The Use of Technology to Support Precision Health in Nursing Science.

PURPOSE: This article outlines how current nursing research can utilize technology to advance symptom and self-management science for precision health and provides a roadmap for the development and use of technologies designed for this purpose. APPROACH: At the 2018 annual conference of the National Institute of Nursing Research (NINR) Research Centers, nursing and interdisciplinary scientists discussed the use of technology to support precision health in nursing research projects and programs of study. Key themes derived from the presentations and discussion were summarized to create a proposed roadmap for advancement of technologies to support health and well-being. CONCLUSIONS: Technology to support precision health must be centered on the user and designed to be desirable, feasible, and viable. The proposed roadmap is composed of five iterative steps for the development, testing, and implementation of technology-based/enhanced self-management interventions. These steps are (a) contextual inquiry, focused on the relationships among humans, and the tools and equipment used in day-to-day life; (b) value specification, translating end-user values into end-user requirements; (c) design, verifying that the technology/device can be created and developing the prototype(s); (d) operationalization, testing the intervention in a real-world setting; and (e) summative evaluation, collecting and analyzing viability metrics, including process data, to evaluate whether the technology and the intervention have the desired effect. CLINICAL RELEVANCE: Interventions using technology are increasingly popular in precision health. Use of a standard multistep process for the development and testing of technology is essential.

Whole blueberry powder modulates the growth and metastasis of MDA-MB-231 triple negative breast tumors in nude mice.

Previous studies in our laboratory demonstrated that blueberry (BB) extract exhibited antitumor activity against MDA-MB-231 triple negative breast cancer (TNBC) cells and decreased metastatic potential in vitro. The current study tested 2 doses of whole BB powder, 5 and 10% (wt:wt) in the diet, against MDA-MB-231 tumor growth in female nude mice. In this study, tumor volume was 75% lower in mice fed the 5% BB diet and 60% lower in mice fed the 10% BB diet than in control mice (P ≤ 0.05). Tumor cell proliferation (Ki-67) was lower in the 5 and 10% BB-fed mice and cell death (Caspase 3) was greater in the 10% BB-fed mice compared to control mice (P ≤ 0.05). Gene analysis of tumor tissues from the 5% BB-fed mice revealed significantly altered expression of genes important to inflammation, cancer, and metastasis, specifically, Wnt signaling, thrombospondin-2, IL-13, and IFNγ. To confirm effects on Wnt signaling, analysis of tumor tissues from 5% BB-fed mice revealed lower ß-catenin expression and glycogen synthase kinase-3ß phosphorylation with greater expression of the ß-catenin inhibitory protein adenomatous polyposis coli compared to controls. A second study tested the ability of the 5% BB diet to inhibit MDA-MB-231-luc-D3H2LN metastasis in vivo. In this study, 5% BB-fed mice developed 70% fewer liver metastases (P = 0.04) and 25% fewer lymph node metastases (P = 0.09) compared to control mice. This study demonstrates the oral antitumor and metastasis activity of whole BB powder against TNBC in mice.

White button mushroom (Agaricus bisporus) exhibits antiproliferative and proapoptotic properties and inhibits prostate tumor growth in athymic mice.

White button mushrooms are a widely consumed food containing phytochemicals beneficial to cancer prevention. The purpose of this research was to evaluate the effects of white button mushroom extract and its major component, conjugated linoleic acid (CLA) on prostate cancer cell lines in vitro and mushroom extract in vivo. In all cell lines tested, mushroom inhibited cell proliferation in a dose-dependent manner and induced apoptosis within 72 h of treatment. CLA inhibited proliferation in the prostate cancer cell lines in vitro. DU145 and PC3 prostate tumor size and tumor cell proliferation were decreased in nude mice treated with mushroom extract, whereas tumor cell apoptosis was increased compared to pair-fed controls. Microarray analysis of tumors identified significant changes in gene expression in the mushroom-fed mice as compared to controls. Gene network analysis identified alterations in networks involved in cell death, growth and proliferation, lipid metabolism, the TCA cycle and immune response. The data provided by this study illustrate the anticancer potential of phytochemicals in mushroom extract both in vitro and in vivo and supports the recommendation of white button mushroom as a dietary component that may aid in the prevention of prostate cancer in men.

Anti-aromatase activity of phytochemicals in white button mushrooms (Agaricus bisporus).

