Abstract
Background/Aim: The aim of this study was to investigate the potential role of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) as tumor markers for endometrial cancer. Patients and Methods: Serum samples were obtained from 40 women with endometrial cancer, 52 with myoma uteri and 52 healthy individuals. Class III, IV of ADH and total ADH activity was measured by a photometric method and class I, II ADH and ALDH activity, by a fluorometric method. Results: The total activity of ADH and ADH class I was significantly higher in the serum of patients with endometrial cancer than in healthy individuals and patients with myoma. The diagnostic sensitivity for ADH I was 69%, specificity 77%, positive predictive value and negative predictive value were 75% and 71% respectively. The area under curve for ADH I was 0.682 and for total ADH was 0.623. Conclusion: The results suggest a potential role of ADH I as a marker for endometrial cancer.
Endometrial cancer comprises approximately 4% of all cancer in women worldwide and is the fourth most common type of cancer in Western countries (1). The development of this type of cancer has been related to exposure to endogenous or exogenous estrogens (2). Several studies have shown a positive association between alcohol intake and estrogen levels in women (3, 4). Alcohol could increase plasma estrogen levels either by promoting the induction of aromatases, which can convert androgens to estrogens, or by impairing of the metabolism of estrogens in the liver (5). Moreover, it has been suggested that an increased risk for cancer is attributed to acetaldehyde, the first metabolite of ethanol (6). Acetaldehyde is highly toxic and may interfere with DNA synthesis and repair. Ethanol may also act through the induction of cytochrome P450 2E1, giving rise to free radical oxygen compounds (7).
Human alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), enzymes responsible for ethanol metabolism, exist in multiple molecular forms that have been grouped into several classes. Acetaldehyde is the primary product of ethanol oxidation, mediated by alcohol dehydrogenase, and is further metabolized by aldehyde dehydrogenase. Many studies have demonstrated that ADH and ALDH play a significant role in gastrointestinal and breast carcinogenesis because they catalyze the oxidation or cause reduction of many biological substances (8).
Expression of I and IV class ADH isoenzymes are also observed in the uterus (9). It was found that class I ADH isoenzymes participate in the metabolism of bioamines and prostaglandins, and together with class IV ADH isoenzymes in retinoic acid (RA) metabolism (10). Retinol is required for normal growth and development, keratinization of the cutis and mucosa, and appropriate resistance to infection (11). In a previous study, we showed that ADH and ALDH activities are present in endometrial cancer cells. In addition, the activity of class I ADH isoenzymes and total ADH were significantly higher in cancer cells than in healthy endometrium. Moreover, the activity of ADH seems to be disproportionately high compared to the activity of ALDH, which suggests an increased ability of cancer cells to metabolize ethanol in the form of acetaldehyde, which may intensify carcinogenesis (12).
In this study, we investigated the activity of ADH and its isoenzymes, and the total activity of ALDH in the sera of patients with endometrial cancer. We also defined diagnostic criteria such as sensitivity, specificity, predictive value for positive (PPV) and negative results (NPV), and receiver-operating characteristics (ROC) curve of tested isoenzymes. We hypothesized that a changed activity of ADH isoenzymes in cancer cells would be reflected in the serum and could thus be helpful for diagnosing endometrial cancer.
Patients and Methods
Patients. Serum samples were obtained before treatment from 40 women (mean age=62 years, range=44-82 years) with endometrial cancer and 52 women (mean age=50 years, range=31-72 years) with myoma uteri. None of the women had received chemotherapy or radiotherapy before tissue collection, and patients were diagnosed as either stage II or III. All patients had a history of occasional alcohol consumption. The control group were serum samples taken from 52 healthy women (mean age=58 years, range=51-67 years).
The research protocol was approved by the Medical University of Białystok's Human Care Committee located in Bialystok, Poland (approval no. R-I-002/179/2009). All patients gave their informed consent for the examination.
Determination of total ADH activity. Total ADH activity was estimated by photometric method using p-nitrosodimethylaniline (NDMA) as a substrate (13). The reaction mixture (2 ml) contained serum (0.1 ml), 1.8 ml of a 26 μM solution of NDMA in 0.1 M of sodium phosphate buffer, pH 8.5 and 0.1 ml of mixture containing 0.25 M n-butanol and 5 mM NAD. The reduction of NDMA was monitored at 440 nm on a Shimadzu UV/VIS 1202 spectrophotometer (Shimadzu Europa GmbH, Duisburg, Germany).
Determination of total ALDH activity. ALDH activity was measured using fluorogenic method based on the oxidation of 6-methoxy-2-naphthaldehyde to fluorescent 6-methoxy-2-naphthoate (14). The reaction mixture contained 60 μl of serum, 60 μl of substrate, 20 μl of 11.4 mM NAD and 2.8 ml of 50 mM of sodium phosphate buffer, pH 8.5. The mixture also contained 50 μl of a 12 mM solution of 4-methylpyrazole as a specific inhibitor of ADH activity. The fluorescence was read at an excitation wavelength of 310 and an emission wavelength of 360 nm on a Shimadzu RF–5301 spectrofluorophotometer (Shimadzu Europa GmbH, Duisburg, Germany).
