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Original Article |

Study on Ceramide Expression and DNA Content in Patients With Healthy Mucosa, Leukoplakia, and Carcinoma of the Larynx FREE

Fang-Lu Chi, MD, PhD; Ya-Sheng Yuan, MD; Shu-Yi Wang, MD; Zheng-Min Wang, MD
[+] Author Affiliations

From the Departments of Otolaryngology (Drs Chi, Yuan, and Z.-M. Wang) and Pathology (Dr S.-Y. Wang), Eye Ear Nose and Throat Hospital, Fudan University, Shanghai, People's Republic of China. The authors have no relevant financial interest in this article.


Arch Otolaryngol Head Neck Surg. 2004;130(3):307-310. doi:10.1001/archotol.130.3.307.
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Published online

Objective  To investigate the expression of ceramide produced by sphingomyelin and DNA content in patients with healthy laryngeal mucosa, leukoplakia, and laryngeal carcinoma.

Design  A retrospective review of the clinical and surgical records of 178 patients with leukoplakia of the larynx; 23 of them developed laryngeal carcinoma.

Setting  University medical center.

Patients  One hundred seventy-eight consecutive patients with leukoplakia of the larynx were identified from the archived pathology files of the Eye Ear Nose and Throat Hospital of Fudan University from January 1, 1990, to December 30, 2001. Among them, 23 developed laryngeal carcinoma. Flow cytometry and immunohistochemistry were performed to test DNA content and ceramide expression in healthy tissue, tissue with leukoplakia, and tissue with laryngeal carcinoma from the same patient.

Results  Among 23 patients with leukoplakia, 20 had aneuploidy and 3 had diploidy. The healthy tissues were all diploids, and the tissues with laryngeal carcinoma were all aneuploids. The expression of ceramide decreased gradually from healthy tissue to tissue with leukoplakia to tissue with laryngeal carcinoma (0, no staining; 1+, weak staining; 2+, mild staining; 3+, moderate staining; 4+, strong staining; and 5+, the highest staining intensity). The expression of ceramide in DNA diploid cells is stronger than in aneuploid cells.

Conclusions  Ceramide, the second messenger in apoptosis, may associate with the progress of leukoplakia to carcinoma of the larynx. The reduction of ceramide may contribute to laryngeal carcinogenesis.

Figures in this Article

The process of programmed death is known as apoptosis. Cellular apoptosis is thought to be a genetically determined process critical in normal development and organ homeostasis, acting on healthy and malignant tissues.1,2 The loss of apoptotic regulation has been hypothesized to result in excessive cellular accumulation or loss, leading to various pathological processes. Investigators are increasingly studying the role of apoptotic dysregulation in diseases of the head and neck.3

Recent research has shown that tumor necrosis factor (TNF)–TNF receptor and Fas–Fas ligand are key molecular determinants of this intracellular death process.4 Ceramide is the second messenger. Ceramides produced by sphingomyelin hydrolysis activate a cycle that is followed by 3 different major cellular responses: down-regulation of cell proliferation, induction of cell differentiation, and apoptosis.5 The process may be blocked when the intracellular ceramide level decreases. Liu et al6 noted that glycosylation of ceramide potentates cellular resistance to TNF-α–induced apoptosis. Sautin et al7 described ceramide-induced apoptosis of human thyroid cancer cells resistant to apoptosis by irradiation. Deshmukh et al8 found that glycosylation of ceramide is the key factor in epithelial proliferation of the renal tubule. However, there have been few studies regarding the significance of ceramide in laryngeal carcinogenesis. The present study investigates the relationship between intracellular ceramide expression and laryngeal carcinogenesis. In addition, DNA ploidy has been analyzed as a prognostic, or predictive, factor for head and neck squamous cell carcinoma. The most important significance of DNA ploidy as a biological variable is to reflect the accumulation of multiple genomic alterations, which indicate the aggressiveness of the cancer cells.9,10 Therefore, we also investigate the relationship between DNA ploidy and intracellular ceramide expression.

PATIENT DATA

One hundred seventy-eight consecutive patients with leukoplakia of the larynx were identified from the archival pathology files of the Eye Ear Nose and Throat Hospital of Fudan University from January 1, 1990, to December 30, 2001; 23 of these patients developed laryngeal carcinoma. We studied these 23 patients. Specimens from these patients included tissues from their healthy laryngeal mucosa, tissues with leukoplakia, and tissues with laryngeal carcinoma. The specimens were obtained using a direct laryngoscope. All of the tumors were squamous cell carcinoma. Sixteen specimens were grade 1, and 7, grade 2. Patient medical records were reviewed, and clinical data were recorded. This well-characterized panel of human tissues has been immunostained and tested by flow cytometry from the original panel tissue.

