Mitochondria targeting and its consequences for cell death in neuroblastoma

Mitochondria are one of the central regulators of several physiological and pathological processes. For instance, homeostatic glycolysis, citric acid cycle, and oxidative phosphorylation are all regulated by mitochondria. In addition, these organelles play a pivotal role in apoptosis, a process of programmed cell death. In detail, the permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic proteins from the intermembrane space are considered as an initiating and the no return step of apoptosis. Therefore, apoptosis induction and targeting mitochondria of cancer cells is a promising strategy for tumor cell elimination. During the studies of this PhD project mitochondria targeting and its consequences for cell death in neuroblastoma (NB) were investigated. In Paper I we demonstrated how low doses of the mitochondrial Complex II inhibitor thenoyltrifluoroacetone (TTFA) can sensitize a panel of chemoresistant NB cells for cisplatin treatment. Increase in cell death was due to TTFAs specific inhibition of the ubiquinonebinding site of succinate dehydrogenase (SDH) that led to the formation of reactive oxygen species (ROS). Excessive ROS production resulted in the cytochrome c release from the intermembrane space of mitochondria and activation of apoptosis. However, the coadministration of TTFA and cisplatin increased cell death only in NB cell lines with functionally active SDH. These results revealed how mitochondria targeting can be used to raise treatment efficacy, but previous testing of SDH activity is needed for successful outcome. Many of therapeutic drugs damage DNA, but their ability to target mitochondria is unknown. The effect of conventionally used chemotherapeutic drugs on mitochondria was tested in Paper II. Obtained results revealed etoposide-triggered suppression of Complex I respiration. Blockage of the electron chain led to the ROS formation, which did not induce apoptosis. However, cell death was detected after removal of glutamine, a precursor of the antioxidant glutathione. Therefore, etoposide also sensitizes mitochondria and can direct the mitochondrial membrane properties to apoptosis. Several tumors, such as NB, are dependent on glutamine and targeting glutamine metabolism offers a rationale for treatment improvement. The effect of glutamine withdrawal on anticancer drug treatment of NB cells was studied in Paper III. We discovered a contrasting effect of drugs in glutamine deprived environment. Glutamine removal inhibited etoposide-induced, but significantly increased cisplatin-induced apoptosis, suggesting the activation of distinct mechanisms. Nevertheless, targeting glutamine metabolism could be considered as part of anticancer therapy, but further studies to understand the mechanism of this finding are needed. Taken together, the findings of this PhD thesis have given new insights into mitochondria targeting for tumor cell elimination. We found new sensitizing effects of TTFA and etoposide that target respiratory chain complexes. Furthermore, the PhD project elucidated the role of glutamine metabolism in the outcome of NB treatment, which can be used as a basis for further studies

Targeting succinate: ubiquinone reductase potentiates the efficacy of anticancer therapy

Mitochondria play a pivotal role in apoptosis: permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic proteins from the intermembrane space of mitochondria are regarded as the key event in apoptosis induction. Here we demonstrate how non-toxic doses of the mitochondrial Complex II inhibitor thenoyltrifluoroacetone (TTFA), which specifically inhibits the ubiquinone-binding site of succinate dehydrogenase (SDH), synergistically stimulated cell death, induced by harmless doses of cisplatin in a panel of chemoresistant neuroblastoma cell lines. Apoptotic cell death was confirmed by cytochrome c release from the mitochondria, cleavage of poly ADP-ribose polymerase, processing of caspase-3, which is an important executive enzyme in apoptosis, and caspase-3-like activity. Methyl malonate, an inhibitor of the SDHA subunit partially reversed apoptosis stimulated by TTFA in SK-N-BE(2) neuroblastoma cells (NB), indicating that sensitization requires oxidation of succinate. In contrast, in IMR-32 NB cells, the same concentrations of TTFA markedly suppressed cisplatin-induced apoptosis. Comparison of oxygen consumption in cisplatin-resistant SK-N-BE(2) and cisplatin-sensitive IMR-32 cells clearly demonstrated impaired Complex II activity in IMR-32 cells. We also found that in SK-N-BE(2) cells co-treatment with cisplatin and TTFA markedly stimulated formation of reactive oxygen species (ROS), whereas in IMR cells, cisplatin-mediated ROS production was attenuated by TTFA, which explains apoptosis suppression in these cells. Thus, functionally active SDH is a prerequisite for the ROS-mediated sensitization to treatment by TTFA

