Evidence for a stromal-epithelial “lactate shuttle” in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts

Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the “reverse Warburg effect,” which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, “energy transfer” or “metabolic-coupling” between the tumor stroma and epithelial cancer cells “fuels” tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.Key words: caveolin-1, oxidative stress, pseudohypoxia, lactate shuttle, MCT1, MCT4, metabolic coupling, tumor stroma, predictive biomarker, SLC16A1, SLC16A3, monocarboxylic acid transporter     

Are intracellular bacteria hallmarks for human tumors?

正There are ten major characteristics that are used to differentiate cancer from normal cells and tissue (Hanahan and Weinberg, 2011); detection of bacterial species associated with cancer cells is not included among these characteristics,and at first glance this might seem to be irrelevant. The     

Evidence for a slow time-scale of interaction for magnetic fields inhibiting tamoxifen’s antiproliferative action in human breast cancer cells

One critical biophysical feature of environmental-level magnetic field (MF) interactions with biological systems is the time-scale of interaction. A recently proposed fast/slow hypothesis states that a fast mechanism can only sense the instantaneous absolute value of the MF, and that a slow mechanism is potentially capable of sensing features such as frequency and relative orientation and magnitude of the field components. Here we applied the fast/slow hypothesis to a breast cancer model system: A 1.2 μT(rms), 60-Hz field inhibits tamoxifen’s (TAM’s) cytostatic action in MCF-7 cells via a MF interaction. We measured the growth of MCF-7 cells treated with TAM over 7 d, within different MFs: a sinusoidal, 60-Hz, 0.2-μT(rms) field; a sinusoidal, 60-Hz, 1.2-μT(rms) field; and a full-wave rectified version of the 1.2-μT(rms) sinusoidal field. A fast mechanism should not be able to distinguish between the latter two exposures. We observe that the rectified 1.2-μT field does not inhibit TAM’s action, but that the 1.2-μT sinusoidal field does. Therefore, the 1.2-μT MF inhibition of TAM’s cytostatic action operates via a relatively slow mechanism, and we predict that there exists a biologically dynamic complex capable of sensing a 1.2-μT, 60-Hz sinusoidal MF with an intrinsic time-scale of 17 ms or longer, the period of the 60-Hz applied field.     

The Inversion Effect in Biological Motion Perception: Evidence for a “Life Detector”?

If biological-motion point-light displays are presented upside down, adequate perception is strongly impaired. Reminiscent of the inversion effect in face recognition, it has been suggested that the inversion effect in biological motion is due to impaired configural processing in a highly trained expert system. Here, we present data that are incompatible with this view. We show that observers can readily retrieve information about direction from scrambled point-light displays of humans and animals. Even though all configural information is entirely disrupted, perception of these displays is still subject to a significant inversion effect. Inverting only parts of the display reveals that the information about direction, as well as the associated inversion effect, is entirely carried by the local motion of the feet. We interpret our findings in terms of a visual filter that is tuned to the characteristic motion of the limbs of an animal in locomotion and hypothesize that this mechanism serves as a general detection system for the presence of articulated terrestrial animals.     

Masting in a temperate tree: Evidence for environmental prediction?

Many plant species produce large fruit crops in some years and then produce few or no fruits in others. Synchronous, inter‐annual variation in plant reproduction is known as ‘masting’ and its adaptive significance has yet to be fully resolved. For 8 consecutive years, I quantified every fruit produced by 22 females of a New Zealand tree species (Dysoxylum spectabile), which has an unusual habit of taking a full calendar year to mature fruits after flowering. Fruit production varied strongly among years and was tightly synchronized among trees. Annual variability in fruit production declined with total reproductive output, indicating trees with lower fecundity exhibited a stronger tendency to mast. Although unrelated to temperature, annual fruit production was positively related to precipitation during annual periods of fruit development, and negatively related to fruit production in the previous year. Seedlings had higher rates of survivorship in a wet, high‐seed year than in a dry, low‐seed year, suggesting that seedlings might be drought sensitive. Therefore, D. spectabile produced large fruit crops during periods of high rainfall prior to fruit maturation, which may enhance survivorship of drought‐intolerant seeds. Results were inconsistent with several hypotheses that are widely believed to be the most likely explanations for masting. Instead, results were consistent with the environmental prediction hypothesis, suggesting that this hypothesis may be more important than previously appreciated.     

