Gene expression profile of ABC transporters and cytotoxic effect of ibuprofen and acetaminophen in an epithelial ovarian cancer cell line in vitro

Perfil da expressão gênica dos transportadores ABC e efeito citotóxico do ibuprofeno e acetaminofen em uma linhagem celular de câncer epitelial de ovário in vitro

Abstracts

PURPOSES:

To determine the basic expression of ABC transporters in an epithelial ovarian cancer cell line, and to investigate whether low concentrations of acetaminophen and ibuprofen inhibited the growth of this cell line in vitro.

METHODS:

TOV-21 G cells were exposed to different concentrations of acetaminophen (1.5 to 15 μg/mL) and ibuprofen (2.0 to 20 μg/mL) for 24 to 48 hours. The cellular growth was assessed using a cell viability assay. Cellular morphology was determined by fluorescence microscopy. The gene expression profile of ABC transporters was determined by assessing a panel including 42 genes of the ABC transporter superfamily.

RESULTS:

We observed a significant decrease in TOV-21 G cell growth after exposure to 15 μg/mL of acetaminophen for 24 (p=0.02) and 48 hours (p=0.01), or to 20 μg/mL of ibuprofen for 48 hours (p=0.04). Assessing the morphology of TOV-21 G cells did not reveal evidence of extensive apoptosis. TOV-21 G cells had a reduced expression of the genes ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 and ABCE1 within the ABC transporter superfamily.

CONCLUSIONS:

This study provides in vitro evidence of inhibitory effects of growth in therapeutic concentrations of acetaminophen and ibuprofen on TOV-21 G cells. Additionally, TOV-21 G cells presented a reduced expression of the ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 and ABCE1 transporters.

Acetaminophen; Apoptosis; ATP-binding cassette transporters; Cell proliferation; Chemoprevention; Drug therapy; Ibuprofen; Ovarian neoplasms

OBJETIVOS:

Determinar a expressão básica dos transportadores ABC em uma linhagem celular do câncer epitelial de ovário, e investigar se o acetaminofen e o ibuprofeno em baixas concentrações são capazes de inibir o crescimento desta linhagem celular in vitro.

MÉTODOS:

A linhagem celular TOV-21 G foi exposta a diferentes concentrações de acetaminofen (1,5 a 15 µg/mL) e ibuprofeno (2,0 a 20 µg/mL), de 24 a 48 horas. O crescimento celular foi avaliado utilizando-se um ensaio de viabilidade celular. A morfologia celular foi determinada por meio da microscopia de fluorescência. O perfil de expressão gênica foi estabelecido por um painel de 42 genes da superfamília de transportadores ABC.

RESULTADOS:

Observou-se um decréscimo significativo no crescimento das células TOV-21 G expostas a 15 µg/mL de acetaminofen durante 24 (p=0,02) e 48 horas (p=0,01), ou a 20 µg/mL de ibuprofeno por 48 horas (p=0,04). Ao avaliar a morfologia das células cultivadas, não foi observada evidência de apoptose extensiva. A linhagem de células estudada subexpressa os genes de ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 e ABCE1 na superfamília de transportadores ABC.

CONCLUSÕES:

Este estudo fornece evidências in vitro referentes aos efeitos inibidores do crescimento de concentrações terapêuticas do acetaminofen e ibuprofeno na linhagem celular testada. Além disso, as células TOV-21 G apresentaram uma expressão reduzida de genes dos transportadores ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 e ABCE1.

Acetaminofen; Apoptose; Transportadores de cassetes de ligação de ATP; Proliferação de células; Quimioprevenção; Quimioterapia; Ibuprofeno; Neoplasias ovarianas

Introduction

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy^{1 , 2}. Given the poor prognosis for women with EOC, it is imperative to continue exploring novel chemo-preventive and chemotherapeutic agents³.

A range of findings, from epidemiological studies of patients to molecular studies of genetically modified mice, has led to a general acceptance that inflammation and cancer are linked^{4 , 5}. Research focusing on colorectal and prostate cancers has provided strong evidence that nonsteroidal anti-inflammatory drugs (NSAID) are effective in both cancer prevention and treatment of established tumors^{6 - 8}. Increasing evidence suggests that the inflammation significantly contributes to the etiology of EOC^{5 , 9}. Collectively, hypotheses attributing the EOC to ovulation, gonadotropin release, and hormonal influences likely are not mutually exclusive as they all converge on the role of inflammation in promoting ovarian tumorigenesis^{9 , 10}. These findings result in the initiation of a number of animal and clinical trials that examine the efficacy of cyclooxygenase (COX) inhibitors in primary and/or secondary prevention of cancer, both alone or as part of a combination therapy regimen for established tumors¹¹.

