Effects of Sodium Butyrate on Cell Death ... - Wiley Online Library

Photochemistry and Photobiology, 2009, 85: 1182–1188

Effects of Sodium Butyrate on Cell Death Induced by Photodynamic Therapy in U373-MG and D54-MG Astrocytoma Cell Lines Roxana Magaly Flores-Ancona1, Fanny Yocelin Garcıá-Go´mez1, Ana Marıá Jimeńez-Betanzos1, Mario Solis-Paredes1, Violeta Castro-Leyva1, Alfredo Cruz-Orea2, Francisco Arenas-Huertero1 and Eva Ramoń-Gallegos*1 1

Laboratorio de Citopatologıá Ambiental, Escuela Nacional de Ciencias Biolo´gicas, Instituto Politećnico Nacional, Me´xico, D.F., Mexico 2 Departamento de Fı´sica, Centro de Investigacioń y Estudios Avanzados-IPN, Me´xico, D.F., Mexico Received 10 June 2008, accepted 19 February 2009, DOI: 10.1111 ⁄ j.1751-1097.2009.00561.x

ABSTRACT The damage induced by end products of photodynamic therapy (PDT) in astrocytoma tumors leads to cytotoxicity and cell death. Chromatin modifiers such as sodium butyrate (NaB) induce several genes involved in apoptosis, among others. The PDT improvement was evaluated by the measurement of its effectiveness in the treatment of U373-MG and D54-MG astrocytoma cell lines exposed to NaB. Cells exposed to 80 lg mL)1 of d-aminolevulinic acid (ALA) as precursor of endogenous photosensitizer (PS), protoporphyrin IX (PpIX), induced 16.67% and 28.9% of mortality in U373-MG and D54-MG, respectively. The mortality increased to 70.62% and 96.7%, respectively, when U373-MG and D54-MG cells were exposed for 24 h to 8 mM NaB prior to ALA-induction. In this condition, re-expression of some genes related to apoptosis in U373-MG, and differentiation in D54-MG were induced. PpIX accumulation was higher than ALA-induction and the acetylation of histone H4 induced by NaB was verified by immunocytochemistry in both cells. It can be concluded that modified chromatin and genes induced by NaB increment the cellular death induced by PDT in astrocytoma cells using PpIX as endogenous PS.

INTRODUCTION Astrocytoma, frequently encountered in children, accounts for the 20% in adult patients as the main cause of morbidmortality in Mexico (1), and for the more frequent solid tumors (2). Low-grade tumors have better prognosis and can be controlled. However, high-grade astrocytomas (anaplastic and glioblastoma) represent a challenge in oncology, because almost all these tumors have no effective responses to combined therapy (surgery, chemotherapy and radiotherapy) (3). Another strategy explored in oncology is photodynamic therapy (PDT), a noninvasive selective therapy that offers distinct advantages for the treatment of some solid tumors (4). Traditionally, PDT involves systemic administration of a photosensitizer (PS) followed by a period that enables the *Corresponding author email: [email protected], [email protected] (Eva Ramoń-Gallegos) 2009 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/09

distribution and localization of active components in target tissues. Subsequently, lesions are exposed to light of an appropriate PS-absorbing wavelength. Photon energy absorption triggers a photochemical reaction, which usually leads to reactive oxygen species formation (5). Oxidative damage induced by the reaction of singlet oxygen with cell components leads to cytotoxicity followed by cell death (6). DNA is one of these cell components that are damaged and participate in cell death (5,6). Protoporphyrin IX (PpIX) can act as an endogenous PS. Kennedy et al. suggested that PpIX accumulation occurs after systemic administration of d-aminolevulinic acid (d-ALA). This has been used as an alternative procedure to the administration of exogenous PSs (7). Exogenous administration of ALA circumvents the first step in heme biosynthesis and the normal regulatory feedback. In subsequent steps, ALA is converted to PpIX; numerous studies have documented that PpIX is accumulated in some tumoral cell lines at rates several times higher than in their normal cell counterparts. ALA can also increase PpIX levels by several orders of magnitude, enough to make PDT more effective, at least in vitro (8). The acetylation and deacetylation of histones of the core proteins of the nucleosomes in chromatin play an important role in the regulation of gene expression. Two classes of enzymes, mainly, control the acetylation status of the histones: histone acetyl transferases and histone deacetylases (HDAC) (9). Different inhibitors of HDAC (HDIs) that induce growth arrest, differentiation and apoptosis in cancerous cells have been identified (10,11). HDIs belong to a heterogeneous class of compounds that includes derivates of fatty acids of short chain, among others (12). HDI sodium butyrate (NaB) and fatty acids of short chain occurs naturally in the body from the acetyl-CoA-dependent catabolic oxidation of long chain saturated fatty acids (13). NaB also has growth inhibitory effect on cancerous cells, which has been attributed to its ability to induce cell cycle arrest, differentiation and apoptosis (14,15). In order to evaluate, in this study, the synergistic effect of NaB and PDT to induce more efficient cell death we proved that 8 mM NaB as a pretreatment (24 h before) to the induction of endogenous PS (PpIX) via ALA exposition in U373-MG and D54-MG astrocytoma cells improved the cellular effects of PDT. Thus, cell mortality was increased

