Griseofulvin Inhibits the Growth of Adrenocortical Cancer Cells In Vitro
| Authors | E. L. Bramann1, H. S. Willenberg1, B. Hildebrandt2, V. Müller-Mattheis3, M. Schott1, W. A. Scherbaum1, M. Haase1 |
| Affiliations | 1 Department of Endocrinology, Diabetes and Rheumatology, University Hospital Duesseldorf, Duesseldorf, Germany 2 Institute of Human Genetics and Anthropology, Heinrich-Heine-University Duesseldorf, Germany 3 Department of Urology, University Hospital Duesseldorf, Duesseldorf, Germany |
Key words
adrenal tumor · proliferation viability · apoptosis
Abstract
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Supernumerary centrosomes and aneuploidy are associated with a malignant phenotype of tumor cells. Centrosomal clustering is a mecha- nism used by cancer cells with supernumerary centrosomes to solve the threatening problem of multipolar spindles. Griseofulvin is an antifungal substance that interferes with the microtubule apparatus and inhibits centrosomal clustering. It has also been demonstrated that griseofulvin inhibits the growth of tumor cells in vitro and in vivo. However, it is not yet known whether treat- ment with griseofulvin inhibits growth of adren- ocortical tumor cells. We studied the viability and antiproliferative effects of griseofulvin on cultured NCI-H295R adrenocortical carcinoma cells using Wst-1-, BrdUrd-, and [3H]-thymidine
assays. For the detection of apoptosis we used a caspase 3/7 cleavage assay and light microscopy techniques. We observed that incubation with griseofulvin for 24-48 h leads to a decrease in the viability and proliferation of NCI-H295R cells in a dose-dependent manner. Significant effects could be observed after incubation with griseof- ulvin as measured by Wst-1-, BrdUrd-, and [3H] dT- uptake assays. Apoptosis of NCI-H295R cells was increased in a dose-dependent manner up to 4.5-fold after incubation with griseofulvin 40 uM for 24h as shown by caspase 3/7 cleavage assay and light microscopy. With regard to new treatment strategies for adrenocortical cancer, griseofulvin, and possibly other agents, which interfere with the microtubule apparatus and inhibit centrosomal clustering, may turn out to be interesting targets for further research.
received 06.08.2012 accepted 20.09.2012
Bibliography
DOI http://dx.doi.org/ 10.1055/s-0032-1327642 Published online: October 30, 2012 Horm Metab Res 2013; 45: 297-300 @ Georg Thieme Verlag KG Stuttgart . New York ISSN 0018-5043
Correspondence
M. Haase M.D. Department of Endocrinology, Diabetes and Rheumatology University Hospital Duesseldorf Moorenstraße 5 40225 Duesseldorf Germany Tel .: +49/211/811 7810
Fax: +49/211/811 7860
Matthias.Haase@uni-duessel dorf.de
Introduction
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It has been shown that the antifungal substance griseofulvin inhibits centrosomal clustering, a cel- lular mechanism that presumably saves the genetic integrity and thus enables cancer cells to survive and proliferate [1,2]. Amplification of centrosomes is a common characteristic of tumor cells and is associated with genetic instability [3-5]. Supernu- merary centrosomes on the one hand support tumorigenesis through promoting aneuploidy and/ or disrupting cell polarity [3]. On the other hand it represents a severe fitness risk for the growth of mature cancer cells due to multipolar mitosis and the genesis of nonviable daughter cells [3]. To avoid the genetic instability, which is thought to be asso- ciated with multipolar mitosis, affected cells use centrosomal clustering to prevent the formation of multipolar spindles. This process of coalescence is probably influenced by microtubule based motor proteins and microtubule binding proteins along the microtubular network, but not all details have been fully understood [2,6].
