E Platinum, a newly synthesized platinum compound, induces autophagy via inhibiting phosphorylation of mTOR in gastric carcinoma BGC-823 cells

Toxicology Letters 210 (2012) 78–86

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E Platinum, a newly synthesized platinum compound, induces autophagy via inhibiting phosphorylation of mTOR in gastric carcinoma BGC-823 cells Chen Hu a,1 , Mei-Juan Zou a,1 , Li Zhao a , Na Lu a , Ya-Jing Sun a , Shao-Hua Gou b , Tao Xi a,∗ , Qing-Long Guo a,∗ a State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China b School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 18 October 2011 Received in revised form 3 January 2012 Accepted 23 January 2012 Available online 31 January 2012 Keywords: E Platinum Autophagy Cell growth mTOR Gastric carcinoma cells

a b s t r a c t A tightly regulated catabolic process named autophagy involves the degradation of intracellular components via lysosomes. Here we investigate the antitumor effect of E Platinum, a newly synthesized derivative of oxaliplatin, in vivo and in vitro. E Platinum exhibits growth inhibition of various tumor cells in a dose-dependent manner, but the mechanism underlying it is unclear. Based on theory introducing autophagy, we preliminarily investigate whether autophagy could contribute to the antitumor activity of E Platinum. Our results showed that autophagy induced by 12.5 ␮M E Platinum in gastric carcinoma BGC-823 cells was significantly characterized by the FITC-fluorescent microtubule associated protein 1 light chain 3 (MAP-LC3), lysosomal-rich/acidic compartments visualized with Lysotracker red (LTR-red) and an accumulation of numerous large autophagic vesicles within the cytoplasm, but not in the control cells. Meanwhile treatment of cells with 12.5 ␮M E Platinum resulted in conversion of water soluble LC3 (LC3-I) to lipidated and autophagosome-associated form (LC3-II) as well as increasing expression of autophagy protein Beclin 1. Activation of predominant lysosomal aspartic protease, LAMP-1 and cathepsin D, was demonstrated. Moreover, RNA interference targeting Beclin 1, inhibition of autophagy by 3-methyladenine (3-MA) and chloroquine significantly suppressed the above process as well as the BGC-823 cells growth inhibition triggered by 12.5 ␮M E Platinum. Studies of mechanism revealed that E Platinum suppressed activation of mTOR and p70S6K by decreasing phosphorylation of Akt, ERK1/2, JNK and p38 involved in mitogen-activated protein kinase signaling. We supported new evidences for E Platinum as a promising antitumor agent, involving with autophagy induction. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Autophagy called ‘self-eating’, is a tightly regulated catabolic process where cytoplasm and organelles are initially sequestered within double-membrane vesicles (autophagosomes), and delivered to the lysosomes for degradation and recycling (autolysosomes) (Rosenfeldt and Ryan, 2009). In unstressed cells, the microtubule associated protein 1 light chain 3 (MAP-LC3) is present in the cytoplasm, while the lipidated form of LC3 (LC3-II) is associated with double-membrane containing organelles in cells undergoing autophagy (Fulda et al., 2010). Given the established role of ATG5 during the recruitment of LC3-II to the membrane, while ATG5-ATG12 complex dissociates from the membrane

∗ Corresponding author. Tel.: +86 25 83271055; fax: +86 25 83271055. E-mail addresses: Xi [email protected] (T. Xi), anticancer [email protected] (Q.-L. Guo). 1 These authors contributed equally to this article. 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2012.01.019

beyond the finish of autophagosome formation, LC3-II remains associated with the membrane (Bommareddy et al., 2009). The biochemical properties of Beclin 1, a tumor suppressor protein, suggest a role in two fundamentally important cell biological pathways: autophagy and apoptosis (Yue et al., 2003). Beclin 1 is the mammalian homolog of the yeast protein ATG6 correlating directly with autophagosome formation (Liang et al., 1999) and is also part of a class III PI-3 kinase complex mediating the localization of autophagy proteins to autophagic vesicles (Nguyen et al., 2007). Recently, increasing evidence shows that autophagy present at a basal level in cells regulates the protein and organelle turnover for cellular homeostasis (Bommareddy et al., 2009; Mehrpour et al., 2010; Salazar et al., 2009a,b). The progression of autophagy includes four different stages: initiation, autophagosome formation, maturation, and degradation (Gutierrez et al., 2004), which eventually results in lysosomal breakdown of cytoplasmic material (Bergmann, 2007). Therefore, when autophagy reaches a high level, cell death will occur because of the overconsumption of critical cellular organelles/components (Bonapace et al., 2010).

