https://orcid.org/0000-0002-8553-4559
Abstract
DCZ3301, a novel aryl-guanidino compound, was previously found to have potent anti-tumor activity in myeloma and B-cell lymphoma. In the present study, we investigated the effects of DCZ3301 on T-cell leukemia/lymphoma cells both in vitro and in vivo via cell proliferation, cell cycle analysis, apoptosis assay, mitochondrial membrane potential (MMP) assay, western blot analysis and tumor xenograft models. We found that DCZ3301 inhibited the viability of T-cell leukemia/lymphoma cells in a dose- and time-dependent manner. DCZ3301-induced G2/M cell cycle arrest, associated with downregulation of CDK1, cyclin B1, and cdc25C. DCZ3301 also induced cell apoptosis by decreasing MMP in T-cell leukemia/lymphoma cells, but had no significant pro-apoptotic effect on normal peripheral blood mononuclear cells (PBMCs). In addition, DCZ3301-induced apoptosis may be mediated by the caspase-dependent pathway and suppressing the phosphoinositide 3-kinase (PI3K)/AKT pathway. Finally, we showed that DCZ3301 treatment effectively inhibited tumor growth, with no significant side effects, in xenograft mouse models. In conclusion, these results suggest that DCZ3301 may be regarded as a new therapeutic strategy for T-cell leukemia/lymphoma patients.
Introduction
T-cell leukemia/lymphomas are uncommon but aggressive non-Hodgkin’s lymphoma (NHL), which account for approximately 30% of NHL in Asia [1,2]. T-cell leukemia/lymphomas are classified into four subtypes: acute, lymphoma, chronic, and smoldering, based on clinicopathologic features and prognosis [3]. Compared with B-cell lymphomas, T-cell leukemia/lymphomas are often more aggressive with worse prognosis [4]. Although combination chemotherapy has significantly improved the prognosis of T-cell leukemia/lymphoma patients over the past decades, the outcome of patients remains poor [5,6]. Thus, novel bio-therapeutic agents should be identified for the therapeutic strategy of T-cell leukemia/lymphoma patients.
Dysregulation of apoptosis is involved in the initiation and progression of malignant cancer [7]. Researchers have found that many agents can trigger tumor cell apoptosis in T-cell leukemia/lymphoma [8–10]. It is well-known that multiple cellular signaling pathways are critically associated with the pathogenesis of lymphoma, including phosphoinositide 3-kinase (PI3K)/AKT [11,12], extracellular signal-regulating kinase1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) [13], and Janus kinase/signal transducers and activators of transcription (JAK/STAT) [14,15]. However, the role of these cellular signaling pathways in T-cell leukemia/lymphoma is still unknown.
DCZ3301 is a novel aryl-guanidino compound, which was identified from a compound library by using cell-based in vitro screening (Fig. 1A). In our previous study, we have observed that DCZ3301 has potent cytotoxicity against multiple myeloma both in vitro and in vivo [16]. In addition, the result of our subsequent study with diffuse large B-cell lymphoma showed that DCZ3301 could inhibit cell proliferation via the STAT3 pathway [17], further indicating that DCZ3301 may be a potent anti-tumor agent. However, the effect of DCZ3301 on T-cell leukemia/lymphoma has never been investigated.
DCZ3301 inhibits the proliferation of T-cell leukemia/lymphoma cellsin vitro (A) The chemical structure of DCZ3301. (B) Jurkat cells were treated with DCZ3301 (0, 2.5, 5, 10, and 20 μM), and (C) HUT78 cells were treated with DCZ3301 (0, 7.5, 15, 30, and 60 μM) in 96-well plates for 24, 48, and 72 h, and then cell viability was analyzed using a CCK8 kit.
In this study, we investigated the anti-tumor activity of DCZ3301 in T-cell leukemia/lymphoma both in vitro and in vivo. Our results showed that DCZ3301 inhibited cell proliferation, induced G2/M cell cycle arrest and cell apoptosis in T-cell leukemia/lymphoma cells, with no cytotoxicity toward normal cells. We also found that administration of DCZ3301 significantly inhibited tumor growth in xenograft mouse models. Taken together, our findings demonstrate that DCZ3301 has anti-tumor activity and it may be a promising therapeutic agent for T-cell leukemia/lymphoma patients.
