The combination of sulfinosine and 8-Cl-cAMP induces synergistic cell growth inhibition of the human neuroblastoma cell line in vitro

Key words: neuroblastoma, sulfinosine, 8-Cl-cAMP, combination, cell cycle arrest, apoptosis

Summary

To identify purine analogs that could be effective in treating neuroblastomas, we tested the anticancer properties of sulfi- nosine, 8-Cl-cAMP and 8-Cl-adenosine in the SK-N-SH cell line. First we examined the effects of these three agents on cell growth inhibition and cell viability by the BrdU and Sulforhodamine B assay. Treatment of SK-N-SH cells with increasing concentrations of these compounds led to a significant inhibition of cell proliferation and decrease of cell vi- ability in a time- and dose-dependant manner at micromolar concentration (<10 µM). Treatment with a combination of sulfinosine and 8-Cl-cAMP resulted in synergistic effects on growth inhibition, cell cycle arrest and induction of apop- tosis. Flow-cytometric analysis showed that 8-Cl-cAMP arrested the cells in the G0/G1 phase and sulfinosine blocked cell cycle progression at the G2/M stage, in contrast to the combined effects of both agents that did not arrest growth at any particular phase of the cell cycle. Further analysis of apoptosis induction demonstrated an increase from 17 to 24% of both early and late apoptotic cells and a very low percentage of necrotic cells. These results indicate that apop- tosis was the predominant type of cell death after treatment of SK-N-SH cells with both substances, as well as with their combinations.

Introduction

Neuroblastomas are neuro-ectodermal tumors of embryonic neural-crest-derived cells [1]. They represent a complex and diverse group of tumors of early childhood whose clinical behavior is reflected by multiple alterations found in differ- ent clinical subgroups [2]. However, improved identification of patients at risk has not led to novel therapeutic strategies or improved survival rates. While treatment of low- and intermediate-risk patients has mostly been satisfactory, it has not been the case with the high-risk group [3]. Thus, the development of new chemotherapeutic agents and the introduction of new combinations of regimes for treating neuroblastomas are desirable.

In the present study we investigated the antitumor effects of three purine analogs, 8-Cl-cAMP, 8-Cl-adenosine (8-Cl- AD) and sulfinosine on the human neuroblastoma cell line. 8-Cl-cAMP is a site-specific cAMP analogue that selec- tively down-regulates PKA I, a signaling protein directly involved in various cellular functions, including cell proliferation, differentiation and neoplastic transformation, and mediation of the mitogenic effects of different oncogenes and growth factors [4]. There are two types of PKA, PKA- I and PKA-II. Both share a common catalytic subunit but contain different R subunits which are referred to as RI and RII respectively [5]. RI/PKA-I is preferentially ex- pressed in transformed cells or during the early stages of oncogenesis, whereas the expression of RII/PKA-II is in- duced in growth-arrested cancer cells after treatment with cAMP analogues or differentiating agents [6, 7]. Thus, the positive and negative signals that are transduced by cAMP may depend on the availability of RI and RII subunits re- spectively. A site-selective cAMP analogue, 8-Cl-cAMP, down-regulates RI/PKA-I and up-regulates RII/PKA-II in several cancer cell lines [6]. 8-Cl-cAMP inhibits the growth of a wide variety of cancer cell types in vitro and in vivo [8–11], and represents the first cAMP analogue to enter clinical trials in over 30 years of research in this field [12]. The actions of cAMP are well known in the regulation of activation of cAMP-dependent PKA [13]. In several cell lines the effect of 8-Cl-cAMP cannot be explained by its action as a cAMP analogue. Data suggests that serum phos- phodiesterase (PDE) and 5r-nucleotidase activity converts 8-Cl-cAMP into 8-Cl-adenosine in the cell culture medium. This product could act as a conventional antimetabolite that inhibits DNA and/or RNA polymerases [14–16].

