The human ALL lines Jurkat and Molt-4 were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China). Jurkat cells and Molt-4 cells were routinely cultured in RPMI-1640 (Gibco) medium supplemented with 10% fetal bovine serum (Gibco) with 1% glutamine-penicillin (Beyotime). Cells were cultured at 37°C with 5% CO₂.

Following institutional guidelines, blood samples were obtained following informed consent from six patients with T-ALL and six healthy patients. Patients were recruited from the Zhejiang Provincial People's Hospital in November and December, 2018. Lymphocytes were isolated from EDTA-treated blood by density gradient centrifugation using Lymphocyte Separation Medium (TBD) as per the manufacturer's instructions. Peripheral anti-coagulated blood (4 ml) was mixed with calcium-magnesium-free phosphate-buffered saline (PBS) (ratio 1:1). Samples were layered onto 5-ml lymphocyte separation medium and centrifuged at 300 × g for 25 min at room temperature. The interface layer was centrifuged at 50 × g for another 10 min at room temperature, and the pellet was collected. The enriched cells were cultured in RPMI-1640 medium supplemented with 20% fetal bovine serum.

The relevant ethics approval was granted by the Zhejiang Provincial People's Hospital Ethics Committee.

GA with a purity of >98% was purchased from Chengdu Must Bio-Technology Co. Ltd. It was dissolved in dimethyl sulfoxide and then stored at −20°C for further analyses.

Cell viability after GA treatment was determined using a Cell Counting Kit-8 (CCK-8) assay (Beyotime). Cells were seeded in 96-well plates (1×10⁵ cells/well) and treated with different concentrations (1.2, 2.4 and 4.8 µM) of GA, and controls were treated with 0.1% DMSO. The plates were incubated for 24, 48 and 72 h. After incubation, 20 µl of CCK-8 solution was added to each well. The plates were further incubated at 37°C in a CO₂ incubator for 3 h. Optical density was analyzed using a microplate reader at 450 nm.

The cell cycle distribution of Jurkat cells after GA treatment (at various concentrations) were analyzed using a Cell Cycle Staining Kit (LianKe) following the manufacturer's instructions. Jurkat cells were suspended and mixed in DNA staining solution (50 µg/ml RNase A for 20 min at room temperature, followed by 50 µg/ml propidium iodide for 10 min in the dark). After the 30 min treatment, distribution of cell cycle stages were detected by flow cytometry (Becton Dickinson). In the present analysis, each phase of the cell cycle was recorded as a percentage.

GA-treated Jurkat cells (at various concentrations) were suspended in 2-ml PBS to obtain a cell density of 1–5×10⁶/ml. The cells were tested using a PI Apoptosis Detection Kit (MultiSciences Biotech Co.) according to the manufacturer's protocol. Briefly, 10-µl PI was added to the suspension, incubated for 20 min at room temperature, and maintained in the dark. The percentage of apoptotic cells was examined by flow cytometry. The results were analyzed using Novo Express software.

Jurkat cells were treated with various concentrations of GA and immersed overnight. Coverslips were treated with 0.2-mg/ml poly-L-lysine for 4 h prior to placing them on coverslips. After 1.5 h of incubation, the cells were placed firmly on the coverslips. These were fixed with 4% PFA (Beyotime) for 10 min at room temperature. Samples were fixed in 0.1% Triton X-100 (Beyotime) and 5% BSA prior to incubation. The primary antibody against p-GSK3β S9 (1:200, Cell Signaling Technology, cat. no. 5558) was added, and the specimens were incubated overnight at 4°C. The next day, the cells were washed with PBS three times and the appropriate fluorescent secondary antibody Alexa Fluor 488 was added (1:1,000, Beyotime, cat. no. A0423). The samples were incubated at room temperature for 1 h. Cells were stained with DAPI (Beyotime) to allow visualization of the nucleus. Finally, images were obtained using a fluorescence microscope (Olympus).

