MKT-077 (FJ-776) is a cationic rhodacyanine dye that demonstrates antiproliferative activity against cancer cell lines (EC50s: 1.4-2.2 μM in vitro) through its ability to inhibit members of the Hsp70 family of molecular chaperones.

体外 MKT-077 摄取实验
a.溶液配制：
1.母液配置：配置成一定浓度的MKT-077 母液，分装后于 -20℃ 或 -80℃ 避光保存。
2.工作液配置：将MKT-077 稀释成 2、4 或 6 μg/ml工作液，根据实验需求选择合适的工作液浓度，尽量现配现用。
b.操作步骤：
1.细胞正常生长至 ∼80–90% 汇合后，消化离心并重悬于不含酚红的 D-MEM（使用不含酚红的介质来限制背景光谱干扰）中，密度为 1×106 个细胞/ml。
2.将 15 mL 细胞悬液置于平衡至 37°C 的水套室中，并与小型磁力搅拌棒轻轻混合。
3.加入不同浓度 MKT-077（2、4 或 6 μg/ml），立即采集 1.5 mL 样品，此后每 30 分钟采集一次，持续 2 小时。
4.离心样品，用 PBS 洗涤，并用 200 μl 乙醇裂解。
5.重新离心样品，并在 495 nm 处用分光光度法分析裂解物。

系统性给药MKT-077显著延缓了小鼠TT异种移植瘤的生长。在化合物治疗结束时，我们发现MKT-077治疗组的瘤重量约为对照组的两倍。这些数据与我们在体外环境中观察到的MKT-077的生长抑制效果一致[1]。来自接受了连续5天每天一次15 mg/kg体重MKT-077的静脉注射的大鼠肝脏中分离出的线粒体，在琥珀酸诱导、ADP刺激的呼吸率显著低于未处理对照组[3]。

MTT assay was performed as previously described. Briefly, cells were seeded in 24 well plates and allowed to attach for 48 hours. After drug treatment, cells were incubated with 400 μL of MTT (0.5 mg/mL) in complete medium for 2 hours at 37℃, switched into 200 μL DMSO, and shaken for 5 minutes at room temperature before measuring absorbance at 540 nm [1].

The 1×10^7 TT cells in 200 μL Hank's balanced salt solution were inoculated subcutaneously into the rear flanks of 6-week-old female athymic nude (nu/nu) mice. Once palpable, tumors were measured using Vernier calipers at intervals indicated in the text. Tumor volumes (TVs) were calculated using the formula: TV=L×W^2×0.5 (L, length; W, width). When TV reached 100 mm^3, mice were sorted into groups of 8 to achieve equal distribution of tumor size in all treatment groups. Group 1 received only the vehicle (1:9 mixture of DMSO/saline) and group 2 received MKT-077 (10 mg/kg body weight/dose). A 200 μL of ether solution was administered by intraperitoneal injection every 2 days (total 10 doses). At the end of the experiments, animals were euthanized by CO2 asphyxiation [1].