0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Original Article |

Antineoplastic Effects of Decitabine, an Inhibitor of DNA Promoter Methylation, in Adrenocortical Carcinoma Cells FREE

Insoo Suh, MD; Julie Weng, BS; Gustavo Fernandez-Ranvier, MD; Wen T. Shen, MD; Quan-Yang Duh, MD; Orlo H. Clark, MD; Electron Kebebew, MD
[+] Author Affiliations

Author Affiliations: Department of Surgery and Helen Diller Comprehensive Cancer Center, University of California, San Francisco.


Arch Surg. 2010;145(3):226-232. doi:10.1001/archsurg.2009.292.
Text Size: A A A
Published online

Hypotheses  Decitabine recovers expression of silenced genes on chromosome 11q13 and has antineoplastic effects in adrenocortical carcinoma (ACC) cells.

Design  NCI-H295R cells were treated with decitabine (0.1-1.0μM) over 5 days. Cells were evaluated at 24-hour intervals for the effects of decitabine on ACC cell proliferation, cortisol secretion, and cell invasion. Expression was quantified for 6 genes on 11q13 (DDB1, MRPL48, NDUFS8, PRDX5, SERPING1, and TM7SF2) that were previously shown to be underexpressed in ACC.

Setting  Academic research.

Study Specimen  Human ACC cell line.

Main Outcome Measures  Adrenocortical carcinoma cell proliferation, cortisol secretion, and cell invasion were measured using immunometric assays. Quantitative reverse transcription–polymerase chain reaction was used to measure gene expression relative to GAPDH.

Results  Decitabine inhibited ACC cell proliferation by 39% to 47% at 5 days after treatment compared with control specimens (P < .001). The inhibitory effect was cytostatic, time dependent, and dose dependent. Decitabine decreased cortisol secretion by 56% to 58% at 5 days after treatment (P = .02) and inhibited cell invasion by 64% at 24 hours after treatment (P = .03). Of 6 downregulated genes on 11q13, decitabine recovered expression of NDUFS8 (OMIM 602141) (P < .001) and PRDX5 (OMIM 606583) (P = .006).

Conclusions  Decitabine exhibits antitumoral properties in ACC cells at clinically achievable doses and may be an effective adjuvant therapy in patients with advanced disease. Decitabine recovers expression of silenced genes on 11q13, which suggests a possible role of epigenetic gene silencing in adrenocortical carcinogenesis.

Figures in this Article

Adrenocortical carcinoma (ACC) is a rare malignant neoplasm involving the adrenal cortex, affecting 2 persons per 1 million per year.1,2 These are aggressive tumors, with a 5-year survival of 50% among patients with surgically resectable disease and with a median survival of less than 1 year among patients with metastases.3,4 In addition to its high mortality, ACC causes significant detriment to the quality of life of affected patients because of tumoral hypersecretion of hormones, particularly cortisol.5 Most patients require systemic adjuvant chemotherapy and mitotane treatment to attempt to limit tumor progression and hormonal hypersecretion.68 However, these agents have poor efficacy in most patients.

The molecular pathogenesis of sporadic ACC is poorly understood, but several factors seem important, including insulin growth factor 2 overexpression.9 In particular, loss of heterozygosity at chromosome 11q13 has been reported in up to 70% to 100% of ACCs,10,11 suggesting that inactivation of genes on 11q13 could contribute to adrenocortical carcinogenesis. Six significantly underexpressed genes on 11q13 in ACC were recently identified using genome-wide expression microarray analysis.12 Although the specific mechanisms behind silencing of these genes are unknown, the concept of using therapies to recover expression of these candidate tumor suppressor genes and to possibly halt cancer progression is promising.

Recently, there is increasing evidence that reversible, or epigenetic, mechanisms in gene silencing have an important role in cancer. In turn, this has prompted enthusiasm for the possible usefulness of drugs that specifically target these epigenetic alterations.13,14 Decitabine (5-aza-2′-deoxycytidine) reverses the classic epigenetic alteration, DNA promoter methylation. It acts by removing inhibitory methyl groups from the cytosine residues of promoter sequences, which presumably restores downstream gene transcription. Compared with high-dose regimens that were used in the 1970s, lower in vitro (≤10μM) and in vivo (15-20 mg/m2/d for 3-5 days) doses of decitabine have demonstrated growth inhibitory properties in several cancer models with fewer adverse effects.15,16 Decitabine is approved by the Food and Drug Administration as first-line treatment of myelodysplastic syndromes.14,17

Although a few studies18,19 previously examined the effect of decitabine on cell proliferation and cortisol secretion in human ACC cells, the current emphasis on lower-dose clinical regimens warranted a more specific study on the functional influence of low-dose decitabine. We set out to determine the effects of low-dose decitabine (0.1μM and 1.0μM) on a human ACC cell line. We examined ACC cell proliferation and cortisol secretion using immunometric assays and estimated invasive potential using a model of cell migration through a synthetic polycarbonate membrane. In addition, we determined the effects of decitabine on expression of underexpressed genes at 11q13.

CELL CULTURE AND REAGENTS

NCI-H295R cells (ATCC, Rockville, Maryland) were grown and maintained in a 1:1 solution of Dulbecco modified Eagle medium (DMEM) and F12 (DMEM:F12) supplemented with premix (ITS+; BD Biosciences, San Jose, California), serum (Nu-Serum I [2.5%], BD Biosciences), and 10 000 U/mL of penicillin-streptomycin in a standard humidified incubator at 37°C in a 5% CO2 atmosphere. Decitabine (Sigma, St Louis, Missouri) was dissolved in dimethyl sulfoxide (DMSO) vehicle and prepared to 2 clinically achievable doses (0.1μM in 0.025% DMSO and 1.0μM in 0.25% DMSO). In addition, DMSO vehicle without decitabine was prepared at analogous doses for control specimens. Treatments were initiated 48 hours after trypsin splitting and reseeding of cells onto new plates.

