Mitochondrial bioenergetics as a cell fate rheostat for responsive to Bcl-2 drugs: New cues for cancer chemotherapy.

Pro-survival BCL-2 proteins prevent the initiation of intrinsic apoptosis (mitochondria-dependent pathway) by inhibiting the pro-apoptotic proteins BAX and BAK, while BH3-only proteins promote apoptosis by blocking pro-survival BCL-2 proteins. Disruptions in this delicate balance contribute to cancer cell survival and chemoresistance. Recent advances in cancer therapeutics involve a new generation of drugs known as BH3-mimetics, which are small molecules designed to mimic the action of BH3-only proteins. Promising effects have been observed in patients with hematological and solid tumors undergoing treatment with these agents. However, the rapid emergence of mitochondria-dependent resistance to BH3-mimetics has been reported. This resistance involves increased mitochondrial respiration, altered mitophagy, and mitochondria with higher and tighter cristae. Conversely, mutations in isocitrate dehydrogenase 1 and 2, catalyzing R-2-hydroxyglutarate production, promote sensitivity to venetoclax. This evidence underscores the urgency for comprehensive studies on bioenergetics-based adaptive responses in both BH3 mimetics-sensitive and -resistant cancer cells. Ongoing clinical trials are evaluating BH3-mimetics in combination with standard chemotherapeutics. In this article, we discuss the role of mitochondrial bioenergetics in response to BH3-mimetics and explore potential therapeutic opportunities through metabolism-targeting strategies.

Shedding Light on the Dentition and Venom Delivery System of the Rear-Fanged Snake, Galvarinus chilensis chilensis (Serpentes: Dipsadidae: Tachymenini) from Chile.

Although the rear-fanged snake Galvarinus chilensis chilensis (formerly named Tachymenis ch. chilensis) causes ophidian accidents with clinical importance in Chile, the anatomical and histological characterizations of the venom delivery system (venom gland and fang) of this species still remain unknown. This study describes the dentition and characteristics of fangs and their ontogenetic variations in G. ch. chilensis. Moreover, histological and histochemistry analyses of the venom glands of this species are presented. Using micro-computed tomography and scanning electron microscopy, the dentitions of neonates, juveniles, and adults were analyzed, and no ontogenetic variations in teeth length and number present in the dentary and maxilla were observed. Moreover, we found three types of basic teeth, with distributional patterns conserved in all ontogenetic categories. The fangs exhibited a groove from the base to the middle. At the end of the groove, prominent ridges are formed. The fang and groove lengths were significantly distinct between ontogenetic categories. No differences between females and males were observed. Histologically, we found that the venom gland is close to the fangs and has a seromucous composition. Our results describe, for the first time, the distributional pattern and characteristics of the dentition and venom delivery system of the poorly studied snake G. ch. chilensis.

A paraguayan toad Rhinella schneideri preparation based on Mbya tradition increases mitochondrial bioenergetics with migrastatic effects dependent on AMPK in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: In Paraguay, healers from the Mbya culture treat cancer with a recipe prepared with the native toad Rhinella schneideri. However, the chemical composition and biological effects of the recipe remain unknown. AIM OF THE STUDY: The aim is to determine the composition of the traditional preparation made using the toad R. schneideri and to evaluate its effect on human breast cancer (BC) cells. MATERIALS AND METHODS: The metabolites contained in the preparation were concentrated using XAD-7 resin, and the concentrate was analyzed by HPLC-MS/MS. The effect of the preparation was assessed in normal (MCF10F) and BC cells (MDA-MB-231 and MCF7). The mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) levels, and cell cycle progression were determined by flow cytometry. The oxygen consumption rate (OCR) was measured by Clark electrode, and fibronectin-dependent migration in normoxia and hypoxia-like conditions were evaluated by transwell assay. RESULTS: From the Amberlite-retained extract from the preparation, 24 compounds were identified, including alkaloids, amino acids, bufadienolides, and flavonoids, among others. The crude extract (CE) did not affect cell cycle progression and viability of BC cell lines. Moreover, it did not make cancer cells more sensitive to the cytotoxic effect of the chemotherapeutics doxorubicin and teniposide. On the other hand, the CE reduced the menadione-induced ROS production and increased NADH, Δψm, and the OCR. Respiratory complexes I and III as well as ATP synthase levels were increased in an AMPK-dependent manner. Moreover, the CE inhibited the migration of BC cells in normoxia and a hypoxia-like condition using CoCl2 as a HIF1α-stabilizing agent. This latter effect involved an AMPK-dependent reduction of HIF1α levels. CONCLUSIONS: The Paraguayan toad recipe contains metabolites from the toad ingredient, including alkaloids and bufadienolide derivatives. The CE lacks cytotoxic effects alone or in combination with chemotherapeutics. However, it increases mitochondrial bioenergetics and inhibits the cancer cell migration in an AMPK-dependent manner in BC cells. This is the first report of the in vitro anticancer effect of a traditional Rhinella sp. toad preparation based on Mbya tradition.