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Photochemistry and Photobiology, 2009, 85 from 16.67% to 70.6% in U373-MG cells and from 28.9% to 96.7% in D54-MG cells.

MATERIAL AND METHODS Cell lines and culture conditions. Human astrocytoma cells U373-MG were kindly gifted by Dr. Antonio Arias-Montanõ (Departamento de Fisiologı´ a, Biofı´ sica y Neurociencias, CINVESTAV-IPN, Me´xico, D.F.) and D54-MG was purchased from the American Type Culture Collection (Rockville, MD) and maintained in a 5% carbon dioxide, 95% regular air, humid atmosphere at 37C. Cells were grown in suspension in DMEM ⁄ F12 1:1 medium supplemented with 2 mm L-glutamine, 10% fetal bovine serum, 1X nonessential amino acids, 100 U mL)1 of penicillin and 100 U mL)1 of streptomycin. These astrocytoma cell lines were chosen due to the differences in their responses as happens in primary and secondary astrocytomas: D54-MG and U373-MG cells were isolated from an astrocytoma grade III and glioblastoma grade IV, respectively; furthermore, they are dissimilar in morphological appearance. Determination of basal PpIX and treatments to induce NaB and PpIX by d-ALA. Piomelli spectrofluorometry assay (16) for free erythrocyte porphyrins as adapted for cultured cells was used to quantify PpIX (8). PpIX was measured in cells 24 h before and after exposure to 80 lg mL)1 of d-ALA (98% pure hydrochloric salt; Sigma). We found that this concentration of ALA produced the major accumulation of induced PpIX. Other conditions were 24 h exposure only to 8 mM of NaB (Research Organics), 24 h exposure to 8 mM of NaB before d-ALA exposure and the simultaneous exposure to both compounds (NaB plus d-ALA) for 24 h. Previous results demonstrated that 8 mM of NaB is the concentration that produces the biggest inhibitory effect on histone deacetylation in these and several cell lines included pediatric tumors (17,18) . The supernatant was removed and the cells were rinsed once in saline phosphate buffer (PBS, pH 7.4), then collected by centrifugation and lysed in 0.2 mL of 5% celite suspension 0.9% NaCl. This was supplemented with 4 mL of 4:1 ethyl acetate ⁄ acetic acid, shaken 10 s on a vortex mixer and centrifuged for 30 s at 176 g. The supernatant was placed in another test tube supplemented with 4 mL of 1.5 N HCl and shaken for 10 s on a vortex mixer. A Pasteur pipette was used to transfer an aliquot of the lower HCl phase to a cuvette and PpIX levels were read directly as lg ⁄ 5 · 105 cells using a Perkin Elmer LS-2B spectrofluorometer calibrated with coproporphyrin I (0.05 lg mL)1; Sigma) stock solution (excitation wavelength 408 nm, emission 608 nm). All assays were performed in triplicate. A blank was prepared in parallel by replacing the cellular suspension with 40 lL of saline solution. Cellular death using PDT with NaB and PpIX. In all conditions described above, U373-MG and D54-MG cells were exposed in 96 well plates and irradiated for 7.15 min with an argon laser at 88.8 mW. Each circular well has an area of 0.5 cm2. The total light dose was 80 J cm)2 at 488 nm wavelength, this energy density was calculated using Eqs. (1) and (2). The used groups were: negative control (cells without NaB, ALA and irradiation), irradiation control (irradiated cells only), treated cells group (cells with NaB and irradiated, cells with ALA and irradiation, and cells with NaB, ALA and irradiation). The last condition was analyzed with cells exposed at the same time to NaB and ALA, and exposed to NaB for 24 h before the ALA. Power density ¼

