|Year : 2012 | Volume
| Issue : 2 | Page : 277-281
In search of malignant transformation: A pilot study
Abhishek Singh Nayyar, Mubeen Khan
Department of Oral Medicine and Radiology, Government Dental College and Research Institute Bangalore, Karnataka, India
|Date of Web Publication||26-Jul-2012|
Abhishek Singh Nayyar
Department of Oral Medicine and Radiology, Government Dental College and Research Institute, Bangalore, Karnataka
Source of Support: None, Conflict of Interest: None
Context: The role of oxygen free radicals in the initiation, promotion and progression of carcinogenesis and the protective role of antioxidant defenses has been the subject of much speculation in the recent past, with conflicting reports in the literature.
Aims: The aim of this study was to measure the concentration of serum total proteins and albumin and advanced oxidation protein products (AOPP) in the sera of patients diagnosed with speckled leukoplakia and well-differentiated oral squamous cell carcinoma.
Materials and Methods: The study consisted of sera analysis of 30 new cases of histologically proven well-differentiated, oral squamous cell carcinoma and 10 patients with speckled leukoplakia aged between 40 and 60 years in addition to 25 healthy controls.
Statistical Analysis Used: One-way analysis of variance (ANOVA) was used to test the difference between groups. To find out which of the two groups' mean was significantly different, post hoc test of Scheffe was used.
Results: The study revealed variations in sera levels of albumin and advanced oxidation protein products to be statistically significant.
Conclusions: The results obtained emphasize the need for more studies with larger sample sizes to be conducted before a conclusive role for sera levels of total protein, albumin and AOPP could be drawn as markers of transition of the various oral precancerous lesions and conditions to frank oral squamous cell carcinoma.
Keywords: Antioxidants, carcinogenesis, free radicals, pre-cancerous, reactive oxygen species, transformation
|How to cite this article:|
Nayyar AS, Khan M. In search of malignant transformation: A pilot study. J Can Res Ther 2012;8:277-81
| > Introduction|| |
Despite tremendous advances in the diagnosis and management of oral cancers, the diagnostic adjuncts that are used to aid an early diagnosis of oral cancers either suffer from a lack of sensitivity in the initial stages of the processes leading to frank oral cancers or suffer from a setback of not being so cost-effective.
In addition, biopsy, which is considered the gold standard in diagnosis of oral cancers, suffers from the reliability of an appropriate site for the obtainment of specimen to be conclusive. The introduction of the concept of field of cancerization has further questioned the significance of biopsy results in the approval or rejection of the reports that come out to be confirmative of either dysplastic or frank cancerous changes seen in the tissue.
The role of biochemical markers, on the other hand, seems to be a convincing enough evidence of the changes taking place in the body at a time when tissue- and cell-level changes are not obvious to be taken as an evidence of frank malignant degenerations.
Hence, the present study is being planned to assess the levels of serum total protein and albumin as plasma's potent antioxidant defenses and advanced oxidation protein products, the markers of oxidant-mediated protein damage, as reliable markers of oxidative stress in the body that could be helpful in the early identification and, even more significantly, in determining the predisposition of the various oral precancerous lesions and conditions into their transformation to frank oral cancers.
| > Materials and Methods|| |
Source of data
The study was conducted for a period of 3 months from January 2010 to March 2010. The study consisted of 30 new cases of clinically diagnosed and histologically proven well-differentiated, oral squamous cell carcinoma and 10 patients with speckled leukoplakia aged between 40 and 60 years in addition to 25 healthy controls.
Method of collection of data
None of the patients were on any therapeutic modality prior to the inclusion in the study or were suffering from any systemic condition, especially hepatic or renal disorders with or without dialysis that could have affected serum total protein, albumin as well as advanced oxidation protein products (AOPP) levels. The sera of the subjects were obtained taking full precautions to prevent hemolysis.
Collection of blood and serum separation
For this, following an overnight fasting period, 5 mL of venous blood was taken from selected patients from the antecubital vein using a sterile disposable syringe in the sitting position between 8 A.M. and 10 A.M. Serum was immediately separated by ultracentrifugation taking full precautions to prevent hemolysis. The supernatant was discarded and the rest of the sample was stored at -20°C.
Assay of serum total protein and albumin in sera
Sera levels of total protein and albumin were assayed with the help of Biuret method. Serum total protein and albumin were expressed as g/dL.
The biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper (II) ion forms a violet-colored complex in an alkaline solution. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.
Assay of AOPP in sera
Sera levels of AOPP were measured by spectrophotometry. The assay was calibrated using chloramine-T and the absorbance was read at 340 nm on a microplate reader. Sera levels of AOPP were expressed as μmol/L of chloramine-T equivalents.
Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement.
Many older spectrophotometers are calibrated by a procedure known as "zeroing." The absorbancy of a reference substance is set as a baseline value; therefore, the absorbancies of all other substances are recorded relative to the initial "zeroed" substance. The spectrophotometer then displays % absorbancy (the amount of light absorbed relative to the initial substance).
Method of statistical analysis
The following methods of statistical analysis have been used in this study:
The results were averaged (mean ±- standard deviation) for continuous data and number and percentage for dichotomous data have been presented in Tables and Figures.
