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CMSXXX10.1177/1203475415589055Journal of Cutaneous Medicine & SurgeryEl Tawdy et al
Original Article
Abstract
Background: Histone deactylases (HDAC) have a role in the pathogenesis of mycosis fungoides (MF) through their actions
on different apoptosis pathways.
Objective: To assess the possible role played by HDAC-2 in MF by estimating the tissue expression of HDAC2 mRNA in
different stages of MF.
Methods: This study included 28 MF patients and 30 controls. The HDAC-2 levels were detected by real-time polymerase
chain reaction (PCR). Correlations of HDAC-2 levels with clinical presentation and different stages of MF were analyzed.
Results: Mean HDAC-2 level was significantly higher in patients (P < .001) than in controls. HDAC-2 highest mean value was
significantly detected in patients with stage IIb, and the lowest mean value was detected in patients with stage Ia (P < .001).
Conclusion: Up-regulation of tissue HDAC-2 in MF patients might develop a new approach in the understanding of the
pathogenesis of MF. Histone deactylases are important targets for molecular cancer therapeutics.
Keywords
apoptosis, cancer therapeutics, cell cycle, HDAC-2, mycosis fungoides
Methods
A 4 mm punch skin biopsy was obtained from lesional skin
Statistical Analysis
of patients and from normal skin of controls. The biopsies Statistical Package for Social Science (SPSS) program
were stored at -70°C for measurement of HDAC-2 mRNA. version 17 was used for analysis of data. Data were sum-
marized as mean, standard deviation (SD), number (N),
and percentage (%). The t test was used for analysis of
Detection of HDAC-2 Gene Expression Level
quantitative data, and 1-way analysis of variance
Using Real-Time PCR (ANOVA) test was used for analysis of more than 2 quan-
RNA extraction and cDNA synthesis. Total RNA was extracted titative variables. Pearson’s correlation was done to test
from skin tissue using Trizol reagent (Invitrogen, Karlsruhe, linear relation between quantitative variables. P value is
Germany). The quantity and purity of extracted RNA was considered significant if <.05, and chi-square test was
assessed by measuring absorbance at 260 nm and the ratio used for analysis of qualitative data.
A260/A280 in a UV-spectrophotometer (NanoDrop Inc,
Wilmington, DE, USA). Only samples with an A260/A280
ratio up to 1.8 were considered valid for real-time PCR.
Results
Reverse transcription of 1 μg of RNA into cDNA was per- This case control study included 28 MF patients and 30
formed using Takara first strand cDNA synthesis kit healthy age- and sex-matched individuals serving as con-
(Takara, Bio Inc, Shiga, Japan), according to manufacturer’s trols. The MF patients were 11 males (39%) and 17 females
instructions. (61%), their age ranged from 25 to 62 years (mean, 34.32 ±
17.31 years). The duration of their disease ranged from 0.08
Real-time PCR. The expressions of target gene (HDAC-2) to 15 years (4.42 ± 3.86 years). Twenty patients (71.4%) pre-
were quantified using the comparative threshold cycle (Ct) sented clinically with patch stage, while 8 patients (28.6%)
method where the amount of target mRNA was normalized presented clinically with plaque stage.
to an internal control (glyceraldehyde-3-phosphate dehydro- According to the staging of MF, 6 patients (21.4%) had
genase [GAPDH]). The real-time PCR was performed using stage Ia, 6 patients (21.4%) had stage Ib, 8 patients (28.6%)
the LightCycler FastStart DNA SYBR-Green I kit (Roche had stage IIa, and 8 patients (28.6%) had stage IIb.
Applied Sciences, RAS, Mannheim, Germany) according The controls group included 30 age- and sex-matched
to the protocol provided in the kit, briefly 10 μL amplifica- healthy individuals (P = .178 and .118, respectively). They
tion mixtures containing equivalent to 8 ng of reverse-tran- were 12 (40%) males and 18 females (60%), with age range
scribed RNA and 300 nM primers; the sequences of PCR from 36 to 55 years (41.80 ± 12.20 years).
42 Journal of Cutaneous Medicine and Surgery 20(1)
The tissue levels of HDAC-2 in MF patients (range, 0.63- Table 2. Tissue Histone Deacetylase Levels (mean ± SD) in
2.70; 1.57 ± 0.59) were statistically significantly higher than Different Mycosis Fungoides (MF) Staging.
those of controls (range, 0.08-0.22; 0.14 ± 0.05) (P < .001). MF Staging Ia (n = 6) Ib (n = 6) IIa (n = 8) IIb (n = 8)
In the patients group, there was a significant positive cor-
relation between age of patients and disease duration (r = Histone 0.84 ± 0.11 1.17 ± 0.19 1.7 ± 0.2 2.3 ± 0.27
0.021; P = .011); however, there were nonsignificant positive deacetylase
correlations between the tissue levels of HDAC-2 with both P valuea <.001*
age of patients (r = 0.109; P = .580) and disease duration (r = a
P < .05 is statistically significant.
