ZGDHu-1 promotes apoptosis of mantle cell lymphoma cells

Mantle cell lymphoma (MCL) is a well-defined aggressive Non-Hodgkin-lymphoma with short survival rates and remains incurable to date. Previously, we demonstrated the antitumor activity of ZGDHu-1(N, N’-di-(m-methylphenyi)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide) in chronic lymphocytic leukemia. In this study, ZGDHu-1 shows potent anti-lymphoma activity in MCL cells. ZGDHu-1 significantly induces cell cycle G2/M phase arrest and apoptosis in MCL cells. ZGDHu-1 reduces the protein levels of Mcl-1, Bcl-XL and cyclin D1. Importantly, ZGDHu-1 inhibits TNFα-induced IkBa phosphorylation, p65 nuclear translocation and NF-kB downstream target gene expression in MCL cells. MCL samples expressing high levels of Bcl-2 and high Bcl-2/Bax ratios tend to be less effective to ZGDHu-1. Together, these results suggest that ZGDHu-1 could inhibit the NF-kB signaling pathway partly, which may lead to the suppression of cell proliferation and the induction of apoptosis in MCL cells. Thus, our studies provide evidence of the potential of ZGDHu-1 in treating mantle cell lymphoma.


INTRODUCTION
Mantle cell lymphoma (MCL) is a highly aggressive form of Non-Hodgkin-lymphomas (NHL) with a median survival of approximately 5 years and accounts for 6-8% of NHL [1][2][3].Although some patients exhibit clinically indolent progression, MCL is generally aggressive, with most patients in stage III or IV at diagnosis [3].Although conventional chemotherapy results in an overall 60-80% response, most patients relapse and succumb to MCL [4].Patient responses to currently available therapies, including monoclonal antibody therapy and high-dose chemotherapy followed by stem cell transplantation, have been limited [5][6][7][8].Thus far, no therapy has been sufficiently effective in extending the overall survival time of patients with MCL.Therefore, novel treatments that maximize therapeutic benefits and minimize toxicities are desperately needed.
MCL is characterized by chromosomal translocation (11; 14) (q13; q32).Cyclin D1 is regulated by NF-κB and regulates the G1-S transition of the cell cycle [9].Though cyclin D1 overexpression has become the hallmark of MCL, it is not sufficient for the development of MCL.Accumulating evidence suggests that MCL is often detrimental to other cellular processes, such as the apoptosis and DNA repair.Apoptosis plays an important role in cancer development.MCL cells evade apoptosis by up-regulating the expression of anti-apoptotic proteins [9].We know that NF-κB activation plays a critical role in MCL cell survival and leads to the overexpression of several anti-apoptotic molecules [9][10][11][12][13].Among these, anti-apoptotic members of the Bcl-2 family appears to be particularly important in the the development of MCL [10].Inhibiting NF-κB activation has been shown to induce cell-cycle arrest and cell death in MCL cells [11][12][13].Therefore, the NF-κB pathway is an attractive target for therapeutic intervention in the onset and progression of MCL.
In this study, we screened 17 primary MCL patients and three different MCL cell lines to investigate the antilymphoma activity of ZGDHu-1.Primary MCL cells from two MCL patients (MCL #3 and MCL #12) and three MCL cell lines were further examined to study ZGDHu-1's molecular mechanism.We found that ZGDHu-1 exerted cytotoxic activity and significantly induced cell cycle G2/M phase arrest and apoptosis in MCL cells.We also demonstrated that ZGDHu-1 decreased IkBa phosphorylation and reduced the expression of Mcl-1, Bcl-XL and cyclin D1 and blocked the TNFα-induced NF-κB signaling pathway in MCL cells.These findings suggest that ZGDHu-1 induces apoptosis by inhibiting the NF-κB signaling pathway in MCL cells partly, thereby providing evidence that ZGDHu-1 is a potential therapeutic molecule that may improve MCL patients' outcomes.

