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Endoplasmic reticulum stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity
Biochemical and Biophysical Research Communications 466 (2015) 369e375
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Endoplasmic reticulum stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity Keren Wu a, Ning Li a, Huaqin Sun b, Tao Xu a, Fa Jin a, Jifeng Nie c, * a
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China Department of Anesthesiology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China c Department of Minimally Invasive, Hospital of Integrated Chinese and Western Medicine in Zhejiang Province, Hangzhou, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 August 2015 Accepted 5 September 2015 Available online 8 September 2015
In the current study, we examined the potential effect of Ginsenoside Rg3 against gallbladder cancer cells, the underlying signaling mechanisms were also studied. We demonstrated that Rg3 exerted potent cytotoxic and pro-apoptotic activity against established and primary human gallbladder cancer cells. Yet it was safe to non-cancerous gallbladder epithelial cells. At the molecular level, we showed that Rg3 induced endoplasmic reticulum (ER) stress activation, the latter was evidenced by C/EBP homologous protein (CHOP) upregulation, inositol-requiring enzyme 1 (IRE1)/PKR-like endoplasmic reticulum kinase (PERK) phosphorylations, and caspase-12 activation in gallbladder cancer cells. Reversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP shRNA knockdown significantly attenuated Rg3-induced cytotoxicity against gallbladder cancer cells. In vivo, we showed that Rg3 oral administration significantly inhibited GBC-SD gallbladder cancer xenograft growth in nude mice, its activity was, however, compromised with co-administration of the ER stress inhibitor salubrinal. Thus, we suggest that ER stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity in vitro and in vivo. © 2015 Elsevier Inc. All rights reserved.
Keywords: Ginseng Rg3 Endoplasmic reticulum stress Gallbladder cancer CHOP Nude mice
1. Introduction Gallbladder cancer is the most frequent malignancy in the bile duct, it is also a highly aggressive and lethal neoplasm with poor prognosis [1,2]. This malignancy is often diagnosed at advanced and/or late stages, possibly due to absence of specific symptoms and clinical signs [1,2]. Clinically, surgical resection is the only curative treatment for qualified gallbladder cancer patients with decent conditions [1,2]. Yet, many post-surgery patients will likely develop recurrence or even metastasis following surgery [1,2]. Therefore, chemotherapy and other adjuvant therapies are vital for gallbladder cancer patients [1,2]. The search of more effective therapeutic agents against this devastating disease is extremely important. Ginseng is one of the most-widely utilized ancient Chinese herbal medicine with many pharmaceutical and therapeutic functions [3,4]. The main molecular components responsible for the
* Corresponding author. Department of Minimally Invasive, Hospital of Integrated Chinese and Western medicine in Zhejiang province, Hangzhou, 310006, China. E-mail address: [email protected] (J. Nie). http://dx.doi.org/10.1016/j.bbrc.2015.09.030 0006-291X/© 2015 Elsevier Inc. All rights reserved.
actions of Ginseng are ginsenosides [3,4]. The level of ginsenoside Rg3 (Rg3) is extremely low in Ginseng, yet Rg3 has displayed multiple functional activities, including hepatoprotection, neuroprotection, immune-stimulating activity, and most importantly, the tumor-suppressing function [3,4]. However, the potential effect of Rg3 in human gallbladder cancer cells, and the associated signaling mechanisms have not been fully studied. Endoplasmic reticulum (ER) regulates synthesis, posttranslational modification, proper folding, and maturation of newly synthesized proteins [5,6]. It also vital for the intracellular calcium homeostasis [5,6]. Many anti-cancer drugs could disrupt proper ER functions, causing pathological ER stress activation [5,6], and induction of several unfolded protein responses (UPR). The latter will lead to up-regulation of ER chaperones and folding enzymes, including C/EBP homologous protein (CHOP), GADD 45, GRP 78 and GRP 94; Further, translation will be inhibited to limit further accumulation of misfolded proteins; Third, misfolded proteins are likely to be degradated inside ER [7]. ER membrane receptors including double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) act as the sensors of stress to UPR [7]. Although the UPR is could be a pro-survival response, in
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the event of prolonged or severe ER stress, the UPR will initiate cell apoptosis [7]. In the current study, we provided evidence to support that pathological ER stress mediates Rg3-induced anti-gallbladder cancer activity in vitro and in vivo. 2. Material and methods 2.1. Chemicals and reagents Ginseng Rg3 and the ER stress inhibitor salubrinal (Sal) [8], as well as ER stress inducers thapsigargin (Tg) and tunicamycin (Tm) were obtained from Sigma (St. Louis, MO). The pan-caspase inhibitor (z-VAD-fmk) and the specific caspase-12 inhibitor (z-ATADfmk) [9] were purchased from Calbiochem (Darmstadt, Germany). Anti-PERK, CHOP, IRE-1 and tubulin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p-IRE1 was obtained from Abcam (Shanghai, China). All other antibodies used in this study were obtained from Cell Signaling Tech (Danvers, MA). Cell culture reagents were from Life Technologies (Gaithersburg, MD). 2.2. Cell lines and culture Mz-ChA-1, QBC939 and GBC-SD human gallbladder cancer cells, purchased from Shanghai Biological Science Institute (Shanghai, China), were maintained in DMEM/RPMI medium, supplemented with 10% FBS (Sigma), penicillin/streptomycin (1:100; Sigma) and 4 mM L-glutamine (Sigma). 2.3. Primary culture of human gallbladder cancer cells and noncancerous epithelial cells Surgery-isolated gallbladder cancer tissues of two patients (male, 54/61 years old, no prior chemotherapy) were thoroughly washed. Non-cancerous surrounding gallbladder epithelial tissues were separated and preserved. Tissues were minced and washed in DMEM with 100 units/penicillinestreptomycin. Single-cell suspensions of primary gallbladder cancer cells (from cancer tissues) and non-cancerous epithelial cells (from epithelial tissues) were achieved by re-suspending tissues in 0.20% (w/v) collagenase for 1 h. Afterwards, individual cell was pelleted and rinsed twice with DMEM. Cells were then cultured in primary cell culture medium: DMEM, 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 20 mM HEPES, 1.0 g/L glucose, 5 mg/mL insulin, 5 mg/ mL human transferrin, 5 ng/mL sodium selenite, 1 vitamins solution, and 1 nonessential amino acids solution. The study was approved by the institutional review board of all authors' institutions, and written informed consent was obtained from all individual participated patients. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. 2.4. Cell viability assay Cells were seeded at 0.5 104 cells per well onto 96microculture-well plates. Following treatment, cell viability was assayed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) reagent according to the protocol provided. The MTT absorbance was measured at 490 nm as an indicator of cell viability. 2.5. Clonogenicity assay Cells (5 103) were suspended in 1 mL of DMEM containing 1% agar (Sigma, St. Louis, MO), 10% FBS and with indicated treatment.
