150 IN VITRO MODEL FOR EFFECT OF ENDOCRINE-DISRUPTING CHEMICALS ON PLACENTAL CATION TRANSPORTER CHANNEL

2016 ◽  
Vol 28 (2) ◽  
pp. 205
Author(s):  
J.-H. Lee ◽  
M. H. Lee ◽  
M. J. Lee ◽  
E.-B. Jeung
Keyword(s):  
Cell Line ◽  
Steroid Hormones ◽  
Endocrine Disrupting Chemicals ◽  
Control Group ◽  
Bewo Cell ◽  
Mrna Levels ◽  
Phenol Red ◽  
Human Trophoblast ◽  
Protein Levels ◽  
Endocrine Disrupting

Calcium, copper, iron, oxygen, and carbon dioxide are essential factors in fetal growth. These molecules are transferred by specific receptors located on the cell membrane or cytoplasm in placenta. Calcium, copper, and iron transfer genes are regulated by oestrogen, placental lactogen, and vitamin D. During pregnancy, expression of these receptors is controlled by the nutritional status of the maternal and fetal environment. Some synthetic plastics contain endocrine-disrupting chemicals (EDC), which have similar structures to steroid hormones or endogenous hormones related to reproduction. These substances disturb action of hormones (e.g. increasing oestrogen or progesterone) by interacting with their receptors or affecting the expression of transporting genes for cations. We used a BeWo cell line (human trophoblast cell line) to test the effect of EDC during pregnancy. The cells were cultured in phenol red-free DMEM supplemented with 5% charcoal dextran-stripped fetal bovine serum for 48 h to ensure the depletion of steroid hormones in the cells. Ethinyl oestradiol (EE), which activates oestrogen receptors, was used as a positive control. Then, EE (10–9, 10–8, and 10–7 M), octylpehnol (OP; 10–7, 10–6, and 10–5 M), nonylphenol (NP; 10–8, 10–7, and 10–6 M), and bisphenol A (BPA; 10–7, 10–6, and 10–5 M) were treated in BeWo cells for 48 h, and the cells were harvested. The mRNA and protein levels for calcium transporting genes (PMCA1 and TRPV6), copper transporting genes (CTR1 and ATP7A), and iron transporting genes (IREG1 and HEPH) were quantified by RT-qPCR, and Western blotting, respectively. Experiments were carried 3 times, and results were statistically analysed by GraphPad Prism6 (GraphPad Software, San Diego, CA, USA). We observed dose-dependent decreases in mRNA levels of PMCA1, TRPV6, ATP7A, and IREG1 compared with control group in OP-, NP-, or BPA-treated groups. Protein levels showed a similar pattern to mRNA levels. Based on our data, we confirmed that these EDC affect metal ion channels such as calcium, copper, and iron transporters during pregnancy.

scholarly journals Daily exposure to phthalates and alkylphenols alters miR biogenesis and expression in mice ovaries

2020 ◽  
Vol 65 (4) ◽  
pp. 175-186
Author(s):  
Daniel Patiño-García ◽  
Leonor Cruz-Fernandes ◽  
Julio Buñay ◽  
Renán Orellana ◽  
Ricardo D Moreno
Keyword(s):  
Endocrine Disrupting Chemicals ◽  
Organ Dose ◽  
Mrna Levels ◽  
Hormone Synthesis ◽  
Female Mice ◽  
Protein Levels ◽  
Endocrine Disrupting ◽  
Relevant Dose ◽  
Hormone Biosynthesis ◽  
Hormone Imbalance

