The human RCC cell lines 786-O, Caki-1, A-498 and ACHN were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% (v/v) penicillin-streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C in a humidified atmosphere containing 5% CO₂. Dauricine was purchased from Solarbio Biotechnology Co., Ltd. (Beijing, China). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and propidium iodide (PI) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Primary antibodies against caspase-8, caspase-3, caspase-9, cleaved PARP, Bcl-2, p21, and Bax were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against cyclin D1, cyclin-dependent kinase 2 (CDK2), CDK4, p-Akt, Akt, p-PI3K, and PI3K were all purchased from Abcam (Cambridge MA, USA), and primary antibodies against GAPDH and donkey anti-rabbit and sheep anti-mouse immunoglobulin were purchased from Sigma-Aldrich. All other chemicals not specifically mentioned here were purchased from Sigma-Aldrich; Merck KGaA.

Cell viability was evaluated by MTT assay. Briefly, cells were seeded in a 96-well plate (5×10³ cells/well) and then treated with various doses of dauricine for 24 h. A total of 50 µl of MTT solution (5 mg/ml) was added, and the cells were incubated for another 4 h. The medium was then removed, and 200 µl of DMSO was added to each well. The absorbance of the solutions was measured on a BioTek microplate reader at 595 nm. The relative cell viability was normalized to the control, which was treated with 0.1% DMSO.

Cells were harvested after treatment with dauricine and fixed in ethanol. The cells were washed with PBS and stained with propidium iodide (BD Biosciences, Franklin Lakes, NJ, USA) for 30 min in PBS supplemented with RNase at room temperature in the dark. The cell cycle distribution was then evaluated using FACSVerseTM (Beckman Coulter Fullerton, CA, USA), and the data were analyzed using FlowJo V10 (Tree Star, Inc., Ashland, OR, USA).

For apoptosis assays, cells were seeded at a density of 1×10⁴ cells/well into 96-well plates at 37°C overnight and treated with various doses of dauricine for 24 h. Cells were subsequently harvested and treated with the Nucleosome ELISA kit (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) according to the manufacturer's instructions.

The activity of caspases was measured using a caspase activation kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Briefly, cell lysates were prepared after treatment with various doses of dauricine and incubated with the supplied reaction buffer and the colorimetric substrates at 37°C for 2 h in the dark. Then, the absorbance of the solutions was measured on a BioTek microplate reader (BioTek Instruments, Winooski, VT, USA) at 405 nm.

Total protein was lysed in RIPA lysis buffer (Invitrogen Life Technologies, Carlsbad, CA, USA) and then quantified with a BCA kit. Equal amounts of proteins were resolved on 12% SDS-PAGE gels and transferred onto PVDF membranes. Membranes were incubated with primary antibodies overnight at 4°C followed by HRP-conjugated secondary antibodies at room temperature for 1 h; signals were visualized by ECL reagent (Pierce, Rockford, IL, USA).

All the experiments were carried out at least three times. The data were analyzed using GraphPad Prism 6.0 software. The results are shown as the mean ± standard deviation (SD), and the differences were measured using one-way analysis of variance (ANOVA) followed by Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the cytotoxicity of dauricine, four different renal carcinoma cell lines (786-O, Caki-1, A-498, and ACHN) were treated with various concentrations of dauricine (0, 5, 10, 15, 20 µM) for 24 h, followed by MTT assay. As shown in Fig. 1, dauricine significantly decreased cell viability in a dose-dependent manner, and the IC₅₀ values of dauricine in different cell lines are listed in Table I. Caki-1 and A-498 cells were the most sensitive and the least sensitive to dauricine, respectively. These two cell lines and 786-O cells were used for the subsequent studies.

MTT assay revealed that dauricine inhibits the viability of RCC cells. To better understand the underlying mechanism, the effects of dauricine on cell cycle were analyzed using flow cytometry. Caki-1, A-498 and 786-O cells were treated with different doses of dauricine (10 and 20 µM) for 24 h. As shown in Fig. 2A, incubation with dauricine triggered cell cycle arrest at the G0/G1 phase as well as a reduction in the S phase in a dose-dependent manner. Moreover, cell cycle-related proteins, such as cyclin D1, CDK2, and CDK4, were decreased, and p21 was increased after treatment with dauricine (Fig. 2B).

To measure apoptosis induced by dauricine, nucleosome ELISA assays were used. As shown in Fig. 3A, dauricine treatment led to an increase in apoptosis in a dose-dependent manner. The activation of caspases plays a very important role in the process of apoptosis. There are two pathways that lead to apoptosis, namely, the extrinsic pathway and the intrinsic pathway. The extrinsic and intrinsic pathways are initiated by caspase-8 and caspase-9, respectively. Activation of both caspase-8 and caspase-9 can lead to the activation of caspase-3, which finally leads to apoptosis. Therefore, we investigated whether dauricine activated any of these caspases using a colorimetric caspase activity assay and western blot analyses. As shown in Fig. 3B and C, activation of caspase-3 and caspase-9 but not caspase-8 was observed after incubation with dauricine. Besides the cleavage of caspase-3, we also observed the expression of pro-caspase-9 was decreased which indicated the activation of caspase-9 (7,8) (Fig. 3C). In addition, cleavage of PARP, which is a substrate of caspase-3, was observed (Fig. 3C). The Bcl-2 family proteins are well-known regulators of the intrinsic apoptotic pathway. We also found that dauricine was able to downregulate the anti-apoptotic Bcl-2 and Mcl-1 proteins in a dose-dependent manner (Fig. 3C). Moreover, the pro-apoptotic protein Bax was increased by dauricine in a dose-dependent manner (Fig. 3C). These data suggest that dauricine induced apoptosis mainly through the intrinsic apoptotic pathway in renal carcinoma cells.

The PI3K/Akt signaling pathway plays a critical role in cell survival and protecting cancer cells from apoptosis (9). Therefore, we investigated whether dauricine could affect the PI3K/Akt signaling pathway. Caki-1, A-498 and 786-O cells were treated with various concentrations of dauricine for 24 h; then, cellular lysates were subjected to western blot analysis. As shown in Fig. 4A, the activation of PI3K and Akt was suppressed by dauricine. To further investigate the role of dauricine in PI3K inhibition, we examined its effect with or without the endotoxin LPS, which is a potent activator of the PI3K/Akt signaling pathway. The cells were incubated with 2 µg/ml LPS or 20 µM dauricine for 24 h. As depicted in Fig. 4B, stimulation with LPS significantly induced Akt activation, and dauricine could repress the activation of Akt. These data suggest that dauricine may exert antitumor effects through the inhibition of PI3K/Akt.