SLC26A7 protein is a chloride/bicarbonate exchanger and its abundance is osmolarity- and pH-dependent in renal epithelial cells.

Acid-secreting intercalated cells of the collecting duct express the chloride/bicarbonate kidney anion exchanger 1 (kAE1) as well as SLC26A7, two proteins that colocalize in the basolateral membrane. The latter protein has been reported to function either as a chloride/bicarbonate exchanger or a chloride channel. Both kAE1 and SLC26A7 are detected in the renal medulla, an environment hyper-osmotic to plasma. Individuals with mutations in the SLC4A1 gene encoding kAE1 and mice lacking Slc26a7 develop distal renal tubular acidosis (dRTA). Here, we aimed to (i) confirm that SLC26A7 can function as chloride/bicarbonate exchanger in Madin-Darby canine kidney (MDCK) cells, and (ii) examine the behavior of SLC26A7 relative to kAE1 wild type or carrying the dRTA mutation R901X in iso- or hyper-osmotic conditions mimicking the renal medulla. Although we found that SLC26A7 abundance increases in hyper-osmotic growth medium, it is reduced in low pH growth conditions mimicking acidosis when expressed at high levels in MDCK cells. In these cells, SLC26A7 exchange activity was independent from extracellular osmolarity. When SLC26A7 protein was co-expressed with kAE1 WT or the R901X dRTA mutant, the cellular chloride/bicarbonate exchange rate was not additive compared to when proteins are expressed individually, possibly reflecting a decreased overall protein expression. Furthermore, the cellular chloride/bicarbonate exchange rate was osmolarity-independent. Together, these results show that (i) in MDCK cells, SLC26A7 is a chloride/bicarbonate exchanger whose abundance is up-regulated by high osmolarity growth medium and (ii) acidic extracellular pH decreases the abundance of SLC26A7 protein.

Hyperpolarizability studies and Hirshfeld surface analysis of two heterocyclic chalcones.

In this work, the nonlinear optical (NLO) properties of two heterocyclic chalcones, (E)-1-(5-chlorothiophen-2-yl)-3-(thiophen-2yl)-2-propen-1-one (CLTT) and (E)-1-(5-methylfuran-2-yl)-3-(5-methylthiophen-2-yl)prop-2en-1-one (2MFT), are investigated. Using an iterative electrostatic embedding approach via the Møller-Plesset perturbation (MP2) theory, the chalcone crystals were simulated and the polarization effects on the isolated molecules are investigated. The electrical parameters of CLTT and 2MFT as dipole moment and linear polarizability were calculated via MP2/6-311++G(d) and the second hyperpolarizability was obtained via DFT/CAM-B3-LYP/6-311++G(d) level. A significant influence of the molecular packing on the chalcone electric parameters was observed. The static linear refractive index and the third-order electric susceptibility of the compounds were calculated and compared with experimental results available for other chalcone derivatives, indicating the CLTT crystal as a promising candidate for NLO applications in photonic and optoelectronic devices. The Hirshfeld surface analysis has been used to quantify the intermolecular interactions of the molecular crystals. Additionally, the solvent medium effects on the electrical properties of the heterocyclic chalcones were also studied.

Pharmacological relevance of endoxifen in a laboratory simulation of breast cancer in postmenopausal patients.

