The human T-ALL cell line Jurkat and the human T-lymphoid cell line H9 were purchased from the American Type Culture Collection. Cells were cultured in complete medium containing 90% RPMI-1640 (HyClone; Cytiva) and 10% FBS (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.) with 1% penicillin-streptomycin mixture (Beijing Solarbio Science & Technology Co., Ltd.) in an incubator with 5% CO₂ and saturated humidity at 37˚C.

Hirsutanol A, a sesquiterpenoid compound isolated and synthesized in the laboratory of Professor Lan Wenjian, School of Pharmacy, Sun Yat-sen University, China, was dissolved in DMSO and stored in a refrigerator at -40˚C until use. p53 inhibitor pifithrin-α (PFTα) was purchased from APeXBIO Technology LLC. The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies Inc. Hoechst 33258 was purchased from Beijing Solarbio Science & Technology Co., Ltd. An apoptosis test kit (Annexin V-FITC Apoptosis Detection Kit I) was purchased from BD Biosciences, and the cell cycle detection kit was purchased from Nanjing KeyGen Biotech Co., Ltd. Rabbit anti-human p53 (1:1,000; cat. no. 2527), p53 upregulated modulator of apoptosis (Puma; 1:1,000; cat. no. 12450), Bcl-2 (1:1,000; cat. no. 4223), Caspase-3 (1:1,000; cat. no. 9662), poly (ADP-ribose) polymerase (PARP; 1:1,000; cat. no. 9532), cleaved caspase-3 (cCaspase-3; 1:1,000; cat. no. 9664) and cleaved poly(ADP-ribose) polymerase (cPARP; 1:1,000; cat. no. 9185) monoclonal antibodies were purchased from Cell Signaling Technology, Inc. The rabbit anti-human GAPDH (1:1,000; cat. no. 10494-1-AP) primary antibody was purchased from ProteinTech Group, Inc. The horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1,000; cat. no. A0208) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

Jurkat cells were collected and resuspended in medium to adjust the cell density to 2x10⁵ cells/ml. This cell suspension was added to a 96-well plate at 90 µl/well. Subsequently, hirsutanol A (10 µl/well) at different concentrations (0.71, 1.42, 2.84, 5.68 and 11.36 µM) was added for incubation. In parallel, a blank group and a control group (containing only cells and culture medium) were used, and each group was set up in 3 duplicate wells. Following incubation for 48 h, 5 µl CCK-8 solution was added to each well, followed by incubation for another 4 h. The optical density (OD) value of each well was measured using an ultraviolet spectrophotometer (Beckman Coulter, Inc.) at a wavelength of 450 nm. Human T lymphocyte H9 cells were treated with a series of hirsutanol A concentrations (2, 4, 8, 16, and 32 µM) for 48 h, and the other experimental procedures were the same as Jurkat cells. In addition, Jurkat cells were treated with 5 µM hirsutanol A, and viability was assessed at different time points (24, 48, and 72 h) after treatment. The cell viability rate was calculated as follows: Cell viability rate (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group). The half-maximal inhibitory concentration (IC₅₀) of hirsutanol A was determined as the concentration of the drug leading to 50% cell inhibition.

Cell apoptosis was assessed using the Annexin V-FITC Apoptosis Detection Kit I. Jurkat cells were collected and resuspended to adjust the cell density to 2x10⁵ cells/ml. This cell suspension was added to a 6-well plate at 2 ml/well and different concentrations (0, 3, 5, 6, and 9 µM) of hirsutanol A and/or PFTα (10 µM) were added. After 48 h of incubation at 37˚C, the cells were collected, washed twice with cold PBS and resuspended with 1X binding buffer. Of this suspension, 100 µl (~1x10⁵ cells/ml) were placed in a 5-ml culture tube and propidium iodide (PI; 5 µl) and Annexin V-FITC (5 µl) were added. Following incubation for 15 min in the dark at room temperature, 200 µl 1X binding buffer was added per tube and apoptotic cells were detected using a BD FACScan™ flow cytometer (BD Biosciences) within 1 h. The results were analyzed using BD FACSuite™ (BD Biosciences). Early and late apoptosis were assessed.

