Cepharanthine

Tetrandrine and cepharanthine induce apoptosis through caspase cascade regulation, cell cycle arrest, MAPK activation and PI3K/Akt/mTOR signal modification in glucocorticoid resistant human leukemia Jurkat T cells

Wencheng Xub,c, Xiaoqin Wangc,d, Yuanchao Tuc,d, Hiroshi Masakia, Sachiko Tanakaa, Kenji Ondaa, Kentaro Sugiyamaa, Haruki Yamadaa, Toshihiko Hiranoa,*

A B S T R A C T

Tetrandrine (TET) and cepharanthine (CEP) are two bisbenzylisoquinoline alkaloids isolated from the traditional herbs. Recent molecular investigations firmly supported that TET or CEP would be a potential candidate for cancer chemotherapy. Prognosis of patients with glucocorticoid resistant T cell acute lymphoblastic leukemia (T- ALL) remains poor; here we examined the anti-T-ALL effects of TET and CEP and the underlying mechanism by using the glucocorticoid resistant human leukemia Jurkat T cell line in vitro. TET and CEP significantly inhibited cell viabilities and induced apoptosis in dose- and time-dependent manner. Further investigations showed that TET or CEP not only upregulated the expression of initiator caspases such as caspase-8 and 9, but also increased the expression of effector caspases such as caspase-3 and 6. As the important markers of apoptosis, p53 and Bax were both upregulated by the treatment of TET and CEP. However, TET and CEP paradoXically increased the expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1, and activated the survival protein NF-κB, leading to high expression of p–NF–κB. Cell cycle arrest at S phase accompanied by increase in the amounts of cyclin A2 and cyclin B1, and decrease in cylcin D1 amount in cells treated with TET or CEP will be another possible mechanism. During the process of apoptosis in Jurkat T cells, treatment with TET or CEP also increased the phosphorylation of JNK and p38. The PI3K/Akt/mTOR signaling pathway modification appears to play sig- nificant role in the Jurkat T cell apoptosis induced by TET or CEP. Moreover, TET and CEP seemed to down- regulate the expressions of p-PI3K and mTOR in an independent way from Akt, since these two drugs strongly stimulated the p-Akt expression. These results provide fundamental insights into the clinical application of TET or CEP for the treatment of patients with relapsed T-ALL.

Keywords:
Tetrandrine Cepharanthine
T cell acute lymphoblastic leukemia Jurkat T cell
Apoptosis

1. Introduction

Acute lymphoblastic leukemia (ALL) is a heterogeneous hemato- logic disease characterized by the proliferation of immature lymphoid cells in the bone marrow, peripheral blood, and other organs [1]. Ap- proXimately 75–80% of ALL develop in children [2]. Optimal use of antileukemic drugs and improved supportive care in contemporary clinical trials have improved the 5-year survival rate of childhood ALL above 85% in developed countries [3]. However, the majority (~80%) of relapses occurs within 2 years of diagnosis in T cell ALL (T-ALL), and the prognosis after relapse is dismal, with a 5-year survival of less than 10% [4].
Tetrandrine (TET, Fig. 1A), isolated from Stephania tetrandra S. Moore, is a bisbenzylisoquinoline alkaloid and approved for treating patients with silicosis and rheumatic arthritis in China [5]. Our pre- vious study revealed that TET inhibited the proliferation of T-cell mitogen-activated human peripheral blood mononuclear cells and en- hanced the glucocorticoid pharmacodynamics by inhibiting mitogen- activated protein kinase (MAPK) [5,6]. Meanwhile, recent investiga- tions firmly supported that TET would be a potential candidate for cancer chemotherapy [7,8]. TET upregulated the caspase cascade pro- tein (cleaved PARP, cleaved caspase-3 and cleaved caspase-9) and in- hibited the phosphorylation of Akt/mTOR, resulted in significant apoptosis on human gastric cancer cell [9]. TET also inhibited pan- creatic cancer tumor growth by downregulating Skp2, an E3 ligase specific for degradation of p27Kip1 during the cell cycle, and indirectly impaired the activities of CDK4/6 to halt deregulated cell cycle [10]. Moreover, one of the clinical trials for TET showed that TET, in com- bination with traditional chemotherapy drugs, had potential-reversing effect for the treatment of refractory and relapsed acute myelogenous leukemia [11]. The above information hints that TET could inhibit the proliferation of both T cells and cancer cells. Sharing the similar che- mical skeleton with TET, cepharanthine (CEP, Fig. 1A) is another bis- benzylisoquinoline alkaloid isolated from the plant Stephania cephar- antha Hayata [12]. From these points of view, we are encouraged to investigate the anti-cancer effects of TET and CEP on human T-lym- phoblastic leukemia cells having features of both T cells and cancer cells.
Since poor response to glucocorticoid is one of the strongest pre- dictors of adverse outcome for the treatment of childhood T-ALL [13], glucocorticoid resistant human leukemia Jurkat T cell line [14], a well- established T-ALL tumor line from the peripheral blood of a 14-year-old boy [15], was used in the present research to examine potential effi- cacies of TET and CEP, and their underlying action mechanisms fo- cusing on caspase cascade, cell cycle, MAPK and PI3K/Akt/mTOR sig- naling pathway.

