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些疗效，但迄今为止，它们已被证明是无效的，这表明需要其他形式的全身治疗。这项提议是开发黑色素瘤全身治疗方法的长期努力的一部分。我们最近的研究表明，给予lonidamine （LND, 100 mg/kg）大大增强了美法兰的活性，现在已经将这一发现扩展到阿霉素治疗。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）的显著降低。LND的一些机制被提出：1)抑制己糖激酶II， 2)干扰线粒体电子传递，3)通过单羧酸转运体（MCT）抑制细胞乳酸输出。我们提出了第四种机制来解释LND治疗后肿瘤的生物能量下降：4)抑制假定的线粒体丙酮酸载体（MPC），这将耗尽肿瘤细胞氧化磷酸化的关键底物，并诱导糖酵解代谢的增加，以补偿氧化ATP产生的减少。我们最近验证了一种称为单体分析的同位素分析的新扩展，并将其应用于灌注的DB1黑色素瘤细胞。我们认为这是第一个被验证的肿瘤能量代谢的代谢网络模型。我们建议验证以下假设：1)选择性肿瘤酸化是由MCT1抑制引起的，2)肿瘤失能是由MPC抑制引起的。在本提案的目的1中，我们建议使用13C磁共振波谱（MRS）和液相色谱-质谱（LC-MS）结合键合单体分析，在灌注的DB1和DB8黑色素瘤细胞以及这些肿瘤系的体内异种移植物中验证这些假设。在Aim 2中，我们将直接测量LND对非洲爪蟾MCT1和MCT4表达的抑制作用，并评估LND对离体肝脏和心脏线粒体以及通透化DB1和DB8细胞的影响。对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，为此我们召集了一个黑色素瘤的领先专家团队，他们建议：1)定义LND的机制（本提案的目的），2)首先将LND纳入melphalan的黑色素瘤热离体肢体灌注（一种已经在临床实践中的方法），3)然后对已经用n -芥子或阿霉素治疗的癌症进行全身治疗。该项目将对阐明一类新型药物的活性机制产生重大影响，如LND抑制肿瘤细胞中的mct和其他关键转运体，从而改变肿瘤微环境，增强传统抗癌药物的活性。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.