Prolactin (PRL) in adipose tissue: regulation and functions.

New information concerning the effects of prolactin (PRL) on metabolic processes warrants reevaluation of its overall metabolic actions. PRL affects metabolic homeostasis by regulating key enzymes and transporters associated with glucose and lipid metabolism in several target organs. In the lactating mammary gland, PRL increases the production of milk proteins, lactose, and lipids. In adipose tissue, PRL generally suppresses lipid storage and adipokine release and affect adipogenesis. A specific case is made for PRL in the human breast and adipose tissues, where it acts as a circulating hormone and an autocrine/paracrine factor. Although its overall effects on body composition are both modest and species-specific, PRL may be involved in the manifestation of insulin resistance.

Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet.

Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA(F/F)) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA(F/F) MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT mice contain significantly more adipocytes than those isolated from PCNA(F/F) mice. This study identifies a critical role for PCNA in adipose tissue development, and for the first time identifies a role of the core DNA replication machinery at the interface between proliferation and differentiation.

Adipogenic effect of calcium sensing receptor activation.

We established that human adipose cells and the human adipose cell line LS14 express the calcium-sensing receptor (CaSR) and that its activation induces inflammatory cytokine production. Also, its expression is enhanced upon exposure to obesity-associated proinflammatory cytokines. We have thus proposed that CaSR activation may be associated with adipose dysfunction. Here, we evaluated a possible effect on adipogenesis. We induced adipose differentiation of primary and LS14 human preadipocytes with or without the simultaneous activation of CaSR, by the exposure to the calcimimetic cinacalcet. Activation of the receptor for 24 h decreased by 40 % the early differentiation marker CCAAT/enhancer-binding protein ß. However, upon longer-term (10 day) exposure to the adipogenic cocktail, cinacalcet exerted the opposite effect, causing a dose-response increase in the expression of the mature adipose markers adipocyte protein 2, adiponectin, peroxisome proliferator-activated receptor γ, fatty acid synthase, and glycerol-3-phosphate dehydrogenase. To assess whether there was a time-sensitive effect of CaSR activation on adipogenesis, we evaluated the 10 day effect of cinacalcet exposure for the first 6, 24, 48 h, 6, and 10 days. Our observations suggest that regardless of the period of exposure, 10 day adipogenesis is elevated by cinacalcet. CaSR activation may interfere with the initial stages of adipocyte differentiation; however, these events do not seem to preclude adipogenesis from continuing. Even though adipogenesis (particularly in subcutaneous depots) is associated with insulin sensitivity and adequate adipose function, the implications of our findings in visceral adipocytes, especially in the context of inflamed AT and overnutrition, remain to be established.

What can we learn from rodents about prolactin in humans?

Prolactin (PRL) is a 23-kDa protein hormone that binds to a single-span membrane receptor, a member of the cytokine receptor superfamily, and exerts its action via several interacting signaling pathways. PRL is a multifunctional hormone that affects multiple reproductive and metabolic functions and is also involved in tumorigenicity. In addition to being a classical pituitary hormone, PRL in humans is produced by many tissues throughout the body where it acts as a cytokine. The objective of this review is to compare and contrast multiple aspects of PRL, from structure to regulation, and from physiology to pathology in rats, mice, and humans. At each juncture, questions are raised whether, or to what extent, data from rodents are relevant to PRL homeostasis in humans. Most current knowledge on PRL has been obtained from studies with rats and, more recently, from the use of transgenic mice. Although this information is indispensable for understanding PRL in human health and disease, there is sufficient disparity in the control of the production, distribution, and physiological functions of PRL among these species to warrant careful and judicial extrapolation to humans.

Dopamine Receptors in Breast Cancer: Prevalence, Signaling, and Therapeutic Applications.