Argon laser power (mW) Irradiation area (cm2 Þ

Energy density ðJ cm2 Þ ¼ Power density ðW cm2 Þ tðsÞ

ð1Þ

ð2Þ

In all the experiments the plates with U373-MG and D54-MG cells were conserved in darkness before and after irradiation. Each experiment was performed with at least two sets of experiments, each performed in quadruplicate. Cell viability determination: neutral red dye retention to assess viability. After exposing cells to the different conditions mentioned above, cell viability was measured. Cellular viability was estimated by means of the neutral-red spectrophotometric assay (8). The medium containing ALA was removed from the wells and replaced with 100 lL of fresh medium per well containing 100 lL of neutral red. The plates

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were then returned to the incubator for 3 h. The medium was subsequently removed, and the cultures were washed rapidly with a mixture of 40% formaldehyde and 10% CaCl2 vol ⁄ vol (4:1). A mixture of 1% vol ⁄ vol acetic acid and 50% vol ⁄ vol ethanol (1:1) was then added to extract the neutral red. The plate was shaken for 60 s and left to stand at room temperature for 15 min. The absorbance of the solubilized dye was subsequently read at 540 nm. Quantification of the extracted dye was correlated with the live cell number. Control wells were prepared in parallel, and these cells were exposed to neutral red, but not to ALA. The percentage of viable cells in the cell population at each concentration of the test agent was calculated by means of the following formula: % Viability ¼

Mean absorbance of treated cells 100 Mean absorbance of control cells

Immunohistochemistry to histone H4 in cell lines pre- and posttreatment to PDT. Histones H3 and H4 are the best substrates to be acetylated by histone acetylases. Also, they have more lysine residues that can be modified (19). So, in order to prove the deacetylation effect of NaB on astrocytoma cell lines, the acetylation of the main substrate of histone acetylases, histone H4 was analyzed. Cells were grown on 96 well plates and were treated as described above in triplicate. In the cells treated without and with PDT, histone H4 was observed using the immunofluorescent technique. The medium was aspirated and cells were washed with PBS (pH 7.4) three times and they were fixed in 1% paraformaldehyde for 30 min at room temperature. Cells were washed three times as mentioned above and they were blocked with horse normal serum (HNS) and permeated with detergent in a solution of HNS 10%-0.3% triton X-100-PBS 1X for 45 min. The liquid was aspirated and cells were washed three times with PBS 1X-0.1% bovine serum albumin (BSA). The acetylated antibody anti-histone H4 was added at a dilution of 1:20 in 10% of HNS in PBS 1X overnight at 4C. The primary antibody solution was discarded and cells were washed with PBS 1X-0.1% BSA as described above. They were then incubated in the dark with ALEXA-Fluorlabeled secondary antibody at a dilution of 1:500 at 10% of HNS in PBS 1X at room temperature for 1 h. Finally, the secondary antibody solution was retired, and cells were washed three times with PBS 1X0.1% BSA. The cells were then examined under a Carl Zeiss microscope. Images were captured by a CCD camera and imported into Advanced Spot Image analysis software package. Five fields were examined, and pictures were taken in each experimental condition with and without PDT induction in both cell lines. Histochemical staining of glial fibrillar acidic protein (GFAP). Histochemical staining was performed in accordance with the Rı´ oHortega technique for microglia. 4 · 104 cells were seeded in 24 wells for 24 h in the presence of 4 and 8 mM NaB and without it. At the end, the cells were washed in PBS 1X for 5 min. The cells were then fixed in formalin 10% for 30 min, washed twice, with ammoniacal water and with double distilled water. Immediately, the cells were incubated in ammoniacal-silver: 75 lL of 10% AgNO3 plus 2.25 mL of 5% NaCO3 plus 3 mL distilled water, for 10 min. Ammoniacal silver was finally prepared with some drops of ammonium hydroxide until dissolving the mixture of AgNO3 and NaCO3. Finally, the silver impregnated in the cells was reduced in 1% formalin for 15 min. The reaction was stopped by replacing the solution with distilled water. RNA extraction and cDNA synthesis. Total RNA was extracted from duplicates of U373-MG and D54-MG cells cultured in 30 mm dishes, incubated with and without 8 mM NaB for 24 h, using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was obtained from 1 lg total RNA using a Reverse Transcription System (PROMEGA) under the conditions specified therein and oligo-dT was used as the primer. cDNA concentrations were normalized for glyceraldehyde-diphosphate dehydrogenase gene messages using a Biometra Tpersonal thermocycler (Whatman). Reverse transcriptase-polymerase chain reactions (RT-PCR) were performed on 20 lL total volumes containing 1· PCR buffer (PROMEGA), 2.0 mM MgCl2, 50 pmol of each primer, 0.5 lM dNTP, 1 U of Taq DNA polymerase (PROMEGA) and 2 lL of each cDNA. The cDNA sequences of the apoptosis genes were obtained from the GenBank Sequence Database at the National Center for Biotechnology Information. The genes were caspase-3 (NM_004346), caspase-9 (BC002452), Bax (NM_004324), Bak-1 (NM-001188), Bcl-2

