The human MCL cell lines Jeko-1 and Granta519 were purchased from American Type Culture Collection (Manassas, VA, USA). The human MCL cell lines Z138, Mino and Rec-1 were provided by Dr Xue Han of Tianjin Medical University Cancer Hospital (Tianjin, China). Human peripheral blood mononuclear cells (PBMCs) were acquired from three healthy normal donors (2 males, 1 female; age, 24, 24 and 28 years, respectively) using Ficoll-Hypaque density gradient centrifugation at 568 × g for 30 min at room temperature. Informed consent was obtained from each healthy donor. The present study was approved by the institutional review board of Shanghai Tenth People's Hospital (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). All cells were maintained in a humidified atmosphere containing 5% CO₂ at 37°C.

DHCE was synthesized from celastrol using the method previously described by Klaic et al (25). DHCE solution (2 mM) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), stored at −20°C and diluted with cell culture medium to obtain various concentrations. Antibodies against caspase-3 (cat. no. 9665, 1:1,000), cleaved caspase-8 (cat. no. 9496, 1:1,000), caspase-9 (cat. no. 9508, 1:1,000), B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) (cat. no. 2772, 1:1,000), Bcl-2 (cat. no. 2872, 1:1,000), Bcl-extra large (Bcl-xL) (cat. no. 2764, 1:1,000), Akt (cat. no. 2920, 1:2,000), phosphorylated (p)-Akt (cat. no. 4060, 1:2,000), p-p70S6K (cat. no. 9205, 1:1,000), p70S6K (cat. no. 2708, 1:1,000), p-4E-BP1 (cat. no. 9456, 1:1,000), 4E-BP1 (cat. no. 9644, 1:1,000), p-mTOR (Ser2481) (cat. no. 2974, 1:1,000), mTOR (cat. no. 2983, 1:1,000) and β-actin (cat. no. 3700, 1:1,000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Cyclin D1 (cat. no. ab134175, 1:1,000), cyclin-dependent kinase 4 (CDK4) (cat. no. ab108357, 1:1,000), CDK6 (cat. no. ab124821, 1:1,000), NF-κB inhibitor α (IκBα) (cat. no. ab32518, 1:1,000), p-IκBα (cat. no. ab92700, 1:1,000), NF-κB-p65 (cat. no. ab32536, 1:50,000) and p-NF-κB-p65 (cat. no. ab76302, 1:10,000) were obtained from Abcam (Cambridge, UK). The pan-caspase inhibitor Z-VAD-FMK was purchased from Selleck Chemicals (Houston, TX, USA). Cell Counting kit-8 (CCK-8) was obtained from Shanghai Yeasen Biotechnology Co., Ltd. (Shanghai, China) and the BD Pharmingen™ Annexin V/propidium iodide (PI) Apoptosis Detection kit was obtained from BD Biosciences (Franklin Lakes, NJ, USA).

Jeko-1, Z138, Mino, Rec-1 and Granta519 cells were plated into 96-well plates at a density of 2×10⁵ cells/ml and were treated with 0.2, 0.4, 0.6, 0.8 and 1.2 µM DHCE for 24, 48 and 72 h. PBMCs were plated into 96-well plates at a density of 3×10⁵ cells/ml and were treated with 1.6 µM DHCE for 48 h. After incubation at 37°C and 5% CO₂, 10 µl CCK-8 solution was added to each well and the cells were incubated for an additional 2 h at 37°C. Absorbance was then measured at 450 nm using a microplate reader.

Double-staining with the BD Pharmingen™ Annexin V/PI Apoptosis Detection kit was used to detect cell apoptosis. Following DHCE (0.4, 0.6 and 0.8 µM) and/or Z-VAD-FMK (50 µM) exposure for 48 h, 1×10⁵ Jeko-1 and Z138 cells were resuspended in 95 µl binding buffer and 5 µl fluorescein isothiocyanate-conjugated Annexin V after centrifugation at 142 × g and 4°C for 5 min. After incubation for 30 min at 4°C in the dark, 500 µl binding buffer and 20 µl PI solution were added to each cell suspension, and the BD FASCanto II flow cytometer (BD Biosciences) was used for analysis by FlowJo 10 (FlowJo LLC, Ashland, OR, USA). Apoptotic cells were identified as Annexin V⁺/PI⁻ (early apoptosis) and Annexin V⁺/PI⁺ (late apoptosis).

Following 0.4 and 0.8 µM DHCE exposure for 36 h, Jeko-1 and Z138 cells at a density of 2×10⁵ cells/ml were washed with cold PBS, and fixed in 70% ice-cold ethanol overnight at −20°C. The ethanol-fixed samples were washed with PBS, and incubated in 500 µl PI/RNase staining buffer (BD Biosciences) for 15 min at room temperature in the dark prior to flow cytometry. Results were analyzed by ModFit LT 3.2 (Verity Software House, Inc., Topsham, ME, USA).

