Requirement of a carbon spacer in benzyl isothiocyanate-mediated cytotoxicity and MAPK activation in head and neck squamous cell carcinoma

Vivian W.Y. Lui1, Abbey L. Wentzel1, Dong Xiao2, Karen L. Lew2, Shivendra V. Singh2 and Jennifer R. Grandis1,2,3

1 Department of Otolaryngology and 2 Pharmacology, University of Pittsburgh School of Medicine and the University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cruciferous vegetable-derived isothiocyanates (ITCs; chemical structure: R-N=C=S) are highly effective in affording protection against chemically induced cancers in animal models. Here, we studied the antitumor effects of benzyl isothiocyanate (BITC; Ph-CH2-N=C=S), the predominant ITC compound in broccoli, on head and neck squamous cell carcinoma (HNSCC) cell lines. Proliferation, apoptosis and immunoblotting assays were used to determine the effects and mechanism of several ITCs on HNSCC cells. The IC50 for BITC (24 h treatment) in two of the HNSCC cell lines was ~22 and 17 µM, respectively. Interestingly, phenyl isothiocyanate (PITC; Ph-N=C=S), which is a close structural analog of BITC but lacks a -CH2- spacer that links the aromatic ring to N=C=S moiety, did not result in significant killing of the HNSCC cells in this dose range. BITC (but not PITC) caused activation of caspase 3 and PARP cleavage. Within 20 min of treatment, BITC (but not PITC) induced a rapid activation of p38 MAPK. In addition, BITC (but not PITC) treatment resulted in the activation of p44/42 MAPK. Co-treatment with a specific p38 MAPK inhibitor, SB203580, or an inhibitor of the MEK/MAPK pathway, U0126, partially rescued cells from BITC-induced killing. Our results show that minor structural differences in ITCs can be crucial for the antiproliferative activity of ITCs and that BITC may be a promising chemopreventive as well as therapeutic agent in HNSCC.

Abbreviations: BITC, benzyl isothiocyanate; HNSCC, head and neck squamous cell carcinoma; ITC, isothiocyanates; PEITC, phenethyl isothiocyanate; PITC, phenyl isothiocyanate


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy that is the sixth most frequent cancer worldwide. Despite advancements in surgery, radiation and chemotherapy, the overall 5-year survival rate has remained <50% over the past 40 years (1,2). This is mainly due to the tendency of these cancers to invade local tissues and the development of a second neoplasm in ~16–40% of HNSCC patients (3). The high rate of multiple primary tumors is believed to be caused by the field effects of carcinogens (e.g. tobacco and alcohol) exposed to the entire upper aerodigestive tract epithelium (4,5). Thus, effective chemoprevention strategies are required to prevent second primary tumor formation.

Organic isothiocyanates (ITCs; chemical structure: R-N=C=S) are present in large quantities in many edible plants, such as broccoli, cabbage and watercress. Some ITCs have been shown to be highly effective in preventing chemically induced cancer in vivo (6). The chemoprotective effect of ITCs was first demonstrated by long-term feeding of Wistar rats with {alpha}-naphthyl-ITC, which significantly reduced the formation of liver tumors induced by ethionine, N-2-fluorenylacetamide and 3'-methyl-4-dimethyl-aminoazobenzene (7,8). Subsequently, the protective effects of various ITCs against chemically induced carcinogenesis were demonstrated in many other animal models including tumors of the breast (9), forestomach (10), lung (1113) and esophagus (14). Recent studies aimed at elucidating the mechanism of action of ITCs showed that some ITCs could induce apoptosis in a variety of cancer cell lines (1518). Specifically, phenyl ethylisothiocyanate and other structurally related ITCs were evaluated as inhibitors of HeLa cell viability where PITC was found to only weakly inhibit cell viability (17). In contrast, the other ITCs induced apoptosis and caspase 3 activity. These findings imply that ITCs may have therapeutic benefits in addition to their well-documented chemopreventive effects.

