Introducing Wound Healing Assays in the Undergraduate Biology Laboratory Using Ibidi Plates.

The wound healing assay is a simple and inexpensive method that allows researchers to experimentally mimic cell growth and migration leading to wound healing. In this assay, a wound is created on a monolayer of cultured mammalian cells and cell migration is monitored. Micrographs are captured at regular intervals during the duration of the experiment. These microscopy images are analyzed to compare cell migration and wound closure under different conditions. Introduction of different cytotoxic treatments into a wound healing assay can provide information as to whether a particular drug or compound of interest has the ability to affect cell migration. This type of analysis is important when assessing the ability of a particular cancer cell line to display invasive and metastatic behaviors. One of the challenges of this assay is to create the original wound in a way that is consistent across plates or treatments, facilitating comparisons across experimental groups. This is a particular challenge when using the wound healing assay in the context of an undergraduate biology class to expose students to a distinct form of mammalian cell culture and help them apply scientific knowledge and research skills. We found an easy way to overcome this obstacle by using ibidi plates. In this article, we provide a simple protocol to use ibidi plates and HeLa cells to set up wound healing assays. This laboratory exercise allows undergraduate students to utilize different skills developed through cell culture experience, such as growing, treating, and imaging mammalian cells.

Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

Syndecan-1 is required to maintain intradermal fat and prevent cold stress.

Homeostatic temperature regulation is fundamental to mammalian physiology and is controlled by acute and chronic responses of local, endocrine and nervous regulators. Here, we report that loss of the heparan sulfate proteoglycan, syndecan-1, causes a profoundly depleted intradermal fat layer, which provides crucial thermogenic insulation for mammals. Mice without syndecan-1 enter torpor upon fasting and show multiple indicators of cold stress, including activation of the stress checkpoint p38α in brown adipose tissue, liver and lung. The metabolic phenotype in mutant mice, including reduced liver glycogen, is rescued by housing at thermoneutrality, suggesting that reduced insulation in cool temperatures underlies the observed phenotypes. We find that syndecan-1, which functions as a facultative lipoprotein uptake receptor, is required for adipocyte differentiation in vitro. Intradermal fat shows highly dynamic differentiation, continuously expanding and involuting in response to hair cycle and ambient temperature. This physiology probably confers a unique role for Sdc1 in this adipocyte sub-type. The PPARγ agonist rosiglitazone rescues Sdc1-/- intradermal adipose tissue, placing PPARγ downstream of Sdc1 in triggering adipocyte differentiation. Our study indicates that disruption of intradermal adipose tissue development results in cold stress and complex metabolic pathology.

MAGE proteins regulate KRAB zinc finger transcription factors and KAP1 E3 ligase activity.

Expression of Melanoma AntiGen Encoding (MAGE) genes, particularly MAGE-A3, has been correlated with aggressive clinical course, the acquisition of resistance to chemotherapy and poor clinical outcomes of melanoma and other malignancies. MAGE proteins bind to KAP1, a gene repressor and ubiquitin E3 ligase which also binds KRAB domain containing zinc finger transcription factors (KZNFs), and MAGE expression may affect KZNF mediated gene regulation. To investigate mechanisms for these effects, we tested the hypothesis that differences in KRAB domain composition affect KZNF poly-ubiquitination and determine whether MAGE expression increases, decreases, or has no effect on KZNFs mediated gene repression. Using an integrated reporter gene responsive to repression by KRAB domain fusion proteins, we found that MAGE-A3 relieved KZNF mediated repression and induced KZNF poly-ubiquitination and degradation in association with expression of the A+B box KRAB domain. In contrast, MAGE-A3 enhanced KAP1 mediated repression of KZNFs expressing A or A+b box KRAB domains but caused no increase in poly-ubiquitination or degradation. MAGE-A3 has no significant impact on KZNFs with KRAB domains containing the Scan box motif. These data support our hypothesis by showing that the effects of MAGE-A3 on gene repression depend on the type of KZNF KRAB domain involved.

Dual inhibition of phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin signaling in human nonsmall cell lung cancer cells by a dietary flavonoid fisetin.