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The mammalian target of rapamycin (mTOR) is one conserved serine/threonine kinase that regulates key point for the function of many carcinogenic and metabolic events, including autophagy (Yang and Klionsky, 2010). In recent years, increasing evidence demonstrates that mTOR inhibition induces catabolic processes, which include autophagy and cell growth suppression (Turcotte et al., 2008). Previous studies reported that activation of mTOR in mammals was regulated by the kinase cascade consisting of PI3K/AKT (Degtyarev et al., 2008) or by decreasing the phosphorylation of some protein kinases such as p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK) (Tang et al., 2008). The phosphorylation of mTOR promotes downstream targets such as p70-S6 kinase (p70S6K) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), which leads to regulation of a diverse array of cellular progression (Bonapace et al., 2010; Feng et al., 2005). It is reported that the phosphorylation level of p70S6K, which is critical for initiating protein translation associated with cell growth and proliferation, is a key event for the deregulation of mTOR (Feng et al., 2005). The interest in platinum-based antitumor drugs has its origin in the 1960s, with the serendipitous discovery by Rosenberg of the inhibition of cell division by Pt complexes (Wong and Giandomenico, 1999). Oxaliplatin (C8 H12 N2 O4 Pt, Fig. 1A), is typically administered with fluorouracil and leucovorin in a combination known as FOLFOX for the treatment of colorectal cancer (Wheate et al., 2010). Oxaliplatin has been compared with other platinum compounds such as Cisplatin and Carboplatin in advanced cancers (gastric, ovarian). It is thought that cytotoxicity of platinum compounds result from inhibition of DNA synthesis in cancer cells (Kabolizadeh et al., 2011). Studies in vivo showed that Oxaliplatin has antitumor activity against colon carcinoma through its (non-targeted) cytotoxic effects (Howells et al., 2010). E platinum (C18 H36 N2 O6 Pt, Fig. 1B), a newly synthesized platinum compound bearing the basic structure of oxaliplatin, may have inhibitory activity against cell growth. The difference between the two chemical structures indicates that they may modulate different biochemical processes. Previous studies suggested that autophagy activation under oxaliplatin therapy stress contributes to HCC tumor cell survival (Ding et al., 2011). Furthermore, oxaliplatin-induced protective autophagy partially prevents apoptosis in gastric cancer MGC803 cells (Xu et al., 2011). However, whether E platinum can induce autophagy process or the autophagy induced by E platinum can suppress the cell growth remained unknown. In our present study, we assessed the antitumor activity of E platinum in vitro and in vivo, and also investigated the autophagyinduce by E platinum in gastric cancer BGC-823 cells via its inhibition of phosphorylation of mTOR signaling. Even more importantly, RNA interference targeting Beclin 1, autophagy inhibitor 3-methyladenine (3-MA) and chloroquine were used to investigate the role autophagy played as a promotion mechanism for tumor cells death, which appeared in contradiction to the earlier conclusion that autophagy-induced by oxaliplatin protected cell death or contributed to cell survival (Ding et al., 2011; Xu et al., 2011). This study demonstrates the functional role of autophagy in cancer cell growth and provides a novel mechanism of the antitumor activity of E Platinum. 2. Materials and methods

