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Inhibition of gastric cancer cell growth by a PI3K-mTOR dual inhibitor GSK1059615
Biochemical and Biophysical Research Communications 511 (2019) 13e20
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Inhibition of gastric cancer cell growth by a PI3K-mTOR dual inhibitor GSK1059615 Songhua Bei 1, Fan Li 1, Huanqin Li, Jian Li, Xiaohong Zhang, Qi Sun**, Li Feng* Endoscopy Center, Minhang Hospital, Fudan University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2019 Accepted 7 February 2019 Available online 11 February 2019
Gastric cancer (GC) is a common malignancy. Developing novel and efficient anti-GC agents is urgent. GSK1059615 is a PI3K (phosphatidylinositol 3-kinase) and mTOR (mammalian target of rapamycin) dual inhibitor. It activity in human GC cells is tested here. In AGS cells and primary human GC cells, GSK1059615 potently inhibited cell growth, survival, proliferation and cell cycle progression. Further, significant apoptosis activation was detected in GSK1059615-treated GC cells. Contrarily in the primary human gastric epithelial cells, GSK1059615 failed to induce significant cytotoxicity and apoptosis. GSK1059615 blocked PI3K-AKT-mTOR cascade activation, inducing microRNA-9 downregulation but LMX1A (LIM homeobox transcription factor 1a) upregulation in GC cells. Significantly, GSK1059615 administration (i.p., daily, at 10 or 30 mg/kg) in nude mice potently inhibited subcutaneous AGS xenograft growth. AKT-mTOR inhibition and LMX1A upregulation were detected in AGS xenograft tissues with GSK1059615 administration. Together, we conclude that GSK1059615 inhibits GC cell growth in vitro and in vivo. © 2019 Elsevier Inc. All rights reserved.
Keywords: Gastric cancer PI3K-AKT-mTOR cascade GSK1059615 microRNA-9-LMX1A cascade
1. Introduction Gastric cancer is a common malignancy worldwide, causing significant cancer-associated human mortalities each year [1]. The prognosis for advanced, recurrent and metastatic GC is not satisfactory, with current treatment options very limited [2,3]. Molecularly-targeted therapies are extremely important for better GC therapy [4,5]. PI3K (phosphatidylinositol 3-kinase)-AKT-mTOR (mammalian target of rapamycin) cascade is often dysregulated and hyperactivated in GC [6], linked to cancer initiation, progression and therapy resistance [5,7,8]. Sustained activation of the upstream receptor tyrosine kinases (RTKs, i.e. EGFR, PDGFR and VEGFR) and/ or genetic mutations of key signaling proteins (including PIK3CA and PTEN) could be the reason of PI3K-AKT-mTOR over-activation in GC [5,7,8]. Contrarily, blockage of the PI3K-AKT-mTOR can result in potent inhibition of GC cell progression in vitro and in vivo [9,10].
* Corresponding author. Endoscopy Center, Minhang Hospital, Fudan University, 170 Xinsong Road, Minhang District, Shanghai, 201199, China. ** Corresponding author. E-mail addresses: [email protected] (Q. Sun), [email protected] (L. Feng). 1 These authors have contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.02.032 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Very recent studies have developed GSK1059615 as a PI3K and mTOR dual inhibitor [11,12]. Unlike other PI3K-AKT-mTOR specific inhibitors, this compound simantanuously blocks PI3K and mTOR activation, thereby absolutely shutting down the whoe PI3K-AKTmTOR cascade [11]. A recent study by Xie et al., has demonstrated that the dual inhibitor blocked PI3K-AKT-mTOR cascade to kill head and neck squamous cell carcinoma (HNSCC) cells [12]. The current study will test its potential activity in human GC cells. 2. Materials and methods 2.1. Chemicals and reagents GSK1059615 was provided by Dr. Xie at Wenzhou Medical University [12]. The mTOR kinase inhibitor AZD-2014 [13] and the AKT specific inhibitor MK-2206 [14] were obtained from Selleck (Beijing, China). Puromycin, cell culture reagents and PCR reagents were from Sigma Chemicals (St. Louis, Mo). LMX1A (LIM homeobox transcription factor 1a) and LMX1B (LIM homeobox transcription factor 1b) antibodies were purchased from Abcam (Cambridge, MA). All other antibodies were purchased from Cell Signaling Tech (Danvers, MA). Lipofectamine 2000 and other transfection reagents were provided by Invitrogen Thermo-Fisher Scientific (Carlsbad, CA).
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2.2. Cell culture
2.9. Annexin V FACS
As previously described [15], AGS cells, from the Cell Bank of the Biological Institute of CAS China (Shanghai, China), were cultured in RPMI-1640 medium plus 10% FBS. Culture of the primary human GC cells, derived from two written-informed consent primary GC patients (“GC-1” and “GC-2” [16]), were described previously [15]. The primary human gastric epithelial cells, derived from two writteninformed donors (“Epi-1” and “Epi-2”), were provided by Dr. Lv [17,18]. The protocols of this study were according to the principles of Declaration of Helsinki, approved by the Ethics Board of Fudan University.
Following treatments, cells were harvested, washed, and stained with Annexin V (5.0 mg/mL, BD Pharmingen) and propidium Iodide (PI, 5 mg/mL, BD Pharmingen). By using a Flow Cytometer (Beckman Coulter, Brea, CA) Annexin V positive cells were gated.
2.3. Cell viability assay As reported [15], at a density of 5,000 cells per well GC cells or the gastric epithelial cells were seeded into 96-well plates. Following the applied GSK1059615 treatment, cell viability was tested by the Cell Counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). Its optical density (OD) was recorded at the test wavelength of 570 nm. 2.4. Soft agar colony formation Cells were trypsinized and re-suspended in 0.5% agarosecontaining complete medium, poured onto a bed of 1.0 mL of 1.0% agarose into the six-well plates. Following GSK1059615 treatment for 10 days the colonies were stained and counted. 2.5. EdU assay
2.10. TUNEL assay TUNEL In Situ Cell Death Detection Kit (Roche, Shanghai, China) was applied to quantify cell apoptosis. At 20,000 cells per well, GC cells were seeded into 12-well plates. Following GSK1059615 treatment, TUNEL and DAPI dyes were added. TUNEL ratio (TUNEL/ DAPI 100%), counting at least 500 cells in five random views for each treatment, was calculated.
