The variation in the climate, the geographical location and the rich ethnomedicinal traditions has made the Darjeeling Himalayas an abode of invaluable repository of traditional medicinal plants. In this study, we explored the 2) and human cervix adenocarcinoma cell line (HeLa). The cytotoxicity was evaluated by performing MTT assay, trypan blue exclusion assay and morphological assessment under phase contrast inverted microscope. For the extracts which tested positive, IC50 (the concentration that inhibited cell growth by 50%) was calculated. The extract(s) were further subjected to Thin Layer Chromatography (TLC) to determine their phytochemical profile.The ethanolic leaf extracts of 30 medicinal plants were tested for their cytotoxicity against human breast adenocarcinoma cell line (MCF 7), human hepatocarcinoma cell line  contains supplementary material, which is available to authorized users. Catharanthus roseus L. [Combretum caffrum [Taxus brevifolia [Cephalotaxus harringtonia [Cancer stands second, after cardiovascular disorders, in the list of diseases responsible for maximum deaths in the world . In 2008oseus L. . Others evifolia  and homoingtonia . In addiingtonia .in vitro cytotoxic activity of ethanolic extracts of 30 ethno-pharmacological important plants against three different human cancer cell lines and characterize the phytochemical constituents with the view of isolating compounds from these plants which may contribute to drug development against cancer.Darjeeling district is situated in the northernmost part of the state of West Bengal in India that lies between 28°31′-27°13′ N latitude and 87°59′ - 88°53′ E longitude in the Eastern Himalayan region of India. The variation in altitude between hills and plains of Darjeeling that ranges between 150 m to 3636 m, has led to diverse climatic conditions that supports one of the richest biodiversity in the world. Thus, this region houses a wealth of varied medicinal plants . The ethFresh leaves of plants as listed in Table 2) and human cervix adenocarcinoma cell line (HeLa) was obtained from National Centre for Cell Science, Pune, India. All cell lines were cultured in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 mg/ml streptomycin, 0.14% sodium bicarbonate and 0.1 mM sodium pyruvate. The cell lines were maintained in CO2 incubator (N-Biotech) at 37°C in a 5% CO2 atmosphere with 95% humidity.The human breast adenocarcinoma cell line (MCF 7), human hepatocarcinoma cell line (HepG2 and HeLa) were seeded in 96-well plates at a density of 5×103 cells/well in 200 μl culture medium. Following 24 h incubation and attachment, the cells were treated with different concentrations of extracts and similar concentration of diluents (DMSO) for further 24 h. After treatment, media was replaced with MTT solution (10 μl of 5 mg/ml per well) prepared in PBS and incubated for 3 h at 37°C in a humidified incubator with 5% CO2. The yellow MTT dye was reduced by succinate dehydrogenase in the mitochondria of viable cells to purple formazan crystals. To solubilize the formazan, 50 μl of isopropanol was added to each well. The plates were gently shaken for 1 min and absorbance was measured at 600 nm, with reference 490 nm, by microtiter plate reader . The percentage of cytotoxicity was calculated as (Y-X)/Y x 100, where Y is the mean optical density of control (DMSO treated cells) and X is the mean optical density of treated cells with plant extracts.Stock solutions at a concentration of 10 mg/ml were prepared by reconstituting the dried alcoholic extracts in dimethyl sulfoxide . The cytotoxic effect of each of the plant leaf extract was evaluated by tetrazolium- dye, MTT, assay ,18 with 2 incubator. The following day, cells were treated with either 30 μg/ml plant leave extract or DMSO alone, serving as control, and incubated again at the same conditions. The morphological changes of cancerous cells under treated and control conditions were compared by monitoring with phase contrast inverted microscope  at 200X magnification. The images were photographed at 8, 16, and 24 h of incubation.Cells were seeded in 35 mm polyvinyl coated cell culture plates and allowed to attach at 37°C for 24 h in CO4 cells/ml. The % cell viability was determined as [ x 100. The percentage of growth inhibition was represented as {cell viability (control) – cell viability (with extract)}.After morphological assessment, the cell viability was simultaneously assessed by Trypan blue dye exclusion assay . For thi3) resulting in the appearance of yellow or greenish fluorescent spot under UV light at 365 nm indicated the presence of flavonoids. A brown or yellowish coloration with Lieberman-Burchard’s reagent reveals the presence of triterpenes and steroids. A 10% vanillin ethanol solution was used for detecting saponins, the presence of which results in blue, violet and sometimes yellow spots. Using a 2% ferric chloride solution, development of chestnut, violet, green and blue colour reveals the presence of polyphenols, whereas a bluish or greenish black colour would indicate a positive test for tannins.Phytochemical screening by means of TLC was carried out for selected plants following the method of Wagner and Bladt . For thi50) regression analysis was performed using Microsoft Excel 2010. Data were analyzed and compared by one-way analysis of variances (ANOVA) by using the software SPSS 15.0 for windows  and differences with p < 0.05 were considered significant.Results were presented as Mean ± SD of triplicates from three independent experiments. To calculate the concentration required to produce 50 % reduction in cell viability (IC2 (hepatocarcinoma) and HeLa (cervix adenocarcinoma), as high rates of these specific cancers were noted in West Bengal. Several studies on screening various folklore medicinal plants to identify their cytotoxic activity against human carcinoma cell lines have been reported [The medicinal plants are the most sought after substitutes as the source of antimicrobial and anticancer chemotherapeutic agents. The use of traditional medicinal plants for varied ailments by different ethnic groups of Darjeeling district has been aptly recognized and it has been suggested that cures for diseases like cancer; AIDS, etc. may lay hidden in this wealth of folklore medicinal plants ,21,22. Wreported –27.2 cells and 2 plant extracts against MCF7 cells was observed. From the total of 30 plant extracts, only five showed > 50 % cytotoxic activity against the different cancerous cell lines at a concentration of 50 μg/ml after 24 h incubation. The possibility that the other plant extracts may also show greater inhibitory activity at either higher concentration or prolonged incubation cannot be ruled out. Nevertheless, we set stringent conditions to identify extracts exhibiting highly potential and effective cytotoxicity. In a nutshell, results of our study implicated that the human cervix adenocarcinoma cell line (HeLa) was sensitive while the other two cell lines, human breast adenocarcinoma (MCF7) and human hepatocarcinoma cell line (HepG2) were comparatively resistant to cytotoxicity of the tested extracts. Similarly, while screening plants used in Thai folklore medicine for cytotoxic activity, Mahavorasirikul et al. observed that sensitivity towards the tested extracts was dependent on the type of cancer cell line used and HepG2 appeared to be the most resistant cell line [Ethanolic leaf extract of each of the listed plants were screened for their cytotoxicity against the three cancer cell lines at a single dose of 50 μg/ml. Our aim was to consider only those plants which exhibited a potent cytotoxic activity (~50%) and to meet this need we assessed the growth inhibitory activity of 50 μg/ml of each extract at an incubation period of 24 h. Preliminary screening results showed that the extent of inhibition varied between the types of cell lines and also the types of extract used. Out of the 30 extracts tested, 9 had almost no effect (<10 %) on the growth of any of the three cell lines (Table ell line . In addiell line .Table 2Artemisia indica (P4), Maesa macrophylla (P19) and Phlogacanthus thyrsiformis (P22) could inhibit the growth of HeLa and MCF7 cells. The extent of inhibition by P4 and P22 was in the range of 50 to 60 %. On the other hand, P19, showed ~ 75 to 80 % growth inhibition of both the cell lines. The extract of Eupatorium odoratum (P16) was cytotoxic to HepG2 (53.11 ± 4.87 % inhibition) and HeLa (78.03 ± 1.63) cells, whereas Eupatorium adenophorum (P15) could inhibit the growth of only HeLa cells by ~61 %  values at the concentration range of 50 μg, 40 μg, 30 μg, 20 μg, and 10 μg. To determine the concentration of the plant extracts that caused a 50% reduction in cell viability (IC50), regression analysis was done. The results obtained are tabulated in Table 50 value of Artemisia indica was found to be 48 μg/ml for both HeLa and MCF7 cell lines. Earlier studies have demonstrated antimicrobial, cytotoxic and antioxidant activities of essential oil from the aerial parts of Artemisia indica [50 value of 32 μg/ml for HeLa cells and 50 μg/ml for HepG2 was obtained with the extract of Eupatorium odoratum. The ability of acetone and ethyl acetate extracts of leaves of Eupatorium odoratum to induce cell death in MCF7 and Vero cell lines have previously been reported [E. odoratum against MCF7 perhaps because we used ethanol as the extracting solvent. Phlogacanthus thyrsiformis is an evergreen shrub (vasaka in hindi) that has been reported to have antimicrobial, analgesic and free radical scavenging properties [P. thyrsiformis with IC50 value of ~50 μg/ml for two cancer cell lines HeLa and MCF7. Eupatorium adenophorum showed an IC50 value of 42 μg/ml against HeLa cell lines. The antioxidant activity of essential oil of E. adenophorum has earlier been reported [The five promising plant extracts obtained after the first screening were further assessed for their ICa indica . An IC50operties -33. Our reported .Table 3Maesa macrophylla showed the most promising inhibitory effect at 24 h incubation with IC50 value of 22.45 μg/ml and 22.66 μg/ml for HeLa and MCF7 cell lines respectively. Maesa macrophylla, belonging to family Myrsinaceae, is found in West Bengal and Uttar Pradesh in India. The bark of the plant is used to treat tonsillitis, the juice of the fruit is applied to treat scabies and the leaves are used to treat fever and boils. The bark of the plant has been reported to possess antimicrobial and antiviral activity [M. macrophylla leaves have revealed the presence of a novel quinone [in vitro cytotoxic activity against carcinoma cells, if the IC50 value at an incubation period between 48 and 72 hours, is less than 20 μg/ml [Maesa macrophylla at 72 h of incubation. Our results revealed an IC50 of 9.55 μg/ml for HeLa and 16.19 μg/ml for MCF7 cell line .Among the five plants, activity . Studies quinone . However20 μg/ml . Based oe Figure . Hence, M. macrophylla, the extract was subjected to TLC and detected using specific reagents [Maesa macrophylla and evaluating their anticancer properties are underway.Phytochemicals are considered as active medicinal chemical constituents of plants . To analreagents . The resreagents . Diosgenreagents -43. The reagents ,45. The 2 cancer cells. The level of inhibition was different for different cell lines and none of the extract could inhibit all the three cancer cell lines under investigation. Only one plant, Maesa macrophylla, showed a highly promising cytotoxic activity against HeLa (IC50 9.55 μg/ml) and MCF7 (IC50 16.19 μg/ml) cell lines. Phytochemical analysis revealed the presence of multiple medicinally active compounds. Identifying the active compound(s) and comparing its cytotoxicity with known anticancer compounds is in progress.This is perhaps the first study that deals with the screening of plants from Darjeeling hills for their cytotoxic activity against cancerous cell lines. The outcome of the present study indicates that five out of the thirty plants tested, exerted a >50% growth inhibitory effect on HeLa, MCF7 and HepG


