- Research article
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New anti-cancer chemicals Ertredin and its derivatives, regulate oxidative phosphorylation and glycolysis and suppress sphere formation in vitro and tumor growth in EGFRvIII-transformed cells
© The Author(s). 2016
- Received: 4 February 2016
- Accepted: 5 July 2016
- Published: 19 July 2016
EGFRvIII is a mutant form of the epidermal growth factor receptor gene (EGFR) that lacks exons 2–7. The resulting protein does not bind to ligands and is constitutively activated. The expression of EGFRvIII is likely confined to various types of cancer, particularly glioblastomas. Although an anti-EGFRvIII vaccine is of great interest, low-molecular-weight substances are needed to obtain better therapeutic efficacy. Thus, the purpose of this study is to identify low molecular weight substances that can suppress EGFRvIII-dependent transformation.
We constructed a new throughput screening system and searched for substances that decreased cell survival of NIH3T3/EGFRvIII spheres under 3-dimensional (3D)-culture conditions, but retained normal NIH3T3 cell growth under 2D-culture conditions. In vivo activity was examined using a mouse transplantation model, and derivatives were chemically synthesized. Functional characterization of the candidate molecules was investigated using an EGFR kinase assay, immunoprecipitation, western blotting, microarray analysis, quantitative polymerase chain reaction analysis, and measurement of lactate and ATP synthesis.
In the course of screening 30,000 substances, a reagent, “Ertredin” was found to inhibit anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII cDNA. Ertredin also inhibited sphere formation in cells expressing wild-type EGFR in the presence of EGF. However, it did not affect anchorage-dependent 2D growth of parental NIH3T3 cells. The 3D-growth-inhibitory activity of some derivatives, including those with new structures, was similar to Ertredin. Furthermore, we demonstrated that Ertredin suppressed tumor growth in an allograft transplantation mouse model injected with EGFRvIII- or wild-type EGFR-expressing cells; a clear toxicity to host animals was not observed. Functional characterization of Ertredin in cells expressing EGFRvIII indicated that it stimulated EGFRvIII ubiquitination, suppressed both oxidative phosphorylation and glycolysis under 3D conditions, and promoted cell apoptosis.
We developed a high throughput screening method based on anchorage-independent sphere formation induced by EGFRvIII-dependent transformation. In the course of screening, we identified Ertredin, which inhibited anchorage-independent 3D growth and tumor formation in nude mice. Functional analysis suggests that Ertredin suppresses both mitochondrial oxidative phosphorylation and cytosolic glycolysis in addition to promoting EGFRvIII degradation, and stimulates apoptosis in sphere-forming, EGFRvIII-overexpressing cells.
- Oxidative phosphorylation
Epidermal growth factor receptor (EGFR) expression and signaling are known to contribute to the development of multiple epithelial malignancies, including glioblastoma, squamous carcinomas of the skin, and breast cancer [1–6]. Thus, EGFR mutations have important implications for drug resistance and prognosis [7–9]. Among these mutations, an intragenic deletion of exons 2 to 7 of the wild-type EGFR (EGFRwt) gene results in an in-frame deletion of 267 amino acid residues from the extra-cellular domain of the 175-kDa EGFRwt. This deletion results in a truncated, about 140-kDa constitutively activated mutant EGFR, with an intracellular domain identical to EGFRwt [10–12]. This mutant EGFR gene was discovered by Shibuya et al. in 1988 [10, 13] and named ⊿EGFR, de2-7 EGFR, or more commonly, EGFRvIII. The EGFRvIII gene has been found in glioblastoma, lung, breast, ovarian, colorectal, head and neck squamous cell carcinoma (HNSCC), and prostate cancer. EGFRvIII signaling has been shown to correlate with a poor prognosis [14, 15]. There is extensive evidence indicating that EGFRvIII is a tumor-specific protein , and aberrant EGFRvIII signaling has been shown to be important in tumor progression. Because it is expressed only in tumor cells, it appears to be a rational and attractive target for cancer therapy [2, 15, 16]. Although the anti-EGFRvIII vaccine has received increased attention, it may not be effective for all EGFRvIII tumor-carrying patients. Therapies involving vaccines are difficult to apply in immune-suppressed cancer patients, and have potential risks such as the induction of autoimmune diseases. Thus, low-molecular-weight substances are required for efficient therapy.
