الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Hepatocellular carcinoma is the most common form of primary liver cancer. Globally, it ranks the fifth most common cancer and in terms of mortality it is the second leading cause of cancer related deaths. In Egypt, HCC represents 70% of all liver tumors. HCC is a complex disease with multiple risk factors, where HBV chronic infection is the most common risk factor accounting for 50-80% of HCC cases. While, HCV chronic infection ranks the second position accounting for 20% of HCC cases worldwide. Other risk factors include ingestion of large amounts of aflatoxins, alcohol, NAFLD and diabetes. The presence of cirrhosis increases the risk for HCC development; however, it is not an essential precursor. The dominance of the risk factor varies geographically, where chronic HBV infection and AFB1 exposure are the major risk factors in the high-risk areas. In contrast, HCV infection, excessive alcohol consumption, and diabetes/obesity are found to have important roles in areas of low-risk of HCC.
Development and progression of HCC is a multistep process governed by the accumulation of genetic and epigenetic alterations responsible for the activation of oncogenes and the inactivation of tumor suppressor genes, resulting in disruption of the fundamental cellular processes. Hepatocarcinogenesis involves several signaling pathways controlling proliferation, differentiation, inflammation, angiogenesis and tumor suppression.
The Ras/Raf/MEK/ERK and PI3K/Akt/mTOR pathways are often activated by aberrant upstream signaling molecules such as growth factors and cytokines. The activation of Ras/MAPK pathway, responsible for cellular proliferation, represents the main mechanism for HCC development specifically HBV and HCV-related HCC, through HBx and HCV core proteins, respectively.
The asymptomatic nature of HCC in early stages results in disease detection in the advanced stages, thus creating a therapeutic challenge which may lead to incurable consequences. Based on BCLC staging system, treatment options are stratified to cover all disease stages from the early stage where transplantation, resection and ablation are the treatment options, to TACE as the treatment of choice for the intermediate stage and finally molecular-targeted therapies for the advanced stages.
Sorafenib is a multikinase inhibitor, targeting Raf kinase, the tyrosine kinase activities of the VEGFR-2/-3 and PDGFR-β. Sorafenib has been approved as the first effective systemic drug for treating advanced HCC showing an increase in the overall survival by 3 months. However, sorafenib response is still far from satisfactory.
Honokiol, a natural compound extracted from Magnolia officinalis, was reported to exhibit antiproliferative and apoptotic effects, through the SHP-1 mediated STAT-3 inhibition and the activation of p38 MAPK and caspase-3 to initiate the apoptotic signal in human hepatoma cell lines. These discoveries brought the focus on HNK as a potential chemotherapeutic agent, either alone or in combination with other therapies, that can inhibit both primary and metastatic tumors, help overcome drug resistance and reduce the toxicity associated with chemotherapeutic agents.
This study focused on HNK-mediated antitumor effects, either alone or in combination with sorafenib, in the treatment of HCC. These effects were evaluated through the measurement of protein expression levels of various growth factors, transcription factors and signaling molecules such as activated Ras, p-Akt, HGF, eIF4E, STAT-3, cell cycle regulator cyclin D1, pro-apoptotic protein Bax, active caspase-3, HIF-1α and VEGF.
Summary
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In this study, HCC HepG2 cell line was used. Cell viability and cytotoxicity assays using MTT reagent, protein expression analysis using ELISA and colorimetric assays were performed.
Growth factors play a critical role in the activation of various signaling pathways that are essential for transduction of proliferation and survival signals from extracellular receptors to the nucleus to modulate various cellular functions favoring HCC growth.
One point of cross-talk is the activated Ras and its stimulation for downstream effectors like Raf and PI3K activating both Ras/Raf/MAPK and PI3K/Akt/mTOR pathway. In the present study, HNK alone produced a significant downregulation in the expression level of Ras-GTP. However, HNK combined with sorafenib produced further reduction in the expression level of the activated Ras, although this decrease was not significant when compared to each drug individually.
Also, HNK alone or combined with sorafenib reduced the expression levels of p-eIF4E, cyclin D1 and HIF-1α. As p38MAPK is regulated by activated Ras, inhibition of Ras activation, by both HNK and sorafenib, could have downregulated p38 MAPK phosphorylation and activation leading to a reduction in its targets such as Mnk-1 resulting in further decrease in p-eIF4E and cyclin D1.