QUANTIFICATION OF ACC CELL PROLIFERATION

Cells were reseeded onto a 96-well plate at a concentration of 1 × 105 cells/200 μL of culture medium. After treatment administration, cells were incubated for 1, 2, 3, 4, and 5 days at 37°C in 5% CO2. For each specimen group and time point, culture medium was aspirated from the well, and the cells were lysed and immediately frozen at −80°C for 24 hours. The plates were thawed at room temperature and prepared for cell count quantification using an assay kit (CyQUANT; Invitrogen, Carlsbad, California) that measures total nucleic acid content from lysed cells. The plates were processed according to the manufacturer's instructions and analyzed on a fluorometric plate reader at 480-nm and 520-nm wavelengths.

CORTISOL SECRETION MEASUREMENT

Cells were reseeded onto a 12-well plate at a concentration of 5 × 105 cells/mL of culture medium. After treatment administration, cells were incubated for 5 days at 37°C in 5% CO2. At 24-hour intervals during the 5-day incubation, 30-μL aliquots of culture medium were taken from each well. To remove any cells in suspension, the aliquots were centrifuged, and the supernatants were aspirated off and stored at −80°C until the time of measurement. Cortisol concentration was measured using an immunometric assay kit (Parameter; R&D Systems, Minneapolis, Minnesota) according to the manufacturer's instructions. The assayed samples were analyzed on a colorimetric plate reader at 480-nm and 520-nm wavelengths.

CELL INVASION ASSAY

Determination of cell invasion was performed with an assay (QCM ECMatrix Cell Invasion Assay; Millipore, Billerica, Massachusetts) that uses a modified Boyden chamber technique. Briefly, cells were starved for 48 hours with serum-free DMEM:F12 before reseeding onto a manufacturer-supplied 96-well upper chamber at a concentration of 1 × 105 cells/100 μL of DMEM:F12. The bottom of the upper chamber consists of a porous polycarbonate membrane with a layer of extracellular matrix (ECM). The upper chamber was placed into a lower 96-well plate with normal serum-containing culture medium, and the cells were incubated for 24 hours at 37°C in 5% CO2. Cells that invaded through the membrane were detached and lysed, and the cell count was quantified using the kit described in the “Quantification of ACC Cell Proliferation” subsection.

RNA PREPARATION AND QUANTITATIVE REVERSE TRANSCRIPTION–POLYMERASE CHAIN REACTION

After treatment and incubation, total RNA was isolated from cells using a reagent (TRIzol, Invitrogen) and purified using a kit (RNeasy Mini Kit; Qiagen, Valencia, California). At a concentration of 125 ng/μL, total RNA was reverse transcribed using a complementary DNA (cDNA) synthesis kit (RT Script; USB, Cleveland, Ohio). Quantitative polymerase chain reaction (PCR) was performed on the cDNA samples to measure expression levels of 6 genes on 11q13 (DDB1 [OMIM 600045], MRPL48 [OMIM 611853], NDUFS8, PRDX5, SERPING1 [OMIM 606860], and TM7SF2 [OMIM 603414]) that were previously found to be underexpressed in ACC.12 The expression level of each gene was normalized to that of the GAPDH housekeeping gene. The PCR primers and probes for the genes were premade (TaqMan Assay-on-Demand kit; Applied Biosystems, Foster City, California). The manufacturer's inventory numbers for the primer-probe sets were Hs01096554_g1 (for DDB1), Hs99999905_m1 (GAPDH), Hs00740658_m1 (MRPL48), Hs00159597_m1 (NDUFS8), Hs00201536_m1 (PRDX5), Hs00163781_m1 (SERPING1), and Hs00162807_m1 (TM7SF2). The PCR reactions were performed (ABI PRISM 7900 Sequence Detection System, Applied Biosystems) with 1 μL of cDNA in a final volume of 20 μL for 12 minutes at 95°C, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Gene expression was quantified using the following equation:

STATISTICAL ANALYSIS

Two-tailed t test was used to compare outcome variables between study specimens vs controls. The outcome variables were cell count, cortisol concentration, and percentage gene expression.

DECITABINE INHIBITS ACC CELL PROLIFERATION IN NCI-H295R CELLS

The untreated control specimen of NCI-H295R cells had a doubling time of 2 days. No significant difference was noted in ACC cell proliferation between the untreated control specimen and the DMSO vehicle control specimen. Decitabine (0.1μM and 1.0μM doses) caused significant time-dependent cytostatic attenuation of ACC cell proliferation (Figure 1A). The differences in ACC cell proliferation were significant at 5 days after treatment, with 39% (P = .02) and 47% (P < .001) decreases in cell counts for the 0.1μM and 1.0μM doses, respectively (Figure 1B). The higher dose of decitabine led to greater inhibition of ACC cell proliferation than the lower dose, although the difference was not statistically significant.

Place holder to copy figure label and caption

Figure 1. Effects of decitabine on cortical secretion in NCI-H295R cells (ATCC, Rockville, Maryland). Cells were treated with decitabine (in dimethyl sulfoxide [DMSO] vehicle) at 0.1μM and 1.0μM doses for 5 days, and cell counts were measured using an assay kit (CyQUANT; Invitrogen, Carlsbad, California). Compared with control specimens, decitabine caused time-dependent (A) and dose-dependent (B) inhibitory effects on adrenocortical carcinoma cell proliferation, with significant 39% (*P = .02) and 47% (†P < .001) decreases in cell counts observed at 5 days after treatment. Serum (10%) is Nu-Serum (BD Biosciences, San Jose, California).