Normal cells that grow in the adherent state undergo apoptosis shortly after losing their adhesion to the substratum, a phenomenon known as “anoikis” [13, 17–20]. However, cancer cells are still able to survive and grow in the absence of adhesion or anchorage to a substratum . For example, glioblastoma cells overexpressing EGFRwt or EGFRvIII have been shown to be anchorage-independent. This “anchorage independence” is believed to be one of the most important oncogenic properties of cancer cells and cancer stem cells [19–22].
In the present study, we describe a high throughput method for the screening of EGFRvIII-cascade inhibitors. By screening 30,000 substances, we identified “Ertredin derivatives” that suppressed anchorage-independent growth in vitro and tumor growth in EGFRvIII-transformed cells.
NIH3T3 cell lines overexpressing human EGFRvIII (de2-7EGFR) (NIH3T3/EGFRvIII) and wild-type EGFR (NIH3T3/EGFRwt) were established using a previously reported method . NIH3T3, NIH3T3/EGFRvIII, and NIH3T3/EGFRwt cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5 % FBS. All cells were cultured with 50 U/mL penicillin/streptomycin at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % air. Viable cell counts were assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay or the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, USA).
AG1478 was purchased from Wako (Osaka, Japan), gefitinib was obtained from AstraZeneca, erlotinib was from ChemieTek (Indianapolis, USA), and KT5720 and LY294002 were from Sigma-Aldrich. The chemical library included 30,000 low-molecular weight compounds supplied by the ChemBridge Screening Libraries (San Diego, CA, USA). Rabbit anti-human EGFR (D38B1), rabbit anti-phospho-EGFR Tyr1068 (D7A5), mouse anti-ubiquitin (P4D1), and HIF-1α monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, USA). Mouse anti-β-actin (AC-15) monoclonal antibody was obtained fom Abcam (Cambridge, UK). Peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were supplied by Jackson Immunoresearch (West Grove, PA, USA).
Anchorage-independent 3D cell culture and screening
In the 3D culture, viable cells were quantitated with the CellTiter-Glo luminescent cell viability assay (Promega). In the 2D culture, cell number was assessed by CellTiter 96 (Promega).
Cells (2 × 105 cells/mL, 10 mL/well) were seeded on ULAS 6-well plates for 3D growth, or were seeded on 12-well cell culture plates at 5 × 104 cells/well for 2D growth. The cells were then transfected for 72 h (for 3D culture) or 24 h (for 2D culture) with 12 pmoles of small interfering RNA (siRNA) using Lipofectamine RNAiMAX transfection reagent (Life Technologies). After transfection, the cells were harvested and lysed for western blotting.
Cells (2 × 105 cells/mL, 0.1 mL) were seeded on ULAS 96-well plates for 3D growth, or were seeded in 96-well cell culture plates at 2 × 103 cells/well for 2D growth. The cells were then transfected for 72 h with 0.12 pmoles siRNA, and cell proliferation was quantitated. NM005228_stealth_2438 (RNA-GGAUCCCAGAAGGUGAGAAAGUUAA), NM005228_stealth_2858 (RNA-CAGAAGGAGGCAAAGUGCCUAUCAA), and M005228_stealth_2874 (RNA-GCCUAUCAAGUGGAUGGCAUUGGAA) were purchased from Life Technologies for EGFR-siRNA. Negative control siRNA (46-2002; Life Technologies) that did not target any mRNA sequence was used as a control.
