Novel mechanism of harmaline on inducing G2/M cell cycle arrest and apoptosis by up-regulating Fas/FasL in SGC-7901 cells

Harmaline (HAR), a natural occurrence β-carboline alkaloid, was isolated from the seeds of Peganum harmala and exhibited potent antitumor effect. In this study, the anti-gastric tumor effects of HAR were firstly investigated in vitro and in vivo. The results strongly showed that HAR could inhibit tumor cell proliferation and induce G2/M cell cycle arrest accompanied by an increase in apoptotic cell death in SGC-7901 cancer cells. HAR could up-regulate the expressions of cell cycle-related proteins of p-Cdc2, p21, p-p53, Cyclin B and down-regulate the expression of p-Cdc25C. In addition, HAR could up-regulate the expressions of Fas/FasL, activated Caspase-8 and Caspase-3. Moreover, blocking Fas/FasL signaling could markedly inhibit the apoptosis caused by HAR, suggesting that Fas/FasL mediated pathways were involved in HAR-induced apoptosis. Interestingly, HAR could also exert on antitumor activity with a dose of 15 mg/kg/day in vivo, which was also related with cell cycle arrest. These new findings provided a framework for further exploration of HAR which possess the potential antitumor activity by inducing cell cycle arrest and apoptosis.


HAR inhibited the proliferation of SGC-7901 cells.
To determine the effect of HAR on tumor cell proliferation, SGC-7901 cells were treated with various concentrations of HAR for 48 h and cell viability was determined using MTT assay. Proliferation of SGC-7901 cells was markedly inhibited by HAR-treatment in a dose dependent manner with a lower IC 50 value of 4.08 ± 0.89 μ M ( Fig. 2A) and no obvious cytotoxicity was observed in mouse fibroblast cells (3T3) (Fig. 2B). To further determine the characteristics of HAR-induced SGC-7901 cell death, morphologic change was observed. In SGC-7901 cells, exposure to HAR for 48 h resulted in morphologic characteristic alterations of apoptosis, including membrane blebbing, nuclear condensation and granular apoptotic bodies (Fig. 2C) compared with the control group.

HAR induced G2/M cell cycle arrest in SGC-7901 cells.
There were some reports that β -carbolines were specific inhibitors of Cyclin dependent kinases. To further investigate the functional mechanism of HAR in inhibiting cell growth, the effect of HAR on the regulation of the cell cycle in SGC-7901 cells was investigated.
Flow cytometry analysis was performed on the SGC-7901 cells treated with or without HAR for different time points. Interestingly, the results clearly showed that HAR-treated SGC-7901 cells were arrested in G2/M phase in a time-dependent manner (Fig. 3). After 48 hours of incubation, HAR arrested SGC-7901 cells at the G2/M phases (21.1% ± 2.04%) as compared to the control (2.2% ± %1.72%), and also the sub-G1 peak was observed by FACS analysis due to the DNA fragmentation in apoptotic cells.

Effects of HAR on the expressions of cell cycle regulatory proteins.
To further characterize the mechanism by which HAR induced G2/M cell cycle arrest, the effects of HAR on the expressions of P21, p-P53, P53, Cyclin B1, p-Cdc2 and p-Cdc25C were examined. As expected, the cellular level of P21, p-P53 and Cyclin B1 dramatically increased after 24 h HAR-treatment and continued to increase up to 48 h. However, HAR-treatment resulted in a remarkably time-dependent decrease in p-Cdc25C expression levels accompanied the positive augment of p-Cdc2 (Fig. 4A). These data strongly indicated that HAR might induce G2/M arrest by altering the expressions of cell cycle related proteins (Fig. 4B-E).