Materials and Methods
Cell culture
The T-cell leukemia cell line Jurkat and T-cell lymphoma cell line HUT78 were purchased from the American Type Culture Collection (Manassas, USA). Jurkat cell line expressing T-cell characteristics and complement receptors was isolated from the peripheral blood of patients with T-cell leukemia [18]. HUT78 cell line was a kind of cutaneous T-cell lymphomas originally derived from the peripheral blood of patients with Sezary syndrome [19]. Normal peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy donors using Lymphoprep (Stemcell Technologies, Vancouver, Canada) by Ficoll–Hypaque density gradient centrifugation. All the cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (Gibco) at 37°C 5% CO2. Blood samples were obtained from healthy donors after informed consent was obtained in accordance with the Declaration of Helsinki protocol and approved by the institutional review board of The 10th People’s Hospital of Shanghai, Tongji University (Shanghai, China).
Reagents
DCZ3301 (purity >98%; Shanghai Institute of Materia Medica, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO; Sigma, St Louis, USA) at a concentration of 16 mM and stored at −20°C. Antibodies against AKT, p-AKT, PI3K, Bcl-2, Bax, and cleaved caspase-8 as well as the horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG) were purchased from Cell Signaling Technology (Danvers, USA). Antibodies against caspase-9, cleaved caspase-3, GAPDH, CDK1, cyclin B1, and cdc25C were purchased from Abcam (Cambridge, USA). The Cell Counting Kit-8 (CCK8) was purchased from Yeasen (Shanghai, China). The Annexin-V/propidium iodide (PI) apoptosis detection kit was purchased from BD Pharmingen (Franklin Lakes, USA). JC-1 Kit for the detection of mitochondrial membrane potential (MMP) was obtained from Beyotime Institute of Biotechnology (Shanghai, China). The pan-caspase inhibitor, Z-VAD-FMK, was purchased from Selleck Chemicals (Houston, USA).
Cell viability assay
Jurkat and HUT78 cells (100 μl) were seeded into 96-well plates at a density of 2 × 105 cells/ml and treated with different concentrations of DCZ3301 for 24, 48, and 72 h. CCK8 was used to detect the cell viability. The half maximal inhibitory concentration (IC50) values were calculated with CalcuSyn software.
Cell cycle analysis
Jurkat and HUT78 cells (1 ml) were cultured in 24-well plates at a density of 2 × 105 cells/ml and treated with DCZ3301 (0, 2, or 4 μM) for 6, 12, and 24 h. Then cells were collected and washed with cold phosphate-buffered saline (PBS), and fixed with 70% ethanol at −20°C overnight. Then fixed-cells were washed with PBS and stained with 300 μl of PI/RNase staining buffer (BD Pharmingen) at room temperature for 15 min and analyzed with a BD FASCCanto II flow cytometer (BD BioScience, San Jose, USA).
Cell apoptosis assays
Jurkat and HUT78 cells (1 ml) were cultured in 24-well plates at a density of 2 × 105 cells/ml and treated with different concentrations of DCZ3301 for 24, 48, and 72 h. Then, cells were collected and washed with PBS, and then stained with Annexin-V/PI to detect apoptosis with a FASCCanto II flow cytometer. Annexin-V+/PI− (early apoptotic) and Annexin-V+/PI+ (late apoptotic) were considered to be apoptotic.
Mitochondrial membrane potential analysis
MMP was detected by flow cytometry using a JC-1 Kit according to the manufacturer’s instructions. Cells were cultured in 24-well plates and treated with DCZ3301 for 48 h, and stained with 2 μM JC-1 followed by incubation at 37°C for 25 min according to the manufacturer’s instructions. Cells were collected and washed with PBS, then analyzed by flow cytometry.
Western blot analysis
Cellular proteins were extracted using lysis buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol). Protein concentrations were detected using the BCA method (Beyotime Insititute of Biotechnology). Proteins (30 μg) were separated by 8%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Hercules, USA) and transferred electrophoretically to membranes. Membranes were blocked with 5% defatted milk and then probed with the primary antibodies overnight at 4°C. Membranes were washed with PBS containing 0.1% Tween 20 (PBST) for three times before incubation with the indicated secondary antibody for 1 h at room temperature. Finally, signals were detected by using the Odyssey two-color infrared laser imaging system (LICOR, Lincoln, USA).
