Carborane‐Based Analogues of 5‐Lipoxygenase Inhibitors Co‐inhibit Heat Shock Protein 90 in HCT116 Cells

Abstract

5‐Lipoxygenase converts arachidonic acid into leukotrienes, which are involved in inflammation and angiogenesis. The introduction of carboranes can improve the pharmacokinetic behavior of metabolically less stable pharmaceutics. Herein we report the syntheses of several carborane‐based inhibitors of the 5‐lipoxygenase pathway. The isosteric replacement of phenyl rings by carboranes leads to improved cytotoxicity toward several melanoma and colon cancer cell lines. For the colon cancer cell line HCT116, the co‐inhibition of heat shock protein 90 was observed.

Borcalein: a Carborane‐Based Analogue of Baicalein with 12‐Lipoxygenase‐Independent Toxicity

“…[11] For example, the introduction of a carborane moiety into indomethacin increased the selectivity toward the cancer-specific isoform of cyclooxygenase, COX-2. [14] As shown previously, [13,15] also other members of the lipoxygenase family were efficiently inhibited by carboranebased compounds, e. g. 5-lipoxygenase (5-LO) by the carborane analogue of Rev-5901 (CarbORev-5901), leading to increased selective cytotoxicity toward aggressive melanoma and colon adenocarcinoma cell lines.…”

“…The most sensitive cell line CT26CL25 was selected and cells were treated with IC 50 concentrations of borcalein or baicalein for 48 h. Afterward, the impact of the compounds on proliferation rate, apoptosis, caspase activity as well as autophagy was assessed as described previously. [13,15,25] In the carboxy fluorescein succinimidyl ester (CFSE) assay, a decreased fluorescence represents a higher proliferation rate of cells. A left shift of the signal on CFSE staining (decreased fluorescence) detected in the non-treated control indicates that CT26CL25 cells were dividing within 48 h (Figure 3A).…”

“…Afterward, cells were treated with IC 50 concentrations of the selected compounds for 48 h, and analysed by flow cytometry (CyFlow® Space Partec and Partec-FloMax® software) as described previously. [13,15] Detection of the cell death by annexin V/propidium iodide staining and caspase activity by apostat staining: Cells were treated with IC 50 doses of baicalein and borcalein for 48 h. Subsequently, to evaluate whether cytotoxic effects are mediated by induction of apoptosis or caspases, annexin V/propidium iodide double-staining, or apostat staining were performed following manufacturer's protocol. Flow cytometry (CyFlow® Space Partec and PartecFloMax® software) was used for analysis as reported previously.…”

“…Flow cytometry (CyFlow® Space Partec and PartecFloMax® software) was used for analysis as reported previously. [13,15] Detection of autophagy by acridine orange staining: After 48 h incubation of the cells, acridine orange was used to detect autophagy. Therefore, cells were incubated with 1 μg/mL acridine orange for 15 min at 37 °C (5 % pCO 2 ), followed by washing, resuspending in PBS, and flow cytometry analysis as mentioned above.…”

“…Determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by DHR staining: Cells were incubated with 1 μM of redox-sensitive dye DHR (dihydrorhodamine 123) for 20 min, and subsequently, treated with IC 50 doses of baicalein and borcalein for 48 h. After washing the cells with PBS, generation of ROS and RNS were detected and analysed by flow cytometry (CyFlow® Space Partec and PartecFloMax® software). [15,25] Measurement of intracellular nitric oxide (NO) by DAF-FM staining: After 48 hours treatment, cells were stained with 2 μM DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) in phenol red free RPMI-1640 medium for 1 h at 37 °C. Then, cells were treated and analysed as indicated previously.…”