Research ArticleExperimental Studies

The Hedgehog Pathway Inhibitor GDC-0449 Alters Intracellular Ca2+ Homeostasis and Inhibits Cell Growth in Cisplatin-resistant Lung Cancer Cells

FEI TIAN, KATHRIN SCHRÖDL, ROSEMARIE KIEFL, RUDOLF MARIA HUBER and ALBRECHT BERGNER
Anticancer Research January 2012, 32 (1) 89-94;

Abstract

Aim: Cisplatin resistance is an important issue in lung cancer. We aimed at investigating if the Hedgehog pathway inhibitor GDC-0449 is effective in cisplatin-resistant cells and if it alters intracellular Ca2+-homeostasis. Materials and Methods: The cytoplasmatic ([Ca2+]cyto) and endoplasmatic ([Ca2+])ER Ca2+ concentration of HCC (adeno carcinoma of the lung) and H1339 (small cell lung carcinoma) cells were measured with the calcium indicator dye Fura-2 AM. The expression of the inositol-1,4,5-trisphosphate receptor (IP3R) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were analyzed using western blot analysis. Results: GDC-0449 inhibited cell growth in cisplatin-naïve and -resistant cells. In both cell types, GDC-0449 increased [Ca2+]cyto and reduced endoplasmatic [Ca2+]ER. Cisplatin failed to considerably alter Ca2+ homeostasis in resistant cells. The effects of GDC-0449 on intracellular Ca2+ homeostasis were not mediated by an altered expression of IP3R or SERCA. Conclusion: GDC-0449 alters intracellular Ca2+ homeostasis and inhibits cell growth in cisplatin-resistant lung cancer cells.

Despite recent therapeutical advances, lung cancer is still the leading cause of cancer death for both men and women (1). Although therapeutical regimens including chemotherapy support quality of life, they frequently fail to increase long-term survival. Cisplatin-based therapy regimens have become the backbone of lung cancer chemotherapy. First-line chemotherapy often leads to encouraging responses, but, throughout course of the treatment, resistance to cisplatin regularly occurs.

The hedgehog (Hh) signaling pathway plays an important role in organ development and body patterning during embryogenesis (2). In adults, activation of the Hh pathway mainly occurs during tissue repair. After binding of Hh ligands such as Sonic, Indian or Desert to the Hh receptor Patched-1, its inhibitory effect on the signal transducer Smoothened (SMO) is relieved. This leads to activation of the transcription factor glioma-associated oncogene (GLI) and thereby to transcription of Hh target genes. Such target genes comprise proliferation and apoptosis regulating proteins such as cyclins or B-cell lymphoma 2 (BCL2). Not surprisingly, therefore, dysregulation of the Hh pathway has been implicated in a variety of cancer types (3). In lung cancer, pathological activation has also been reported both in small-cell lung cancer (SCLC) (4) and non-small cell lung cancer (NSCLC) (5, 6).

Molecular therapies target specific cellular structures that are essential for malignant cell growth. Therefore, the detection of molecular therapies is a main focus of current anti-cancer research (7, 8). GDC-0449 is a low-molecular Hh pathway inhibitor that binds to and inhibits SMO (9). It is the first systemic SMO inhibitor that has entered clinical trials. GDC-0449 is proven to be both effective and of low toxicity in several types of solid tumor (10-12) In particular, it appears to have antitumor activity in advanced basal cell carcinoma (13). Recently, we showed that GDC-0449 is effective in lung cancer cell lines and that in combination with cisplatin it has an additional effect (14). A phase II clinical trial on the combination of GDC-049 with cisplatin and etoposide in SCLC has been initiated (15). However, no data on the effect of GDC-0449 on cisplatin-resistant lung cancer cells are available.

Calcium is a ubiquitous signal molecule that is involved in almost all cellular pathways (16, 17). This is particularly true for proliferation and apoptosis, and an imbalance of cell growth and cell death finally leads to cancer. In apoptosis, calcium from the extracellular space or released from the endoplasmic reticulum (ER) leads to an increase in the mitochondrial Ca2+ concentration, which in turn opens the permeability transition pore, followed by an efflux of cytochrome c. Cytochrome c amplifies the Ca2+ release from the ER and activates the intrinsic pathway of apoptosis via caspase 9 (18-20). In a previous study, we showed that cisplatin-resistance in lung cancer cells was mediated by a lower ER Ca2+ content ([Ca2+]ER) caused by altered expression of inositol-1,4,5-trisphosphate receptors (IP3R) and sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) (21).