Graphic Jump Location
DECITABINE LEADS TO DECREASED CORTISOL SECRETION IN NCI-H295R CELLS

Cortisol concentrations did not differ significantly between the untreated control specimen and the DMSO vehicle control specimen of NCI-H295R cells. Treatment with either dose of decitabine led to a significant decrease in cortisol concentration at 5 days after treatment, with 58% (P = .001) and 56% (P = .02) decreases for the 0.1μM and 1.0μM doses, respectively (Figure 2A and B). However, only the 0.1μM dose caused a significant decrease in cortisol concentration relative to cell concentration (calculated by dividing cortisol concentration by absolute cell count) (P = .03) (Figure 2C).

Place holder to copy figure label and caption

Figure 2. Effects of decitabine on cortisol concentration in NCI-H295R cells (ATCC, Rockville, Maryland). Cells were treated with decitabine (in dimethyl sulfoxide [DMSO] vehicle) at 0.1μM and 1.0μM doses for 5 days, and cortisol concentrations in cell media were measured using an immunometric assay kit (Parameter; R&D Systems, Minneapolis, Minnesota). Media taken from decitabine-treated cells had decreased cortisol concentration over time (A), with significant decreases at 5 days after treatment of 58% (*P = .001) and 56% (†P = .02) for the 0.1μM and 1.0μM doses, respectively (B). The inhibitory effect of decitabine on cortisol concentration per cell (C) was significant only with the lower 0.1μM dose (‡P = .03).

Graphic Jump Location
DECITABINE INHIBITS NCI-H295R CELL INVASION

The inhibitory effects of decitabine on the invasive potential of NCI-H295R cells were observed at 24 hours after treatment. Compared with controls, decitabine-treated cells showed significantly attenuated cell invasion through the ECM at 24 hours (Figure 3). As with ACC cell proliferation, the effects of decitabine on cell invasion were dose dependent, with a 64% decrease in cell invasion at 24 hours using the 1.0μM dose (P = .03).

Place holder to copy figure label and caption

Figure 3. Effects of decitabine on NCI-H295R cell (ATCC, Rockville, Maryland) invasion. Cells were starved of serum for 48 hours and then placed in a modified Boyden chamber for 24 hours. Cells that invaded through the extracellular matrix barrier were counted using an assay kit (CyQUANT; Invitrogen, Carlsbad, California). Decitabine had a dose-dependent inhibitory effect on NCI-H295R cell invasion, with a 64% decrease in cell invasion using the 1.0μM dose (*P = .03). DMSO indicates dimethyl sulfoxide.

Graphic Jump Location
DECITABINE RECOVERS EXPRESSION OF UNDEREXPRESSED GENES ON 11q13

As already described, quantitative reverse transcription–PCR (RT-PCR) analysis was performed for 6 underexpressed genes at 11q13 on NCI-H295R cells after decitabine treatment. The RT-PCR was performed at 3 days after decitabine (1.0μM) treatment (along with respective controls). After decitabine treatment, expression was significantly altered in 4 of 6 genes (DDB1, NDUFS8, PRDX5, and TM7SF2). Of these, only NDUFS8 (P < .001) and PRDX5 (P = .006) showed significantly recovered expression after decitabine treatment (Table).

Table Graphic Jump LocationTable. Quantitative RT-PCR Analysis of 4 Genes on Chromosome 11q13 That Are Underexpressed at Baseline in ACC and Differentially Expressed in NCI-H295R Cells at 3 Days After Decitabine (1.0μM) Treatment

Treatment strategies for advanced-stage or recurrent ACC predominantly rely on adjuvant chemotherapeutic regimens that include mitotane. Mitotane remains the most significant single agent shown to affect the disease course of patients with metastatic ACC, with useful clinical remissions of 10 months in up to 30% of patients.6 Perhaps most important, mitotane leads to significantly diminished hormonal hypersecretion in 80% of patients with functional tumors, thereby improving the quality of life for affected patients.5 Furthermore, these effects seem optimal at lower-dosing regimens (≤3 g/d of mitotane), which have the dual benefit of achieving therapeutic serum concentrations (10-14 mg/L) and minimizing adverse reactions.20 However, 70% of patients with ACC do not respond to mitotane treatment, and many patients develop debilitating neurologic and gastrointestinal toxic effects, which attest to the limitations of mitotane therapy in controlling the spread of this aggressive cancer. The use of mitotane in combination with other agents, most notably cisplatin, has only slightly improved outcomes.7

Recently, mounting evidence about the role of epigenetic mechanisms in carcinogenesis has prompted interest in whether agents that reverse these processes can be effective anticancer treatments. Epigenetics refers to reversible changes in gene expression that do not fundamentally mutate the genomic DNA sequence. Hypermethylation of DNA promoter sequences is a classic example of these changes, which usually cause silencing of genes downstream of the affected region.13 Theoretically, the reversal of these changes could lead to reexpression of silenced tumor suppressor genes and inhibit cancer cell progression. Indeed, the relevance of activated DNA methyltransferase to adrenocortical carcinogenesis was first established in mouse models more than a decade ago.21

Decitabine is an inhibitor of DNA methyltransferase, which effectively removes methyl groups from silenced promoter sequences. Decitabine has a dual dose-dependent mechanism of action. Lower doses (≤5μM to 10μM) of decitabine inhibit methylation and reactivate gene expression, whereas higher doses (≥10μM to 100μM) induce cytotoxic effects via covalent trapping of DNA methyltransferase into DNA.16 Even at low doses, decitabine has been shown to inhibit in vitro growth in several human cancers, including colorectal carcinoma, melanoma, renal cell carcinoma, lung carcinoma, and hepatocellular carcinoma.2225 Its benefits have been most apparent in hematologic malignant neoplasms, especially myelodysplastic syndrome, for which decitabine has recently been approved by the Food and Drug Administration as first-line therapy.17

We hypothesized that decitabine treatment of NCI-H295R cells would have a significant antineoplastic effect. To this aim, we examined the effects of decitabine on ACC cell proliferation, cortisol secretion, and cell invasion, which are 3 hallmarks causing clinical morbidity and mortality in this disease. Using 2 lower doses (0.1μM and 1.0μM), we examined the effects of decitabine on ACC cells at daily intervals for up to 5 days. Decitabine cytostatically inhibited ACC cell proliferation in a time-dependent and dose-dependent manner. Cortisol secretion was also attenuated at 5 days after treatment, although only the lower 0.1μM dose seemed to act via a mechanism independent of its growth inhibitory effects. As opposed to the long incubation time that is necessary to observe differences in cell count (probably because of a long doubling time for NCI-H295R cells), the effects of decitabine on invasive behavior were seen after only 24 hours. These findings support the idea that the functional effects of decitabine can be immediate, regardless of the basal growth rate of the target cell.

