Role of human pregnane X receptor in tamoxifen- and 4-hydroxytamoxifen-mediated CYP3A4 induction in primary human hepatocytes and LS174T cells.

Previously we observed that the antiestrogens tamoxifen and 4-hydroxytamoxifen (4OHT) induce CYP3A4 in primary human hepatocytes and activate human pregnane X receptor (PXR) in cell-based reporter assays. Given the complex cross-talk between nuclear receptors, tissue-specific expression of CYP3A4, and the potential for tamoxifen and 4OHT to interact with a myriad of receptors, this study was undertaken to gain mechanistic insights into the inductive effects of tamoxifen and 4OHT. First, we observed that transfection of the primary cultures of human hepatocytes with PXR-specific small interfering RNA reduced the PXR mRNA expression and the extent of CYP3A4 induction by tamoxifen and 4OHT by 50%. Second, in LS174T colon carcinoma cells, which were observed to have significantly lower PXR expression relative to human hepatocytes, neither tamoxifen nor 4OHT induced CYP3A4. Third, N-desmethyltamoxifen, which did not induce CYP3A4 in human hepatocytes, also did not activate PXR in LS174T cells. We then used cell-based reporter assay to evaluate the effects of other receptors such as glucocorticoid receptor GR alpha and estrogen receptor ER alpha on the transcriptional activation of PXR. The cotransfection of GR alpha in LS174T cells augmented PXR activation by tamoxifen and 4OHT. On the other hand, the presence of ER alpha inhibited PXR-mediated basal activation of CYP3A4 promoter, possibly via competing for common cofactors such as steroid receptor coactivator 1 and glucocorticoid receptor interacting protein 1. Collectively, our findings suggest that the CYP3A4 induction by tamoxifen and 4OHT is primarily mediated by PXR but the overall stoichiometry of other nuclear receptors and transcription cofactors also contributes to the extent of the inductive effect.

Sulfasalazine-induced reduction of glutathione levels in breast cancer cells: enhancement of growth-inhibitory activity of Doxorubicin.

BACKGROUND: We previously showed that the anti-inflammatory drug, sulfasalazine (salicylazosulfapyridine, SASP), can arrest proliferation of MCF-7 and MDA-MB-231 mammary cancer cells by inhibiting uptake of cystine via the x(c-) cystine/glutamate antiporter. Here we examined SASP with regard to reduction of cellular glutathione (GSH) levels and drug efficacy-enhancing ability. METHODS: GSH levels were measured spectrophotometrically. Cellular drug retention was determined with 3H-labeled methotrexate, and drug efficacy with a colony formation assay. RESULTS: Incubation of the mammary cancer cells with SASP (0.3-0.5 mM) led to reduction of their GSH content in a time- and concentration-dependent manner. Similar to MK-571, a multidrug resistance-associated protein inhibitor, SASP increased intracellular accumulation of methotrexate. Preincubation of cells with SASP (0.3 mM) significantly enhanced the potency of the anticancer agent doxorubicin (2.5 nM). CONCLUSIONS: SASP-induced reduction of cellular GSH levels can lead to growth arrest of mammary cancer cells and enhancement of anticancer drug efficacy.

Stress and prolactin effects on bone marrow myeloid cells, serum chemokine and serum glucocorticoid levels in mice.

OBJECTIVE: Current evidence supports the conclusion that prolactin (PRL) is not an obligate immunoregulatory hormone and influences the immune system predominantly during stress conditions. In this study, we examined the impact of PRL on the psychogenic stress-induced responses of myeloid cells. METHODS: Seven-week-old PRL+/- (normal) and PRL-/- (deficient) mice were exposed to a predator for 1 h/day on 3 consecutive days. Another group of PRL-deficient mice received either 1 pituitary graft (hyperprolactinemic) or sham surgery at 5 weeks of age, while PRL-normal mice only received sham surgery. Two weeks later, these mice were also subjected to predator exposure. One day after the last predator exposure session, all mice were killed and the bone marrow and blood harvested. RESULTS: Significant differences in the myeloid cells between PRL-normal and PRL-deficient mice only occurred in stressed conditions. The median serum corticosterone levels were consistently higher in PRL-deficient mice. The implantation of a pituitary graft lowered the corticosterone levels to those observed in PRL-normal mice. The absolute number of immature neutrophils as well as the numbers of granulocyte macrophage, monocyte/macrophage and granulocyte colonies were significantly higher in the stressed PRL-deficient mice; however, only the increased number of immature neutrophils was reversed by pituitary grafting. CONCLUSIONS: Our findings support previous observations that PRL influences myeloid cells of the bone marrow most profoundly in stressed conditions. However, the mechanism by which PRL influences bone marrow myeloid cells during stress cannot be explained solely by its effect on serum corticosterone.