To date, 48 ATP-binding cassette (ABC) transporters that are divided into seven families (ABC A-G) have been identified in the human genome¹². Several of these transporters can actively efflux a wide range of anticancer drugs and reduce intracellular drug concentrations. This phenomenon eventually confers cross-resistance to various chemother apeutics drugs, resulting in multidrug resistance (MDR) acquisition^{13 , 14}.

The objective of this study was to determine the basic expression pattern of ABC transporters in an EOC cell line, and to investigate whether clinically relevant concentrations of acetaminophen and ibuprofen can inhibit the growth of EOC cells in vitro.

Methods

Cell culture

The human ovarian adenocarcinoma cell line TOV-21 G was obtained from the American Type Culture Collection (ATCC # CRL-11730), and spread according to their recommendations. These cells were maintained in a 1:1 mixture of culture medium 199 (Sigma # M2520) and MCDB 131 (Sigma # M8537), containing 10% fetal bovine serum (FBS) at 37ºC in a humidified atmosphere of 5% CO₂.

Cytotoxicity

Cell viability was determined through the use of 3-[4,5-dimethylthiazol-2-yl]-2,55-diphenyltetrazolium bromide (MTT) colorimetric assay; in which MTT, a nontoxic pale yellow substrate, was converted to a dark blue formazan product by living cells. The formazan accumulation can be spectrophotometrically measured and it is directly proportional to the number of viable cells. All experiments were performed in quadruplicate. Cells were plated at 1x10⁵ cells/well in 96-well plates and incubated at 37ºC, for 24 hours. The cells were washed in the following day with PBS and treated with acetaminophen at concentrations ranging from 1.5 to 15 μg/mL, or ibuprofen at 2.0 to 20 μg/mL. Control wells contained no drug. After incubation periods of 24 and 48 hours, 10 μL of MTT tetrazolium solution was added to each well and the cells were incubated at 37ºC for three hours. Plates were then centrifuged and the supernatant was removed. The formazan was solubilized with 50 μL of DMSO, and absorbance at 550 nm was measured. The results of the treated cultures are expressed as the percentage of viable cells compared with untreated controls.

Fluorescence staining

Cell morphology was established after exposure to concentrations of 1.5, 7.5 and 15 µg/mL of acetaminophen or 2, 10 and 20 µg/mL of ibuprofen for 48 hours through staining with DAPI, MitoTracker Orange and Live/Dead using a fluorescence microscope (Axiovert 200, Zeiss).

TOV-21G cells were treated with acetaminophen and ibuprofen, as previously described, fixed for one hour with 4% paraformaldehyde in PBS and then stained with DAPI solution for five minutes. DAPI is a fluorescent stain that binds strongly to DNA. When stained with DAPI, the DNA can be visualized with blue-white fluorescence. The cells were visually assessed for any morphological features of apoptosis, such as cell shrinkage, chromatin condensation, and formation of apoptotic bodies. To label mitochondria, cells were incubated with 50 nM MitoTracker Orange dye in pre-warmed medium, during 60 minutes at 37ºC. This dye passively diffuses across the plasma membrane and accumulates in active mitochondria. The Live/Dead assay provides a two-color fluorescence cell viability assay, which is based on the simultaneous labeling of live and dead cells with two probes. Cells were washed with Hanks' balanced saline solution and stained with Live/Dead solution (1 µM Calcein AM and 2 µM Ethidium homodimer-1 in phosphate-buffered saline) for 10 minutes at room temperature in the dark. These labels allow the visualization of two recognized parameters of cell viability - intracellular esterase activity and plasma membrane integrity. The cell must be viable and functional so this fluorescence can happen. Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually non-fluorescent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is retained within live cells, producing an intense uniform green fluorescence in live cells. EthD-1 enters the cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells.

Real-time polymerase chain reaction

The quantitative polymerase chain reaction (PCR) was used to measure the expression level of human ABC transporter superfamily genes in TOV-21 G cells using a pre-made panel from Roche Applied Science. Each multi-well plate contains assays for 42 different ABC transporter genes in duplicate. Seven referential genes served as PCR controls and allowed for the quantification of the relative expression of target genes. The assays were carried out using a LightCycler^(r) 480 instrument and 96 multi-well plates containing target-specific primers and a FAM-labeled Universal Probe Library hydrolysis probe, which has RNA. Total RNA was isolated from TOV-21 G cells using TRIzol^(r) reagent. Two micrograms of total RNA were used to generate the first strand cDNA through reverse transcription.

