Healthy non-tumorigenic, MCF-10A and breast cancer, MDA-MB-231, Hs578T, T47D and MCF-7 cell lines were electrically characterized using the open-ended coaxial probe technique from 200 MHz to 13.6 GHz. The dielectric constant, dielectric loss and conductivity between breast non-tumorigenic and breast cancer cells lines were analyzed and their differences determined. Our results showed that the four breast cancer cell lines analyzed exhibited higher dielectric properties when compared to healthy cells. Interestingly, we found that breast and colon cancer cells have different dielectric properties as well, thus suggesting that each type of cancer has a unique microwave signature. This study shows that microwave characterization of breast cancer cell lines is reliable with potential in biomedical applications such as designing electromagnetic models for detection of tumorous cells in healthy tissues.Here we investigated the feasibility of using microwave spectroscopy for characterization of normal and breast cancer cell lines cultured  Cancer originates when certain genetic abnormalities arise in a cell or group of cells. Such cells do not respond to normal signaling system of the body and hence divide and multiply uncontrollably forming bulk cancerous tissues. Cancer can be classified depending on the type of cell in which they originate. Breast, lung and colorectal cancers are the most common cancer types with highest incidence rates worldwide.2. The larger dielectric properties of cancerous tissues arise mainly from its increased water content and can contribute to the increased scattering of microwaves. An extensive amount of studies has been carried out to electrically characterize living tissues using microwave spectroscopy techniques17. Some of the earliest dielectric studies using open ended coaxial probe method were performed on animal tissues. Joines et al. measured electrical properties of rat tissues from 30 MHz to 2 GHz3, whereas Stuchly et al. characterized ex vivo cat tissue samples from 0.01 to 1 GHz4. The dielectric studies were later extended to human tissues. Joines et al. reported dielectric characterization of different types of human tissues ex vivo from 50 to 900 MHz5. It was estimated that the contrast in permittivity and conductivity between malignant tissues and normal tissues was 6.4:1 and 3.8:1, respectively. Lazebnik et al. performed the most extensive study to characterize breast tissues from 0.5 to 2 GHz12. His experiments unveiled the variation of breast tissue dielectric properties with respect to breast heterogeneity and adipose content. Martellosio et al. successfully evaluated breast tissue dielectric properties over a broad frequency range up to 50 GHz17.The increased incidence of cancer is a subject of major concern worldwide. Timely diagnosis of cancer can help in more effective treatment and reduction in mortality rate. Microwave based cancer diagnosis and treatment is one of the most relevant areas of research nowadays. Such research stems from the basic knowledge about the considerable contrast in the dielectric parameters of normal and cancerous tissues18. Cancer cell lines are frequently used as in vitro models of cancer and serve as powerful tools in learning cancer biology and testing cancer treatments. The main advantage of using cell lines is that when grown in a standard medium, they provide an unlimited supply of self-replicating homogeneous cell population. To a good extent, cell lines preserve the genomic properties of the original tissues from which they have been derived21. The degrees to which the cell lines represent their parent tissues are still being investigated. In this paper, we have studied the present the electrical signatures of different cancer cell lines dielectric properties of breast cell lines and determined the difference in electrical properties of normal and cancerous breast cells.In our studies, we experimented a different approach in which dielectric measurements are performed on cultured cell lines as reported inHuman breast cancer cells MDA-MB-231(HTB-26), Hs578T (HTB126), normal mammary epithelial cell MCF-10A (CRL-10317) and colon cancer cell line HT-29 were obtained from ATCC. Human breast cancer cells MCF-7 (300273) and T47D (300353) were obtained from Cell line service (CLS)-GmbH. MDA-MB-231, MCF-7 and HT-29 cells were maintained in DMEM  complemented with 10% fetal bovine serum (FBS). T47D cells were maintained in RPMI  complemented with 10% fetal bovine serum (FBS). MCF-10A were maintained in DMEM-F12 (In vitrogen) complemented with 15% FBS and 20 ng/mL human EGF recombinant protein (Sigma-Aldrich). All media were supplemented , 100 U/ml penicillin/streptomycin .5 cells/well and placed in 37 °C incubator with 5% CO2 for 24 hours to allow cells to form homogeneous confluent monolayer. Prior to measurement, cells were washed three times in phosphate-buffered saline solution (PBS) at room temperature and then resuspended in 1 ml cell culture media.For sample preparation, cells were seeded in triplicate in 12-well plates at a density of 4 × 10The open-ended coaxial probe (DAK-3.5 Dielectric Probe) developed by Speag was employed to determine the dielectric properties of cell line samples. An R&S®ZVL vector network analyzer 9 KHz to 13.6 GHz) and a Dielectric Assessment Kit software (DAK-3.5) were the key components of the measuring set up  to be measured was kept under the probe in such a way that the probe tip was slightly immersed in it without any air bubbles on the probe surface, and the probe kept at very close proximity from the base of the cell line plate, at about 0.75 mm. The prepared sets of samples were analyzed with and without culture media. For analysis without media, the solution was carefully removed by means of a sterile pipette. To avoid any contamination, the probe was wiped with ethanol in between measurements. It was also ensured that the probe or the cable was not moved during the measurements so as to avoid any kind of distortions in the amplitude and phase of the reflected waves through the cable. All measurements were acquired at room temperature in the 200 MHz-13.6 GHz frequency band. VNA computes the reflection coefficients at the probe tip and the DAK software converts it into dielectric parameters.ε′ is its real part representing the energy stored and ε″ is its imaginary part representing losses.ω is the angular frequency and εo is the free space permittivity.Microwave dielectric characterization by open ended coaxial cable method serves as a low cost, easy and fast technique to identify the dielectric properties of biological samples. The way biological samples interact with microwave radiation is governed by its dielectric properties, which in turn are the result of cellular or molecular level chemical constituents of the samples. The interaction of microwaves with biological matter is attributed to two different processes: energy storage caused by dipole polarization in response to external applied field and energy dissipation when the dipoles do not align instantly with varying field. The dielectric properties are therefore represented in terms of complex relative permittivity as23:fr is the relaxation frequency at which the dipole molecules fail to align with the field resulting in larger variations in real and imaginary parts of permittivity. At this particular frequency, real part tends to decrease and imaginary part shows a maximum value.Water, a polar molecule being the major chemical constituent of biological tissues contributes mainly to the interaction with electromagnetic radiation. The dielectric behavior of water molecules can be reasonably approximated using the well-known Debye relationIn addition to water molecules, cells contain other species such as free ions, carbohydrates, proteins and other organic chemical compounds. Ionic species contribute to energy dissipation and hence their behavior can be explained by adding an additional term in Equation  as5\docuSimilarly, the other species are also polar in nature and behave differently in an electric field. The effect of these species can be modeled by adding to these equations, appropriate parameters specific to their behavior.Biological cell characterization using microwaves is indeed more reliable as the waves with these frequencies completely penetrate the cell membranes, leading to accurate identification of the dielectric nature of intra cellular contents.p < 0.05, **p < 0.005 and ***p < 0.001).Statistical analysis were done using SPSS version 21 software. All statistical analysis were performed using one-way ANOVA test followed by Tukey’s HSD test. Significance for all statistical comparisons was set at p < 0.05  and two less invasive and metastatic (T47D and MCF-7) breast cancer cell lines along with the non-tumorigenic breast cell line (MCF-10A) have been characterized 16. The α-dispersion falls in the lower frequency range (kHz range) and is related to ionic diffusions at cell membranes. The β-dispersion which arises from cell membrane polarization occurs at the medium frequency range (lower MHz range). Finally, the With three replicates per sample, a total of 39 readings per experiment were taken from the samples including the culture medium. Experiment was repeated three times at different days and with freshly thawed cells each time. The dielectric data corresponding to each sample were analyzed and averaged. The permittivity and conductivity values of the five cell lines evaluated with and without the culture media are shown in Figs In this study, β-dispersion and most of the ε′ = 50 at 13.6 GHz. This type of behavior clearly indicates the high cytoplasm conductivity of the cell upon wave penetration. Hence, each cancer cell line can be differentiated based on their respective inflection point and dielectric responses. The point of inflection representing the frequency at which change in the behavior of each cell line is noted was 6.0, 4.7, 4.7 and 4.3 GHz for T47D, MCF-7, MDA-MB-231 and Hs578t cells, respectively. Dielectric constant of the non-tumorigenic cell line MCF-10 A, on the other hand, had much lower value, did not show inflection point and varied over the measurement spectrum at a relatively lower rate than that of cancer cell lines. Also, dielectric loss as well as conductivity exhibited a similar trend with respect to cancer cell line type but showed larger values to increase in frequency except for the non-tumorigenic cell line the values of which decreased at higher frequencies. As it is shown in Table 25. The frequency variation of dielectric parameters follows the Cole–Cole pattern23. The interactions of biological cells with microwaves are translated to relaxations and resonances of dielectric parameters. Our results are consistent with other published results for bulk tissues13.Between 4–7 GHz, the dielectric response was dominated by broad asymmetric peaks (points of inflection) in the spectra of dielectric constants of cancer cell lines. These inflection points denoted critical transition points after which the dielectric response reversed its trend, with T47D showing the highest value followed by MCF-7, MDA-MB-231 and Hs578T. In the On the other hand, in the absence of culture medium, cancer cell lines displayed lower dielectric values varying at a slower rate over the measurement frequency spectrum. The non-tumorigenic cell line exhibited the least dielectric response with slight or no variation with respect to frequency. Based on the results obtained from cell lines without the media, the contrast in the dielectric constant and conductivity of cancerous and normal cell lines was determined. The dielectric and conductivity ratio, at three specific frequencies, between normal and cancerous cells are summarized in Table Next, we analyzed the dielectric properties of the human colon cancer cell line HT-29. Figure In this study, we determined the dielectric response of breast and colon cell lines. The comparison of dielectric properties of breast cell lines revealed a significant dielectric contrast between normal and cancer cells. The dielectric response exhibited relaxations and resonances, which can be attributed to the dielectric relaxations of cells. Our analysis shows that microwave characterization of cell lines is reliable and has a strong potential in biomedical applications like cancer detection and designing electromagnetic models of healthy and tumorous tissues.


Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1–3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere‐like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved  when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer. The pig, in particular, conveys great similarity to humans in their anatomy, physiology, and genomics, therefore, allowing them to exhibit symptoms of human pathologies more accurately and reliably than other animal models  between hypoxic (HYP) and control (CON) cultured fibroblast donors and was not different amongst cell lines (p = 0.44). While the total proportion of embryos cleaved was not different between donor fibroblast oxygen culture treatments (p = 0.75), HYP reconstructed clones had a higher (p = 0.03) proportion cleave earlier (within the first 24 hr) compared to control in both experiments  however some of these measures were lower in the cell line used for preliminary in vitro experiments (p < 0.01).Fusion of reconstructed embryos was not significantly different p = 0.0 betweenp < 0.01; Table p < 0.0001; See Table p ≤ 0.01; 24%) and similar amongst the other cell lines used . In utero, the homozygous KO line had lower survival than the heterozygous KO line  but not different from the Ossabaw line . Ossabaw and the heterozygous KO line did not have significantly different survival in utero (p = 0.05). From 446 embryos transferred into 12 surrogates, 6 surrogates became pregnant carrying 20 HYP and 9 CON fetuses  and cell lines . Survival probability was increased when clones were from HYP versus CON donors , epigenetic reprogramming, degree of pluripotency, and antioxidant treatment of cells  were cultured under hypoxic conditions the metabolism of the donor cells would become more blastomere‐like and thus it might improve reprogramming and subsequent cloning efficiency. Therefore we designed these experiments to test our hypothesis and see if this mechanism (hypoxic culture) would be effective and worth pursuing further. It was desirable that this ‘reprogramming’ induction be through a simple mechanism that could be applied to any cell line, such as altering the metabolism during culture via hypoxia. Previously our laboratory reported that oxygen restriction in porcine fetal fibroblasts (the same line used in in vitro experiments in the current study) induced expression of In this study, we demonstrate that measures of in vitro development were improved when fibroblasts cultured in hypoxia were used as donors for SCNT. Clones from HYP fibroblasts consistently cleaved earlier and produced more blastocysts with higher cell numbers Table , 2 regarThe improvement in blastocyst production from HYP donors is modest (5.5%) however this translated to a 1.5‐fold increase in cell number and 2.1‐fold increase in the probability of survival (fetuses attained). Overall these changes lead to a 2.5‐fold increase in system efficiency . In this study, our Day 35 pregnancy rate was 50% (6 of 12 surrogate gilts became pregnant) which is higher than previous experiments in our laboratory involving the same personnel . The homozygous SIGLEC1 KO line produced few Day 5 blastocysts, limiting the number of embryos we could equally transfer from each treatment. Of the two replicates, two of the four gilts became pregnant and whereas they were not transferred on the same day, between the two gilts all combinations of the homozygous and heterozygous KO lines and donor treatments were represented. Due to the decreased blastocyst production however we decided to use a different cell line (Ossabaw) in combination with the SIGLEC1 heterozygous KO line, a line proven to be ‘clonable’ and yield live animals which has been used for editing genes by the National Swine Research and Resource Center . According to another study from Kuhholzer, Hawley, Lai, Kolber‐Simonds, and Prather, . In this study, in similar terms, of the 223 embryos from HYP donors, there were 20 fetuses (~9%); the caveat being future studies must be conducted which carry HYP clones to term to ascertain this rate of efficiency holds true. However, at the present, we thought that these results were novel and worth reporting to the scientific community to open further exploration into mechanisms and effectiveness of hypoxic culture on donor cells. From these experiments, we propose that the clones from donor cells cultured in hypoxia may be developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer. Implications of our results may be a method for improved efficiency for cloning of pigs.4Chemicals and materials were purchased from Sigma‐Aldrich, St. Louis, MO unless otherwise specified.4.1All procedures performed in studies involving animals were approved and conducted in accordance with the ethical standards of the University of Missouri Institutional Animal Care and Use Committee at the University of Missouri in Columbia, Missouri. This study does not contain any experiments with human participants.4.2Porcine fetal fibroblast cell lines collected from dorsal regions  used in the study were established from d 35 pregnancies and utilized in previous experiments in our laboratory , a system of four nitrogen tanks connected by one gas line was assembled to ensure adequate nitrogen gas was available to incubators. The regulators of nitrogen tanks were set to sequentially empty the tanks one by one as culture experiments required a large quantity of nitrogen gas, and at least one of the tanks had to be exchanged for a full tank daily. During this, oxygen concentration of incubators was regularly monitored by a handheld gas monitor  which was professionally tested by Utility Lab Services  and proved to have an error of ±0.05% oxygen. Therefore the low oxygen treatment may have been as high as 1.25% or as low as 1.15% oxygen. Incubators were maintained at 38.5°C with a humidified atmosphere of 5.5% carbon dioxide during experiments.In experiments where the modular incubator chamber  was used (four replicates of embryo transfer experiments), petri dishes of sterile water were placed in the bottom of the chamber below the grate to provide humidity. Vented cell culture flasks were placed inside chambers, which were then sealed and flooded with mixed gas for several minutes to displace gas within and achieve hypoxic conditions. Afterward, air ports were sealed and chambers were placed in the standard incubators with controls.g) and either diluted and replated (Day 5) or approximately 2,000 cells/treatment were collected and placed in droplets to be used for selection during SCNT (Day 7).Cells from all treatments were plated at the same initial densities between treatments and either passaged on Day 5 of growth and plated at the same density (if needed) or had gas‐equilibrated media changed and allowed to grow 7 days consecutively. Passaging was done quickly, and dissociation reagents were kept cold in an effort to decrease the metabolism of cells and prevent the metabolism of oxygen as much as possible. Media that cells were plated in had been equilibrated to desired oxygen concentrations by incubating media in the incubators in petri dishes or within modular incubator chambers for an hour or more. At passaging, flasks were briefly rinsed with phosphate buffered saline (PBS) + 0.01 M ethylenediaminetetraacetic acid, and fibroblast cells were dissociated from flasks by brief incubation (37°C) with 1× TrypLE Express . Fibroblasts were pelleted  by two DC pulses  at 1.2 kV/cm for 30 µsec using a BTX Electro Cell Manipulator . After electric pulse fusion, fused embryos were also chemically activated with 200 µM thimerosal (in 1× PVA‐TL Hepes) for 10 min in the dark and 8 mM dithiothreitol (in 1× PVA‐TL Hepes) for 30 min  were surgically transferred into the oviductal ampullary‐isthmic junction of surrogate gilt recipients (3–5 days postobserved estrus). Surgical embryo transfer technique was conducted as previously established within our laboratory  at 35 of embryonic development. The reproductive tract was excised from the cervix to ovaries and opened along the entire length of uterine horns for fetal collection. Tissue was excised from fetuses and washed in 1× PBS then frozen until DNA extraction and genotyping. To establish which donor cell treatment survived better in utero, fetuses were genotyped Figure  via poly et al., . When ba4.6p < 0.05.For in vitro developmental measures, nine replicates  were used in analyses. Three replicates of the same donor cell line were used to compare cell number within blastocysts for donor cell oxygen culture treatments . Embryo transfer data consisted of 446 total blastocysts transferred into 12 surrogates (6 replicates of surrogate pairs). Data distributions and Shapiro–Wilk test of normality values were generated using the Univariate procedure of SAS ; log or square root transformations were made before statistical analysis where necessary to meet assumptions for analyses. All data were analyzed using a MIXED procedure in SAS for main effects of donor cell treatment and cell line; interactions were assessed however this effect was removed from analyses when found to be insignificant. For embryo transfer data, blastocysts transferred were nested within surrogate gilt and a random effect of replicate was used (mainly because it corresponds to the time/season differences for this study duration). Survival probability was considered the number of favorable outcomes (viable fetuses) from the total (embryos transferred). Analyzed variables were considered statistically different if the The authors declare that there are no conflict of interests.


