Date of Award

6-2021

Document Type

Thesis

Degree Name

Master of Science in Molecular Biology and Biotechnology

Department

Biology

First Advisor

Dr. Mohammed Ayoub

Abstract

Hepatocellular carcinoma (HCC) is one of the most commonly occurring cancers worldwide. The risk factors include obesity, hepatitis, alcoholism, smoking etc. One of the biggest associated challenges is the current treatment strategies. The therapeutic options are limited with economical and accessibility challenges, especially with increased HCC incidence. Sorafenib, a multikinase inhibitor, is one of the first FDA approved drugs for HCC. But drug resistance and cancer relapse are a common drawback for this treatment. The need for better treatment is crucial now more than ever. The use of saffron as a potential natural therapeutic option is not a novel strategy. It has been long used as an analgesic, anti-inflammatory and anti-spasmodic in folk medicine. Recently, saffron ant its bioactive constituents have shown anti-cancer properties through different mechanisms. In this study, Safranal was tested against the hepatocellular carcinoma cell line (HepG2) for its effectiveness on cell viability and autophagy. The cells exhibited a decreased survival on treatment with Safranal which was associated with induced autophagy. This was confirmed by SDS-PAGE and western blot showing an increase in the expression of the major autophagic protein markers such as Beclin-1 and Atg 12. These effects were also supported by microscopic analysis where phagosome formation was evident. Moreover, the combination of Safranal with Sorafenib showed antagonistic effect on Safranal inhibiting cell viability. the effect of Safranal and Sorafenib alone and their combination on the canonical kinase, ERK1/2, controlling the survival pathway, was also investigated. Safranal had no effect on AKT expression level but inhibited the phosphorylated form of AKT which is consistent with the inhibition of cell survival and the induction of autophagy. These results have provided with a possibility of Safranal being an effective chemotherapeutic against HCC. The combination treatment led to more complex observations that require further investigation in the future. Also, Safranal’s effect on other cancerous cell lines and the other possible mechanisms by which Safranal affects HCC can be potential areas to broach in the future.