FLOW CYTOMETRIC ANALYSIS

Paraffin-stored blocks were found by flow cytometry. A suspension of isolated nuclei for the DNA ploidy analysis was prepared as described by Vindelov et al.11 The suspensions were analyzed with a flow cytometer (Epics MCL XL; Beckman Coulter, Miami, Fla), which was interfaced to a computer system multiplus (Phoenix Flow Systems, San Diego, Calif). Diploidy and aneuploidy were defined as described by Tsutsui et al.12,13 Diploidy was defined as a single G0/G1 peak on the DNA histogram. Aneuploidy was defined as a clearly distinct additional G0/G1 peak with a small G2/M peak. The DNA index of the aneuploid peak was calculated as the ratio of the DNA content of the aneuploid peak to the DNA content of the diploid peak. A multivariate analysis was performed using the Cox proportional hazards regression model. P≤.05 was regarded as statistically significant.

IMMUNOHISTOCHEMISTRY

Well-characterized commercially available mouse monoclonal antibody to ceramide (MID 15B4; ALEXIS Biochemicals, San Diego, Calif) was used for immunohistochemistry analysis. Tissue within a block was cut in 5-µm thickness; and sections underwent deparaffinization, were washed in 0.02M phosphate-buffered saline, and were then incubated in primary antibody (dilution, 1:10) overnight in the refrigerator. After washing, sections were incubated in a second antibody (Envision; DakoCytomation Inc, Carpinteria, Calif) (dilution, 1:200) for 2 hours, then stained with 3′,3-diaminobenzidine hydrochloride for half an hour, and observed under the light microscope. The ceramide staining was scored as the percentage of cell staining positive per high-power field. The overall intensity of the staining reaction against ceramide was scored as follows: 0, no staining; 1+, weak staining; 2+, mild staining; 3+, moderate staining; 4+, strong staining; and 5+, the highest staining intensity. For the negative control, we used phosphate-buffered saline to replace the primary antibody. The staining of the immunohistochemistry analysis was blindly assessed by 2 pathologists in the Fudan University Medical Center.

Immunohistochemistry staining intensity was subjected to statistical evaluation, performed with a statistical computer program (SigmaStat; Jandel Scientific Software, San Rafael, Calif). A Kruskal-Wallis analysis of variance, a Mann-Whitney rank sum test, and a Spearman rank correlation test were performed where appropriate. P≤.05 was considered significant.

HISTOGRAM OF DNA PLOIDY

Diploidy has a single G0/G1 peak on the DNA histogram (Figure 1). Aneuploidy has a clearly distinct additional G0/G1 peak with a smaller G2/M peak (Figure 2). This indicates that the aneuploid cell has gone into the mitosis stage of the cell cycle. Cellular proliferation has begun.

Place holder to copy figure label and caption
Figure 1.

Histogram of DNA diploidy. Diploidy has a single G0/G1 peak on the DNA histogram (ie, the cells stay in the G0/G1 stage).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Histogram of DNA aneuploidy. Aneuploidy has a clearly distinct additional G0/G1 peak with a smaller G2/M peak (ie, the cells have gone into mitosis).

Graphic Jump Location
RELATIONSHIP BETWEEN DNA PLOIDY AND CARCINOGENESIS

All 20 patients with leukoplakia who had aneuploidy developed laryngeal carcinoma; of the 158 patients with leukoplakia who had diploidy, only 3 developed laryngeal carcinoma. This indicates that the rate of carcinomatous change in aneuploid cells is higher than in diploid cells (P<.01).

OVERALL STATUS OF DNA PLOIDY

The overall status of DNA ploidy in healthy tissues, tissues with leukoplakia, and tissues with carcinoma of the larynx is as follows. All 23 healthy tissues had diploidy, while all 23 tissues with laryngeal carcinoma had aneuploidy. Finally, among 23 patients with leukoplakia whose disease progressed to laryngeal carcinoma, 20 had aneuploidy and 3 had diploidy.