The mechanism of the anticarcinogenic activities of ITCs is not completely understood. The chemopreventive effects of ITCs were originally thought to be due to the inhibition of carcinogen activation or enhancement of detoxification of activated carcinogenic species through the induction of Phase II enzymes such as glutathione transferases. Other studies have implicated additional potential mechanisms of the antitumor activation of ITCs. We, and others, have shown that PEITC induced apoptosis in prostate cancer and cervical carcinoma cell lines partly via MEK/MAPK and caspase 3-dependent pathways, respectively (15,17). PEITC has been shown to inhibit epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation via the induction of apoptosis (19).

Although some ITCs have been shown to have chemopreventive effects, not all ITCs have the same or even comparable potency against the same types of tumors. In fact, allyl ITC has even been reported to function as a carcinogen in male rats (20). It has been shown that the intrinsic structural differences of various ITCs can be an important factor (21). An increased understanding of the structure–activity relationship of ITCs is critical in order to optimize their chemopreventive and antitumor activities. Some studies have attempted to investigate the effects of the R group on the antitumor activity of ITCs. For instance, an increase in the alkyl chain length in arylalkyl ITCs augmented their inhibitory activity against lung tumors in an NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] induced lung neoplasia model (22,23). Although it is generally believed that increased alkyl chain length or the addition of an aromatic ring enhances the potency of ITCs, this is not always true. For example, although PEITC [Ph-(CH2)2-N=C=S] is more potent than AITC in inhibiting the growth of HL60 cells, both AITC and PEITC have similar potencies in inducing cytotoxicity in ML-1 cells (24). Therefore, the structure–activity relationship of various ITC agents needs to be determined for each tumor system.

The potential chemopreventive and therapeutic effects of ITCs have not been examined previously in HNSCC. In the present study, we investigated the effects of BITC (Ph-CH2-N=C=S), as well as its structural analog, PITC (Ph-N=C=S, lacking a -CH2- spacer), on HNSCC lines. We demonstrated, for the first time, that BITC is highly effective in inducing apoptosis in five different HNSCC lines. In addition, our data indicate that minor structural differences between BITC and PITC are crucial for the anti-proliferative activity, as well as the activation of p38 MAPK and MEK/MAPK pathways by BITC. Finally, activation of p38 MAPK and MEK/MAPK pathways were found to contribute to BITC-induced apoptosis in some, but not all HNSCC cell lines. This observed cell line-specific effect indicates that a diverse and more complicated mechanism is likely to be involved in ITC-mediated antitumor activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and reagents
BITC, PITC, DMSO and MTT were purchased from Sigma-Aldrich Corporation (St Louis, MO). Antibodies against p38 MAPK, phosopho-p38 MAPK, p44/42 MAPK, phospho-p44/42 MAPK and cleaved PARP were from New England Biolabs (Beverly, MA), whereas the antibody against pro-caspase 3 was from BD Pharmingen (San Diego, CA) and the anti-actin antibody was from Oncogene Research Products (Boston, MA). Secondary antibodies, goat-anti-mouse and goat-anti rabbit IgG-horseradish peroxidase conjugates were purchased from Bio-Rad Laboratories, (Hercules, CA). The inhibitors, U0126 and SB203580 were purchased from New England Biolabs (Beverly, MA) and Transduction Laboratories (Lexington, KY) respectively.

Cell culture
All HNSCC lines (1483, UM-22B, PCI-15B and PCI-37A) were of human origin and have been described previously (2527). UPCI:SCC103 cell line was a kind gift from Dr S.Gollin (Graduate School of Public Health, University of Pittsburgh, PA). Cells were maintained in DMEM with 12% heat-inactivated fetal calf serum (Invitrogen, Carlsbad, CA) and 1x penicillin/streptomycin mix (Invitrogen) at 37°C with 5% CO2.