Arabic Abstract

ﺳﺮطﺎن اﻟﻜﺒﺪ ھﻮ أﺣﺪ أﻛﺜﺮ أﻧﻮاع اﻟﺴﺮطﺎﻧﺎت ﺷﯿﻮﻋًﺎ ﻓﻲ ﺟﻤﯿﻊ أﻧﺤﺎء اﻟﻌﺎﻟﻢ. ﺗﻌﺘﺒﺮ اﻟﺴﻤﻨﺔ واﻟﺘﮭﺎب اﻟﻜﺒﺪ وإدﻣﺎن اﻟﻜﺤﻮل واﻟﺘﺪﺧﯿﻦ ﻣﻦ اﻟﻌﻮاﻣﻞ اﻟﻤﺆدﯾﺔ ﻟﻺﺻﺎﺑﺔ. ﺧﯿﺎرات اﻟﻌﻼج اﻟﺤﺎﻟﯿﺔ ﻣﺤﺪودة وﻟﯿﺴﺖ اﻗﺘﺼﺎدﯾﺔ وﻻ ﯾﻤﻜﻦ اﻟﻮﺻﻮل إﻟﯿﮭﺎ ﺑﺴﮭﻮﻟﺔ ﻓﻲ ﺟﻤﯿﻊ أﻧﺤﺎء اﻟﻌﺎﻟﻢ، ﺧﺎﺻﺔ ﻓﻲ اﻟﺤﺎﻻت اﻟﺘﻲ ﯾﺰﯾﺪ ﻓﯿﮭﺎ ﻣﻌﺪل اﻹﺻﺎﺑﺔ ﺑﺴﺮطﺎن اﻟﻜﺒﺪ. ﯾﻌﺪ ﺳﻮراﻓﯿﻨﯿﺐ، ﻣﺜﺒﻂ اﻟﻌﺪﯾﺪ ﻣﻦ اﻻﻧﺰﯾﻤﺎت، أﺣﺪ اﻷدوﯾﺔ اﻷوﻟﻰ اﻟﻤﻌﺘﻤﺪة ﻣﻦ ﻗِﺒﻞ إدارة اﻷﻏﺬﯾﺔ واﻟﻌﻘﺎﻗﯿﺮ ﻟﻌﻼج ﺳﺮطﺎن اﻟﻜﺒﺪ. إﻻ أن ﻣﻘﺎوﻣة ھﺬا اﻟﻌﻘﺎر وﻋﻮدهة اﻟﻤﺮض ﻣﻦ أﺧﻄﺮ ﺳﻠﺒﯿﺎت ھﺬا اﻟﻌﻘﺎر. ﻣﻤﺎ ﺟﻌﻞ اﻟﺤﺎﺟﺔ اﻟﻰ ﻋﻼج أﻓﻀﻞ أﻣﺮ ﺑﺎﻟﻎ اﻷھﻤﯿﺔ اﻵن أﻛﺜﺮ ﻣﻦ أي وﻗﺖ ﻣﻀﻰ. اﺳﺘﺨﺪام اﻟﺰﻋﻔﺮان ﻛﻤﺮﻛﺐ ﻋﻼﺟﻲ ﻟﯿﺲ ﺑﻤﻔﮭﻮم ﺟﺪﯾﺪ ﻓﻠﻄﺎﻟﻤﺎ اﺳﺘﺨﺪم ﻛﻤﺴﻜﻦ وﻣﻀﺎد ﻟﻼﻟﺘﮭﺎﺑﺎت وﻣﻀﺎد ﻟﻠﺘﺸﻨﺞ ﻓﻲ اﻟﻄﺐ اﻟﺸﻌﺒﻲ. وﻓﻲ اﻵوﻧﺔ اﻷﺧﯿﺮة أظﮭﺮت ﻣﻜﻮﻧﺎت اﻟﺰﻋﻔﺮان ﺧﺼﺎﺋﺺ ﻣﻀﺎدة ﻷﻧﻮاع ﻣﺨﺘﻠﻔﺔ ﻣﻦ اﻟﺴﺮطﺎن ﻣﻦ ﺧﻼل آﻟﯿﺎت ﺟﺰﯾﺌﯿة ﻣﺨﺘﻠﻔﺔ. ﻓﻲ ھﺬه اﻟﺪراﺳﺔ، ﺗﻤﺖ دراﺳﺔ ﻓﺎعلية اﻟﺴﺎﻓﺮاﻧﺎل ﻓﻲ ﺣﯿﻮﯾﺔ واﻻﻟﺘﮭﺎم اﻟﺬاﺗﻲ ﻟﺨﻼﯾﺎ ﺳﺮطﺎن اﻟﺨﻼﯾﺎ اﻟﻜﺒﺪﯾﺔ. أظﮭﺮت اﻟﺨﻼﯾﺎ اﻧﺨﻔﺎﺿاً ﻓﻲ ﻣﻌﺪل اﻟﺒﻘﺎء ﻋﻠﻰ ﻗﯿﺪ اﻟﺤﯿﺎة ﻋﻨﺪ اﻟﻌﻼج ﺑﺎﺳﺘﺨﺪام اﻟﺴﺎﻓﺮاﻧﺎل اﻟﺬي