SNX-7081 and SNX-2112 were prepared in our laboratory with purities of >98.0% according to a previously described procedure (20,21), dissolved in 10 mM dimethyl sulfoxide (DMSO) as a stock solution and stored at −20°C. Antibodies against IKKα, Raf-1, CHK1 and GSK3β were purchased from Epitomics (Burlingame, CA, USA), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Millipore (Billerica, MA, USA).

Eleven cell lines, including eight human cancer cell lines and three human normal cell lines, were used in the present study (Table I). Primary HDF cells were isolated from the foreskins of newborns using a previously reported procedure (22,23). All other cell lines were purchased from the Cell Bank of the China Science Academy (Shanghai, China). CML K562 cells, A375 melanoma cells, the normal human diploid fibroblast (HDF) and the normal human liver L-02 cells were cultured in RPMI-1640 medium. MCF-7 breast cancer cells, Hep-2 laryngeal epidermoid carcinoma, HepG2 liver carcinoma, A549 lung adenocarcinoma, HeLa cervical carcinoma and SW-620 colon carcinoma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). The normal human fibroblastic cell line (MRC-5) was cultured in Minimum Essential Medium (MEM). Cells were maintained at 37°C in a 5% CO₂ atmosphere. All the media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) supplemented with 50 U/ml penicillin and streptomycin.

Exponentially growing cells were seeded in 96-well culture plates (5–10×10³ cells/well) and allowed to adhere overnight. Cancer cells were incubated with SNX-7081 or SNX-2112 at various concentrations (0–10 μM) for 72 h. HDF, MRC5 and L-02 normal human cells were incubated with drugs at various concentrations (0–50 μM) for 72 h, along with an equal volume of DMSO as the solvent control. MTT solution (10 μl) was added to each well (0.5 mg/ml) for an additional 4-h incubation (37°C, 5% CO₂). The precipitated formazan was dissolved in 100 μl of DMSO. A 96-well multi-scanner autoreader (M450; Bio-Rad Laboratories, Hercules, CA, USA) was used to measure the absorbance of each well at 570 nm, with a reference wavelength of 630 nm. The IC₅₀ values, defined as the drug concentration that caused 50% inhibition of absorbance compared with the control cells treated with DMSO only, were calculated using the PrismPad program.

Cell cycle distribution was determined by DNA staining with propidium iodide (PI) (13). Briefly, cancer cell lines were cultured and treated in 6-well culture plates (6×10⁵ cells/well) with SNX-7081 or SNX-2112 (1.0 μM) for 48 h. The cells were then washed in phosphate-buffered saline (PBS) and fixed in 70% ethanol overnight. The cells were collected and resuspended in PBS containing 50 μg/ml PI, 0.1 mg/ml RNase, and 5% Triton X-100 and incubated at 37°C for 30 min. Subsequently, the cells were analyzed on a flow cytometer (Becton-Dickinson, San Jose, CA, USA), and the percentages of cells present in different phases of the cell cycle were analyzed using the CellQuest software (Becton-Dickinson).

Apoptosis was measured by flow cytometry after staining with Annexin V-FITC and PI according to the instructions of the Annexin V-FITC/PI staining kit (Nanjing KeyGen Biotech., Co., Ltd., Nanjing, China). Briefly, cancer cells were cultured in the presence of the indicated concentrations of SNX-7081 or SNX-2112 (1.0 μM) for 48 h, harvested, washed twice and resuspended in 500 μl of 1X binding buffer containing Annexin V-FITC and PI. Samples were incubated at room temperature for 10 min and analyzed by FACS.

K562 cells were incubated with 1.0 μM SNX-7081 or SNX-2112 for 0, 6, 12, 24 or 48 h. Whole-cell lysates were prepared by washing the cells with PBS and subjecting them to lysis with RIPA buffer for 30 min on ice. Total protein concentrations of whole-cell lysates were determined using the BCA protein assay kit. Equal amounts of protein samples were loaded onto 8–12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gels. After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore), examined with primary antibodies and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Specific protein bands were visualized using the chemiluminescence method and imaged by autoradiography. Any differences in protein loading were normalized to the corresponding levels of the GAPDH control.

The affinity of SNX-7081 and SNX-2112 against Hsp90 was determined by the MOE docking assay. The crystal structure of Hsp90 was taken from the Protein Data Bank (PDB code: 3R92). The two compounds were converted to 3D structures, and energy was minimized in MOE. The binding site of Hsp90 was minimized using the AMBER 99 force field in MOE with the default parameter. The two compounds were docked, employing Triangle Matcher as the placement method and the function London dG as the first scoring function. The refinement was set to force field (AMBER 99), and the docked poses were energy minimized in the receptor pocket (24). The conformations of lowest energy were given, and a lower scoring value indicated a more favorable binding.

Data were evaluated by the Welch t-test when only two value sets were compared. One-way ANOVA followed by the Dunnett’s test was used for more group comparisons if ≥3 experiments were involved. Results were expressed as means ± SD with a significance at ^*P<0.05 or ^**P<0.01.