White button mushrooms (Agaricus bisporous) are a potential breast cancer chemopreventive agent, as they suppress aromatase activity and estrogen biosynthesis. Therefore, we evaluated the activity of mushroom extracts in the estrogen receptor-positive/aromatase-positive MCF-7aro cell line in vitro and in vivo. Mushroom extract decreased testosterone-induced cell proliferation in MCF-7aro cells but had no effect on MCF-10A, a nontumorigenic cell line. Most potent mushroom chemicals are soluble in ethyl acetate. The major active compounds found in the ethyl acetate fraction are unsaturated fatty acids such as linoleic acid, linolenic acid, and conjugated linoleic acid. The interaction of linoleic acid and conjugated linoleic acid with aromatase mutants expressed in Chinese hamster ovary cells showed that these fatty acids inhibit aromatase with similar potency and that mutations at the active site regions affect its interaction with these two fatty acids. Whereas these results suggest that these two compounds bind to the active site of aromatase, the inhibition kinetic analysis indicates that they are noncompetitive inhibitors with respect to androstenedione. Because only conjugated linoleic acid was found to inhibit the testosterone-dependent proliferation of MCF-7aro cells, the physiologically relevant aromatase inhibitors in mushrooms are most likely conjugated linoleic acid and its derivatives. The in vivo action of mushroom chemicals was shown using nude mice injected with MCF-7aro cells. The studies showed that mushroom extract decreased both tumor cell proliferation and tumor weight with no effect on rate of apoptosis. Therefore, our studies illustrate the anticancer activity in vitro and in vivo of mushroom extract and its major fatty acid constituents.

Kaurene diterpenes from Laetia thamnia inhibit the growth of human cancer cells in vitro.

Four ent-kaurene diterpenes were isolated from the leaves of Laetia thamnia L.: ent-kaur-16-en-19-oic acid (1a), ent-3beta-hydroxykaur-16-ene (2), ent-kaur-16-en-3alpha,19-diol (3a), and ent-17-hydroxykaur-15-en-19-oic acid (4). The methyl ester (1b) of compound 1a and the acetate diester (3b) of compound 3a were prepared, and all compounds were evaluated for cytotoxicity against human prostate (22Rv1, LNCaP), colon (HT29, HCT116, SW480, SW620), and breast (MCF-7) tumor cells at concentrations ranging from 6 to 50microg/mL. The kaurenes showed activity in all cell lines tested, with the prostate cells demonstrating the most sensitivity as follows: 22 Rv1 cells towards 1a (IC(50) 5.03microg/mL) and 1b (IC(50) 6.81microg/mL), and LNCaP towards 2 (IC(50) 12.83microg/mL) and 4 (IC(50) 17.63microg/mL).

Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro.

Berry fruits are widely consumed in our diet and have attracted much attention due to their potential human health benefits. Berries contain a diverse range of phytochemicals with biological properties such as antioxidant, anticancer, anti-neurodegerative, and anti-inflammatory activities. In the current study, extracts of six popularly consumed berries--blackberry, black raspberry, blueberry, cranberry, red raspberry and strawberry--were evaluated for their phenolic constituents using high performance liquid chromatography with ultraviolet (HPLC-UV) and electrospray ionization mass spectrometry (LC-ESI-MS) detection. The major classes of berry phenolics were anthocyanins, flavonols, flavanols, ellagitannins, gallotannins, proanthocyanidins, and phenolic acids. The berry extracts were evaluated for their ability to inhibit the growth of human oral (KB, CAL-27), breast (MCF-7), colon (HT-29, HCT116), and prostate (LNCaP) tumor cell lines at concentrations ranging from 25 to 200 micro g/mL. With increasing concentration of berry extract, increasing inhibition of cell proliferation in all of the cell lines were observed, with different degrees of potency between cell lines. The berry extracts were also evaluated for their ability to stimulate apoptosis of the COX-2 expressing colon cancer cell line, HT-29. Black raspberry and strawberry extracts showed the most significant pro-apoptotic effects against this cell line. The data provided by the current study and from other laboratories warrants further investigation into the chemopreventive and chemotherapeutic effects of berries using in vivo models.

Pomegranate juice, total pomegranate ellagitannins, and punicalagin suppress inflammatory cell signaling in colon cancer cells.