Reproductive hormone imbalance in infertile women is correlated to high levels of phthalates and alkylphenols, which are among endocrine-disrupting chemicals (EDCs). Previous studies have shown that they interfere with gene expression by deregulating levels of microRNAs (miRs), small non-coding RNAs targeting mRNAs encoding enzymes in the hormone biosynthesis pathway. However, this effect depends on the target organ, dose and whether or not they are alone or in mixtures. Our goal was to study whether the biosynthesis, and a specific group of miRs targeting mRNAs encoding enzymes in steroid hormone biosynthesis, are deregulated in the ovaries of female mice chronically exposed to a mixture of three phthalates (DEHP+DBP+BBP) and two alkylphenols (NP+OP) at a human environmentally relevant dose. We performed qPCR and Western blot assays along with a bioinformatics approach and found that this mixture modified the biogenesis machinery of miRs, inducing an increase in the mRNA levels of Drosha and Dicer1 and DROSHA protein levels. In addition, we found changes in the precursor and mature forms of miR-96-5p, miR-200b-3p, miR-365-3p, miR-378a-3p and miR-503-5p which target steroidogenic pathway enzymes. Finally, using primary granulosa cell culture, we confirmed that miR-200b-3p targets Cyp19a1, transcript encoding CYP19A1, the enzyme that produces estradiol (E2). These results indicate that chronic exposure to phthalates and alkylphenols mixture alters the biogenesis of ovary miRs and increases the expression of miRs implicated in the control of steroidal hormone synthesis in female mice, thus contributing to reproductive pathologies.

YAP1 Overexpression Is Associated with Kidney Dysfunction in Lupus Nephritis

Pathobiology ◽  
2021 ◽  
pp. 1-12
Author(s):  
Ying Xie ◽  
Yuanyuan Ruan ◽  
Huimei Zou ◽  
Yixin Wang ◽  
Xin Wu ◽  
...  
Keyword(s):  
Lupus Nephritis ◽  
Epithelial Mesenchymal Transition ◽  
Kidney Tissue ◽  
Mouse Kidney ◽  
Experimental Comparison ◽  
Control Group ◽  
Mrna Levels ◽  
Mesenchymal Transition ◽  
Protein Levels

<b><i>Objective:</i></b> The goal of the present study was to determine the expression of yes-associated protein 1 (YAP1) in renal tissues of mice with lupus nephritis (LN) and elucidate its role in the progression of renal fibrosis. <b><i>Methods:</i></b> C57BL/6 mice and MRL/lpr mice were selected for experimental comparison. Mouse kidney tissues were removed and sectioned for hematoxylin and eosin staining, Masson’s trichome staining, Sirius staining, and immunohistochemistry. The mRNA and protein levels of YAP1 in mouse kidney tissues were detected, and the correlation between YAP1 and fibronectin (FN) mRNA levels was analyzed. Mouse renal epithelial cells were used for in vitro experiments. After transfection and stimulation, the cells were divided into 4 groups, namely the C57BL/6 serum group (group 1), the MRL/lpr serum group (group 2), the MRL/lpr serum + siRNA-negative control group (group 3), and the MRL/lpr serum + siRNA-YAP1 group (group 4). Epithelial-mesenchymal transition (EMT) markers in each group were detected by Western blotting and immunofluorescence staining. Serum creatinine, blood urea nitrogen, and urinary protein levels were detected and assessed for their correlation with YAP1 mRNA levels by Spearman’s analysis. <b><i>Results:</i></b> Compared to C57BL/6 mice, MRL/lpr mice exhibited obvious changes in fibrosis in renal tissues. In addition, YAP1 expression was significantly higher in the renal tissues of MRL/lpr mice than in those of C57BL/6 mice, and YAP1 mRNA levels were positively correlated with those of FN. YAP1 silencing in lupus serum-stimulated cells could effectively relieve serum-induced EMT. Finally, we observed that YAP1 mRNA levels in mouse kidney tissue were significantly and positively correlated with the degree of renal function injury. <b><i>Conclusion:</i></b> YAP1 expression in the kidney tissues of LN mice was higher than that observed in normal mice, indicating that YAP1 may play an important role in the occurrence and development of LN.

scholarly journals OctylPhenol (OP) Alone and in Combination with NonylPhenol (NP) Alters the Structure and the Function of Thyroid Gland of the Lizard Podarcis siculus