0.023; P = .906).
Comparing the tissue levels of HDAC-2 in relation to the
sex difference in MF patients revealed a nonsignificant dif-
ference (P = .961) between their mean value in male patients
(1.58 ± 0.65) when compared to female patients (1.57 ±
0.59).
Comparing the tissue levels of HDAC-2 in relation to the
different clinical presentations in MF patients revealed that
their mean value was higher in patients with plaque stage (1.79
± 0.69) than in patients with patch stage (1.49 ± 0.56). However,
this finding was statistically nonsignificant (P = .232).
On comparing the tissue levels of HDAC-2 in relation to
the different staging of MF in the patient group, we found a
statistically significant difference (P < .001) with the high-
est mean value detected in patients with stage IIb and the
lowest mean value detected in patients with stage Ia (Table 2, Figure 1. Mean tissue histone deacetylase levels in different
mycosis fungoides (MF) staging.
Figure 1).
Romidepsin and Vorinostat have been FDA approved for 6. Liu T, Kuljaca S, Tee A, Marshall GM. Histone deacetylase
treatment of refractory MF cases. Both inhibit HDAC I and inhibitors: multifunctional anticancer agents. Cancer Treat
II isoforms.9,18 Histone deactylase-2 belongs to class I Rev. 2006;32:157-165.
HDAC, thus its inhibition might contribute to the reported 7. Nevala H, Karenko L, Vakeva L, Ranki A. Proapoptotic
and antiapoptotic markers in cutaneous T-cell lymphoma
therapeutic efficacy of those drugs.
skin infiltrates and lymphomatoid papulosis. Br J Dermatol.
This study lacks the immunohostochemical or the mRNA
2001;145:928-937.
in situ hybridization analysis for proper HDAC-2 localiza- 8. Winter M, Moser MA, Meunier D, et al. Divergent roles of
tion. Further studies on HDACs are recommended to delin- HDAC1 and HDAC2 in the regulation of epidermal develop-
eate more their future promising impact on treating several ment and tumorigenesis. EMBO J. 2013;32:3176-3191.
types of malignancies. Also, comparing the expression of 9. Poligone B, Lin J, Chung C. Romidepsin: evidence for its
HDAC in MF (as neoplastic lymphocytes) and psoriasis (as potential use to manage previously treated cutaneous T cell
inflammatory lymphocytes) is our ongoing research in order lymphoma. Core Evid. 2011;6:1-12.
to estimate the role of HDAC in neoplastic versus non-neo- 10. Olsen E, Vonderheid E, Pimpinelli N, et al. Revisions to the
plastic, inflammatory cells and assess their levels semiquan- staging and classification of mycosis fungoides and Sezary syn-
titatively. Comparing involved skin to normal skin in MF drome: a proposal of the International Society for Cutaneous
Lymphomas (ISCL) and the cutaneous lymphoma task force
patients is also recommended for better understanding of the
of the European Organization of Research and Treatment of
possible role played by HDAC in MF.
Cancer (EORTC). Blood. 2007;110:1713-1722.
11. Livak KJ, Schmittgen TD. Analysis of relative gene expression
Declaration of Conflicting Interests data using real-time quantitative PCR and the 2(-Delta Delta
The author(s) declared no potential conflicts of interest with respect C(T)) method. Methods. 2001;25:402-408.
to the research, authorship, and/or publication of this article. 12. Jain S, Zain J. Romidepsin in the treatment of cutaneous T-cell
lymphoma. Blood Med. 2011;2:37-47.
13. Bi G, Jiang G. The molecular mechanism of HDAC inhibitors
Funding in anticancer effects. Cell Mol Immunol. 2006;3:285-290.
The author(s) received no financial support for the research, author- 14. Wagner JM, Hackanson B, Lubbert M, Jung M. Histone
ship, and/or publication of this article. deacetylase (HDAC) inhibitors in recent clinical trials for can-
cer therapy. Clin Epigenetics. 2010;1:117-136.
15. Chang CC, Lin BR, Chen ST, et al. HDAC2 promotes cell
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