Nuclear Receptor Expression and Function in Human Lung Cancer Pathogenesis.

Lung cancer is caused by combinations of diverse genetic mutations. Here, to understand the relevance of nuclear receptors (NRs) in the oncogene-associated lung cancer pathogenesis, we investigated the expression profile of the entire 48 NR members by using QPCR analysis in a panel of human bronchial epithelial cells (HBECs) that included precancerous and tumorigenic HBECs harboring oncogenic K-rasV12 and/or p53 alterations. The analysis of the profile revealed that oncogenic alterations accompanied transcriptional changes in the expression of 19 NRs in precancerous HBECs and 15 NRs according to the malignant progression of HBECs. Amongst these, peroxisome proliferator-activated receptor gamma (PPARγ), a NR chosen as a proof-of-principle study, showed increased expression in precancerous HBECs, which was surprisingly reversed when these HBECs acquired full in vivo tumorigenicity. Notably, PPARγ activation by thiazolidinedione (TZD) treatment reversed the increased expression of pro-inflammatory cyclooxygenase 2 (COX2) in precancerous HBECs. In fully tumorigenic HBECs with inducible expression of PPARγ, TZD treatments inhibited tumor cell growth, clonogenecity, and cell migration in a PPARγ-sumoylation dependent manner. Mechanistically, the sumoylation of liganded-PPARγ decreased COX2 expression and increased 15-hydroxyprostaglandin dehydrogenase expression. This suggests that ligand-mediated sumoylation of PPARγ plays an important role in lung cancer pathogenesis by modulating prostaglandin metabolism.

Combined therapeutic potential of nuclear receptors with receptor tyrosine kinase inhibitors in lung cancer.

Cancer heterogeneity is a big hurdle in achieving complete cancer treatment, which has led to the emergence of combinational therapy. In this study, we investigated the potential use of nuclear receptor (NR) ligands for combinational therapy with other anti-cancer drugs. We first profiled all 48 NRs and 48 biological anti-cancer targets in four pairs of lung cell lines, where each pair was obtained from the same patient. Two sets of cell lines were normal and the corresponding tumor cell lines while the other two sets consisted of primary versus metastatic tumor cell lines. Analysis of the expression profile revealed 11 NRs and 15 cancer targets from the two pairs of normal versus tumor cell lines, and 9 NRs and 9 cancer targets from the primary versus metastatic tumor cell lines had distinct expression patterns in each category. Finally, the evaluation of nuclear receptor ligand T0901317 for liver X receptor (LXR) demonstrated its combined therapeutic potential with tyrosine kinase inhibitors. The combined treatment of cMET inhibitor PHA665752 or EGFR inhibitor gefitinib with T0901317 showed additive growth inhibition in both H2073 and H1993 cells. Mechanistically, the combined treatment suppressed cell cycle progression by inhibiting cyclinD1 and cyclinB expression. Taken together, this study provides insight into the potential use of NR ligands in combined therapeutics with other biological anti-cancer drugs.

The Advent of Lifestyle Medicine.

The fact that lifestyle is closely associated with the pathogenesis of chronic diseases has been known for more than three decades. Smoking may cause lung cancer, and a lifestyle of fast food consumption and little exercise can cause metabolic diseases. The importance of lifestyle changes in terms of a new medical paradigm to solve chronic diseases is becoming popular in modern times. Lifestyle medicine is a medicine based on personal lifestyle. To apply it to patients and ordinary people, physicians have to cooperate with experts in many fields such as nutrition, exercise, psychology, etc. In addition, patients must be partners in the treatment rather than passive recipients. The advent of lifestyle medicine has been caused by changes in disease patterns. In the past, acute diseases like infectious disease were prevalent; however, in the late 20(th) century, chronic diseases such as metabolic diseases, cancers, neurological disease, etc. increased in occurrence. As lifestyle is closely related with these diseases, the attitudes toward medicine need to be changed. Recently, the concept of "Lifestyle Medicine" was proposed, and we predict it will be an important field in future medicine.

Functional Expression of P2Y Receptors in WERI-Rb1 Retinoblastoma Cells.