PTEN inactivation is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in nucleus. Poly (ADP-ribose) polymerase (PARP) plays key roles in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in endometrial cancer cell lines.PTEN were analyzed in cells with wild-type PTEN.The response to olaparib was evaluated using a clonogenic assay with SF50 values  in 16 endometrial cancer cell lines. The effects of PTEN on the sensitivity to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN + (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and γH2AX, and induction of cleaved PARP. The effects of siRNA to t test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and γH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN + cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN + cells. In addition, knocking down PTEN by siRNA did not alter the sensitivity to olaparib in 2 cell lines with wild-type PTEN.The SF50 values were 100 nM or less in four (25%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in four (25%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 746 ± 838 nM; Wild-type [n = 4]: 215 ± 85 nM, p = 0.26 by Student’s Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. Inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer. BRCA 1/2 mutations in breast and ovarian cancers [PTEN, ATM, RAD51[Homologous recombination (HR) is a critical step for DNA repair, and certain types of cancers are HR defective, including BRCA1/2 deficiency -5. Tumor cancers ,7. HowevTM, RAD51-10. TherK-Ras (10–20%), PIK3CA (25–36%), AKT (2%), and PTEN (34–56%) [PTEN locus at chromosome 10q23.31 is also associated with the inactivation of PTEN [Endometrial cancer is the fourth most common malignancy among women in the United States . In endo(34–56%) -15. Addi of PTEN -18.In addition to a negative regulator of the PI3K/AKT signaling pathway, PTEN contributes to maintaining genomic stability and DNA repair processes by regulating the expression of RAD51, a key protein in HR DNA repair . The lacDedes and coworkers showed that PTEN-deficient endometrial cell lines, which fail to elicit RAD51 to DNA damage sites, are sensitive to PARP inhibitors . HoweverThe purpose of this study is to clarify the anti-tumor effect of olaparib on a panel of endometrial cancer cell lines and to assess the association among PTEN status, HR repair, and sensitivity to olaparib in endometrial cancer cells.We used 16 endometrial cancer cell lines  was provided by AstraZeneca . Olaparib was solved in DMSO, and the concentration of DMSO in each assay was 0.1%.Cells were plated at approximately 30% confluence in 100-mm plates and incubated for 24 h before transfection with small interfering RNA (siRNA) duplexes at the concentrations indicated, using Lipofectamine 2000 RNAiMAX  and Opti-MEM medium . The target sequence of siRNA specific for PTEN was described previously . A negatThe mutational status of PTEN exons 1–9) was analyzed by PCR and direct sequencing as described previously . The mut–9 was an380), AKT , phospho-AKT (Ser473), PARP, cleaved PARP , RAD51 , and β-actin  were used for western blotting, as recommended by the manufacturer. Proteins were visualized using an ECL western blot detection kit .Cells were lysed as described previously ,25. AntiImmunocytochemistry was performed as described previously . PrimaryCell cycle analysis was performed by flow cytometry, as previously described . The celCells were seeded in six-well plates at a concentration of 2,000 cells per well with olaparib (10 nM to 100 μM) or IR (2 Gy to 6 Gy). Cells were continuously exposed to olaparib with media during the incubation. After 14–21 days of incubation, the cells were fixed with methanol and stained with Giemsa (Wako). All experiments were repeated three times and the SF50 (surviving fractions at 50%) values, which indicate the concentration required to inhibit cell survival to 50%, were calculated by proliferation curves.Cells were irradiated using a Shimadzu PANTAK HF-350 X-ray generator .t-test, and a p-value of <0.05 was considered to denote a statistically significant difference.Data are expressed as the means ± standard deviations of three independent determinations. The significance of differences between the two samples was analyzed using Student’s PTEN mutations were detected in 12 of the 16 (75%) endometrial cancer cell lines . Among the 12 cell lines with PTEN mutations, the expression of PTEN was observed in the HEC-116, HEC-88, HEC-151, and HEC-251 cell lines  by introducing wild-type PTEN into a parental HEC-6 (PTEN null) cell line. The expression of exogenous PTEN was confirmed Figure B and thet test). Additionally, the lack of PTEN expression did not increase the sensitivity to olaparib  did not affect the sensitivity in clonogenic assay  for 24 h induced foci formation of both γH2AX and RAD51 in the nuclei Figure A. The nuParental HEC-6 and HEC-6-PTEN + cells were exposed to IR at 10 Gy as a DNA damaging agent. The expression of PTEN, as well as the phosphorylation level of PTEN (Ser-380), was increased in HEC-6 PTEN + cells within 24 h following IR exposure  or IR  was performed by flow cytometry in the HEC-6 and HEC-6-PTEN + cells. The sub-G1 population was markedly increased in both cell lines by either olaparib or IR Figure . AlthougInhibiting PARP is a promising strategy in cancer cells, especially in cells with a deficiency in HR repair. Although BRCA1/2 play key roles in HR repair, the association of other tumor suppressor genes with HR repair is still debatable. In phase II clinical trials conducted in high-grade serous ovarian carcinomas, olaparib has been reported to be effective in certain patients without BRCA1/2 mutations ,28. The et al. reported that PTEN-deficient cells were more sensitive to a PARP inhibitor than PTEN-wild type endometrial cancer cells [PTEN mutations did not result in high sensitivity to olaparib. One of the 4 PTEN wild-type cells and three of the 12 PTEN mutant cells were classified as sensitive (SF50 ≦ 100 nM). Moreover, all the 4 resistant cell lines  were PTEN mutant. The report by Dedes et al. included only 2 PTEN wild-type cell lines (HEC-1B and EFE-184), whereas 4 PTEN-wild type cell lines were included in our study. All the SF50 values in these 4 cell lines were 340 nM or lower. In addition, they did not include 3 of the 4 “resistant” (and PTEN mutant) cell lines in this study  [Clonogenic assays revealed that sensitivity to olaparib is greatly distinct among endometrial cell lines, with SF50 values ranging from 8 nM to 2,500 nM. The high ratio (25%) of sensitive cells with SF50 values <100 nM suggests that endometrial cancers are good candidates for PARP inhibitors such as olaparib. Recently, the possibility of a relationship between PTEN status and sensitivity to PARP inhibitors has received much attention ,22,30. Der cells . HoweverHEC-108) . The conTherefore, we further examined the relationship between PTEN and HR-related proteins in endometrial cancer cells. RAD51 mediates the formation of DNA joints that link homologous DNA molecules . SeveralOne of the earliest events in the signal transduction cascade initiating DSBs is the phosphorylation of serine 139 of histone H2AX (γH2AX) and RAD51 filament formation on DNA ,35. Our PTEN mutations.We also examined whether the response to IR-induced DNA damage is affected by PTEN status. IR directly produces DNA DSBs . After IOur study has some limitations. Predictive biomarkers for olaparib are still unknown. Further studies are warranted to elucidate whether PTEN inactivation is a biomarker for resistance to olaparib. The role of PTEN might be distinct when a PARP inhibitor was administered in combination with another drug (or irradiation), as the combination of a PARP inhibitor with cisplatin or irradiation was reported to be effective in PTEN deficient cells ,40. AddixIn conclusion, our findings have noteworthy implications: PTEN inactivation is not a good biomarker of olaparib treatment and PARP inhibitors might still provide a promising therapeutic strategy in certain endometrial carcinomas.All the authors declare no competing interests.AM performed the experiments and wrote the manuscript. KO (corresponding author) supervised the experiments and wrote the manuscript. YI wrote the manuscript. OH-W, TK, TK, TF, KI, KS, YU, RK, KN, YM, TA, SN, TY, KK, YO, and TF contributed reagents, materials, experimental techniques, and data analysis. AE, NH, and KM contributed experiments using IR. HK established 11 HEC endometrial cancer cell lines. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/179/prepubResponse to olaparib was not influenced by PTEN expression in endometrial cancer cells. (A) PTEN was knocked down by siRNAspecific to PTEN at 10nM (si PTEN) in two PTEN-wild type cell lines (HEC-1B: left and HEC-50B: middle). Non-silencing siRNA (si CT) was used as a control. Parental cells are also used as a control (CT). Cells were continuously exposed to olaparib for 14 days of incubation with media. Suppression of PTEN expression was examined by Western blotting. In clonogenic assay, the surviving fraction was not significantly affected by knocking down PTEN in these two cell lines. (B) HA-tagged wild-type PTEN expression plasmid (pcDNA3-HA-PTEN) was transfected into PTEN-null AN3CA cells (ad PTEN). pcDNA3 plasmid with HA-tag alone (ad CT) was used as a control. Cells were continuously exposed to olaparib for 14 days of incubation with media. Exogenous PTEN expression was confirmed by Western blotting. In clonogenic assay, the surviving fraction was not significantly affected by introduction of wild-type PTEN in AN3CA cells.Click here for file


