Synthesis and Different Effects of Biotinylated PAMAM G3 Dendrimer Substituted with Nimesulide in Human Normal Fibroblasts and Squamous Carcinoma Cells

Squamous cell carcinoma (SCC) remains a main cause of mortality in patients with neck and head cancers, with poor prognosis and increased prevalence despite of available therapies. Recent studies have identified a role of cyclooxygenases, particularly inducible isoform cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2 (PGE2) in cancer cell proliferation, and its inhibition become a target for control of cancer development, particularly in the view of recognized additive or synergic action of COX-2 inhibitors with other forms of therapy. Nimesulide (N), the selective COX-2 inhibitor, inhibits growth and proliferation of various types of cancer cells by COX-2 dependent and independent mechanisms. In the presented study, the conjugates of biotinylated third generation poly(amidoamine) dendrimer (PAMAM) with covalently linked 18 (G3B18N) and 31 (G3B31N) nimesulide residues were synthesized and characterized by NMR spectroscopy. Biological properties of conjugates were evaluated, including cytotoxicity, proliferation, and caspase 3/7 activities in relation to COX-2/PGE2 axis signaling in human normal fibroblast (BJ) and squamous cell carcinoma (SCC-15). Both conjugates exerted a selective cytotoxicity against SCC-15 as compared with BJ cells at low 1.25–10 µM concentration range and their action in cancer cells was over 250-fold stronger than nimesulide alone. Conjugates overcome apoptosis resistance and sensitized SCC-15 cells to the apoptotic death independently of COX-2/PGE2 axis. In normal human fibroblasts the same concentrations of G3B31N conjugate were less effective in inhibition of proliferation and induction of apoptosis, as measured by caspase 3/7 activity in a manner depending on increase of PGE2 production by either COX-1/COX-2.


Introduction
Squamous cell carcinoma (SCC-15) is a malignant tumor of squamous epithelium that may occur in many different organs, including skin. Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity, accounting for over 90% of the malignant neoplasms in this amide-bonded biotin by reaction of G3 with N-hydroxysuccinimido-biotin (NHS-biotin) as before to give monosubstituted G3 B [50].
The 21 mM solution of G3 B in dimethylsulfoxide (dmso) was further used to obtain conjugates with nimesulide (N). N was covalently attached to G3 B as follows: 0.4769 g of N (1.55 mmoles) was dissolved in 6.5 mL of chloroform and 300 µL of triethylamine was added. To this solution 0.3429 g (1.70 mmoles) of p-nitrophenylchloroformate (NPCF) was added in portions as solid with vigorous stirring at room temperature for 4 h. The product of substitution was purified by extraction in chloroform -water system, isolated from chloroform phase and purified chromatographically on silicagel with chloroform:ethyl acetate 7:1 v/v. The product was eluted as first fraction and identified by the 1 H NMR spectroscopy (see Scheme 3 for atom numbering). 1  Next, to a 1 mL of G3 B in dmso (21 µmoles) the solution of activated N was added 3.00 mL (synthesis 1, containing 684 µmoles of activated N) or 1.85 mL (synthesis 2; 410 µmoles of activated N). The mixtures were kept for 12 h, and then the solutions were dialyzed using nitrocellulose dialysis tube against water for four days. Solutions with precipitate were separated from dialysis tube, then solvents were removed under reduced pressure with rotary evaporator at temperature <50 • C and dried overnight at high vacuum (3 mm Hg).
The products were analyzed by 1 H NMR spectroscopy in dmso-d 6 . For comparison, the 1 H NMR spectrum of N in dmso was recorded and chemical shifts of 1 H resonances assigned in accordance with literature data [51].
Biomolecules 2019, 9, 437 5 of 20 All assays were performed as triplicates of three independent experiments. Cell morphology was observed under Nikon TE2000S Inverted Microscope (Tokyo, Japan) with phase contrast after treatment.

Cytotoxicity
Cells were seeded in flat-bottom 96-well plates at a density of 1 × 10 4 cells/well (BJ) or 2 × 10 4 (SCC-15) and allowed to attach for 24 h. Working solutions of dendrimers (0.625-10 µM) or N (25-800 µM) were prepared in culture media. The DMSO concentration were 0.4% (dendrimer conjugates) or 0.2% (N), that had no significant effect on treated cell lines. After 24 h exposure to dendrimers or N alone, the neutral red (NR) assay was performed as described [52].

Proliferation Assay
Cells were seeded in flat-bottom 96-well plates at a density of 5 × 10 3 cells/well (BJ) or 1 × 10 4 (SCC-15) and, after 24 h, treated with either dendrimer conjugate at 0.625-5 µM concentration range for 96 h. After removing the medium, cells were washed with PBS and fixed with 3.7% formaldehyde in PBS for 10 min. at room temperature (RT). Subsequently, nuclear DNA was labeled with 600 nM DAPI (30 min., RT, in darkness) and fluorescence was measured at 360/460 nm with Infinite M200 PRO Multimode Microplate Reader (TECAN Group Ltd., Switzerland). Results are triplicates of three independent experiments.

Western Blot
Western blot analysis of the COX-2 protein level in cell lysates were prepared as described [53].