After washing with PBS, cells were treated with lysis buffer [50 mM HEPES (pH 7.2), 10 % glycerol, 150 mM NaCl, 1 % TritonX-100, 1 mM EGTA, 100 M PMSF, 100 units/mL of aprotinin, 10 mM Na3VO4, 100 mM NaF] . Protein abundance in the cell lysate was assessed with the BCA Protein Assay Kit-Reducing Agent Compatible (Thermo Fisher Scientific, Waltham, U.S.A.). After addition of one-third the volume of the NuPAGE LDS Sample Buffer (4X) (Life Technologies) to the cell lysate, an appropriate amount of protein from the mixture was applied to 10 % SDS-PAGE. The proteins in the gel were transferred to a PVDF membrane using iBlot (Life Technologies). The protein was detected using a primary antibody and peroxidase-conjugated secondary antibody, and visualized with the ECL Prime Western Blotting Detection Reagent. The images were acquired using the ImageQuant LAS 4000 Mini (GE Healthcare Life Sciences) and processed by Adobe Photoshop.
E1 (original Ertredin), E7, and E8 (a new substance), were synthesized as shown in Additional file 1. E2 was obtained from Enamine (Kiev, Ukraine), and E3, E5, and E15 were from Life Chemicals (Niagara-on-the-Lake, Canada). E4 and E14 were obtained from Pharmeks (Moscow, Russia), E6 was from UkrOrgSynthesis (Kiev, Ukraine), and E9-13 were from ChemBridge Screening Libraries.
Mouse transplantation model
NIH3T3/EGFRvIII (1 × 105) cells or NIH3T3/EGFRwt (1 × 106) cells were suspended in 100 μL of 62.5 % v/v Matrigel (BD Biosciences) in DMEM and transplanted subcutaneously into female Balb/c nude mice at 7–8 weeks of age. Either Ertredin derivatives or gefitinib were dissolved in a vehicle consisting of saline, 10 % w/w DMSO, and 1 % w/w Tween80. After cell transplantation, 30 mg/kg Ertredin or I-Ertredin was injected intraperitoneally once daily from day 1–17. Gefitinib at 100 or 200 mg/kg was orally administrated to mice transplanted with these cells as a positive control.
Five to 6 mice were included in the vehicle-injected group, 4–5 in the Ertredin-or I-Ertredin-injected group, and 3–5 in the gefitinib-injected group. We measured the long and short diameters of the tumors every 2 or 3 days, and calculated tumor volume with the following equation: tumor volume = (long diameter) × (short diameter)2 / 2. On day 17 or 18, the tumors were extirpated, weighed, and photographed using a Nikon COOLPIX 990. The images were processed by Preview (MacOS). For TUNEL staining, parts of the tumors were fixed using 4 % paraformaldehyde phosphate buffer solution (Wako), followed by paraffin embedding.
Total RNA templates for cDNA synthesis were isolated from 5 mg tumor tissue homogenate by using NucleoSpinRNAXS (Macherey-Nagel, Düren, Germany). cDNA synthesis was performed using the SuperScript Vilo cDNA Synthesis Kit (Thermo Fisher Scientific Inc.). For the differential RT-PCR products of EGFRvIII or EGFRwt, we used a forward primer sequence corresponding to an element in exon 1, and reverse primer sequence in exon 11 . For common RT-PCR products, a forward primer sequence corresponding to an element in exon 13, and reverse primer sequence corresponding to exon 21 were used . The primers were supplied by Sigma-Aldrich. AccuPower PCR PreMix (Bioneer, Daejeon, Korea) was used in PCR assays and the reaction proceeded under the following conditions: 94 °C for 2 min, 50 °C for 30 min, 30 cycles at 94 °C for 30 s, 60 °C for 60 s, and 72 °C for 30 s. After PCR by iCycler (Bio-Rad Laboratories, Hercules, U.S.A.), the reaction mixture was applied to 2 % agarose gel for electrophoresis with ethidium bromide. The bands were detected using an UV light trans-illuminator.
Cellular caspase activity was assessed by using the Caspase-Glo 3/7 Assay (Promega). The assay was performed according to the manufacturer’s instructions, and the detection of caspase-3/7 cleavage of the luminogenic substrate containing the DEVD sequence was developed using the ARVO SX1420 multi-label counter (Perkin Elmer).