Contrasting effects of glutamine deprivation on apoptosis induced by conventionally used anticancer drugs

Tumor cells dependence on glutamine offers a rationale for their elimination via targeting of glutamine metabolism. The aim of this work was to investigate how glutamine deprivation affects the cellular response to conventionally used anticancer drugs. To answer this question, neuroblastoma cells were pre-incubated in a glutamine-free medium and treated with cisplatin or etoposide. Obtained results revealed that glutamine withdrawal affected cellular response to therapeutic drugs in a different manner. Glutamine deprivation suppressed etoposide-induced, but markedly stimulated cisplatin-induced apoptosis. Suppression of etoposide-induced cell death correlated with a downregulation of p53 expression, which, among other functions, regulates the expression of death receptor 5, one of the activators of caspase-8. In contrast, stimulation of cisplatin-induced cell death involved reactive oxygen species-mediated downregulation of FLIP-S, an inhibitor of caspase-8. As a result, the activity of caspase-8 was stimulated causing cleavage of the pro-apoptotic protein Bid, which is involved in the permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic factors, such as cytochrome c from mitochondria. Thus, suppression of glutamine metabolism can sensitize tumor cells to treatment and could be utilized for anti-cancer therapy. However, it should be done cautiously, since adverse effects may occur when combined with an inappropriate therapeutic drug

Linseed, Baru, and Coconut Oils: NMR-Based Metabolomics, Leukocyte Infiltration Potential In Vivo, and Their Oil Characterization. Are There Still Controversies?

Different fatty acid proportions produce potential inflammatory and metabolic changes in organisms. However, the evidence for how each fatty acid mediates the metabolic pathway, and its lipid stability remains controversial. To resolve this controversy, the present study investigated the metabolic effects of cold-pressed linseed (LG), coconut (CG), and baru (BG) oils in comparison to those of soybean oil (SG) in mice, in terms of their oil characterization and stability. The quality analysis showed less oxidative behavior among PUFA-rich oils (SO, BO, and LO, with induction periods lower than 2 h compared to 39.8 h for CG), besides the high contents of tocopherols and carotenoids in SG and LG. In the experimental study, CG presented higher triglyceride (257.93 ± 72.30) and VLDL-cholesterol levels (51.59 ± 14.46, p < 0.05), while LG reduced LDL levels (59.29 ± 7.56, p < 0.05) when compared to SG (183.14 ± 22.06, 36.63 ± 4.41 and 131.63 ± 29.0, respectively). For visceral fats, the adiposity index was lower for BG (7.32 ± 3.13) and CG (9.58 ± 1.02, p < 0.05) in relation to SG (12.53 ± 2.80), and for leukocyte recruitment, CG presented lower polymorphonuclear (PMN) (p < 0.0001) and mononuclear (MN) (p < 0.05) cell infiltration, demonstrating anti-inflammatory potential. In NMR-based metabolomics, although CG presented higher values for the glucose, lactate, and LDL/VLDL ratio, this group also evidenced high levels of choline, a lipotropic metabolite. Our study emphasized the controversies of saturated fatty acids, which impair serum lipids, while alfa-linolenic acid presented cardioprotective effects. However, coconut oil also has a positive immunomodulatory pathway and was found to reduce visceral bodyfat in mice. Therefore, for future applications, we suggest a combination of lauric and al-fa-linolenic acid sources, which are present in coconut and linseed oil, respectively. This combination could be less obesogenic and inflammatory and exert cardioprotective action

Odanacatib for the treatment of postmenopausal osteoporosis : Results of the LOFT multicentre, randomised, double-blind, placebo-controlled trial and LOFT Extension study

