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Tiron and trolox potentiate the autophagic cell death induced by magnolol analog Ery5 by activation of Bax in HL-60 cells

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Abstract

This study describes the mechanism of trolox and tiron induced potentiation of cytotoxicity caused by Ery5, an analog of magnolol, in human myeloid leukemia HL-60 cells. Ery5 induced cytotoxicity in HL-60 cells by involving activation of bax and cleavage of caspase 3, which contributed towards activation of both apoptotic and autophagic pathways. Trolox and tiron, even at non-toxic concentrations, contributed to the cytotoxicity of Ery5 by activation of autophagic proteins like ATG7, ATG12 and LC3-II. Z-VAD-fmk mediated reduction in the cytotoxicity and expression of autophagic proteins, further suggested that autophagy induced by Ery5 is largely dependent upon caspases. Interestingly, Ery5 induced autophagy was accompanied by the downregulation of PI3K/AKT pathway whereas, trolox and tiron strongly enhanced this effect. In addition to that treatment of cells with Ery5, trolox and tiron individually, displayed a marked upregulation of Bax. The involvement of Bax in trolox and tiron induced enhancement of the cytotoxicity of Ery5 was confirmed, when siRNA induced silencing of Bax led to increased viability of the cells and exerted a strong inhibitory effect on LC3-II accumulation and p62 degradation in case of cells treated by the combination of Ery5 with trolox or tiron. Additionally, an important role of PARP in Ery5 mediated cell death has been suggested by PARP silencing experiments, however, potentiation of autophagic cytotoxicity by trolox and tiron did not seem to be dependent on PARP-1. Therefore, Bax seems to play a vital role in trolox and tiron mediated potentiation of autophagic cell death by Ery5 in HL-60 cells.

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Abbreviations

AO:

Acridine orange

ATG:

Autophagy-related gene

BH3:

Bcl2 homology 3

LC3:

Microtubule-associated protein light chain 3

MTT:

3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide

PARP-1:

Poly(ADP-ribose) polymerase-1

PI:

Propidium iodide

Rh-123:

Rhodamine-123

ROS:

Reactive oxygen species

siRNA:

Small interfering RNA

Tiron:

4,5-Dihydroxy-1,3-benzene disulfonic acid-disodium salt

Trolox:

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

zVAD (zVAD fmk):

Benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone

SEM:

Scanning electron microscopy

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Acknowledgments

We are thankful to Council of Scientific and Industrial Research, India for financial assistance to carry out this research work. We are also grateful to University Grants Commission, India, for providing research fellowship to Suresh Kumar.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Cancer Pharmacology, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu, 180001, India

    Suresh Kumar, Ajay Kumar, Anup Singh Pathania, Santosh Kumar Guru, Parduman Raj Sharma, Shashi Bhushan, Ajit Kumar Saxena & Fayaz Malik

  2. Organic Division-I, Indian Institute of Chemical Technology, Hyderabad, 500007, India

    Srinivas Jada & H. M. Sampath Kumar

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  1. Suresh Kumar
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Kumar, S., Kumar, A., Pathania, A.S. et al. Tiron and trolox potentiate the autophagic cell death induced by magnolol analog Ery5 by activation of Bax in HL-60 cells. Apoptosis 18, 605–617 (2013). https://doi.org/10.1007/s10495-013-0805-y

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