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The conversion of many drugs into toxic or less toxic metabolites depends on the first-pass effect of the liver. Toxic metabolites are associated with drug-induced liver injury, often causing liver failure. Thus, therapies often have to be interrupted despite a promising drug effect.
The development of an improved drug metabolism model consisting of liver-derived CYP3A4-overexpressing cells and tumour cells should be used to study drugs in vitro for their efficacy on tumour cells considering the first-pass effect.
Two different co-culture system approaches were tested (transwell insert-based and liver cell supernatant transfer) with HepG2 CYP3A4 cells, originally derived from a tumor characterized by genetic instability, serving as an in vitro liver cell model for establishment. PANC-1 cells (pancreatic cancer) or MCF-7 cells (breast cancer) were used as prototypical tumour cell lines. For cell treatment, three PANC-1 or MCF-7 active drugs MG-132 (MG), Taxol (TX) and Tamoxifen (TAM), were used as these drugs are inactivated (MG and TX) or activated (TAM) by the liver first-pass effect. Three widely used cytotoxicity assays (XTT-, CellTiter-Glo™ 2.0-, and trypan blue exclusion assay) were used to determine acute cytotoxicity to both liver and tumour cells. For co-culture validation, HepG2 CYP3A4 cells were replaced by CYP3A4-overexpressing immortalized primary-like hepatocytes, the FH3 CYP3A4 cells, representing the human liver cell model more closely.
Finally, in a pilot project, primary colon carcinoma cells (pCC-cells) were tested along with HepG2 CYP3A4 and MG.
The use of co-cultures with HepG2 CYP3A4 showed, that first-pass metabolism of MG resulted in reduced cytostatic effects in PANC-1 cells. The TX-induced cytotoxic effect on PANC-1 and MCF-7 cell lines is only slightly attenuated by the first-pass effect. Activation of TAM with FH3 CYP3A4 liver cells in indirect co-culture with transwell inserts most efficiently enhanced the cytotoxic effect on MCF-7 cells. In contrast, the use of HepG2 CYP3A4 proved to be insufficient.
PCC-cells were affected by MG in MC in a dose-dependent manner. Using co-culture systems with HepG2 CYP3A4 cells, colon cancer cells were clearly protected from MG up to a concentration of 2 μM.
In conclusion, first-pass effect could be simulated in vitro in MG- and TX-treated PANC-1 cells and TX-treated MCF-7 cells as well as on MG-treated pCC-cells using HepG2 CYP3A4 cells. Obtained results with PANC-1 and MCF-7 could be validated with FH3 CYP3A4 cells. However, demonstration of the first-pass effect using different end-point measurement methods yielded different results and further studies are needed for definite conclusions.
Lens epithelium derived growth factor (LEDGF), is known to be overexpressed in different solid cancers and cancer cell lines but is also involved in acquired immunodeficiency syndrome (AIDS) and diverse inflammatory diseases. Due to its chromatin-binding ability, it acts as a transcriptional coactivator, promoting anti-apoptotic pathways leading to increased tumor aggressiveness and chemotherapy resistance. In this thesis, the role of LEDGF in laryngeal cancer cells was investigated. Thus, a knock-out of LEDGF in HEp-2 cells was successfully generated by CRISPR/Cas9 technology. Moreover, LEDGF-deficient HEp-2 cells exhibited a decreased proliferation and migration, as well as an increased chemotherapy sensitivity towards topoisomerase II inhibitor etoposide. The DNA damage response was in more detail investigated, where LEDGF depleted cells showed a drastic reduction in the recruitment of downstream damage-response related proteins, more precisely replication protein A subunit of 32kDa (RPA32), and an increased amount of DNA fragmentation, highlighting the role of LEDGF in homology-directed DNA repair.
In addition, a efficient method was established to allow genomic tagging of LEDGF at its C-terminus with EGFP which enables the monitoring of LEDGF in living cells. Therefore, an efficient Cas9-mediated DNA DSB induction was supplemented with a DNA template containing homology arms to the C-terminus as well as the EGFP sequence to allow homology-directed repair. Subsequently, EGFP-expressing cells were FACS sorted and single cells expanded. Almost all test clones showed a homozygous genotype for LEDGF-EGFP fusion which exhibited the typical nuclear pattern of LEDGF.