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Superoxide activates mTOR–eIF4E–Bax route to induce enhanced apoptosis in leukemic cells

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Abstract

Mammalian target of rapamycin (mTOR) is a central kinase that regulates cell survival, proliferation and translation. Reactive oxygen species (ROS) are second messengers with potential in manipulating cellular signaling. Here we report that two ROS generating phytochemicals, hydroxychavicol and curcumin synergize in leukemic cells in inducing enhanced apoptosis by independently activating both mitogen activated protein kinase (MAPK) (JNK and P38) and mTOR pathways. Low level transient ROS generated after co-treatment with these phytochemicals led to activation of these two pathways. Both mTOR and MAPK pathways played important roles in co-treatment-induced apoptosis, by knocking down either mTOR or MAPKs inhibited apoptosis. Activation of mTOR, as evident from phosphorylation of its downstream effector eukaryotic translation initiation factor 4E-binding protein 1, led to release of eukaryotic translation initiation factor 4E (eIF4E) which was subsequently phosphorylated by JNK leading to translation of pro-apoptotic proteins Bax and Bad without affecting the expression of anti-apoptotic protein Bcl-xl. Our data suggest that mTOR and MAPK pathways converge at eIF4E in co-treatment-induced enhanced apoptosis and provide mechanistic insight for the role of mTOR activation in apoptosis.

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Abbreviations

ROS:

Reactive oxygen species

JNK:

c-Jun-N-terminal kinase

FITC:

Fluorescein isothiocyanate

GM-CSF:

Granulocyte-macrophage colony-stimulating factor

mTOR:

Mammalian target of rapamycin

MAPK:

Mitogen activated protein kinase

eIF4E:

Eukaryotic translation initiation factor 4E

4E-BP1:

Eukaryotic translation initiation factor 4E-binding protein 1

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Acknowledgments

We thank Dr. Carlo Gambacorti-Passerini (Instituto Nazionale Tumori, Milan, Italy) for providing KCL22 cell line. We are also thankful to Mr. Surjendu Bikash Debnath for technical assistance and Dr. Anupam Banerjee for confocal microscopy. This work is financially supported by Council of Scientific and Industrial Research (CSIR), New Delhi, India.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Nabendu Biswas

    Present address: Department of Biotechnology, Presidency University, Kolkata, India

Authors and Affiliations

  1. Division of Cancer Biology and Inflammatory Disorder, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), 4, Raja S.C. Mullick Road, Jadavpur, Kolkata, 700032, India

    Jaydeep Chaudhuri, Avik Acharya Chowdhury, Nabendu Biswas, Anirban Manna & Santu Bandyopadhyay

  2. Division of Chemistry, CSIR-IICB, Kolkata, India

    Saurav Chatterjee, Tulika Mukherjee & Parasuraman Jaisankar

  3. The Institute of Hematology and Transfusion Medicine, Medical College, Kolkata, India

    Utpal Chaudhuri

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Fig. 1

a Viability of normal cell lines were assessed by MTT after indicated treatment for 48 h. b Viability of PBMC isolated from one normal donor and two CML patients was determined by Trypan blue dye exclusion. **p < 0.01. (TIFF 8448 kb)

Fig. 2

a Bar diagram representation of annexin V positive cells of Fig. 1C, 1D and 1E. Data represent mean ± SD of three experiments. (TIFF 2565 kb)

Fig. 3

a K562 cells were treated with graded concentrations of HCH as indicated and were subjected to MTT assay for determination of viability. *p < 0.01, **p < 0.001. b Whole cell lysates of K562 cells treated with varying concentrations of HCH alone were subjected to immunoblotting. (TIFF 8440 kb)

Fig. 4

Intracellular flow cytometry analysis of pro-apoptotic and anti-apoptotic proteins in K562 cells which were transfected with mTOR siRNA followed by incubation with HCH and Cur simultaneously. Open histograms represent staining with isotype matched control antibodies. Filled histograms represent staining with specific antibodies. Values in the boxes represent specific mean fluorescence intensity after subtracting control values. (TIFF 8439 kb)

Fig. 5

a K562 cells were pretreated with NAC (5 mM) and MnTBAP (100 μM) for 1 h before treatment with HCH plus Cur in combination. MitoSOX™ Red fluorescence was then measured by flow cytometry 30 min after co-treatment. b K562 cells were fractionated into mitochondrial and cytosolic fractions after indicated treatments. Fractions were immunoblotted for cytochrom c. (TIFF 8440 kb)

Fig. 6

Intracellular flow cytometry analysis for pro-apoptotic and anti-apoptotic proteins in K562 cells after transfection with eIF4E siRNA followed by treatment with HCH plus Cur. (TIFF 328 kb)

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Chaudhuri, J., Chowdhury, A.A., Biswas, N. et al. Superoxide activates mTOR–eIF4E–Bax route to induce enhanced apoptosis in leukemic cells. Apoptosis 19, 135–148 (2014). https://doi.org/10.1007/s10495-013-0904-9

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