1184 Roxana Magaly Flores-Ancona et al. (NM_138761), and glyceraldehyde phosphate dehydrogenase, GADPH (AF261085). The primers were: Casp3-F: 5¢-GATCATACATGGAAGCGAA-3¢, Casp3-R: 5¢-TTCTGAATGTTTCCCTGAG-3¢; Casp9-F: 5¢-ATGATCGAGGACATCCAG-3¢, Casp9-R: 5¢-TGTCCTCTAAGCAGGAGA-3¢; Bax-F: 5¢-AAGCTGAGCGAGTGTCTCA-3¢, Bax-R: 5¢-AACATGTCAGCTGCCACTC-3¢; Bak-1-F: 5¢-TTCTGAGGAGCAGGTAGC-3¢, Bak-1-R: 5¢-GGTTGCAGAGGTAAGGTG-3¢: Bcl-2-F: 5¢-TGTGGAGAGCGTCAACC-3¢, Bcl-2-R: 5¢-CAGAGACAGCCAGGAGA-3¢; GADPH-F: 5¢-TCCCATCACCATCTTCCAG3¢, GADPH-R: 5¢-ATGAGTCCTTCCACGATACC-3¢. Amplification conditions were: initial denaturation at 94C for 5 min, followed by denaturation at 94C for 30 s, annealing at that temperature for each primer for 30 s, extension at 72C for 30 s, for 30 cycles, and a final extension at 72C for 5 min. The products were analyzed by electrophoresis using silver staining of polyacrylamide gel electrophoresis. Semiquantitative RT-PCR conditions were previously confirmed by amplifying the products at 20, 25, 30, 35 and 40 cycles with the GAPDH gene control (results not shown). It was therefore decided to perform the final analysis of amplified products at 30 cycles (nonsaturable conditions). Densitometry. The intensity of each band was determined by densitometry using Image J software. Results are expressed as densitometric units (DU) of the ratio between mRNA levels of each gene: Casp3, Casp9, Bax, Bcl-2 and Bak; and GAPDH gene control in the presence and absence of 8 mM of NaB in each cell line. Statistical analysis. Two-way analysis of variance (ANOVA) was performed for analyzers between treatment and cell lines. In both cases, the level of significance was set at P < 0.05. The tests were performed with Sigma Stat v2.03 for Windows (Jandel Scientific).

