Department of Laboratory Medicine and Li Ka Shing Knowledge Institute, St Michael's Hospital, Toronto, Canada.
PLoS One. 2010 Nov 3;5(11):e13831. doi: 10.1371/journal.pone.0013831.
Cancer stem cells (CSCs) have increased resistance to cancer chemotherapy. They can be enriched as drug-surviving CSCs (D-CSCs) by growth with chemotherapeutic drugs, and/or by sorting of cells expressing CSC markers such as aldehyde dehydrogenase-1 (ALDH). CSCs form colonies in agar, mammospheres in low-adherence cultures, and tumors following xenotransplantation in Scid mice. We hypothesized that tranilast, a non-toxic orally active drug with anti-cancer activities, would inhibit breast CSCs.
METHODOLOGY/FINDINGS: We examined breast cancer cell lines or D-CSCs generated by growth of these cells with mitoxantrone. Tranilast inhibited colony formation, mammosphere formation and stem cell marker expression. Mitoxantrone-selected cells were enriched for CSCs expressing stem cell markers ALDH, c-kit, Oct-4, and ABCG2, and efficient at forming mammospheres. Tranilast markedly inhibited mammosphere formation by D-CSCs and dissociated formed mammospheres, at pharmacologically relevant concentrations. It was effective against D-CSCs of both HER-2+ and triple-negative cell lines. Tranilast was also effective in vivo, since it prevented lung metastasis in mice injected i.v. with triple-negative (MDA-MB-231) mitoxantrone-selected cells. The molecular targets of tranilast in cancer have been unknown, but here we demonstrate it is an aryl hydrocarbon receptor (AHR) agonist and this plays a key role. AHR is a transcription factor activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic hydrocarbons and other ligands. Tranilast induced translocation of the AHR to the nucleus and stimulated CYP1A1 expression (a marker of AHR activation). It inhibited binding of the AHR to CDK4, which has been linked to cell-cycle arrest. D-CSCs expressed higher levels of the AHR than other cells. Knockdown of the AHR with siRNA, or blockade with an AHR antagonist, entirely abrogated the anti-proliferative and anti-mammosphere activity of tranilast. Thus, the anti-cancer effects of tranilast are AHR dependent.
CONCLUSION/SIGNIFICANCE: We show that tranilast is an AHR agonist with inhibitory effects on breast CSCs. It is effective against CSCs of triple-negative breast cancer cells selected for anti-cancer drug resistance. These results suggest it might find applications in the treatment of breast cancer.
癌症干细胞（CSCs）对癌症化疗有较强的抵抗力。它们可以通过与化疗药物一起生长而被富集为耐药性癌症干细胞（D-CSCs），或者通过分选表达 CSC 标志物（如乙醛脱氢酶-1（ALDH）的细胞来进行分选。CSCs 在琼脂中形成集落，在低粘附培养物中形成类乳腺球体，在 Scid 小鼠异种移植后形成肿瘤。我们假设曲尼司特是一种具有抗癌活性的非毒性口服药物，能够抑制乳腺癌 CSCs。
方法/结果：我们检测了乳腺癌细胞系或通过米托蒽醌培养这些细胞生成的 D-CSCs。曲尼司特抑制集落形成、类乳腺球体形成和干细胞标志物表达。米托蒽醌选择的细胞富含表达干细胞标志物 ALDH、c-kit、Oct-4 和 ABCG2 的 CSCs，并且能够有效地形成类乳腺球体。曲尼司特在药理学相关浓度下显著抑制 D-CSCs 的类乳腺球体形成，并使已形成的类乳腺球体解离。它对 HER-2+和三阴性细胞系的 D-CSCs 均有效。曲尼司特在体内也有效，因为它可以防止静脉注射米托蒽醌选择的三阴性（MDA-MB-231）细胞的小鼠发生肺转移。曲尼司特在癌症中的分子靶点一直不清楚，但在这里我们证明它是一种芳香烃受体（AHR）激动剂，这是关键所在。AHR 是一种转录因子，可被 2,3,7,8-四氯二苯并-p-二恶英（TCDD）、多环芳烃和其他配体激活。曲尼司特诱导 AHR 向核内易位，并刺激 CYP1A1 表达（AHR 激活的标志物）。它抑制 AHR 与 CDK4 的结合，这与细胞周期停滞有关。D-CSCs 表达的 AHR 水平高于其他细胞。用 siRNA 敲低 AHR 或用 AHR 拮抗剂阻断，完全消除了曲尼司特的抗增殖和抗类乳腺球体活性。因此，曲尼司特的抗癌作用依赖于 AHR。
结论/意义：我们表明，曲尼司特是一种 AHR 激动剂，对乳腺癌 CSCs 具有抑制作用。它对用于抗癌药物耐药性选择的三阴性乳腺癌细胞的 CSCs 有效。这些结果表明，它可能在乳腺癌的治疗中得到应用。
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