P2Y receptors are metabotropic G-protein-coupled receptors, which are involved in many important biologic functions in the central nervous system including retina. Subtypes of P2Y receptors in retinal tissue vary according to the species and the cell types. We examined the molecular and pharmacologic profiles of P2Y purinoceptors in retinoblastoma cell, which has not been identified yet. To achieve this goal, we used Ca(2+) imaging technique and western blot analysis in WERI-Rb-1 cell, a human retinoblastoma cell line. ATP (10 µM) elicited strong but transient [Ca(2+)](i) increase in a concentration-dependent manner from more than 80% of the WERI-Rb-1 cells (n=46). Orders of potency of P2Y agonists in evoking [Ca(2+)](i) transients were 2MeS-ATP>ATP>>UTP=αß-MeATP, which was compatible with the subclass of P2Y(1) receptor. The [Ca(2+)](i) transients evoked by applications of 2MeS-ATP and/or ATP were also profoundly suppressed in the presence of P2Y(1) selective blocker (MRS 2179; 30 µM). P2Y(1) receptor expression in WERI-Rb-1 cells was also identified by using western blot. Taken together, P2Y(1) receptor is mainly expressed in a retinoblastoma cell, which elicits Ca(2+) release from internal Ca(2+) storage sites via the phospholipase C-mediated pathway. P2Y(1) receptor activation in retinoblastoma cell could be a useful model to investigate the role of purinergic [Ca(2+)](i) signaling in neural tissue as well as to find a novel therapeutic target to this lethal cancer.

Repression of human telomerase reverse transcriptase using artificial zinc finger transcription factors.

Telomerase activation is a key step in the development of human cancers. Expression of the catalytic subunit, human telomerase reverse transcriptase (hTERT), represents the limiting factor for telomerase activity. In this study, we have used artificial zinc finger protein (ZFP) transcription factors (TF) to repress the expression of hTERT in human cancer cell lines at the transcriptional level. We have constructed four-fingered ZFPs derived from the human genome which binds 12-bp recognition sequences within the promoter of the hTERT gene and fused them with a KRAB repressor domain to create a potent transcriptional repressor. Luciferase activity was decreased by >80% in all of the transcriptional repressors with luciferase reporter assay. When they were transfected into the telomerase-positive HEK293 cell line, a decrease of mRNA level and telomerase activity together with shortening of telomere length was observed. Actual growth of HEK293 cells was also inhibited by transfection of artificial ZFP-TFs. The repression was maintained for 100 days of culture. The repression of telomerase expression by artificial ZFP-TFs targeting the promoter region of the hTERT presents a new promising strategy for inhibiting the growth of human cancer cells.

Calcium mobilization by activation of M(3)/M(5) muscarinic receptors in the human retinoblastoma.

Activation of muscarinic acetylcholine receptors (mAChR) is one of the most important signal transduction pathways in the human body. In this study, we investigated the role of mAChR activation in relation to its subtypes in human retinoblastoma cell-lines (WERI-Rb-1) using Ca(2+) measurement, real-time PCR, and Western Blot techniques. Acetylcholine (ACh) produced prominent [Ca(2+)](i) transients in a repeated manner in WERI-Rb-1 cells. The maximal amplitude of the [Ca(2+)](i) transient was almost completely suppressed by 97.3 +/- 0.8% after atropine (1 microM) pretreatment. Similar suppressions were noted after pretreatments with thapsigargin (1 microM), an ER Ca(2+)-ATPase (SERCA) inhibitor, whereas the ACh-induced [Ca(2+)](i) transient was not affected even in the absence of extracellular calcium. U-73122 (1 microM), a PLC inhibitor, and xestospongin C (2 microM), an IP(3)-receptor antagonist, elicited 11.5 +/- 2.9% and 17.8 +/- 1.9% suppressions, respectively. The 50% inhibitory concentration of (IC(50)) values for blockade of a 100 microM ACh response by pirenzepine and 4-DAMP were 315.8 and 9.1 nM, respectively. Moreover, both M(3) and M(5) mAChRs were prominent in quantitative real-time-PCR. Taken together, the M(3)/M(5) subtypes appear to be the major contributor, leading to intracellular calcium mobilization from the internal store via an IP(3)-dependent pathway in the undifferentiated retinoblastoma cells.

On the role of pore helix in regulation of TRPV5 by extracellular protons.

