scholarly journals Evaluation of the effects of acepromazine and xylazineon viability in an equine dermal cell line

2020 ◽  
Vol 14 (4) ◽  
pp. 259-264
Author(s):  
Jéssica Grace da Silveira ◽  
Bruno Egídio Cappelari ◽  
Ana Paula Muterle Varela ◽  
Thais Fumaco Teixeira ◽  
Giovana Dantas de Araujo
Keyword(s):  
Veterinary Medicine ◽  
Cell Viability ◽  
Cell Line ◽  
Mtt Assay ◽  
Tissue Damage ◽  
The Other ◽  
Drug Concentrations ◽  
Dermal Cell

Xylazine and acepromazine are drugs used exclusively in veterinary medicine. Xylazineis used as a sedative, analgesic, and tranquilizer while acepromazine is used as a sedative, pre-anesthetic, and anesthetic adjuvant. In vitrodrug toxicity experimentation is essential to predict possible damage associated with treatment. This study was carried out to evaluate and compare the in vitroeffects of acepromazine and xylazine on cell viability. Equine Dermis cells lines were used to examine different drug concentrations (0.02 mg/mL, 0.01 mg/mL, 0.005 mg/mL and 0.0025 mg/mL). An MTT assay was carried out to reveal cell viability. Both tested drugs reduced the viability of ED cells at 0.02 and 0.01 mg/mL. At 0.005 mg/mL, only acepromazine presented an effect. These results corroborate previous studies with xylazine. On the other hand, this is thefirst report about acepromazine and cell viability. Previous studies suggest that the mechanisms involved in reducing cell viability are apoptosis for xylazine and the activation of the autophagic pathway for acepromazine. Both mechanisms have been seen in other drugs of the same classes. These findings reveal that both acepromazine and xylazine cause concentration-dependent cytotoxicity in vitro. Future experiments could further elucidate the mechanisms by which this effect happens and thus circumvent therisk of potential tissue damage in vivo.

scholarly journals Libidibia ferreaFruit Crude Extract and Fractions Show Anti-Inflammatory, Antioxidant, and Antinociceptive EffectIn Vivoand Increase Cell ViabilityIn Vitro

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Tamires Rocha Falcão ◽  
Aurigena Antunes de Araújo ◽  
Luiz Alberto Lira Soares ◽  
Iuri Brilhante de Farias ◽  
Wliana Alves Viturino da Silva ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Cell Viability ◽  
Gallic Acid ◽  
Cell Line ◽  
Crude Extract ◽  
Leukocyte Migration ◽  
Total Glutathione ◽  
Anti Inflammatory

Background.Libidibia ferrea(L. ferrea)is found throughout the northeastern region of Brazil, where it has been used in folk medicine with beneficial effects on many inflammatory disorders.Purpose. This study investigated the phytochemical composition of the crude extract and fractions ofL. ferreafruit and evaluated its anti-inflammatory and antinociceptive activitiesin vivoand effect on cell viabilityin vitro.Methods. Characterization of polyphenols present in crude extract (CE), hydroalcoholic fractions of 20-80% ethanol (CE20, CE40, CE60, and CE80), aqueous fraction (AqF), and ethyl acetate (EAF) fractions ofL. ferreafruit was performed by chromatographic analysis.Anti-inflammatory activity was evaluated by using a carrageenan-induced peritonitis model submitted to a leukocyte migration assay and myeloperoxidase activity (MPO) analysis. Total glutathione and malondialdehyde (MDA) levels were assessed to evaluate the oxidative stress level. Antinociceptive activity was evaluated by acetic acid-induced abdominal writhing and hot plate test.In vitrocell viability was determined by using MTT assay in a mouse embryonic fibroblast cell line (3T3 cells).Results. Chromatography revealed the presence of ellagic acid content in EAF (3.06), CE (2.96), and CE40 (2.89). Gallic acid was found in EAF (12.03), CE 20 (4.43), and CE (3.99).L. ferreacrude extract and all fractions significantly reduced leukocyte migration and MPO activity (p<0.001).L. ferreaantioxidant effect was observed through high levels of total glutathione and reduction of MDA levels (p<0.001). Acetic acid-induced nociception was significantly inhibited after administration ofL. ferreacrude extract and all fractions (p<0.001). Crude extract and all fractions significantly increased the viability of the 3T3 cell line (p<0.05).Conclusions. The appropriate extraction procedure preserves the chemical components ofL. ferreafruit, such as gallic acid and ellargic acid. Crude extract and fractions ofL. ferreafruit exhibited anti-inflammatory, antioxidant, antinociceptive activitiesin vivoand enhanced cell viabilityin vitro.