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The final concentration of 5% glucose solution, the solvent, did not exceed 0.1% (v/v) throughout the study; 3-methyladenine (3-MA, Sigma, M9281) and chloroquine (Sigma, C6628) were diluted to 5 mM and 20 ␮M, respectively, before use. Primary antibodies to MAP-LC3 (SC-28266), Beclin 1 (SC-10086), AKT (SC-8312), p-AKT (SC-7985), P38 (SC-7149), p-P38 (SC-101758), p-ERK1/2 (SC-23759), JNK (SC-7345), p-JNK (SC-6254), p70S6K (SC-8418), p-p70S6K (SC-11759), cathepsin D (SC-13148) and LAMP-1 (SC-20011) were obtained from Santa Cruz Biotechnology. The primary antibody to ␤-actin (BM0627) was from Boster Biological Technology Ltd. Primary antibodies for ERK1/2 (BS-1112), mTOR (BS-3611), and p-mTOR (BS-4706) were from Bioworld Technology Co. Ltd. The secondary antibodies are: (1) anti-mouse IgG: IRDyeTM 800-conjugated anti-mouse IgG (Rockland Immunochemicals, 610-132-121); (2) anti-rabbit IgG: Alexa Fluor 680 goat anti-rabbit IgG (Invitrogen, A21076); (3) anti-goat IgG: Alexa Fluor 680 rabbit anti-goat IgG (Invitrogen, A21088). 2.2. Cell culture The human hepatocellular carcinoma HepG2 and BEL-7402 cells, human colon carcinoma HCT116, HT29 and SW1116 cells, human gastric carcinoma MGC-803, BGC-823 and MKN-45 cells were purchased from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. All the cells were grown in 90% RPMI-1640 medium supplemented with 10% heat-inactivated calf serum (CS) or fetal bovine serum (FBS) containing both 100 units/mL penicillin and 100 ␮g/mL streptomycin. Exponentially growing cultures were maintained in a humidified atmosphere of 5% CO2 at 37 ◦ C. 2.3. MTT assay MTT (Sigma, 88417) was dissolved in 10 mM phosphate-buffered saline (PBS) to a concentration of 5 mg/mL. Various kinds of tumor cell lines were plated in 96well culture plates (5 × 103 per well). After 24 h of incubation, the cells were treated with E Platinum (0, 0.195313, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 ␮M) for 24 h. Subsequently, 20 ␮L of MTT solution (5 mg/mL) was transferred to each well to yield a final assay volume of 220 ␮L/well. Plates were incubated for 4 h at 37 ◦ C and 5% CO2 . After incubation, supernatants were removed, and 100 ␮L DMSO was added to ensure total solubility of formazan crystals. Plates were placed on an orbital shaker for 2 min and the absorbance was recorded at 562 nm. Cell viability was determined based on mitochondrial conversion of MTT to formazan. Inhibition ratio (%) was calculated using the following equation: Inhibitory ratio (%) = (1 − average absorbance of treated group/average absorbance of control group) × 100. IC50 was taken as the concentration that caused 50% inhibition of cell viability and calculated by the Logit method. 2.4. Trypan blue exclusion assay The survival ratio was determined by trypan blue exclusion test. Cells seeded on a six-well plate and treated with 12.5 ␮M E Platinum for 0, 6, 12, 18, 24, 30 and 36 h. When harvested and stained with trypan blue (0.4% in PBS), the number of viable cells was determined by counting the trypan blue-excluding cells under a microscope with a cell-counting chamber. The survival ratio was calculated as: survival ratio (%) = (the number of viable cells/the total number of cells) ×100. 2.5. Tumor xenograft This experiment was conducted in accordance with the guideline issued by the State Food and Drug Administration (SFDA, China). The animals were kept and fed in accordance with the guidelines established by the National Science Council of China. Human gastric carcinoma BGC-823 cells were injected subcutaneously (s.c.) into the right axillary fossa of the nude mice (4 × 106 in 200 ␮L) and tumors were allowed to develop for 20 days until they reached 100–300 mm3 . The mice were randomly divided into four groups (each group contained six mice): 0.9% normal saline control group, 10 mg/kg oxaliplatin positive control group, 2.5 mg/kg, 5 mg/kg and 10 mg/kg E Platinum groups. E Platinum, oxaliplatin and vehicle treatments were given intravenous (i.v.) once every other day for a total for 21 days. Tumor size was measured once every other day in two perpendicular dimensions with vernier callipers and converted to tumor volume (TV) using the formula: (ab2 )/2, where a and b referred to the longer and shorter dimensions respectively. The body weight of the animals was measured twice a week at the same time when tumor dimensions were measured. The general body parameters and the mortality were monitored daily. At the end of treatment, all mice were sacrificed and tumors were excised and weighed.

2.1. Reagents and antibodies 2.6. Immunofluorescence and confocal fluorescence microscopy E Platinum (>96%, supplementary document for the NMR spectra) was a newly synthesized platinum compound bearing the basic structure of oxaliplatin by Dr. Shao-Hua Gou (Southeast University, China) according to the protocols reported previously with slight modifications (Ashfield et al., 2004). It was dissolved at a concentration of 10 mM in 5% glucose solution as a stock solution, stored at −20 ◦ C, and diluted with RPMI-1640 medium (Gibco, 23400-021) before each experiment.