Breast cancer (BC) is the most common malignancy among women, with over one million cases occurring annually worldwide. Although therapies against estrogen receptors and HER2 have improved response rate and survival, patients with advanced disease, who are resistant to anti-hormonal therapy and/or to chemotherapy, have limited treatment options for reducing morbidity and mortality. These limitations provide major incentives for developing new, effective, and personalized therapeutic interventions. This review presents evidence on the involvement of dopamine (DA) and its type 1 receptors (D1R) in BC. DA is produced in multiple peripheral organs and is present in the systemic circulation in significant amounts. D1R is overexpressed in ~ 30% of BC cases and is associated with advanced disease and shortened patient survival. Activation of D1R, which signals via the cGMP/PKG pathway, results in apoptosis, inhibition of cell invasion, and increased chemosensitivity in multiple BC cell lines. Fenoldopam, a peripheral D1R agonist that does not penetrate the brain, dramatically suppressed tumor growth in mouse models with D1R-expressing BC xenografts. It is proposed that D1R should serve as a novel diagnostic/prognostic factor through the use of currently available D1R detection methods. Fenoldopam, which is FDA-approved to treat renal hypertension, could be repurposed as an effective therapeutic agent for patients with D1R-expressing tumors. Several drugs that interfere with the cGMP/PKG pathway and are approved for treating other diseases should also be considered as potential treatments for BC.

Suppression of Breast Cancer by Small Molecules That Block the Prolactin Receptor.

Prolactin (PRL) is a protein hormone which in humans is secreted by pituitary lactotrophs as well as by many normal and malignant non-pituitary sites. Many lines of evidence demonstrate that both circulating and locally produced PRL increase breast cancer (BC) growth and metastases and confer chemoresistance. Our objective was to identify and then characterize small molecules that block the tumorigenic actions of PRL in BC. We employed three cell-based assays in high throughput screening (HTS) of 51,000 small molecules and identified two small molecule inhibitors (SMIs), named SMI-1 and SMI-6. Both compounds bound to the extracellular domain (ECD) of the PRL receptor (PRLR) at 1-3 micromolar affinity and abrogated PRL-induced breast cancer cell (BCC) invasion and malignant lymphocyte proliferation. SMI-6 effectively reduced the viability of multiple BCC types, had much lower activity against various non-malignant cells, displayed high selectivity, and showed no apparent in vitro or in vivo toxicity. In athymic nude mice, SMI-6 rapidly and dramatically suppressed the growth of PRL-expressing BC xenografts. This report represents a pre-clinical phase of developing novel anti-cancer agents with the potential to become effective therapeutics in breast cancer patients.

Obesity-associated proinflammatory cytokines increase calcium sensing receptor (CaSR) protein expression in primary human adipocytes and LS14 human adipose cell line.

Obesity-associated health complications are thought to be in part due to the low-grade proinflammatory state that characterizes this disease. The calcium sensing receptor (CaSR), which is expressed in human adipose cells, plays an important role in diseases involving inflammation. To assess the relevance of this protein in adipose pathophysiology, we evaluated its expression in adipocytes under obesity-related proinflammatory conditions. As in primary adipose cells, we established that LS14, a recently described human adipose cell line, expresses the CaSR. Differentiated LS14 and primary adipose cells were exposed overnight to cytokines typically involved in obesity-related inflammation (interleukin (IL)1beta, IL6 and tumor necrosis factor (TNF)alpha). The cytokines increased CaSR abundance in differentiated adipocytes. We incubated LS14 cells with medium previously conditioned (CM) by adipose tissue from subjects with a wide range of body mass index (BMI). Cells exposed to CM from subjects of higher BMI underwent a greater increase in CaSR protein, likely resulting from the greater proinflammatory cytokines secreted from obese tissue. Our observations that proinflammatory factors increase CaSR levels in adipocytes, and the reported ability of CaSR to elevate cytokine levels, open new aspects in the study of obesity inflammatory state pathophysiology, providing a potential novel therapeutic prevention and treatment target.

Prolactin confers resistance against cisplatin in breast cancer cells by activating glutathione-S-transferase.

Resistance to chemotherapy is a major obstacle for successful treatment of breast cancer patients. Given that prolactin (PRL) acts as an anti-apoptotic/survival factor in the breast, we postulated that it antagonizes cytotoxicity by chemotherapeutic drugs. Treatment of breast cancer cells with PRL caused variable resistance to taxol, vinblastine, doxorubicin and cisplatin. PRL prevented cisplatin-induced G(2)/M cell cycle arrest and apoptosis. In the presence of PRL, significantly less cisplatin was bound to DNA, as determined by mass spectroscopy, and little DNA damage was seen by gamma-H2AX staining. PRL dramatically increased the activity of glutathione-S-transferase (GST), which sequesters cisplatin in the cytoplasm; this increase was abrogated by Jak and mitogen-activated protein kinase inhibitors. PRL upregulated the expression of the GSTmu, but not the pi, isozyme. A GST inhibitor abrogated antagonism of cisplatin cytotoxicity by PRL. In conclusion, PRL confers resistance against cisplatin by activating a detoxification enzyme, thereby reducing drug entry into the nucleus. These data provide a rational explanation for the ineffectiveness of cisplatin in breast cancer, which is characterized by high expression of both PRL and its receptor. Suppression of PRL production or blockade of its actions should benefit patients undergoing chemotherapy by allowing for lower drug doses and expanded drug options.