We examined the impact of acute GVHD on the kidney in allogeneic BMT rats and compared them with those in Lewis-to-Lewis syngeneic BMT control and non-BMT control rats. In syngeneic BMT and non-BMT control rats, acute GVHD did not develop by day 28. In allogeneic BMT rats, severe acute GVHD developed at 21–28 days after BMT in the skin, intestine, and liver with decreased body weight (>20%), skin rush, diarrhea, and liver dysfunction. In the kidney, infiltration of donor-type leukocytes was by day 28. Mild inflammation characterized by infiltration of CD3+ T-cells, including CD8+ T-cells and CD4+ T-cells, and CD68+ macrophages to the interstitium around the small arteries was noted. During moderate to severe inflammation, these infiltrating cells expanded into the peritubular interstitium with peritubular capillaritis, tubulitis, acute glomerulitis, and endarteritis. Renal dysfunction also developed, and the serum blood urea nitrogen (33.9±4.7 mg/dL) and urinary N-acetyl-β-D-glucosaminidase (NAG: 31.5±15.5 U/L) levels increased. No immunoglobulin and complement deposition was detected in the kidney. In conclusion, the kidney was a primary target organ of acute GVHD after BMT. Acute GVHD of the kidney was characterized by increased levels of urinary NAG and cell-mediated injury to the renal microvasculature and renal tubules.Allogeneic hematopoietic cell or bone marrow transplantation (BMT) causes graft-versus-host-disease (GVHD). However, the involvement of the kidney in acute GVHD is not well-understood. Acute GVHD was induced in Lewis rats (RT1 Allogeneic hematopoietic cell transplantation (HCT) is a clinical treatment for a variety of conditions, including hematologic disorders, metabolic storage diseases, immune deficiencies, and is used as a rescue technique after cancer treatment In patients with HCT, multiple factors have been linked to the development of renal impairments, including preexisting renal injury, direct effects of conditioning chemotherapy and irradiation, complications of the infused cryopreserved cells, tumor lysis syndrome, calcineurin inhibitor used for graft-versus-host disease (GVHD) prophylaxis, and infection and its treatment a, RT1Aa) into lethally irradiated Lewis rat  recipients without immunosuppression In the present study, to clarify whether acute GVHD develops in the kidney, we used the major histocompatibility complex–disparate rat allogeneic bone marrow transplantation (BMT) model. We used the already established rat GVHD model, which involves transplantation of bone marrow cells (BMCs) from DA rats . We used inbred male DA and Lewis rats  that weighed 190–220 g and 220–270 g, respectively. All animals received humane care in compliance with the Guideline by the Committee of Nippon Medical School.7 BMCs from the DA or Lewis rats were then injected into Lewis rat recipients via the tail vein.BMC suspensions were harvested from DA and Lewis rats by flushing the marrow from the femurs and tibias with cold RPMI 1640  supplemented with 2.5% fetal bovine serum and 25 mM HEPES. Recipient Lewis rats were irradiated with a dose of 10 Gy  prior to BMT. After 2–3 h, 6.0×10In this model, acute GVHD developed by day 21 to day 28 in allogeneic BMT rats. The growth of transplanted BMCs, body weight, degree of acute GVHD, liver and renal functions, pathology, and cytokines milieu were evaluated by day 28 in allogeneic BMT rats (n = 5 at each time point), Lewis-to-Lewis syngeneic BMT control rats (n = 5 at each time point), and non-BMT control rats (n = 3 at each time point).a (donor-type cells), CD6+ T-cells, CD8+ T-cells, CD4+ T-cells, and CD68+ macrophages. Peripheral blood mononuclear cells were treated with anti-mouse CD16/32 Ab (clone 2.4G2) to block the Fc-receptors followed by direct or indirect staining of fluorochrome-conjugated antibodies , and flow cytometry was conducted to assess the expression of RT1Atibodies . Dead ceThe degree of systemic GVHD was assessed using a standard scoring system that incorporated five clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity To study the histological features of GVHD, the skin, liver, intestine, and kidney tissues were fixed in 20% buffered formalin and embedded in paraffin for light microscopic examination. Tissues were stained with hematoxylin and eosin (H&E) and periodic and acid-Schiff (PAS) staining for histopathological examination, and naphthol AS-D chloroacetate esterase staining to detect neutrophils. The primary antibodies used for immunohistochemistry are indicated in In each kidney sample, the number of infiltrating CD3+ T-cells, CD8+ T-cells, and CD68+ macrophages was examined in 40 randomly selected interstitial fields at ×200 magnification, by an investigator blinded to the clinical or histological findings.a,b antibody (donor type of RT1Aa) and phycoerythrin (PE)-conjugated anti-rat CD45 antibody (leukocyte common antigen) was performed. To detect CD8+ T-cells or CD4+ T-cells, double stain with FITC-conjugated anti-rat CD3 antibody and PE-conjugated anti-rat CD8α antibody or anti-rat CD4 antibody was performed. To evaluate the expression of MHC class II in renal tubules, immunostaining with PE-conjugated anti-rat RT1B antibody was performed. In each kidney sample, more than 100 cross-sections of renal tubules were graded semiquantitatively based on the specimens stained for rat RT1B, using the following grading system: absence of MHC class II staining; 0, mild increase of MHC class II staining; 1, moderate increase of MHC class II staining; 2, marked increase of MHC class II staining; 3.Immunofluorescence study was also performed using frozen tissues. The deposition of IgM, IgG, and C3 was evaluated using the indirect method. To detect the donor type of leukocytes in the kidney, double stain with fluorescein isothiocyanate (FITC)-conjugated anti-rat RT1AReal-time reverse transcription-polymerase chain reaction  was performed as described previously t-test using an analytical software program  or ANOVA determined by analysis of variance which performed Fisher's one. Differences were considered significant at the 95% confidence intervals (P<0.05).The results were expressed as mean ± standard deviation. Differences were evaluated using either the Student After 10 Gy irradiation and allogeneic or syngeneic BMT, circulating leukocytes markedly decreased (by>95%) on day 7, but recovered by day 14 . In alloIn syngeneic BMT rats, circulating CD6+ and CD8+ T-cell levels gradually recovered by day 28 . In alloAfter DA to Lewis allogeneic BMT, the body weight of Lewis recipient rats gradually decreased by day 28 by>20% compared with that of pretransplantation . By contIn conjunction with the development of acute GVHD in the skin, liver, and digestive duct, renal function abnormalities developed by day 28. Serum BUN and urinary NAG levels increased on day 28 , indicatPathology of the kidney with acute GVHD indicated mononuclear cell infiltration to the interstitium . Acute GOn day 28, infiltrating cells in the kidney were characterized by a large number of CD3+ T-cells and ED1+ macrophages in the interstitium . CD3+ T-The occurrence of GVHD is a major complication after HCT. In the present study, although the skin, liver, and digestive tract were found to be the main target organs of GVHD, we demonstrated that the kidney could also be a direct target organ of acute GVHD following BMT. Pathological examination revealed infiltration of mononuclear cells, CD3+ T-cells, mainly CD8+ T-cells, CD4+ T-cells, and macrophages, in the renal interstitium with peritubular capillaritis, tubulitis, acute glomerulitis, and endarteritis. These findings suggest the development of T cell-mediated injury in the renal microvasculature and renal tubules during acute GVHD of the kidney. Increased urinary NAG level was an early marker of acute GVHD in the kidney, whereas serum Cr and urinary protein levels were stable. Acute GVHD in rat BMT model in the present study was different condition from the clinical human GVHD after HCT, because human GVHD is induced in rich stem cell transplantation with a very close degree of HLA matching between donor and recipient, and use the immunosuppressant or conditioning regimens to prevent GVHD. Therefore, further studies are needed to evaluate the acute GVHD of the kidney in human GVHD after clinical HCT.After allogeneic HCT, GVHD is a major cause of morbidity and mortality, responsible for 15–40% of mortality On the other hand, acute renal disorders after HCT are considered to be mediated by multiple factors Recently, several studies have reported that GVHD can involve renal insufficiency In the present study, we found significant infiltration of donor leukocytes in the kidney, and that infiltration of CD3+ T-cells, CD8+ T-cells, CD4+ T-cells, and macrophages mediated renal inflammation with peritubular capillaritis, tubulitis, acute glomerulitis, and endarteritis in allogeneic BMT recipients with systemic acute GVHD. Our findings of acute GVHD in the kidney were quite similar to pathological findings, as acute T cell-mediated rejection of the kidney in allogeneic renal transplantation GVHD is caused by host-reactive T-cells derived from the donor bone marrow itself, or from the peripheral blood that contaminates the BM during its preparation In the GVHD pathophysiology, both cellular factors (such as donor T-cells and macrophages) and soluble factors (cytokines) play a role in the development of acute GVHD. Based on the cytokine profile, the Th1 cytokines  have been implicated in the pathophysiology of acute GVHD Based on laboratory findings, serum BUN and urinary NAG levels increased in acute renal GVHD in the present model. Inflammatory damage to the renal tubules from GVHD may be associated with an increase in the urinary NAG levels. We speculate that urinary NAG levels may be an early marker of renal GVHD that can be detected when serum Cr and urinary protein levels are stable. Further studies are needed to clarify the occurrence of acute renal GVHD after clinical HCT, the correlation between acute renal GVHD and urinary NAG levels in human GVHD, and useful pre-emptive therapy to improve transplant outcome after clinical HCT.In summary, the kidney may be a target organ of GVHD, and the increased urinary NAG levels after BMT may indicate the development of acute GVHD of the kidney. As the number of HCTs increases every year, hematologists, nephrologists, oncologists, and pathologists should work together to identify patients with acute GVHD of the kidney to both prevent its development and initiate therapy early to improve outcomes after HCT.