Adrenocortical carcinoma (ACC) is a rare malig- nant disease with a poor prognosis despite sur- gery, radiation, and pharmacotherapy [7-10]. Few agents have been proven to be of some ben- efit in the treatment, for example, platinum- based cytotoxic chemotherapy, streptozotocin, and mitotane [11-13]. However, despite best clinical care, the overall survival rate remains lower than 50% at 5 years [14]. The malignant phenotype may be supported by the genetic insta- bility and centrosomal amplification that is also typical for adrenocortical carcinomas [15, 16]. Griseofulvin is an established antifungal agent that has recently been identified as an inductor of multipolar spindles and mitoses in tumor cells with supernumerary centrosomes via the inhibi- tion of centrosomal clustering [2]. The induction of multipolar mitoses was associated with apop- tosis as well as with the inhibition of prolifera- tion in different cancer cell lines [2]. The mechanism of action seems to be the disruption of the interphase microtubule network via an inhibition of microtubule polymerization, result-
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ing in the dispersion of supernumerary centrosomes throughout the cytoplasm [2]. Griseofulvin has been tested in several stud- ies as a new option in cancer therapy and it has been described that griseofulvin diminishes proliferation of malignant cells in several cell lines alone or in combination with established chemotherapeutical treatment strategies [17,18]. Moreover, it has also been described that griseofulvin blocks proliferation in cells that are transformed by polyoma virus or by SV40. This observation has been thought to be a result of the altered micro- tubule network in transformed cells [19].
Therefore, we posed the question whether griseofulvin may also be an effective agent in the treatment of adrenocortical carci- noma. We studied here the effects of griseofulvin on the viabil- ity, proliferation and apoptosis of adrenocortical carcinoma cells in vitro.
Material and Methods
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Cell culture
For in vitro studies, we employed the NCI-H295R adrenocortical cancer cell line. The medium used was RPMI1640 + l -glutamine (Invitrogen, Karlsruhe, Germany) supplemented with 2% fetal bovine serum, insulin (66nM), hydrocortisone (10nM), apo- transferrin (10µg/ml), ß-estradiol (10nM), sodium-selenite (30nM), penicillin (100U/ml), and streptomycin (100µg/ml) at 37℃ in a humidified atmosphere of 95% air and 5% CO2, as described before [20]. Medium was changed every 3 days and cells were subcultured at confluence using accutase (PAA Labo- ratories, Cölbe, Germany). We seeded the NCI-H295R cells in 96-well microtiter culture plates for the wst-1 assay, the BrdUrd assay, the apoptosis detection, and for measuring the tritiated thymidine ([3H]dT) uptake. Griseofulvin was obtained from Sigma-Aldrich (Sigma-Aldrich, Munich, Germany) and dissolved in DMSO and added to the cell culture medium at a final concen- tration of 100 nM, 1 µM, 10uM, 40 µM, or 100 uM. A DMSO-con- taining vehicle medium served as control. The control contained 0.12% DMSO and was equal to the maximum final concentration of DMSO used for the incubation with griseofulvin.
Chromosomal analysis
NCI-H295R cells were analyzed for genetic aberrations. Chromo- some preparations were obtained after colcemid treatment using standard hypotonic pretreatment procedures with KCl. Fif- teen trypsin G-banded cells were analyzed and karyotyped for detecting numerical and structural chromosome aberrations.
Wst-1 assay
To assess cell viability we applied the wst-1 assay (Roche, Mann- heim, Germany) as described by the manufacturer’s instruc- tions. In short, the cells were seeded in 96-well microtiter plates and cultured with or without griseofulvin at the concentrations given above. After 24h of incubation, the tetrazolium salt wst-1 was added to the medium for 4h. The change of the tetrazolium salt into a red formazan dye that correlates with the viability of the cells was detected by the measurement of absorbance at 440 nm using a microplate reader. A control without cells was used to measure the background absorbance of the medium, which was subtracted from the results.
BrdUrd assay
To determine cell proliferation more directly through measuring the DNA-synthesis we applied the colorimetric BrdUrd Assay (Cell Proliferation ELISA, BrdUrd by Roche) as indicated by the manufacturer’s instructions. Again, we seeded NCI-H295R-cells in 96 well microtiter plates and cultured with or without grise- ofulvin at the indicated concentrations. After 12 h, the cells were labeled with BrdUrd and the plates incubated at 37 ℃ for another 12h. The cell medium was then carefully removed and the cells dried and affixed. Subsequently we located the BrdUrd label in the DNA with the peroxidase-conjugated BrdUrd antibody. The microplate was then washed thoroughly 3 times with 1 x PBS. Afterwards the bound peroxidase-conjugated BrdUrd antibody was quantified with peroxidase substrate tetramethylbenzidine. Finally the BrdUrd Absorbance was measured at 440 nm using an ELISA plate reader. A control without cells was used to measure the background absorbance of the medium and was subtracted from the results.