Functional, immunological characterization, and anticancer activity of BaMtx: A new Lys49- PLA2 homologue isolated from the venom of Peruvian Bothrops atrox snake (Serpentes: Viperidae).

Bothorps atrox is responsible for most of the ophidism cases in Perú. As part of the envenoming, myotoxicity is one of the most recurrent and destructive effects. In this study, a myotoxin, named BaMtx, was purified from B. atrox venom to elucidate its biological, immunological, and molecular characteristics. BaMtx was purified using CM-Sephadex-C-25 ion-exchange resin and SDS-PAGE analysis showed a unique protein band of 13 kDa or 24 kDa under reducing or non-reducing conditions, respectively. cDNA sequence codified a 122-aa mature protein with high homology with other Lys49-PLA2s; modeled structure showed a N-terminal helix, a ß-wing region, and a C-terminal random coil. This protein has a poor phospholipase A2 enzymatic activity. BaMtx has myotoxic (DMM = 12.30 ± 0.95 µg) and edema-forming (DEM = 26.00 ± 1.15 µg) activities. Rabbit immunization with purified enzyme produced anti-BaMtx antibodies that reduced 50.28 ± 10.15% of myotoxic activity and showed significant cross-reactivity against B. brazili and B pictus venoms. On the other hand, BaMtx exhibits mild anti-proliferative and anti-migratory effects on breast cancer cells, affecting the ROS and NADH levels, which may reduce mitochondrial respiration. These results contribute to the understanding of B. atrox Lys49-PLA2 effects and establish the anticancer potential de BaMtx.

Extracellular Matrix Signals as Drivers of Mitochondrial Bioenergetics and Metabolic Plasticity of Cancer Cells During Metastasis.

The role of metabolism in tumor growth and chemoresistance has received considerable attention, however, the contribution of mitochondrial bioenergetics in migration, invasion, and metastasis is recently being understood. Migrating cancer cells adapt their energy needs to fluctuating changes in the microenvironment, exhibiting high metabolic plasticity. This occurs due to dynamic changes in the contributions of metabolic pathways to promote localized ATP production in lamellipodia and control signaling mediated by mitochondrial reactive oxygen species. Recent evidence has shown that metabolic shifts toward a mitochondrial metabolism based on the reductive carboxylation, glutaminolysis, and phosphocreatine-creatine kinase pathways promote resistance to anoikis, migration, and invasion in cancer cells. The PGC1a-driven metabolic adaptations with increased electron transport chain activity and superoxide levels are essential for metastasis in several cancer models. Notably, these metabolic changes can be determined by the composition and density of the extracellular matrix (ECM). ECM stiffness, integrins, and small Rho GTPases promote mitochondrial fragmentation, mitochondrial localization in focal adhesion complexes, and metabolic plasticity, supporting enhanced migration and metastasis. Here, we discuss the role of ECM in regulating mitochondrial metabolism during migration and metastasis, highlighting the therapeutic potential of compounds affecting mitochondrial function and selectively block cancer cell migration.

Aspirin-triggered resolvin D1 reduces parasitic cardiac load by decreasing inflammation in a murine model of early chronic Chagas disease.