Phytochemicals from fruits such as the pomegranate (Punica granatum L) may inhibit cancer cell proliferation and apoptosis through the modulation of cellular transcription factors and signaling proteins. In previous studies, pomegranate juice (PJ) and its ellagitannins inhibited proliferation and induced apoptosis in HT-29 colon cancer cells. The present study examined the effects of PJ on inflammatory cell signaling proteins in the HT-29 human colon cancer cell line. At a concentration of 50 mg/L PJ significantly suppressed TNFalpha-induced COX-2 protein expression by 79% (SE = 0.042), total pomegranate tannin extract (TPT) 55% (SE = 0.049), and punicalagin 48% (SE = 0.022). Additionally, PJ reduced phosphorylation of the p65 subunit and binding to the NFkappaB response element 6.4-fold. TPT suppressed NFkappaB binding 10-fold, punicalagin 3.6-fold, whereas ellagic acid (EA) (another pomegranate polyphenol) was ineffective. PJ also abolished TNFalpha-induced AKT activation, needed for NFkappaB activity. Therefore, the polyphenolic phytochemicals in the pomegranate can play an important role in the modulation of inflammatory cell signaling in colon cancer cells.

The Spectrum of Caregiving in Palliative Care for Serious, Advanced, Rare Diseases: Key Issues and Research Directions.

Rare diseases are often life-limiting conditions, the majority of which require constant caregiving needs. The realization of a spectrum of palliative care throughout the trajectory of rare diseases could ensure individualized and caregiver-focused approaches to the care of patients and families. In June 2015, the National Institute of Nursing Research (NINR), the lead institute at the National Institutes of Health for end-of-life research, in conjunction with the National Center for Advancing Translational Sciences, Office of Rare Diseases Research (ORDR) held an interdisciplinary workshop on the unique challenges of caregiving and palliative care in adult and pediatric rare diseases. The panel identified gaps in current knowledge, and afforded suggestions for research opportunities in palliative care science to improve the care of individuals with serious, advanced, rare diseases and their caregivers. This meeting provided an in-depth opportunity to incorporate new concepts into palliative and end-of-life care for individuals with a range of rare diseases and their caregivers. This report presents a summary of the workshop.

In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice.

Pomegranate (Punica granatum L.) fruits are widely consumed as juice (PJ). The potent antioxidant and anti-atherosclerotic activities of PJ are attributed to its polyphenols including punicalagin, the major fruit ellagitannin, and ellagic acid (EA). Punicalagin is the major antioxidant polyphenol ingredient in PJ. Punicalagin, EA, a standardized total pomegranate tannin (TPT) extract and PJ were evaluated for in vitro antiproliferative, apoptotic and antioxidant activities. Punicalagin, EA and TPT were evaluated for antiproliferative activity at 12.5-100 microg/ml on human oral (KB, CAL27), colon (HT-29, HCT116, SW480, SW620) and prostate (RWPE-1, 22Rv1) tumor cells. Punicalagin, EA and TPT were evaluated at 100 microg/ml concentrations for apoptotic effects and at 10 microg/ml concentrations for antioxidant properties. However, to evaluate the synergistic and/or additive contributions from other PJ phytochemicals, PJ was tested at concentrations normalized to deliver equivalent amounts of punicalagin (w/w). Apoptotic effects were evaluated against the HT-29 and HCT116 colon cancer cell lines. Antioxidant effects were evaluated using inhibition of lipid peroxidation and Trolox equivalent antioxidant capacity (TEAC) assays. Pomegranate juice showed greatest antiproliferative activity against all cell lines by inhibiting proliferation from 30% to 100%. At 100 microg/ml, PJ, EA, punicalagin and TPT induced apoptosis in HT-29 colon cells. However, in the HCT116 colon cells, EA, punicalagin and TPT but not PJ induced apoptosis. The trend in antioxidant activity was PJ>TPT>punicalagin>EA. The superior bioactivity of PJ compared to its purified polyphenols illustrated the multifactorial effects and chemical synergy of the action of multiple compounds compared to single purified active ingredients.

Total cranberry extract versus its phytochemical constituents: antiproliferative and synergistic effects against human tumor cell lines.