The aim of the present study was to investigate if GDC-0449 is effective on cisplatin-resistant cells and if its effects may be mediated by altered intracellular Ca2+ homeostasis.

Materials and Methods

Materials. Cell culture reagents were obtained from Life Technologies (Eggenstein, Germany). Other reagents were bought from Sigma-Aldrich (Deisenhofen, Germany) unless stated otherwise. The human adeno carcinoma cell line HCC and the human small-cell lung carcinoma cell line H1339 were purchased from DSMZ, Braunschweig, Germany.

Survival curves. HCC and H1339 cells were seeded in 25 cm2 cell culture flasks and cultured for 24 h. Cells were exposed to GDC-0449 (25 μM and 50 μM) for 4 days and cell viability was evaluated by trypan blue exclusion cell counting. For cisplatin treatment, cells were 3 h exposed to 1 μM cisplatin for 3 h followed by incubation with medium with Dimethylsulfoxide (detergent for GDC-0449, used as control) for 4 days. This approach was chosen because after application of cisplatin to humans a relevant plasma concentration of unbound cisplatin (active form) lasts for only 3 h (22).

Ca2+ imaging. For quantification of changes of the [Ca2+]cyto, cells were loaded for 90 min at 37°C with the calcium indicator dye Fura-2 AM (10 μM, Molecular Probes, Eugene, USA) in cell culture medium. After loading, cells were incubated for another 30 min in Phosphate Buffered Saline (with Ca2+ and Mg2+) to allow complete dye de-esterification and were examined with a fluorescence microscope (Axiovert 200 M, Carl Zeiss, Jena, Germany). Images were obtained at both excitatory wavelength 340 nm and 380 nm with a digital CCD camera (AxioCamMRm; Carl Zeiss Vision, Munich, Germany). For each image, regions of interest (ROIs) were defined in the cytoplasm of the cells, and the average fluorescence intensity was calculated using custom written macros in the image analysis software Scion. Ca2+ concentrations were calculated according to Grynkiewicz (23). The ER Ca2+ content was measured using an indirect approach. One micromolar cyclopiazonic acid was applied during Fura-2 loading to inhibit SERCA, which pumps Ca2+ into the ER, leading to a net Ca2+ efflux out of the ER. The increase of the cytoplasmatic Ca2+ concentration was utilized as an estimate of ER Ca2+ concentration. In order to prevent Ca2+ entry by store-operated channels, the incubation solution was substituted by PBS without Ca2+ and Mg2+ immediately prior to imaging.

Western blot analysis. HCC and H1339 cells were washed twice with ice-cold phosphate-buffered saline (10 mM, pH 7.4) and scraped in a 0.02% EDTA solution. Whole protein extraction was carried out according to the protocol from the complete lysis-M protein extraction reagent set. Lysis buffer (500 μl) was used for 107 cells for high protein concentration and efficient protein extraction. Protein concentration was measured with a non-interfering protein assay kit and a standard curve from BSA standard samples.

Protein (50 μg) from each sample was diluted with NuPAGE reducing buffer, sample buffer and ddH2O to 50 μl and was heated by a thermomixer at 70°C for 10 min for denaturation. Electrophoresis voltage was set at constant 150 V and with a run time of 90 min. Western transfer was performed at constant 30 V for 60 min. The membranes were blocked in blocking buffer at room temperature for 3 h and were afterwards incubated with specific antibodies for 16 h at 4°C. Staining was performed using specific antibodies (rabbit anti-SERCA 1/2/3, dilution 1:50; rabbit anti-IP3R, dilution 1:250) and secondary antibodies (goat anti-rabbit IgG horseradish peroxidase (HRP), dilution 1:10000). ß-Actin staining was used as loading control (mouse anti-human β-actin HRP, dilution 1:5000, all from Santa Cruz Biotechnology,). Antibody complexes were visualized using Hyperfilm ECL chemiluminescence (Amersham Biosciences, UK) and evaluated using Image-J analysis software (National Institutes of Health).

Statistics. One-way or two-way ANOVA (combined with pairwise multiple comparisons) were performed using Sigma Stat software (Jandel Scientific, Chicago, USA). A p-value of less than 0.05 was considered statistically significant.