Statistical analysis

In order to compare the mRNA expression of different ABC transporters, the results of gene expression were analyzed based on the cycle threshold (Ct) values normalized to beta-2-micro-globulin and 18 S probable dimethyladenosine transferase precursor genes. The Ct values of control genes were subtracted from those of the transporters to calculate a ΔCt value (e.g., Ct of transporter - CtB2M). The Student's t-test was performed to analyze the statistical significance of MTT values, and a one-way ANOVA was executed for pair-wise comparisons. Statistical analyses were done using GraphPad Software, Prism 4.0 (GraphPad Software, San Diego, CA, USA). Differences were considered statistically significant if p<0.05.

Results

Cell proliferation

Acetaminophen at concentrations of 1.5, 7.5 and 15 µg/L decreased TOV-21 G cell proliferation in 3.5 (p=0.10), 3.5 (p=0.08), and 7.2% (p=0.02), respectively, at 24 hours compared to untreated control cells considered to be of 100% proliferation (Figure 1A). After 48 hours, acetaminophen decreased cell proliferation in 7.6 (p=0.12), 5.2 (p=0.43), and 12.3% (p=0.01), as seen in Figure 1B.

Figure 1.
Cell viability in ovarian cancer cell lines exposed to acetaminophen in different time intervals. Bars represent the means of experiments performed in quadruplicate. Error bars indicate the standard deviation. Cell growth is expressed as a percentage of control. TOV-21 G cells showed a proliferation decrease at concentrations of 1.5, 7.5 and 15 μg/mL for drug exposure at (A) 24 and (B) 48 hours

When the cellular response was analyzed to 2, 10 and 20 µg/L of ibuprofen at 24 hours, decreases in cellular growth of 0.3 (p=0.96), 4.9 and 3.9% (p=0.05), respectively, were seen compared to controls (Figure 2A). These decreases were more apparent after 48 hours of ibuprofen exposure, at which time cellular growth was diminished to 7.8 (p=0.16), 9.52 (p=0.12), and 9.5% (p=0.04) compared to controls (Figure 2B).

Figure 2.
Cell viability in ovarian cancer cell lines exposed to ibuprofen in different time intervals. Bars represent the means of experiments performed in quadruplicate. Error bars indicate the standard deviation. Cell growth is expressed as a percentage of control. TOV-21 G cells showed a proliferation decrease at concentrations of 2, 10 and 20 μg/mL for drug exposure at (A) 24 and (B) 48 hours

Morphological changes

The morphological characteristics of cells were evaluated through fluorescent staining. Forty-eight hours after treatment, cells that were treated with acetaminophen and ibuprofen did not show any morphological signs of apoptosis than did the controls (Figures 3 and 4A to D). The Live/Dead assay revealed that 48 hours of treatment with acetaminophen at 15 µg/mL or ibuprofen at 20 µg/mL did not increase the number of dead cells (Figures 3 and 4E to H).

Figure 3.
Representative images of DAPI (A to D), Live/Dead (E to H) and MitoTracker (I to L) staining of TOV-21 G cells treated with 1.5, 7.5 and 15 μg/mL of acetaminophen. The white circles indicate red dead cells

Figure 4.
Representative images of DAPI (A to D), Live/Dead (E to H) and MitoTracker (I to L) staining of TOV-21 G cells treated with 2, 10 and 20 μg/mL of ibuprofen. The white circles indicate red dead cells

MitoTracker Orange is an orange-fluorescent dye that stains mitochondria in live cells. Cells were considered MitoTracker Orange positive if a bright punctate orange fluorescence of the mitochondria was observed, and negative if cells exhibited a diffuse orange cytoplasmic staining. By 48 hours, cells treated with acetaminophen or ibuprofen showed no morphological changes in their mitochondria (Figures 3 and 4I to L).

Real-time polymerase chain reaction

TOV-21 G cells had reduced expression of the ABCA1, ABCC3, ABCC4, ABCD3, ABCD4, and ABCE1 transporter genes. The expression of ABCC4 (ΔCtB2M=-0.77 and ΔCt18S=-10.48) was not altered as much as that of ABCA1 (ΔCtB2M=-10.73 and ΔCt18S=-24,43), which was significantly under-expressed (Figure 5).

