Robert G. Canada

Terbium fluorescence studies of cisplatin binding to GH3/B6 pituitary tumor cells.

Terbium (Tb3+) fluorescence was used to investigate the interaction of cisplatin with GH3/B6 pituitary tumor cells. The binding of cisplatin to GH3/B6 cells quenched the fluorescence intensity of bound Tb3+. The IC50 for cisplatin inhibition of Tb3+-GH3/B6 fluorescence was determined to be 190 microM. Cisplatin was found to non-competitively inhibit the cellular binding of Tb3+, causing a dramatic decrease in the maximum number of high-affinity Tb3+ binding sites (by 33%), without markedly affecting their binding affinity. The half-life for the cellular binding of cisplatin was calculated to be 2.7 min. It was suggested that the plasma membrane of GH3/B6 cells contain a specific protein receptor for binding cisplatin.

Terbium luminescence studies of binding of adriamycin and cisplatin to tumorigenic cells

Abstract The luminescent properties of terbium ions are used to investigate the interaction of adriamycin and cisplatin with GH3/B6 pituitary tumor cells. Clinically relevant concentrations of adriamycin were found to quench the intensity (IC50 = 0.6 μM) and excited-state lifetime (τ/τ0 = 0.73) of the Tb3+—GH3/B6 complex. Inspection of the Tb3+—GH3/B6 emission spectrum and the visible absorption spectrum of adriamycin strongly strongly suggests that the quenching of Tb3+ luminescence by adriamycin is due to dipole-dipole resonant energy transfer; and, according to Forsters theory (R0 = 33.6 A), the adriamycin receptor site is located ca. 40 A away from the bound probe, at the lipid/protein interface. The quenching of Tb3+ luminescence by cisplatin is best explained by a static energy-exchange mechanism; in that the cisplatin receptor site is contiguous with the Tb3+ binding site at the outer surface of the membrane. The data suggest that, in the plasma membrane of tumorigenic cells, the adriamycin and ciplatin receptor sites are intimately associated with the same calcium-binding protein.

Binding of terbium and cisplatin to C13* human ovarian cancer cells using time-resolved terbium luminescence

Terbium (Tb3+) has been shown to increase the cellular accumulation and cytotoxicity of cisplatin in cisplatin-resistant human breast and ovarian cancer cells. Time-resolved Tb3+ luminescence was used to describe the binding of cisplatin to cisplatin-resistant C13* cells. A high-affinity Tb3+ binding site was identified in the plasma membrane of the C13* cells (n=105+/-2 fmol/cell and Kd=36. 3+/-5.2 microM). The binding of Tb3+ is suggested to occur through a cation-pi interaction with tryptophan residues in the plasma membrane, resulting in an enhancement of the intensity and lifetime of Tb3+. Stern-Volmer quenching analysis revealed that the Tb3+ binding site is not readily accessible to the aqueous environment. The quenching of the Tb3+-C13* intensity by cisplatin occurred by static quenching processes, involving both a direct electron-exchange interaction as well as an indirect dipole-dipole resonant energy transfer mechanism. Formation of the Tb3+-C13*-cisplatin complex does not interfere with the high-affinity binding of Tb3+; cisplatin and Tb3+ bind within 5 to 10 A of each other. A specific terbium/cisplatin binding protein is suggested to play a role in the cellular accumulation and cytotoxicity of cisplatin. Therefore, the transport of cisplatin across the plasma membrane must also involve a facilitated diffusion process. Our results indicate that the binding of Tb3+ to the plasma membrane may be potentially useful in the reversal of cisplatin resistance.