One-way Analysis of Variance (ANOVA) was used to test the difference between groups.
To find out which of the two groups' mean was significantly different, post hoc test of Scheffe was used.
For comparison of two variances, S a 2 and S b 2 , estimated for two groups, N a and N b subjects, respectively, F test was used, wherein:
with N a -1 and N b -1 degrees of freedom.
In the above test, P-values less than 0.05 were taken to be statistically significant.
The data was analysed using SPSS (version 10.5).
The normality of data was checked using the Kolmogorov-Smirnov and Shapiro-Wilk tests for significance before the statistical analysis was performed [Table 1].
| > Results|| |
While the mean values of serum total protein were much the same in controls (8.236 ± 1.5025 g/dL) as against the cases diagnosed with speckled leukoplakia (9.85 ± 3.6788 g/dL) and well-differentiated, oral squamous cell carcinoma (7.8 ± 3.1500 g/dL) [Table 2], there were observed great variations in the minimum (1.6 g/dL) to the maximum values (18.2 g/dL) for well-differentiated, oral squamous cell carcinoma. The P-value however came out to be statistically insignificant, implying the role of various other factors in protein metabolism in cancer patients [Table 2].
|Table 2: Depicting mean serum total protein in study groups along with standard deviation and P-values|
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Serum albumin levels however came out to be statistically significant (P < 0.001) [Table 3], with serum albumin levels as low as 1.7 g/dL in frank oral squamous cell carcinoma as against a minimum of 3 g/dL in the control group. The mean values of serum albumin came out to be 4.956 ± 1.0579 g/dL in the control group [Table 3].
|Table 3: Depicting mean serum albumin in study groups along with standard deviation and P-values|
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Advanced oxidation protein products, also, revealed marked variations in controls and the patients diagnosed with frank squamous cell carcinomas (0.42563 ± 0.2010 μmol/L) with P-value < 0.001 [Table 4]. The mean value of serum advanced oxidation protein products was found to be 0.0788 ± 0.0279 μmol/L in the control group [Table 4]. Sera levels of advanced oxidation protein products shot up to 0.918 μmol/L in the frank oral squamous cell carcinoma group as against a minimum of 0.041 μmol/ L in the control group [Table 4]. The mean value of serum advanced oxidation protein products in patients diagnosed with speckled leukoplakia also came out to be significantly high (0.368 ± 0.0978 μmol/L) [Table 4].
|Table 4: Depicting mean serum advanced oxidation protein products in study groups along with standard deviation and|
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The level of significance came out to be less than 0.001 in case of sera levels of albumin in between the controls and well-differentiated, oral squamous cell carcinoma patients [Table 5]. The study also revealed statistically significant results in between the controls and the patients diagnosed with speckled leukoplakia and controls and the patients afflicted with well-differentiated, squamous cell carcinomas being less than 0.001 [Table 5].
|Table 5: Depicting comparison of serum total protein, albumin and advanced oxidation protein products in study groups along with the mean difference, standard error and signifi cance|
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The results arrived at confirmed the results obtained from other studies in relation to sera levels of total protein and albumin while again being in concordance with the results obtained in the published studies in relation to advanced oxidation protein products in relation to general body cancers. The statistically insignificant results obtained in the patients diagnosed with speckled leukoplakia and oral squamous cell carcinoma groups were explained on the basis of multiple, confounding factors that play a significant role in protein metabolism in cancer patients.
| > Discussion|| |
Oral cancer has a much higher prevalence in the elderly age group, with this higher prevalence among the elderly population explained on the basis of an age-related increase in the magnitude of the attack of the oral carcinogens as free radicals including the so-called reactive oxygen and nitrogen species causing various DNA mutations and aberrations. It may also result from an age-related reduction in the body's defense mechanisms, including the body's antioxidant defenses. ,, Ti is only when this equilibrium is disturbed that the damage to the DNA is brought about and cancer evolves. ,
Most free radicals are said to be highly reactive and short lived. , Sun has proposed that free radicals are involved in both the initiation and the promotion stages of carcinogenesis. These free radicals have been shown to cause DNA damage, activate procarcinogens and alter the cellular antioxidant defense mechanisms. 
Plasma is known to contain a wide range of important antioxidants including albumin, ascorbic acid and uric acid. In contrast, concentrations of enzymes such as superoxide dismutase, reduced glutathione and catalase, all of which are known to be important intracellular antioxidants, are low in plasma. While ascorbate is an important extracellular antioxidant, albumin, via its thiol groups, provides quantitatively almost 10-folds greater antioxidant protection against the various reactive oxygen and nitrogen species held responsible for the genetic damage, eventually leading to the development of cancers. ,
The analysis of changes in serum total protein in malignancy is in itself a means of studying abnormality in the protein metabolism in this condition. Until recently, radical induced damage to proteins was considered to be mainly a chain-terminating process. It was thought that the products of damage produced on the protein, as a result of protein scission, cross-linking and chemical modification of side chains, were relatively inert, with the intermediaries subsequently degraded by intra- and extracellular enzymes. It has recently been demonstrated however that these intermediaries are capable of initiating further chemical reactions, thereby leading to the depletion of important cellular reductants such as ascorbates and glutathione via redox reactions. , Serum total protein in our study came out to be statistically insignificant, implying the role of the several complex factors that may play a role in protein metabolism in cancer patients as held by the numerous other studies conducted earlier in this regard. 
