Anti-breast cancer agents, quinolines, targeting gap junction

Abstract

Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). The process of cancer formation causes a disruption in cell homeostasis, affecting the ability to respond to extracellular signals, as well as triggering some intracellular events which alter gap junctional intercellular communication (GJIC). Previous research has shown that the first two generations of substituted quinolines have anti-cancer effects in human breast cancer cells. This report presents the synthesis and bioactivities of third generation substituted quinolines. Scrape load/dye transfer studies showed that 100 nM of PQ15, a third generation substituted quinoline, causes a 4.5-fold increase of gap junction activity in T47D breast cancer cells. Furthermore, a significant decrease of cell proliferation and viability was observed in the presence of 200 nM PQ15 compared to control. The expression of α-survivin was reduced to <40% in the treatment of 200 nM PQ15 compared to solvent alone. Alpha-survivin expression is upregulated in human cancers and associated with resistance to chemotherapy, suggesting that α-survivin prolongs the survival of cancer cells. Thus, it has been shown that substituted quinolines stimulate gap junction activity, decrease alpha survivin expression, and subsequently inhibit cancer cell growth. Our findings demonstrate that PQ15 has a promising role in exerting anti-cancer activity in human breast cancer cells.

Figure 1

Chemical Structures of PQ1, PQ11, and PQ15

Figure 2

Gap Junction Activity of PQ15 in T47D breast cancer cells. T47D cells were treated with 0, 100, 200, and 1000 nM PQ15 for 3 hours (A, D, E and F). Cells were treated with 200 nM TPA alone (C), 100 nM PQ15 and 200 nM TPA (G), or 200 nM PQ15 and 200 nM TPA (H) for 3 hours. Controls are media alone (A) or solvent, DMSO (B). Scrape load/dye transfer assay was performed as described in Materials and Methods. Lucifer yellow was used as a gap junctional dye and rhodamine-dextran was used to mark the cutting site. Fluorescence green indicates the passages of dye from the cutting site, showing increase of GJIC. Graphical presentation of three experiments shows the distance of dye transfer of T47D cells.

Figure 3

Effect of PQ15 on Cell Proliferation. 1×10⁴ T47D breast cancer cells were treated with 0, 10, 100, 200, and 1000 nM of PQ15 for 48 hours. Controls are media alone and DMSO as solvent. Total number of cells was counted and expressed in percent of control. The graphical presentation of three independent samples was calculated and indicated with a statistical significance, *p<0.05.

Figure 4

Effect of PQ15 on Cell Viability. 1×10⁴ T47D breast cancer cells were treated with various concentrations of PQ15 for 48 hours. A cell suspension was mixed with trypan blue dye and then visually examined viable cells. The graphical presentation of three independent samples was measured with a statistical significance, *p<0.05.

Figure 5

Effect of PQ15 on Connexin 43 and Survivin Expression. T47D cells were treated with 10, 100, 200 and 1000 nM PQ15 for 24 hours. Treatment with DMSO was used as control. Western blot analysis was performed. Nitrocellulose membrane was blotted with the active form of Cx43 (43 kDa), phosphorylated Cx43 (Serine 368) and α-survivin (16.5 kDa) antibodies. Actin acts as loading control. Note: pCx43 antibodies specifically recognize the phosphorylation of Cx43 at Serine 368.