Mechanism of Activity of Lonidamine

描述（由申请人提供）：黑色素瘤仍然是最致命的人类癌症之一，没有有效的方法来治疗这种播散性疾病。虽然靶向治疗已经显示出一定的疗效，但迄今为止，它们被证明是非治愈性的，这表明需要其他形式的全身治疗。这项建议是长期努力的一部分，以开发系统治疗黑色素瘤的方法。我们最近已经表明，氯尼达明（LND，100毫克/公斤）的管理，大大提高了美法仑的活性，现在已经扩展到多柔比星治疗这一发现。作为最终临床转化的关键一步，我们现在将研究LND活性的详细机制。我们和其他人已经表明，LND产生肿瘤特异性细胞内酸化和肿瘤能量状态（NTP/Pi）的大幅下降。已经提出了许多LND的机制：1）抑制己糖激酶II，2）干扰线粒体电子传递，3）抑制细胞乳酸盐通过单羧酸转运蛋白（MCT）输出。我们提出了第四种机制来解释用LND治疗后肿瘤的生物能量下降：4）抑制推定的线粒体丙酮酸载体（MPC），这将耗尽肿瘤细胞的氧化磷酸化的关键底物，并诱导糖酵解代谢的增加以补偿减少的氧化磷酸化ATP产生。我们最近验证了一种新的扩展同位素分析称为cumomer分析，并将其应用于灌注DB 1黑色素瘤细胞。我们认为这是第一个经过验证的肿瘤能量代谢代谢网络模型。我们提出了以下假设：1）选择性肿瘤酸化是由抑制MCT 1引起的，2）肿瘤去酸化是由抑制MPC引起的。在本提案的目标1中，我们建议使用13 C磁共振光谱（MRS）和液相色谱-质谱（LC-MS）结合结合键合的cumomer分析来测试灌注的DB 1和DB 8黑色素瘤细胞和这些肿瘤细胞系的体内异种移植物中的这些假设。在目标2中，我们将直接测量LND对非洲爪蟾中表达的MCT 1和MCT 4的抑制作用，还将评估LND对分离的肝脏和心脏线粒体以及透化的DB 1和DB 8细胞的作用。之前综述的所有批评都得到了解决，包括临床翻译的关键问题，我们组建了一个黑色素瘤领先专家团队，他们建议：1）定义LND的机制（本提案的目的），2）首先将LND合并到使用美法仑的黑素瘤的高温隔离肢体灌注中（一种已经在临床实践中的方法），和3）然后进行已经用N-木霉素或多柔比星治疗的癌症的全身治疗。该项目将对阐明一类新型药物的活性机制产生重大影响，这些药物如LND抑制肿瘤细胞中的MCT和其他关键转运蛋白，从而改变肿瘤微环境以增强常规抗癌药物的活性。它还将为肿瘤代谢和癌症药物活性机制的研究开发新的代谢组学方法。

DESCRIPTION (provided by applicant): Melanoma remains one of the deadliest of human cancers with no effective method for treating the disseminated disease. While targeted therapies have shown some efficacy, they have thus far proven to be noncurative, which points to the need for other forms of systemic therapy. This proposal is part of a long-term effort to develop methods for systemic therapy of melanoma. We have recently shown that administration of lonidamine (LND, 100 mg/kg) substantially enhances the activity of melphalan and have now extended this finding to doxorubicin treatment. As a key step towards eventual clinical translation, we will now examine the detailed mechanism of LND activity. We and others have shown that LND produces tumor specific intracellular acidification and a substantial decrease in tumor energy status (NTP/Pi). A number of mechanisms have been proposed for LND: 1) inhibition of hexokinase II, 2) interference with mitochondrial electron transport, 3) inhibition of cellular lactate export through the monocarboxylate transporters (MCT). We are proposing a fourth mechanism to explain the bioenergetic decline of the tumor following treatment with LND: 4) inhibition of the putative mitochondrial pyruvate carrier (MPC), which would deplete the tumor cells of a key substrate for oxidative phosphorylation and induce an increase in glycolytic metabolism to compensate for decreased oxidative ATP production. We have recently validated a novel extension of isotopomer analysis called cumomer analysis and applied it to perfused DB1 melanoma cells. We believe that this is the first validated metabolic network model of tumor energy metabolism. We propose to test the hypotheses that 1) selective tumor acidification results from inhibition of MCT1, and 2) tumor de-energization is caused by inhibition of the MPC. In Aim 1 of this proposal, we propose to use 13C magnetic resonance spectroscopy (MRS) and liquid chromatography-mass spectrometry (LC-MS) in conjunction with bonded cumomer analysis to test these hypotheses in perfused DB1 and DB8 melanoma cells and in in vivo xenografts of these tumor lines. In Aim 2 we will directly measure the inhibitory effect of LND on MCT1 and MCT4 expressed in Xenopus laevis and will also evaluate the effect of LND on isolated liver and cardiac mitochondria and on permeabilized DB1 and DB8 cells. All of the criticisms of the previous review have been addressed including the critical issue of clinical translation for which we assembled a team of leading experts on melanoma who recommended: 1) define the mechanism of LND (the aim of this proposal), 2) first incorporate LND into hyperthermic isolated limb perfusion of melanoma with melphalan (a method already in clinical practice), and 3) then proceed to systemic therapy of cancers that are already treated with N-mustards or doxorubicin. This project will have a major impact on elucidating the mechanism of activity of a new class of drugs that like LND inhibit MCTs and other key transporters in tumor cells and thereby modify the tumor microenvironment to augment the activity of conventional anticancer agents. It will also develop novel metabolomic methods for the study of tumor metabolism and mechanisms of cancer drug activity.