Statistical Analysis
To estimate differences between G3 B18N , G3 B31N , PAMAM G3 and N treated and non-treated samples the statistical analysis was performed using non-parametric Kruskal-Wallis test or Mann-Whitney U test to estimate differences between G3 B18N and G3 B31N in appropriate concentrations for the same cell line. p < 0.05 was considered as statistically significant. Calculations were performed using Statistica PL 12.5 version software (StatSoft).

Bioconjugate Synthesis
p-Nitrophenylchloroformate (NPCF), the convenient reagent to provide one-atom linkers has been used to couple PEG with anticancer drug hydroxycamptothecin (HPTC) by formation of active carbonate with hydroxyl groups of PEG or HPTC [55] and conjugate PEG with PAMAM G5 and G6 dendrimers to construct effective non-viral gene delivery carriers [56]. Similarly, the hydroxyl group of block copolymer (BE) obtained from 1,2-butylene oxide and ethylene oxide were activated with NPCF to give NP-carbonate-BE [57]. The p-nitrophenyl substituent of the latter was then leaving group when reacted with amine group-ended PAMAM dendrimer to give linear-dendritic copolymer linked by amide.
Nimesulide (N) was demonstrated to react also with acyl chlorides, like acetate-or benzoil chlorides at ambient temperature, to obtain corresponding N-acetylated sulfonamide derivatives within couple of hours with > 70% yield [58]. We have applied NPCF to activate N by formation of N-(p-nitrophenylcarbonate)nimesulide, which was further used to conjugate N into PAMAM G3 dendrimer via carbonyl linker (Scheme 1).
Biomolecules 2019, 9, x FOR PEER REVIEW 6 of 20 NPCF to give NP-carbonate-BE [57]. The p-nitrophenyl substituent of the latter was then leaving group when reacted with amine group-ended PAMAM dendrimer to give linear-dendritic copolymer linked by amide. Nimesulide (N) was demonstrated to react also with acyl chlorides, like acetate-or benzoil chlorides at ambient temperature, to obtain corresponding N-acetylated sulfonamide derivatives within couple of hours with > 70% yield [58]. We have applied NPCF to activate N by formation of N-(p-nitrophenylcarbonate)nimesulide, which was further used to conjugate N into PAMAM G3 dendrimer via carbonyl linker (Scheme 1). The conjugates were evaluated by 1 H NMR spectroscopy. Extensive dialysis of dimethylsulfoxide solutions of conjugates with water enabled to remove low molecular side products, like p-nitrophenol which was released at the final stage of synthesis. The products were better soluble in DMSO than in water. Based upon integral intensity of 1 H resonances in the spectra of nimesulide containing conjugates the average number of 18 and 31 moieties of N, and an average of biotin was present in G3 B18N and G3 B31N , respectively, as determined by 1 H NMR. The NMR spectra of N and G3 B18N , and G3 B31N conjugates are presented at Figure 1. The conjugates were evaluated by 1 H NMR spectroscopy. Extensive dialysis of dimethylsulfoxide solutions of conjugates with water enabled to remove low molecular side products, like p-nitrophenol which was released at the final stage of synthesis. The products were better soluble in DMSO than in water. Based upon integral intensity of 1 H resonances in the spectra of nimesulide containing conjugates the average number of 18 and 31 moieties of N, and an average of biotin was present in G3 B18N and G3 B31N , respectively, as determined by 1 H NMR. The NMR spectra of N and G3 B18N , and G3 B31N conjugates are presented at Figure 1.

Cytotoxicity of G3 B18N and G3 B31N Conjugates as Compared with Nimesulide Alone and PAMAM G3
Cytotoxicity estimated by NR assay revealed that both investigated cell lines were sensitive for either conjugate in concentration dependent manner ( Figure 2).