The cell cycle was assessed using the cell cycle detection kit. Jurkat cells were collected and resuspended to adjust the cell density to 2x10⁵ cells/ml. This cell suspension was added to a 6-well plate at 2 ml/well and different concentrations (0, 3, 5, 6, and 9 µM) of hirsutanol A and/or PFTα (10 µM) were added. After 48 h of incubation, the cells were collected, washed with PBS and fixed with 70% cold ethanol at 4˚C overnight. Following centrifugation (300 x g at 4˚C and 5 min) and washing with PBS, 400 µl PI/RNase A staining solution (PI/RNase A ratio, 1:9; prepared in advance) was added, followed by incubation at room temperature in the dark for 30 min. Subsequently, the cell cycle was assessed using a BD FACScan™ flow cytometer. The results were analyzed using ModFit LT 4.0 (Verity Software House, Inc.).

Jurkat cells were collected and resuspended to adjust the cell density to 2x10⁵ cells/ml. The suspension was added to a 6-well plate at 2 ml/well. Following the addition of different concentrations (0, 3, 6, and 9 µM) of hirsutanol A, the cells were incubated for 48 h. Subsequently, the cells were collected, Hoechst 33258 working solution (5 µg/ml) was added to fully cover the cells and the cells were then placed in an incubator at 37˚C for 30 min. The staining solution was discarded via centrifugation (300 x g at room temperature for 5 min) and the cells were washed with PBS twice, followed by observation via fluorescence microscopy (maximum excitation wavelength, 352 mm; maximum emission wavelength, 461 mm). Each sample was viewed from five fields with roughly the same number of cells.

JC-1 buffer (1X) was prepared from 5X JC-1 buffer (Beijing Solarbio Science & Technology Co., Ltd) and distilled water at a ratio of 1:4 and maintained in an ice bath for later use. Jurkat cells were collected and resuspended to adjust the cell density to 2x10⁵ cells/ml. This cell suspension was transferred to a 6-well plate at 2 ml/well and the cells were treated with different concentrations (0, 3, 6, and 9 µM) of hirsutanol A for 48 h. The cells were then collected and resuspended in medium, JC-1 staining solution was added and then the suspension was inverted several times to mix. The cells were cultured in the incubator at 37˚C for 30 min. Following centrifugation (300 x g at 4˚C and 5 min), the supernatant was discarded and the cells were washed twice with JC-1 buffer (1X). The cells were then observed under a fluorescence microscope. Each sample was viewed from five fields with roughly the same number of cells.

Jurkat cells treated with different concentrations (0, 3, 5 and 6 µM) of hirsutanol A and/or PFTα (10 µM) were collected and washed twice with PBS. Then, lysis buffer (80:10:10:1 ratio of RIPA lysate (Beyotime Institute of Biotechnology), protease inhibitor, phosphatase inhibitor and PMSF) was added, and cells were lysed on ice for 30 min. Following centrifugation at 4,000 x g for 10 min at 4˚C, the supernatant was obtained and the protein concentration was measured via the bicinchoninic acid method. The supernatant was mixed with SDS-PAGE protein loading buffer (5X) and the mixture was placed in a metal bath at 100˚C for 5 min to denature the protein. Protein samples (20 µg/lane) were separated via 10% SDS-PAGE (the current and time were appropriately adjusted according to different proteins), followed by transfer onto Immobilon^® polyvinylidene difluoride membranes (Sigma-Aldrich; Merck KGaA) and blocking with 5% skimmed milk at room temperature for 1 h. The membrane was incubated with the aforementioned primary antibodies at 4˚C overnight and then incubated with a secondary antibody for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence kit (EMD Millipore) and images were captured on a chemiluminescence imager (Vilber Lourmat). GAPDH was used as the loading control. Protein expression was semi-quantified using ImageJ version 2.0 software (National Institutes of Health).