MTT assay
The effect of BITC or PITC on the survival of HNSCC cells was determined by MTT assay. Cells (1.5 x 104 cells) were plated in a 48-well plate, and allowed to attach overnight. Medium was replaced with fresh complete medium containing desired concentrations of BITC and PITC. Stock solutions of BITC and PITC were first prepared in DMSO:serum-free DMEM (1:1) and then serially diluted with complete DMEM. Stock solution of the vehicle control (DMSO) was prepared similarly. In some cases, inhibitors were added simultaneously with the BITC or DMSO control. Following a 24-h incubation at 37°C, medium was removed and MTT solution (5 mg/ml in 1x PBS) was added to the cells and incubated at 37°C for 2–4 h. At the end of the incubation, MTT solution was removed from the wells and DMSO (150 µl) was added to each well in order to solubilize the blue MTT-formazan product. Absorbance of the solubilized product was measured at 550 nm using a Kinetic Microplate reader (Molecular Devices, Sunnyvale, CA). The absorbance was proportional to the survival of the cells. Percentage killing was calculated as: (A550 DMSO-A550 ITC) x 100%/A550 DMSO. Statistical analysis (unpaired t-test) was performed using GraphPad Prism Software (GraphPad Software, San Diego, CA).

Western blotting
HNSCC cells were treated with desired concentrations of BITC, PITC or DMSO. At different time points, cells were lysed with a lysis solution containing 1% Nonidet-P40, 150 mM NaCl, 1 mM EDTA, 10 mM sodium phosphate buffer (pH 7.2), 0.25 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin and 10 µg/ml aprotinin for 5 min at 4°C. The lysate cleared by centrifugation, 1200 r.p.m for 15 min. Supernatant was collected for protein quantification using the Protein Assay Solution (Bio-Rad Laboratories). Proteins were resolved on a 10% SDS–PAGE gel and transferred onto the Protran membrane (Schleicher & Schuell, Keene, NH) using the semi-dry transfer machine (Bio-Rad Laboratories). The membrane was blocked overnight with a solution containing 5% non-fat dry milk, 0.2% Tween-20 in 1x PBS. The membrane was incubated with the primary antibody for 2 h at the following dilutions: p38 MAPK, Phospho-p38 MAPK, p44/42 MAPK and Phospho-p44/42 MAPK (1:1000 dilution), cleaved PARP (1:1000), Pro-caspase 3 (1:1000 dilution) and actin (1:15000 dilution). The membrane was then washed with the Blotto solution (0.6% dry milk powder, 0.9% NaCl, 0.5% Tween-20 and 50 mM Tris, pH 7.4) three times for 10 min each. The membrane was then incubated with the secondary antibody for 1 h and then washed three times for 10 min each. The membrane was quickly rinsed with a rinsing solution and the immunoreactive bands were visualized using Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer's instructions.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dose-dependent cytotoxicity of BITC against HNSCC
The effect of BITC on the survival of HNSCC cells was investigated using MTT assay, and the data are summarized in Figure 1 and Table I. Two HNSCC cell lines with different growth kinetics were chosen. 1483 cells have a doubling time of ~24 h, whereas UM-22B cells proliferate more rapidly, with a doubling time of ~6 h. As shown in Figure 1, a 24 h exposure to BITC resulted in a concentration-dependent effect on the survival in these two representative HNSCC cell lines. The IC50 of BITC for 1483 and UM-22B were ~22 and 17 µM, respectively. Although PITC (Ph-N=C=S) is a close structural analog of BITC (Ph-CH2-N=C=S), it did not result in effective killing of 1483 and UM-22B cell lines in this dose range. Only at very high doses could the cytotoxic effects of PITC be observed. When compared with BITC, the IC50 of PITC for 1483 and UM-22B cells was 31- (674 µM) and 40-fold higher (704 µM) than that of BITC, indicating the relatively low potency of PITC in HNSCC cells. This significant difference in cytotoxic activity between BITC and PITC on HNSCC cells was further confirmed using three additional HNSCC cell lines, PCI-15B, UPCI:SCC103 and PCI-37A (Table I). At 30 µM, BITC resulted in ~70% killing of all HNSCC lines tested, while no significant effect was observed with PITC. These results suggest that HNSCC cells are highly susceptible to the cytotoxic effects of BITC.