Sulfinosine or SF (2-amino-9-beta-D-ribofuranosylp urine-6-sulfina mide) is a relatively unexplored anti- neoplastic agent which is active against six solid tumors and four strains of experimental leukemia [17, 18]. Sulfino- sine has considerable antitumor activity against non-small cell lung carcinoma (NSCLC) and small cell lung carci- noma (SCLC) cell lines. Apparently it inhibits cell growth and induces apoptosis in vitro [19]. Although a derivative of 6-thioguanosine, SF penetrates the central nervous sys- tem more readily than 6-thioguanosine and is more effec- tive in the treatment of L1210 leukemia, being curative for some mice [18]. Sulfinosine was observed to be more effective against experimental leukemias and tumors resis- tant to 6-mercaptopurine, 6-thioguanosine than other clin- ically used chemotherapeutic agents [18]. The metabolism of sulfonamide derivatives involves the cells’ glutathione system. The observation that SF readily forms adducts with sulfhydryl compounds (glutathione and cysteine) may be the reason why SF lowers glutathione levels and indirectly induces death of tumor cells [18]. These findings stimulated the investigation of the antitumor potential of SF on human neuroblastomas.

In cancer therapy combinations of two or more drugs and/or adjustments of the dosage and administration sched- ule can improve the efficacy of treatment [20]. Combination studies were necessary to compare the anticancer potency of a single agent to the potency of a combination of different agents, and whether the latter simultaneously induces cell growth inhibition and cell death. It has been demonstrated that 8-Cl-cAMP at a dose causing mild or no growth inhibi- tion, synergistically increased the growth-inhibitory effects of either paclitaxel or cisplatin in a number of cell lines, including human breast, lung, ovarian, colon and head car- cinomas and melanoma. A similar effect was also observed with docetaxel and carboplatin [20]. Also, the low doses of 8-Cl-cAMP acted synergistically with 9-cis-RA, 13-cis- RA and all-trans-RA in inhibiting the viability of Ewing’s sarcoma CHP-100 cells [4].

The induction of apoptosis and inhibition of cell prolifer- ation by purine analogs is the focus of our research [11, 19, 21, 22]. Little is known about the antitumor effects of SF and 8-Cl-AD in human neuroblastoma cell lines. With regard to 8-Cl-cAMP, only a few studies have described its dual ef- fect on the inhibition of cell proliferation and induction of apoptosis in SH-SY5Y cells [23–25]. In the present work we chose human SK-N-SH cells that have previously been used as a target cell line in cell mediated cytotoxicity assays. In order to elucidate the antiproliferative and cytotoxic ac- tivities of SF, 8-Cl-cAMP and 8-Cl-AD, we first examined the antitumor effects of these three agents on human SK-N- SH cells. Subsequently we investigated whether sulfinosine together with 8-Cl-cAMP, exert a cooperative inhibitory ef- fect on cell growth, induce apoptosis and promote cell cycle arrest. Our results suggest that combinations of sulfinosine with 8-Cl-cAMP are more effective than either compound alone.

Materials and methods

Drugs. Sulfinosine ([R,S]-2-9H-b-D-ribofuranosylpur ine- 6-sulfinamide) was synthesized from 6-thioguanosine ac- cording to the published procedure [17] (Figure 1A). 8-Cl-cAMP (8-Chloroadenosine 3r5r-cyclic phosphate) was directly synthesized by chlorination of adenosine 3r5r-cyclic phosphate as described [26] (Figure 1B). 8-Cloro-adenosine (8-Cl-AD) was purchased from the BI- OLOG Life Science Institute, Bremen, Germany (Fig. 1C). Sulfinosine (SF) was freshly diluted in water and filter- sterilized every time prior to treatment, while aliquots of 8-Cl-cAMP and 8-Cl-AD were thawed from 20◦C each time prior to treatment.

Chemicals. Eagle Minimum Essential Medium, propidium iodide and a penicillin/streptomycin solution were purchased from Sigma (St. Luis, MO). Trypsin was obtained from Amersham International Ltd. (Buckinghamshire, United Kingdom) and prepared as a 0.25% Trypsin-EDTA solution. Fetal bovine serum was purchased from Invitro- gen Life Technologies. Cell Proliferation ELISA, BrdU and Annexin-V-Fluos were obtained from Roche Applied Sci- ence and Sulforhodamine B was obtained from Sigma (St. Luis, MO).

