Zebrafish as an in vivo model of drug screens for liver cancer: the role of phenothiazines
Author(s)
Advisor
Karakayalı, Özlen KonuDate
2021-08Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
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Abstract
Hepatocellular carcinoma (HCC) is one of the most dangerous cancer types and sorafenib
(SFB) is a commonly used drug against HCC, being the only FDA-approved medication until
2018. However, its limited efficacy and severe side effects emphasize the importance of
developing alternative approaches. Regulation of cholesterol has been implicated in the
progression of various cancers including HCC. Phenothiazines, which are in use as
antipsychotic drugs and with effects on cholesterol biosynthesis, have drawn recent attention
as anti-cancer drugs against different cancers. The elucidation of how phenothiazines exert
their effects at cellular level might pave the way for proposing better strategies for HCC
treatment. Zebrafish serves as a good cancer model for in vivo validations, since it enables
efficient tumor formation and tracking. Its conserved genes and similar organ system to
human make it even better of a model to reveal both molecular and morphological alterations
caused by drugs. Based on previous cell viability studies from our lab, the combination of
trifluoperazine (TFP) with SFB was found to exhibit synergism in Hep3B cells, while it was
antagonistic in SkHep1 cells. Moreover, RNA-seq analysis on Hep3B cells has demonstrated
that steroid biosynthesis and cholesterol metabolism was among the modulated pathways.
Therefore, in the present thesis, the regulation of cholesterol levels in response to
phenothiazine derivatives was investigated in HCC cell lines SkHep1 and Hep3B. The combinatorial approach was examined on Huh7 cells in terms of the expression of cholesterol
biosynthesis genes based on RT-qPCR. After showing the ability of phenothiazine derivatives
and TFP-SFB combinations to modulate cellular cholesterol, zebrafish drug screens were
performed to determine applicable derivative and combination doses. Assessment of safe
doses for TFP, SFB and their combination directed us for xenograft studies, which resulted in
enhanced Hep3B cell survival by the combination of low dose (6 μM) TFP and 1 μM of SFB.
This finding was further validated based on Alu-based DNA quantifiation, a powerful method
we have established. With the help of RNA-seq analysis results, orthologous zebrafish genes,
involved in cholesterol biosynthesis were identified and used in cross-species testing. The
impact of TFP on liver vasculature was evaluated using transgenic (Fli1a:EGFP) zebrafish
and found insignificant. Overall, the findings presented in this thesis highlight that TFP alone
or in combination was able to change cellular cholesterol levels and tumor growth.
Keywords
Hepatocellular carcinomaPhenothiazines
Sorafenib
Drug repurposing
Cholesterol
Zebrafish
In vivo drug screening
Xenograft