Results

HCC (adenocarcinoma of the lung) and H1339 (small cell lung carcinoma) cells were exposed to the Hh-pathway inhibitor GDC-0449 (25 μM or 50 μM) or cisplatin. GDC-0449 concentration dependently reduced cell growth (Figure 1). This effect was less pronounced compared to the effect of cisplatin.

An increase in [Ca2+]cyto can lead to apoptosis (19). We therefore investigated if GDC-0449 or cisplatin increased [Ca2+]cyto. In both cell lines, [Ca2+]cyto was significantly elevated after exposure to 50 μM GDC-0449, and to 1 μM cisplatin, with the increase being higher after exposure to GDC-0449 (Figure 2). An increase in [Ca2+]cyto can result from an Ca2+ influx from the extracellular space or from Ca2+ release from the ER. Recently, we have shown that in lung cancer cell lines, Ca2+ release from the ER is the major source of [Ca2+]cyto (21). Analyzing the [Ca2+]ER after exposure to 50 μM GDC-0449 or 1 μM cisplatin, we found that it was significantly decreased in both cell lines (Figure 3). The decrease was significantly more pronounced after exposure to GDC-0449 compared to cisplatin.

Recently, we were also able to create cisplatin-resistant cell clones by repeatedly exposing cells to cisplatin (21). Overcoming cisplatin resistance is an important clinical issue. We therefore investigated the effectiveness of GDC-0449 in cisplatin-resistant cells. Exposing cisplatin-resistant HCC and H1339 cells to GDC-0049, we found that GDC-0449 significantly inhibited cell growth (Figure 4).

Next, we measured the [Ca2+]cyto in cisplatin-resistant HCC and H1339 cells after exposure to 50 μM GDC-0449. Despite cisplatin-resistance, GDC-0449 caused a substantial increase in [Ca2+]cyto relative to the control (Figure 5). Analyzing the [Ca2+]ER, we found it to be significantly decreased after exposure to GDC-0449 compared to both the control and cisplatin-treated cells (Figure 6).

Figure 1.
Figure 1.

HCC (A) and H1339 (B) cells were exposed to 25 μM GDC-0449, 50 μM GDC-0449 or 1 μM cisplatin and the cell number was assessed. Error bars are not shown for clarity (n=3, *p<0.05 versus all other groups).

The IP3R mediates Ca2+ release from the ER (24). Sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) force calcium against the concentration gradient from the cytoplasm into the ER. In a previous study, we showed that the expression of IP3R and/or SERCA is altered in cisplatin-resistant cells (21). The increase in [Ca2+]cyto in cisplatin-resistant cells triggered by GDC-0449, could have been caused by an increased expression of IP3R or reduced expression of SERCA. Nevertheless, we found the expression of IP3R and SERCA in cisplatin-resistant cells to be unchanged after exposure to GDC-0449 (Figure 7).

Discussion

In this study, we showed that the inhibiting Hh pathway via the use of GDC-0449 reduces cell growth in lung cancer cells while increasing [Ca2+]cyto and reducing [Ca2+]ER. In cisplatin-resistant cells, GDC-0449 also inhibits cell growth and similarly increases [Ca2+]cyto, as well as reducing [Ca2+]ER. Cisplatin fails to considerably alter the intracellular Ca2+ homeostasis in cisplatin-resistant cells. The effects of GDC-0449 on [Ca2+]ER and [Ca2+]cyto in cisplatin-resistant cells are not mediated by altered expression of IP3R or SERCA.

Figure 2.
Figure 2.

HCC (A) and H1339 (B) cells were exposed to 50 μM GDC-0449 or 1 μM cisplatin and the [Ca2+]cyto was measured using fluorescence microscopy. In both cell lines, [Ca2+]cyto increased with the increase being higher after exposure to GDC-0449 (n=60, *p<0.01 versus controls, #p<0.01 versus cisplatin).

So far, only few studies have investigated a possible role of the intracellular Ca2+ homeostasis in cisplatin resistance. Liang et al. reported that in cisplatin-resistant cells of the human lung adenocarcinoma cell line A549, the resting [Ca2+]cyto was decreased compared to non-resistant cells (25). Tsunoda et al. postulated for the cisplatin-resistance of bladder cancer cells a role for the inositol-1,4,5-trisphosphate receptor (26). The authors showed that IP3R expression was down-regulated in resistant cells as well as after exposure of non-resistant cells to cisplatin. Recently, Splettstoesser and colleagues showed that in Hela-S3, but not in human osteoblastoma cells, cisplatin induced Ca2+ influx from the extracellular space involving IP3R on the plasma membrane (27).