The effects of terbium on the accumulation of cisplatin in human ovarian cancer cells

In this investigation, we report a relationship between the terbium (Tb3+) binding protein and the accumulation of cisplatin in human ovarian cancer cells. The number of Tb3+ binding sites in cisplatin-resistant C13+ cells is significantly greater by 79% than those in cisplatin-sensitive 2008 cells. Exposure to Tb3+ also increased the cellular accumulation of cisplatin. The accumulation of cisplatin as a function of the Tb3+ concentration in the C13+ cells (0.85%/microM Tb3+) was significantly greater than the accumulation of cisplatin in 2008 cells with respect to Tb3+ (0.46%/microM Tb3+). The number of Tb3+ binding sites in revertant RH4 cells was similar to that in 2008 cells. The RH4 cells were less sensitive to the stimulatory effects of Tb3+ than the C13+ cells. Our results show that the Tb3+ binding protein correlates with cisplatin resistance, and the receptor binding of Tb3+ increases the accumulation of cisplatin in cisplatin-resistant cells.

Interactions of adriamycin with a calcium binding site

Terbium (Tb3+) luminescence has been used to investigate the interactions of adriamycin with a specific calcium binding protein, in the plasma membrane of GH3/B6 pituitary tumor cells. The luminescence intensity and lifetime of the Tb3+-GH3/B6 complex was quenched in the presence of adriamycin. According to Stern-Volmer analysis, the quenching of Tb3+-GH3/B6 luminescence was by both membrane bound adriamycin (Ka = 3.7 x 10(5) M-1) and free adriamycin (kq = 7.3 x 10(7) M-1 s-1). The data suggests that, the calcium binding site at the outer surface of the membrane is collisionally accessible to freely diffusing adriamycin; and, that the toxin receptor site is located near the bound metal ion.

The effects of terbium on the cellular accumulation of cisplatin in MDA-MB-231 human breast tumor cells

Abstract Purpose: Cisplatin (DDP) is an effective antitumor agent limited in its efficacy by the development of tumor cell resistance. The defective accumulation of DDP has been shown to be a prominent feature in many DDP-resistant cell lines. In an effort to circumvent this problem, we examined the cellular accumulation of DDP in the presence of terbium (Tb3+). We also examined the effects of verapamil on the cellular accumulation of DDP in order to delineate the specific interaction of Tb3+ and DDP. All experiments were performed on DDP-sensitive or DDP-resistant MDA-MB-231 human breast tumor cells. Methods: The cellular accumulation of DDP and verapamil were determined by electrothermal atomic absorption spectrophotometry. Time-resolved luminescence spectroscopy was used to obtain equilibrium binding constants for the Tb3+/MDA cell complexes. Results: We found that 100 μM Tb3+ increased DDP accumulation in the parent MDA cell line, 5.7-fold resistant MDA/A13 and 10-fold resistant MDA/CH cells by 56.2±7.4, 71.9±9.4 and 50.8±9.4%, respectively (P<0.0001 for all MDA cell types). In contrast, 20 μM verapamil had no significant effect on DDP accumulation in the MDA cell lines. In addition, a positive correlation between the membrane binding of Tb3+ and the cellular accumulation of DDP was found to exist in the parent cell line and sublines (r=0.9). Conclusions: In agreement with earlier studies, the plasma membrane of MDA cell lines contain a specific Tb3+-binding protein. Our findings suggest that the Tb3+-binding protein may be intimately associated with the accumulation of DDP in breast tumor cells.

Physicochemical characteristics of the terbium-adriamycin complex and its effects on the sinus node automaticity.

The physicochemical characteristics of the terbium-adriamycin complex (terbomycin) were studied. Perturbations in the visible absorption spectrum of adriamycin by terbium (Tb3+) was indicative of formation of the terbomycin complex. The absorption maximum of free adriamycin at 479 nm shifted towards the absorption maximum of terbomycin at 539 nm. The binding of Tb3+ to adriamycin was negligible at acidic pH. At alkaline pH, the affinity of Tb3+ for adriamycin increased. The stoichiometry of binding was estimated to be 0.5; one Tb3+ ion per two adriamycin molecules. Thermodynamic analysis revealed that the spontaneous formation of terbomycin was due to an increase in the entropy of the system. The effects of adriamycin, Tb3+ and terbomycin on sinus node automaticity were studied using sinus node from rats, superfused with modified mammalian Tris-Tyrodes solution (37 degrees C). The sinus node rate was monitored with intracellular microelectrodes. 25 microM Tb3+ increased the sinus node rate. Adriamycin (50 microM) depressed sinus node automaticity. Terbomycin also reduced the sinus node rate. There was no difference between the effects of adriamycin and terbomycin. The chronotropic effect of terbomycin persisted in the presence of atropine.