BACKGROUND: Tamoxifen is metabolically activated via a CYP2D6 enzyme system to the more potent hydroxylated derivatives 4-hydroxytamoxifen and endoxifen. This study addresses the pharmacological importance of endoxifen by simulating clinical scenarios in vitro. METHODS: Clinical levels of tamoxifen metabolites in postmenopausal breast cancer patients previously genotyped for CYP2D6 were used in vitro along with clinical estrogen levels (estrone and estradiol) in postmenopausal patients determined in previous studies. The biological effects on cell growth were evaluated in a panel of estrogen receptor-positive breast cancer cell lines via cell proliferation assays and real-time polymerase chain reaction (PCR). Data were analyzed with one- and two-way analysis of variance and Student's t test. All statistical tests were two-sided. RESULTS: Postmenopausal levels of estrogen-induced proliferation of all test breast cancer cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 11 ± 1.74, P < .001; T47D = 7.52 ± 0.72, P < .001; BT474 = 1.75 ± 0.23, P < .001; ZR-75-1 = 5.5 ± 1.95, P = .001. Tamoxifen and primary metabolites completely inhibited cell growth regardless of the CYP2D6 genotype in all cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 1.57 ± 0.38, P = .54; T47D = 1.17 ± 0.23, P = .79; BT474 = 0.96 ± 0.2, P = .98; ZR-75-1 = 0.86 ± 0.67, P = .99). Interestingly, tamoxifen and its primary metabolites were not able to fully inhibit the estrogen-stimulated expression of estrogen-responsive genes in MCF-7 cells (P < .05 for all genes), but the addition of endoxifen was able to produce additional antiestrogenic effect on these genes. CONCLUSIONS: The results indicate that tamoxifen and other metabolites, excluding endoxifen, completely inhibit estrogen-stimulated growth in all cell lines, but additional antiestrogenic action from endoxifen is necessary for complete blockade of estrogen-stimulated genes. Endoxifen is of supportive importance for the therapeutic effect of tamoxifen in a postmenopausal setting.

Simulation with cells in vitro of tamoxifen treatment in premenopausal breast cancer patients with different CYP2D6 genotypes.

BACKGROUND AND PURPOSE: Tamoxifen is a prodrug that is metabolically activated by 4-hydroxylation to the potent primary metabolite 4-hydroxytamoxifen (4OHT) or via another primary metabolite N-desmethyltamoxifen (NDMTAM) to a biologically active secondary metabolite endoxifen through a cytochrome P450 2D6 variant system (CYP2D6). To elucidate the mechanism of action of tamoxifen and the importance of endoxifen for its effect, we determined the anti-oestrogenic efficacy of tamoxifen and its metabolites, including endoxifen, at concentrations corresponding to serum levels measured in breast cancer patients with various CYP2D6 genotypes (simulating tamoxifen treatment). EXPERIMENTAL APPROACH: The biological effects of tamoxifen and its metabolites on cell growth and oestrogen-responsive gene modulation were evaluated in a panel of oestrogen receptor-positive breast cancer cell lines. Actual clinical levels of tamoxifen metabolites in breast cancer patients were used in vitro along with actual levels of oestrogens observed in premenopausal patients taking tamoxifen. KEY RESULTS: Tamoxifen and its primary metabolites (4OHT and NDMTAM) only partially inhibited the stimulant effects of oestrogen on cells. The addition of endoxifen at concentrations corresponding to different CYP2D6 genotypes was found to enhance the anti-oestrogenic effect of tamoxifen and its metabolites with an efficacy that correlated with the concentration of endoxifen; at concentrations corresponding to the extensive metabolizer genotype it further inhibited the actions of oestrogen. In contrast, lower concentrations of endoxifen (intermediate and poor metabolizers) had little or no anti-oestrogenic effects. CONCLUSIONS AND IMPLICATIONS: Endoxifen may be a clinically relevant metabolite in premenopausal patients as it provides additional anti-oestrogenic actions during tamoxifen treatment.

Influence of the length and positioning of the antiestrogenic side chain of endoxifen and 4-hydroxytamoxifen on gene activation and growth of estrogen receptor positive cancer cells.

Tamoxifen has biologically active metabolites: 4-hydroxytamoxifen (4OHT) and endoxifen. The E-isomers are not stable in solution as Z-isomerization occurs. We have synthesized fixed ring (FR) analogues of 4OHT and endoxifen as well as FR E and Z isomers with methoxy and ethoxy side chains. Pharmacologic properties were documented in the MCF-7 cell line, and prolactin synthesis was assessed in GH3 rat pituitary tumor cells. The FR Z-isomers of 4OHT and endoxifen were equivalent to 4OHT and endoxifen. Other test compounds used possessed partial estrogenic activity. The E-isomers of FR 4OHT and endoxifen had no estrogenic activity at therapeutic serum concentrations. None of the newly synthesized compounds were able to down-regulate ER levels. Molecular modeling demonstrated that some compounds would each create a best fit with a novel agonist conformation of the ER. The results demonstrate modulation by the ER complex of cell replication or gene transcription in cancer.