Determination of class I and II ADH isoenzymes. Class I and II ADH isoenzyme activity was measured using fluorogenic substrates (4-methoxy-1-naphthaldehyde for class I and 6-methoxy-2-naphthaldehyde for class II) in a reduction reaction according to Wierzchowski et al. (15). The assays were performed in a reaction mixture containing serum (60 μl), substrate (150 μl of 300 μM), NADH (100 μl of 1 mM) and 0.1 M of sodium phosphate buffer, pH 7.6 (2.69 ml) using the conditions previously described (16). The measurements were performed on a Shimadzu RF–5301 spectrofluorophotometer at an excitation wavelength of 316 nm for both substrates and emission of 370 nm for class I and 360 nm for class II isoenzymes.
Determination of class III ADH isoenzyme. The assay mixture for class III ADH contained serum (100 μl), formaldehyde as a substrate (100 μl of 1 mM), glutathione (100 μl of 1 mM) and NAD (240 μl of 1.2 mM) in 0.1 mol NaOH-pyrophosphate buffer pH 8.0 (17). The final volume was 2 ml. The reduction of NAD was monitored at 340 nm and 25°C on a Shimadzu UV/VIS 1202 spectrophotometer.
Determination of class IV ADH isoenzyme. The assay mixture for class IV of ADH activity contained serum (50 μl), m–nitrobenzaldehyde as substrate (132 μl of 80 μM) and NADH (172 μl of 86 μM) in 0.1 M sodium phosphate buffer pH 7.5 (18). The oxidation of NADH was monitored at 340 nm and 25°C on a Shimadzu UV/VIS 1202 spectrophotometer.
Calculation of diagnostic values. The diagnostic criteria, sensitivity, specificity, PPV and NPV and the ROC curve, were determined using GraphRoc Program for Windows (University of Turku, Turku, Finland) (19) as follows.
Statistical analysis. Preliminary statistical analysis (Chi-square test) revealed that the distribution of ADH and ALDH activities did not follow a normal distribution. Consequently, the Wilcoxon test was used for statistical analysis. Data are presented using median, range and mean values. Statistically significant differences are defined as comparisons resulting in p<0.05.
Results
The activities of total ADH, ALDH and ADH isoenzymes in the sera of patients with endometrial cancer are presented in Table I. The total activity of ADH was significantly higher (about 34%) in the serum of patients with endometrial cancer than in healthy individuals. The activity of total ADH was also significantly higher (about 30%) in the serum of patients with cancer in comparison to women with myoma uteri. The total ADH activity was not statistically higher in the group with myoma than in the controls. The median total activity of ADH was 0.883 U/l in the endometrial-cancer group, 0.573 U/l in myoma and 0.562 U/l in the control group. The analysis of ALDH activity did not indicate significant differences between the tested groups.
The comparison of ADH isoenzyme activities showed that the highest difference was exhibited by class I ADH. The median activity of this class of isoenzymes in the cancer group was higher by about 38% (1.943 mU/l) in comparison to the controls (1.274 mU/l) and about 34% in comparison to the myoma uteri group (1.286 mU/l). The increase of ADH I activity was statistically significant in both cases. The activity of ADH I was higher in patients with myoma uteri than in the control group but the difference was not statistically significant.
Activity of ADH isoenzymes (mU/l) and ALDH (mU/l) in the sera of patients with endometrial cancer and healthy women.
Diagnostic criteria for total alcohol dehydrogenase (ADH) and its isoenzyme class I (ADH I) for endometrial cancer.
The other tested classes of ADH isoenzymes had higher activities in the serum of patients with cancer but the differences were not statistically significant (p>0.05). We found that the activities of ADH class II, III and IV were not statistically different in patients with myoma uteri in comparison to healthy women, although there was a tendency towards increased activity in myoma.
Table II shows the diagnostic criteria for total ADH and ADH I. The sensitivity (69%) and specificity (77%) of ADH I were higher than the values for total ADH. Both the PPV and NPV were also the highest for ADH I.
The relationship between diagnostic sensitivity and specificity is illustrated by a ROC curve (Figure 1). It shows that the area under the ROC curve for ADH I (0.682) was higher than that of total ADH (0.623).
Discussion
Endometrial cancer is one of the most common types of cancer of the lower female genital tract. Early detection of cancer in this organ is very important. Therefore, it is necessary to find markers that may be helpful for diagnosing endometrial cancer. The best tumor markers should be characterized by high sensitivity and high specificity.
Ohno et al. revealed the expression of ADH I mRNA in the uterus (9). In our previous study, we found the activity of human ADH in healthy endometrium and also in endometrial cancer cells. The activity of human ADH was significantly higher in cancer tissues than in healthy ones, however, the activity of ALDH was not different between these tissue types. We also found higher activity of ADH I and lower ALDH activity in endometrial cancer tissues, which might suggest that cancerous cells have a greater capability for ethanol oxidation and lesser ability to remove acetaldehyde as compared to healthy tissue cells. This may lead to accumulation of carcinogenic acetaldehyde in the endometrium, contributing to the development of endometrial cancer (12). Formation and degradation of acetaldehyde in the body is regulated by the activity of ADH and ALDH, so changes in the activity of these enzymes could be a factor involved in pathogenesis of endometrial cancer.