Epidermal growth factor receptor cross-talks with ligand-occupied estrogen receptor-alpha to modulate both lactotroph proliferation and prolactin gene expression.

Both estrogen (E2) and EGF regulate lactotrophs, and we recently demonstrated that EGF phosphorylates S118 on estrogen receptor-alpha (ERalpha) and requires ERalpha to stimulate prolactin (PRL) release. However, the interactions between ligand-occupied ERalpha and activated ErbB1 and its impact on lactotroph function are unknown. Using rat GH3 lactotrophs, we found that both E2 and EGF independently stimulated proliferation and PRL gene expression. Furthermore, their combination resulted in an enhanced stimulatory effect on both cell proliferation and PRL gene expression. Inhibitors of ER as well as ErbB1 blocked the combined effects of E2 and EGF. Pretreatment with UO126 abolished the combined effects, demonstrating Erk1/2 requirement. Although bidirectionality in ER-ErbB1 cross-talk is a well-accepted paradigm, interestingly in lactotrophs, ErbB1 kinase inhibitor failed to block the effect of E2 on proliferation and stimulation of PRL gene expression, suggesting that ER does not require ErbB1 to mediate its effects. Furthermore, E2 did not affect the ability of EGF to induce c-Fos expression or modulate AP-1 activity. However, both E2 and EGF combine to enhance S118 phosphorylation of ERalpha, leading to enhanced E2-mediated estrogen response element transactivation. Taken together, our results suggest that, in lactotrophs, activated ErbB1 phosphorylates ERalpha to enhance the stimulatory effect of E2, thereby providing the molecular basis by which EGF amplifies the response of E2.

Bisphenol A at low nanomolar doses confers chemoresistance in estrogen receptor-alpha-positive and -negative breast cancer cells.

BACKGROUND: Resistance to chemotherapy is a major problem facing breast cancer patients, and identifying potential contributors to chemoresistance is a critical area of research. Bisphenol A (BPA) has long been suspected to promote carcinogenesis, but the high doses of BPA used in many studies generated conflicting results. In addition, the mechanism by which BPA exerts its biological actions is unclear. Although estrogen has been shown to antagonize anticancer drugs, the role of BPA in chemoresistance has not been examined. OBJECTIVE: The objective of our study was to determine whether BPA at low nanomolar concentrations opposes the action of doxorubicin, cisplatin, and vinblastine in the estrogen receptor-alpha (ERalpha)-positive T47D and the ERalpha-negative MDA-MB-468 breast cancer cells. METHODS: We determined the responsiveness of cells to anticancer drugs and BPA using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Specific ERalpha and ERbeta inhibitors and real-time polymerase chain reaction were used to identify potential receptor(s) that mediate the actions of BPA. Expression of antiapoptotic proteins was assessed by Western blotting. RESULTS: BPA antagonizes the cytotoxicity of multiple chemotherapeutic agents in both ERalpha-positive and -negative breast cancer cells independent of the classical ERs. Both cell types express alternative ERs, including G-protein-coupled receptor 30 (GPR30) and members of the estrogen-related receptor family. Increased expression of antiapoptotic proteins is a potential mechanism by which BPA exerts its anticytotoxic effects. CONCLUSIONS: BPA at environmentally relevant doses reduces the efficacy of chemotherapeutic agents. These data provide considerable support to the accumulating evidence that BPA is hazardous to human health.

Activation of the cGMP/protein kinase G system in breast cancer by the dopamine receptor-1.

