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

Abstract

BackgroundAberrant 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.MethodsThe 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.ResultsOur 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.ConclusionsTogether, 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2200-x) contains supplementary material, which is available to authorized users.

The Antibiotic Drug Tigecycline: A Focus on its Promising Anticancer Properties

“…Supporting this concept, Alam et al (2016a) demonstrated that enhanced mitochondrial aerobic respiration is necessary for many types of cancer cells to gain tumorigenic and drug-resistant potential, such as non-small cell lung cancer (NSCLC) and breast cancer. Inhibition of mitochondrial respiration by Hedgehog inhibitors, such as cyclopamine tartrate, could strongly interfere with cell proliferation and induce apoptosis in NSCLC (Alam et al, 2016b). In addition, studies have demonstrated that based on the context, the mitochondrial mass can serve as either a pro-survival or pro-death modulator in tumor development and progression (Joshi et al, 2016).…”

WW45, a Gli1 binding protein, negatively regulated Hedgehog signaling in lung cancer

“…Cyclopamine, the inhibitor for Smo, has shown anti-cancer activity [17]. In this study, we found that over-expression of WW45 promoted the ubiquitination and degradation of Gli1.…”

The Bioenergetic Role of Mitochondria in Lung Cancer

“…Cyclopamine tartrate inhibits SMO, thereby inhibiting Hedgehog signaling. In NSCLC HCC4017 cells, studies have demonstrated intensified oxygen consumption compared to benign HBEC cells from the same patient [20]. Low levels of glucose also contribute to higher oxygen consumption rates, and low levels of glutamine lower oxygen consumption rates.…”

“…Consequently, when the mitochondria of tumor cells are tampered with to inhibit oxidative phosphorylation, these tumor cells become significantly more susceptible to cytotoxic drugs [16,19]. Agents such as cyclopamine tartrate, a water-soluble derivative of a molecule found in corn lilies [20]; metformin, an antidiabetic drug [18]; BAY 87-2243, a lead structure [15]; and microRNA-126, a microRNA [21] have shown potential to interfere with normal mitochondrial function in cancer cells. These molecules interact with various aspects of mitochondrial function, such as interfering with Hedgehog signaling and inhibiting Complex I. Cyclopamine tartrate functions by inhibiting Hedgehog signaling [20].…”