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Dasatinib synergizes with doxorubicin to block growth, migration, and invasion of breast cancer cells.

Pichot CS, Hartig SM, Xia L, Arvanitis C, Monisvais D, Lee FY, Frost JA, Corey SJ - Br. J. Cancer (2009)

Bottom Line: We also tested the effects of these drugs on the actin cytoskeleton and associated signalling pathways.Importantly, combination treatment with doxorubicin resulted in synergistic growth inhibition in all cell lines and blocked the migration and invasion of the highly metastatic, triple-negative MDA-MB-231 cell line.The observed synergy between dasatinib and doxorubicin warrants the re-evaluation of dasatinib as an effective agent in multi-drug regimens for the treatment of invasive breast cancers.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX, USA.

ABSTRACT

Background: Src family kinases control multiple cancer cell properties including cell cycle progression, survival, and metastasis. Recent studies suggest that the Src inhibitor dasatinib blocks these critical cancer cell functions.

Methods: Because the molecular mechanism of action of dasatinib in breast cancers has not been investigated, we evaluated the effects of dasatinib as a single agent and in combination with the commonly used chemotherapeutic doxorubicin, on the proliferation, viability, and invasive capacity of breast cancer cells lines earlier categorised as dasatinib-sensitive (MDA-MB-231) and moderately resistant (MCF7 and T47D). We also tested the effects of these drugs on the actin cytoskeleton and associated signalling pathways.

Results: The cell lines tested varied widely in sensitivity to growth inhibition (IC(50)=0.16-12.3 microM), despite comparable Src kinase inhibition by dasatinib (IC(50)=17-37 nM). In the most sensitive cell line, MDA-MB-231, dasatinib treatment induced significant G(1) accumulation with little apoptosis, disrupted cellular morphology, blocked migration, inhibited invasion through Matrigel (P<0.01), and blocked the formation of invadopodia (P<0.001). Importantly, combination treatment with doxorubicin resulted in synergistic growth inhibition in all cell lines and blocked the migration and invasion of the highly metastatic, triple-negative MDA-MB-231 cell line.

Conclusion: The observed synergy between dasatinib and doxorubicin warrants the re-evaluation of dasatinib as an effective agent in multi-drug regimens for the treatment of invasive breast cancers.

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Related in: MedlinePlus

Doxorubicin and dasatinib synergize to block migration and invasion of MDA-MB-231 cells. (A) MDA-MB-231 cells were grown to 80% confluence in glass chamber slides and treated for 6 h before being streaked with a sterile pipette tip. Phase contrast images were taken at 0 and 6 h after wounding. All bright field images were obtained at × 10 magnification and wound width quantified (in pixels) using the NIH Image J software. (B) Cells were pre-treated with dasatinib for 4 h before transfer to Matrigel invasion chambers in serum-free, dasatinib-containing media. Cells were allowed to invade for 16 h towards media containing 10% FCS plus dasatinib. Data represent the average number of invading cells per membrane. (C) MDA-MB-231 cells were plated on FITC-gelatin in media containing DMSO or 100 nM dasatinib and allowed to adhere and invade for 20 h. Cells were then stained for cortactin, and invadopodia counted as co-localisations of cortactin staining and degraded FITC-signal (indicated by white arrows) in 10 random fields per sample. Results represent the average number of invadopodia per cell. (D) Wound healing and Matrigel invasion assays were repeated as in panels A and B, with dasatinib-, doxorubicin-, or combination (100 nM each drug)-treated cells. Error bars represent s.d. *Indicates P<0.05; **indicates P<0.01.
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fig5: Doxorubicin and dasatinib synergize to block migration and invasion of MDA-MB-231 cells. (A) MDA-MB-231 cells were grown to 80% confluence in glass chamber slides and treated for 6 h before being streaked with a sterile pipette tip. Phase contrast images were taken at 0 and 6 h after wounding. All bright field images were obtained at × 10 magnification and wound width quantified (in pixels) using the NIH Image J software. (B) Cells were pre-treated with dasatinib for 4 h before transfer to Matrigel invasion chambers in serum-free, dasatinib-containing media. Cells were allowed to invade for 16 h towards media containing 10% FCS plus dasatinib. Data represent the average number of invading cells per membrane. (C) MDA-MB-231 cells were plated on FITC-gelatin in media containing DMSO or 100 nM dasatinib and allowed to adhere and invade for 20 h. Cells were then stained for cortactin, and invadopodia counted as co-localisations of cortactin staining and degraded FITC-signal (indicated by white arrows) in 10 random fields per sample. Results represent the average number of invadopodia per cell. (D) Wound healing and Matrigel invasion assays were repeated as in panels A and B, with dasatinib-, doxorubicin-, or combination (100 nM each drug)-treated cells. Error bars represent s.d. *Indicates P<0.05; **indicates P<0.01.