Despite recent advances in the detection and treatment of breast cancer, many shortcomings remain, providing incentives to search for new therapeutic targets. This review provides information on the expression and actions of dopamine receptor-1 (D1R) in breast cancer. D1R is overexpressed in a significant number of primary breast tumors, characterized by having an aggressive phenotype and predicting a shorter survival time for patients. Activation of D1R in breast cancer cells by selective agonists caused suppression of cell viability, stimulation of apoptosis, inhibition of cell invasion, and an increase in chemosensitivity. Instead of being linked to the cAMP/PKA system as expected, D1R in breast cancer is linked to the activation of the cGMP/protein kinase G (PKG) pathway. Fenoldopam, a peripheral D1R agonist that does not penetrate the brain, dramatically suppressed the growth of breast cancer xenografts in immune-deficient mice. A new imaging system for detecting D1R-expressing tumors and metastases was also developed. The review offers a novel concept that D1R can serve as a biomarker for prognosis in advanced breast cancer and its agonists can be used as effective and personalized therapeutics in a subpopulation of patients with D1R-expressing breast tumors. Several drugs, some of which are FDA-approved, that bypass the D1R and directly activate the cGMP/PKG apoptotic system, are also identified.

Insulin stimulates interleukin-6 expression and release in LS14 human adipocytes through multiple signaling pathways.

IL-6 is an important cytokine that regulates both immune and metabolic functions. Within adipose tissue, preadipocytes produce significant amounts of IL-6, but little is known about the factors or mechanisms that regulate IL-6 production in these cells. Using LS14, a newly developed human adipocyte cell line, our objective was to determine the mechanisms by which insulin stimulates IL-6 production and release in preadipocytes. Insulin increased IL-6 gene expression and secretion in a time- and dose-dependent manner. Insulin decreased cyclic AMP (cAMP) but increased cyclic GMP (cGMP) levels, and IL-6 expression/release was stimulated by a cGMP analog. The stimulatory effect of insulin and cGMP was abrogated by a specific inhibitor of protein kinase G (cyclic GMP-dependent protein kinase). Both insulin and cGMP rapidly induced phosphorylation of cAMP response element binding protein. Insulin also activated the MAPK signaling pathway, and its blockade prevented the insulin-stimulated increases in IL-6 cell content and release, but not IL-6 gene expression. Although inhibition of the proteosome increased IL-6 cell content and release, proteosome activity was unaffected by insulin. These data suggest that the stimulatory effects of insulin on IL-6 release involve several interrelated components: transcription, intracellular releasable pool, and secretion, which are differentially regulated and, thus, determine the size of the releasable pool of IL-6. Insulin-induced IL-6 gene expression is mediated by cGMP/cyclic GMP-dependent protein kinase/cAMP response element binding protein, whereas MAPK is involved in the insulin-stimulated IL-6 synthesis/release.

Prolactin release by adipose explants, primary adipocytes, and LS14 adipocytes.

BACKGROUND: Prolactin (PRL) is a multifunctional hormone produced in humans by both pituitary and extrapituitary sites, including adipose tissue. OBJECTIVES: Our objectives were to: 1) compare PRL secretion by sc and visceral adipose explants and mature adipocytes from obese and nonobese patients; and 2) examine the effects of insulin and selected cytokines on PRL gene expression and release from primary adipocytes and LS14 adipocytes. DESIGN AND SUBJECTS: Adipose tissue was obtained from morbidly obese [body mass index (BMI) > 40 kg/m(2)] and nonobese (BMI <30 kg/m(2)) patients. Explants and isolated mature adipocytes were incubated for 10 d. Primary adipocytes or LS14 cells were used before or after differentiation and incubated with the test compounds for 24 h. PRL release was analyzed by a bioassay, and PRL expression was determined by real-time PCR. RESULTS: PRL release from explants and mature adipocytes increased in a time-dependent manner indicating removal from inhibition. Visceral explants from obese patients showed higher PRL release than that from sc explants; both types of explants from nonobese patients released similar amounts of PRL. Analysis of data from 50 patients revealed an inverse relationship between PRL release from sc depots and BMI. Insulin suppressed PRL expression and release from differentiated adipocytes but moderately stimulated PRL release from nondifferentiated cells. The cAMP elevating compound forskolin increased PRL release in both cell types. CONCLUSIONS: PRL should be recognized as an important adipokine whose release is regulated by insulin and is affected by obesity in a depot-specific manner.

Bisphenol A at environmentally relevant doses inhibits adiponectin release from human adipose tissue explants and adipocytes.

