Another Form of Modified, Highly-Active 6-Phosphofructo-1-Kinase in Cancer Cells

Enhanced glycolytic flux is a hallmarks of cancer cells. Posttranslational modification of the key regulatory enzyme of glycolysis, 6-Phosphofructo-1- Kinase (Pfk1) might trigger metabolic flux deregulation. In the cancer cells the human 85 kDa muscle type nPfk-M enzyme can be proteolytically cleaved to form highly-active 47 kDa shorter fragments that retain activity but become resistant to feed-back inhibition. In several tumorigenic cell lines, no native 85 kDa liver type nPfk-L isoforms could be either found and only 70 kDa shorter fragments were detected by immune-blotting. To learn more about the cancer-specific modified sfPfk-L enzyme, the truncated human sfPfk-L gene encoding 70 kDa fragments was inserted into the pfk null yeast S.cerevisiae cell. The recombinant modified enzyme showed higher affinity toward the substrate fructose-6-phosphate, reduced sensitivity toward the citrate and ATP inhibition in respect to the recombinant native PFK-L enzyme. Partially purified cancer-specific sfPfk-L fragments lacking the C-portion of the enzyme showed some instability under the diluted conditions in the buffer in respect to the tetrameric native nPfk-L enzyme. Growth characteristics of the yeast transformant encoding short sfPfk-L enzymes were similar to those encoding shorter sfPfk-M enzymes. No growth of the transformant with the sfPfk-L gene was observed on glucose but it grew faster than the transformant with the native human nPfk-L enzyme in a narrow ecological niche with low maltose concentration and 10 mM of ethanol in the medium. Similar to modified 47 kDa sfPfk-M fragments, also the short 70 kDa nPfk- Lfragments might cause deregulation of the glycolytic flux in the yeast and in the cancer cells. In yeast, deregulated metabolic flux unbalances redox potential that results in reduced growth rate. However, the cancer cells beat the redox unbalance by rapid re-oxidation of redundant NADH that results in lactate formation while the growth rate remains high.

LDH-A Knockdown: Changes in The LDH Isoenzyme Profile and Variability in Glioma Response

Background: Lactate metabolism in tumors is now recognized as a major energy source and a major gluconeogenic precursor for many tumors, as well as shown to exhibit signaling properties. There is less information on the role of the LDH/lactate axis in brain tumors, although lactate formation in gliomas is associated with poor survival. Methods: Three murine glioma cell lines (GL261, CT2A, and ALTS1C1) were transduced to knockdown (KD) expression of the murine LDH-A gene. The effects of the LDH-A KD were compared to those in control (NC) cells and tumors. Results: Differences in the expression of LDH-A and LDH-B mRNA, protein, and enzymatic activity were observed in the six cell lines. LDH zymography showed a major difference in LDH subunit distribution between GL261 LDH-A KD and NC tumors, whereas little or no effect of LDH-A KD was observed in CT2A and ALTS1C1 tumors. Tumors LDH-A and LDH-B immunohistochemistry and a Weka segmentation analysis were consistent with isoenzyme patterns and the above analyses. An “inverse” LDH-A/LDH-B staining relationship (high vs low) was observed in many local GL261 tumor regions. In contrast, CT2A tumors showed a more “direct” local LDH-A/LDH-B staining relationship. LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice (GL261 NC tumors had a prolonged growth delay). In nude mice, both LDH-A KD and NC GL261 tumors grew more rapidly than GL261 NC tumors in C57BL/6 mice. No differences between NC and KD cell proliferation (in vitro) and tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors, consistent with the absence of a difference in their LDH isoenzyme profiles. Conclusions: These results show the combined impact of a genetic alteration (LDH-A depletion) on the LDH isoenzyme profile, expression of LDH-A vs LDH-B and LDH enzymatic activity, and the immune system (C57BL/6 vs nude mice) on the growth of s.c. located tumors.

A retrospective analysis of CO2 derived goal-directed perfusion variables under normothermic conditions: do we need to re-evaluate threshold values?

