Effects of exogenous nitric oxide on oral squamous cell carcinoma: An in vitro study

J Oral Maxillofac Surg 60:905-910, 2002

Effects of Exogenous Nitric Oxide on Oral Squamous Cell Carcinoma: An In Vitro Study Zheng-Jun Shang, DDS, MD, PhD,* Jin-Rong Li, DDS,† and Zu-Bing Li, MS‡ Purpose:

Nitric oxide (NO) is a newly found unstable free radical gas, serving as an important mediator, messenger, and signal transduction molecule and involved in a variety of pathophysiologic processes. Recently, NO has been reported to have cytotoxic effects on several tumor cells as an effector molecule of activated macrophage. The objective of this study was to investigate the effects of exogenous NO on oral squamous cell carcinoma cell line and to try to clarify the possible mechanisms by which it kills tumor cells. Methods: TSCCa cell line, established from a patient with oral squamous cell carcinoma of the tongue, was exposed to various concentrations of exogenous NO that were released from an NO donor, sodium nitroprusside (SNP), for 48 hours. Nitrite/nitrate levels in the culture supernatant were determined with a commercial available NO kit. Both morphologic and ultrastructural changes were evaluated by reverse phase contrast microscopy or transmission electron microscopy. The DNA was harvested from SNPtreated or untreated TSCCa cells and assessed by agarose gel electrophoresis. Results: SNP released NO into medium in a dose-dependent manner. NO had a concentrationdependent cytotoxicity against TSCCa cells. NO induced tumor cell death through apoptosis, which was characterized by incompleteness of nuclear membrane, disappearance of nucleole and nuclear condensation, chromatin margination, or chromatin homogenization. Agarose gel electrophoresis showed a typical internucleosomal DNA cleavage pattern (DNA ladder), a reliable indicator of apoptosis. Conclusions: Our results suggested that NO had a tumoricidal potential against oral cancer cells. NO might exert its cyotoxicity as an effector molecule of activated microphage through at least apoptosis. © 2002 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 60:905-910, 2002 It has been reported that oral cancer accounts for 3% of all new cancers in populations, affecting more than 200,000 people worldwide.1,2 The most prevalent form of oral cancer is oral squamous cell carcinoma (OSCC). Its high incidence is due to comprehensive interaction of many factors. Although many measures exist to treat it, the survival rate is still undesirable,

with a rate 54% and 33% for whites and blacks, respectively.2 A higher mortality rate for oral cancer has been reported in Hungary and Hong Kong, with more than 14:100,000 population.2 Therefore, it is urgent to find new effective treatments to control this disease and to enhance life quality. It is well known that host defense mechanism plays an important role in the development and growth of tumors3 and that the incidence of malignancy is increased in populations with compromised immunity.4 There are several types of immunocytes that have a role in host response to cancer. Among these immunocytes, macrophage is considered to be one of the most privotal tumoricidal cells. Activated macrophages could exclude neoplastic cells in vivo by direct macrophage-to-tumor cell or through secretion of several kinds of soluble cytokines such as tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1), prostaglandins (PGs), and others.5-9 Recently, many studies have shown that activated macrophages could also produce large amounts of nitric oxide (NO) and that

Received from the Third Department of Oral and Maxillofacial Surgery, College and Hospital of Stomatology, Wuhan University, Wuhan, People’s Republic of China. *PhD Candidate and Lecturer. †Professor. ‡Professor and Chairman. Address correspondence and reprint requests to Dr Shang: Department of Oral and Maxillofacial Surgery, College and Hospital of Stomatology, Wuhan University, 237 Luoyu Rd, Wuhan 430079, People’s Republic of China; e-mail: [email protected] © 2002 American Association of Oral and Maxillofacial Surgeons

0278-2391/02/6008-0010$35.00/0 doi:10.1053/joms.2002.33860

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906 activated macrophages exerted their cytotoxicity in an NO-dependent manner.10-12 There also is evidence that NO could mediate cellular apoptosis.13,14 Thus, NO, an unstable free radical gas, has been regarded as a key molecule in the field of neoplastic research. Until now, however, there was relatively little knowledge about the behavior of NO in OSCC and the role of NO in carcinogenesis and tumor cell growth remained unclear and controversial.15,16 The purpose of the present study was to investigate whether NO, released from sodium nitroprusside (SNP), was cytotoxic to the TSCCa cell line established from a patient with OSCC of the tongue and to try to clarify its possible mechanisms of killing tumor cells as a effector molecule of activated macrophages.