Cytotoxicity of G3 B18N and G3 B31N Conjugates as Compared with Nimesulide Alone and PAMAM G3
Cytotoxicity estimated by NR assay revealed that both investigated cell lines were sensitive for either conjugate in concentration dependent manner ( Figure 2).
The PAMAM cytotoxicity is influenced by generation, surface chemistry and dosage. The cytotoxicity of cationic PAMAM dendrimers is attributed to the interaction of surface cationic charge with negatively charged biological membranes that results in membrane damage via disruption of membrane structure and nanohole formation [59]. Many extensive studies have been performed in vitro using various models including lipid bilayers, liposomes, and Langmuir monolayers to study PAMAM dendrimer-membrane interactions [60][61][62]. It has been shown that low generation (<G5) of amine-terminated PAMAM dendrimers intercalate or adsorb to membrane surfaces rather than remove lipids. They are flexible and flatten against the membrane increasing the number of charge-charge interactions [63]. The PAMAM cytotoxicity is influenced by generation, surface chemistry and dosage. The cytotoxicity of cationic PAMAM dendrimers is attributed to the interaction of surface cationic charge with negatively charged biological membranes that results in membrane damage via disruption of membrane structure and nanohole formation [59]. Many extensive studies have been performed in vitro using various models including lipid bilayers, liposomes, and Langmuir monolayers to study PAMAM dendrimer-membrane interactions [60][61][62]. It has been shown that low generation (<G5) of amine-terminated PAMAM dendrimers intercalate or adsorb to membrane surfaces rather than remove lipids. They are flexible and flatten against the membrane increasing the number of chargecharge interactions [63].
Review of PAMAM dendrimer toxicity and surface modifications for its reduction is given by Janaszewska et al. [37]. In general, cationic dendrimers were cytotoxic (72 h incubation), displaying IC50 values = 50-300 µg/mL dependent on dendrimer-type, cell-type and generation [64]. In vitro investigations of the cytotoxicity of native G3 dendrimers revealed that it differs very much depending on cell type. Well recognized is high neurotoxicity of cationic PAMAM dendrimers. G4 PAMAM with unmodified positively charged surface significantly reduced hippocampal neurons viability at 1 µM concentration [65]. G3 PAMAM affected human neural progenitor cell viability and neuronal differentiation at 10 µg/mL concentration [66]. Introduced chemical modifications has been shown to reduce of PAMAM dendrimer neurotoxicity [65][66][67].
Published data concerning the low generation PAMAM cationic dendrimers cytotoxicity for cancer cell lines amounted to vary different values with IC50 equal to 402 µM for human hepatocellular carcinoma (HepG2), 13.24 µM for human prostate cancer (DU145), 35 µM for murine melanoma cells (B16F10) [64,68]. PAMAM G3 were non-toxic at 20 µM concentration for human breast cancer (MCF-7) and at 60 µM for epithelial lung carcinoma (A549) cell lines [40]. Our earlier Review of PAMAM dendrimer toxicity and surface modifications for its reduction is given by Janaszewska et al. [37]. In general, cationic dendrimers were cytotoxic (72 h incubation), displaying IC50 values = 50-300 µg/mL dependent on dendrimer-type, cell-type and generation [64]. In vitro investigations of the cytotoxicity of native G3 dendrimers revealed that it differs very much depending on cell type. Well recognized is high neurotoxicity of cationic PAMAM dendrimers. G4 PAMAM with unmodified positively charged surface significantly reduced hippocampal neurons viability at 1 µM concentration [65]. G3 PAMAM affected human neural progenitor cell viability and neuronal differentiation at 10 µg/mL concentration [66]. Introduced chemical modifications has been shown to reduce of PAMAM dendrimer neurotoxicity [65][66][67].
This diversity is due to complexity of mechanisms responsible for dendrimer cytotoxicity. The advanced studies on that issue, considering the neurotoxicity of higher generations (>4) of cationic PAMAM dendrimers, has been published and reviewed. This include apoptosis, mitochondrial activity, neuronal differentiation and gene expression due to oxidative stress and DNA damage [66,69]. Similar observations have been made for human colon cell line (SW480) and immortalized keratinocytes (HaCaT) with much higher sensitivity of HaCaT cells [70,71]. Wide range of the PAMAM G3 dendrimer cytotoxicity observed in various types of cells reveal the problem of its individual evaluation, depending on potential therapeutic target.
