الفهرس 
Introduction and aim of the workOnly 14 pages are availabe for public view
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Abstract
Aims: This study was performed to investigate the role of tuftelin1 (TUFT1) as an early diagnostic biomarker in hepatocellular carcinoma (HCC) and to evaluate its potential therapeutic effect via intervention of the signaling pathway responsible for its oncogenic role in HCC progression by dantrolene sodium (DAN). In addition, to explore the role of DAN in chemo-sensitization of hepatic tissue to sorafenib (SF). Materials &Methods: The study was conducted on 180 Wistar albino adult male rats divided into two main categories: Saline treated category consisted of 70 rats injected intrapritoneally (i.p.) with a single dose of saline (0.6 – 0.8 ml per rat), the vehicle in which diethyl nitrosamine (DENA) was dissolved, followed by continuation on drinking water till the end of induction period where they were further allocated into normal control group (10 rats) as well as drugs control groups including three regimen doses of DAN (1.1, 2.2, and 6.6 mg/kg), and the same three doses of DAN plus SF (SF+DAN). Another category was DENA treated one consisted of 110 rats’ injected i.p. with a single dose of DENA (200 mg/kg). Two weeks later, phenobarbital sodium (0.05%) was added to drinking water for the remaining period of induction. They were further allocated into 2 sets: Staging set consisted of 3 groups (10 rats per each) including DENA8W group where rats were sacrificed after 8 weeks of DENA injection, DENA16W group in which rats were sacrificed after 16 weeks of DENA injection, and DENA24W group where rats were sacrificed after 24 weeks of DENA injection. Another one was treatment set, where induction of HCC in this group was continued for 18 weeks. At the end of 18th week, rats were randomly allocated into 8 groups (10 rats per each). One group was left as HCC model without any additional treatment (HCC group) and other ones were treated with either SF (HCC+SF group), DAN at three different dose levels including HCC+DAN (1.1, 2.2, and 6.6 mg/kg) groups or combination of SF and either of the three different doses of DAN including HCC+SF+DAN (1.1, 2.2, and 6.6 mg/kg). At the end of each experiment, serum and liver tissues were collected. Hepatic content of TUFT1 protein was assayed via western blot and immunohistochemistry (IHC). Serum amino transferases (ALT, AST) were measured spectrophomerically. In addition, serum alph-fetoprotein (AFP), and hepatic content of phosphatidylinositol-3-kinase (PI3K), protein 53 (p53), vascular endothelial growth factor (VEGF), Cyclin D1, as well as matrix metalloproteinase-9 (MMP-9) were assessed using ELISA technique. Hepatic and serum Ca+2 were also computed colorimetrically. Furthermore, Ki-67 expression was measured by IHC. Results: In staging experiment, the three studied stages showed significant elevations (p <0.05) in all measured parameters including serum ALT, AST, and AFP as well as elevated hepatic contents of TUFT1, Ca+2, PI3K, p53, VEGF, Cyclin D1, and MMP-9, in addition to histological changes when compared to control group. Moreover, the three stages exhibited significant differences, when compared to each other, in TUFT1 expression either on western blot or IHC results, as well as, hepatic Ca+2 content, PI3K, p53, VEGF, Cyclin D1, and MMP-9 expressions. The distinction between the three stages was also clarified at histopathological level, where the early stage showed the lowest values for all mentioned parameters in addition to mild changes in histological pattern, while the late stage exhibited the highest values in all parametrs beside the great anaplastic alterations showed in histopathological analysis. Concerning treatment experiments, rats treated with either of the three doses of DAN showed significant reductions (p <0.05) in all evaluated parameters as compared to those treated with DENA alone (HCC group). Furthermore, in comparing the potency between the three doses, the sub-therpeutic dose exhibited the lowest potency while the high dose revealed the highest potency. For this reason, Ki-67 expression was assessed in rats treated with this high dose, where it also showed anti-proliferaticve activity. Moreover, rats treated with SF in combination with either of the three doses of DAN recorded significant attenuations of HCC induced elevation of all parameters mentioned above compared to those treated with SF alone. More importantly, in comparing the potency of the three combination regimens, the regimen involved SF and the sub-therapeutic dose of DAN exhibited the lowest potency. On the other hand, the regimen involved sorafenib and the high dose of DAN displayed the highest potency, in addition, it showed greater anti-proliferative activity when compared to that of SF. Conclusions: Our study verified that TUFT1 protein can serve as an early diagnostic biomarker in HCC as well as the degree of its expression can be correlated with HCC stage when assayed along with other HCC related biomarkers such as AFP. Moreover, DAN, at either of three given doses, exerted antineoplastic effect as well as it augmented the antineoplastic activity of SF via intervention of TUFT1/Ca+2/PI3K pathway, manifested by ameliorating apoptosis, proliferation, cell cycle progression, angiogenesis, and metastasis.
Keywords: Hepatocellular carcinoma; Tuftelin-1; Intracellular Ca+2; Dantrolene sodium; Sorafenib.