The cell suspension was then added on top of a pre-solidified 1% agar in a 100 mm culture dish. The drug-containing medium was replaced every two days. After ten days of incubation, the left surviving colonies were manually counted. 2.6. Trypan blue staining assay Following treatment, the percentage (%) of “dead” cells was calculated by the number of the trypan blue stained cells, divided by the total cell number ( 100%), which was recorded by an automated cell counter (Merck Millipore, Shanghai, China). 2.7. Apoptosis detection by ssDNA ELISA assay Cell apoptosis was quantified by an ApoStrand™ ELISA apoptosis detection kit (BIOMOL International, PA) according to the manufacturer's instructions [10]. This assay is based on the sensitivity of DNA in apoptotic cells to formamide denature and the detection of the denatured DNA with an antibody to single-stranded DNA (ssDNA) [10]. ELISA OD was utilized as a quantitative indicator of cell apoptosis. 2.8. Apoptosis assay by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) staining Cell apoptosis was detected by ApopTag fluorescence cell apoptosis kit (Millipore Corporation, Billerica, MA) according to the manufacturer's instruction. The number of TUNEL-positive cells was counted in ten randomly selected fields of view (100 magnification) from each treatment. TUNEL percentage (vs. total Hoechst nuclei) was recorded. 2.9. Annexin V flow cytometry analysis Cell apoptosis was also tested by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) according to attached procedure (FACSCalibur, BD Biosciences, Shanghai, China). Annexin V positive cells were gated as the apoptotic cells. 2.10. Caspase-12 activity assay As previously reported [9,11], after treatment, 20 mg of cytosolic extracts were added to caspase assay buffer (312.5 mM HEPES, 31.25% sucrose, 0.3125% CHAPS) with ATAD-7-amido-4-(trifluoromethyl) coumarin (AFC) (10 mg/mL) (Calbiochem, Darmstadt, Germany) as the substrate [9,11]. The amount of AFC liberated from ATAD-AFC was measured using a spectrofluorometer (ThermoLabsystems, Helsinki, Finland) with excitation of 380 nm and emission wavelength of 460 nm. 2.11. Western blots Cells were lysed with the lysis buffer from Biyuntian (Wuxi, China); Proteins (40 mg/sample) were resolved by SDS-PAGE, and were transferred to PVDF membranes. The latter were incubated sequentially in TBS containing 0.05% Tween-20 and 5% nonfat dry milk as follows: no addition, 30 min at room temperature (blocking); primary antibody, overnight at 4 C; and secondary antibody (Amersham), 1 h at room temperature. Bound secondary antibody was detected by ECL system. Western blot results were quantified by Image J software from NIH website.
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2.12. CHOP shRNA-knockdown through lentiviral infection
the Institutional Animal Care and Use Committee (IACUC).
The two non-overlapping human CHOP shRNAs and one scramble non-sense shRNA containing lentiviral particles were gifts from Dr. Zhang's group [9]. Cells were seeded in a six-well plate in the growth medium. The lentiviral particles (15 mL/mL medium) were added to the cells. After 24 h, the medium was replaced by fresh growth medium, and cells were further cultured for additional 72 h. The expression of CHOP and the equal loading (tubulin) in the infected cells was detected by Western blots.
2.14. Statistical analysis All results were expressed as mean ± SD. Statistical analyses were performed using SPSS 16.0 software. The significance of difference between groups was estimated by one-way ANOVA, followed by Bonferroni's post hoc test for independent samples. Differences were considered significant at p < 0.05. 3. Results
2.13. Tumor xenograft study Male, 7/8-week-old nude mice were randomly divided into four groups (Vehicle control, Rg3, Sal, Rg3 þ Sal, n ¼ 10/group). GBC-SD cells (5 106/mouse) were mixed with PBS (200 mL/mouse) and inoculated into the right flank of each mouse. When the tumors reached a volume of 50e70 mm3, mice were given a daily oral dose of 20 mg/kg Rg3 or the vehicle (200 mL PBS, control group), and/or intraperitoneally once daily of 1 mg/kg Sal, for 21 days. Tumor dimensions were measured every 5 days using a digital caliper, and tumor volume was calculated using the formula: V ¼ length width2 0.5. The weight of each mice was also recorded as a general measurement of health. The selected doses of Rg3 and salubrinal were based on previous publications [12e14]. At the end of the experiment, the mice were killed and the tumors were excised and weighed. The animal protocols were approved by
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3.1. Rg3 exerts potent cytotoxic effect against human gallbladder cancer cells GBC-SD cells, the established human gallbladder cancer cell line [15], were stimulated with applied concentrations of Rg3, cells were further cultured. MTT cell viability results in Fig. 1a demonstrated that Rg3 dose-dependently inhibited GBC-SD cell survival. Further, the effect by Rg3 was also time-dependent (Fig. 1a). Rg3 took at least 48 h to exert its anti-survival effect (Fig. 1a). Rg-3-induced cytotoxic effect was also confirmed by the colony formation assay (Fig. 1b) and trypan blue staining assay (Fig. 1c). As demonstrated, Rg3, in a dosedependent manner, decreased the number of viable GBC-SD colonies (Fig. 1b), while increasing the number of trypan blue stained (“dead”) cells (Fig. 1c). The similar anti-survival activity by Rg3 was also observed in two other established human gallbladder cancer
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Fig. 1. Rg3 exerts potent cytotoxic effect against human gallbladder cancer cells. Human gallbladder cancer cell lines, GBC-SD (aec), QBC939 (d) and Mz-ChA-1 (e), as well as patient-derived primary gallbladder cancer cells (f, “Cancer cells”) or the surrounding non-cancerous gallbladder epithelial cells (f, “Epithelial cells”), were either left untreated (“C”) or treated with applied concentrations of Ginseng Rg3 (“Rg3”) for indicated time point, cell survival was tested by MTT assay (a, def) or colony formation assay (b, for GBC-SD cells); Cell death was examined by trypan blue staining assay (c, for GBC-SD cells). The values were expressed as the means ± SD. For each assay, n ¼ 6. The experiments in this figure were repeated four times, and similar results were obtained. *P < 0.05 vs. “C” group.