Cystoisospora was created in 1977  cells  cells . Developexamined . DevelopC. canis was examined in MDCK cells and primary cells from embryonic canine kidneys, and embryonic canine intestine as well as embryonic bovine trachea and kidney (C. canis sporozoites using bovine turbinate and African green monkey kidney (CV-1) cells provided remarkedly different findings of no asexual multiplication but rather MZTC formation in both host cell types (C. belli MZTC in the tissues of humans (C. canis in mice (C. ohioensis in mice (C. felis in mice (C. canis using two canine, four human, one monkey, and one bovine cell lines (C. canis isolates by different research groups. It suggests that two different genetic types of C. canis exist and that one divides by endodyogeny in culture and the other produces MZTC. How this would translate to in vivo development and pathogenicity in dogs is also an interesting area needing research.Development of sporozoites of d kidney . Developll types . Examinaf humans , 18, C.  in mice , C. ohio in mice . The orgll lines . No asexll lines  and Houkll lines  with drall lines . AdditioC. suis in five types of mammalian host cells in culture indicated that development was by endodyogeny (C. suis development of Type I meronts by endodyogeny in these cell cultures (in vitro (C. suis was observed in a swine testicular cell line (The initial studies on the development of odyogeny  and stagodyogeny . Similarodyogeny . Howeverodyogeny . The ultcultures  was examcultures . Notablyin vitro . Completell line . Developell line . Oocystsell line . The stuell line .C. suis conforms to all other reported features of the genus.No evidence of MZTC formation has been reported in cell cultures. Similarly, studies in pigs and potential paratenic hosts have been negative , 29. ThiC. belli in human and other mammalian cell cultures (C. belli as are present in C. suis (in vitro but otherwise the structural findings were similar to those reported for C. suis. Stages of C. belli obtained from macrophage cell cultures were infectious for RMK cells upon subinoculation and development by endodyogeny occurred (Four studies have examined development of sporozoites obtained from oocysts of cultures –33. The cultures . The aut C. suis  while unoccurred  but MZTCCystoisospora species from mammals and to provide insight into future research directions. Development will occur in many host cell types but studies with C. suis illustrate that selection of the proper host cell type can lead to complete development thus increasing the utility of an in vitro system. Studies with C. canis suggest that genetic differences in Cystoisospora species exist that are reflected by the phenotype of development expressed in vitro. I look forward to seeing what advances in our knowledge of this group are revealed in the future.I have attempted to point out the similarities and important differences in the development of The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


G12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis  revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells.In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the Kras Pancreatic cancer (PaCa) is the fourth leading cause of all cancer death in the United States of America and Germany. Smoking, chronic pancreatitis, heavy alcohol consumption or genetic predispositions, such as Lynch syndrome, are only a small variety of risk factors. Common symptoms, including weight loss, abdominal or back pain, mean that identification during the early stages can be difficult. With its disastrous five-year survival rate of about 8%, the chances of survival after diagnosis are remote. Besides chemotherapy or radiation therapy as therapeutic options to alleviate pain or extend survival, the only potentially curative prospect remains resection ,2,3,4. TIn about 90% of PaCa cases, a Kras mutation is detectable . Kras isIn recent years, whole-genome sequencing led to the discovery of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and CRISPR-associated (Cas) genes in various bacteria and archaea . The ideG12D (c.35G>A) mutation. After transfection, puromycin selection and single-cell cloning, we produced about five to seven clones (Kras clones). Western blotting was performed to detect the knock-out effects introduced with the CRISPR/Cas9 gene editing system. In all tested cell lines, the results showed a knock-out with no protein expression of mutant protein KrasG12D. However, a normal total Kras protein expression level was observed for Panc-1 wildtype (WT) cell lines, negative controls  and Kras clones. In the SUIT-2 cell line, WT and Kras clones resembled each other, whereas the protein expression in the negative controls was higher. Total Kras expression in the TB32047 clones was lower than in the WT and negative controls  and a murine cell line (TB32047) were used to perform a CRISPR/Cas9-based knock-out of the mutated allele. All cell lines contained a heterozygous KRAScontrols .G12D. All knock-out clones had indel mutations in the sequence of KrasG12D. Most clones  had small indels (< 20 bp), while Panc-1 clones 2.7 and 2.14 had single-nucleotide insertions. Only three clones, one from each cell line , had large indels (>20 bp)  see .G12D protein knock-out, the effect on key signal transduction pathways was analyzed. Specifically, the (non-)phosphorylated proteins Erk, Akt, Stat3, AMPKα and c-myc were evaluated . Approximately half of the DEGs (218) were up-regulated, while the other half (199) were down-regulated (see Lamb1 (log2 fold-change = −5) and Slc27a6  data from eight TB32047 samples  were analyzed to determine RNA expression changes in the Krasples see A. A totaples see . PrincipPC1, see B. In addated see C. Among As part of the G-protein family, Kras is involved in the regulation of cell proliferation and survival. Inactivation of its GTPase activity, as a result of mutation in the protein, leads to hyperactive effector signaling. Therefore, mutated Kras plays a major role in and is the driver of PaCa initiation. Mouse models have shown that an elevated frequency of activation in Kras leads to precursor lesions and the onset of PaCa ,18,19. HG12D using the CRISPR/Cas9 gene editing system in all of the three analyzed Kras heterozygous cell lines . Interestingly, the cell cultivation showed no apoptosis or growth arrest, only a decreased growth rate compared to the wildtype could be observed. This could be due to knocking-out mutated Kras, which can result in a slower growth ability.We were able to successfully knock-out KrasWe verified the knock-outs using DNA sequencing and western blotting (WB). However, we observed no changes in the expression level of total Kras. These results suggest that Kras is not important for maintaining PaCa cells in vitro .G12D does not affect one common pathway of signal transduction, but that each cell line displays different effects. Others state that PI3K/Akt hyperactivation is important in PaCa cells lacking Kras [Furthermore, we looked for changes in key signaling pathways, such as mitogen-activated protein kinase (MAPK) or PI3K/Akt, which are known to be the major effector pathways of Kras activation. Our work indicates that the knock-out of Krasing Kras . But PanIf the clones are not using MAPK or PI3K/Akt pathway to survive, how can they resist apoptosis? Besides Erk and Akt, we analyzed the signal transduction proteins Stat3, AMPKα and c-myc. Stat3 has an anti-apoptotic effect induced by TGF-beta and is engaged in cell survival, whereas c-myc regulates cell cycle progression, cell growth, and apoptosis . Our resLamb1) and solute carrier family 27, member 6 (Slc27a6). As part of a subunit which assembles to a heterotrimeric isoform, Lamb1 facilitates cell differentiation, motility, adhesion and—as a ligand of the laminin receptor—it enables the ability of tumor cells to invade and build vessels [G12D knock-out these two necessary genes are down-regulated, which could negatively affect intensive angiogenesis, invasion and energy supply and therefore provide a useful tool to combat PaCa.Through RNA sequencing, we gained insight into the changed gene expression of edited TB32047 clones. We identified 417 differentially expressed genes. Two heavily down-regulated genes were Laminin B1 . Panc-1 cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 . The SUIT-2 cell line was purchased from Japanese Collection of Research Bioresources Cell Bank . SUIT-2 cells were cultured in minimum essential medium . The TB32047 cell line was obtained with courtesy from Prof. Dave Tuveson, Cold Spring Harbor Laboratory. TB32047 cells were cultured in Dulbecco’s modified Eagle’s medium . Every culture medium was enriched with a final concentration of 10% fetal bovine serum (FBS) . All cell lines were grown under sterile conditions to avoid contamination. For cultivation the cells were incubated in monolayer culture at 37 °C, 5% CO2 and 80% air humidity. All cells were harvested by treatment with 0.25% trypsin-ethylenediaminetetraacetic acid .The human cell lines Panc-1 and SUIT-2 and the murine cell line TB32047 were used in this study. The Panc-1 cell line was bought from ATCCG12D was knocked out by the CRISPR/Cas9 gene editing system. The following primers were synthesized by Eurofins Genomics: Hs_KRASm_sg1-forward (5′-CACCGTAGTTGGA GCTGATGGCGT-3′), Hs_KRASm_sg1-reverse (5′-AAACACGCCATCAGCTCCAATAC-3′), Hs_KRASm_sg2-forward (5′-CACCGCTTCTGGTAGTTGGAGCTGA-3′), Hs_KRASm_sg2-reverse (5′-AAACTCAGCTCCAACTACCACAAGC-3′). Cloning was performed using pSpCas9(BB)-2A- Puro (PX459) V2.0 vector, which was a gift from Feng Zhang  [® 10G electrocompetent cells . Plasmid construction was performed according to protocol and confirmed by sequencing. The above-mentioned cell lines were transfected with KrasG12D knock-out plasmid by Lipofectamine® 3000 Transfection reagent  for 24 h. After one day the transfected cells were selected with puromycin (concentration 5 ng/μL). Mouse epidermal growth factor (mEGF) was added to the clone culture medium. Thereafter, single-cell cloning was performed in a 96-well plate to grow single clones. After growth, western blot and sequencing were performed to detect the knock-out.In this study, Krase_62988) . The lig® BCA Protein Assay Kit. Gel electrophoresis was performed with Bolt™ 4–12% Bis-Tris Plus Gels  and proteins were transferred to a nitrocellulose membrane. After blocking with 5% milk, the following primary antibodies were used: Akt (pan) , phospho-Akt (Ser 473) , AMPKα , phospho- AMPKα (Thr172) , c-myc , p44/42 MAPK (Erk1/2) , phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) , Ras , Ras (G12D Mutant Specific) , Stat3 , phospho-Stat3 (Tyr705) . β-Actin , glyceraldehyde 3-phosphate dehydrogenase  and Na,K-adenosine triphosphate (ATP)ase  served as loading controls. As secondary antibodies, horseradish peroxidase (HRP)-linked anti-mouse immunoglobulin G (IgG)  and HRP-linked anti-rabbit IgG  were utilized. The signals were detected with an Amersham Imager 600 with