Extracellular matrix proteins protect human HT1080 cells against the antimigratory effect of doxorubicin

Abstract

In solid tumors, the cell microenvironment appears to be a key determinant in the emergence of drug resistance, a major obstacle to the successful use of antitumor drugs. Our aim was to determine whether type I collagen and fibronectin, proteins of the extracellular matrix, were able to influence the antimigratory properties induced by the antitumor drug doxorubicin. These properties were investigated at doxorubicin concentrations of 10 and 20 nM, which do not affect cell proliferation on a 24 h drug exposure. Using videomicroscopy, we found that these subtoxic doses of doxorubicin were sufficient to inhibit individual tumor cell motion on two-dimensional plastic surfaces. Such a drug treatment induced a dramatic disturbance of actin stress fiber formation and of vinculin distribution in 80% of cells. In contrast, on extracellular matrix proteins, cell speed was unaffected by drug and perturbation of both actin network and vinculin distribution was detected in only 50% of cells, suggesting a protective effect of the microenvironment. In addition, the phosphorylation of focal adhesion kinase and GTPase RhoA was less affected by doxorubicin with cells cultured on extracellular matrix proteins. In conclusion, our findings indicate that the cell microenvironment prevents drug-dependent inhibition of cell migration in vitro. A n increasing body of evidence indicates that the tumor cell microenvironment is a key determinant of cell response to cytotoxic drugs. The environmental causes that contribute to cancer cell survival after initial therapy, allowing resistant cells to proliferate are multifactorial. They include particularly limited drug penetration, tumor cell adaptation to hypoxia, acidic extracellular pH, and direct contact between cancer cells and the ECM or adjacent cells.(1) Concerning ECM proteins, fibronectin or collagen type I and IV have been shown to have the potential to confer resistance to diverse chemotherapeutic agents in cancer cell lines originated from both hematopoietic and solid tumors. (2,3) This has led to the concept of CAM-DR, a form of de novo drug resistance which may play an important role in the acquisition of a multidrug resistance phenotype. (4,5) Studies on the impact of the microenvironment on drug response have focused exclusively on the cytotoxic or proapoptotic effects induced by an extensive panel of well-known chemotherapeutics, either microtubule-disturbing agents such as vinca-alcaloids, taxoids, or DNA-damaging compounds such as nitrogen mustards and anthracyclines. (6,7) However, with chemotherapeutics such as anthracyclines, beyond their well-known antitumor activity, it has been recently demonstrated that they may display potent anti-invasive effects. Indeed, doxorubicin has been shown to inhibit cell escape from multicellular spheroids with cells of prostate, colon, breast, or lung carcinoma.(8) Using the highly mobile fibrosarcoma cell line HT1080 it has been shown that DA-125, a new analog of doxorubicin, inhibits the invasion of these cells by downregulating MMP.(9...

Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy

“…More recently, Harisi et al described the effect on induced doxorubicin-resistance by the extra cellular matrix in human osteosarcoma cells 9 . This observation was confirmed in 2008 by Fourre et al, demonstrating the protective effect of the extracellular matrix on the HT1080 cell line, against the action of doxorobucin 10 . Reduced radiation induced toxicity when cells are grown in a 3D environment has also been recently demonstrated 11 .…”

Chemotherapeutic efficiency of drugs in vitro: Comparison of doxorubicin exposure in 3D and 2D culture matrices

“…The results clearly indicate that the collagen matrix affects the effective concentration of the exposure solution, reducing it by a factor of ~1.6 which in part would contribute to the observed increase in viability previously perceived as a drug resistance (Fourre et al, 2008;Harisi et al, 2007) of cells to doxorubicin when grown in a 3D environment. Correspondingly the effective concentration of the cytotoxic assay solution is in itself reduced by a factor of 1.78 coupled with a different effective surface area of the cell exposed to the dye solution and therefore different uptake rate; result in the reduced cytotoxic response recorded.…”

“…The in vitro cytotoxicity assessment of the effects of the chemotherapeutic agent DOX in cells cultures in a 2D compared to a 3D environment indicate a significant difference between the cultures and potentially an increased resistance (Fourre et al, 2008;Harisi et al, 2007) of the cells to the drug in the 3D environment. However, a similar study of the same cell line , cultured in the same two environments, demonstrated that, although the cytotoxicity study gave similar indications of reduced toxic response of the two cultures, no differences in cell population viability were apparent in a flow cytometric screening measurement.…”

“…For example, it has been shown that antibodies against B1-integrins exhibit different behaviour when tested in 2D compared to 3D models (Wang et al, 1998); induced doxorubicin-resistance by the extra cellular matrix in human osteosarcoma and HT1080 cells has been demonstrated (Fourre et al, 2008;Harisi et al, 2007); and reduced radiation induced toxicity when cells are grown in a 3D environment has also been reported (Sowa et al, 2010).…”

“…The use of collagen gels or other commercially developed systems are reported to provide the cells with a microenvironment or matrix which is analogous to the extra cellular matrix (ECM) experienced by cells in vivo and is widely considered to be a better representation of the cancerous cell phenotype when compared to those grown in conventional 2D culture (Petersen et al, 1992, Prestwich, 2008, Weaver et al, 1995. The effects on cells grown in such systems compared to 2D systems is widely documented and it has been reported that cells can exhibit different behaviour when cultured in 3D when compared to 2D, such as toxicological response, phenotype, metabolism and composition (Sowa et al;2010;Wang et al, 1998;Fourre et al, 2008;Harisi et al) .…”