Nitric oxide mediates increased P-glycoprotein activity in interferon-{gamma}-stimulated human intestinal cells.

Patients with refractory inflammatory bowel disease (IBD) exhibit increased expression of intestinal P-glycoprotein (P-gp) as well as elevated luminal IFN-gamma and nitric oxide (NO) levels. Using the in vitro Caco-2 cell culture model, we investigated whether these pathological mediators associated with the etiology of IBD affect functional activity of intestinal efflux systems. IFN-gamma reduced cellular uptake of cyclosporin A (CysA) but not methotrexate (MTX) in a time- and concentration-dependent manner. Simultaneously, P-gp expression increased by approximately twofold. Coincubation with the inducible NO synthase inhibitor l-N(6)-(1-iminoethyl)lysine (l-NIL) dramatically reduced production of intracellular NO in response to IFN-gamma stimulus. The presence of l-NIL also abrogated the cytokine-mediated increase in P-gp expression and function suggesting that NO is required for IFN-gamma-mediated activation of this efflux system. Exposure of Caco-2 cells to the chemical NO donor S-nitroso-N-acetylpenicillamine (SNAP) produced a concentration-dependent decrease in intracellular CysA accumulation that was paralleled by an increase in P-gp expression. Both IFN-gamma and SNAP enhanced DNA binding of NF-kappaB, whereas inclusion of l-NIL dramatically decreased this cytokine-induced effect on NF-kappaB binding. These results suggest that NO mediates IFN-gamma-induced increase in expression and function of intestinal P-gp in the human Caco-2 cell culture model by altering DNA binding of NF-kappaB, which may enhance transcription of the ABCB1 gene encoding for this efflux system.

Induction of CYP3A4 by efavirenz in primary human hepatocytes: comparison with rifampin and phenobarbital.

The antiretroviral agent efavirenz enhances the systemic clearance of coadministered drugs that are cytochrome P450 (CYP) 3A4 substrates. The mechanism of the apparent increase in CYP3A4 activity by efavirenz and the magnitude of change relative to other known inducers are not known. The authors tested the hypothesis that increased enzymatic activity by efavirenz entails CYP3A4 induction and activation of the human pregnane X receptor (hPXR), a key transcriptional regulator of CYP3A4. Employing primary cultures of human hepatocytes, they compared the CYP3A4 inductive effects of efavirenz (1-10 microM) to rifampin (10 microM) and phenobarbital (2 mM). A cell-based reporter assay was employed to assess hPXR activation. The authors observed that efavirenz caused a concentration-dependent CYP3A4 induction and hPXR activation. Based on the CYP3A4 activity assay, the average magnitude of induction by efavirenz (5-10 microM) was approximately 3- to 4-fold. In comparison, phenobarbital (2 mM) and rifampin (10 microM) caused a 5- and 6-fold induction, respectively.

Differences in the induction of cytochrome P450 3A4 by taxane anticancer drugs, docetaxel and paclitaxel, assessed employing primary human hepatocytes.

