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.

In vitro MKT-077 uptake experiment
a. Solution preparation:
1. Stock solution preparation: Prepare a certain concentration of MKT-077 stock solution, store it at -20℃ or -80℃ in the dark after aliquoting.
2. Working solution preparation: Dilute MKT-077 to 2, 4 or 6 μg/ml working solution. Select the appropriate working solution concentration according to the experimental requirements and try to prepare it before use.
b. Operation steps:
1. After the cells grow normally to ∼80–90% confluence, digest and centrifuge and resuspend them in D-MEM without phenol red (using phenol red-free medium to limit background spectral interference) at a density of 1×106 cells/ml.
2. Place 15 mL of cell suspension in a water-jacketed chamber equilibrated to 37°C and gently mix with a small magnetic stirring bar.
3. Add different concentrations of MKT-077 (2, 4, or 6 μg/ml) and collect 1.5 mL samples immediately and every 30 minutes thereafter for 2 hours.
4. Centrifuge samples, wash with PBS, and lyse with 200 μl of ethanol.
5. Re-centrifuge samples and analyze lysates spectrophotometrically at 495 nm.

Systemic administration of MKT-077 significantly delayed the growth of TT xenografts in mice throughout the treatment. At the end of the drug treatment, we found that tumor weights were about two-timed less in MKT-077-treated group than in the control group. These data are consistent with the growth inhibitory effects of MKT-077 observed in the in vitro setting above [1]. The succinate-induced, ADP-stimulated respiratory rate in mitochondria isolated from the liver of rats treated with a bolus i.v. injection of 15 mg MKT-077 1kg body weight each day for 5 days is significantly lower than that of untreated controls [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].