scholarly journals Jianpi Yangwei decoction promotes apoptosis and suppresses proliferation of 5-fluorouracil resistant gastric cancer cells in vitro and in vivo

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Huijuan Tang ◽  
Wenjie Huang ◽  
Qiang Yang ◽  
Ying Lin ◽  
Yihui Chen ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Drug Resistance ◽  
Cell Line ◽  
Real Time ◽  
Mtt Assay ◽  
Xenograft Tumor ◽  
Rt Pcr ◽  
Induced Apoptosis

Abstract Background The exploration of new therapeutic agents targeting 5-Fu resistance may open a new opportunity to gastric cancer treatment. The objective is to establish a 5-Fu resistant gastric cancer cell line and observe the effect of Jianpi Yangwei decoction (JPYW) on its apoptosis and drug-resistance related proteins. Methods MTT assay was used to measure the effect of JPYW on the BGC823 cells proliferation, and the apoptosis was observed by flow cytometry and Hoechst fluorescence staining. The BGC823 xenograft tumor nude mice models were established, the apoptosis was detected by Tunel method. BGC-823/5-Fu was established by repeated low-dose 5-Fu shocks, the drug resistance index and proliferation were detected by the MTT assay; MDR1 mRNA was detected by real-time RT-PCR; Western blot was used to detect the ratio of p-AKT to AKT; The BGC823/5-Fu xenograft tumor nude mice models were established and apoptosis was measured. The expressions of MRP1, MDR1, ABCG2, AKT, p-AKT, caspase-3 and bcl-2 were detected by immunohistochemistry and the AKT mRNA expression was detected by real-time RT-PCR. Results JPYW induced apoptosis in BGC823 cells; Drug-resistant cell line BGC-823/5-Fu was sucessfully established; JPYW induced apoptosis of BGC823/5-Fu cells, down-regulated the expression of MRP1, MDR1 and ABCG2 in vitro and in vivo, and further decreased MDR1 expression when combined with pathway inhibitor LY294002 (P < 0.05); JPYW down-regulated the ratio of p-AKT to AKT in vitro in a dose-dependent manner, the same as after the combination with LY294002 (P < 0.05). Conclusion JPYW can induce apoptosis of BGC823 and BGC823/5-Fu cells, and down-regulate the expression of MDR1, MRP1, ABCG2 in vitro and in vivo. Its in vitro effect is related to the PI3K/AKT signaling pathway.

Rational Combinations Including a Novel Syk Inhibitor, Fostamatinib Disodium (FosD) in Diffuse Large B Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 283-283
Author(s):  
Randall M Rossi ◽  
Valerie Grose ◽  
Polly Pine ◽  
Richard I Fisher ◽  
Craig T. Jordan ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Viability ◽  
B Cell ◽  
Cell Line ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
In Vivo Studies

