Dp44mT

Iron chelator

Dp44mT 在 SK-N-MC、SK-Mel-28 和 MCF-7 细胞中显示出显着的抗增殖作用，IC50 分别为 30 nM、60 nM 和 60 nM，而对正常 MRC-5 成纤维细胞没有影响。在 SK-N-MC 神经上皮瘤和 M109 细胞中，Dp44mT 减少细胞从 Fe-Tf 中吸收的 Fe 量并引发细胞凋亡。 [1] 当拓扑异构酶 topo2 被 MDA-MB-231 细胞中的 Dp44mT 特异性靶向时，会导致 DNA 损伤。 [2] 通过窃取溶酶体 P-糖蛋白 (Pgp) 的控制，Dp44mT（一种 Pgp 底物）也能消除多药耐药性。 [3]

Dp44mT (0.4 mg/kg, iv) 以剂量依赖性方式抑制携带 M109 肿瘤的 CD2F1 小鼠的肿瘤生长。 [1]

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值得注意的是，Dp44mT在仅5天后显著（P<0.01）将小鼠的肿瘤重量降低到对照组的47%，而动物重量或血液学指标没有明显变化。末端脱氧核糖核苷酸转移酶（TdT）介导的dUTP缺口末端标记（TUNEL）染色显示，用Dp44mT治疗的小鼠肿瘤发生凋亡。[1]

对于 DNA 拓扑异构酶 II 测定，单链寡核苷酸的 5' 末端或 pBluescript SK(-) 噬菌粒 DNA 的 161 bp 片段用 [32P]ATP 和 T4 多核苷酸激酶标记。为了去除未掺入的标签，然后通过 Mini Quick Spin DNA 柱（对于 pSK 片段）或 Oligo 柱（对于寡核苷酸）离心标记混合物。将反应混合物加热至 95 °C，并在 10 mM Tris-HCl (pH 7.8)、100 mM NaCl 和 1 mM EDTA 中冷却至室温过夜，然后与寡核苷酸的互补链退火。在存在或不存在 Dp44mT 的情况下，将 DNA 底物（10 pmol/反应）与 500 ng top2a 或 top2h 在 10 L 反应缓冲液中于 25°C 孵育指定时间。添加SDS（终浓度0.5%）以终止反应。样品在变性浓度为 16%（对于 pSK DNA）或 20%（对于寡核苷酸）（7 M 尿素）的聚丙烯酰胺变性凝胶上进行分离。 PhosphorImager[1] 用于成像和定量。

二-2-吡啶基酮-4,4，-二甲基-3-氨基硫脲（Dp44mT）是一种具有选择性抗癌活性的铁螯合剂。我们研究了Dp44mT杀死乳腺癌症细胞的机制，无论是作为单一药物还是与阿霉素联合使用。与健康的乳腺上皮细胞（MCF-12A）相比，单独的Dp44mT在乳腺癌症细胞系MDA-MB-231中诱导选择性细胞杀伤。在纳摩尔浓度下，它诱导G（1）细胞周期阻滞并减少癌症细胞克隆生长。Dp44mT，而不是铁螯合剂脱羧酶，诱导DNA双链断裂，定量为S139磷酸化组蛋白灶（γ-H2AX）和MDA-MB-231细胞中诱导的彗星尾。在低浓度Dp44mT存在下，阿霉素诱导的细胞毒性和DNA损伤均显著增强。螯合剂引起DNA拓扑异构酶IIα（top2alpha）的选择性中毒，通过体外DNA切割试验和细胞拓扑异酶-DNA复合物形成进行测量。与同源top2alpha（+/-）或top2beta（-/-）细胞相比，杂合Nalm-6 top2alph敲除细胞（top2alpha-+/-）对Dp44mT诱导的细胞毒性具有部分抗性。通过在HeLa细胞中敲除top2alpha和top2beta小干扰RNA，证实了top2alph的特异性。结果表明，Dp44mT对乳腺癌症细胞具有细胞毒性，至少部分是由于对top2α的选择性抑制。因此，Dp44mT可能作为癌症的一种机制独特的治疗方法，因为它具有螯合铁和抑制top2α活性的双重能力。[2]

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在这项研究中，研究人员研究了新型抗肿瘤药物二-2-吡啶基酮4,4-二甲基-3-氨基硫脲（Dp44mT）如何克服MDR。利用四种不同的细胞类型来评估Pgp增强药物溶酶体靶向以克服MDR的效果。为了评估Dp44mT克服耐药性的机制，细胞研究利用了Pgp抑制剂、Pgp沉默、促溶酶体药物、增殖试验、免疫印迹、Pgp ATP酶活性试验、放射性标记的药物摄取/排出、罗丹明123保留试验、溶酶体膜通透性评估和DCF（2'，7'-二氯荧光素）氧化还原研究。使用BALB/cnu/nu异种移植物小鼠模型研究了Dp44mT在表达Pgp的MDR细胞与药物敏感细胞中的抗肿瘤活性和选择性。我们证明Dp44mT是由溶酶体Pgp药物泵运输的，导致溶酶体靶向Dp44mT，从而增强MDR细胞的细胞毒性。溶酶体Pgp和pH值被证明对增加Dp44mT介导的溶酶体损伤和随后在耐药细胞中的细胞毒性至关重要，Dp44mT被证明是Pgp底物。事实上，Pgp依赖性溶酶体损伤和Dp44mT的细胞毒性被Pgp抑制剂、Pgp沉默或使用溶酶体运动碱增加溶酶体pH值所消除。在体内，Dp44mT与小鼠中不表达Pgp的肿瘤相比，能有效靶向化疗耐药的人Pgp表达异种移植物肿瘤。本研究强调了一种新的Pgp劫持策略，即独特的双吡啶基缩氨基硫脲系列缩氨基硫脲，通过利用溶酶体Pgp转运活性来克服MDR。[3]

