Cyclopamine tartrate, an inhibitor of Hedgehog signaling, strongly interferes with mitochondrial function and suppresses aerobic respiration in lung cancer cells

Previously, we showed that the rate of oxygen consumption in NSCLC HCC4017 cells is intensified compared to the nonmalignant HBEC cells representing normal lung epithelial cells from the same patient [18], as shown in Fig. 1a. Additionally, we measured oxygen consumption rates in five other NSCLC cell lines and found that they were substantially increased in all NSCLC cell lines (see Fig. 1a). Furthermore, we examined the effect of glucose depletion, glutamine depletion, and CycT on the rates of oxygen consumption in these NSCLC cell lines. We found that glucose depletion generally enhances oxygen consumption rates, while glutamine depletion diminishes the rates (see Fig. 1b-g). Glucose and glutamine are two critical fuels for cancer cells [19, 20]. When glucose is limiting, the cells use glutamine, and vice versa. Thus, these results showed that in the absence of glucose, glutamine supports intensified oxygen consumption in cancer cells. In the absence of glutamine, even in the presence of glucose, oxygen consumption was substantially reduced (Fig. 1b-g). Notably, CycT diminished the rates of oxygen consumption in cancer cells largely to the same extent as glutamine depletion. Likewise, another SMO inhibitor SANT1 [21, 22] diminished oxygen consumption in NSCLC cells, as expected. These results indicate that CycT can cause the same effect as glutamine depletion on cancer cell metabolism and aerobic respiration.

The data shown above revealed a strong effect of CycT on aerobic respiration. Thus, we further examined the effect of CycT on NSCLC cell proliferation. We found that CycT diminishes the proliferation and survival of NSCLC cells, although the sensitivity of different cell lines to CycT varies (see Additional file 1: Fig. S1). We also tested whether CycT causes apoptosis in NSCLC cells by using Annexin V and propidium iodide (PI) staining. We found that CycT indeed causes apoptosis in NSCLC cells, albeit with varying efficacy in different NSCLC cell lines. For example, after 24 h of treatment with CycT, H1299 cells were mostly apoptotic, as detected by Annexin V staining (Fig. 2a). PI staining further showed that a fraction of these apoptotic H1299 cells were in the late apoptotic stage. A549 cells, as shown by the proliferation rates in Additional file 1: Fig. S1, were more resistant to CycT (see Fig. 2b). After 24 h of treatment, only a fraction of the cells showed signs of apoptosis, as detected by Annexin V staining. No A549 cells were in late apoptotic stage (see Fig. 2b). Nonetheless, our results showed that CycT can cause apoptosis in NSCLC cells. Notably, another SMO inhibitor SANT1, like CycT, also exerted similar effects on NSCLC cells (Fig. 2a and b).

Heme is a central factor in aerobic respiration and oxidative phosphorylation [23]. Previously, we have shown that limiting intracellular heme levels strongly diminishes mitochondrial respiration and NSCLC cell proliferation and migration [18]. Therefore, we examined whether CycT impacts heme synthesis and metabolism. We found that CycT does not significantly affect the rate of heme synthesis in NSCLC cells (data not shown). Likewise, we found that CycT does not significantly affect the protein levels of the rate-limiting heme synthetic enzyme ALAS1 and the degradation enzyme HO1 (see Fig. 3a and b). For a control, we showed that CycT reduces the level of the Hh signaling target Gli1 (Fig. 3c), as expected. Furthermore, we found that CycT treatment reduced the levels of phosphorylated p44/42 MAPK. The activation of p44/42 MAPK signaling pathway has been shown to be critical for Hh signaling previously [24]. These results show that CycT does not affect aerobic respiration by impacting heme metabolism.

