Targeting Mitochondria in Melanoma

Drastically elevated glycolytic activity is a prominent metabolic feature of cancer cells. Until recently it was thought that tumor cells shift their entire energy production from oxidative phosphorylation (OXPHOS) to glycolysis. However, new evidence indicates that many cancer cells still have functional OXPHOS, despite their increased reliance on glycolysis. Growing pre-clinical and clinical evidence suggests that targeting mitochondrial metabolism has anti-cancer effects. Here, we analyzed mitochondrial respiration and the amount and activity of OXPHOS complexes in four melanoma cell lines and normal human dermal fibroblasts (HDFs) by Seahorse real-time cell metabolic analysis, immunoblotting, and spectrophotometry. We also tested three clinically approved antibiotics, one anti-parasitic drug (pyrvinium pamoate), and a novel anti-cancer agent (ONC212) for effects on mitochondrial respiration and proliferation of melanoma cells and HDFs. We found that three of the four melanoma cell lines have elevated glycolysis as well as OXPHOS, but contain dysfunctional mitochondria. The antibiotics produced different effects on the melanoma cells and HDFs. The anti-parasitic drug strongly inhibited respiration and proliferation of both the melanoma cells and HDFs. ONC212 reduced respiration in melanoma cells and HDFs, and inhibited the proliferation of melanoma cells. Our findings highlight ONC212 as a promising drug for targeting mitochondrial respiration in cancer

Combination of metronomic cyclophosphamide and dietary intervention inhibits neuroblastoma growth in a CD1-nu mouse model

BACKGROUND MYCN-amplification in high-grade Neuroblastoma (NB) tumors correlates with increased vascularization and therapy resistance. This study combines an anti-angiogenic approach with targeting NB metabolism for treatment. METHODS AND RESULTS Metronomic cyclophosphamide (MCP) monotherapy significantly inhibited NB growth and prolonged host survival. Growth inhibition was more pronounced in MYCN-amplified xenografts. Immunohistochemical evaluation of this subtype showed significant decrease in blood vessel density and intratumoral hemorrhage accompanied by blood vessel maturation and perivascular fibrosis. Up-regulation of VEGFA was not sufficient to compensate for the effects of the MCP regimen. Reduced Bcl-2 expression and increased caspase-3 cleavage were evident. In contrast non MYCN-amplified tumors developed resistance, which was accompanied by Bcl-2-up-regulation. Combining MCP with a ketogenic diet and/or calorie-restriction significantly enhanced the anti-tumor effect. Calorie-restricted ketogenic diet in combination with MCP resulted in tumor regression in all cases. CONCLUSIONS Our data show efficacy of combining an anti-angiogenic cyclophosphamide dosing regimen with dietary intervention in a preclinical NB model. These findings might open a new front in NB treatment

Lithium and Not Acetoacetate Influences the Growth of Cells Treated with Lithium Acetoacetate

The ketogenic diet (KD), a high-fat/low-carbohydrate/adequate-protein diet, has been proposed as a treatment for a variety of diseases, including cancer. KD leads to generation of ketone bodies (KBs), predominantly acetoacetate (AcAc) and 3-hydroxy-butyrate, as a result of fatty acid oxidation. Several studies investigated the antiproliferative effects of lithium acetoacetate (LiAcAc) and sodium 3-hydroxybutyrate on cancer cells in vitro. However, a critical point missed in some studies using LiAcAc is that Li ions have pleiotropic effects on cell growth and cell signaling. Thus, we tested whether Li ions per se contribute to the antiproliferative effects of LiAcAc in vitro. Cell proliferation was analyzed on neuroblastoma, renal cell carcinoma, and human embryonic kidney cell lines. Cells were treated for 5 days with 2.5, 5, and 10 mM LiAcAc and with equimolar concentrations of lithium chloride (LiCl) or sodium chloride (NaCl). LiAcAc affected the growth of all cell lines, either negatively or positively. However, the effects of LiAcAc were always similar to those of LiCl. In contrast, NaCl showed no effects, indicating that the Li ion impacts cell proliferation. As Li ions have significant effects on cell growth, it is important for future studies to include sources of Li ions as a control