Lung cancer is one of the most commonly occurring malignancies. It has been reported that mammalian target of rapamycin (mTOR) is phosphorylated in lung cancer and its activation was more frequent in tumors with overexpression of phosphatidylinositol 3-kinase (PI3K)/Akt. Therefore, dual inhibitors of PI3K/Akt and mTOR signaling could be valuable agents for treating lung cancer. In the present study, we show that fisetin, a dietary tetrahydroxyflavone inhibits cell growth with the concomitant suppression of PI3K/Akt and mTOR signaling in human nonsmall cell lung cancer (NSCLC) cells. Using autodock 4, we found that fisetin physically interacts with the mTOR complex at two sites. Fisetin treatment was also found to reduce the formation of A549 cell colonies in a dose-dependent manner. Treatment of cells with fisetin caused decrease in the protein expression of PI3K (p85 and p110), inhibition of phosphorylation of Akt, mTOR, p70S6K1, eIF-4E and 4E-BP1. Fisetin-treated cells also exhibited dose-dependent inhibition of the constituents of mTOR signaling complex such as Rictor, Raptor, GßL and PRAS40. There was an increase in the phosphorylation of AMPKα and a decrease in the phosphorylation of TSC2 on treatment of cells with fisetin. We also found that treatment of cells with mTOR inhibitor rapamycin and mTOR-siRNA caused decrease in phosphorylation of mTOR and its target proteins which were further downregulated on treatment with fisetin, suggesting that these effects are mediated in part, through mTOR signaling. Our results show that fisetin suppressed PI3K/Akt and mTOR signaling in NSCLC cells and thus, could be developed as a chemotherapeutic agent against human lung cancer.

Disruption of androgen and estrogen receptor activity in prostate cancer by a novel dietary diterpene carnosol: implications for chemoprevention.

Emerging data are suggesting that estrogens, in addition to androgens, may also be contributing to the development of prostate cancer (PCa). In view of this notion, agents that target estrogens, in addition to androgens, may be a novel approach for PCa chemoprevention and treatment. Thus, the identification and development of nontoxic dietary agents capable of disrupting androgen receptor (AR) in addition to estrogen receptor (ER) could be extremely useful in the management of PCa. Through molecular modeling, we found that carnosol, a dietary diterpene, fits within the ligand-binding domain of both AR and ER-alpha. Using a time-resolved fluorescence resonance energy transfer assay, we found that carnosol interacts with both AR and ER-alpha and additional experiments confirmed that it functions as a receptor antagonist with no agonist effects. LNCaP, 22Rv1, and MCF7 cells treated with carnosol (20-40 mumol/L) showed decreased protein expression of AR and ER-alpha. Oral administration of carnosol at 30 mg/kg 5 days weekly for 28 days to 22Rv1 PCa xenografted mice suppressed tumor growth by 36% (P = 0.028) and was associated with a decrease in serum prostate-specific antigen by 26% (P = 0.0042). These properties make carnosol unique to any known antiandrogen or antiestrogen investigated thus far for the simultaneous disruption of AR and ER-alpha. We suggest that carnosol may be developed or chemically modified through more rigorous structure-activity relationship studies for a new class of investigational agents-a dual AR/ER modulator.

Fisetin induces autophagic cell death through suppression of mTOR signaling pathway in prostate cancer cells.

The mammalian target of rapamycin (mTOR) kinase is an important component of PTEN/PI3K/Akt signaling pathway, which is frequently deregulated in prostate cancer (CaP). Recent studies suggest that targeting PTEN/PI3K/Akt and mTOR signaling pathway could be an effective strategy for the treatment of hormone refractory CaP. Here, we show that the treatment of androgen-independent and PTEN-negative human CaP PC3 cells with fisetin, a dietary flavonoid, resulted in inhibition of mTOR kinase signaling pathway. Treatment of cells with fisetin inhibited mTOR activity and downregulated Raptor, Rictor, PRAS40 and GbetaL that resulted in loss of mTOR complexes (mTORC)1/2 formation. Fisetin also activated the mTOR repressor TSC2 through inhibition of Akt and activation of AMPK. Fisetin-mediated inhibition of mTOR resulted in hypophosphorylation of 4EBP1 and suppression of Cap-dependent translation. We also found that fisetin treatment leads to induction of autophagic-programmed cell death rather than cytoprotective autophagy as shown by small interfering RNA Beclin1-knockdown and autophagy inhibitor. Taken together, we provide evidence that fisetin functions as a dual inhibitor of mTORC1/2 signaling leading to inhibition of Cap-dependent translation and induction of autophagic cell death in PC3 cells. These results suggest that fisetin could be a useful chemotherapeutic agent in treatment of hormone refractory CaP.

Lupeol inhibits proliferation of human prostate cancer cells by targeting beta-catenin signaling.