BACKGROUND: Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in Latin America and is widely distributed worldwide because of migration. In 30% of cases, after years of infection and in the absence of treatment, the disease progresses from an acute asymptomatic phase to a chronic inflammatory cardiomyopathy, leading to heart failure and death. An inadequate balance in the inflammatory response is involved in the progression of chronic Chagas cardiomyopathy. Current therapeutic strategies cannot prevent or reverse the heart damage caused by the parasite. Aspirin-triggered resolvin D1 (AT-RvD1) is a pro-resolving mediator of inflammation that acts through N-formyl peptide receptor 2 (FPR2). AT-RvD1 participates in the modification of cytokine production, inhibition of leukocyte recruitment and efferocytosis, macrophage switching to a nonphlogistic phenotype, and the promotion of healing, thus restoring organ function. In the present study, AT-RvD1 is proposed as a potential therapeutic agent to regulate the pro-inflammatory state during the early chronic phase of Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 wild-type and FPR2 knock-out mice chronically infected with T. cruzi were treated for 20 days with 5 µg/kg/day AT-RvD1, 30 mg/kg/day benznidazole, or the combination of 5 µg/kg/day AT-RvD1 and 5 mg/kg/day benznidazole. At the end of treatment, changes in immune response, cardiac tissue damage, and parasite load were evaluated. The administration of AT-RvD1 in the early chronic phase of T. cruzi infection regulated the inflammatory response both at the systemic level and in the cardiac tissue, and it reduced cellular infiltrates, cardiomyocyte hypertrophy, fibrosis, and the parasite load in the heart tissue. CONCLUSIONS/SIGNIFICANCE: AT-RvD1 was shown to be an attractive therapeutic due to its regulatory effect on the inflammatory response at the cardiac level and its ability to reduce the parasite load during early chronic T. cruzi infection, thereby preventing the chronic cardiac damage induced by the parasite.

FRI-1 Is an Anti-Cancer Isoquinolinequinone That Inhibits the Mitochondrial Bioenergetics and Blocks Metabolic Shifts by Redox Disruption in Breast Cancer Cells.

Since breast cancer (BC) cells are dependent on mitochondrial bioenergetics for promoting proliferation, survival, and metastasis, mitochondria highlight as an important target for anticancer drug discovery. FRI-1, methyl 1, 3-dimethyl-5, 8-dioxo-5, 8-dihydro-4-isoquinolinecarboxylate, was previously described as a selective cytotoxic compound on cancer cell lines, however, details on the mechanism of action remain unknown. In this work, we describe that FRI-1 inhibits mitochondrial bioenergetics, producing apoptosis in MCF7 and MDA-MB-231 BC cell lines. FRI-1 decreases the maximal oxygen consumption rate (OCR), Δψm, NADH, and ATP levels, with a notable increase of mitochondrial reactive oxygen species (ROS) production, promoting AMPK activation with pro-survival effects. Moreover, FRI-1 inhibits the metabolic remodeling to glycolysis induced by oligomycin. In isolated tumoral mitochondria, FRI-1 increases Complex I and III-dependent OCR state 2, and this is sensitive to rotenone and antimycin A inhibitor additions, suggesting a redox cycling event. Remarkably, α-ketoglutarate and lipoic acid supplementation reversed and promoted, respectively, the FRI-1-induced apoptosis, suggesting that mitochondrial redox disruption affects 2-oxoglutarate dehydrogenase (OGDH) activity, and this is involved in their anticancer mechanism. Consistent with this, the combination of FRI-1 and CPI-613, a dual inhibitor of redox-sensible tricarboxylic acid (TCA) cycle enzymes PDH and OGDH, produced extensive BC cell death. Taken together, our results suggest that FRI-1 exhibits anticancer effects through inhibition of mitochondrial bioenergetics by redox disruption in BC cells.

The Parotoid Gland Secretion from Peruvian Toad Rhinella horribilis (Wiegmann, 1833): Chemical Composition and Effect on the Proliferation and Migration of Lung Cancer Cells.