Background Odanacatib, a cathepsin K inhibitor, reduces bone resorption while maintaining bone formation. Previous work has shown that odanacatib increases bone mineral density in postmenopausal women with low bone mass. We aimed to investigate the efficacy and safety of odanacatib to reduce fracture risk in postmenopausal women with osteoporosis. Methods The Long-term Odanacatib Fracture Trial (LOFT) was a multicentre, randomised, double-blind, placebo-controlled, event-driven study at 388 outpatient clinics in 40 countries. Eligible participants were women aged at least 65 years who were postmenopausal for 5 years or more, with a femoral neck or total hip bone mineral density T-score between −2·5 and −4·0 if no previous radiographic vertebral fracture, or between −1·5 and −4·0 with a previous vertebral fracture. Women with a previous hip fracture, more than one vertebral fracture, or a T-score of less than −4·0 at the total hip or femoral neck were not eligible unless they were unable or unwilling to use approved osteoporosis treatment. Participants were randomly assigned (1:1) to either oral odanacatib (50 mg once per week) or matching placebo. Randomisation was done using an interactive voice recognition system after stratification for previous radiographic vertebral fracture, and treatment was masked to study participants, investigators and their staff, and sponsor personnel. If the study completed before 5 years of double-blind treatment, consenting participants could enrol in a double-blind extension study (LOFT Extension), continuing their original treatment assignment for up to 5 years from randomisation. Primary endpoints were incidence of vertebral fractures as assessed using radiographs collected at baseline, 6 and 12 months, yearly, and at final study visit in participants for whom evaluable radiograph images were available at baseline and at least one other timepoint, and hip and non-vertebral fractures adjudicated as being a result of osteoporosis as assessed by clinical history and radiograph. Safety was assessed in participants who received at least one dose of study drug. The adjudicated cardiovascular safety endpoints were a composite of cardiovascular death, myocardial infarction, or stroke, and new-onset atrial fibrillation or flutter. Individual cardiovascular endpoints and death were also assessed. LOFT and LOFT Extension are registered with ClinicalTrials.gov (number NCT00529373) and the European Clinical Trials Database (EudraCT number 2007-002693-66). Findings Between Sept 14, 2007, and Nov 17, 2009, we randomly assigned 16 071 evaluable patients to treatment: 8043 to odanacatib and 8028 to placebo. After a median follow-up of 36·5 months (IQR 34·43–40·15) 4297 women assigned to odanacatib and 3960 assigned to placebo enrolled in LOFT Extension (total median follow-up 47·6 months, IQR 35·45–60·06). In LOFT, cumulative incidence of primary outcomes for odanacatib versus placebo were: radiographic vertebral fractures 3·7% (251/6770) versus 7·8% (542/6910), hazard ratio (HR) 0·46, 95% CI 0·40–0·53; hip fractures 0·8% (65/8043) versus 1·6% (125/8028), 0·53, 0·39–0·71; non-vertebral fractures 5·1% (412/8043) versus 6·7% (541/8028), 0·77, 0·68–0·87; all p<0·0001. Combined results from LOFT plus LOFT Extension for cumulative incidence of primary outcomes for odanacatib versus placebo were: radiographic vertebral fractures 4·9% (341/6909) versus 9·6% (675/7011), HR 0·48, 95% CI 0·42–0·55; hip fractures 1·1% (86/8043) versus 2·0% (162/8028), 0·52, 0·40–0·67; non-vertebral fractures 6·4% (512/8043) versus 8·4% (675/8028), 0·74, 0·66–0·83; all p<0·0001. In LOFT, the composite cardiovascular endpoint of cardiovascular death, myocardial infarction, or stroke occurred in 273 (3·4%) of 8043 patients in the odanacatib group versus 245 (3·1%) of 8028 in the placebo group (HR 1·12, 95% CI 0·95–1·34; p=0·18). New-onset atrial fibrillation or flutter occurred in 112 (1·4%) of 8043 patients in the odanacatib group versus 96 (1·2%) of 8028 in the placebo group (HR 1·18, 0·90–1·55; p=0·24). Odanacatib was associated with an increased risk of stroke (1·7% [136/8043] vs 1·3% [104/8028], HR 1·32, 1·02–1·70; p=0·034), but not myocardial infarction (0·7% [60/8043] vs 0·9% [74/8028], HR 0·82, 0·58–1·15; p=0·26). The HR for all-cause mortality was 1·13 (5·0% [401/8043] vs 4·4% [356/8028], 0·98–1·30; p=0·10). When data from LOFT Extension were included, the composite of cardiovascular death, myocardial infarction, or stroke occurred in significantly more patients in the odanacatib group than in the placebo group (401 [5·0%] of 8043 vs 343 [4·3%] of 8028, HR 1·17, 1·02–1·36; p=0·029, as did stroke (2·3% [187/8043] vs 1·7% [137/8028], HR 1·37, 1·10–1·71; p=0·0051). Interpretation Odanacatib reduced the risk of fracture, but was associated with an increased risk of cardiovascular events, specifically stroke, in postmenopausal women with osteoporosis. Based on the overall balance between benefit and risk, the study's sponsor decided that they would no longer pursue development of odanacatib for treatment of osteoporosis