ZGDHu-1 induces cell cycle G2/M phase arrest in MCL cells
Because ZGDHu-1 inhibits MCL cell proliferation, it is likely that ZGDHu-1 causes MCL cell cycle arrest.To test this hypothesis, we treated two random primary MCL cells (MCL #3 and MCL #12) and three MCL cell lines with ZGDHu-1 (100 -200 ng/ml) for 48 h and measured the cell cycle distribution.Indeed, following ZGDHu-1 treatment, the percentage of cells in the G2/M phase significantly increased in a dose-dependent manner in primary MCL cells and three MCL cell lines (Figure 2A-2B and Supplementary Figure 2).The percentages of cells in the G01 and S phase were not significantly changed.We also showed that ZGDHu-1 increased the fraction of cells in the sub-G1 phase (Figure 2A-2B and Supplementary Figure 2).Together, these data suggested that ZGDHu-1 could induce cell cycle G2/M phase arrest and apoptosis in MCL cells.
To further confirm whether caspase-dependent apoptosis plays a prominent role in ZGDHu-1-induced apoptosis, we treated MCL cells with ZGDHu-1 in the presence of the pan-caspase inhibitor Z-VAD.fmk(200 mM) and observed that ZGDHu-1-induced Annexin V + PI -apoptosis, ZGDHu-1-induced caspase-3 activation as well as the cleavage of PARP was largely abrogated (Figure 3B-3C,Figure 3F-3H and Supplementary Figure 3A, 3C).But the adding of Z-VAD.fmk did not inhibit the dissipation of ∆Ψm (Figure 3D-3E and Supplementary Figure 3B).Together, these results strongly suggested that ZGDHu-1 induced the caspase-dependent apoptosis of MCL cells.

ZGDHu-1 induces changes in ROS in MCL cells
Because reactive oxygen species (ROS) plays a key role in apoptosis, we used DHR staining and flow cytometry to examine whether ROS levels were affected by ZGDHu-1 in MCL cells.ZGDHu-1 (100 -200 ng/ ml, 48 h) significantly induced ROS generation of MCL cells in a dose-dependent manner (Figure 4A-4B).We also tested whether the ROS scavenger glutathione (GSH) could suppress ZGDHu-1-induced apoptosis in primary MCL cells and three MCL cell lines.Pretreatment with GSH (100 μM) for 2 h could significantly block ZGDHu-1-induced ROS generation (Figure 4A-4B), and partly inhibited the pro-apoptotic effect of ZGDHu-1 on MCL cells (Figure 4C-4D) and ZGDHu-1-induced the dissipation of ∆Ψm in MCL cells (Figure 4E).Taken together, these results suggested that ZGDHu-1 can promote apoptosis of MCL cells by inducing ROS generation.

ZGDHu-1 decreases the expression levels of multiple cell cycle and apoptosis regulators in MCL cells
Because ZGDHu-1 induced cell cycle G2/M phase arrest and apoptosis, we examined the protein levels of several cell cycle and apoptosis regulators to investigate the apoptosis mechanism triggered by ZGDHu-1 in MCL cells.In primary MCL cells and three MCL cell lines, ZGDHu-1 (100 -200 ng/ml, 48 h) decreased the protein levels of cyclin D1, but not cyclin B1, cyclin E and CDK2, in dose-dependent manners (Figure 5A).Likewise, ZGDHu-1 decreased the protein levels of Mcl-1 and Bcl-XL, but not Bcl-2 and Bax, in dose-dependent manners in MCL #3 primary MCL cells and three MCL cell lines (Figure 5A).ZGDHu-1 decreased the Bcl-XL protein levels in dose-dependent manners in MCL #12 primary MCL cells (Figure 5A).
To confirm that the Mcl-1 and Bcl-XL protein loss were due to decrease in the transcription level, Mcl-1 and Bcl-XL mRNA levels following ZGDHu-1 treatment were measured using qRT-PCR and a-tubulin as a control.ZGDHu-1 (100 -200 ng/ml, 48 h) caused a rapid reduction of Mcl-1 mRNA and Bcl-XL mRNA in Rec-1 (Figure 5B), Jeko-1 (Figure 5C), MAVER-1 (Figure 5D) and 17 primary cells (Figure 5E).These results confirmed that the losses of Mcl-1 and Bcl-XL protein were due to decrease on the transcription level.