Abstract Abstract 283 Certain malignant B-cells rely upon B-cell receptor-mediated survival signals. Spleen tyrosine kinase (Syk) initiates and amplifies the B-cell receptor-mediated signal. We and others have demonstrated that fostamatinib disodium (FosD: a prodrug of R406, a potent and specific inhibitor of Syk) induces apoptosis in lymphoma cell lines and primary tumors. A recent clinical trial has demonstrated significant clinical activity of FosD in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia, and minimal overlap in toxicities with conventional agents. Given this background, future development in B-cell NHL will include rational combinations of FosD and currently available therapies. Therefore, we conducted in vitro and in vivo studies of rational combinations including FosD, in anticipation of clinical trial development. First, using a human DLBCL cell line of GCB genotype, (OCI-Ly19), we analyzed in vitro the combination of R406 with the following agents: fludarabine, rapamycin, rituximab, bendamustine and bortezomib. Increased cytotoxicity was observed using in vitro culture assays with the addition of fludarabine, rapamycin, or rituximab to R406. Cell viability at 72 hours was 25% with R406 alone, 27% for fludarabine alone, and only 9% for the fludarabine/R406. At 48 hours, cell viability was 49% using R406 alone, 31% using rituximab alone, and 21% for rituximab/R406. At 120 hours using primary lymphoma cells (DLCL27), there were no viable cells treated with the rapamycin/FosD combination, compared with rapamycin alone (7%) or FosD alone (25%) The addition of bortezomib or bendamustine to FosD resulted in only a minimal additive increase in cytotoxicity. Results with all combinations were similar with the OCI-Ly10 human DLBCL line of ABC genotype. We then performed in vivo studies by subcutaneous transplantation of the DLBCL cell line OCI-Ly19, (engineered to express luciferase allowing for real time in vivo imaging) into immune deficient NOD/SCID mice which reproducibly formed tumors. Recipient animals were separated into uniform cohorts when the tumors were less than or equal to 500 mm3 in size. The animals were then simultaneously treated with FosD (n=7; 3 gm/kg ad. lib.; translates into 2-5 micromolar R406 systemically throughout the 24h period) and either bortezomib, (n=6; 0.4 mg/kg weekly IP), or rituximab, (n=13; 3 mg/kg, 2x weekly IP). Analysis of the OCI-Ly19 tumor volumes at day 46 showed a median of 2364 mm3 with bortezomib alone compared with 1823 mm3 with bortezomib and FosD. When FosD was combined with rituximab the most significant cytotoxicity was observed: (p=0.01; median tumor volume of 497 mm3 following the combination) in comparison to either FosD alone (3150 mm3) or rituximab alone (1764 mm3). We conclude that the addition of FosD appears to increase activity against NHL of several drugs, including fludarabine and rapamycin. These agents have significant activity in indolent and mantle cell NHL as well as CLL. Moreover, there is no evidence that FosD impedes rituximab responses in vitro or in vivo; in fact we have suggested possible synergy with the combination of rituximab and FosD. Based upon the documented single agent activity of FosD in humans, and this data, clinical trials are now indicated using these promising combinations in NHL and CLL. Disclosures: Pine: Rigel: Employment. Friedberg:Rigel: Research Funding.

scholarly journals In vivo and in vitro toxicity evaluation of liposome-encapsulated sirolimus

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Murilo Batista Abud ◽  
Ricardo Noguera Louzada ◽  
David Leonardo Cruvinel Isaac ◽  
Leonardo Gomes Souza ◽  
Ricardo Gomes dos Reis ◽  
...  
Keyword(s):  
Cell Viability ◽  
Mtt Assay ◽  
Tunel Assay ◽  
In Vitro Toxicity ◽  
In Vivo Experiments ◽  
Significant Difference ◽  
The Difference ◽  
New Formulation

Abstract Background To evaluate the in vivo and in vitro toxicity of a new formulation of liposome-encapsulated sirolimus (LES). Methods In vitro experiments were done using ARPE-19 and HRP cells. An MTT assay was used to determine cell metabolic activity and a TUNEL assay for detecting DNA fragmentation. In vivo experiments were conducted on New Zealand albino rabbits that received intravitreal injections of empty liposomes (EL) or different concentrations of LES. Histopathological and immunohistochemical analyses were performed on the rabbit’s eyes following injection. Results Eighteen eyes of nine rabbits were used. MTT assay cell viability was 95.04% in group 1 (12.5 µL/mL LES). 92.95% in group 2 (25 µL/mL LES), 91.59% in group 3 (50 µL/mL LES), 98.09% in group 4 (12.5 µL/mL EL), 95.20% on group 5 (50 µL/mL EL), 98.53% in group 6 (50 µL/mL EL), and 2.84% on group 8 (50 µL/mL DMSO). There was no statistically significant difference among groups 1 to 7 in cell viability (p = 1.0), but the comparison of all groups with group 8 was significant (p < 0.0001). The TUNEL assay comparing two groups was not statistically significant from groups 1 to 7 (p = 1.0). The difference between groups 1 to 7 and group 8 (p < 0.0001) was significant. Histopathological changes were not found in any group. No activation of Müller cells was detected. Conclusion A novel formulation of LES delivered intravitreally did not cause in vitro toxicity, as evaluated by MTT and TUNEL assays, nor in vivo toxicity as evaluated by histopathology and immunohistochemistry in rabbit eyes.