Five cell lines were established from a Singaporean patient of Chinese origin with breast ductal carcinoma in situ (DCIS). These five cell lines express exogenous human telomerase reverse transcriptase (hTERT) which confers the ability to proliferate indefinitely.Cells were isolated from the DCIS excision and transfected with a plasmid expressing hTERT, a catalytic subunit of telomerase. Five immortalized colonies were propagated and characterized by karyotyping, array comparative genomic hybridization (CGH), immunostaining and Western blots for biomarkers, in vitro anchorage independent growth, in vivo mouse tumorigenicity, drug sensitivity, species authentication and virology safety testing.Array CGH analysis showed that the cell lines harbored different specific genetic aberrations. Common mutations observed in most breast cancer cell lines such as the general loss of heterozygosity (LOH) throughout chromosome X and chromosome 17 are also observed in our cell lines. The cell lines were further characterized as human breast cells that are estrogen- and progesterone-receptor positive, and sensitive to tamoxifen. The cell lines showed anchorage-independent growth in the soft agar assay and can grow in common culture medium without supplementation with growth factor, therefore demonstrating transformed characteristics. Four of the cell lines can engraft and form measureable tumors after 50 days when injected subcutaneously into immune-deficient (SCID) mice. The weak tumorigenicity of these cell lines corresponded well with their non-malignant growth origin. The cell lines were authenticated to be of human origin based on DNA fingerprint of the cells. The cell lines were free from contamination of 20 viruses and mycoplasma in the virological safety test panel.Unlike most available breast cell lines, our cell lines are derived from primary breast cancer tissues that represent earlier grades or tumor progression stages. They would be useful as study models for basic and clinical research programs directed at early diagnosis and intervention. There are many benefits in using primary cell cultures with little or no laboratory-induced transformation. The behaviors of these cells are hence more truly reflective of the organ of origins. However, the slow population doubling time and the finite lifespan of these primary cells hinder large scale experimentation that require continuous supply of cells with consistent behavior. Primary cultures require supplementation of growth factors and bovine pituitary extract which are costly and suffer from batch to batch variations. There is also ethical pressure on scientists to reduce the use of animal in laboratory research.. Cell lines continue to be used as models for medical research due to their ease of use and storage, and consistent cell behavior.On the other hand, immortalized cell lines are the in vitro equivalent of cancerous cells as they maintained many characteristics of the cancer tissue from which it was derived. Cancer occurs when a somatic cell which normally cannot divide undergoes alterations which cause de-regulation of the normal cell cycle controls leading to uncontrolled proliferation. Immortalized cell lines have undergone similar alterations allowing a cell type which would normally not divide to proliferate in vitroThe most commonly used breast cancer cell line in the world is MCF-7. This, together with other commonly used breast cell lines such as the MDA-MB-series, are not derived from primary breast tumors but from tumor metastases in pleural effusions . Cells We have established five sub-lines derived from primary tissue obtained from a Singapore female patient with ductal carcinoma in situ, a common type of non-invasive breast cancer. The five sub-lines were obtained following over-expression of the human telomerase reverse transcriptase (hTERT) in primary cells. hTERT alone can immortalize cells without causing cancer-associated changes or altering phenotypic properties . EstabliThe Cancer Cell Isolation kit (Panomics) was used to isolate cancer cells from tissue obtained from a Singapore female with breast ductal carcinoma in situ. The resulting cell line was named ETCC001. ETCC001 cells were grown and maintained in M171 media. To verify the species origin of ETCC001, the cells were sent to IDEXX Laboratories, Minnesota, USA for STR DNA fingerprinting and PCR species evaluation. The alleles for nine different STR markers were established  depicted a normal karyotype  was transfected into ETCC001 cells. The pGRN145 plasmid contains a hygromycin resistance gene. Transfected ETCC001 cells were seeded in 15-cm plates. After two weeks, five hygromycin-resistant clones were obtained and named ETCC006, ETCC007, ETCC008, ETCC010 and ETCC011. There was a dramatic increase of telomerase activity in ETCC006, ETCC007, ETCC008, ETCC010 and ETCC011, compared to ETCC001 Figure A. The clOver-expression of the TERT component of telomerase confers replicative immortality such that cells over-expressing it would continue to proliferate indefinitely . To deteThe average population doubling time of ETCC001 and the cell lines are about 20 hours  is commonly used as an epithelial cell marker and it was used to determine whether the origin of cell lines are epithelial cells . The celTo confirm the results of the Western blots and to check for other markers for the epithelial cells, immunofluorescence assays were done Figure B. The epAPC (5q21) and TERT (5p15) gene copy numbers observed in ETCC006, ETCC007 and ETCC011. Significant LOH was observed in chromosome 17 in ETCC006, ETCC007, ETCC010 and ETCC011. Figure TP53 (17p13) and copy number neutral LOH in MAP2K4 (17p11) in all cell lines except ETCC008. Copy number neutral LOH in BRCA1 (17q21) is observed in ETCC007, ETCC010 and ETCC011. The gene for a regulatory subunit of protein kinase A, PRKAR1A (17q23), was loss or LOH in ETC007, ETCC010 and ETCC011.Genomic analysis was performed on the cells (ETCC001 at passage 100 and the hTERT-expressing cell lines at passage 50–60) to determine if they contained genomic mutations commonly found in breast cancer. Digital karyograms documenting the gene gain, gene loss and loss of heterozygosity (LOH) are shown in Figure These cell lines showed differences in their growth pattern and acceptance rate. ETCC001 and ETCC008 did not show any tumor growth and were observed to be non-tumorigenic up to 90 days. ETCC006, ETCC007, ETCC010 and ETCC011 cell lines are tumorigenic and vary in their acceptance rate and tumor growth pattern. Mice injected with ETCC011 and ETCC007 exhibited overall good acceptance (8/8) and take rate (100%) whereas the animals injected with ETCC010 and ETCC006 exhibited lower take rate of 87.5% (7/8) and 75% (6/8) respectively. Tumor volume and body weight data are represented in Figure The cells were treated with compounds commonly used for breast cancer therapy in the clinics. In general, the cell lines were not sensitive to small molecule inhibitors paclitaxel, 5-fluorouracil, gemcitabine, cisplatin and camptothecin Table . The celIn this study, we have established 5 cell lines from primary cells from breast cancer tissue obtained from a Chinese female patient of Singapore origin. The primary cell line, ETCC001, is confirmed to be of human origin and free from inter-species contamination.8 discrimination rate for unrelated individuals).Cell lines are widely used in many aspect of laboratory research. It has been reported that 18% of new cell lines deposited at the German Cell Line Bank were found to be cross-contaminated . It is iBreast tumor biopsies are heterogeneous and it is possible that the derived cell lines could be contaminated with fibroblasts and normal epithelial cells. In order to rule out these possibilities, we have performed magnetic sorting for fibroblasts and surface marker analyses of the cell lines. The cell lines exhibit typical epithelial markers (pan-CK) as well as epithelial cancer markers CK19 and EMA ,8.Human mammary epithelial cells have been found to stop dividing at 55–60 population doublings. Transfection of these cells with hTERT are found to extend cell division to over 250 population doublings . In our We report here the establishment of breast cell lines from non-invasive breast DCIS that express both ER and PR. Most common breast cell lines such as the MDA-MB-XXX series, BT20, SkBr3 and ZR75.1 do not express ER and PR. On the other hand, there are fewer choices of cell lines positive for the expression of ER and PR, examples being MCF-7 and T47D. Even though MCF7 and T47D express both ER and PR, they are of metastatic origin and are derived from lung pleural effusion  and BC-0BRCA1 wild-type allele inherited from the unaffected parent through LOH represents the event that initiates the tumorigenesis process observed in primary breast and ovarian tumors [BRCA1 copy number neutral LOH in three of the derived cell lines. Chromosome 17 is also a chromosome that suffers most LOH and/or loss of genes in all the derived cell lines. Besides BRCA1, other important cancer causal genes on chromosome 17 such as TP53, MAP2K4, ERBB2 and PRKAR1A [MAP2K4 and BRCA1 have also been reported in several breast cancer cell lines [Karyotyping with GTL-banding of metaphase cells shows that ETCC001 is heteroploid with multiple chromosomal abnormalities. Our CGH analysis also shows chromosomal variations among the cell lines. We observed LOH on the X-chromosomes on ETCC001 and all its derived cell lines. LOH on the X-chromosome have been reported to be associated with human breast cancer ,22 and on tumors . We have PRKAR1A  are all ll lines . This maOne of the definitive assays to determine whether cells are transformed is anchorage-independent growth. All cell lines, except ETCC001, can form colonies in soft agar. This suggests that ETCC001 is untransformed while the cell lines over-expressing hTERT are transformed. hTERT levels do not correlate with the ability of the cells to form colonies in soft agar and tumorigenicity in SCID mice. It is likely that the changes in gene copy number observed in the CGH study could have contributed to their ability to grow in the soft agar. Extended number of cell doublings through bypassing cellular senescence would allow accumulation of genetic changes favoring cell survival. Extension of lifespan by telomerase was reported to select for c-myc over-expression in human mammary epithelial cells . We haveOur hTERT-transformed cell lines can form colonies in soft agar but are only weakly tumorigenic in the SCID mice. The weak tumorigenicity in SCID mice is representative of its origin as a