The human cell lines Jurkat (acute T cell leukemia; STCC10904G) and CEM/C1 (T lymphoblast; STCC10910G) were purchased from Wuhan Servicebio Technology Co., Ltd, Wuhan, China . CEM-C7H2 (acute T lymphoblastic leukemia; GD-C60404492) and HL-7702 (normal liver; GD-C60306615) were purchased from Shanghai Guandao Bioengineering Co., Ltd, Shanghai, China. All cell lines were validated by STR profiling and tested negative for mycoplasma. All cells were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 5% FBS (Biological Industries, Beijing, China) in a 37 °C humidified incubator with 5% CO${}_2$. The following antibodies were used in this study: $\beta$-actin (af7018, Affinity, Changzhou, China), Caspase-3 (9662S, Cell Signaling Technology (CST), Boston, MA, USA), Cleaved-Caspase-9 (7237S, CST), PARP (9542S, CST), Bcl-2 (4223S, CST), Bim (2933S, CST), GR (12041S, CST), P-GR (4161S, CST), NOTCH1 (4147, CST), CDK2 (ab32147, Abcam, Boston, MA, USA), Cyclin E1 (ab33911, Abcam), C-Myc (ab32072, Abcam), JAK2 (ab108596, Abcam), P-JAK2 (381556, ZEN-BIO), STAT3 (ab68153, Abcam), P-STAT3 (ab76315, Abcam), PI3K (4249S, CST), P-PI3K (4228S, CST), AKT (4685S, CST), and P-AKT (11054, SAB, College Park, MD, USA).

A solution of Methyl 3,5-dichloro-2-oxoindoline-3-carboxylate (1.0 g, 3.85 mmol) and N-benzyl-2-(1H-indol-3-yl) ethan-1-amine (0.96 g, 3.85 mmol) in 10 mL of MeCN was prepared. To this solution, DIPEA (2.01 mL, 11.54 mmol) was slowly added at 0 °C. The resulting mixture was then stirred at room temperature for 2 hours. Finally, the mixture was concentrated under reduced pressure. The crude product was purified by flash column chromatography (DCM/MeOH = 300:1–150:1) to obtain the product (1.15 g, yield 63%). Methyl 3-((2-(1H-indol-3-yl) ethyl) (benzyl) amino)-5-chloro-2-oxoindoline-3-carboxylate: white solid, m. p: 153.2–154.5 °C ${}^1$H nuclear magnetic resonance (NMR, 600 MHz, CDCl3) $\delta$ 8.56 (br. s., 1 H), 7.85 (br. s., 1 H), 7.57 (d, J = 7.31 Hz, 2 H), 7.55 (d, J = 2.07 Hz, 1 H), 7.37 (t, J = 7.55 Hz, 2 H), 7.21–7.29 (m, 3 H), 7.11 (td, J = 7.35, 1.51 Hz, 1 H), 6.94–7.00 (m, 2 H), 6.80 (d, J = 8.26 Hz, 1 H), 6.67 (s, 1 H), 4.13 (s, 2 H), 3.75 (s, 3 H), 2.96–3.00 (m, 1 H), 2.86–2.87 (m, 1 H), 2.58 (td, J = 9.70, 5.72 Hz, 2 H); ${}^{13}$C NMR (150 MHz, CDCl3) $\delta$ 176.9, 169.3, 140.2, 140.1, 136.1, 130.3, 128.7, 128.4, 128.3, 128.2, 127.3, 127.2 , 126.0, 121.8, 121.7, 119.0, 118.5, 113.5, 111.8, 111.1, 76.6, 55.7, 53.4, 52.5, 25.3. High-resolution mass spectrometry (HRMS) (ESI) m/z calcd. for C${}_{27}$H${}_{24}$O${}_3$N${}_3$Cl [M + Na]${}^{+}$: 496.13984, found: 496.13843. Purity ($>$98%).

Cells (1.5 $\times$ 10${}^4$ cells/well) were treated with different concentrations of both LWX-473 (2, 4, 8, 12, 16 µM), with or without Dexamethasone (DEX) (100 µM), in 96-well plates for 24, 48, and 72 h. After incubation, 10 µL of MTT (5 mg/mL) was added to each well and incubated for 4 h. Later, 100 µL of DMSO was added to dissolve the formazan crystals and quantified at 570 nm using a Synergy2 modular Multi-Mode Reader (BioTek, Winooski, VT, USA). The cell viability rates were evaluated using the following equation: survival rate (%) = (OD of treatment – OD of control)/OD of control $\times$ 100. Before the addition of the MTT reagent, the cellular morphology of the control or treated cells was recorded using a Nikon inverted microscope [13].