K. Wu et al. / Biochemical and Biophysical Research Communications 466 (2015) 369e375
in Rg3-induced activity. We thus examined activity of caspase-12, an indicator or ER stress-mediated apoptosis [9,16,17], in Rg3treated cells. Results in Fig. 2a demonstrated that Rg3 dosedependently induced caspase-12 activation in GBC-SD cells. Subsequent apoptosis activation was confirmed by three independent assays: including ssDNA ELISA assay (Fig. 2b), TUNEL staining assay (Fig. 2c) and Annexin V FACS assay (Fig. 2d). The pro-apoptosis activity by Rg3 was again dose-dependent (Fig. 2aec). Importantly, the caspase-12 inhibitor z-ATAD-fmk (“ATAD”) or the pan caspase inhibitor z-VAD-fmk (“VAD”) remarkably inhibited Rg3 (100 mM)-induced caspase-12 activation, and following GBC-SD cell apoptosis (Fig. 2aed). As a result, Rg3-induced GBC-SD cell viability reduction (Fig. 2e) and death (Fig. 2f) were largely attenuated by these caspase inhibitors. Note
cell lines: QBC939 (Fig. 1d) and Mz-ChA-1 (Fig. 1e). The potential effect of Rg3 against the patient-derived primary gallbladder cancer cells was also analyzed. Significantly, as shown in Fig. 1f, Rg3 at 50 mM significantly inhibited survival of primary cancer cells. On the other hand, same Rg3 treatment was non-cytotoxic against gallbladder epithelial cells (non-cancerous cells, Fig. 1f). These results suggest that Rg3 was cytotoxic only in cancer cells. These results demonstrate that Rg3 exerts potent cytotoxic effect against primary and established human gallbladder cancer cells. 3.2. Rg3 activates caspase-12-dependent apoptosis in gallbladder cancer cells The current study was set to understand the role of ER stress
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Fig. 2. Rg3 induces caspase-12-dependent apoptosis in gallbladder cancer cells. GBC-SD cells were either left untreated (“C”), treated with applied concentrations of Ginseng Rg3 (“Rg3”) or plus the caspase-12 inhibitor z-ATAD-fmk (“ATAD”, 50 mM) and the pan caspase inhibitor z-VAD-fmk (“VAD”, 50 mM) for indicated time point, caspase-12 activity was tested (a); Cell apoptosis was tested by ssDNA ELISA assay (b), TUNEL staining assay (c) or Annexin V FACS assay (d); Cell survival and cell death were tested by MTT assay (e) and Trypan blue staining assay (f), respectively. Patient-derived primary gallbladder cancer cells (f, “Cancer cells”) or the surrounding non-cancerous epithelial cells (f, “Epithelial cells”), pre-treated with z-ATAD-fmk (“ATAD”, 50 mM) or z-VAD-fmk (“VAD”, 50 mM), were stimulated with Rg3 (50 mM) for indicated time, cell apoptosis was tested by ssDNA ELISA assay (g), and cell death was tested by trypan blue staining assay (h, for “Cancer cells”). The values were expressed as the means ± SD. For each assay, n ¼ 6. The experiments in this figure were repeated four times, and similar results were obtained. *P < 0.05 vs. “C” group. #P < 0.05.
K. Wu et al. / Biochemical and Biophysical Research Communications 466 (2015) 369e375
that similar results were also obtained in two other established human gallbladder cancer cell lines: QBC939 and Mz-ChA-1 (Data not shown). At the meantime, Rg3 induced significant apoptosis in primary human gallbladder cancer cells (tested by ssDNA ELISA assay,Fig. 2g), but was non-effective in gallbladder epithelial cells (Fig. 2g). Similarly, the caspase-12 inhibitor and the pan caspase inhibitor significantly inhibited Rg3-exerted cytotoxicity against primary cancer cells (Fig. 2h). Note that the caspase inhibitors alone had no effect on survival or apoptosis of above cells (Data not shown). These results indicate a critical role of caspase-12dependent apoptosis in mediating Rg3-exerted cytotoxicity against gallbladder cancer cells.
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3.3. ER stress activation mediates Rg3-induced cytotoxicity in human gallbladder cancer cells Above results have shown that caspase-12-dependent apoptosis participates in Rg3-induced cytotoxicity, indicating a role of ER stress [9,16,17] in Rg3's actions. We thus tested the effect of Rg3 on ER stress activation. Western blot results showed that Rg3 dosedependently induced CHOP expression as well as PERK and IRE1 phosphorylations in GBC-SD cells (Fig. 3a), confirming ER stress activation [9]. Significantly, the ER stress inhibitor salubrinal (Sal) [8] dramatically inhibited Rg3-induced viability reduction (Fig. 3b) and apoptosis activation (Fig. 3c) in GBC-SD cells. Similar results were also obtained in QBC939 cells and Mz-ChA-1 cells (Data not
Fig. 3. ER stress activation mediates Rg3-induced cytotoxicity against human gallbladder cancer cells. GBC-SD cells or primary human gallbladder cancer cells were either left untreated (“C”), or treated with applied concentrations of Ginseng Rg3 (“Rg3”) for applied time, expression of listed ER-stress-associated proteins was tested by Western blots (a and h). GBC-SD cells or primary human gallbladder cancer cells, pre-treated with/out the ER stress inhibitor salubrinal (“Sal”, 10 mM) for 2 h, were stimulated with applied Rg3 or ER stress inducers thapsigargin (Tg, 5 mM) and tunicamycin (Tm, 5 mM), cell survival and cell apoptosis were tested by MTT assay (b, for GBC-SD cells) and TUNEL staining assay (c and g, for GBC-SD cells), cell death was tested by trypan blue staining assay (g and i). Expression of CHOP and tubulin (Equal loading) in GBC-SD cells, infected with scramble-shRNA (“SCR shRNA”) or CHOP-shRNA (1/2) was shown (d). Above cells were treated with Rg3 (100 mM) for applied time, cell survival and apoptosis were tested (e and f). The values were expressed as the means ± SD. For each assay, n ¼ 4. CHOP expression (vs. tubulin) and PERK/IRE1 phosphorylations were quantified. The experiments in this figure were repeated three times, and similar results were obtained. *P < 0.05 vs. “C” group. #P < 0.05 vs. Rg3 only group (b, c, e and f). #P < 0.05 (h).