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Fig. 1. BITC induced decreased survival of HNSCC lines in a dose-dependent manner. 1483 cells and UM-22B cells (1.5 x 104) were treated with increasing concentrations of BITC or PITC for 24 h prior to MTT assay. The percentage of HNSCC survival following treatment with BITC or PITC is shown. Three to twelve samples are represented by each data point. The graphed point represents the mean value and the error bars represent the SEM.

 

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Table I. Effect of BITC versus PITC in the killing of five HNSCC lines

 
BITC induces PARP cleavage and caspase 3 activation
To determine whether BITC-induced cell killing is linked to caspase 3 activation, the effects of this compound on pro-caspase 3 expression and PARP cleavage (a target for activated caspase 3) were determined. Caspase 3 is an active cell death protease involved in the execution of apoptosis and is activated in response to various apoptotic stimuli (28). Data on densitometric scanning for expression of pro-caspase 3, after correction for differences in protein loading, in BITC- or PITC-treated UM-22B and 1483 cells relative to DMSO treated control cells are summarized in Figure 2A. In UM-22B cells, a statistically significant reduction in the level of pro-caspase 3 was observed as early as 5 h after BITC treatment. The level of pro-caspase 3 in BITC-treated UM-22B cells was reduced by >90% at 16 and 24 h time points when compared with the control. The level of pro-caspase 3 was also reduced in BITC treated 1483 cells. On the other hand, a similar treatment of cells with PITC did not result in activation of caspase 3 in either cell line (Figure 2 and data not shown). Consistent with these observations, BITC treatment induced PARP cleavage in both cells lines (Figure 2B). The degree of PARP cleavage increased with time, with the maximal cleavage at ~16 h following BITC exposure. However, neither PITC nor DMSO control treatment induced PARP cleavage. These results indicate that caspase 3 activation is induced by BITC treatment in HNSCC.



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Fig. 2. BITC, but not PITC, treatment causes caspase 3 activation (A) and cleavage of PARP (B) in HNSCC cells. HNSCC cells (1483) were treated with DMSO or 30 µM of BITC or, PITC for different time intervals as indicated (1–24 h). Protein extracts were collected and western blotting was performed for pro-caspase 3, cleaved PARP or ß-actin as indicated. Cumulative results are shown from two independent experiments.

 
BITC, but not PITC, induces activation of p38 MAPK
We, and others, have demonstrated previously that ITC-induced apoptosis involved activation of c-Jun-N-terminal kinase (JNK1/2) or p44/42 MAPK (or ERK1/2) in other cancer cells (15,29). However, a unifying mechanism for ITC-induced apoptosis or antitumorigenic activity has not been reported. To elucidate the mechanism of BITC-induced apoptosis of HNSCC cells, we investigated the effects of BITC on the intracellular regulators of apoptosis or oxidative stress signaling. Within an hour of exposure to BITC, p38 MAPK was activated (phosphorylation at Thr 180/Tyr 182) in both 1483 and UM-22B (Figure 3A). In fact, p38 MAPK phosphorylation could be detected as early as 10 and 30 min upon BITC treatment in 1483 and UM-22B cells, respectively (Figure 3B). A longer time-course experiment was performed to determine the duration of p38 MAPK activation by BITC in these cell lines. In 1483 cells, the immediate p38 MAPK response peaked at ~1–3 h and then rapidly declined at 5 h, reaching a minimum level at 16 h. However, in UM-22B cells, the BITC-induced phosphorylation of p38 MAPK was maintained at a very high level from 1 to 9 h and then started to decline at 16 h. At 24 h of exposure to BITC, p38 MAPK activation was still detectable. Protein expression levels of p38 MAPK were not significantly affected by the treatment and therefore served as a loading control for immunoblotting. In contrast, PITC did not result in the activation of p38 MAPK in both HNSCC cell lines when compared with DMSO control.