2. Materials and methods

2.1. Drugs and reagents

RPMI 1640 and FBS were purchased from Gibco BRL (Grand Island, NY, USA). TET and CEP were obtained from Sigma Aldrich and Cayman Chemical Company respectively. Camptothecin (CPT) was provided by Wako Pure Chemical Industries, Ltd., Japan. Phospho–NF–κB (dilution 1:1000, #sc-136,548) and β-actin (dilution 1:5000, #66009-1-Ig) an- tibodies were purchased from Santa Cruz Biotechnology and from commercial vendors.

2.2. Cell cultures

Human leukemic Jurkat T-cells were grown in RPMI 1640 medium supplemented with 10% FBS, 100,000 IU/L penicillin and 100 mg/L streptomycin at 37 °C in the presence of 5% CO2 as we described before [16].

2.3. Cell viability measured by WST-8 assay

Cellular proliferation was assessed using WST-8 assay. Jurkat T cells at a cell density of 1.5 × 105 cells/mL were seeded in 96-well plates. TET or CEP was subsequently added into the corresponding wells to adjust the final concentrations of 3, 5, 10 and 15 μM. The cells were incubated with ethanol as a control. After 48 h treatment at 37 °C, 10 μL of WST-8 solution was added to each well, followed by additional 3 h incubation. Optical density value was measured at 450 nm absorbance (ref. 650 nm). Percentages of cell viability in the reagent-treated group, as compared to control (ethanol-treated group), were presented [17].

2.4. Hoechst 33,342 fluorescent staining

In order to observe the influence of drugs on the morphology of Jurkat T cells, cells were treated with TET or CEP for 48 h and then harvested to stain with Hoechest 33,342 (#17535, AAT Bioquest, Inc, CA, USA). Briefly, after collecting, cells were fiXed with 4% paraf- ormaldehyde, followed with PBS washing. Then, the cells were con- tinued to incubate with Hoechst 33,342 for 10 min at room tempera- ture. The images of nuclear alteration-related apoptosis were captured with a fluorescence microscope (Biozero BZ-8000 Series, Keyence, Japan) [16].

2.5. Assessment of apoptosis

Jurkat T cells were seeded in 24-well plates and treated with serial concentrations of TET or CEP (0.3, 3, 5, 10 and 15 μM). 10 nM of CPT group was set up as a positive control for apoptotic cells. After 48 h incubation at 37 °C, cells were harvested and washed by PBS twice. Then, the cells were co-stained using FITC Annexin V Apoptosis Detection Kit (BD Pharmingen™). Fluorescence of the cells was im- mediately determined by a flow cytometer (FACSCanto™ II, BD Biosciences, CA, US) [17].

2.6. Cell cycle analysis

Jurkat T cells were seeded in 6-well plates and incubated with RPMI 1640 medium containing blank solvent, CPT (10 nM), CEP (5, 10 and 15 μM) and TET (5, 10 and 15 μM) for 48 h, respectively. Then, the cells were harvested and washed with PBS, and subsequently fiXed in 70% ethanol at 4 °C for 1 h, and stained with PI (50 μg/mL, #P4864, Sigma Chemical Co.) and RNase A solution (0.25 mg/mL, #R5500, Sigma Chemical Co.). DNA content of cells was determined by flow cytometry (FACSCanto™ II, BD Biosciences, CA, US). Data were analyzed by ModFit LT™ (Version 3.1, Verity Software House, Topsham, ME, USA) [17].