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shown). To further confirm the role of ER stress in Rg3-exerted actions, shRNA strategy was applied to selectively knockdown CHOP [9]. CHOP plays a key role in ER stress-induced apoptosis [18,19]. Western blot results showed that the two non-overlapping CHOP shRNAs [9] significantly downregulated CHOP expression in GBC-SD cells (Fig. 3d). Consequently, Rg3-induced cytotoxicity and apoptosis were remarkably attenuated with CHOP silencing (Fig. 3e and f). Notably, ER stress inducers thapsigargin (Tg) and tunicamycin (Tm) mimicked Rg30 actions, and induced significant apoptosis and cytotoxicity in GBC-SD cells (Fig. 3g). Significantly, in primary human gallbladder cancer cells, Rg3 induced similar CHOP expression and PERK/IRE1 phosphorylations (Fig. 3g), again confirming ER stress activation. Sal similarly inhibited Rg-3-induced cytotoxicity against primary cancer cells (Fig. 3h). Together, these results demonstrate that CHOP induction and ER stress activation are required for Rg3-induced cytotoxicity against gallbladder cancer cells.
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4. Discussions Existing evidences have demonstrated that Rg3 inhibits growth of several cancer cell lines, yet the underlying mechanism is not fully understood [14,20,21]. In the current study, we showed that Rg3 induced significant pro-apoptotic and cytotoxic activity against established and primary human gallbladder cancer cells. Interestingly, same Rg3 treatment was safe to the non-cancerous gallbladder epithelial cells. At the molecular level, we suggest that pathological ER stress activation could be the key signaling mechanism responsible for actions in gallbladder cancer cells. Several components of ER stress are strongly associated with cell apoptosis. For example, when facing unsolved ER stress, CHOP is upregulated through all three arms of UPR [22]. High level of CHOP then changes the balance between pro-survival and proapoptotic Bcl-2 family members, eventually leading to cell apoptosis [23,24]. CHOP dictates pro-apoptotic Bim upregulation and ER translocation to facilitate mitochondrial apoptosis pathway [24]. CHOP could also up-regulate TRAIL receptor to activate extrinsic apoptotic pathway [19]. Here, we showed that CHOP expression was significantly increased following Rg3 treatment in both primary and established human gallbladder cancer cells. CHOP silencing through targeted shRNAs dramatically attenuated Rg3-induced cytotoxicity and apoptosis in gallbladder cancer cells. These results confirm that CHOP expression could be a key ER stress mediator of Rg3-induced anti-gallbladder cancer cell activity. Aside from CHOP, existing evidence has also implied other ER
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Finally, we studied the in vivo activity of Rg3. Results in Fig. 4a demonstrated that xenografted tumors were established after GBCSD cell inoculation in nude mice. Significantly, oral administration of Rg3 (20 mg/kg, daily, 21 days) dramatically inhibited GBC-SD xenograft growth (Fig. 4a). The weights of Rg3-treated GBC-SD tumors were significantly lower than that of vehicle (PBS) group (Fig. 4b). Remarkably, the in vivo activity by Rg3 was attenuated with co-administration of the ER stress inhibitor Sal (1 mg/kg, daily, i.p.) (Fig. 4a and b), indicating that ER stress is also required for the in vivo activity by Rg3. Sal alone showed no effect on GBC-SD xenograft growth (Fig. 4a and b). There was no significant difference in the mice body weights of each group, indicating the general safe to heath of applied regimens (Fig. 4c). Thus, we demonstrate that Rg3 inhibits GBC-SD cell growth in vivo, and its activity was inhibited with Sal co-administration.
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3.4. Rg3 inhibits GBC-SD cell growth in vivo, and its activity was compromised with salubrinal co-administration
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Fig. 4. Rg3 inhibits GBC-SD cell growth in vivo, and its activity was compromised with salubrinal co-administration. GBC-SD cell bearing nude mice (male, 6/7 weeks old) were given the vehicle (“Ctrl”, 200 mL PBS, oral gavage, daily), Rg3 (20 mg/kg, oral gavage, daily), and/or salubrinal (“Sal”, 1 mg/kg, intraperitoneally, daily), for a total of 21 days, tumor volumes (a) and mice body weights (c) were recorded every 5 days. At the end of the experiment, the mice were killed, and the tumors were excised and weighed (b). The experiments in this figure were repeated twice, and similar results were obtained. *P < 0.05 vs. “Ctrl” group. #P < 0.05 vs. Rg3 only group.
stress-mediated apoptosis mechanisms [5,7]. For instance, phosphorylated IRE1 under ER stress recruits adapter proteins (i.e. TRAF2 and ASK1), causing subsequent recruitment of pro-caspase12, the latter has been proposed as a key mediator of ER stressinduced apoptosis [9,11,17,25]. Here, we demonstrated that Rg3 induced IRE1 phosphorylation and subsequent caspase-12 activation in gallbladder cancer cells. Significantly, the caspase-12 inhibitor z-ATAD-fmk along with the pan caspase inhibitor z-VADfmk dramatically inhibited Rg3-induced cytotoxicity against
K. Wu et al. / Biochemical and Biophysical Research Communications 466 (2015) 369e375
gallbladder cancer cells, confirming an important role of caspase-12 activation in Rg3's activity. One important finding of this study is that oral administration of a single dose of Rg3 (20 mg/kg, daily) could remarkably inhibit GBC-SD xenograft growth in nude mice. More importantly, such activity by Rg3 was attenuated with co-administration of Sal. Thus, ER stress activation is likely also important for the in vivo activity of Rg3. In summary, the results of this study imply that pathological ER stress activation mediates Rg3-induced anti-gallbladder cancer cell activity in vitro and in vivo. Rg3 could be further studied as a promising anti-gallbladder cancer agent.
[9]
[10]
[11]
[12]
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Acknowledgments [14]
This work is supported by the National Science Foundation of China. [15]
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.09.030.