BGC-823 cells were treated with 12.5 ␮M E Platinum for 0, 6, 12, 18, 24, and 36 h. 3 MA at 5 mM or 20 ␮M chloroquine was pretreated 2 h before E Platinum treatment for 36 h. Cells were fixed with 4% paraformaldehyde in PBS at 1-h intervals, permeabilized with 0.5% Triton X-100, and blocked with 2% BSA for 30 min. Incubation with primary antibodies (diluted 1:50) against MAP-LC3 was done overnight at 4 ◦ C. After

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Fig. 1. The chemical structure of oxaliplatin and E Platinum and viability inhibition of cancer cells by E Platinum. (A) The chemical formula of oxaliplatin (C8 H12 N2 O4 Pt, MW = 395.27). (B) The chemical formula of E Platinum (C18 H36 N2 O6 Pt, MW = 571.57). (C) IC50 values of different cancer cell lines containing colon carcinoma cell line (SW1116, HCT116, HT29), gastric carcinoma cell line (MGC-803, BGC-823, MKN-45), or hepatocellular carcinoma cell line (HepG2, BEL7402) by E Platinum. Different cancer cells were treated for 24 h with increasing concentrations of E Platinum, and the inhibitory ratio was determined using MTT assay as described. IC50 values represent the concentration required to inhibit cell viability by 50% relative to untreated cells. Results are the mean ± SD of at least three independent experiments, each performed in triplicate. (D) survival ratio for gastric carcinoma BGC-823 cells treated with E Platinum. Bars derived from the trypan-blue excluding test represent the survival ratio under 12.5 ␮M E Platinum for 6, 12, 18, 24, 30 and 36 h. Results shown are representative of three independent experiments. *P < 0.05; **P < 0.01, 0 h versus E Platinum -treated cells. washing, cells were exposed to fluorescein-5-isothiocyanate (FITC)-conjugated antibody (1:1000, Invitrogen, Carlsbad, CA). Lysosomal-rich/acidic compartments were visualized with Lysotracker Red (Beyotime, C1046), used at a final concentration of 50 nM and added 1 h before imaging. Lysosomal-dependent proteolysis was visualized with DQ Green BSA (Invitrogen, D12050), at 10 ␮g/mL and added 1 h before imaging. After washing, the nuclei were stained with 4 , 6-diamidino2-phenylindole (DAPI, Sigma) 10 min before imaging. Laser scanning biological microscope FV10-ASW [Ver 2.1] (Olympus Corp, MPE FV1000) was used for colocalization analysis. 2.7. Western blot analysis After washing twice with PBS, the cultured cells were collected and lysed in lysis buffer (100 mM Tris–Cl, pH 6.8, 4% (m/v) SDS, 20% (v/v) glycerol, 200 mM bmercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 g/mL aprotinin). The lysates were centrifuged at 13,000 × g for 15 min at 4 ◦ C. The concentration of total proteins was measured using the BCA assay method with Varioskan spectrofluorometer and spectrophotometer (Thermo) at 562 nm. Protein samples were separated with 15% SDS-PAGE and transferred onto the polyvinyldifluoride (PVDF) membranes (Millipore, IPVH 304 F0). Immune complexes were formed by incubation of proteins with primary antibodies overnight at 4 ◦ C followed by IRDyeTM 800 conjugated second antibody for 1 h at 37 ◦ C. Immunoreactive protein bands were detected with an Odyssey Scanning System (LI-COR Biosciences, 9201-03). 2.8. Transmission electron microscopy For experiment in vitro, cells were treated with 12.5 ␮M E Platinum for 0, 6, 12, 18, 24 and 36 h. Cells were directly fixed with 1% glutaraldehyde and postfixed with 2% osmium tetroxide. The cell pellets or sections were embedded in epon resin. Representative areas were chosen for ultrathin sectioning and viewed with a JEM 1010 transmission electron microscope (JEOL) operating at 80 kV. 2.9. Statistical analysis All data were expressed as the mean ± SD. The data shown in the study were obtained in at least three independent experiments performed in a parallel manner unless otherwise indicated. Statistical analysis was performed using an unpaired,

two-tailed Student’s t-test. All comparisons were made relative to untreated controls and significance of difference was indicated as *P < 0.05 and **P < 0.01.