Materials and Methods MAIN REAGENTS AND INSTRUMENTS

The main reagents and instruments included RPMI 1640 (GIBCO BRL, Grand Island, NY), fetal bovine serum (FBS; purchased from Sigma Chemical, St Louis, MO), penicillin and streptomycin (North China Parmaceutical Factory, Tianjing, People’s Republic of China), SNP (Lot 990318; Experimental Pharmaceutical Factory, Beijing Institute of Pharmaceutical Industry, Beijing, China), NO kit (Peiking Bangding Biotechnology Co Ltd, Peiking, China), Elx808 Unitra Microplate Reader system (Bio-Tek Instruments, Inc, Winooski, VT), inverted phase microscopy (Nikon, Instech Co, Ltd, Kanagawa, Japan), transmission electron microscopy (H-600; Hitachi Ltd, Tokyo, Japan), UV-VIS recording spectrophotometer (UV-2401PC; Shimadzu Corporation, Suzhow, China), automatic CO2 cupboard (Precision), phosphate-buffered saline (PBS), 10% sodium dodecyl sulfate (SDS), and crystal violet (obtained from our laboratory), and proteinase K and RNase (kind gifts of Dr Min Zhao). CELL LINE AND CELL CULTURE

The TSCCa cell line was established by Dr Jin Huixi from a patient with OSCC of the tongue at our hospital. TSCCa cells were cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated FBS, 100 u/mL penicillin, and 100 ␮g/mL streptomycin. The cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. CRYSTAL VIOLET ASSAY OF CYTOTOXICITY

Cytotoxic effects of SNP were determined with crystal violet assay according to Sumitani et al11 and Ohashi et al.17 Briefly, TSCCa cells were seeded at a density of 5 ⫻ 104 cells per well in 96-well microplates and incubated until they attained confluence. Then, the cells were exposed to SNP at various con-

EFFECTS OF NITRIC OXIDE ON TSCCa CELLS

centrations. After incubation for 48 hours, the medium was removed, and the cells were washed twice with PBS. One hundred microliters of 0.2% crystal violet solution was added to the pellets, and the plates were incubated for 30 minutes at room temperature. After 3 washes with distilled water, 100 ␮L of 1% SDS was added for 3 minutes. The optical density (OD) value was measured at 630 nm with an Elx808 Unitra Microplate Reader system. The inhibition rate (IR) of SNP against TSCCa cells was calculated according to the following formula. IR ⫽ (1 ⫺ OD experiment/OD control) ⫻ 100% DETERMINATION OF NO LEVELS IN CULTURE SUPERNATANT

The measurement of NO itself is difficult because of its short half-life in biologic systems ranging from 0.1 to 0.5 second.16 At the present, many are inclined to assay nitrite/nitrate indirectly for its presence. In this experiment, 100-␮L aliquots of culture supernatant were aspirated from each well of 96-well microplates for NO detection. After reducing nitrates to nitrites using nitrate reductase, total nitrite levels in the culture supernatant were determined with a commercially available NO kit according to the manufacturer’s instructions. The OD value at 530 nm was read with a UV-VIS recording spectrophotometer. NaNO2 was used as standard in this experiment. NO levels in the medium were calculated from the following formula. NO in vivo ⫽ OD sample/OD standard ⫻ 100 ( ␮ mol/L) NO levels were given as the mean value of the triplicate. MORPHOLOGIC AND ULTRASTRUCTURAL OBSERVATIONS ON SNP-TREATED TSCCa CELLS

TSCCa cells were treated with 2.5 mmol/L SNP for 48 hours. Then, morphologic changes were observed with reverse phase microscopy and graphed. After digestion with 0.25% trypsin, the TSCCa cells were centrifugated twice at a speed of 1,000 rpm, and the supernatant was removed. After fixation with 2.5% glutaraldehyde and gradient elution, the pellets were embedded with epoxy resin. Then, ultrathin sections were cut and stained for transmission electron microscopy (TEM) observation. DNA EXTRACTION AND AGAROSE GEL ELECTROPHORESIS

After incubation with 2.5 mmol/L SNP for 48 hours, TSCCa cells were pelleted at 4°C, resuspended in 1 mL of lysis solution (0.2 mmol/L Tris/HCl, pH 8.0, 20 mmol/L EDTA 䡠 Na2, 100 ␮g/mL proteinase K, and 0.5% SDS) and incubated for 5 hours at 50°C. After RNase treatment (100 ␮g/mL) for 1 hour at 37°C,

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DNA was extracted with phenol-chloroform mixture (vol/vol) and precipitated with 3 mmol/L sodium acetate (1:10 vol) and ice-cold ethanol (2.5 vol) for 1 hour at room temperature. DNA samples were mixed with loading buffer (30% glycerol, 0.1% bromphenol blue), loaded onto 1.5% agarose gels, and electrophoresed for about 1 hour at 50 V. The gels were stained with ethidium bromide, and the DNA bands were visualized under 312-nm light.