2.11. Western blotting Quantified cellular lysates or tissue lysates were resolved by SDS-PAGE (10%) gels, transferred to the PVDF membranes. The blots were blocked (in 10% milk in PBST), incubated with designated primary and secondary antibodies. To visualize the immunoreactive bands, an ECL kit (Roche, Shanghai, China) was utilized. By using the ImageJ software (NIH) the total gray of each band was quantified.
2.12. qPCR assay 5
Cells were seeded into the six-well tissue plates (1 10 cells per well). An EdU (5-ethynyl-20-deoxyuridine) Kit (Ribo-Bio, Guangzhou, China) was applied to test cell proliferation. Following GSK1059615 treatment EdU (5 mM) was added. Cell nuclei were costained with DAPI for 10 min, visualized under a fluorescent microscope (Leica). For each treatment at least 500 cells from five random views (1:100 magnification) were included to calculate EdU ratio (EdU/DAPI 100%).
Following the applied treatment, 500 ng total RNA of each treatment was first reversely transcribed by the Reverse Transcriptase M-MLV (Promega, Madison, WI). The quantitative realtime PCR (“qPCR”) was performed by a SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). LMX1A mRNA was normalized to GAPDH. miR-9 expression was normalized to U6 RNA. Primers for qPCR were described early [15].
2.6. BrdU ELISA 2.13. Tumor xenograft assay GC cells were seeded into 96-well plates at 3 103 cells per well. Following GSK1059615 treatment, cells were incubated with BrdU (5 mM, Cell Signaling Tech). By applying a BrdU ELISA kit (Cell Signaling Tech) BrdU incorporation was tested. At the test wavelength of 405 nm the ELISA OD was recorded. 2.7. Cell cycle analysis Following GSK1059615 treatment, AGS were stained with 5 mM propidium iodide (PI, Sigma), analyzed under a Flow Cytometer (Beckman Coulter, Brea, CA). G1, S and G2 percentages were recorded. 2.8. Caspase-3/-9 activity Caspase-3/-9 activity was determined by Caspase-3/-9 colorimetric assay kits (R&D Systems, Shanghai, China). Briefly, cells with the GSK1059615 treatment were lysed. Cell lysates (40 mg per treatment) were incubated with the Caspase-3 substrate (AcDEVD-pNA) or the Caspase-9 substrate (Ac-LEHD-pNA) for 1 h at room temperature. Caspase-3/-9 colorimetric activity was measured by quantifying the released pNA at tested wavelength of 405 nm.
The nude mice (5e7 week age, 18e20 g in weight) were purchased from the Experimental Animal Center of Soochow University (Suzhou, China), maintained under standard conditions. For each mouse, six million AGS cells (in 100 mL DMEM and 100 mL Matrigel) were inoculated via s.c. injection to the right flank. Within 20e22 days the established xenograft tumors were established, with volume of each tumor close to 100 mm3. The mice were randomly assigned into three groups (n ¼ 10 per group). Tumor volumes were recorded every 7 days for a total of 35 days, using the modified ellipsoid formula: (p/6) AB2, where A represents the longest, and B represents the shortest perpendicular axis of an tumor mass. The animal procedure was approved by the IACUC of Fudan University.
2.14. Statistical analyses All values were expressed as mean ± standard deviation (SD). Statistical analyses were performed by the SPSS 18.0 (SPSS Co., Chicago, IL). The statistical significance was estimated by one-way ANOVA, with Bonferroni's post hoc test. Differences were considered significant at P < 0.05. IC-50 was calculated by SPSS 18.0.
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3. Results 3.1. GSK1059615 inhibits human GC cell growth, survival, proliferation and cell cycle progression To study the potential activity of GSK1059615 on GC cell functions, AGS cells [15] were cultured in complete medium (with 10% FBS), treated with GSK1059615 at applied concentrations (0.1e3 mM) [12]. Testing cell growth, by a simple cell counting assay, confirmed that GSK1059615 dose-dependently inhibited AGS cell growth (Fig. 1A). Furthermore, CCK-8 cell viability assay results, in Fig. 1B, demonstrated that GSK1059615 inhibited AGS cell survival, in dose- and time-dependent manners. At the lowest concentration, 0.1 mM, GSK1059615 was ineffective on AGS cell growth and survival (Fig. 1A and B). The IC-50 of GSK1059615, or the concentration resulting in 50% reduction of cell viability, was close to 1 mM (Fig. 1B). This concentration was selected for following experiments. Further studies show that GSK1059615 (1 mM) potently inhibited AGS cell soft agar colony formation (Fig. 1C). Additionally, AGS cell proliferation was significantly inhibited by GSK1059615 (1 mM) treatment, evidenced by decreased EdU staining (Fig. 1D and E) and inhibited BrdU incorporation (Fig. 1F). Furthermore, cell cycle analyses by PI-FACS confirmed that GSK1059615 disrupted AGS cell cycle progression, resulting in G1-S arrest (Fig. 1G and H). In the primary human GC cells (“GC-1/-2”, derived from two GC patients [15]), following GSK1059615 (1 mM) treatment the CCK-8 viability (Fig. 1I) and the EdU ratio (Fig. 1J) were both inhibited, indicating cell survival and proliferation inhibition. Conversely, the same GSK1059615 (1 mM) treatment failed to significantly affect the function of primary human gastric epithelial cells (“Epi-1/-2”) (Fig. 1I and J). Collectively, these results show that GSK1059615 inhibits human GC cell growth, survival, proliferation and cell cycle progression. 