The role of MEK1/2 and MEK5 in melatonin-mediated actions on osteoblastogenesis, osteoclastogenesis, bone microarchitecture, biomechanics, and bone formation.

Melatonin, the primary hormone involved in circadian entrainment, plays a significant role in bone physiology. This study aimed to assess the role of MEK1/2 and MEK5 in melatonin-mediated actions in mouse and human mesenchymal stem cells (MSCs) and on bone using small-molecule inhibitors and CRISPR/Cas9 knockout approaches. Consistent with in vitro studies performed in mMSCs and hMSCs, nightly (25 mg/kg, i.p., 45 days) injections with PD184352 (MEK1/2 inhibitor) or Bix02189 (MEK5 inhibitor) or SC-1-151 (MEK1/2/5 inhibitor) demonstrated that MEK1/2 and MEK5 were the primary drivers underlying melatonin's actions on bone density, microarchitecture (i.e., trabecular number, separation, and connectivity density), and bone mechanical properties (i.e., ultimate stress) through increases in osteogenic (RUNX2, BMP-2, FRA-1, OPG) expression and decreases in PPARγ. Furthermore, CRISPR/Cas9 knockout of MEK1 or MEK5 in mMSCs seeded on PLGA scaffolds and placed into critical-size calvarial defects in Balb(c) mice (male and female) revealed that treatment with melatonin (15 mg/L; p.o., nightly, 90 days) mediates sex-specific actions of MEK1 and MEK5 in new bone formation. This study is the first to demonstrate a role for MEK1/2 and MEK5 in modulating melatonin-mediated actions on bone formation in vivo and in a sex-specific manner.

Melatonin effects on bone: Implications for use as a therapy for managing bone loss.

Melatonin is the primary circadian output signal from the brain and is mainly synthesized in pinealocytes. The rhythm and secretion of melatonin are under the control of an endogenous oscillator located in the SCN or the master biological clock. Disruptions in circadian rhythms by shift work, aging, or light at night are associated with bone loss and increased fracture risk. Restoration of nocturnal melatonin peaks to normal levels or therapeutic levels through timed melatonin supplementation has been demonstrated to provide bone-protective actions in various models. Melatonin is a unique molecule with diverse molecular actions targeting melatonin receptors located on the plasma membrane or mitochondria or acting independently of receptors through its actions as an antioxidant or free radical scavenger to stimulate osteoblastogenesis, inhibit osteoclastogenesis, and improve bone density. Its additional actions on entraining circadian rhythms and improving quality of life in an aging population coupled with its safety profile make it an ideal therapeutic candidate for protecting against bone loss in susceptible populations. The intent of this review is to provide a focused discussion on bone loss and disorders of the bone as it relates to melatonin and conditions that modify melatonin levels with the hope that future therapies include those that include melatonin and correct those factors that modify melatonin levels like circadian disruption.

Melatonin receptor ligands: A pharmaco-chemical perspective.

Melatonin MT1 and MT2 receptor ligands have been vigorously explored for the last 4 decades. Inspection of approximately 80 publications in the field revealed that most melatonergic ligands were structural analogues of melatonin combining three essential features of the parent compound: an aromatic ring bearing a methoxy group and an amide side chain in a relative arrangement similar to that present in melatonin. While several series of MT2 -selective agents-agonists, antagonists, or partial agonists-were reported, the field was lacking MT1 -selective agents. Herein, we describe various approaches toward the development of melatonergic ligands, keeping in mind that most of the molecules/pharmacophores obtained were essentially melatonin copies, even though diverse tri- or tetra-cyclic compounds were explored. In addition to lack of structural diversity, only few studies examined the activity of the reported melatonergic ligands in vivo. Moreover, an extensive pharmacological characterization including biopharmaceutical stability, pharmacokinetic properties, specificity toward other major receptors to name a few remained scarce. For example, many of the antagonists described were not stable in vivo, were not selective for the melatonin receptor subtype of interest, and were not fully characterized from a pharmacological standpoint. Indeed, virtual screening of large compound libraries has led to the recent discovery of potent and selective melatonin receptor agonists and partial agonists of new chemotypes. Having said this, the melatonergic field is still lacking subtype-selective melatonin receptor antagonists "active" in vivo, which are critical to our understanding of melatonin and melatonin receptors' role in basic physiology and disease.

Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.

G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT1), its associated G protein, and ß-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT1 signal-transduction pathway inhibiting stress-mediated cytochrome c release and caspase activation. These findings coupled with our observation that mitochondrial MT1 overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, "automitocrine," analogous to "autocrine" when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand-receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.

Pharmacological, Mechanistic, and Pharmacokinetic Assessment of Novel Melatonin-Tamoxifen Drug Conjugates as Breast Cancer Drugs.

Tamoxifen is used to prevent and treat estrogen receptor-positive (ER+) breast cancer (BC); however, its chronic use can increase uterine cancer risk and induce tamoxifen resistance. Novel melatonin-tamoxifen drug conjugates may be promising to treat BC and may help offset the adverse effects of tamoxifen usage alone due to the presence of melatonin. We synthesized and screened five drug conjugates (C2, C4, C5, C9, and C15 linked) for their effects on BC cell (MCF-7, tamoxifen-resistant MCF-7, mouse mammary carcinoma, MDA-MB-231, and BT-549) viability, migration, and binding affinity to melatonin receptor 1 (MT1R) and estrogen receptor 1 (ESR1). C4 and C5 demonstrated the most favorable pharmacological characteristics with respect to binding profiles (affinity for ESR1 and MT1R) and their potency/efficacy to inhibit BC cell viability and migration in four phenotypically diverse invasive ductal BC cell lines. C4 and C5 were further assessed for their actions against tamoxifen-resistant MCF-7 cells and a patient-derived xenograft triple-negative BC cell line (TU-BcX-4IC) and for their mechanisms of action using selective mitogen-activated protein kinase kinase MEK1/2, MEK5, and phosphoinositide 3-kinase (PI3K) inhibitors. C4 and C5 inhibited tamoxifen-resistant MCF-7 cells with equal potency (IC50 = 4-8 µM) and efficacy (∼90% inhibition of viability and migration) but demonstrated increased potency (IC50 = 80-211 µM) and efficacy (∼140% inhibition) to inhibit migration versus cell viability (IC50 = 181-304 mM; efficacy ∼80% inhibition) in TU-BcX-4IC cells. Unique pharmacokinetic profiles were observed, with C4 having greater bioavailability than C5. Further assessment of C4 and C5 demonstrates that they create novel pharmacophores within each BC cell that is context specific and involves MEK1/2/pERK1/2, MEK5/pERK5, PI3K, and nuclear factor κB. These melatonin-tamoxifen drug conjugates show promise as novel anticancer drugs and further preclinical and clinical evaluation is warranted.

Relationship between melatonin and bone resorption rhythms in premenopausal women.

Although evidence exists for a daily rhythm in bone metabolism, the contribution of factors such as melatonin levels, the light-dark cycle, and the sleep-wake cycle is difficult to differentiate given their highly correlated time courses. To examine these influences on bone resorption, we collected 48-h sequential urine samples under both ambulatory (8-h sleep:16-h wake) and constant routine (CR) (constant wake, posture, nutrition and dim light) conditions from 20 healthy premenopausal women. Urinary 6-sulphatoxymelatonin (aMT6s; ng/h) and the bone resorption marker amino-terminal cross-linked collagen I telopeptide (NTx; bone collagen equivalents nM/h) were assayed and fit by cosinor models to determine significant 24-h rhythms and acrophase. Most participants had significant 24-h aMT6s rhythms during both ambulatory and CR conditions (95 and 85%, respectively), but fewer had significant 24-h NTx rhythms (70 and 70%, respectively). Among individuals with significant rhythms, mean (± SD) aMT6s acrophase times were 3:57 ± 1:50 and 3:43 ± 1:25 h under ambulatory and CR conditions, respectively, and 23:44 ± 5:55 and 3:06 ± 5:15 h, respectively, for NTx. Mean 24-h levels of both aMT6s and NTx were significantly higher during CR compared with ambulatory conditions (p < 0.0001 and p = 0.03, respectively). Menstrual phase (follicular versus luteal) had no impact on aMT6s or NTx timing or 24-h levels. This study confirms an endogenous circadian rhythm in NTx with a night-time peak when measured under CR conditions, but also confirms that environmental factors such as the sleep-wake or light-dark cycles, posture or meal timing affects overall concentrations and peak timing under ambulatory conditions, the significance of which remains unclear.