Cells and cell culture. Human SK-N-SH cell line (HTB 11) was purchased from American Type Culture Collection (Rockville, MD). SK-N-SH cells were maintained in Eagle Minimum Essential Medium supplemented with 10% FBS, L-glutamine (2 mM) and (10 000 units/ml) penicillin and (10 mg/ml) streptomycin solution at 37◦C in a humidified 5% CO2 atmosphere. The cells were subcultured at 72 h intervals using 0.25% trypsin/EDTA and seeded into fresh medium at a density of 13 000 cells/cm2.

Cell proliferation by BrdU labeling. Cells grown in 75 cm2 tissue flasks were trypsinised, seeded into flat-bottom 96- well tissue culture plates (8 000 cells/well) and incubated overnight. The cells were then treated with SF (0.5, 1, 2.5, 5, 10 and 25 µM), 8-Cl-cAMP (0.5, 1, 2.5, 5, 10 and 25
µM) and 8-Cl-AD (0.5, 1, 2.5, 5, 10 and 25 µM) for 72 h. Untreated control and treated cells were incubated with the thymidine analog bromodeoxy-uridine (BrdU) 4 h prior to the end of the incubation period. After fixation the cells were processed according to the manufacturer’s protocol (Roche Applied Science). The relative incorporation of BrdU was determined by absorbance reading at 450 nm, with cor- rection at 670 nm (LKB 5060–006 Micro Plate Reader, Austria).

Chemosensitivity by the sulforhodamine B (SRB) assay. Briefly, the cells in 96-well plates were fixed in 50% trichlo- racetic acid (TCA) (50 µl/well) for 1h at 4◦C, rinsed in tap- water and stained with 0.4% (w/v) Sulforhodamine B in 1% acetic acid (100µ rl/well) for 30 min at room temperature. The cells were then rinsed 3 times with 1% acetic acid to re- move unbound stain. The protein-bound stain was extracted using 200 µl 10 mM Tris-base (pH 10.5) per well. The optical density was read at 540 nm, with correction at 670 nm (LKB 5060–006 Micro Plate Reader, Austria). The re- sults were expressed as % growth inhibition (I) determined according to the following equitation: IC50 values were determined by the Forecast function using Microsoft Excel.

Flow cytometry. Unsynchronized cells were plated at a den- sity of 200 000 cells/ 100 mm dish and incubated for 72 h with SF, 8-Cl-cAMP and their combinations. After 72 h the attached and floating cells were trypsinized and collected by centrifugation at 1 000 g for 5 min and fixed in 70% ethanol for 24–72h at 20 C. After fixation the cells were resuspended in 0.5 ml phosphate buffered saline (PBS) and counted using a hemocytometer. The cells were pretreated with 50 µg/ml RNAse A at 37◦C for 30 min and incubated with 50 µg/ml propidium iodide at 4◦C for at least 1 h prior to flow cytometric analysis. Flow cytometric analysis was performed on the FACSCalibur flow cytometer (Bec- ton Dicinson, San Jose, CA, USA) and a minimum of 10 000 events were collected for each experiment sample. Cell cycle distribution was determined automatically in Mod- FIT (Verity Softwasre House, Inc). The validity of the data analysis model was verified using the R.C.S. value (reduced Chi-square, R.C.S. < 15%).

Cell death detection. The percentages of apoptotic, necrotic and viable cells were determined by flow cytometric anal- ysis (FACSCalibur flow cytometer, Becton Dicinson, San Jose, CA, USA) using Annexin-V-Fluos and propidium io- dide. Unsynchronized SK-N-SK cells were plated at a den- sity of 200 000 cells/100mm dish and incubated for 72 h with SF, 8-Cl-cAMP and their combinations. After 72 h, the attached and floating cells were trypsinized and collected by centrifugation at 1 000 g for 5 min. The cell pellet was re- suspended in 100 µM incubation buffer containing 10 mM HEPES/NaOH, 140 mM NaCl, 5 mM CaCl2, supplemented with Annexin-V-Fluos. After the incubation period (10–20 min at 15–25◦C), additional 400 µl of incubation buffer was added. Propidium iodide (final concentration 1 µg/ml) was added to the cells 5 min prior to FACS analysis.