An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo

1994 ◽  
Vol 107 (4) ◽  
pp. 813-825 ◽  
Author(s):  
M.R. Shanks ◽  
D. Cassio ◽  
O. Lecoq ◽  
A.L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Membrane Proteins ◽  
Cell Line ◽  
Hybrid Cell ◽  
The Other ◽  
Hepatocyte Polarity ◽  
Rat Hepatoma

Studies of hepatocyte polarity, an important property of liver epithelial cells, have been hampered by the lack of valid in vitro models. We report here that a new polarized hepatoma-derived hybrid cell line, called WIF-B, has improved characteristics to those of its parent, WIF12-1. This latter line originated from the fusion of non-polarized rat hepatoma Fao cells with human fibroblasts (WI-38) and selection for a polarized phenotype. We generated the WIF-B line by growing WIF12-1 cells as unattached aggregates for three weeks and selecting for survivors. Karyotype analysis showed a broad chromosome pattern in the initial WIF-B population, but this pattern stabilized after a few passages. The growth and phenotypic properties of these cells were quite different from those of their polarized WIF12-1 parent. WIF-B cells attained a 4-fold higher maximal density in monolayer culture, survived at this density for &gt; 5 days rather than 1 day, and exhibited two to three times more apical structures during this period (80 to 95%). We compared several parameters of liver differentiation in the WIF-B cells with those of a related hybrid clone, WIF12-E, which is extinguished for most liver-specific functions, and with the common hepatoma parent, Fao. By immunoblot analysis, the levels of expression of eight plasma membrane proteins were higher in the WIF-B cells than in either of the other two cell lines and ranged from 10 to 200% of those in vivo. Two plasma membrane proteins were not detected in WIF12-E cells. By immunofluorescence, the apical membrane proteins in WIF-B displayed different cellular localizations than in either of the other two cell lines. In WIF-B cells, apical proteins were confined to a plasma membrane region that we have identified as the apical domain by several criteria (Ihrke, G., Neufeld, E.D., Meads, T., Shanks, M.R., Cassio, D., Laurent, M., Schroer, T.A., Pagano, R. E. and Hubbard, A. L. J. Cell Biol., 123, 1761–1765). The same molecules were distributed over the entire plasma membrane of Fao and WIF12-E cells and also (for Fao cells) in intracellular punctate structures that did not colocalize with the majority of structures containing a secretory protein, albumin. Our results indicate that the WIF-B cells are more highly differentiated than any of their ancestors (Fao or WIF12-1 cells) and thus, are promising candidates for in vitro studies of hepatocyte polarity.

scholarly journals Screening for 15 pathogenic viruses in human cell lines registered at the JCRB Cell Bank: characterization of in vitro human cells by viral infection

2018 ◽  
Vol 5 (5) ◽  
pp. 172472 ◽  
Author(s):  
Setsuko Shioda ◽  
Fumio Kasai ◽  
Ken Watanabe ◽  
Kohei Kawakami ◽  
Azusa Ohtani ◽  
...  
Keyword(s):  
Viral Infection ◽  
Cell Line ◽  
Cell Lines ◽  
Human Cell ◽  
The Other ◽  
Pcr Analysis ◽  
Human Cell Lines ◽  
Cell Bank

Human cell lines have been used in a variety of research fields as an in vitro model. These cells are all derived from human tissue samples, thus there is a possibility of virus infection. Virus tests are routinely performed in clinical practice, but are limited in cell lines. In this study, we investigated 15 kinds of viruses in 844 human cell lines registered at the Japanese Collection of Research Bioresources (JCRB) Cell Bank. Our real-time PCR analysis revealed that six viruses, EBV, HTLV-1, HBV, B19V, HHV-6 and HHV-7, were detected in 43 cell lines. Of them, 20 cell lines were transformed by intentional infection in vitro with EBV or HTLV-1. Viruses in the other 23 cell lines and one EBV transformed cell line are derived from an in vivo infection, including five de novo identifications of EBV, B19V or HHV-7 carriers. Among them, 17 cell lines were established from patients diagnosed with virus-associated diseases. However, the other seven cell lines originated from in vivo cells unrelated to disease or cellular tropism. Our approach to screen for a set of 15 viruses in each cell line has worked efficiently to identify these rare cases. Virus tests in cell lines contribute not only to safety assessments but also to investigation of in vivo viral infection which can be a characteristic feature of cell lines.

scholarly journals Corrosion Study of Implanted Tin Electrodes Using Excessive Electrical Stimulation in Minipigs