Our findings about ACC cell proliferation are in agreement with the results of other studies.18,19,26 To our knowledge, we are the first group to demonstrate inhibition of ACC cell invasion by decitabine. Our modified Boyden chamber technique used a commercially available kit with a 2-dimensional barrier composed of reconstituted basement membrane and ECM components. Despite its widespread acceptance, this method for determining cell invasion has well-known limitations. First, despite improved standardization of preparation techniques by manufacturers, reconstituted matrices may contain “contaminants” (such as metalloproteinases) that could affect experimental results. Second, individual tumor cells display significant variability in their ability to adhere to and migrate through ECM, which may not be taken into account by quantifying cells in aggregate. Third, with its 2-dimensional design and components within reconstituted matrices, the Boyden chamber model does not necessarily recapitulate the tumor microenvironment. Newer techniques offer 3-dimensional models or tissue-based cell invasion barriers, which may address these limitations but need to be validated in future experiments.27

At first glance, our findings about cortisol secretion seem to contradict those in a prior study by Liu et al,18 who found that decitabine increased cortisol secretion in NCI-H295R cells, possibly via selective regulation of steroidogenic gene expression. However, 2 primary differences between our studies may explain these divergent results. First, Liu et al used a higher dose of decitabine (10μM). The differing mechanism of action at higher doses (as already described) could at least partially account for the discrepancies in our results. Indeed, our results show that only the lower 0.1μM dose led to decreased cortisol secretion independent of decreased cell count. Furthermore, our studies seem to agree that the relative trend of cortisol secretion decreases in proportion to decitabine dose. We speculate that the lower dose of decitabine may affect cortisol expression and secretion, while the higher dose of decitabine may have only a cytotoxic effect, reducing cell count and cortisol secretion.

Second, our preparation of decitabine (unlike that by Liu et al) involved the use of DMSO vehicle to prevent rapid degradation of the unstable molecule. In theory, the benefit of our approach was longer duration of decitabine action, which may be more clinically relevant to longer-course treatment protocols used in patients. The disadvantage was the incorporation of another drug into our experiments and the associated possibility of a confounding effect, despite our use of DMSO concentrations well below thresholds that are known to have functional effects in other in vitro models.2830 We believe that we effectively controlled for this factor by standardizing decitabine to proper DMSO vehicle control specimens in our experiments.

In addition to demonstrating the functional effects of low-dose decitabine on NCI-H295R cells, we also sought to evaluate its effects on several genes at 11q13. This chromosomal region seems relevant in adrenocortical carcinogenesis, with studies10,11 showing that loss of heterozygosity at 11q13 is found in 70% to 100% of sporadic ACCs. A previous microarray-based study12 identified 6 genes on 11q13 that were underexpressed in ACC and demonstrated high diagnostic accuracy for distinguishing benign from malignant tumors. We tested the effects of decitabine on these genes using quantitative RT-PCR, with the hypothesis that decitabine could recover gene expression if methylation had a role in silencing any of these genes. For these experiments, we used decitabine at 1.0μM because of the marked effects on ACC cell proliferation and cell invasion that we observed at this dose, along with the indirect inhibitory effects on cortisol secretion. Of 6 genes, NDUFS8 and PRDX5 showed significantly recovered expression after decitabine treatment, suggesting that hypermethylation may have a gene silencing role in ACC. However, future studies using methylation-specific techniques are needed to definitively establish methylation patterns of these genes in tumor samples.

The clinical relevance of NDUFS8 and PRDX5 remains unknown. NDUFS8 encodes a subunit protein of a critical enzyme in the mitochondrial respiratory chain, and mutations of this gene are associated with Leigh disease.31,32PRDX5 encodes the antioxidant enzyme peroxiredoxin, which has genome-protective properties in response to oxidative stressors.33 Underexpression of neither NDUFS8 nor PRDX5 has been shown in other cancer models,3438 suggesting that dysregulation of these genes may be specific to adrenocortical carcinogenesis. However, further study is needed to determine the functional effects of modifying expression of these genes individually in the absence of other confounding factors. Moreover, the mechanisms behind the seemingly paradoxical downregulation of gene expression after decitabine treatment (such as that of DDB1 and TM7SF2 in our experiments), as well as the ways in which restoration of other genes interacts with these inhibitory effects, are unknown and require further investigation.

A cautionary note must be given about attempts to mold the results of our in vitro experiments to clinical relevance. The effects of decitabine on ACC cells are undoubtedly different depending on whether the drug is administered via culture medium or via in vivo intravenous or subcutaneous routes. Decitabine's success in inhibiting hematogenous cancers both in patients as well as in cell cultures may in part be due to similar immersive drug exposures in the bloodstream and in culture medium. This similarity may not necessarily translate as well for solid organ tumors. Indeed, early evidence in the 1980s showed a disappointing lack of demethylating agent activity on solid organ cancers.16 Nevertheless, we believe that decitabine holds promise as a therapy for patients with ACC for several reasons. First, the earlier findings were limited by adverse effects secondary to higher drug doses and by limited treatment durations.16 More recent trials13,17 in other cancers reported superior results using low-dose and longer-duration drug regimens. Second, studies18,19,21,26,39,40 using in vitro and in vivo models demonstrated the relevance of DNA promoter methylation in adrenocortical carcinogenesis. Third, decitabine has already been approved for use in humans, which should theoretically streamline its path to clinical trials.