[16]
[17]
Conflict of interests The authors declare that they have no conflict of interests. [18]
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Endoplasmic reticulum stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity Keren Wu a, Ning Li a, Huaqin Sun b, Tao Xu a, Fa Jin a, Jifeng Nie c, * a
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China Department of Anesthesiology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China c Department of Minimally Invasive, Hospital of Integrated Chinese and Western Medicine in Zhejiang Province, Hangzhou, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 August 2015 Accepted 5 September 2015 Available online 8 September 2015
In the current study, we examined the potential effect of Ginsenoside Rg3 against gallbladder cancer cells, the underlying signaling mechanisms were also studied. We demonstrated that Rg3 exerted potent cytotoxic and pro-apoptotic activity against established and primary human gallbladder cancer cells. Yet it was safe to non-cancerous gallbladder epithelial cells. At the molecular level, we showed that Rg3 induced endoplasmic reticulum (ER) stress activation, the latter was evidenced by C/EBP homologous protein (CHOP) upregulation, inositol-requiring enzyme 1 (IRE1)/PKR-like endoplasmic reticulum kinase (PERK) phosphorylations, and caspase-12 activation in gallbladder cancer cells. Reversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP shRNA knockdown significantly attenuated Rg3-induced cytotoxicity against gallbladder cancer cells. In vivo, we showed that Rg3 oral administration significantly inhibited GBC-SD gallbladder cancer xenograft growth in nude mice, its activity was, however, compromised with co-administration of the ER stress inhibitor salubrinal. Thus, we suggest that ER stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity in vitro and in vivo. © 2015 Elsevier Inc. All rights reserved.
Keywords: Ginseng Rg3 Endoplasmic reticulum stress Gallbladder cancer CHOP Nude mice
1. Introduction Gallbladder cancer is the most frequent malignancy in the bile duct, it is also a highly aggressive and lethal neoplasm with poor prognosis [1,2]. This malignancy is often diagnosed at advanced and/or late stages, possibly due to absence of specific symptoms and clinical signs [1,2]. Clinically, surgical resection is the only curative treatment for qualified gallbladder cancer patients with decent conditions [1,2]. Yet, many post-surgery patients will likely develop recurrence or even metastasis following surgery [1,2]. Therefore, chemotherapy and other adjuvant therapies are vital for gallbladder cancer patients [1,2]. The search of more effective therapeutic agents against this devastating disease is extremely important. Ginseng is one of the most-widely utilized ancient Chinese herbal medicine with many pharmaceutical and therapeutic functions [3,4]. The main molecular components responsible for the
* Corresponding author. Department of Minimally Invasive, Hospital of Integrated Chinese and Western medicine in Zhejiang province, Hangzhou, 310006, China. E-mail address: [email protected] (J. Nie). http://dx.doi.org/10.1016/j.bbrc.2015.09.030 0006-291X/© 2015 Elsevier Inc. All rights reserved.
actions of Ginseng are ginsenosides [3,4]. The level of ginsenoside Rg3 (Rg3) is extremely low in Ginseng, yet Rg3 has displayed multiple functional activities, including hepatoprotection, neuroprotection, immune-stimulating activity, and most importantly, the tumor-suppressing function [3,4]. However, the potential effect of Rg3 in human gallbladder cancer cells, and the associated signaling mechanisms have not been fully studied. Endoplasmic reticulum (ER) regulates synthesis, posttranslational modification, proper folding, and maturation of newly synthesized proteins [5,6]. It also vital for the intracellular calcium homeostasis [5,6]. Many anti-cancer drugs could disrupt proper ER functions, causing pathological ER stress activation [5,6], and induction of several unfolded protein responses (UPR). The latter will lead to up-regulation of ER chaperones and folding enzymes, including C/EBP homologous protein (CHOP), GADD 45, GRP 78 and GRP 94; Further, translation will be inhibited to limit further accumulation of misfolded proteins; Third, misfolded proteins are likely to be degradated inside ER [7]. ER membrane receptors including double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) act as the sensors of stress to UPR [7]. Although the UPR is could be a pro-survival response, in
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the event of prolonged or severe ER stress, the UPR will initiate cell apoptosis [7]. In the current study, we provided evidence to support that pathological ER stress mediates Rg3-induced anti-gallbladder cancer activity in vitro and in vivo. 2. Material and methods 2.1. Chemicals and reagents Ginseng Rg3 and the ER stress inhibitor salubrinal (Sal) [8], as well as ER stress inducers thapsigargin (Tg) and tunicamycin (Tm) were obtained from Sigma (St. Louis, MO). The pan-caspase inhibitor (z-VAD-fmk) and the specific caspase-12 inhibitor (z-ATADfmk) [9] were purchased from Calbiochem (Darmstadt, Germany). Anti-PERK, CHOP, IRE-1 and tubulin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p-IRE1 was obtained from Abcam (Shanghai, China). All other antibodies used in this study were obtained from Cell Signaling Tech (Danvers, MA). Cell culture reagents were from Life Technologies (Gaithersburg, MD). 2.2. Cell lines and culture Mz-ChA-1, QBC939 and GBC-SD human gallbladder cancer cells, purchased from Shanghai Biological Science Institute (Shanghai, China), were maintained in DMEM/RPMI medium, supplemented with 10% FBS (Sigma), penicillin/streptomycin (1:100; Sigma) and 4 mM L-glutamine (Sigma). 2.3. Primary culture of human gallbladder cancer cells and noncancerous epithelial cells Surgery-isolated gallbladder cancer tissues of two patients (male, 54/61 years old, no prior chemotherapy) were thoroughly washed. Non-cancerous surrounding gallbladder epithelial tissues were separated and preserved. Tissues were minced and washed in DMEM with 100 units/penicillinestreptomycin. Single-cell suspensions of primary gallbladder cancer cells (from cancer tissues) and non-cancerous epithelial cells (from epithelial tissues) were achieved by re-suspending tissues in 0.20% (w/v) collagenase for 1 h. Afterwards, individual cell was pelleted and rinsed twice with DMEM. Cells were then cultured in primary cell culture medium: DMEM, 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 20 mM HEPES, 1.0 g/L glucose, 5 mg/mL insulin, 5 mg/ mL human transferrin, 5 ng/mL sodium selenite, 1 vitamins solution, and 1 nonessential amino acids solution. The study was approved by the institutional review board of all authors' institutions, and written informed consent was obtained from all individual participated patients. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. 2.4. Cell viability assay Cells were seeded at 0.5 104 cells per well onto 96microculture-well plates. Following treatment, cell viability was assayed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) reagent according to the protocol provided. The MTT absorbance was measured at 490 nm as an indicator of cell viability. 2.5. Clonogenicity assay Cells (5 103) were suspended in 1 mL of DMEM containing 1% agar (Sigma, St. Louis, MO), 10% FBS and with indicated treatment.