TUNEL (TdT-mediated dUTP nick-end labeling) staining
TUNEL staining of spheres was carried out essentially by method described previously [26, 27]. Briefly, cells (3 × 105 cells/mL) were seeded on 100-mm ultra-low attachment dishes; after 3 days, the spheres were harvested, washed with PBS, and fixed with 4 % paraformaldehyde phosphate buffer solution followed by paraffin embedding. Thin sections from the paraffin block were assayed using the TUNEL method with an apoptosis in situ detection kit (Wako).
In vitro EGFR kinase assay
Direct inhibition of EGFR kinase by Ertredin and AG1478 was investigated with the Kinase Enzyme System (Promega). In this assay, we used the C-terminal kinase recombinant protein of human EGFR expressed in SF9 cells for the enzyme, and poly-(Glu4, Tyr1) for the substrate, as described in the manufacturer’s protocol.
Cells (3 × 105 cells/mL) were seeded on 10 cm ULAS (Ultra-Low attachment surface) dishes and cultured overnight. Sphere formation was observed, and MG132, a proteasome inhibitor, was added to the medium. After 2 h of cultivation, the indicated chemical agents were added. After cultivation for 17 h, the spheres were harvested and washed with PBS, followed by suspension in lysis buffer. Cells in the suspension were disrupted via repeated passages through a 23-gauge syringe. Cellular debris was pelleted by centrifugation at 10,000 × g for 10 min at 4 °C. The protein in the supernatant was quantitated, and a primary antibody was added to 1 mg/mL of cellular protein suspension at a 1/100 dilution. After 1 h of incubation at 0 °C, 20 μL of Protein A/G PLIUS-Agarose (Santa Cruz Biotechnology, Inc. Dallas, USA) was added to each mixture and the mixture was incubated at 4 °C overnight with mixing. The agarose beads were collected by centrifugation at 1000 × g for 30 s at 4 °C and washed 3 times with 1 mL lysis buffer. After the final wash, the beads were resuspended in sample buffer and the suspension was applied to 10 % SDS-PAGE for western immunoblot analysis after boiling for 5 min.
ATP synthesis in mitochondria
Cells under 2D-cell culture conditions (2000) were seeded in 384-well plates with the galactose-supplemented culture medium (10 mM galactose, 0 mM glucose, 0 % serum) and cultured for 1 h. After the cells attached to the bottom of each well, the test substances were added. Cells under 3D-cell culture conditions (20,000) were seeded on in 96-well Clear Black Round Bottom ULAS plates (Corning) and cultured for 4 days. The medium was then changed to galactose-supplemented culture medium after washes; the cells were then cultured for 1 h, followed by the addition of test substances for 2 h. ATP was measured using the Mitochondrial ToxGloAssay (Promega).
The total RNA was isolated from cells by NucleoSpinRNA (Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions. RNA samples were quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, U.S.A.) and the quality was confirmed with an Experion System (Bio-Rad Laboratories). The cRNA was amplified, labeled, and hybridized to a 60 K Agilent (Santa Clara, U.S.A.) 60-mer oligomicroarray, according to the manufacturer’s instructions. All hybridized microarray slides were scanned by an Agilent scanner. Relative hybridization intensities and background hybridization values were calculated using Agilent Feature Extraction Software (126.96.36.199). The raw signal intensities of two samples were log2-transformed and normalized by quantile algorithm with ‘preprocessCore’ library package  on Bioconductor software . To identify up- or down-regulated genes, we calculated Z-scores  and ratios (non-log scaled fold-change) from the normalized signal intensities of each probe for comparison between the control and experimental sample. We then established criteria for the regulated genes: up-regulated genes had a Z-score ≥ 2.0 and ratio ≥ 1.5-fold, down-regulated genes had a Z-score ≤ −2.0 and ratio ≤ 0.66.