Biological effects of melatonin on osteoblast/osteoclast cocultures, bone, and quality of life: Implications of a role for MT2 melatonin receptors, MEK1/2, and MEK5 in melatonin-mediated osteoblastogenesis.

The Melatonin Osteoporosis Prevention Study (MOPS) demonstrated that nightly melatonin resulted in a time-dependent decrease in equilibrium ratios of serum osteoclasts and osteoblasts in perimenopausal women. This study examines mechanisms related to the ratios of osteoblasts and osteoclasts using coculture models (transwell or layered) of human mesenchymal stem cell (MSC) and human peripheral blood monocytes (PBMCs). Human MSC/PBMC cocultures exposed to melatonin in osteogenic (OS+) medium for 21 days induced osteoblast differentiation and mineralization; however, only in layered cocultures did melatonin inhibit osteoclastogenesis. Melatonin effects were mediated through MT2 melatonin receptors, MEK1/2, and MEK5. In layered but not transwell cocultures, melatonin increased OPG:RANKL ratios by inhibiting RANKL, suggesting that contact with osteoclasts during osteoblastogenesis inhibits RANKL secretion. Melatonin modulated expression of ERK1/2, ERK5, ß1 integrin, GLUT4, and IRß that was dependent upon the type of coculture; however, in both cultures, melatonin increased RUNX2 and decreased PPARγ expression, indicating a role for metabolic processes that control osteogenic vs adipogenic cell fates of MSCs. Furthermore, melatonin also has osteoblast-inducing effects on human adipose-derived MSCs. In vivo, one-year nightly melatonin (15 mg/L) given to neu female mice in their drinking water increased pErk1/2, pErk5, Runx2, and Opg and Rankl levels in bone consistent with melatonin's already reported bone-enhancing effects. Finally, analysis of daily logs from the MOPS demonstrated a significant improvement in mood and perhaps sleep quality in women receiving melatonin vs placebo. The osteoblast-inducing, bone-enhancing effects of melatonin and improvement in quality of life suggest that melatonin is a safe and effective bone loss therapy.

Oxyprenylated Phenylpropanoids Bind to MT1 Melatonin Receptors and Inhibit Breast Cancer Cell Proliferation and Migration.

Oxyprenylated compounds (i.e., ferulic acid and coumarin derivatives) demonstrate neuroprotection and anticancer properties as reported in previous studies. We have tested the affinity of oxyprenylated ferulic acid (1-4) and umbelliferone derivatives (5-11) to melatonin receptors as well as their antiproliferation and antimigratory properties against breast cancer (BC) cell lines. All the compounds except for ferulic acid, boropinic acid, and umbelliferone had binding affinities to melatonin receptors in the nM to µM range, and both auraptene and umbellinprenin reduced BC cell proliferation and migration in phenotypically diverse BC including triple negative.

Melatonin effects on bone: potential use for the prevention and treatment for osteopenia, osteoporosis, and periodontal disease and for use in bone-grafting procedures.

An important role for melatonin in bone formation and restructuring has emerged, and studies demonstrate the multiple mechanisms for these beneficial actions. Statistical analysis shows that even with existing osteoporotic therapies, bone-related disease, and mortality are on the rise, creating a huge financial burden for societies worldwide. These findings suggest that novel alternatives need to be developed to either prevent or reverse bone loss to combat osteoporosis-related fractures. The focus of this review describes melatonin's role in bone physiology and discusses how disruption of melatonin rhythms by light exposure at night, shift work, and disease can adversely impact on bone. The signal transduction mechanisms underlying osteoblast and osteoclast differentiation and coupling with one another are discussed with a focus on how melatonin, through the regulation of RANKL and osteoprotegerin synthesis and release from osteoblasts, can induce osteoblastogenesis while inhibiting osteoclastogenesis. Also, melatonin's free-radical scavenging and antioxidant properties of this indoleamine are discussed as yet an additional mechanism by which melatonin can maintain one's bone health, especially oral health. The clinical use for melatonin in bone-grafting procedures, in reversing bone loss due to osteopenia and osteoporosis, and in managing periodontal disease is discussed.

