Identification and Analysis of the Active Phytochemicals from the Anti-Cancer Botanical Extract Bezielle

Bezielle is a botanical extract that has selective anti-tumor activity, and has shown a promising efficacy in the early phases of clinical testing. Bezielle inhibits mitochondrial respiration and induces reactive oxygen species (ROS) in mitochondria of tumor cells but not in non-transformed cells. The generation of high ROS in tumor cells leads to heavy DNA damage and hyper-activation of PARP, followed by the inhibition of glycolysis. Bezielle therefore belongs to a group of drugs that target tumor cell mitochondria, but its cytotoxicity involves inhibition of both cellular energy producing pathways. We found that the cytotoxic activity of the Bezielle extract in vitro co-purified with a defined fraction containing multiple flavonoids. We have isolated several of these Bezielle flavonoids, and examined their possible roles in the selective anti-tumor cytotoxicity of Bezielle. Our results support the hypothesis that a major Scutellaria flavonoid, scutellarein, possesses many if not all of the biologically relevant properties of the total extract. Like Bezielle, scutellarein induced increasing levels of ROS of mitochondrial origin, progressive DNA damage, protein oxidation, depletion of reduced glutathione and ATP, and suppression of both OXPHOS and glycolysis. Like Bezielle, scutellarein was selectively cytotoxic towards cancer cells. Carthamidin, a flavonone found in Bezielle, also induced DNA damage and oxidative cell death. Two well known plant flavonoids, apigenin and luteolin, had limited and not selective cytotoxicity that did not depend on their pro-oxidant activities. We also provide evidence that the cytotoxicity of scutellarein was increased when other Bezielle flavonoids, not necessarily highly cytotoxic or selective on their own, were present. This indicates that the activity of total Bezielle extract might depend on a combination of several different compounds present within it

LRRK2 deficiency induced mitochondrial Ca2+ efflux inhibition can be rescued by Na+/Ca2+/Li+ exchanger upregulation

Variants of leucine-rich repeat kinase 2 (lrrk2) are associated with an increased risk in developing Parkinson’s disease (PD). Mitochondrial dysfunction and specifically mitochondrial Ca2+ handling has been linked to the pathogenesis of PD. Here we describe for the second time a mitochondrial Ca2+ efflux deficiency in a model displaying alterations in a PD-associated risk protein. LRRK2 deletion, inhibition and mutations led to an impaired mitochondrial Ca2+ extrusion via Na+/Ca2+/Li+ exchanger (NCLX) which in turn lowered mitochondrial permeability transition pore (PTP) opening threshold and increased cell death. The mitochondrial membrane potential was found not to be the underlying cause for the Ca2+ extrusion deficiency. NCLX activity was rescued by a direct (phosphomimetic NCLX mutant) and indirect (protein kinase A) activation which in turn elevated the PTP opening threshold. Therefore, at least two PD-associated risk protein pathways appear to converge on NCLX controlling mitochondrial Ca2+ extrusion and therefore mitochondrial health. Since mitochondrial Ca2+ overload has been described in many neurological disorders this study warrants further studies into NCLX as a potential therapeutic target

Targeted apoptosis in ovarian cancer cells through mitochondrial dysfunction in response to Sambucus nigra agglutinin

Ovarian carcinoma (OC) patients encounter the severe challenge of clinical management owing to lack of screening measures, chemoresistance and finally dearth of non-toxic therapeutics. Cancer cells deploy various defense strategies to sustain the tumor microenvironment, among which deregulated apoptosis remains a versatile promoter of cancer progression. Although recent research has focused on identifying agents capable of inducing apoptosis in cancer cells, yet molecules efficiently breaching their survival advantage are yet to be classified. Here we identify lectin, Sambucus nigra agglutinin (SNA) to exhibit selectivity towards identifying OC by virtue of its specific recognition of α-2, 6-linked sialic acids. Superficial binding of SNA to the OC cells confirm the hyper-sialylated status of the disease. Further, SNA activates the signaling pathways of AKT and ERK1/2, which eventually promotes de-phosphorylation of dynamin-related protein-1 (Drp-1). Upon its translocation to the mitochondrial fission loci Drp-1 mediates the central role of switch in the mitochondrial phenotype to attain fragmented morphology. We confirmed mitochondrial outer membrane permeabilization resulting in ROS generation and cytochrome-c release into the cytosol. SNA response resulted in an allied shift of the bioenergetics profile from Warburg phenotype to elevated mitochondrial oxidative phosphorylation, altogether highlighting the involvement of mitochondrial dysfunction in restraining cancer progression. Inability to replenish the SNA-induced energy crunch of the proliferating cancer cells on the event of perturbed respiratory outcome resulted in cell cycle arrest before G2/M phase. Our findings position SNA at a crucial juncture where it proves to be a promising candidate for impeding progression of OC. Altogether we unveil the novel aspect of identifying natural molecules harboring the inherent capability of targeting mitochondrial structural dynamics, to hold the future for developing non-toxic therapeutics for treating OC