PURPOSE: The induction of cytochrome P450 (CYP) 3A4 by drugs and other xenobiotics is a common cause of serious drug interactions. The aim of this study was to comparatively examine the effects of paclitaxel and docetaxel, two structurally related taxane anticancer agents, on the activity and expression of hepatic CYP3A4. METHODS: Employing primary cultures of human hepatocytes from multiple donors, we investigated the differences in the magnitude of CYP3A4 induction and relative accumulation of paclitaxel and docetaxel. The CYP3A4 activity of intact hepatocytes was measured as the rate of testosterone 6beta-hydroxylation. The CYP3A4-specific immunoreactive protein and mRNA levels were measured employing Western blot and Northern blot analysis, respectively. Furthermore, employing cell-based reporter gene assay in CV-1 cells, we evaluated the capacity of paclitaxel and docetaxel to activate human pregnane X receptor (hPXR), an orphan nuclear receptor that plays a key role in the transcriptional regulation of CYP3A4. RESULTS: In concurrence with previous reports, we observed that paclitaxel potently induced CYP3A4 activity and expression in hepatocytes treated for 48-96 h. However, docetaxel did not increase the activity or the CYP3A4 immunoreactive protein levels for treatment periods up to 96 h. A marginal increase in the CYP3A4 mRNA levels was observed in cells treated with higher levels (5 and 10 microM) of docetaxel. Furthermore, while paclitaxel effectively activated hPXR (the half-maximal effective concentration, EC50, being about 5.2 microM), docetaxel weakly activated hPXR, and moreover the activation occurred only at high concentrations relative to paclitaxel. A comparison of the cellular concentrations of paclitaxel and docetaxel, in the cell culture models employed for evaluating CYP3A4 induction and hPXR activation, revealed that the intracellular paclitaxel levels were three-fold higher than that of docetaxel. Thus, it appears that both pharmacokinetic (drug concentration) and pharmacodynamic differences (hPXR activation) may account for the observed differences in CYP3A induction by paclitaxel and docetaxel. CONCLUSION: Our studies suggest that docetaxel has markedly reduced propensity to cause drug interactions that may entail hepatic CYP3A4 induction.

Identification of HRPAP20: a novel phosphoprotein that enhances growth and survival in hormone-responsive tumor cells.

The prolactin (PRL)-dependent rat Nb2 T lymphoma is a valuable model for investigation of molecular mechanisms that underlie tumor progression in hormone-dependent cancers. mRNA differential display was used to screen for novel gene products expressed in hormone-stimulated or differentiating agent-treated Nb2 sublines. From numerous transcripts identified, DNA sequencing and GenBank analysis revealed a novel 289-bp fragment. Using 5'-rapid amplification of complementary ends-PCR, this fragment was used to clone a unique 2117-bp cDNA, designated HRPAP20 (hormone-regulated proliferation-associated protein), in rat lymphoma cells. Computer-assisted sequence analysis revealed a single open reading frame that encoded a putative 20.2-kDa protein. The effect of hormone stimulation to alter expression of HRPAP20 was evaluated by Northern blot analysis of total RNA obtained from PRL-stimulated, lactogen-dependent Nb2-11 cells. Quiescent cells, synchronized in the G(0)-G(1) phase of cell cycle, exhibited reduced HRPAP20 expression compared with exponentially proliferating cultures. The addition of mitogenic concentrations of PRL to stationary cells increased HRPAP20 mRNA accumulation within 4-6 h, corresponding to G(1) cell cycle progression. Immunoblot analysis showed that PRL also increased HRPAP20 protein levels within 4 h. In addition, PRL stimulated serine phosphorylation of the HRPAP20 protein with a similar kinetic pattern. Stable transfection of the HRPAP20 cDNA into Nb2-11 cells significantly (P < 0.01) increased proliferation in the absence of hormonal stimulation and inhibited apoptosis induced by lactogen deprivation (P < 0.001). In the hormone-independent and highly malignant Nb2-SFJCD1 subline, the constitutive expression of HRPAP20 was markedly reduced by exposure of the cells to dietary differentiating agents (butyrate, retinoic acid, and vitamin D(3)). After removal of these substances, PRL stimulated its expression in a manner similar to that observed in PRL-dependent Nb2-11 cells. HRPAP20 expression was also evaluated in MCF-7 cells. Its expression was detectable in quiescent cultures; addition of PRL significantly (P < 0.05) increased HRPAP20 during G(1) cell cycle progression. Exposure of the cells to butyrate or retinoic acid reduced HRPAP20 expression, similar to the effects of these substances in the malignant rat lymphoma. Stable transfection of HRPAP20 into MCF-7 cells significantly (P < 0.006) increased proliferation in the absence of hormone stimulation and augmented survival in the absence of serum (P < 0.05). We conclude that HRPAP20 is a phosphoprotein that is required for proliferation and survival of hormone-dependent tumor cells.

Dose-dependent induction of cytochrome P450 (CYP) 3A4 and activation of pregnane X receptor by topiramate.