Mentions: Based on the observed morphology changes in MDA-MB-231 cells following dasatinib treatment, we investigated the functional effects of dasatinib on cell migration and invasion. MCF7 and T47D cells were not tested because they are resistant to migration along the edge of a wound, are not invasive, and responded poorly to dasatinib. To measure effects on cell migration we used streak assays, where the ability of cells to repopulate a wounded area in a monolayer can be measured. We observed that untreated MDA-MB-231 cells were highly motile and repopulated the denuded area within 24 h. However, following dasatinib treatment, migration into the wounded area was greatly abrogated with a clear dose-dependent association (Figure 5A, P<0.05). To measure cell invasion, we used a Boyden chamber assay with Matrigel-coated filters. In these assays invasion through the Matrigel by MDA-MB-231 cells was decreased by 88% with 50 nM dasatinib treatment (Figure 5B; P<0.01). To confirm that these effects were due to cytoskeletal defects and not a result of inhibited cell proliferation, we studied the formation of invadopodia in individual invading cells. Invadopodia are actin-rich, finger-like projections that protrude into the extracellular matrix, secreting proteinases to degrade the matrix and allow for invasion of a cell through the barrier (Weaver, 2006). By plating MDA-MB-231 cells on a fluorescently-labeled gelatin substrate, we are able to visualise discrete points of matrix degradation and cytoskeletal extensions, which are identified as active invadopodia (Clark et al, 2007). Dasatinib treatment reduced the average number of invadopodia per cell from 5.4 to 0.4, indicating a near complete blockage of cytoskeletal remodelling activity and invasive capacity (Figure 5C; P<0.05).


Dasatinib synergizes with doxorubicin to block growth, migration, and invasion of breast cancer cells.

Pichot CS, Hartig SM, Xia L, Arvanitis C, Monisvais D, Lee FY, Frost JA, Corey SJ - Br. J. Cancer (2009)