ZGDHu-1 blocks TNFα-induced NF-κB activation and the expression of anti-apoptotic proteins
It is well known that the NF-κB signaling pathway inhibits apoptosis by inducing the expression of antiapoptotic proteins Mcl-1, Bcl-2 and Bcl-XL.NF-κB is constitutively active and levels of nuclear p65 and IkBa phosphorylation increase in MCL cells compared to normal B cells [19][20].Traditionally, NF-κB is composed of two subunits and is normally sequestered in the cytoplasm by its inhibitor protein, IκB [21].NF-κB activation can induce IκB phosphorylation in NF-κB exposed cells, targeting them for rapid degradation via a proteasome pathway, which releases NF-κB to the nucleus, where it binds to specific sequences in the promoter regions of genes [22].To investigate the mechanism by which ZGDHu-1 inhibits the expression of anti-apoptotic proteins, we tested whether ZGDHu-1 inhibits the NF-κB signaling pathway.Initially, we examined the effect of ZGDHu-1 on IkBa phosphorylation, a major target molecule of the NF-κB signaling pathway.The results indicated significant dosedependent down-regulation of IkBα phosphorylation and nuclear p65 following ZGDHu-1 treatment in primary MCL cells and three MCL cell lines (Figure 5A).But ZGDHu-1 did not change the protein levels of Bcl-2, Bax and cytoplasmic p65 of primary MCL cells and three MCL cell lines (Figure 5A).
We further evaluated whether ZGDHu-1 inhibited TNFα-induced p65 nuclear translocation in three MCL cell lines.As expected, TNFα (10 ng/ml) activated the NF-κB signaling pathway in three MCL cell lines, as indicated by the phosphorylation of IkBa, the degradation of IkBa and the induction of Mcl-1 and Bcl-XL in Rec-1 (Figure 6A), Jeko-1 (Figure 6B) and MAVER-1 (Figure 6C).ZGDHu-1 (200 ng/ml, 24 h) almost completely blocked the phosphorylation of IkBa, the degradation of IkBa and the induction of Mcl-1 and Bcl-XL proteins in Rec-1 (Figure 6A), Jeko-1 (Figure 6B).ZGDHu-1 did not affect the protein levels of Bcl-2, Bax and p65 in three MCL cell lines (Figure 6A-6C).P65 nuclear translocation is a symbol for NF-κB activation [21].We further used western blot analysis to evaluate whether ZGDHu-1 inhibits TNFα-induced p65 nuclear translocation in MCL cells.As indicated in Figure 6D, ZGDHu-1 significantly inhibited TNFα-induced p65 nuclear translocation at 60 to 120 min in three MCL cell lines.Overall, these results suggest that ZGDHu-1 suppresses cell growth and induces apoptosis by inhibiting the NF-κB signaling pathway in MCL cells.

Bcl-2 expression confers resistance to ZGDHu-1
We know Mcl-1, Bcl-XL, Bcl-2 and Bax play controlling roles in the survival of MCL cells [23].Our results indicated that ZGDHu-1 reduced Mcl-1 and Bcl-XL protein levels in primary MCL cells.Next, we evaluated putative differences in the basal mRNA relative levels of these proteins in correlation with sensitivity to ZGDHu-1 in primary MCL cells (Table 1, Figure 7A).Interestingly, we observed that Bcl-2 mRNA levels and Bcl-2/Bax ratios were inversely correlated with ZGDHu-1 sensitivity (Figure 7C, 7F), thus indicating ZGDHu-1 less effective in Bcl-2 high primary MCL cells.However, no significant association was observed between Mcl-1 mRNA levels (Figure 7B), Bax mRNA levels (Figure 7D), Bcl-XL mRNA levels (Figure 7E) and ZGDHu-1 sensitivity.
As we observed that high levels of Bcl-2 conferred less effective to ZGDHu-1, we postulated whether ZGDHu-1 could less effective in Bcl-2 high MCL cell lines.To prove our surmise, we treated the representative Bcl-2 high cell line MAVER-1 and Bcl-2 low cell line REC-1 with ZGDHu-1 (Table 2).As expected, the results indicated Bcl-2 low cell line REC-1 was sensitizer to ZGDHu-1 than Bcl-2 high cell line MAVER-1 (Figure 1C and Figure 3C,  3E, 3G).