3. Results 3.1. E Platinum inhibits cell viability in tumor cells To investigate the potential inhibition of cell growth by E Platinum in human cancer cells, we first performed MTT assay. The viability inhibitory effects of E Platinum on cancer cell lines containing colon carcinoma cell lines (SW1116, HCT116 and HT29), gastric carcinoma cell lines (MGC-803, BGC-823 and MKN-45), and hepatocellular carcinoma cell line (HepG2 and BEL7402) were evaluated and the IC50 values of E Platinum as shown in Fig. 1C were 2.6089 ± 0.2545, 2.9872 ± 0.3088, 3.2292 ± 0.3392, 11.1800 ± 1.1360, 12.3293 ± 1.0901, 14.7546 ± 1.2795, 34.9780 ± 2.7962 and 74.9664 ± 4.4654 ␮M for 24 h treatment, respectively. MTT assay showed that E Platinum inhibited the viability of colon carcinoma cells more potently than that of gastric carcinoma or hepatocellular carcinoma cells (Fig. 1C), suggesting that colon carcinoma cells possessed a relatively higher sensitivity to E Platinum similar to oxaliplatin (Howells et al., 2010). It was also observed that the degree of inhibition was, to some extent, correlated with exposure time at a given E Platinum concentration (12.5 ␮M). The survival ratio values of E Platinum on gastric carcinoma BGC-823 cells were (88.95 ± 3.50), (79.65 ± 3.45), (71.14 ± 3.49), (54.26 ± 3.33), (37.09 ± 2.10), and (15.45 ± 3.01) % obtained for 6, 12, 18, 24, 30 and 36 h treatment, respectively (Fig. 1D). The results above illustrate the increased effect of 12.5 ␮M of E Platinum was able to obtain more than 50% inhibition of gastric carcinoma BGC-823 cancer cells after 24 h.

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Fig. 2. E Platinum inhibits the growth of transplanted tumors in vivo. (A) The transplanted tumors was segregated and weighed after 10 mg/kg oxaliplatin and 2.5, 5 and 10 mg/kg E Platinum treatment for 21 days. (B) Mean of tumor volume measured at the indicated number of days after implant. During the 21-day treatment, tumor volumes were estimated by measurements taken by external calipers (mm3 ). (C) The body weight of nude mice for 21-day treatment.

3.2. E Platinum inhibits the growth of transplanted tumors in vivo Tumor xenografts transplanted by human gastric carcinoma BGC-823 cells were used to evaluate the antitumor effect of E Platinum in vivo. The weight of tumors was significantly reduced for groups treated with 2.5, 5, and 10 mg/kg E Platinum and 10 mg/kg oxaliplatin (Fig. 2A). Tumor inhibition rates of 38.20%, 55.39%, 69.64% and 71.79% were observed. Furthermore, tumor volume in E Platinum- or oxaliplatin-treated mice was less than that in negative control mice (Fig. 2B). Values of T/C in the 2.5, 5, and 10 mg/kg E Platinum and 10 mg/kg oxaliplatin group were 72.37%, 60.25%, 39.56% and 38.66%, respectively, indicating that E Platinum inhibited tumor growth in a dose-dependent manner during the 21-day treatment. Meanwhile, in contrast with mice treated with 0.9% normal saline, 10 mg/kg oxaliplatin treatment exhibited significant inhibition of nude mice weight. In contrast, weight inhibition was observed less in the 2.5, 5, and 10 mg/kg E Platinum-treated mice (Fig. 2C), indicating that E Platinum may work with lower toxicity as well as obvious antitumor effect in vivo. 3.3. E Platinum induces autophagy initiated with formation of autophagosome in BGC-823 cells Cells were analyzed by confocal fluorescence microscopy. As shown in Fig. 3A, treatment of BGC-823 cells with 12.5 ␮M E Platinum displayed an increase in not only the number but also the size of MAP-LC3-positive points starting from 12 h, which indicated that E Platinum treatment firstly induced the formation of the autophagosome. The autophagosomes would be expected to undergo acidification after maturation and finally, fuse with lysosomes so that their content is digested by lysosomal hydrolases. The MAP-LC3-positive cells ratio in each of the 200 cells after treatment of 12.5 ␮M E Platinum were 1.25%, 3.50%, 8.00%, 18.75%, 30.00%, and 45.25% for 0, 6, 12, 18, 24 and 36 h, respectively. In addition, the ratio decreased significantly (1.50%) in cells pretreated with autophagy inhibitor 5 mM 3-MA 2 h before treatment of 12.5 ␮M E Platinum

