XKA at >95% purity was isolated from the fermentation broth of Streptomyces sp. CC8-201 and purified by the previously described method (7). It was prepared into a 5 mM solution with methanol and stored at −20°C before use. Dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), triciribine hydrate (TCN) and propidium iodide (PI) were all purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). In addition, 2,7-dichlorfluorescin diacetate (H₂DCFDA), RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM) were obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Human hepatoblastoma HepG2, non-small lung cancer A549, breast cancer MCF-7, prostate carcinoma PC-3, colon carcinoma HCT 116, cervical cancer HeLa and human neuroblastoma SH-SY5Y cell lines were all purchased from Shanghai Cell Bank, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The STR profiles of all the cell lines were confirmed by the provider. Concerning the HepG2 cell line it is misidentified according to: (https://www.ncbi.nlm.nih.gov/pubmed/19751877). This cell line was thought to be derived from a hepatocellular carcinoma (HCC) tumor but it has since been shown to be from hepatoblastoma. It has not affected the outcomes of this study.

All the cells were cultured in RPMI-1640 or DMEM supplemented with 10% fetal bovine serum (FBS; Tianjin Haoyang Biotech Co., Tianjin, China). The cells were incubated at 37°C with 5% CO₂ in a humidified atmosphere.

Cell viability was assessed by an MTT assay. Cells were seeded in a 96-well plate for 24 h at a cell density of 3×10³/well and then treated with XKA for 72 h. The final concentration of methanol in the culture media was <0.1%. MTT method was performed as previously described (13). The control group was set as 100%, and IC₅₀ values were assessed using the non-linear regression analysis.

Western blot assay was performed as previously described (14). The antibodies against cleaved caspase-3 (1:1,000; cat. no. 9664), caspase-9 (1:1,000; cat. no. 9505), AKT (1:1,000; cat. no. 9272), p-AKT (1:1,000; cat. no. 9271) and PARP-1 (1:1,000; cat. no. 9542) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibodies against p53 (1:2,000; cat. no. sc-126) and β-actin (1:5,000; cat. no. sc-1616) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

The apoptotic cells were stained with Annexin V-FITC/PI apoptosis kit (BD Biosciences, San Jose, CA, USA), following the manufacturer's protocol. The fluorescence intensities were assessed using a BD FACSCalibur flow cytometer (BD Biosciences).

Cells were transfected with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions at a concentration of 100 pmol siRNA/gene. The sequences of AKT and p53 siRNA were as follows: AKT, 5′-ACGAGUUUGAGUACCUGAA-3′ and p53, 5′-GACTCCAGTGGTAATCTAC-3′.

Following treatment with 2 µM XKA for different time-points, the cells were stained with 5 mM H₂DFFDA for 1 h at 37°C. The cells were harvested, and then flow cytometry was performed with excitation and emission wavelengths of 488 and 530 nm, respectively.

To determine the type of cell death, cells (5×10⁴ cells/well) were cultured in 24-well plates and then treated with XKA. Cells were washed with PBS twice and stained with 50 µg/ml PI for 15 min. The cells were observed and imaged under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

AKT1 assay kit was purchased from BioVision, Inc. (Mountain View, CA, USA). The assay was performed according to the manufacturer's protocol. In brief, the activity of the immunoprecipitated AKT1 was assessed using exogenously recombinant GSK3α as the substrate. The bands of phosphorylated GSK3α were determined by western blot analysis.

Data are expressed as the means ± standard deviations (SD). One-way analysis of variance (ANOVA) was used for the inter-group comparison. Statistical analysis was performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant result.

In order to assess the cytotoxic activity of XKA, seven human tumor cell lines were used for the MTT assay. As displayed in Fig. 1, the growth-inhibitory activity of XKA on PC-3, SH-SY5Y and A549 cells was concentration-dependent. The IC₅₀ values of XKA on the seven cell lines have been listed in a previous study (7), with a range of 0.43 to 2.77 µM. The cell line most susceptible to XKA was PC-3.

