Tumor Biol. DOI 10.1007/s13277-016-4977-2
ORIGINAL ARTICLE
Platinum-zoledronate complex blocks gastric cancer cell proliferation by inducing cell cycle arrest and apoptosis Hui Yang 1,2 & Ling Qiu 1,2 & Li Zhang 2 & Gaochao Lv 2 & Ke Li 2 & Huixin Yu 2 & Minhao Xie 1,2 & Jianguo Lin 1,2
Received: 23 November 2015 / Accepted: 4 February 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016
Abstract A series of novel dinuclear platinum complexes based on the bisphosphonate ligands have been synthesized and characterized in our recent study. For the purpose of discovering the pharmacology and action mechanisms of this kind of compounds, the most potent compound [Pt(en)]2ZL was selected for systematic investigation. In the present study, the inhibition effect on the human gastric cancer cell lines SGC7901 and action mechanism of [Pt(en)]2ZL were investigated. The traditional 3-[4,5-dimethyl-2-thiazolyl]-2,5diphenyl-2-tetrazolium bromide (MTT) assay and colony formation assay were carried out to study the effect of [Pt(en)]2ZL on the cell viability and proliferation capacity, respectively. The senescence-associated β-galactosidase staining and immunofluorescence staining were also performed to assess the cell senescence and microtubule polymerization. Fluorescence staining and flow cytometry (FCM) were used to monitor the cell cycle distribution and apoptosis, and Western blot analysis was applied to examine the expression of several apoptosis-related proteins. The results demonstrated that [Pt(en)]2ZL exhibited remarkable Electronic supplementary material The online version of this article (doi:10.1007/s13277-016-4977-2) contains supplementary material, which is available to authorized users. * Minhao Xie
[email protected] * Jianguo Lin
[email protected]
1
Department of Radiation Oncology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China
2
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China
cytotoxicity and anti-proliferative effects on the SGC7901 cells in a dose- and time-dependent manner, and it also induced cell senescence and abnormal microtubule assembly. The cell apoptosis and cell cycle arrest induced by [Pt(en)]2ZL were also observed with the fluorescence staining and FCM. The expressions of cell cycle regulators (p53, p21, cyclin D1, cyclin E, and cyclin-dependent kinase (CDK)2) and apoptosis-related proteins (Bcl-2, Bax, caspase-3, poly ADP ribose polymerase (PARP), and survivin) were regulated by the treatment of [Pt(en)]2ZL, resulting in the cell cycle arrest and apoptosis. Therefore, [Pt(en)]2ZL exerted antitumor effect on the gastric cancer via inducing cell cycle arrest at G1/S phase and apoptosis. Keywords Platinum-zoledronate complex . Human gastric cancer cell line SGC7901 . Cell cycle arrest . Apoptosis
Introduction Gastric cancer is one of the cancers with the highest incidence and mortality worldwide, accounting for 8 % of the total cancer cases and 10 % of the total cancer deaths [1]. Up to now, surgery is still the main treatment method for gastric cancer, especially for those patients in early stage [2]. Nevertheless, for those in late stage with metastasis, surgery is palliative but not curative. In these cases, systematic chemotherapy is widely accepted, which often leads to good responses, such as improvement in the quality of life and increase in the survival rate. To date, fluorouracil- and platinum-based drug combination chemotherapy is considered as a standard first-line treatment in patients with gastric cancer [3]. Classic platinumbased anti-carcinogenic agents including cisplatin (CDDP) cause DNA damages by binding to two adjacent guanosine residues and therefore forming intrastrand cross-links and
Tumor Biol.