2021 ◽  
Vol 80 (3) ◽  
pp. 567-578 ◽  
Author(s):  
Rosaria Sciarrillo ◽  
Mariana Di Lorenzo ◽  
Salvatore Valiante ◽  
Luigi Rosati ◽  
Maria De Falco
Keyword(s):  
Thyroid Gland ◽  
Endocrine Disrupting Chemicals ◽  
Thyroid Stimulating Hormone ◽  
Control Group ◽  
Type Ii ◽  
Endocrine Disrupting ◽  
Pituitary Thyroid Axis ◽  
Podarcis Siculus ◽  
Thyroid Axis ◽  
By Products

Abstract Different environmental contaminants disturb the thyroid system at many levels. AlkylPhenols (APs), by-products of microbial degradation of AlkylPhenol Polyethoxylates (APEOs), constitute an important class of Endocrine Disrupting Chemicals (EDCs), the two most often used environmental APs being 4-nonylphenol (4-NP) and 4-tert-octylphenol (4-t-OP). The purpose of the present study was to investigate the effects on the thyroid gland of the bioindicator Podarcis siculus of OP alone and in combination with NP. We used radioimmunoassay to determine their effects on plasma 3,3′,5-triiodo-L-thyronine (T3), 3,3′,5,5′-L-thyroxine (T4), thyroid-stimulating hormone (TSH), and thyrotropin-releasing hormone (TRH) levels in adult male lizards. We also investigated the impacts of AP treatments on hepatic 5′ORD (type II) deiodinase and hepatic content of T3 and T4. After OP and OP + NP administration, TRH levels increased, whereas TSH, T3, and T4 levels decreased. Lizards treated with OP and OP + NP had a higher concentration of T3 in the liver and 5′ORD (type II) activity, whereas T4 concentrations were lower than that observed in the control group. Moreover, histological examination showed that the volume of the thyroid follicles became smaller in treated lizards suggesting that that thyroid follicular epithelial cells were not functionally active following treatment. This data collectively suggest a severe interference with hypothalamus–pituitary–thyroid axis and a systemic imbalance of thyroid hormones. Graphic Abstract

Expression and Significance of Lipin1 and AMPKα in Hepatic Insulin Resistance in Diet-Induced Insulin Resistance Rats

2012 ◽  
Vol 120 (02) ◽  
pp. 84-88 ◽  
Author(s):  
S. Chen ◽  
X. Zhuang ◽  
Y. Liu ◽  
A. Sun ◽  
C. Chen
Keyword(s):  
Insulin Resistance ◽  
High Fat Diet ◽  
Diet Group ◽  
Hepatic Insulin Resistance ◽  
Control Group ◽  
Mrna Levels ◽  
Ampk Signaling ◽  
Protein Levels ◽  
Obese Rats ◽  
Male Wistar Rats

AbstractLipin1, a lately indentified adipokine, may link obesity with insulin resistance and diabetes. The present study aimed to investigate the changes and significance of lipin1 expression and lipin1-AMPK signaling in diet-induced hepatic insulin resistance.24 4-week-old Male Wistar rats were randomly divided into 2 groups: (1) control group (CO), (2) high-fat diet group (HF). Insulin sensitivity was evaluated by hyperinsulinemic-euglycemic clamp technique. The mRNA levels of α1 and α2 subunit of AMPKα as well as Lipin1 were measured using Real-time RT-PCR. The activities of AMPKα and Akt were evaluated by detection of p-AMPKα (Thr-172) and p-Akt (ser473) by Western blot.After treatment of 4 months, HF group showed significantly increased levels of body weight, fasting plasma glucose and insulin levels; Plasma and liver total cholesterol (TC), triglycerides (TG) levels were also markedly elevated; Lipin1 expression at both mRNA and protein levels were significantly deceased. Compared with CO group, the mRNA and protein levels of AMPKα1 and AMPKα2 were not changed, whereas the p-AMPK (Thr-172) and p-AKT (ser473) levels in liver were significantly decreased in HF group.These findings indicated that the decrease in lipin1 expression and AMPKα activation may contribute to hepatic insulin resistance in diet-induced obese rats.

scholarly journals Combined effects of low-dose gambogic acid and NaI131 in drug-resistant non-small-cell lung cancer cells