for 36 h (Fig. 3B). To further confirm the progression of autophagy, the up-regulation of Beclin 1 expression and the conversion from soluble form of LC3 (LC3-I) d to the lipidated and autophagosomeassociated form (LC3-II) after treatment of 12.5 ␮M E Platinum were along with the occurrence of MAP-LC3-positive dots in a timedependent manner (Fig. 3C). The above induction by 12.5 ␮M E Platinum for 6, 12, 18, 24 and 36 h with the LC3-II/LC3-I ratio (2.58, 7.75, 11.75, 18.59, 43.89, compared to 0 h) also decreased in present of 5 mM 3-MA to 1.39 for 36 h (Fig. 3C and D). 3.4. E Platinum drived autophagosome–lysosome fusion and triggered the activity of autolysosome in BGC-823 cells The large lysosomes subsequently recruit multiple autophagosomes. In order to analyze these possibilities, endo-lysosomes were detected in BGC-823 cells treated with 12.5 ␮M E Platinum, which send signals in the acidic environment of autolysosomes (labeled with lysotracker Red). Alternatively, to independently demonstrate the efficiency of E Platinum on lysosomal activity, cells were assayed for the ability to process DQ-BSA (a derivative of BSA whose green fluorescence is quenched unless cleaved by proteolytic enzymes). In addition, emission of DQ-BSA was monitored at the lysosomes by colocalization with lysotracker Red. As shown in Fig. 4A, DQ-BSA was efficiently cleaved in the presence of E Platinum. The proteolyzed DQ-BSA (as arbitrary fluorescence units) of BGC-823 cells after treatment of 12.5 ␮M E Platinum for 0, 6, 12, 18, 24 and 36 h were 22.06%, 36.32%, 44.22%, 48.07%, 51.93% and 60.98%, respectively (Fig. 4B). The lysosomotropic agent chloroquine (20 ␮M) decreased lysosomes activity with the proteolyzed DQ-BSA of 6.45% (Fig. 4A and B). Autophagy is a key function of the lysosomal compartment, so the lysosomal marker LAMP1 and cathepsin D, the predominant lysosomal aspartic protease, were examined by a Western blot. Inhibitory effects were observed using chloroquine (Fig. 4C). These results showed that vacuoles assumed to be autophagosomes are expected to undergo acidification after maturation and finally, fuse with lysosomes so that

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Fig. 3. E Platinum induces autophagy initiated with formation of autophagic vacuoles in BGC-823 cells. (A) Confocal fluorescence images of BGC-823 cells showing endogenous MAP-LC3 levels at different time points after E Platinum treatment. Nuclei (blue) were labeled by DAPI. MAP-LC3 expression (green) was detected using an anti-MAP-LC3 polyclonal antibody. Goat anti-rabbit IgG/FITC were used as secondary antibody. Confocal microscopy images were obtained. Bar = 50 ␮m. (B) Quantification of the percentage of cells with focal MAP-LC3 at the indicated times after E Platinum treatment. Error bars correspond to SD of three independent experiments, counting 200 cells each. *P < 0.05; **P < 0.01, 0 h versus E Platinum-treated cells. (C) Effect of E Platinum on expression of LC3 lipidation and Beclin 1 levels in cells were analyzed by Western blotting, cells were treated with 12.5 ␮M E Platinum for the indicated times. (D) Ratio of LC3-II/LC3-I was quantified. Results shown are representative of three independent experiments. *P < 0.05; **P < 0.01, 0 h versus E Platinum-treated cells. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)

their content is digested by lysosomal hydrolases. The appearance of autophagosome–lysosome fusion was initially observed by 18 h and the activity of autolysosome reached a peak by 36 h. Transmission electron microscopy (TEM) images in Fig. 5 revealed an accumulation of numerous large autophagic vesicles within the cytoplasm of E Platinum-treated BGC-823 cells, and both doublemembrane and single-membrane vesicles containing intact and disintegrating materials were observed in treated cells, but not in the control cells (Fig. 5A). Meanwhile, images revealed a significantly increased accumulation of autophagosome–autolysosome in BGC-823 cells with treatment of E Platinum from 6 to 36 h (Fig. 5B–F). 3.5. E Platinum inhibited the phosphorylation of mTOR and p70S6K The mechanism of E Platinum induced autophagy in BGC-823 cells is not well understood, which led to further investigation of the biochemical process. Inhibition of mTOR is considered to be the key step in the early triggering of autophagy (Maiuri et al., 2007). Therefore effects of E Platinum on the expression of mTOR and its phosphorylation product p-mTOR (Ser2448) were examined since mTOR specifically phosphorylates the p70S6 kinase at Thr-389. A Western blot is used to determine the phosphorylation of p70S6 kinase and ␤-actin was used as internal standard (Brown et al., 1995). As shown in Fig. 6A and B, when treated with 12.5 ␮M E