non-invasive breast ductal carcinoma in situ. Cells derived from certain epithelial tissues have been reported to have in vitro anchorage-independent phenotype that is not associated with tumorigenicity . These eSensitivities toward tamoxifen and doxorubicin have decreased in the hTERT-expressing cell lines compared to the primary ETCC001 cell line. This could be due to more genetic aberrations in the hTERT-expressing cell lines leading to loss of tumor suppressors and/or activation of oncogenes as discussed above. However, this does not imply a loss of the DCIS identity as we still observed weak tumorigenicity in SCID mice with these hTERT-expressing cell lines which is consistent with the non-invasive nature of DCIS in early phase breast cancer.We have established five cell lines from a patient with breast ductal carcinoma in situ. The cell lines were derived from cells that have been stably transformed with hTERT. A compendium of genetic, physical and molecular characterizations has demonstrated that the five cell lines carry distinct genetic changes. The cell lines also express estrogen and progesterone receptors, CK19, EMA and other surface biomarkers, confirming that they are of breast epithelial origin. The cell lines together with the information that we have acquired will provide a rich resource in drug discovery and cell physiology studies for early stage breast cancer.2.Tissue was obtained from breast ductal carcinoma in situ (DCIS) of a 47 year-old female of Chinese origin. The Cancer Cell Isolation Kit (Panomics) was used to obtain primary cells from the tissue. Fibroblasts were removed using the MAC Separator (Miltenyi Biotech). Primary cells obtained were labeled as ETCC001 and were grown in M171 media (Life Technologies) with mammary epithelial growth supplement (MEGS). Cell lines arising from ETCC001 after hTERT transformation were grown in RPMI supplemented with 10% fetal bovine serum (FBS), L-glutamine and Penicillin/Streptomycin (Life Technologies). Cell cultures were maintained in a humidified incubator at 37°C with 5% atmospheric CO3 hygromycin-resistant cells were seeded in a 15-cm petri dish and grew for at least 2 weeks. Single cell colonies were then amplified. Telomerase activity in these cell colonies was measured using the TRAPeze Telomerase Detection Kit (Millipore). Heat inactivated samples were used as negative controls for the telomerase activity detection.pGRN145 vector containing hTERT sequence (ATCC) and hygromycin resistant gene was used to transfect ETCC001 primary cells. Transfection was performed using Lipofectamine 2000 (Life Technologies). 30 μg/ml of Hygromycin (Life Technologies) was used to select for positively transfected cells. 1 × 10ETCC001 cells in metaphase were fixed and stained for Giemsa/Trypsin/Leishman (GTL)-banding to obtain karyograms . Cell pellets were also sent to IDEXX Laboratories Inc  for further authentication and virological safety testing. Short tandem repeat (STR) analysis were done to establish cell line identity, inter-species . The cell lines were tested for contaminations by polymerase chain reaction (PCR) screening for 18 types of virus, and mycoplasma.Western blot and Immunofluorescence staining were performed to detect breast and epithelial cell markers. Primary antibodies used were mouse anti-EMA (Dako), rabbit anti-Pan-Ck (Abcam), rabbit anti-Ck19 (Abcam), mouse anti-estrogen receptor (ER) α (Abcam), rabbit anti-ERβ (Abcam) and mouse anti-progesterone receptor (PR) (Abcam). Secondary antibodies used in Western blot analysis were horse-radish peroxidase conjugated goat anti-rabbit and anti-mouse antibodies (Dako). Secondary antibodies used in immunofluorescence study were Alexa Fluor 594 goat anti-rabbit (Life Technologies) and Alexa Fluor 488 goat anti-mouse (Life Technologies). Antibody concentrations used in Western blot and immunofluorescence staining were as recommended by the antibody manufacturers.5 cells were seeded in T-25 flasks and placed in the IncuCyte. Cell growth was monitored until confluency was achieved. Analysis was performed with the IncuCyte software which auto-generates growth curve according to cell confluency.Cell doubling time was determined using the IncuCyte real-time cell analyzer (Essen Bioscience). The Incucyte uses cell confluency as a measure of cell growth. 1 × 106 cells were sent to Origen Labs (Singapore) for CGH array hybridization using the Affymetrix SNP 6.0 platform. Data analysis was performed with Affymetrix Chromosome Analysis Suite.Cell pellets containing 1 × 102 for 2 to 3 weeks. After overnight staining with tetrazolium dye, MTT (Sigma) at 70 μl per well at 5 mg/ml, the colonies were counted using the GelCount® instrument (Oxford Optronix).Soft agar colony formation assay was done in 24-well plates. Each well contained 0.6 ml of 0.6% agar (Sigma) in complete medium in the bottom layer, 0.5 ml of 0.36% agar in complete medium with cells in the middle layer, and covered with 0.5 ml medium. The cells were cultured at 37°C with 5% atmospheric CO6 cells from ETCC001 and the five hTERT-expressing cell lines were injected subcutaneously into the right flank of each female SCID mice (age 6–8 weeks). Eight mice were injected for each cell lines. All the animals were observed for clinical signs, body weight, tumor volume & mortality. Clinical signs, body weight and tumor volume were recorded twice weekly throughout the experiment. The mice were sacrificed at the end of the experiment. A piece of each palpable tumor was snap frozen or fixed in 10% Neutral-buffered formalin solution for histopathology study by hematoxylin and eosin (H&E) staining. The study was carried out at the A-STAR Biological Resource Center - an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility. All animals were handled humanely with due regard for their welfare. The study was designed to use the fewest possible number of animals. The study design was reviewed and approved by the Institutional Animal Ethics Committee.10 × 1050) was determined. Error bars denote standard deviation (SD).Tamoxifen, paclitaxel, 5-fluorouracil, gemcitabine, cisplatin, doxorubicin and camptothecin were purchased from Calbiochem, and herceptin was purchased from Roche. Cells were seeded in 50 μl medium in 96-well plates at 8000 cells/well and incubated overnight. Compounds (50 μl) were added to cells and incubated for 48 hours.100 μl of CellTiter-Glo (Promega) was added to the cells. After 10 minutes of incubation at room temperature, luminescence was measured with Safire II plate reader (Tecan). Data was analyzed with Graphpad Prism software and the half maximal inhibitory concentration (IC


The application of human embryonic stem cell (hESC) derivatives to regenerative medicine is now becoming a reality. Although the vast majority of hESC lines have been derived for research purposes only, about 50 lines have been established under Good Manufacturing Practice (GMP) conditions. Cell types differentiated from these designated lines may be used as a cell therapy to treat macular degeneration, Parkinson’s, Huntington’s, diabetes, osteoarthritis and other degenerative conditions. It is essential to know the genetic stability of the hESC lines before progressing to clinical trials. We evaluated the molecular karyotype of 25 clinical-grade hESC lines by whole-genome single nucleotide polymorphism (SNP) array analysis. A total of 15 unique copy number variations (CNVs) greater than 100 kb were detected, most of which were found to be naturally occurring in the human population and none were associated with culture adaptation. In addition, three copy-neutral loss of heterozygosity (CN-LOH) regions greater than 1 Mb were observed and all were relatively small and interstitial suggesting they did not arise in culture. The large number of available clinical-grade hESC lines with defined molecular karyotypes provides a substantial starting platform from which the development of pre-clinical and clinical trials in regenerative medicine can be realised. Since the derivation of human embryonic stem cells (hESCs) from blastocysts in 19981www.mrc.ac.uk/research/facilities/stem-cell-bank; stemcells.nih.gov).In recent years, there have been advances in the derivation of hESC lines whereby fully defined media devoid of animal-derived products is used68911121315161718202122in vitro fertilization (IVF) units to provide GMP-compliant embryos for hESC line derivation and further funded the Human Embryonic Stem Cell Co-ordinators (hESCCO) network, subsequently the National Clinical hESC Forum. This allowed the derivation centres to work with the Human Fertilisation and Embryology Authority and the UK Stem Cell Bank to establish common parameters for patient consent, screening and embryo procurement for the derivation of clinical-grade hESC lines. Ultimately this farsighted policy has yielded a cohort of hESC lines which have benefited from the shared implementation of GMP-compliant IVF laboratory standards, hESC derivation procedures and ethical principles for donor consentingRemarkably, 38 of these lines have been derived among five different centres in the United Kingdom through funding from the Medical Research Council (MRC), Scottish Enterprise, the North West Development Agency and the Juvenile Diabetes Research Foundation. The MRC launched an initiative in 2005 to provide infrastructure funding to UK in vitro differentiationThe value of a large number of different cell lines as starting material for clinical applications is three-fold: (a) different hESC lines have varying propensities to generate specific cell lineages during BCL2L1, within this region has been shown to be a driver of growth advantage36in vitro differentiation from genetically healthy PSCs can also lead to genomic instabilityIn accordance with GMP standards applicable to the sourcing and the application of raw materials used during production processes, such as hESC/iPSC cell derivation and differentiation into desired cell types, there is emerging evidence that equal scrutiny should be undertaken to account for the genetic health of cells at all stages of production32634de novo genomic alteration that arose during development of the blastocyst or during establishment and maintenance of the cell line.However, most CNVs are benign and relatively large duplications and deletions (>100 kb) are common in healthy individualsAs well as CNVs, copy-neutral loss of heterozygosity (CN-LOH) represent another form of genomic structural variation characterised by a stretch of homozygosity along part of a chromosome43In this study, we sought to examine the genetic integrity of 25 