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Tian, Fei (2013): Effect of the Hedgehog Pathway inhibitor GDC-0449 in lung cancer cells and lung cancer stem cells. Dissertation, LMU München: Medizinische Fakultät



The cancer stem cell hypothesis implicates that tumor cell population is heterogeneous with relatively well-differentiated cells and poorly-differentiated cells. Only the small population of tumourigenic poorly-differentiated CSCs can escape the normal limits of self-renewal and has the ability to proliferate and maintain the malignant growth of the tumor. One characteristic of stem cell is that the ability to exclude DNA dyes, such as Hoechst 33342 via the over-expression of ATP-binding cassette transporters (ABC transporters) on the cell membrane. It makes the detecting of the stem cell possible. After the Hoechst 33342 staining, stem cells extrude this dye and show a typical profile of low fluorescence in Hoechst red versus Hoechst blue bivariate dot plots. These low Hoechst 33342 stained cells are named as side population (SP) cells. This characteristic has enabled purification and characterization of CSCs when carried out alone or in combination with stem cell surface markers. The CSC hypothesis could have a fundamental influence on cancer therapy. CSCs have shown significant substantial resistance to conventional chemotherapy in contrast to the differentiated cancer cells. It is essential to design a complete therapy strategy first to reduce or minimize proliferating cell mass and then to differentiate or eliminate CSCs, so that the relapses of metastatic cancers could be prevented. This work aimed at investigating if Hedgehog pathway inhibitor GDC-0449 is effective in the lung cancer cell lines HCC (adeno-carcinoma) and H1339 (small cell lung carcinoma) and also the cisplatin resistant lung cancer cells, and if possible effects of GDC-0449 are mediated via SPs. Furthermore, the effect of GDC-0449 on the calcium homeostasis was also investigated. GDC-0449 showed dose-dependent inhibitory effects on cell growth in both HCC and H1339 cells. The combination of GDC-0449 and cisplatin gave an additional inhibitory effect. GDC- 0449 could also inhibit the cell growth in cisplatin resistant HCC and H1339 cells. SP cells as cancer stem-cell-like cells could be found in HCC and H1339 cells. Only the SP cells showed the repopulation ability but not the non-SP cells. GDC-0449 could inhibit the SP cell fraction in both HCC and H1339 cells. So the inhibitory effect of GDC-0449 on cell growth may be mediated via SP. GDC-0449 affected the expression of the Hh pathway components in both HCC and H1339 cells. In HCC cells, GDC-0449 inhibited the activity of the Hh pathway and the down- regulation of Shh, Patched and Gli-1 could be shown. In H1339 cells, GDC-0449 could also inhibit the pathway activity and decrease the expression of Gli-1 in an autocrine pattern due to the over-expression of Shh. The inhibition of Hh pathway increased the expression of Bmi-1 to compensate the loss of Hh pathway function. The Hh pathway activity could be detected only in SP cells from HCC and H1339 cells. The application of GDC-0449 on HCC and H1339 naïve and cisplatin resistant cells increased [Ca2+]c by decreasing [Ca2+]ER. GDC-0449 induced Ca2+ release from ER into cytoplasm in HCC and H1339 naïve and cisplatin resistant cells. The Ca2+ overload could lead to apoptosis, which was related to the cell growth inhibitory effect of GDC-0449 in lung cancer cells. The expression of SERCA and IP3R was not detectably influenced by GDC-0449. The effect of GDC-0449 on lung cancer cell Ca2+ -regulating machinery was not via the alternation of the expression of ER Ca2+ regulating channels.