2.7. Western blot analysis

Whole cell protein was extracted by RIPA buffer containing Protease and Phosphatase Inhibitor (#A32961, Thermo Scientific). Western blot was performed using standard procedures, as we described previously [16]. Membranes were incubated with primary antibodies against in- dividual proteins overnight at 4 °C followed by an appropriate sec- ondary antibody (Anti-mouse IgG, HRP-linked, #7076, Cell Signaling Technology, Inc.) at a dilution of 1:1000 for 1 h at room temperature. After incubation with regents of an ECL or ECL Prime Western Blotting detection kit (#RPN2109 and RPN2232, GE Healthcare), the mem- branes were then analyzed in a luminescent image analyzer (Fujifilm; LAS-3000; Fujifilm, Tokyo, Japan). Quantitative densitometry data of the images were evaluated by ImageJ software (version 1.52e, National Institutes of Health, USA; http://imagej.nih.gov/ij).

2.8. Statistical analysis

Differences in the percentages of viable cells, early or late apoptotic cells and percentage of cells in cell-cycle phases were analyzed with Bonferroni Multiple Comparison Tests. Statistical analyses for the ex- pressions several proteins were performed using Dunnett’s Multiple Comparison Test. These analyses were processed by using GraphPad PRISM 5.0 (GraphPad Software Inc., San Diego, CA). In each case, two- sided p values < 0.05 were considered to be significant. 3. Results 3.1. TET and CEP inhibit the proliferation of Jurkat T cells and change the cell morphology To investigate the cytotoXicity of TET and CEP against glucocorti- coid resistant human leukemia cells, Jurkat T cells, were exposed to serial concentrations of TET and CEP (3, 5, 10 and 15 μM). The result of cell viability was provided in Fig. 1B. Both CEP and TET inhibited the proliferation of Jurkat T cells significantly in a dose- and time-depen- dent manner. After the cells were stained by Hoechst 33,342, the cell number decreased obviously in the cells treated by TET or CEP at concentrations of 10 and 15 μM compared with control group (Fig. 1C). Images from the fluorescence microscope also showed that 10 nM of camptothecin (CPT) used as a positive drug, 10 and 15 μM of TET and CEP largely changed the cell morphology with abnormal nuclear of karyorrhexis, chromatin condensation and fragmentation (Fig. 1C). 3.2. TET and CEP induce apoptosis in Jurkat T cells To explore whether apoptosis was involved in the cytotoXicity of TET and CEP, Annexin V-FITC/PI dual staining assay was performed. Percentages of apoptotic cells were summarized in Fig. 2B–D and ty- pical dot-plot diagram was shown as Fig. 2A. Comparing with control group, the percentage of viable cells treated by 10 nM CPT was 42.1 ± 4.4% (mean ± SD, P < 0.05, Fig. 2B). Both TET and CEP inhibited the cell growth dose-dependently. 15 μM of CEP and TET showed the strongest cytotoXic effects, and the mean ± SD values of viable cells were 8.4 ± 6.9 and 7.4 ± 2.4%, respectively (P < 0.001, Fig. 2B). Consequently, the percentages of the early and the late apoptotic cells increased significantly after treating with 10 nM CPT or higher doses of TET and CEP (Fig. 2C and D). 3.3. TET and CEP regulate the expression of proteins related to caspase cascades To further investigate the mechanism underlying the TET or CEP- induced apoptosis, the expressions of proteins related to caspase cas- cades were examined. Complicated regulation networks were divided into apoptotic markers and anti-apoptotic factors. Apoptotic markers in the cells, such as caspase-3/6/8/9, p53 and Bax, were upregulated by TET or CEP (Fig. 3A–I). As shown in Fig. 3A, the expression levels of caspase-3 significantly increased following treatment with 10 and 15 μM TET (P < 0.01). Meanwhile, TET and CEP tended to increase the expression of caspase-8 dose-dependently (Figs. 3D), and 15 μM of TET showed a significant up-regulation (P < 0.05). A similar increasing tendency was observed in the ex-pressions of caspase-9 and caspase-6 after the cells were exposed to CEP or TET (Fig. 3B and E). However, the drug treatment seemed to show little influence on the expression of caspase-7 (Fig. 3C). CPT at 10 nM did not upregulate the expression of caspase-3/6/7/8/9, whereas CPT treatment induced the formation of cleaved PARP (89 kDa), which leading to a significant downregulation of full length PARP (Fig. 3F). 