Melatonin enhances the effect of ABT-737 in acute monocytic leukemia THP-1 cells

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Abstract

Melatonin (N-acetyl-5-methoxytryptamine, MEL) is a hormone synthesized by the pineal gland. Due to its oncostatic effect, it can be considered as an antitumor agent and used for combination therapy. ABT-737, a Bcl-2 inhibitor, promotes cell death after treatment with agents that induce pro-apoptotic signals. In the present study, the combined effect of MEL and ABT-737 on changes in proliferative and mitotic activity, mitochondrial membrane potential, intracellular production of reactive oxygen species (ROS) and cytosolic Ca2+ was studied. Moreover, changes in the expression of anti- and pro-apoptotic proteins (Bcl-2 and Bax), autophagy markers (LC3A/B (I, II)), endoplasmic reticulum stress markers (chaperones BIP and PDI, CHOP) were studied under these conditions. The effect of MEL together with ABT-737 led to an increase in the level of cytosolic Ca2+, intracellular production of ROS, and a decrease in the membrane potential of mitochondria. Under these conditions, the content of Bcl-2 increased, while the level of Bax decreased. The activation of CHOP stimulated autophagy and led to a decrease in the expression of BIP and PDI chaperones. These results suggest that MEL is able to enhance the effects of other chemotherapeutic agents and can be used in strategies in the treatment of cancer.

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About the authors

A. I. Lomovsky

Institute of Theoretical and Experimental Biophysics

Email: ovkres@mail.ru
Russian Federation, Pushchino, Moscow Region, 142290

Yu. L. Baburina

Institute of Theoretical and Experimental Biophysics

Email: ovkres@mail.ru
Russian Federation, Pushchino, Moscow Region, 142290

R. S. Fadeev

Institute of Theoretical and Experimental Biophysics

Email: ovkres@mail.ru
Russian Federation, Pushchino, Moscow Region, 142290

M. I. Kobyakova

Institute of Theoretical and Experimental Biophysics; Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences

Email: ovkres@mail.ru

Research Institute of Clinical and Experimental Lymphology

Russian Federation, Pushchino, Moscow Region, 142290; Novosibirsk, 630117

Ya. V. Lomovskaya

Institute of Theoretical and Experimental Biophysics

Email: ovkres@mail.ru
Russian Federation, Pushchino, Moscow Region, 142290

R. R. Krestinin

Institute of Theoretical and Experimental Biophysics

Email: ovkres@mail.ru
Russian Federation, Pushchino, Moscow Region, 142290

L. D. Sotnikova

Institute of Theoretical and Experimental Biophysics

Email: ovkres@mail.ru
Russian Federation, Pushchino, Moscow Region, 142290

O. V. Krestinina

Institute of Theoretical and Experimental Biophysics

Author for correspondence.
Email: ovkres@mail.ru
Russian Federation, Pushchino, Moscow Region, 142290

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2. Fig. 1. Concentration dependence of the cytotoxic effects of MEL (a) and ABT-737 (b) in THP-1 cells. The proportion of viable cells was determined 24 hours after the addition of substances by the intensity of resazurin reduction. Cells were seeded in a 96-well plate at a density of 5 × 103 cells per well and treated with substances at different concentrations. Data are presented as mean ± standard deviation (n = 6).

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3. Fig. 2. Viability and mitotic activity of THP-1 cells after 24 hours of incubation with MEL and ABT-737. a – Change in proliferation. b – Changes in the mitotic index in the presence of MEL and CIT. The number of viable cells in the intact culture (control, without drug treatment) was taken to be 100%. Data are presented as mean ± standard deviation (n = 6). *p < 0.05 – significant change compared to control, #p < 0.05 – significant change compared to AVT-737.

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4. Fig. 3. Effect of MEL on the viability of human monocytes. The cells were incubated with MEL for 24 hours. The number of living cells in the intact culture (control, without drug treatment) was taken as 100%. Data are presented as mean ± standard deviation (n = 6).

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5. Fig. 4. Effect of MEL and its combination with ABT-737 on the content of Bcl-2 and Bax proteins in THP-1 cells. Cells were incubated with the drugs for 24 hours. a – Immunostaining with antibodies to Bcl-2 and Bax. GAPDH was used as a protein loading control. b – Bcl-2/GAPDH ratio diagram. c – Bax/GAPDH ratio diagram. The protein level in the cell lysate without additives (100%) was used as a control. Data are presented as mean ± standard deviation (n = 4). *p < 0.05 – Significant change compared to control, #p < 0.05 – significant change compared to ABT-737.

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6. Fig. 5. Effect of MEL and ABT-737 on changes in cytosolic Ca2+ and CHOP content in THP-1 cells. Cells were incubated with the drugs for 24 hours. a – Change in cytosolic Ca2+ content. Fluorescence intensity of intact cells was used as a control, data are presented as mean ± standard deviation (n = 5). b – Immunostaining with antibodies to CHOP and GAPDH was used as a control for protein load. Data are presented as mean ± standard deviation (n = 4). *p < 0.05 – significant change compared to the corresponding control, #p < 0.05 – significant change compared to ABT-737.

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7. Fig. 6. Effect of MEL and ABT-737 on changes in ΔΨm, intracellular ROS production and autophagy markers (LC3A/B-I/II) in THP-1 cells. The cells were incubated with the drugs for 24 hours. a – Change in ΔΨm in THP-1 cells. b – Change in intracellular ROS production; data are presented as mean ± standard deviation (n = 6). c – Immunostaining with antibodies to LC3A/B-I/II, GAPDH was used as a protein load control. Data are presented as mean ± standard deviation (n = 4). *p < 0.05 – significant change compared to the corresponding control, #p < 0.05 – significant change compared to ABT-737.

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8. Fig. 7. Effect of MEL and its combination with ABT-737 on the content of BIP and PDI in THP-1 cells. Cells were incubated with the drugs for 24 hours. a – Immunostaining with antibodies to BIP and PDI, GAPDH was used as a protein load control. b – Diagram showing the ratio of BIP to GAPDH. c – Diagram showing the ratio of PDI to GAPDH. The protein level in the cell lysate without any additives served as a control (100%). Data are presented as mean ± standard deviation (n = 4). *p < 0.05 – significant change compared to control, #p < 0.05 – significant change compared to AVT-737.

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