Clinical potential of Thymoquinone in colorectal cancer: identification of molecular targets and efficacy in combination therapy

Being the most common cancer in Germany and having an important impact worldwide, colorectal cancer (CRC) has been studies extensively to achieve an appropriate therapy for this endemic disease. This PhD thesis aimed to study the effect of the naturally derived drug Thymoquinone (TQ) against CRC. In a first place we investigated the effect of TQ in combination along with traditional chemotherapy. Therefore we explored the ability of TQ, when combined with 5-Fluorouracil (5-FU) to chemosensitize apoptosis-resistant, p53-mutant HT29 colorectal cancer cells. The combined treatment induced a remarkable reduction in cell viability, an increased apoptosis rate, and an inhibition of anchorage-independent growth. Cell cycle analysis suggests that, upon combined treatment, the heavily damaged HT29 cells exit the S-phase and enter the M-phase due to the deficient G2/M checkpoint control. We observed as well cytoskeletal reorganization after single and combination treatment. Due to the high number of multinucleated cells we hypothesize that mitotic catastrophe could be at the origin of the cell death observed after combining TQ and 5-FU. In concordance with in vitro data, in vivo combination of TQ and 5-FU led to a significant reduction of relative tumor size and a higher survival rate. In a second place we examined the possible role of TQ in targeted therapy. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ. By the mean of a kinome array, we identified 50 proteins showing a ≥2 fold-upregulation after TQ treatment. In silico analysis revealed several candidates belonging to the AKT-MEK-ERK1/2 pathway. The oncogenic p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAKThr212 followed by late increase in pPAKThr423. TQ induced early increase of pERK1/2 and triggered the formation of an ERK1/2-PAK1 complex. Modeling confirmed that TQ binds in the vicinity of Thr212 accompanied by conformational changes in ERK2-PAK1 binding, thus abolishing the accessibility of Thr212 to ERK2. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAKThr423 and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with the kinase-dead dominant negative K299R mutant. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAKThr423, finally inhibiting MEKERK1/ 2 signaling and disrupting its prosurvival function. In conclusion, TQ revealed to be beneficial in both combinational and targeted therapies against CRC. Combined with 5-FU, TQ possess the potential to overcome drug resistance in HT29 cells. On the other hand, we showed that TQ can affect classical oncogenic targets belonging to known cancer related signaling pathways. For the first time we show that TQ directly binds to a kinase like PAK1 and induces changes in its conformation and its scaffold function. Finally we strongly recommend the combination of TQ with available therapies in future anti-CRC treatments.