G3 B18N conjugate was significantly cytotoxic against SCC-15 cells at 5 µM concentration and against BJ cells at 10 µM concentration (about 70% and 55% of cell viability, respectively). It has to be pointed out, that at 10 µM G3 B18N concentration was nearly three times more toxic for cancer (20% viability) than fibroblasts (55% viability). G3 B31N conjugate with higher amount of N decreased remarkably SCC-15 cells viability at 1.25 µM and BJ cells at 2.5 µM concentration to 15% of control. At higher concentrations of G3 B31N , no viable cells were seen. These results were confirmed by corresponding morphological changes observed in cultured cells (Figure 3). G3 B18N caused shrinking and aggregation of SCC-15 cells with reduction of NR accumulation at 10 µM concentration, whereas only small depletion of the dye accumulation was observed in BJ cells without loss of adhesion and noticeable changes in morphology. Severe morphology disturbances were visible in both cell lines treated with G3 B31N at 2.5 µM and dying cell morphology including fragmentation, degradation and loss of cell adhesion were seen at higher conjugate concentrations.
investigations of IC50 for native cationic PAMAM G3 reveal value 12.68 µM for SCC-15 cell line [49]. Less data are available for cationic PAMAM low generation cytotoxicity estimations against nontransfected cells. In human neural progenitor cells, a 10 µg/mL concentration significantly inhibited cell viability [66]. G4 dendrimers significantly reduced hippocampal neurons viability at 1 µM [65]. In our earlier studies, IC50 of G3 PAMAM for normal BJ fibroblasts was equal to 5.64 µM. This diversity is due to complexity of mechanisms responsible for dendrimer cytotoxicity. The advanced studies on that issue, considering the neurotoxicity of higher generations (>4) of cationic PAMAM dendrimers, has been published and reviewed. This include apoptosis, mitochondrial activity, neuronal differentiation and gene expression due to oxidative stress and DNA damage [66,69]. Similar observations have been made for human colon cell line (SW480) and immortalized keratinocytes (HaCaT) with much higher sensitivity of HaCaT cells [70,71]. Wide range of the PAMAM G3 dendrimer cytotoxicity observed in various types of cells reveal the problem of its individual evaluation, depending on potential therapeutic target.
G3 B18N conjugate was significantly cytotoxic against SCC-15 cells at 5 µM concentration and against BJ cells at 10 µM concentration (about 70% and 55% of cell viability, respectively). It has to be pointed out, that at 10 µM G3 B18N concentration was nearly three times more toxic for cancer (20% viability) than fibroblasts (55% viability). G3 B31N conjugate with higher amount of N decreased remarkably SCC-15 cells viability at 1.25 µM and BJ cells at 2.5 µM concentration to 15% of control. At higher concentrations of G3 B31N , no viable cells were seen. These results were confirmed by corresponding morphological changes observed in cultured cells (Figure 3). G3 B18N caused shrinking and aggregation of SCC-15 cells with reduction of NR accumulation at 10 µM concentration, whereas only small depletion of the dye accumulation was observed in BJ cells without loss of adhesion and noticeable changes in morphology. Severe morphology disturbances were visible in both cell lines treated with G3 B31N at 2.5 µM and dying cell morphology including fragmentation, degradation and loss of cell adhesion were seen at higher conjugate concentrations. To compare the biological effects of the investigated nimesulide conjugated PAMAM dendrimers with N alone, the cytotoxicity assay with NR was performed ( Figure 2B). The obtained results showed that the G3 BN18 was over 295-fold, and G3 B31N was 266-fold more cytotoxic for the SCC-15 cells as compared to N alone (Figure 2A,B) In BJ cells, the differences were over 40-and 70-fold for G3 BN18 and G3 B31N , respectively. Calculated selectivity index (ratio of IC 50 of cancer to IC 50 of normal cells) revealed that dendrimer conjugates, particularly G3 BN18 , were more selective against cancer cells than N alone, with a selectivity index equal to 0.41 (Table 1).  (G3 B18N and G3 B31N , respectively). Moreover, our earlier study revealed that native PAMAM G3 dendrimer exhibited higher toxicity against normal fibroblasts (BJ) than squamous carcinoma cells (SCC-15) IC 50 value estimated with NR assay after 24 h incubation was equal 5.64 µM for BJ cells and 12.68 µM for SCC-15 with selectivity index 2.25 [49]. G3 PAMAM dendrimer substituted with one biotin molecule showed similar cytotoxicity than native PAMAM G3 dendrimer (no significant differences). Thus, the biotinylation and substitution of PAMAM G3 with 18 or 31 residues of N changed its cytotoxicity profile and selectivity. This resulted in a higher cytotoxicity of G3 B18N against SCC-15 cells as compared to normal fibroblasts, with SI equal to 0.41 (Table 1).

