Rac3 induces a molecular pathway triggering breast cancer cell aggressiveness: differences in MDA-MB-231 and MCF-7 breast cancer cell lines.

BACKGROUND: Rho GTPases are involved in cellular functions relevant to cancer. The roles of RhoA and Rac1 have already been established. However, the role of Rac3 in cancer aggressiveness is less well understood. METHODS: This work was conducted to analyze the implication of Rac3 in the aggressiveness of two breast cancer cell lines, MDA-MB-231 and MCF-7: both express Rac3, but MDA-MB-231 expresses more activated RhoA. The effect of Rac3 in cancer cells was also compared with its effect on the non-tumorigenic mammary epithelial cells MCF-10A. We analyzed the consequences of Rac3 depletion by anti-Rac3 siRNA. RESULTS: Firstly, we analyzed the effects of Rac3 depletion on the breast cancer cells' aggressiveness. In the invasive MDA-MB-231 cells, Rac3 inhibition caused a marked reduction of both invasion (40%) and cell adhesion to collagen (84%), accompanied by an increase in TNF-induced apoptosis (72%). This indicates that Rac3 is involved in the cancer cells' aggressiveness. Secondly, we investigated the effects of Rac3 inhibition on the expression and activation of related signaling molecules, including NF-κB and ERK. Cytokine secretion profiles were also analyzed. In the non-invasive MCF-7 line; Rac3 did not influence any of the parameters of aggressiveness. CONCLUSIONS: This discrepancy between the effects of Rac3 knockdown in the two cell lines could be explained as follows: in the MDA-MB-231 line, the Rac3-dependent aggressiveness of the cancer cells is due to the Rac3/ERK-2/NF-κB signaling pathway, which is responsible for MMP-9, interleukin-6, -8 and GRO secretion, as well as the resistance to TNF-induced apoptosis, whereas in the MCF-7 line, this pathway is not functional because of the low expression of NF-κB subunits in these cells. Rac3 may be a potent target for inhibiting aggressive breast cancer.

Induction of acquired drug resistance in endothelial cells and its involvement in anticancer therapy.

BACKGROUND: Multidrug resistance (MDR) is one of the major problems in the treatment of cancer. Overcoming it is therefore expected to improve clinical outcomes for cancer patients. MDR is usually characterized by overexpression of ABC (ATP-binding cassette) protein transporters such as P-gp, MRP1, and ABCG2. Though the importance of ABC transporters for cancer cells is recognized, few studies have looked at its implications for the endothelial cells that are essential to tumor angiogenesis. This study investigated the expression and functions of these ABC transporters in endothelial cells in vitro and their potential contribution to cancer growth in mice. METHODS: Human micro vessel endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC) were exposed to increasing doses of Doxorubicin (Dox) to induce ABC gene expression. Cell viability was then quantified by (3)H-thymidine and MTS assay. Flow cytometry, qPCR, and western blot were used to detect mRNA and the protein expression of P-gp, MRP1, and ABCG2. The intracellular accumulation of Rhodamine 123 (Rho) was used to evaluate drug efflux function and the inhibitors for P-gp, ABCG2, and MRP1 were used to verify their respective roles in vitro. In an attempt to evaluate drug resistance in endothelial cells in vivo, athymic mice were treated with Dox for 15 days before a MDA-MB-435 tumor graft to observe subsequent changes in the inhibition curves of tumor growth in response to Dox treatment. Furthermore, endothelial cells from multiple sites in these mice were also isolated to estimate their P-gp expression by flow cytometry. RESULTS: Drug resistance in HMEC-1 and HUVEC was successfully induced by the addition of Dox to the culture media. Two stabilized subcell lines of HMEC1 (HMECd1 and HMECd2) showed 15- and 24-fold increases in resistance. Tests also showed that these induced endothelial cells were cross-resistant to the structurally unrelated drugs Daunorubicin, Vinblastine, and Etoposide. P-gp protein levels increased four and six fold in HMECd1 and HMECd2 as revealed by western blot. The qPCR demonstrated 3.4- and 7.2-fold increases in P-gp, and a slight increase in ABCG2, gene expression. The Rho accumulation within these cells was inversely correlated with the expression levels of P-gp. The inhibitors of P-gp, but not of ABCG2 or MRP1, were able to block the induced endothelial cell resistance to Dox. Furthermore, we also showed that injecting Dox into healthy mice induced an increase in P-gp expression in endothelial cells. Using these pretreated mice in a tumor growth experiment, we observed a dramatic diminution in the therapeutic efficiency of Dox treatment, suggesting implications for drug resistance in mice endothelial cells supporting tumor growth. CONCLUSIONS: ABC transporter expression can be induced in endothelial cells in vitro. This study also indicates that P-gp plays an important role in the acquisition of resistance to Dox in endothelial cells and that this reduces the efficiency of chemotherapy.