Tumor xenograft models
Male BALB/C nude mice (5 weeks old) were purchased from Shanghai Laboratory Animal Center (Shanghai, China). Mice were maintained under a 12 h light–dark cycle at 22°C, and fed a standard diet with free access to water. Jurkat cells (2 × 107) in 100 μl serum-free culture medium were subcutaneously injected into the upper flank region of the nude mice. After the tumor volume (TV) reached appropriate size, mice were randomly divided into control group and treatment group: the vehicle-treated group (DMSO and saline) and 30 mg/kg DCZ3301-treated group (dissolved in DMSO and saline solution). Mice were administrated with DMSO and saline or DCZ3301 by intraperitoneal injection for 14 days. Tumor size and body weight were assessed every other day. TV was calculated using the following formula: TV = a × b2/2 (a = long diameter; b = low diameter). At the end of the experiment, mice were sacrificed, and the tumors were weighed and imaged. All animal studies were approved by the Animal Care and Use Committee of The 10th People’s Hospital of Shanghai, Tongji University. This study was also approved by the Science and Technology Commission of Shanghai Municipality (ID: SYXK 2007-0006) under the permit number 2011-0111.
Statistical analysis
Data are presented as the mean ± standard deviation (SD). Student’s t-test was performed as appropriate using SPSS v20.0 software (IBM, Armonk, USA). Significance was established at a P-value of <0.05.
Results
DCZ3301 inhibits the proliferation of T-cell leukemia/lymphoma cells in vitro
To determine whether DCZ3301 has anti-proliferation activity in T-cell leukemia/lymphoma cells, we first detected the viability of T-cell leukemia/lymphoma cell lines Jurkat and HUT78, treated with different doses of DCZ3301 for 24, 48, and 72 h, using CCK8 assay. It was found that the viability of Jurkat and HUT78 cells was markedly inhibited by DCZ3301 treatment in a time- and dose-dependent manner (Fig. 1B,C). Using the CalcuSyn software, IC50 values for Jurkat and HUT78 cells were 3.02 μM and 9.42 μM, respectively, after DCZ3301 treatment for 48 h.
DCZ3301 induces cell cycle arrest in T-cell leukemia/lymphoma cells in vitro
The anti-proliferation activity of DCZ3301 was further determined by detecting the cell cycle of T-cell leukemia/lymphoma cells by using flow cytometry analysis. Our data showed that DCZ3301 treatment induced obvious accumulation of Jurkat and HUT78 cells in the G2/M phase in a time-dependent manner (Fig. 2A,B). To further investigate the mechanism of DCZ3301-induced cell cycle arrest, we evaluated the protein levels of CDK1, cyclin B1, and cdc25C by western blot analysis in Jurkat cells. Results showed that DCZ3301 decreased the expression of CDK1, cyclin B1, and cdc25C (Fig. 2C).
DCZ3301 causes G2/M cell cycle arrest in T-cell leukemia/lymphoma cellsin vitro (A) Jurkat cells were treated with 2 μM DCZ3301, and HUT78 cells were treated with 4 μM DCZ3301 for 6, 8, and 12 h, and then cell cycle was analyzed by PI staining using flow cytometry. (B) The percentage of the cell population in G2/M phase. Data are presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01. (C) Western blot analysis was used to evaluate the protein levels of CDK1, cyclin B1, and cdc25C in Jurkat cells after treatment with DCZ3301 (0, 2, 4, and 8 μM) for 24 h.
DCZ3301 induces cell apoptosis and decreases mitochondrial membrane potential in T-cell leukemia/lymphoma cells in vitro
We next detected the effects of DCZ3301 on apoptosis in T-cell leukemia/lymphoma cells using Annexin-V/PI double staining and flow cytometry analysis. Our data showed that DCZ3301 induced a marked percentage of apoptotic cells in Jurkat and HUT78 cells after treatment with different doses of DCZ3301 for 24, 48, and 72 h (Fig. 3A,B), these results were consistent with the results of CCK8 assay. Mitochondrial dysfunction is involved in the development of cell apoptosis [20,21]. Therefore, we evaluated MMP level of T-cell leukemia/lymphoma cells using JC-1 MMP kit and flow cytometry analysis. Our data indicated that MMP was significantly decreased in Jurkat and HUT78 cells after treatment with DCZ3301 for 48 h (Fig. 3C).
DCZ3301 induces cell apoptosis and decreases mitochondrial membrane potential in T-cell leukemia/lymphoma cellsin vitro Jurkat cells were treated with DCZ3301 (0, 2, 4, and 8 μM) and (B) HUT78 cells were treated with DCZ3301 (0, 4, 8, and 12 μM) in 24-well plates for 24, 48, and 72 h, then apoptosis was detected by Annexin-V/PI double staining followed by flow cytometry. Data are presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***p < 0.001, compared with the 0 μM group. (C) Jurkat cells were treated with DCZ3301 (0, 2, 4, and 8 μM) and HUT78 cells were treated with DCZ3301 (0, 4, 8, and 12 μM) in 24-well plates for 48 h, then the level of MMP was analyzed using a JC-1 MMP kit, followed by flow cytometry analysis. Data are presented as the mean ± SD from three independent experiments. *P < 0.05, compared with the 0 μM group.