clinical-grade hESC lines utilising whole-genome SNP genotyping analysis. While karyology is sufficient for establishing genetic normalcy within current regulatory standards, advances in technology and an increasing cytogenetic knowledge base demand higher resolution investigation of cell lines and cell products designed for clinical use.Human ES cell lines were cultured in feeder-free conditions prior to the isolation of genomic DNA . The DNA333648424950http://dgv.tcag.ca) to determine whether the CNVs have been previously observed in healthy individuals41Of the 15 unique CNVs detected, 12 were heterozygous duplications, 2 were heterozygous deletions, and 1 was a homozygous deletion . We askeMLLT4, KIF25, FRMD1—was represented on the DGV and has been reported in the healthy population at a frequency of over 1 in 50 individuals  in healthy individuals3951395153543951AKAP13, and this CNV was not found in the sibling hESC line, Man12. One of the CNVs detected in MasterShef7, a 315 kb duplication present on chromosome 14q21.3, contains a single gene, MDGA, and this CNV is also present on the DGVAmongst the 15 large hESC CNVs, we found 10 had clear evidence of being present in healthy individuals. For example, a duplication of 267 kb on chromosome 6q27 observed in MasterShef3 containing 3 protein-encoding genes—ividuals  485153. A3 genes . AlthougBoth heterozygous deletions and the homozygous deletion were found to be naturally occurring in the human population. Two unrelated cell lines, KCL031 and RC9 hESCs harboured the same 120 kb deletion on chromosome 8q24.23 . This CN39485153555639485155Cadherin-18, that was present in two sibling cell lines, KCL032 and KCL033 CCDC85A in the CN-LOH of RC11 is predicted, but not validated, to be an imprinted gene on this database.In addition to the 15 CNVs identified, our analysis detected 3 regions of CN-LOH greater than 1 MB among three different hESC lines, KCL040, MasterShef5, and RC11 . All thr6163While none of the CNV and CN-LOH regions observed in the hESCs appear to harbour genomic anomalies associated with culture adaptation at the passages reported here , we have36Our molecular karyotypic evaluation of 25 clinical-grade hESC lines has established a valuable platform for the development and manufacture of cell therapy products for clinical application in regenerative medicine.Approval for the use of all hESC lines used in this study was granted by the MRC Steering Committee for the UK Stem Cell Bank and for the Use of Stem Cell Lines. The following clinical-grade hESC lines were kindly provided by the following derivation centres: The University of Sheffield (12 lines) MasterShef2, MasterShef3, MasterShef4, MasterShef5, MasterShef7, MasterShef8, MasterShef10, MasterShef11, MasterShef12, MasterShef13, MasterShef14 and Shef6; King’s College London (8 lines) KCL031, KCL032, KCL033 KCL034, KCL037, KCL038, KCL039, and KCL040; Roslin Cells Ltd (3 lines) RC9, RC11, and RC17; and Central Manchester NHS Foundation Trust/The University of Manchester (2 lines) Man11 and Man12. Most of the cell samples were provided as frozen cell pellets, which were directly processed for genomic DNA isolation. However, Man11 and Man12, sibling hESC lines, were provided as cryopreserved hESC lines, and Shef6 was obtained directly from the UK Stem Cell Bank (UKSCB Accession No. R-05-031). These three lines were thawed and cultured in Essential 8 media (Life Technologies) on Laminin-521 substrate (Biolamina) for less than 10 passages before pelleting by centrifugation for genomic DNA isolation. Human ES cell morphology and pluripotent marker, NANOG, expression were maintained during this expansion . NANOG iGenomic DNA was isolated from cell pellets using the MasterPure™ Complete DNA and RNA Purification Kit (Epicentre) according to the manufacturer’s instructions. Briefly, cell pellets were lysed with Tissue and Cell Lysis Solution, followed by Proteinase K and RNase A treatment. Proteins were precipitated with MPC Protein Precipitation Reagent and removed by centrifugation. Genomic DNA was precipitated with isopropanol, pelleted by centrifugation and then resuspended in TE buffer to a final concentration of 50 ng/μl. Purity was checked by spectrophotometry using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific).All samples were subjected to HLA typing at the Histocompatibility and Immunogenetics (H&I) Laboratory of the Scottish National Blood Transfusion Service (SNBTS). The HLA typing data  was usedhttp://dgv.tcag.ca) to identify naturally-occurring structural variations in the human genome41https://decipher.sanger.ac.uk/) to determine if they were associated with any clinical syndromeshttp://www.geneimprint.com) to determine if the genomic variants occurred in known imprinted regionsGenomic DNA samples were assayed using the Illumina HumanCytoSNP-12 v2.1 BeadChip, at either AROS  or the Wellcome Trust Clinical Research Facility . The data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE68508. Genotyping was initially assessed using GenomeStudio genotyping module . KaryoStudio  was employed to perform automatic normalisation and to identify genomic aberrations utilising default settings of the built-in cnvPartition algorithm  to generate B-allele frequency and smoothened Log R ratio plots for detected regions. These parameters are designed to detect CNVs greater than 75 kb and CN-LOH regions larger than 1 MB with a confidence value greater than 35. All identified duplication and deletions were first cross-matched to the Database of Genomic Variants .


In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Thus, pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport.Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPARα, PPARγ, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPARα, and PPARγ represent potential anti-cancer drug targets, we here investigated the effects of 39 pirinixic acid derivatives on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability and, subsequently, the effects of selected compounds on drug-resistant neuroblastoma cells. Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anti-cancer effects and the effects on 5-LO, mPGES1, PPARα, or PPARγ. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATP-binding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2μM with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calcein-AM but not of doxorubicin, rhodamine 123, or JC-1.  Pirinixic acid WY-14,643) was discovered as a peroxisome proliferator-activated receptor (PPAR)α agonist in 1974 4,643 was. In the 5-LO, mPGES1, PPARα, and PPARγ are also potential drug targets for anti-cancer therapies –17. Howe50) in the tested concentration range up to 100μM, compounds that displayed IC50s between 10μM and 100μM, and compounds that displayed IC50s below 10μM. Although there were substantial similarities between the effects of most compounds on the viability of both cell lines, there were also some substantial differences  showed IC50 values between 10μM and 100μM in UKF-NB-3 cells but IC50s >100μM in PC-3 cells. Five compounds  displayed IC50 values <100μM and >10μM in PC-3 cells but IC50 values >100μM in UKF-NB-3. Three compounds  had IC50 values <10μM in UKF-NB-3 cells and IC50 values >10μM and <100μM in PC-3 cells. Notably, there was no significant difference between the IC50 value of YS80 in UKF-NB-3 cells (9.87 ± 1.19μM) and in PC-3 cells (11.61 ± 7.83μM) although they were classified into different categories  included HZ49 (fold change >7.5), HZ51 (fold change 3.9), HZ53 (fold change 2.3), and LP123 (fold change 28.5). Compounds that were >2 times more effective in PC-3 than in UKF-NB-3 cells (fold change IC50 UKF-NB-3/IC50 PC-3 >2) included HZ37 (fold change >5.6), HZ74 (fold change >4.7), and LP119 (fold change >3.2). The lowest IC50 value was determined for LP123 in UKF-NB-3 cells (2.04 ± 0.69μM)  Figure .rCDDP1000), doxorubicin (UKF-NB-3rDOX20), and vincristine (UKF-NB-3rVCR10). In addition, we determined the effects of these compounds on the viability of Be(2)-C cells. Be(2)-C is a clonal sub-line of the neuroblastoma cell line SK-N-BE(2) that was isolated from a neuroblastoma patient after repeated courses of chemotherapy and radiotherapy [Next we compared the effects of pirinixic acid, HZ51, LP117, LP123, YS71, and YS80 on the viability of the cell line UKF-NB-3 and its sub-lines with acquired resistance to cisplatin  more resistant to HZ51 and LP117 than UKF-NB-3 cells. All other fold changes (IC50 resistant UKF-NB-3 sub-line/UKF-NB-3) were >0.5 and <2 -C2 Figure .50 67.64 ± 31.39μM; HZ37, IC50 >100μM; HZ59, IC50 >100μM; LP117, IC50 29.36 ± 12.42μM; YS71, IC50 26.51 ± 1.94μM; YS80, IC50 9.87 ± 1.19μM; YS81, >100μM, Figure rVCR10. In the absence of vincristine, these compounds had exerted similar effects on the viability of UKF-NB-3 and UKF-NB-3rVCR10 cells (IC50 UKF-NB-3rVCR10/IC50 UKF-NB-3 >0.5 and <2): HZ25, IC50 >100μM; HZ37, IC50 >100μM; HZ59, IC50 > 100μM; LP117, IC50 33.39 ± 9.11μM; YS71, IC50 33.25 ± 2.30μM; YS80, IC50 17.16 ± 0.80μM; YS81, >100μM  influenced rhodamine 123 accumulation in UKF-NB-3rVCR10 cells in the investigated concentrations , LP123 , and YS80  were the only compounds that displayed IC50 values below 10μM in at least one of the cell lines. Further research will have to show whether some of these compounds may exert promising anti-cancer activity in a broader range of models.In this study, we screened a panel of pirinixic acid derivatives for effects on the viability of the neuroblastoma cell line UKF-NB-3 and the prostate carcinoma cell line PC-3. HZ53 , doxorubicin- (UKF-NB-3rDOX20), and vincristine-resistant (UKF-NB-3rVCR10) sub-lines of UKF-NB-3 [50 resistant cell line/IC50 UKF-NB-3 < 2. All four resistant cell lines showed enhanced resistance to LP123 as indicated by fold changes IC50 resistant cell line/IC50 UKF-NB-3 > 2. HZ51 was similarly effective to UKF-NB-3 in all resistant cell lines but UKF-NB-3rDOX20, while UKF-NB-3rDOX20 and Be(2)-C cells were resistant to LP117. These findings suggest that the investigated pirinixic acid derivatives differ in the mechanisms underlying their effects on cancer cell viability.HZ51, LP117, LP123, YS71, and YS80 were characterized by varying activity profiles in a set of drug-resistant neuroblastoma cell lines consisting of the cisplatin- , UKF-NB-3 sub-lines adapted to doxorubicin (UKF-NB-3rDOX20) or paclitaxel (UKF-NB-3rPCL10), and the intrinsically ABCB1-expressing neuroblastoma cell line UKF-NB-4 [rDOX20 or UKF-NB-3rVCR10 cells to the non-ABCB1 substrate cisplatin. These data, together with the finding that LP117 stimulates ABCB1 ATPase