Effects of HAR on the expressions of extrinsic apoptotic related proteins. Since HAR showed the higher sensitivity and selectivity on SGC-7901 cells than other cell lines based on our previous study, the main apoptosis-related protein levels were further evaluated. Interestingly, it found that both z-DEVD-fmk (Caspase-3 inhibitor) and z-IETD-fmk (Caspase-8 inhibitor) increased the cell viability compared with cells treated by HAR only (Fig. 5A). It also found that the expressions of Fas/FasL were up-regulated after HAR-treatment accompanied by the positive augments of the activated Caspase-8 and Caspase-3 ( Fig. 5B-F). Moreover, Caspase-3 activity assay was applied to determine whether Caspase-3 involved in the HAR-induced apoptosis. As presented in Fig. 5G,H, the activity of Caspase-3 was significantly increased after HAR treatment in a time and dose dependent manner. To further confirm the death receptor (extrinsic) apoptotic pathway contributed to cell death treated by HAR, the additional experiments were carried out. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited the cell death caused by HAR (Fig. 5I). In addition, activated Caspase-3 and Caspase-8 were also downregulated after blocking Fas/FasL signaling (Fig. 5J). These experiments demonstrated that the Fas death receptor pathway contributed to HAR-induced apoptosis. Antitumor activity and toxicity of HAR in vivo. Based on the in vitro findings, the efficacy of HAR in SGC-7901 cells in nude mice was evaluated and three different doses of HAR (5, 15, 30 mg/kg/day) were studied. Compared with the control group, both median (15 mg/kg/day) and high doses (30 mg/kg/day) could significantly inhibit the growth of human gastric tumors in nude mice in a dose dependent manner (P < 0.001) (Fig. 6A). Meanwhile, body weight loss was observed after high dose treatment. Based on the therapeutic efficacy of HAR in vivo model, the expressions of p21, p-Cdc2 and p-Cdc25C were examined in the tumor samples. As shown in Fig. 6B, HAR treatment significantly increased the expressions of p21, p-Cdc2 accompanying by the reduction of p-Cdc25C. These results strongly suggested that HAR might induce apoptosis through cell cycle arrest in vivo. Regarding the toxicity in high dose treatment, the adverse effects, particularly in liver, were examined. The liver enzymes AST and ALT in serum level were normally used as biomarkers of hepatotoxicity. In this study, no alteration of the ALT or AST in serum level was observed, except high dose group. Moreover, the contents of serum WBC, RBC, HGB and PLT also had no change in the low and median dose treatment group (Table 1). Therefore, the modest HAR-treatment without induced apparent systemic toxicity, and strengthened its use for gastric tumor. However, the high dose treatment could result in the WBC reduction and enhancing the level of ALT.

Discussions
Gastric cancer is a leading cause of death worldwide. Although surgery is the mainstay of any curative treatment, recurrences and metastases are still observed in approximately two-thirds of patients 18 . HAR is the one of major alkaloids in P. harmala seeds which exerts main function. In the present study, we demonstrated a novel molecular mechanism through which HAR inhibited gastric cancer cell proliferation in vitro and in vivo.
HAR showed a significantly inhibition in cell proliferation of SGC-7901 cells in a dose dependent manner. Also SGC-7901 cells underwent apoptosis after HAR treatment based on the typical morphologic change and sub-G1 fraction resulting from DNA fragmentation. Identification of the sub-G1 subpopulation may be used as an index of apoptotic cells in SGC-7901 cells treated by HAR.
Moreover, the results showed that HAR exerted a strong anti-proliferative effect against gastric cancer cells by inducing G2/M arrest. As we know, cell cycle progression is regulated by Cyclin-dependent kinases (Cdks) and Cyclins. Cyclin B-Cdc2 plays a main role in G2/M phase transition. Cyclin dependent kinase inhibitor (CDKI), Cip1/p21, also regulates the transitions of G2/M phase by binding and inhibiting the activity of Cdc2-Cyclin B1 complex. The ability of p21 to promote cell cycle inhibition may also depend on its ability to mediate p53-dependent gene repression, as p21 is both necessary and sufficient for p53-dependent repression of genes regulating cell cycle progression 19,20 . Cdc25C is another critical regulator of Cdc2-Cyclin B1 kinase activity and control cell cycle progression by dephosphorylating and activating CDKs [21][22][23] . Consistent with these notions, we observed an increase in the protein level of inhibitory p21 in SGC-7901 cell. An increased phosphorylation of Cdc2 accompanied by a decreased p-Cdc25C protein was also observed in SGC-7901 cells treated with HAR. The increased Cdc2 phosphorylation was likely due to reduced p-Cdc25C protein level which prevented it from dephosphorylating Cdc2.