This study investigated if current predictive values for increased lactate formation: VCO2i > 60 ml min−1 m−2, respiratory quotient (RQ) > 0.90, and DO2/VCO2 < 5.0, are valid under normothermic conditions. CO2 derived parameters were analyzed in 91 patients undergoing normothermic CABG and related to increase of blood lactate concentrations during bypass. In this study population, 85 patients (93%) had a median VCO2i above 60 ml min−1 m−2 and 53 patients (58%) had a DO2/VCO2 ⩽ 5.0. Eighteen patients (20%) had a median RQ ⩾ 0.90, but RQ remained with a maximum value of 0.94 below the biological threshold of 1.0. Increase of lactate concentrations remained without clinical significance and showed weak correlations with VCO2i (rs = 0.277, p = 0.008) and RQ (rs = 0.346, p = 0.001). The cohort was separated for the different CO2 variables by their median value to compare increase in lactate concentration. Patients with a high VCO2i (⩾70 ml min−1 m−2) and a high RQ (⩾0.82) showed significant higher increase in lactate concentration compared to patients with VCO2i < 70 ml min−1 m−2 (p = 0.004), and a RQ < 0.82 (p = 0.012). Groups separated by a median DO2/VCO2 of 4.8 did not show a difference in increase of lactate concentration in blood. In summary, specific CO2 derived threshold values for the prediction of lactate formation, which have been reported in other studies, cannot be confirmed with our findings. However, a CO2 production ⩾70 ml min−1 m−2 and a RQ ⩾ 0.82 in this study population were correlated with increased lactate formation. Because CO2 production during bypass depends on patient temperature, a different cutoff value, that may take into account the influence of demographic variables, should be determined during normothermic CPB.

Kinetics of Alkyl Lactate Formation from the Alcoholysis of Poly(Lactic Acid)

Alkyl lactates are green solvents that are successfully employed in several industries such as pharmaceutical, food and agricultural. They are considered prospective renewable substitutes for petroleum-derived solvents and the opportunity exists to obtain these valuable chemicals from the chemical recycling of waste poly(lactic acid). Alkyl lactates (ethyl lactate, propyl lactate and butyl lactate) were obtained from the catalysed alcoholysis reaction of poly(lactic acid) with the corresponding linear alcohol. Reactions were catalysed by a Zn complex synthesised from an ethylenediamine Schiff base. The reactions were studied in the 50–130 °C range depending on the alcohol, at autogenous pressure. Arrhenius temperature-dependent parameters (activation energies and pre-exponential factors) were estimated for the formation of the lactates. The activation energies (Ea1, Ea2 and Ea−2) for alcoholysis in ethanol were 62.58, 55.61 and 54.11 kJ/mol, respectively. Alcoholysis proceeded fastest in ethanol in comparison to propanol and butanol and reasonable rates can be achieved in temperatures as low as 50 °C. This is a promising reaction that could be used to recycle end-of-life poly(lactic acid) and could help create a circular production economy.

Targeting UCP2 Suppresses the FAK Signaling and Progression of Human Head and Neck Cancer Cells

Background: New adjuvant therapies for human head and neck (H&N) cancer to improve the quality of life of the patients are in great demand. Our early studies have demonstrated that uncoupling protein 2 (UCP2) is upregulated in the tumor tissues of H&N cancer compared to the adjacent normal tissues; however, the role of UCP2 in H&N cancer has not been studied. Objective: In this manuscript, we aim to examine whether UCP2 contributes to H&N cancer progression in vitro. Methods: We generated UCP2 stable knockdown H&N cancer cells and detected the effects of UCP2 inhibition on cell proliferation, migration, invasion, 3D spheroid formation, and the sensitivity to a chemodrug treatment. Results: Knockdown of UCP2 suppressed the progression of H&N cancer in vitro, which might be mediated via the following mechanism: 1) increased the G1 phase whereas decreased the S phase of the cell cycle, which could be mediated by suppression of the G1/S regulators including CDK4/6 and cyclin D1. 2) Decreased mitochondrial oxygen consumption, ATP production, and lactate formation, which is consistent with the downregulation of c-Myc. 3) FAK may serve as the upstream signaling molecule, and its action was mediated by Akt and ERK. Conclusions: Our studies first demonstrate that targeting UCP2 may suppress H&N cancer progression in vitro.

Salbutamol-induced severe lactic acidosis in acute asthma

Selective beta-adrenoceptor agonists are worldwide prescribed to manage bronchial obstruction. However, they expose to a potential risk of hyperlactatemia and lactic acidosis even with normal doses. The mechanism still poorly understood and suggested that salbutamol diverts the metabolism of pyruvate acid from Krebs cycle toward lactate formation. We report the case of a 42-year-old patient, admitted to intensive care unit for acute severe asthma. He presented a transient lactic acidosis over the first 48 h, following an excessive use of salbutamol. The metabolic acidosis caused tachypnea, as a compensatory mechanism, leading to respiratory failure. The diagnosis of salbutamol-induced lactic acidosis must be made by elimination and only accepted after deleting the other causes. The main clinical character is the worsening of dyspnea despite regression of bronchospasm. It is transient and usually normalizes within 24–48 h after stopping or decreasing doses of salbutamol.