Several reports have shown that the expression of ADH genes and ADH activities are regulated by sex hormones. Estrogen treatment induced the expression of ADH I mRNA in rat kidney and rat liver (19, 20). Sex steroid hormones play a key role in the development of endometrial cancer. The first hypothesis describing the relationship between sex steroid hormones and endometrial cancer risk states that the risk is increased among women who have high circulating levels of bioavailable estrogens and low levels of progesterone (21). Androgens have been hypothesized to increase endometrial cancer risk, likely through the aromatization of androgens into estrogens, but alternatively might reduce risk by reducing the proliferative effects of estrogens in the endometrium (22, 23). Some evidence suggests that alcohol intake positively influences endogenous estrogens (3, 24). Alcohol may increase endogenous levels of estrogen in women, possibly by delaying its metabolism and prolonging its half-life (25). Alcohol consumption-related increases in estrogen levels may in turn be partially responsible for the associated decrease in risk for osteoporosis, as well as for increased risk of breast and endometrial cancer (26).
Receiver-operating characteristics (ROC) curves for alcohol dehydrogenase class I (ADH I) and total alcohol dehydrogenase (ADH). AUC (area under curve); SE (standard error of the mean).
In our study, we found that total activity of ADH was significantly higher in the serum of patients with endometrial cancer than in the control group and in the group of patients with myoma. But the analysis of ALDH activity did not indicate significant differences between the tested groups. The activity of ADH I was significantly higher in the sera of patients with cancer. It is commonly accepted that changes in enzyme activity in neoplastic tissues are reflected by an increase of the corresponding enzyme activity in the serum. Hence an elevated serum level of ADH I could be caused by the release of these isoenzymes from endometrial cancer cells, which is consistent with ADH activity studies in colorectal cancer. The activities of total ADH and class I of ADH were found to be significantly higher in colorectal cancer than in healthy colon mucosa (27). Therefore, the activity of ADH I isoenzymes is significantly elevated in the sera of patients with colorectal cancer (28).
We did not find a statistically significant difference in the activity of ADH I in patients with myoma uteri in comparison with the control group, similarly to the study of Shveiky et al. They found reduced expression of ADH I in myoma, both at the level of genes and proteins (29).
Another cancer type whose pathogenesis associated with sex steroid hormones is breast cancer. Many lines of breast cancer cells are dependent on estrogen and progesterone for growth. In the serum of patients with breast cancer, the activity of ADH I is elevated as in endometrial cancer. Moreover, the serum activity of ADH I isoenzymes appeared to have a tendency to increase with advancement of the disease. The activity of ADH I was significantly higher in the sera of patients with stage IV breast cancer as compared to healthy controls, however, this was a result of isoenzymes being released from organs damaged by metastatic disease (30). The activity of ADH I was lower in breast cancer cells than in normal parenchyma, but the ratio of ADH to ALDH activity was still higher in cancer cells (31). This would suggest that cancer cells have a higher capability for oxidation of exogenous ethanol than for removal of acetaldehyde.
Tumor markers are substances expressed in different biological fluids which are synthesized and excreted by tumor tissues. Higher levels of ADH in patients with endometrial cancer, which might result from enzyme release of cancer cells, could be helpful in the diagnosis of this cancer type. The diagnostic criteria for disease markers are sensitivity, specificity and AUC. In our study, the sensitivity of ADH I was 69% but the one for the total activity of ADH was 60%. The sensitivity of ADH I is higher and for total ADH is equal to that of the classic endometrial cancer marker – Cancer Antigen 125 (CA 125) (32). It is interesting that there were no significant differences between the activity of ADH in the sera of patients with myoma uteri and of healthy women. However, some data have shown that CA 125 concentration could be increased in patients with benign changes in the uterus (33). Several previous analyses have shown elevated concentrations of circulating cytokines, such as macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF), but their sensitivity in endometrial cancer is much lower than that for ADH I and total ADH. The sensitivity of M-CSF is 51%, but the sensitivity of G-CSF and SCF is only 24% (32).
The most important criterion for tumor markers is the sensitivity/specificity diagram, the ROC curve. The area under the ROC curve indicates the clinical usefulness of tested markers. An AUC larger than 0.5 means that the study parameter has diagnostic utility. In this study, the ADH I AUC was 0.682 and the one for total ADH was 0.623, which suggests the possibility of using these isoenzymes as markers of endometrial cancer.
In conclusion, we can state that the activity of class I ADH isoenzymes and the total activity of ADH were elevated in the sera of patients with endometrial cancer as compared to the healthy control and the group of patients with myoma uteri. This is the first study, as far as we are aware of, to examine all the diagnostic criteria for ADH and ALDH in patients with endometrial cancer. These results suggest a potential role for ADH isoenzymes (especially ADH I) as markers of gynecological cancer, which may also be used for differentiation of cancer from benign lesions. Further investigations and confirmation by a prospective study should be necessary.
- Received July 9, 2013.
- Revision received July 21, 2013.
- Accepted July 22, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
