Development of flexible-heteroarotinoids for kidney cancer

G₁ cell cycle arrest in both cancer and normal cells. Flow cytometric analysis of cell cycle distribution of Caki-1 cells (A) or HK-2 normal kidney cells (B) treated with 10 μmol/L SHetA2 (C) or the same volume of solvent for 24 h before fixation and PI staining. Cyclocheximide (CHX) and a proteasome inhibitor (MG132) inhibited the SHetA2-induced G₁ cell cycle arrest (C). Western blot analysis showed that Cyclin D1 protein levels are decreased in association with the cell cycle arrest (D).

Glutathione and NAC in apoptosis and G₁ arrest. Flow cytometric analysis of PI-stained cells showed that reduced glutathione (GSH; 5 mmol/L) and NAC (5 mmol/L) could prevent G₁ arrest in Caki-1 cancer cells (A) but not HK-2 normal cells (B) treated with 5 μmol/L SHetA2 for 24 h. C, flow cytometric analysis showed that apoptosis was not inhibited by the reduced glutathione (10 mmol/L) or NAC (10 mmol/L) in Caki-1 cells treated with 10 μmol/L SHetA2 for 20 h. Columns, mean and standard error of a representative experiment of two independent experiments done in quadruplicate; bars, SE. P values represent significant differences in the indicated columns determined by posttests using One-Way ANOVA analysis.

Induction of tubule differentiation. Top, H&E-stained sections of Caki-1 organotypic cultures grown in the absence (A and B) or presence of 1 μmol/L SHetA2 (C and D) for 2 wk before histochemical analysis. Middle, H&E-stained sections of xenograft tumors from untreated control mice (E) showing that most of the malignant cells are arranged singly and separate one from the other, compared with the more orderly arrangement of cells in tumors from mice treated with oral SHetA2 (60 mg/kg/d) 5 d per week for 30 d at the University of Oklahoma (F and G) or every day for 30 d at the RAID program (H). In some areas of the treated tumors, collections of tumor cells formed cords that were similar to tubules but no lumen was present (identified by a rectangle in F). In other areas of the treated tumors, a few tubules with lumens were present (circled in G and H). Neither cords nor lumens were present in the control tumors. I, rt-PCR analysis of E-Cadherin expression in tumors from the RAID xenografts in (bottom; H). To calculate fold induction, the expression levels in the individual treated tumors were compared with the average expression observed in three separate untreated tumors. J, real-time rtPCR analysis of E-Cadherin in Caki-1 cells treated with SHetA2 for the indicated times. Results are representative of two independent experiments. K, Western blot analysis of E-Cadherin expression in cell cultures treated with SHetA2. 24C, control cultures treated with solvent only for 24 h.

Repression of NF-κB activity. Caki-1 renal cancer cells were cotransfected with pNF-κB-Luc reporter and pRLTK transfection control plasmids and treated with DMSO or 10 μmol/L SHetA2 with or without 20 ng/mL tumor necrosis factor α (TNFα) for 4 or 8 h (A) or 10 μmol/L SHetA2 only (B) for various amounts of time before lysing the cells and evaluating for luciferase activity. c, control treated with solvent only. Tumor necrosis factor α was added to last 30 min of treatment. The firefly luciferase (NF-κB reporter) was normalized to the renilla luciferase (transfection control) activity.