2020 ◽  
Author(s):  
Jing Huang ◽  
Ming Ding ◽  
Ying Wu ◽  
Shuhua Han ◽  
Yan Xie ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Low Dose ◽  
Small Cell ◽  
Cyclin B ◽  
Gambogic Acid ◽  
Control Group ◽  
Mrna Levels ◽  
Drug Resistant ◽  
Small Cell Lung ◽  
Protein Levels

Abstract Background Radioactive seed is a method for treating drug-resistant, late-stage non-small cell lung cancer (NSCLC), but has undesirable side effects. Gambogic acid (GA), an ingredient of traditional Chinese medicine, exerts broad-spectrum antitumour activities via several pathways. This study aimed to elucidate the mechanism involved in the combined effect of low-dose GA and NaI131 to sensitize the antitumour activity of NaI131 in drug-resistant NSCLC cells. Methods Human NSCLC cell line A549 and drug-resistant cell lines A549/DDP and A549/Taxol were treated with NaI131, low-dose GA or a combination of both; control group of each cell line was treated with phosphate-buffered saline. Following treatment, cell proliferation, apoptosis, cell cycle, and levels of expression of apoptosis-related proteins namely CDK1, Cyclin B, mtp53, HSP90, and Bax, Bcl-2 respectively, and P-glycoprotein 1 (P-gp) known to confer resistance to chemotherapy, were detected using western blotting and immunofluorescence. mRNA levels of mtp53 and HSP90 were measured using qRT-PCR. Results Compared to the control group, A549, A549/DDP, and A549/Taxol cells treated with NaI131, GA or combination of drugs exhibited G2/M arrest, increased percentage of total apoptotic cells, significantly reduced protein levels of CDK1, Cyclin B, mtp53, HSP90, Bcl-2 and P-gp, increased protein levels of Bax and decreased mRNA levels of mtp53 and HSP90. The changes in the combination group were significantly different from the other groups. Conclusion In NSCLC cell lines, low-dose GA could enhance the effect of NaI131 on G2/M arrest, promote cell apoptosis, reduce drug-resistance and hence could be explored as a potential radionuclide sensitizer.

Coordinate expression of amplified metallothionein I and II genes in cadmium-resistant Chinese hamster cells

1985 ◽  
Vol 5 (12) ◽  
pp. 3525-3531
Author(s):  
J K Griffith
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Recombinant Dna ◽  
Chinese Hamster ◽  
Single Copy ◽  
Ovary Cell ◽  
Gene Copy ◽  
Mrna Levels ◽  
Protein Levels ◽  
Copy Numbers

Recombinant DNA probes complementary to Chinese hamster metallothionein (MT)-1 and MT-2 mRNAs were used to compare MT gene copy numbers, zinc-induced MT mRNA levels, and uninduced MT mRNA levels in cadmium-resistant (Cdr) Chinese hamster ovary cell lines. Quantitative hybridization analyses determined that the MT-1 and MT-2 genes are each present at approximately single-copy levels in the genome of cell line Cdr2C10 and are coordinately amplified approximately 7, 3, and 12 times over the Cdr2C10 value in the genomes of cell lines Cdr20F4, Cdr30F9, and Cdr200T1, respectively. The maximum zinc-induced MT-1 mRNA concentrations in cell lines Cdr20F4, Cdr30F9, and Cdr200T1 were equal to 1, 3, and 15 times that measured in Cdr2C10, respectively. Similarly, the maximum zinc-induced MT-2 mRNA concentrations were equal to 1, 3, and 14 times that measured in Cdr2C10, respectively, and in each instance they were 90 to 150 times greater than their respective concentrations in uninduced cells. Thus, relative MT gene numbers are closely correlated with both zinc-induced and uninduced MT mRNA levels in Cdr2C10, Cdr30F9, and Cdr200T1, but not in Cdr20F4. Each of the latter two lines possesses structurally altered chromosomes whose breakpoints are near the MT locus. Nonetheless, the ratio of the levels of MT-1 to MT-2 mRNAs was constant in each of the four cell lines, including Cdr20F4. These results demonstrate that MT-1 and MT-2 mRNAs are induced coordinately in each Cdr cell line. Therefore, the coordination of the induction of MT-1 and MT-2 mRNA is independent of MT gene amplification, MT gene rearrangement, and the relative inducibilities of amplified MT genes. However, MT mRNA and protein levels each indicate that MT-1 and MT-2 expression is non-coordinate in uninduced cells. Thus, regulation of MT expression may involve two different mechanisms which are differentially operative in induced and uninduced cells.

