Development of an In Vitro Model to Screen CYP1B1-Targeted Anticancer Prodrugs

Cytochrome P450 1B1 (CYP1B1) is an anticancer therapeutic target due to its overexpression in a number of steroid hormone–related cancers. One anticancer drug discovery strategy is to develop prodrugs specifically activated by CYP1B1 in malignant tissues to cytotoxic metabolites. Here, we aimed to develop an in vitro screening model for CYP1B1-targeted anticancer prodrugs using the KLE human endometrial carcinoma cell line. KLE cells demonstrated superior stability of CYP1B1 expression relative to transiently transfected cells and did not express any appreciable amount of cognate CYP1A1 or CYP1A2, which would have compromised the specificity of the screening assay. The effect of two CYP1B1-targeted probe prodrugs on KLE cells was evaluated in the absence and presence of a CYP1B1 inhibitor to chemically “knock out” CYP1B1 activity (CYP1B1 inhibited). Both probe prodrugs were more toxic to KLE cells than to CYP1B1-inhibited KLE cells and significantly induced G0/G1 arrest and decreased the S phase in KLE cells. They also exhibited pro-apoptotic effects in KLE cells, which were attenuated in CYP1B1-inhibited KLE cells. In summary, a KLE cell–based model has been characterized to be suitable for identifying CYP1B1-targeted anticancer prodrugs and should be further developed and employed for screening chemical libraries.

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L6E9 Myoblasts Are Deficient of Myostatin and Additional TGF-βMembers Are Candidates to Developmentally Control Their Fiber Formation

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/326909 ◽

2010 ◽

Vol 2010 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Stefania Rossi ◽

Elena Stoppani ◽

Massimiliano Gobbo ◽

Anna Caroli ◽

Alessandro Fanzani

Keyword(s):

Developmental Regulation ◽

Fiber Formation ◽

Dominant Negative ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

Knock Out ◽

Fiber Size ◽

Vitro Model ◽

Activin Receptors

This work provides evidence that the robust myoblast differentiation observed in L6E9 cells is causally linked to deficiency of myostatin, which, conversely, has been found to be expressed in C2C12 cells. However, despite the absence of endogenous myostatin, L6E9 myoblasts expressed functional Activin receptors type II (ActRIIs) and follistatin as well as the highly related TGF-βmembers Activins and GDF11, suggesting that in this cell line the regulation of fiber size might be under the control of multiple regulators regardless of myostatin. In line with this hypothesis, delivery of a dominant-negative ActRIIb form or the increase of follistatin, as obtained via Trichostatin treatment or stable transfection of a short human follistatin form, enhanced the L6E9 cell differentiation and further increased the size of myotubes, suggesting that L6E9 myoblasts provide a spontaneous myostatin knock-out in vitro model to study TGF-βligands involved in developmental regulation of fiber size.

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G1 Arrest and Caspase-Mediated Apoptosis in HL-60 Cells by Dichloromethane Extract of Centrosema pubescens

The American Journal of Chinese Medicine ◽

10.1142/s0192415x10008536 ◽

2010 ◽

Vol 38 (06) ◽

pp. 1143-1159 ◽

Cited By ~ 9

Author(s):

Subramanireddy Ramadevi Mani ◽

Baddireddi Subhadra Lakshmi

Keyword(s):

Cell Cycle ◽

Mononuclear Cells ◽

Tumor Biology ◽

G1 Phase ◽

G1 Arrest ◽

Synergistic Activity ◽

Peripheral Blood Mononuclear ◽

Normal Peripheral Blood ◽

Vitro Model

Cell division and apoptosis are two crucial components of tumor biology and the importance of increased cell proliferation and reduced cell death have made them valid therapeutic targets. The plant kingdom is a relatively underexploited cache of novel drugs, and crude extracts of plants are known for their synergistic activity. The present study assessed the anti-proliferative activity of the medicinal plant Centrosema pubescens Benth. Centrosema pubescens dichloromethane extract (CPDE) inhibited the proliferation of HL-60 (promyelocytic acute leukaemia) cells with an IC 50 value of 5 μg/ml. Further studies also showed that CPDE induces growth arrest at the G1 phase and specifically down-regulates the expressions of cyclin E and CDK2 and up-regulates p27(CKI) levels. These events apparently lead to the induction of apoptosis, which was demonstrated qualitatively by a DNA fragmentation assay and propidium iodide staining. Quantitative assessment of the effective arrest of the cell cycle and of apoptosis was confirmed by flow cytometry. CPDE exhibited negligible cytotoxicity even at the highest dose tested (100 μg/ml) in both normal peripheral blood mononuclear cells and in an in vitro model (HL-60). Our results strongly suggest that CPDE arrests the cell cycle at the G1 phase and triggers apoptosis by caspase activation.