Evidence for a membrane carbonic anhydrase IV anchored by its C-terminal peptide in normal human pancreatic ductal cells

The high concentration of HCO₃ ^– ions (150 mM) in the human pancreatic ducts raises the question of the membrane proteins responsible for their secretion in addition to the Cl^–/HCO₃ ^– exchanger. In this study, we investigated the expression of carbonic anhydrase IV (CA IV), a possible candidate. Experiments were carried out on specimens of normal human pancreas obtained from brain-dead donors (n=9) as well as on isolated human ductal cells. Two antibodies were generated: CA IV NH₂ antibody directed against the NH₂ terminal of human glycosyl phosphatidylinositol (GPI)-anchored CA IV and CA IV COOH antibody directed against the COOH terminal of the same protein before its association with a GPI in the rough endoplasmic reticulum. A 35-kDa CA IV was detected in the homogenates of human pancreas. Immunocytochemistry demonstrated the expression of CA IV in centroacinar cells and in intercalated, intralobular, and interlobular ductal cells. The immunoreactivity observed with the CA IV COOH antibody was mainly localized on luminal membranes of ductal cells. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase C indicated that the CA IV expressed in pancreatic ducts was not GPI-anchored. Its detection in the same extracts by the CA IV COOH antibody indicated that it was anchored by a hydrophobic segment at the carboxy terminal. Taken together, these results suggest that normal human pancreatic ductal cells express a 35-kDa CA IV anchored in their luminal plasma membrane by a hydrophobic segment of the COOH terminus. In view of its localization and its mode of anchorage in luminal plasma membranes, this CA IV may participate in the maintenance of luminal pH.The first two authors have contributed equally to this work     

Evidence for a Müllerian mimetic radiation in Asian pitvipers

Müllerian mimicry, in which toxic species gain mutual protection from shared warning signals, is poorly understood in vertebrates, reflecting a paucity of examples. Indirect evidence for mimicry is found if monophyletic species or clades show parallel geographic variation in warning patterns. Here, we evaluate a hypothesis of Müllerian mimicry for the pitvipers in Southeast Asia using a phylogeny derived from DNA sequences from four combined mitochondrial regions. Mantel matrix correlation tests show that conspicuous red colour pattern elements are significantly associated with sympatric and parapatric populations in four genera. To our knowledge, this represents the first evidence of a Müllerian mimetic radiation in vipers. The putative mimetic patterns are rarely found in females. This appears paradoxical in light of the Müllerian prediction of monomorphism, but may be explained by divergent selection pressures on the sexes, which have different behaviours. We suggest that biased predation on active males causes selection for protective warning coloration, whereas crypsis is favoured in relatively sedentary females.     

Evidence for PHI-immunoreactive nerve fibres in the human skin: coexistence with VIP?

Using indirect immunofluorescence methodology, PHI-like immunoreactivity was found in a certain subpopulation of nerve fibres and terminals of the human skin. The immunoreactive fibres were mainly seen close to and around blood vessels and sweat glands, and they were of a fine-calibre type with smooth preterminal axons and a sparse plexus of varicosities at their terminal field. Furthermore, they were also observed around hair follicles, though more rarely around sebaceous glands. Finally, single PHI immunoreactive fibres could be seen in the close vicinity of the erector pili muscles. These fibres in all probability represent peripheral branches of the autonomic nervous system. Single (somatic?) immunoreactive fibers were, however, also found in the apical parts of the dermis, close to the epidermal-dermal junctional zone. The occurrence of VIP was also analysed and found to be similar to that of PHI. Thus, the present data point to a probable coexistence of PHI and VIP, a possibility that should be taken into account when discussing functional effects of VIP in human skin.     

Is there a role for culture in human behavioral ecology?

Most research in human behavioral ecology has been acultural, which raises the question of how best to incorporate the concept of culture into this approach. A necessary step in this direction is to pare the culture concept down to its ideational elements, excluding behavior and its material products (Durham 1991; Geertz 1973; Keesing 1974). The cultural and reproductive success hypothesis, though empirically successful (Irons 1993), is not a model for all of culture because of widespread discrepancies between behavior and culture to which it does not call attention. Cultural transmission models are also weakened by such discrepancies, but, more importantly, such models are most relevant to phenomena different from those central to human behavioral ecology. A better way to incorporate culture into human behavioral ecology is to see it as the context of human action and as a tool people use in social manipulation. The study of signal systems is a key to an understanding of social manipulation and to the incorporation of culture into human behavioral ecology. Examples of the manipulation of culture for reproductive benefit include Yanomamö kin term manipulation (Chagnon 1988), incest rules (Thornhill 1990, 1991), and the derogation of sexual competitors (Buss and Dedden 1990). The human behavioral ecological study of social manipulation in cultural contexts needs to be expanded. Two phenomena that might shed light on such manipulation are the Rashomon effect and the audience effect.     

