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

Abstract

Background: Aberrant Hedgehog (Hh) signaling is associated with the development of many cancers including prostate cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, and basal cell carcinoma. The Hh signaling pathway has been one of the most intensely investigated targets for cancer therapy, and a number of compounds inhibiting Hh signaling are being tested clinically for treating many cancers. Lung cancer causes more deaths than the next three most common cancers (colon, breast, and prostate) combined. Cyclopamine was the first compound found to inhibit Hh signaling and has been invaluable for understanding the function of Hh signaling in development and cancer. To find novel strategies for combating lung cancer, we decided to characterize the effect of cyclopamine tartrate (CycT), an improved analogue of cyclopamine, on lung cancer cells and its mechanism of action.

Methods: The effect of CycT on oxygen consumption and proliferation of non-small-cell lung cancer (NSCLC) cell lines was quantified by using an Oxygraph system and live cell counting, respectively. Apoptosis was detected by using Annexin V and Propidium Iodide staining. CycT's impact on ROS generation, mitochondrial membrane potential, and mitochondrial morphology in NSCLC cells was monitored by using fluorometry and fluorescent microscopy. Western blotting and fluorescent microscopy were used to detect the levels and localization of Hh signaling targets, mitochondrial fission protein Drp1, and heme-related proteins in various NSCLC cells.

Results: Our findings identified a novel function of CycT, as well as another Hh inhibitor SANT1, to disrupt mitochondrial function and aerobic respiration. Our results showed that CycT, like glutamine depletion, caused a substantial decrease in oxygen consumption in a number of NSCLC cell lines, suppressed NSCLC cell proliferation, and induced apoptosis. Further, we found that CycT increased ROS generation, mitochondrial membrane hyperpolarization, and mitochondrial fragmentation, thereby disrupting mitochondrial function in NSCLC cells.

Conclusions: Together, our work demonstrates that CycT, and likely other Hh signaling inhibitors, can interrupt NSCLC cell function by promoting mitochondrial fission and fragmentation, mitochondrial membrane hyperpolarization, and ROS generation, thereby diminishing mitochondrial respiration, suppressing cell proliferation, and causing apoptosis. Our work provides novel mechanistic insights into the action of Hh inhibitors in cancer cells.

Fig. 1

a The rates of oxygen consumption are intensified in various NSCLC cell lines. b-g CycT, like glutamine depletion, strongly diminished oxygen consumption rates in NSCLC H1299 (b), H1395 (c), Calu-3 (d), A549 (e), HCC4017 (f), and H460 (g) cells. NSCLC cell lines were cultured in their normal medium or in medium lacking glucose or glutamine, or treated with CycT, as indicated. h SMO inhibitor SANT1, like CycT, can diminish oxygen consumption in NSCLC cells. H1299 cells were treated with CycT or SANT1. The rates of oxygen consumption were measured. The data shown were averages of at least three independent measurements. For statistical analysis, the values were compared to that in nontumorigenic HBEC lung cells (a) or those in normal culture medium (in B-G), by using Welch 2-sample t-test. *, p value < 0.05; **, p value < 0.005

Fig. 2

CycT and SANT1 induce apoptosis in H1299 (a) and A549 (b) NSCLC cell lines. The NSCLC cells were treated with CycT or SANT1 for 24 h. Then cells were subjected to apoptosis assay by using Annexin V-FITC and Propidium Iodide (PI) staining. The images of cells were captured with bright field microscopy (BF) or with fluorescent microscopy with a FITC or rhodamine (for PI) filter

Fig. 3

The effect of CycT treatment on the levels of ALAS1 (a), HO1 (b), Gli1 (c), and phospho-p44/p42 MAPK (d). The NSCLC A549 cells were cultured and treated with CycT for 24 h [lane 2 in (a) and (b)] or without CycT [lane 1 in (a) and (b)]. In (c) and (d), cells were treated without (lane 1) or with CycT for 1 (lane 2), 3 (lane 3), 6 (lane 4), 12 (lane 5), and 24 (lane 6) hours as indicated. Protein extracts were prepared and the levels of the proteins were detected by Western blotting. The protein level of vinculin was used for normalization. For statistical analysis, the levels in treated cells were compared to the levels in untreated cells, by using Welch 2-sample t-test. *, p value < 0.05

Fig. 4

CycT treatment increases ROS production in NSCLC H1299 (a), A549 (b), and H460 (c) cells. d SANT1 treatment also increases ROS production in NSCLC H1299 cells. NSCLC cells were treated with CycT or SANT1 for the indicated time periods. Then cells were incubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min. Fluorescence intensity was measured and normalized according to cell density. For statistical analysis, the levels in CycT-treated cells were compared to the levels in untreated cells, by using Welch 2-sample t-test. *, p value < 0.05; **, p value < 0.005

Fig. 5

The effect of CycT treatment on mitochondrial membrane potential in NSCLC H1299 (a), A549 (b), and H460 (c) cells. d SANT1 treatment also increases mitochondrial membrane potential in NSCLC H1299 cells. NSCLC cells were treated with CycT or SANT1 for the indicated time periods. Then mitochondrial membrane potential in these cells was measured by using JC-1 staining. Mitochondrial membrane potential was expressed as the ratio of aggregates/monomer, which was calculated by dividing red fluorescence intensity with green fluorescence intensity. For statistical analysis, the levels in CycT-treated cells were compared to the levels in untreated cells, by using Welch 2-sample t-test. *, p value < 0.05; **, p value < 0.005

Fig. 6

CycT treatment causes mitochondrial fragmentation in NSCLC H1299 (a), A549 (b), and H460 (c) cells. NSCLC cells were treated with CycT for 24 h, and then stained with MitoTracker Red. Fluorescent images were acquired and shown here. The scale bar indicates 10 μm