DISCUSSION
In this study, we found that ZGDHu-1 showed great cytotoxicity in 17 primary MCL cells and three different MCL cell lines and identified ZGDHu-1 as a potent antilymphoma compound to MCL.Some differences in  sensitivity to ZGDHu-1 were observed among primary MCL cells, but not in B cells from healthy donors [16].ZGDHu-1 inhibited MCL cell proliferation by decreasing the protein levels of cyclin D1 and inducing cell cycle G2/M phase arrest.More importantly, ZGDHu-1 induced apoptosis of MCL cells by decreasing the anti-apoptotic protein levels of Mcl-1 and Bcl-XL.We demonstrated that ZGDHu-1 inhibited the activation of the TNFα-induced NF-κB signaling pathway and the induction of these antiapoptotic proteins.
The intracellular redox status, which depends on both the GSH levels and ROS generation, is important in stabilizing mitochondrial functions.The mitochondrial dysfunction associated with ROS induced the activation of pro-apoptotic proteins, then led to caspase-dependent or caspase-independent apoptosis [24].ZGDHu-1 significantly induced ROS generation of MCL cells and GSH could suppress ZGDHu-1-induced apoptosis in MCL cells.These results suggested ZGDHu-1 could regulate mitochondria by elevating the ROS levels and affecting the intracellular redox status.Furthermore, our data also showed that ZGDHu-1 decrease the ∆Ψm, induced the cleavage of caspase-3 and PARP in MCL cells.Taken together, these findings showed that ZGDHu-1 ultimately activated the mitochondrial apoptotic pathway and caused caspase-dependent apoptosis in MCL cells, which consistent with previous reports [16,25].
Cyclins and the CDK family play important roles in cell-cycle regulation and cell replication.Coupled with CDK, cyclin D1 regulates the G1-S transition of the cell cycle and is involved in the regulation of MCL cell proliferation.In this study, our results show that ZGDHu-1 significantly decreased cyclin D1 protein levels and induced cell cycle G2/M phase arrest in MCL cells.The suppression of cyclin D1 by ZGDHu-1 resulted in the suppression of MCL cell proliferation.Because NF-κB is  well known to mediate antiapoptotic effects, elicits cellcycle arrest and contributes to the stabilities of cyclin D1 in MCL cells [9], we examined whether suppression of NF-κB by ZGDHu-1 could lead to apoptosis.
To further elucidate the mechanism of ZGDHu-1-induced apoptosis in MCL cells, we investigated the important apoptotic regulators which are known to be regulated by NF-κB [26][27].NF-κB activity is commonly elevated in CLL [28], MCL [9,29], and other solid tumors [30].The activation of the NF-κB signaling pathway is appreciated as a key mechanism for cell proliferation [31][32] and apoptosis [33][34].We demonstrated that ZGDHu-1 inhibited the TNFα-induced expression of Bcl-XL and Mcl-1, IκBa phosphorylation and nuclear p65 levels.In the absence of exogenous TNFα, ZGDHu-1 in MCL cells also inhibited IκBa phosphorylation and nuclear p65 levels and ultimately suppressed the expression of proliferation (cyclin D1) and survival (Bcl-xL, Mcl-1) molecules.These findings suggested that ZGDHu-1 induced apoptosis most likely through the inhibition of NF-κB signaling pathway in MCL cells.
Our results indicated that ZGDHu-1 induced cell cycle G2/M phase arrest and caused caspasedependent apoptosis in MCL cells, in consistent with similar experiments performed with CLL cells [16].In CLL, ZGDHu-1-induced apoptosis is promoted by antiapoptotic Bcl-2.Herein, we noticed that Bcl-2 was highly constitutively expressed in MCL patients, in accordance with other studies [35].However, we did not observe Bcl-2 down-regulation in primary MCL cells following ZGDHu-1 treatment, suggesting a distinct mechanism for apoptosis induction in MCL cells.In this study, Mcl-1 and Bcl-XL reductions caused by ZGDHu-1 may account for the observed apoptosis.Nevertheless, although similar reductions in Mcl-1 mRNA levels were observed among all primary MCL cases, a variable degree of cytotoxic response to ZGDHu-1 could be found, suggesting that other mechanisms may be involved in ZGDHu-1-induced apoptosis of MCL cells.Remarkably, we found that high Bcl-2 expression levels and Bcl-2/Bax ratios correlated to reduced responses to ZGDHu-1 in primary MCL cells.Furthermore, Next we continue to study whether the targeting Bcl-2 can improve the sensitivity of ZGDHu-1 in Bcl-2 high MCL cells.
In conclusion, this is the first report evaluating the effects of a novel tetrazine compound ZGDHu-1 on MCL.Our results show that ZGDHu-1 can potently inhibit cell proliferation and induce apoptosis in MCL cells through the inhibition of NF-κB regulated anti-apoptotic genes expression in vitro.In addition, results show the antilymphoma activity of ZGDHu-1 in MCL cells was on the targeting NF-κB pathway.Our research thus provides evidence and rationale regarding the potentially therapeutic effects of ZGDHu-1 and the possibility that treatment with this molecule may improve the outcomes of MCL patients.