The growth inhibitory effects of SNX-7081 and SNX-2112 on human cancer lines originating from bone marrow, colon, skin, larynx, breast, liver, cervix and lung were comopared. Cells were exposed to various concentrations (0–10 μM) for 72 h and cell viability was quantified by the MTT assay. SNX-7081 and SNX-2112 significantly inhibited the growth of eight human cancer cells in a dose-dependent manner. The cell viability of SNX-7081 was significantly lower than that of SNX-2112 at the low concentration (<1 μM) in K562, A375, MCF-7, Hep-2, HepG2 and A549 cells (Fig. 1A). In addition, SNX-7081 exhibited similar or weaker effects to SNX-2112 in SW-620 and HeLa cells (Fig. 1B). The IC₅₀ values of SNX-7081 and SNX-2112 for these cells were always ~1 μM (Table I). For the remaining experiments, 1 μM was applied as the optimal concentration.

The cytotoxic effects of two agents on three human normal cell lines (L-02, HDF and MRC5 cells) were determined by the MTT assay. The cells were exposed to SNX-7081 or SNX-2112 at concentrations of 3.13–50.0 μM for 72 h. The IC₅₀ value could not be determined even at the high concentration of 50.0 μM (Table I and Fig. 2), suggesting that SNX-7081 cytotoxicity was lower as compared to SNX-2112 in L-02, MRC5 and HDF cells. Therefore, SNX-7081 and SNX-2112 were provided with acceptable cytotoxicity toward normal cells and high selectivity in cancer cells.

To explore the anti-proliferative mechanism of SNX-7081 and SNX-2112, we assessed their effects on the cell cycle distribution in the K562, Hep-2, A549, SW620 and HeLa cancer cell lines using flow cytometry. Cells treated with SNX-7081 and SNX-2112 (1 μM) for 48 h, were subjected to flow cytometric analysis after PI staining. SNX-7081 more effectively increase the cell amount of G2/M phase in K562, Hep-2 and A549 cells (Fig. 3A), while SNX-2112 induces a more significant G2/M arrest in SW-620 and HeLa cells (Fig. 3B). The finding suggested that SNX-7081-induced inhibitory effects may be mediated by cell cycle arrest.

To examine whether apoptosis is involved in the inhibition of cancer cell proliferation by SNX-2112 or SNX-7081, cancer cells were treated with 1 μM SNX-2112 or SNX-7081 for 48 h and subjected to Annexin V- and PI-double staining for flow cytometric analysis. The degree of apoptosis was calculated as the sum of the percentages of cells in the lower right and upper right quadrants. SNX-7081 and SNX-2112 clearly induced apoptosis in all six cell lines. SNX-7081 induced more apoptosis in K562, Hep-2 and A549 cells (Fig. 4A), but less apoptosis in SW-620 and HeLa cells as compared to that of SNX-2112 (Fig. 4B). The data indicated that SNX-7801-induced apoptosis in some cancer cells was more potent than SNX-2112.

The basic mechanism of Hsp90 inhibitors involves binding to the ATP pocket and suppressing the ATPase activity of Hsp90, resulting in the degradation of its client proteins (25). It was previously demonstrated that SNX-2112 led to downregulation of the Hsp90 client proteins Bcr-abl and Akt in K562 cells (13). In the present study, we compared the effect of SNX-7081 and SNX-2112 on the expression of other essential Hsp90 client proteins, including IKKα, GSK3, CHK1 and Raf-1, which are crucial for the growth of cancer cells. Western blot analysis showed that the levels of these proteins were reduced in a time-dependent manner following exposure to the compounds (Fig. 5). After treatment with 1 μM of SNX-7081 for 48 h, the expression of IKKα, CHK1, GSK3 and Raf-1 was significantly decreased to 11.7, 21.8, 5.8 and 10.4%, respectively. SNX-7081 exerted stronger inhibitory effects on the expression levels of Hsp90 client proteins than SNX-7081. These data support the principle that SNX-7081 and SNX-2112 act via the inhibition of Hsp90 chaperone function, and the SNX-7081 was more potent in suppressing Hsp90 client proteins.

To determine the binding affinity of SNX-7081 and SNX-2112 to Hsp90, the inhibitors were docked into the ATP site of Hsp90, using MOE. A lower scoring value suggests a more favorable binding. The ligand-receptor interaction maps for SNX-7081 and SNX-2112 are shown in Fig. 6. We found two hydrogen bond residues (Phe-138 and Trp-163), two side chain donor molecules (Lys-58 and Thr-184), and one side chain acceptor molecule (Asp-93) that bound to SNX-7081. SNX-7081 interacted with five residues in the pocket, while SNX-2112 only interacted with three residues. The scoring value of SNX-7081 was −33.53 kcal/mol, which was lower than the −30.94 kcal/mol on SNX-2112. It was evident that SNX-7081 was the highly-scored compound, suggesting that SNX-7081 had a stronger binding affinity to Hsp90 than SNX-2112.