Results EFFECTS OF EXOGENOUS NO ON TSCCa CELLS

To investigate the cytotoxicity of exogenous NO against oral cancer cells, TSCCa cells were exposed to various concentrations of SNP for 48 hours, and then cytotoxicity and nitrite levels in the supernatant were examined. As illustrated in Fig 1, SNP released NO within the culture supernatant in a dose-dependent pattern. SNP also exhibited concentration-dependent cytotoxic effects on TSCCa cells (Fig 2). However, SNP seemed to have no cytotoxicity against TSCCa cells until the SNP level exceeded 1 mmol/L. According to Figs 1 and 2, our results suggested that the inhibition of TSCCa cells was attributed to the generation of NO from SNP and that lower concentrations of NO might have no cytotoxic effects on TSCCa cells. MORPHOLOGICAL AND ULTRASTRUCTURAL CHANGES OF SNP-TREATED TSCCa CELLS

After incubation with 2.5 mmol/L SNP for 48 hours, TSCCa cells presented remarkably morphologic changes. Under reverse phase microscopy, numerous TSCCa cells appeared to have a polygonal or short spindle shape and were arranged like paving in the absence of SNP. With the presence of SNP, however,

FIGURE 2. Cytotoxic effects of various concentrations of SNP on TSCCa cells.

the number of TSCCa cells decreased, with many cells floating in the medium, which made the micrograph slightly unclear. At the same time, round TSCCa cells obviously increased and exhibited an irregular spheroid shape (Figs 3A, B). On TEM examination, SNP-treated TSCCa cells showed typical changes of apoptosis (programmed cell death) (Figs 4B, C). These changes were characterized by an incomplete nuclear membrane, high nuclear condensation (or named as apoptotic body), chromatin margination or chromatin homogenization, disappearance of nucleolei and microvilli around the cytomembrane, vacuolar or reticular degeneration of cytoplasm, and obvious reduction in cell organs, especially mitochondrion. These changes might reflect that TSCCa cells were undergoing apoptosis. In addition, the SNP-treated cells became round. However, no such information was observed in the TSCCa cells without exposure to SNP (Fig 4A). There are large amounts of microvilli located around the cytomembrane, an obvious sign of nuclear division, and abundant mitochondrion and other cell organs, indicating a vigorous metabolism. AGAROSE GEL ELECTROPHORESIS OF DNA FROM SNP-TREATED TSCCa CELLS

FIGURE 1. NO levels in the medium supernatant when TSCCa cells were incubated with various concentrations of SNP for 48 hours.

To confirm that the ultrastructural changes resulted from apoptosis, the DNA was harvested from TSCCa cells that were untreated with SNP and treated with 2.5 mmol/L SNP for 48 hours. Agarose gel electrophoresis showed that typical internucleosomal DNA cleavage pattern (DNA ladder) was in agreement with apoptosis (Fig 5). This finding strongly supported the microscopic evidence of cell death and more rigorously showed the connection between NO and apoptosis.

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EFFECTS OF NITRIC OXIDE ON TSCCa CELLS

FIGURE 3. Photomicrographs showing morphologic changes of TSCCa cells in the control (A) and experiment (B) group (reverse phase microscope original magnification ⫻20).

Discussion NO was first identified as an endothelium-derived relaxing factor (EDRF) in 1987.15,18,19 Endogenous NO is synthesized from amino acid L-arginine by 3 isoforms of NO synthase (NOS). These NOS isoforms include neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), with each isoenzyme being the product of a distinct gene.12,15,16,20-22 As a striking mediator, messenger, and signal transduction molecule, NO has been reported to have many biologic activities and to participate in a variety of pathophysiologic processes, including the regulation of vascular tone, as a neuromodulator in the central nervous system, smooth muscle relaxation, mediation of the cytotoxic effects of macrophages, and activity during the process of angiogenesis.12,15-18,21 In recent years, several authors have found that NOS was overexpressed in gynecologic cancer, breast cancer, brain tumor, gastric cancer, lung cancer, and head and neck cancer.3,23,24 However, this was not sufficient to understand the true role of NO in carcinogenesis, and

the mechanisms by which NO killed tumor cells were poorly understood. NO is an unstable free radical gas with a short half-life in biologic systems, ranging from 0.1 to 0.5 second. This makes it difficult to distinguish the effects of NO itself on cancer cells. Recently, some authors have tried to study the effects of NO on tumor cells by using interferon-␣ and IL-1 as stimuli of endogenous NO production.15,22 This also had an undesirable method because interference was present, so the conclusions might be ambiguous. Therefore the NO donor is very important for biofunctional investigation on NO. The NO donor that is frequently used is a chemical donor. In the current study, we used SNP as an exogenous NO donor to investigate whether NO has cytotoxicity against TSCCa cells. It was well known that the biologic activity of SNP is mediated by releasing NO spontaneously.11,16,25 Thus, we proposed that the use of SNP had at least 2 advantages: easy regulation of NO concentration and absence of additional disturbance of the cell culture

