Effect of Chloroquine on Expression of Apoptosis and Autophagy Genes in MOLT-3 and IMR-32 Cells

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Abstract

The goal of the study was to compare an influence of chloroquine (CQ) on expression of apoptosis and autophagy genes in the cells of two tumor cells – leukemia MOLT-3 and neuroblastoma IMR-32 cultured in complete growth and serum-free RPMI-1640 and DMEM media, respectively, for 24 and 48 hours. The viability of cells was evaluated by MTT method, gene expression – by real time PCR. For MTT test, the cells were incubated with 10–100 µМ CQ. The expression of apoptosis (CASP3, BAX, BCL2) and autophagy (ULK1, BECN1, MAP1LC3B) genes was studied using 30 and 50 µМ CQ, which exerted considerable inhibitory effect on viability of cells of both lines, but did not promote their complete death. The sensitivity of both cell lines to CQ was higher in serum-free medium, however, the expression of apoptosis and autophagy genes substantially differed between them. In MOLT-3 cells, mRNA levels of pro-apoptotic genes CASP3 and BAX increased after 24-h incubation in serum-free medium, whereas in IMR-32 cells the expression of these genes increased only after 48-h in the presence of higher CQ concentration. In the cells of both lines 24-h CQ treatment resulted in enhanced expression of anti-apoptotic gene BCL2. In MOLT-3 cells, the absence of nutrients different combinations stimulated the genes of all three autophagy stages ULK1, BECN1 и MAP1LC3B, but none of applied treatment schemes did not affect the expression of ULK1 and MAP1LC3B genes in IMR-32 cells. Overall, 24-h culture with CQ under conditions of serum starvation appears to be more optimal for modulation of autophagy in MOLT-3 cells. In IMR-32 cells, CQ does not exert considerable influence on expression of autophagy genes, and their decreased viability is associated with activation of other mechanisms.

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About the authors

E. S. Prokopenko

Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS; Saint Petersburg State University

Author for correspondence.
Email: nagalak@mail.ru
Russian Federation, Saint Petersburg; Saint Petersburg

T. V. Sokolova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS

Email: nagalak@mail.ru
Russian Federation, Saint Petersburg

O. V. Nadey

Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS

Email: nagalak@mail.ru
Russian Federation, Saint Petersburg

A. D. Trubnikova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS

Email: nagalak@mail.ru
Russian Federation, Saint Petersburg

N. I. Agalakova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS

Email: nagalak@mail.ru
Russian Federation, Saint Petersburg

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2. Fig. 1. Viability of MOLT-3 and IMR-32 cells after culturing with 10-100 μM CQ in complete and serum-free media for 24 and 48 h. Mean values ± SE (n = 8 - 15) for each cell line are presented. The viability level of cells growing under control conditions was taken as 100% and is shown as a red dashed line. CQ (10-100) - Chloroquine at different concentrations. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control (one-factor ANOVA analysis for multiple comparisons with Dunnett's a posteriori test).

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3. Fig. 2. Effect of 30 and 50 μM CQ on relative CASP3 gene expression in MOLT-3 and IMR-32 cells in complete and serum-free RPMI-1640 and DMEM media, respectively, after 24- and 48-h culturing. Data were normalised relative to the CNOT4 reference gene. Medians and interquartile ranges are shown (n = 5 - 7). Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p ˂ 0.05, ** p ˂ 0.01 compared to control (Kraskell-Wallis test for multiple comparisons with Dunn's a posteriori test).

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4. Fig. 3. Changes in BAX gene expression in MOLT-3 and IMR-32 cells after 24- and 48-h incubation with different concentrations of CQ in media with and without serum. Medians and interquartile ranges are shown (n = 5 - 7). Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p ˂ 0.05, ** p < 0.01 compared to control (Kraskell-Wallis test with Dunn's posterior test).

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5. Fig. 4. Effect of CQ on BCL2 gene expression in MOLT-3 and IMR-32 cell lines after 24- and 48-h incubation in complete and serum-free media. Medians and interquartile ranges are shown (n = 5 - 7). Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p ˂ 0.05, ** p < 0.01 compared to control for cells of each line (Kraskell-Wallis test with Dunn's posterior test).

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6. Fig. 5. ULK1 autophagy gene mRNA levels after treatment of MOLT-3 and IMR-32 cells with different concentrations of CQ in media with and without serum for 24 and 48 h. Medians and interquartile ranges are shown (n = 5 - 7). Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p ˂ 0.05 compared to control (Kraskell-Wallis test with Dunn's posterior test).

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7. Fig. 6. Effect of 30 and 50 μM CQ on BECN1 gene expression in MOLT-3 and IMR-32 cell lines after 24- and 48-h incubation in complete and serum-free media. Medians and interquartile ranges are shown (n = 5 - 7). Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p ˂ 0.05, ** p < 0.01 compared to control for cells of each line (Kraskell-Wallis test with Dunn's posterior test).

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8. Fig. 7. MAP1LC3B autophagy gene expression in MOLT-3 and IMR-32 cells after incubation with different concentrations of CQ in different media. Medians and interquartile ranges (n = 5 - 7) are shown. Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p < 0.05, ** p < 0.01 compared to control for cells of each line (Kraskell-Wallis test with Dunn's posterior test).

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