For apoptotic detection, cells (20 $\times$ 10${}^4$ cells/well) were inoculated into a six-well plate and treated with different concentrations of LWX-473 (4, 8, 12 µM) and/or DEX (100 µM) for 48 h. Afterward, the cells were collected, washed in PBS, and stained using the Annexin V FITC/PI apoptosis detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The stained cells were then analyzed using a Flow cytometer (ACEA NovoCyte, Miami, FL, USA) [14].

For the cell cycle analysis, Cells (20 $\times$ 10${}^4$ cells/well) were treated with different concentrations of LWX-473 (4, 8, 12 µM) and/or DEX (100 µM) for 48 h. The cells were then collected and fixed with 70% ethanol overnight at –20 °C. Afterward, the cells were washed twice with PBS and incubated in the dark with Triton X-100, RNase A, and PI for 30 min at 37 °C. Finally, the cells were analyzed using a flow cytometer (ACEA NovoCyte) [15].

The cells (2 $\times$ 10${}^4$ cells/well) were seeded into a 6-well plate and treated with DMSO, DEX (100 µM), LWX-473 (8 µM), and DEX + LWX-473 (100 + 8 µM) for 48 h. Afterward, the cells were collected and washed twice with precooled PBS. They were then stained with the Hoechst 33258 Staining Kit (Beyotime, China). Finally, a suitable volume of PBS was used to resuspend the cells, which were then dropped and mounted on a slide for observation of apoptotic bodies using a Nikon fluorescence microscope [14].

The mitochondrial membrane potential (MMP) variations were detected using the MMP assay kit with JC-1 (Beyotime, China) following the manufacturer’s protocol. Briefly, the collected cells were incubated with JC-1 dye solution for 20 min. Afterward, the cells were collected and washed (600 g, 4 °C) in JC-1 dye buffer (1$\times$) for 3–4 min. The cells were then resuspended in an appropriate amount of JC-1 dye buffer (1$\times$) and observed under a Nikon fluorescence microscope [16].

Cells (5 $\times$ 10${}^5$/dish) were treated with DMSO, LWX-473 (8 µM), DEX (100 µM), and LWX-473 + DEX (8 + 100 µM) for 48 h. After incubation, the cells were collected and washed. An appropriate amount of cell lysate (RIPA:PMSF = 100:1) was added, and the mixture was incubated at 4 °C for 30 min. Cell debris was removed by centrifugation at 12,000 rpm for 15 min at 4 °C. Protein quantification was performed using the BCA assay (Solarbio, Beijing, China). The protein samples were then mixed with 5$\times$ loading buffer and boiled at 100 °C for 3–5 min. Equal amounts of protein from each sample were separated by sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h and then incubated overnight with primary antibodies at 4 °C. After incubation with the secondary antibodies (H + L; DyLightTM 800 4X PEG Conjugate; CST, 5151, Boston, MA, USA), the membranes were scanned using an Odyssey scanner (Li-Cor Biosciences, Lincoln, NE, USA). Densitometry analyses of the western blot bands were conducted using Image Studio software (Vesion 3.1, texas instruments, Dallas, TX, USA) [16, 17].

All statistical analyses were performed using the ANOVA test, followed by post-hoc analysis using GraphPad Prism9 Software (GraphPad Software, Inc., San Diego, CA, USA). All experiments were conducted in triplicates with three independent experiments. The data were expressed as mean $\pm$ SD, and a p-value (*p 0.05, **p 0.01, ***p 0.001, ****p 0.0001) was considered statistically significant.

Natural indole compounds have shown promise in addressing drug resistance [18, 19]. However, these compounds are naturally produced in small amounts. To overcome this limitation, a new indole derivative, methyl 3-((2-(1H-indol-3-yl)ethyl)(benzyl)amino)-5-chloro-2-oxoindoline-3-carboxylate (LWX-473), was designed and synthesized using a previously reported method. The synthesis involved the reaction of methyl 3,5-dichloro-2-oxoindoline-3-carboxylate with N-benzyl-2-(1H-indol-3-yl)ethan-1-amine, as depicted in Fig. 1A–D.

Fig. 1.