Platelet-rich preparations to improve healing. Part I: workable options for every size practice.

Numerous studies have demonstrated that platelet-rich preparations applied to surgical sites, injuries, or wounds are a safe and effective way to promote soft tissue healing and bone growth. Various protocols have been developed for preparing platelet-rich preparations, with subtle but important differences between them. Unfortunately, only a minority of clinicians use platelet-rich preparations, such as platelet-rich plasma and platelet-rich fibrin, in their practice, possibly due to confusion about the different methods and their advantages and disadvantages. Therefore, the different types of preparations are described to help guide the selection of the best method for any size practice. Classic methods generally require large volumes of blood and can be expensive, complicated, and time-intensive. Simpler protocols have been developed recently, which require relatively inexpensive equipment and small blood volumes and, thus, may be more applicable for small clinical practices. Platelet-rich preparations accelerate healing at earlier time points to reduce discomfort and the potential for adverse outcomes, including infection, poor wound closure, and delays in forming strong bone for subsequent procedures (such as implants). However, platelet-rich preparations may also improve long-term outcomes in patients expected to have impaired healing, such as with lifestyle choices (eg, smoking), medications (eg, steroids), diseases (eg, diabetes, osteoporosis, atherosclerosis), and aging, by supplementing the deficient wound environment to restore proper healing. Therefore, both large and small clinical practices would benefit from utilizing platelet-rich preparations to enhance healing in their patients.

Platelet-rich preparations to improve healing. Part II: platelet activation and enrichment, leukocyte inclusion, and other selection criteria.

Multiple platelet-rich preparations have been reported to improve wound and bone healing, such as platelet-rich plasma (PRP) and platelet rich fibrin (PRF). The different methods employed during their preparation are important, as they influence the quality of the product applied to a wound or surgical site. Besides the general protocol for preparing the platelet-rich product (discussed in Part 1 of this review), multiple choices need to be considered during its preparation. For example, activation of the platelets is required for the release and enmeshment of growth factors, but the method of activation may influence the resulting matrix, growth factor availability, and healing. Additionally, some methods enrich leukocytes as well as platelets, but others are designed to be leukocyte-poor. Leukocytes have many important roles in healing and their inclusion in PRP results in increased platelet concentrations. Platelet and growth factor enrichment reported for the different types of platelet-rich preparations are also compared. Generally, TGF-ß1 and PDGF levels were higher in preparations that contain leukocytes compared to leukocyte-poor PRP. However, platelet concentration may be the most reliable criterion for comparing different preparations. These and other criteria are described to help guide dental and medical professionals, in large and small practices, in selecting the best procedures for their patients. The healing benefits of platelet-rich preparations along with the low risk and availability of simple preparation procedures should encourage more clinicians to incorporate platelet-rich products in their practice to accelerate healing, reduce adverse events, and improve patient outcomes.

High affinity nuclear and nongenomic estradiol binding sites in the human and mouse lens.