In conclusion, low-dose decitabine exhibits significant antineoplastic effects in human ACC cells, possibly by recovering expression of NDUFS8 and PRDX5. Considering that decitabine is already approved by the Food and Drug Administration for hematologic malignant neoplasms and because ACC is an orphan disease for which there is no effective chemotherapy to date for locally advanced and metastatic ACC, future studies evaluating the clinical efficacy of decitabine should be considered based on our results.

Correspondence: Electron Kebebew, MD, Department of Surgery and Helen Diller Comprehensive Cancer Center, University of California, San Francisco, PO Box 1674, San Francisco, CA 94143-1674.

Accepted for Publication: May 15, 2009.

Author Contributions:Study concept and design: Suh, Duh, Clark, and Kebebew. Acquisition of data: Suh, Weng, and Fernandez-Ranvier. Analysis and interpretation of data: Suh, Weng, Shen, Duh, Clark, and Kebebew. Drafting of the manuscript: Suh, Fernandez-Ranvier, and Kebebew. Critical revision of the manuscript for important intellectual content: Weng, Shen, Duh, Clark, and Kebebew. Statistical analysis: Suh, Weng, and Kebebew. Obtained funding: Kebebew. Administrative, technical, and material support: Weng and Kebebew. Study supervision: Shen, Duh, Clark, and Kebebew.

Financial Disclosure: None reported.

Funding/Support: This study was supported in part by grants from the American Cancer Society, grant NCI R21 from the National Institutes of Health, the Hellman Family Grant, the Mount Zion Health Fund, and the Helen Diller Comprehensive Cancer Center.

Previous Presentation: This study was presented at the 80th Annual Meeting of the Pacific Coast Surgical Association; February 15, 2009; San Francisco, California.