Antiproliferative Activity
Antineoplastic agents kill cancer cells with a high rate of proliferation. However, this therapy also affects normal, rapidly dividing cells of the skin, hair follicles, or digestive tract, causing undesirable and damaging side effects [75]. Therefore, it is a matter of importance to compare the effect of investigated anti-cancer drugs with both cancer and normal human cells.
After 96 h incubation, both studied bioconjugates indicated anti proliferative action selective against SCC-15 cells, as estimated by fluorescence assay of nuclear DNA content with DAPI dye (Figure 4).
In proliferation assay the G3 B18N exerted high selectivity against cancer cells. Significant inhibition of proliferation of SCC-15 cells was seen at 2.5 µM concentration with decrease of cell number to about 30% and at 5 µM to about 50% of control, with no significant changes noticed in normal fibroblasts. G3 B31N inhibited proliferation at 1.25 µM concentration for both SCC-15 and BJ cells. At 2.5 µM concentration, also cytotoxic effect was seen in SCC-15 cells (number of cells was below of that in 0-FBS culture) and antiproliferative action against fibroblasts (about 50% of control). In proliferation assay the G3 B18N exerted high selectivity against cancer cells. Significant inhibition of proliferation of SCC-15 cells was seen at 2.5 µM concentration with decrease of cell number to about 30% and at 5 µM to about 50% of control, with no significant changes noticed in normal fibroblasts. G3 B31N inhibited proliferation at 1.25 µM concentration for both SCC-15 and BJ cells. At 2.5 µM concentration, also cytotoxic effect was seen in SCC-15 cells (number of cells was below of that in 0-FBS culture) and antiproliferative action against fibroblasts (about 50% of control).
Antiproliferative properties of N against various types of cancer cells were observed by the others, but estimated drug concentrations were rather high, with IC50 in range of 30-800 µM [24,76]. N conjugated with hyaluronic acid decreased proliferation of colorectal cancer cells (HT-29, HCT-15) with IC50 about 400 µM as compared to 1600 µM for N alone [31]. Hida et al. have shown the selective antitumor action of N against non-small cell lung cancer as compared with normal human epithelial cells and poor response to N treatment of the human squamous cell lung carcinoma-derived PC-10 cells. In OSCC KB cells the N treatment at 100 µM for 72 h resulted in 43.3% of proliferation inhibition, with estimated IC50 equal to 130 µM. However, no induction of DNA fragmentation at nimesulide concentrations of up to 200 µM after 24 h incubation was observed [76]. In two head-and-neck carcinoma cell lines (SCC9 and SCC25), N (50-600 µM) inhibited cell proliferation without affecting colony-forming ability [10].
The highly potentiated effect of nimesulide conjugated with biotinylated PAMAM G3 dendrimer as inhibitor of proliferation of SCC-15 cells is clearly visible. It has to be considered that cancer cell growth and proliferation may be affected by local interactions of dendrimer conjugates surface molecules with cell membranes, particularly with receptors of cell surviving and death signals acting as stimulants or inhibitors. This would depend on dendrimer size, surface molecule charge, reactivity, and concentration. For small nanoparticles (< 10 nm), it has been found that rareearth fluoride nanoparticles at low concentration stimulate proliferation of three human cell lines-A549, SW837, and MCF-7-through activation of EGFR and integrin signaling [77]. Native PAMAM G3 dendrimer molecule has about 3.2 nm diameter. At higher concentration of the conjugates, the inhibitory effect may appear. Currently, a new concept of cell fate regulation including interaction with adhesion and extracellular matrix-based mechanisms and new mechanisms of mechanotransduction has been proposed that may explain this experimental data [78,79].  [76]. In two head-and-neck carcinoma cell lines (SCC9 and SCC25), N (50-600 µM) inhibited cell proliferation without affecting colony-forming ability [10].
The highly potentiated effect of nimesulide conjugated with biotinylated PAMAM G3 dendrimer as inhibitor of proliferation of SCC-15 cells is clearly visible. It has to be considered that cancer cell growth and proliferation may be affected by local interactions of dendrimer conjugates surface molecules with cell membranes, particularly with receptors of cell surviving and death signals acting as stimulants or inhibitors. This would depend on dendrimer size, surface molecule charge, reactivity, and concentration. For small nanoparticles (<10 nm), it has been found that rare-earth fluoride nanoparticles at low concentration stimulate proliferation of three human cell lines-A549, SW837, and MCF-7-through activation of EGFR and integrin signaling [77]. Native PAMAM G3 dendrimer molecule has about 3.2 nm diameter. At higher concentration of the conjugates, the inhibitory effect may appear. Currently, a new concept of cell fate regulation including interaction with adhesion and extracellular matrix-based mechanisms and new mechanisms of mechanotransduction has been proposed that may explain this experimental data [78,79].

