背景:补骨脂素加紫外线A(PUVA)光化学疗法是一种用于炎性和肿瘤性皮肤病的联合治疗方法,例如真菌病(MF),皮肤T细胞淋巴瘤(CTCL)最常见的类型。然而,30%的MF患者对PUVA没有足够的反应,需要更积极的治疗。
目的:该项目的目的是研究抑制共济失调性毛细血管扩张症和Rad3相关激酶(ATR)是否可以增强光疗的功效。
方法:CTCL细胞系(MyLa2000、SeAx和Mac2a)用作体外细胞模型。ATR和Chk1被小分子拮抗剂VE-821、VE-822或Chir-124或小干扰RNA(siRNA)抑制。通过流式细胞术评估细胞周期和活力。
结果:ATR和Chk1的小分子抑制剂可有效地使所有细胞系对PUVA敏感,重要的是,也是UVA,其本身不会引起凋亡反应。VE-821/2阻断了ATR通路的激活,并从UVA和PUVA引起的G2/M阻滞中释放细胞,但不影响其他化疗药物(依托泊苷,吉西他滨,多柔比星)或过氧化氢。用siRNA敲除ATR和Chk1也阻断ATR途径并从G2/M阻断释放细胞,但如使用小分子抑制剂所观察到的,不使细胞对UVA敏感。后者表明,VE-821/2或Chir-124与UVA之间的协同作用不仅是由ATR激酶的特异性阻断引起的,而且是不依赖ATR的光敏作用。通过在UVA照射之前和之后施用VE-821/2或Chir-124进一步验证了这一假设。以及比较它们与其他ATR和Chk1抑制剂(AZD6738和MK8776)的活性。我们发现只有VE-821/2和Chir-124激酶抑制剂与UVA有协同作用,并且仅在用UVA治疗之前应用。
结论:小分子ATR和Chk1抑制剂可有效地使淋巴瘤细胞对UVA辐射敏感,并诱导显著的凋亡反应。有趣的是,这种作用是由于这些化合物的双重(激酶抑制和光敏)作用模式。
BACKGROUND: Psoralen plus ultraviolet A (PUVA) photochemotherapy is a combination treatment used for inflammatory and neoplastic skin diseases such as mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL). However, 30% of MF patients do not respond sufficiently to PUVA and require more aggressive therapies.
OBJECTIVE: The aim of this project was to investigate whether inhibition of Ataxia Telangiectasia and Rad3 related kinase (ATR) may enhance efficacy of phototherapy.
METHODS: CTCL cell lines (MyLa2000, SeAx and Mac2a) served as in vitro cell models. ATR and Chk1 were inhibited by small molecule antagonists VE-821, VE-822 or Chir-124, or by small interfering RNAs (siRNAs). Cell cycle and viability were assessed by flow cytometry.
RESULTS: Small molecule inhibitors of ATR and Chk1 potently sensitized all cell lines to PUVA and, importantly, also to UVA, which by itself did not cause apoptotic response. VE-821/2 blocked ATR pathway activation and released the cells from the G2/M block caused by UVA and PUVA, but did not affect apoptosis caused by other chemotherapeutics (etoposide, gemcitabine, doxorubicine) or by hydrogen peroxide. Knockdown of ATR and Chk1 with siRNA also blocked the ATR pathway and released the cells from G2/M block but did not sensitize the cells to UVA as observed with the small molecule inhibitors. The latter suggested that the synergism between VE-821/2 or Chir-124 and UVA was not solely caused by specific blocking of ATR kinase but also ATR-independent photosensitization. This hypothesis was further verified by administrating VE-821/2 or Chir-124 before and after UVA irradiation, as well as comparing their activity with other ATR and Chk1 inhibitors (AZD6738 and MK8776). We found that only VE-821/2 and Chir-124 kinase inhibitors had synergistic effect with UVA, and only if applied before treatment with UVA.
CONCLUSIONS: Small molecule ATR and Chk1 inhibitors potently sensitize lymphoma cells to UVA radiation and induce a prominent apoptotic response. Interestingly, this effect is due to the dual (kinase inhibiting and photosensitizing) mode of action of these compounds.