Cell culture models and novel gene therapeutic strategies for colorectal cancer

The development of relevant cellular model systems for colorectal
cancer is of utmost importance for an improved in vitro assessment
of therapeutic strategies against colorectal cancer. Recently
published low passage colon cancer cell lines that closely reflect
the characteristics of the respective parental in vivo tumor cells
represent very promising cell culture models and were therefore
used for the investigations in the present thesis. To provide an in
vitro model system that also recapitulates the three-dimensional
structure of in vivo tumors, these low passage cell lines were
cultivated as multicellular spheroids. Compared to monolayer
cultures the multicellular spheroids exhibited a wide variety of
changes in their expression patterns. The differential expression
includes proteins that are involved in growth signaling
(15-hydroxyprostaglandin dehydrogenase), protein biosynthesis
(acidic ribosomal protein P0), and regulation of the cyto- or
nucleoskeleton (acidic calponin and LMNA protein). These proteins
were identified by 2D electrophoresis and subsequent MALDI-TOF mass
spectrometry. Both methods were established in the lab in the
context of this work. Chemotherapy with 5-fluorouracil (5-FU)
represents the traditional treatment of colorectal cancer. However,
in many patients the efficiency of this therapeutic strategy is
often limited by the development of chemoresistance against 5-FU.
Therefore, it was an aim of this thesis to detect novel proteins
involved in 5-FU chemoresistance that were previously not ascribed
to resistance against this chemotherapeutic drug. A chemoresistant
subline of a colon cancer cell line was generated by long-term
treatment with 5-FU and served as a model for the investigation of
5-FU chemoresistance. This subline exhibited resistance against
both 5-FU-induced inhibition of proliferation and apoptosis.
Differences in the expression of cytokeratin 18, heat shock protein
27 and aldehyde dehydrogenase 1B1 between the chemoresistant
subline and parental cells were detected by 2D electrophoresis.
These findings imply that the cytoskeleton plays a role in the
development of chemoresistance against 5-FU. Furthermore, processes
located to the mitochondria seem to be involved in this resistance,
since heat shock protein 27 and aldehyde dehydrogenase 1B1 are
associated with this subcellular organelle. The biological
relevance of the findings made in the present PhD thesis has to be
determined in further studies. Gene therapy represents a promising
alternative strategy for the treatment of colorectal cancer. A
novel nonviral gene transfer system was developed by combination of
DNA with the polycation PEI25br and the cationic lipids DOCSPER or
DOSPER to form lipopolyplexes. These lipopolyplexes enabled
enhanced gene transfer in vitro and are promising for in vivo
applications, since the established lipopolyplexes preserved their
small size at physiological conditions; a property essential for a
successful in vivo application. Furthermore, the lipopolyplexes
exhibited the capability to efficiently transfect three-dimensional
multicellular spheroids. The potential of lipopolyplexes for
therapeutic applications was further increased by the utilization
of the artificial promoter CTP4 which enables highly specific gene
expression in cancer cells with mutations in the Wnt signaling
pathway by transcriptional targeting. In addition to its high
specificity, this promoter enabled high gene expression levels that
were comparable to expression levels obtained by the strong, but
unspecific CMV promoter. The efficiency of the CTP4 promoter was
demonstrated in seven low passage colon cancer cell lines and also
in multicellular spheroids. The transcriptional targeted
lipopolyplexes not only enabled high tumor specific expression of
reporter genes like luciferase or EGFP but also the expression of a
therapeutic gene, interleukin-2 (IL-2). Furthermore, tumor specific
expression of cytotoxic protease 2A in combination with IL-2 was
possible by using a novel bicistronic construct. The expression of
the rhinoviral protease 2A led to efficient reduction of overall
cap-dependent gene expression levels and therefore also the
proliferation of the transfected cells, while continued IL-2
expression was guaranteed by an IRES element enabling
cap-independent gene expression in the presence of protease 2A. In
summary, the present results provide a promising basis for the
development of novel potent strategies in the treatment of
colorectal cancer.