保留膀胱的放疗在膀胱癌（BCa）患者中具有较高的接受度，但治疗过程中出现继发耐受（ARR）是临床放疗失败的重要原因之一。经常报道 COX-2 在各种癌症中高表达并与放射抗性相关。本研究探讨了蒲公英甾醇（Tara）作为放射增敏剂的可行性，并探讨了Tara对COX-2的靶向作用及其潜在机制。采用MTT法检测了Tara对BCa细胞的毒性，并对细胞进行了毒性试验。通过克隆形成测定比较对 IR 或 Tara  IR 的反应。接下来，采用小RNA干扰系统（siRNA）降低BCa细胞中内源性COX-2的表达，并分别使用微球形成和transwell小室实验研究不同处理下BCa细胞的干细胞样特征和运动能力。同时通过qRT-PCR、western blot、ELISA等方法测定一系列炎症相关分子和干细胞特征分子的表达量。在体内研究中，BCa 细胞被皮下注射到每只雄性小鼠的右侧腹部。然后将这些小鼠分组并接受不同的处理：Tara、IR、IR  Tara 和未处理的对照组。每两天测量每个肿瘤的体积，并通过免疫组织化学（IHC）染色检测靶蛋白。结果表明，由于COX-2敲低或Tara处理，COX-2下降可以大大增强BCa细胞的放射敏感性并显着降低其迁移、侵袭和微球形成能力，同时JAK2、phos-STAT3、MMP2和MMP9表达减少。然而，Tara不能进一步降低COX-2缺陷型BCa细胞中上述细胞分子的表达。相应地，Tara治疗不能进一步增强siCOX-2 BCa细胞对IR的反应。我们的数据支持Tara可以通过靶向COX-2/PGE2来提高BCa细胞的放射敏感性。其机制可能涉及调节STAT3磷酸化、DNA损伤反应蛋白激活以及MMP2/MMP9的表达。

Radiotherapy with bladder preservation is highly acceptable among patients bearing bladder cancer (BCa), but the occurrence of secondary tolerance (ARR) during treatment is one of the important reasons for the failure of clinical radiotherapy. COX-2 has been frequently reported to be highly expressed and associated with radio-resistance in various cancers. In this study, the feasibility of Taraxasterol (Tara) as a radiosensitizer was investigated, and the target effect of Tara on COX-2 and its underlying mechanism were explored.The toxicity of Tara toward BCa cells was detected with the MTT method and cells in response to IR or Tara + IR were compared by clone formation assay. Next, a small RNA interference system (siRNA) was employed to decrease endogenous COX-2 expression in BCa cells, and the stem cell-like features and motion abilities of BCa cells under different treatments were investigated using microsphere formation and transwell chamber assay, respectively. Meanwhile, the expression of a series of inflammation-related molecules and stem cell characteristic molecules was determined by qRT-PCR, western blot and ELISA method. In vivo studies, BCa cells were subcutaneously injected into the right flank of each male mouse. Those mice were then grouped and exposed to different treatment: Tara, IR, IR + Tara and untreated control. The volumes of each tumor were measured every two days and target proteins were detected with immunohistochemical (IHC) staining.The results show that COX-2 decline, due to COX-2 knocking-down or Tara treatment, could greatly enhance BCa cells' radiosensitivity and significantly decrease their migration, invasion and microsphere formation abilities, companied with the reduce of JAK2, phos-STAT3, MMP2 and MMP9 expression. However, Tara could not further reduce the expression of an above molecule of cells in COX-2-deficient BCa cells. Correspondingly, Tara treatment could not further enhance those siCOX-2 BCa cells response to IR.Our data support that Tara can improve the radiosensitivity of BCa cells by targeting COX-2/PGE2. The mechanism may involve regulating STAT3 phosphorylation, DNA damage response protein activation, and expression of MMP2/MMP9.