To further investigate the mode by which CycT causes NSCLC cell death, we measured ROS generation in CycT-treated and untreated NSCLC cells. We found that CycT causes a substantial increase in ROS generation in NSCLC cells, including H1299 (Fig. 4a), A549 (Fig. 4b), and H460 (Fig. 4c) cells. In addition, we found that another SMO inhibitor SANT1 increased ROS generation in NSCLC cells (Fig. 4d). Because of the dominant role of mitochondria in oxygen metabolism, mitochondria are mainly responsible for the generation of cellular ROS. Therefore, we also examined the effect of CycT on mitochondrial function. First, we measured and compared mitochondrial membrane potential in CycT-treated and CycT-untreated cells. We found that CycT increases mitochondrial membrane potential substantially in NSCLC cells (Fig. 5a, b and c). This effect of CycT appeared to be stronger in H1299 (Fig. 5a) and H460 (Fig. 5c) cells than in A549 cells (Fig. 5b). Likewise, another SMO inhibitor SANT1 increased mitochondrial membrane potential in NSCLC cells (Fig. 5d).

Mitochondrial morphology is closely linked to mitochondrial function [25]. Changes in mitochondrial morphology regulate many mitochondrial functions, such as the respiratory activity of the electron transport chain and apoptosis [26]. We therefore examined the effect of CycT on mitochondrial morphology in NSCLC cells by using MitoTracker Red. Fig. 6 shows that CycT treatment caused mitochondrial fragmentation in NSCLC cells, H1299 (Fig. 6a), A549 (Fig. 6b), and H460 (Fig. 6c) cells. SANT1 exerted similar effects on the NSCLC cells (not shown). Because Drp1 is the main protein promoting mitochondrial fission and fragmentation [27], we also examined the effect of CycT on Drp1 distribution. Additional file 2: Fig. S2 shows that in CycT-treated NSCLC H1299 (Additional file 2: Fig. S2A) and A549 (Additional file 2: Fig. S2B) cells, Drp1 were indeed selectively localized to various mitochondrial fission sites: The localization pattern of Drp1 completely coincided with the MitoTracker Red staining pattern. These results demonstrated that CycT can induce Drp1 to promote mitochondrial fission and fragmentation.

HBEC30KT and HCC4017 cell lines representing normal and NSCLC cells [44, 45] were provided by Dr. John Minna’s lab (UTSW) as a gift. They were developed from the same patient and were maintained in ACL4 supplemented with 2 % FBS under 5 % CO₂ at 37 °C [45]. All other NSCLC cell lines, H1395, H1299, Calu-3, A549 and H460 were purchased from ATCC, and were maintained according to ATCC procedures. All tissue culture media, including those lacking glucose or glutamine, were purchased from Invitrogen Life Technologies. HBEC30KT, HCC4017, H1299 and A549, were authenticated by using the services provided by Genetica DNA Laboratories, and they were found to be 100 % match. Other NSCLC cell lines were used for measurements immediately following the purchase. Cyclopamine tartrate (CycT) was provided by Logan Natural Products (Plano, Texas). SANT1 was purchased from Santa Cruz Biotechnology. For measuring the effect of CycT or SANT1 on lung cell proliferation, cells were seeded in 48-well plate at a density of 10⁴ cells/well. After culturing for 24 h, cells were treated with the indicated concentrations for 24 h or the indicated time points. At the indicated times, the number of live cells was counted by using trypan blue staining and a hemocytometer. Polyclonal anti-ALAS1, anti-HO1, anti-Gli1 and anti-Drp1 were purchased from Santa Cruz Biotechnology. Monoclonal anti-vinculin antibody was purchased from Sigma-Aldrich. Polyclonal anti-phospho-p44/42 MAPK antibody was purchased from Cell Signaling Technology.

NSCLC cells (∼70 % confluence) were maintained in medium with 25 μM CycT or 50 μM SANT1, or in medium lacking glucose or glutamine for 24 h. Then oxygen consumption was measured, as described previously [46]. Briefly, cells with about 80 % confluence were trypsinized and resuspended in fresh, air-saturated medium. For each measurement, 10⁶ cells (in 350 μl) were introduced in the chamber of an Oxygraph system (Hansatech Instruments), with a Clark-type electrode placed at the bottom of the respiratory chamber. During measurements, the chamber was thermostated at 37 °C by a circulating water bath. An electromagnetic stirrer bar was used to mix the contents of the chamber. Each measurement was replicated at least three times. Standard deviations were calculated, and p values were calculated by using Welch 2-sample t-test (R program).