CERAMIDE STAINING INTENSITIES

Positive labeling was observed in the cytoplasm of tested tissue (Figure 3). Strong ceramide expression was observed in healthy mucosa (mean ±SD, 4.0 ± 0.3); in contrast, weak ceramide expression was observed in tissues with laryngeal carcinoma (mean ± SD, 0.1 ± 0.1). Tissues with leukoplakia showed an intermediate level of ceramide expression (mean ± SD, 1.8 ± 0.2). A significant difference (P<.01) in staining intensities was found among healthy tissues (Figure 3), tissues with leukoplakia (Figure 4), and tissues with carcinoma of the larynx (Figure 5).

Place holder to copy figure label and caption
Figure 3.

Strong ceramide expression was observed in healthy mucosa of the larynx.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Weak ceramide expression was observed in tissues with leukoplakia of the larynx.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.

Ceramide expression was almost negative in tissues with carcinoma of the larynx.

Graphic Jump Location
COMPARISON OF STAINING INTENSITIES BETWEEN DIPLOID AND ANEUPLOID CELLS

More intense ceramide staining was shown in diploid cells, and weaker ceramide expression was observed in aneuploid cells (P = .01). Therefore, the expression of ceramide in aneuploid cells is less than in diploid cells.

A model of neoplastic development has been hypothesized to involve a disruption in the balance between cell proliferation and cell death.14 The role of apoptosis in the regulation of tissue growth is to gain increased attention as a contributing factor in neoplastic growth.14

The induction of apoptosis can be initiated by such signals as cytokines, hormones, toxin exposure, and the withdrawal of various survival factors. Ceramide, produced by sphingomyelin, is the second messenger in apoptosis mediated by TNF–TNF receptor and Fas–Fas ligand. Ceramide is a key factor in this process.6 The decrease of ceramide, such as the glycosylation of ceramide, has been hypothesized to result in excessive cellular accumulation, leading to various pathological processes, such as inflammation proliferation and precancerous lesions.5 To understand the function of ceramide from precancerous lesions to carcinoma of the larynx, we investigated the expression of ceramide. In our retrospective study, ceramide expression gradually decreased from healthy laryngeal mucosa to tissues with leukoplakia to tissues with carcinoma of the larynx. A significant difference in ceramide expression has been found between each of them (P<.05). Because of the decrease of ceramide expression, the process of apoptosis is blocked. The balance of proliferation and apoptosis breaks. Therefore, healthy mucosa progressed into precancerous lesions and leukoplakia progressed into laryngeal cancer. Similar results were reported in a study15 of breast cancer and colon carcinoma.

DNA ploidy has been analyzed as a prognostic, or predictive, factor for head and neck squamous cell carcinoma. The most important significance of DNA ploidy is as a biological variable that indicates the aggressiveness of cancer cells.9,10 We found that the incidence of carcinomatous change in aneuploid cells is much higher than in diploid cells (P<.01). DNA aneuploidy may be a marker in the early stage of carcinomatous change. The expression of ceramide in aneuploid cells is less than in diploid cells. The staining intensity was as follows: diploid cells in healthy tissues (n = 20), 4.0 ± 0.2; diploid cells in tissues with leukoplakia (n = 3), 3.1 ± 0.2; and aneuploid cells in tissues with leukoplakia (n = 20), 1.5 ± 01. Reduction of ceramide expression in patients with laryngeal carcinoma resulted in the blockage of apoptosis.

Several studies have shown that sphingolipids control the balance in cells between growth and proliferation and cell death by apoptosis. Sphingosine-1-phosphate and glucosylceramide induce the proliferation process; and ceramide, a metabolic intermediate between the 2, induces apoptosis.16 In cancers, the balance seems to have come undone and it should be possible to kill the cells by enhancing the processes that lead to ceramide accumulation. The 2 control systems are intertwined, modulated by many different agents affecting the activities of the enzymes in ceramide–glucosylceramide–sphingosine-1-phosphate interdependence.16 It has been proposed that successful cancer chemotherapy requires the use of many agents to elevate ceramide levels adequately. Inherent or acquired drug resistance, which frequently characterizes cancer cells, is caused by multiple mechanisms, including dysfunctional metabolism of the lipid second messenger, ceramide.17 Ceramide, the basic structural unit of the sphingolipids, plays a role in activating cell death signals initiated by cytokines, chemotherapeutic agents, and ionizing radiation.17

Recent research about the metabolism of ceramide suggests that this agent may have an important influence on the effectiveness of various cancer therapeutic agents. In particular, the cytotoxic effect of chemotherapy is decreased when the generation of ceramide is impaired, but is increased when the degradation of ceramide is blocked.17 It has been proposed that successful cancer chemotherapy requires the use of many agents to elevate the ceramide level adequately.