FIGURE 4. Photomicrographs showing ultrastructural changes of TSCCa cells in the presence or absence of SNP. Cells were treated with no SNP (A) (transmission electron microscopy original magnification ⫻5,000) and 2.5 mmol/L SNP for 48 hours (B and C) (transmission electron microscopy original magnification ⫻12,000, ⫻3,500).

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FIGURE 5. Agarose gel electrophoresis of DNA from SNP-treated (left) or untreated (right) TSCCa cells.

model. Our results showed that SNP served as a successful NO donor for studying the biologic activity of NO in tumor biology because increasing concentrations of SNP gave a dose-related release of NO evaluated by nitrite/nitrate levels in the culture supernatant. Crystal violet assay revealed that exogenous NO that was chemically derived from SNP inhibited the growth of TSCCa cells. However, lower concentrations of SNP appeared to have no effects on the TSCCa cells. This might be explained by the dual role of NO in carcinogenesis: appropriate lower levels can promote the development and growth of tumor cells, and excessive NO production may, in contrast, be noxious to the tumor cells. The dual protumor and antitumor action of NO was recently shown to be dependent on the local concentration of the molecules.26 The extent of NO production may be related to the stage of cancer cells and may vary with various types of cancer cells. Our present experiment also showed that SNP has a dose-dependent cytotoxicity against TSCCa cells and that the cytotoxicity of SNP was attributed to its automatic release of NO into the culture supernatant. These results were almost identical to studies about cytotoxic potential of NO against different kinds of cells by using S-nitroso-Nacetyl-DL-penicillamine (SNAP), SNP, and 3-morpholinosydnonimine (SIN-1), all known as powerful NOdonating reagents.11,15,17 As described earlier, NO derived from SNP brought about severe damage to TSCCa cells. The vital TSCCa cells reduced and became round, with numerous dead cells floating in the medium. TEM showed that typical characteristics of apoptosis could be found in SNP-treated cells, and the cells with absence of SNP exhibited almost no change in appearance. These results were supported by some other studies and

thus were within our anticipated outcome. Albina et al27 first pointed out that NO could induce apoptosis, after they studied the effects of endogenous or exogenous NO on macrophages. Fehsel et al13 found the same phenomenon in mice chymocytes in vitro and in vivo. In human cancers, Ohashi et al17 and Sumitani et al11 concluded that there were apoptotic changes in tumor cells treated with NO donors. In this in vitro study, agarose gel electrophoresis showed characteristic DNA ladder fragmentation pattern in agreement with apoptosis of TSCCa cell line when exposed to 2.5 mmol/L of SNP for 48 hours. A similar result was found in human pancreatic tumor cell line (HA-hpc2); the typical DNA cleavage pattern, however, was absent when scavenger carboxy-PTIO was added into the medium.15 Activated macrophages can produce large amounts of NO that destroy or prevent the division of tumor cells through inhibition of DNA replication and prevention of mitochondrial respiration. NO was shown to account for the macrophage cytotoxic activities against tumor cells and to be related to tumor regression.28-30 Our results, together with these studies, might infer that NO is a key effector molecule of the activated macrophages. In our experience, mechanisms that have been proposed to explain the cytotoxicity of NO against various cells included its reaction with superoxide to generate cytotoxic peroxynitrite, inhibition of their synthesis of DNA, direct DNA damage of target cells, induction of programmed cell death, and suppression of c-myc and c-myb proto-oncogene expression.11,15,31 Taken together, NO could at least inhibit or kill tumor cells through induction of apoptosis. In conclusion, we postulated that endogenous NO might be a critical effector molecule of the activated macrophages for elimination of cancer cells and inhibition of tumor metastasis, although further studies are necessary. Among many mechanisms by which NO killed tumor cells, induction of cell apoptosis was important. It is also necessary to develop a novel therapeutic option based on NO donor drugs for the treatment of patients with oral squamous cell carcinoma. Acknowledgment The authors sincerely thank Dr Min Zhao, MD, PhD, Department of Molecular Biology, Medical School of Wuhan University, for his kind gift of several reagents. The authors also gave their sincere thanks to professor Hao-Lin Liang, Director of the Department of Electron Microscopy, Medical School of Wuhan University.

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