Novel KCNJ10 Gene Variations Compromise Function of Inwardly Rectifying Potassium Channel 4.1.

TheKCNJ10gene encoding Kir4.1 contains numerous SNPs whose molecular effects remain unknown. We investigated the functional consequences of uncharacterized SNPs (Q212R, L166Q, and G83V) on homomeric (Kir4.1) and heteromeric (Kir4.1-Kir5.1) channel function. We compared these with previously characterized EAST/SeSAME mutants (G77R and A167V) in kidney-derived tsA201 cells and in glial cell-derived C6 glioma cells. The membrane potentials of tsA201 cells expressing G77R and G83V were significantly depolarized as compared with WTKir4.1, whereas cells expressing Q212R, L166Q, and A167V were less affected. Furthermore, macroscopic currents from cells expressing WTKir4.1 and Q212R channels did not differ, whereas currents from cells expressing L166Q, G83V, G77R, and A167V were reduced. Unexpectedly, L166Q current responses were rescued when co-expressed with Kir5.1. In addition, we observed notable differences in channel activity between C6 glioma cells and tsA201 cells expressing L166Q and A167V, suggesting that there are underlying differences between cell lines in terms of Kir4.1 protein synthesis, stability, or expression at the surface. Finally, we determined spermine (SPM) sensitivity of these uncharacterized SNPs and found that Q212R-containing channels displayed reduced block by 1 µmSPM. At 100 µmSPM, the block was equal to or greater than WT, suggesting that the greater driving force of SPM allowed achievement of steady state. In contrast, L166Q-Kir5.1 channels achieved a higher block than WT, suggesting a more stable interaction of SPM in the deep pore cavity. Overall, our data suggest that G83V, L166Q, and Q212R residues play a pivotal role in controlling Kir4.1 channel function.

Paraoxon and Pyridostigmine Interfere with Neural Stem Cell Differentiation.

Acetylcholinesterase (AChE) inhibition has been described as the main mechanism of organophosphate (OP)-evoked toxicity. OPs represent a human health threat, because chronic exposure to low doses can damage the developing brain, and acute exposure can produce long-lasting damage to adult brains, despite post-exposure medical countermeasures. Although the main mechanism of OP toxicity is AChE inhibition, several lines of evidence suggest that OPs also act by other mechanisms. We hypothesized that rat neural progenitor cells extracted on embryonic day 14.5 would be affected by constant inhibition of AChE from chronic exposure to OP or pyridostigmine (a reversible AChE blocker) during differentiation. In this work, the OP paraoxon decreased cell viability in concentrations >50 µM, as measured with the MTT assay; however, this effect was not dose-dependent. Reduced viability could not be attributed to blockade of AChE activity, since treatment with 200 µM pyridostigmine did not affect cell viability, even after 6 days. Although changes in protein expression patterns were noted in both treatments, the distribution of differentiated phenotypes, such as the percentages of neurons and glial cells, was not altered, as determined by flow cytometry. Since paraoxon and pyridostigmine each decreased neurite outgrowth (but did not prevent differentiation), we infer that developmental patterns may have been affected.

Chemical profile and in vivo hypoglycemic effects of Syzygium jambos, Costus speciosus and Tapeinochilos ananassae plant extracts used as diabetes adjuvants in Puerto Rico.

BACKGROUND: The increasing numbers of people who use plant-based remedies as alternative or complementary medicine call for the validation of less known herbal formulations used to treat their ailments. Since Puerto Rico has the highest rate of Type 2 diabetes within all the states and territories of the United States, and Puerto Ricans commonly use plants as diabetes adjuvants, it is important to study the plants' physiological effects, and identify their bioactive compounds to understand their role in modulation of blood glucose levels. We present the phytochemical profiles and hypoglycemic effects of Tapeinochilus ananassae, Costus speciosus and Syzygium jambos. METHODS: Phytochemicals in methanolic and aqueous extracts were analyzed by thin layer chromatography (TLC). Alkaloids (Bromocresol green, λ=470 nm), flavonoids (AlCl3, λ=415 nm), saponins (DNS, λ=760 nm), tannins (FeCl3/K4Fe(CN)6, λ=395 nm) and phenolics (Folin-Ciocalteau, λ=765 nm) were quantified. Male C57BLKS/J (db/db) and C57BL/J (ob/ob) genetically obese mice were orally gavaged with aqueous extracts of lyophilized plant decoctions for 10 wks. RESULTS: Our results show that T. ananassae had significantly greater amounts of flavonoids and tannins, while S. jambos showed the greatest concentration of phenolics and C. speciosus exhibited higher amounts of alkaloids. C57BLKS/J db/db treated with plant extracts show better glucose modulation when the extracts are administered in complement with an insulin injection. Finally, C57BL/J ob/ob mice on T. ananassae and S. jambos treatments show better blood glucose modulation over time. CONCLUSION: These results document for the first time the chemical profile of T. ananassae and provide evidence for a potential anti-diabetic efficacy of T. ananassae and S. jambos.

Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase.

The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC(50) of 1.1 µM for Rac inhibition by EHop-016 is ∼100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 µM. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 µM), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.

Dietary grape polyphenol resveratrol increases mammary tumor growth and metastasis in immunocompromised mice.

BACKGROUND: Resveratrol, a polyphenol from grapes and red wine has many health beneficial effects, including protection against cardiovascular and neurodegenerative diseases and cancer. However, our group and others have provided evidence for a dual cancer promoting or inhibitory role for resveratrol in breast cancer, dependent on estrogenic or antiestrogenic activities. Moreover, much of the inhibitory effects of resveratrol have been reported from studies with high non-physiological concentrations. METHODS: We investigated the effects of a range of concentrations (0.5, 5, 50 mg/kg body weight) of resveratrol on mammary tumor development post-initiation, using immunocompromised mice. RESULTS: Our findings suggest promotion of mammary tumor growth and metastasis by resveratrol at all concentrations tested in tumors derived from the low metastatic estrogen receptor (ER)α(-), ERß(+) MDA-MB-231 and the highly metastatic ER(-) MDA-MB-435 cancer cell lines. Additionally, the activity of the migration/invasion regulator Rac, which we have previously shown to be regulated by resveratrol in vitro, was measured in tumors from resveratrol treated mice. Our results show a significant induction of tumoral Rac activity and a trend in increased expression of the Rac downstream effector PAK1 and other tumor promoting molecules following resveratrol treatment. CONCLUSION: Taken together, our findings implicate low concentrations of resveratrol in potential promotion of breast cancer. Therefore, this study illuminates the importance of further delineating resveratrol's concentration dependent effects, particularly in breast cancer, before it can be tested in the clinic or used as a dietary supplement for breast cancer patients.

Development of Competency-based Online Genomic Medicine Training (COGENT).

The fields of genetics and genomics have greatly expanded across medicine through the development of new technologies that have revealed genetic contributions to a wide array of traits and diseases. Thus, the development of widely available educational resources for all healthcare providers is essential to ensure the timely and appropriate utilization of genetics and genomics patient care. In 2020, the National Human Genome Research Institute released a call for new proposals to develop accessible, sustainable online education for health providers. This paper describes the efforts of the six teams awarded to reach the goal of providing genetic and genomic training modules that are broadly available for busy clinicians.

Ganoderma lucidum (Reishi) inhibits cancer cell growth and expression of key molecules in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most lethal and least understood form of advanced breast cancer. Its lethality originates from its nature of invading the lymphatic system and absence of a palpable tumor mass. Different from other metastatic breast cancer cells, IBC cells invade by forming tumor spheroids that retain E-cadherin-based cell-cell adhesions. Herein we describe the potential of the medicinal mushroom Ganoderma lucidum (Reishi) as an attractive candidate for anti-IBC therapy. Reishi contains biological compounds that are cytotoxic against cancer cells. We report the effects of Reishi on viability, apoptosis, invasion, and its mechanism of action in IBC cells (SUM-149). Results show that Reishi selectively inhibits cancer cell viability although it does not affect the viability of noncancerous mammary epithelial cells. Apoptosis induction is consistent with decreased cell viability. Reishi inhibits cell invasion and disrupts the cell spheroids that are characteristic of the IBC invasive pathology. Reishi decreases the expression of genes involved in cancer cell survival and proliferation (BCL-2, TERT, PDGFB), and invasion and metastasis (MMP-9), whereas it increases the expression of IL8. Reishi reduces BCL-2, BCL-XL, E-cadherin, eIF4G, p120-catenin, and c-Myc protein expression and gelatinase activity. These findings suggest that Reishi is an effective anti-IBC therapeutic.