Synthesis and characterization of LWX-473. (A) The synthesis of compound LWX-473. (B) The ${}^1$H NMR of compound LWX-473. (C) The HRMS of compound LWX-473. (D) The ${}^{13}$C NMR of compound LWX-473. LWX-473, methyl 3-((2-(1H-indol-3-yl)ethyl)(benzyl)amino)-5-chloro-2-oxoindoline-3-carboxylate; NMR, nuclear magnetic resonance; HRMS, high-resolution mass spectrometry.

The T-cell leukemic Jurkat cells have been reported to exhibit resistance to glucocorticoids (GCs) due to qualitative (GR mutations) or quantitative defects in GR expression [20]. To investigate the resistance of Jurkat cells to DEX-induced cell death, we treated the cells with different doses of DEX for varying durations and observed their response. The results showed that even at a high dose of 100 µM, DEX was unable to induce cell death in Jurkat cells (Fig. 2A–C). To assess the sensitivity of the novel indole derivative LWX-473 to DEX, we treated Jurkat cells with LWX-473 alone, DEX alone, or a combination of LWX-473 and DEX for different durations (24, 48, and 72 h). The results demonstrated that LWX-473 inhibited the growth of Jurkat cells in a time- and dose-dependent manner. Notably, the co-treatment of LWX-473 with DEX exhibited a more pronounced growth inhibition compared to LWX-473 alone at 48 and 72 h. These findings suggested a synergistic growth inhibition effect of LWX-473 and DEX on Jurkat, CEM/C1, and CEM-C7H2 cells (Fig. 2D–F; Supplementary Fig. 1). Morphological changes such as cellular loss, membrane bubbling, and cell death, leading to a loss of cell viability, further contributed to the synergistic effect of LWX-473 and DEX in Jurkat cells (Fig. 2G). Importantly, LWX-473 displayed minimal toxicity on non-tumorous human normal liver cells HL7702, indicating its potential selectivity towards cancer cells (Fig. 2H).

Fig. 2.

Effect of LWX-473 combined with Dexamethasone on the proliferation of Jurkat cells. (A) Flow cytometric analysis of indicated Dexamethasone (DEX) concentrations on the proliferation of Jurkat cells. (B) Densitometry analysis of apoptotic effects of different DEX concentrations on Jurkat cells at 48 h. (C) Cell viability effect of different concentrations of DEX on Jurkat cells at indicated times. (D–F) The effect of various concentrations of LWX-473 on the proliferation of Jurkat cells in the presence of DEX (100 µM) for 24 (D), 48 (E), and 72 h (F) of incubation. (G) Microscopic images (20$\times$) showing the changes in cell density and morphology of Jurkat cells treated with DEX or/and LWX-473 at the indicated time points. scale bar = 100 µm. (H) Proliferation of human non-tumorous liver cells, HL7702 with different concentrations of LWX-473 compound. * p 0.05, ** p 0.01, *** p 0.001, **** p 0.0001 vs. control group. DMSO, dimethylsulfoxide.

Glucocorticoid resistance in leukemia cells allows them to evade normal apoptosis processes and survive. Therefore, we investigated whether LWX-473 could induce apoptosis in Jurkat cells. We observed that LWX-473 could induce apoptosis in a dose-dependent manner. Moreover, when combined with DEX, LWX-473 exhibited a more pronounced induction of apoptosis, overcoming the drug resistance effect of DEX (Fig. 3A,B). Hoechst staining further confirmed these findings, as treatment with DEX or LWX-473 alone did not significantly induce apoptosis, whereas the combination of DEX and LWX-473 resulted in significant apoptosis and cellular fragmentation, characterized by the presence of apoptotic bodies (Fig. 3C). Furthermore, we examined the levels of various apoptotic markers and found that the combination of DEX and LWX-473 significantly increased the levels of cleaved PARP, cleaved caspase3, cleaved caspase9, BIMEL, and BIMSL. Additionally, it significantly reduced the levels of NOTCH1, c-Myc, and Bcl-2, promoting the apoptotic effects, compared to the control (Fig. 3D,E). These results suggest that the combination of DEX and LWX-473 enhances apoptosis in Jurkat cells by modulating key apoptotic proteins.

Fig. 3.