Figure 5.
Gene expression profile of different ABC transporters normalized to a beta-2-micro-globulin precursor gene (ΔCt B2M=Cttransporter- CtB2M) and an 18 S gene (ΔCt 18S=Cttransporter-Ct18S)

Discussion

Testing different drugs on cell lines is a critical component of the antineoplastic drug development. MTT assays allow for testing different concentrations and durations of drug exposure. Limited data are available regarding the effects of NSAIDS on cell lines of EOC^{15 , 16}. The published literature includes a wide range of drugs, doses and schedules, making it challenging to reconcile the differences and to understand how well a drug may work in the clinical practice^{17 - 19}.

From a clinically relevant perspective, ibuprofen is one of the most widely used analgesic, antipyretic and anti-inflammatory drugs nowadays²⁰. It has relatively low risks for gastrointestinal, hepato-renal and other rare adverse drug reactions compared with other NSAIDs and coxibs²⁰. Indeed, it has been described as "the mildest NSAID with the fewest side effects that has been in clinical use for a long time"²⁰. Patients have been maintained on high doses of ibuprofen for years, without serious adverse effects¹⁷. Advanced age has a minimal influence on the pharmacokinetics of ibuprofen, and dosage apparently does not need to be adjusted for age²¹. The drug has, in general, predictable and reliable kinetic properties²⁰. The maximum serum concentration (C_max) following the ingestion of 400, 600 and 800 mg doses is 15.4, 17.1 and 24.2 µg/mL, respectively²⁰.

Acetaminophen is one of the most popular and widely used drugs for the treatment of pain and fever, and does not produce gastrointestinal damage or untoward cardiorenal effects²². Despite much research, definitive proof that the analgesic and antipyretic effects of acetaminophen are dependent on COX inhibition is still lacking²². Indeed, inhibition of a third form of COX, COX-3, is one of the more recent proposals that have been put forward to explain the unusual effects of acetaminophen^{22 , 23}. Plasma concentrations of 10 to 20 µg/mL have an assumed role to be therapeutic for acetaminophen²⁴.

To our knowledge, this is the first report describing the effect of ibuprofen and acetaminophen on the growth of an EOC cell line in vitro, using therapeutically relevant drug concentrations. Acetaminophen at a concentration of 15 µg/mL showed a significant inhibition of an EOC cell line after 24 and 48 hours of treatment. Ibuprofen inhibited growth at 20 µg/mL, but only 48 hours after exposure. The anti-proliferative effect exerted by ibuprofen on the EOC cell line studied here is in accordance with previous observations¹. The finding that therapeutic levels of acetaminophen and ibuprofen have effects on EOC cells, suggests the possibility of a chemo-preventive/chemotherapeutic strategy using these agents in dosages and schedules that will minimize the side effects, while yielding optimal ovarian cancer prevention/treatment.

This study demonstrated that acetaminophen and ibuprofen at therapeutics levels do not activate apoptosis in EOC cells. The mechanism(s) underlying the decrease in cell proliferation remains to be elucidated. It has been previously proposed that ibuprofen anti-proliferative actions are independent of COX and that p75NTR, a recently identified tumor suppressor, may in part be responsible for ibuprofen anticancer properties^{1 , 25 , 26}. Previous studies suggest that acetaminophen is a tyrosine kinase substrate and intracellular glutathione depletion, reactive oxygen species formation and mitochondrial toxicity contributed to acetaminophen selective toxicity in melanoma cell lines^{27 , 28}.

Measuring ABC transporter gene expression may be useful in predicting anticancer drug response. Real-time PCR is the most reliable and sensitive gene expression profiling technology for analyzing a panel of genes. Many studies have been carried out assessing the expression profile of ABC transporters. Thus, most of these investigations analyze only one or a small group of ABC transporters. In contrast, our method allows for the complete analysis of 42 ABC transporters. In this study, we focused on determining the basic expression profile of ABC transporters in TOV-21 G cells, an EOC cell line. Our data demonstrated that ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 and ABCE1 were severely down-regulated. Yasui et al. found that ABCC4, ABCD3, ABCD4 and ABCE1 were amplified among 19 of the examined resistant cell lines²⁹. ABCA1 is a major regulator of HDL metabolism, yet its mechanistic involvement in cancer cell proliferation and progression is unclear³⁰. Data indicate that ABCC4, which was not altered largely, can release prostaglandins from cells. In addition to inhibiting prostaglandin synthesis, some NSAIDs might also act by inhibiting this release³¹. Therefore, an effect on ABCC4 may underlie the potentially beneficial effects of NSAIDs.