3.2. GSK1059615 induces apoptosis activation in GC cells Activation of apoptosis could be a primary reason of GC cell inhibition by GSK1059615. As demonstrated, in AGS cells GSK1059615 (1 mM) treatment potently increased the activity of Caspase-3 (Fig. 2A) and Caspase-9 (Fig. 2B). Further, analyzing apoptosis-associated proteins, by a Western blotting assay, confirmed that GSK1059615 induced cleavages of Caspase-3, Caspase-9 and PARP (poly (ADP-ribose) polymerase) in AGS cells (Fig. 2C). Additionally, in GSK1059615-treated AGS cells Annexin V positive staining was significantly increased (Fig. 2D and E). TUNEL ratio was increased as well by GSK1059615 treatment (Fig. 2F). In the primary human GC cells (“GC-1/-2”), treatment with GSK1059615 significantly increased Caspase-3 activity (Fig. 2G) and Annexin V ratio (Fig. 2H). Both were however not significantly changed in the primary human gastric epithelial cells (“Epi-1/-2”) following GSK1059615 treatment (Fig. 2G and H). These results show that GSK1059615 induces apoptosis activation in GC cells. 3.3. GSK1059615 blocks PI3K-AKT-mTOR activation and changes miR-9-LMX1A expression in GC cells GSK1059615 is a novel PI3K and mTOR dual inhibitor [12]. By testing phosphorylated (“p-”) PI3K p85 (Tyr-458), we demonstrated that GSK1059615 blocked PI3K activation in AGS cells (Fig. 3A) and primary GC cells (“GC-1”, Fig. 3B). Activation of mTOR complex 1 (mTORC1) and mTORC2, tested by p-p70S6K1 (Thr-389) and p-AKT (Ser-473) respectively [19], was blocked by GSK1059615 (1 mM) as well in AGS cells (Fig. 3A) and primary human GC cells (Fig. 3B). Furthermore, p-AKT (Thr-308) was also blocked by
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GSK1059615, suggesting complete AKT inhibition [19] (Fig. 3A and B). These results imply that GSK1059615 blocks PI3K-AKT-mTOR cascade in GC cells. In the primary human gastric epithelial cells (“Epi-1”) basal activation of PI3K-AKT-mTOR was significantly lower (when compared to GC cells, Fig. 3C). This could be at least one reason of the ineffectiveness of this compound in the epithelial cells (Figs. 1 and 2). LMX1A is evolutionary conserved transcription factor [20]. It is downregulated in human GC tissues, acting as a tumor suppressor [21,22]. Our previous study has shown that microRNA-9 (“miR-9”) upregulation could account for its target LMX1A downregulation in human GC tissues and cells [15].Conversely, miR-9 inhibition increased LMX1A expression, inhibiting GC cell progression [15]. We therefore analyzed the potential effect of GSK1059615 on miR-9-LMX1A cascade. In AGS cells GSK1059615 (1 mM) treatment decreased miR-9 expression (Fig. 3D), while increasing LMX1A mRNA (Fig. 3E) and protein expression (Fig. 3F). LMX1B protein expression was unchanged (Fig. 3F). Similarly in GSK1059615treated primary GC cells (“GC-1”), miR-9 downregulation (Fig. 3G) and LMX1A upregulation (Fig. 3H and I) were detected. Significantly, the CCK-8 cell viability assay results, in Fig. 3J and K, demonstrated that at same concentration (1 mM) of GSK1059615 was more potent in killing AGS cells and primary GC cells (“GC-1”) than the mTOR kinase inhibitor AZD-2014 and the AKT specific inhibitor MK-2206. Notably, AZD-2014 and MK-2206 had no significant effect on miR-9-LMX1A expression in GC cells (Data not shown). 3.4. GSK1059615 administration inhibits subcutaneous AGS xenograft growth in nude mice In order to study the anti-cancer activity of GSK1059615 in vivo, AGS cells were s.c. injected to the right flanks of nude mice. Xenograft tumors were established within three weeks, with tumor volumes close to 0.1 cm3 (or “Day-0”). The tumor-bearing mice were randomly assigned into three groups, receiving GSK1059615 administration or the vehicle control. Tumor growth curve, in Fig. 4A, demonstrated that GSK1059615 administration (i.p. daily, 10 and 30 mg/kg, for 21 consecutive days) significantly inhibited AGS xenograft growth in nude mice. The estimated tumor growth was calculated, using the formula: (Tumor volume at Day-35 subtracting Tumor volume at Day-0) ÷ 35 (Days). The results, in Fig. 4B, confirmed that AGS xenograft growth was largely inhibited by GSK1059615 administration. At Day-35, tumors of all three groups were isolated and weighted individually. AGS tumors from GSK1059615-treated mice were significantly lighter than those from the vehicle-treated mice (Fig. 4C). The mice body weights were not significantly different between the three groups (Fig. 4D), and experimental mice were well-tolerated to GSK1059615 treatments, showing no apparent toxicities. To testing signaling changes in AGS xenografts, at Day-7 and Day-14, one tumor of each group was isolated, and signaling proteins in fresh tumor lysates were tested by Western blotting assays. As demonstrated, GSK1059615 administration potently inhibited phosphorylation of AKT (at both Ser-473 and Thr-308 residues) and p70S6K1 (at Thr-389) in tumor tissues, suggesting AKT-mTOR inhibition (Fig. 4E and F). LMX1A expression levels were however increased by GSK1059615 (Fig. 4E and F). These signaling changes in vivo were consistent with in vitro findings (see Fig. 3). 4. Discussion The results of the current study indicate that GSK1059615 could be a promising anti-GC agent. In AGS cells and primary human GC
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Fig. 1. GSK1059615 inhibits human GC cell growth, survival, proliferation and cell cycle progression. AGS cells (AeH), the primary human GC cells (“GC-1”/“GC-2”, I and J) or the primary human gastric epithelial cells (“Epi-1”/“Epi-2”, I and J) were treated with vehicle control (“Veh”, 0.1% DMSO) or GSK1059615 (“GSK”, 0.1e3 mM), cells were further maintained in GSK1059615-containing medium for applied time periods, cell growth (A), viability (B and I), cell proliferation (C-F and J) and cell cycle progression (GeH) were tested by the assays mentioned in the text. Data were presented as mean ± standard deviation (SD) (Same for all Figures). For each assay, n ¼ 5 (five replicate wells/dishes). *P < 0.05 vs. “Veh” treatment. Experiments in this figure were repeated four times, and similar results were obtained. Bar ¼ 100 mm (D).