Platinum for 6, 12, 18, 24 and 36 h, the phosphorylation levels of both mTOR and p70S6K were reduced in a time-dependent manner, while the total steady state protein level remained unchanged. 3.6. Influence of E Platinum on mTOR-related signaling pathways A Western blot was performed to evaluate the molecular mechanism in which E Platinum inhibited the phosphorylation of mTOR and p70S6K, which triggered autophagy progression. The effects of E Platinum on the related downstream signaling molecules Akt, ERK1/2, JNK and p38 MAPK were investigated and ␤-actin was used as internal standard. In Fig. 6C–F, treatment with 12.5 ␮M E Platinum effectively inhibited phosphorylation of Akt, ERK1/2, and p38 MAPK in a time-dependent manner. In all cases, the total steady state protein levels of Akt, ERK1/2, and p38 MAPK remained unchanged. These results suggest that E Platinum targets mTOR, which leads to an induction of autophagy signal transduction. 4. Discussion In this study, we show that E Platinum, a newly synthesized platinum compound of potential antitumor agents, induces autophagy of cancer cells that is responsible for the cell growth inhibition activity of this platinum compound which has a similar structure to oxaliplatin. During the progression of autophagy, the cytoplasm or cell organelles were originally sequestered within

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Fig. 4. E Platinum-triggered autophagy is driven by autophagosome–autolysosome formation. (A) Confocal visualization of lysosomal-dependent proteolysis upon cleavage and release of the fluorescent moiety of DQ-BSA (green) in E Platinum-treated BGC-823 cells. Chloroquine is added to monitor DQ-BSA emission in cells with blocked lysosomal activity. Cells were simultaneously imaged in the presence of Lysotracker red (LTRred) to visualize the lysosomal compartment. Bar = 50 ␮m. (B) The DQ-BSA-Lysotracker colocalization was estimated in a minimum of 150 cells in three independent experiments, and it is expressed (as arbitrary fluorescence units) with respect to control treated cells. Data are indicated as means ± SD. *P < 0.05; **P < 0.01, 0 h versus E Platinum-treated cells. (C) Western blotting analysis of cell lysates collected at the indicated time points from the experiment shown in A. Arrowheads indicate the positions for LAMP-1, CathD 43, and CathD 28. CathD 43, the 43–50 kD forms of cathepsin D precursors. CathD 28, the 28 kD cathepsin D heavy chain. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)

double-membrane structures (Pan et al., 2008). The autophagosomes undergo acidification after maturation (Meijer and Codogno, 2004; Yang et al., 2005) and subsequently fuse with lysosomes (called autolysomes) where the autophagosomes content is digested by lysosomal hydrolases (Turcotte et al., 2008). The above sequence of events is strongly supported by the results from our present studies. Recently, oxaliplatin, which bears the basic structure of E platinum, has been reported to induce autophagy of various kinds of cancer cells. Autophagy was functionally activated in hepatocellular carcinoma cell lines and xenografts after oxaliplatin treatment (Ding et al., 2011). Their previous studies concluded that suppression of autophagy using either pharmacologic inhibitors or RNA interference of essential autophagy gene enhanced cell death induced by oxaliplatin in hepatocellular carcinoma cells (Ding et al., 2011) or significantly enhanced the inhibition of cell proliferation and the induction of cell apoptosis in gastric cancer cells (Xu et al., 2011). However, our current studies showed that the autophagy induced by 12.5 ␮M E Platinum may contribute to cell growth inhibition in the gastric carcinoma BGC-823 cells. Firstly, BGC-823 cells exposed to E Platinum displayed cytoplasmic structures staining with the FITC-fluorescent MAP-LC3 and lysosomal-rich/acidic compartments were visualized

with Lysotracker Red, that was originally detected among the larger vacuoles compared with the punctate staining observed for LC3. Because 3-MA and chloroquine act as autophagy inhibitor and lysosomotropic agent, respectively, we imply them to monitor the action which can be observed after autophagosome and fusion with lysosomes (autolysosomes) (Weihua et al., 2008), this staining pattern suggests that these large vacuoles are associated with the acidic components of autolysosomes. Secondly, transmission electron microscopy (TEM) images showed large numbers of autophagic vacuoles in E Platinum treated cells, but not in untreated cells. Double-membrane containing cellular organelles was observed in E Platinum treated BGC-823 cells at higher magnification. Thirdly, the selective autophagy gene Beclin 1 expression and conversion of the soluble form of LC3 (LC3-I) to the lipidated and autophagosome-associated form (LC3-II) were analyzed by Western blotting. This conversion was supported by the occurrence of MAP-LC3-positive dots in E Platinum treated cells. Finally, xenograft tumor growth was inhibited by E Platinum. Overall the results indicate that E Platinum activated the autophagic process in vitro in cancer cells and inhibited tumor xenograft models in vivo. Significant progress has been achieved over years in elucidating the molecular regulators of autophagy (Rouschop and Wouters,