Cranberries (Vaccinium macrocarpon Ait.) are an excellent dietary source of phytochemicals that include flavonol glycosides, anthocyanins, proanthocyanidins (condensed tannins), and organic and phenolic acids. Using C-18 and Sephadex Lipophilic LH-20 column chromatography, HPLC, and tandem LC-ES/MS, the total cranberry extract (TCE) has been analyzed, quantified, and separated into fractions enriched in sugars, organic acids, total polyphenols, proanthocyanidins, and anthocyanins (39.4, 30.0, 10.6, 5.5, and 1.2% composition, respectively). Using a luminescent ATP cell viability assay, the antiproliferative effects of TCE (200 microg/mL) versus all fractions were evaluated against human oral (KB, CAL27), colon (HT-29, HCT116, SW480, SW620), and prostate (RWPE-1, RWPE-2, 22Rv1) cancer cell lines. The total polyphenol fraction was the most active fraction against all cell lines with 96.1 and 95% inhibition of KB and CAL27 oral cancer cells, respectively. For the colon cancer cells, the antiproliferative activity of this fraction was greater against HCT116 (92.1%) than against HT-29 (61.1%), SW480 (60%), and SW620 (63%). TCE and all fractions showed >/=50% antiproliferative activity against prostate cancer cells with total polyphenols being the most active fraction (RWPE-1, 95%; RWPE-2, 95%; 22Rv1, 99.6%). Cranberry sugars (78.8 microg/mL) did not inhibit the proliferation of any cancer cell lines. The enhanced antiproliferative activity of total polyphenols compared to TCE and its individual phytochemicals suggests synergistic or additive antiproliferative interactions of the anthocyanins, proanthocyanidins, and flavonol glycosides within the cranberry extract.

Pomegranate ellagitannin-derived compounds exhibit antiproliferative and antiaromatase activity in breast cancer cells in vitro.

Estrogen stimulates the proliferation of breast cancer cells and the growth of estrogen-responsive tumors. The aromatase enzyme, which converts androgen to estrogen, plays a key role in breast carcinogenesis. The pomegranate fruit, a rich source of ellagitannins (ET), has attracted recent attention due to its anticancer and antiatherosclerotic properties. On consumption, pomegranate ETs hydrolyze, releasing ellagic acid, which is then converted to 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one ("urolithin") derivatives by gut microflora. The purpose of this study was to investigate the antiaromatase activity and inhibition of testosterone-induced breast cancer cell proliferation by ET-derived compounds isolated from pomegranates. A panel of 10 ET-derived compounds including ellagic acid, gallagic acid, and urolithins A and B (and their acetylated, methylated, and sulfated analogues prepared in our laboratory) were examined for their ability to inhibit aromatase activity and testosterone-induced breast cancer cell proliferation. Using a microsomal aromatase assay, we screened the panel of ET-derived compounds and identified six with antiaromatase activity. Among these, urolithin B (UB) was shown to most effectively inhibit aromatase activity in a live cell assay. Kinetic analysis of UB showed mixed inhibition, suggesting more than one inhibitory mechanism. Proliferation assays also determined that UB significantly inhibited testosterone-induced MCF-7aro cell proliferation. The remaining test compounds also exhibited antiproliferative activity, but to a lesser degree than UB. These studies suggest that pomegranate ET-derived compounds have potential for the prevention of estrogen-responsive breast cancers.

Blueberry phytochemicals inhibit growth and metastatic potential of MDA-MB-231 breast cancer cells through modulation of the phosphatidylinositol 3-kinase pathway.

Dietary phytochemicals are known to exhibit a variety of anticarcinogenic properties. This study investigated the chemopreventive activity of blueberry extract in triple-negative breast cancer cell lines in vitro and in vivo. Blueberry decreased cell proliferation in HCC38, HCC1937, and MDA-MB-231 cells with no effect on the nontumorigenic MCF-10A cell line. Decreased metastatic potential of MDA-MB-231 cells by blueberry was shown through inhibition of cell motility using wound-healing assays and migration through a polyethylene terephthalate membrane. Blueberry treatment decreased the activity of matrix metalloproteinase-9 and the secretion of urokinase-type plasminogen activator while increasing tissue inhibitor of metalloproteinase-1 and plasminogen activator inhibitor-1 secretion in MDA-MB-231 conditioned medium as shown by Western blotting. Cell signaling pathways that control the expression/activation of these processes were investigated via Western blotting and reporter gene assay. Treatment with blueberry decreased phosphatidylinositol 3-kinase (PI3K)/AKT and NFkappaB activation in MDA-MB-231 cells, where protein kinase C and extracellular signal-regulated kinase (ERK) were not affected. In vivo, the efficacy of blueberry to inhibit triple-negative breast tumor growth was evaluated using the MDA-MB-231 xenograft model. Tumor weight and proliferation (Ki-67 expression) were decreased in blueberry-treated mice, where apoptosis (caspase-3 expression) was increased compared with controls. Immunohistochemical analysis of tumors from blueberry-fed mice showed decreased activation of AKT and p65 NFkappaB signaling proteins with no effect on the phosphorylation of ERK. These data illustrate the inhibitory effect of blueberry phytochemicals on the growth and metastatic potential of MDA-MB-231 cells through modulation of the PI3K/AKT/NFkappaB pathway.

