positive ionization mode by multiple reaction monitoring (MRM), respectively. All calibration curves showed acceptable linearity (r ≥ 0.9950). The RSDs of intra- and inter-day precisions of low, mid and high concentrations were ≤ 8.88%. The repeatabilities (RSDs ≤ 7.17%) and stabilities (RSD ≤ 4.79%) of the samples were qualified. The recoveries were found in the range of 93.07 ± 3.86 to 103.98 ± 2.98% with the RSD varying between 1.30 and 7.86%. The final rapid, sensitive, precise, accurate and reliable UHPLC-QQQ-MS/MS method was used for the simultaneous quantification of 19 constituents in WDD and its commercial preparations. The strategy of combining the contents of the 19 chemicals in a daily dose of the WDD preparations with the hierarchical cluster analysis and the 3D principal component analysis was employed to effectively distinguish the WDD preparations provided by the different suppliers, which represents a contribution to the evaluation and control of the quality of WDD (or other decoctions consisting of the same herbs) and the preparations of WDD in other dosage forms such as tablets and granules.


Sign in / Sign up

Export Citation Format

Share Document