Evaluation of the Pink Luminous Breast LED-Based Technology Device as a Screening Tool for the Early Detection of Breast Abnormalities.

Breast cancer is the leading cause of sex-specific female cancer deaths in the United States. Detection at earlier stages contributes to decreasing the mortality rate. The mammogram is the "Gold Standard" for breast cancer screening with an estimated sensitivity of 86.9% and a specificity of 88.9%. However, these values are negatively affected by the breast density considered a risk factor for developing breast cancer. Herein, we validate the novel LED-based medical device Pink Luminous Breast (PLB) by comparison with the mammogram using a double blinded approach. The PLB works by emitting a LED red light with a harmless spectrum of 640-800 nanometers. This allows the observation of abnormalities represented by dark or shadow areas. In this study, we evaluated the sensitivity and specificity of the PLB device as a screening tool for the early detection of breast abnormalities. Our results show that the PLB device has a high sensitivity (89.6%) and specificity (96.4%) for detecting breast abnormalities comparable to the adjusted mammogram values: 86.3 and 68.9%, respectively. The percentage of presence of breast density was 78.2% using PLB vs. 72.9% with the mammogram. Even with higher findings of breast density, the PLB is still capable of detecting 9.4% of calcifications compared to 6.2% in mammogram results and the reported findings for cysts, masses, or tumor-like abnormalities was higher using the PLB (6.5%) than the mammogram (5.6%). A 100% of the participants felt comfortable using the device without feeling pain or discomfort during the examination with 100% acceptability. The PLB positive validation shows its potential for routine breast screening at non-clinical settings. The PLB provides a rapid, non-invasive, portable, and easy-to-use tool for breast screening that can complement the home-based breast self-examination technique or the clinical breast examination. In addition, the PLB can be conveniently used for screening breasts with surgical implants. PLB provides an accessible and painless breast cancer screening tool. The PLB use is not intended to replace the mammogram for breast screening but rather to use it as an adjunct or complemental tool as part of more efficient earlier detection strategies contributing to decrease mortality rates.

Soy and Frequent Dairy Consumption with Subsequent Equol Production Reveals Decreased Gut Health in a Cohort of Healthy Puerto Rican Women.

The U.S. Hispanic female population has one of the highest breast cancer (BC) incidence and mortality rates, while BC is the leading cause of cancer death in Puerto Rican women. Certain foods may predispose to carcinogenesis. Our previous studies indicate that consuming combined soy isoflavones (genistein, daidzein, and glycitein) promotes tumor metastasis possibly through increased protein synthesis activated by equol, a secondary dietary metabolite. Equol is a bacterial metabolite produced in about 20-60% of the population that harbor and exhibit specific gut microbiota capable of producing it from daidzein. The aim of the current study was to investigate the prevalence of equol production in Puerto Rican women and identify the equol producing microbiota in this understudied population. Herein, we conducted a cross-sectional characterization of equol production in a clinically based sample of eighty healthy 25-50 year old Puerto Rican women. Urine samples were collected and evaluated by GCMS for the presence of soy isoflavones and metabolites to determine the ratio of equol producers to equol non-producers. Furthermore, fecal samples were collected for gut microbiota characterization on a subset of women using next generation sequencing (NGS). We report that 25% of the participants were classified as equol producers. Importantly, the gut microbiota from equol non-producers demonstrated a higher diversity. Our results suggest that healthy women with soy and high dairy consumption with subsequent equol production may result in gut dysbiosis by having reduced quantities (diversity) of healthy bacterial biomarkers, which might be associated to increased diseased outcomes (e.g., cancer, and other diseases).

Implications of ER stress, the unfolded protein response, and pro- and anti-apoptotic protein fingerprints in human monocyte-derived dendritic cells treated with alcohol.