Phytochemicals for breast cancer prevention by targeting aromatase.

Aromatase is a cytochrome P450 enzyme (CYP19) and is the rate limiting enzyme in the conversion of androgens to estrogens. Suppression of in situ estrogen production through aromatase inhibition is the current treatment strategy for hormone-responsive breast cancers. Drugs that inhibit aromatase have been developed and are currently utilized as adjuvant therapy for breast cancer in post-menopausal women with hormone dependent breast cancer. Natural compounds have been studied extensively for important biologic effects such as antioxidant, anti-tumor and anti-viral effects. A significant number of studies have also investigated the aromatase inhibitory properties of a variety of plant extracts and phytochemicals. The identification of natural compounds that inhibit aromatase could be useful both from a chemopreventive standpoint and in the development of new aromatase inhibitory drugs. This review will discuss whole food extracts and the common classes of phytochemicals which have been investigated for potential aromatase inhibitory activity. We will review reported aromatase inhibition, kinetic data and possible structural variations that may inhibit or enhance the interaction of phytochemicals with the aromatase enzyme.

Mechanisms of nuclear vitamin D receptor resistance in Harvey-ras-transfected cells.

The hormone 1,25 dihydroxyvitamin D (1,25(OH)(2)D) binds to the nuclear vitamin D receptor (nVDR), which heterodimerizes with retinoid X receptor alpha (RXRalpha), and this complex interacts with specific response elements [vitamin D response elements (VDREs)] to regulate gene transcription. Previous results show a significant reduction in 1,25(OH)(2)D-induced nVDR transcriptional activity in fibroblast (C3H10T1/2) cells transfected with the Harvey ras gene (ras cells) compared with parental cells. The purpose of this study was to investigate the mechanisms by which the H-ras gene interferes with nVDR transcriptional activity. Similar to the ras cells, transcriptional activity of the nVDR was reduced following induction of the H-ras gene for 9 days. The ras cells expressed similar protein levels of RXRalpha with the parent cells, and overexpression of the wild-type RXRalpha plasmid did not restore 1,25(OH)(2)D-mediated nVDR activity in ras cells. Inhibiting activation of extracellular signal-regulated kinase (ERK1/2) had no effect on nVDR activity in ras cells. Furthermore, the binding of nVDR to VDREs was reduced in 1,25(OH)(2)D-treated ras cells. In addition, neither treatment of ras cells with an inhibitor (ketoconazole) of the 1,25(OH)(2)D degradative enzyme, 24-hydroxylase, nor the protein kinase C inhibitors, bisindoylmaleimide I and Gö 6976, had an effect on nVDR activity. In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 resulted in a 1.6-fold significant increase in the nVDR activity in the ras cells. Taken together, these results indicate that PI3K may, at least in part, mediate the suppression of the 1,25(OH)(2)D regulation of nVDR transcriptional activity by the H-ras gene, leading to reduced ability to associate with response elements.

Eugenia jambolana Lam. berry extract inhibits growth and induces apoptosis of human breast cancer but not non-tumorigenic breast cells.

