Oxidative stress induces a cellular redox imbalance that has been found to be present in various cancer cells as compared with normal cells, the redox imbalance thus may be related to oncogenic stimulation [23
]. The disturbance of the pro-oxidant/antioxidant balance, resulting from increased free radical production, antioxidant enzyme inactivation, and excessive antioxidant consumption, is the causative factor in oxidative damage [24
]. There are potentially different types of DNA lesions resulting from ROS and reactive nitrogen species, which could be mutagenic and involved in the etiology of cancer [25
Xanthine oxidase (XO: EC 220.127.116.11) is an enzyme playing a part in purine metabolism. It catalyzes the conversion reactions of hypoxanthine to xanthine and xanthine to uric acid, the last reaction in the purine catabolism, with byproduct of toxic superoxide radical. In this regard, it is a key enzyme between purine and free radical metabolism. It was reported that XO is an endogenous source of ROS and reactive nitrogen species (RNS) that can induce oxidative stress and inflect tissue injury [26
]. There is growing evidence that superoxide radicals generated by XO are primarily responsible for the cellular deterioration associated with several conditions [28
The data reported in the literature on oxidant, antioxidant molecule, and enzymes in different human cancer types are controversial. Our findings show significant increase in XO activity of bilharzial bladder cancer and non-bilharzial bladder cancer patients compared to normal healthy ones. Kokoglu et al. [29
] found higher XO activity in tumoral brain tissues and suggested that the levels of XO in brain tissues could be used as a biochemical marker for the differentiation of tumoral tissues from normal ones. Our findings are consistent with Kaynar et al. [30
] who found an increase in XO activity in patients with small cell and non-small cell lung cancer. Also, Ozturk et al. [31
] observed a significant increase in XO activity in patients with colorectal cancer compared to control group. The authors attributed this increase in this enzyme to an unbalancement alteration in oxidant-antioxidant status due to the cancer process. In addition, Gülec et al. [28
] found that XO activity increased in bladder cancer patients suggested that oxidative stress might be increased in cancerous changes and process, and may affect the course of the disease. The burst of XO-mediated free oxygen radical generation in the cancerous tissue can be triggered by a large increase in substrate formation which occurs secondary to the rapid turn-our of adenine nucleotides during cancer process. On the other hand, as proposed by Durak et al. [32
] high XO activity may be an attempt to lower salvage pathway activity for purines, which is vital for rapid DNA synthesis in cancerous bladder tissue. Moreover, Mohamed et al. [33
] found a significant increase in XO activity in mice infected with S. mansoni
Given the evidence that supports a relationship between abnormal glucose metabolism and cancer risk, serum fructosamine, one of the markers of abnormal glucose metabolism, is a glycated protein resulting from spontaneous, non enzymatic condensation of glucose and proteins, produces an unstable ketoamine, which is generally referred to as fructosamine due to its structural similarities to fructose [34
]. As albumin is the most abundant protein in serum and contains multiple lysine residues, measurement of fructosamine is mainly the determination of glycated albumin [36
]. This non enzymatic modification of protein is considered by several authors as a possible common mechanism involved in the progression of many pathological conditions such as myocardial and renal fibrosis, colorectal adenoma and chronic renal failure [35
The current investigation revealed that there was a highly significant increase in fructosamine level in bilharzial bladder cancer and non-bilharzial bladder cancer patients compared to normal ones. This data is in line with Misciagna et al. [35
] who reported that fructosamine is associated with colorectal adenoma. Also, Platek et al. [39
] observed a 60% risk increase for breast cancer in women with high levels of fructosamine, suggesting that fructosamine, as an indicator of glucose consumption, may be a predictor of breast cancer. Recently, Pasanisi et al. [40
] found that fructosamine was not associated with recurrence of breast cancer. Mohamed et al. [33
] reported an increase in serum fructosamine of S. mansoni
infected mice, this results was in accordance with our result in case of bilharzial baldder cancer patients. Our results are important because this is the first study investigating serum fructosamine in relation to bladder cancer and may be used as a useful marker for bladder carcinoma risk.