SignalFire™ (Elite) ECL Reagent . All western blot assays were performed more than three times to validate the reliability of the results.To analyze the protein expression of the genetically modified cell clones, the cells were lysed using radioimmunoprecipitation assay (RIPA) buffer. The protein concentration was determined photometrically using Pierce® Tissue  was used according to the manufacturer’s protocol. The DNA concentration was determined using the NanoDrop 2000 . PCR products for sequencing were amplified using Platinum™ SuperFi™ PCR Master Mix . PCR fragments were displayed using gel electrophoresis. The following primers were used for the human cell lines: hKras_f1 (AAGCGTCGATGGAGGAGTTT) and hKras_r1 (ACCCTCTCAC GAAACTCTGA); and for the murine cell line Mm_Kras_f1 (ACTCTGTACATCTGTAGTCACTG) and Mm_Kras_r1 (GCACGCAGACTGTAGAGCA) were used. PCR fragments were cloned into pMiniT 2.0 with the NEB® PCR Cloning Kit . For each single cell clone 10 bacteria colonies were picked and plasmid DNA was isolated . Plasmid DNA was sequenced by Eurofins Genomics and the resulting sequences were aligned using Snapgene .To isolate DNA from the cell lines, NucleoSpin® RNA Plus kit  was used to isolate RNA from the cell lines according to the manufacturer’s protocol. The RNA concentration and pureness were determined using a 2100 Bioanalyzer using the Agilent RNA 6000 Nano Kit .The NucleoSpin2 fold-change ≥ 2 or ≤ −2 were considered differentially expressed. Normalized rlog-transformed gene counts were calculated using the DESeq2’s rlog function.Profiling of whole-genome transcriptomic patterns was performed with 500 ng of high-quality total RNA per sample by RNA sequencing at the next generation sequencing (NGS) core unit of the Medical Faculty of the FAU Erlangen. Libraries of the samples were generated using the Illumina stranded mRNA library kit. After sequencing on an Illumina HiSeq-2500 platform, the sequences were assigned to their sample of origin according to the used indices. The quality of reads was assessed using FastQC v0.11.4 . Adapterhttp://david.abcc.ncifcrf.gov/) [Functional and pathway analysis was performed with the Database for Annotation and Integrated Discovery  ,35. In pG12D in human and mouse cell lines. This leads to cell clones that exhibit different aspects of Kras signal transduction.In conclusion, the CRISPR/Cas gene editing system can be used to knock-out Kras


Corticosteroids are the first line therapy for acute graft-versus-host disease (GVHD). However, the outcome of steroid refractory GVHD (SR-GVHD) is poor due to a lack of effective treatments. The development of therapies for SR-GVHD is limited by an incomplete understanding of its pathophysiology partly because of the absence of clinically relevant animal models of SR-GVHD. Here we addressed the need for a SR-GVHD animal model by developing both MHC matched multiple minor histocompatibility antigens (miHAs) mismatched and MHC mismatched haploidentical murine models of SR-GVHD. We demonstrate that animals can develop SR-GVHD regardless of whether steroids are initiated early or late post allogeneic bone marrow transplantation . In general, we observed increased GVHD specific histopathological damage of target organs in SR-GVHD animals relative to steroid responsive animals. Interestingly, we found no significant differences in donor T cell characteristics between steroid refractory and responsive animals suggesting that donor T cell independent mechanisms may play more prominent roles in the pathogenesis of SR-GVHD than was considered previously. Many agents2 have shown encouraging phase II response rates, but none have demonstrated a long term survival advantage including three tested in randomized controlled trials3. The lack of effective SR-GVHD treatments is likely compounded by our incomplete understanding of its pathophysiology. There are many reasons why SR-GVHD pathogenesis is poorly understood, but one reason may be the lack of clinically relevant animal models. Herein we generated murine SR-GVHD models, which surprisingly suggest that donor T cell independent mechanisms may play a more important role in SR-GVHD pathogenesis than previously appreciated.Acute graft-versus-host disease (GVHD) is a major complication of allogeneic cell transplantation . For decades, steroids have remained the first-line treatment for acute GVHD; however, 30–50% of patients develop steroid refractory GVHD (SR-GVHD), which portends an extremely poor prognosis5 so as to better reflect the prednisolone-equivalent dose used clinically. Surprisingly, despite dexamethasone treatment, there were no significant differences in overall survival (OS), GVHD clinical scores, or the percentages of body weight (BW) lost between treated and untreated control groups in our model  matched, we first utilized the major histocompatibility (MHC) matched multiple minor histocompatibility antigens (miHAs) mismatched C3H.sw→B6 BMT model. Recipient B6 WT animals were lethally irradiated and transplanted with splenic T cells along with bone marrow (BM) cells from either syngeneic B6 or allogeneic C3H.sw donors. Dexamethasone (DEX) was delivered from day +7 to day +21 at a dose of 0.1 mg/kg Fig.  which wadel Fig. . To deteBecause we couldn’t see any therapeutic responses in DEX treated animals, we speculated that treated animals have different responses to DEX in spite of a uniform genetic background and housing conditions. Interestingly, we observed three phenotypes in response to DEX: 1) progressive GVHD characterized by severe peak GVHD clinical scores, progressive BW loss and early mortality; 2) stable GVHD characterized by moderate GVHD clinical scores, moderate BW loss, and minimal late mortality; and 3) steroid-responsive GVHD characterized by near complete normalization of GVHD clinical scores, near complete normalization of BW loss, and no mortality Fig. . We wereTo eliminate strain dependent factors, we also examined whether the differential steroid response predicted by our scoring systems holds in an MHC mismatched haploidentical B6→B6D2F1 model. We found a similar differential response to corticosteroids; albeit with a slightly different distribution among the groups and greater mortality in the St and Res groups Fig. .To test whether our scoring system specifically separates DEX treated animals relative to control untreated animals, we applied our scoring system to 15 untreated control animals shown in Fig. To account for any confounding effect of radiation toxicity, we next sought to determine whether steroid refractoriness can be modelled at a later time-point. To accomplish this, we utilized the same C3H.sw → B6 model except that DEX was administered from day +21, at which point there was already approximately 20% mortality among allogeneic recipients, to day +35 Fig. . We agai7, we determined whether these cytokines correlated with treatment responses. To our surprise, serum levels of IFN-γ, and IL-17A were not significantly altered in any early or late DEX-treated animals , proinflammatory cytokine production , and CD4+CD25+Foxp3+ regulatory T cells (Tregs) by FACS. However, donor CD4+ and CD8+ T cell expansion in the spleen was similar among these groups at day 21 after allo-BMT , memory (CD44+CD62L+), naïve (CD44−CD62L+) T cells, or Tregs in the spleen, but these subsets were similar among all allogeneic groups . Collectively, these data suggest that our SR-GVHD models effectively predict survival and therapeutic responses. Unexpectedly, our data also suggest that SR-GVHD may be mediated by a donor T cell independent mechanism.In this study, we establish steroid refractory murine BMT models using two different clinically relevant BMT models, and two different treatment intervention strategies, early and late DEX treatment. In addition, we used corticosteroid doses that correspond to clinically relevant doses. All of the experiments presented here are required for establishing appropriate models to eventually decipher SR-GVHD pathogenesis and to develop SR-GVHD treatments.9 in Table 10. Therefore, our GVHD scoring system may not be specific nor correlate with histological GVHD grade but this system is used worldwide and consistently reflects murine GVHD severity. Recently, Naserian, et al. proposed a new simple, reproducible, and efficient clinical grading system for murine GVHD relying on the binary evaluation of five visual parameters, including diarrhea, and demonstrated correlation with histological grade11. Future work will seek to assess if Nasarian’s criteria improve on our model of SR-GVHD.Certainly, our models, like any model system have several limitations including the need to be continually optimized to reflect clinical BMT as clinical practice evolves. Nevertheless, we summarize the main differences between our criteria for murine SR-GVHD and clinical SR-GVHD13 following steroid treatment. T cell exhaustion could certainly help explain the clinical differences among our groups. However, why there would be differential exhaustion following steroid treatment in genetically and environmentally homogenous animals is not readily apparent. Nevertheless, our data suggest that immunoregulatory aspects in the context of steroid refractoriness need to be further explored. In addition, these data also suggest that T cell independent mechanisms of tissue tolerance may contribute to SR-GVHD and should be further explored15.To our surprise, we found that donor T cell characteristics were not significant among steroid responsive, refractory, or stable groups. Future studies will seek to elucidate the critical cellular and molecular mechanisms of glucocorticoid response and resistance. One potential explanation for different responses to DEX is differences in pharmacokinetics between groups, which we will explore in future studies. It would also helpful to assess whether donor derived T cells from newly generated bone marrow or those from peripheral expansion are more affected by glucocorticoid treatment. Another consideration to assess is whether donor T cells exhaustion is different among our groups as there are conflicting reports in this regardIn conclusion, we established clinically relevant steroid refractory GVHD murine models. Although we have not shown any critical mechanisms, developing a model is the key first steps. We propose using this model to better understand the pathophysiology of SR-GVHD and to develop new therapeutic and prophylactic GVHD strategies.b, CD45.2+), B6 Ly5.2 , B6D2F1 (H-2b/d), and C3H.sw (H-2b) mice were purchased from the Jackson Laboratory . All animals were cared for according to regulations reviewed and approved by the University Committee on Use and Care of Animals of the University of Michigan, based on University Laboratory Animal Medicine guidelines.C57BL/6  utilizing CD90.2 microbeads (Miltenyi Biotec), and bone marrow (BM) was harvested from donor femurs and tibias. B6 and B6D2F1 animals were used as recipients and received 10–11 Gy (137Cs source) on day −1 and 3 × 106 CD90.2+ T cells