15 μM of CEP also downregulated the expression of full length PARP significantly (P < 0.05), but we did not observe the cleavage type (Fig. 3F). Whereas, the effects of TET on full length PARP were not statistically significant (Fig. 3F). As shown in Fig. 3G and I, Lamin A-C and Bax were influenced by the treatment of TET or CEP, and 10 μM of TET maximally increased the expression levels of these two pro-apoptotic proteins significantly (P < 0.05, Fig. 3G and I). Furthermore, CEP treatment stimulated the expression of p53 in a dose-dependent manner, and 15 μM of CEP significantly increased the amount of p53 (P < 0.05, Fig. 3H). TET at 5–15 μM tended to stimulate the expression of p53, though the effects were not statistically significantly (Fig. 3H). ParadoXically, anti-apoptotic factors, such as Bcl-2, Mcl-1 and p–NF–κB, were also enhanced by the treatment with CPT, TET and CEP (Fig. 4A–D). As shown in Fig. 4A, TET or CEP dose-dependently in- creased the expression of Bcl-2, and 15 μM of TET showed a significant increase in the amount of this protein (P < 0.05). Meanwhile, both TET and CEP largely upregulated the expression levels of Mcl-1 and p–NF–κB significantly in a dose-dependent manner (Fig. 4C and D). None of the three agents changed the expression of Bcl-XL (Fig. 4B). 3.4. TET and CEP trigger the cell cycle arrest in Jurkat T cells To evaluate whether TET or CEP mediated inhibition of cell growth correlated with cell cycle arrest, Jurkat T cells were treated with serial concentrations of TET or CEP (5, 10 and 15 μM). As shown in Fig. 5A–B, 10 nM of CPT as a positive control, largely increased cell population at S phase (P < 0.001), followed with a significant decrease of cell popu- lation at G0/G1 phase (P < 0.001). Interestingly, TET or CEP showed similar results that they triggered cell cycle arrest and leaded the cell growth to stop at S phase in a dose-dependent manner, and thus de- creasing the percentage of cells at G0/G1 phase. None of these three agents influenced the population of cells at G2/M phase largely. 3.5. TET and CEP regulate the expression of cell cycle-related proteins We continued to examine the influences of TET and CEP on cell cycle-related proteins. As shown in Fig. 6B and 10 nM of CPT sig- nificantly increased the expression level of cyclin B1 (P < 0.01), and TET or CEP showed the similar regulation effect in a dose-dependent manner. All of these three drugs enhanced the expression of cyclin A2, and 15 μM of TET possessed the strongest efficacy (P < 0.05, Fig. 6A). In contrast, cyclin D1 protein expression was effectively downregulated by TET or CEP (Fig. 6C). 3.6. TET and CEP activate MAPK MAPK activation maintains the proliferation of T cells. To in- vestigate the molecular basis for the effects of TET and CEP against Jurkat T cell growth, we examined the influence of TET or CEP on MAPK activation. As shown in Fig. 7A, both TET and CEP apparently stimulated the phosphorylation of p38 in a dose-dependent manner (Fig. 7A). 15 μM of CEP changed the ratio of p-p38 and p38 significantly (P < 0.05). As shown in Fig. 7B and 10 nM of CPT significantly in- creased the phosphorylation of JNK (P < 0.05), and TET or CEP also tended to increase the phosphorylation of JNK dose-dependently, though the effects were not significant. However, none of these three drugs significantly influenced the expression of p-ERK (Fig. 7C). 3.7. TET and CEP modify PI3K/Akt/mTOR signaling pathway To further investigate the molecular mechanism underlying the in- hibitory effects of TET and CEP on cell survival and growth in the Jurkat T cells, we evaluated the effect of TET or CEP on PI3K/Akt/ mTOR signaling pathway. As shown in Fig. 8A and 10 nM of CPT in- hibited the expression of p-PI3K significantly (P < 0.05). Similarly, both TET and CEP appeared to decrease the p-PI3K expression dose- dependently (Fig. 8A). All of these three agents largely downregulated the expression of mTOR, and TET or CEP showed the inhibitory effect in a dose-dependent manner (Fig. 8C). However, CPT and TET or CEP paradoXically increased the expression of p-Akt1, and the upregulating effects of 10 and 15 μM TET were statistically significant (P < 0.01 and 0.05, respectively). 