Status of salivary human beta defensin-2 in oral potentially malignant disorders and oral cancer: Quest for a novel non-invasive biomarker
Ananya Madiyal1, Subhas G. Babu2, Suchetha Kumari N3, Sharmila K. P4, Prajna U5,
Deesha Kumari6, Mohammed Faizal Asan7
1,2AB Shetty Memorial Institute of Dental Sciences (ABSMIDS), Nitte (Deemed to be University),
Department of Oral Medicine and Radiology.
3KS Hegde Medical Academy (KSHEMA), Nitte (Deemed to be University), Department of Biochemistry.
4,5KS Hegde Medical Academy (KSHEMA), Nitte (Deemed to be University), Central Research Laboratory.
6AB Shetty Memorial Institute of Dental Sciences (ABSMIDS), Nitte (Deemed to be University),
Department of Public Health Dentistry.
7Indus Group of Dental Diagnostics, Andhra Pradesh.
*Corresponding Author E-mail: ananyamadiyal@gmail.com
ABSTRACT:
Background: Oral cancer ranks sixth among all the types of cancers globally and contributes to significant mortality and morbidity. Inflammation is known to play an important role in tumorigenesis. Human Beta Defensins are a type of AMP that play a role as chemo attractive, antimicrobial, and antitumor agents and also act as immunomodulators. They have also been demonstrated in cancer cell lines. Beta defensins act as tumor suppressor genes by manipulating the tumor microenvironment. The existing literature on human beta defensin-2 activity is scarce. There exists no literature on the comparison of the level of salivary human beta defensin-2 between subjects with oral potentially malignant disorders and oral cancer. Saliva contains constituents that reflect the physiologic state of the body. This can be utilized for rapid and atraumatic diagnosis of diseases owing to its non-invasive nature of collection. Aims and Objectives: The aim of the present study was to assess the level of human beta defensin-2 in the saliva of subjects with oral potentially malignant disorders and oral cancer and compare them with levels in healthy subjects. Materials and methods: The study sample included 75 subjects who were divided into three groups consisting of healthy subjects, subjects with oral potentially premalignant disorders and subjects with oral cancer. Results: The mean salivary Human beta defensin-2 level in subjects with oral cancer was significantly higher than in healthy controls and subjects with oral potentially malignant disorders. The level was highest in the oral cancer group and least in the control group. This difference among the 3 groups was statistically significant. In the group with premalignant disorders, the variation in the level of salivary human beta defensin-2 according to the type of lesion was not statistically significant. Conclusion: This study highlights the diagnostic role of hBD-2 in saliva. The presence of Human beta defensin-2 in the saliva of healthy controls points to its role in the maintenance of mucosal integrity. Elevation in the level of hBD-2 in oral potentially malignant disorders and a further increase in oral cancer indicate the potential use of hBD-2 as a biomarker in early diagnosis of oral cancer. Use of saliva as the diagnostic fluid aids in establishing a non-invasive and atraumatic means of diagnosis.
KEYWORDS: Neoplasm, Malignancy, Leukoplakia, Biomarker, Defensin, Saliva.
INTRODUCTION:
Both smoking and smokeless forms of tobacco play a major role in the etiology of oral cancer.5 Smokeless tobacco causes continued exposure of the oral mucosa to carcinogens along with micro-abrasion of the epithelial lining.6,7 The metabolic products of alcohol like aldehydes have a well-known carcinogenic effect. Alcohol also increases the penetration of other carcinogens into the oral cavity by altering the mucosal permeability. Combined with the use of tobacco products, alcohol may cause oral mucosal lesions. Potentially malignant disorders along with oral carcinomas cause significant morbidity and mortality in patients and decrease their quality of life.8
Inflammation is the reaction to cell injury.9 It is known to play an important role in tumorigenesis.6 The innate and adaptive arms of immunity help in the prevention of disease progression. They are augmented by physical and chemical barriers that produce Antimicrobial Peptides (AMP). These AMPs have been found in neutrophils, epithelium, as well as saliva.10 Human Beta Defensins (hBD) are a type of AMP that play a role as chemo attractive, antimicrobial, and antitumor agents and also act as immunomodulators.11 hBD are expressed in numerous tissues and influence the adaptive immune response as chemokines. They also protect the mucosal surface by playing a role in wound healing.12 hBD are vital for tissue defense and local homeostasis in the oral cavity. They are expressed in response to pathogens, proinflammatory mediators, and tumor necrosis factors.10
hBD have also been demonstrated in cancer cell lines.13 Human Beta Defensin-2 (hBD-2) is known to induce angiogenesis, cause migration of endothelial cells and recruit immature dendritic cells and memory T cells to the tumor site where it facilitates their maturation and activation thereby acting as a tumor suppressor.14 Dysregulation of hBD-2 mRNA is seen in various cancers in the human body. It may be upregulated or downregulated based on the anatomic location and cancer type.13 Beta defensins act as tumor suppressor genes by manipulating the tumor microenvironment. They have shown a direct cytotoxic effect towards cancer cells and regulate tumor growth and metastasis.14
Timely diagnosis of oral cancer can help prevent financial, psychological, and functional loss to the patient.4,15A necessity for early and easy detection of oral cancer has led to research on various biomarkers in tissues and body fluids.16 Saliva contains constituents that reflect the physiologic state of the body. This can be utilized for rapid and atraumatic diagnosis of diseases owing to its non-invasive nature of collection.17 A recent surge of research has been seen in the area of salivary proteomic, metabolomic, transcriptomic, and genomic biomarkers.