along with 5 × 106 BM cells from either syngeneic (B6 or B6D2F1) or allogeneic (C3H.sw or B6) donors on day 0.BMTs were performed as previously described using well established models19. Histopathological analysis of the liver, gastrointestinal (GI) tract, lung, and skin, which are the primary GVHD target organs, was performed as described previously20 utilizing a semiquantitative scoring system implemented by a single pathologist (Chen Liu)20. After scoring, the codes were broken, and the data compiled.We monitored survival after allo-HCT daily and assessed the degree of clinical GVHD weekly, as described previously22. Briefly, to analyze donor T cell or myeloid cell expansion and activation markers, splenocytes from transplanted animals were resuspended in FACS wash buffer  in phosphate buffered saline (PBS)) and stained with conjugated monoclonal antibodies (moAbs). The following antibodies were used: FITC-conjugated moAbs to mouse CD45.1 (clone SF1–1.1) and CD229.1 (clone 30C7); PerCP-Cy5.5-conjugated moAbs to mouse CD4 (clone GK1.5), CD8 (clone 53–6.7) and CD11c (clone N418); PE-conjugated moAbs to mouse CD69 (clone H1.2F3), CD62L (clone MEL-14), and CD11b (clone M1/70); and APC-conjugated moAbs to mouse CD44 (clone IM7) and CD25 (clone PC61). All antibodies were purchased from Biolegend  except the FITC anti-CD229.1, which was purchased from BD bioscience . After staining, the cells were washed twice and fixed with 2% formaldehyde as described previously21. Cells were analyzed using a BD Accuri C6 flow cytometer (BD Bioscience). For intracellular cytokine staining, cells were permeabilized after fixation and stained with PE-conjugated IFN-γ  and granzyme B , APC-conjugated IL-17A  and perforin  according to manufacturer’s protocol.FACS analyses were performed as previously describedTM  or ELISA MAXTM . Assays were performed according to each manufacturer’s protocol and read at 450 nm using a microplate reader . All samples and standards were run in duplicate.Concentrations of IFN-γ and IL-17A were measured in the serum by ELISA with specific anti-mouse MoAbs for capture and detection utilizing BD OptEIAin vitro data, and the Wilcoxon rank test for survival data. A p value <0.05 was considered statistically significant.Statistical significance was determined using the Mann-Whitney U test for Supplementary information


Despite high cure rates, about 20% of patients with advanced germ cell tumors (GCTs) fail cisplatin-based chemotherapy. High levels of DNA methylation have been identified in GCTs and linked to cisplatin resistance. Here, we examined the effects of DNA hypomethylating 5-azacitidine (5-aza) on two embryonal carcinoma cell lines  and their cisplatin-resistant isogenic derivatives. Effects on cell viability and cisplatin sensitivity were assessed by the trypan blue exclusion method. Western blotting was used to examine induction of apoptosis 5-aza and results were validated by flow cytometry. Single agent treatment with 5-aza strongly impacted viability and induced apoptosis at low nanomolar concentrations, both in cisplatin-sensitive and -resistant cell lines. 5-aza exerted an immediate apoptotic response, followed by a prolonged inhibitory effect on cell viability and cell-cycle progression. Sequential treatment with 5-aza and cisplatin reduced cellular survival of the cisplatin-resistant sublines already at nanomolar concentrations, suggesting a partial restoration of cisplatin sensitivity by the compound. 5-aza demonstrated anti-tumor activity as a single agent at low nanomolar concentrations in GCT cells, irrespective of cisplatin-sensitivity. 5-aza may also have the potential at least to partially restore cisplatin-sensitivity in non-seminoma cells, supporting the hypothesis that combining DNA demethylating agents with cisplatin-based chemotherapy may be a valid therapeutic approach in patients with refractory GCTs. Germ cell tumors (GCTs) are the most common solid malignancy among men up to the age of 45. Histologically, GCTs are classified into undifferentiated, primordial germ cell-like seminomas and more differentiated non-seminomas. Based on the extraordinary sensitivity towards cisplatin-based chemotherapy, GCTs are recognized as a model of a curable disease, even in advanced, metastatic stages . HoweverEpigenetic modifications play a significant role in carcinogenesis and in response of cancer cells to chemotherapy through regulating the expression of genes involved in cell replication, tumor suppression, DNA repair and apoptosis . EpigeneKLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs.Chemical DNA demethylation can restore the expression of silenced genes, which can be achieved, for example, by the use of the first generation DNMT inhibitor (DNMTI) 5-azacitidine (5-aza), which is approved by the Food and Drug Administration (FDA) for the treatment of myelodysplastic syndromes. The cytotoxic activity of 5-aza as a ribonucleoside, which gets incorporated into both DNA and RNA, is based on broad effects on both DNA methylation patterns and the cell’s transcriptome and gene expression profile ,14. In aTP53, and 2102Ep, which has wild type TP53, in an in vitro model system of acquired cisplatin-resistance using isogenic, resistant sublines NCCIT-R and 2102Ep-R.Here, we examined the effect of 5-aza, which has not been examined in non-seminomatous GCT cells so far, on two distinct embryonal carcinoma (EC) cell lines, e.g., NCCIT bearing a mutant 50 values ranging from 18 to 23 nM  polymerase 1 (PARP1). The cisplatin sensitive EC cells treated with their respective IC50s of cisplatin for 72 h served as controls of apoptosis induction. In all four cell lines we detected a strong apoptotic response upon 72 h of treatment with the respective IC50 doses of 5-aza as single agent as evidenced by increased caspase-3 and PARP1 cleavage  or 2102Ep (0.8 µM) or a supra-lethal dose of 5 µM representing the IC50 of both resistant sublines NCCIT-R and 2102Ep-R. With respect to a possible impact of the timing of cisplatin exposure after 5-aza treatment, two different schedules were tested. Cisplatin was applied either 24 h after re-plating (immediate sequence), or after a 168 h drug-free period following 5-aza exposure (delayed sequence) to assess possible prolonged effects of 5-aza on cisplatin sensitivity  was significantly less effective in the isogenic cisplatin-resistant sublines with a mean relative viability of 38% vs. 63% (p ≤ 0.0001) for NCCIT/-R and 46% vs. 69% (p = 0.0007) for 2102Ep/-R. Moreover, cisplatin demonstrated a dose-dependent toxicity in the resistant sublines after 48 h of treatment with either the aforementioned doses or a supra-lethal dose, showing a 14% reduction of the mean viability for NCCIT-R (p = 0.0091) and a 30% for 2102Ep-R (p = 0.001)  b,c. Toge50 doses of 5-aza for all cell lines, decreased cell viability to an unexpectedly high extent compared to the experiments shown in Treatment with 10 nM 5-aza alone did not significantly impact cell viability when compared to untreated controls, confirming the results in 50 of the corresponding sensitive cell lines, ranging from 14% (p = 0.012) to 18% (p = 0.014) in NCCIT-R cells, and from 15% (p = 0.019) to a borderline significant 13% (p = 0.058) in 2102Ep-R cells for immediate and delayed sequencing, respectively. Importantly, combined treatment decreased cell viability in the resistant sublines almost to levels achieved by cisplatin alone in the sensitive cell lines (p = 0.21). Except for the cisplatin-resistant, TP53-mutant subclone NCCIT-R , combining 10nM of 5-aza with a supra-physiological dose of cisplatin (5µM) did not add a relevant cytotoxic effect compared to 5µM of cisplatin alone , which did not show significant impact on cell growth  are thought to be one mechanism leading to cisplatin resistance in GCTs, mediated by an altered apoptotic response to treatment [50 values for the sensitive lines NCCIT and 2102Ep. The IC50 of the resistant sublines was much higher at supra-physiological 5 µM as reported before, which thus appropriately reflects the clinical burden of cisplatin-resistant disease [Both cell lines used in this project originate from embryonal carcinomas, a common chemo-sensitive subtype of non-seminomatous GCTs. The cell line NCCIT is derived from a primary mediastinal germ cell tumor with p53-deficiency, as it is frequently found in these rare tumors. reatment ,18. Impo disease . Thus, sRASSF1A, HIC1 and MGMT promoters [Epigenetics refers to gene expression alterations without changes in the DNA sequence. The most intensively studied epigenetic mechanisms comprise DNA methylation, regulation by microRNAs, and covalent, post-translational histone tail modifications, i.e., methylation and acetylation . The difromoters ,11,12. Aberrant DNA hypermethylation is a reversible epigenetic alteration, which can be therapeutically targeted with the aim of achieving global DNA hypomethylation and re-expression of tumor-suppressor genes ,21. NANOG or SOX2, and Myc target genes in NT2/D1 cells [NANOG and OCT3/4, or hypermethylation of distinct putative drivers of cisplatin-resistance, i.e., CFLAR, together with decreased cisplatin resistance upon 5-aza treatment in the seminoma cell line TCam-2 [TP53-mutation status, which is why our results add another important aspect to the complexity by showing a sustained strong apoptotic response and a  restoration of cisplatin sensitivity irrespective of the cell lines TP53 mutational status . This observation supports the hypothesis that the effects of DNMT inhibition may be multi-facetted, which could mean that the majority of cisplatin-resistant GCTs may be amenable to 5-aza (and/or other DNMTI) treatment irrespective of key clinical aspects related to cisplatin-resistance, particularly p53 pathway aberrations [Overall, our results, where single agent 5-aza was equally effective in both p53-deficient (NCCIT) and proficient 2102Ep) isogenic in vitro models of cisplatin-resistant GCTs, are in line with those reported for the treatment of the embryonal carcinoma cell line NT2/D1 and its resistant subline NT2/D1-R1 with either decitabine or guadecitabine. Moreover, sensitivity towards therapeutic DNA demethylation with either decitabine or guadecitabine was functionally dependent on a high expression of DNMT3B ,21. This02Ep isogD1 cells ,23. On te TCam-2 . Taken trrations .In general, GCTs consistently displayed an extraordinary sensitivity towards DNA demethylating drugs at low nanomolar concentrations, which is why DNA demethylation, either alone or in combination with other cytotoxic substances, holds promise as a new treatment strategy in refractory GCTs.Being the first and clinically the most often used epigenetic drug so far, 5-aza is currently recommended as first-line treatment of high-risk myelodysplastic syndromes and acute myeloid leukemia in patients unsuitable for more aggressive chemotherapy. Repeated cycles of 5-aza have proven to be highly effective and well tolerated in such patients ,25,26. A2 daily, days 1 to 