PURPOSE: In clinical studies, topiramate (TPM) was shown to cause a dose-dependent increase in the clearance of ethinyl estradiol. We hypothesized that this interaction results from induction of hepatic cytochrome P450 (CYP) 3A4 by TPM. Accordingly, we investigated whether TPM induces CYP3A4 in primary human hepatocytes and activates the human pregnane X receptor (hPXR), a nuclear receptor that serves as a regulator of CYP3A4 transcription. METHODS: Human hepatocytes were treated for 72 h with TPM (10, 25, 50, 100, 250, and 500 microM) and known inducers, phenobarbital (PB; 2 mM), and rifampicin (10 microM). The rate of testosterone 6beta-hydroxylation by hepatocytes served as a marker for CYP3A4 activity. The CYP3A4-specific protein and mRNA levels were determined by using Western and Northern blot analyses, respectively. The hPXR activation was assessed with cell-based reporter gene assay. RESULTS: Compared with controls, TPM (50-500 microM)-treated hepatocytes exhibited a considerable increase in the CYP3A4 activity (1. 6- to 8.2-fold), protein levels (4.6- to 17.3-fold), and mRNA levels (1.9- to 13.3-fold). Comparatively, rifampicin (10 microM) effected 14.5-, 25.3-, and a 20.3-fold increase in CYP3A4 activity, immunoreactive protein levels, and mRNA levels, respectively. TPM (50-500 microM) caused 1.3- to 3-fold activation of the hPXR, whereas rifampicin (10 microM) caused a 6-fold activation. CONCLUSIONS: The observed induction of CYP3A4 by TPM, especially at the higher concentrations, provides a potential mechanistic explanation of the reported increase in the ethinyl estradiol clearance by TPM. It also is suggestive of other potential interactions when high-dose TPM therapy is used.

Prolactin-regulated pim-1 transcription: identification of critical promoter elements and Akt signaling.

Previously we showed that the distal element (DE) (-427 to -336 bp) within the pim-1 promoter appeared to regulate its prolactin (PRL)-induced transcription. To determine which specific DE sequences conferred PRL responsiveness, seven 12-bp deletion mutants ligated upstream of the chloramphenicol acetyltransferase gene were transfected into FDC/Nb2 cells. Results from promoter/ reporter studies showed that sequential 12-bp deletions of the DE significantly (p < 0.001) reduced PRL responsiveness. An additional site, nuclear factor-1 (-224 to -217), was also mutated by deletion or point mutation; both abrogated promoter activation by PRL (p < 0.0001). In other experiments, PRL signaling to pim-1 expression was investigated in FDC/Nb2 cells stably expressing the wild-type (WT) Jak2 cDNA or a carboxy-terminal kinase-deficient Jak2 mutant and in cells infected with adenoviral constructs of WT-Akt or dominant negative Akt. Altered Jak2 did not affect PRL-stimulated pim-1 expression while inhibition of Akt attenuated its transcription. We conclude that the DE and NF-1 half-site mediate PRL responsiveness of the pim-1 promoter. Moreover, the accumulated evidence does not support a role for the Jak2/Stat signaling pathway but, instead, implicates that Akt activation was a component of PRL-induced pim-1 transcription.

Prolactin suppresses glucocorticoid-induced thymocyte apoptosis in vivo.

The hypothesis that prolactin (PRL) functions as an immunomodulator was based on studies showing lymphocyte PRL receptors, and its effects on growth, differentiation, and apoptosis in lymphoid cells. However, studies of PRL (PRL-/-) and PRL receptor knockout mice indicated that PRL was not required for immune system development or function under basal conditions. Because PRL maintains survival in glucocorticoid (GC)-treated Nb2-T lymphocytes in vitro, and PRL and GCs are elevated during stress, we investigated whether PRL protected T cells in vivo from GC-induced apoptosis. Adrenalectomized mice [PRL -/-, undetectable PRL; pituitary grafted PRL-/- (PRL-/-Graft), elevated PRL; and PRL+/-, normal PRL] were treated with dexamethasone (DEX) or PBS. Thymocytes and splenocytes were isolated and annexin V labeling of phosphatidylserine, DNA fragmentation, and caspase-3 activation were assessed as indices of apoptosis. Total thymocytes and CD4+ and CD8+ T cells obtained from DEX-treated PRL-/- mice exhibited significantly increased annexin V binding. In contrast, binding was not altered by DEX in PRL-/-Graft thymocytes. In addition, DEX induced classic DNA fragmentation in PRL-/- thymocytes. Elevated serum PRL reduced this effect. Thymocytes from DEX-treated PRL-/- mice exhibited increased caspase-3 activation, which was inhibited in cells from PRL-/-Graft mice. Finally, elevated expression of X-linked inhibitor of apoptosis, XIAP, was observed in thymi from DEX-treated PRL -/-Graft mice. This is the first demonstration that elevated PRL antagonizes apoptosis in thymocytes exposed to GCs in vivo. These observations suggest that, under conditions of increased GCs, such as during stress, elevated PRL functions physiologically to maintain survival and function of T-lymphocytes.