Schteingart  DEDoherty  GMGauger  PG  et al.  Management of patients with adrenal cancer: recommendations of an international consensus conference. Endocr Relat Cancer 2005;12 (3) 667- 680
PubMed Link to Article
Sidhu  SSywak  MRobinson  BDelbridge  L Adrenocortical cancer: recent clinical and molecular advances. Curr Opin Oncol 2004;16 (1) 13- 18
PubMed Link to Article
Paton  BLNovitsky  YWZerey  M  et al.  Outcomes of adrenal cortical carcinoma in the United States. Surgery 2006;140 (6) 914- 920
PubMed Link to Article
Bilimoria  KYShen  WTElaraj  D  et al.  Adrenocortical carcinoma in the United States: treatment utilization and prognostic factors. Cancer 2008;113 (11) 3130- 3136
PubMed Link to Article
Luton  JPCerdas  SBillaud  L  et al.  Clinical features of adrenocortical carcinoma, prognostic factors, and the effect of mitotane therapy. N Engl J Med 1990;322 (17) 1195- 1201
PubMed Link to Article
Lubitz  JAFreeman  LOkun  R Mitotane use in inoperable adrenal cortical carcinoma. JAMA 1973;223 (10) 1109- 1112
PubMed Link to Article
Bukowski  RMWolfe  MLevine  HS  et al.  Phase II trial of mitotane and cisplatin in patients with adrenal carcinoma: a Southwest Oncology Group study. J Clin Oncol 1993;11 (1) 161- 165
PubMed
Pommier  RFBrennan  MF An eleven-year experience with adrenocortical carcinoma. Surgery 1992;112 (6) 963- 971
PubMed
Soon  PS McDonald  KLRobinson  BGSidhu  SB Molecular markers and the pathogenesis of adrenocortical cancer. Oncologist 2008;13 (5) 548- 561
PubMed Link to Article
Kjellman  MRoshani  LTeh  BT  et al.  Genotyping of adrenocortical tumors: very frequent deletions of the MEN1 locus in 11q13 and of a 1-centimorgan region in 2p16. J Clin Endocrinol Metab 1999;84 (2) 730- 735
PubMed Link to Article
Schulte  KMMengel  MHeinze  M  et al.  Complete sequencing and messenger ribonucleic acid expression analysis of the MEN I gene in adrenal cancer. J Clin Endocrinol Metab 2000;85 (1) 441- 448
PubMed Link to Article
Fernandez-Ranvier  GGWeng  JYeh  RF  et al.  Candidate diagnostic markers and tumor suppressor genes for adrenocortical carcinoma by expression profile of genes on chromosome 11q13. World J Surg 2008;32 (5) 873- 881
PubMed Link to Article
Baylin  SB DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol 2005;2(suppl 1)S4- S11
PubMed Link to Article
Issa  JP DNA methylation as a therapeutic target in cancer. Clin Cancer Res 2007;13 (6) 1634- 1637
PubMed Link to Article
de Vos  Dvan Overveld  W Decitabine: a historical review of the development of an epigenetic drug. Ann Hematol 2005;84(suppl 1)3- 8
PubMed Link to Article
Kurkjian  CKummar  SMurgo  AJ DNA methylation: its role in cancer development and therapy. Curr Probl Cancer 2008;32 (5) 187- 235
PubMed Link to Article
Kantarjian  HIssa  JPRosenfeld  CS  et al.  Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study. Cancer 2006;106 (8) 1794- 1803
PubMed Link to Article
Liu  JLi  XDVaheri  AVoutilainen  R DNA methylation affects cell proliferation, cortisol secretion and steroidogenic gene expression in human adrenocortical NCI-H295R cells. J Mol Endocrinol 2004;33 (3) 651- 662
PubMed Link to Article
Utriainen  PLiu  JKuulasmaa  TVoutilainen  R Inhibition of DNA methylation increases follistatin expression and secretion in the human adrenocortical cell line NCI-H295R. J Endocrinol 2006;188 (2) 305- 310
PubMed Link to Article
Huang  HFojo  T Adjuvant mitotane for adrenocortical cancer: a recurring controversy. J Clin Endocrinol Metab 2008;93 (10) 3730- 3732
PubMed Link to Article
Ramchandani  SMacLeod  ARPinard  Mvon Hofe  ESzyf  M Inhibition of tumorigenesis by a cytosine–DNA, methyltransferase, antisense oligodeoxynucleotide. Proc Natl Acad Sci U S A 1997;94 (2) 684- 689
PubMed Link to Article
Hsi  LCXi  XWu  YLippman  SM The methyltransferase inhibitor 5-aza-2-deoxycytidine induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells. Mol Cancer Ther 2005;4 (11) 1740- 1746
PubMed Link to Article
Reu  FJBae  SICherkassky  L  et al.  Overcoming resistance to interferon-induced apoptosis of renal carcinoma and melanoma cells by DNA demethylation. J Clin Oncol 2006;24 (23) 3771- 3779
PubMed Link to Article
Zhou  XPopescu  NCKlein  GImreh  S The interferon-alpha responsive gene TMEM7 suppresses cell proliferation and is downregulated in human hepatocellular carcinoma. Cancer Genet Cytogenet 2007;177 (1) 6- 15
PubMed Link to Article
Momparler  RLBouffard  DYMomparler  LFDionne  JBelanger  KAyoub  J Pilot phase I-II study on 5-aza-2′-deoxycytidine (decitabine) in patients with metastatic lung cancer. Anticancer Drugs 1997;8 (4) 358- 368
PubMed Link to Article
Gao  ZHSuppola  SLiu  JHeikkila  PJanne  JVoutilainen  R Association of H19 promoter methylation with the expression of H19 and IGF-II genes in adrenocortical tumors. J Clin Endocrinol Metab 2002;87 (3) 1170- 1176
PubMed Link to Article
Yuan  KSingh  RKRezonzew  GSiegal  GP In vitro matrices for studying tumor cell invasion.  In: Wells  A, ed. Cell Motility in Cancer Invasion and Metastasis. Dordrecht, Germany: Springer-Verlag; 2006:25-54
Sawai  MTakase  KTeraoka  HTsukada  K Reversible G1 arrest in the cell cycle of human lymphoid cell lines by dimethyl sulfoxide. Exp Cell Res 1990;187 (1) 4- 10
PubMed Link to Article
Takase  KSawai  MYamamoto  K  et al.  Reversible G1 arrest induced by dimethyl sulfoxide in human lymphoid cell lines: kinetics of the arrest and expression of the cell cycle marker proliferating cell nuclear antigen in Raji cells. Cell Growth Differ 1992;3 (8) 515- 521
PubMed
Qi  WDing  DSalvi  RJ Cytotoxic effects of dimethyl sulphoxide (DMSO) on cochlear organotypic cultures. Hear Res 2008;236 (1-2) 52- 60
PubMed Link to Article
Lescuyer  PMartinez  PLunardi  J YY1 and Sp1 activate transcription of the human NDUFS8 gene encoding the mitochondrial complex I TYKY subunit. Biochim Biophys Acta 2002;1574 (2) 164- 174
PubMed Link to Article
Procaccio  VWallace  DC Late-onset Leigh syndrome in a patient with mitochondrial complex I NDUFS8 mutations. Neurology 2004;62 (10) 1899- 1901
PubMed Link to Article
Kropotov  ASerikov  VSuh  J  et al.  Constitutive expression of the human peroxiredoxin V gene contributes to protection of the genome from oxidative DNA lesions and to suppression of transcription of noncoding DNA. FEBS J 2006;273 (12) 2607- 2617
PubMed Link to Article
De Simoni  SGoemaere  JKnoops  B Silencing of peroxiredoxin 3 and peroxiredoxin 5 reveals the role of mitochondrial peroxiredoxins in the protection of human neuroblastoma SH-SY5Y cells toward MPP+Neurosci Lett 2008;433 (3) 219- 224
PubMed Link to Article
Kropotov  AGogvadze  VShupliakov  O  et al.  Peroxiredoxin V is essential for protection against apoptosis in human lung carcinoma cells. Exp Cell Res 2006;312 (15) 2806- 2815
PubMed Link to Article
Midorikawa  YTsutsumi  STaniguchi  H  et al.  Identification of genes associated with dedifferentiation of hepatocellular carcinoma with expression profiling analysis. Jpn J Cancer Res 2002;93 (6) 636- 643
PubMed Link to Article
Sensi  MNicolini  GZanon  M  et al.  Immunogenicity without immunoselection: a mutant but functional antioxidant enzyme retained in a human metastatic melanoma and targeted by CD8+ T cells with a memory phenotype. Cancer Res 2005;65 (2) 632- 640
PubMed
Shiota  MIzumi  HMiyamoto  N  et al.  Ets regulates peroxiredoxin1 and 5 expressions through their interaction with the high-mobility group protein B1. Cancer Sci 2008;99 (10) 1950- 1959
PubMed
Szyf  MSlack  AD Mechanisms of epigenetic silencing of the c21 gene in Y1 adrenocortical tumor cells. Endocr Res 2000;26 (4) 921- 930
PubMed Link to Article
Wong  IHChan  JWong  JTam  PK Ubiquitous aberrant RASSF1A promoter methylation in childhood neoplasia. Clin Cancer Res 2004;10 (3) 994- 1002
PubMed Link to Article

Figures

Place holder to copy figure label and caption

Figure 1. Effects of decitabine on cortical secretion in NCI-H295R cells (ATCC, Rockville, Maryland). Cells were treated with decitabine (in dimethyl sulfoxide [DMSO] vehicle) at 0.1μM and 1.0μM doses for 5 days, and cell counts were measured using an assay kit (CyQUANT; Invitrogen, Carlsbad, California). Compared with control specimens, decitabine caused time-dependent (A) and dose-dependent (B) inhibitory effects on adrenocortical carcinoma cell proliferation, with significant 39% (*P = .02) and 47% (†P < .001) decreases in cell counts observed at 5 days after treatment. Serum (10%) is Nu-Serum (BD Biosciences, San Jose, California).