An increased understanding of the mechanisms underlying drug resistance may lead to the development of more successful therapeutic protocols²⁹. Hence, the expression profile of ABC transporter genes in cell lines used for drug screening is highly important for the antineoplastic drug discovery.

These findings ensure further studies on the physiologic role, clinical relevance, and potential use of ABCC4 as a therapeutic target. Due to the limitations of in vitro testing, these results should be confirmed in animal models for safety and efficacy. Using more than one cell type may help avoid making decisions based on tissue-specific responses.

Finally, the therapeutic concentrations of acetaminophen and ibuprofen directly inhibit EOC cell viability, which may significantly contribute to their antineoplastic effects. These results should encourage further research regarding the potential benefit of acetaminophen and NSAIDs in chemoprevention or as adjuvant therapies in EOC treatment.

References

• ¹
  Andrews P, Zhao X, Allen J, Li F, Chang M. A comparison of the effectiveness of selected non-steroidal anti-inflammatory drugs and their derivatives against cancer cells in vitro. Cancer Chemother Pharmacol. 2008;61(2):203-14.
• ²
  Kurman RJ, Shih IM. The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory. Am J Surg Pathol. 2010;34(3):433-43.
• ³
  Elit L, Oliver TK, Covens A, Kwon J, Fung MF, Hirte HW, et al. Intraperitoneal chemotherapy in the first-line treatment of women with stage III epithelial ovarian cancer: a systematic review with metaanalyses. Cancer. 2007;109(4):692-702.
• ⁴
  Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature. 2008;454(7203):436-44.
• ⁵
  Trabert B, Pinto L, Hartge P, Kemp T, Black A, Sherman ME, et al. Pre-diagnostic serum levels of inflammation markers and risk of ovarian cancer in the prostate, lung, colorectal and ovarian cancer (PLCO) screening trial. Gynecol Oncol. 2014;135(2):297-304.
• ⁶
  Gupta RA, Dubois RN. Colorectal cancer prevention and treatment by inhibition of cyclooxygenase-2. Nat Rev Cancer. 2001;1(1):11-21.
• ⁷
  Shebl FM, Sakoda LC, Black A, Koshiol J, Andriole GL, Grubb R, et al. Aspirin but not ibuprofen use is associated with reduced risk of prostate cancer: a PLCO study. Br J Cancer. 2012;107(1):207-14.
• ⁸
  Murphy MA, Trabert B, Yang HP, Park Y, Brinton LA, Hartge P, et al. Non-steroidal anti-inflammatory drug use and ovarian cancer risk: findings from the NIH-AARP Diet and Health Study and systematic review. Cancer Causes Control. 2012;23(11):1839-52.
• ⁹
  Shan W, Liu J. Inflammation: a hidden path to breaking the spell of ovarian cancer. Cell Cycle. 2009;8(19):3107-11.
• ¹⁰
  Brasky TM, Liu J, White E, Peters U, Potter JD, Walter RB, et al. Non-steroidal anti-inflammatory drugs and cancer risk in women: results from the Women's Health Initiative. Int J Cancer. 2014;135(8):1869-83.
• ¹¹
  Li W, Wang J, Jiang HR, Xu XL, Zhang J, Liu ML, et al. Combined effects of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on ovarian carcinoma in vivo. Int J Mol Sci. 2011;12(1):668-81.
• ¹²
  Leonard GD, Fojo T, Bates SE. The role of ABC transporters in clinical practice. Oncologist. 2003;8(5):411-24.
• ¹³
  Auner V, Sehouli J, Oskay-Oezcelik G, Horvat R, Speiser P, Zeillinger R. ABC transporter gene expression in benign and malignant ovarian tissue. Gynecol Oncol. 2010;117(2):198-201.
• ¹⁴
  Wu CP, Hsieh CH, Wu YS. The emergence of drug transporter-mediated multidrug resistance to cancer chemotherapy. Mol Pharm. 2011;8(6):1996-2011.
• ¹⁵