Metals ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 389 ◽  
Author(s):  
Suzan Meijs ◽  
Kristian Rechendorff ◽  
Søren Sørensen ◽  
Nico Rijkhoff
Keyword(s):  
Electrical Stimulation ◽  
Tissue Damage ◽  
Charge Injection ◽  
The Other ◽  
Electrochemical Tests ◽  
Implanted Electrodes ◽  
Electrode Degradation ◽  
Lower Charge

(1) Background: Titanium nitride (TiN) electrodes have been used for implantable stimulation and sensing electrodes for decades. Nevertheless, there still is a discrepancy between the in vitro and in vivo determined safe charge injection limits. This study investigated the consequences of pulsing implanted electrodes beyond the in vivo safe charge injection limits. (2) Methods: The electrodes were implanted for a month and then pulsed at 20 mA and 50 mA and 200 Hz and 400 Hz. Afterwards, the electrodes were investigated using electrochemical and analytical methods to evaluate whether electrode degradation had occurred. (3) Results: Electrochemical tests showed that electrodes that pulsed at 20 mA and 200 Hz (lowest electrical dose) had a significantly lower charge injection capacity and higher impedance than the other used and unused electrodes. (4) Conclusions: The electrodes pulsed at the lowest electrical dose, for which no tissue damage was found, appeared to have degraded. Electrodes pulsed at higher electrical doses for which tissue damage did occur, on the other hand, show no significant degradation in electrochemical tests compared to unused implanted and not implanted electrodes. It is thus clear that the tissue surrounding the electrode has an influence on the charge injection properties of the electrodes and vice versa.

scholarly journals Piperazine-Derived α1D/1A Antagonist 1- Benzyl-N- (3-(4- (2-Methoxyphenyl) Piperazine-1-yl) Propyl) -1H- Indole-2- Carboxamide Induces Apoptosis in Benign Prostatic Hyperplasia Independently of α1-Adrenoceptor Blocking

2021 ◽  
Vol 11 ◽  
Author(s):  
Qing Xiao ◽  
Qi-Meng Liu ◽  
Ru-Chao Jiang ◽  
Kai-Feng Chen ◽  
Xiang Zhu ◽  
...  
Keyword(s):  
Benign Prostatic Hyperplasia ◽  
Cell Viability ◽  
Cell Line ◽  
Prostate Volume ◽  
Prostatic Hyperplasia ◽  
Subtype Selectivity ◽  
Prostate Growth ◽  
Apoptotic Induction

Previous studies have indicated that α1D/1A antagonist naftopidil (NAF) suppresses prostate growth by decreasing cell proliferation without affecting apoptosis and prostate volume in benign prostatic hyperplasia (BPH). A NAF-derived α1D/1A antagonist 1- benzyl-N-(3-(4-(2-methoxyphenyl) piperazine-1-yl) propyl)-1H-indole-2- carboxamide (HJZ-12) has been reported from our laboratory, which exhibits high subtype-selectivity to both α1D- and α1A- AR (47.9- and 19.1- fold, respectively) with respect to a1B-AR in vitro. However, no further study was conducted. In the present study, a pharmacological evaluation of HJZ-12 in BPH was performed on an estrogen/androgen-induced rat BPH model and human BPH-1 cell line. In vivo, HJZ-12 exhibited better performance than NAF in preventing the progression of rat prostatic hyperplasia by not only decreasing prostate weight and proliferation (similar to NAF) but also, shrinking prostate volume and inducing prostate apoptosis (different from NAF). In vitro, HJZ-12 exhibited significant cell viability inhibition and apoptotic induction in BPH-1 cell line, without presenting cell anti-proliferation properties. Intriguingly, the role of HJZ-12 on cell viability and apoptosis was an α1-independent action. Furthermore, RNA-Seq analysis was applied to screen out six anti-apoptotic genes (Bcl-3, B-lymphoma Mo-MLV insertion region 1 [Bmi-1], ITGA2, FGFR3, RRS1, and SGK1). Amongst them, Bmi-1 was involved in the apoptotic induction of HJZ-12 in BPH-1. Overall, HJZ-12 played a remarkable role in preventing the progression of prostatic hyperplasia through α1-independent apoptotic induction, indicating that it will be a multi-target effective candidate for BPH treatment.

scholarly journals Analysis Of Candidate Genes Expected To Be Essential For Melanoma Surviving