activity, suggest that LP117 and other pirinixic derivatives interfere with ABCB1 function, possibly being substrates.A range of pirinixic acid derivatives sensitized ABCB1-expressing UKF-NB-3UKF-NB-4 ,24 but din silico docking studies indicated a notable difference in the interaction profile of doxorubicin with known ABCB1 binding sites compared to vincristine, vinorelbine, paclitaxel, and actinomycin D that may contribute to the discrepancies observed. LP117 and verapamil differed in their interactions with rhodamine 123. Only verapamil caused rhodamine 123 accumulation in ABCB1-expressing cells in the tested concentration range of up to 10μM. In accordance, in silico docking studies revealed recognizable disparities in the ABCB1 binding site interaction profiles of LP117 and verapamil.Most strikingly, LP117 sensitized ABCB1-expressing cells to ABCB1 substrates including vincristine, vinorelbine, actinomycin D, and paclitaxel but not to doxorubicin. Among the three fluorescent ABCB1 substrates, LP117 only caused cellular accumulation of calcein-AM but not of rhodamine 123 or JC-1 in the investigated concentration range. This suggests that LP117 interferes with ABCB1-mediated drug transport in a substrate-specific fashion. Such a substrate-specific interaction with ABCB1 function appears to be a common feature of the investigated pirinixic acid derivatives since HZ25, HZ37, HZ59, YS71, and YS80 all sensitized ABCB1-expressing cells to vincristine but did not cause rhodamine 123 accumulation in ABCB1-expressing cells. Verapamil also displayed stronger effects on the sensitivity of ABCB1-expressing cells to vincristine and paclitaxel than to doxorubicin. Indeed, rDOX20 (doxorubicin-resistant), UKF-NB-3rPCL10 (paclitaxel-resistant), and UKF-NB-3rVCR10 (vincristine-resistant). Verapamil sensitized UKF-NB-3rDOX20 cells to paclitaxel and vincristine in a similar fashion but displayed more pronounced effects on paclitaxel in UKF-NB-3rPCL10 cells and more pronounced effects on vincristine in UKF-NB-3rVCR10 cells. This may indicate that cancer cell adaptation to ABCB1 substrates may not only result in increased ABCB1 expression but potentially also to further changes that increase the specificity of ABCB1 to certain substrates.Our data shed further light on the complexity of substrate interactions with ABCB1 adding to previous reports that had suggested that the mode and/or strength of ABCB1 interaction may differ among ABCB1 substrates –30. NotaMore targeted approaches combining pharmacologically active ABCB1 substrates with tailored ABCB1 inhibitors would offer novel therapeutic opportunities. High ABCB1 levels represent an important cancer cell resistance mechanism –34. HoweIn conclusion, we provide evidence that pirinixic acid derivatives interfere with ABCB1-mediated drug transport in a substrate-specific manner.The structures of the investigated compounds are shown in The prostate carcinoma cell line PC-3 was obtained from DSMZ  and the MYCN-amplified neuroblastoma cell lines Be(2)-C and IMR-32 from ATCC . The alveolar rhabdomyosarcoma cell line Rh30 was kindly provided by Dr. P.J. Houghton . The MYCN-amplified neuroblastoma cell lines UKF-NB-2, UKF-NB-3, and UKF-NB-4 were established from stage 4 neuroblastoma patients ,22,35.http://www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html): IMR-32rVCR10 (vincristine), UKF-NB-2rVCR10, UKF-NB-3rCDDP1000 (cisplatin), UKF-NB-3rDOX20 (doxorubicin), UKF-NB-3rPCL10 (paclitaxel), UKF-NB-3rVCR10, Rh30rVCR10. Parental chemosensitive cell lines had been adapted to growth in the presence of anti-cancer drugs by continuous exposure to increasing drug concentrations as described previously [The following drug-adapted cell lines were derived from the resistant cancer cell line (RCCL) collection -2,5-diphenyltetrazolium bromide (MTT) dye reduction assay after 120 h incubation modified as previously described ,24,35,36The ABCB1-ATPase activity was determined using membrane preparations  and an established kit  following the manufacturer's instruction.The ABCB1-mediated efflux of fluorescent ABCB1 substrates was determined as described previously . Cells wProtonate3D protocol in MOE which consisted of capping termini and chain breaks, and protonation and charge correction using AMBER99 for charge assignment. The structure was minimized using the ligX protocol under “tethered” conditions to avoid any considerable displacements from the original crystal structure. The optimized structure was used first to validate our docking protocol by re-docking the co-crystallized ligand  at the three respective locations in the co-crystallized structure. This exercise resulted in very low Root-mean-square deviation (RMSD) values between the docked and the co-crystallized structures (RMSD < 1Å), which indicates the ability of achieving a near-native ligand pose. All the compounds used in the cell sensitization experiments were docked into the binding sites of the Abcb1 structure. All ligands were minimized using self-consistent field (SCF) method with PM6 Hamiltonian for partial charge assignment. The compounds were then docked at several different binding sites suggested by the literature for substrate or ATP binding. This included the M-site, R-site, H-site, elbow helix-2, verapamil, and the ATP 1 and ATP 2 binding sites. The amino acid residues defining these binding sites are presented in Molecular docking experiments were performed using the most recently solved crystal structure of murine holo Abcb1 (PDB code: 4Q9J), the holo structure with the highest resolution to date (3.6 Å), complexed with three cyclic peptide structures , one of which is located at a possibly new binding site at the elbow helix-2 region . All docResults are expressed as mean ± S.D. of at least three experiments. Comparisons between two groups were performed using Student's t-test. Three and more groups were compared by ANOVA followed by the Student-Newman-Keuls test. P values lower than 0.05 were considered to be significant.


Rumex hastatus is evident from the previous reports. Recently the R. hastatus has been evaluated for anticancer potential against HepG2, MCF7 or LNCaP cell lines with considerable cytotoxicity. We also reported the anti-tumor and anti-angiogenic potentials of R. hastatus. The current study has been arranged to evaluate cytotoxic potential of this plant against HeLa and NIH/3T3 cell lines and sort out the most active fraction of R. hastatus along with the identification of bioactive compounds responsible for cytotoxicity.The importance of Rumex genus and the renowned ethnopharmacological and biological potentials of R. hastatus was performed following  MTT calorimetric assay. Four concentrations  of each sample were used against both cell lines. Two cell lines i.e. HeLa and NIH/3T3 were used in the assay. Furthermore, chemical characterization of chloroform fraction was performed by GC-MS analysis.The cytotoxic potential of methanolic extract and sub-fractions of R. hastatus were active against HeLa and NIH/3T3 cell lines. Among all the fractions, chloroform fraction was dominant in activity against both cell lines. The observed IC50 values of chloroform fraction were 151.52 and 53.37 μg/ml against HeLa and NIH/3T3 respectively. The GC-MS analysis of chloroform fraction revealed the identification of 78 compounds with the identification of bioactive ones like ar-tumerone, phytol, dihydrojasmone, sitostenone etc.The current investigational study demonstrates that all the solvent fractions of Rumex hastatus D. Don possess strong cytotoxic potential. Moreover, the observed IC50 values and GC-MS analysis of chloroform fraction reveal that most of the bioactive compounds are in chloroform fraction. It can be further deduce that the chloroform fraction is a suitable target for the isolation of compounds having potential role in cancer therapy.It can be concluded from our results that  Rumex hymenosepalus has been reported with the isolation of antitumor compounds, i.e. leucodelphinidin and leucopelargonidin [The leading research teams around the world are in continuous struggle to explore novel aspects to facilitate life. The facilitation of life also encompasses decreased morbidity and mortality . One of rgonidin . Severalrgonidin .Rumex hastatus is one of the most important species which has been used traditionally for the treatment of various ailments like rheumatism, tonsillitis, piles etc [R. hastatus has been evaluated for anticancer potential against HepG2, MCF7 or LNCaP cell lines with considerable cytotoxicity [R. hastatus has been evaluated for anticholinesterase, antioxidant, anti-tumor, anti-angiogenic, phytotoxic and antibacterial potentials [R. hastatus, the current study was designed to explore cytotoxic potential of this plant against cell lines and to find out the bioactive phytoconstituents responsible for anticancer activity using GC-MS analysis.iles etc –17. Prevtoxicity . Previoutentials –22. BaseR. hastatus were collected from the surrounding area of University of Malakand, Pakistan. The plant’s name was confirmed by Dr. Ali Hazrat, Plant Taxonomist, Department of Botany, Shaheed Benazir Bhutto University, Sheringal Dir (U), KPK, Pakistan, and deposited with voucher specimen No. 1015SA. The plant’s material was shade dried, powdered and subjected to maceration process. Afterwards, it was filtered and the filtrate was evaporated under reduced pressure using rotary evaporator at 40 °C [n-hexane (Rh.Hex), chloroform (Rh.Chf), ethyl acetate (Rh.EtAc) and aqueous fraction (Rh.Aq) respectively [The aerial parts of mature plant of at 40 °C , 24. Simectively , 26.n-pentane, v/v) of 1.0 μl were injected manually in the splitless mode.Samples were subjected to GC analysis using an Agilent USB-393752 gas chromatograph  with HHP-5MS 5 % phenylmethylsiloxane capillary column  equipped with an FID detector. The initial temperature of the oven was retain at 70 °C for 1 min, followed by increase at the rate of 6 °C/min to 180 °C for 5 min and finally at the rate of 5 °C/min to 280 °C for 20 min. The temperature of injector and detector were set at 220 and 290 °C, correspondingly. Helium was used as carrier gas at a flow rate of 1 ml/min, and diluted samples  with a HHP-5MS 5 % phenylmethylsiloxane capillary column  outfitted with an Agilent HP-5973 mass selective detector in the electron impact mode (Ionization energy: 70 eV) working under the same experimental conditions as described for GC.Compounds were recognized by comparison of their retention times with those of authentic compounds in the literature under the same set of conditions. Further identification were