Since Rhinella sp. toads produce bioactive substances, some species have been used in traditional medicine and magical practices by ancient cultures in Peru. During several decades, the Rhinella horribilis toad was confused with the invasive toad Rhinella marina, a species documented with extensive toxinological studies. In contrast, the chemical composition and biological effects of the parotoid gland secretions (PGS) remain still unknown for R. horribilis. In this work, we determine for the first time 55 compounds from the PGS of R. horribilis, which were identified using HPLC-MS/MS. The crude extract inhibited the proliferation of A549 cancer cells with IC50 values of 0.031 ± 0.007 and 0.015 ± 0.001 µg/mL at 24 and 48 h of exposure, respectively. Moreover, it inhibited the clonogenic capacity, increased ROS levels, and prevented the etoposide-induced apoptosis, suggesting that the effect of R. horribilis poison secretion was by cell cycle blocking before of G2/M-phase checkpoint. Fraction B was the most active and strongly inhibited cancer cell migration. Our results indicate that the PGS of R. horribilis are composed of alkaloids, bufadienolides, and argininyl diacids derivatives, inhibiting the proliferation and migration of A549 cells.

An acylhydroquinone derivative produces OXPHOS uncoupling and sensitization to BH3 mimetic ABT-199 (Venetoclax) in human promyelocytic leukemia cells.

Since cancer cells have different mitochondrial bioenergetic requirements than non-cancerous cells, therapeutic inhibition of its mitochondrial functionality continues to be an important target for anticancer drug discovery. In this study, a series of acylhydroquinones with different acyl-chain length, and their chlorinated derivatives, in the aromatic ring, synthesized by Fries rearrangement under microwave irradiation, were evaluated for their anticancer activity in two leukemia cell lines. Findings from the primary and secondary screening of the 18 acylhydroquinones, tested at 5 µM on acute promyelocytic leukemia HL-60 and acute lymphoblastic leukemia CEM cells lines, identified an acylchlorohydroquinone (12) with a highly selective anti-proliferative effect toward HL-60 cells. This compound induced S-phase arrest in the cell cycle progression of HL-60 cells with insignificant toxicity on leukemic CEM cells and non-cancerous Hs27 cells. In HL-60 leukemic cells, 12 triggered increased mitochondrial NADH oxidation, increased respiration in presence of oligomycin (state 4o), mitochondrial depolarization, and ROS production, suggesting an uncoupling of OXPHOS. This provoked a metabolic adaptation dependent on AMPK/ACC/autophagy axis, having the mitochondrial ß-oxidation a pro-survival role since the combination of 12 and etomoxir, a carnitine palmitoyl-transferase (CPT) inhibitor promoted extensive HL-60 cell death. Finally, 12-induced metabolic stress sensitized to HL-60 cells to cell death by the FDA-approved anti-leukemic drug ABT-199, a BH3 mimetic. Therefore, our results suggest that acylchlorohydroquinone is a promising scaffold in anti-promyelocytic leukemia drug research.

Simvastatin Improves Cardiac Function through Notch 1 Activation in BALB/c Mice with Chronic Chagas Cardiomyopathy.

Chagas disease, caused by the protozoan Trypanosoma cruzi, endemic in Latin America but distributed worldwide because of migration. Without appropriate treatment, the disease progresses from an acute asymptomatic phase to a chronic, progressive inflammatory cardiomyopathy causing heart failure and death. Despite specific trypanocidal therapy, heart damage progression cannot be stopped or reversed. Statins, as part of their pleiotropic actions, can modulate chagasic myocarditis by inducing the production of 15-epi-lipoxin A4 (15-epi-LXA4), a proresolution lipid mediator in inflammation. Furthermore, several reports suggest that simvastatin activates the Notch pathway after stroke in cerebral endothelial cells, enhancing blood flow by promoting angiogenesis. Thus, statins are an attractive therapeutic strategy for modulating the Notch pathway to reverse the chronic heart damage induced by T. cruzi BALB/c mice chronically infected with T. cruzi were treated with 1 mg/kg/day simvastatin or 25 µg/kg/day 15-epi-LXA4 for 20 days. During the treatment period, cardiac function was evaluated by echocardiography. At 80 days postinfection, the heart tissues were assessed for Notch 1 activity. T. cruzi infection activated the Notch 1 pathway, and simvastatin (but not 15-epi-lipoxin A4) produced a further increase in that activity, correlating with improvement in the ejection fraction and histopathologic findings typical of T. cruzi infection, including improvements in inflammation and fibrosis. Moreover, simvastatin increased the number of isolectin B4-positive cells, suggesting active angiogenesis in the chronically infected hearts without alteration of the parasitic load. Simvastatin, probably acting through the Notch 1 pathway, decreases inflammation, improving cardiac function in mice chronically infected with T. cruzi.