Doxorubicin and dasatinib synergize to block migration and invasion of MDA-MB-231 cells. (A) MDA-MB-231 cells were grown to 80% confluence in glass chamber slides and treated for 6 h before being streaked with a sterile pipette tip. Phase contrast images were taken at 0 and 6 h after wounding. All bright field images were obtained at × 10 magnification and wound width quantified (in pixels) using the NIH Image J software. (B) Cells were pre-treated with dasatinib for 4 h before transfer to Matrigel invasion chambers in serum-free, dasatinib-containing media. Cells were allowed to invade for 16 h towards media containing 10% FCS plus dasatinib. Data represent the average number of invading cells per membrane. (C) MDA-MB-231 cells were plated on FITC-gelatin in media containing DMSO or 100 nM dasatinib and allowed to adhere and invade for 20 h. Cells were then stained for cortactin, and invadopodia counted as co-localisations of cortactin staining and degraded FITC-signal (indicated by white arrows) in 10 random fields per sample. Results represent the average number of invadopodia per cell. (D) Wound healing and Matrigel invasion assays were repeated as in panels A and B, with dasatinib-, doxorubicin-, or combination (100 nM each drug)-treated cells. Error bars represent s.d. *Indicates P<0.05; **indicates P<0.01.
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fig5: Doxorubicin and dasatinib synergize to block migration and invasion of MDA-MB-231 cells. (A) MDA-MB-231 cells were grown to 80% confluence in glass chamber slides and treated for 6 h before being streaked with a sterile pipette tip. Phase contrast images were taken at 0 and 6 h after wounding. All bright field images were obtained at × 10 magnification and wound width quantified (in pixels) using the NIH Image J software. (B) Cells were pre-treated with dasatinib for 4 h before transfer to Matrigel invasion chambers in serum-free, dasatinib-containing media. Cells were allowed to invade for 16 h towards media containing 10% FCS plus dasatinib. Data represent the average number of invading cells per membrane. (C) MDA-MB-231 cells were plated on FITC-gelatin in media containing DMSO or 100 nM dasatinib and allowed to adhere and invade for 20 h. Cells were then stained for cortactin, and invadopodia counted as co-localisations of cortactin staining and degraded FITC-signal (indicated by white arrows) in 10 random fields per sample. Results represent the average number of invadopodia per cell. (D) Wound healing and Matrigel invasion assays were repeated as in panels A and B, with dasatinib-, doxorubicin-, or combination (100 nM each drug)-treated cells. Error bars represent s.d. *Indicates P<0.05; **indicates P<0.01.
Mentions: Based on the observed morphology changes in MDA-MB-231 cells following dasatinib treatment, we investigated the functional effects of dasatinib on cell migration and invasion. MCF7 and T47D cells were not tested because they are resistant to migration along the edge of a wound, are not invasive, and responded poorly to dasatinib. To measure effects on cell migration we used streak assays, where the ability of cells to repopulate a wounded area in a monolayer can be measured. We observed that untreated MDA-MB-231 cells were highly motile and repopulated the denuded area within 24 h. However, following dasatinib treatment, migration into the wounded area was greatly abrogated with a clear dose-dependent association (Figure 5A, P<0.05). To measure cell invasion, we used a Boyden chamber assay with Matrigel-coated filters. In these assays invasion through the Matrigel by MDA-MB-231 cells was decreased by 88% with 50 nM dasatinib treatment (Figure 5B; P<0.01). To confirm that these effects were due to cytoskeletal defects and not a result of inhibited cell proliferation, we studied the formation of invadopodia in individual invading cells. Invadopodia are actin-rich, finger-like projections that protrude into the extracellular matrix, secreting proteinases to degrade the matrix and allow for invasion of a cell through the barrier (Weaver, 2006). By plating MDA-MB-231 cells on a fluorescently-labeled gelatin substrate, we are able to visualise discrete points of matrix degradation and cytoskeletal extensions, which are identified as active invadopodia (Clark et al, 2007). Dasatinib treatment reduced the average number of invadopodia per cell from 5.4 to 0.4, indicating a near complete blockage of cytoskeletal remodelling activity and invasive capacity (Figure 5C; P<0.05).

Bottom Line: We also tested the effects of these drugs on the actin cytoskeleton and associated signalling pathways.Importantly, combination treatment with doxorubicin resulted in synergistic growth inhibition in all cell lines and blocked the migration and invasion of the highly metastatic, triple-negative MDA-MB-231 cell line.The observed synergy between dasatinib and doxorubicin warrants the re-evaluation of dasatinib as an effective agent in multi-drug regimens for the treatment of invasive breast cancers.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX, USA.

ABSTRACT

Background: Src family kinases control multiple cancer cell properties including cell cycle progression, survival, and metastasis. Recent studies suggest that the Src inhibitor dasatinib blocks these critical cancer cell functions.

Methods: Because the molecular mechanism of action of dasatinib in breast cancers has not been investigated, we evaluated the effects of dasatinib as a single agent and in combination with the commonly used chemotherapeutic doxorubicin, on the proliferation, viability, and invasive capacity of breast cancer cells lines earlier categorised as dasatinib-sensitive (MDA-MB-231) and moderately resistant (MCF7 and T47D). We also tested the effects of these drugs on the actin cytoskeleton and associated signalling pathways.

Results: The cell lines tested varied widely in sensitivity to growth inhibition (IC(50)=0.16-12.3 microM), despite comparable Src kinase inhibition by dasatinib (IC(50)=17-37 nM). In the most sensitive cell line, MDA-MB-231, dasatinib treatment induced significant G(1) accumulation with little apoptosis, disrupted cellular morphology, blocked migration, inhibited invasion through Matrigel (P<0.01), and blocked the formation of invadopodia (P<0.001). Importantly, combination treatment with doxorubicin resulted in synergistic growth inhibition in all cell lines and blocked the migration and invasion of the highly metastatic, triple-negative MDA-MB-231 cell line.

Conclusion: The observed synergy between dasatinib and doxorubicin warrants the re-evaluation of dasatinib as an effective agent in multi-drug regimens for the treatment of invasive breast cancers.

Show MeSH
Related in: MedlinePlus