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Fig. 3. BITC, but not PITC, induced activation of p38 MAPK phosphorylation in HNSCC cells. (A) 1483 and UM-22B cells were treated with the vehicle (DMSO) or 30 µM of BITC or PITC for 1, 3, 5, 9, 16 and 24 h. Phosphorylation of p38 MAPK was determined by western blotting using antibodies specific for phosphorylated p38 MAPK. The levels of p38 MAPK were unchanged and therefore serve as a control for loading. (B) A short time-course study demonstrated immediate activation of p38 MAPK by BITC, but not PITC, in both HNSCC cell lines. Immunoblotting for p38 MAPK demonstrates equal loading.

 
BITC-induced MEK/MAPK activation
Our previous study in prostate cancer cells showed that activation of p44/42 MAPK (ERK-1/2) was partially involved in the PEITC-induced cell death in a human prostate carcinoma cell line, PC-3 (15). Therefore, we investigated if BITC-induced death of HNSCC cells involved the activation of p44/42 MAPK. In 1483 cells, BITC induced a strong and sustained activation (phosphorylation) of p44/42 MAPK (Thr 202/ Tyr 204) for up to 24 h (Figure 4A). Although PITC also induced activation of p44/42 MAPK slightly above that of the DMSO control, the phosphorylation was transient and reduced back to the basal levels at ~5 h. In UM-22B cells, a strong activation of p44/42 MAPK was induced by BITC, but not PITC (Figure 4B). Immunoblotting for unphosphorylated p44/42 MAPK demonstrated equal loading under all conditions.



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Fig. 4. BITC-induced MEK/MAPK phosphorylation in HNSCC cells. (A) 1483 and (B) UM-22B cells were treated with DMSO or 30 µM of BITC or PITC for 1, 3, 5, 9, 16 and 24 h. Phosphorylation of p44/42-MAPK (MEK/MAPK) was determined by western blotting using antibodies specific for phosphorylated p44/42 MAPK. The levels of p44/42-MAPK was not changed by BITC or PITC treatment and therefore served as a loading control.

 
The requirement of p38 MAPK and MEK/MAPK in BITC-induced cytotoxicity is cell line-specific
To determine if the activation of both p38 MAPK and p44/42 MAPK are responsible for mediating the killing of HNSCC cells, the effect of specific inhibitors of these pathways on BITC-induced cell killing was examined. SB203580 is a specific inhibitor of p38 MAPK, while U0126 is a potent specific inhibitor of MEK-1/2 (the upstream kinases of p44/42 MAPK). As shown in Figure 5A, the p38 MAPK inhibitor alone, SB203580, did not have any effect on the growth of 1483 cells when administered with DMSO as a control (P value = 0.43; n = 6). However, when SB203580 was added together with BITC, the BITC-mediated killing was significantly reduced from 88.68 ± 1.25% to 46.73 ± 7.39% (P value = 0.0002; n = 6). Similarly, co-treatment with the specific MEK-1/2 inhibitor, U0126, also significantly reduced the BITC-mediated killing of 1483 cells from 80.00 ± 3.87 to 59.76 ± 4.43% (P value = 0.006; n = 6). U0126 (10 mM) alone also had a minor inhibitory effect on 1483 cells (P value = 0.04, n = 6). When both inhibitors were added simultaneously, no synergistic inhibition of the BITC-mediated cell death was observed (data not shown). Our results indicate that both p38 MAPK and p44/42 MAPK pathways are involved in the BITC-induced cell killing in 1483 cells. However, neither U0126 nor SB203580 affected the killing of UM-22B induced by BITC (data not shown), even though both the p38 MAPK and MEK/MAPK pathways are activated by BITC in UM-22B cells.