The cell suspension was then added on top of a pre-solidified 1% agar in a 100 mm culture dish. The drug-containing medium was replaced every two days. After ten days of incubation, the left surviving colonies were manually counted. 2.6. Trypan blue staining assay Following treatment, the percentage (%) of “dead” cells was calculated by the number of the trypan blue stained cells, divided by the total cell number ( 100%), which was recorded by an automated cell counter (Merck Millipore, Shanghai, China). 2.7. Apoptosis detection by ssDNA ELISA assay Cell apoptosis was quantified by an ApoStrand™ ELISA apoptosis detection kit (BIOMOL International, PA) according to the manufacturer's instructions [10]. This assay is based on the sensitivity of DNA in apoptotic cells to formamide denature and the detection of the denatured DNA with an antibody to single-stranded DNA (ssDNA) [10]. ELISA OD was utilized as a quantitative indicator of cell apoptosis. 2.8. Apoptosis assay by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) staining Cell apoptosis was detected by ApopTag fluorescence cell apoptosis kit (Millipore Corporation, Billerica, MA) according to the manufacturer's instruction. The number of TUNEL-positive cells was counted in ten randomly selected fields of view (100 magnification) from each treatment. TUNEL percentage (vs. total Hoechst nuclei) was recorded. 2.9. Annexin V flow cytometry analysis Cell apoptosis was also tested by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) according to attached procedure (FACSCalibur, BD Biosciences, Shanghai, China). Annexin V positive cells were gated as the apoptotic cells. 2.10. Caspase-12 activity assay As previously reported [9,11], after treatment, 20 mg of cytosolic extracts were added to caspase assay buffer (312.5 mM HEPES, 31.25% sucrose, 0.3125% CHAPS) with ATAD-7-amido-4-(trifluoromethyl) coumarin (AFC) (10 mg/mL) (Calbiochem, Darmstadt, Germany) as the substrate [9,11]. The amount of AFC liberated from ATAD-AFC was measured using a spectrofluorometer (ThermoLabsystems, Helsinki, Finland) with excitation of 380 nm and emission wavelength of 460 nm. 2.11. Western blots Cells were lysed with the lysis buffer from Biyuntian (Wuxi, China); Proteins (40 mg/sample) were resolved by SDS-PAGE, and were transferred to PVDF membranes. The latter were incubated sequentially in TBS containing 0.05% Tween-20 and 5% nonfat dry milk as follows: no addition, 30 min at room temperature (blocking); primary antibody, overnight at 4 C; and secondary antibody (Amersham), 1 h at room temperature. Bound secondary antibody was detected by ECL system. Western blot results were quantified by Image J software from NIH website.
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2.12. CHOP shRNA-knockdown through lentiviral infection
the Institutional Animal Care and Use Committee (IACUC).
The two non-overlapping human CHOP shRNAs and one scramble non-sense shRNA containing lentiviral particles were gifts from Dr. Zhang's group [9]. Cells were seeded in a six-well plate in the growth medium. The lentiviral particles (15 mL/mL medium) were added to the cells. After 24 h, the medium was replaced by fresh growth medium, and cells were further cultured for additional 72 h. The expression of CHOP and the equal loading (tubulin) in the infected cells was detected by Western blots.
2.14. Statistical analysis All results were expressed as mean ± SD. Statistical analyses were performed using SPSS 16.0 software. The significance of difference between groups was estimated by one-way ANOVA, followed by Bonferroni's post hoc test for independent samples. Differences were considered significant at p < 0.05. 3. Results
2.13. Tumor xenograft study Male, 7/8-week-old nude mice were randomly divided into four groups (Vehicle control, Rg3, Sal, Rg3 þ Sal, n ¼ 10/group). GBC-SD cells (5 106/mouse) were mixed with PBS (200 mL/mouse) and inoculated into the right flank of each mouse. When the tumors reached a volume of 50e70 mm3, mice were given a daily oral dose of 20 mg/kg Rg3 or the vehicle (200 mL PBS, control group), and/or intraperitoneally once daily of 1 mg/kg Sal, for 21 days. Tumor dimensions were measured every 5 days using a digital caliper, and tumor volume was calculated using the formula: V ¼ length width2 0.5. The weight of each mice was also recorded as a general measurement of health. The selected doses of Rg3 and salubrinal were based on previous publications [12e14]. At the end of the experiment, the mice were killed and the tumors were excised and weighed. The animal protocols were approved by
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3.1. Rg3 exerts potent cytotoxic effect against human gallbladder cancer cells GBC-SD cells, the established human gallbladder cancer cell line [15], were stimulated with applied concentrations of Rg3, cells were further cultured. MTT cell viability results in Fig. 1a demonstrated that Rg3 dose-dependently inhibited GBC-SD cell survival. Further, the effect by Rg3 was also time-dependent (Fig. 1a). Rg3 took at least 48 h to exert its anti-survival effect (Fig. 1a). Rg-3-induced cytotoxic effect was also confirmed by the colony formation assay (Fig. 1b) and trypan blue staining assay (Fig. 1c). As demonstrated, Rg3, in a dosedependent manner, decreased the number of viable GBC-SD colonies (Fig. 1b), while increasing the number of trypan blue stained (“dead”) cells (Fig. 1c). The similar anti-survival activity by Rg3 was also observed in two other established human gallbladder cancer
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Fig. 1. Rg3 exerts potent cytotoxic effect against human gallbladder cancer cells. Human gallbladder cancer cell lines, GBC-SD (aec), QBC939 (d) and Mz-ChA-1 (e), as well as patient-derived primary gallbladder cancer cells (f, “Cancer cells”) or the surrounding non-cancerous gallbladder epithelial cells (f, “Epithelial cells”), were either left untreated (“C”) or treated with applied concentrations of Ginseng Rg3 (“Rg3”) for indicated time point, cell survival was tested by MTT assay (a, def) or colony formation assay (b, for GBC-SD cells); Cell death was examined by trypan blue staining assay (c, for GBC-SD cells). The values were expressed as the means ± SD. For each assay, n ¼ 6. The experiments in this figure were repeated four times, and similar results were obtained. *P < 0.05 vs. “C” group.