ﺗﺴﺒﺐ ﻓﻲ اﻻﻟﺘﮭﺎم اﻟﺬاﺗﻲ. ﺗﻢ ﺗﺄﻛﯿﺪ ذﻟﻚ ﻣﻦ ﺧﻼل ﺗﺠﺎرب معملية دقيقة اﻟﺘﻲ اظﮭﺮت زﯾﺎدة ﻓﻲ اﻧﺘﺎج ﺑﺮوﺗﯿﻨﺎت اﻟﺒﻠﻌﻤﺔ اﻟﺮﺋﯿﺴﯿﺔ ﻣﺜﻞ Beclin-1 و Atg 12، وﻛﺬﻟﻚ ﻣﻦ ﺧﻼل اﻟﺘﺤﻠﯿﻞ اﻟﻤﺠﮭﺮي ﻟﺘﺸﻜﯿﻞ اﻟﺒﻠﻌﻤﺔ. ﻋﻼوة ﻋﻠﻰ ذﻟﻚ، ﻓﺈن اﻟﺠﻤﻊ ﺑﯿﻦ اﻟﺴﺎﻓﺮاﻧﺎل وﺳﻮراﻓﯿﻨﯿﺐ أظﮭﺮ ﺗﺄﺛﯿﺮاً ﻣﻌﺎﻛﺴاً ﻋﻠﻰ ﺣﯿﻮﯾﺔ اﻟﺨﻼﯾﺎ اﻟﻤﺜﺒﻄﺔ لـ ﻟﺴﺎﻓﺮاﻧﺎل. ﻟﻘﺪ ﺑﺤﺜﻨﺎ أﯾﻀًﺎ ﻓﻲ ﺗﺄﺛﯿﺮ اﻟﺴﺎﻓﺮاﻧﺎل وﺳﻮراﻓﯿﻨﯿﺐ وﻣﺰﯾﺠﮭﻤﺎ ﻋﻠﻰ اﻧﺰﯾﻤﺎت ERK1/2، واﻟﺘﺤﻜﻢ ﻓﻲ ﻣﺴﺎرات اﻟﺘﻜﺎﺛﺮ واﻟﺒﻘﺎء، ﻋﻠﻰ اﻟﺘﻮاﻟﻲ. ﻟﻢ ﯾﻜﻦ ﻟﻠﺴﺎﻓﺮاﻧﺎل أي ﺗﺄﺛﯿﺮ ﻋﻠﻰ ﻣﺴﺘﻮى ﺗﻌﺒﯿﺮ AKT وﻟﻜﻨﮫ أﻋﺎق ﺑﺸﻜﻞ ﻛﺒﯿﺮ اﻟﺸﻜﻞ اﻟﻔﺴﻔﻮري ﻟـ AKT واﻟﺬي ﯾﺘﻮاﻓﻖ ﻣﻊ ﺗﺜﺒﯿﻂ ﺑﻘﺎء اﻟﺨﻠﯿﺔ وﺗﺤﺮﯾﺾ اﻻﻟﺘﮭﺎم اﻟﺬاﺗﻲ. وﻗﺪ أﺗﺎﺣﺖ ھﺬه اﻟﻨﺘﺎﺋﺞ إﻣﻜﺎﻧﯿﺔ أن ﯾﻜﻮن اﻟﺴﺎﻓﺮاﻧﺎل ﻋﻼﺟًﺎ ﻛﯿﻤﯿﺎﺋﯿًﺎ ﻓﻌﺎﻻً ﺿﺪ ﺳﺮطﺎن اﻟﻜﺒﺪ. أدى اﻟﻌﻼج اﻟﻤﺮﻛﺐ إﻟﻰ ﻣﻼﺣﻈﺎت أﻛﺜﺮ ﺗﻌﻘﯿﺪًا ﺗﺘﻄﻠﺐ ﻣﺰﯾﺪًا ﻣﻦ اﻟﺘﺤﻘﯿﻖ ﻓﻲ اﻟﻤﺴﺘﻘﺒﻞ. ﻛﻤﺎ ﯾﻤﻜﻦ أن ﯾﻜﻮن ﺗﺄﺛﯿﺮ اﻟﺴﺎﻓﺮاﻧﺎل ﻋﻠﻰ ﺧﻄﻮط اﻟﺨﻼﯾﺎ اﻟﺴﺮطﺎﻧﯿﺔ اﻷﺧﺮى واﻵﻟﯿﺎت اﻟﻤﺤﺘﻤﻠﺔ اﻷﺧﺮى اﻟﺘﻲ ﯾﺆﺛﺮ ﺑﮭﺎ اﻟﺴﺎﻓﺮاﻧﺎل ﻋﻠﻰ ﺳﺮطﺎن اﻟﻜﺒﺪ ﻣﺠﺎﻻت ﺑﺤﺜﯿﮫ ﻣﺤﺘﻤﻠﺔ ﻟﻠﺘﻄﺮق إﻟﯿﮭﺎ ﻓﻲ اﻟﻤﺴﺘﻘﺒﻞ.

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