cells, GSK1059615 suppressed GC cell growth, survival, proliferation and cell cycle progression, while inducing significant apoptosis activation. Importantly, GSK1059615 was non-cytotoxic when added to the primary human gastric epithelial cells. At the molecular level, GSK1059615 blocked the whole PI3K-AKT-mTOR cascade in human GC cells. In vivo, GSK1059615 daily administration significantly inhibited subcutaneous AGS xenograft growth in nude
mice, resulting in potent AKT-mTOR inhibition in AGS xenograft tissues. In GC and many other human cancers, co-current activation of multiple oncogenic cascades can work synergistically or separately to promote cancer cell progression [23,24]. Therefore, inhibition of one cascade often resulted in moderate or even negligible anticancer activity [23,24]. mTOR is the central player in the PI3K-
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Fig. 2. GSK1059615 induces apoptosis activation in GC cells. AGS cells (AeF), the primary human GC cells (“GC-1”/“GC-2”, G and H) or the primary human gastric epithelial cells (“Epi-1”/“Epi-2”, G and H) were treated with vehicle control (“Veh”, 0.1% DMSO), or GSK1059615 (1 mM), cells were further maintained in GSK1059615-containing medium for applied time periods, relative Caspase-3/-9 activity (A, B and G) and expression of listed proteins (C) were tested; Cell apoptosis was tested by Annexin V-FACS (D, E, and H) and TUNEL staining assay (F). Expression of listed proteins were quantified, and normalized to the loading control Tubulin (C). For each assay, n ¼ 5 (five replicate wells/dishes). *P < 0.05 vs. “Veh” treatment. Experiments in this figure were repeated four times, and similar results were obtained.
AKT-mTOR cascade. Two distinct mTOR complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [25], have been identified thus far. Both are critically involved in GC progression [5,25,26]. Our results show that GSK1059615 blocked mTORC1 (pS6K1) and mTORC2 (p-AKT Ser-473) in established and primary GC cells. The dual inhibitor also blocked PI3K-AKT activation in GC cells. This possibly explain the superior anti-GC cell activity by this compound, which was more efficiently than mTOR kinase inhibitor AZD-2014 and the AKT inhibitor MK-2206. It might also be the reason of the ineffectiveness of GSK1059615 in gastric epithelial cells, where basal activation of PI3K-AKT-mTOR cascade is extremely low. LMX1A, a primary member of LIM-homeodomain (LIM-HD) family protein and a tumor suppressor, is downregulated in human GC tissues and cells [21,27]. Our previous study has shown that miR-9 upregulation might account for the
downregulation of its target LMX1A in human GC tissues [15]. Contrarily, miR-9 inhibition induced increased expression of LMX1A, thus inhibiting GC cell progression in vitro [15]. In the present study we show that GSK1059615 treatment induced miR-9 downregulation, while increasing LMX1A expression in established and primary GC cells. Furthermore, LMX1A protein levels were significantly increased in GSK1059615-administrated AGS tumor tissues. These are possibly the unique actions by GSK1059615, as expression of the miR-9-LMX1A axis was unchanged following treatment of other PI3K-AKT-mTOR inhibitors (AZD-2014 and MK-2206). Therefore, targeting miR-9-LMX1A axis could be another key advantage of GSK1059615 in suppressing GC cells. The underlying mechanisms may warrant further studies. Together, GSK1059615 inhibits GC cell growth in vitro and in vivo.
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Fig. 3. GSK1059615 blocks PI3K-AKT-mTOR activation and changes miR-9-LMX1A expression in GC cells. AGS cells (A and D-F), the primary human GC cells (“GC-1”, B and G-I) or the primary gastric epithelial cells (“Epi-1”, C) were treated with vehicle control (“Veh”, 0.1% DMSO), or GSK1059615 (1 mM), cells were further maintained for applied time periods, total cell lysates were achieved, listed proteins and genes were tested by Western blotting (A-C, F and I) and qPCR (D, E, G and H) assays, respectively. AGS cells (J) or the primary human GC cells (“GC-1”) (K) were treated with 1 mM of GSK1059615 (“GSK”), AZD-2014 (“AZD”) or MK-2206 (“MK”) for 72 h, cell viability was tested. Expression of listed proteins were quantified, and normalized to the loading controls (A-C, F and I). For each assay, n ¼ 5 (five replicate wells/dishes). *P < 0.05 vs. “Veh” treatment. #P < 0.05 vs. “GSK” treatment (J and K). Experiments in this figure were repeated four times, and similar results were obtained.
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Fig. 4. GSK1059615 administration inhibits subcutaneous AGS xenograft growth in nude mice. AGS xenograft-bearing nude mice were i.p. injected with GSK1059615 (“GSK”, daily, 10 or 30 mg/kg, for 21 consecutive days) or the saline vehicle control (“Vehicle”), tumor volumes (A) and mice body weights (D) were recorded every seven days for a total of 35 days; Estimated daily tumor growth was calculated as described (B); At Day-35, tumors were separated and weighted individually (C). At Day-7 and Day-14, one tumor of each group was isolated, and expression of the listed proteins in fresh tumor lysates was tested by Western blotting (E and F). Expression of listed proteins were quantified, and normalized to the loading controls (E and F). For each assay, n ¼ 10 (Ten mice per group). *P < 0.05 vs. group of “Vehicle” (AeC).
