Limits...
Vascular disrupting agent drug classes differ in effects on the cytoskeleton.

Kim S, Peshkin L, Mitchison TJ - PLoS ONE (2012)

Bottom Line: CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 µM, showed none of these effects.DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4.We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America. Sujeong_Kim@hms.harvard.edu

ABSTRACT
Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 µM, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.

Show MeSH

Related in: MedlinePlus

DMXAA does not induce any morphological changes in HUVEC cells whereas CA4 treated cells immediately contract.(A) Images were collected from phase contrast time-lapse movies at indicated times. Original time-lapse imaging was taken every 30 sec for an hour in the presence of 500 µM of DMXAA, 100 nM of CA4 or without drug (control). Elapsed time indicated in hours:minutes. (B) Cell edges were drawn to measure the number of pixels within the cell edges. Individual cell areas were summed to measure total cell surface area at each time point. The total cell area for each time point was normalized by time point 0. At each condition, Cell surface areas were averaged from at least 3 different stage positions. Error bars were calculated as the standard deviation from the results of 3 independent experiments. (C) Images were collected from phase contrast time-lapse movies at 12 hours after drug treatment. Asynchronously grown cells were treated with 500 µM of DMXAA, 100 nM of CA4 or without drug (control). (D) From the time-lapse image, mitotic cells were counted after 12 hours of drug treatment. At least 200 cells from 3 different stage positions were counted at each condition. Error bars were calculated as the standard deviation from the results of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3404093&req=5

pone-0040177-g001: DMXAA does not induce any morphological changes in HUVEC cells whereas CA4 treated cells immediately contract.(A) Images were collected from phase contrast time-lapse movies at indicated times. Original time-lapse imaging was taken every 30 sec for an hour in the presence of 500 µM of DMXAA, 100 nM of CA4 or without drug (control). Elapsed time indicated in hours:minutes. (B) Cell edges were drawn to measure the number of pixels within the cell edges. Individual cell areas were summed to measure total cell surface area at each time point. The total cell area for each time point was normalized by time point 0. At each condition, Cell surface areas were averaged from at least 3 different stage positions. Error bars were calculated as the standard deviation from the results of 3 independent experiments. (C) Images were collected from phase contrast time-lapse movies at 12 hours after drug treatment. Asynchronously grown cells were treated with 500 µM of DMXAA, 100 nM of CA4 or without drug (control). (D) From the time-lapse image, mitotic cells were counted after 12 hours of drug treatment. At least 200 cells from 3 different stage positions were counted at each condition. Error bars were calculated as the standard deviation from the results of 3 independent experiments.

Mentions: To compare the effects of CA4 and DMXAA on endothelial cells we performed time-lapse imaging of HUVEC cells before drug, and in drug for 30 min (Fig. 1). Live cell microscopy can sensitively report effects on cytoskeleton, adhesion, proliferation and the signaling pathways that control them. CA4 caused rapid contraction and loss of cell-cell interaction starting within minutes of drug addition (Fig. 1A middle panel) as previously described [13]. This response was evident from retraction of cell margins and formation of thin retraction fibers. Retraction fibers terminated at the cell body in characteristic phase-dense structures (yellow arrows), similar to those previously characterized in cells rounding up for mitosis [24]. DMXAA and none treated control HUVEC cells showed no signs of retraction (Fig. 1A lower panel and upper panel). These differential effects on retraction were quantified by image analysis which revealed time-dependent retraction in CA4 but not DMXAA treated cells (Fig. 1B). Total cell surface area was decreased about 20% in CA treated cells after 30 min whereas control and 500 µM of DMXAA had no effect (Fig. 1B).


Vascular disrupting agent drug classes differ in effects on the cytoskeleton.

Kim S, Peshkin L, Mitchison TJ - PLoS ONE (2012)

DMXAA does not induce any morphological changes in HUVEC cells whereas CA4 treated cells immediately contract.(A) Images were collected from phase contrast time-lapse movies at indicated times. Original time-lapse imaging was taken every 30 sec for an hour in the presence of 500 µM of DMXAA, 100 nM of CA4 or without drug (control). Elapsed time indicated in hours:minutes. (B) Cell edges were drawn to measure the number of pixels within the cell edges. Individual cell areas were summed to measure total cell surface area at each time point. The total cell area for each time point was normalized by time point 0. At each condition, Cell surface areas were averaged from at least 3 different stage positions. Error bars were calculated as the standard deviation from the results of 3 independent experiments. (C) Images were collected from phase contrast time-lapse movies at 12 hours after drug treatment. Asynchronously grown cells were treated with 500 µM of DMXAA, 100 nM of CA4 or without drug (control). (D) From the time-lapse image, mitotic cells were counted after 12 hours of drug treatment. At least 200 cells from 3 different stage positions were counted at each condition. Error bars were calculated as the standard deviation from the results of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3404093&req=5

pone-0040177-g001: DMXAA does not induce any morphological changes in HUVEC cells whereas CA4 treated cells immediately contract.(A) Images were collected from phase contrast time-lapse movies at indicated times. Original time-lapse imaging was taken every 30 sec for an hour in the presence of 500 µM of DMXAA, 100 nM of CA4 or without drug (control). Elapsed time indicated in hours:minutes. (B) Cell edges were drawn to measure the number of pixels within the cell edges. Individual cell areas were summed to measure total cell surface area at each time point. The total cell area for each time point was normalized by time point 0. At each condition, Cell surface areas were averaged from at least 3 different stage positions. Error bars were calculated as the standard deviation from the results of 3 independent experiments. (C) Images were collected from phase contrast time-lapse movies at 12 hours after drug treatment. Asynchronously grown cells were treated with 500 µM of DMXAA, 100 nM of CA4 or without drug (control). (D) From the time-lapse image, mitotic cells were counted after 12 hours of drug treatment. At least 200 cells from 3 different stage positions were counted at each condition. Error bars were calculated as the standard deviation from the results of 3 independent experiments.
Mentions: To compare the effects of CA4 and DMXAA on endothelial cells we performed time-lapse imaging of HUVEC cells before drug, and in drug for 30 min (Fig. 1). Live cell microscopy can sensitively report effects on cytoskeleton, adhesion, proliferation and the signaling pathways that control them. CA4 caused rapid contraction and loss of cell-cell interaction starting within minutes of drug addition (Fig. 1A middle panel) as previously described [13]. This response was evident from retraction of cell margins and formation of thin retraction fibers. Retraction fibers terminated at the cell body in characteristic phase-dense structures (yellow arrows), similar to those previously characterized in cells rounding up for mitosis [24]. DMXAA and none treated control HUVEC cells showed no signs of retraction (Fig. 1A lower panel and upper panel). These differential effects on retraction were quantified by image analysis which revealed time-dependent retraction in CA4 but not DMXAA treated cells (Fig. 1B). Total cell surface area was decreased about 20% in CA treated cells after 30 min whereas control and 500 µM of DMXAA had no effect (Fig. 1B).

Bottom Line: CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 µM, showed none of these effects.DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4.We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America. Sujeong_Kim@hms.harvard.edu

ABSTRACT
Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 µM, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.

Show MeSH
Related in: MedlinePlus