Estrogen is reported to be protective against cataracts in women and animal models. Immunodetection methods have identified the classic estrogen receptors (ER), ERα and ERß, in human lens epithelial cells and their RNAs have been detected in the rat and human lens. To verify that estrogen binding occurs in the lens, sensitive [(125)I]-17ß-estradiol binding analyses were performed on subcellular lens fractions from women (ages 39-78 years). The presence of high affinity estradiol binding sites in the nuclear, cytoplasmic, and membrane fractions indicate the lens is able to respond to estrogens, even up to age 78, although fewer binding sites were detected in the postmenopausal women. Additionally, due to the importance of mouse models in estrogen action and lens research, lenses from intact female mice were also analyzed. Both the C57BL/6 and FVB/N mouse strains also possessed high affinity binding sites in all three lens fractions. Furthermore, transcripts for ERα, ERß, and G protein-coupled estrogen receptor (GPER; previously called GPR30) that bind estradiol with high affinity were expressed in the human and mouse lenses. These data provide the first evidence of GPER expression in the lens. Its role, functions, and subcellular location are currently unknown, but a G-shift assay in the membrane fractions of human and mouse lenses did not show evidence that estradiol induced classic G protein-coupled receptor activation. All three receptor transcripts were also detected in the lens capsule region isolated from female C57BL/6 mice, which is mainly comprised of epithelial cells. In contrast, only ERα and GPER were expressed in the cortex/nuclear region, which is primarily composed of differentiating and organelle-free fiber cells. No significant differences in specific estradiol binding and receptor RNA expression were observed in the lenses between male and female C57BL/6 mice. These findings indicate that the lens is an estrogen target tissue in both sexes. The identification of GPER, in addition to ERα and ERß, in the lens also adds to the complexity of possible estrogen responses in the lens. Accordingly, the protective effects of estrogen in women and animals may be mediated by all three estrogen receptors in the lens. In addition, the similarities in binding and receptor RNA expression in the lenses of both species suggest that mice can be used to model estrogen action in the human lens.

Pharmacist and Student Knowledge and Perceptions of Herbal Supplements and Natural Products.

We aimed to collect parallel perspectives from pharmacists and pharmacy students on their use, knowledge, attitudes, and perceptions about herbal supplements/natural products. Two cross-sectional descriptive survey questionnaires-one focusing on pharmacists and the other focusing on pharmacy students-were administered from March to June 2021 via Qualtrics. The surveys were sent out to preceptor pharmacists and pharmacy students currently enrolled at a single U.S. school of pharmacy. The questionnaires were composed of five main sections, including (1) demographics; (2) attitudes/perceptions; (3) educational experience; (4) resource availability; and (5) objective knowledge of herbal supplements/natural products. Data analysis primarily utilized descriptive statistics with relevant comparisons across domains. A total of 73 pharmacists and 92 pharmacy students participated, with response rates of 8.8% and 19.3%, respectively. A total of 59.2% of pharmacists and 50% of pharmacy students stated they personally used herbal supplements/natural products. Most respondents (>95% for both groups) considered vitamins/minerals safe, although a lower percentage agreed on this for herbal supplements/natural products (60% and 79.3% for pharmacists and pharmacy students, respectively). Patient inquiries in the pharmacy setting were most seen for vitamin D, zinc, cannabidiol, and omega-3. A total of 34.2% of pharmacists reported having training in herbal supplements/natural products as a required part of their Pharm.D. training, and 89.1% of pharmacy students desired to learn more. The median score on the objective knowledge quiz was 50% for pharmacists and 45% for pharmacy students. Ultimately, herbal supplements/natural products are recognized by pharmacists/pharmacy students as a consistent and embedded part of pharmacy practice, although there is a need to enhance knowledge and skills in this area.

A novel calcium aluminate-melatonin scaffold enhances bone regeneration within a calvarial defect.

Over 500,000 bone graft or bio-implant procedures are performed annually in the United States. It has been reported that osseous autograft procurement may result in donor site complications and bio-implant allografts have been associated with disease transmission. Ceramic scaffolds are only osteoconductive, limiting their clinical use. The objective of this study was to create a bone filler substitute with regenerating properties similar to natural bone. Therefore, melatonin and platelet-rich plasma (PRP) were utilized for their known osteoinductive properties. It was hypothesized that melatonin and/or PRP would enhance the osteoinductive and osteoconductive properties of calcium aluminate (CA) scaffolds to promote bone regeneration in a model of calvarial defects. The biocompatibility of CA and CA-Mel scaffolds was tested in vitro and in vivo. Data show that CA-Mel scaffolds, in comparison with CA scaffolds, enhanced the adhesion, viability, and proliferation of normal human osteoblasts cells but not that of NIH3T3 fibroblasts. Data also showed that human adult mesenchymal stem cells grown on CA or CA-Mel scaffolds showed a time-dependent induction into osteoblasts over 14days revealed through scanning electron microscopy and by alkaline phosphatase analyses. Implantation of CA-Mel scaffolds into critical size calvarial defects in female, ovariectomized rats showed that the CA-Mel scaffolds were biocompatible, allowed for tissue infiltration, and showed evidence of scaffold biodegradation by 3 and 6months. Bone regeneration, assessed using fluorochrome labeling at 3 and 6months, was greatest in animals implanted with the CA-Mel scaffold. Overall, results from this study show that CA-Mel scaffolds were osteoconductive and osteoinductive.