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Inhibition of gastric cancer cell growth by a PI3K-mTOR dual inhibitor GSK1059615 Songhua Bei 1, Fan Li 1, Huanqin Li, Jian Li, Xiaohong Zhang, Qi Sun**, Li Feng* Endoscopy Center, Minhang Hospital, Fudan University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2019 Accepted 7 February 2019 Available online 11 February 2019
Gastric cancer (GC) is a common malignancy. Developing novel and efficient anti-GC agents is urgent. GSK1059615 is a PI3K (phosphatidylinositol 3-kinase) and mTOR (mammalian target of rapamycin) dual inhibitor. It activity in human GC cells is tested here. In AGS cells and primary human GC cells, GSK1059615 potently inhibited cell growth, survival, proliferation and cell cycle progression. Further, significant apoptosis activation was detected in GSK1059615-treated GC cells. Contrarily in the primary human gastric epithelial cells, GSK1059615 failed to induce significant cytotoxicity and apoptosis. GSK1059615 blocked PI3K-AKT-mTOR cascade activation, inducing microRNA-9 downregulation but LMX1A (LIM homeobox transcription factor 1a) upregulation in GC cells. Significantly, GSK1059615 administration (i.p., daily, at 10 or 30 mg/kg) in nude mice potently inhibited subcutaneous AGS xenograft growth. AKT-mTOR inhibition and LMX1A upregulation were detected in AGS xenograft tissues with GSK1059615 administration. Together, we conclude that GSK1059615 inhibits GC cell growth in vitro and in vivo. © 2019 Elsevier Inc. All rights reserved.
Keywords: Gastric cancer PI3K-AKT-mTOR cascade GSK1059615 microRNA-9-LMX1A cascade
1. Introduction Gastric cancer is a common malignancy worldwide, causing significant cancer-associated human mortalities each year [1]. The prognosis for advanced, recurrent and metastatic GC is not satisfactory, with current treatment options very limited [2,3]. Molecularly-targeted therapies are extremely important for better GC therapy [4,5]. PI3K (phosphatidylinositol 3-kinase)-AKT-mTOR (mammalian target of rapamycin) cascade is often dysregulated and hyperactivated in GC [6], linked to cancer initiation, progression and therapy resistance [5,7,8]. Sustained activation of the upstream receptor tyrosine kinases (RTKs, i.e. EGFR, PDGFR and VEGFR) and/ or genetic mutations of key signaling proteins (including PIK3CA and PTEN) could be the reason of PI3K-AKT-mTOR over-activation in GC [5,7,8]. Contrarily, blockage of the PI3K-AKT-mTOR can result in potent inhibition of GC cell progression in vitro and in vivo [9,10].
* Corresponding author. Endoscopy Center, Minhang Hospital, Fudan University, 170 Xinsong Road, Minhang District, Shanghai, 201199, China. ** Corresponding author. E-mail addresses: [email protected] (Q. Sun), [email protected] (L. Feng). 1 These authors have contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.02.032 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Very recent studies have developed GSK1059615 as a PI3K and mTOR dual inhibitor [11,12]. Unlike other PI3K-AKT-mTOR specific inhibitors, this compound simantanuously blocks PI3K and mTOR activation, thereby absolutely shutting down the whoe PI3K-AKTmTOR cascade [11]. A recent study by Xie et al., has demonstrated that the dual inhibitor blocked PI3K-AKT-mTOR cascade to kill head and neck squamous cell carcinoma (HNSCC) cells [12]. The current study will test its potential activity in human GC cells. 2. Materials and methods 2.1. Chemicals and reagents GSK1059615 was provided by Dr. Xie at Wenzhou Medical University [12]. The mTOR kinase inhibitor AZD-2014 [13] and the AKT specific inhibitor MK-2206 [14] were obtained from Selleck (Beijing, China). Puromycin, cell culture reagents and PCR reagents were from Sigma Chemicals (St. Louis, Mo). LMX1A (LIM homeobox transcription factor 1a) and LMX1B (LIM homeobox transcription factor 1b) antibodies were purchased from Abcam (Cambridge, MA). All other antibodies were purchased from Cell Signaling Tech (Danvers, MA). Lipofectamine 2000 and other transfection reagents were provided by Invitrogen Thermo-Fisher Scientific (Carlsbad, CA).
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2.2. Cell culture
2.9. Annexin V FACS
As previously described [15], AGS cells, from the Cell Bank of the Biological Institute of CAS China (Shanghai, China), were cultured in RPMI-1640 medium plus 10% FBS. Culture of the primary human GC cells, derived from two written-informed consent primary GC patients (“GC-1” and “GC-2” [16]), were described previously [15]. The primary human gastric epithelial cells, derived from two writteninformed donors (“Epi-1” and “Epi-2”), were provided by Dr. Lv [17,18]. The protocols of this study were according to the principles of Declaration of Helsinki, approved by the Ethics Board of Fudan University.
Following treatments, cells were harvested, washed, and stained with Annexin V (5.0 mg/mL, BD Pharmingen) and propidium Iodide (PI, 5 mg/mL, BD Pharmingen). By using a Flow Cytometer (Beckman Coulter, Brea, CA) Annexin V positive cells were gated.
2.3. Cell viability assay As reported [15], at a density of 5,000 cells per well GC cells or the gastric epithelial cells were seeded into 96-well plates. Following the applied GSK1059615 treatment, cell viability was tested by the Cell Counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). Its optical density (OD) was recorded at the test wavelength of 570 nm. 2.4. Soft agar colony formation Cells were trypsinized and re-suspended in 0.5% agarosecontaining complete medium, poured onto a bed of 1.0 mL of 1.0% agarose into the six-well plates. Following GSK1059615 treatment for 10 days the colonies were stained and counted. 2.5. EdU assay
2.10. TUNEL assay TUNEL In Situ Cell Death Detection Kit (Roche, Shanghai, China) was applied to quantify cell apoptosis. At 20,000 cells per well, GC cells were seeded into 12-well plates. Following GSK1059615 treatment, TUNEL and DAPI dyes were added. TUNEL ratio (TUNEL/ DAPI 100%), counting at least 500 cells in five random views for each treatment, was calculated.
2.11. Western blotting Quantified cellular lysates or tissue lysates were resolved by SDS-PAGE (10%) gels, transferred to the PVDF membranes. The blots were blocked (in 10% milk in PBST), incubated with designated primary and secondary antibodies. To visualize the immunoreactive bands, an ECL kit (Roche, Shanghai, China) was utilized. By using the ImageJ software (NIH) the total gray of each band was quantified.
