Crucial role of type 1, but not type 3, inositol 1,4,5-trisphosphate (IP(3)) receptors in IP(3)-induced Ca(2+) release, capacitative Ca(2+) entry, and proliferation of A7r5 vascular smooth muscle cells.

Stimulation of G protein- or tyrosine kinase-coupled receptors regulates cell proliferation through intracellular Ca(2+) ([Ca(2+)](i)) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP(3)) mediates vasopressin (VP)-evoked Ca(2+) release from intracellular stores and showed that types 1 (IP(3)R(1)) and 3 (IP(3)R(3)) IP(3) receptors were expressed. Using antisera selective for IP(3)R(1) or IP(3)R(3) and another that interacted equally well with both subtypes, together with membranes from SF:9 cells expressing only single IP(3)R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP(3)R(1) and 19% IP(3)R(3). To elucidate the contributions of IP(3)R(1) and IP(3)R(3) to Ca(2+) signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (-90 to +9) corresponding to the two IP(3)R subtypes were selected. Mild inhibition of IP(3)R(1) (71+/-8% of control level) slightly attenuated the IP(3)-evoked Ca(2+) release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca(2+) entry (CCE) and proliferation. Moderate inhibition (34+/-6%) strongly decreased both IICR and CCE and further blocked proliferation. Complete inhibition almost abolished IICR and CCE and arrested proliferation entirely. Complete inhibition of IP(3)R(3) expression slightly attenuated IICR without affecting CCE or proliferation. In cells microinjected with a low dose of heparin, VP-induced CCE was more susceptible than IICR to mild inhibition of both IP(3)R(1) and IP(3)R(3). A high dose of heparin had a similar effect to complete inhibition of IP(3)R(1) expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP(3)R(1), but not IP(3)R(3), is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.

Plasmalemmal pH-gradients in drug-sensitive and drug-resistant MCF-7 human breast carcinoma xenografts measured by 31P magnetic resonance spectroscopy.

31p Magnetic resonance spectroscopy (MRS) was employed to investigate tumor pH in xenografts of drug-sensitive and drug-resistant MCF-7 human breast carcinoma cells. Measured extracellular pH values were found to be lower than the intracellular pH in all three tumor types investigated. The magnitude of this acid-outside plasmalemmal pH gradient increased with increasing tumor size in tumors of two drug-resistant variants of MCF-7 cells, but not in tumors of the parent (drug-sensitive) cells. The partitioning of weak-base or weak-acid drug molecules across the plasma membrane of a tumor cell is dependent upon the acid-dissociation constant (pKa) of the drug as well as the plasmalemmal pH gradient. A large acid-outside pH gradient, such as those seen in MCF-7 xenografts, can exert a protective effect on the cell from weak-base drugs such as anthracyclines and Vinca alkaloids, which have pKa values of 7.5 to 9.5. The possibility of enhancing the therapeutic efficacy of weak-base drugs by dietary or metabolic manipulation of the extracellular pH, in order to reduce or reverse the plasmalemmal pH gradient, deserves investigation.

Early increases in breast tumor xenograft water mobility in response to paclitaxel therapy detected by non-invasive diffusion magnetic resonance imaging.

An important goal in cancer chemotherapy is to sensitively and quantitatively monitor the response of individual patients' tumors to successful, or unsuccessful, therapy so that regimens can be altered iteratively. Currently, tumor response is monitored by frank changes in tumor morphology, yet these markers take long to manifest and are not quantitative. Recent studies suggest that the apparent diffusion coefficient of water (ADCw), measured noninvasively with magnetic resonance imaging, is sensitively and reliably increased in response to successful CTx. In the present study, we investigate the combination chemotherapy response of human breast cancer tumor xenografts sensitive or resistant to Paclitaxel by monitoring changes in the ADCw. Our results indicate that there is a clear, substantial, and early increase in the ADCw after successful therapy in drug sensitive tumors and that there is no change in the ADCw in p-glycoprotein-positive tumors, which are resistant to Paclitaxel. The mechanism underlying these changes is unknown yet is consistent with apoptotic cell shrinkage and a concomitant increase in the extracellular water fraction.

Diaryl chalcogenides as selective inhibitors of thioredoxin reductase and potential antitumor agents.

Thioredoxin reductase is a selenocysteine containing flavoenzyme that catalyzes the NADPH dependent reduction of the redox protein thioredoxin. Thioredoxin is over-expressed by a number of human tumors. Experimental studies have shown that thioredoxin is responsible for the growth and transformed phenotype of some human cancer cells. Thus, thioredoxin reductase presents an attractive target for anticancer drug development to regulate the activity of the thioredoxin system. We have examined a series of 12 organoselenium compounds and 16 organotellurium compounds, mostly of the diaryl chalcogenide type, as inhibitors of human thioredoxin reductase and have investigated the cytotoxicity and antitumor activity of some of the compounds. The organoselenium compound Ebselen was found to be a competitive inhibitor of human thioredoxin reductase (Ki 2.8 microM), while a number of organotellurium compounds were found to be noncompetitive inhibitors (Kis 2.3 to 35.2 microM). Human glutathione reductase was not appreciably inhibited by any of the compounds, except for one dinitro organotellurium compound that caused inhibition with an IC50 of 0.5 microM and an over 20-fold selectivity compared to thioredoxin reductase. The compounds inhibited the growth of human cancer cells in culture with IC50s as low as 2 microM Some organotellurium compounds when administered daily by intraperitoneal injection to mice caused up to 50% inhibition of the growth of MCF-7 human breast cancer xenografts but the relative insolubility of the compounds was a limiting factor in their use.