scholarly journals A Theacrine-Based Supplement Increases Cellular NAD+ Levels and Affects Biomarkers Related to Sirtuin Activity in C2C12 Muscle Cells In Vitro

Nutrients ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3727
Author(s):  
Petey W. Mumford ◽  
Shelby C. Osburn ◽  
Carlton D. Fox ◽  
Joshua S. Godwin ◽  
Michael D. Roberts
Keyword(s):  
Cell Line ◽  
Mitochondrial Biogenesis ◽  
C2c12 Myoblast ◽  
Mrna Levels ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Protein Levels ◽  
Myoblast Cell Line ◽  
Rodent Cell ◽  
Myoblast Cell

There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h (n = 6 treatment wells per time point). Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD+) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD+ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.

Modification of biophysical properties of lung epithelial Na+ channels by dexamethasone

2000 ◽  
Vol 279 (3) ◽  
pp. C762-C770 ◽  
Author(s):  
A. Lazrak ◽  
A. Samanta ◽  
K. Venetsanou ◽  
P. Barbry ◽  
S. Matalon
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Alveolar Epithelial Cell ◽  
A549 Cells ◽  
Alveolar Epithelium ◽  
Housekeeping Gene ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Open Probability ◽  
Protein Levels

There is considerable interest in identifying the basic mechanisms by which dexamethasone alters ion transport across the adult alveolar epithelium. Herein, we incubated synchronized A549 cells, a human alveolar epithelial cell line, with dexamethasone (1 μM) for 24–48 h. When normalized to HPRT (a housekeeping gene), A549 β- and γ-subunit mRNA levels for the human amiloride-sensitive epithelial sodium channel (hENaC), assessed by RT-PCR, increased by 1.6- and 17-fold respectively, compared with control values ( P < 0.05). These changes were abolished by actinomycin D, indicating transcriptional regulation. Western blotting studies revealed that dexamethasone also increased expression of β- and γ-hENaC protein levels. In contrast, α-hENaC mRNA increased by onefold ( P > 0.05) and α-hENaC protein level was unchanged. Incubation of A549 cells with dexamethasone increased their whole cell amiloride-sensitive sodium currents twofold and decreased the K 0.5 for amiloride from 833 ± 69 to 22 ± 5.4 nM (mean ± SE; P < 0.01). Single channel recordings in the cell-attached mode showed that dexamethasone treatment increased single channel open time and open probability threefold and decreased channel conductance from 8.63 ± 0.036 to 4.4 ± 0.027 pS (mean ± SE; P < 0.01). We concluded that dexamethasone modulates the amiloride-sensitive Na+ channels by differentially regulating the expression of β- and γ-subunits at the mRNA and protein levels in the human A549 cell line, with little effect on α-hENaC subunit.

Cytotoxicity Assessment of Quassinoids Neosergeolide and Isobrucein B toward Hematopoietic and Solid Malignancies Identifies STAT3 Activation To Be Predictive of Antitumor Response.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1394-1394
Author(s):  
Mitsuteru Hiwatari ◽  
Jingqiu Dai ◽  
Wei Liu ◽  
Yu-Dong Zhou ◽  
Dale G. Nagle ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cytotoxic Activity ◽  
Cell Line ◽  
Cell Lines ◽  
Strong Support ◽  
Luciferase Reporter ◽  
Control Group ◽  
Antitumor Effects ◽  
Protein Levels ◽  
Ic50 Values