Total RNA templates for cDNA synthesis were isolated from approximately 2–3 × 106 cells by using the NucleoSpinRNAII (Macherey-Nagel). cDNA synthesis was performed using the SuperScript Vilo cDNA Synthesis Kit. Quantitative PCR was performed using the SYBR Premix Ex Taq™ II (TAKARA, Kusatsu, Japan) and Thermal Cycler Dice Real Time System (TAKARA). The standard protocol (95 °C for 30 s, for one cycle; 95 °C for 5 s and 60 °C for 30 s, for 40 cycles; 95 °C for 15 s, 60 °C for 30 s, 95 °C for 15 s, for one cycle) was used for all reactions. Relative quantification was carried out with glucuronidase beta (Gusb) as an endogenous control. The primer sets were as follows: GLUT1 (NM_011400.3, forward: TGTGGGCATGTGCTTCCAGTA, reverse: GCCTTTGGTCTCAGGGACTTTG), HK1 (NM_001146100.1, forward: AGAGGCCTAGACCACCTGAATGTAA, reverse: ACTGTTTGGTGCATGATTCTGGAG), HK2 (NM_013820.3, forward: GGCCAACTTCATGGACAAGCTAC, reverse: CCACGCCACTGGACTTGAAC), PFK1 (NM_008826.4, forward: CAGTCCGGTCACAGAACTCAAG, reverse: GCATCAGCCGCAGATTCA), PKM (NM_001253883.1, forward: ATGCCTGGGCTGAGGATGTC, reverse: ACTACACGCATGGTGTTGGTGAA), LDHA (NM_001136069.2, forward: GAACTGGGCACTGACGCAGA, reverse: CCAATGGCCCAGGATGTGTA), PDK1 (NM_172665.5,forward: GGAAGTCCATCTCATCGAAAGCA, reverse: AAAGCCGCCTAGCGTTCTCA), MCT1 (NM_009196.4, forward: GGCTTGGTGACCATTGTGGA, reverse: TGATGCCCATGCCAATGAA), GUSB (NM_010368.1, forward: CTGTGACCGATACGGGATTGTG, reverse: ACCTCTAGGTGGTGCCGAAGTG).
Cells (2 × 106) were seeded in ULAS 6 well plates, and test substances were added after 1 day of culture. After 1 day of treatment, the cells were harvested, and lactate was quantitated by L-Lactate Assay Kit (Abcam).
Sphere formation and growth of cells in 3D-culture induced by EGFRvIII expression
Throughput screening for inhibitors suppressing anchorage-independent growth induced by EGFRvIII
Ertredin derivatives inhibited NIH3T3/EGFRvIII cells in 3D culture
Ertredin (E1 in Fig.3c), its fluoro-analog (E7), and a novel iodo-derivative (E8) named I-Ertredin, were synthesized and used for biological study. All the synthetic compounds demonstrated 3D sphere-inhibitory activity similar to the original Ertredin. With regard to 2D growth, none of the Ertredin derivatives produced cytotoxicity in NIH3T3 cells (Fig. 3c). When the Br located in the phenyl ring was substituted by Cl (E6 in Fig. 3c), or I (E8), 3D inhibitory potency was retained, whereas fluorination (E7) or methylation (E4) decreased the activity, and hydrogenation (E9) removed inhibitory activity completely.
The inhibitory activities of E2, E3, E5, and E10-12 on NIH3T3/EGFRvIII cell growth in 3D-culture was remarkable; these compounds differ from Ertredin (E1) in the amino groups of their phenyl ring. When the secondary amine was changed to a tertiary amine (E13, E14), the inhibitory activity in 3D-culture was no longer observed.
The original compound Ertredin was previously reported to inhibit mDia-1-mediated actin assembly by targeting mDia1 FH2 at IC50 = 2.5 μM in vitro . However, there are no published data showing the effect of the compound on mDia-1 protein on the cell level; the concentrations required for in vitro inhibition of mDia1 activity were very high, in the order of 2 μM. Moreover, replacing 5-Br with Cl or F in this compound was reported to remove the suppression activity on mDia-1  while retaining the inhibitory activity of these compounds on anchorage-independent growth (Fig. 3c). Therefore, the activity of this compound (Ertredin) on mDia1 suppression observed in vitro may be unrelated to its inhibitory activity on anchorage-independent growth for the cells shown here.