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in Rg3-induced activity. We thus examined activity of caspase-12, an indicator or ER stress-mediated apoptosis [9,16,17], in Rg3treated cells. Results in Fig. 2a demonstrated that Rg3 dosedependently induced caspase-12 activation in GBC-SD cells. Subsequent apoptosis activation was confirmed by three independent assays: including ssDNA ELISA assay (Fig. 2b), TUNEL staining assay (Fig. 2c) and Annexin V FACS assay (Fig. 2d). The pro-apoptosis activity by Rg3 was again dose-dependent (Fig. 2aec). Importantly, the caspase-12 inhibitor z-ATAD-fmk (“ATAD”) or the pan caspase inhibitor z-VAD-fmk (“VAD”) remarkably inhibited Rg3 (100 mM)-induced caspase-12 activation, and following GBC-SD cell apoptosis (Fig. 2aed). As a result, Rg3-induced GBC-SD cell viability reduction (Fig. 2e) and death (Fig. 2f) were largely attenuated by these caspase inhibitors. Note
cell lines: QBC939 (Fig. 1d) and Mz-ChA-1 (Fig. 1e). The potential effect of Rg3 against the patient-derived primary gallbladder cancer cells was also analyzed. Significantly, as shown in Fig. 1f, Rg3 at 50 mM significantly inhibited survival of primary cancer cells. On the other hand, same Rg3 treatment was non-cytotoxic against gallbladder epithelial cells (non-cancerous cells, Fig. 1f). These results suggest that Rg3 was cytotoxic only in cancer cells. These results demonstrate that Rg3 exerts potent cytotoxic effect against primary and established human gallbladder cancer cells. 3.2. Rg3 activates caspase-12-dependent apoptosis in gallbladder cancer cells The current study was set to understand the role of ER stress
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Fig. 2. Rg3 induces caspase-12-dependent apoptosis in gallbladder cancer cells. GBC-SD cells were either left untreated (“C”), treated with applied concentrations of Ginseng Rg3 (“Rg3”) or plus the caspase-12 inhibitor z-ATAD-fmk (“ATAD”, 50 mM) and the pan caspase inhibitor z-VAD-fmk (“VAD”, 50 mM) for indicated time point, caspase-12 activity was tested (a); Cell apoptosis was tested by ssDNA ELISA assay (b), TUNEL staining assay (c) or Annexin V FACS assay (d); Cell survival and cell death were tested by MTT assay (e) and Trypan blue staining assay (f), respectively. Patient-derived primary gallbladder cancer cells (f, “Cancer cells”) or the surrounding non-cancerous epithelial cells (f, “Epithelial cells”), pre-treated with z-ATAD-fmk (“ATAD”, 50 mM) or z-VAD-fmk (“VAD”, 50 mM), were stimulated with Rg3 (50 mM) for indicated time, cell apoptosis was tested by ssDNA ELISA assay (g), and cell death was tested by trypan blue staining assay (h, for “Cancer cells”). The values were expressed as the means ± SD. For each assay, n ¼ 6. The experiments in this figure were repeated four times, and similar results were obtained. *P < 0.05 vs. “C” group. #P < 0.05.
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that similar results were also obtained in two other established human gallbladder cancer cell lines: QBC939 and Mz-ChA-1 (Data not shown). At the meantime, Rg3 induced significant apoptosis in primary human gallbladder cancer cells (tested by ssDNA ELISA assay,Fig. 2g), but was non-effective in gallbladder epithelial cells (Fig. 2g). Similarly, the caspase-12 inhibitor and the pan caspase inhibitor significantly inhibited Rg3-exerted cytotoxicity against primary cancer cells (Fig. 2h). Note that the caspase inhibitors alone had no effect on survival or apoptosis of above cells (Data not shown). These results indicate a critical role of caspase-12dependent apoptosis in mediating Rg3-exerted cytotoxicity against gallbladder cancer cells.
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3.3. ER stress activation mediates Rg3-induced cytotoxicity in human gallbladder cancer cells Above results have shown that caspase-12-dependent apoptosis participates in Rg3-induced cytotoxicity, indicating a role of ER stress [9,16,17] in Rg3's actions. We thus tested the effect of Rg3 on ER stress activation. Western blot results showed that Rg3 dosedependently induced CHOP expression as well as PERK and IRE1 phosphorylations in GBC-SD cells (Fig. 3a), confirming ER stress activation [9]. Significantly, the ER stress inhibitor salubrinal (Sal) [8] dramatically inhibited Rg3-induced viability reduction (Fig. 3b) and apoptosis activation (Fig. 3c) in GBC-SD cells. Similar results were also obtained in QBC939 cells and Mz-ChA-1 cells (Data not
Fig. 3. ER stress activation mediates Rg3-induced cytotoxicity against human gallbladder cancer cells. GBC-SD cells or primary human gallbladder cancer cells were either left untreated (“C”), or treated with applied concentrations of Ginseng Rg3 (“Rg3”) for applied time, expression of listed ER-stress-associated proteins was tested by Western blots (a and h). GBC-SD cells or primary human gallbladder cancer cells, pre-treated with/out the ER stress inhibitor salubrinal (“Sal”, 10 mM) for 2 h, were stimulated with applied Rg3 or ER stress inducers thapsigargin (Tg, 5 mM) and tunicamycin (Tm, 5 mM), cell survival and cell apoptosis were tested by MTT assay (b, for GBC-SD cells) and TUNEL staining assay (c and g, for GBC-SD cells), cell death was tested by trypan blue staining assay (g and i). Expression of CHOP and tubulin (Equal loading) in GBC-SD cells, infected with scramble-shRNA (“SCR shRNA”) or CHOP-shRNA (1/2) was shown (d). Above cells were treated with Rg3 (100 mM) for applied time, cell survival and apoptosis were tested (e and f). The values were expressed as the means ± SD. For each assay, n ¼ 4. CHOP expression (vs. tubulin) and PERK/IRE1 phosphorylations were quantified. The experiments in this figure were repeated three times, and similar results were obtained. *P < 0.05 vs. “C” group. #P < 0.05 vs. Rg3 only group (b, c, e and f). #P < 0.05 (h).