Figure 3.
Figure 3.

HCC (A) and H1339 (B) cells were exposed to 50 μM GDC-0449 or 1 μM cisplatin and the [Ca2+]ER was measured using fluorescence microscopy. In both cell lines, [Ca2+]ER decreased with the decrease being larger after exposure to GDC-0449 (n=60, *p<0.01 versus controls, #p<0.05 versus cisplatin).

In our study, we found that cisplatin increased [Ca2+]cyto and reduced [Ca2+]ER in naïve cells. It is, therefore, reasonable to assume that the increase in [Ca2+]cyto was caused by Ca2+ release from the ER. An elevated [Ca2+]cyto leads to an increase in the mitochondrial Ca2+ concentration, which in turn opens the permeability transition pore. This is followed by an efflux of cytochrome c, activating the intrinsic pathway of apoptosis (18). In resistant cells, cisplatin had almost no effect on the Ca2+ homeostasis. These data are compatible with our previous findings that cisplatin-resistant cells avoid apoptosis by avoiding cytoplasmic Ca2+ overload (21). But GDC-0449 altered the intracellular Ca2+ homeostasis in cisplatin-resistant cells in the same way as in cisplatin-naïve cells. Accordingly, GDC-0449 also inhibited cell growth despite cisplatin resistance.

Figure 4.
Figure 4.

Cisplatin-resistant HCC (A) and H1339 (B) cells were exposed to 1 μM cisplatin, 25 μM GDC-0449 or 50 μM GDC-0449 and the cell number was assessed. Error bars are not shown for clarity. (n=3, *p<0.05 versus all other groups).

The Hh pathway leads to altered transcription of Hh target genes. Such target genes comprise proliferation and apoptosis-regulating proteins such as cyclins and BCL2. As GDC-0449 altered the intracellular Ca2+ homeostasis, we were interested to see if the expression of Ca2+-regulating proteins was changed. Because we had previously shown that the expression of IP3R and SERCA is different in cisplatin-resistant lung cancer cells compared to non-resistant cells (21), we investigated the expression of these proteins after exposure to GDC-0449. However, no influence of GDC-0449 was detected. Therefore, the link between the Hh pathway and the Ca2+-regulating machinery of the cell has yet to be established.

Figure 5.
Figure 5.

Cisplatin-resistant HCC (A) and H1339 (B) cells were exposed to 50 μM GDC-0449 or 1 μM cisplatin and the [Ca2+]cyto was measured using fluorescence microscopy. In both cell lines, [Ca2+]cyto increased after exposure to GDC-0449 (n=60, *p<0.01 versus all other groups).

In a phase II clinical trial the effect of combination of GDC-049 with cisplatin and etoposide in SCLC is currently under investigation (15). This approach seems particularly promising given our data on the SCLC cell line H1339. With cisplatin being quite effective in SCLC, the addition of GDC-0449 may be able to inhibit the survival of cisplatin-resistant cells and therefore prevent relapse of the disease.

We believe that GDC-0449 is a promising substance which should be clinically tested for effectiveness in lung cancer. Nevertheless, future studies have to address the mechanisms of how GDC-0449 alters intracellular Ca2+ homeostasis.

Figure 6.
Figure 6.

Cisplatin-resistant HCC (A) and H1339 (B) cells were exposed to 50 μM GDC-0449 or 1 μM cisplatin and the [Ca2+]ER was measured using fluorescence microscopy. In both cell lines, [Ca2+]ER decreased after exposure to GDC-0449 (n=60, *p<0.01 versus all other groups).

Figure 7.
Figure 7.

Cisplatin-resistant HCC and H1339 cells were exposed to 50 μM GDC-0449 and the expressions of SERCA and IP3R were analysed using Western blot analysis. No difference compared to controls was observed (n=3, representative blots are shown).

  • Received September 6, 2011.
  • Revision received November 19, 2011.
  • Accepted November 20, 2011.

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The Hedgehog Pathway Inhibitor GDC-0449 Alters Intracellular Ca2+ Homeostasis and Inhibits Cell Growth in Cisplatin-resistant Lung Cancer Cells
FEI TIAN, KATHRIN SCHRÖDL, ROSEMARIE KIEFL, RUDOLF MARIA HUBER, ALBRECHT BERGNER
Anticancer Research Jan 2012, 32 (1) 89-94;
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