The data were analyzed with SPSS 19.0 software (SPSS, Inc.) and expressed as the mean ± standard deviation of at least three independent experiments. Differences between two groups were assessed using the paired Student's t-test. Differences amongst multiple groups were evaluated using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The antiproliferative effect of hirsutanol A on the T-ALL cell line Jurkat was examined using a CCK-8 assay. Jurkat cells were treated with a series of hirsutanol A concentrations from 0.71 to 11.44 µM for 48 h. As presented in Fig. 2A, hirsutanol A markedly reduced the viability of Jurkat cells in a concentration-dependent manner, with an IC₅₀ value of 5.16 µM. Therefore, three concentrations (3, 6 and 9 µM) were selected in the subsequent experiments. Furthermore, human T lymphocyte H9 cells were treated with a series of hirsutanol A concentrations from 2 to 32 µM for 48 h, displaying an IC₅₀ value of 16.22 µM (Fig. 2C). These results indicated that hirsutanol A was less toxic to the normal cell line compared with the T-ALL cell line. Consistent with the dose effect of hirsutanol A, the viability of Jurkat cells was also inhibited in a time-dependent manner. Jurkat cells were then treated with 5 µM hirsutanol A, and cytotoxicity was assessed at different time points after treatment. At 24 h, the cell viability rate of Jurkat cells was 89.24±2.58%, which declined to 21.83±0.61% at 72 h (Fig. 2B). Thus, these results indicated that hirsutanol A inhibited the viability of T-ALL Jurkat cells in a dose- and time-dependent manner.

It is well known that cell cycle arrest is a key intracellular event contributing to reduced cell proliferation (21). It was therefore assessed whether hirsutanol A altered the cell cycle progression of Jurkat cells via flow cytometric analysis. As presented in Fig. 3, after 48 h of hirsutanol A treatment, the population of Jurkat cells in the G₂ phase increased significantly, whereas there was no significant change in the proportion of cells in the S phase compared with the control group, suggesting that hirsutanol A blocked the cell cycle of Jurkat cells in the G₂ phase.

To further clarify whether the inhibitory effect of hirsutanol A on Jurkat cell viability resulted from the induction of apoptosis, Jurkat cells were treated with a series of concentrations of the compound for 48 h, stained with Hoechst 33258 and then visualized under a fluorescence microscope. As presented in Fig. 4A, with increasing hirsutanol A concentrations, Jurkat cells exhibited typical morphological characteristics of apoptosis, including nuclear fragmentation and chromatin condensation. The induction of apoptosis was further demonstrated via flow cytometric analysis of cells stained with Annexin V-FITC/PI. The results indicated that, compared with the control group, hirsutanol A significantly induced the apoptosis of Jurkat cells in a dose-dependent manner (Fig. 4B and C). The percentages of total apoptotic cells following hirsutanol A treatment for 48 h were 15.94±0.33% at 3 µM, 47.53±0.74% at 6 µM and 81.25±1.42% at 9 µM, compared with 5.72±0.31% in the control group.

The mitochondrial pathway is one of the most classical pathways of cell apoptosis (22). In the early stage of cell apoptosis, the mitochondrial structure undergoes certain crucial changes, one of which is loss of the mitochondrial membrane potential (23,24). Therefore, in the present study, changes in the mitochondrial membrane potential of Jurkat cells after 48 h of hirsutanol A treatment at different concentrations were detected via JC-1 staining. As presented in Fig. 4D, the green fluorescence was markedly increased in a hirsutanol A dose-dependent manner, indicating that cells lost their mitochondrial membrane potential following hirsutanol A treatment.

Consistent with the aforementioned results of hirsutanol A inducing apoptosis, compared with the control group, the expression levels of cCaspase-3 and cPARP, well-known apoptotic markers (25), increased significantly following hirsutanol A treatment in a dose-dependent manner (Fig. 4E and F). Furthermore, the expression levels of Puma were significantly increased, whereas the expression of Bcl-2 was significantly decreased after hirsutanol A treatment compared with the control group. As hypothesized, compared with the control group, the expression of p53 in Jurkat cells increased significantly after hirsutanol A treatment in a concentration-dependent manner.

As p53 is one of the master regulators of cell proliferation and cell death (26), whether the induction of apoptosis of Jurkat cells by hirsutanol A was dependent on p53 was assessed. For this, Jurkat cells were treated with the p53 inhibitor PFTα in combination with hirsutanol A. As presented in Fig. 5, apoptosis induced by hirsutanol A was significantly inhibited from 48.92±0.83 to 19.24±0.46% by PFTα. Consistently, the expression levels of proapoptotic proteins (p53, Puma, cCaspase-3 and cPARP) were significantly decreased in the group treated with hirsutanol A + PFTα compared with those in the group treated with hirsutanol A alone, whereas the expression of the antiapoptotic protein Bcl-2 was significantly upregulated (Fig. 5). Collectively, the present results indicated that hirsutanol A inhibited the proliferation of Jurkat cells by inducing cell cycle arrest and p53-dependent apoptosis.