Abstract Quassinoids are natural product compounds known to possess tumor cytotoxicity and antimalarial activity. Neosergiolide and isobrucein B are two quassinoids previously isolated from roots and stems of Picrolemma sprucei. In screening studies to identify inhibitors that target STAT3, we discovered neosergeolide and isobrucein B as active compounds. Approximately 5000 plant-derived extracts were screened using a cell line that stably expresses a STAT3-dependent luciferase reporter and NPM-ALK, which constitutively induces STAT3 transcriptional activity. Of 25 total hits, a P. sprucei extract was potent and selective for STAT3 inhibition, and bioassay-guided isolation identified neosergeolide and isobrucein B as the inhibitory compounds. Western blot analysis confirmed that neosergeolide and isobrucein B not only inhibit the tyrosine phosphorylation and activation of STAT3 but also decrease total STAT3 protein levels via a mechanism due in part to enhanced proteasome-mediated degradation. Small-molecule proteasome inhibitors such as MG132 and ALLN reversed the ability of the two quassinoids to decrease STAT3 protein levels; furthermore, simultaneous incubation of various hematopoietic malignancy cell lines with either neosergeolide or isobrucein B and MG132 or ALLN antagonized the cytotoxic activity of the quassinoids. Assessment of neosergiolide and isobrucein B antitumor effects using an XTT assay revealed both compounds to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with T-leukemias/lymphomas being especially responsive. For example, mycosis fungoides (MF)- and Sezary syndrome (SS)-derived cell lines, as well as non-MF/SS cutaneous T-cell lymphoma (CTCL) lines, were potently inhibited by both quassinoids (neosergiolide IC50 values: MAC-1, 11.6 nM; MAC-2A, 6.9 nM; Hut-78, 6.6 nM; HH, 4.3 nM; MJ, 7.0 nM; isobrucein B IC50 values: MAC-1, 31.9 nM; MAC-2A, 72.3 nM; Hut-78, 23.5 nM; HH; 20.3 nM; MJ, 13.5 nM). Non-hematopoietic cell lines representing various solid tumors also exhibited potent cytotoxic responses to the quassinoids (e.g., gastric carcinoma line AGS [neosergiolide IC50: 16.9 nM; isobrucein B IC50: 114.9 nM]). With rare exceptions, the cytotoxicity of the quassinoids against a specific tumor cell line correlated with STAT3 activation status; for example, breast cancer line MCF7 with inactive STAT3 was resistant to both quassinoids even at the maximum concentration tested (6.25 μM), whereas breast cancer lines MDA-MB-468 and MDA-MB-435s with activated STAT3 were inhibited by both compounds at low concentrations (neosergiolide IC50: MDA-MB-435s, 31.3 nM; MDA-MB-468, 29.9 nM; isobrucein B IC50: MDA-MB-435s, 209.3 nM; MDA-MB-468, 356.8 nM). The in vitro antitumor activity of the two quassinoids could also be demonstrated in vivo. For example, isobrucein B (1.0 mg/kg IP once q 3d x 5 doses) could be safely administered and potently inhibited the growth in SCID mice of the CD30+ primary CTCL MAC-1 cell line; mice at treatment day 16 showed average subcutaneous tumor volumes of 3839 ± 863 (s.e.) mm3 in the vehicle-control group and 913 ± 349 (s.e.) mm3 in the isobrucein B group (P=0.008, t-test). These results provide strong support for STAT3 targeting in antitumor drug discovery and suggest that quassinoids may have utility in such an approach.

Establishment of Novel Leukemic Cell Lines Resistant to Clofarabine by Dual Mechanisms of Decreased Active Metabolite and Increased Antiapoptotic Factor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1356-1356
Author(s):  
Hiroko Shigemi ◽  
Takahiro Yamauchi ◽  
Takanori Ueda
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Cell Lines ◽  
Active Metabolite ◽  
Nucleoside Analogs ◽  
Leukemic Cell ◽  
Mrna Levels ◽  
Protein Levels ◽  
Sports Science ◽  
Bcl2 Protein