Statistical analysis. The determination of a significant dif- ference between data sets was performed using STATIS- TICA 6.0 software. Values for inhibitory concentrations obtained by the BrdU and Sulforhodamine B assay, were tested by one-way analysis of variance (ANOVA). If a sta- tistical significance was found, the Tukey honest significant difference (HSD) test was used to determine which groups differed from the control. Statistical significance was ac- cepted if p < 0.1 (∗), p < 0.05 (∗∗) and pp < 0.005 (∗∗∗).

Median effect analysis. The nature of the interaction ob- served between SF and 8-Cl-cAMP was analyzed using the Calcusyn software which uses the combination index (CI) method of Chou and Talalay [27, 28] based on the mul- tiple drug effect equation. This analysis requires: (a) that each drug alone has a dose-effect relationship, and (b) that at least three or more data points for each single drug are available in each experiment. The non-constant ratio combi- nation design was chosen to assess the effect of both drugs in combination. The advantage of this method is the au- tomatic construction of a fraction affected-CI table, graph and classic isobologram by software. A value of CI < 1 indicates a more pronounced additive effect (synergism: the smaller the value, the greater the degree of synergy); CI 1 indicates an additive and CI > 1 an antagonistic effect.Each CI ratio represented here is the mean value derived from two separate experiments.

Results

The effects of sulfinosine, 8-Cl-cAMP and 8-Cl-Adenosine on cell proliferation and viability in SK-N-SH cells

Cell proliferation was evaluated by BrdU incorporation af- ter treatment for 72 h with either SF or 8-Cl-cAMP or 8- Cl-AD at six different concentrations (0.5, 1, 2.5, 5, 10, 25 µM). At all of the examined concentrations the in- hibition of cell growth was significant compared to the control cells. The obtained results (Figure 2A) indicate a dose-dependent increase in the inhibition of cell pro- liferation. The effects of SF, 8-Cl-cAMP and 8-Cl-AD on the proliferation of SK-N-SH cells were compared by calculating the inhibitory concentrations (IC50) that were obtained from the BrdU test 72 h after treatment. The IC50 values for SF, 8-Cl-cAMP and 8-Cl-AD were 7.2 µM, 2.5 µM and 2.5 µM respectively.

A sulforhodamine B (SRB) assay was used to evaluate the viability of neuroblastoma cells 72 h after incubation with SF, 8-Cl-cAMP or 8-Cl-AD. The viability index represents the viability of treated cells compared to the viability of untreated cells. As the cells were treated with increasing concentrations of SF, 8-Cl-cAMP or 8-Cl-AD (0.5, 1, 2.5,5 and 10 µM), the number of viable cells decreased in a dose-dependent manner (Figure 2B). The IC50 values were 9 µM, 7.5 µM and 21.3 µM for SF, 8-Cl-cAMP and 8-Cl- AD respectively. At all of the concentrations the reduction in the number of viable cells was significant compared to the control cells. The above experiment implies the existence of different IC50 values for 8-Cl-cAMP (7.5 µM) and 8-Cl- AD (21.3 µM) and suggests that SK-N-SH cells were more sensitive to 8-Cl-cAMP, especially at lower concentrations.