In conclusion, ceramide, the second messenger in apoptosis, may associate with the progress of precancerous lesions to laryngeal cancer. Elevating the intracellular content of ceramide will be a new way to interfere with carcinogenesis.

Corresponding author and reprints: Fang-Lu Chi, MD, PhD, Department of Otolaryngology, Eye, Ear, Nose, and Throat Hospital, Fudan University, 83 Fenyang Rd, Shanghai 200031, People's Republic of China (e-mail: chifanglu@Yahoo.com.cn).

Submitted for publication February 7, 2003; final revision received June 3, 2003; accepted August 12, 2003.

This study was supported by Fund Z-43 from the Shanghai Science and Technology Committee, Shanghai, People's Republic of China.

Searle  JCollins  DJHarmon  BKerr  JF The spontaneous occurrence of apoptosis in squamous carcinomas of the uterine cervix. Pathology.1973;5:163-169.
PubMed
Wyllic  AH The biology of cell death in tumours. Anticancer Res.1985;5:131-136.
PubMed
Mehta  SBlackinton  DOmar  I  et al Combined cytotoxic action of paclitaxel and ceramide against the human Tu138 head and neck squamous carcinoma cell line. Cancer Chemother Pharmacol.2000;46:85-92.
PubMed
Galibert  LTometsko  MEAnderson  DMCosman  DDougall  WC The involvement of multiple tumor necrosis factor receptor (TNFR)–associated factors in the signaling mechanisms of receptor activator of NF-κB, a member of the TNFR superfamily. J Biol Chem.1998;273:34120-34127.
PubMed
Di Nardo  ABenassi  LMagnoni  CCossarizza  ASeidenari  SGiannetti  A Ceramide 2 (N-acetyl sphingosine) is associated with reduction in Bcl-2 protein levels by Western blotting and with apoptosis in cultured human keratinocytes. Br J Dermatol.2000;143:491-497.
PubMed
Liu  YYHan  TYGiuliano  AE  et al Glycosylation of ceramide potentates cellular resistance to tumor necrosis factor-α–induced apoptosis. Exp Cell Res.1999;252:464-470.
PubMed
Sautin  YTakamura  NShklyaev  S  et al Ceramide-induced apoptosis of human thyroid cancer cells resistant to apoptosis by irradiation. Thyroid.2000;10:733-740.
PubMed
Deshmukh  GDRadin  NSGattone  VH  et al Abnormalities of glycosphingolipid, sulfatide, and ceramide in the polycystic (cpk/cpk) mouse. J Lipid Res.1994;35:1611-1618.
PubMed
Watanabe  MKuwano  HTanaka  S  et al Flow cytometric DNA analysis is useful in detecting multiple genetic alterations in squamous cell carcinoma of the esophagus. Cancer.1999;85:2322-2328.
PubMed
Schackney  SEBerg  GSimon  SR  et al Origins and clinical implications of aneuploidy in early bladder cancer. Cytometry.1995;22:307-316.
PubMed
Vindelov  LLChristensen  IJNissen  NI A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry.1983;3:323-327.
PubMed
Tsutsui  SOhno  SMurakami  SHachitanda  YOda  S Prognostic value of DNA ploidy in 653 Japanese women with node-negative breast cancer. Int J Clin Oncol.2001;6:177-182.
PubMed
Tsutsui  SKuwano  HMori  M  et al A flow cytometric analysis of DNA content in primary and metastatic lesions of esophageal squamous cell carcinoma. Cancer.1992;70:2586-2591.
PubMed
Green  DRMartin  SJ The killer and the executioner: how apoptosis controls malignancy. Curr Opin Immunol.1995;7:694-703.
PubMed
Burow  MEWeldon  CBTang  Y  et al Differences in susceptibility to tumor necrosis factor alpha–induced apoptosis among MCF-7 breast cancer variants. Cancer Res.1998;58:4940-4946.
PubMed
Radin  NS Killing cancer cells by poly-drug elevation of ceramide levels: a hypothesis whose time has come? Eur J Biochem.2001;268:193-204.
PubMed
Senchenkov  ALitvak  DACabot  MC Targeting ceramide metabolism—a strategy for overcoming drug resistance. J Natl Cancer Inst.2001;93:347-357.
PubMed

Figures

Place holder to copy figure label and caption
Figure 1.

