Effect of the Hedgehog Pathway inhibitor GDC-0449 in lung cancer cells and lung cancer stem cells

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.