Jurkat cells were plated in 6-well plates and treated with different concentrations (1.2, 2.4 and 4.8 µM) of GA, and controls were treated with 0.1% DMSO. After treatment, the cells were collected and suspended with PBS at a density of 1.5×10⁶ cells/ml, followed by co-staining with monodansylcadaverine (MDC) plus DAPI for another 30 min at 37°C. Approximately 10–20 µl of the cell suspension was placed onto a glass slide and covered with a cover glass. The MDC staining was examined and photographed using a fluorescence microscope.

Lysed cells in RIPA lysis buffer (Beyotime) were maintained on ice for 30 min and centrifuged at 14,000 × g for 10 min at 4°C. The protein concentration in the supernatant was verified by BCA Protein Assay (Thermo Scientific Inc). Total proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were blocked in 5% de-fatted milk for 1 h at room temperature and then incubated with primary antibodies at 4°C overnight. The matched secondary antibody (1:3,000, Beyotime, cat. nos. A0208, A0192) was incubated for 2 h at room temperature. Protein levels were detected using an enhanced chemiluminescence reagent and quantified by ImageJ software. The antibodies used in the study were as follows: Anti-CyclinB (1:1,000, Abcam, cat. no. ab32053), anti-CDK1 (1:500, Beyotime, cat. no. AF0111), anti-Beclin1 (1:1,000, Thermo, cat. no. MA5-15825), and anti-LC3-II (1:1,500, Sigma, cat. no. 610832); anti-Bax (1:1,000, Abcam, cat. no. ab32503), anti-Bcl-2 (1:1,000, Abcam, ab182858), anti-cleaved caspase3 (1:1,000, Cell Signaling Technology, cat. no. 9661S), anti-cleaved PARP (1:1,000, Abcam, cat. no. ab191217) anti-p-GSK3β S9 (1:1,000, Cell Signaling Technology, cat. no. 5558), and anti-Atg7 (1:1,000, Cell Signaling Technology, cat. no. 8558s); anti-β-catenin (1:1,000, Cell Signaling Technology, cat. no. 19807S), anti-c-Myc (1:1,000, Cell Signaling Technology, cat. no. 2278s), and anti-CyclinD1 (1:1,000, Abcam, cat. no. ab134175); and anti-β-actin (1:3,000, Beyotime, cat. no. AF5001).

Jurkat cells were seeded in 6-well plates and transfected using Lipofectamine 3000 (Invitrogen) with recombinant β-catenin and blank plasmids. The procedure was performed in accordance with the manufacturer's instructions. After 24 h, the cells were treated with GA (2.5 µM). Observations were made after 48 h using an inverted fluorescence microscope. The expression of marker proteins in stably transfected cells were analyzed by western blot analysis.

Data are presented as mean ± standard deviation. Data were analyzed using SPSS21.0. The data were collected from at least three independent experiments. Significance of the difference between two groups means of data sets was determined using the Student's t-test. Multiple comparisons test was analyzed by one-way ANOVA (Dunnett's correction). Results with P<0.05 and P<0.01 were considered statistically significant.

To detect the anti-proliferative effects of GA in vitro, the human leukemia cell lines Jurkat and Molt-4 cells were exposed to increasing concentrations of GA. GA reduced the viability of Jurkat and Molt-4 cells in a dose-dependent manner (Fig. 1A-B). The IC50 values of GA for Jurkat and Molt-4 were 2.23 and 3.03 µM, respectively. Jurkat and Molt-4 cells were treated with GA (1.2, 2.4 and 4.8 µM) for 0, 24, 48 and 72 h. At the higher concentrations (2.4 and 4.8 µM), GA inhibited the growth of cells significantly, and the inhibition rate increased with time. These results suggested that GA cytotoxicity is dose- and time-dependent in leukemia cells.

To test whether GA could induce Jurkat cell cycle arrest, cell cycle distribution was performed. The results demonstrated that the exposure of cells to GA at concentrations of 2.4 and 4.8 µM led to a remarkable increase in G2/M phase, with a corresponding decrease in the S phase compared with exposure of cells to control or 1.2-µM GA (Fig. 1C). To determine the underlying mechanisms causing G2/M phase arrest in Jurkat cells, we examined the effect of GA on the expression of CyclinB and CDK1 (key regulators of G2/M transition). Western blot results revealed that the protein levels of CyclinB and CDK1 were significantly decreased by GA (Fig. 1D). Taken together, these results indicate that GA may inhibit the proliferation of Jurkat cells by inducing cell cycle arrest at the G2/M phase.