2.12. qPCR assay 5
Cells were seeded into the six-well tissue plates (1 10 cells per well). An EdU (5-ethynyl-20-deoxyuridine) Kit (Ribo-Bio, Guangzhou, China) was applied to test cell proliferation. Following GSK1059615 treatment EdU (5 mM) was added. Cell nuclei were costained with DAPI for 10 min, visualized under a fluorescent microscope (Leica). For each treatment at least 500 cells from five random views (1:100 magnification) were included to calculate EdU ratio (EdU/DAPI 100%).
Following the applied treatment, 500 ng total RNA of each treatment was first reversely transcribed by the Reverse Transcriptase M-MLV (Promega, Madison, WI). The quantitative realtime PCR (“qPCR”) was performed by a SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). LMX1A mRNA was normalized to GAPDH. miR-9 expression was normalized to U6 RNA. Primers for qPCR were described early [15].
2.6. BrdU ELISA 2.13. Tumor xenograft assay GC cells were seeded into 96-well plates at 3 103 cells per well. Following GSK1059615 treatment, cells were incubated with BrdU (5 mM, Cell Signaling Tech). By applying a BrdU ELISA kit (Cell Signaling Tech) BrdU incorporation was tested. At the test wavelength of 405 nm the ELISA OD was recorded. 2.7. Cell cycle analysis Following GSK1059615 treatment, AGS were stained with 5 mM propidium iodide (PI, Sigma), analyzed under a Flow Cytometer (Beckman Coulter, Brea, CA). G1, S and G2 percentages were recorded. 2.8. Caspase-3/-9 activity Caspase-3/-9 activity was determined by Caspase-3/-9 colorimetric assay kits (R&D Systems, Shanghai, China). Briefly, cells with the GSK1059615 treatment were lysed. Cell lysates (40 mg per treatment) were incubated with the Caspase-3 substrate (AcDEVD-pNA) or the Caspase-9 substrate (Ac-LEHD-pNA) for 1 h at room temperature. Caspase-3/-9 colorimetric activity was measured by quantifying the released pNA at tested wavelength of 405 nm.
The nude mice (5e7 week age, 18e20 g in weight) were purchased from the Experimental Animal Center of Soochow University (Suzhou, China), maintained under standard conditions. For each mouse, six million AGS cells (in 100 mL DMEM and 100 mL Matrigel) were inoculated via s.c. injection to the right flank. Within 20e22 days the established xenograft tumors were established, with volume of each tumor close to 100 mm3. The mice were randomly assigned into three groups (n ¼ 10 per group). Tumor volumes were recorded every 7 days for a total of 35 days, using the modified ellipsoid formula: (p/6) AB2, where A represents the longest, and B represents the shortest perpendicular axis of an tumor mass. The animal procedure was approved by the IACUC of Fudan University.
2.14. Statistical analyses All values were expressed as mean ± standard deviation (SD). Statistical analyses were performed by the SPSS 18.0 (SPSS Co., Chicago, IL). The statistical significance was estimated by one-way ANOVA, with Bonferroni's post hoc test. Differences were considered significant at P < 0.05. IC-50 was calculated by SPSS 18.0.
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3. Results 3.1. GSK1059615 inhibits human GC cell growth, survival, proliferation and cell cycle progression To study the potential activity of GSK1059615 on GC cell functions, AGS cells [15] were cultured in complete medium (with 10% FBS), treated with GSK1059615 at applied concentrations (0.1e3 mM) [12]. Testing cell growth, by a simple cell counting assay, confirmed that GSK1059615 dose-dependently inhibited AGS cell growth (Fig. 1A). Furthermore, CCK-8 cell viability assay results, in Fig. 1B, demonstrated that GSK1059615 inhibited AGS cell survival, in dose- and time-dependent manners. At the lowest concentration, 0.1 mM, GSK1059615 was ineffective on AGS cell growth and survival (Fig. 1A and B). The IC-50 of GSK1059615, or the concentration resulting in 50% reduction of cell viability, was close to 1 mM (Fig. 1B). This concentration was selected for following experiments. Further studies show that GSK1059615 (1 mM) potently inhibited AGS cell soft agar colony formation (Fig. 1C). Additionally, AGS cell proliferation was significantly inhibited by GSK1059615 (1 mM) treatment, evidenced by decreased EdU staining (Fig. 1D and E) and inhibited BrdU incorporation (Fig. 1F). Furthermore, cell cycle analyses by PI-FACS confirmed that GSK1059615 disrupted AGS cell cycle progression, resulting in G1-S arrest (Fig. 1G and H). In the primary human GC cells (“GC-1/-2”, derived from two GC patients [15]), following GSK1059615 (1 mM) treatment the CCK-8 viability (Fig. 1I) and the EdU ratio (Fig. 1J) were both inhibited, indicating cell survival and proliferation inhibition. Conversely, the same GSK1059615 (1 mM) treatment failed to significantly affect the function of primary human gastric epithelial cells (“Epi-1/-2”) (Fig. 1I and J). Collectively, these results show that GSK1059615 inhibits human GC cell growth, survival, proliferation and cell cycle progression. 3.2. GSK1059615 induces apoptosis activation in GC cells Activation of apoptosis could be a primary reason of GC cell inhibition by GSK1059615. As demonstrated, in AGS cells GSK1059615 (1 mM) treatment potently increased the activity of Caspase-3 (Fig. 2A) and Caspase-9 (Fig. 2B). Further, analyzing apoptosis-associated proteins, by a Western blotting assay, confirmed that GSK1059615 induced cleavages of Caspase-3, Caspase-9 and PARP (poly (ADP-ribose) polymerase) in AGS cells (Fig. 2C). Additionally, in GSK1059615-treated AGS cells Annexin V positive staining was significantly increased (Fig. 2D and E). TUNEL ratio was increased as well by GSK1059615 treatment (Fig. 2F). In the primary human GC cells (“GC-1/-2”), treatment with GSK1059615 significantly increased Caspase-3 activity (Fig. 2G) and Annexin V ratio (Fig. 2H). Both were however not significantly changed in the primary human gastric epithelial cells (“Epi-1/-2”) following GSK1059615 treatment (Fig. 2G and H). These results show that GSK1059615 induces apoptosis activation in GC cells. 