Zhang Yajing, Feng Yangchun, Sun Xiaojie
Department of Laboratory Affiliated Cancer Hospital of Xinjiang Medical University Urumqi Xinjiang 830011 China.
Department of Blood Transfusion Affiliated Cancer Hospital of Xinjiang Medical University Urumqi Xinjiang 830011 China.
Chronic Dis Transl Med. 2022 Mar 31;8(2):124-133. doi: 10.1002/cdt3.12. eCollection 2022 Jun.
As erythropoietin (EPO) has been used to treat anemia in cancer patients, negative controversy has continued. Unfortunately, its effects on non-small-cell lung carcinoma (NSCLC) cell lines are uncertain and the phenomenon of inducing immune escape of tumor cells remains to be explored. This study aimed to provide an important basis for the application of exogenous EPO in the treatment of tumor-associated anemia.
Cells were cultured in 1% O, 5% CO, and 94% N to simulate a hypoxic environment of the tumor. A549 cell line (lower expression EPOR) and NCI-H838 cell line (higher expression EPOR) were treated with 2 and 8 U/ml recombinant human EPO (rhEPO). CCK-8 method was used to determine the logarithmic growth phase of the cells and to detect cell proliferation. The expression levels of VEGF, HIF-1α, and PD-L1 were determined by western blot. One-way ANOVA was used for statistical analysis between groups, with < 0.05 indicating a significant difference.
Hypoxia itself could decrease the survival rate of NSCLC cells. Under the hypoxic condition, rhEPO induced tumor cells proliferation, especially in the NCI-H838 cell line, where 2 U/ml rhEPO increased the total number of surviving cells (Hypoxia + rhEPO 2 U/ml vs. Hypoxia, < 0.05). Western blot analysis showed that hypoxia upregulated the expression of VEGF, HIF-1α, and PD-L1 in NSCLC cell lines (Normoxia vs. Hypoxia, < 0.05), but may not be dependent on the expression levels of EPOR. RhEPO decreased the expression levels of VEGF and HIF-1α. In the A549 cell line, it depended on the concentration of rhEPO and was particularly obvious in HIF-1α (Hypoxia vs. Hypoxia + rhEPO 2 U/ml vs. Hypoxia + rhEPO 8 U/ml, < 0.05). A low concentration of rhEPO may not reduce VEGF expression. In the NCI-H838 cell line, the effect of rhEPO on VEGF was more obvious, but it may be independent of rhEPO concentrations. The downregulation of PD-L1 expression by rhEPO was only presented in the A549 cell line and required higher rhEPO concentrations (Hypoxia + rhEPO 8 U/ml vs. Hypoxia&Hypoxia + rhEPO 2 U/ml, < 0.05).
The effect of prolonged high concentrations of rhEPO under hypoxic conditions resulted in accelerated cells proliferation of non-small-cell lung cancer and was independent of EPOR expression levels on the cell lines surface. Hypoxia resulted in increased expression of VEGF, HIF-1α, and PD-L1 on the NSCLC cell lines. Under normoxic conditions, rhEPO did not affect the expression of VEGF, HIF-1α, and PD-L1; but under hypoxic conditions, the application of rhEPO reduced the expression of VEGF, HIF-1α, and PD-L1, producing an impact on the biological behavior of tumor cells.
由于促红细胞生成素（EPO）已被用于治疗癌症患者的贫血，负面争议一直存在。不幸的是，其对非小细胞肺癌（NSCLC）细胞系的影响尚不确定，肿瘤细胞诱导免疫逃逸的现象仍有待探索。本研究旨在为外源性EPO在肿瘤相关性贫血治疗中的应用提供重要依据。
将细胞置于1%氧气、5%二氧化碳和94%氮气的环境中培养，以模拟肿瘤的缺氧环境。用2和8 U/ml重组人促红细胞生成素（rhEPO）处理A549细胞系（EPOR表达较低）和NCI-H838细胞系（EPOR表达较高）。采用CCK-8法测定细胞的对数生长期并检测细胞增殖。通过蛋白质免疫印迹法测定VEGF、HIF-1α和PD-L1的表达水平。采用单因素方差分析进行组间统计分析，P<0.05表示差异有统计学意义。
缺氧本身可降低NSCLC细胞的存活率。在缺氧条件下，rhEPO诱导肿瘤细胞增殖，尤其是在NCI-H838细胞系中，2 U/ml rhEPO可增加存活细胞总数（缺氧+rhEPO 2 U/ml与缺氧组相比，P<0.05）。蛋白质免疫印迹分析表明，缺氧上调了NSCLC细胞系中VEGF、HIF-1α和PD-L1的表达（常氧与缺氧组相比，P<0.05），但可能不依赖于EPOR的表达水平。rhEPO降低了VEGF和HIF-1α的表达水平。在A549细胞系中，这取决于rhEPO的浓度，在HIF-1α中尤为明显（缺氧组与缺氧+rhEPO 2 U/ml组与缺氧+rhEPO 8 U/ml组相比，P<0.05）。低浓度的rhEPO可能不会降低VEGF的表达。在NCI-H838细胞系中，rhEPO对VEGF的影响更明显，但可能与rhEPO浓度无关。rhEPO对PD-L1表达的下调仅出现在A549细胞系中，且需要更高的rhEPO浓度（缺氧+rhEPO 8 U/ml与缺氧组及缺氧+rhEPO 2 U/ml组相比，P<0.05）。
缺氧条件下长时间高浓度rhEPO导致非小细胞肺癌细胞增殖加速，且与细胞系表面EPOR表达水平无关。缺氧导致NSCLC细胞系中VEGF、HIF-1α和PD-L1表达增加。在常氧条件下，rhEPO不影响VEGF、HIF-1α和PD-L1的表达；但在缺氧条件下，rhEPO的应用降低了VEGF、HIF-1α和PD-L1的表达，对肿瘤细胞的生物学行为产生影响。