DCZ3301 treatment induces caspase activation and suppresses the PI3K/AKT pathway
To further explore the molecular mechanism of DCZ3301-induced apoptosis in T-cell leukemia/lymphoma cells, we evaluated caspase-3, caspase-8, and caspase-9 activities and mitochondrial apoptotic pathway proteins (Bcl-2 and Bax) by western blot analysis in Jurkat cells. As shown in Fig. 4A, DCZ3301 induced the cleavage of caspase-3, caspase-8, and caspase-9, suggesting that DCZ3301 induces apoptosis in T-cell leukemia/lymphoma cells through both extrinsic and intrinsic pathways. Importantly, the downregulation of Bcl-2 and upregulation of Bax further confirmed the disruption of mitochondrial integrity (Fig. 4A). In addition, we observed that pan-caspase inhibitor, Z-VAD-FMK, significantly abrogated DCZ3301-induced apoptosis in Jurkat cells (Fig. 4B,C). PI3K/AKT pathway is associated with cellular transformation, tumorigenesis, and cancer progression, therefore we evaluated its activity by western blot analysis after DCZ3301 treatment in Jurkat cells. As shown in Fig. 4A, treatment with DCZ3301 significantly decreased the expression of PI3K and p-AKT, with no significant change in total AKT level. These findings suggested that DCZ3301-triggered apoptosis was caspase-dependent and mediated by suppression of PI3K/AKT pathway. In addition, we found that DCZ3301 had no significant pro-apoptotic effect on normal PBMCs, even at the high concentration of 40 μM (Fig. 4D,E).
DCZ3301 treatment induces caspase activation and suppresses the PI3K/AKT pathway (A) Jurkat cells were treated with DCZ3301 (0, 2, 4, and 8 μM) for 48 h, then western blot analysis was performed to detect the protein levels of cleaved caspase-3, capase-8, and caspase-9, Bcl-2, and Bax as well as PI3K and p-AKT. (B) Jurkat cells were pretreated with or without Z-VAD-FMK for 2 h and then treated with 2 μM DCZ3301 for 48 h, stained with Annexin-V/PI and evaluated by flow cytometry. (C) The percentage of Annexin-V positive cells. Data are presented as the mean ± SD from three independent experiments. **P < 0.01, compared with the DCZ3301 group. (D) PBMCs were treated with DCZ3301 (0, 20, and 40 μM) for 48 h, and apoptosis was detected by Annexin-V/PI double staining, followed by flow cytometry. (E) The percentage of Annexin-V positive cells. Data are presented as the mean ± SD from three independent experiments.
DCZ3301 inhibits tumor growth in vivo
To examine the anti-tumor activity of DCZ3301 in vivo, we established a human T-cell leukemia/lymphoma xenograft models. Five weeks old male BALB/c nude mice were injected subcutaneously with 2 × 107 Jurkat cells. After the TV reached appropriate size, mice were treated with DCZ3301 (30 mg/kg), or DMSO and saline by intraperitoneal injection for a total of 14 days. At the end of the experiment, the mice were sacrificed and tumors were imaged and weighed. Tumor growth and tumor weight in DCZ3301-treated groups were obviously inhibited compared with the vehicle-treated group (Fig. 5A–C). Moreover, we measured the body weight of vehicle-treated and DCZ3301-treated mice. The results showed that there was no significant change in the mouse body weight between these two groups, indicating that DCZ3301 was well tolerated (Fig. 5D).
DCZ3301 inhibits tumor growthin vivo Jurkat (2 × 107) cells were subcutaneously injected into the upper flank of 5-week-old male nude mice. Mice were treated with vehicle or 30 mg/kg DCZ3301 for 14 days via intraperitoneal injection after tumor formation. (A) Tumor samples were collected and imaged using a high-definition digital camera. (B) Tumor weight was evaluated in 14 days. Data are presented as the mean ± SD. ***P < 0.001, compared with the control group, n = 4 mice/group. (C) Tumor volume was measured every other day. Data are presented as the mean ± SD. **P < 0.01, n = 4 mice/group. (D) The body weight of each mouse was measured every other day. Data are presented as the mean ± SD (n = 4 mice/group).