Abstract Abstract 1356 Clofarabine(2-Chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine,2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, CAFdA) is a relatively new purine nucleoside analog. Upon administration, CAFdA is incorporated into leukemic cells by human Equilibrative Nucleoside Transporters (hENT) 1 and 2, and human Concentrative Nucleoside Transporter (hCNT) 3. Inside the cell, the agent is phosphorylated to CAFdA monophosphate by cytosolic deoxycytidine kinase (dCK) and mitochondrial deoxyguanosine kinase (dGK), and then to an intracellular active metabolite CAFdA triphosphate (CAFdATP). CAFdATP inhibits ribonucleotide reductase and is incorporated into DNA, thereby terminating DNA synthesis as an antimetabolite. Moreover, CAFdA induces apoptosis via direct mitochondrial damage. Clinical studies suggest that CAFdA is effective against both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). CAFdA therapy should be optimized based on the mechanistic understanding, because pharmacological determinants that correlate to the drug sensitivity may predict clinical efficacy of CAFdA as biological surrogate markers. Here, we have established two novel leukemic cell line variants that were resistant to CAFdA, and elucidated the mechanism of the drug resistance. The study focused on factors that were involved in the intracellular CAFdATP production and in the induction of apoptosis. To develop resistant variants, HL-60 cells were treated with escalating concentrations of CAFdA with the initial concentration at 1/100 of the concentration that inhibited 50% cell growth. After 7 months of the repeated passage, one cell line resistant to CAFdA (HL/CAFdA20) was cloned by the limiting dilution method. A part of this clone was further maintained with the drug for the subsequent 4 months to develop another variant (HL/CAFdA80). The 2 variants were 20- and 80-fold more CAFdA-resistant than HL-60 cells, respectively. They were cross-resistant to similar nucleoside analogs such as cladribine, gemcitabine, and cytarabine. Compared with HL-60 cell line, mRNA levels of the transporters (hENT1, hENT2, hCNT3) and protein levels of kinases (dCK, dGK), and the subsequent production of intracellular CAFdATP were all reduced in both CAFdA-resistant variants. Real time RT-PCR demonstrated that mRNA levels of hENT1, hENT2, and hCNT3 were 53.9%, 41.8%, 18.3% in HL/CAFdA20 cells, and 30.8%, 41.6%, 31.5% in HL/CAFdA80 cells, respectively, compared to the parental cells. The values of the initial uptake of CAFdA into the cell at 40 seconds after administration of tritiated CAFdA are 0.2 pmol/107cells in HL/CAFdA20 cells, and 0.1 pmol/107cells in HL/CAFdA80 cells, compared with 0.6 pmol/107 cells in parental HL60 cells. Western blotting revealed that protein levels of kinases were also reduced in these resistant variants with the greater reduction in HL/CAFdA80 cells. The subsequent production of CAFdATP after 4-h incubation with 10 μM CAFdA was 20 pmpl/107 cells in HL/CAFdA20 cells, 3 pmol/107cells in HL/CAFdA80 cells, and 63 pmol/107cells in HL-60 cells. The decreased CAFdATP production led to the attenuated incorporation of the drug into both mitochondrial and nuclear DNA. Concerning apoptosis, antiapoptotic Bcl2 protein overexpressed in the 2 resistant variants. The two variants were resistant to mitochondria-related apoptosis induced by CAFdA, in part due to the enhanced Bcl2 expression. A Bcl2 inhibitor ABT737 synergized the cytotoxic effect and the growth inhibition effect of CAFdA in both variants and HL-60, but the synergism was more profound in the resistant cell lines. The Combination Index values were 0.27 in HL/CAFdA20 cells, and 0.21 in HL/CAFdA80 cells, compared with 0.63 in HL-60 cells. This suggested the contribution of the enhanced Bcl2 protein to the mechanism of drug resistance. In conclusion, the mechanism of cellular resistance to CAFdA in the 2 variants was multifactorial, but primarily includes the reduced CAFdATP production and the increased antiapoptotic factor. It is noted that the decreased dGK level and Bcl2 overexpression were not reported previously in the context of CAFdA resistance. We suggest combination of CAFdA and ABT737 might be effective to CAFdA resistant and refractory leukemia. (This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. No.23501307) Disclosures: No relevant conflicts of interest to declare.

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