Fas/FasL death receptor plays an important role in cell apoptosis. Upon binding to the FasL, the Fas trimerizes and induces apoptosis through the cytoplasmic death domain (DD) that interacts with signaling adaptors like Fas-associated death domain (FADD). FADD carries a death effector domain (DED) and it recruits the DED containing procaspase-8 protein which is in inactive state. Procaspase-8 is proteolytically activated to Caspase-8. FADD also helps in the activation of Caspase-10. Upon activation, Caspase-8 and Caspase-10 cleave and activate downstream effector Caspases, including Caspase-3, 6 and 7. But there was also reported that harmol activated a key element of the Fas signaling pathway independently of Fas/FADD activation 24 . Therefore, Fas, FasL, and two key downstream effector Caspases, activated Caspase-8 and Caspase-3 were detected to reveal whether Fas/ FasL signaling pathway involved in inducing apoptosis of SGC-7901 cells upon treatment of HAR. Surprised, up-regulation of Fas, FasL, activated Caspase-8 and caspase-3 was observed in SGC-7901 cells. The Caspase-3 activity was also detected in gastric tumor cells. As expected, increased Caspase-3 activity was also observed in a time or dose dependent manner. Blocking Fas/FasL signaling using an anti-Fas antibody significantly blocked Caspase-3 activation, and Caspase-8 activation. These results implied that Fas/FasL signaling pathway was initiated and involved in SGC-7901 cell apoptosis (Fig. 7). However, it was not known if intrinsic apoptotic pathway also contributed to SGC-7901 cells apoptosis treated by HAR. It was still needed to be verified further in the future.
The strong tumor inhibition properties, as well as the Fas/FasL-mediated apoptotic action of HAR, prompted us to evaluate its efficacy and safety in vivo. In our current experiments, HAR suppressed tumor growth at a dose of 15 mg/kg/day without any significant changes of the mice body weight and the possible mechanism was cell cycle arrest owning to the up-regulating P21 and p-Cdc2. Side effects, such as hair loss, lethargy, dysphoria, or other macroscopical visceral pathogenic changes were not observed. However, it was reported that HAR could significantly reduced the viability of four human cultured non-transformed (CCD18Lu) and transformed (HeLa, C33A and SW480) cells in a dose-dependent manner 25 . In this study, only high does of HAR (30 mg/kg/day) treatment showed toxicity based on the body weight loss, the serum WBC reduction and the increasing of ALT level. Therefore, enhanced in vivo studies and narrow research was still required to draw a line between cytotoxic and antitumor efficiency of HAR.