4. Discussion The WST-8 assay data suggested that 10 and 15 μM TET or CEP inhibited the proliferation of Jurkat T cells significantly (Fig. 1B), and the inhibitory tendency was consistent with the results obtained from the flow cytometer analysis by use of Annexin-V and PI staining (Fig. 2B). Images obtained from fluorescence microscopy revealed that the cell morphology was largely changed to show karyorrhexis, chro- matin condensation and fragmentation (Fig. 1C), which indicated that cell apoptosis seriously occurred. This finding was also certified by the results of apoptosis analysis in Fig. 2C and D. Both TET and CEP showed cytotoXic effect on Jurkat T cells by inducing apoptosis, which were consistent with the observations of previous reports [18,19]. Caspase cascades are pivotal components of apoptosis. Caspases are expressed in cells as inactive zymogens, which are also known as pro- caspases and are activated via proteolytic cleavage. Wu et al. revealed that CEP treatment induced activation of capase-3/8/9 accompanied by cleavage of PARP in Jurkat T cells [19]. CEP-induced apoptosis was completely blocked by a caspase inhibitor Z-VAD-fmk [19]. Similarly, TET-induced apoptotic DNA damage in T cells requires activated cas- pase-3, and this effect of TET could be also inhibited by caspase in- hibitors Z-VAD-fmk and Z-DEAD-fmk [20]. Thus, the present study continuously observed the effects of TET and CEP on procaspases and other caspase cascades. Our present investigation showed that TET or CEP not only upregulated the expression of initiator caspases such as caspase-8 and 9 (Fig. 3D and E), but also increased the expression of effector caspases such as caspase-3 and 6 (Fig. 3A and B). However, TET or CEP seemed to show little effect on caspase-7 (Fig. 3C). Compared with CPT, TET or CEP treatment did not largely stimulate the cleavage of PARP, but downregulated the expression amount of full length PARP (Fig. 3F). The above different phenomenon would be related to the different activities of CPT, TET and CEP on caspase cascades, since CPT had almost no effects on the expression of caspase-3/6/7/8/9 (Fig. 3A–E). Lamins and p53, as the downstream proteins of caspase cascades, were also influenced by the treatment of TET and CEP (Fig. 3 G and H). However, apoptosis induced by these alkaloids did not re- quire functional p53 since Jurkat T cells have a mutated p53 [21,22]. Bax is one of the members of Bcl-2 family, which induces apoptosis through mitochondrial stress [23–25]. TET or CEP strongly stimulated the expression of Bax at 5 and 10 μM, while they decreased the expression at 15 μM (Fig. 3I). EXcessive stimulation of p-Akt1 by 15 μM TET or CEP, as shown in Fig. 8B, may account for the paradoXical results, since Akt inhibits a conformational change in the pro-apoptotic Bax protein and its translocation into mitochondria [26]. However, all of these three agents seem to enhance the expression of survival pro- tein, p–NF–κB, and anti-apoptotic proteins of Bcl-2 family such as Bcl-2 and Mcl-1 (Fig. 4A–D). However, our analysis using the Annexin V staining (Fig. 2) showed that very high percentage of cells underwent apoptosis. The paradoXical phenomenon suggested that both caspase- independent and dependent apoptotic signaling pathways are im- plicated in the action of TET or CEP, and this proposed explanation was consistent with the finding of Lai et al. [20]. Similar to the most of common human tumors, cell cycle arrest would be an important way to regulate T-ALL cell growth and pro- liferation [17]. In the present study, we found that CPT, TET and CEP largely arrested the cell cycle progression at S phase in a dose-depen- dent manner, accompanied by a significant decrease of cell population at G0/G1 phase (Fig. 5A and B). S phase is a crucial event in the cell cycle that allows for proper replication of DNA without accumulating genetic abnormalities [27]. Topoisomerase Ⅰ relaxes the DNA supercoil form during the DNA replication process, and we