الفهرس 
Introduction and Aim of the WorkOnly 14 pages are availabe for public view
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Abstract
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide; in Egypt in particular. HCC is the fifth commonest malignancy and the third cause of mortality from cancer (Poon et al., 2002). Unfortunately, the overall response rate of liver cancer treatment is unsatisfactory mainly due to late diagnosis and poor treatment efficacy, resistance to chemotherapeutic drugs and metastasis to other organs (Cappuro et al.,2003). HCC responds to chemotherapy in a poor or nonexistent manner; given the fact that HCC is a highly aggressive tumor that often shows resistance to chemotherapy. Surgery remains the standard treatment (Wei et al., 2013). However, immunotherapy-based treatment represents a new and promising approach for therapy (Wei et al., 2013).
Glypican-3 (GPC-3), a member of glypican family with high expression in HCC selectivity, emerged to be a promising diagnostic marker as well as a new target for antibody-based therapy of HCC (Cappuro et al., 2008). The study investigated GPC-3 as well as one of its pathways including; GPC-3, sulfatase-2 (SULF2); insulin like growth factor (IGF-II), heparan sulphate proteoglycan (HSPG) and caspase-3. The study was divided into three main aspects; clinical, experimental and in vitro study.
In the clinical study, the aim was to investigate GPC-3 as a possible diagnostic marker. Also, it aimed to investigate the mentioned pathway in human. 74 patients including; HCC and cirrhotic patients were recruited in addition to 10 healthy subjects as control for HCC. Statistical analysis of ELISA measurements showed that HCC patients have significant high levels of GPC-3, SULF2 and IGF-II and significant low levels of HSPG when compared to cirrhotic patients and control.
Oxidative imbalance in HCC patients was also detected. Where, a significant increase in oxidative stress markers; malondialdhyde (MDA) level, and a significant decrease in biological antioxidant agents; glutathione (GSH) concentration and superoxide dismutase (SOD) activity, were detected when compared to both cirrhotic patients and control.
A distortion in liver function of HCC patients seen by the measurment of liver function markers including; a significant increase in alanine aminotansferase (ALT) activity and total bilirubin concentration, and significant decrease in albumin concentration when compared with control and significant increase in ALT activity and albumin level and significant decrease in total bilirubin compared with cirrhotic patients.
HCC patients showed a significant decrease in Hb concentration when compared with control group, while showed a significant increase in Hb concentration when compared to cirrhotic patients. Moreover, HCC patients showed significant decrease in RBCs and platelets count when compared with control group. HCC patients showed significant increase in WBCs count when compared with either cirrhotic or control group. While, there was no Insignificance difference was found between both HCC and cirrhotic patients and insignificance difference in RBCs and platelets count, when compared to cirrhotic patients.
The clinical study concluded that GPC-3 can be used as a diagnostic marker for HCC, even of with or without cirrhotic origin. HCC is highly heterogeneous. Where, several markers were significantly increased or reduced from normal levels that promoted carcinogenesis and hepatic degeneration including; SULF2, HSPG, IGF-II and oxidative stress.
In the experimental study, the aim was to investigate GPC-3 as a possible target for immunotherapy by antiglypican-3 (antiGPC-3) and to block the previously investigated pathway in human subjects. Thiocetamide-inducing HCC model in rats was applied. 40 Sprague Dawley adult male rats weighing 180-200 g were randomly divided into 4 equal groups; normal and antiGPC-3 control, HCC and antiGPC-3 treated HCC group. AntiGPC-3, antibody that targets GPC-3, was used in thiocetamide-induced HCC rats; in a dose of 1.2 µg/kg once weekly.
Statistical analysis of antiGPC-3 effects on several markers including; significant decrease in GPC-3 and SULF2 level and gene expression of both caspase-3 and IGF-II; and significant increase in HSPG concentration in antiGPC-3 treated HCC group as compared to HCC group.
The oxidative imbalance was partially ameliorated by antiGPC-3. Where, a significant decrease in MDA and superoxide anion levels and a significant increase in GSH concentration and SOD activity in antiGPC-3 treated HCC group as compared to HCC group were detected.
The liver function tests showed that antiGPC-3 was able to reduce the elevated ALT activity and total bilirubin, and increase albumin concentration in antiGPC-3 when compared to HCC group, indicating the hepatoprotective effect of antiGPC-3, also confirmed by the morphological liver staining with (H & E).
The experimental study concluded that antiGPC-3 (in a dose of 1.2 µg/ml once weekly) is effective in blocking GPC-3 and reduce its level as well as the subsequent pathway involved; SULF-2, HSPG levels, caspase-3 (apoptotic marker) and IGF-II. This action against the pathway caused a protection against HCC development (confirmed by the significant reduction of alpha fetoprotein in antiGPC-3 treated HCC compared to the HCC group); an increase in animal survival (up to 90% in antiGPC-3 treated HCC group compared to only 40 % in HCC group), as well as avoided the cellular damage in hepatocytes (confirmed by the improved liver function test markers, morphological analysis and reduced apoptosis seen in the gene expression of caspase-3).
In the in vitro study, the aim was to confirm the antiGPC-3 antitumor activity and cell cytotoxicity to cancerous cells of HCC. Also, the study investigated whether the decrease in enzyme level of SULF2 by its own substrate (GPC-3) was a direct cause of antiGPC-3. HepG2, human HCC cell line, was used after culturing with three different doses (5, 10 and 20 µg/ml). The result showed that all three doses significantly possessed antitumor and cell cytotoxicity in a dose-dependent manner (proved by MTT assay and lactate dehydrogenase activity). In addition, all three doses significantly reduced GPC-3 in a dose-dependent manner. While, antiGPC-3 increased HSPG in all three doses significantly against hepG2 without treatment, however, the dose of 20 µg/ml showed the highest elevation in the level of HSPG. Moreover, antiGPC-3 was not able to reduce neither SULF-2 nor oxidative stress (measured through hydrogen peroxide level and SOD activity). AntiGPC-3 was able to show antitumor activity through the increase of caspase-3 activity in treated cells by all three doses against untreated cells of hepG2.
The in vitro study concluded that antiGPC-3 was not able to directly affect SULF2 level through blockage of the substrate, GPC-3, but rather indirectly through the improvement of the status of the liver thought antiGPC-3 hepatoprotective effect, thus preventing the elevation of SULF2.
The study recommends the following:
1. The consideration of using GPC-3 as a diagnostic marker for HCC, to allow a more selective and reliable diagnostic tool for HCC.
2. Further investigation of the use of antiGPC-3 as an effective antitumor agent against HCC.
3. AntiGPC-3 should be studied further in combination with other antitumor natural compound for optimum therapy strategy.
4. AntiGPC-3 should be studied further in combination with other certified chemotherapies, to reduce their doses and thus their side effects.
5. Further investigation of the interaction, direct or indirect, of GPC-3 on other markers including; SULF-2 and IGF-II.