Moreover, the PI3K/Akt/mTOR pathway regulates cellular proliferation, survival, invasion, metabolism and angiogenesis. Similarly, PI3K is a downstream effector for the activated Ras, which activates Akt protein regulating its downstream targets such as mTORC-1. As demonstrated in the current results, inhibition of p-Akt by HNK monotherapy or combined with sorafenib consequently may inhibit mTORC-1 and its downstream targets like p70S6K.
Activation of STAT-3, through phosphorylation, leads to STAT-3 dimerization and translocation to the nucleus where they bind to the DNA and induce transcription of many genes namely; cyclin D1, HIF-1α and VEGF. HNK alone reduced the expression level of p-STAT-3 through inhibition of non-receptor tyrosine kinases, c-Src, JAK-1/2 and induction of SHP-1. However, sorafenib treatment resulted in further decrease in p-STAT-3 level which is mediated through increase in SHP-1 activity and inhibition of Akt mediated phosphorylation of STAT-3 (Tyr705) residue and inhibition of MEK/ERK-regulated phosphorylation of (Tyr727) subunit. The downregulation of p-STAT-3 upon treatment of HepG2 cell line with combined HNK and sorafenib was mainly due to sorafenib-mediated effects.
In the present study, the expression level of various targets for the Ras/MAPK and the PI3K/Akt/mTOR pathways were evaluated. Starting from the eIF4E as an essential factor for the initiation of translation of several genes, among them are cyclin D1, as a cell cycle regulator, HIF-1α and VEGF as markers for angiogenesis.
The level of p-eIF4E was reduced upon treatment with HNK alone or combined with sorafenib, and this was probably due to the inhibition of Ras/Raf/Mnk -1 signaling or PI3K/Akt/mTOR pathway inhibition.
The expression of cyclin D1 was reduced by HNK alone or in combination with sorafenib. The combination produced more pronounced decrease in cyclin D1 expression level, as compared to each drug monotherapy. This could be attributed to the dual inhibition of cyclin D1 regulators such as c-Fos, STAT-3 and eIF4E by both HNK and sorafenib.
Summary
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Although the expression level of HIF-1α, under normoxic conditions, was markedly reduced upon HNK treatment, yet sorafenib was superior to HNK in decreasing HIF-1α expression through its multiple tyrosine kinase inhibitory activity and the inhibition of RTKs mediated activation for the Ras/MAPK and the PI3K/Akt/mTOR pathway at multiple levels, as observed in this study. Therefore, the pronounced downregulation for the HIF-1α expression level upon combined therapy might be mediated mainly through sorafenib.
The expression level of VEGF was downregulated in the present study upon incubation with HNK alone or combined with sorafenib. The combination showed more inhibitory effect on VEGF expression level, as compared to either drug monotherapy. This was presumably due to the induction of SHP-1 by both HNK and sorafenib in addition to other mechanisms.
For better understanding of the effect of HNK monotherapy or combined with sorafenib on apoptosis, the proapoptotic protein Bax expression and caspase-3 activity were measured.
The present data revealed that exposure of HepG2 cell line to HNK alone resulted in an upregulation in the expression level of Bax protein, and an increase in caspase-3 activity through activation of p38 MAPK pathway. Moreover, HNK combined with sorafenib produced further upregulation in Bax protein expression and caspase-3 activity. Sorafenib was found to activate Ras/Raf/JNK pathway, upregulate PUMA and increase Bax protein expression and caspase-3 activity. Additionally, sorafenib induces Bim and t-Bid proteins that translocate to the nucleus and induce Bax protein expression. Besides activation of the sensitizer protein Bad that neutralizes the anti-apoptotic members of the Bcl-2 family, thus activating Bax, caspase-9 and its downstream effector caspase-3. The obtained results indicated that Bax protein upregulation was mediated through upregulation of Bax expression by both HNK and sorafenib. However, sorafenib was superior to HNK in the induction of caspase-3 activity.
In summary, this study showed that HNK alone demonstrated several anti-tumor effects including downregulation of activated Ras, p-Akt, p-STAT-3, p-eIF4E, cyclin D1 expression, HIF-1α expression and VEGF, in addition to upregulation of the apoptotic protein Bax and caspase-3 activity. Combination of HNK and sorafenib had more favorable effects on the evaluated parameters and showed antitumor effects surpassing each drug alone. These results highlighted the beneficial antitumor effects of HNK as a chemotherapeutic agent either alone or in combination with sorafenib to help to overcome drug resistance and reduce toxicity associated with sorafenib.