Tumor suppression by Ertredin in vivo
Ertredin treatment by intraperitoneal injection at 30 mg/kg body weight/day efficiently suppressed tumorigenicity equal to that of a positive control, gefitinib, which was administered orally at 100 mg/kg/day or 200 mg/kg/day (Fig. 4b). After injection with Ertredin, the mice carrying tumors did not lose body weight, suggesting no clear toxicity to host animals (Fig. 4c). On the other hand, the tumor-bearing mice treated with gefitinib did lose body weight (Fig. 4c). As shown in Additional file 2, I-Ertredin also suppressed the tumorigenic activity of NIH3T3/EGFRvIII cells without remarkable toxicity. In these tumors transplanted with NIH3T3/EGFRvIII or NIH3T3/EGFRwt cells, we confirmed the expression of EGFRvIII or EGFRwt mRNAs using RT-PCR (Additional file 3).
Ertredin induced apoptosis but did not inhibit EGFR kinase
It is known that the EGFRvIII protein is ubiquitinated by Cbl and degraded in proteasome-dependent manner, similar to activated EGFR [35, 36]. Ertredin was found to up-regulate ubiquitination of EGFRvIII via anti-hEGFR immunoprecipitation (Fig. 6c, lane 2), compared to control cells or cells treated with AG1478 (lane 3). The amount of EGFRvIII after Ertredin treatment decreased to one-half of the control, however, ubiquitination of total cellular proteins was unaltered with or without Ertredin (lane 4–5). These results suggest that digestion of EGFRvIII in proteasomes is activated in cells treated with Ertredin, but in a limited manner. Ertredin suppressed 3D cell growth and induced a remarkable level of apoptosis at concentrations less than 0.1 μM; however, only 50 % inhibition of the EGFRvIII protein level was observed even at 1 μM. Thus, targets for Ertredin, other than the ubiquitination and degradation pathway, may exist within EGFRvIII-cells in 3D-culture.
Ertredin suppresses mitochondrial oxidative phosphorylation and cytosolic glycolysis
We carried out further screening in other sources, including 30,000 microorganism metabolites. Through this screening, we found that Piericidin and other Streptomyces metabolites suppressed the anchorage-independent growth of EGFRvIII-overexpressing cells (data not shown). Piericidin is known to be an NADH dehydrogenase inhibitor. Some of other hits also showed potent mitochondrial inhibition. Respiratory chain inhibitors were previously shown to inhibit a 3D culture of T47D breast cancer cells . Therefore, we examined the effect of Ertredin on oxidative phosphorylation. We also investigated the suppressive potency of Rotenone and Antimycin against NIH3T3/EGFRvIII 3D growth, which are well known to be respiratory chain complex I and complex III inhibitors, respectively.
By comparing Fig. 7b–d with e–g, we can see a similarity between the suppression of sphere growth and the decrease in mitochondrial ATP synthesis by Ertredin in NIH3T3/EGFRvIII under 3D conditions. On the other hand, the growth under 2D conditions was not affected by suppression of the mitochondrial ATP synthesis. These results suggest that the inhibition of mitochondrial ATP synthesis is important for the inhibition of growth in NIH3T3/EGFRvIII under 3D conditions.
We also examined the gene expression of glycolytic pathway enzymes under 2D conditions. Surprisingly, neither Ertredin nor Rotenone suppressed the expression of glycolysis-related enzymes at detectable levels (Fig. 8b). Thus, these results indicate that the suppression of the gene expression in glycolysis enzymes with Ertredin occurs specifically under 3D conditions.
Glycolytic enzymes are known to be positively regulated by hypoxia-inducible factor (HIF)-1α; thus, we investigated HIF-1α levels in cells treated with Ertredin under 3D and 2D conditions (Additional file 4). Western blotting indicated that HIF-1α was not decreased in cells treated with Ertredin under 3D conditions. In contrast, Ertredin and Rotenone slightly decreased HIF-1α under 2D conditions. These results suggest that Ertredin suppresses the expression of glycolytic pathway genes independently from HIF-1α under 3D conditions.