Graphic Jump Location
Place holder to copy figure label and caption

Figure 2. Effects of decitabine on cortisol concentration in NCI-H295R cells (ATCC, Rockville, Maryland). Cells were treated with decitabine (in dimethyl sulfoxide [DMSO] vehicle) at 0.1μM and 1.0μM doses for 5 days, and cortisol concentrations in cell media were measured using an immunometric assay kit (Parameter; R&D Systems, Minneapolis, Minnesota). Media taken from decitabine-treated cells had decreased cortisol concentration over time (A), with significant decreases at 5 days after treatment of 58% (*P = .001) and 56% (†P = .02) for the 0.1μM and 1.0μM doses, respectively (B). The inhibitory effect of decitabine on cortisol concentration per cell (C) was significant only with the lower 0.1μM dose (‡P = .03).

Graphic Jump Location
Place holder to copy figure label and caption

Figure 3. Effects of decitabine on NCI-H295R cell (ATCC, Rockville, Maryland) invasion. Cells were starved of serum for 48 hours and then placed in a modified Boyden chamber for 24 hours. Cells that invaded through the extracellular matrix barrier were counted using an assay kit (CyQUANT; Invitrogen, Carlsbad, California). Decitabine had a dose-dependent inhibitory effect on NCI-H295R cell invasion, with a 64% decrease in cell invasion using the 1.0μM dose (*P = .03). DMSO indicates dimethyl sulfoxide.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable. Quantitative RT-PCR Analysis of 4 Genes on Chromosome 11q13 That Are Underexpressed at Baseline in ACC and Differentially Expressed in NCI-H295R Cells at 3 Days After Decitabine (1.0μM) Treatment