Enhancement of cisplatin cytotoxicity by terbium in cisplatin-resistant MDA/CH human breast cancer cells.

Purpose: The development of cisplatin resistance is a major problem in the treatment of cancer patients with cisplatin chemotherapy. The membrane binding of terbium (Tb3+) has been shown to increase the cellular accumulation of cisplatin in breast cancer cells. Therefore, the ability of Tb3+ to modulate the cytotoxicity of cisplatin was investigated in cisplatin-sensitive (MDA) and cisplatin-resistant (MDA/CH) MDA-MB-231 human breast cancer cells. Methods: The cytotoxic parameters of cisplatin were determined using live cell microfluorometry and median effect analysis. Results: MDA/CH cells (IC50 = 142 ± 9 μM) were found to be approximately 3.3-fold more resistant to cisplatin than MDA cells (IC50 = 43.5 ± 3.0 μM). In both cell lines, the IC50 value for cisplatin was reduced two-fold in the presence of 80 μM Tb3+, thus indicating that the cytotoxicity of cisplatin is increased by Tb3+. The cytotoxic activity of cisplatin alone was observed to be 5.7 and 1.6 times more potent than that of Tb3+ alone in MDA and MDA/CH cells, respectively. Combination index analyses revealed that the interaction between cisplatin and Tb3+ was only synergistic at very low indices of cell death in MDA cells. However, in MDA/CH cells, the two drugs were synergistic up to intermediate levels of cell death. Conclusions: Our results suggest that the enhancement of cisplatin cytotoxicity by Tb3+ is more effective in cisplatin-resistant MDA/CH cells than in cisplatin-sensitive MDA cells. Therefore, terbium is potentially useful in cisplatin combination therapy for breast cancer patients, especially for those patients who have developed resistance to the drug.

Terbium fluorescence characteristics of cultured neurons using ultraviolet microspectrofluorometry

The fluorescence emission intensity of terbium is enhanced upon the binding of Tb3+ to cultured mouse spinal cord and dorsal root ganglion neurons, via nonradiative resonant energy transfer from membrane proteins. The relative fluorescence intensities of Tb3+ bound to dorsal root ganglion neurons were considerably greater than that of Tb3+ bound to large multipolar spinal cord neurons. The cell bodies of the dorsal root ganglion neurons were completely covered in a dense fluorescent blanket, where as the fluorescence from the spinal cord soma presented a discontinuous pattern. The neurites of the spinal cord neuron were speckled with bright patches of Tb3+ fluorescence. A high concentration of Ca2+ reduced the relative fluorescence intensity of the Tb3+-neuron complex. It is suggested that Tb3+ binds to Ca2+-binding sites on the surface membrane of neurons.

Synergism between dipyridamole and cisplatin in human breast cancer cells in vitro

Cisplatin is very effective in the treatment of metastatic breast cancer. However, the development of cellular resistance is a serious problem in cisplatin chemotherapy. In the present work, the effects of dipyridamole (DPM) on the cellular accumulation and cytotoxicity of cisplatin was studied in cisplatinsensitive (MDA/S) and cisplatinresistant (MDA/R) human breast cancer cells. In the presence of 30 µM DPM, the IC50 of cisplatin was reduced by 39% for both cell lines. Combination index analysis revealed that cisplatin and dipyridamole interact synergistically in MDA/R cells. In the MDA/S cells, the cellular accumulation of cisplatin increased by 57 ± 8% in the presence of 30 µM DPM. In the MDA/R cells, the cellular accumulation of cisplatin remained the same with or without 30 µM DPM. The results suggest that the enhancement of cisplatin cytotoxicity by DPM in MDA/S cells may be related to a DPM-induced increase in cisplatin accumulation, but the enhanced cytotoxicity in MDA/R cells employs a mechanism that does not involve an increase in the cellular accumulation of cisplatin.