Melatonin osteoporosis prevention study (MOPS): a randomized, double-blind, placebo-controlled study examining the effects of melatonin on bone health and quality of life in perimenopausal women.

The purpose of this double-blind study was to assess the effects of nightly melatonin supplementation on bone health and quality of life in perimenopausal women. A total of 18 women (ages 45-54) were randomized to receive melatonin (3mg, p.o., n=13) or placebo (n=5) nightly for 6months. Bone density was measured by calcaneal ultrasound. Bone turnover marker (osteocalcin, OC for bone formation and NTX for bone resorption) levels were measured bimonthly in serum. Participants completed Menopause-Specific Quality of Life-Intervention (MENQOL) and Pittsburgh Sleep Quality Index (PSQI) questionnaires before and after treatment. Subjects also kept daily diaries recording menstrual cycling, well-being, and sleep patterns. The results from this study showed no significant change (6-month-baseline) in bone density, NTX, or OC between groups; however, the ratio of NTX:OC trended downward over time toward a ratio of 1:1 in the melatonin group. Melatonin had no effect on vasomotor, psychosocial, or sexual MENQOL domain scores; however, it did improve physical domain scores compared to placebo (mean change melatonin: -0.6 versus placebo: 0.1, P<0.05). Menstrual cycling was reduced in women taking melatonin (mean cycles melatonin: 4.3 versus placebo: 6.5, P<0.05), and days between cycles were longer (mean days melatonin: 51.2 versus placebo: 24.1, P<0.05). No differences in duration of menses occurred between groups. The overall PSQI score and average number of hours slept were similar between groups. These findings show that melatonin supplementation was well tolerated, improved physical symptoms associated with perimenopause, and may restore imbalances in bone remodeling to prevent bone loss. Further investigation is warranted.

Effects on bone by the light/dark cycle and chronic treatment with melatonin and/or hormone replacement therapy in intact female mice.

In this study, the effects of the light/dark cycle, hormone replacement therapy (HRT), and nocturnal melatonin supplementation on osteogenic markers and serum melatonin levels were examined in a blind mouse model (MMTV-Neu transgenic mice). Melatonin levels in this mouse strain (FVB/N) with retinal degeneration (rd-/-) fluctuate in a diurnal manner, suggesting that these mice, although blind, still perceive light. Real-time RT-PCR analyses demonstrated that Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA levels coincide with melatonin levels. The effect of chronic HRT (0.5 mg 17ß-estradiol + 50 mg progesterone in 1800 kcal of diet) alone and in combination with melatonin (15 mg/L drinking water) on bone quality and density was also assessed by histomorphometry and microcomputed tomography, respectively. Bone density was significantly increased (P < 0.05) after 1 yr of treatment with the individual therapies, HRT (22% increase) and nocturnal melatonin (20% increase) compared to control. Hormone replacement therapy alone also increased surface bone, decreased trabecular space, and decreased the number of osteoclasts without affecting osteoblast numbers compared to the control group (P < 0.05). Chronic HRT + melatonin therapy did not significantly increase bone density, even though this combination significantly increased Bglap mRNA levels. These data suggest that the endogenous melatonin rhythm modulates markers important to bone physiology. Hormone replacement therapy with or without nocturnal melatonin in cycling mice produces unique effects on bone markers and bone density. The effects of these therapies alone and combined may improve bone health in women in perimenopause and with low nocturnal melatonin levels from too little sleep, too much light, or age.