3.3. GSK1059615 blocks PI3K-AKT-mTOR activation and changes miR-9-LMX1A expression in GC cells GSK1059615 is a novel PI3K and mTOR dual inhibitor [12]. By testing phosphorylated (“p-”) PI3K p85 (Tyr-458), we demonstrated that GSK1059615 blocked PI3K activation in AGS cells (Fig. 3A) and primary GC cells (“GC-1”, Fig. 3B). Activation of mTOR complex 1 (mTORC1) and mTORC2, tested by p-p70S6K1 (Thr-389) and p-AKT (Ser-473) respectively [19], was blocked by GSK1059615 (1 mM) as well in AGS cells (Fig. 3A) and primary human GC cells (Fig. 3B). Furthermore, p-AKT (Thr-308) was also blocked by
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GSK1059615, suggesting complete AKT inhibition [19] (Fig. 3A and B). These results imply that GSK1059615 blocks PI3K-AKT-mTOR cascade in GC cells. In the primary human gastric epithelial cells (“Epi-1”) basal activation of PI3K-AKT-mTOR was significantly lower (when compared to GC cells, Fig. 3C). This could be at least one reason of the ineffectiveness of this compound in the epithelial cells (Figs. 1 and 2). LMX1A is evolutionary conserved transcription factor [20]. It is downregulated in human GC tissues, acting as a tumor suppressor [21,22]. Our previous study has shown that microRNA-9 (“miR-9”) upregulation could account for its target LMX1A downregulation in human GC tissues and cells [15].Conversely, miR-9 inhibition increased LMX1A expression, inhibiting GC cell progression [15]. We therefore analyzed the potential effect of GSK1059615 on miR-9-LMX1A cascade. In AGS cells GSK1059615 (1 mM) treatment decreased miR-9 expression (Fig. 3D), while increasing LMX1A mRNA (Fig. 3E) and protein expression (Fig. 3F). LMX1B protein expression was unchanged (Fig. 3F). Similarly in GSK1059615treated primary GC cells (“GC-1”), miR-9 downregulation (Fig. 3G) and LMX1A upregulation (Fig. 3H and I) were detected. Significantly, the CCK-8 cell viability assay results, in Fig. 3J and K, demonstrated that at same concentration (1 mM) of GSK1059615 was more potent in killing AGS cells and primary GC cells (“GC-1”) than the mTOR kinase inhibitor AZD-2014 and the AKT specific inhibitor MK-2206. Notably, AZD-2014 and MK-2206 had no significant effect on miR-9-LMX1A expression in GC cells (Data not shown). 3.4. GSK1059615 administration inhibits subcutaneous AGS xenograft growth in nude mice In order to study the anti-cancer activity of GSK1059615 in vivo, AGS cells were s.c. injected to the right flanks of nude mice. Xenograft tumors were established within three weeks, with tumor volumes close to 0.1 cm3 (or “Day-0”). The tumor-bearing mice were randomly assigned into three groups, receiving GSK1059615 administration or the vehicle control. Tumor growth curve, in Fig. 4A, demonstrated that GSK1059615 administration (i.p. daily, 10 and 30 mg/kg, for 21 consecutive days) significantly inhibited AGS xenograft growth in nude mice. The estimated tumor growth was calculated, using the formula: (Tumor volume at Day-35 subtracting Tumor volume at Day-0) ÷ 35 (Days). The results, in Fig. 4B, confirmed that AGS xenograft growth was largely inhibited by GSK1059615 administration. At Day-35, tumors of all three groups were isolated and weighted individually. AGS tumors from GSK1059615-treated mice were significantly lighter than those from the vehicle-treated mice (Fig. 4C). The mice body weights were not significantly different between the three groups (Fig. 4D), and experimental mice were well-tolerated to GSK1059615 treatments, showing no apparent toxicities. To testing signaling changes in AGS xenografts, at Day-7 and Day-14, one tumor of each group was isolated, and signaling proteins in fresh tumor lysates were tested by Western blotting assays. As demonstrated, GSK1059615 administration potently inhibited phosphorylation of AKT (at both Ser-473 and Thr-308 residues) and p70S6K1 (at Thr-389) in tumor tissues, suggesting AKT-mTOR inhibition (Fig. 4E and F). LMX1A expression levels were however increased by GSK1059615 (Fig. 4E and F). These signaling changes in vivo were consistent with in vitro findings (see Fig. 3). 4. Discussion The results of the current study indicate that GSK1059615 could be a promising anti-GC agent. In AGS cells and primary human GC
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Fig. 1. GSK1059615 inhibits human GC cell growth, survival, proliferation and cell cycle progression. AGS cells (AeH), the primary human GC cells (“GC-1”/“GC-2”, I and J) or the primary human gastric epithelial cells (“Epi-1”/“Epi-2”, I and J) were treated with vehicle control (“Veh”, 0.1% DMSO) or GSK1059615 (“GSK”, 0.1e3 mM), cells were further maintained in GSK1059615-containing medium for applied time periods, cell growth (A), viability (B and I), cell proliferation (C-F and J) and cell cycle progression (GeH) were tested by the assays mentioned in the text. Data were presented as mean ± standard deviation (SD) (Same for all Figures). For each assay, n ¼ 5 (five replicate wells/dishes). *P < 0.05 vs. “Veh” treatment. Experiments in this figure were repeated four times, and similar results were obtained. Bar ¼ 100 mm (D).