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Glucocorticoids and rituximab in vitro: synergistic direct antiproliferative and apoptotic effects

Blood ◽

10.1182/blood.v100.5.1765.h81702001765_1765_1773 ◽

2002 ◽

Vol 100 (5) ◽

pp. 1765-1773 ◽

Cited By ~ 93

Author(s):

Andrea L. Rose ◽

Barbara E. Smith ◽

David G. Maloney

Keyword(s):

Cell Lines ◽

Negative Impact ◽

G1 Arrest ◽

Specific Lysis ◽

Non Hodgkin Lymphoma ◽

Mediate Cytotoxicity ◽

Increased Sensitivity ◽

Anti Cd20 ◽

Apoptotic Effects

Rituximab, a chimeric human immunoglobulin G1(IgG1) anti-CD20 monoclonal antibody has been shown to mediate cytotoxicity in malignant B cells via several mechanisms in vitro. These include direct antiproliferative and apoptotic effects, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). Glucocorticoids (GCs) are often administered in conjunction with rituximab in chemotherapeutic regimens or as premedication to reduce infusion-related symptoms. The effects of GCs on CDC and ADCC, and the direct apoptotic and antiproliferative effects of rituximab are unknown. Therefore, we evaluated these mechanisms in 9 B-cell non-Hodgkin lymphoma (B-NHL) cell lines using rituximab and GCs. Rituximab and dexamethasone induced synergistic growth inhibition in 6 B-NHL cell lines. Dexamethasone and rituximab induced significant G1 arrest in 9 of 9 cell lines. The combination of rituximab and dexamethasone resulted in supra-additive increases in phosphatidylserine exposure and hypodiploid DNA content in 5 and 3 B-NHL cell lines, respectively. CDC and ADCC were neither impaired nor enhanced when dexamethasone and rituximab were administered concurrently. However, preincubation of both effector and tumor cells with dexamethasone reduced specific lysis in ADCC assays in 4 B-NHL cell lines. Preincubation of tumor cell lines with dexamethasone significantly increased cell sensitivity to CDC in 3 B-NHL cell lines. We conclude that the addition of dexamethasone to rituximab results in supra-additive cytotoxicity with respect to its direct antiproliferative and apoptotic effects, induces a cell-dependent increased sensitivity to rituximab-induced CDC, and has minimal negative impact on ADCC when used simultaneously with rituximab.

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Cytoplasmic sequestration of wild-type p53 protein impairs the G1 checkpoint after DNA damage.

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.1126 ◽

1996 ◽

Vol 16 (3) ◽

pp. 1126-1137 ◽

Cited By ~ 169

Author(s):

U M Moll ◽

A G Ostermeyer ◽

R Haladay ◽

B Winkfield ◽

M Frazier ◽

...

Keyword(s):

Cell Lines ◽

P53 Protein ◽

G1 Arrest ◽

Wild Type ◽

Suppressor Function ◽

Wild Type P53 ◽

Vitro Model ◽

Dna Strand ◽

Cytoplasmic Structures

Wild-type p53 protein is abnormally sequestered in the cytoplasm of a subset of primary human tumors including neuroblastomas (NB) (U. M. Moll, M. LaQuaglia, J. Benard, and G. Riou, Proc. Natl. Acad. Sci. USA 92:4407-4411, 1995; U. M. Moll, G. Riou, and A. J. Levine, Proc. Natl. Acad. Sci.USA 89:7262-7266, 1992). This may represent a nonmutational mechanism for abrogating p53 tumor suppressor function. To test this hypothesis, we established the first available in vitro model that accurately reflects the wild-type p53 sequestration found in NB tumors. We characterized a series of human NB cell lines that overexpress wild-type p53 and show that p53 is preferentially localized to discrete cytoplasmic structures, with no detectable nuclear p53. These cell lines, when challenged with a variety of DNA strand-breaking agents, all exhibit impaired p53-mediated G1 arrest. Induction analysis of p53 and p53-responsive genes show that this impairment is due to suppression of nuclear p53 accumulation. Thus, this naturally occurring translocation defect compromises the suppressor function of p53 and likely plays a role in the tumorigenesis of these tumors previously thought to be unaffected by p53 alterations.

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An in vitro model for investigation of vitamin A effects on wound healing

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000692 ◽

2021 ◽

pp. 1-6

Author(s):

Hoda Keshmiri Neghab ◽

Mohammad Hasan Soheilifar ◽

Gholamreza Esmaeeli Djavid

Keyword(s):

Wound Healing ◽

Endothelial Cell ◽

Vitamin A ◽

In Vitro Model ◽

Cellular Proliferation ◽

Endothelial Cell Migration ◽

Normal Fibroblast ◽

Anti Inflammatory ◽

Vitro Model

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inﬂammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p < 0.05). Similarly, it stimulated angiogenesis by promoting endothelial cell migration up to approximately 4 fold and interestingly tube formation up to 8.5 fold (p < 0.01). Furthermore, vitamin A treatment was shown to decrease the level of nitric oxide production in a dose-dependent effect (p < 0.05), exhibiting the anti-inflammatory property of vitamin A in accelerating wound healing. These results may reveal the therapeutic potential of vitamin A in diabetic wound healing by stimulating regeneration, angiogenesis, and anti-inﬂammation responses.

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