7, every 4 weeks, with a reported peak plasma concentration 750 ± 403 ng/mL occurring 0.5 h after infection with a half-life of 41 ± 8 min. A slightly different but also intense dosage was applied in the phase II trial by Roth et al.  evaluating 5-aza as a single agent in 17 refractory GCC patients, where no responses were seen [The standard dosage to treat hematological malignancies is a subcutaneous injection of 75 mg/mere seen . With re50 determined in EC cell lines in this study, as well as those reported for other DNMTIs in the literature, are much lower and more in the range found in hematological neoplastic cells than the doses causing anti-neoplastic effects in solid cancer models when using other entities, such as pancreatic, hepatocellular, or breast cancer cells [Hypothetically, much lower doses may thus be effective in vivo to  overcome cisplatin-resistance in GCTs and to reduce toxic side effects at the same time. Interestingly, the ICer cells ,30,31. LPresumably due to the multifactorial origin of platinum-resistance, targeted approaches using single agents failed to achieve clinically meaningful responses in refractory GCTs so far. Combined therapeutic approaches may thus be more promising . InteresThe extragonadal embryonal carcinoma cell line NCCIT was purchased from the American Type Culture Collection . The gonadal embryonal carcinoma cell line 2102Ep was a generous gift from P.W. Andrews . Cells were routinely checked for contamination with mycoplasma and inspected microscopically for stable phenotype. All the cell lines used in the current research have been tested and authenticated.L-Glutamine (2 mM) , and 50 IU/mL penicillin and 50 µg/mL Streptomycin . For both cell lines, isogenic, cisplatin-resistant subclones were developed by repeated exposure to sublethal, increasing cisplatin-doses as described before [2. Cells were passaged when reaching 80–90% confluency, and only used for assays in passages 3–7. Both cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM)/F12, 1:1 nutrient mixture medium  with 10% fetal bovine serum supplement  plus d before ,35. Cell5-azacitidine (5-aza) was purchased from Selleckchem . For the treatment, 5-aza was freshly dissolved in phosphate-buffered saline (PBS) and added daily to the culturing medium at the indicated concentrations for 72 h with drug-containing media replenishment each day. Cisplatin solution was a generous gift from Medac Pharma . Cisplatin treatment was performed at the indicated concentrations and time points for 48 h. 5 cells/well were seeded in 6-well format in complete growth medium (2 mL/well). Following the plating, the cells were incubated overnight to allow proper adhesion before drug treatment. For determination of the IC50 for 5-aza, cells were treated with different doses of the drug in triplicate for 72 h. Cell viability was assessed by Trypan blue staining method combined with the automatic cell counting using Beckman Coulter Vi-CELL . For combined treatment, two approaches were applied: (i) cells were treated with 5-aza at either a fixed-dose of 10 nM, or the respective IC50 over 72 h. Cells were then washed with PBS, trypsinized, pelleted using centrifugation at 300× g, and resuspended in 6 mL complete growth medium. One quarter of the cell suspension volume was re-plated in a 6-well, incubated for 24 h, and treated with cisplatin at either the IC50 or a fixed-dose of 5 µM (IC50 of the resistant subclones NCCIT-R and 2102Ep-R) for another 48 h. (ii) In order to assess possible long-term effects of low-dose 5-aza, cells were treated in the same way except for a prolonged drug-free period of 7 days (168 h) between the end of 5-aza treatment and onset of cisplatin exposure. Cell viability was assessed by both the trypan blue staining assay and a semi-automated cell counting using a Beckman Coulter Vi-CELL . Each experiment was performed in triplicates and repeated independently at least two times.For assessment of cell viability, 1.5 × 103 cells of each cell line were treated with or without 5-aza and then analyzed by flow cytometry  after propidium iodide (PI) staining as described before [For cell cycle analysis, 5 × 10d before  Cells wev/v), 0,25% NaDesoxycholat (10%) (w/v), 1mM Phenylmethylsulfonylfluorid (PMSF), 1mM Natriumorthovanadat (Na3VO4), 10 mL Complete Protease Inhibitors (Aprotinin/Leupeptin)). Equal amounts of total protein (30μg) were electrophoresed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), all as described before [Whole cell extracts were isolated using RIPA buffer . The statistical significance of differences in cell viability and in the cell cycle phases distribution was calculated using two-sided, unpaired student’s In summary, we report here that 5-aza not only effectively affects cell viability and strongly induces apoptosis at low nanomolar concentrations as a single agent, but also achieves a substantial re-sensitization towards cisplatin in an in vitro EC model of cisplatin-resistance supporting the evaluation of low-dose DNMTIs in cisplatin-resistant GCTs in the setting of a clinical study.


The globalization of problem-based learning (PBL) in health professions education has been both celebrated and criticized. Using a critical narrative review approach, underpinned by our archive of global PBL literature and a targeted literature search, we analyze these dominant global discourses of PBL in health professions education. More precisely, we explore what is missed when the globalization of PBL is theorized either as a positive consequence of standardization, or a problematic spread of Western educational ideals and values around the world. We make visible how two dominant global discourses, a universalist and culturalist discourse, have emerged in the global proliferation of PBL. We also discuss the limitations of the two discourses by demonstrating how they either ignore contextual and cultural diversity or see it as problematic. We then turn to a perspective that has been marginalized in the PBL literature that emphasizes the global origins of PBL, transcending the dichotomy between West and non-West. We make a case for relating to PBL as a plural construct in order to learn from the cultural and situational nuances of educational activities labeled PBL around the world. We argue that PBL as a singular and universal concept has no global future, yet versions of PBL may continue to thrive locally. Finally, we propose avenues for future research that may help elucidate the global and local values that underpin our curricula, as well as the socio-political factors that perpetuate neo-colonialist views and practices in the uptake and implementation of PBL approaches across the globe. The globalization of health professions education has resulted in more collaboration between educational institutions and a greater flux of students and teachers across the globe . In other words, individuals and organizations that embody dominant conversations related to PBL are regarded as legitimate authorities on how to practice PBL, and in turn contribute to the proliferation of these conversations. At least in health professions education, PBL has been a dominant conversation topic worldwide for 50 years, as evidenced by the number of citations related to PBL Azer . These cPrevious research on the globalization of health professions education distinguishes between discourses around universalism and homogenization  and discourses around culturalism and hybridization  . In practice, this enables institutions “who have historically been in a position to build capacity and experience in PBL and share their knowledge and research worldwide, such as Maastricht University, to continue being looked at for expertise (…), possibly at the expense of others who did not have such a head start or who have a different educational message that is less aligned with mainstream global discourse”  rather than educational effectiveness per se  and instead be shaped by the global dissemination of best practices, underpinned by research on the science of learning. PBL, as a term, has come to mean many different things. It is often used simply as a short form to signify an appreciation for the idea that learning can be self-directed and can move away from large group lectures. For this reason, each institution may find it useful to define whether their approach is reminiscent of PBL, how they interpret PBL, and how they shape and implement it locally. At a global level, institutions may share their experiences with other institutions as part of a global conversation for improving education. We believe this ultimate goal of improving education is best served if this conversation includes participants bringing in a rich diversity of educational approaches and perspectives, and focuses on how we can deliver high quality graduates rather than how we label our approach or who is allowed to use that label. This means recognizing and encouraging the plurality of approaches that has developed, not only as part of the PBL movement, but as part of student-centered and community-oriented education more broadly. To be effective, power relations must also be considered when organizing and participating in global sharing events and practices, considering that those currently in power have more access to resources, and greater capacity to control how we disseminate and share knowledge about PBL approaches.Thus, the term PBL as a singular and universal concept has no global future. Globally, we can only talk about PBL in the plural sense, which we may emphasize by using plural expressions such as ‘PBL approaches’ or ‘PBL strategies’. This subtle change in terminology has implications for how the term is currently used worldwide  because these situations often require a singular, shared understanding across contexts and institutions. Rather than “nailing down definitions”, the potential value of exploring the meaning of educational concepts, including the philosophies and values that underpin them in different contexts worldwide, has been emphasized previously in terms of opening up alternative ways of seeing and knowing what may be better in other contexts (Naidu and Kumagai In addition to functioning as a platform for sharing local experiences and interpretations of PBL, how and why it might function in different contexts, and opening up to approaches of student-centered education more broadly, research on PBL approaches may focus on the factors that draw institutions worldwide to constructs of PBL. Specifically, future research should analyze why people and institutions are drawn to PBL constructs, what they see as added value of using the term, how they take decisions regarding its adoption and implementation, and what forces guide these decisions. These avenues of research may help elucidate the global and local values that underpin our curricula, as well as the socio-political factors that perpetuate neo-colonialist views and practices in the uptake and implementation of PBL approaches across the globe.