Patients
Seventeen MCL patients (12 males and 5 females) aged 59-83 years (with a median age of 73 years) were enrolled in this study.The biological characteristics of these cases are shown in Table 1.Patients with MCL were identified on the basis of morphologic, immunophenotypic, and molecular criteria according to World Health Organization (WHO) lymphoma classification.Only those patients who had not received previous treatments within the last 6 months were included in the study.All 17 patients were collected prior to the commencement of any treatment.Age-matched controls were obtained from 10 healthy donors.Ethical approval for this project, including informed consent from patients, was granted based on the guidelines of the Zhejiang Provincial People's Hospital research ethics committee.

Lymphocyte purification and culture
Primary MCL cells were obtained from peripheral blood samples of MCL patients who were diagnosed according to the WHO criteria.Lymphocytes were isolated using Ficoll-Hypaque gradient centrifugation according to the manufacturer's instructions.After 1 h of incubation at 37°C in 5% CO 2 , adhesive mononuclear cells were removed.T lymphocytes were removed using anti-CD3 dynabeads (Dynal, Merseyside, UK).Purification of B lymphocytes was assessed by flow cytometry with anti-CD19 antibodies.This cell preparation contained approximately 95% CD19 positive cells (Figure 1B).

Hoechst 33258 staining
For Hoechst 33258 staining, MCL cells were seeded into 6-well plates at 5×10 5 cells per well.After ZGDHu-1 treatment at the indicated times, the cells were washed with serum-free RPMI 1640 and subsequently with 1× phosphate-buffered saline (PBS; DingGuo Biotechnology Co., Ltd., Beijing, China), after which they were fixed with fixative (methanol:glacial acetic acid 3:1) for 5 min at 4°C and stained with 10 μg/ml of Hoechst 33258 (Applygen Tech Inc, Beijing, China) for 10 min.Morphological changes associated with apoptosis was observed using fluorescence microscopy (Nikon Y-THS, Japan) with 350to 370-nm excitation wavelengths and emission detection at 465 nm.

Cell viability assay
Cell viability was determined as described using PI stainning and flow cytometer [18].The effects of ZGDHu-1 on the viability of MCL cells were assayed using MTT assay.The optical density was measured using a microplate reader M680 (Bio-Rad, Hercules, CA, USA) and reference and test wavelengths of 630 nm and 570 nm, respectively.All experiments were performed in triplicate and repeated at least three times.The cell viability was expressed as a percentage of the DMSO-treated control samples.