Apoptosis
Inhibition of apoptosis is the canonical marker of cancer and has been a target for the development of anticancer strategies. Activation of the specific cysteine proteases (caspases) plays a central role in the cascade reactions realizing apoptotic program [80]. The ability of biotinylated PAMAM G3 dendrimers conjugated with N to induction of apoptosis was evaluated by measuring the activity of executive caspases 3 and 7 ( Figure 5). Both dendrimer conjugates revealed concentration dependent, pro-apoptotic effect with higher caspase 3/7 activity seen in fibroblasts. Significant G3 B18N stimulatory effect was seen at 5-10 µM concentrations. At 10 µM concentration, caspase activity was doubled in BJ and increased by 60% in SCC-15 cells ( Figure 5).

Apoptosis
Inhibition of apoptosis is the canonical marker of cancer and has been a target for the development of anticancer strategies. Activation of the specific cysteine proteases (caspases) plays a central role in the cascade reactions realizing apoptotic program [80]. The ability of biotinylated PAMAM G3 dendrimers conjugated with N to induction of apoptosis was evaluated by measuring the activity of executive caspases 3 and 7 ( Figure 5). Both dendrimer conjugates revealed concentration dependent, pro-apoptotic effect with higher caspase 3/7 activity seen in fibroblasts. Significant G3 B18N stimulatory effect was seen at 5-10 µM concentrations. At 10 µM concentration, caspase activity was doubled in BJ and increased by 60% in SCC-15 cells ( Figure 5). The effect of G3 B31N conjugate, carrying the higher load of N, was much more pronounced, particularly for fibroblasts. In BJ cells, an increase of caspase activity was observed at 2.5 µM concentration (about three-fold of control), with further increase to 5-6 fold of control at 5-10 µM concentrations, respectively ( Figure 5). A smaller but significant effect was seen in SCC-15 cells. At 2.5 µM concentration, an increase of caspase activity amounted to 150%, and at higher concentrations, it was doubled, as compared with control ( Figure 5). Induction of apoptosis by N has been recognized in many types of cancer cells including human pancreatic cancer (PANC-1), lung cancer (A549), gastric cancer (SGC-7901), and squamous carcinoma cells (KB, SCC9, SCC15) at rather high concentrations (in the range of 100-400 µM) [13,26,81]. In OSCC KB cells, N at 100 µM concentration was not able to induce apoptosis as detected by the poly(ADP-ribose) polymerase (PARP) fragmentation and DNA fragmentation assays ,and up to 200 µM did not alter the basal level of caspase-3/7 activity [74]. Both dendrimer conjugates indicated significant induction of apoptosis measured as activity of caspases 3/7 at 2.5-10 µM concentrations with much higher effect in fibroblasts. However, a matter of consideration is validation of the use of executive caspase 3/7 assay as measure of apoptosis [82]. Executive 3/7 caspases have been recognized as executioners of apoptosis, which made them a target in anti-cancer therapy, but their function in proper organism development, tissue homeostasis, and post-injury recovery has to be remembered. Many investigations revealed non-apoptotic functions of that enzymes [83]. New evidence reveals that activation of caspases in many cancers plays a pivotal role in maintaining their tumorigenicity and metastasis [84][85][86]. Particularly important is a phenomenon termed apoptosis-induced proliferation (AiP) causing proliferation cells by apoptotic caspases activated in stress-induced dying The effect of G3 B31N conjugate, carrying the higher load of N, was much more pronounced, particularly for fibroblasts. In BJ cells, an increase of caspase activity was observed at 2.5 µM concentration (about three-fold of control), with further increase to 5-6 fold of control at 5-10 µM concentrations, respectively ( Figure 5). A smaller but significant effect was seen in SCC-15 cells. At 2.5 µM concentration, an increase of caspase activity amounted to 150%, and at higher concentrations, it was doubled, as compared with control ( Figure 5). Induction of apoptosis by N has been recognized in many types of cancer cells including human pancreatic cancer (PANC-1), lung cancer (A549), gastric cancer (SGC-7901), and squamous carcinoma cells (KB, SCC9, SCC15) at rather high concentrations (in the range of 100-400 µM) [13,26,81]. In OSCC KB cells, N at 100 µM concentration was not able to induce apoptosis as detected by the poly(ADP-ribose) polymerase (PARP) fragmentation and DNA fragmentation assays, and up to 200 µM did not alter the basal level of caspase-3/7 activity [74]. Both dendrimer conjugates indicated significant induction of apoptosis measured as activity of caspases 3/7 at 2.5-10 µM concentrations with much higher effect in fibroblasts. However, a matter of consideration is validation of the use of executive caspase 3/7 assay as measure of apoptosis [82]. Executive 3/7 caspases have been recognized as executioners of apoptosis, which made them a target in anti-cancer therapy, but their function in proper organism development, tissue homeostasis, and post-injury recovery has to be remembered. Many investigations revealed non-apoptotic functions of that enzymes [83]. New evidence reveals that activation of caspases in many cancers plays a pivotal role in maintaining their tumorigenicity and metastasis [84][85][86]. Particularly important is a phenomenon termed apoptosis-induced proliferation (AiP) causing proliferation cells by apoptotic caspases activated in stress-induced dying cells and its role in tissue regeneration and cancerogenesis [87][88][89]. The obtained results reveal activation of caspases 3/7 by tested dendrimer conjugates at low 2.5-10 µM concentrations in both fibroblasts (to higher degree) and squamous carcinoma cell line (to lower degree). However, mechanisms of that phenomenon seem to be different. Smaller inhibitory effect on BJ cell viability and proliferation and higher stimulation of caspase 3/7 activity may be due to AiP effect. In SCC-15 opposite effects were seen that may be result of prevalence of pro-apoptotic effect of N dendrimer conjugates. More information concerning the mechanisms of OSCC cell death induced by investigated conjugates nimesulide requires investigations of other death pathways including autophagy and necrosis (for review, see [90]).

COX-2 Expression and Prostaglandin E2 Production
In order to evaluate the role of COX-2/PGE 2 axis in anticancer properties of investigated conjugates the COX-2 expression and prostaglandin E2 production were estimated. Analysis of COX-2 protein expression revealed two bands recognized by specific anti-COX-2 antibody, similar to that seen by the others with OSCC samples, and is due to different glycosylation profile of COX-2 protein [91,92]. The basal level of COX-2 protein was found in normal human proliferating fibroblasts [53,93]. Animal studies reveal that COX-2 is constitutively expressed in various tissues and organs (the renal medulla and pelvis, the gastrointestinal tract, lung, thymus, and brain) in the absence of inflammation signaling pathways [94].
As was mentioned in the Introduction, high expression of COX-2 protein is a marker of a majority of malignances. Immunohistochemical studies of the expression of COX-2 in human oral squamous cell carcinoma (OSCC), premalignant lesions, and normal oral epithelium reveal the high level of COX-2 protein in OSCC and dysplasia compared to normal mucosa [91,95]. No effect of G3 BN18 treatment on COX-2 protein level was observed in SCC-15 cells at all tested concentrations, whereas G3 BN31 significantly induced enzyme expression at 5-10 µM concentrations to about twice the control level ( Figure 6). In fibroblasts, G3 BN18 exerted limited effect on COX-2 expression with an increase of up to 170% of control at 2.5 µM concentration and without/small effect (120%) at 5-10 µM concentration. More remarkable changes were observed after G3 BN31 treatment that induced of COX-2 protein expression, up to three-and five-fold of control level at 2.5-10 µM concentration ( Figure 6). cells and its role in tissue regeneration and cancerogenesis [87][88][89]. The obtained results reveal activation of caspases 3/7 by tested dendrimer conjugates at low 2.5-10 µM concentrations in both fibroblasts (to higher degree) and squamous carcinoma cell line (to lower degree). However, mechanisms of that phenomenon seem to be different. Smaller inhibitory effect on BJ cell viability and proliferation and higher stimulation of caspase 3/7 activity may be due to AiP effect. In SCC-15 opposite effects were seen that may be result of prevalence of pro-apoptotic effect of N dendrimer conjugates. More information concerning the mechanisms of OSCC cell death induced by investigated conjugates nimesulide requires investigations of other death pathways including autophagy and necrosis (for review, see [90]).