Malignant tumors inhabit a complex carbohydrate metabolism which differs from that of non-neoplastic cells with two main paradigms: 1) malignant cells produce large amounts of lactate even in the presence of sufficient oxygen for aerobic glycolysis. 2) intermediates of the tricarboxylic acid cycle (TCA) are used for fatty, amino and nucleic acid synthesis [41
]. Thus, the extensive glucose uptake of cancer cells is needed not only for energy supply but also to provide the components for cellular growth and a high amount of reducing equivalents such as NADPH[41
]. To accomplish this, high levels of pyruvate are needed which can be introduced either into the TCA, converted into acetyl-COA or degraded to lactate by LDH[41
]. By degradation of pyruvate to lactate by LDH, the pool of reductive equivalents on the one hand and the availability of citric acid cycle intermediates for fatty and amino acid synthesis on the other hand is raised. The overexpression of LDH5
in tumor cells supports this theory of a glucose metabolism optimized for cellular growth within malignant tumors.
Results in the present study showed a highly significant increase in LDH activity in both serum and bladder carcinoma tissues of bilharzial bladder and non-bilharzial bladder patients compared to healthy ones. The highest significance was observed in grade III of the disease. These results are in harmony with Kayser et al. [44
] who found that immunohistochemical analysis for LDH5
expression in non-small cell lung cancer (NSCLC) is specific to differentiate malignant neoplasia from healthy lung tissue. The authors reported that LDH5
overexpression in cancer cells induces an upregulated glycolytic metabolism and induced dependence on the presence of oxygen. In addition, Wu et al. [45
] found that the level of LDH was significantly higher in patients with liver metastasis of colorectal cancer than those without liver metastasis.
Another glycolytic enzyme was also estimated in the present study, which is pyruvate kinase enzyme, catalyzing the conversion of phosphoenol pyruvate into pyruvate. Our results revealed significant increase in the activity of PK enzyme in bilharzial and non-bilharzial bladder tissues when compared to the unaffected normal ones. In accordance with our data Shonk et al. [46
] studied the activities of different glycolytic enzymes in carcinoma of rectum and colon and in the corresponding non-malignant counterparts. They observed that glycolytic enzyme activities of the neoplastic tissues of rectum and colon are much higher than the activites in the corresponding normal rectum and colon. Also, Ramo Rao [47
] stated that the glycolytic enzyme pyruvate kinase requires potassium for its maximum activity i.e., the enzyme activity correlates with potassium concentration in tissues or blood. In support, Reddy et al. [48
] expected that the levels of potassium which may be a cofactor for pyruvate kinase should be higher in carcinoma tissues. Recently, Rautray et al. [49
] observed that a potassium level in the carcinoma blood of gallbladder is lower than those in the normal blood samples. Potassium is a major intracellular cation which is also excreted in gastrointestinal tract, sativa, gastric juice, bile, pancreatic and intestinal juices, its deficiency causes acidosis, renal damage and cardiac arrest.
The metabolism of healthy and functionally active cells is optimized for productivity in terms of fulfilling their duty to synthesize, degrade, transport or contract. In contrast to this, malignant tumor cells do not meet these demands but only strive for cellular growth and mitosis [44
]. According to this theory high amounts of ATP as demanded within healthy cells are not only not required but act contra - productive for the synthesis of basic elements for cellular growth such as proteins, nucleic acids and fatty acids. Protein is a critical reservoir of metabolic fuel and may become seriously depleted during tumor growth [50
]. The metabolism of protein and amino acids in cancer patients is closely linked to glucose metabolism and is regulated by a number of the same hormones and metabolites [51
Our findings in the current study showed a slight increase (P < .025) in total protein concentration in serum of bilharzial bladder cancerous patients compared to healthy ones while, there was non-significant decrease in serum total protein of bladder cancer patients when compared to healthy ones. These results are in harmony with Lai et al. [50
] who found that in cancer patients, increased hepatic protein synthesis and gluconeogenesis might lead to a slight decrease in plasma free amino acids levels and a slight increase in plasma protein levels. On contrary to our results, Rossi Fancelli et al. [52
] reported that whole - body protein breakdown has been demonstrated to increase in cancer patients and correlated with gluconeogenesis in malnourished cancer patients.
On the other hand, Sauer and Dauchy, [53
] found that fructose - 1, 6 - disphosphatase, the enzyme involved in the conversion of pyruvate to glucose is elevated in the liver, suggesting that liver protein is utilized for gluconeogenesis in cancer patients. Recently Kojima et al. [54
] detected an elevated proteins in serum of gastric cancer patients with peritoneal dissemination compared with serum samples from patients with gastric cancer in general.
