化疗是急性髓系白血病（AML）的主要治疗选择，但白血病细胞对化疗药物的获得性耐药往往导致AML治疗困难和疾病复发。高降钙素受体样（CALCRL）表达与 AML 患者较差的预后密切相关。因此，本研究通过在具有低 CALCRL 表达的 AML 细胞系 HL-60 和 Molm-13 中进行 CALCRL 过表达构建体来进行。结果表明，在HL-60和Molm-13中过表达CALCRL可以赋予AML细胞耐药特性，并减少柔红霉素（DNR）等引起的DNA损伤和细胞周期G0/G1期阻断效应。 CALCRL 的过度表达也减少了 DNR 诱导的细胞凋亡。从机制上讲，癌症临床研究数据库分析了 AML 患者中 XRCC5 和 CALCRL 之间的显着正相关性。因此，结合RT-PCR和Western blot研究进一步证实，过表达CALCRL后XRCC5和PDK1基因和蛋白的表达水平显着上调。相反，XRCC5/PDK1 下游通路 AKT/PKCε 蛋白的磷酸化水平显着上调。在反应研究中，用 XRCC5 siRNA 转染过表达的 CALCRL 细胞可显着上调 AML 对 DNR 的药物敏感性。下游通路PDK1蛋白和AKT/PKCε磷酸化蛋白的表达水平受到明显抑制，凋亡相关蛋白Bax和cleaved caspase-3的表达上调。动物实验表明，裸鼠过表达CALCRL后，DNR对HL-60细胞生长和骨髓侵袭次数的抑制作用明显逆转。然而，XCRR5 shRNA慢病毒感染CALCRL过表达的HL-60细胞会减弱CALCRL过表达的作用，并上调DNR诱导的凋亡相关蛋白的表达。本研究为AML患者CALCRL高表达与化疗预后不良的关系提供了初步解释。为后续研究中DNR结合分子靶点的精准治疗提供了更多的实验基础。版权所有©2023 Wolters Kluwer Health, Inc.保留所有权利。

Chemotherapy is the main treatment option for acute myeloid leukemia (AML), but acquired resistance of leukemic cells to chemotherapeutic agents often leads to difficulties in AML treatment and disease relapse. High calcitonin receptor-like (CALCRL) expression is closely associated with poorer prognosis in AML patients. Therefore, this study was performed by performing CALCRL overexpression constructs in AML cell lines HL-60 and Molm-13 with low CALCRL expression. The results showed that overexpression of CALCRL in HL-60 and Molm-13 could confer resistance properties to AML cells and reduce the DNA damage and cell cycle G0/G1 phase blocking effects caused by daunorubicin (DNR) and others. Overexpression of CALCRL also reduced DNR-induced apoptosis. Mechanistically, the Cancer Clinical Research Database analyzed a significant positive correlation between XRCC5 and CALCRL in AML patients. Therefore, the combination of RT-PCR and Western blot studies further confirmed that the expression levels of XRCC5 and PDK1 genes and proteins were significantly upregulated after overexpression of CALCRL. In contrast, the phosphorylation levels of AKT/ PKCε protein, a downstream pathway of XRCC5/PDK1, were significantly upregulated. In the response study, transfection of overexpressed CALCRL cells with XRCC5 siRNA significantly upregulated the drug sensitivity of AML to DNR. The expression levels of PDK1 protein and AKT/PKCε phosphorylated protein in the downstream pathway were inhibited considerably, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were upregulated. Animal experiments showed that the inhibitory effect of DNR on the growth of HL-60 cells and the number of bone marrow invasions were significantly reversed after overexpression of CALCRL in nude mice. However, infection of XCRR5 shRNA lentivirus in HL-60 cells with CALCRL overexpression attenuated the effect of CALCRL overexpression and upregulated the expression of apoptosis-related proteins induced by DNR. This study provides a preliminary explanation for the relationship between high CALCRL expression and poor prognosis of chemotherapy in AML patients. It offers a more experimental basis for DNR combined with molecular targets for precise treatment in subsequent studies.Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.