BACKGROUND: The incidence of obesity has risen dramatically over the last few decades. This epidemic may be affected by exposure to xenobiotic chemicals. Bisphenol A (BPA), an endocrine disruptor, is detectable at nanomolar levels in human serum worldwide. Adiponectin is an adipocyte-specific hormone that increases insulin sensitivity and reduces tissue inflammation. Thus, any factor that suppresses adiponectin release could lead to insulin resistance and increased susceptibility to obesity-associated diseases. OBJECTIVES: In this study we aimed to compare a) the effects of low doses of BPA and estradiol (E(2)) on adiponectin secretion from human breast, subcutaneous, and visceral adipose explants and mature adipocytes, and b) expression of putative estrogen and estrogen-related receptors (ERRs) in these tissues. METHODS: We determined adiponectin levels in conditioned media from adipose explants or adipocytes by enzyme-linked immunosorbant assay. We determined expression of estrogen receptors (ERs) alpha and beta, G-protein-coupled receptor 30 (GPR30), and ERRs alpha, beta, and gamma by quantitative real-time polymerase chain reaction. RESULTS: BPA at 0.1 and 1 nM doses suppressed adiponectin release from all adipose depots examined. Despite substantial variability among patients, BPA was as effective, and often more effective, than equimolar concentrations of E(2). Adipose tissue expresses similar mRNA levels of ERalpha, ERbeta, and ERRgamma, and 20- to 30-fold lower levels of GPR30, ERRalpha, and ERRbeta. CONCLUSIONS: BPA at environmentally relevant doses inhibits the release of a key adipokine that protects humans from metabolic syndrome. The mechanism by which BPA suppresses adiponectin and the receptors involved remains to be determined.

LS14 cells: a model for chemoresistance in liposarcoma.

Liposarcoma, a malignancy of adipose tissue, is the most common soft tissue sarcoma. Patients whose primary tumor cannot be resected or those who have developed metastasis, have poor prognosis since liposarcomas are highly resistant to chemotherapy. We recently generated a spontaneously immortalized cell line, named LS14, from a patient with metastatic liposarcoma. Our goal was to compare the responsiveness of LS14 and SW872 liposarcoma cells to anti-cancer drugs and explore mechanisms of chemoresistance. Using complementary assays for cell viability and number we found that SW872 cells responded robustly to relatively low concentrations of doxorubicin, cisplatin and vinblastine. This reduction in cell viability was due to apoptosis, as evident by phosphatidylserine exposure and caspase 3 cleavage. In contrast, only a high dose of doxorubicin or combination therapy effectively reduced LS14 cell viability and induced apoptosis. LS14 cells showed a higher expression of Bcl-2 and Bcl-xL, but a lower expression of survivin and Bax, than SW872 cells, suggesting that anti-apoptotic proteins contribute to chemoresistance in LS14 cells. Although LS14 cells did not form colonies in soft agar, they generated large tumors and metastases in SCID mice, establishing their tumorigenicity in vivo. In conclusion, LS14 cells are much more resistant to chemotherapy than SW872 cells, making them an excellent model for exploring the efficacy and mechanism of action of anti-cancer drugs in liposarcomas.

Focus on prolactin as a metabolic hormone.

New information about the effects of prolactin (PRL) on metabolic processes warrants re-evaluation of the overall metabolic actions of PRL. PRL affects metabolic homeostasis by regulating key enzymes and transporters that are associated with glucose and lipid metabolism in several target organs. In the lactating mammary gland, PRL increases the production of milk proteins, lactose and lipids. In adipose tissue, PRL generally suppresses lipid storage and adipokine release. PRL supports the growth of pancreatic islets, stimulates insulin secretion and increases citrate production in the prostate. A specific case is made for PRL in the human breast and adipose tissue, where it acts as a circulating hormone and an autocrine or paracrine factor. Although the overall effects of PRL on body composition are modest and species specific, PRL might be involved in the manifestation of insulin resistance.

Soluble guanylate cyclase stimulators increase sensitivity to cisplatin in head and neck squamous cell carcinoma cells.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive and often fatal disease. Cisplatin is the most common chemotherapeutic drug in the treatment of HNSCC, but intrinsic and acquired resistance are frequent, and severe side effects occur at high doses. The second messenger cyclic GMP (cGMP) is produced by soluble guanylate cyclase (sGC). We previously reported that activation of the cGMP signaling cascade caused apoptosis in HNSCC cells, while others found that this pathway enhances cisplatin efficacy in some cell types. Here we found that sGC stimulators reduced HNSCC cell viability synergistically with cisplatin, and enhanced apoptosis by cisplatin. Moreover, the sGC stimulators effectively reduced viability in cells with acquired cisplatin resistance, and were synergistic with cisplatin. The sGC stimulator BAY 41-2272 reduced expression of the survival proteins EGFR and ß-catenin, and increased pro-apoptotic Bax, suggesting a potential mechanism for the anti-tumorigenic effects of these drugs. The sGC stimulator Riociguat is FDA-approved to treat pulmonary hypertension, and others are being studied for therapeutic use in several diseases. These drugs could provide valuable addition or alternative to cisplatin in the treatment of HNSCC.