COX-2 Expression and Prostaglandin E2 Production
In order to evaluate the role of COX-2/PGE2 axis in anticancer properties of investigated conjugates the COX-2 expression and prostaglandin E2 production were estimated. Analysis of COX-2 protein expression revealed two bands recognized by specific anti-COX-2 antibody, similar to that seen by the others with OSCC samples, and is due to different glycosylation profile of COX-2 protein [91,92]. The basal level of COX-2 protein was found in normal human proliferating fibroblasts [53,93]. Animal studies reveal that COX-2 is constitutively expressed in various tissues and organs (the renal medulla and pelvis, the gastrointestinal tract, lung, thymus, and brain) in the absence of inflammation signaling pathways [94].
As was mentioned in the Introduction, high expression of COX-2 protein is a marker of a majority of malignances. Immunohistochemical studies of the expression of COX-2 in human oral squamous cell carcinoma (OSCC), premalignant lesions, and normal oral epithelium reveal the high level of COX-2 protein in OSCC and dysplasia compared to normal mucosa [91,95]. No effect of G3 BN18 treatment on COX-2 protein level was observed in SCC-15 cells at all tested concentrations, whereas G3 BN31 significantly induced enzyme expression at 5-10 µM concentrations to about twice the control level ( Figure 6). In fibroblasts, G3 BN18 exerted limited effect on COX-2 expression with an increase of up to 170% of control at 2.5 µM concentration and without/small effect (120%) at 5-10 µM concentration. More remarkable changes were observed after G3 BN31 treatment that induced of COX-2 protein expression, up to three-and five-fold of control level at 2.5-10 µM concentration ( Figure 6). The stimulation of COX-2 expression by its inhibitors was reported by the others. Ko at al. observed that four COX-2 inhibitors, celecoxib, NS-398, nimesulide, and meloxicam increased COX-2 protein expression in the OCSC KB cells [74]. Nimesulide treatment (100 µM for 24 h) doubled the level of COX-2 protein. Similarly, stimulated expression of COX-2 was seen at 72-h treatment with 100 µM NS398 in OSCC cancer samples and cell lines [91]. Induction of enzyme protein expression by its inhibitors is a recognized phenomenon in cancer cells. The increase of dihydrofolate reductase and thymidylate synthase protein expression is considered to be part of the mechanism of methotrexate and 5FU resistance in human lymphoma cells [96][97][98].
Estimation of PGE 2 production and the effect of dendrimer conjugates reveal a different pattern in investigated cell lines. In fibroblasts, both G3 BN18 and G3 BN31 significantly decreased PGE 2 production at 1.25 µM concentration, which confirmed the inhibitory action of dendrimer-bound nimesulide on COX-2 activity. At higher concentrations, both conjugate effects become smaller, and the level of PGE 2 production, although lower than in control, become non-significant. This allows us to consider that nimesulide conjugates in BJ cells decrease PGE 2 production despite a significant increase of COX 2 protein expression (Figures 6 and 7). The stimulation of COX-2 expression by its inhibitors was reported by the others. Ko at al. observed that four COX-2 inhibitors, celecoxib, NS-398, nimesulide, and meloxicam increased COX-2 protein expression in the OCSC KB cells [74]. Nimesulide treatment (100 µM for 24 h) doubled the level of COX-2 protein. Similarly, stimulated expression of COX-2 was seen at 72-h treatment with 100 µM NS398 in OSCC cancer samples and cell lines [91]. Induction of enzyme protein expression by its inhibitors is a recognized phenomenon in cancer cells. The increase of dihydrofolate reductase and thymidylate synthase protein expression is considered to be part of the mechanism of methotrexate and 5FU resistance in human lymphoma cells [96][97][98].
Estimation of PGE2 production and the effect of dendrimer conjugates reveal a different pattern in investigated cell lines. In fibroblasts, both G3 BN18 and G3 BN31 significantly decreased PGE2 production at 1.25 µM concentration, which confirmed the inhibitory action of dendrimer-bound nimesulide on COX-2 activity. At higher concentrations, both conjugate effects become smaller, and the level of PGE2 production, although lower than in control, become non-significant. This allows us to consider that nimesulide conjugates in BJ cells decrease PGE2 production despite a significant increase of COX 2 protein expression ( Figure 6; Figure 7). In SCC-15 cells, the basal level of PGE2 was very low, and dendrimer conjugate treatment was without effect up to a 2.5 µM concentration. However, increased PGE2 production was observed at higher concentrations-at 5 µM with G3 BN31 and at 10 µM with both conjugates. That was parallel with a significant increase of COX-2 protein level (Figures 6 and 7). This is in contrary to observations of Ko et al., who found that at concentration range 0.5-50 µM the COX-2 inhibitors (celecoxib, NS-398, meloxicam, and nimesulide) significantly suppressed PGE2 production. Nimesulide after 1 h treatment at 0.5 µM concentration decreased PGE2 production to 20% of control in OSCC KB cells [74]. COX-2-selective inhibitor NS398 dramatically reduced PGE2 secretion in oral carcinoma-derived cell lines at 10 and 20 µM concentrations, but growth inhibition was not observed after 72 h treatment [91].