The transient receptor potential channel TRPV5 is localized to the apical membrane of the distal renal tubule and plays an important role in the regulation of transepithelial Ca(2+) reabsorption in kidney. We have previously reported that extracellular protons inhibit TRPV5 by binding to glutamate-522 (E522) in the extracellular domain of the channel. We suggested that E522 is an extracellular "pH sensor" and its titration by extracellular protons inhibits TRPV5 via conformational change(s) of the pore helix. We now report that mutation of a pore helix residue glutamate-535 to glutamine (E535Q) enhances the sensitivity of the channel to inhibition by extracellular protons (i.e., shifting the apparent pKa for inhibition by extracellular protons to the more alkaline extracellular pH). The enhancement of extracellular proton-mediated inhibition of E535Q mutant is also dependent on E522. We have also reported that intracellular acidification enhances the sensitivity of TRPV5 to inhibition by extracellular protons. We now find that modulation of the extracellular proton-mediated inhibition by intracellular acidification is preserved in the E535Q mutant. These results provide further support for the idea that pore helix is involved in the regulation of TRPV5 by extracellular protons. Inhibition of TRPV5 by extracellular protons may contribute to hypercalciuria in diseases associated with high acid load.

Conformational changes of pore helix coupled to gating of TRPV5 by protons.

The transient receptor potential channel TRPV5 constitutes the apical entry pathway for transepithelial Ca2+ transport. We showed that TRPV5 was inhibited by both physiological intra- and extracellular acid pH. Inhibition of TRPV5 by internal protons was enhanced by extracellular acidification. Similarly, inhibition by external protons was enhanced by intracellular acidification. Mutation of either an extra- or an intracellular pH sensor blunted the cross-inhibition by internal and external protons. Both internal and external protons regulated the selectivity filter gate. Using the substituted cysteine accessibility method, we found that intracellular acidification of TRPV5 caused a conformational change of the pore helix consistent with clockwise rotation along its long axis. Thus, rotation of pore helix caused by internal protons facilitates closing of TRPV5 by external protons. This regulation by protons likely contributes to pathogenesis of disturbances of Ca2+ transport in many diseased states. Rotation of pore helix may be a common mechanism for cross-regulation of ion channels by extra- and intracellular signals.

Mechanism and molecular determinant for regulation of rabbit transient receptor potential type 5 (TRPV5) channel by extracellular pH.

The transient receptor potential type 5 (TRPV5) channel is present in kidney and intestine and important for transepithelial (re)absorption of calcium in these tissues. We report that in whole-cell patch clamp recording extracellular acidification inhibited rabbit TRPV5 with apparent pKa approximately 6.55. The two extracellular loops between the fifth and sixth transmembrane segments of TRPV5 presumably form part of the outer opening of the pore and likely are important in binding and regulation by external protons. We found that mutation of glutamate 522 to glutamine (E522Q) decreased the sensitivity of the channel to extracellular acidification. Mutations of other titratable amino acids within the two extracellular loops to non-titratable amino acids had no effect on pH sensitivity. Substitutions of aspartate or other titratable amino acids for glutamate 522 conferred an increase in pH sensitivity. The pH sensitivity mediated by glutamate 522 was independent of extracellular or intracellular Mg2+. Single channel analysis revealed that extracellular acidification reduced single channel conductance as well as open probability of the wild type channel. In contrast to wild type channel, extracellular acidification did not reduce open probability for E522Q mutant. Methanethiosulfonate reagents inhibited the activity of glutamine 522 to cysteine mutant channel with a reaction rate constant approaching that with free thiols in solution, suggesting that glutamate 522 is located on the surface of the channel. These data suggest that glutamate 522 of the rabbit TRPV5 is a "pH sensor," and extracellular protons inhibit TRPV5 likely by altering conformation of the channel protein.

Iontophoresis and sonophoresis stimulate epidermal cytokine expression at energies that do not provoke a barrier abnormality: lamellar body secretion and cytokine expression are linked to altered epidermal calcium levels.

We performed this study to identify whether the expression of epidermal cytokines is altered by changes in epidermal calcium content, independent of skin barrier disruption. Iontophoresis and sonophoresis with the energies that do not disrupt the skin barrier, but induce changes in the epidermal calcium gradient, were applied to the skin of hairless mice. Immediately after iontophoresis and sonophoresis, immersion in a solution containing calcium was carried out, and iontophoresis in either high- or low-calcium solutions was performed. The biopsy specimens were taken for real-time quantitative RT-PCR to detect changes in mRNA level of interleukin-1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta in the epidermis and for immunohistochemical stain with primary antibodies to IL-1alpha and TNF-alpha. The expression of each cytokine mRNA increased in the epidermis treated with iontophoresis and sonophoresis compared to a nontreated control as well as in tape-stripped skin used as a positive control and was lower after immersion in a high-calcium solution than in low-calcium solution. IL-1alpha and TNF-alpha immunohistochemical protein staining increased with iontophoresis at low calcium. These studies suggest that changes in epidermal calcium can directly signal expression of epidermal cytokines in vivo, independent of changes in barrier function.