الفهرس 
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Abstract
Bladder cancer is one of the most common urinary system malignancies worldwide occurring in both sexes. It is more frequent in men than women, and the incidence and mortality rate of bladder cancer greatly increased in the recent years. Cigarette smoking is the main risk factor for the development of urothelial tumor. Other risk factors may include exposure to arsenic in drinking water, occupational exposure to aromatic amines, and genetic alterations.
A large number of chemotherapeutic agents including natural and synthetic compounds have been identified as having some potential cancer therapeutic value such as inhibiting mutagenesis, hyperproliferation or induce apoptosis or differentiation. Current scientific interest in the management of cancer is directed towards the utilization of naturally occurring compounds for chemotherapeutics.
The current investigation was designed to evaluate the therapeutic activity of myrtenal in N-butyl-N-(4- hydroxybutyl)-nitrosamine (BBN)-induced bladder carcinoma model with the objective of elucidating the modulatory role in the inflammatory and apoptotic proteins and anti-oxidant property in animal models as well as the antioxidant potential in the applied animal model.
Study design
Fifty male Wistar rats, weighing 120-150 g were randomly allocated into 5 equally- sized groups as follows:
group I Normal Control (NC); Rats were fed normal diet for 20 consecutive weeks serving as normal controls.
group II Myrtenal (Mrt); Rats were orally treated with myrtenal (80 mg/kg b.w./d) dissolved in corn oil for 20 consecutive weeks (Ayyasamy and Leelavinothan, 2016).
group III Corn oil (CO); Rats were orally treated with corn oil (1 ml/kg b.w./d) for 20 consecutive weeks.
group IV bladder carcinogenesis model (BBN+γ- IRR); Rats were exposed to γ-ionizing radiation (1 Gy/week, for a total of 8 Gy) concurrent with BBN-containing water for 8 consecutive weeks then BBN-water was replaced by tap water for the rest of the experiment (12 weeks).
group V Myrtenal protected group (BBN+γ- IRR+Mrt); Rats were exposed to γ-ionizing radiation (1 Gy/week, for a total of 8 Gy) concurrent with BBN- containing water for 8 consecutive weeks besides oral administration of myrtenal (80 mg/kg b.w./d) dissolved in corn oil for 8 weeks then BBN-water was replaced by tap water and rats were still given myrtenal orally for the rest of experimental period (12 weeks).
Body weight was recorded weekly and the general health of rats was observed regularly.
Biochemical studies
At the end of the experiment (week 20th), the animals were fasted overnight and blood samples were withdrawn from rats by heart puncture after light ether anesthesia. Collected blood samples were allowed to coagulate at 37oC for 15 min, then centrifuged at 3000 rpm for another 15 minutes. Serum portions were separated and used to determine the levels of tumor necrosis factor (TNF-α), Interleukin-6 (IL-6) and nitric oxide (NO). Urinary bladders were divided into three parts: First part was placed in ice- cold saline (0.9 N NaCl) and homogenized to yield ultimately a 10% (w/v) whole tissue homogenates for analysis of malondialdehyde (MDA), myeloperoxidase enzyme (MPO), and caspase-3 levels. Molecular Investigation
The second part was used for the determination of gene expression levels of cyclooxygenase-2 (COX-2), proapototic protein (Bax), and antiapoptotic protein (Bcl-2), nuclear factor κB (NF- κB) and signal transducer and activator of transcription 3 (STAT3) by real time-PCR.
Histopathological examination
The third bladder part was fixed in 10% neutral formalin overnight, embedded in paraffin, and cut into 4- 5µm sections for histological analysis following hematoxylin and eosin staining.
Results of the present study demonstrated that administration of BBN with exposure to gamma radiation has resulted in non-invasive bladder carcinoma appeared as a mushroom-like growth that is only in the inner layer of the bladder lining. A severely atypical papillary hyperplasia and a non-invasive papillary transitional cell carcinoma were seen. The cancer cells look more abnormal and grow slightly more quickly (Grade 2). These histological findings proved the credibility of the applied experimental carcinogenesis model. On the other hand, urinary bladder mucosa of myrtenal treated animals showed improvement characterized by largely delicate papillae and some of them fused with each other. The cancer cells look a lot like normal bladder cells (Grade 1).
The biochemical outcomes of the present study supported the histopathological findings and indicated that the tumorigenic effect of BBN and γ-radiation was associated with a significant reduction in the average final body weight
and the weight gain of animals combined with a significant increase in the bladder relative weights. This was associated with an increase in the oxidative stress (MDA & NO) and inflammatory (TNF-α, IL-6 and MPO) biomarkers. On the other hand, exposure to BBN and γ-radiation altered the balance between proapoptotic and antiapoptotic biomarkers which was elicited by the significant increase in the caspase-3 level and the mRNA expression of Bcl-2 together with down regulation in the expression of Bax and Bax/Bcl-2 ratio as compared to normal group. Additionally, exposure to BBN and γ-ionizing radiation significantly upregulated the mRNA expression of COX-2 and NF-κB.
In contrast, animals received myrtenal together with BBN and γ-IRR manifested a considerable lesser reduction in their body weight and weight gain. Also, the increase in bladder relative weight was considerably lightened through treatment of tumored animals with myrtenal. In addition, administration of myrtenal to BBN-treated rats exhibited downregulation in the expressions of COX-2, NF-κB and STAT-3 associated with suppression of inflammatory cytokines levels of TNF-α and IL-6 as well as biomarkers of oxidative damage (MDA & NO). Further, myrtenal treatment
caused a significant increase in caspase-3 activity and Bax/Bcl-2 ratio.
Conclusion
The powerful anti-inflammatory, antioxidant and pro- apoptotic properties of myrtenal in the present study were able to ameliorate the histology and the biochemical outcomes in the BBN-induced bladder carcinoma. Present findings, in addition to benefits described in other pathologies, indicated myrtenal as a potential adjuvant natural compound for the prevention of tumor progression of bladder cancer. However, it is necessary to further characterize the signaling pathways that regulate these processes in order to further potentiate the therapeutic applications of myrtenal in various cancers.