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shown). To further confirm the role of ER stress in Rg3-exerted actions, shRNA strategy was applied to selectively knockdown CHOP [9]. CHOP plays a key role in ER stress-induced apoptosis [18,19]. Western blot results showed that the two non-overlapping CHOP shRNAs [9] significantly downregulated CHOP expression in GBC-SD cells (Fig. 3d). Consequently, Rg3-induced cytotoxicity and apoptosis were remarkably attenuated with CHOP silencing (Fig. 3e and f). Notably, ER stress inducers thapsigargin (Tg) and tunicamycin (Tm) mimicked Rg30 actions, and induced significant apoptosis and cytotoxicity in GBC-SD cells (Fig. 3g). Significantly, in primary human gallbladder cancer cells, Rg3 induced similar CHOP expression and PERK/IRE1 phosphorylations (Fig. 3g), again confirming ER stress activation. Sal similarly inhibited Rg-3-induced cytotoxicity against primary cancer cells (Fig. 3h). Together, these results demonstrate that CHOP induction and ER stress activation are required for Rg3-induced cytotoxicity against gallbladder cancer cells.
a. 2500 Ctrl
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4. Discussions Existing evidences have demonstrated that Rg3 inhibits growth of several cancer cell lines, yet the underlying mechanism is not fully understood [14,20,21]. In the current study, we showed that Rg3 induced significant pro-apoptotic and cytotoxic activity against established and primary human gallbladder cancer cells. Interestingly, same Rg3 treatment was safe to the non-cancerous gallbladder epithelial cells. At the molecular level, we suggest that pathological ER stress activation could be the key signaling mechanism responsible for actions in gallbladder cancer cells. Several components of ER stress are strongly associated with cell apoptosis. For example, when facing unsolved ER stress, CHOP is upregulated through all three arms of UPR [22]. High level of CHOP then changes the balance between pro-survival and proapoptotic Bcl-2 family members, eventually leading to cell apoptosis [23,24]. CHOP dictates pro-apoptotic Bim upregulation and ER translocation to facilitate mitochondrial apoptosis pathway [24]. CHOP could also up-regulate TRAIL receptor to activate extrinsic apoptotic pathway [19]. Here, we showed that CHOP expression was significantly increased following Rg3 treatment in both primary and established human gallbladder cancer cells. CHOP silencing through targeted shRNAs dramatically attenuated Rg3-induced cytotoxicity and apoptosis in gallbladder cancer cells. These results confirm that CHOP expression could be a key ER stress mediator of Rg3-induced anti-gallbladder cancer cell activity. Aside from CHOP, existing evidence has also implied other ER
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Finally, we studied the in vivo activity of Rg3. Results in Fig. 4a demonstrated that xenografted tumors were established after GBCSD cell inoculation in nude mice. Significantly, oral administration of Rg3 (20 mg/kg, daily, 21 days) dramatically inhibited GBC-SD xenograft growth (Fig. 4a). The weights of Rg3-treated GBC-SD tumors were significantly lower than that of vehicle (PBS) group (Fig. 4b). Remarkably, the in vivo activity by Rg3 was attenuated with co-administration of the ER stress inhibitor Sal (1 mg/kg, daily, i.p.) (Fig. 4a and b), indicating that ER stress is also required for the in vivo activity by Rg3. Sal alone showed no effect on GBC-SD xenograft growth (Fig. 4a and b). There was no significant difference in the mice body weights of each group, indicating the general safe to heath of applied regimens (Fig. 4c). Thus, we demonstrate that Rg3 inhibits GBC-SD cell growth in vivo, and its activity was inhibited with Sal co-administration.
Tumor net weights (gram)
3.4. Rg3 inhibits GBC-SD cell growth in vivo, and its activity was compromised with salubrinal co-administration
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day 5
day 10 day 15 day 20 day 25 day 30 day 35
Fig. 4. Rg3 inhibits GBC-SD cell growth in vivo, and its activity was compromised with salubrinal co-administration. GBC-SD cell bearing nude mice (male, 6/7 weeks old) were given the vehicle (“Ctrl”, 200 mL PBS, oral gavage, daily), Rg3 (20 mg/kg, oral gavage, daily), and/or salubrinal (“Sal”, 1 mg/kg, intraperitoneally, daily), for a total of 21 days, tumor volumes (a) and mice body weights (c) were recorded every 5 days. At the end of the experiment, the mice were killed, and the tumors were excised and weighed (b). The experiments in this figure were repeated twice, and similar results were obtained. *P < 0.05 vs. “Ctrl” group. #P < 0.05 vs. Rg3 only group.
stress-mediated apoptosis mechanisms [5,7]. For instance, phosphorylated IRE1 under ER stress recruits adapter proteins (i.e. TRAF2 and ASK1), causing subsequent recruitment of pro-caspase12, the latter has been proposed as a key mediator of ER stressinduced apoptosis [9,11,17,25]. Here, we demonstrated that Rg3 induced IRE1 phosphorylation and subsequent caspase-12 activation in gallbladder cancer cells. Significantly, the caspase-12 inhibitor z-ATAD-fmk along with the pan caspase inhibitor z-VADfmk dramatically inhibited Rg3-induced cytotoxicity against
K. Wu et al. / Biochemical and Biophysical Research Communications 466 (2015) 369e375
gallbladder cancer cells, confirming an important role of caspase-12 activation in Rg3's activity. One important finding of this study is that oral administration of a single dose of Rg3 (20 mg/kg, daily) could remarkably inhibit GBC-SD xenograft growth in nude mice. More importantly, such activity by Rg3 was attenuated with co-administration of Sal. Thus, ER stress activation is likely also important for the in vivo activity of Rg3. In summary, the results of this study imply that pathological ER stress activation mediates Rg3-induced anti-gallbladder cancer cell activity in vitro and in vivo. Rg3 could be further studied as a promising anti-gallbladder cancer agent.
[9]
[10]
[11]
[12]
[13]
Acknowledgments [14]
This work is supported by the National Science Foundation of China. [15]
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.09.030.
[16]
[17]
Conflict of interests The authors declare that they have no conflict of interests. [18]
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