The intracellular ROS levels were measured by using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (Cayman Chemical), which is oxidized into highly fluorescent 2,7- dichlorofluorescein in the presence of intracellular ROS. Cells were seeded in 96-well plates with black walls and clear bottoms at a density of 10,000 cells per well. Cells were treated with 25 μM CycT or 50 μM SANT1 for the indicated times. Cells were then incubated for 30 min with 10 μM DCFH-DA dissolved in fresh media. Cells were then rinsed twice with PBS, and each well was filled with 100 μl PBS. Fluorescence was detected by using a fluorescent plate reader (Bio Tek, Synergy Mx microplate reader) with the excitation and emission wavelengths at 490 and 535 nm, respectively.

Changes in the mitochondrial membrane potential (ΔΨm) were measured quantitatively by staining with the cationic dye JC-1(Molecular probes), which accumulates in the mitochondria, showing green fluorescence at lower membrane potential and forming J-aggregates with red fluorescence at higher membrane potential. Cells were seeded in 96 well black wall and clear bottom plates at a density of 10,000 cells per well. Cells were treated with 25 μM CycT or 50 μM SANT1 for the indicated times. Cells were then incubated with 200 μl of fresh medium containing 2 μg/μl of JC-1 dye for 30 min in the dark. The cells were washed twice with PBS, and the plates were immediately read with a fluorescent plate reader (Bio Tek, Synergy Mx microplate reader) with excitation and emission wavelengths set at 540 and 595 nm, respectively, for red fluorescence; and 485 and 535 nm, respectively, for green fluorescence.

NSCLC cells were treated, collected, and lysed by using the RIPA buffer (Cell Signaling Technology) containing the protease inhibitor cocktail. Protein concentrations were determined by using the BCA assay kit (Thermo Scientific). 50 μg of proteins from each treatment condition were electrophoresed on 9 % SDS–Polyacrylamide gels, and then transferred onto the Immuno-Blot PVDF Membrane (Bio-Rad). The membranes were probed with polyclonal antibodies, followed by detection with a chemiluminescence Western blotting kit (Roche Diagnostics). The signals were detected by using a Carestream image station 4000MM Pro, and quantitation was performed by using the Carestream molecular imaging software version 5.0.5.30 (Carestream Health, Inc.). Antibodies used were purchased from Santa Cruz Biotechnology and Sigma-Aldrich.

Mitochondria were visualized by using Mito Tracker Red CMXRos (Molecular probes), which passively diffuses across the plasma membrane and accumulates in active mitochondria. Cells were grown on chamber slides and treated with CycT for 24 h. Cells were then stained with 200 nM of CMXRos in complete growth medium for 30 min at 37 °C, washed with prewarmed PBS three times, and fixed with 4 % formaldehyde in PBS for 10 min. After washing twice with PBS the slides were covered, and fluorescent images were captured with a multi-channel Zeiss Axio Observer Z1 fluorescent microscope with a Zeiss 40X Oil immersion lens and with a high speed AxioCam MRm Rev3 monochrome camera.

Indirect immunofluorescence staining with Drp1 antibodies (purchased from Santa Cruz Biotechnology) was performed by following the procedures provided by the antibody manufacturer. FITC and DAPI fluorescent images were captured by using a multi-channel Zeiss Axio Observer Z1 fluorescent microscope. Apoptosis was detected by using the ApoAlert Annexin V-FITC Apoptosis Kit (Clontech). Cells were seeded in a 96-well black wall clear bottom plate at the density of 10,000 cells per well. After one day, cells were treated with 25 μM CycT or SANT1 in fresh medium. Twenty four hours post treatment, apoptosis assay was performed according to manufacturer’s protocol. Fluorescent images were captured using a multi-channel Zeiss Axio Observer Z1 fluorescent microscope with a Zeiss 20X lens and with a high speed AxioCam MRm Rev3 monochrome camera.

There was not any ethics approval or consent required for the use of human derived cell lines in this study.