7ACC1 (D 142) selectively affects a single part of the MCT symporter translocation cycle, leading to strict inhibition of lactate influx. This singular activity is associated with antitumor effects less prone to resistance and side effects.

7-(Diethylamino)coumarin-3-carboxylic acid compounds on lactate influx using oxidative cancer cells known to maintain in vitro their capacity to take up lactate as an energetic fuel, and the lack of effects on lactate efflux using highly glycolytic cells. Accordingly, in oxidative human cancer cervix cells, SiHa and Hela, which express both MCT1 and MCT4 isoforms, a potent inhibition of both lactate influx and cell proliferation was obtained with 7-(Diethylamino)coumarin-3-carboxylic acid, whereas the bona fide MCT1/MCT2 inhibitor AR-C155858 failed to do so. The effects of 7-(Diethylamino)coumarin-3-carboxylic acid were confirmed in MCT1/4-expressing pharynx squamous FaDu tumor cells. These observations strongly suggest that 7-(Diethylamino)coumarin-3-carboxylic acid compounds are inhibitors of lactate entry through both MCT1 and MCT4 preventing any compensatory effects when MCT1, the main path for lactate uptake, is inhibited.

7-(Diethylamino)coumarin-3-carboxylic acid developed to selectively interfere with lactate fluxes in the lactate-rich tumor microenvironment.?The pharmacologic properties of two compounds of this family, including their effects on lactate influx and efflux and antitumor activity, were investigated using human cancer cell lines and mouse xenograft models.?Contrary to the reference MCT1 inhibitor AR-C155858, 7-(Diethylamino)coumarin-3-carboxylic acid unexpectedly inhibited lactate influx but not efflux in tumor cells expressing MCT1 and MCT4 transporters.?7-(Diethylamino)coumarin-3-carboxylic acid delayed the growth of cervix SiHa tumors, colorectal HCT116 tumors, and orthoptopic MCF-7 breast tumors.?MCT target engagement was confirmed by the lack of activity of 7-(Diethylamino)coumarin-3-carboxylic acid on bladder UM-UC-3 carcinoma that does not express functional MCT.7-(Diethylamino)coumarin-3-carboxylic acid?also inhibited SiHa tumor relapse after treatment with cisplatin.?Finally, we found that contrary to AR-C155858, 7-(Diethylamino)coumarin-3-carboxylic acid did not prevent the cell entry of the substrate-mimetic drug 3-bromopyruvate (3BP) through MCT1, and contributed to the inhibition of tumor relapse after 3BP treatment.

Usage:
I. Solution preparation
1. Stock solution: Dissolve 7ACC1 in an appropriate organic solvent (such as DMSO), usually prepared as a 1–10 mM stock solution.
2. Working solution: Dilute the stock solution to the required working concentration, usually 0–50 µM, using an experimental buffer (such as PBS, pH 7.4) according to experimental needs.
II. Application steps
Cell culture experiment
(1) Cell type: 7ACC1 is mainly used in the study of renal cancer cells, breast cancer cells and other cancer cell lines.
(2) Treatment method: Add 7ACC1 to the cell culture medium, culture the cells for 24–96 hours, and adjust the concentration as needed.
(3) Cell function detection:
a. Proliferation detection: MTT or CCK-8 method can be used to detect cell proliferation.
b. Migration and invasion detection: The migration and invasion ability of cells can be evaluated by scratch test or Transwell test.
c. MCT-1/MCT-4 expression detection: Western blot or qPCR method was used to detect the protein or mRNA expression level of MCT-1/MCT-4.
d. Lactate concentration detection: Lactate determination kit was used to detect the lactate content in cell culture medium.
Animal experiment
(1) Treatment method: 7ACC1 was injected or administered to experimental mice by intraperitoneal injection or oral administration. The dose was usually 10–50 mg/kg according to the experimental design.
(2) Tumor inhibition experiment: The inhibitory effect of 7ACC1 on tumor growth was evaluated by measuring tumor size.
(3) Immunohistochemical analysis: Immunohistochemical analysis was performed on tumor tissue to detect the expression level of MCT-1/MCT-4 and lactate accumulation.
3. Calibration and control
(1) Control group: An untreated cell or animal group was set as a control to verify the effect of 7ACC1.
(2) Standard curve: In the lactate concentration detection experiment, a standard curve is established to calibrate the relationship between lactate concentration and fluorescence signal.
Notes
(1) Solubility: 7ACC1 has good solubility in DMSO. When using, avoid leaving undissolved solids in the solution.
(2) Storage conditions: 7ACC1 should be stored at -20°C in a dark environment and avoid repeated freezing and thawing.
(3) Cytotoxicity: Although 7ACC1 has an inhibitory effect on cancer cells, it may be toxic to normal cells at high concentrations. The concentration should be adjusted according to experimental requirements.
(4) Photosensitivity: 7ACC1 may be sensitive to light. Avoid direct light during the experiment.

Eight-week-old NMRI female nude mice (Elevage Janvier) were injected subcutaneously with 2 × 106 SiHa cells, 2 × 10^6 HCT-11^6 cells, or 5 × 10^6 UM-UC-3 cells. An orthotopic breast cancer model was also used with MCF-7 tumor cells injected into the mammary fat pad of mice; a 17β-estradiol pellet had first been subcutaneously implanted in these mice as previously described . When tumors reached a mean diameter of 5 mm, 7-(Diethylamino)coumarin-3-carboxylic acid compounds (3 mg/kg) or AR-C155858 (3 mg/kg) were daily injected intraperitoneally; in some experiments, 7-(Diethylamino)coumarin-3-carboxylic acid treatment was combined with cisplatin (5 mg/kg) injected intraperitoneally at days 0 and 7 (7-(Diethylamino)coumarin-3-carboxylic acid administered daily except at days 0 and 7) or 3BP(3 mg/kg) injected i.p. from day 0 to 4 and day 7 to 11 (7-(Diethylamino)coumarin-3-carboxylic acid administered together with 3BP). Cisplatin and 3BP were also administered alone and control mice were injected with vehicle (dimethyl sulfoxide). Tumor sizes were tracked with an electronic calliper and determined using the formula: (length × width^2 × π)/6.