  Sutcliffe S, Grubb Iii RL, Platz EA, Ragard LR, Riley TL, Kazin SS, et al. Non-steroidal anti-inflammatory drug use and the risk of benign prostatic hyperplasia-related outcomes and nocturia in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. BJU Int. 2012;110(7):1050-9.
• ¹⁶
  Lang Kuhs KA, Hildesheim A, Trabert B, Kemp TJ, Purdue MP, Wentzensen N, et al. Association between regular aspirin use and circulating markers of inflammation: a study within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Cancer Epidemiol Biomarkers Prev. 2015;24(5):825-32.
• ¹⁷
  Konstan MW, Byard PJ, Hoppel CL, Davis PB. Effect of high-dose ibuprofen in patients with cystic fibrosis. N Engl J Med. 1995;332(13):848-54.
• ¹⁸
  Valle BL, D'Souza T, Becker KG, Wood WH 3rd, Zhang Y, Wersto RP, et al. Non-steroidal anti-inflammatory drugs decrease E2F1 expression and inhibit cell growth in ovarian cancer cells. PLoS One. 2013;8(4):e61836.
• ¹⁹
  Baandrup L, Faber MT, Christensen J, Jensen A, Andersen KK, Friis S, et al. Nonsteroidal anti-inflammatory drugs and risk of ovarian cancer: systematic review and meta-analysis of observational studies. Acta Obstet Gynecol Scand. 2013;92(3):245-55.
• ²⁰
  Rainsford KD. Ibuprofen: pharmacology, efficacy and safety. Inflammopharmacology. 2009;17(6):275-342.
• ²¹
  Albert KS, Gillespie WR, Wagner JG, Pau A, Lockwood GF. Effects of age on the clinical pharmacokinetics of ibuprofen. Am J Med. 1984;77(1A):47-50.
• ²²
  Bertolini A, Ferrari A, Ottani A, Guerzoni S, Tacchi R, Leone S. Paracetamol: new vistas of an old drug. CNS Drug Rev. 2006;12(3-4):250-75.
• ²³
  Duncan K, Uwimpuhwe H, Czibere A, Sarkar D, Libermann TA, Fisher PB, et al. NSAIDs induce apoptosis in nonproliferating ovarian cancer cells and inhibit tumor growth in vivo. IUBMB Life. 2012;64(7):636-43.
• ²⁴
  Stocker ME, Montgomery JE. Serum paracetamol concentrations in adult volunteers following rectal administration. Br J Anaesth. 2001;87(4):638-40.
• ²⁵
  Khwaja F, Allen J, Lynch J, Andrews P, Djakiew D. Ibuprofen inhibits survival of bladder cancer cells by induced expression of the p75NTR tumor suppressor protein. Cancer Res. 2004;64(17):6207-13.
• ²⁶
  Quann EJ, Khwaja F, Zavitz KH, Djakiew D. The aryl propionic acid R-flurbiprofen selectively induces p75NTR-dependent decreased survival of prostate tumor cells. Cancer Res. 2007;67(7):3254-62.
• ²⁷
  Vad NM, Yount G, Moore D, Weidanz J, Moridani MY. Biochemical mechanism of acetaminophen (APAP) induced toxicity in melanoma cell lines. J Pharm Sci. 2009;98(4):1409-25.
• ²⁸
  Trabert B, Ness RB, Lo-Ciganic WH, Murphy MA, Goode EL, Poole EM, et al. Aspirin, nonaspirin nonsteroidal anti-inflammatory drug, and acetaminophen use and risk of invasive epithelial ovarian cancer: a pooled analysis in the Ovarian Cancer Association Consortium. J Natl Cancer Inst. 2014;106(2):djt431.
• ²⁹
  Yasui K, Mihara S, Zhao C, Okamoto H, Saito-Ohara F, Tomida A, et al. Alteration in copy numbers of genes as a mechanism for acquired drug resistance. Cancer Res. 2004;64(4):1403-10.
• ³⁰
  Fukuchi J, Hiipakka RA, Kokontis JM, Hsu S, Ko AL, Fitzgerald ML, et al. Androgenic suppression of ATP-binding cassette transporter A1 expression in LNCaP human prostate cancer cells. Cancer Res. 2004;64(21):7682-5.
• ³¹
  Reid G, Wielinga P, Zelcer N, van der Heijden I, Kuil A, de Haas M, et al. The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs. Proc Natl Acad Sci U S A. 2003;100(16):9244-9.

Publication Dates

• Publication in this collection
  June 2015

History

• Received
  03 Feb 2015
• Accepted
  18 May 2015