Jeko-1 and Z138 cells (1×10⁴ cells/well) were mixed with RPMI-1640 media containing 10% FBS and 0.33% agar, and were layered on top of a base layer of 0.5% agar in each well of 6-well plates. After culturing for 2 weeks, cells were incubated with 0.5% crystal violet for 1 h at room temperature. Subsequently, images of cell colonies were captured using a digital camera after staining with extra crystal violet. The number of cell colonies was semi-quantified using ImageJ 1.51 software (National Institutes of Health, Bethesda, MD, USA).

Jeko-1 and Z138 cells were plated into a 6-well plate at a density of 4×10⁵ cells/well, and were incubated with or without 0.8 µM DHCE at 37°C for 48 h. Subsequently, all cells were incubated with 50 µM EdU (Guangzhou RiboBio Co., Ltd., Guangzhou, China) at 37°C for 1 h. After being fixed with 4% paraformaldehyde at room temperature for 30 min, the cells were treated with 0.5% Triton X-100 at room temperature for 10 min and washed three times with PBS. Thereafter, the cells were exposed to 100 µl 1X Apollo reaction cocktail at 37°C for 30 min and incubated with DAPI at room temperature to stain the cell nuclei for 5 min. A confocal laser-scanning microscope was used to detect stained cells.

Jeko-1 and Z138 cells were plated into a 6-well plate at a density of 4×10⁵ cells/well, and were incubated with or without 0.8 µM DHCE at 37°C for 48 h. After centrifugation at 142 × g and 37°C for 5 min and washing with PBS, cells were fixed in 4% paraformaldehyde at 37°C for 30 min and permeabilized with 0.1% Triton X-100 at 37°C for 10 min. After washing, cells were incubated with TUNEL (Beyotime Institute of Biotechnology, Shanghai, China) reaction mixture for 60 min at 37°C. Prior to plating on coverslips, cells were stained with DAPI for 5 min at room temperature in the dark. The cells were imaged under a fluorescence microscope (Zeiss Axiovert 25; Zeiss GmbH, Jena, Germany).

Jeko-1 and Z138 cells were incubated in lysis buffer [100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol] for protein extraction. Protein concentration was determined using the bicinchoninic acid method. Samples containing 30 µg protein were separated by 10 or 12.5% SDS-PAGE and were then transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk or 5% bovine serum albumin [Anlite (Shanghai) Pharmaceutical Technology Co., Ltd., Shanghai, China] at room temperature for 1 h, and were then incubated overnight at 4°C with primary antibodies. Subsequently, the membranes were probed with secondary antibodies [goat ant-mouse immunoglobulin G (IgG), cat. no. 926-32210, 1:1,000; goat ant-rabbit IgG, cat. no. 926-32211, 1:1,000; LI-COR Biosciences, Lincoln, NE, USA) for 60 min at room temperature, and the blots were detected using the Odyssey two-color infrared laser imaging system (LI-COR Biosciences).

A total of 8 male nude mice (athymic, BALB/C nu/nu; age, 6 weeks; weight, 17–20 g) were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All mice were maintained in an air-conditioned room at 24°C with a 12-hour light/dark cycle and 45% relative humidity. All mice had free access to water and food. All animal studies were approved by the institutional review board of the Shanghai Tenth People's Hospital (ID: SYXK 2011–0111). Human MCL Jeko-1 cells (10×10⁶) were suspended in 100 µl serum-free culture medium and were subcutaneously injected into the upper flank region of nude mice. When tumors were measurable, mice were randomly divided into the control and DHCE groups. In the DHCE group, mice were administered 3 mg/kg DHCE (DHCE was dissolved in 5% DMSO, 15% Tween-80 and saline), whereas in the control group, mice received 100 µl vehicle (5% DMSO, 15% Tween-80 and saline) daily for 19 consecutive days. Tumor size and mouse weight were measured, and the tumor volume was calculated as (length x width²) × 0.5. At the end of treatment, mice were sacrificed by cervical dislocation following intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg). Hematoxylin-eosin (26) and TUNEL staining of the tumor tissue sections were then performed.

Jeko-1 and Z138 cells were treated for 24 h with DHCE (0.4, 0.6, 0.8 and 1.0 µM) and/or bort-ezomib (8, 12, 16 and 20 nM) (Sigma-Aldrich; Merck KGaA). Combination index (CI) values were calculated using the Chou-Talalay equation (27): CI values <1 indicated synergism; CI values equal to 1 indicated an additive effect; and CI values >1 indicated antagonism.