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Fig. 5. Requirement of p38 MAPK and MEK/MAPK activation in BITC-induced cytotoxicity in 1483 cells. Cell killing induced by BITC was significantly abrogated when 1483 cells were treated with specific pharmacological inhibitors of p38 MAPK (SB203580, 10 mM) (A) or MEK/MAPK inhibitor (U0126, 10 mM) (B) in the presence of BITC. The P values are shown (n = 6).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The development of effective chemopreventive agents is critical to improve the survival rate of many cancers including HNSCC. The chemopreventive effects of ITCs have not been carefully examined in this tumor system. In this study, we demonstrated, for the first time, that BITC is highly cytotoxic against HNSCC cells, implicating the potential of such an agent for the prevention or treatment of HNSCC. Although the anticarcinogenic activity of ITCs has been studied previously in a number of in vitro and in vivo models, their effects on HNSCC has not been investigated. In a related study, Morse et al. (11) showed that PEITC could not abrogate the formation of nasal cavity tumors but was able to inhibit lung tumor formation, induced by a tobacco-specific carcinogen, NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] in rats. In our in vitro models, PEITC could also induce HNSCC apoptosis, although to a lesser extent compared with BITC (data not shown).

Both BITC and PITC are naturally occurring isothiocyanates (30). Inhibition of carcinogen activation and/or induction of Phase II enzymes is believed to be an important mechanism for anticarcinogenic activity of ITCs in chemically induced carcinogenesis models. In fact, many ITCs can inhibit Phase I enzymes (cytochrome P-450) that are involved in the bioactivation of carcinogens (31,32) and also can induce Phase II detoxification enzymes, which accelerate the inactivation of carcinogens (3335). Phase II detoxification enzymes such as glutathione S-transferases (GSTs) catalyze the conjugation of glutathione with reactive chemical species generating conjugates that are eventually eliminated via transport or through mercapturic acid pathway. A recent study demonstrated that the treatment of rat liver epithelial cells with BITC resulted in the immediate production of reactive oxygen intermediates (ROIs) and correlated induction of a GST (GSTP1), suggesting a role of ROIs or possible redox regulation in the induction of GST by BITC (36). An in vivo study suggested that many ITCs, including BITC (but not PITC), could inhibit the activation of the tobacco-specific carcinogen [nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone; NNK] by inhibiting the activity of Phase I enzymes in hamster liver (37). Other in vivo studies also showed that various ITC agents demonstrated differential abilities to inhibit the formation of DNA adducts in NNK-induced lung tumorigenesis models (12,13). It is also possible that the rate of intracellular accumulation and metabolism of ITCs is structure-dependent.

However, whether changes in Phase I and II enzyme levels are actually contributing to the observed chemopreventive effects of ITCs remain largely unknown. Recent in vitro studies revealed that additional mechanisms are probably involved. Direct induction of apoptosis has been observed in cell lines derived from prostate cancer, cervical carcinoma, colon cancer, leukemia and normal epidermis (15,1719,24,38). Although the detailed mechanism of apoptosis induction has not been completely elucidated, involvement of MEK/MAP kinase, c-Jun N-terminal kinase 1 (JNK1), caspase-3, Bid and p53 have been implicated. Different pathways appear to be responsible for the induction of apoptosis in different systems. For example, activation of JNKs has been shown to be associated with the induction of apoptosis by PEITC in HeLa, Jurkat and 293 cells, but not in the PC-3 human prostate cancer cell line (20,36). Similarly, the requirement of p53 for PEITC-induced apoptosis is not absolutely essential as was first described in mouse embryonic fibroblasts (37). In fact, we, and others have recently shown that PEITC can also induce apoptosis in p53-deficient cells (15,24). These findings suggest that the mechanism(s) of apoptosis induction by ITCs can involve multiple pathways and are probably cell line- or tumor system-specific. This is consistent with the in vivo findings that certain ITCs which demonstrate a potent anticarcinogenic effect in one cancer model will be ineffective in another model.