Novel benzoate-lipophilic cations selectively induce cell death in human colorectal cancer cell lines.

INTRODUCTION: Colorectal cancer (CRC) is a critical health issue worldwide. The high rate of liver and lung metastasis associated with CRC creates a significant barrier to effective and efficient therapy. Tumour cells, including CRC cells, have metabolic alterations, such as high levels of glycolytic activity, increased cell proliferation and invasiveness, and chemo- and radio-resistance. However, the abnormally elevated mitochondrial transmembrane potential of these cells also provides an opportunity to develop drugs that selectively target the mitochondrial functions of tumour cells. METHODS: In this work, we used a new batch of benzoic acid esters with cytotoxic activities attached to the triphenylphosphonium group as a vehicle to target tumour mitochondria and improve their activity. We evaluated the cytotoxicity, selectivity, and mechanism of action of these derivatives, including the effects on energy stress-induced apoptosis and metabolic behaviour in the human CRC cell lines HCT-15 and COLO-205. RESULTS: The benzoic acid derivatives selectively targeted the tumour cells with high potency and efficacy. The derivatives induced the uncoupling of the oxidative phosphorylation system, decreased the transmembrane potential, and reduced ATP levels while increasing AMPK activation, thereby triggering tumour cell apoptosis in both tumour cell lines tested. CONCLUSION: The benzoic acid derivatives studied here are promising candidates for assessing in vivo models of CRC, despite the diverse metabolic characteristics of these tumour cells.

Complex Mitochondrial Dysfunction Induced by TPP+-Gentisic Acid and Mitochondrial Translation Inhibition by Doxycycline Evokes Synergistic Lethality in Breast Cancer Cells.

The mitochondrion has emerged as a promising therapeutic target for novel cancer treatments because of its essential role in tumorigenesis and resistance to chemotherapy. Previously, we described a natural compound, 10-((2,5-dihydroxybenzoyl)oxy)decyl) triphenylphosphonium bromide (GA-TPP+C10), with a hydroquinone scaffold that selectively targets the mitochondria of breast cancer (BC) cells by binding to the triphenylphosphonium group as a chemical chaperone; however, the mechanism of action remains unclear. In this work, we showed that GA-TPP+C10 causes time-dependent complex inhibition of the mitochondrial bioenergetics of BC cells, characterized by (1) an initial phase of mitochondrial uptake with an uncoupling effect of oxidative phosphorylation, as previously reported, (2) inhibition of Complex I-dependent respiration, and (3) a late phase of mitochondrial accumulation with inhibition of α-ketoglutarate dehydrogenase complex (αKGDHC) activity. These events led to cell cycle arrest in the G1 phase and cell death at 24 and 48 h of exposure, and the cells were rescued by the addition of the cell-penetrating metabolic intermediates l-aspartic acid ß-methyl ester (mAsp) and dimethyl α-ketoglutarate (dm-KG). In addition, this unexpected blocking of mitochondrial function triggered metabolic remodeling toward glycolysis, AMPK activation, increased expression of proliferator-activated receptor gamma coactivator 1-alpha (pgc1α) and electron transport chain (ETC) component-related genes encoded by mitochondrial DNA and downregulation of the uncoupling proteins ucp3 and ucp4, suggesting an AMPK-dependent prosurvival adaptive response in cancer cells. Consistent with this finding, we showed that inhibition of mitochondrial translation with doxycycline, a broad-spectrum antibiotic that inhibits the 28 S subunit of the mitochondrial ribosome, in the presence of GA-TPP+C10 significantly reduces the mt-CO1 and VDAC protein levels and the FCCP-stimulated maximal electron flux and promotes selective and synergistic cytotoxic effects on BC cells at 24 h of treatment. Based on our results, we propose that this combined strategy based on blockage of the adaptive response induced by mitochondrial bioenergetic inhibition may have therapeutic relevance in BC.

Antiproliferative and proapoptotic activities of aza-annulated naphthoquinone analogs.