The combined effects of Sulfinosine and 8-Cl-cAMP

As both SF and 8-Cl-cAMP were effective in inhibiting cell proliferation, we next examined the interactive effects of 8-Cl-cAMP and SF on growth inhibition. To that end, SK-N-SH cells were simultaneously treated with 2.5 µM, 5 µM and 7.2 µM SF and 0.5, 1, 2.5, 5 or 10 µM 8-Cl-cAMP (Figure 3 A, B and C). SF at the 2.5 µM concentration induced cell growth inhibition for 26%, while 1 µM 8-Cl- cAMP inhibited cell growth for 15%. Their combination
led to a 41% growth inhibition. This result clearly shows that the effect was more pronounced when the substances were applied together rather than separately. A similar re- sult was obtained by combining 2.5 µM SF and 2.5 µM 8-Cl-cAMP. This combination inhibited cell proliferation for 75% (Figure 3B). When higher concentrations of 8-Cl- cAMP (5 or 10 µM) were applied together with 2.5 µM SF their combinations exerted a similar effect as when 8- Cl-cAMP was given alone. However, the combination of 5 µM SF (inhibits proliferation by 33%) and 1 %M 8-Cl- cAMP exerted 50% growth inhibition (Figure 2B). Simi- larly, the simultaneous treatment of cells with 5 µM SF and 2.5 µM 8-Cl-cAMP resulted in a 76% growth inhibition and thus exerted a more pronounced effect than treatments with each substance by itself. Finally, the effects of simultane- ous treatments of neuroblastoma cells with 7.2 µM SF (the IC50 value of sulfinosine) and with increasing concentra- tions of 8-Cl-cAMP demonstrated that 8-Cl-cAMP induces a greater growth-inhibitory effect than SF on SK-N-SH cells in a dose-dependent manner (Figure 3C).

The interaction between SF and 8-Cl-cAMP on the de- crease of cell viability was also observed. As in previous experiments 2.5 µM SF (which decreases the cell viability index to 0.8) was combined with 0.5, 1, 2.5, 5 and 10 µM 8- Cl-cAMP (Figure 4A). The combination of 2.5 µM SF and
2.5 µM 8-Cl-cAMP decreased cell viability to 0.4. This ef- fect of the combination of compounds was more pronounced than when the substances were applied separately (Figure 4A). At higher concentrations 8-Cl-cAMP in combination with 2.5 µM SF yielded similar results as the previous com- bination, decreasing the viability to 0.4. The combination of 5 µM SF (which decreased the cell viability index to 0.7) with increasing concentrations of 8-Cl-cAMP also exhib- ited a cooperative effect and decreased the viability index to 0.5–0.4 (Figure 4B). Similar results were observed when 7.2 µM SF was combined with different concentrations of 8- Cl-cAMP. The viability index (0.4–0.3) pointed to a gradual decrease of cell viability by these combinations (Figure 4C).

Analysis of the effects of a simultaneous exposure to Sulfinosine and 8-Cl-cAMP

The results of the BrdU and SRB tests were subjected to computerized synergism/antagonism Calcusyn software analysis [18, 19]. As shown in Table 1, synergistic growth inhibition (CI < 1) was observed at all of the examined com- binations of 2.5 µM SF and 8-Cl-cAMP, except at the combinations of 0.5 or 1 µM 8-Cl-cAMP and 2.5 µM SF, which exerted an antagonistic effect (CI > 1). As shown in Table 1, synergistic growth inhibition was observed when 2.5 µM SF was combined with 2.5 µM or 5 µM 8-Cl-cAMP. The other combinations of 2.5 µM SF and 8-Cl-cAMP exerted either moderate synergistic or antagonistic effects. The com- binations of 5 µM SF and 2.5 µM and 5 µM 8-Cl-cAMP synergistically inhibited cell proliferation (CI<1), while the combinations of 0.5, 1 or 10 µM 8-Cl-cAMP and 5 µM SF were either antagonistic or slightly synergistic (CI > 1). Except for 0.5 µM 8-Cl-cAMP, all other concentrations of 8-Cl-cAMP and 7.2 µM SF synergistically inhibited cell growth.

The results of the viability test when subjected to syner- gism/antagonism analysis pointed to a moderate synergistic effect of the combination of 2.5 µM SF and 0.5 µM 8- Cl-cAMP whereas other combinations induced strong syn- ergistic effects. A variable degree of synergism was also observed when 5 µM SF was combined with increasing concentrations of 8-Cl-cAMP (CI < 1). The combination index for all of the investigated combinations of 7.2 µM SF and 8-Cl-cAMP indicated a strong synergistic effect on the decrease of cell viability.

