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Modulation of cell death in human colorectal and breast cancer cells through a manganese chelate by involving GSH with intracellular p53 status

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Abstract

Chemotherapy is central to current treatment modality especially for advanced and metastatic colorectal and breast cancers. Targeting the key molecular events of the neoplastic cells may open a possibility to treat cancer. Although some improvements in understanding of colorectal and breast cancer treatment have been recorded, the involvement of glutathione (GSH) and dependency of p53 status on the modulation of GSH-mediated treatment efficacy have been largely overlooked. Herein, we tried to decipher the underlying mechanism of the action of Mn-N-(2-hydroxyacetophenone) glycinate (MnNG) against differential p53 status bearing Hct116, MCF-7, and MDA-MB-468 cells on the backdrop of intracellular GSH level and reveal the role of p53 status in modulating GSH-dependant abrogation of MnNG-induced apoptosis in these cancer cells. Present study discloses that MnNG targets specifically wild-type-p53 expressing Hct116 and MCF-7 cells by significantly depleting both cytosolic, mitochondrial GSH, and modulating nuclear GSH through Glutathione reductase and Glutamate–cysteine ligase depletion that may in turn induce p53-mediated intrinsic apoptosis in them. Thus GSH addition abrogates p53-mediated apoptosis in wild-type-p53 expressing cells. GSH addition also overrides MnNG-induced modulation of phase II detoxifying parameters in them. However, GSH addition partially replenishes the down-regulated or modulated GSH pool in cytosol, mitochondria, and nucleus, and relatively abrogates MnNG-induced intrinsic apoptosis in p53-mutated MDA-MB-468 cells. On the contrary, although MnNG induces significant cell death in p53-null Hct116 cells, GSH addition fails to negate MnNG-induced cell death. Thus p53 status with intracellular GSH is critical for the modulation of MnNG-induced apoptosis.

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Abbreviations

ABC transporter:

ATP-binding cassette transporter

ATP:

Adenosine triphosphate

BSO:

DL-buthionine (S,R) sulfoximine

CAT:

Catalase

CRC:

Colorectal cancer

Dox:

Doxorubicin

EA:

Ethacrynic acid

EAC/Dox:

Doxorubicin resistant Ehrlich ascites carcinoma

EAC/S:

Doxorubicin sensitive Ehrlich ascites carcinoma

GPx:

Glutathione peroxidase

GST:

Glutathione-S-transferase

GSH:

Reduced glutathione

MnNG:

Manganese N-(2-hydroxy acetophenone) glycinate

MRP:

Multidrug resistance proteins

P-gp:

P-glycoprotein

PFT-α:

Pifithrin-α

SOD:

Superoxide dismutase

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Acknowledgements

This investigation received financial support from the Council of Scientific and Industrial Research (CSIR), New Delhi, No. 09/030/(0069)/2012 EMR-I and Indian Council of Medical Research (ICMR), New Delhi, No. 74/10/2014-PERS. (EMS). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

Conceived and designed the experiments: SKC, KB. Performed the experiments: KB, SD. Analyzed the data: KB. Contributed reagents/materials/analysis tools: SM, SM, JB. Wrote the paper: KB, SKC.

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Authors and Affiliations

  1. Department of In Vitro Carcinogenesis and Cellular Chemotherapy, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata, 700 026, India

    Kaushik Banerjee, Satyajit Das, Jaydip Biswas & Soumitra Kumar Choudhuri

  2. Division of Molecular Medicine, Bose Institute, Kolkata, India

    Saikat Majumder & Subrata Majumdar

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  1. Kaushik Banerjee
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  2. Satyajit Das
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Correspondence to Soumitra Kumar Choudhuri.

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11010_2016_2896_MOESM1_ESM.tif

Supplementary Fig. 1: MnNG does not induce extrinsic apoptosis in differential p53 status bearing cancer cells. (A) Hct116 p53WT and Hct116 p53−/− cells were either treated with MnNG (0.8 µM) or treated with rIFN-γ for indicated time were labeled with anti FasR antibody. Immunofluorescence analysis was performed by flow cytometry.. Similarly, (B) both MCF-7 p53WT and MDA-MB-468 p53Mut cells were either treated with MnNG or treated with rIFN-γ for indicated time periods were labeled with anti FasR antibody. Here we also represent the data of three independent experiments. Supplementary material 1 (TIFF 3360 kb)

Appendix: Materials and methods for Supplementary Fig. 1

Appendix: Materials and methods for Supplementary Fig. 1

Determination of FasR expression by Facs analysis

CD95 and FasR expression of cancer cells were assessed following MnNG treatment. Appropriate untreated (MnNG untreated) and positive (human recombinant IFN-γ treated) controls were maintained for comparison. After drug (MnNG) and IFN-γ treatment, cancer cells were rinsed twice with PBS and were incubated with anti FasR primary antibody at room temperature (RT) for 45 min. Following incubation, cells were washed thrice with PBS and then with 3% FBS. Cells were then incubated with FITC-conjugated secondary antibody at RT for 30 min. Negative controls were incubated with secondary antibodies only. Final volume was adjusted to 500 ml with PBS, and labeling was analyzed by flow cytometry by using a FACS or fluorescence-activated cell sorter and CELLQuest software (BD Biosciences, San Jose, CA). A minimum of 104 cells were counted for each sample. The gate was set to exclude approximately 99.5% of the negative control cells. At least duplicate independent measurements of the effects of each treatment were performed.

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Banerjee, K., Das, S., Majumder, S. et al. Modulation of cell death in human colorectal and breast cancer cells through a manganese chelate by involving GSH with intracellular p53 status. Mol Cell Biochem 427, 35–58 (2017). https://doi.org/10.1007/s11010-016-2896-6

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