Summary: IgA nephropathy is the most common type of primary glomerulonephritis worldwide. At least 25% of patients may progress to kidney failure requiring dialysis or transplantation. Treatment of IgA nephropathy using generalized immunosuppression is controversial, with concerns regarding the balance of safety and efficacy in a nonspecific approach. This review describes the recent scientific evidence, and a current clinical trial, investigating whether spleen tyrosine kinase (SYK) may be a novel and selective therapeutic target for IgA nephropathy. SYK, a cytoplasmic tyrosine kinase, has a pivotal role as an early intermediate in intracellular signal transduction cascades for the B-cell receptor and the immunoglobulin Fc receptor, and thus is critical for B-cell proliferation, differentiation, and activation, and for mediating proinflammatory responses after Fc-receptor engagement in various cell types. In renal biopsy specimens of patients with IgA nephropathy, increased expression and phosphorylation of SYK were detected, and this correlated with the histologic features of mesangial and endocapillary proliferation. In cell culture studies, patient-derived IgA1 stimulated mesangial cell SYK activation, cell proliferation, and cytokine production, and these responses were attenuated by pharmacologic or molecular inhibition of SYK. A global, randomized, double-blind, placebo-controlled trial investigating the safety and efficacy of fostamatinib  in the treatment of patients with IgA nephropathy is ongoing, which may provide important evidence of the safety and efficacy of targeting this pathway in clinical disease. Spleen tyrosine kinase (SYK) is an immunoreceptor-associated protein tyrosine kinase with numerous biological functions. First identified in 1991  domains and a C-terminal kinase domain A.3 The Slinker-kinase sandwich .26More recent attention has focused on the role of SYK in signaling for nonclassic immunoreceptors. SYK has been implicated in integrin signaling in myeloid cells, which is thought to be dependent on the association of integrins with ITAM-containing adapter proteins such as FcRγ chain and DAP12.Given its important role in immunoreceptor signaling and in mediating immune and inflammatory responses, SYK function has been studied in a number of in vivo models of allergic and autoimmune disease. Studies using bone marrow chimeras generated using SYK-deficient progenitor cells, for example, confirmed that SYK deficiency in the hematopoietic compartment conferred resistance to a passively transferred antibody-dependent model of inflammatory arthritis.20,A number of pharmaceutical companies are working to develop compounds to inhibit SYK for use in allergic and autoimmune disease.38Current understanding of IgA nephropathy proposes a multihit model of disease pathogenesis.Evidence for the latter phenomenon initially was provided by immunohistochemical analysis of renal biopsy specimens from patients with IgA nephropathy.The functional importance of SYK in IgA nephropathy initially was investigated using a cell culture model, wherein human glomerular mesangial cells were stimulated with IgA1 purified from patients with IgA nephropathy.The identity of the specific mesangial cell receptor(s) for IgA1 in patients with IgA nephropathy has been controversial, and a number of putative candidates have been suggested. The transferrin receptor (CD71) perhaps is the best characterized of these, being expressed on human mesangial cells, and up-regulated and co-localized with IgA1 deposits in IgA nephropathy.The specific role of SYK in myeloid cells expressing the classic FcαR1 (CD89) in the pathogenesis of IgA nephropathy is less well studied. Historical data have suggested that glomerular and tubulointerstitial mononuclear phagocytic cell infiltration is associated with a worse prognosis in IgA nephropathyA number of in vivo models of human IgA nephropathy have been described, including spontaneous, inducible, and genetically modified systems.Nevertheless, important lessons about the potential role of antibody-mediated inflammation have been learned in models of anti–glomerular basement membrane (GBM) antibody-mediated glomerulonephritis in rodents, which are reliably characterized by significant glomerular inflammation and progression to renal failure. In the passive-transfer model of anti-GBM glomerulonephritis in Wistar Kyoto rats (nephrotoxic nephritis), treatment with fostamatinib  was effective in reducing macrophage infiltration, fibrinoid necrosis, and crescent formation, with a reduction in proteinuria and serum creatinine.In the active immunization model of anti-GBM glomerulonephritis , disease is induced by immunization with the α3-chain of type IV collagen (the Goodpasture autoantigen), resulting in the development of autoantibody and crescentic glomerulonephritis. In this model, early treatment with fostamatinib reduced the production of autoantibody and the severity of glomerulonephritis.It is challenging to attribute the importance of a specific molecule or cytokine to the pathogenesis of glomerulonephritis, given that multiple complex, inter-related pathways may be involved. Taken together, however, the available preclinical data imply that SYK makes a significant contribution to the pathogenesis of IgA nephropathy, in particular via its role in mediating proinflammatory responses after the deposition of IgA1- and IgG-containing immune complexes in the renal mesangium. Emerging evidence suggests that the interaction of IgA1 with various receptors on mesangial cells results in SYK activation, although the potential of signaling via other receptors  has yet to be explored specifically in IgA nephropathy. In addition, the role of SYK in the production of abnormal IgA1, and the autoantibodies directed against it, by B cells and plasma cells, has not been explicitly studied in IgA nephropathy, although data from other experimental immune models have suggested this may be a potential benefit, and other B-cell–directed therapies are undergoing clinical evaluation in IgA nephropathy. It is possible that a reduction in disease severity observed in the animal models of glomerulonephritis may be achieved by selective reduction (rather than complete abrogation) of SYK activity on these multiple pathwaysConversely, some of the therapeutic effects that have been observed when using small-molecule inhibitors may be attributed to off-target effects. R406 (the active metabolite of fostamatinib) is a potent inhibitor of SYK, although with a significant off-target effect on Flt3 and vascular endothelial growth factor receptor.A proof-of-principle global clinical trial of fostamatinib was performed to study the efficacy and safety of fostamatinib in the treatment of patients with IgA nephropathy (NCT02112838). This was a randomized, double-blind, placebo-controlled, clinical trial that included a run-in period with optimized control of hypertension and proteinuria with angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker for 90 days before randomization. The patients then were randomized to groups receiving fostamatinib 100 mg twice daily, 150 mg twice daily, or placebo for 24 weeks. This study completed global recruitment in February 2018, and results are expected later in 2018. Both functional  and histopathologic end points will be evaluated because enrolled patients will be offered a repeat renal biopsy at the end of the treatment period. The results of this clinical trial will be pivotal in understanding whether SYK inhibition may be a safe and effective treatment of IgA nephropathy.