Treatment with sulfinosine, together with 8-Cl-cAMP induces cell cycle arrest in SK-N-SH cells

Since detailed studies of the effects of SF and 8-Cl-cAMP on neuroblastoma cell cycle kinetics are lacking, one of the aims of this study was to examine whether the growth in- hibitory effect of these substances could be the result of a disturbance of cell-cycle kinetics. Flow cytometric analysis of cell cycle distribution of SK-N-SH cells treated either with SF (2.5, 5, 7.2 and 25 µM) or 8-Cl-cAMP (2.5, 5, 10 and 25 µM) and their combinations was performed (Figure 5). 8-Cl-cAMP induced the accumulation of cells in G0/G1 in a dose-dependent manner (Figure 5A). The number of cells arrested in the G0/G1 phase of the cell cycle was 84– 86% after treatment with 8-Cl-cAMP. This was more than in untreated cells where 70.45% of cells accumulated in G0/G1 (Table 2). At the same time, a significant decrease of the percentage of treated cells in S and G2/M phases was observed compared to untreated cells (Table 2). Treatment with SF arrested the SK-N-SH cells preferentially in the G2/M phase of the cell cycle (Figure 5B). As shown in Table 2, the SK-N-SH cell line exhibited a significant G2/M arrest (21–28%) after exposure to increasing concentrations of SF compared to the control cells (18.48% of cells in G2/M). Cytofluoromeric analysis of the effects of combinations of SF and 8-Cl-cAMP demonstrated a significant cell cycle disturbance compared to the controls (Figure 5C). Interest- ingly, the combined treatment of SF and 8-Cl-cAMP did not induce growth arrest at any particular phase of the cell cycle (Table 3). As shown in Figure 5C, cell cycle profiles indi- cate a significantly lower number of analyzed cells, probably due to a decreased number of viable cells as a result of the synergistic effect of SF and 8-Cl-cAMP on cell viability.

Induction of apoptosis in SK-N-SH cell

The potent growth inhibition and reduction in the number of viable cells after treatment with SF and 8-Cl-cAMP sug- gested that these substances induced cell death. To elucidate which type of cell death was predominant in SK-N-SH cells, 72 h after exposure to SF, 8-Cl-cAMP and their combina- tions, Annexin-V-fluos/propidium iodide staining was used. In this procedure the viable cells are negative for both dyes, apoptotic cells only bind annexin-V and necrotic cells are stained by annexin-V and propidium iodide. The results of this analysis showed that when compared to untreated cells, the number of viable cells decreased after treatments either with each substance alone or with their combinations (Table 4). At the same time, the percentage of necrotic cells also decreased from 6.12% in untreated cells to 2–5% and 3–4% after the treatment with different concentrations of SF and 8-Cl-cAMP respectively. SK-N-SH cells treated with SF and 8-Cl-cAMP exhibited an elevated percentage of early and late apoptotic cells when compared to the control. The number of apoptotic cells was dose-dependent. The increase in the percentage of apoptotic cells was even more signifi- cant after the simultaneous treatment of cells with different combinations of SF and 8-Cl-cAMP. The combination of 7.2 µM SF and 2.5, 5 and 10 µM 8-Cl-cAMP yielded 17, 21 and 24% of both early and late apoptotic cells respectively. These results indicate that apoptosis was the predominant type of cell death after the treatment of SK-N-SH cells with both substances, as well as with their combinations.

Discussion

The aim of this study was to investigate the antitumor ef- fects of three purine analogs, sulfinosine, 8-Cl-cAMP and 8-Cl-adenosine that have different modes of action on the human neuroblastoma SK-N-SH cell line. Another goal of this study was to investigate whether sulfinosine and 8-Cl- cAMP exert cooperative antitumor effects on the SK-N-SH cell line.

