Research sector 'Membranology and Cytology', ESC 'Institute of biology'
Contributions
Type: Publication Only
Background
Maleimide derivative (MI-1, 1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1?-pyrrole-2,5-dione synthesized in Taras Shevchenko National University of Kyiv, Ukraine) is a competitive inhibitor of PDK1, VEGF?R1,2,3, EGF(h), Src(h), Syk(h) and other protein kinases. MI-1 inhibits the proliferation of the colon cancer cells (HCT-116, SW-620) in vitro and in vivo decreases the number of colon tumors and normalizes an increased number of monocytes and platelets in blood of rat with 1,2-dimethylhydrazine-induced colon carcinogenesis. MI-1 inhibits proliferation and mitotic activity, activates apoptosis, causes shift of cells from G2/M+S to Go/G1 stage of human monocytic cells U-937.
Aims
The aim of this study was to investigate the effects of MI-1 on the morphofunctional state of U-937 in combination with agonist protein kinase C phorbol-13-miristat-13-acetat.
Methods
Cells U-937 were incubated (5 % CO2, 100 humidity, 37 oC) in 96 well plates in RPMI-1640 («Sigma», USA) with 10 % FBS («Sigma»), 2 mM Glutamine and 40 μg/ml gentamicin. MI-1 at final concentration of 0.008 mM or 0.016 mM in combination with 100 nM phorbol-12-myristate-13-acetate (PMA) were added to cell cultures and incubated for 24 or 48 hours. The number of live and dead cells was calculated in hemocytometer with 0.1% trypan blue staining. Percent of apoptotic, mitotic or necrotic cells was calculated per 1000 cells in cytospin prepared specimens after Pappenheim’s stained. SPSS 16.0 One-Way ANOVA followed by Hochberg and DunnettT3 tests were used. Mean and SD are presented.
Results
MI-1 at 0.008 mM with 100 nM PMA tended to reduce proliferation of U937 by 22% (0,67±0,18; p=0.064), at 0.016 mM and PMA reduced proliferation by 35% (0,56±0,12; p=0.004) in comparison with PMA only (PMA-control 0,86±0,13) after 24 hours exposure. Number of dead cells didn’t differ between the indicated groups (0,08±0,03, p=0.944; 0,12±0,03, p=0,12 vs. PMA-control 0,07±0,05).
Extension of treatment for 48 hours with 0.008 mM of MI-1 and PMA reduced proliferation by 40% (0,57±0,13; p<0.001), with 0.016 mM - by 62% (0,36±0,09; p<0.001) vs. PMA-control (0,95±0,13). Number of dead cells at 0.008 mM of MI-1 (0,08±0,04; p=0.94) didn’t differ from the control (0,10±0,04), at 0.016 mM - increased (0,32±0,09; p=0.008).
MI-1 at 0.008 mM and PMA tended to increase by 26% (2,57±0,32%; p=0.245) and at 0.016 mM increased the number of apoptotic cells in 8,5 times (17,17±2,52%, p=0.018) vs. PMA only (2,03±0,31%). The number of apoptotic cells was increased after 48 hours exposure of MI-1 and PMA (10,17±0,15%, p=0.001; 10,73±0,64%, p<0.001 respectively) vs. PMA-control (2,00±0,53%).
MI-1 at both investigated concentration with PMA decreased the number of mitotic cells (1,90±0,10%, p=0.005; 1,60±0,10%, p=0.001 respectively) vs. PMA-control (2,50±0,10%) after 24 hours exposure. 48 hours exposure of MI-1 at 0.008 mM and PMA reduced by 20% (1,07±0,15% p=0.526), at 0,016 mM - by 47% of the mitotic cells (0,70±0,10%, p=0.125) vs. PMA-control (1,33±0,31%).
The number of necrotic cells after 24 exposure of MI-1 at 0.008 mM and PMA (2,63±0,15%, p=0.001) lower from the PMA-control (5,10±0,36) and tended to reduce at 0.016 mM (4,27±0,31%, p=0.060) but after 48 hours exposure tended to increase (1,90±0,36% p=0.592, 2,17±0,40% p=0.060) vs. PMA (0,87±0,31%).
Summary
MI-1 in combination with PMA reinforces oppression proliferation activity of U-937, reduces mitotic activity and activates apoptosis through inhibiting of VEGFR-, EGFR- and of non-receptor PDK1-, Src- and Syk- kinases that are involved in hematopoiesis.