1,4-Naphthoquinone derivatives have been widely documented with regard to their biological properties, and particularly their anticancer activities. In the 9,10-anthraquinone family, aza-annulation involving one of the carbonyl oxygen atoms has afforded more potent, possibly less toxic analogues. We recently carried out different modifications on the naphthoquinone skeleton to generate 3-chloro-2-amino- and 3-chloro-2-(N-acetamido)-1,4-naphthoquinone and 3,4-dihydrobenzo[f]quinoxalin-6(2H)-one derivatives. These three series of compounds were now tested against normal human fibroblasts and six human cancer cell lines. Some of the dihydrobenzoquinoxalinone derivatives were not only more potent than their 1,4-naphthoquinone counterparts, but also exhibited 10- to 14-fold selectivity between bladder carcinoma and normal cells and were equipotent with the non-selective reference drug used (etoposide). The fusion of an additional azaheterocycle to the 1,4-naphthoquinone nucleus modulates both the activity, selectivity and mechanism of action of the compounds. The electrochemical properties of selected compounds were evaluated in an attempt to correlate them with cytotoxic activity and mechanism of action. Finally, 3D-QSAR CoMFA and CoMSIA models were built on the AGS, J82, and HL-60 cell lines. The best models had values of r2pred = 0.815; 0.823 and 0.925. The main structural relationships found, suggest that acetylation and alkylation of the amino group with large groups would be beneficial for cytotoxic activity.

Derivatives of alkyl gallate triphenylphosphonium exhibit antitumor activity in a syngeneic murine model of mammary adenocarcinoma.

We previously demonstrated that alkyl gallates coupled to triphenylphosphine have a selective and efficient antiproliferative effect by inducing mitochondrial uncoupling in vitro due to the increased mitochondrial transmembrane potential of tumor cells. Therefore, in this work, the in vivo antitumor activities of alkyl gallate triphenylphosphonium derivatives (TPP+C8, TPP+C10 and TPP+C12) were evaluated in a syngeneic murine model of breast cancer. We found that TPP+C10 increased the cytosolic ADP/ATP ratio and significantly increased the AMP levels in a concentration-dependent manner in TA3/Ha murine mammary adenocarcinoma cells. Interestingly, TPP+C10 induced a decrease in the levels of cellular proliferation markers and promoted caspase-3 activation in tumor-bearing mice. Additionally, TPP+C10 inhibited tumor growth in the syngeneic mouse model. Importantly, 30days of intraperitoneal (i.p.) administration of the combination of TPP+C10 (10mg/kg/48h) and the antibiotic doxycycline (10mg/kg/24h) completely eliminated the subcutaneous tumor burden in mice (n=6), without any relapses at 60days post-treatment. This enhancement of the individual activities of TPP+C10 and doxycycline is due to the uncoupling of oxidative phosphorylation by TPP+C10 and the inhibition of mitochondrial biogenesis by doxycycline, as demonstrated by loss of mitochondrial mass and overexpression of PGC1-α as an adaptive response. Moreover, i.p. administration of TPP+C10 (10mg/kg/24h) to healthy mice did not produce toxicity or damage in organs important for drug metabolism and excretion, as indicated by hematological, biochemical and histological assessments. These findings suggest that the combination of TPP+C10 with doxycycline is a valuable candidate therapy for breast cancer management.

Destabilization of mitochondrial functions as a target against breast cancer progression: Role of TPP(+)-linked-polyhydroxybenzoates.

Mitochondrion is an accepted molecular target in cancer treatment since it exhibits a higher transmembrane potential in cancer cells, making it susceptible to be targeted by lipophilic-delocalized cations of triphenylphosphonium (TPP(+)). Thus, we evaluated five TPP(+)-linked decyl polyhydroxybenzoates as potential cytotoxic agents in several human breast cancer cell lines that differ in estrogen receptor and HER2/neu expression, and in metabolic profile. Results showed that all cell lines tested were sensitive to the cytotoxic action of these compounds. The mechanism underlying the cytotoxicity would be triggered by their weak uncoupling effect on the oxidative phosphorylation system, while having a wider and safer therapeutic range than other uncouplers and a significant lowering in transmembrane potential. Noteworthy, while the TPP(+)-derivatives alone led to almost negligible losses of ATP, when these were added in the presence of an AMP-activated protein kinase inhibitor, the levels of ATP fell greatly. Overall, data presented suggest that decyl polyhydroxybenzoates-TPP(+) and its derivatives warrant future investigation as potential anti-tumor agents.