Gastrointestinal cancer cells treatment with bevacizumab activates a VEGF autoregulatory mechanism involving telomerase catalytic subunit hTERT via PI3K-AKT, HIF-1α and VEGF receptors.
Gastrointestinal cancer cells treatment with bevacizumab activates a VEGF autoregulatory mechanism involving telomerase catalytic subunit hTERT via PI3K-AKT, HIF-1α and VEGF receptors.
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DOI：
10.1371/journal.pone.0179202
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Hilal G
中科院分区：
文献类型：
--
作者：
Mahfouz N;Tahtouh R;Alaaeddine N;El Hajj J;Sarkis R;Hachem R;Raad I;Hilal G
靶向血管生成已被认为是一种有前途的治疗选择的大量恶性肿瘤，包括胃肠道癌症。贝伐珠单抗是一种抗血管内皮生长因子（抗VEGF）用于此目的。然而，治疗效果在很大程度上受到质疑。负责癌细胞永生的端粒酶活性在85-95%的人类癌症中被检测到，并且被认为是VEGF的潜在调节剂。本研究的目的是探讨VEGF和hTERT在胃肠道肿瘤中的相互关系，并探讨细胞对端粒酶和VEGF联合抑制的反应。AGS（胃癌）、Caco-2（结直肠癌）和HepG 2/C3 A（肝细胞癌）用端粒酶抑制剂BIBR-1232（10μM）和考司他丁（10μM）、贝伐单抗（5 ng/ml或100μg/ml的Avastin®）或两种类型抑制剂的组合治疗。RT-PCR检测VEGF、hTERT mRNA表达及端粒酶活性。通过ELISA定量VEGF水平。使用hTERT siRNA敲低端粒酶，并使用表达野生型hTERT（hTERT-WT）或显性阴性hTERT（hTERT-DN）的构建体在端粒酶阴性细胞系Saos-2（骨肉瘤）中过表达hTERT。使用ECMatrix™（EMD Millipore）评估HUVEC的管形成。我们的研究结果表明，端粒酶调节VEGF的表达和分泌，通过其催化亚基hTERT在AGS，Caco 2，和HepG 2/C3 A，独立于其催化活性。有趣的是，贝伐单抗（100μg/ml）抑制VEGF使AGS中hTERT表达增加42.3%，Caco 2中增加94.1%，HepG 2/C3 A中增加52.5%，并使AGS中端粒酶活性增加30倍，Caco 2中增加10.3倍，HepG 2/C3 A中增加8倍。进一步的研究表明，VEGF上调hTERT表达的机制涉及PI 3 K/AKT/mTOR通路和HIF-1α。此外，贝伐单抗治疗通过hTERT增加癌细胞和人脐静脉内皮细胞（HUVEC）中的VEGFR 1和VEGFR 2表达。因此，贝伐单抗与端粒酶抑制剂的组合降低了癌细胞的VEGF表达和分泌，抑制了VEGFR 1和VEGFR 2上调，并减少了HUVEC的管形成。综上所述，我们的结果表明，贝伐单抗治疗激活了涉及hTERT和VEGF受体的VEGF自身调节机制，并且抑制该途径可以改善肿瘤细胞对抗VEGF治疗的反应。
Targeting 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. AGS (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). Our 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. Taken 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.