We demonstrated that Ertredin suppressed both mitochondrial ATP synthesis and cytosolic glycolysis under 3D conditions. Ertredin may efficiently inhibit total energy metabolism. Both Ertredin and Rotenone suppress mitochondrial ATP synthesis and the gene expression of many glycolysis enzymes in 3D-cultured cells. These results suggest that at least one of the targets of Ertredin within the cell is very similar to that of Rotenone. Ertredin inhibits mitochondrial ATP synthesis under both 2D and 3D conditions but it suppresses cell survival and growth under 3D-culture conditions compared to 2D conditions. A possible explanation is firstly that Ertredin suppresses glycolytic pathway preferentially under 3D conditions (Fig. 8d), and secondly that 3D-sphere growth is more sensitive to mitochondrial inhibition as shown previously (33).
Ertredin was found to be safe and capable of suppressing tumors in mice. The mechanical analysis suggests that Ertredin is multifunctional. Inhibition of glycolysis by Ertredin may suppress the Warburg effect, a common metabolic alteration of most tumor cells. And almost total inhibition of energy metabolism by Ertredin may initiate apoptosis in cancer cells without affecting normal cells. Because Ertredin has these unique characteristics, it could potentially lead to the development of new cancer therapeutic agents. We are currently studying the effect of Ertredin and its derivatives on human tumors.
We introduced a high throughput method in the identification of inhibitors of anchorage-independent growth of NIH3T3/EGFRvIII cells. Over the screening of 30,000 substances, we identified Ertredin that inhibited the growth of allograft tumors in vivo. Ertredin promoted apoptosis in 3D-spheres as well as tumors of mice transplanted with NIH3T3/EGFRvIII cells. Functional characterization revealed that Ertredin suppressed not only mitochondrial oxidative phosphorylation but also cytosolic glycolysis under 3D-culture conditions. In addition, an increase in EGFRvIII ubiquitination and degradation were observed in NIH3T3/EGFRvIII cells treated with Ertredin. Our evidence suggests that the multi-effects of Ertredin induce apoptosis in NIH3T3/EGFRvIII cells under 3D-culture conditions and tumors in vivo.
2D, 2-dimensional; 2DG, 2-Deoxy-D-glucose; 3D, 3-dimensional; DMSO, Dimethyl sulfoxide; EGFR, epidermal growth factor receptor; EGFRvIII, EGFR variant III; EGFRwt, wild-type EGFR; FBS, Fetal bovine serum; HIF, Hypoxia-inducible factors; PBS, phosphate buffered saline; PI3 kinase, Phosphoinositide 3-kinase; PKA, protein kinase A; RT-PCR, reverse transcriptase-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; siRNA, small interfering RNA; TUNEL, TdT-mediated dUTP nick-end labeling; ULAS, Ultra-Low attachment surface.
We wish to acknowledge staff of Department of Intellectual Property and Public Relations for all through this work. We thank to Cell Innovator for performing Microarray analysis.
This work was supported by Microbial Chemistry Research Foundation.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files. Microarray data (GSE 76959) were deposited in Gene Expression Omnibus (GEO).
SA developed the screening system, found the substance, and carried out functional analysis in vitro. CN screened microorganism metabolites, isolated substances, and analyzed mitochondrial function. HA contributed to the chemical synthesis of Ertredin. TK participated in analogue synthesis. YK participated in the chemical synthesis of I-Ertredin. HK contributed to the chemical synthesis of Ertredin for in vivo experiments. TW participated in chemical synthesis design and coordination, and helped draft the manuscript. SO and HI carried out in vivo experiments. MK participated in the design of the study and helped draft the manuscript. MS participated in coordination and helped with the chemical synthesis. AK participated in the design of the study and participated in coordination. MS conceived of the study, participated in its design, and helped draft the manuscript. All authors read and approved the final manuscript.
No additional information is needed about authors.
Conflicts of interest
The authors have no conflicts of interest directly relevant to the content of this article.
Consent to publish
Ethics approval and consent to participate
All animal experiments were approved by the Institutional Committee for Animal Experiments in Institute of Microbial Chemistry and performed in accordance with relevant guidelines and regulations to minimize animal suffering.
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