The complexity of the COX-2 effect on cancer cell death was revealed by studies of Totzke et al. Nimesulide effects were investigated in TNF-resistant HeLa H21 and TNF-sensitive HeLa D98 cells In SCC-15 cells, the basal level of PGE 2 was very low, and dendrimer conjugate treatment was without effect up to a 2.5 µM concentration. However, increased PGE 2 production was observed at higher concentrations-at 5 µM with G3 BN31 and at 10 µM with both conjugates. That was parallel with a significant increase of COX-2 protein level (Figures 6 and 7). This is in contrary to observations of Ko et al., who found that at concentration range 0.5-50 µM the COX-2 inhibitors (celecoxib, NS-398, meloxicam, and nimesulide) significantly suppressed PGE 2 production. Nimesulide after 1 h treatment at 0.5 µM concentration decreased PGE 2 production to 20% of control in OSCC KB cells [74]. COX-2-selective inhibitor NS398 dramatically reduced PGE 2 secretion in oral carcinoma-derived cell lines at 10 and 20 µM concentrations, but growth inhibition was not observed after 72 h treatment [91].
The complexity of the COX-2 effect on cancer cell death was revealed by studies of Totzke et al. Nimesulide effects were investigated in TNF-resistant HeLa H21 and TNF-sensitive HeLa D98 cells and MCF-7 cells transfected with COX-2. Nimesulide sensitized cancer cells to extrinsic death receptor-induced apoptotic pathway (TNF, CD95, and TRAIL receptors), independently of COX-2 presence and PGE 2 production. The phenomenon that the COX-2 expression may be not accompanied by COX-2 activity and PGE 2 secretion is supported by many investigations, and various mechanisms are recognized. This includes COX-2 itself and its inhibitors effects on various signaling pathways in normal and cancer cells, including inhibition of various apoptotic signals or modulation of cell cycle checkpoints [15,[99][100][101][102]. Particularly interesting are observations that various selective COX-2 inhibitors decrease the proliferation rate and induce apoptosis in a manner not related to the COX-2/PGE 2 axis [103].
The phenomenon observed in SCC-15 cells that, despite the inhibitory action of G3 B18N and particularly G3 B31N conjugates on proliferation rate at low concentrations (1.25-5 µM), only a small increase of caspase 3/7 activity and no increase of PGE 2 production was seen, is in agreement with other data, showing that much lower concentrations of N affects the death of cancer cells than that required for COX-2 inhibition [74]. Thus, it allowed us to assume that nimesulide-conjugated dendrimers executed their anti-cancer activity mainly by COX-2/PGE 2 independent signaling pathways.
Inhibitory effect of conjugates on PGE 2 production in fibroblasts, despite the increased caspase 3/7 activity and high COX-2 protein expression, may indicate that at low concentrations dendrimer conjugates inhibit COX-2 activity. However, the G3 B31N , with higher amount of nimesulide, exerted a much lower inhibitory effect up to a 2.5 µM concentration and was without effect at 5-10 µM. This may be explained by recent investigations concerning the functional interchangeability of two COX isoforms. Inhibition of COX-2 by coxibs, beside the inhibition of COX-2-mediated inflammatory prostaglandins causes also shifts prostanoids synthesis toward the COX-1-mediated pathway in human arthritis cartilage [104]. The functional interchangeability of these two COX isoforms is also confirmed by [105] and [106] with an animal cell model. Similarly, recently published observations by [107] concerning genomic, lipidomic, and metabolomic analysis of wild-type and cyclooxygenase-null mice lung fibroblasts revealed increased COX-1 activity and production of PGE 2 in COX-2 −/− cells and showed the compensation of various eicosanoids at the genomic and metabolic levels.

Conclusions
Substitution of biotinylated PAMAM G3 dendrimers with 18 (G3 B18N ) or 31 (G3 B31N ) nimesulide residues remarkably increased activity of this drug compared to its native form. IC 50 estimated for G3 B18N was 6.0 µM and 14.5 µM in SCC-15 and BJ cells, respectively, with SI = 0.41 as compared to IC 50 equal to 430 µM and 590 µM for nimesulide alone. Our data suggest that biotinylated dendrimer conjugated with nimesulide at low concentrations (1.25-10 µM) inhibits proliferation, overcomes apoptosis resistance, and selectively sensitizes squamous carcinoma cells SCC-15 to the apoptotic pathway independently of the COX-2/PGE 2 axis. In normal human fibroblasts (BJ), the same concentrations of G3 B31N conjugate was less effective in inhibition of proliferation and increased apoptosis as measured by caspase 3/7 activity in a manner depending on the increase of PGE 2 production by either COX-1/COX-2. In conclusion, the nimesulide dendrimer conjugates are potential candidates for local squamous cell carcinoma treatment, but it has to be taken into consideration that their effects are concentration-and cell type (normal/cancer)-dependent.