The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-d-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC₅₀ 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells.

Gene expression signatures associated with breast cancer metastases suggest that metabolic re-wiring is important for metastatic growth in lungs, bones, and other organs. However, since pathway fluxes depend on additional factors such as ATP demand, allosteric effects, and post-translational modification, flux analysis is necessary to conclusively establish phenotypes. In this study, the metabolic phenotypes of breast cancer cell lines with low (T47D) or high (MDA-MB-231) metastatic potential, as well as lung (LM)- and bone (BoM)-homing lines derived from MDA-MB-231 cells, were assessed by ¹³C metabolite labeling from [1,2-¹³C] glucose or [5-¹³C] glutamine and the rates of nutrient and oxygen consumption and lactate production. MDA-MB-231 and T47D cells produced 55 and 63%, respectively, of ATP from oxidative phosphorylation, whereas LM and BoM cells were more glycolytic, deriving only 20–25% of their ATP from mitochondria. ATP demand by BoM and LM cells was approximately half the rate of the parent cells. Of the anabolic fluxes assessed, nucleotide synthesis was the major ATP consumer for all cell lines. Glycolytic NADH production by LM cells exceeded the rate at which it could be oxidized by mitochondria, suggesting that the malate-aspartate shuttle was not involved in re-oxidation of these reducing equivalents. Serine synthesis was undetectable in MDA-MB-231 cells, whereas 3–5% of glucose was shunted to serine by LM and BoM lines. Proliferation rates of T47D, BoM, and LM lines tightly correlated with their respiration-normalized NADPH production rates. In contrast, MDA-MB-231 cells produced NADPH and GSH at higher rates, suggesting this line is more oxidatively stressed. Approximately half to two-thirds of NADPH produced by T47D, MDA-MB-231, and BoM cells was from the oxidative PPP, whereas the majority in LM cells was from the folate cycle. All four cell lines used the non-oxidative PPP to produce pentose phosphates, although this was most prominent for LM cells. Taken together, the metabolic phenotypes of LM and BoM lines differed from the parent line and from each other, supporting the metabolic re-wiring hypothesis as a feature of metastasis to lung and bone.

J. Neurochem. (2011) 119, 1137–1150. Mitochondrial outer membrane Bax oligomers are critical for cytochrome c release, but the role of resident mitochondrial proteins in this process remains unclear. Membrane‐associated Bax has primarily been studied using 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS) as the solubilizing agent, as it does not induce conformational artifacts, although recent evidence indicates it may have other artifactual effects. The objective of this study was to investigate digitonin as an alternative detergent to assess Bax oligomeric state, and possible interaction with voltage‐dependent anion channel (VDAC)1 in cerebellar granule neurons. VDAC1 co‐immunoprecipitated with Bax in digitonin extracts from healthy and apoptotic neurons. Two‐dimensional blue native‐SDS–PAGE revealed five Bax and VDAC1 oligomers having similar masses from 120 to 500 kDa. The levels of two VDAC1 oligomers in Bax 1D1 immunodepleted extracts negatively correlated with levels of co‐precipitated VDAC1, indicating the co‐precipitated VDAC1 was derived from these oligomers. Immunodepletion with the 6A7 antibody modestly reduced the levels of Bax oligomers from apoptotic but not healthy neurons. A sixth 170 kDa oligomer containing exclusively 6A7 Bax and no VDAC1 was identified after apoptosis induction. CHAPS failed to solubilize VDAC1, and additionally yielded no distinct oligomers. We conclude that digitonin is a potentially useful detergent preserving Bax–VDAC1 interactions that may be disrupted with CHAPS.