Given the dramatic cytotoxic effects of BITC on HNSCC cells, we attempted to elucidate the mechanism of BITC induced-cell killing in HNSCC cells. Similar to our previous study in prostate cancer cells, we found that both the p38 MAPK and MEK/MAPK pathways were immediately activated upon the addition of BITC to two different HNSCC lines, 1483 and UM-22B. However, JNK activation was not observed (data not shown). In 1483 cells, inhibition of p38 MAPK or MEK/MAPK partially reduced the BITC-induced cell killing suggesting that both pathways are involved in this process. This is different from what we observed in prostate cancer cells, where the activation of p38 MAPK did not contribute to PEITC-induced apoptosis and cell killing (15). This could be due to the difference between BITC and PEITC or possibly the difference between the HNSCC and prostate cancer cells. However, in UM-22B cells, neither of these pathways was required for the cell killing effect of BITC, although both pathways are activated in these cells. In addition, inhibition of both pathways together did not result in additive or synergistic inhibition of BITC-induced cell killing of both HNSCC cell lines (data not shown). It appears that BITC can activate multiple MAPK signaling pathways in HNSCC cells; however, not all pathways contribute to cell killing to the same extent in different cell lines. Our results are consistent with previous studies demonstrating that the mechanism underlying antiproliferative activity of ITCs may be cell line-specific.

There are several potential explanations for the apparently different mechanisms of apoptosis induction and cell killing in different cell lines/tumor models. For instance, the two HNSCC cell lines examined demonstrated different growth kinetics: 1483 cells have a >3-fold longer doubling time (~24 h) compared with UM-22B cells (6 h). Therefore, the regulation of intracellular pathways as well as the rate of metabolism or excretion of xenobiotics would likely be different. In fact, the kinetics of p38 MAPK, MEK/MAPK and PARP activation are clearly different in these two HNSCC lines. As multiple pathways are probably involved in anti-proliferative effect of ITCs, the presence and relative abundance of different apoptosis executioners or regulators (such as the levels or activity of pro- and anti-apoptotic proteins) could be very different in the two cell lines. For instance, 1483 cells have a point mutation in exon 5 of p53 (Dr Gary L.Clayman, personal communication) while UM-22B cells have a mutant p53 in codon 220, changing a tyrosine to a cysteine residue (Dr Thomas Carey, personal communication). Further studies are needed to determine whether the difference in p53 levels or mutations contribute to the effects of different intracellular signaling pathways that regulate apoptosis in HNSCC, where p53 mutation is known to be very common.

It is known that different ITC agents demonstrate distinct activity profiles against different cancer models. However, the structure–activity relationship of ITC in HNSCC and other cancers as well as the mechanism of differences in potency of different ITC agents are unknown. We studied the effects of two structurally closely related ITC agents, namely BITC and PITC, on HNSCC cells. Both BITC and PITC are known to inhibit the induction of mammary tumor formation (by 7, 12-dimethylbenz[a]anthracene) in rats (13). In the present study, we showed that BITC (Ph-CH2-N=C=S) effectively inhibited the proliferation of all HNSCC lines tested. However, its structural analog, PITC, which lacks a -CH2- spacer linking the aromatic ring to N=C=S moiety, did not result in significant killing of HNSCC cells in a comparable dose range. Consistent with the differences in their ability to affect cell proliferation, BITC, but not PITC, stimulated activation of p38 MAPK and MEK/MAPK in HNSCC cells. Our results imply that a minor structural difference between BITC and PITC could lead to the triggering of different molecular events that may be involved in cell killing. Our results demonstrated that BITC might serve as an effective chemopreventive or therapeutic agent for the treatment of HNSCC. Even though the activity of BITC in an animal model of HNSCC has yet to be tested, 1(4.5-dimethylthiazol-2-yl)-3,5-diphenyformuzan, a naturally occurring structural analog of BITC, is highly effective in suppressing the growth of PC-3 prostate cancer xenografts (unpublished observations). Further studies are required to evaluate the detailed mechanisms of apoptosis induction by various ITC agents in HNSCC as well as their structure–activity relationship in this tumor system.


    Notes
 
3 To whom correspondence should be addressed Email: jgrandis{at}pitt.edu. Back


    Acknowledgments
 
This work was supported by grants CA77308 (to J.R.G.) and CA55589, CA76348 (to S.V.S.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 1, 2003; revised July 15, 2003; accepted July 23, 2003.