The existing literature on hBD-2 activity is scarce. Most of these studies have been done on tissue samples. There exists no literature on the comparison of the level of salivary human beta defensin-2 between subjects with oral potentially malignant disorders and oral cancer. The results of the present study could be useful in determining hBD-2 as a salivary biomarker in the diagnosis and prognosis of oral potentially malignant disorders and oral cancer. Use of saliva as the diagnostic fluid aids in establishing a non-invasive and atraumatic means of diagnosis.
MATERIALS AND METHODS:
The authors designed and conducted a cross sectional study on subjects reporting to a Dental Hospital over a period of two years. The sample population consisted of semi-urban and rural subjects.
The study sample included 75 subjects in the age group of 20 to 70 years who were divided into the following groups: The first group was of healthy subjects, Group II was subjects with oral potentially premalignant disorders and Group III consisted of subjects with oral cancer. Subjects with history of any systemic diseases, malignancies, any other oral mucosal lesions, pregnant women, subjects who are on any medications, and subjects with oral potentially malignant disorders and oral cancer without adverse oral habits were excluded from the study.
Sample collection and estimation:
Informed consent was obtained from each subject before commencement of the study. Institutional Ethics Committee provided the ethical clearance. Spit technique was used to collect two-hours post-prandial saliva sample. Supernatant was obtained by centrifugation (10 minutes at 300 rotations per minute) and stored at -200C.
Estimation of Human beta defensin-2 was done using the GENLISA ELISA (Krisgen Biosystems) kits.
RESULTS:
Demographic data analysis of the study groups:
The age of the subjects ranged from 20 to 70 years. The mean age in Group I was 32.64+10.21 years, in Group II was 46.47+11.97 years and in Group III was 51.96+13.36 years. 40% of the subject in Group I, 93.3% in Group II and 80% in Group III were males while females comprised of 60% in Group I, 6.7% in Group II and 20% in Group III (Table 1).
Table 1: Table showing distribution of study subjects according to age and gender
|
|
Group 1 - Control Group |
Group 2 - Oral potentially malignant disorders |
Group 3- Oral cancer |
|
|
Age |
32.64+ 10.206 |
46.47+ 11.968 |
51.96+ 13.359 |
|
|
Gender |
Male |
10 (40%) |
28 (93.3%) |
20 (80%) |
|
|
Female |
15 (60%) |
2 (6.7%) |
5 (20%) |
Analysis of salivary human beta defensin-2 levels in the study groups:
The mean salivary Human beta defensin-2 level in Group I was 6.4+2.4 ng/ml, while in Group I and Group II it was 7.56+3.2 ng/ml and 10.25+3.95 ng/ml respectively. The salivary human beta defensin-2 level was highest in the oral cancer group and least in the control group. This difference in the salivary human beta defensin-2 levels among the 3 groups was statistically significant (p=0.000) (Table 2).
Tukey’s post hoc analysis showed that the difference in the level of salivary human beta defensin-2 in the oral cancer group was significantly different from the control group and Oral potentially malignant disorders group (p=0.000 and p=0.008 respectively) (Table 3).
No significant difference was observed in the level of salivary human beta defensin-2 between males and females in all the 3 groups. In the group with premalignant disorders, the variation in the level of salivary human beta defensin-2 according to the type of lesion was not statistically significant (Table 4, 5).