Time-dependent effects of imatinib in human leukaemia cells: a kinetic NMR-profiling study

The goal of this study was to evaluate the time course of metabolic changes in leukaemia cells treated with the Bcr-Abl tyrosine kinase inhibitor imatinib. Human Bcr-Abl+ K562 cells were incubated with imatinib in a dose-escalating manner (starting at 0.1 μM with a weekly increase of 0.1 μM imatinib) for up to 5 weeks. Nuclear magnetic resonance spectroscopy and liquid-chromatography mass spectrometry were performed to assess a global metabolic profile, including glucose metabolism, energy state, lipid metabolism and drug uptake, after incubation with imatinib. Initially, imatinib treatment completely inhibited the activity of Bcr-Abl tyrosine kinase, followed by the inhibition of cell glycolytic activity and glucose uptake. This was accompanied by the increased mitochondrial activity and energy production. With escalating imatinib doses, the process of cell death rapidly progressed. Phosphocreatine and NAD+ concentrations began to decrease, and mitochondrial activity, as well as the glycolysis rate, was further reduced. Subsequently, the synthesis of lipids as necessary membrane precursors for apoptotic bodies was accelerated. The concentrations of the Kennedy pathway intermediates, phosphocholine and phosphatidylcholine, were reduced. After 4 weeks of exposure to imatinib, the secondary necrosis associated with decrease in the mitochondrial and glycolytic activity occurred and was followed by a shutdown of energy production and cell death. In conclusion, monitoring of metabolic changes in cells exposed to novel signal transduction modulators supplements molecular findings and provides further mechanistic insights into longitudinal changes of the mitochondrial and glycolytic pathways of oncogenesis

Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms

Immediate <it>in vivo</it> target-specific cancer cell death after near infrared photoimmunotherapy

<p>Abstract</p> <p>Background</p> <p>Near infrared (NIR) photoimmunotherapy (PIT) is a new type of cancer treatment based on a monoclonal antibody (mAb)-NIR phthalocyanine dye, (IR700) conjugate. <it>In vitro</it> cancer-specific cell death occurs during NIR light exposure in cells previously incubated with mAb-IR700 conjugates. However, documenting rapid cell death <it>in vivo</it> is more difficult.</p> <p>Methods</p> <p>A luciferase-transfected breast cancer cell (epidermal growth factor receptor+, MDA-MB-468luc cells) was produced and used for both <it>in vitro</it> and <it>in vivo</it> experiments for monitoring the cell killing effect of PIT. After validation of cytotoxicity with NIR exposure up to 8 J/cm²<it>in vitro</it>, we employed an orthotopic breast cancer model of bilateral MDA-MB-468luc tumors in female athymic mice, which subsequently received a panitumumab-IR700 conjugate <it>in vivo</it>. One side was used as a control, while the other was treated with NIR light of dose ranging from 50 to 150 J/cm². Bioluminescence imaging (BLI) was performed before and after PIT.</p> <p>Results</p> <p>Dose-dependent cell killing and regrowth was successfully monitored by the BLI signal <it>in vitro</it>. Although tumor sizes were unchanged, BLI signals decreased by >95% immediately after PIT <it>in vivo</it> when light intensity was high (>100 J/cm²), however, in mice receiving lower intensity NIR (50 J/cm²), tumors recurred with gradually increasing BLI signal.</p> <p>Conclusion</p> <p>PIT induced massive cell death of targeted tumor cells immediately after exposure of NIR light that was demonstrated with BLI <it>in vivo</it>.</p

Calcium signalling-dependent mitochondrial dysfunction and bioenergetics regulation in respiratory chain Complex II deficiency

International audienceDespite advanced knowledge on the genetic basis of oxidative phosphorylation (OXPHOS)-related diseases, the molecular and/or cellular determinants for tissue specific dysfunction are not completely understood. Here, we report the cellular events associated with mitochondrial respiratory Complex II deficiency occurring prior to cell death. Mutation or chronic inhibition of Complex II determined a large increase of basal and agonist-evoked Ca2+ signals in the cytosol and the mitochondria, in parallel with mitochondrial dysfunction characterized by membrane potential (∆ψmit) loss, [ATP] reduction and increased Reactive Oxygen Species (ROS) production. cytosolic and mitochondrial Ca2+ overload are linked to increased ER Ca2+ leakage, and to SERCA2b and PMCA proteasome-dependent degradation. Increased [Ca2+]mit is also contributed by decreased mitochondrial motility and increased ER-mitochondria contact sites. Interestingly, increased intracellular [Ca2+] activated on the one hand a compensatory Ca2+-dependent glycolytic ATP production and determined on the second hand mitochondrial pathology. These results revealed the primary role for Ca2+ signalling in the control of mitochondrial dysfunction and cellular bioenergetics outcomes linked to respiratory chain Complex II deficiency