Casearia capitellata, Baccaurea motleyana, Phyllanthus pulcher and Strobilanthus crispus), indigenous to Malaysia, were extracted in different solvents, sequentially. The obtained 28 extracts were evaluated for their in vitro anticancer properties, using the MTS assay, on four human cancer cell lines: colon (HT-29), breast (MCF-7), prostate (DU-145) and lung (H460) cancers. The best anticancer activity was observed for the ethyl acetate (EA) extract of Casearia capitellata leaves on MCF-7 cell lines with IC50 2.0 μg/mL and its methanolic (MeOH) extract showed an outstanding activity against lung cancer cell lines. Dichloromethane (DCM) extract of Phyllanthus pulcher aerial parts showed the highest anticancer activity against DU-145 cell lines, while significant activity was exhibited by DCM extract of Phyllanthus pulcher roots on colon cancer cell lines with IC50 value of 8.1 μg/mL. Total phenolic content (TPC) ranged over 1–40 mg gallic acid equivalents (GAE)/g. For all the samples, highest yields of phenolics were obtained for MeOH extracts. Among all the extracts analyzed, the MeOH extracts of Strobilanthus crispus leaves exhibited the highest TPC than other samples (p < 0.05). This study shows that the nature of phenol determines its anticaner activity and not the number of phenols present.Different parts of four edible medicinal plants ( Plant based drugs have a long history in both traditional and modern societies as herbal remedies or crude drugs, or as purified compounds approved by the Food and Drug Administration and similar regulatory agencies ,2,3. DruInvestigations about natural products have recently regained prominence with the rapidly increasing understanding of their biological significance and increasing recognition of the origin and function of their structural diversity.Cancer is a leading cause of death worldwide. Breast and prostate cancer are two of the most common malignancies and contribute significantly to the societal and economic burden of cancer. About 10 million new cases are diagnosed and over 6 million deaths occur worldwide annually (excluding non-melanoma skin cancers) . ImportaPhyllanthus pulcher (Euphorbiaceae), Casearia capitellata (Flacourtiaceae), Strobilanthes crispus (Acanthaceae) and Baccaurea motleyana (Phyllanthaceae) for their TPC and anticancer potential. Plants selected for this study were chosen because of their use in local traditional cuisine. Information on the traditional culinary purposes of these plants was collected through a structured literature review. The present paper deals with the screening of the following medicinal plants: P. pulcher from genus Phyllanthus is found throughout South East Asia and is locally  known as Naga Buana (Dragon of the World). The plant is used extensively in Malay traditional medicines: as a decoction for stomach ache, the leaves to treat tooth ache, poultices are applied to the nose for ulceration, to the skins to treat boils, and to the abdomen of children with kidney problems [C. capitellata from the family of Flacourtiaceae is a lowland rain forest treeendemic to Malaysia. Casearia species are traditionally used in Malaysia as antiseptic, cicatrizant and topical anasthetic agent [Strobilanthes is a genus of about 250 species of flowering plants in the family Acanthaceae, mostly native to tropical Asia, but with a few species extending north into temperate regions of Asia. Traditionally, the leaves of S. crispus were boiled with water and the filtrates were used in traditional medicine in Malaysia and Indonesia as antidiabetic, diuretic, antilytic, and laxative. S. crispus has been proven to be possessing high antioxidant and antihyperglycemic properties [Baccaurea motleyana (Family: Phyllanthaceae) is a species of fruit tree, which grows wild in parts of Southeast Asia and is cultivated for its fruit in Thailand and Peninsular Malaysia. The fruits are approximately 2 to 5 centimeters long and about too wide and grow in strands. Each fruit has velvety pinkish, yellow, or brown skin, which wrinkles at ripening and is filled with whitish pulp containing 3 to 5 seeds. The pulp is sweet to acidic in taste. The fruits are eaten fresh or processed into beverages and wine. The tree is also used for shade and low-quality wood [The study shows problems . C. capiic agent . They haic agent , antiinfic agent , antihypic agent , and hypic agent  activitiic agent ,20. Strooperties ,22. Baccity wood .A survey of the literature revealed that no studies on the anticancer potential of extracts from these plants have been undertaken on four human cancer cell lines: breast cancer cell line (MCF-7), prostate cancer cell line (DU-145), colon cancer cell line (HT-29) and lung cancer cell line (H460). It is known that different cell lines might exhibit different sensitivities towards an anticancer compound, so the use of more than one cell lines was therefore considered necessary in the evaluation of anticancer effects. Bearing this in mind, four human cell lines of different histological origin were used in the present study. The study is aimed to evaluate anticancer potential of sequentially prepared extracts from different parts of these plants on above said cell lines. The aim of this study was so to investigate the anticancer activity of these extracts against a panel of tumor cell lines. The TPC from these extracts was also determined. The findings of the study will be helpful in understanding anticancer and antioxidant potential of these plants and significance for the development of new anticancer drugs.i.e., 2.0 to 40%. However, the highest yield was obtained for methanolic extracts. It is in accordance with earlier observations [The yield of extracts, from different parts of all plants prepared sequentially in different solvents, is shown in rvations , reportiS. crispus leaves contained the highest amount of phenolics (40 mg/g) followed by P. pulcher roots (32 mg/g), C. apitellata (30.4 mg/g), B. motleyana fruit (21 mg/g), S. crispus flowers (17 mg/g), P. pulcher aerial parts (15 mg/g), whereas the lowest level was found in B. motleyana peel (12 mg/g) (S. crispus leaves (40 mg/g) as compared to S. crispus flowers (17 mg/g), while P. pulcher roots (32 mg/g) contained more phenols than P. pulcher aerial parts (15 mg/g). Whereas B. motleyana fruits (21 mg/g) contained more phenols in comparison to B. motleyana peel (12 mg/g).Total phenolic contents were determined spectrophotometrically using Folin-Ciocalteu reagent and results are presented in 12 mg/g) . Phenolsi.e., solvent employed. Having used several solvents, it was found that the best yields of phenolic compounds, for all the samples, were obtained in high polar medium, i.e., MeOH extracts. This is in agreement with a recent study on Pistacia vera, where it was found that the yield in total phenols depends on the method and the choice of solvent, and that the highest amount was obtained in highly polar extracts  [The variation among TPC may be assumed due to the presence of different types of phenols and other constituents in different parts/plants; selectively extractable depending upon the extraction medium,  powder) .The variations in amounts of phenolics in these plant extracts could be partly due to the differences in growing conditions. Under field conditions, the phenolic compositions of plant tissues are reported to be varying considerably with seasonal, genetic, and agronomic factors . In addi50 values) of plant extracts on the growth of MCF-7, HT-29, DU-145 and H460 cell lines, measured by MTS assay, are given in All the four cell lines, used in this assay, were capable of attachment to form a homogeneous monolayer on plastic substratum of culture wells, which is ideal for MTS assay. The MTS test is a simple bioassay used for the primary screening of crude plant extracts and isolated compounds. For each cell line, there was a linear relationship between cell number and absorbance; measured at 550 nm in both control and drug-treated wells. After 72 h of treatment, the anticancer activity of the plant extracts was determined. The anticancer activity . The different botanical taxa studied in this work are shown in Breast cancer (MCF-7), prostate cancer (DU-145), colon cancer (HT-29) and lung cancer (H460) cell lines were obtained from American Type Culture Collection . Sample absorbance was recorded by a Shimadzu UV-1700 pharma spectrophotometer at 765 nm.The plant material was subjected to dryness at room temperature, cut into small pieces and was then ground to a fine powder using an electric mill. The cold extraction method using organic solvents was employed to obtain crude extracts from all the plants. Dried powdered plant materials were sequentially extracted with hexane, dichloromethane (DCM), ethyl acetate (EA) and methanol (MeOH). The extraction process was conducted thrice to ensure the complete extraction of required materials and the combined filtrate, for individual sample, was concentrated using a rotary evaporator. Total phenolic contents (TPC) were determined using Folin-Ciocalteu reagent as described earlier  with sli2 atmosphere with 95% humidity. The optimal plating density of cell lines was determined to be 1 × 103 to ensure exponential growth throughout the experimental period and to ensure a linear relationship between absorbance at 550 nm and cell number when analyzed by the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cytotoxicity assay to determine the anticancer effects of all plant extracts against a panel of cancer cell lines. The MTS cytotoxic assay has been considered a valuable tool in studying cytotoxicity induced by chemical agents, for many years. It uses a tetrazolium salt, which is reduced to a coloured formazan product by mitochondrial dehydrogenases in living cells. It is frequently used and is reported to be suitable for in vitro drug screening tests [5 cells per well and in a volume of 180 µL of culture media. The plates were incubated at 37 °C (5% CO2 and 95% air) overnight to allow attachment onto the wells, before the addition of plant extracts in replicates of 4 wells for each concentration to give final concentrations in the range of 0.1–100 µg/mL. Following incubation at 37 °C in an atmosphere of 5% CO2 and 95% air for 72 h, 50 µL of MTS (2 mg/mL in PBS) were added into each well containing the cells and medium above (blank). The plates were re-incubated for 1–4 h to allow metabolism of MTS by cellular mitochondrial dehydrogenases. The viable cells converted soluble yellow MTS into insoluble purple formazan. Following the incubation period, the medium containing MTS was aspirated from each well and 100 µL of DMSO was added to dissolve formazan in order to read the absorbance of soluble formazan at 550 nm, corresponding to number of living cells. The absorbance was determined at 550 nm using a 96-well microplate reader. The percentage of cell viability was calculated using the formula:Cells were grown in RPMI-1640 culture medium supplemented with 10% heat-inactivated foetal bovine serum, 100 IU/mL of penicillin and 100 μg/mL of streptomycin. Subconfluent cells were trypsinised using trypsin-EDTA after every 3–4 days and cells were placed in 3 mL of medium and syringed to obtain a suspension of single cells. The cell lines were maintained at 37 °C in 5% COng tests . Cells w50).Dose-response curves (% of viability Vs concentration) were constructed to obtain 50% inhibitory concentrations (ICp < 0.05. The inhibitory concentration 50% (IC50) was calculated from the dose–response curves  obtained by plotting the percentage of inhibition versus the concentrations.All the experiments were conducted in triplicate and statistical analyses was done according to SPSS 12.0. Data were expressed as means ± S.D. Statistical analysis was performed by one-way analysis of variance, followed by Duncan’s multiple range tests to determine the significance of differences among the means and multiple comparisons of unpaired data. Differences were considered significant at C. capitellaat, P. pulcher and S. crispus, can be an interesting source of anticancer compounds. Further studies identifying and isolating the key phenolic compounds may allow for a potential biomedical application in the therapy of cancer diseases.In conclusion, this work reveals that selected Malaysian flora species, including