Table 2: Table showing One-Way ANOVA analysis
|
|
N |
Mean |
Std. Deviation |
Std. Error |
95% Confidence Interval for Mean |
Sum of squares |
Mean Square |
F |
P value |
|
|
Lower Bound |
Upper Bound |
|||||||||
|
Group 1 |
25 |
6.3976 |
2.40581 |
0.4811 |
5.4045 |
7.3907 |
197.021 |
98.510 |
9.370 |
0.000* |
|
Group 2 |
30 |
7.5587 |
3.19864 |
0.5839 |
6.3643 |
8.7531 |
||||
|
Group 3 |
25 |
10.2548 |
3.94691 |
0.7893 |
8.6256 |
11.8840 |
||||
Table 3: Table showing Post Hoc Tukey Test
|
Tukey HSD |
||||||
|
(I) Group |
(J) Group |
Mean Difference (I-J) |
Std. Error |
Sig. |
95% Confidence Interval |
|
|
Lower Bound |
Upper Bound |
|||||
|
Group 1 |
Group 2 |
-1.16107 |
0.87803 |
0.387 |
-3.2594 |
0.9373 |
|
Group 3 |
-3.85720* |
0.91708 |
0.000* |
-6.0489 |
-1.6655 |
|
|
Group 2 |
Group 1 |
1.16107 |
0.87803 |
0.387 |
-0.9373 |
3.2594 |
|
Group 3 |
-2.69613* |
0.87803 |
0.008* |
-4.7945 |
-0.5978 |
|
|
*The mean difference is significant at the 0.05 level. |
||||||
Table 4: Table showing difference in Human beta defensin-2 levels between genders in each study group
|
|
Gender |
N |
Mean |
Std. Deviation |
Std. Error Mean |
95% Confidence Interval of the Difference |
Mean Difference |
t |
df |
P value |
|
|
Lower |
Upper |
||||||||||
|
Group 1 |
Male |
10 |
6.2650 |
1.8548 |
0.58656 |
-2.29428 |
1.85228 |
-.22100 |
-0.221 |
23 |
0.827 |
|
Female |
15 |
6.4860 |
2.7729 |
0.71598 |
|||||||
|
Group 2 |
Male |
28 |
7.6786 |
3.2787 |
0.61962 |
-3.03206 |
6.62920 |
1.79857 |
0.763 |
28 |
0.452 |
|
Female |
2 |
5.8800 |
0.6505 |
0.46000 |
|||||||
|
Group 3 |
Male |
20 |
9.9485 |
4.0913 |
0.91485 |
-5.64905 |
2.58605 |
-1.53150 |
-0.769 |
23 |
0.449 |
|
Female |
5 |
11.480 |
3.4077 |
1.52399 |
|||||||
Table 5: Table showing difference in Human beta defensin-2 between premalignant lesions
|
Group 2 |
N |
Mean |
Std. Deviation |
Std. Error |
95% Confidence Interval for Mean |
Sum of squares |
Mean Square |
F |
P value |
|
|
Lower Bound |
Upper Bound |
|||||||||
|
OSMF |
9 |
8.2478 |
3.51612 |
1.17204 |
5.5450 |
10.9505 |
36.252 |
12.084 |
1.206 |
0.327 |
|
Leukoplakia |
17 |
7.3294 |
3.08626 |
0.74853 |
5.7426 |
8.9162 |
||||
|
OSMF+Leukoplakia |
2 |
9.7100 |
2.95571 |
2.09000 |
-16.8460 |
36.2660 |
||||
|
Tobacco pouch keratosis |
2 |
4.2550 |
0.64347 |
0.45500 |
-1.5263 |
10.0363 |
||||
DISCUSSION:
While clinical examination and chairside investigations such as biochemical investigations, chemiluminescence, exfoliative cytology, vital staining, brush biopsy, and fluorescence imaging help in detecting oral cancer, biopsy remains the gold standard.18,19 Oral self-examination, screening programs, and opportunistic screening can help in decreasing diagnostic delay. However, Fuller et al found that oral cytology had better diagnostic value than clinical examination alone.20Recent advances in molecular biology have led to the discovery of biomarkers. Biomarkers act as molecular indicators of disease, prognosis, as well as response to treatment. In the multistep model of tumorigenesis, the various genotypic and phenotypic alterations can be identified in body fluids that drain the tumor.21 Detection of biomarkers in biological fluids helps in early diagnosis and prompt treatment.22,23
Saliva, with its non-invasive and inexpensive sampling technique is gaining popularity in the diagnosis of various diseases.24 Saliva has intimate contact with oral lesions which increases its sensitivity and specificity during diagnosis of mucosal diseases. Cristaldi et al stated that oral fluid could be a potential source of diagnostic and prognostic biomarkers for OSCC owing to the presence of DNA, RNA, cytokines, tissue derived cells, and circulating cells.22
Despite recent advances in the diagnosis and treatment of oral cancer, the World Health Organization reports a 5-year mortality rate of 45%.25Since oral cancers remain asymptomatic until advanced stages, early diagnosis becomes essential in decreasing the morbidity and mortality associated with it. Biomarkers for oral cancer diagnosis can detect altered proteins, altered mRNA transcripts or alteration in the DNA.21
Defensins are antimicrobial peptides which are positively charged and have a molecular weight in the range of 3.5 kDA – 6.5 kDa. They are broadly classified into alpha, beta, and delta defensins based on the disulfide bond binding patterns as well as their size. The beta-defensins are six in number and have been mapped to chromosome 8p.21 Defensins show an antimicrobial property through their ability to disintegrate cellular membranes.25 Along with their microbial barrier action through the innate arm of immunity, they also demonstrate chemotactic effect via cell-mediated immunity.26