Numerous studies suggest that 8-Cl-cAMP, a potent ana- log of cAMP, induces growth inhibition in vitro and in vivo in a broad spectrum of human carcinomas, including neu- roblastoma cell lines [23–25, 29]. Although 8-Cl-cAMP has been used in clinical trials the mechanism of its ac- tion remains controversial. One potential mode of action is through modulation of protein kinase A (PKA) activity. Ac- cording to another suggested mechanism the conversion of 8-Cl-cAMP by serum enzymes to 8-Cl-AD occurs. In this study we examined the antitumor properties of both analogs together with sulfinosine in order to compare their antipro- liferative and cytotoxic effects on human SK-N-SH cells. We found that 72 h after treatment with 8-Cl-cAMP and 8- Cl-AD the proliferation of SK-N-SH cells was inhibited in a concentration-dependent manner. At a 2.5 µM concentra- tion both 8-Cl-cAMP and 8-Cl-AD induced 50% growth in- hibition (IC50) in SK-N-SH cells. However, neuroblastoma cells showed a different sensitivity to 8-Cl-cAMP and 8-Cl- AD according to the SRB test. With regard to the inhibitory concentrations, 8-Cl-AD had an almost 3-fold higher IC50 (21.3 µM) compared to 8-Cl-cAMP (7.2 µM). These results suggest that the concentrations at which growth inhibition occurs for 8-Cl-cAMP (2.5 µM) and 8-Cl-AD (2.5 µM) are lower than their respective IC50 values (7.2 and 21.3 µM, re- spectively), as obtained by the SRB test. This findings point to their antiprolifertive effects in the neuroblastoma cell line.

It is still not clear whether 8-Cl-cAMP exerts its effect “directly” or, at least in part as a prodrug for 8-Cl-AD. Several studies have shown that 8-Cl-cAMP exerts dual anticancer activity on human SH-SY5Y cells through the inhibition of cell proliferation and the induction of apop- tosis [23, 24]. A study was carried out in order to clarify the overall cellular effects of 8-Cl-cAMP and 8-Cl-AD on SK-NDZ human neuroblastoma cells in a gene expression study using a cDNA microarray [25]. The obtained results indicated that the two drugs shared common gene expres- sion down-regulation pathways, but used distinct pathways for the up-regulation of different gene clusters. Based on these findings it was suggested that the antitumor activity of 8-Cl-cAMP resulted at least in part through 8-Cl-adenosine [15]. The pharmacokinetics of 8-Cl-cAMP was examined in patients with different solid tumors [20, 30]. During the Phase I trial it was reported that the effective plasma con- centrations of 8-Cl-cAMP was 2–5 µM. This concentration was not toxic and was shown to be effective in studies against human cancer cell lines in vitro. Unfortunately, the protocol of this study did not consider potential formation of 8-Cl-AD in the plasma and tumors of 17 patients [30].

The antitumor potential of sulfinosine, a “sulfonamide-type” compound, was for the first time investigated with respect to cell growth, apoptosis and cell cycle progression on neuroblastomas. Sulfinosine has not been sufficiently examined as an antineoplastic agent. Its antitumor activ- ity was previously described in human lung carcinoma cell lines where the concentration of SF used (<10 µM) was sufficient to reduce cell growth and viability to 50% com- pared to untreated cells [19]. Our results confirmed that SF is also a powerful inhibitor of SK-N-SH cell growth and cell viability and that it acts in a dose-dependent manner. In this study we found that sulfinosine inhibited proliferation and cell viability at IC50 values of 7.2 and 9 µM, respectively. Considering that sulfinosine was significantly efficacious in the neuroblastoma cell line, the purpose of the study was to assess the cytotoxic interaction between 8-Cl-cAMP and sulfinosine and to define a potential role of this combination for future clinical trials. Therefore, the most important result of this study is the observation that 8-Cl-cAMP enhances the growth inhibitory effect of sulfinosine on SK-N-SH cells in a dose-dependent manner. Comparison of growth inhibition by sulfionosine and 8-Cl-cAMP alone versus a combina- tion of the two agents revealed a substantial enhancement of growth inhibition at clinically relevant concentrations. Based on the present study it is clear that these drugs act synergistically and have a potential for combined use in chemotherapy. The data presented in this study also point to a synergistic effect of sulfinosine and 8-Cl-cAMP on the viability of SK-N-SH cells. Of great importance is the ob- servation that even at a concentration of sulfinosine as low as 2.5 µM (which is three times lower than its IC50 value), in combination with increasing concentrations of 8-Cl-cAMP, synergistically decreases cell viability. The data presented here revealed a highly synergistic effect of 8-Cl-cAMP and sulfinosine after their simultaneous administration to neuroblastomas.