BACKGROUND: Dendritic cells (DCs) are responsible for the activation of T cells and B cells. There is accumulating evidence that psychoactive substances such as alcohol can affect immune responses. We hypothesize that this occurs by modulating changes in proteins triggering a process known as unfolded protein response (UPR). This process protects cells from the toxic effects of misfolded proteins responsible for causing endoplasmic reticulum (ER) stress. Although much is known about ER stress, little is understood about the consequences of ethanol use on DC's protein expression. METHODS: In this study, we investigated alterations in the proteins of human monocyte-derived dendritic cells (MDDC) treated with 0.1% of alcohol by two-dimensional (2D) gel electrophoresis followed by liquid chromatography-tandem mass spectrometry, protein identification, and confirmation at the gene expression level by qRT-PCR. RESULTS: Proteomes of related samples demonstrated 32 differentially expressed proteins that had a 2-fold or greater change in expression (18 spots were up-regulated and 14 were down-regulated), compared to the control cultures (untreated cells). Alcohol significantly changed the expression of several components of the UPR stress-induced pathways that include chaperones, ER stress, antioxidant enzymes, proteases, alcohol dehydrogenase, cytoskeletal and apoptosis-regulating proteins. qRT-PCR analyses highlighted the enhanced expression of UPR and antioxidant genes that increased (18 hours) with alcohol treatment. CONCLUSION: Results of these analyses provide insights into alcohol mechanisms of regulating DC and suggest that alcohol induced specifically the UPR in DC. We speculate that activation of a UPR by alcohol may protect the DC from oxidant injury but may lead to the development of alcohol-related diseases.

Prevalence of drug resistance and associated mutations in a population of Hiv-1+ Puerto Ricans in 2005.

This is a continuation of our efforts to maintain a record of the evolution of HIV-1 infection in Puerto Rico by monitoring the expression levels of antiretroviral resistance-associated mutations. Samples from 2005 were analyzed (458: 270 males, 137 females, 51 anonymous), using the TRUGENE HIV-1 Genotyping Kit and the OpenGene DNA Sequencing System. Results show that 60.1% of males and 50.2% of females had HIV-1 with resistance to at least one medication. The average number of HIV mutations in males was 6.27, while the average number of HIV mutations in females was 5.49. The highest levels of resistance were to Zalcitabine, Lamivudine, and Stavudine. The reverse transcriptase mutations with the highest frequency of expression were M184V, K103N and D67N. Protease mutations with the highest rate of expression were L63P, M361 and L90M. Significant differences between men and women were recorded in the levels of HIV-1 expressed mutations and resistance.

Prevalence of drug resistance and associated mutations in HIV-positive Puerto Ricans: sex variations.

INTRODUCTION: A cross sectional study was conducted from 2002-2004 to record the evolution of HIV-1 infection in Puerto Rico by monitoring the expression of antiretroviral resistance-associated mutations. METHODS: Samples were analyzed by using the TRUGENE HIV-1 Genotyping Kit and the OpenGene DNA Sequencing System. RESULTS: Mutations in the HIV-1 virus were detected in 92.7% of men and 94.8% of women. Of these, 75.1% of men and 72.4% of women had HIV-1 with resistance to at least one medication. The average number of HIV mutations was 6.1 in men and 5.3 in women. In 2002 and 2003, strains were most frequently resistant to the antiretroviral drugs zalcitabine, lamivudine and didanosine, while in 2004, strains were most frequently resistant to zalcitabine, lamivudine, and efavirenz. The most prevalent mutations in the reverse transcriptase gene were M184V, K103N, T215Y, and M41L. The most prevalent mutations in the protease gene were L63P, M361, L90M, A71V, and L101. CONCLUSIONS: Significant differences between men and women were recorded in the levels of HIV-1 expressed mutations and resistance. When comparing these results with data from 2000 and 2001, results indicate that expression of resistant mutations has remained constant.

Ganoderma lucidum extract (GLE) impairs breast cancer stem cells by targeting the STAT3 pathway.

The aggressive nature of triple negative breast cancer (TNBC) may be explained in part by the presence of breast cancer stem cells (BCSCs), a subpopulation of cells, which are involved in tumor initiation, progression, metastasis, recurrence, and therapy resistance. The signal transducer and activator of transcription 3 (STAT3) pathway participates in the development and progression of BCSCs, but its role in TNBC remains unclear. Here, we report that Ganoderma lucidum extract (GLE), a medicinal mushroom with anticancer activity, acts on BCSCs in vitro and in TNBC pre-clinical animal tumor models by downregulating the STAT3 pathway. We show that GLE significantly reduces TNBC cell viability, and down-regulates total and phosphorylated STAT3 expression. This is consistent with the reduction of OCT4, NANOG and SOX2 expression, reduction in the BCSC population by loss of the ALDH1 and CD44+/CD24- population, the deformation of mammospheres, and the strong reduction in animal tumor volume and tumor weight. Analysis of the BCSC compartment in tumors revealed that GLE decreases the STAT3 pathway and the expression of OCT4, NANOG, and SOX2 in BCSCs. These findings demonstrate that the anti-cancer activity of GLE targets BCSCs of TNBC through the downregulation of the STAT3 pathway.