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Fig. 5. Transmission electron images of BGC-823 cells after treatment with E Platinum. Cells were treated with 12.5 ␮M E Platinum for 0, 6, 12, 18, 24 and 36 h. Cells were directly fixed with 1% glutaraldehyde and postfixed with 2% osmium tetroxide. The cell pellets or sections were embedded in epon resin. Representative areas were chosen for ultrathin sectioning and viewed with a JEM 1010 transmission electron microscope (JEOL) operating at 80 kV. (A) Control, (B–F), 6, 12, 18, 24 and 36 h, red arrows, initial AVs with phagophore (isolation membrane); yellow arrows, degradative autolysosomes; asterisks, later empty vacuoles. Bars: 1 ␮m. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)

2009) as reviewed previously (Feng et al., 2005). The mTOR pathway was principally examined in autophagy regulation because recent studies indicated that inhibition of the mTOR pathway was consistently associated with triggering autophagy in cancer cells (Degtyarev et al., 2008). The inactivated mTOR was demonstrated by reduced phosphorylation of its downstream target p70S6 kinase at Thr-389 using Western blotting analysis. The protein kinase Akt positively regulates the activity of the mTOR complex by phosphorylating and inhibiting TSC2 and PRAS40 (a well-established Akt substrate within the mTOR complex) (Fuhler et al., 2009). Akt inhibition decreases mTOR activity and promotes autophagy. The inhibitory effect of E Platinum on the phosphorylation of AKT was detected in a time-dependent manner in our present studies. In addition, a previous study testified that mTOR pathway could be regulated by MAPK pathway (Carracedo et al., 2008). The phosphorylation of ERK1/2, JNK and p38 involved in the mitogenactivated protein kinase signaling pathway in BGC-823 cells treated with E Platinum was monitored. The suppression of these kinase activations has been related to inhibition of mTOR. E Platinum markedly suppressed the phosphorylation of ERK1/2, JNK, and p38 MAPK, as well as Akt, which indicated that this inhibitory effect leads to autophagy. This negative effect of E Platinum on mTOR phosphorylation and its signal transduction may be able, at least in part, to promote potent autophagy-induction activity. E Platinum was further investigated in order to explain the mechanisms of action for those kinases and the effect on their downstream targets. Autophagy is implicated in various physiological processes including protein and organelle turnover, response to starvation, cellular differentiation, cell death, and pathogenesis (Sarkar et al., 2009). It has been defined as an intracellular bulk protein

degradation system where most long-lived proteins and some cytoplasmic organelles are digested (Sarkar and Rubinsztein, 2008; Weihua et al., 2008). Therefore, autophagy has been considered either an adaptive response to enhance cell survival or an initiation of the cell death process (Meijer and Codogno, 2004; Sarkar et al., 2009). Thus, the present results clearly show that induction of autophagy is involved in the process in which E Platinum promotes the inhibition of cell growth. In order to determine whether autophagy induced by E Platinum was responsible in BGC-823 cells, the autophagic cells were measured for 36 h after treating cells with 3-MA and chloroquine to inhibit autophagy. The rate of autophagic cells was partially inhibited by 3-MA and chloroquine, indicating that E Platinum-induced autophagy precedes cell growth inhibition in BGC-823 cells. A majority of existing chemotherapeutic agents such as oxaliplatin are limited in clinical application because their cytotoxicity also affects healthy cells (Edinger and Thompson, 2003). Therefore, it is imperative to explore new compounds, which can work with higher therapeutic indexes as well as lower toxicity (Cao et al., 2006). The autophagic process took place from approximately 12 h after E Platinum treatment of BGC-823 cells. A new route that links the activation of autophagy to cell growth inhibition was identified (Martinet and De Meyer, 2009). Identification of the mTOR signaling transduction pathway will initially promote the understanding of the molecular facts that lead to activation of autophagy-mediated cell growth inhibition by antitumor agents and could contribute to the design of new therapeutic strategies for inhibiting tumor growth. The first evidence indicating that E Platinum induces autophagy via inhibition of mTOR signaling in human gastric carcinoma BGC-823 cells was presented. Although the detailed mechanisms, which mediate the activation of those kinases connected with mTOR remain to be elucidated, this finding

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Fig. 6. E Platinum induced autophagy via inhibiting the phosphorylation of mTOR and the related signaling transduction pathway. Cells were incubated for 6, 12, 18, 24 and 36 h in the presence of 12.5 ␮M E Platinum. After cell lysis, the levels of mTOR, p70S6K, Akt, ERK1/2, JNK, and p38 MAPK and their phosphorylated forms were analyzed by Western blotting with antibodies against various mTOR signaling proteins (A–F). Results shown are representative of at least three independent experiments.

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