Data are expressed as the means ± standard deviation. Statistical analysis was conducted using an unpaired Student's t-test or one-way analysis of variance followed by least significant difference test for multiple comparisons. All statistical analyses were performed using SPSS version 20.0 statistical analysis software (IBM Corp., Armonk, NY, USA). P≤0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1A, DHCE is a synthesized dihydro-analog compound with a molecular weight of 452.6. In the present study, five MCL cell lines (Jeko-1, Z138, Granta519, Mino and Rec-1) were treated with various doses of DHCE. After 48 h of DHCE exposure, cell viability was measured using a CCK-8 assay. MCL cell viability was decreased in a dose-dependent manner (Fig. 1B), and the 48 h half maximal inhibitory concentration values of DHCE in Jeko-1, Z138, Mino, Rec-1 and Granta519 cells were 0.65±0.10, 0.45±0.04, 0.42±0.01, 0.35±0.03 and 0.91±0.08 µM, respectively. Jeko-1 and Z138 cells were selected for subsequent analyses, as they have previously been used in numerous studies regarding MCL (28,29). Furthermore, a time-course study of DHCE further demonstrated that cell viability was inhibited in a time-dependent manner (Fig. 1C). Conversely, at concentrations as high as 1.6 µM, DHCE conferred minimal cytotoxic effects on normal PBMCs obtained from three healthy volunteers (Fig. 1D). To investigate the inhibitory effects of DHCE on tumorigenic potential, anchorage-independent colony formation in soft agar was compared between the control and treatment groups. DHCE treatment reduced the ability of Jeko-1 and Z138 cells to form tumorspheres in a dose-dependent manner, and colony formation was almost completely inhibited among Z138 cells treated with 0.8 µM DHCE (Fig. 1E). To further explore the effects of DHCE on cell proliferation, the integration of EdU was measured, in order to detect the levels of DNA synthesis; cell nuclei were stained with DAPI. The results demonstrated that DHCE suppressed DNA synthesis in MCL cells, thus indicating that DHCE inhibited proliferation of MCL cells (Fig. 1F). These findings suggested that DHCE may decrease cell viability and inhibit cell proliferation in a dose- and time-dependent manner; however, DHCE is not cytotoxic to normal cells.

The cyclin D1-CDK4 and cyclin D1-CDK6 complexes are critical for the G₁/S checkpoint, and regulate the progression of cells from G₁ phase to S phase (30). To evaluate the effects of DHCE on cell cycle progression, Jeko-1 and Z138 cells were treated with DHCE and analyzed by flow cytometry. The results demonstrated that following treatment with DHCE, the percentage of Jeko-1 and Z138 cells in the G₀/G₁ phase of the cell cycle was significantly increased (Fig, 2A). The present study also analyzed the expression levels of cell cycle regulatory proteins, and indicated that DHCE suppressed the expression of cyclin D1, CDK4 and CDK6 (Fig. 2B). Taken together, these data suggested that DHCE may induce G₀/G₁ phase arrest in a dose-dependent manner by inhibiting the expression of cell cycle regulatory proteins.

Evasion of apoptosis has a key role in tumorigenesis and chemotherapeutic resistance (31). To investigate whether apoptosis is associated with the mechanism underlying the cytotoxic effects of DHCE, Jeko-1 and Z138 cells were incubated with various concentrations of DHCE for 48 h. DHCE markedly increased the percentage of apoptotic cells in a dose-dependent manner, compared with in the control group (Fig. 3A). Furthermore, Jeko-1 and Z138 cells were simultaneously treated with DHCE and a pan-caspase inhibitor, Z-VAD-FMK. The results demonstrated that Z-VAD-FMK induced a significant reduction in DHCE-induced cell apoptosis (Fig. 3B; data of Z138 cells not shown), thus indicating that DHCE-mediated apoptosis is dependent upon caspase activity. These findings demonstrated that DHCE may induce MCL cell apoptosis in a dose-dependent manner.

A series of morphological alterations are triggered when apoptosis is activated, including cell shrinkage, aberrant chromatin compaction, DNA hydrolysis, nuclear fragmentation and the formation of apoptotic bodies (32). To directly confirm that DHCE promoted the apoptosis of MCL cells, a confocal laser-scanning microscope was used to detect the binding of TUNEL reagent with DNA fragments. Compared with in the control group, MCL cells treated with DHCE for 48 h exhibited chromatin fragmentation (Fig. 3C), which is a typical morphological characteristic of apoptotic cells. These cytological observations further confirmed that DHCE promotes the apoptosis of MCL cells.