2020 ◽  
Author(s):  
Irina Krivosheeva ◽  
Alexandra Filatova ◽  
Sergei Moshkovskii ◽  
Ancha Baranova ◽  
Mikhail Skoblov
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Melanoma Cell ◽  
Melanoma Cell Line ◽  
Computational Models ◽  
Target Genes ◽  
The Other ◽  
Other Hand ◽  
Before And After

Abstract Cancers may be treated by selective targeting of the genes vital for their survival. A number of attempts have led to discovery of several genes essential for surviving of tumor cells of different types. In this work, we tried to analyze genes that were previously predicted to be essential for melanoma surviving. Here we present the results of transient siRNA-mediated knockdown of the four of such genes, namely, UNC45A, STK11IP, RHPN2 and ZNFX1, in melanoma cell line A375, then assayed the cells for their viability, proliferation and ability to migrate in vitro. In our study, the knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating. Possible explanation for such counterintuitive results may include insufficiency of the predicting computational models or the necessity of a multiplex knockdown of the genes.AimsTo examine the hypothesis of essentiality of hypomutated genes for melanoma surviving we have performed knockdown of several genes in melanoma cell line and analyzed cell viability and their ability to migrate.MethodsKnockdown was performed by siRNAs transfected by Metafectene PRO. The levels of mRNAs before and after knockdown were evaluated by RT-qPCR analysis. Cell viability and proliferation were assessed by MTT assay. Cell migration was assessed by wound healing assay.ResultsThe knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating. ConclusionOur results do not confirm initial hypothesis that the genes predicted essential for melanoma survival as a matter of fact support the survival of melanoma cells.

The preclinical anti-angiogenic and pro-apoptotic activity of lenalidomide in urothelial carcinoma (UC).

2013 ◽  
Vol 31 (6_suppl) ◽  
pp. 294-294
Author(s):  
Weiguo Jian ◽  
Jonathan M. Levitt ◽  
Keith S. Chan ◽  
Seth P. Lerner ◽  
Guru Sonpavde
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Cell Line ◽  
Immune Modulation ◽  
Human Subjects ◽  
Low Grade ◽  
Angiogenic Activity ◽  
Apoptotic Activity

294 Background: Lenalidomide (Len) is an immunomodulatory drug (IMiD) approved for hematologic conditions and demonstrates immune modulation, anti-angiogenic activity and direct anti-tumor cytotoxicity. A rationale can be made to evaluate the preclinical activity of Len in UC. Methods: The in vitro anti-tumor activity of Len was evaluated in 4 human (5637, TCC-SUP, RT4, RT112) and 1 murine (MB49) cell line. Anti-proliferative activity activity (MTT assay), apoptosis (Annexin-FITC immunohistochemistry [IHC], flow cytometry) and cell viability by colony forming assay were measured. In vivo activity of daily oral Len 10 mg/kg or placebo orally for 5 days a week for up to 4 weeks was examined in syngeneic immunocompetent C57BL/6 mice bearing subcutaneous (SC) MB49-Luc25 tumors and RT4 subcutaneous xenografts. Tumors underwent immunohistochemistry (IHC) for microvessel density (CD31), apoptosis (cleaved caspase [cc]-3) and CD3+/CD20+ lymphocyte infiltration. Cereblon, a molecular target of Len was analyzed by IHC. Results: In vitro cultures for 3 days with daily repletion of Len showed significant pro-apoptotic activity (flow cytometry) at low micromolar concentrations attainable in human subjects (2.2 µM) against RT4 cells, a superficially invasive human UC cell line. Long-term cultures of RT4 cells for 2 weeks with daily repletion of Len significantly reduced cell viability and colony forming ability. Cereblon expression was numerically lower in sensitive RT4 cells compared to resistant 5637 cells (p=NS). In the immunocompetent model in vivo, Len did not decrease tumor size, or increase cc-3 and CD3+/CD20+ lymphocytes, but post-Len tumors exhibited decreased CD31 (p<0.05). In RT4 xenografts, Len significantly decreased the size of tumors and CD31, and increased cc-3 (all p<0.05). Cereblon expression increased in Len treated RT4 xenografts (p=0.024). Conclusions: Lenalidomide demonstrated selective preclinical activity against superficially invasive low grade human UC cells attributable to direct tumor cell apoptosis and anti-angiogenic activity. Clinical evaluation in patients with low grade or non-invasive UC and further study of cereblon as a predictive biomarker may be warranted.

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