LWX-473 combined with DEX induces apoptosis of Jurkat cells. (A) Flow cytometric analysis to detect the effect of LWX-473 with or without DEX on apoptosis of Jurkat cells. (B) Apoptotic effects of different combinational concentrations on Jurkat cells at 48 h of incubation. (C) Jurkat cells treated with various indicated combinations were stained with Hoechst (20$\times$) and analyzed for apoptotic bodies. scale bar = 200 µm. (D) Western blot images showing variations in indicated protein levels after the intervention of different indicated drug combinations, $\beta$-actin was used as loading control. (E) Graph showing variations in protein expression levels of incited proteins after the indicated cotreatments. Data were represented as mean $\pm$ standard deviation (SD), * p 0.05, *** p 0.001, **** p 0.0001 vs. the control group.

As an early event in apoptosis, there is a decrease in mitochondrial membrane potential (MMP) [21]. To assess the effect of the combination of LWX-473 and DEX on MMP, we used JC-1, a fluorescent probe to detect MMP. In Fig. 4A, compared to the other groups, the combination group exhibited stronger green fluorescence (JC-1 monomers) and weaker red fluorescence (JC-1 aggregates), indicating a significant decrease in MMP (Fig. 4B). Furthermore, it is known that DEX can induce G1 phase cell cycle arrest in cancer cells [22]. To investigate whether the co-administration of LWX-473 and DEX affects the cell cycle of Jurkat cells, we treated the cells with different concentrations of LWX-473 and DEX individually. The results demonstrated that LWX-473 could dose-dependently induce G1 phase cell cycle arrest in Jurkat cells (Fig. 4C,D). Additionally, we examined G1 phase-related proteins using western blot analysis. When LWX-473 was combined with DEX, the levels of CDK2 and cyclin E1 proteins were significantly downregulated (Fig. 4E,F) in Jurkat cells, indicating the blockade of the cell cycle in the G1 phase.

Fig. 4.

LWX-473 combined with DEX induced mitochondrial membrane potential (MMP) changes and G1 phase cell cycle arrest of Jurkat cells. (A) Cells treated with indicated combinations for 48 h were stained with JC-1 and analyzed by fluorescent microscope (40$\times$) to differentiate the JC-1 monomers and polymers. scale bar = 100 µm. (B) Cells treated at indicated concentrations were analyzed for green and red fluorescence stating mitochondrial damage. (C) Flow cytometric analysis to detect the effect of LWX-473 (4, 8,12 µM) with or without DEX (100 µM) on the cell cycle of Jurkat cells. (D) Densitometry of the cells undergoing arrest at various stages of the cell cycle 48 h of incubation. (E) Western blot images showing variations in indicated protein levels after the intervention of different indicated drug combinations, $\beta$-actin was used as the loading control. (F) Graph showing variations in protein expression levels of incited proteins after the indicated cotreatments. Data were represented as mean $\pm$ SD, * p 0.05, ** p 0.01, *** p 0.001, **** p 0.0001 vs. the control group.

Inhibition of the JAK/STAT pathway has been shown to enhance the sensitivity of DEX-induced apoptosis. To investigate the effect of LWX-473 and DEX on this pathway, we analyzed the protein expression of phosphorylated JAK2, STAT3, PI3K, and AKT. We observed that the combination of LWX-473 and DEX significantly reduced the phosphorylation of JAK2. Neither DEX nor LWX-473 alone had an impact on the expression of phosphorylated JAK2 protein compared to the control (Fig. 5A). Furthermore, we found that co-treatment with LWX-473 and DEX significantly reduced the phosphorylation of PI3K and AKT proteins, indicating their involvement in the regulation of this pathway (Fig. 5B). These findings suggest that the combinational treatment of LWX-473 and DEX can overcome glucocorticoid resistance in Jurkat cells through the JAK2/STAT3/PI3K/AKT signaling pathway. Additionally, during co-treatment, there was an increased expression of phosphorylated GR (Ser211), confirming the escape of cells from the chemoresistance of glucocorticoids in Jurkat cells.

Fig. 5.

LWX-473 enhances glucocorticoid DEX sensitivity of Jurkat cells by inhibiting JAK2/STAT3/PI3K/AKT pathway. (A) Western blot images showing variations in indicated protein levels after the intervention of different indicated drug combinations, $\beta$-actin was used as the loading control. (B) Graph showing variations in protein expression levels of incited proteins after the indicated cotreatments. (C) Signal pathway diagram of LWX-473 overcoming glucocorticoid resistance in Jurkat cells. Data were represented as mean $\pm$ SD, * p 0.05, ** p 0.01, *** p 0.001, **** p 0.0001 vs. the control group.