FACS
Primary MCL cells were stained with anti-CD19-PerCP CY 5.5 and anti-active caspase-3-PE.The stained cells were analyzed using NAVIOS FACS.Then, 10,000 cells were counted for each sample and CD19 + cell population was gated for analysis as follows.The effects of ZGDHu-1 on apoptosis in MCL cells were evaluated via annexin V/PI assay, mitochondrial membrane potentials (ΔΨm), active caspase-3 and cell cycle analysis.The annexin V/PI assay was performed according to the manufacturer's instructions, and only annexin V-positive (+) and PI-negative (-) cells were defined as apoptotic.ΔΨm was measured using JC-1 dye.The intracellular accumulation of ROS was assessed using DHR fluorescent dye.The percentage of active caspase-3-positive cells was calculated using NAVIOS FACS.

Cell cycle analysis
A DNA Prep™ reagent Kit was used to evaluate alterations in the cell cycles of MCL cells.After the designated treatments, MCL cells were harvested and washed with PBS solution.500 μl of DNA Prep LPR was added, and after 8 s, 500 μl of DNA Prep stain was added and then remained for 15 min at room temperature.Finally, the cells were analyzed by NAVIOS FACS, and the fractions of the cell population in the G1/G0 (G01), S, and G2/M phases were quantified via Wincycle 32 software.The sub-G1 fraction was determined from the total event count.

Protein extraction and western blot analysis
Following incubation with different concentrations ZGDHu-1, MCL cells were lysed, and proteins were extracted and quantitated using a bicinchoninic protein assay kit (DingGuo Biotechnology Co., Ltd.).The supernatant constituted the cytoplasmic protein fraction.The pellet was washed once with Cytoplasm Lysis Buffer and extracted using Nuclear Lysis Buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS, 1% P-8340(Sigma, St. Louis, MO)).The proteins were loaded into wells of an 8 or 12% SDS-PAGE, electrophoresed and transferred onto a nitrocellulose membrane (DingGuo Biotechnology Co., Ltd.).The membrane was incubated with the appropriate primary antibody and then washed and incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Inc.).

Real-time quantitative polymerase chain reaction (qRT-PCR)
Following incubation with ZGDHu-1, TRIzol reagent (Takara, Dalian, China) was used to extract total RNA from cells according to the manufacturer's instructions.Reverse transcription to complementary DNA (cDNA) was achieved using a PrimeScript RT reagent kit with a gDNA eraser (Takara).Amplification reactions were performed using a SYBR Premix Ex Taq II kit (Takara) and a LightCycler 480 real-time PCR system (Roche Diagnostics, Mannheim, Germany).The Mcl-1, Bcl-2, Bcl-XL and Bax expression levels were determined in duplicate using predesigned Assay-On-Demand probes (Invitrogen, Beijing, China) on a LightCycler 480 realtime PCR system.Each gene's relative expression level was quantified using the comparative cycle threshold (Ct) method (ΔΔCt) and b-actin as the endogenous control.The expression level was given in arbitrary units, using control (untreated cells) or Jeko-1 cells as a reference.

Statistical analysis
All data were collected from three independent experiments.Values, expressed as the means ± standard deviations (SD), were analyzed using Student's t test, and P values less than 0.05 were considered statistically significant.

Figure 1 :
Figure 1: Identification of ZGDHu-1 as a potent anti-lymphoma compound in MCL cells.A. The chemical structure of ZGDHu-1 B. Purity of isolated primary MCL cells C. Six primary MCL cells were cultured with 0 -1000 ng/ml ZGDHu-1 for 72 h.Cell viability was measured with MTT assay.

Figure 2 :
Figure 2: ZGDHu-1 induces cell cycle G2/M phase arrest in MCL cells.A. MCL cells were cultured in 0.05% DMSO drugfree medium (control) or 100 -200 ng/ml ZGDHu-1 for 48 h.The cell cycle distribution was analyzed using the Wincycle32 software.The percentage of cells in the subG1 phase is depicted in each plot.B. Quantifications of the proportions of cells in subG1, G01, S, and G2/M phases are listed for each experiment.ZGDHu-1 (100 -200 ng/ml, 48 h) significantly increased the percentage of G2/M phase of MCL cells compared to DMSO controls.Values represent means ± SD for three separate experiments, each performed in triplicate.