Tritiated thymidine ([3H]dT) uptake assay
Proliferation of cells was determined measuring the incorporation of [3H]dT. Briefly, cells were seeded at a density of 2×104 cells/well in a 96 well plate and subsequently incubated with or without gri- seofulvin as indicated. After 24h of incubation, cells were pulsed with [3H]dT (Amersham Pharmacia) 1 uCi for a further 24h. At the end of the incubation period cells were transferred to a 96-well uni- filter microplate (PerkinElmer, Massachusetts, USA), washed exten- sively, dried, and covered with scintillation mixture. [3H]dT uptake was analyzed by measuring radioactivity using a scintillation coun- ter (Trilux, Wallach, Germany).
Apoptosis assay
To determine the fraction of apoptotic cells we applied the Apo- toxglo Assay (Apotoxglo Triplex Assay G6320, Promega), based on the manufacturer’s instructions. In this case, we only used the last test component to measure the caspase-Glo 3/7 cleavage with luminescence, which corresponds to the amount of apop- totic cells. The NCI-H295R cells were seeded in 96 well micro- titer plates and the cells incubated with griseofulvin for 24h at the concentrations indicated above. The Caspase Glo 3/7 reagent was then added, incubated, and subsequently the cleavage of the substrate was measured through luminescence detection with a luminometer (Lumat LB 9507, Berthold Technologies). Three wells of each sample were pooled before the measurement of the luminescence. A control without cells was used to measure the background luminescence and was subtracted from the results of each experiment. The apoptosis positive control was set up with paclitaxel, a substance well known for its antiprolif- erative and proapoptotic effects on adrenocortical NCI H295 cells [21,22].
Statistical analysis
The results are given as the mean +standard error of the mean (SEM) of at least 3 separate experiments each performed in trip- licate, with the control mean adjusted to 100%. Statistical analy- sis was performed using a Kruskal-Wallis statistic followed by Dunn’s post-test applying GraphPad Prism 5 software (Graph- Pad Software, La Jolla, USA). A p-value of <0.05 was determined as significant.
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Results
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Cytogenetic analysis of the NCI-H295R cells
A wide range of chromosome numbers were observed in the NCI-H295R cell line; with 89-103 chromosomes the cell line is near tetraploid. Numerical and structural abnormalities were observed. The chromosomes 17 and 18 were present in up to 8 copies. Most of the structural aberrations were present in a dif- ferent number of copies. The structural rearrangements were mostly 2- or 3-break rearrangements.
Effects of griseofulvin on the viability and proliferation of NCI-H295R
Exposure to griseofulvin for 28 h decreased the viability of NCI- H295R cells in a dose-dependent manner as measured by metabolism of wst-1-salt. The incubation with griseofulvin 10uM reduced viability to 93% in comparison to the control.
Exposure of NCI-H295R cells to griseofulvin 40 uM and 100 uM reduced viability to approximately 69 and 72%, respectively.
Using the BrdUrd-Assay, a decrease in the proliferation to 76% at 10uM griseofulvin and to 63% at 100uM griseofulvin could be detected after exposure of NCI-H295R cells for 24h when compared with the control.
The [3H]dT uptake assay results indicated a nearly equally strong effect of griseofulvin on the proliferation of NCI-H295R cells after exposure for 48 h. Concentrations of 10 uM griseofulvin decreased the [3H]dT uptake to approximately 81 % of the basal uptake. Con- centrations of griseofulvin 100uM led to a decrease to 57% as compared to the [3H]dT uptake of the untreated control ( Fig. 1).
Assessment of apoptosis
Exposure to griseofulvin for 24h increased the caspase 3/7 cleavage activity, correlating with the level of apoptosis, in NCI- H295R cells in a dose-dependent manner. A concentration of 10uM griseofulvin lead to an increase in the rate of apoptosis of
WST-1 Metabolism in %
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Uptake of BrdUrd in %
[ H]dT uptake in %
Caspage cleavage activity in %
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Control
Griseofulvin 100 nM
Griseofulvin 1 µM
Griseofulvin 10 µM
Griseofulvin 40 µM
Griseofulvin 100 µM
Control
Griseofulvin 100 nM
Griseofulvin 1 µM
Griseofulvin 10 µM
Griseofulvin 40 µM
Griseofulvin 100 µM
Control
Griseofulvin 100 nM
Griseofulvin 1 µM
Griseofulvin 10 µM
Griseofulvin 100 µM
Control
Griseofulvin 1 µM
Griseofulvin 10 µM
Griseofulvin 40 µM
Griseofulvin 60 µM
Griseofulvin 100 µM
Paclitaxel 10 LM
a
b
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NCI-H295R cells to 161% as compared to the control. After expo- sure to griseofulvin, 40 uM caspase activity raised to 459% in com- parison to the control. At concentrations of griseofulvin 100 uM we detected a level of caspase activity of 435% ( Fig. 1, 2).