It is widely known that apoptosis signaling pathways consist of extrinsic and intrinsic pathways. Upon receiving specific signals instructing the cells to undergo apoptosis, caspase proteases lead to the cleaving of cellular components including structural proteins in the cytoskeleton and nuclear proteins, such as DNA repair enzymes, through extrinsic and intrinsic pathways (31). DHCE markedly activated caspase-8 and caspase-3 (Fig. 3D), which are associated with the extrinsic pathway of apoptosis. In addition, DHCE suppressed the expression of the ant-apoptotic proteins Bcl-2 and Bcl-xL, and upregulated the expression of Bax, which is a proapoptotic protein of the intrinsic pathway (Fig. 3D) (33). Increased procaspase-9 substrate cleavage was also detected (Fig. 3D). These findings suggested that DHCE may activate the extrinsic and intrinsic pathways of apoptosis in a dose-dependent manner.

mTORC1 phosphorylates p70S6K and 4E-BP1, which are the two best-characterized downstream targets of mTORC1, thus regulating protein translation and contributing to cell growth and proliferation. To address the potential involvement of mTORC1 in DHCE-mediated inhibition of MCL cell proliferation, the present study measured the protein expression levels of p70S6K and 4E-BP1. Western blot analysis of Jeko-1 and Z138 cells demonstrated that phosphorylation of p70S6K and 4E-BP1 was inhibited by DHCE in a dose-dependent manner (Fig. 4A). Although DHCE treatment decreased the total protein expression of 4E-BP1 after 24 h, phosphorylation of 4E-BP1 was markedly inhibited compared with the control. These results indicated that DHCE-mediated inhibition of cell proliferation and growth may result from the suppression of mTORC1/p70S6K/4E-BP1 activity (Fig. 4B).

In contrast to mTORC1, mTORC2 modulates cell survival and apoptosis. Unlike rapamycin, a highly potent and selective inhibitor of mTORC1, which has little or no apoptotic effects on MCL cells (34), DHCE strongly induced apoptosis of Jeko-1 and Z138 cells (Fig. 3). Previous studies demonstrated that Akt regulated the activation of NF-κB, and that NF-κB can be stimulated downstream of mTORC2 by either Akt-dependent or Akt-independent mechanisms (35–38). The present western blot analysis results demonstrated that treatment of MCL cells with DHCE downregulated the protein expression levels of NF-κB target genes, including Bcl-2, Bcl-xL and cyclin D1. Therefore, it was hypothesized that DHCE may induce apoptosis of MCL cells by suppressing mTORC2/Akt/NF-κB signaling. Treatment of cells with DHCE resulted in a marked, dose-dependent inhibition of Akt (Ser473) and mTOR (Ser2481) phosphorylation in Jeko-1 and Z138 cells compared with in the control group (Fig. 4C). DHCE treatment exerted only a minimal effect on total mTOR and total Akt protein expression. Subsequently, the effects of DHCE on NF-κB activity were assessed and demonstrated that DHCE treatment resulted in decreased IκBα and NF-κB-p65 phosphorylation. These findings suggested that DHCE may block the activity of mTORC2/Akt/NF-κB, thus leading to apoptosis and inhibition of cell survival (Fig. 4B).

The present study further examined the therapeutic efficacy of DHCE using a xenograft mouse model. Nude mice bearing subcutaneous Jeko-1 cell xenograft tumors were administered daily intraperitoneal injections of either DHCE (3 mg/kg) or a vehicle control. The diameter and volume of the largest tumor in these mice was 1.98 cm and 3.20 cm³, respectively. DHCE treatment markedly decreased tumor size (Fig. 5A) and significantly decreased tumor volume (Fig. 5B); however, body weight was not significantly altered between the DHCE-treated and vehicle-treated mice (Fig. 5C). The results of H&E staining demonstrated that necrosis in the tumor samples of the DHCE-treated group was increased compared with in the vehicle control group. TUNEL staining revealed that DHCE treatment increased apoptosis compared with in the vehicle control group (Fig. 5D). DHCE treatment was well tolerated; no symptoms of poor health or abnormal behaviors were observed in DHCE-treated or vehicle-treated mice, and microscopic examination of individual organs revealed no evidence of tissue damage in either treatment group (data not shown). Consistent with the in vitro observations, these in vivo findings demonstrated that DHCE may inhibit MCL tumor growth in a xenograft model without exerting lethal toxicity.

Combination chemotherapy is a rational strategy for the treatment of MCL (4). Combination treatment with DHCE and bortezomib inhibited growth in both cell lines to a greater degree than treatment with DHCE or bortezomib alone. In the present study, CI values were <1.0, indicating synergism of DHCE and bortezomib (Fig. 6). Combined treatment of MCL cells with DHCE and bortezomib exhibited clear synergistic effects on the inhibition of cell proliferation.