BackgroundTargeting angiogenesis has been considered a promising treatment of choice for a large number of malignancies, including gastrointestinal cancers. Bevacizumab is an anti-vascular endothelial growth factor (anti-VEGF) being used for this purpose. However, treatment efficacy is largely questioned. Telomerase activity, responsible for cancer cell immortality, is detected in 85–95% of human cancers and is considered a potential regulator of VEGF. The aim of our study was to investigate the interrelationship between VEGF and hTERT in gastrointestinal cancers and to explore cell response to a combined inhibition of telomerase and VEGF.MethodsAGS (gastric cancer), Caco-2 (colorectal cancer) and HepG2/C3A (hepatocellular carcinoma), were treated with telomerase inhibitors BIBR-1232 (10μM) and costunolide (10μM), with bevacizumab (Avastin® at 5 ng/ml or 100μg/ml) or with a combination of both types of inhibitors. VEGF and hTERT mRNA levels, and telomerase activity were detected by RT-PCR. VEGF levels were quantified by ELISA. Telomerase was knocked down using hTERT siRNA and hTERT was overexpressed in the telomerase negative cell line, Saos-2 (osteosarcoma), using constructs expressing either wild type hTERT (hTERT-WT) or dominant negative hTERT (hTERT-DN). Tube formation by HUVECs was assessed using ECMatrix™ (EMD Millipore).ResultsOur results showed that telomerase regulates VEGF expression and secretion through its catalytic subunit hTERT in AGS, Caco2, and HepG2/C3A, independent of its catalytic activity. Interestingly, VEGF inhibition with bevacizumab (100μg/ml) increased hTERT expression 42.3% in AGS, 94.1% in Caco2, and 52.5% in HepG2/C3A, and increased telomerase activity 30-fold in AGS, 10.3-fold in Caco2 and 8-fold in HepG2/C3A. A further investigation showed that VEGF upregulates hTERT expression in a mechanism that implicates the PI3K/AKT/mTOR pathway and HIF-1α. Moreover, bevacizumab treatment increased VEGFR1 and VEGFR2 expression in cancer cells and human umbilical vein endothelial cells (HUVECs) through hTERT. Thus, the combination of bevacizumab with telomerase inhibitors decreased VEGF expression and secretion by cancer cells, inhibited VEGFR1 and VEGFR2 upregulation, and reduced tube formation by HUVECs.ConclusionsTaken together, our results suggest that bevacizumab treatment activates a VEGF autoregulatory mechanism involving hTERT and VEGF receptors and that an inhibition of this pathway could improve tumor cell response to anti-VEGF treatment.

Alpha-fetoprotein (AFP) is a diagnostic marker for hepatocellular carcinoma (HCC). A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. Apoptosis was determined through the Annexin V assay using flow cytometry. The concentrations of AFP were measured using ELISA kits. The AFP mRNA expression was evaluated using RT-PCR, and cell migration was evaluated using a Boyden chamber assay. The in vivo effect of costunolide on AFP production was tested in NSG mice. Telomerase inhibition by costunolide and BIBR 1532 at 5 and 10 μM decreased AFP mRNA expression and protein secretion by HepG2/C3A cells. The same pattern was obtained with cells treated with hTERT siRNA. This treatment exhibited no apoptotic effect. The AFP mRNA expression and protein secretion by PLC/PRF/5 was decreased after treatment with BIBR1532 at 10 μM. In contrast, no effect was obtained for PLC/PRF/5 cells treated with costunolide at 5 or 10 μM. Inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP concentration. In contrast, the MAPK/ERK pathway appeared to not be involved in HepG2/C3A cells, whereas ERK inhibition decreased the AFP concentration in PLC/PRF/5 cells. Modulation of the AFP concentration was also obtained after the inhibition or activation of PKC. Costunolide (30 mg/kg) significantly decreased the AFP serum concentration of NSG mice bearing HepG2/C3A cells. Both the inhibition of telomerase and the inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP production of HepG2/C3A and PLC/PRF/5 cells, suggesting a relationship between telomerase and AFP expression through the PI3K/Akt/mTOR pathway

Backgroundp53 is a tumor suppressor and key regulator of glycolysis in cancer cells, however highly mutated in tumors. In ovarian cancer, studies concerning p53 mutations focus on the DNA binding domain since the majority of hotspot mutations affects this region. Yet, mutations in other regions such as the proline rich domain may also affect the protein’s expression and activity. The aim of this study is to investigate the effect of various positions of mutations in TP53 gene on glycolysis, apoptosis and transcription of p53 target genes.MethodsMutations frequency and their effect on p53 expression were assessed by PCR-SSCP, sequencing and immunohistochemistry on 30 ovarian cancer biopsies. Six tumors were cultured, as well as SK-OV-3, OVCAR-3 and Igrov-1. SK-OV-3 cells were transfected with 2 TP53 mutants. p53 transcriptional activity was assayed by qPCR, apoptosis by flow cytometry and glycolysis by glucose and lactate measurements, with quantification of glycolytic enzymes expression.ResultsOur results showed a high frequency of the P72R mutant, associated with p53 overexpression in the ovarian biopsies. However, P72R mutant cells showed similar apoptosis and glycolysis as WT cells. DNA binding domain mutations decreased the transcriptional activity of the protein and increased glucose consumption and lactate production.ConclusionDespite the overexpression of the P72R mutated protein in the biopsies, it showed a similar apoptotic activity and glucose regulation ability as WT p53. Knowing that p53 expression status is used for chemotherapeutic approaches and prognosis in ovarian cancer, the results obtained highlight the importance of locating TP53 mutations.Electronic supplementary materialThe online version of this article (10.1186/s12935-018-0514-2) contains supplementary material, which is available to authorized users.

Aerobic glycolysis, known as the "Warburg effect”, is one of several hallmarks of cancer cells. The conversion of phosphoenolpyruvate (PEP) to pyruvate can be down regulated by the re-expression of the embryonic isoform 2 of pyruvate kinase (PKM2). This mechanism allows the accumulation of glycolytic intermediates for the biosynthesis of macromolecules, such as proteins, lipids and nucleic acids. PKM2 is favored by the well-known PI3K/Akt/mTOR proliferative pathway. This pathway is induced by high glucose levels, and the mTOR kinase is the central activator of the Warburg effect. In this study, we investigated the role of glucose restriction (GR) and mTOR inhibition  in reversing the Warburg effect in MDA-MB 231 and MCF-7 breast cancer cell lines. PKM2 expression was measured by western blot. Lactate production by cells was determined by a colorimetric assay. The concentration of glucose in the supernatant of cells was measured using the Trinder method. ATP level  was evaluated by using a Colorimetric/Fluorometric ATP Assay Kit. Our results showed that MDA-MB 231 cells increased glucose consumption when the glucose concentration was 0 g/L (P <0.01). In MCF-7 cells, glucose deprivation reduced lactate secretion by 80% (P =0.0001) but tripled glucose consumption (P = 0.0041). ATP concentration increased approximately when MCF-7 cells were deprived of glucose (P = 0.02). GSK1059615 does not significantly modulate lactate secretion and glucose uptake in both cell lines. Glucose restriction contribute to the reduction of the Warburg effect through mTOR inhibition and regulation of PKM2 kinases.

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