Discussion and Conclusions
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Although there are several therapeutic regimes to treat adreno- cortical carcinoma, the prognosis of this disease remains poor and none of the established cytotoxic agents acts exclusively on malignant cells. Therapy, therefore, frequently leads to severe side effects and sometimes even to discontinuation of treatment. Recently, griseofulvin has been identified as an inhibitor of centro- somal clustering that is able to interfere relative specifically with the expansion of tumor cells which contain supernumerary centro- somes [2]. Further studies suggest that griseofulvin leads to growth inhibition by interfering with the microtubule apparatus [18].
Our data demonstrate significant effects of griseofulvin on the viability, proliferation, and apoptosis of adrenocortical carci- noma cells starting at doses of 10 uM after exposure for 24-48 h in vitro. However, this study does not compare the effects of gri- seofulvin on adrenocortical cancer cells and normal adrenocor- tical cells. Thus, further studies are necessary to evaluate the specificity of the observed effects.
Although the pharmacokinetics of griseofulvin differ in vivo from the simple conditions in vitro, peak concentrations that are neces- sary for growth inhibition of NCI-H295R cells may also be reached in vivo. For the treatment of fungal infections a typical single dose consists of 250-500 mg griseofulvin (depending on the size of the griseofulvin particles), leading to a peak serum concentration of 0.5-2.0µg/ml equivalent to 1.4-5.7 1M after 4h of oral adminis- tration [23]. We have chosen the concentrations of griseofulvin in our experiments with regard to these observations. However, a half maximal inhibition of proliferation and viability could not be reached using these concentrations. Furthermore, the distribu- tion of griseofulvin may vary between different tissues.
Nevertheless, there are studies that demonstrate antitumor effects of griseofulvin in animals in vivo. For example, griseoful- vin alone or in combination with nocodazole inhibits the growth of colon adenocarcinoma xenografts in athymic mice [17]. Another study demonstrated that vinblastine and griseofulvin have synergistic effects on MCF-7 cells [18]. Interestingly, Røn- nest et al. reported a collection of griseofulvin analogues, 18 of which demonstrated an increased activity as compared to grise- ofulvin that might prove another option in therapy [24].
With regard to new treatment strategies for adrenocortical can- cer, griseofulvin and possibly other agents, which interfere with the microtubule apparatus and inhibit centrosomal clustering, may turn out to be interesting targets for further research.
Conflict of Interest
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The authors have no conflict of interest.
References
1 Quintyne NJ, Reing JE, Hoffelder DR, Gollin SM, Saunders WS. Spindle multipolarity is prevented by centrosomal clustering. Science 2005; 307: 127-129
2 Rebacz B, Larsen TO, Clausen MH, Rønnest MH, Löffler H, Ho AD, Krämer A. Identification of griseofulvin as an inhibitor of centrosomal cluster- ing in a phenotype-based screen. Cancer Res 2007; 67: 6342-6350
3 Kwon M, Godinho SA, Chandhok NS, Ganem NJ, Azioune A, Thery M, Pell- man D. Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes. Genes Dev 2008; 22: 2189-2203
4 Nigg EA. Origins and consequences of centrosome aberrations in human cancers. Int J Cancer 2006; 119: 2717-2723
5 Pihan GA, Purohit A, Wallace J, Knecht H, Woda B, Quesenberry P, Doxsey SJ. Centrosome defects and genetic instability in malignant tumors. Can- cer Res 1998; 58: 3974-3985
6 Hinchcliffe EH, Sluder G. “It takes two to tango”: Understanding how centrosome duplication is regulated throughout the cell cycle. Genes Develop 2001; 15: 1167-1181