Untargeted Metabolomics Reveals Molecular Effects of Ketogenic Diet on Healthy and Tumor Xenograft Mouse Models

The application of ketogenic diet (KD) (high fat/low carbohydrate/adequate protein) as an auxiliary cancer therapy is a field of growing attention. KD provides sufficient energy supply for healthy cells, while possibly impairing energy production in highly glycolytic tumor cells. Moreover, KD regulates insulin and tumor related growth factors (like insulin growth factor-1, IGF-1). In order to provide molecular evidence for the proposed additional inhibition of tumor growth when combining chemotherapy with KD, we applied untargeted quantitative metabolome analysis on a spontaneous breast cancer xenograft mouse model, using MDA-MB-468 cells. Healthy mice and mice bearing breast cancer xenografts and receiving cyclophosphamide chemotherapy were compared after treatment with control diet and KD. Metabolomic profiling was performed on plasma samples, applying high-performance liquid chromatography coupled to tandem mass spectrometry. Statistical analysis revealed metabolic fingerprints comprising numerous significantly regulated features in the group of mice bearing breast cancer. This fingerprint disappeared after treatment with KD, resulting in recovery to the metabolic status observed in healthy mice receiving control diet. Moreover, amino acid metabolism as well as fatty acid transport were found to be affected by both the tumor and the applied KD. Our results provide clear evidence of a significant molecular effect of adjuvant KD in the context of tumor growth inhibition and suggest additional mechanisms of tumor suppression beyond the proposed constrain in energy supply of tumor cells.(VLID)439383

Metabolic reprogramming related to whole-chromosome instability in models for Hürthle cell carcinoma

Abstract Hürthle cell carcinoma (HCC) is a recurrent subtype of non-medullary thyroid cancer. HCC is characterized by profound whole-chromosome instability (w-CIN), resulting in a near-homozygous genome (NHG), a phenomenon recently attributed to reactive oxygen species (ROS) generated during mitosis by malfunctioning mitochondria. We studied shared metabolic traits during standard and glucose-depleted cell culture in thyroid cancer cell lines (TCCLs), with or without a NHG, using quantitative analysis of extra and intracellular metabolites and ROS production following inhibition of complex III with antimycin A. We found that the XTC.UC1 and FTC-236 cell lines (both NHG) are functionally impaired in complex I and produce significantly more superoxide radicals than SW579 and BHP 2–7 (non-NHG) after challenge with antimycin A. FTC-236 showed the lowest levels of glutathione and SOD2. XTC.UC1 and FTC-236 both exhibited reduced glycolytic activity and utilization of alternative sources to meet energy demands. Both cell lines also shared low levels of α-ketoglutarate and high levels of creatine, phosphocreatine, uridine diphosphate-N-acetylglucosamine, pyruvate and acetylcarnitine. Furthermore, the metabolism of XTC.UC1 was skewed towards the de novo synthesis of aspartate, an effect that persisted even in glucose-free media, pointing to reductive carboxylation. Our data suggests that metabolic reprogramming and a subtle balance between ROS generation and scavenging/conversion of intermediates may be involved in ROS-induced w-CIN in HCC and possibly also in rare cases of follicular thyroid cancer showing a NHG

Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model

<div><p>Introduction</p><p>Neuroblastoma is a malignant pediatric cancer derived from neural crest cells. It is characterized by a generalized reduction of mitochondrial oxidative phosphorylation. The goal of the present study was to investigate the effects of calorie restriction and ketogenic diet on neuroblastoma tumor growth and monitor potential adaptive mechanisms of the cancer’s oxidative phosphorylation system.</p><p>Methods</p><p>Xenografts were established in CD-1 nude mice by subcutaneous injection of two neuroblastoma cell lines having distinct genetic characteristics and therapeutic sensitivity [SH-SY5Y and SK-N-BE(2)]. Mice were randomized to four treatment groups receiving standard diet, calorie-restricted standard diet, long chain fatty acid based ketogenic diet or calorie-restricted ketogenic diet. Tumor growth, survival, metabolic parameters and weight of the mice were monitored. Cancer tissue was evaluated for diet-induced changes of proliferation indices and multiple oxidative phosphorylation system parameters (respiratory chain enzyme activities, western blot analysis, immunohistochemistry and mitochondrial DNA content).</p><p>Results</p><p>Ketogenic diet and/or calorie restriction significantly reduced tumor growth and prolonged survival in the xenograft model. Neuroblastoma growth reduction correlated with decreased blood glucose concentrations and was characterized by a significant decrease in Ki-67 and phospho-histone H3 levels in the diet groups with low tumor growth. As in human tumor tissue, neuroblastoma xenografts showed distinctly low mitochondrial complex II activity in combination with a generalized low level of mitochondrial oxidative phosphorylation, validating the tumor model. Neuroblastoma showed no ability to adapt its mitochondrial oxidative phosphorylation activity to the change in nutrient supply induced by dietary intervention.</p><p>Conclusions</p><p>Our data suggest that targeting the metabolic characteristics of neuroblastoma could open a new front in supporting standard therapy regimens. Therefore, we propose that a ketogenic diet and/or calorie restriction should be further evaluated as a possible adjuvant therapy for patients undergoing treatment for neuroblastoma.</p></div

Immunohistochemical (IHC) staining of mitochondrial OXPHOS complexes I-V and VDAC in SH-SY5Y xenograft tumors.

<p>IHC staining of NB sections were scored on a scale from 0–3 as described in the methods section. The CR-KD group showed a significant decrease in complex I staining. All other evaluated parameters were unaffected by dietary changes. Voltage-dependent ion channel (VDAC) protein levels are used as a surrogate marker of mitochondrial mass. Statistics: ANOVA (p <0.05) followed by two-tailed Dunnett’s test correcting for multiple comparisons. Diet groups are compared to the corresponding SD group.</p

Ketogenic diet and calorie restriction reduce tumor growth and prolong survival in a NB xenograft model.

<p>After establishing tumors on the right flank of CD-1nu mice, the mice were randomized to diet groups as indicated. Tumor volume was measured twice weekly. A) For SH-SY5Y xenografts at day 19, the tumors of all diet groups showed significant growth inhibition compared to the SD group (CR-SD p = 0.001, KD p<0.001, CR-KD p<0.001). B) At day 33, SK-N-BE(2) tumor growth was significantly inhibited by CR (CR-SD p = 0.040, CR-KD p = 0.004). Inhibition of tumor growth was less pronounced in the KD group (p = 0.918). C) SH-SY5Y and D) SK-N-BE(2) show the results of Kaplan-Meier survival analysis of the corresponding treatment groups. Survival of mice with SH-SY5Y tumors at day 22 on SD was 0% compared to 75% on CR-SD (p<0.001), 50% on KD (p<0.001) and 100% on CR-KD (p<0.001). Survival of mice with SK-N-BE(2) xenografts at day 33 on SD was 36% compared to 83% on CR-SD (p = 0.017), 73% on KD (p = 0.09) and 100% on CR–KD (p<0.001). A, B) Data points for tumor growth curves represent mean values ± SEM of the corresponding diet group (n = 8–11). Statistics: ANOVA (p<0.05) followed by two-tailed Dunnett’s test correcting for multiple comparisons. C, D) Survival is expressed by the Kaplan–Meier method and differences between groups were determined in a univariate analysis with the log-rank test. Death is coded: tumor volume above 3000 mm³, tumor ulceration or impaired health condition. Diet groups are compared to the corresponding SD. * p≤0.05; ** p≤0.01; *** p≤0.001.</p