It is known that apoptosis is programmed cell death. Thus, we examined whether GA induces apoptosis in cancer cells. Using a flow cytometric analysis the presence of apoptosis in GA-treated cells was identified. GA treatment induced significant apoptosis in Jurkat cells in a dose-dependent manner (Fig. 2A). Apoptosis was detected by western blot analysis (Fig. 2B). It was noted that GA treatment upregulated the expression of c-PARP, c-Caspase-3, and Bax but downregulated the expression of Bcl-2 compared with control treatment. Overall, these results demonstrated that GA upregulated the expression of pro-apoptotic proteins, thus causing apoptosis in Jurkat cells.

We observed whether autophagic vacuoles were formed when Jurkat cells were treated with GA by MDC staining (Fig. 2C). The number of MDC-positive fluorescent points in GA-treated cells was markedly greater than that in the control group. These results demonstrated that the inhibitory effect of GA on cell viability is related to the induction of autophagy. To investigate whether GA regulated the process of autophagy, the protein expression of autophagy-related markers were assessed by western blot analysis (Fig. 2D). Significant upregulation of Atg7, Beclin-1, and LC3-II occurred in GA-treated groups. These results suggest that GA may encourage autophagy of Jurkat cells.

To identify other mechanisms involved in the inhibitory effect of GA, we focused on the Wnt pathway (Fig. 3A). The protein levels of β-catenin, as well as downstream proteins CyclinD1 and c-Myc, were significantly decreased in a dose-dependent manner after GA treatment. GSK3β, a highly effective kinase within the β-catenin destruction complex, phosphorylated β-catenin and promoted its destruction by proteasomes. Therefore, GA suppressed the expression of p-GSK3β S9 and subsequently promoted β-catenin degradation to reduce β-catenin nuclear import. Immunostaining results showed that after GA treatment (Fig. 3B), the expression of p-GSK3β S9 decreased. Our findings indicated that GA inhibits the growth of Jurkat cells by regulating the Wnt/β-catenin pathway.

Overexpression of β-catenin inhibited the apoptosis of Jurkat cells. To further confirm the role of β-catenin in GA-induced regulation of Jurkat cell proliferation and apoptosis, we transfected the cells with plasmid-carrying β-catenin. We established that the overexpression of β-catenin protects Jurkat cells from apoptosis. Flow cytometry results showed that after β-catenin plasmid transfection, a proportion of the apoptosis (induced by GA) recovered (Fig. 4A). Western blot results revealed that the transfection of β-catenin plasmid reversed the increase of Bax and c-caspase-3 and decreased Bcl-2 induced by GA (2.4 µM) (Fig. 4B). These results confirmed the hypothesis that the overexpression of β-catenin significantly inhibited the apoptosis of Jurkat cells. After transfection of β-catenin plasmid into GA-treated Jurkat cells, the protein levels of β-catenin, CyclinD1, GSK-3β, p-GSK3β S9, and c-Myc were measured using western blot analysis (Fig. 4C). The results showed that the expression of GSK3β significantly decreased in GA-treated Jurkat cells transfected with β-catenin compared with that in cells transfected with the negative control. In the negative control group, the expression of β-catenin, p-GSK3β S9, CyclinD1, and c-Myc are significantly increased.

GA toxicity was then examined. The results showed that GA is toxic to Jurkat cells. To investigate the potential of GA for cancer therapy, we evaluated the effects of GA on lymphocyte cells from healthy individuals and patients with T-ALL. Apoptotic assays revealed that cell viability of healthy individuals decreased slightly when treated with GA (Fig. 5). Conversely, GA treatment induced significant apoptosis in lymphocyte cells from patients with T-ALL.