Polyamines preserve connexin 43-mediated gap junctional communication during intracellular hypercalcemia and acidosis.

Changes in the regulation, formation, and gating of connexin-based gap junction channels occur in various disorders. It has been shown that H and Ca are involved in the regulation of gap junctional communication. Ischemia-induced intracellular acidification and Ca overload lead to closure of gap junctions and inhibit an exchange by ions and small molecules throughout the network of cells in the heart, brain, and other tissues. In this study, we examined the role of the polyamines in the regulation of connexin 43 (Cx43)-based gap junction channels under elevated intracellular concentrations of hydrogen ([H]i) and calcium ([Ca]i) ions. Experiments, conducted in Novikoff and A172 human glioblastoma cells, which endogenously express Cx43, showed that polyamines prevent downregulation of Cx43-mediated gap junctional communication caused by elevated [Ca]i and [H]i, accompanying ischemic and other pathological conditions. siRNA knockdown of Cx43 significantly reduces gap junctional communication, indicating that Cx43 gap junctions are the targets for spermine regulation.

HIV-1 Gp120 clade B/C induces a GRP78 driven cytoprotective mechanism in astrocytoma.

HIV-1 clades are known to be one of the key factors implicated in modulating HIV-associated neurocognitive disorders. HIV-1 B and C clades account for the majority of HIV-1 infections, clade B being the most neuropathogenic. The mechanisms behind HIV-mediated neuropathogenesis remain the subject of active research. We hypothesized that HIV-1 gp120 clade B and C proteins may exert differential proliferation, cell survival and NeuroAIDS effects in human astrocytoma cells via the Unfolded Protein Response, an endoplasmic reticulum- based cytoprotective mechanism. The differential effect of gp120 clade B and C was evaluated using for the first time a Tandem Mass Tag isobaric labeling quantitative proteomic approach. Flow cytometry analyses were performed for cell cycle and cell death identification. Among the proteins differentiated by HIV-1 gp120 proteins figure cytoskeleton, oxidative stress, UPR markers and numerous glycolytic metabolism enzymes. Our results demonstrate that HIV-1 gp120 B induced migration, proliferative and protective responses granted by the expression of GRP78, while HIV-1 gp120 C induced the expression of key inflammatory and pro-apoptotic markers. These novel findings put forward the first evidence that GRP78 is a key player in HIV-1 clade B and C neuropathogenic discrepancies and can be used as a novel target for immunotherapies.

Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells.

Curcumin, an extract from the turmeric rhizome (Curcuma longa), is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12) and Poly (ADP-ribose) polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses.

A decade of viral mutations and associated drug resistance in a population of HIV-1+ Puerto Ricans: 2002-2011.

Puerto Rico has one of the highest rates of HIV/AIDS seen for any US state or territory, and antiretroviral therapy has been a mainstay of efforts to mitigate the HIV/AIDS public health burden on the island. We studied the evolutionary dynamics of HIV-1 mutation and antiretroviral drug resistance in Puerto Rico by monitoring the population frequency of resistance-associated mutations from 2002 to 2011. Whole blood samples from 4,475 patients were analyzed using the TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System in the Immunoretrovirus Research Laboratory at Universidad Central del Caribe. Results show that 64.0% of female and 62.9% of male patients had HIV-1 mutations that confer resistance to at least one antiretroviral medication. L63P and M184V were the dominant mutations observed for the protease (PRO) and reverse transcriptase (RT) encoding genes, respectively. Specific resistance mutations, along with their associated drug resistance profiles, can be seen to form temporal clusters that reveal a steadily changing landscape of resistance trends over time. Both women and men showed resistance mutations for an average of 4.8 drugs over the 10-year period, further underscoring the strong selective pressure exerted by antiretrovirals along with the rapid adaptive response of HIV. Nevertheless, both female and male patients showed a precipitous decrease for overall drug resistance, and for PRO mutations in particular, over the entire course of the study, with the most rapid decrease in frequency seen after 2006. The reduced HIV-1 mutation and drug resistance trends that we observed are consistent with previous reports from multi-year studies conducted around the world. Reduced resistance can be attributed to the use of more efficacious antiretroviral drug therapy, including the introduction of multi-drug combination therapies, which limited the ability of the virus to mount rapid adaptive responses to antiretroviral selection pressure.