done through the spectral data obtained from the Wiley and NIST libraries and further confirmed by comparisons of the fragmentation pattern of the mass spectra with data published in the literature , 28.R. hastatus was assayed in 96-well flat-bottomed micro plates following the standard MTT  colorimetric assay [2 flasks and incubated in 5 % CO2 incubator at 37 °C. Growing cells were harvested exponentially and counted with haemocytometer followed by dilution with a particular medium. Cell culture was prepared having the concentration of 6 x 104 cells/ml and transferred (100 μl/well) into 96-well plates. After overnight incubation, medium was discarded and 200 μl of fresh medium was added with various concentrations of plant samples (1–30 μM). After 48 h, 200 μl MTT (0.5 mg/ml) was added to each well and incubated additionally for 4 h. Afterward, 100 μL of DMSO was added to each well. The extent of MTT reduction to formazan within cells was figured out by measuring the absorbance at 570 nm, employing a micro plate reader . The samples causing 50 % growth inhibition for both cell lines were recorded as IC50. The percent inhibition was calculated by the formula given below;Cytotoxic activity of various samples of ic assay . BrieflyThe results i.e., Percent inhibition were processed via Soft- Max Pro software .P < 0.05 were considered significant.All the tests were performed in triplicate and values were expressed as means ± S.E.M. Multiple group comparison was performed by Two way ANOVA followed by Bonferroni post test in which the R. hastatus were assay against both cell lines. All the samples were found active against both cell lines with chloroform fraction more dominant as shown in Table 50 value of 151.52 μg/ml. Similarly, the second highest activity has been demonstrated by ethyl acetate fraction i.e., 79.66 ± 0.89, 66.32 ± 1.30, 40.93 ± 0.49 and 29.83 ± 1.36 % cytotoxic activity at concentrations of 500, 250, 125 and 62.5 μg/ml against HeLa cell line with IC50 value of 166.50 μg/ml. The methanolic extract and aqueous fraction demonstrated moderate cytotoxic potentials with IC50 values of 347.33 and 369.68 μg/ml respectively. Among all the samples of R. hastatus, the least activity was shown by that of n-hexane fraction with IC50 of 572.61 μg/ml.The MTT assay was carried out against two types of cell lines, i.e., HeLa and NIH/3T3. The crude methanolic extract and sub-fractions of 50 value of 53.37 μg/ml. Similarly, the ethyl acetate fraction revealed the second highest activity against NIH/3T3 cell line i.e., 72.76 ± 0.78, 59.00 ± 0.57, 46.86 ± 0.85 and 31.43 ± 0.81 % at concentrations of 500, 250, 125 and 62.5 μg/ml with IC50 value of 158.73 μg/ml. The IC50 calculated for the rest of the samples were 174.52, 237.62 and 439.26 μg/ml for methanolic extract, aqueous and n-hexane fractions respectively. The cytotoxic potential of all the test samples of R. hastatus against NIH/3T3 cell line has been summarized in Table 50 value <0.1 μg/ml against both cell lines.In NIH/3T3 cell line assay, again the chloroform fraction was found dominant exhibiting 82.13 ± 0.88, 70.66 ± 0.49, 64.02 ± 1.11 and 51.43 ± 0.61 % cytotoxic potential at concentrations of 500, 250, 125 and 62.5 μg/ml with ICBased on the high potency in both cell lines assays, the chloroform fraction was subjected to GC-MS analysis. A total of 78 phytoconstituents were identified by the GC-MS analysis. The identified compounds contain important bioactive compounds responsible for the cytotoxic potential of the plant. The parameters of some compounds found in GC-MS analysis have been summarized in the Table It is evident that area wise the highest percentage has been exhibited by linoleic acid ethyl ester with retention time 31.979 (96.29 %) followed by hexadecanoic acid, ethyl ester with retention time 28.475 (94.9 %). A summary of all identified compounds in the chloroform fraction has been shown in Table The GC-MS chromatogram of the chloroform fraction is shown in Fig. Polygonum hydropiper has been demonstrated with significant cytotoxicity against NIH/3T3 cell line following MTT assay [R. hastatus showed 78 compounds summarized in Table HeLa is a type of immortal cell line obtained from cervical cancer cells and for the very first time this cell line has been taken from late Henrietta Lacks in 1951 and abbreviated for her name . SimilarTT assay . As thisTT assay . SimilarTT assay . The GC-TT assay . In our R. hastatus has been reported to induce programmed cell death in human lymphoid leukemia Molt 4B cells [R. hastatus contain ethyl α-d-glucopyranoside, which may confer the possible anticancer potential to this plant. Anthracenedione has also been reported to possess anticancer properties [R. hastatus also possess nonivamide, which is skin permeation enhancer and used in various ointments etc [20 alcohol present in R. hastatus and C20 aliphatic alcohols has been employed in the treatment of hyperproliferative skin disordersone [R. hastatus was the most active one against two types of cell lines. The regression and correlation analysis shows that this plant has a parallel cytotoxic potential against both the cell lines as depicted in the Fig. 2 value of 0.881. The current study can also be correlated with the previous cytotoxic activity of R. hastatus against brine shrimps in which the chloroform fraction was the most active fraction [Phytol present in 4B cells . Dihydro4B cells . Ethyl-αoperties . Silane ents etc . Similarrdersone . Aristolrdersone . Likewisrdersone , 45. Anordersone . The curfraction . Based oRumex hastatus is a potential source of cytotoxic compounds. Moreover, the chloroform fraction is the active one among other solvent fractions of R. hastatus. Based on the GC-MS analysis of chloroform fraction, we can conclude that the chloroform fraction of R. hastatus is a rich source of bioactive compounds responsible for cytotoxicity.Based on our current results, we can conclude that


BRCA1 or BRCA2 genes, are sensitive to PARP inhibition. Microsatellite instability (MSI) accounts for 10-15% of colorectal cancer (CRC) and is hypothesized to lead to HR defects due to altered expression of Mre11, a protein required for double strand break (DSB) repair. Indeed, others have reported that PARP inhibition is efficacious in MSI CRC.Cells with homologous recombination (HR) deficiency, most notably caused by mutations in the in vitro and in vivo. We compiled a large panel of MSI and MSS CRC cell lines and evaluated the anti-proliferative activity of niraparib. In addition to testing single agent cytotoxic activity of niraparib, we also tested irinotecan  activity alone and in combination with niraparib in vitro and in vivo.Here we examine the response to niraparib, a potent PARP-1/PARP-2 inhibitor currently under clinical evaluation, in MSI versus microsatellite stable (MSS) CRC cell lines ¸ suggesting that the MSI phenotype does not sensitize CRC cell lines to PARP inhibition. Moreover, even the most sensitive MSI cell lines had niraparib EC50s greater than 10 fold higher than BRCA-deficient cell lines. However, MSI lines were more sensitive to SN-38 than MSS lines, consistent with previous findings. We have also demonstrated that combination of niraparib and irinotecan was more efficacious than either agent alone in both MSI and MSS cell lines both in vitro and in vivo, and that niraparib potentiates the effect of irinotecan regardless of MSI status.In contrast to earlier reports, MSI CRC cell lines were not more sensitive to niraparib than MSS CRC cell linesOur results support the clinical evaluation of this combination in all CRC patients, regardless of MSI status. BRCA1 and BRCA2 genes and other genes involved in HR  and put at 95°C for 5 minutes. Protein concentration was assessed in a 96 well format by BCA Protein assay Kit . 30 μg of protein extract was loaded onto 10% Tris-Glycine SDS-PAGE gels and run at 100 Volts for 90 minutes. The gels were transferred onto PVDF membrane and incubated overnight at 4°C with primary antibody,1:1000 rabbit Anti-MRE11 Antibody 2 Fragment (donkey) . Membranes were incubated for 1 hour in secondary antibody and developed using Pierce Supersignal West Dura Extended Duration Substrate .MSI- DLD-1, HCT-8, HCT-15, HCT116, LOVO, LS174T, LS180, LS411N, RKO, RKOE6, SNUC2A, SW48. MSS- COLO205, HT29, NCI-H-508, SK-CO-1, SW403, SW480, SW620, SW837, SW948, SW1116, SW1417, SW1463, T84. To carry out 7 day monotherapy proliferation assays with the cell lines, 500–32,000 cells (cell line-dependent) were seeded in 96-well clear tissue culture plates (190 μl/well) in an appropriate tissue culture medium supplemented with FBS. The plate was incubated for 4 hours at 37°C, and niraparib was added in a 9 point titration, 3-fold dilutions starting at 10 μM for niraparib and starting at either 10 μM, 1 μM, or 100nM for SN-38 . The cells were then incubated for 7 days at 37°C, 5% CO2  and the cell viability was assessed by WST-1 assay (Roche) as described by the Manufacturer. To carry out 7 day combination proliferation assays with the cell lines, 500–32,000 cells (cell line-dependent) were seeded in 96-well clear tissue culture plates (180 μl/well) in an appropriate tissue culture medium supplemented with FBS. The plate was incubated for 4 hours at 37°C, and niraparib was added at 125 nM, 250 nM, or 1 μM and SN-38 was added in an 8 point titration, 3-fold dilutions, starting at 100 nM. The cells were then incubated for 7 days at 37°C, 5% CO2  and the cell viability was assessed by WST-1 assay (Roche) as described by the Manufacturer. The number of living cells was determined by reading the plate at 450 nm on a spectrophotometer. The signal produced is directly proportional to the cell number as the cells convert tetrazolium salt due into a formazan end product. Each experiment was run in duplicate. Cell growth was expressed as the percentage growth with respect to vehicle treated cells. The concentration required to inhibit cell growth by 50% (EC50) was determined using the four-parameter fit in SoftMax Pro 5.2. The Wilcoxon rank sum test was performed to determine statistical significance. Excess Volume HSA combination index was calculated using a MATLAB algorithm as described previously . Th. Th18]. 8 monotheo Figure . In tumoin vitro and in vivo preclinical results demonstrate that in CRC cell lines, MSI does not render cells more sensitive to niraparib, but that combination of niraparib with irinotecan enhances the efficacy of irinotecan in both MSI and MSS CRC cell lines. Our data suggests that both MSI and MSS patient populations will benefit from the combination of niraparib and irinotecan.Our