found that CPT sig- nificantly inhibited the expression of topoisomerase Ⅰ in Jurkat T cells (data not shown). Our present data suggested that CPT caused DNA damage and activated the cell cycle checkpoint with a cell cycle arrest at S phase, as has been suggested by other researchers [28]. In contrast, TET or CEP, sharing the similar cell cycle arrest with CPT, did not in- fluence the expression of topoisomerase Ⅰ in Jurkat T cell nucleus (data not shown). As we have known, transition from one cell cycle phase to another occurs in an orderly fashion and is regulated by different cel- lular proteins, such as cyclin A/B/D [29]. Further investigation re- vealed that both CPT and TET or CEP upregulated the expressions of cyclin A2 and B1 but downregulated the expression of cyclin D1, which might contribute to their similar effects on cell cycle arrest (Fig. 6A–C). Activated mitogen-activated protein kinase (MAPK) has been re- ported to play a major role in promoting and maintaining T lymphocyte populations [30,31]. Unlike the normal T cells, activated MAPK may contribute to the apoptotic process of malignant Jurkat T cells. As shown in Fig. 7A, p38 was activated by the treatment with TET or CEP apparently and dose-dependently, which was accompanied by the PI3K/Akt/mTOR signaling pathway controls multiple cellular re- sponses, including metabolic regulation, cell growth, and survival [34]. In human T-ALL, constitutive activation of the PI3K/Akt/mTOR signal transduction pathway is achieved by deletions or mutations targeting PTEN in about 15% of cases [34–36]. Our data showed that both CPT and TET or CEP inhibited the expression amounts of p-PI3K and mTOR, whereas the treatment by these agents resulted in the high expression of p-Akt1 paradoXically (Fig. 8A–C). Although Akt was viewed as a major downstream effector of PI3K, at least in physiological processes, several studies suggested that PI3K and Akt act independently in cancers [37]. While PI3K is a major regulator of Akt activation in response to a variety of ligands, recent studies highlighted that diverse groups of tyrosine (Ack1/TNK2, Src, PTK6) and serine/threonine (TBK1, IKBKE, DNAPKcs) kinases also activate Akt directly to promote growth, pro- liferation and oncogenic transformation [38]. Thus, TET and CEP seemed to regulate the expressions of p-PI3K and mTOR in an in- dependent way on Akt. On the other hand, downregulation of mTOR expression always associated with the activation of autophagy, which may maintain the tumor homeostasis and lead to drug resistance. Wong et al. revealed that TET could induce autophagic cell death in mTOR- dependent way in MCF-7 cells [39]. Subsequently, TET was also re- ported to suppress proliferation and induce autophagy in MDA-MB- 231 cells by inhibiting the PI3K/AKT/mTOR pathway [40]. Moreover, autophagy induction enhances TET-induced apoptosis via the AMPK/mTOR pathway in human bladder cancer cells [41]. Although all these findings supported that autophagy induction by TET or CEP may lead to tumor suppression, the anti-cancer properties and molecular mechan- isms of TET or CEP are likely to be cell-type specific and remain to be further investigated. TET and CEP, bisbenzylisoquinoline alkaloids isolated from the traditional herbs, are commercially distributed in China and Japan re- spectively, for more than 20 years, which certifies the safety of these two compounds [5,12]. Meanwhile, with the advantage of high toler- ance and prominent P-glycoprotein inhibitory effect, TET has been re- gistered as CBT-1® in USA and was studied in the clinical trials in as- sociation with doXorubicin for treating patients with advanced solid tumors [42]. The present study showed the apoptotic effects of TET and CEP induced by interactional signaling pathways of caspase cascade, cell cycle, MAPK and PI3K/Akt/mTOR signaling pathway in Jurkat T cells. However, these signaling also control the proliferation, survival and death of normal human immune cells. Previous studies already revealed that the potential of this class of compounds also leads to immunosuppression/toXicity on normal human T cells [20,43–45]. 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