Mechanism of Action of G3139

描述（由申请人提供）：最近令人兴奋的临床前和II期临床试验数据，采用G3139（又名Oblimersen），一种靶向bcl-2 mRNA前六个密码子的18聚体I反义硫代磷酸寡脱氧核苷酸，表明这种新的药物与细胞毒性化疗联合治疗相对大量的人类肿瘤可能具有临床活性。然而，关于该药剂的确切作用机制仍存在重大问题。我们和其他人已经确定了G3139的四种不同的可能的作用机制：1）下调bcl-2，推测随后特异性增加化疗敏感性; 2）寡核苷酸的非特异性作用，包括不相关的切割，导致与机制1的协同作用; 3）免疫效应机制的CpG基序调节的刺激，可能涉及Toll样受体，和4）局部（在肿瘤水平）CpG调节的活性氧（ROS）的产生导致细胞生长速率的降低。为了证明哪些机制负责G3139在体外和体内的抗肿瘤作用，我们设计了四个具体目标：具体目标1：我们将确定G3139和相关寡核苷酸在前列腺和膀胱细胞中产生的ROS和H2 O2如何影响这些细胞的生长和活力。我们将向MnROS和H2 O2中加入小分子，向外部培养基中加入过氧化氢酶以阻断H2 O2对细胞生长的影响，并用腺病毒-MnSOD载体感染细胞， 减少ROS的产生。我们还将确定H2 O2的细胞内浓度，并确定这些寡核苷酸以bcl-2依赖性和非依赖性方式诱导脂质过氧化的能力。我们还将检查G3139存在下的线粒体功能（例如，线粒体电位、ATP产生、氧消耗）。具体目标二：我们将在G3139的3'和5'末端使用新型2 '-O，4'-C-亚甲基连接的双环核糖核苷（LNA），以增加寡聚物的稳定性和对其靶点的亲和力。除了与G3139相比下调bcl-2表达的能力之外，还将确定最佳gapmer。具体目标3：我们将使用PC 3细胞异种移植在SCID小鼠中评估免疫刺激下调bcl-2表达的作用，以及局部活性氧的产生在G3139及其LNA gap-mer同系物的抗肿瘤作用中的作用。最后，在具体目标4中，我们将对G3139和相关寡核苷酸处理的前列腺癌和膀胱癌细胞进行Affyrin基因芯片分析，以特异性地确定哪些基因受到G3139处理的影响。

DESCRIPTION (provided by applicant): Recent exciting pre-clinical and phase II clinical trial data employing G3139 (a.k.a. Oblimersen), an 18mer I antisense phosphorothioate oligodeoxynucleotide targeted to the first six codons of the bcl-2 mRNA, indicates that this novel agent may be clinically active in combination with cytotoxic chemotherapy in the treatment of a relatively large number of human tumors. However, significant questions remain as to the precise mechanism of action of this agent. We and others have ascertained four distinct possible mechanisms of action of G3139: 1) Downregulation of bcl-2 with postulated subsequent specific increase in chemosensitivity; 2) Non-specific effects of the oligonucleotide, including irrelevant cleavage, leading to synergy with mechanism 1; 3) CpG motif-modulated stimulation of immune effector mechanisms, possibly involving Toll-like receptors, and 4) Local (at the level of the tumor) CpG-modulated production of reactive oxygen species (ROS) leading a diminution in the rate of cell growth. In order to demonstrate which mechanisms are responsible for the antitumor effects of G3139 both in vitro and in vivo, we have devised four Specific Aims: Specific Aim 1: We will determine how the production of ROS and H202 by G3139 and related oligos in prostate and bladder cells serves to affect the growth and viability of these cells. We will add small molecules to scavenge ROS and H202, add catalase to the external media to block the effects of H202 on cell growth, and infect cells with an adenoviral-MnSOD vector to reduce ROS production. We will also determine the intracellular concentration of H202, and determine the ability of these oligos to induce lipid-peroxidation in a bcl-2 dependent and independent manner. We will also examine mitochondrial function in the presence of G3139 (e.g., mitochondrial potential, ATP production, oxygen consumption). Specific Aim 2: We will employ novel 2'-O,4'-C-methylene-linked bicyclic ribonucleosides (LNAs) at the 3' and 5' termini of G3139 to increase oligo stability and affinity for its target. The optimal gapmer will be determined, in addition to its ability to downregulate expression of bcl-2 compared to G3139. Specific Aim 3: We will use PC3 cell xenografts in SCID mice to evaluate the roles of immunostimulation downregulation of bcl-2 expression, and the local production of reactive oxygen species in the antitumor effect of G3139 and its LNA gap-mer homolog. Finally, in Specific Aim 4, we will perform Affymetrix gene-chip analysis of G3139 and related oligo-treated prostate and bladder carcinoma cells in order to specifically determine which genes are affected by G3139 treatment.