In a previous study, it was demonstrated that selective inhibition of AKT kinase activity required the 3,6-dihydro-2H-pyran ring of PNQ (11). XKA possesses this basic structure and possibly exerts an inhibitory activity of AKT. The AKT1 activity assay kit was used to verify this hypothesis. The reaction conditions of the assay kit were optimized to 4 h for the reaction time and 10 nM AKT1 for the following determinations (Fig. 2A and B). The suppression of AKT1 activity by TCN was concentration-dependent. In contrast to XKA, there were small changes of the phosphorylated bands from the concentrations of 1 to 5 µM (Fig. 2C). The IC₅₀ values for inhibiting AKT1 activity by TCN was 1.8 µM (Fig. 2D). However, the IC₅₀ values for inhibiting AKT1 activity by XKA was over 5 µM.

In order to clarify whether AKT is a target of XKA, AKT protein levels from PC-3, SH-SY5Y and A549 cells were detected by western blotting and quantified (Fig. 3A and B). The highest AKT content and potent inhibition of XKA in PC-3 cells indicated that AKT involves XKA action on tumor cells. RNA interference was used to confirm the relationship. AKT protein levels were markedly knocked down in PC-3 cells (Fig. 3C). The XKA susceptibility was significantly reduced and the IC₅₀ value rised to 1.5 µM, indicating that AKT is a bona fide target of XKA.

In order to decipher the difference between XKA and TCN, AKT phosphorylation was detected by western blotting following exposure to both drugs for 24 h. The phosphorylated AKT disappeared after treatment with 10 µM TCN, while the total AKT protein levels remained unchanged when the cells were treated with TCN from 10 to 100 µM (Fig. 4). Conversely, 0.5 µM XKA treatment significantly reduced phosphorylated AKT levels and the degradation of AKT was obvious following exposure to 1 µM XKA. The results demonstrated that suppression of the AKT activity by XKA is related to protein degradation, not kinase inhibition.

In order to explore the mechanism of the potent inhibition of XKA towards tumor cells, ROS was determined with flow cytometry. ROS generation markedly increased after the PC-3 cells were treated with 2 µM XKA for 1 h and higher at 6 h treatment (Fig. 5), indicating that the XKA treatment triggered oxidant response.

ROS generation triggered by 2 µM XKA verified that the cells were damaged. The ratio of apoptotic cells was detected with Annexin V/PI staining in the XKA-treated PC-3 cells. Approximately 12.4% apoptotic cells were detected after the PC-3 cells were treated with XKA for 24 h (Fig. 6A) and it did not increase after exposure to XKA for 48 h, indicating that the majority of cells did not undergo typical apoptosis.

To confirm the Annexin V/PI staining result, PARP-1 and p53 levels were detected by western blotting in PC-3 cells following exposure to 2 µM XKA for 48 h (Fig. 6B). The cleaved PARP-1 fragment was not observed and degradation of p53 occurred at 6-h treatment. The results further ruled out the possibility that the XKA-induced cell death was apoptosis.

In order to identify the type of cell death, PC-3 cells were incubated with 5 µM XKA from 1 to 48 h and PI-stained cells were counted. With the extension of treatment time, the red fluorescent cells increased significantly. At 48 h of treatment, the cell ratio stained with PI reached 89% (Fig. 7).

In order to search other molecules influencing XKA action, p53 protein levels from seven cell lines were detected. The highest level of p53 protein was observed in A549 and HCT-116 cell lines (data not shown), indicating that p53 may be a protein related with the action of XKA.

Knockdown of p53 in the A549 cells was performed and MTT assay was used for determining the susceptibility to XKA. The sensitivity to XKA increased after the reduction of p53 protein levels. The IC₅₀ value decreased to 1.2 µM in the p53 siRNA-treated cells (Fig. 8).