On the contrary of our results, Alexandrakis et al. [55
] found a significant reduction in serum total protein in malignant neoplasms and cirrhotics. Also, Haitel et al. [56
] stated that transitional cell carcinoma and squamous cell carcinoma of the bladder differ in terms of protein expression and prognosis. In addition Ali et al. [57
] found a significant reduction in serum total protein content in bladder cancer, and bilharzial bladder cancer patients compared to the control healthy group and the authors recorded that the decrease was more in bilharzial bladder cancer group than the two other groups.
The data obtained in the current study of diagnostic marker enzymes revealed a marked increase in the activities of aspartate and alanine aminotransferases in serum and tissues of bilharzial bladder cancer and non-bilharzial bladder cancer versus healthy controls and the highest levels in activities of enzymes were obvious in grade III of the bladder cancer.
The increased of AST and ATL levels were more specified extra cellular release and decreased protein content. Several reports confirmed the elevation in AST and ALT activities as a result of S.mansoni
infection which may reflect a decrease in hepatic cells, where Salah et al. [58
] and Ahmed [59
] indicated the decrease in hepatic enzymes activities may be due to the release of the enzymes from the necrotic tissue or due to increased cell membrane permiability as a result of relative anoxia and irritation by toxic or metabolic wastes of the worms.
The extra cellular release of the transaminases leads to their increase in serum [60
], while the reduction of enzymes activities relative to lower liver protein content was either due to their release in the bloodstream or due to their decrease synthesis, since transaminases can serve as an index of metabolic aerobity degree, and AST can provide Krebs cycle intermediates, while ALT can be correlated with lactate production [61
]. The authors also reported that the elaborated ROS causing damage of cellular membrane, and hence, AST and ALT are sub-cellular enzymes localized in mitochondria and cytoplasm, their release to the bloodstream was increased and their synthesis at the same time decreased.
In accordance with our findings, Ali et al. [57
] who recorded more significant elevation values of AST, ALT in bilharzial and non bilharzial bladder cancer patients compared to normal group. Recently, Wu et al. [45
] found that the levels of ALT and AST were significantly higher in patiens wih liver metastasis of colorectal cancer than in those without liver metastasis.
The amino acids can be classified into two major categories. The essential amino acids are obtained solely from the diet, whereas the body can, under some circumstances, produce non-essential amino acids from other sources. In certain cancers the amino acid profile yields useful data about the disease from which treatment approaches may be better assessed. Colorectal cancer patients exhibit a characteristic amino acid profile: significantly lower taurine, glutamine, valine, and tyrosine in plasma; significantly lower taurine, glutamic acid, methionine and ornithine intracellularly; and elevated valine, isoleucine, leucine, tyrosine, and phenylalanine intracellularly. Obtaining the amino acid profile may help to construct an appropriate dietary manipulation program aimed at modifying intracellular amino acid concentration[62
]. Likewise, squamous cell carcinoma of the head and neck exhibit a profile that is marked by decreased taurine, alanine, asparagine, aspartic acid, glycine, histidine, ornithine, phenylalanine, serine, and threonine, with a marked increase in levels of cystine. These features are noted regardless of the stage of the cancer and nutritional status. Thus, serum amino acid levels in serum and other tissues may become a useful cancer marker cancer and provide valuable prognostic information [63
Remarkable difference in amino acid profiles between the colorectal cancer patients and the control subjects is found in this study and most of the differences are statistically significant in respect of non-essential amino acids. The difference in essential amino acids concentration was noticeably insignificant except for methionine and theronine. Furthermore, some studies showed that the concentration of amino acids in subjects might be correlated with the same diseases[64
The current investigation showed a significant increase in hydroxyproline level in serum of bilharzial bladder cancer and non-bilharzial bladder cancer versus control healthy ones. The increase was more in bilharzial bladder cancer group than the two other groups. In accordance with our data, Proenza et al. [65
] demonstrated an increased levels of hydroxyproline in breast cancer. Also, Mohamed et al. [33
] reported a marked increase in the level of liver hydroxyproline in infected mice versus normal healthy ones. Because collagen is the main element of extracelleular matrix proteins, hydroxyproline, is an amino acid characteristic of collagen metabolism, used as a marker to express the extent of liver fibrosis, as it is the major alteration associated with morbidity [66
]. Similar result was obtained by some authors who emphasized that elevated liver hydroxyproline content was associated with S. mansoni
] and this may be attributed to that S. mansoni
egg granulomas contain factors reposible for the elevation of free L-hydroxyproline content in the fibrotic liver [69
Although IgE is well known for its role in atopic disease and parasitic infections, new evidence suggests that IgE is a pleiotropic Ig molecule with many more functions. Previous studies have shown that IgE possesses antiviral activity, in that IgE anti-HIV-1 antibodies were able to elicit viral clearance and killing through inhibiting virus production [70
Data in the present work showed a significant increase in IgE level in bilharzial and non-bilharzial bladder cancer compared to normal group. It is well established that helminthes infestation induces a very high level of serum IgE which is present at both the initial and later stages of schistosomiasis [71
]. In western countries where the incidence of parasitic disease is very low, a raised IgE may also occur in some patients with cancer [72
In accordance with our findings, Ottesen et al. [73
] indicated that IgE is directed mainly against egg antigen in patients with acute bilharziasis, but it is directed equally to cercarial, egg and adult worm antigens in patients with chronic infestation. In addition, Pidcock et al. [74
] reported that the well established increase in IgE level in patients with schistosomiasis, was also found in bilharzial bladder cancer, indicating that humeral immunity persists in cancer - bearing patients. Patients with cancer not associated with parasitic infestation also had significant increase in their serum level of IgE when compared to healthy Egyptian controls, but 41% of these non-bladder cancer patients showed IgE responses to previous parasitic infestations suggesting that only immunological response to cancer would be on the background of a variable non-specific increase of IgE.