cells, GSK1059615 suppressed GC cell growth, survival, proliferation and cell cycle progression, while inducing significant apoptosis activation. Importantly, GSK1059615 was non-cytotoxic when added to the primary human gastric epithelial cells. At the molecular level, GSK1059615 blocked the whole PI3K-AKT-mTOR cascade in human GC cells. In vivo, GSK1059615 daily administration significantly inhibited subcutaneous AGS xenograft growth in nude
mice, resulting in potent AKT-mTOR inhibition in AGS xenograft tissues. In GC and many other human cancers, co-current activation of multiple oncogenic cascades can work synergistically or separately to promote cancer cell progression [23,24]. Therefore, inhibition of one cascade often resulted in moderate or even negligible anticancer activity [23,24]. mTOR is the central player in the PI3K-
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Fig. 2. GSK1059615 induces apoptosis activation in GC cells. AGS cells (AeF), the primary human GC cells (“GC-1”/“GC-2”, G and H) or the primary human gastric epithelial cells (“Epi-1”/“Epi-2”, G and H) were treated with vehicle control (“Veh”, 0.1% DMSO), or GSK1059615 (1 mM), cells were further maintained in GSK1059615-containing medium for applied time periods, relative Caspase-3/-9 activity (A, B and G) and expression of listed proteins (C) were tested; Cell apoptosis was tested by Annexin V-FACS (D, E, and H) and TUNEL staining assay (F). Expression of listed proteins were quantified, and normalized to the loading control Tubulin (C). For each assay, n ¼ 5 (five replicate wells/dishes). *P < 0.05 vs. “Veh” treatment. Experiments in this figure were repeated four times, and similar results were obtained.
AKT-mTOR cascade. Two distinct mTOR complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [25], have been identified thus far. Both are critically involved in GC progression [5,25,26]. Our results show that GSK1059615 blocked mTORC1 (pS6K1) and mTORC2 (p-AKT Ser-473) in established and primary GC cells. The dual inhibitor also blocked PI3K-AKT activation in GC cells. This possibly explain the superior anti-GC cell activity by this compound, which was more efficiently than mTOR kinase inhibitor AZD-2014 and the AKT inhibitor MK-2206. It might also be the reason of the ineffectiveness of GSK1059615 in gastric epithelial cells, where basal activation of PI3K-AKT-mTOR cascade is extremely low. LMX1A, a primary member of LIM-homeodomain (LIM-HD) family protein and a tumor suppressor, is downregulated in human GC tissues and cells [21,27]. Our previous study has shown that miR-9 upregulation might account for the
downregulation of its target LMX1A in human GC tissues [15]. Contrarily, miR-9 inhibition induced increased expression of LMX1A, thus inhibiting GC cell progression in vitro [15]. In the present study we show that GSK1059615 treatment induced miR-9 downregulation, while increasing LMX1A expression in established and primary GC cells. Furthermore, LMX1A protein levels were significantly increased in GSK1059615-administrated AGS tumor tissues. These are possibly the unique actions by GSK1059615, as expression of the miR-9-LMX1A axis was unchanged following treatment of other PI3K-AKT-mTOR inhibitors (AZD-2014 and MK-2206). Therefore, targeting miR-9-LMX1A axis could be another key advantage of GSK1059615 in suppressing GC cells. The underlying mechanisms may warrant further studies. Together, GSK1059615 inhibits GC cell growth in vitro and in vivo.
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Fig. 3. GSK1059615 blocks PI3K-AKT-mTOR activation and changes miR-9-LMX1A expression in GC cells. AGS cells (A and D-F), the primary human GC cells (“GC-1”, B and G-I) or the primary gastric epithelial cells (“Epi-1”, C) were treated with vehicle control (“Veh”, 0.1% DMSO), or GSK1059615 (1 mM), cells were further maintained for applied time periods, total cell lysates were achieved, listed proteins and genes were tested by Western blotting (A-C, F and I) and qPCR (D, E, G and H) assays, respectively. AGS cells (J) or the primary human GC cells (“GC-1”) (K) were treated with 1 mM of GSK1059615 (“GSK”), AZD-2014 (“AZD”) or MK-2206 (“MK”) for 72 h, cell viability was tested. Expression of listed proteins were quantified, and normalized to the loading controls (A-C, F and I). For each assay, n ¼ 5 (five replicate wells/dishes). *P < 0.05 vs. “Veh” treatment. #P < 0.05 vs. “GSK” treatment (J and K). Experiments in this figure were repeated four times, and similar results were obtained.
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Fig. 4. GSK1059615 administration inhibits subcutaneous AGS xenograft growth in nude mice. AGS xenograft-bearing nude mice were i.p. injected with GSK1059615 (“GSK”, daily, 10 or 30 mg/kg, for 21 consecutive days) or the saline vehicle control (“Vehicle”), tumor volumes (A) and mice body weights (D) were recorded every seven days for a total of 35 days; Estimated daily tumor growth was calculated as described (B); At Day-35, tumors were separated and weighted individually (C). At Day-7 and Day-14, one tumor of each group was isolated, and expression of the listed proteins in fresh tumor lysates was tested by Western blotting (E and F). Expression of listed proteins were quantified, and normalized to the loading controls (E and F). For each assay, n ¼ 10 (Ten mice per group). *P < 0.05 vs. group of “Vehicle” (AeC).
Disclosure statement The authors declare that they have no competing interests. Acknowledgement This project was supported by Minhang District University Building Project (2017MWDXK03), by Minhang District Talent Development Special Fund Project (2017), and Minhang District Subject Pilot Talent Project. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.032. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2018, CA Canc. J. Clin. 68 (2018) 7e30. [2] K.S. Choi, M. Suh, Screening for gastric cancer: the usefulness of endoscopy, Clin. Endosc. 47 (2014) 490e496. [3] D. Hashim, P. Boffetta, C. La Vecchia, M. Rota, P. Bertuccio, M. Malvezzi, E. Negri, The global decrease in cancer mortality: trends and disparities, Ann. Oncol. 27 (2016) 926e933. [4] M.A. Shah, Gastrointestinal cancer: targeted therapies in gastric cancer-the dawn of a new era, Nat. Rev. Clin. Oncol. 11 (2014) 10e11. [5] R. Wadhwa, S. Song, J.S. Lee, Y. Yao, Q. Wei, J.A. Ajani, Gastric cancer-molecular and clinical dimensions, Nat. Rev. Clin. Oncol. 10 (2013) 643e655. [6] T. Matsuoka, M. Yashiro, The role of PI3K/Akt/mTOR signaling in gastric
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