Defensins have been demonstrated in the genitourinary tract, gastrointestinal tract, oral cavity as well as salivary glands. Saliva contains various types of antimicrobial peptides (AMP). Defensins, also grouped under AMP, have been demonstrated in human saliva. 27 In cases of oral squamous cell carcinoma, defensins control induction and transcription of apoptosis. Human beta-defensin 2 plays a role in keratinocyte death thereby promoting tumor progression.28
Although literature on hBD in saliva is scarce, research has been done on tissue samples. Most of the studies used immunohistochemistry and liquid chromatography to estimate the level of Human beta-defensin 2 in tissues. In the present study, the mean Human beta defensin-2 concentration in saliva of healthy controls was 6.4±2.4 mg/dl. Salem et al suggested that the small amount of expressions of hBD-2 observed in samples of normal oral mucosa could be attributed to its role in maintaining mucosal health since it is a part of the innate arm of immunity.29
In the premalignant group we observed that the level of Human beta-defensin 2 was increased to 7.6±3.2 mg/dl and in the oral cancer group the level was further increased to 10.2±3.9 mg/dl. It is hypothesized that stimulation of hBD-2 occurs due to the loss of epithelial integrity in potentially malignant and malignant lesions. Human beta-defensin 2, being an antimicrobial peptide, mediates immune cell chemotaxis and production of cytokines thereby mounting an inflammatory response.29 Since hBD are antimicrobial peptides, oral carcinomas which showed a downregulation of the expression of hBD might be susceptible to bacterial infection.13
Although research has been carried out regarding the expression of hBD-2 in cell lines and tissue samples, there appears to be a dearth in knowledge regarding the level of hBD-2 in body fluids. Saliva, owing to its non-invasive sampling technique must be explored as a potential source of biomarkers in the diagnosis and prognosis of diseases. Although we found one study byKucukkolbasi et al where the salivary level of hBD was estimated, they mainly concentrated their research on oral inflammatory disorders.30 There have been no studies that compare and correlate the levels of Human beta defensin-2 in saliva of patients with oral potentially malignant disorders and oral cancer. A potential for therapeutic use of such antimicrobial peptides exists due to their role in augmenting innate immunity. Therefore, further investigation is necessary to harness the possible use of hBD-2 in the treatment of oral mucosal diseases as well as in immunocompromised individuals.
Ghosh et al found that the dysregulation of hBD-2 was specific to each cancer type and generalization of results may not be appropriate.11 Although the present study is the first of its kind to shed light on the level of Human beta defensin-2 in the saliva of patients with oral potentially malignant disorders and oral cancer, it is evident that further research in this area is necessary in order to arrive at a definite consensus.
CONCLUSION:
Clinicians are always on the lookout for accurate and easy surveillance methods to monitor patients’ health. With a proven correlation with serum values, saliva is considered as the ‘mirror of the body’. It accurately mimics the physiologic and pathologic states of the body. Liquid biopsy using oral fluid has the advantage of being non-invasive, relatively inexpensive, and rapid thereby helping with patient acceptance. With the continued development of newer modalities of assessment, it might soon be possible to utilize salivary diagnostics for personalized medicine delivery and as an accurate prognostic marker. In the present scenario, establishment of sensitivity and specificity of salivary parameters poses a major hurdle in salivary diagnostics. An accurate roadmap is necessary for future researchers in order to extract the full potential of saliva as a source of biomarkers in various health conditions.
The presence of Human beta defensin-2 in the saliva of healthy controls points to its role in the maintenance of mucosal integrity. Elevation in the level of hBD-2 in oral potentially malignant disorders and a further increase in oral cancer indicate the potential use of hBD-2 as a biomarker in early diagnosis of oral cancer. Research in tissue levels of hBD-2 is already underway. However, this study highlights the diagnostic role of hBD-2 in saliva. More research is required with saliva as the source of Human beta defensin-2 in order to establish its role as a biomarker in oral potentially malignant disorders and oral cancer.
Ethics and Consent:
Informed consent was obtained from the subjects before commencement of the study. Institutional Ethics Committee provided the ethical clearance.
CONFLICT OF INTEREST:
None.
FUNDING INFORMATION:
The research was funded through an intramural grant by Nitte (Deemed to be University)
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Received on 22.06.2023 Modified on 18.09.2023
Accepted on 29.11.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(4):1799-1804.
DOI: 10.52711/0974-360X.2024.00286