Induction of cytochrome P450 3A by paclitaxel in mice: pivotal role of the nuclear xenobiotic receptor, pregnane X receptor.

Paclitaxel, a taxane anti-microtubule agent, is known to induce CYP3A in rat and human hepatocytes. Recent studies suggest that a member of the nuclear receptor family, pregnane X Receptor (PXR), is a key regulator of the expression of CYP3A in different species. We investigated the role of PXR activation, in vitro and in vivo, in mediating Cyp3a induction by paclitaxel. Pregnenolone 16 alpha-carbonitrile (PCN), an antiglucocorticoid, was employed as a positive control for mouse PXR (mPXR) activation in vitro, and Cyp3a induction in vivo. In cell based reporter gene assays paclitaxel and PCN activated mPXR with an EC(50) of 5.6 and 0.27 microM, respectively. Employing PXR wild-type and transgenic mice lacking functional PXR (-/-), we evaluated the expression and activity of CYP3A following treatment with paclitaxel and PCN. Paclitaxel significantly induced CYP3A11 mRNA and immunoreactive CYP3A protein in PXR wild-type mice. Consistent with kinetics of CYP3A induction, the V(max) of testosterone 6 beta-hydroxylation in microsomal fraction increased 15- and 30-fold in paclitaxel- and PCN-treated mice, respectively. The Cyp3a induction response was completely abolished in paclitaxel- and PCN-treated PXR-null mice. This suggests that paclitaxel-mediated CYP3A induction in vivo requires an intact PXR-signaling mechanism. Our study validates the use of PXR activation assays in screening newer taxanes for potential drug interactions that may be related to PXR-target gene induction.

Extracellular glucose concentration alters functional activity of the intestinal oligopeptide transporter (PepT-1) in Caco-2 cells.

The objective of this study was to determine the effect of different cell culture media glucose concentrations on the functional activity of PepT-1 in Caco-2 cells. Uptake kinetics of Gly-Sar into Caco-2 cells that were maintained in iso-osmotic media containing 25 or 5.5 mM glucose were determined in the presence and absence of amino acid-selective chemical modifiers and dithiothreitol. Inhibition of Gly-Sar uptake into Caco-2 cells was measured in the presence of dipeptides and xenobiotics exhibiting various binding affinities for the PepT-1. The effect of extracellular glucose on PepT-1 gene expression was assessed using comparative RT-PCR. Long-term exposure of Caco-2 cells to 25 mM glucose reduced maximum transport capacity for Gly-Sar uptake without altering PepT-1 gene expression. In contrast, binding affinity of Gly-Sar and other dipeptides or xenobiotics was not significantly changed. Chemical modification of Lys and Tyr residues decreased V(max), while Cys modification increased the maximum transport capacity of the carrier. Preincubation of Caco-2 cells with dithiothreitol restored PepT-1 activity in cells maintained at 25 mM glucose. In conclusion, cell culture media containing 25 mM glucose decreases maximum transport capacity of PepT-1 in Caco-2 cells without affecting substrate recognition, at least in part, mediated via an oxidative pathway.

Suppression of cystine uptake by sulfasalazine inhibits proliferation of human mammary carcinoma cells.