Keyword(s): Apoptosis, Monocyte, Proliferation, Protein kinase C
Session topic: Publication Only
Type: Publication Only
Background
Maleimide derivative (MI-1, 1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1?-pyrrole-2,5-dione synthesized in Taras Shevchenko National University of Kyiv, Ukraine) is a competitive inhibitor of PDK1, VEGF?R1,2,3, EGF(h), Src(h), Syk(h) and other protein kinases. MI-1 inhibits the proliferation of the colon cancer cells (HCT-116, SW-620) in vitro and in vivo decreases the number of colon tumors and normalizes an increased number of monocytes and platelets in blood of rat with 1,2-dimethylhydrazine-induced colon carcinogenesis. MI-1 inhibits proliferation and mitotic activity, activates apoptosis, causes shift of cells from G2/M+S to Go/G1 stage of human monocytic cells U-937.
Aims
The aim of this study was to investigate the effects of MI-1 on the morphofunctional state of U-937 in combination with agonist protein kinase C phorbol-13-miristat-13-acetat.
Methods
Cells U-937 were incubated (5 % CO2, 100 humidity, 37 oC) in 96 well plates in RPMI-1640 («Sigma», USA) with 10 % FBS («Sigma»), 2 mM Glutamine and 40 μg/ml gentamicin. MI-1 at final concentration of 0.008 mM or 0.016 mM in combination with 100 nM phorbol-12-myristate-13-acetate (PMA) were added to cell cultures and incubated for 24 or 48 hours. The number of live and dead cells was calculated in hemocytometer with 0.1% trypan blue staining. Percent of apoptotic, mitotic or necrotic cells was calculated per 1000 cells in cytospin prepared specimens after Pappenheim’s stained. SPSS 16.0 One-Way ANOVA followed by Hochberg and DunnettT3 tests were used. Mean and SD are presented.
Results
MI-1 at 0.008 mM with 100 nM PMA tended to reduce proliferation of U937 by 22% (0,67±0,18; p=0.064), at 0.016 mM and PMA reduced proliferation by 35% (0,56±0,12; p=0.004) in comparison with PMA only (PMA-control 0,86±0,13) after 24 hours exposure. Number of dead cells didn’t differ between the indicated groups (0,08±0,03, p=0.944; 0,12±0,03, p=0,12 vs. PMA-control 0,07±0,05).
Extension of treatment for 48 hours with 0.008 mM of MI-1 and PMA reduced proliferation by 40% (0,57±0,13; p<0.001), with 0.016 mM - by 62% (0,36±0,09; p<0.001) vs. PMA-control (0,95±0,13). Number of dead cells at 0.008 mM of MI-1 (0,08±0,04; p=0.94) didn’t differ from the control (0,10±0,04), at 0.016 mM - increased (0,32±0,09; p=0.008).
MI-1 at 0.008 mM and PMA tended to increase by 26% (2,57±0,32%; p=0.245) and at 0.016 mM increased the number of apoptotic cells in 8,5 times (17,17±2,52%, p=0.018) vs. PMA only (2,03±0,31%). The number of apoptotic cells was increased after 48 hours exposure of MI-1 and PMA (10,17±0,15%, p=0.001; 10,73±0,64%, p<0.001 respectively) vs. PMA-control (2,00±0,53%).
MI-1 at both investigated concentration with PMA decreased the number of mitotic cells (1,90±0,10%, p=0.005; 1,60±0,10%, p=0.001 respectively) vs. PMA-control (2,50±0,10%) after 24 hours exposure. 48 hours exposure of MI-1 at 0.008 mM and PMA reduced by 20% (1,07±0,15% p=0.526), at 0,016 mM - by 47% of the mitotic cells (0,70±0,10%, p=0.125) vs. PMA-control (1,33±0,31%).
The number of necrotic cells after 24 exposure of MI-1 at 0.008 mM and PMA (2,63±0,15%, p=0.001) lower from the PMA-control (5,10±0,36) and tended to reduce at 0.016 mM (4,27±0,31%, p=0.060) but after 48 hours exposure tended to increase (1,90±0,36% p=0.592, 2,17±0,40% p=0.060) vs. PMA (0,87±0,31%).
Summary
MI-1 in combination with PMA reinforces oppression proliferation activity of U-937, reduces mitotic activity and activates apoptosis through inhibiting of VEGFR-, EGFR- and of non-receptor PDK1-, Src- and Syk- kinases that are involved in hematopoiesis.
Keyword(s): Apoptosis, Monocyte, Proliferation, Protein kinase C
Session topic: Publication Only