Our results are in good correlation with another published study in which 8-Cl-cAMP was used together with standard chemotherapeutic agents such as paclitaxel, docetaxel, cisplatin and carboplatin. This work pointed to new approaches for the treatment of malignant dis- eases. Combinations of these compounds could improve the eradication rates and minimize undesireable side ef- fects [4, 20]. 8-Cl-cAMP is currently under investiga- tion as a single chemotherapeutic agent in clinical Phase II trials. It exhibts growth inhibition at micromolar con- centrations in patients and in various cell lines in vitro [12].

In order to obtain insight into the mechanism by which sulfinosine and 8-Cl-cAMP exert their growth inhibitory ef- fects, we studied the effects of their combinations and of sin- gle agent-treatments on the cell-cycle kinetics and apoptosis induction. Cell-cycle distribution analysis revealed that the growth inhibitory effects of 8-Cl-cAMP on SK-N-SH cells involved G0/G1 cell cycle arrest and induction of apopto- sis, as judged by a marked shift of the cells in a sub-G0/G1 fraction (from 70% in the control to 85% in G0/G1; Table 2). In contrast, the results of the experiments using flow- cytometric analysis demonstrated that SK-N-SH cells were persistently arrested in G2/M after exposure to gradually increasing concentrations of sulfinosine. These results sug- gest that some G2/M events seem to be important and nec- essary for apoptosis induction with sulfinosine. The combi- nation of sulfinosine and 8-Cl-cAMP promoted a synergis- tic cytotoxicity but also induced changes in the cell cycle of SK-N-SH cells and significantly increased the number of apoptotic cells. These changes were detected by flow- cytometric analysis of SK-N-SH cells which discriminates between and quantifies viable, early and late apoptotic and necrotic cells based on their size and fluorescent staining by annexin and propidium iodide. The simultaneous treat- ment of SK-N-SH cells with a combination of 8-Cl-cAMP and sulfinosine resulted in synergistic cytotoxicity with in- creased apoptotic activity. The results of our experiments demonstrated that the combination of compounds induced apoptosis at a very high rate, between 17–24%, with a very low percentage of necrotic cells. In addition, apoptotic cell death induced by 8-Cl-cAMP and sulfinosine after a single treatment was detected as well. The molecular mechanism responsible for the described cooperative effects of 8-Cl- cAMP and sulfinosine could be explained by the previous studies on the mechanism of action of both compounds. The 8-Cl-cAMP-induced apoptosis demonstrated in our study could be due to the ability of the agent to mediate its ac- tivity through PKA. 8-Cl-cAMP has also been reported to act through the conversion to its 8-Cl-AD metabolite in several systems, including mouse epithelial cell lines, hu- man glioma, lung, etc. [16]. Nevertheless, the exact mechanism of 8-Cl-cAMP and 8-Cl-AD-mediated apoptosis in neuroblastomas needs to be clarified. Our study established that sulfinosine possessed considerable antitumor activity in SK-N-SH cells. The metabolism of sulfonamide derivatives involves the glutathione system in tumor cells. The observa- tion that sulfinosine readily forms adducts with sulfhydryl compounds (glutathione and cysteine) could explain why sulfinosine lowers glutathione levels and indirectly induces apoptosis in tumor cells.

To summarize, our studies suggest that sulfinosine, 8- Cl-cAMP and 8-Cl-AD are effective antineoplastic agents in SK-N-SH cells. Simultaneous treatment with 8-Cl-cAMP and sulfinosine results in cytotoxic activities that induce cell growth inhibition, cell cycle arrest and apoptosis. Further studies of the role of these purine analogs in the induction 8-Bromo-cAMP of apoptosis and signaling molecules could provide the basis for neuroblastoma therapy.