7 Fassnacht M, Hahner S, Polat B, Koschker AC, Kenn W, Flentje M, Allolio B. Efficacy of adjuvant radiotherapy of the tumor bed on local recur- rence of adrenocortical carcinoma. J Clin Endocrinol Metab 2006; 91: 4501-4504
8 Kirschner LS. Emerging treatment strategies for adrenocortical carci- noma: a new hope. J Clin Endocrinol Metab 2006; 91: 14-21
9 Libè R, Fratticci A, Bertherat J. Adrenocortical cancer: pathophysiol ogy and clinical management. Endocr Relat Cancer 2007; 14: 13-28
10 Schteingart DE, Doherty GM, Gauger PG, Giordano TJ, Hammer GD, Korobkin M, Worden FP. Management of patients with adrenal cancer: recommendations of an international consensus conference. Endocr Relat Cancer 2005; 12: 667-680
11 Berruti A, Terzolo M, Sperone P, Pia A, Della Casa SD, Gross DJ, Carnaghi C, Casali P, Porpiglia F, Mantero F, Reimondo G, Angeli A, Dogliotti L. Etoposide, doxorubicin and cisplatin plus mitotane in the treatment of advanced adrenocortical carcinoma: a large prospective phase II trial. Endocr Relat Cancer 2005; 12: 657-666
12 Khan TS, Imam H, Juhlin C, Skøgseid B, Grondal S, Tibblin S, Wilander E, Oberg K, Eriksson B. Streptozocin and o,p’DDD in the treatment of adrenocortical cancer patients: long-term survival in its adjuvant use. Ann Oncol 2000; 11: 1281-1287
13 Terzolo M, Angeli A, Fassnacht M, Daffara F, Tauchmanova L, Conton PA, Rossetto R, Buci L, Sperone P, Grossrubatscher E, Reimondo G, Bollito E, Papotti M, Saeger W, Hahner S, Koschker AC, Arvat E, Ambrosi B, Loli P, Lombardi G, Mannelli M, Bruzzi P, Mantero F, Allolio B, Dogliotti L, Berruti A. Adjuvant mitotane treatment for adrenocortical carcinoma. N Eng J Med 2007; 356: 2372-2380
14 Allolio B, Fassnacht M. Clinical review: Adrenocortical carcinoma: clinical update. J Clin Endocrinol Metab 2006; 91: 2027-2037
15 Dohna M, Reincke M, Mincheva A, Allolio B, Solinas-Toldo S, Lichter P. Adrenocortical carcinoma is characterized by a high frequency of chromosomal gains and high-level amplifications. Genes Chromo- somes Cancer 2000; 28: 145-152
16 Roshani L, Fujioka K, Auer G, Kjellman M, Lagercrantz S, Larsson C. Aberrations of centrosomes in adrenocortical tumors. Int J Oncol 2002; 20: 1161-1165
17 Ho YS, Duh JS, Jeng JH, Wang YJ, Liang YC, Lin CH, Tseng CJ, Yu CF, Chen RJ, Lin JK. Griseofulvin potentiates antitumorigenesis effects of nocodazole through induction of apoptosis and G2/M cell cycle arrest in human colorectal cancer cells. Int J Cancer 2001; 91: 393-401
18 Rathinasamy K, Jindal B, Asthana J, Singh P, Balaji PV, Panda D. Grise- ofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine. BMC Cancer 2010; 10: 213
19 Kamech N, Seif R. Effect of microtubule disorganizing or overstabilizing drugs on the proliferation of rat 3T3 cells and their virally induced transformed derivatives. Cancer Res 1988; 48: 4892-4896
20 Haase M, Schott M, Bornstein SR, Malendowicz LK, Scherbaum WA, Wil- lenberg HS. CITED2 is expressed in human adrenocortical cells and regulated by basic fibroblast growth factor. J Endocrinol 2007; 192: 459-465
21 Fallo F, Pilon C, Barzon L, Pistorello M, Pagotto U, Altavilla G, Boscaro M, Sonino N. Paclitaxel is an effective antiproliferative agent on the human NCI-H295 adrenocortical carcinoma cell line. Chemotherapy 1998; 44: 129-134
22 Berruti A, Sperone P, Ferrero A, Germano A, Ardito A, Priola AM, De Francia S, Volante M, Daffaral F, Generali D, Leboulleux S, Perotti S, Baudin E, Papotti M, Terzolo M. Phase II study of weekly paclitaxel and sorafenib as second/third-line therapy in patients with adrenocortical Carcinoma. Eur J Endocrinol 2012; 166: 451-458
23 Arida AI, Al-Tabakha MM, Hamoury HA. Improving the high variable bioavailability of griseofulvin by SEDDS. Chem Pharm Bull 2007; 55: 1713-1719
24 Rønnest MH, Rebacz B, Markworth L, Terp AH, Larsen TO, Krämer A, Clausen MH. Synthesis and structure-activity relationship of griseof- ulvin analogues as inhibitors of centrosomal clustering in cancer cells. J Med Chem 2009; 52: 3342-3347
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