Lupeol, a dietary triterpene, was shown to decrease serum prostate-specific antigen levels and inhibit the tumorigenicity of prostate cancer (CaP) cells in vivo. Here, we show that Lupeol inhibits the proliferative potential of CaP cells and delineated its mechanism of action. Employing a focused microarray of human CaP-associated genes, we found that Lupeol significantly modulates the expression level of genes such as ERBB2, tissue inhibitor of metalloproteinases-3, cyclin D1 and matrix metalloproteinase (MMP)-2 that are known to be associated with proliferation and survival. A common feature of these genes is that all of them are known to either regulate or act as downstream target of beta-catenin signaling that is highly aberrant in CaP patients. Lupeol treatment significantly (1) reduced levels of beta-catenin in the cytoplasmic and nuclear fractions, (2) modulated expression levels of glycogen synthase kinase 3 beta (GSK3beta)-axin complex (regulator of beta-catenin stability), (3) decreased the expression level and enzymatic activity of MMP-2 (downstream target of beta-catenin), (4) reduced the transcriptional activation of T Cell Factor (TCF) responsive element (marker for beta-catenin signaling) in pTK-TCF-Luc-transfected cells and (5) decreased the transcriptional activation of MMP-2 gene in pGL2-MMP-2-Luc-transfected cells. Effects of Lupeol treatment on beta-catenin degradation were significantly reduced in CaP cells where axin is knocked down through small interfering RNA transfection and GSK3beta activity is blocked. Collectively, these data suggest the multitarget efficacy of Lupeol on beta-catenin-signaling network thus resulting in the inhibition CaP cell proliferation. We suggest that Lupeol could be developed as an agent for chemoprevention as well as chemotherapy of human CaP.

A plant flavonoid fisetin induces apoptosis in colon cancer cells by inhibition of COX2 and Wnt/EGFR/NF-kappaB-signaling pathways.

Overexpression of cyclooxygenase 2 (COX2) and uncontrolled wingless and Int (Wnt)-signaling pathway have long been suggested to play crucial roles in colorectal cancer. Studies show that selective COX2 inhibitors possess great potential as chemopreventive agents for colon cancer. Recent studies suggest that targeting COX2 and epidermal growth factor receptor (EGFR) may provide better therapeutic strategy than inhibiting either single target and that this may alleviate the problem of COX2 inhibitor-associated side effects. Therefore, there have been intensive efforts to develop novel dietary substances that target COX2 and EGFR activation. Fisetin is a naturally occurring flavonoid commonly found in various vegetables and fruits. We found that the treatment of COX2-overexpressing HT29 human colon cancer cells with fisetin (30-120 microM) resulted in induction of apoptosis, downregulation of COX2 protein expression without affecting COX1 and inhibited the secretion of prostaglandin E2. Treatment of cells with fisetin also inhibited Wnt-signaling activity through downregulation of beta-catenin and T cell factor 4 and decreased the expression of target genes such as cyclin D1 and matrix metalloproteinase 7. Fisetin treatment of cells also inhibited the activation of EGFR and nuclear factor-kappa B (NF-kappaB). Finally, the formation of colonies in soft agar was suppressed by fisetin treatment. Taken together, we provide evidence that the plant flavonoid fisetin can induce apoptosis and suppress the growth of colon cancer cells by inhibition of COX2- and Wnt/EGFR/NF-kappaB-signaling pathways. We suggest that fisetin could be a useful agent for prevention and treatment of colon cancer.

Lupeol inhibits growth of highly aggressive human metastatic melanoma cells in vitro and in vivo by inducing apoptosis.

PURPOSE: Poor prognosis of metastatic melanoma mandates the development of novel strategies for its treatment and prevention. In this study, the effect of lupeol, a diet-based triterpene, was determined on the growth and tumorigenicity of human melanoma cells in vitro and in vivo. EXPERIMENTAL DESIGN: Normal human melanocytes, and human metastatic (451Lu) and nonmetastatic (WM35) cells were treated with lupeol; its effect on growth, proliferation, and apoptosis were evaluated. Further athymic nude mice bearing 451Lu cell-originated tumors were administered with lupeol thrice a week, and its effect on tumor growth and surrogate biomarkers was evaluated. RESULTS: Lupeol significantly decreased the viability of 451Lu and WM35 melanoma cells but had only a marginal effect on normal human melanocyte cells at similar doses. Lupeol treatment of 451Lu cells caused (a) G(1)-S phase cell cycle arrest and apoptosis; (b) down-regulation of Bcl2 and up-regulation of Bax; (c) activation of caspase-3 and induction of poly(ADP)ribose polymerase cleavage; (d) decreased expression of cyclin D1, cyclin D2, and cdk2; and (e) increased expression of p21 protein. Next, lupeol significantly reduced 451Lu tumor growth in athymic nude mice and modulated the expression of proliferation markers, apoptotic markers, and cell cycle regulatory molecules in tumor xenografts. CONCLUSION: Our findings showed the anticancer efficacy of lupeol with mechanistic rationale against metastatic human melanoma cells. We suggest that lupeol, alone or as an adjuvant to current therapies, could be useful for the management of human melanoma.

Dietary agents for chemoprevention of prostate cancer.