In humans, albumin is the most abundant plasma protein, accounting for about 55-60% of the measured serum proteins. It consists of a single polypeptide chain of 585 amino acids with a molecular weight of around 66,500 Da. The mature, circulating molecule is arranged in a series of alfa-helices, folded and held by 17 disulphide bridges. Albumin synthesis takes place only in the liver and is secreted into the portal circulation as soon as it is formed. The rate of synthesis varies with nutritional and disease states. Among the numerous plasma proteins that possess antioxidant properties owing to their rich concentrations of free thiol groups, albumin is unusual in having a free sulfhydryl group in addition. ,
With normal concentrations lying between 3.5 and 5.5 g/dL, the serum level of albumin is related mainly to its synthesis and catabolism. In fact, only a small number of factors are known to result in variation in serum albumin. In addition, it has been reported that serum albumin decreases with age and cigarette smoking, with the usual half-life of albumin being 20 days. ,,
Several lines of evidence suggest strongly that a reduced serum albumin concentration, although within the normal range, is associated with increased mortality risk.  From studies performed with healthy subjects and patients, it has been reported that the estimated increase in the odds of death ranges from 24% to 56% for each 2.5 g/L decrement in serum albumin concentration. The serum albumin level thus appears to be an independent predictor of mortality risk, with a direct protective effect of the albumin molecule being suggested by the persistence of the association after adjustment for other risk factors. Albumin may thus represent quantitatively the most important component that plays a determinant role in the efficient antioxidant defense organisms have developed to protect against oxidative attack. ,
Albumin in our study came out to be statistically significant, with values varying from a minimum of 2 g/dL to 5.1 g/dL in patients diagnosed with speckled leukoplakia to as low as 1.7 g/dL in patients afflicted with frank oral squamous cell carcinoma. This is in concordance with the observations of the several studies conducted in the past that laid emphasis on the protective role of albumin as one of the most abundant extracellular antioxidants available in the plasma of patients diagnosed with frank squamous cell carcinomas. The exact role of albumin in assessing the prognosis is, therefore, warranted by larger, follow-up studies correlating the level of serum albumin in these groups of patients with the overall 5-year survival rates.
Also, cells can generally remove oxidized proteins by proteolysis. This in only when the normal physiology of the cells is disturbed that the products of oxidative metabolism of proteins are retained and lead to damaging actions of oxidized proteins during aging and, sometimes, culminating into a number of oxidative stress-mediated pathologies, including cancers. ,
Advanced oxidant protein products, first described by Witko-Sarsat et al. (1996), have further been hypothesized to activate the endothelial cells and, to a lesser extent, fibroblasts to generate reactive oxygen species. Furthermore, advanced oxidation protein products generated by different oxidation patterns lead to the production of either NO or H2 O2 , suggesting their role in the generation of different types of reactive oxygen species that set a cascade of reactions with a potential to damage cellular micromolecules, eventually turning out into frank oral squamous cell carcinoma. 
The level of advanced oxidation protein products in our study ranged from a minimum of 0.242 μmol/L to 0.534 μmol/L in patients diagnosed with speckled leukoplakia to as high as 0.918 μmol/L in patients diagnosed with histologically proven well-differentiated, oral squamous cell carcinoma. The results obtained however could not be compared with the observations of other studies as the AOPP levels have been assessed in relation to other cancers of the body in the studies conducted in the past. This study deserves the credit of being the first of its kind in assessing the sera levels of AOPP as one of the important diagnostic markers in patients diagnosed with oral cancers.
| > Conclusion|| |
Reactive oxygen and nitrogen stresses have long been implicated in the genesis of oral cancers. There is enough literature available that shows convincing evidence in the use of antioxidants as chemopreventive agents to halt the transformation of various oral precancerous lesions and conditions into frank oral cancers. The results obtained emphasize the need for more studies to be conducted in this regard for the assessment of sera levels of total protein, albumin and advanced oxidation protein products to accept their utility and to assess their role in the pathogenesis and their impact on the prognosis of oral cancers, providing a scientific ground for the use of diverse chemopreventive strategies in controlling damage at the genetic and molecular levels to prevent the ongoing transition of various oral precancerous lesions and conditions into frank malignant degenerations.
| > Acknowledgement|| |
We thank all the people who directly and indirectly contributed for the study as the study required intense efforts from the people outside our Department including the cancer wards and the Department of Clinical Biochemistry, Bangalore Medical College and Research Institute and Associated Hospitals. We feel highly indebted to Dr. Vijayalakshmi K. R., Associate Professor, Department of Oral Medicine and Radiology, Government Dental College and Research Institute, Bangalore and Dr.Anitha M., Assistant Professor, Department of Clinical Biochemistry, Bangalore Medical College and Research Institute and Associated Hospitals for their continuous support and encouragement while we were carrying-out this study.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]