The prolactin-deficient mouse has an unaltered metabolic phenotype.

Prolactin (PRL), best recognized for its lactogenic activity, is also involved in the regulation of metabolic homeostasis in both mammalian and nonmammalian species. Although several mouse models have been used to study the metabolic functions of PRL, a clear-cut consensus has not emerged given the limited and often conflicting data. To clarify the role of PRL in metabolic homeostasis in males and nonlactating females, we used the PRL-deficient mouse. Our objectives were to compare: 1) weight gain, 2) body composition, 3) serum lipid profile, 4) circulating leptin and adiponectin levels, and 5) glucose tolerance in PRL knockout, heterozygous, and wild-type mice maintained on standard chow, high-fat, or low-fat diets. In addition, we compared the lipolytic actions of PRL using adipose tissue explants from mice, rats, and humans. We are reporting that PRL deficiency does not affect the rate of weight gain, body composition, serum lipids, or adiponectin levels in either sex on any diet. Glucose tolerance was slightly impaired in very young PRL knockout male pups but not in adults or in females at any age. Leptin was elevated in male, but not female, PRL knockout mice maintained on a low-fat diet. PRL did not affect lipolysis in adipose tissue explants from mice but significantly inhibited glycerol release from both rat and human adipose explants in a dose-dependent manner. We conclude that PRL deficiency has negligible gross metabolic effects in mice.

LS14: a novel human adipocyte cell line that produces prolactin.

Adipose tissue is an integral component within the endocrine system. Adipocytes produce numerous bioactive substances, and their dysregulation has serious pathophysiological consequences. We previously reported that human adipose tissue from several depots produces significant amounts of prolactin (PRL). To study locally produced PRL, we sought an acceptable in vitro model. Consequently, we developed an adipocyte cell line derived from a metastatic liposarcoma. The cell line, designated LS14, has been in continuous culture for 2 yr. These cells exhibit many properties of primary preadipocytes, including the ability to undergo terminal differentiation, as judged by morphological alterations, lipid accumulation, and increase in glycerol-3-phosphate dehydrogenase. LS14 cells express many adipose-associated genes, such as adipocyte fatty acid-binding protein (aP(2)), hormone-sensitive lipase, lipoprotein lipase, preadipocyte factor 1, adiponectin, leptin, and IL-6. Similar to primary adipocytes, LS14 cells also produce and respond to PRL, thus making them an attractive model to study adipose PRL production and function. The expression of PRL was confirmed at the transcriptional level by RT-PCR, and PRL secretion was determined by the Nb2 bioassay. Addition of exogenous PRL to LS14 cells resulted in a dose-dependent inhibition of IL-6 release. In summary, we have established a novel human adipocyte cell line with many characteristics of primary adipocytes. The LS14 cells open up new avenues for research on human adipocyte biology and add to the repertoire of nonpituitary, PRL-producing cell lines.

The cyclic GMP/protein kinase G pathway as a therapeutic target in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease with high mortality. Treatments, which can result in significant morbidity, have not substantially changed in three decades. The second messenger cyclic GMP (cGMP), which targets protein kinase G (PKG), is generated by guanylate cyclases (GCs), and is rapidly hydrolyzed by phosphodiesterases (PDEs). Activation of the cGMP/PKG pathway is antineoplastic in several cancer types, but its impact on HNSCC has not been fully exploited. We found differential expression of critical components of this pathway in four HNSCC cell lines. Several activators of soluble GC (sGC), as well as inhibitors of PDE5, increased intracellular cGMP, reduced cell viability, and induced apoptosis in HNSCC cells. The apoptotic effects of the sGC activator BAY 41-2272 and the PDE5 inhibitor Tadalafil (Cialis) were mediated by PKG. Furthermore, Tadalafil substantially reduced the growth of CAL27-derived tumors in athymic mice. Several drugs which either activate sGC or inhibit PDE5 are approved for treatment of nonmalignant conditions. These drugs could be repurposed as novel and effective therapeutics in patients with head and neck cancer.