Prostate cancer (CaP) is the leading cause of cancer-related deaths in American men, responsible for over 29,000 deaths in the year 2007. Chemoprevention is a plausible and cost-effective approach to reduce cancer morbidity and mortality through inhibition of precancerous events before the occurrence of clinical disease. Indeed, CaP is an ideal candidate disease for chemopreventive intervention as it is typically diagnosed in the elderly population with a relatively slower rate of growth and progression. The potential of dietary substances to act as chemopreventive agents against CaP is increasingly appreciated. Further, epidemiological studies have identified significant correlations between CaP incidence and dietary habits. It is hoped that, combining the knowledge based on agents with targets, we will be able to build an armamentarium of naturally occurring chemopreventive substances that could prevent or slow down the development and progression of CaP. In this review, we have summarized the findings from clinical and preclinical studies on dietary agents including green tea, pomegranate, lupeol, fisetin, and delphinidin that are currently being investigated in our laboratory for their chemopreventive potential against CaP.

Carnosol, a dietary diterpene, displays growth inhibitory effects in human prostate cancer PC3 cells leading to G2-phase cell cycle arrest and targets the 5'-AMP-activated protein kinase (AMPK) pathway.

PURPOSE: This study examines the anti-cancer effect of carnosol in human prostate cancer PC3 cells and its role in modulating multiple signaling pathways associated with carcinogenesis. METHODS: PC3 cells were treated with carnosol and were evaluated using a flow cytometry, a protein array and Western blot analysis to identify signaling pathways targeted by carnosol. RESULTS: Using an MTT assay we found that carnosol (10-70 microM) decreases cell viability in a time and dose-dependent manner. Further analysis using flow cytometry as well as biochemical analysis identified G2-phase cell cycle arrest. To establish a more precise mechanism, we performed a protein array that evaluated 638 proteins involved in cell signaling pathways. The protein array identified 5'-AMP-activated protein kinase (AMPK), a serine/threonine protein kinase involved in the regulation of cellular energy balance as a potential target. Further downstream effects consistent with cancer inhibition included the modulation of the mTOR/HSP70S6k/4E-BP1 pathway. Additionally, we found that carnosol targeted the PI3K/Akt pathway in a dose dependent manner. CONCLUSIONS: These results suggest that carnosol targets multiple signaling pathways that include the AMPK pathway. The ability of carnosol to inhibit prostate cancer in vitro suggests carnosol may be a novel agent for the management of PCa.

Isoelectric point-based prefractionation of proteins from crude biological samples prior to two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pI) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in the presence or absence of epigallocatechin-3-gallate was achieved in fewer than 30 min using an anion-exchange resin and two expressly designed buffers. The procedure was carried out in a centrifuge tube and standard instrumentation was used. The cell lysates were prefractionated into two fractions: proteins with pI values above 7 and between 4 and 7, respectively. The fractions were then analyzed by 2-DE, selecting appropriate pH ranges for the IPG strips, and the gels were compared with those of unprefractionated cell lysates. Protein loading capacity was optimized and resolution and visualization of the less abundant and differentially expressed proteins were greatly improved.

Autolytic processing at Glu586-Ser587 within the cysteine-rich domain of human adamalysin 19/disintegrin-metalloproteinase 19 is necessary for its proteolytic activity.

We investigated the regulation of the proteolytic activity of human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19). It was processed at Glu(586)(P1)-Ser(587)(P1') site in the cysteine-rich domain as shown by protein N-terminal sequencing. This truncation was autolytic as illustrated by its R199A/R200A or E346A mutation that prevented the zymogen activation by furin or abolished the catalytic activity. Reagents that block furin-mediated activation of pro-hADAM19, decRVKR-CMK, and brefeldin A abrogated this processing. The sizes of the side chains of the P1 and P1' residues are critical for the processing of hADAM19. The amount of processing product in the E586Q or S587A mutant with a side chain almost the same size as that in the wild type was almost equal. Conversely, very little processing was observed when the size of the side chain was changed significantly, such as in the E586A, E586G, or S587F mutants. Two mutants with presumably subtle structural distinctions from wild type hADAM19, E586D and S587T, displayed rare or little processing and had very low capacities to cleave alpha2-macroglobulin and a peptide substrate. Therefore, this processing is necessary for hADAM19 to exert its proteolytic activities. Moreover, a new peptide substrate, Ac-RPLE-SNAV, which is identical to the processing site sequence, was cleaved at the E-S bond by soluble hADAM19 containing the catalytic and disintegrin domains. This enzyme cleaved the substrate with K(m), k(cat), and k(cat)/K(m) of 2.0 mm, 2.4/min, and 1200 m(-1) min(-1), respectively, using a fluorescamine assay. Preliminary studies showed that a protein kinase C activator, phorbol 12-myristate 13-acetate, promoted the cellular processing of hADAM19; however, three calmodulin antagonists, trifluoperazine, W7, and calmidazolium, impaired this cleavage, indicating complex signal pathways may be involved in the processing.