Morinda citrifolia (Noni) Juice Augments Mammary Gland Differentiation and Reduces Mammary Tumor Growth in Mice Expressing the Unactivated c-erbB2 Transgene.

Morinda citrifolia (noni) is reported to have many beneficial properties, including on immune, inflammatory, quality of life, and cancer endpoints, but little is known about its ability to prevent or treat breast cancer. To test its anticancer potential, the effects of Tahitian Noni Juice (TNJ) on mammary carcinogenesis were examined in MMTV-neu transgenic mice. Mammary tumor latency, incidence, multiplicity, and metastatic incidence were unaffected by TNJ treatment, which suggests that it would not increase or decrease breast cancer risk in women taking TNJ for its other benefits. However, noni may be useful to enhance treatment responses in women with existing HER2/neu breast cancer since TNJ resulted in significant reductions in tumor weight and volume and in longer tumor doubling times in mice. Remarkably, its ability to inhibit the growth of this aggressive form of cancer occurred with the mouse equivalent of a recommended dose for humans (<3 oz/day). A 30-day treatment with TNJ also induced significant changes in mammary secondary ductule branching and lobuloalveolar development, serum progesterone levels, and estrous cycling. Additional studies investigating TNJ-induced tumor growth suppression and modified reproductive responses are needed to characterize its potential as a CAM therapy for women with and without HER2(+) breast cancer.

Mesenchymal Stem Cell and Monocyte Co-cultures.

Transwell co-cultures are critical to study cell-to-cell communication through the release of factors between different cells allowing for the simultaneous assessment of treatment effects on one cell type (e.g., Cell A) and their impact on another cell type (e.g., Cell B). This allows for the simultaneous assessment of two different cell types and the factors they secrete under the same treatment conditions, which minimizes interexperimental variability, demonstrates causation rather than association, and enhances the translatability of the findings to the in vivo condition. Here we describe transwell co-cultures of human mesenchymal stem cells (MSCs) and peripheral blood monocytes or pre-osteoclasts to assess osteoblast-mediated actions on osteoclastogenesis.

Gefitinib-Tamoxifen Hybrid Ligands as Potent Agents against Triple-Negative Breast Cancer.

Anticancer drug conjugates may benefit from simultaneous action at two targets potentially overcoming the drawbacks of current cancer treatment, such as insufficient efficacy, high toxicity, and development of resistance. Compared to a combination of two single-target drugs, they may offer an advantage of pharmacokinetic simplicity and fewer drug-drug interactions. Here, we report a series of compounds connecting tamoxifen or endoxifen with the EGFR-inhibitor gefitinib via a covalent linkage. These hybrid ligands retain both ER antagonist activity and EGFR inhibition. The most potent analogues exhibited single-digit nanomolar activities at both targets. The amide-linked endoxifen-gefitinib drug conjugates 17b and 17c demonstrated the most favorable anti-cancer profile in cellular viability assays on MCF7, MDA-MB-231, MDA-MB-468, and BT-549 breast cancer cells. Most importantly, in TNBC cells 17b and 17c displayed nanomolar IC50-values (380 nM - 970 nM) and were superior in their anti-cancer activity compared to their control compounds and combinations thereof.

N-Acetyl-5-arylalkoxytryptamine analogs: probing the melatonin receptors for MT(1) -selectivity.

A series of melatonin analogs obtained by the replacement of the ether methyl group with larger arylalkyl and aryloxyalkyl substituents was prepared in order to probe the melatonin receptors for MT(1) -selectivity. The most MT(1) -selective agents 11 and 15 were substituted with a Ph(CH(2) )(3) or a PhO(CH(2) )(3) group. Compounds 11 and 15 displayed 11.5-fold and 11-fold higher affinity for the MT(1) receptors than for the MT(2) subtype. Interestingly, in our binding assay 11 and 15 have shown considerably higher MT(1) -affinity and selectivity than the reference ligand, the dimeric agomelatine 1a.