RESULTS Table 1 illustrates intracellular concentrations of PpIX in different conditions in astrocytoma cells. The basal levels of PpIX between both cell lines show that D54-MG has more PS than U373-MG. Postinduction by d-ALA (80 lg mL)1) intracellular levels of PpIX increased in both cell lines, but only in U373-MG was it statistically significant (P < 0.05) compared to basal levels (control). The treatment with NaB did not affect the levels, but when astrocytoma cells were treated simultaneously with d-ALA plus NaB, the concentrations of PpIX increased and only in U373-MG were they significant (P < 0.05). This effect was also observed mainly in U373MG cells when they were previously treated for 24 h with NaB prior to d-ALA administration. In addition, the differences in this cellular line were significant against the levels of PpIX in control U373-MG cells (P < 0.05). In conclusion, the treatment with d-ALA in both cells lines induced an increase in the levels of the endogenous PS, PpIX. After verifying that the d-ALA treatment increased PpIX levels, the next step was to prove whether the PDT induction with laser could induce cell death in both astrocytoma cell lines in all experimental conditions. Table 2 shows the mortality index postinduction of PDT in all treatments in both astroTable 1. Intracellular concentration of PpIX in different conditions on astrocytoma cells (expressed as lg ⁄ 2.5 · 106 cells). Condition Control d-ALA NaB NaB + d-ALA NaB 24 h before d-ALA

U373-MG 0.12 0.59 0.02 1.25 0.69

± ± ± ± ±

0.04 0.14* 0.01 0.021* 0.09*

* Significant differences vs control (P < 0.05).

D54-MG 0.36 0.46 0.43 0.54 0.44

± ± ± ± ±

0.10 0.19 0.03 0.01 0.12

Table 2. Induction of cellular death by PDT in astrocytoma cells exposed to d-ALA and NaB in different conditions. Condition No irradiation Irradiation d-ALA NaB NaB + d-ALA NaB 24 h before d-ALA

U373-MG

D54-MG

0 0.06 ± 0.01 16.67 ± 0.01 0 40.73 ± 0.0* 70.62 ± 0.07*

0 4.45 ± 0.01 28.89 ± 0.01 0 72.30 ± 0.01* 96.70 ± 3.32*

Values are expressed as percentage of mortality according to 0% in control cells. *Significant differences vs no irradiation (P < 0.05).

cytoma cell lines. Without any treatment, the PDT effect induced discrete mortality in both cell lines and in the NaB treatment no mortality was induced. Only in conditions where cells were treated with both d-ALA and NaB, the mortality index increased in comparison with basal conditions when cells were treated with both chemicals, mainly with NaB for 24 h before d-ALA induction of PpIX. These induced mortalities were very high, 70.62% and 96.7% for U373-MG and D54MG cells, respectively, if they are contrasted with the mortality (0.06% and 4.45% for U373-MG and D54-MG cells, respectively) of the cells induced by PDT without any treatment. These differences were statistically significant (P < 0.05). These results prove that the effectiveness of PDT was higher when the cells were treated with NaB before the ALA treatment. As histone H4 has several lysine residues to be acetylated, it was chosen for evaluating the acetylation effect of HDIs. In order to verify only that NaB treatment was effective in both astrocytoma cell lines, the acetylated histone H4 was observed by immunocytochemistry (Fig. 1). Basal U373-MG cells had more histone H4 acetylated than D54-MG (Fig. 1a,b). Any condition with NaB treatment induced more histone H4 acetylated (Fig. 1c–f). These results verify the effect of NaB treatment in increasing the acetylation of histone H4 and the inhibitor effect of NaB on histone deacetylase activity in the astrocytoma cell lines. As NaB increases and improves cell death, mainly with 24 h previous treatment with NaB, we analyzed the expression of genes related to apoptosis: The expression of caspase-3, caspase-9, Bax, Bak-1 and Bcl-2 was evaluated in both astrocytoma cell lines. Table 3 shows that U373-MG cells only expressed Bax and Bak-1 genes in control conditions. However, when cells were treated for 24 h with NaB 8 mM this one induced the re-expression of caspase-3, caspase-9 and Bcl-2. Furthermore, an increase in Bax and Bak-1 was observed (Fig. 2a). D54-MG cells expressed caspase-3, caspase-9 in basal conditions and NaB did not induce more expression; however, NaB repressed Bax and Bcl-2 expression (Fig. 2b). These results demonstrate the positive effect of NaB treatment 24 h prior to PDT, in the improvement of the expression of all five genes related to apoptosis in U373-MG cells. In order to verify that NaB treatment induced cellular responses cell differentiation was evaluated (Fig. 3). In fact, the morphology between both cell lines was dissimilar preserving a fusiform astrocytic-shape in U373-MG and a more regular epithelial-like shape in D54-MG cells (Fig. 3a,d). These morphologies correlate with the presence of GFAP in

Photochemistry and Photobiology, 2009, 85

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Figure 1. Immunohistochemistry to histone H4 acetylated in U373-MG (a–e) and D54-MG cells (f–j). Cells were not induced (a, f), induced with 80 mM d-ALA (b, g), treated with 8 mM NaB (c, h), treated with d-ALA plus NaB at the same time (d, i) and treated for 24 h with NaB, before exposing them to d-ALA (e, j).