BACKGROUND: Malignant progression of lymphoma cells is associated with acquisition of the cystine/glutamate antiporter, xc-, enhancing cystine uptake. Recently, we showed that sulfasalazine (SASP) is a specific xc- inhibitor. Here, we investigated xc- in mammary cancer cell lines. MATERIALS AND METHODS: Expression and function of xc- were evaluated by RT-PCR and 35S-cystine uptake analysis. RESULTS: Xc- expression was elevated 4-fold (p < 0.001) in cells of the most malignant line, MDA-MB-231, associated with increased 35S-cystine uptake (p < 0.001). Proliferation was inhibited by 0.2-0.5 mM SASP. 2-Mercaptoethanol (60 microM), a cystine uptake enhancer, completely prevented SASP-mediated growth inhibition in MDA-MB-231 cultures, but only partially in 184A1 and MCF-7 cultures. SASP-induced growth arrest was reversible and not cell cycle-specific. CONCLUSION: The results suggest: (i) malignant progression of human mammary cancer may be associated with acquisition of xc- expression potentially leading to increased growth autonomy and drug resistance, (ii) xc- may act as a therapeutic target.

Effects of prolactin deficiency on myelopoiesis and splenic T lymphocyte proliferation in thermally injured mice.

The importance of prolactin (PRL) in mammopoiesis and milk production is undisputed. However, previous studies investigating the role of PRL in immune function have yielded inconsistencies. These inconsistencies have led to our hypothesis that the immunomodulatory effects of PRL are only manifest under conditions in which the organism is subjected to stress. Thermal injury is a well-known stressor. The goal of this study was to determine whether the lack of PRL enhanced the negative effects of thermal injury-induced immune alterations utilizing a mouse model in which the PRL gene had been disrupted. Mice received either sham or burn treatment, and were sacrificed 4 days later. The immune parameters studied were the capacity of bone marrow cells to form granulocyte-macrophage colony forming units (GM-CFU) in the presence of granulocyte-macrophage colony stimulating factor, and the ability of the splenic T lymphocytes to proliferate in response to phytohemagglutin (PHA). As shown by others, our results reveal that burn increased the number of GM-CFU compared to sham controls; however, this elevation was only significant in the PRL-/- mice. Thermal injury increased PHA-stimulated proliferation of splenic T lymphocytes, however this increase was only significant in the PRL+/- group. We conclude that under conditions of a controlled stress event (thermal injury) [a] the increase in the GM-CFU is exaggerated in the absence of PRL, and [b] the enhancement of PHA-induced proliferation of splenic lymphocytes required PRL. This study supports the hypothesis that the immunomodulatory effects of PRL are manifest when the organism is subjected to stress.

Induction of cytochrome P450 3A4 in primary human hepatocytes and activation of the human pregnane X receptor by tamoxifen and 4-hydroxytamoxifen.

Tamoxifen is a widely utilized antiestrogen in the treatment and chemoprevention of breast cancer. Clinical studies document that tamoxifen administration markedly enhances the systemic elimination of other drugs. Additionally, tamoxifen enhances its own clearance following repeated dosing. The mechanisms that underlie these clinically important events remain unresolved. Here, we report that tamoxifen and its metabolite 4-hydroxytamoxifen markedly induce cytochrome P450 3A4, a drug-metabolizing enzyme of central importance, in primary cultures of human hepatocytes. Tamoxifen and 4-hydroxytamoxifen (1-10 microM) significantly increased the CYP3A4 expression and activity (measured as the rate of testosterone 6beta-hydroxylation). Maximal induction was achieved at the 5 microM level. At this level, tamoxifen and 4-hydroxytamoxifen caused a 1.5- to 3.3-fold (mean, 2.1-fold) and 3.4- to 17-fold (mean, 7.5-fold) increase in the CYP3A4 activity, respectively. In comparison, rifampicin treatment resulted in a 6- to 16-fold (mean, 10.5-fold) increase. We also observed corresponding increase in the CYP3A4 immunoreactive protein and mRNA levels. Furthermore, tamoxifen and 4-hydroxytamoxifen efficaciously activated the human pregnane X receptor (hPXR; also known as the steroid xenobiotic receptor), a key regulator of CYP3A4 expression. The efficacy of tamoxifen and 4-hydroxytamoxifen relative to rifampicin for hPXR activation was approximately 30 and 60%, respectively. Our results indicate that the mechanism of tamoxifen-mediated alteration in drug clearance pathways in humans may involve CYP3A4 induction by the parent drug and/or its metabolite. Furthermore, the CYP3A4 induction may be a result of hPXR activation. These findings have important implications for optimizing the use of tamoxifen and in the development of newer antiestrogens.