IgE was reported to have the major role in mast cells stimulation which has a central role in the induction of chronic inflammation [75
] and the progression of hepatic fibrosis by producing fibrogenic inflammatory mediators as well as the components of the extracellular matrix proteins (ECMPs) [76
]. Elevated IgE levels have been reported in Hodgkin's lymphoma (HL) and advanced stage of HL disease has also been found to be correlated with elevated IgE [78
It has been postulated that the association between HL and high serum IgE levels is linked to suppressor lymphocyte dysfunction (CD8+) and is distinct from the increase in allergen - specific IgE, which is associated with atopy [79
]. Recently, Fu et al. [80
] reported the presence of elevated IgE and its receptor SCD23 in serum obtained from pancreatic cancer patients versus controls, whereas other Ig isotypes (IgG, IgM, IgA) did not differ between patient and control populations. Also, Chang et al. [81
] reported that high maternal IgE levels were positively associated with childhood leukemia, suggesting that maternal immune function may play a crucial role in the etiology of childhood leukemia.
The current work was also extended to study the inflammatory cytokine, tumor necrosis factor - alpha (TNF-α
) in bilharzial and non-bilharzial bladder cancer patients. The data revealed a significant increase in TNF-α
level in bilharzial and non-bilharzial bladder cancer patients versus normal controls. These results are in harmony with Wahl and Kleinman, [82
] who found an increase in TNF-α
in prostate cancer patients and they attributed this increase in TNF-α
level to the increase of the inducible nitric oxide synthase (iNOS) which is one of the major responses of inflammatory component in a neoplastic transformation. As a result nitric oxide (NO) species have been implicated not only in direct damage to cellular components like DNA and proteins, but can also cause changes to the antioxidant defense of a cell along with increased ROS and could be one of the driving factors for promoting prostate cancer [83
]. Therefore, in addition to ROS generated by inflammatory cells, uncontrolled tumor cell proliferation in an environment rich in growth factors, activated stroma and tumor associated neo-vascualization, can potentiate and/or promote the development of prostate cancer [84
Beside, it was reported that TNF-α
essentially functions as a trophic factor for maintaining adult schistosome viability, it is expressed during egg deposition and has a crucial role in the modulation of granulomatous reaction induced by the eggs [85
]. Torben and Hailu [86
] stated that increased level of this inflammatory cytokine after egg excretion may be an indication of its effect in complications of schistosomiasis, it capable of inducing tissue injury and fibrosis through inducing ROS production, lipid peroxidation [87
], collagen synthesis other fibrogenic risk factors [88
In summary, our results add to exicisting evidence by showing the complexity of the association of bilharzias and bladder cancer. This association seems to be dependent on the repeated infestion of the bladder with S.haematobium parasite. This study compared the profile of the studied biochemical parameters among bilharzial and non bilharzial bladder cancer and normal control subjects. This work is believed to highlight the essential biochemical markers that can be important candidates for bladder cancer diagnosis. In this study, we found that serum fructosamine, as an indicator of glucose consumption, may be a predictor of bladder cancer, also the amino acid, hydroxylproline, as an indicator of liver fibrosis, may be a predictor of bladder cancer risk, in addition, IgE and TNF-α are also may be used as a new immunological markers of bladder cancer.