Table 3. Expression ratio of genes involved in apoptosis of astrocytoma cell lines (control and induced by 24 h of NaB 8 mM) calculated over the GADPH gene expression as control.

U373-MG U373-MG + NaB D54-MG D54-MG + NaB

Casp-3

Casp-9

Bax

Bak-1

Bcl-2

0 0.43* 0.8 0.9

0 0.40* 0.7 0.71

0.6 0.83* 1.72 0

0.46 0.6 0 0

0 0.40* 0.85 0

*Significant differences vs control (P < 0.05).

U373-MG cells (Fig. 3b) and its absence in D54-MG cells (Fig. 3e). The 24 h treatment with 8 mM NaB induced an increase in positive reaction to GFAP in both cell lines, mainly in D54-MG (Fig. 3f). These results verify the induction of GFAP, a marker of differentiation, induced by NaB in both astrocytoma cell lines mainly in D54-MG.

DISCUSSION Anticancer therapy is a prime example of the clinical application of both PDT and HDIs. The primary effects of PDT and HDIs have been evaluated separately, but never have they been combined as in this study. One important characteristic of HDIs is their selectivity in terms of altering gene expression in transformed cells (20). On the other hand, PpIX represents a selective PS in tumoral cells, which can be increased via d-ALA induction (21). In this way, both strategies can be synergistic in their effects on cell death as the results of this study prove. The recent development of HDIs has allowed investigations towards cellular processes and its response to external agents, such as ionizing radiation (22). Here, we report the ability of NaB to sensibilize the astrocytoma cells vs PDT. Some reports have found radiosensitization by HDIs (23,24) and they are consistent with our observations, in this case by using PDT. Our study for the first time compares the effects of HDI and NaB on the effectiveness of PDT in astrocytoma cells: U373MG and D54-MG. Due to the morphological differences in

these astrocytoma cell lines, they represent a good model to evaluate biological responses to this kind of therapy. Some authors have reported that the presence of the p53 protein may further augment the radiosensitization of cancer cells by other HDIs such as trichostatin-A (25). NaB is reported to radiosensitize human tumor cells by affecting their ability to repair the DNA damage induced by ionizing radiation. NaB suppressed the growth of WT p53-containing cells more efficiently, inducing apoptosis. After gamma irradiation, tumoral cells are arrested in the G2 ⁄ M phase both with and without the p53 protein. When some HDIs are present in cells during irradiation, cells are arrested in the G1 phase, leading to apoptosis (26). D54-MG cells are of the p53wt type and this could explain the high mortality induced by PDT, mainly in NaB exposure (24 h previously) to PpIX induced by d-ALA. Several proteins participate in PDT-induced cell death. One is the p53 protein. In fact, Zawacka-Pankau et al. showed that PpIX induces p53 and that several p53 target genes are activated upon p53 accumulation such as Bak, p21 and PUMA (27). Furthermore, PpIX inhibits the interaction of MDM2 with wt-p53 in its N-terminal and the proteasome degradation of the p53 protein (28). This could also occur in PDTinduced cell-death in D54-MG cells which are p53wt. Finally, activation of apoptosis via caspases could also be a mechanism of cell death induced by PDT in D54-MG, due to the expression of Casp-3 and Casp-9 in these cells as demonstrated by Karmakar et al. in U87MG cells at 0.5 and 1 mM of ALA which induced Casp-3 activation (29). Activation of apoptosis via caspases can be verified because in this work, D54-MG cells activate Casp-3 and Casp-9 gene transcription and they maintain their expression in NaB incubation. In addition, NaB induced differentiation in D54MG cells; this biological response is important to improve the effect of PDT (29,30). The addition of HDIs would not be sufficient to reactivate the silenced genes. DNA methylation also seems to have a more dominant repressor effect than histone deacetylation (19). This could operate in D54-MG cells treated with NaB, which does not induce the Bak-1 gene. Sometimes, methylation of histone H3, specifically in lysine 9,