Evidence for γ-actin as a Z disc component in skeletal myofibers

We investigated the targeting of the γ-actin isoform in skeletal myofibers. For this purpose we used expression vectors to produce green fluorescent protein (GFP-) as well as myc-tagged γ-actin in rat flexor digitorum brevis myofibers. We found that the γ-actin fusion proteins accumulated into Z discs but not beneath the sarcolemma. Instead, the GFP-tagged skeletal muscle-specific α-actin isoform was preferentially incorporated into the pointed ends of thin contractile filaments. The localization pattern of the γ-actin fusion proteins was completely different from that of the dystrophin glycoprotein complex on the sarcolemma. The results emphasize the role of γ-actin as a Z disc component but fail to reveal an actin-based sub-sarcolemmal cytoskeleton in skeletal muscle cells.     

Exocortis Disease: Evidence for a New Species of “Infectious” Low Molecular Weight RNA in Plants

THE anomalous properties of the infectious agent of exocortis disease in citrus (CEV), supposedly a virus, have been attributed^1,2 to infectious RNA existing as free nucleic acid. Although this model is consistent with the low sedimentation coefficient and the susceptibility to RNAase, the infectious molecule does not cosediment with either single stranded (ss) or double stranded (ds) RNA markers after equilibrium sedimentation in Cs₂SO₄. Resistance of the 8–16S moiety to inactivation by heat or diethylpyrocarbonate further suggests the presence of ds regions₃. A low molecular weight “infectious” RNA species, undetected in healthy tissue, has been implicated as the causal agent of exocortis disease.     

Evidence for the presence of volume-sensitive KCl transport in ‘young’ human red cells

‘Young’ human red cells are shown to possess a specific K⁺ pathway which is dependent on Cl⁻ and sensitive to cell volume. This system was latent in ‘mature’ cells but was revealed by high hydrostatic pressure. This suggests the pathway is functionally active in ‘young’ cells but becomes masked with cell maturation.     

Evidence that human α-thrombin is a monovalent cation-activated enzyme

The activity of human α-thrombin (EC 3.4.21.5) on small peptide substrates was enhanced by NaCl or KCl while tetramethylammonium chloride ((CH₃)₄NCl) or choline chloride (HO(CH₂)₂N(CH₃)₃Cl) which were used as ionic strength controls were without effect. The steady-state kinetic parameters of thrombin amidolysis of several peptidyl p-nitroanilide substrates were measured. Na⁺ enhanced thrombin activity by decreasing the K_m,app (0.2 to 0.7-fold) of all substrates, as well as increasing thombin turnover (3.4 to 4.5-fold) of some substrates. The average K_A for Na⁺for the four substrates examined was 3.5 × 10^?2m. A comparison of the effects of Na⁺ vs K⁺ on thrombin hydrolysis of a single substrate indicated that both cations similarly decreased the K_m,app (0.2 to 04.-fold) and increased thek_cat,app (3.1 to 3.4-fold) except that higher K⁺ concentrations (K_A = 2.8 × 10^?1M) were required. The rate of inactivation of thrombin by the active site-directed inhibitor N-p-tosyl-lysine chloromethyl ketone under pseudo-first-order conditions was enhanced 3-fold by saturating NaCl. Also, the fibrinogen clotting activity of thrombin was enhanced by NaCl compared to the choline chloride control. Spectral studies demonstrated that thrombin titration by Na⁺ caused a positive ultraviolet difference spectrum with maxima at 281.5 and 288.5 nm (Δ?_288.5 = +1067). The K_m for Na⁺ was 2.3 × 10^?2m which agrees with the kinetically determined K_A for Na⁺. The results are consistent with Na⁺ binding to thrombin causing a conformational change in the active site. It is concluded that human α-thrombin is a monovalent cation-activated enzyme.