References

Schteingart  DEDoherty  GMGauger  PG  et al.  Management of patients with adrenal cancer: recommendations of an international consensus conference. Endocr Relat Cancer 2005;12 (3) 667- 680
PubMed Link to Article
Sidhu  SSywak  MRobinson  BDelbridge  L Adrenocortical cancer: recent clinical and molecular advances. Curr Opin Oncol 2004;16 (1) 13- 18
PubMed Link to Article
Paton  BLNovitsky  YWZerey  M  et al.  Outcomes of adrenal cortical carcinoma in the United States. Surgery 2006;140 (6) 914- 920
PubMed Link to Article
Bilimoria  KYShen  WTElaraj  D  et al.  Adrenocortical carcinoma in the United States: treatment utilization and prognostic factors. Cancer 2008;113 (11) 3130- 3136
PubMed Link to Article
Luton  JPCerdas  SBillaud  L  et al.  Clinical features of adrenocortical carcinoma, prognostic factors, and the effect of mitotane therapy. N Engl J Med 1990;322 (17) 1195- 1201
PubMed Link to Article
Lubitz  JAFreeman  LOkun  R Mitotane use in inoperable adrenal cortical carcinoma. JAMA 1973;223 (10) 1109- 1112
PubMed Link to Article
Bukowski  RMWolfe  MLevine  HS  et al.  Phase II trial of mitotane and cisplatin in patients with adrenal carcinoma: a Southwest Oncology Group study. J Clin Oncol 1993;11 (1) 161- 165
PubMed
Pommier  RFBrennan  MF An eleven-year experience with adrenocortical carcinoma. Surgery 1992;112 (6) 963- 971
PubMed
Soon  PS McDonald  KLRobinson  BGSidhu  SB Molecular markers and the pathogenesis of adrenocortical cancer. Oncologist 2008;13 (5) 548- 561
PubMed Link to Article
Kjellman  MRoshani  LTeh  BT  et al.  Genotyping of adrenocortical tumors: very frequent deletions of the MEN1 locus in 11q13 and of a 1-centimorgan region in 2p16. J Clin Endocrinol Metab 1999;84 (2) 730- 735
PubMed Link to Article
Schulte  KMMengel  MHeinze  M  et al.  Complete sequencing and messenger ribonucleic acid expression analysis of the MEN I gene in adrenal cancer. J Clin Endocrinol Metab 2000;85 (1) 441- 448
PubMed Link to Article
Fernandez-Ranvier  GGWeng  JYeh  RF  et al.  Candidate diagnostic markers and tumor suppressor genes for adrenocortical carcinoma by expression profile of genes on chromosome 11q13. World J Surg 2008;32 (5) 873- 881
PubMed Link to Article
Baylin  SB DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol 2005;2(suppl 1)S4- S11
PubMed Link to Article
Issa  JP DNA methylation as a therapeutic target in cancer. Clin Cancer Res 2007;13 (6) 1634- 1637
PubMed Link to Article
de Vos  Dvan Overveld  W Decitabine: a historical review of the development of an epigenetic drug. Ann Hematol 2005;84(suppl 1)3- 8
PubMed Link to Article
Kurkjian  CKummar  SMurgo  AJ DNA methylation: its role in cancer development and therapy. Curr Probl Cancer 2008;32 (5) 187- 235
PubMed Link to Article
Kantarjian  HIssa  JPRosenfeld  CS  et al.  Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study. Cancer 2006;106 (8) 1794- 1803
PubMed Link to Article
Liu  JLi  XDVaheri  AVoutilainen  R DNA methylation affects cell proliferation, cortisol secretion and steroidogenic gene expression in human adrenocortical NCI-H295R cells. J Mol Endocrinol 2004;33 (3) 651- 662
PubMed Link to Article
Utriainen  PLiu  JKuulasmaa  TVoutilainen  R Inhibition of DNA methylation increases follistatin expression and secretion in the human adrenocortical cell line NCI-H295R. J Endocrinol 2006;188 (2) 305- 310
PubMed Link to Article
Huang  HFojo  T Adjuvant mitotane for adrenocortical cancer: a recurring controversy. J Clin Endocrinol Metab 2008;93 (10) 3730- 3732
PubMed Link to Article
Ramchandani  SMacLeod  ARPinard  Mvon Hofe  ESzyf  M Inhibition of tumorigenesis by a cytosine–DNA, methyltransferase, antisense oligodeoxynucleotide. Proc Natl Acad Sci U S A 1997;94 (2) 684- 689
PubMed Link to Article
Hsi  LCXi  XWu  YLippman  SM The methyltransferase inhibitor 5-aza-2-deoxycytidine induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells. Mol Cancer Ther 2005;4 (11) 1740- 1746
PubMed Link to Article
Reu  FJBae  SICherkassky  L  et al.  Overcoming resistance to interferon-induced apoptosis of renal carcinoma and melanoma cells by DNA demethylation. J Clin Oncol 2006;24 (23) 3771- 3779
PubMed Link to Article
Zhou  XPopescu  NCKlein  GImreh  S The interferon-alpha responsive gene TMEM7 suppresses cell proliferation and is downregulated in human hepatocellular carcinoma. Cancer Genet Cytogenet 2007;177 (1) 6- 15
PubMed Link to Article
Momparler  RLBouffard  DYMomparler  LFDionne  JBelanger  KAyoub  J Pilot phase I-II study on 5-aza-2′-deoxycytidine (decitabine) in patients with metastatic lung cancer. Anticancer Drugs 1997;8 (4) 358- 368
PubMed Link to Article
Gao  ZHSuppola  SLiu  JHeikkila  PJanne  JVoutilainen  R Association of H19 promoter methylation with the expression of H19 and IGF-II genes in adrenocortical tumors. J Clin Endocrinol Metab 2002;87 (3) 1170- 1176
PubMed Link to Article
Yuan  KSingh  RKRezonzew  GSiegal  GP In vitro matrices for studying tumor cell invasion.  In: Wells  A, ed. Cell Motility in Cancer Invasion and Metastasis. Dordrecht, Germany: Springer-Verlag; 2006:25-54
Sawai  MTakase  KTeraoka  HTsukada  K Reversible G1 arrest in the cell cycle of human lymphoid cell lines by dimethyl sulfoxide. Exp Cell Res 1990;187 (1) 4- 10
PubMed Link to Article
Takase  KSawai  MYamamoto  K  et al.  Reversible G1 arrest induced by dimethyl sulfoxide in human lymphoid cell lines: kinetics of the arrest and expression of the cell cycle marker proliferating cell nuclear antigen in Raji cells. Cell Growth Differ 1992;3 (8) 515- 521
PubMed
Qi  WDing  DSalvi  RJ Cytotoxic effects of dimethyl sulphoxide (DMSO) on cochlear organotypic cultures. Hear Res 2008;236 (1-2) 52- 60
PubMed Link to Article
Lescuyer  PMartinez  PLunardi  J YY1 and Sp1 activate transcription of the human NDUFS8 gene encoding the mitochondrial complex I TYKY subunit. Biochim Biophys Acta 2002;1574 (2) 164- 174
PubMed Link to Article
Procaccio  VWallace  DC Late-onset Leigh syndrome in a patient with mitochondrial complex I NDUFS8 mutations. Neurology 2004;62 (10) 1899- 1901
PubMed Link to Article
Kropotov  ASerikov  VSuh  J  et al.  Constitutive expression of the human peroxiredoxin V gene contributes to protection of the genome from oxidative DNA lesions and to suppression of transcription of noncoding DNA. FEBS J 2006;273 (12) 2607- 2617
PubMed Link to Article
De Simoni  SGoemaere  JKnoops  B Silencing of peroxiredoxin 3 and peroxiredoxin 5 reveals the role of mitochondrial peroxiredoxins in the protection of human neuroblastoma SH-SY5Y cells toward MPP+Neurosci Lett 2008;433 (3) 219- 224
PubMed Link to Article
Kropotov  AGogvadze  VShupliakov  O  et al.  Peroxiredoxin V is essential for protection against apoptosis in human lung carcinoma cells. Exp Cell Res 2006;312 (15) 2806- 2815
PubMed Link to Article
Midorikawa  YTsutsumi  STaniguchi  H  et al.  Identification of genes associated with dedifferentiation of hepatocellular carcinoma with expression profiling analysis. Jpn J Cancer Res 2002;93 (6) 636- 643
PubMed Link to Article
Sensi  MNicolini  GZanon  M  et al.  Immunogenicity without immunoselection: a mutant but functional antioxidant enzyme retained in a human metastatic melanoma and targeted by CD8+ T cells with a memory phenotype. Cancer Res 2005;65 (2) 632- 640
PubMed
Shiota  MIzumi  HMiyamoto  N  et al.  Ets regulates peroxiredoxin1 and 5 expressions through their interaction with the high-mobility group protein B1. Cancer Sci 2008;99 (10) 1950- 1959
PubMed
Szyf  MSlack  AD Mechanisms of epigenetic silencing of the c21 gene in Y1 adrenocortical tumor cells. Endocr Res 2000;26 (4) 921- 930
PubMed Link to Article
Wong  IHChan  JWong  JTam  PK Ubiquitous aberrant RASSF1A promoter methylation in childhood neoplasia. Clin Cancer Res 2004;10 (3) 994- 1002
PubMed Link to Article

Correspondence

CME
Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.
Submit a Comment

Multimedia

Some tools below are only available to our subscribers or users with an online account.

Web of Science® Times Cited: 7

Related Content

Customize your page view by dragging & repositioning the boxes below.

Articles Related By Topic
Related Collections
PubMed Articles