Characterization of a Dual Rac/Cdc42 Inhibitor MBQ-167 in Metastatic Cancer.

The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42 homolog) regulate cell functions governing cancer malignancy, including cell polarity, migration, and cell-cycle progression. Accordingly, our recently developed Rac inhibitor EHop-016 (IC50, 1,100 nmol/L) inhibits cancer cell migration and viability and reduces tumor growth, metastasis, and angiogenesis in vivo Herein, we describe MBQ-167, which inhibits Rac and Cdc42 with IC50 values of 103 and 78 nmol/L, respectively, in metastatic breast cancer cells. Consequently, MBQ-167 significantly decreases Rac and Cdc42 downstream effector p21-activated kinase (PAK) signaling and the activity of STAT3, without affecting Rho, MAPK, or Akt activities. MBQ-167 also inhibits breast cancer cell migration, viability, and mammosphere formation. Moreover, MBQ-167 affects cancer cells that have undergone epithelial-to-mesenchymal transition by a loss of cell polarity and inhibition of cell surface actin-based extensions to ultimately result in detachment from the substratum. Prolonged incubation (120 hours) in MBQ-167 decreases metastatic cancer cell viability with a GI50 of approximately 130 nmol/L, without affecting noncancer mammary epithelial cells. The loss in cancer cell viability is due to MBQ-167-mediated G2-M cell-cycle arrest and subsequent apoptosis, especially of the detached cells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis in immunocompromised mice by approximately 90%. In conclusion, MBQ-167 is 10× more potent than other currently available Rac/Cdc42 inhibitors and has the potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42. Mol Cancer Ther; 16(5); 805-18. ©2017 AACR.

Immune cells under altered gravity conditions.

Considerable research over the past three decades has been devoted to characterizing changes in the response of cells of the immune system to stimuli while exposed to altered gravity conditions. A consistent finding shows that mammalian cells subjected to conditions of spaceflight and the microgravity environment of space manifest a number of alterations in structure and function. Changes in cells flown on the space shuttle include reduction in growth activation and decline in growth rate in the total population. Other changes include chromosomal aberration, inhibited locomotion, altered cytokine production, changes in protein kinase C distribution, and increased apoptosis. Human lymphocytes respond poorly to mitogenic stimulation in microgravity and lymphoid cell lines are growth arrested. Recent research into the gene expression of immune cells under altered gravity conditions has provided insight into the possible pathways involved in adapting to gravity changes. Here we synthesize the findings of research related to changes in the immune response as a result of exposure to altered gravity conditions.

Ganoderma lucidum Combined with the EGFR Tyrosine Kinase Inhibitor, Erlotinib Synergize to Reduce Inflammatory Breast Cancer Progression.

The high incidence of resistance to Tyrosine Kinase Inhibitors (TKIs) targeted against EGFR and downstream pathways has increased the necessity to identify agents that may be combined with these therapies to provide a sustained response for breast cancer patients. Here, we investigate the therapeutic potential of Ganoderma lucidum extract (GLE) in breast cancer, focusing on the regulation of the EGFR signaling cascade when treated with the EGFR TKI, Erlotinib. SUM-149, or intrinsic Erlotinib resistant MDA-MB-231 cells, and a successfully developed Erlotinib resistant cell line, rSUM-149 were treated with increasing concentrations of Erlotinib, GLE, or their combination (Erlotinib/GLE) for 72h. Treatment effects were tested on cell viability, cell proliferation, cell migration and invasion. To determine tumor progression, severe combined immunodeficient mice were injected with SUM-149 cells and then treated with Erlotinib/GLE or Erlotinib for 13 weeks. We assessed the protein expression of ERK1/2 and AKT in in vitro and in vivo models. Our results show that GLE synergizes with Erlotinib to sensitize SUM-149 cells to drug treatment, and overcomes intrinsic and developed Erlotinib resistance. Also, Erlotinib/GLE decreases SUM-149 cell viability, proliferation, migration and invasion. GLE increases Erlotinib sensitivity by inactivating AKT and ERK signaling pathways in our models. We conclude that a combinatorial therapeutic approach may be the best way to increase prognosis in breast cancer patients with EGFR overexpressing tumors.