Histogram of DNA diploidy. Diploidy has a single G0/G1 peak on the DNA histogram (ie, the cells stay in the G0/G1 stage).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Histogram of DNA aneuploidy. Aneuploidy has a clearly distinct additional G0/G1 peak with a smaller G2/M peak (ie, the cells have gone into mitosis).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Strong ceramide expression was observed in healthy mucosa of the larynx.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Weak ceramide expression was observed in tissues with leukoplakia of the larynx.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.

Ceramide expression was almost negative in tissues with carcinoma of the larynx.

Graphic Jump Location

Tables

References

Searle  JCollins  DJHarmon  BKerr  JF The spontaneous occurrence of apoptosis in squamous carcinomas of the uterine cervix. Pathology.1973;5:163-169.
PubMed
Wyllic  AH The biology of cell death in tumours. Anticancer Res.1985;5:131-136.
PubMed
Mehta  SBlackinton  DOmar  I  et al Combined cytotoxic action of paclitaxel and ceramide against the human Tu138 head and neck squamous carcinoma cell line. Cancer Chemother Pharmacol.2000;46:85-92.
PubMed
Galibert  LTometsko  MEAnderson  DMCosman  DDougall  WC The involvement of multiple tumor necrosis factor receptor (TNFR)–associated factors in the signaling mechanisms of receptor activator of NF-κB, a member of the TNFR superfamily. J Biol Chem.1998;273:34120-34127.
PubMed
Di Nardo  ABenassi  LMagnoni  CCossarizza  ASeidenari  SGiannetti  A Ceramide 2 (N-acetyl sphingosine) is associated with reduction in Bcl-2 protein levels by Western blotting and with apoptosis in cultured human keratinocytes. Br J Dermatol.2000;143:491-497.
PubMed
Liu  YYHan  TYGiuliano  AE  et al Glycosylation of ceramide potentates cellular resistance to tumor necrosis factor-α–induced apoptosis. Exp Cell Res.1999;252:464-470.
PubMed
Sautin  YTakamura  NShklyaev  S  et al Ceramide-induced apoptosis of human thyroid cancer cells resistant to apoptosis by irradiation. Thyroid.2000;10:733-740.
PubMed
Deshmukh  GDRadin  NSGattone  VH  et al Abnormalities of glycosphingolipid, sulfatide, and ceramide in the polycystic (cpk/cpk) mouse. J Lipid Res.1994;35:1611-1618.
PubMed
Watanabe  MKuwano  HTanaka  S  et al Flow cytometric DNA analysis is useful in detecting multiple genetic alterations in squamous cell carcinoma of the esophagus. Cancer.1999;85:2322-2328.
PubMed
Schackney  SEBerg  GSimon  SR  et al Origins and clinical implications of aneuploidy in early bladder cancer. Cytometry.1995;22:307-316.
PubMed
Vindelov  LLChristensen  IJNissen  NI A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry.1983;3:323-327.
PubMed
Tsutsui  SOhno  SMurakami  SHachitanda  YOda  S Prognostic value of DNA ploidy in 653 Japanese women with node-negative breast cancer. Int J Clin Oncol.2001;6:177-182.
PubMed
Tsutsui  SKuwano  HMori  M  et al A flow cytometric analysis of DNA content in primary and metastatic lesions of esophageal squamous cell carcinoma. Cancer.1992;70:2586-2591.
PubMed
Green  DRMartin  SJ The killer and the executioner: how apoptosis controls malignancy. Curr Opin Immunol.1995;7:694-703.
PubMed
Burow  MEWeldon  CBTang  Y  et al Differences in susceptibility to tumor necrosis factor alpha–induced apoptosis among MCF-7 breast cancer variants. Cancer Res.1998;58:4940-4946.
PubMed
Radin  NS Killing cancer cells by poly-drug elevation of ceramide levels: a hypothesis whose time has come? Eur J Biochem.2001;268:193-204.
PubMed
Senchenkov  ALitvak  DACabot  MC Targeting ceramide metabolism—a strategy for overcoming drug resistance. J Natl Cancer Inst.2001;93:347-357.
PubMed

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