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Figure 2. RT-PCR expression profile of the genes involved in apoptosis in U373-MG cells (a) and D54-MG cells (b). 8 mM sodium butyrate treatment for 24 h is indicated in each lane (NaB), and its corresponding gene expression ⁄ repression or downregulation is illustrated as a PCR-amplified product. Arrows indicate molecular weight based in the 100 bp ladder. GADPH = glyceraldehyde phosphate dehydrogenase gene; casp-3 = caspase-3; casp-9 = caspase-9. PCR products resolved in polyacrylamide-gel electrophoresis and silver staining.

Figure 3. Cristal violeta stain and histochemical stain to GFAP protein by ammoniacal silver in astrocytoma cell lines. Cristal violeta stain in U373-MG (a) and D54-MG (b). GFAP protein stained histochemically in control (b, e) and NaB 8 mM treated for 24 h (c, f) in U373-MG cells (a–c) and D54-MG cells (d–f).

is more related with euchromatic gene silencing (31). This mechanism could operate in D54-MG and explain the noninduction of Bax and Bcl-2 genes post-NaB treatment, but it has to be proved. This fact is in agreement with previous reports that pointed out that PpIX interacts with wild-type p53 protein

in vitro and induces cell death of human colon cancer cells in a p53-dependent and -independent manner (27). The mortality of U373-MG cells was not as high as D54-MG because U373MG cells have p53mut, and they cannot respond to PDT until NaB arrests cells probably in G1 phase, the better gap to

Photochemistry and Photobiology, 2009, 85 apoptosis (26). This hypothesis has to be proved and whether apoptosis is caused in both astrocytoma cells exposed to NaB and treated by d-ALA and PDT remains to be determined. At least, NaB can induce several genes to perform apoptosis (32,33). This gene activation can be via the sensitization of cell death in both cell lines, but is more evident in U373-MG cells. The silencing of HDAC-4 led to radiosensitization and to the abrogation of the G2 DNA damage checkpoint. TSA abrogated radiation-induced G2-M arrest and increased apoptosis (25). In U373-MG cells, NaB can improve the cell death by PDT maybe by inhibition of histone deacetylase-4 (HDAC-4). This mechanism also has to be proved in both cells, mainly in U373-MG that could explain the improved response to PDT in the presence of NaB. However, in U373-MG cells the re-expression of Casp-3, Casp-9 and Bcl-2, and the increase in the expression of Bcl-2 and Bak-1 could be a mechanism operating in this astrocytoma cell line. There is another possibility of inducing cellular death via PpIX interaction with receptors of the mitochondria membrane (peripheral benzodiazepine receptor) and of inducing apoptosis after PDT treatment mainly by releasing cytochrome c from mitochondria (34,35). This could happen in U373-MG cells because they expressed Bak gene and it interacts with and accelerates the opening of the mitochondrial voltage-dependent anion channel, which leads to a loss in membrane potential and the release of cytochrome c (36). On the other hand, the differentiation induced by NaB is another way of inducing cell death, probably in a synergistic way. The results of this study indicate that intracellular accumulation of PpIX occurs in these two cell lines with exposure to d-ALA, mainly in U373-MG cells. This was also documented to occur in other cell lines, including HeLa, Calo and normal cervical cells (8), MCF-7 and H-MESO-1 (37), glioma, neuroblastoma and normal cerebellar granule cells (38), B16 melanoma (39) and human bladder cancer (40). All these results illustrate the potential of application of PDT in several kinds of cancer and the feasibility to improve its effectiveness by NaB treatment as in astrocytoma cells. In summary, the results presented here imply how PpIX produced by d-ALA induction and NaB would be expected to be beneficial as regards PDT. The results presented here may provide the basis for integrating such drugs as HDIs and NaB into anticancer PDT in astrocytoma cells. At present, we are studying the mechanisms involved in the cell death postexposition to NaB and PpIX induction, by PDT in astrocytoma cells. Acknowledgements—Financing for this study was provided by SIP and through projects 20070480 under the direction of Eva RamońGallegos. F.A.-H. is EDI and COFAA fellow. E.R.G. is SNI, COFAA and EDI fellow.

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