The ripe purple berries of the native Indian plant Eugenia jambolana Lam., known as Jamun, are popularly consumed and available in the United States in Florida and Hawaii. Despite the growing body of data on the chemopreventive potential of edible berry extracts, there is paucity of such data for Jamun fruit. Therefore our laboratory initiated the current study with the following objectives: (1) to prepare a standardized Jamun fruit extract (JFE) for biological studies and (2) to investigate the antiproliferative and pro-apoptotic effects of JFE in estrogen dependent/aromatase positive (MCF-7aro), and estrogen independent (MDA-MB-231) breast cancer cells, and in a normal/nontumorigenic (MCF-10A) breast cell line. JFE was standardized to anthocyanin content using the pH differential method, and individual anthocyanins were identified by high performance liquid chromatography with ultraviolet (HPLC-UV) and tandem mass spectrometry (LC-MS/MS) methods. JFE contained 3.5% anthocyanins (as cyanidin-3-glucoside equivalents) which occur as diglucosides of five anthocyanidins/aglycons: delphinidin, cyanidin, petunidin, peonidin and malvidin. In the proliferation assay, JFE was most effective against MCF-7aro (IC(50) = 27 microg/mL), followed by MDA-MB-231 (IC(50) = 40 microg/mL) breast cancer cells. Importantly, JFE exhibited only mild antiproliferative effects against the normal MCF-10A (IC(50) > 100 microg/mL) breast cells. Similarly, JFE (at 200 microg/mL) exhibited pro-apoptotic effects against the MCF-7aro (p

Analysis of the interactions of botanical extract combinations against the viability of prostate cancer cell lines.

Herbal medicines are often combinations of botanical extracts that are assumed to have additive or synergistic effects. The purpose of this investigation was to compare the effect of individual botanical extracts with combinations of extracts on prostate cell viability. We then modeled the interactions between botanical extracts in combination isobolographically. Scutellaria baicalensis, Rabdosia rubescens, Panax-pseudo ginseng, Dendranthema morifolium, Glycyrrhiza uralensis and Serenoa repens were collected, taxonomically identified and extracts prepared. Effects of the extracts on cell viability were quantitated in prostate cell lines using a luminescent ATP cell viability assay. Combinations of two botanical extracts of the four most active extracts were tested in the 22Rv1 cell line and their interactions assessed using isobolographic analysis. Each extract significantly inhibited the proliferation of prostate cell lines in a time- and dose-dependent manner except S. repens. The most active extracts, S. baicalensis, D. morifolium, G. uralensis and R. rubescens were tested as two-extract combinations. S. baicalensis and D. morifolium when combined were additive with a trend toward synergy, whereas D. morifolium and R. rubescens together were additive. The remaining two-extract combinations showed antagonism. The four extracts together were significantly more effective than the two-by-two combinations and the individual extracts alone. Combining the four herbal extracts significantly enhanced their activity in the cell lines tested compared with extracts alone. The less predictable nature of the two-way combinations suggests a need for careful characterization of the effects of each individual herb based on their intended use.

1,25-dihydroxycholecalciferol inhibits apoptosis in C3H10T1/2 murine fibroblast cells through activation of nuclear factor kappaB.

1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] is important in the regulation of cell growth, differentiation, and apoptosis. Previous results from our laboratory demonstrate that 1,25(OH)(2)D(3) inhibits vitamin E succinate (VES) mediated apoptosis in untransformed C3H10T1/2 mouse fibroblast cells. The current work investigated cell survival signaling pathways that may be activated by 1,25(OH)(2)D(3), leading to protection from apoptosis. Results showed that nuclear factor kappaB (NFkappaB) transcriptional activity was significantly increased 1.8-fold over vehicle controls by 1,25(OH)(2)D(3) after 4 h of treatment. Protein kinase B/AKT, a downstream effector of phosphoinositide 3-kinase (PI3K), was activated 4-fold and 8-fold at 2 and 4 h, respectively, after treatment with 1,25(OH)(2)D(3). Pretreatment with two PI3K inhibitors, LY294002 and wortmannin, abolished the activation of NFkappaB by 1,25(OH)(2)D(3), suggesting that this pathway is essential for NFkappaB transcriptional activation. Additionally, the use of a p-21 activated kinase (PAK1) inhibitory construct (PAK(R299)) demonstrated that PAK1 was also required for NFkappaB transcriptional activation by 1,25(OH)(2)D(3). Inhibition of NFkappaB activity with transfection of the NFkappaB inhibitory construct (IkappaB(Ala32)) abolished the protective effect of 1,25(OH)(2)D(3) on VES-mediated apoptosis. In summary, NFkappaB transcriptional activation was essential to 1,25(OH)(2)D(3) protection from VES-mediated apoptosis and 1,25(OH)(2)D(3) regulated NFkappaB activity through PI3K and PAK pathways.