The effect of oxygen levels on cytosolic free NAD/NADH ratios in Hela cells.

<p>Hela cells were incubated in complete RPMI-1640 medium containing 12 mM glucose supplemented with 20 mM lactate under oxygen levels between 21% to 1%. After 24-hour incubation, glucose consumption and lactate generation by Hela cells, cell growth, and intracellular lactate and pyruvate were measured. (A) Cell proliferation; (B) Glucose consumption; (C) Lactate generation; (D) L/G ratio; (E) Intracellular pyruvate; (F) Intracellular lactate; (G) Intracellular L/P ratio; (H) Cytosolic free NAD/NADH ratio estimated from the corresponding L/P ratio (see description in text). Data are mean±SD. Data were confirmed by 2 independent experiments.</p

The effect of extracellular lactate concentration on intracellular lactate concentration and intracellular L/P ratio in Bcap-37 cells.

<p>Bcap-37 cells were incubated in complete RPMI-1640 medium containing 12 mM glucose supplemented with 20 mM lactate. At the indicated time point, intracellular lactate and pyruvate were measured. (A) Intracellular lactate; (B) Intracellular pyruvate; (C) Intracellular L/P ratio. Data are mean ± SD. Data were confirmed by 2 independent experiments.</p

The effect of oxygen levels on intracellular concentration of lactate and pyruvate in Hela cells.

<p>Hela cells were incubated in complete RPMI-1640 medium containing 12 mM glucose. After 24-hour incubation, glucose consumption and lactate generation by Hela cells, cell growth, and intracellular lactate and pyruvate were measured. (A) Cell proliferation; (B) Glucose consumption; (C) Lactate generation; (D) L/G ratio; (E) Intracellular pyruvate; (F) Intracellular lactate; (G) Intracellular L/P ratio; (H) Cytosolic free NAD/NADH ratio erroneously estimated from the corresponding L/P ratio. Data are mean±SD. Data were confirmed by 2 independent experiments.</p

Intracellular lactate concentration and L/P ratio are highly labile in Bcap-37 cells.

<p>Bcap-37 cells were incubated in complete RPMI-1640 medium containing 12 mM glucose and supplemented with 6 mM glucose every 24 hours. At the indicated time point, intracellular lactate and pyruvate, glucose consumption and lactate generation by Bcap-37 cells, and cell growth were monitored. (A) Intracellular lactate; (B) Intracellular pyruvate; (C) Intracellular L/P ratio; (D) Cytosolic free NAD/NADH ratio erroneously estimated from the corresponding L/P ratio; (E) Cell proliferation curves; (F) Glucose consumption; (G) Lactate generation; (H) L/G ratio (the generated lactate divided by the consumed glucose between 2 time points). Data are mean±SD. Data were confirmed by 2 independent experiments.</p

Cytosolic free NAD/NADH ratio estimated at the conversion at equilibrium in Bcap-37 cells.

<p>Bcap-37 cells were incubated in complete RPMI-1640 medium containing 12 mM glucose supplemented with or without lactate (x axis). After 24-hour incubation, glucose consumption and lactate generation by Bcap-37 cells, cell growth, and intracellular lactate and pyruvate were measured. (A) Cell proliferation; (B) Glucose consumption; (C) Lactate generation; (D) L/G ratio; (E) Intracellular pyruvate; (F) Intracellular lactate; (G) Intracellular L/P ratio; (H) Cytosolic free NAD/NADH ratio estimated from the corresponding L/P ratio(see corresponding text). Data are mean±SD. Data were confirmed by 3 independent experiments.</p

Additional file 1: of A novel SLC6A8 mutation associated with intellectual disabilities in a Chinese family exhibiting creatine transporter deficiency: case report

Figure S1. A. Gesell developmental scale evaluated the proband as severely developmentally delayed. B. Urine creatine was significantly increased (0.805 mg/ml) in the proband (peak height 25,000, upper left) compared with the normal control (NC) value of 0.160 ± 0.177 mg/ml (peak height 3,200, below left). The internal standard peak is the reference for the test at right. C. Proton magnetic resonance spectroscopy (MRS), examination using a 3.0-T system on the brain showed normal brain creatine peak (left part) and brain MRI showed normal corpus callosum in the proband’s mother (right part). (TIF 5633 kb

Anticancer Agent Shikonin Is an Incompetent Inducer of Cancer Drug Resistance

<div><h3>Purpose</h3><p>Cancer drug resistance is a major obstacle for the success of chemotherapy. Since most clinical anticancer drugs could induce drug resistance, it is desired to develop candidate drugs that are highly efficacious but incompetent to induce drug resistance. Numerous previous studies have proven that shikonin and its analogs not only are highly tumoricidal but also can bypass drug-transporter and apoptotic defect mediated drug resistance. The purpose of this study is to investigate if or not shikonin is a weak inducer of cancer drug resistance.</p> <h3>Experimental Design</h3><p>Different cell lines (K562, MCF-7, and a MDR cell line K562/Adr), after repeatedly treated with shikonin for 18 months, were assayed for drug resistance and gene expression profiling.</p> <h3>Results</h3><p>After 18-month treatment, cells only developed a mere 2-fold resistance to shikonin and a marginal resistance to cisplatin and paclitaxel, without cross resistance to shikonin analogs and other anticancer agents. Gene expression profiles demonstrated that cancer cells did strongly respond to shikonin treatment but failed to effectively mobilize drug resistant machineries. Shikonin-induced weak resistance was associated with the up-regulation of βII-tubulin, which physically interacted with shikonin.</p> <h3>Conclusion</h3><p>Taken together, apart from potent anticancer activity, shikonin and its analogs are weak inducers of cancer drug resistance and can circumvent cancer drug resistance. These merits make shikonin and its analogs potential candidates for cancer therapy with advantages of avoiding induction of drug resistance and bypassing existing drug resistance.</p> </div

IC₅₀ profile of all the parental cells and the drug-resistant cells to conventional anticancer drugs.

<p>The inhibitory effect of conventional anticancer drugs and shikonin on drug-sensitive and -resistant cells was determined by MTT assay as described in the Material and Methods section. Numbers in parenthesis are the drug-resistance fold (IC₅₀ of drug-resistant cells/IC₅₀ of the parental drug-sensitive cells). n.d., not determined.</p>*<p>, p<0.05;</p>**<p>, p<0.01;</p>***<p>, P<0.001.</p

DT diaphorase is not involved in shikonin induced drug resistance.

<p>MCF-7 and MCF-7/Shk cells were treated with shikonin in the presence or absence of 20 µM dicumarol.</p

IC₅₀ profile of all the parental cells and the drug-resistant cells to shikonin analogues.

<p>The inhibitory effect of shikonin analogues on drug-sensitive and -resistant cells was determined by MTT assay as described in the Material and Methods section.</p>*<p>, p<0.05, statistical significance in comparison to parental cell line K562, K562/Adr or MCF-7.</p><p>Numbers in parenthesis are the drug-resistance fold (IC₅₀ of drug-resistant cells/IC₅₀ of the parental drug-sensitive cells).</p