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DFO、311、3-氨基吡啶-2-甲醛缩氨基硫脲、阿霉素和 DpT 系列螯合剂 (0-25 μM) 在有细胞和无细胞的情况下在 37°C 下孵育 72 小时。使用 MTT 测定，研究了螯合剂对细胞增殖的影响。

Inhibition of M109 lung cancer cell growth by iron chelators in vivo CD2F1 mice were subcutaneously implanted with 1 × 105 M109 cells. Dp44mT was dissolved in propylene glycol, as described above. An established protocol6 was used to test antitumor activity of the chelators in the M109 cancer model. Four days after engraftment, the tumors were palpable and the ligands were administered intravenously, twice a day for 5 days, followed by a rest period of 2 days when the animal was not injected.6 On the 12th day after tumor implantation, mice were killed using methoxyflurane, and blood was obtained by cardiac puncture. Blood indices were measured using a Sysmex Blood Counter. Mouse body weight changes during the treatment period were recorded. The tumor grew subcutaneously as a well-circumscribed mass surrounded by fibrous membranes and was simple to excise. Tumors were weighed and fixed for histologic examination. Experimental groups consisted of 8 mice for the control and each treatment. 3-AP was used as antitumor agent control because it is a chelator with potent antitumor activity with a mechanism of action similar to that of the chelators under investigation (ie, it binds Fe).[1]

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Dissolved in Propylene glycol; 0.4 mg/kg, twice daily; i.v. injection

CD2F1 mice bearing M109 tumors

[1]. Blood . 2004 Sep 1;104(5):1450-8.

[2]. Cancer Res . 2009 Feb 1;69(3):948-57.

[3]. J Biol Chem . 2015 Apr 10;290(15):9588-603.

Aroylhydrazone and thiosemicarbazone iron (Fe) chelators have potent antitumor activity. The aim of the current study was to examine the antitumor effects and mechanisms of action of a novel series of Fe chelators, the di-2-pyridyl thiosemicarbazones. Of 7 new chelators synthesized, 4 showed pronounced antiproliferative effects. The most active chelator was Dp44mT, which had marked and selective antitumor activity-for example, an IC(50) of 0.03 microM in neuroepithelioma cells compared with more than 25 microM in mortal fibroblasts. Indeed, this antiproliferative activity was the greatest yet observed for an Fe chelator. Efficacy was greater than it was for the cytotoxic ligand 311 and comparable to that of the antitumor agent doxorubicin. Strikingly, Dp44mT significantly (P <.01) decreased tumor weight in mice to 47% of the weight in the control after only 5 days, whereas there was no marked change in animal weight or hematologic indices. Terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining demonstrated apoptosis in tumors taken from mice treated with Dp44mT. This chelator caused a marked increase of caspase-3 activity in murine Madison-109 (M109) cells. Caspase activation was at least partially mediated by the release of mitochondrial holo-cytochrome c (h-cytc) after incubation with Dp44mT. In conclusion, Dp44mT is a novel, highly effective antitumor agent in vitro and in vivo that induces apoptosis.[1]

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Our investigation demonstrated that novel DpT analogs, in particular di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT; Figure 1), showed selective antitumor activity. In addition, Dp44mT strikingly decreased tumor growth in mice while not markedly affecting animal weight or a range of hematologic indices. This chelator also induced tumor cell apoptosis that was at least partially mediated through the release of mitochondrial h-cytc into the cytosol and the activation of caspase-3, -8, and -9. The release of h-cytc could be mediated by the imbalance of Bcl-2 and Bax expression induced by incubation of cells with Dp44mT.[1]

C14H15N5S

285.37

285.104

C, 58.93; H, 5.30; N, 24.54; S, 11.23

152095-12-0

10334137

Light yellow to yellow solid powder

1.2±0.1 g/cm3

438.4±43.0 °C at 760 mmHg

218.9±28.2 °C

0.0±1.1 mmHg at 25°C

1.635

1.21

92.54

1

4

3

20

336

0

S=C(N/N=C(C1=NC=CC=C1)\C2=NC=CC=C2)N(C)C

XOBIGRNRXCAMJQ-UHFFFAOYSA-N

InChI=1S/C24H20N4O4S/c1-30-24-26-23(16-7-12-20-21(13-16)32-15-31-20)28(27-24)18-10-8-17(9-11-18)25-22(29)14-33-19-5-3-2-4-6-19/h2-13H,14-15H2,1H3,(H,25,29)

3-(dipyridin-2-ylmethylideneamino)-1,1-dimethylthiourea

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.76 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (8.76 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: Propylene glycol : 1 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。