Figure 3 :Figure 3 (
Figure 3: ZGDHu-1 induces apoptosis in MCL cells.A. Apoptotic cell morphology was measured by the Hoechst33258 staining.The morphologies of primary MCL cells from two MCL patients (MCL #3 and MCL #12) changed dramatically after ZGDHu-1 (100 -200 ng/ml) treatment for 48 h.B. MCL cells were cultured in 0.05% DMSO drug-free medium or 100 -200 ng/ml ZGDHu-1 in the absence or presence of 200 mM Z-VAD.fmk for 48 h, stained with Annexin V/PI and analyzed by flow cytometry.Representative histograms of Annexin-V/PI of primary MCL cells and three MCL cell lines are displayed.C. Quantitative data pertaining to panel B. Percentages of Annexin V-positive cells are shown.Values represent means ± SD, each performed in triplicate.* P<0.05 between ZGDHu-1 and control; # P<0.05 between ZGDHu-1 and ZGDHu-1+Z-VAD.fmk.(Continued )

Figure 4 :
Figure 4: Effects of ZGDHu-1 on ROS levels in MCL cells.A. MCL cells were cultured in 0.05% DMSO drug-free medium or 100 -200 ng/ml ZGDHu-1 in the absence or the presence of 100 μM GSH for 48 h.DHR staining and flow cytometry were used for ROS generation.Representative ROS MFI is displayed from two MCL patients (MCL #3 and MCL #12).B. Quantitative data pertaining to panel A. Data revealed the effect of ZGDHu-1 on ROS MFI of primary MCL cells and three MCL cell lines in the absence or the presence of 100 μM GSH for 48 h.* P<0.05 between ZGDHu-1 and control; # P<0.05 between ZGDHu-1 and ZGDHu-1+GSH.C. Representative histograms of Annexin-V/PI are displayed from two MCL patients (MCL 3# and MCL 12#) following exposure to ZGDHu-1 in the absence or the presence of 100 μM GSH for 48 h.D. Quantitative data pertaining to panel C. Data reveals the effect of GSH on ZGDHu-1-induced apoptosis in primary MCL cells and three MCL cell lines for 48 h.* P<0.05 between ZGDHu-1 and control; # P<0.05 between ZGDHu-1 and ZGDHu-1+GSH.E. Data reveals the effect of GSH on ZGDHu-1-induced changes in the ΔΨm of MCL cells for 48 h.* P<0.05 between ZGDHu-1 and control.# P<0.05 between ZGDHu-1 and ZGDHu-1+GSH.

Figure 6 (
Figure 6 (Continued ): D. Three MCL cell liness were first treated with or without ZGDHu-1 (200 ng/ml) for 24 h.Then, TNFα (10 ng/ ml) was added for 60 and 120 min.The nuclear and cytoplasmic fractions were collected for p65 detection.Tubulin and Histone H3 were used as cytoplasmic and nuclear protein controls.

Figure 7 :
Figure 7: Bcl-2 expression inversely correlates with ZGDHu-1 sensitivity.A. Mcl-1, Bcl-2, Bax and Bcl-XL mRNA relative levels in primary MCL and three MCL cell lines were detected by qRT-PCR using β-actin as a loading control.B. Correlation between Mcl-1 mRNA relative levels and ZGDHu-1 cytotoxicity in primary MCL cells.C. Correlation between Bcl-2 mRNA relative levels and ZGDHu-1 cytotoxicity in primary MCL cells.D. Correlation between Bax mRNA relative levels and ZGDHu-1 cytotoxicity in primary MCL cells.E. Correlation between Bcl-XL mRNA relative levels and ZGDHu-1 cytotoxicity in primary MCL cells.F. Correlation between Bcl-2/Bax ratio and ZGDHu-1 cytotoxicity in primary MCL cells.