Morgan Deri, Berggren Kiersten L, Millington Grace, Smith Hanna, Spiess Colby, Hixon Michael, Woolbright Benjamin L, Taylor John A, Kimple Randall J, Chen Ronald, Shen Xinglei, Gan Gregory N
Department of Radiation Oncology, University of Kansas Medical Center, Kansas City, KS, USA.
The University of New Mexico, School of Medicine, Albuquerque, NM, USA.
World J Oncol. 2024 Dec;15(6):871-881. doi: 10.14740/wjon1945. Epub 2024 Dec 11.
Bladder cancer patients unable to receive cystectomy or who choose to pursue organ-sparing approach are managed with definitive (chemo)radiotherapy. However, this standard of care has not evolved in decades and disease recurrence and survival outcomes remain poor. Identifying novel therapies to combine with radiotherapy (RT) is therefore paramount to improve overall patient outcomes and survival. One approach is to find cellular mechanisms that can be targeted to increase the radiosensitivity of bladder cancer. The stress-activated kinase directly downstream from p38 mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase activated protein kinase 2 (MAPKAPK2 or MK2), has been shown to enhance cancer-mediated inflammation, mesenchymal gene expression, and tumor growth. Here we examined the impact that MK2 knockdown (KD) has on bladder cancer cell radiosensitivity.
We utilized short hairpin RNA (shRNA) KD of MK2 using lentiviral transfection in the bladder cancer cell lines, T24 and HTB9. We compared the growth of KD cells to wild type using colony formation assays, proliferation assays and cell counts to determine differences in cell growth. Apoptosis was examined by annexin-based flow cytometry and western blots. Flow cytometry was also used for cell cycle analysis.
KD clones showed a greater than 90% inhibition of MK2 expression as determined by western blot. Clonogenic assays exhibited an increase in radiosensitivity among the MK2 KD bladder cancer cells. These data were supported with proliferation assays that displayed a greater reduction in cell number following RT in MK2 KD bladder cancer cells. Annexin V binding in bladder cancer cells suggested increased apoptosis in MK2 KD cells. This was confirmed by comparing the amount of cleaved caspase products for the caspases 3 and 8 to scrambled control (SCR), and the release of cytochrome C into the cytosol. Both cell types showed disruptions in the cell cycle but at different points in the cycle.
These results show that MK2 controls irradiation-induced apoptosis in bladder cancer cells.
无法接受膀胱切除术或选择保留器官方法的膀胱癌患者采用根治性（化疗）放疗进行治疗。然而，这种治疗标准几十年来一直没有进展，疾病复发和生存结果仍然很差。因此，确定与放疗（RT）联合的新疗法对于改善患者总体预后和生存率至关重要。一种方法是找到可靶向的细胞机制以增加膀胱癌的放射敏感性。p38丝裂原活化蛋白激酶（MAPK）直接下游的应激激活激酶，丝裂原活化蛋白激酶激活的蛋白激酶2（MAPKAPK2或MK2），已被证明可增强癌症介导的炎症、间充质基因表达和肿瘤生长。在此，我们研究了MK2基因敲低（KD）对膀胱癌细胞放射敏感性的影响。
我们使用慢病毒转染在膀胱癌细胞系T24和HTB9中利用短发夹RNA（shRNA）敲低MK2。我们使用集落形成试验、增殖试验和细胞计数比较KD细胞与野生型细胞的生长情况，以确定细胞生长的差异。通过基于膜联蛋白的流式细胞术和蛋白质印迹法检测细胞凋亡。流式细胞术还用于细胞周期分析。
通过蛋白质印迹法测定，KD克隆显示MK2表达抑制超过90%。克隆形成试验显示MK2基因敲低的膀胱癌细胞放射敏感性增加。增殖试验也支持这些数据，该试验显示MK2基因敲低的膀胱癌细胞在放疗后细胞数量减少更多。膀胱癌细胞中膜联蛋白V结合表明MK2基因敲低的细胞凋亡增加。通过比较半胱天冬酶3和8的裂解产物与乱序对照（SCR）的量以及细胞色素C释放到细胞质中的情况，证实了这一点。两种细胞类型在细胞周期中均显示出紊乱，但处于周期的不同点。
这些结果表明MK2控制膀胱癌细胞中辐射诱导的细胞凋亡。