Taken together, the induction of P21 at early stage, along with the increased p-Cdc2 as a result of the downregulation of Cdc25c, lies at the base of the mechanism through which HAR induced G2/M arrest and the activation Fas/FasL pathway (Fig. 7). Extraction and purification of HAR from P. harmala. The dried seeds of P. harmala (11 kg) were powdered and then extracted three times with 70% aqueous methanol under reflux. After filtration and evaporation of the solvent under reduced pressure, the residues (2.3 kg) were suspended in water and acidified to pH 1.0 with 10% hydrochloric acid. Lipophilic impurities were removed with CHCl 3 extraction and the aqueous fraction was alkalized to pH 7.0 with concentrated NH 3 •H 2 O. The crude alkaloids were extracted four times with CHCl 3 and n-BuOH sequentially. The CHCl 3 fraction (200 g) was separated into 15 fractions (C1-C15) by silica gel column  Cell culture. Human gastric carcinoma SGC-7901 and mouse fibroblast 3T3 cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μ g/ mL streptomycin and maintained in a humidified atmosphere containing 5% CO 2 at 37 °C. HAR was dissolved in methanol and diluted by DMEM keeping a maximum concentration of methanol 0.1% in the culture medium.  High does 8.2 ± 0.8 * 8.6 ± 0.4 134.0 ± 6.5 913.5 ± 26.0 68.9 ± 9.5 * 69.7 ± 6.7 Table 1 Flow cytometry cell cycle propidium iodide (PI) assay. SGC-7901 cells were stained by PI according to the manufacturer's protocol. Briefly, SGC-7901 cells in DMEM containing 10% FBS were seeded into 10 cm dish with 2 × 10 5 cells for each dish and incubated overnight. Then the cells were treated with 5 μ M HAR for 0, 12, 24, 36 or 48 h. After incubation, a suspension of cells with a density of 10 5 cells per treatment was fixed in 3.7% ice-cold paraformaldehyde for 30 min, followed by labeling with PI. The labeled cells were analyzed using a Flow Cytometer.
Western Blot analysis. SGC-7901 cells were washed with cold PBS after 5 μ M HAR treatment in different time points as indicated, and then the cells were lysated in a RIPA buffer that contained a mixture of protease inhibitors or the solid tumors were cut into small pieces and then were lysated in a RIPA buffer that contained a mixture of protease inhibitors. Twenty micrograms of total protein from each sample were run on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were incubated with appropriately diluted primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies against the corresponding species. Labeling was detected using the ECL system (Amersham Biosciences, Pittsburgh, PA, USA).

Caspase-3 activity assay.
Caspase-3 activity assay is a fluorescent assay that detects the activity of Caspase-3 in cell lysates. It contains a fluorogenic substrate (N-Acetyl -Asp-Glu-Val-Asp-7-amino-4-methylcoumarin or Ac-DEVDAMC) special for Caspase-3. SGC-7901 cells were seeded into a 96-well plate before the experimental day and then the cells were treated with 5 μ M HAR in different time courses or the cells were treated by different concentrations of HAR for 48 h. After incubation, both floating and adherent treated-cells were then collected and lysated in ice-cold lysis buffer for 10 min in a 1.5 mL tube. After centrifuged for 10 min at 4 °C, the supernatant was transferred to another tube and then mixed 200 μ L of substrate solution B and 25 μ L lysate solutions in a black plate appropriate for fluorescent assay. The plate was incubated at 37 °C for 1 h in the dark and it was read on a fluorescence plate reader according to the assay protocol. Data were presented as mean ± SD of three independent experiments.
Mouse experiments and tumor xenograft model. Healthy male nude mice (Balb/c, 6-8 weeks of age) were bought from Guangdong Medical Experimental Animal Center. All animal experiment protocols were approved by the Animal Ethics Committee of Wuhan University. Mice were injected subcutaneously with SGC-7901 cells (1 × 10 7 cells/mouse) to the right back. After one week, animals were randomly divided into four groups (10 mice for each group). Mice were treated with either 5% CMC-Na or HAR (low dose group: 5 mg/kg/day; median dose group: 15 mg/kg/day; high dose group: 30 mg/kg/day) dissolved in 5% CMC-Na by oral and were weighted every other day. Finally, mice were sacrificed after 10 days treatment. Twenty-four hours before the last treatment, animals were anesthetized after exposure to ether in desiccators kept in a well-functioning hood. Blood samples were collected by heart puncture, and serum samples were tested using Healife automatic biochemical analyzer. The whole solid tumor tissues were detached from the back of mice and then were frozen in liquid nitrogen for Western Blot analysis after weight. All of the animals were treated according to protocols approved by the Animal Ethics Committee of Wuhan University. And the study was approved by the Animal Ethics Committee of Wuhan University.
Statistical analysis. Data were presented as mean ± standard deviation from three experiments in three different biological replicates, and the significance was evaluated by one-way analysis of variance (ANOVA).