Lionel Cazin

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 ³H-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.

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.

Infiltration by macrophages (Mφ) indicates a poor prognosis in breast cancers, in particular by inducing angiogenesis. Our study aimed 1) to investigate the mechanism by which cooperation between Mφ and aggressive breast cancer cells (MDA-MB-231) induces angiogenesis; 2) to examine the effect of tetrathiomolybdate (TM) on this angiogenic activity.

In frog pituitary melanotrophs, GABA induces a transient stimulation followed by prolonged inhibition of hormone secretion. This biphasic effect is inconsistent with the elevation of cytosolic calcium and the inhibition of electrical activity also provoked by GABA in single melanotrophs. In the present study, standard patch‐clamp configurations and gramicidin‐perforated patches were used to investigate the physiological GABA_A receptor‐mediated response and intracellular chloride concentration ([Cl⁻]_i) in cultured frog melanotrophs. In the gramicidin‐perforated patch configuration, 1 μM GABA caused a depolarization associated with an action potential discharge and a slight fall of membrane resistance. In contrast, at a higher concentration (10 μm) GABA elicited a depolarization accompanied by a transient volley of action potentials, followed by a sustained inhibitory plateau and a marked fall of membrane resistance. Isoguvacine mimicked the GABA‐evoked responses, indicating a mediation by GABA_A receptors. In gramicidin‐perforated cells, the depolarizing excitatory effect of 1 μm GABA was converted into a depolarizing inhibitory action when 0.4 μm allopregnanolone was added to the bath solution. After gaining the whole‐cell configuration, the amplitude and/or direction of the GABA‐evoked current (I_GABA) rapidly changed before stabilizing. After stabilization, the reversal potential of I_GABA followed the values predicted by the Nernst equation for chloride ions when [Cl⁻]_i was varied. In gramicidin‐perforated cells, the steady‐state I–V relationships of 10 μm GABA‐ or isoguvacine‐evoked currents yielded reversal potentials of −37.5 ± 1.6 (n= 17) and −38.6 ± 2.0 mV (n= 8), respectively. These values were close to those obtained by using a voltage‐ramp protocol in the presence of Na⁺, K⁺ and Ca²⁺ channel blockers. The current evoked by 1 μm GABA also reversed at these potentials. We conclude that, in frog pituitary melanotrophs, chloride is the exclusive charge carrier of I_GABA. In intact cells, the reversal potential of I_GABA is positive to the resting potential because of a relatively high [Cl⁻]_i (26.5 mm). Under these conditions, GABA induces a chloride efflux responsible for a depolarization triggering action potentials. However, GABA at a high concentration or in the presence of the potentiating steroid allopregnanolone exerts a concomitant shunting effect leading to a rapid inhibition of the spontaneous firing.

The effects of the neuroactive steroid pregnanolone (5β‐pregnan‐3α‐ol‐20‐one) on the electrical response to GABA were investigated in cultured frog pituitary melanotrophs using the patch‐clamp technique. Low concentrations of pregnanolone (0.01–1 μm) in the extracellular solution enhanced the current evoked by submaximal concentrations of GABA_A receptor agonists and prolonged the GABA‐induced inhibition of the spontaneous action potentials in a dose‐dependent manner. Pregnanolone augmented the opening probability of the single GABA‐activated channels but did not modify the conductance levels. Pregnanolone (1 μm) shifted the GABA dose–response curve towards the low GABA concentrations, reducing the EC₅₀ from 4.2 to 1.8 μm. Internal cell dialysis with pregnanolone (1 or 10 μm) did not alter the GABA‐evoked current. Pregnanolone accelerated the desensitization of both the current and conductance increases caused by GABA. High concentrations of pregnanolone (30 μm) markedly and reversibly diminished the current evoked by 10 μm GABA. At high concentrations (10–30 μm), pregnanolone induced an outward current which reversed at the chloride equilibrium potential. It is concluded that, in frog pituitary melanotrophs, pregnanolone exerts a dual inverse modulation and a direct activation of the GABA_A receptor–channel depending on the concentrations of both GABA and steroid. Pregnanolone acts on an extracellular site on the GABA_A receptor inducing conformational changes of the receptor–channel complex, resulting in a desensitized less‐conducting state.