Discussion
DCZ3301 was characterized as a novel aryl-guanidino inhibitor by using cell-based in vitro screening via compound library searching. We have synthesized this compound and found that it has potent anti-tumor activity in multiple cancer cell lines, especially in hematological cancers [16,17]. Although major progress has been made in the treatment of T-cell leukemia/lymphoma, the outcome of patients remains poor [22]. Thus, in our present study, we investigated the effects of DCZ3301 on human T-cell leukemia/lymphoma both in vitro and in vivo.
In our in vitro study, we found that DCZ3301 significantly inhibited the viability of T-cell leukemia/lymphoma cells and had a dose- and time-dependent anti-proliferation effect in Jurkat and HUT78 cells. Multiple anti-tumor agents could activate the cell cycle checkpoints, resulting in cell cycle arrest and cell death [23]. Our data showed that DCZ3301 could markedly induce an accumulation of cells in the G2/M phases in T-cell leukemia/lymphoma cells. Cyclin-dependent kinases (CDKs) and cyclins play an important role in cell cycle progression [24]. The results of western blot analysis indicated that DCZ3301-induced G2/M phase arrest is associated with downregulation of CDK1, cyclin B1, and cdc25C protein expression, further suggesting that DCZ3301-induced cell cycle arrest is dependent on the cdc25C-degradation pathway.
Most of the cytotoxic anti-tumor drugs have been shown to induce cell apoptosis. Similarly, we found that DCZ3301 could induce cell apoptosis in T-cell leukemia/lymphoma cells, while no significant apoptosis was observed in normal PBMCs treated with DCZ3301. This finding indicated that DCZ3301 may be a safe therapeutic agent. The increase of mitochondrial membrane permeability often leads to the release of proteins, which triggers cell death and early apoptosis [25]. We further examined the level of MMP, an indicator of mitochondrial membrane permeability. Results obtained from flow cytometry analysis showed that treatment with DCZ3301 obviously decreased the level of MMP. In addition, the results of western blot analysis confirmed that DCZ3301-induced caspase-3, caspase-8, and caspase-9 cleavage, as well as decreased the protein levels of Bcl-2 and increased Bax in Jurkat cells, further demonstrating that DCZ3301 induces apoptosis via both extrinsic and intrinsic apoptotic pathways. Moreover, we found that pretreatment with a pan-caspase inhibitor Z-VAD-FMK impaired the effect of DCZ3301-induced cell apoptosis, indicating that DCZ3301-triggered cell apoptosis is caspase-dependent. PI3K/AKT is a main intracellular signaling pathway, which modulates cell proliferation, apoptosis, autophagy and differentiation [26]. Several series of studies have reported that the induction of cell apoptosis is mediated by suppression of the PI3K/AKT pathway [27,28]. Our western blot analysis results showed that treatment with DCZ3301 could inhibit the activation of the PI3K/AKT pathway by downregulating the protein expression of PI3K and phosphorylation of AKT, while no obvious difference was found in total AKT. Taken together, our findings suggest that DCZ3301-induced apoptosis of T-cell leukemia/lymphoma cells is caspase-dependent and is regulated by the PI3K/AKT pathway.
To further confirm the anti-tumor activity of DCZ3301 in T-cell leukemia/lymphoma, we next established a xenograft mouse model in vivo. In our in vivo study, we found that treatment with 30 mg/kg DCZ3301 for 14 days significantly inhibited tumor growth, with no apparent toxicity in DCZ3301-treated group mice, indicating that DCZ3301 is well tolerated in mice.
In summary, our present study demonstrated the anti-tumor activity of DCZ3301 in human T-cell leukemia/lymphoma cells both in vitro and in vivo. Our findings suggest that DCZ3301 treatment inhibits cell proliferation, induces G2/M cell cycle arrest and apoptosis in T-cell leukemia/lymphoma cells. DCZ3301 also decreases MMP level, enhances caspase activation, and suppresses PI3K/AKT pathway activation. In addition, DCZ3301 also inhibits tumor growth in the xenograft mouse model in vivo. Our study demonstrates that DCZ3301 may be used as a promising therapeutic agent in T-cell leukemia/lymphoma patients. However, further investigation is needed to understand the underlying mechanism of DCZ3301 in T-cell leukemia/lymphoma.
Funding
This work was supported by the grants from the National Natural Science Foundation of China (Nos. 81670194, 81570190, 81529001 and 81600174).