finally leading to cell death [4]. However, patients receiving conventional platinum-based drugs often suffer from some side effects, such as normal tissue toxicity and increasing levels of resistance [5]. Therefore, development of novel therapeutic agents with superior anti-cancer profiles holds much promise for gastric cancer patients. Recently, a series of novel platinum complexes with different carrier ligands have been designed and evaluated in our lab [6] in order to overcome the intrinsic and acquired resistance of this devastating disease. The mechanisms of suppressing tumorigenesis usually include carcinogen metabolism, induction of DNA damage, suppression of cell cycle progression, induction of apoptosis, etc. [7]. Among them, cell cycle arrest and apoptosis can be caused by multicellular organisms to eradicate cells in diverse physiological and pathological settings [8]. Accumulating evidence suggests that the efficiency of anti-tumor agents is related to these cellular mechanisms. In this context, it is noteworthy that the induction of apoptosis and the cell cycle arrest seem to have become significant factors in determining the efficiency of new chemotherapeutic agents. The tumor suppressor gene p53, which can be mutated, deleted, or rearranged in more than half of all human tumors, plays a crucial role in the pathogenesis of cancer [9]. It has been noted that p53 may induce the cell cycle arrest and apoptosis in response to the DNA damage [10]. In normal cells, the cell cycle consists of G1, S, G2, and M phases. Specifically, cell cycle progression is regulated by cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) [11]. On the one hand, the disorder of tumor cell cycle progression is governed by altering activation of various cyclin/CDK complexes, such as p53-p21-cyclin D branch. P53 regulates cell cycle checkpoints by promoting transcription of CKI p21. In addition, p21 causes G1 phase arrest by binding and inactivating cyclin/CDK complex [12, 13]. The synthesis of cyclin D is initiated during G1 phase and drives the transition from G1 phase to S phase [14]. On the other hand, cyclin E binds to CDK2 in G1 phase, which is required for the transition from G1 phase to S phase that determines the cell division [15]. At present, uncontrolled production of cyclin D is frequently observed in various cancers and associated with tumorigenesis and metastasis. Additionally, dysregulation cyclin E activity impairs maturation due to increased cell proliferation, apoptosis, or senescence [15, 16]. It has been reported that many anti-cancer compounds selectively induce cell arrest at G1, S, or G2/M phase in different cancer cell lines [17]. Cells arrested at the G1/S phase can die through the p53dependent downstream mitochondrial-mediated apoptotic pathway. As a p53 downstream target, Bax is a proapoptotic member of the Bcl-2 family and can be upregulated during p53-mediated apoptosis. Another important group of proteins involved in the apoptotic cell death is a class of cysteine proteases known as caspases. Caspase-3 is activated in the intrinsic apoptotic cell death after Bcl-2 family alters the
mitochondrial membrane permeability [18]. Poly ADP ribose polymerase (PARP) cleaved and activated by caspase-3 during apoptotic processes can deplete the ATP of a cell in an attempt to repair the damaged DNA. PARP can be inactivated by caspase cleavage and leads to cell apoptosis [19, 20]. The expression of survivin is a member of the inhibitor of apoptosis proteins (IAP) family and it is linked to p53 protein. Furthermore, the survivin protein can inhibit the apoptosis induced by caspase activation and Bcl-2 family, and it can also interact with CDK to regulate the cell cycle [21, 22]. Recently, a series of novel platinum complexes based on the bisphosphonate ligands have been synthesized and characterized by our group [6]. Their cytotoxic activities were evaluated in vitro against various human cancer cell lines, including cancers of gastric, liver, esophagus, and breast. Compared with other complexes, [Pt(en)]2ZL (Fig. 1) showed superior potent anti-cancer activity at low concentrations against most of the human tumor cell lines in vitro [6]. However, its exact mechanism of action inducing cell death remains to be unexplored. In the present study, it was observed that [Pt(en)]2ZL can inhibit cell proliferation, induce cell senescence, and facilitate microtubule polymerization. Further examinations revealed that cell cycle arrest and apoptosis may be involved in the molecular mechanism of anti-cancer effects of [Pt(en)]2ZL in human moderately differentiated gastric cancer cell line SGC7901. This study can help to obtain a detailed understanding of the action mechanism of the new compound [Pt(en)]2ZL (1) at the molecular level and will be instructive for the further design and synthesis of novel more potent platinum-based anti-cancer agents.
Materials and methods Chemicals, reagents, and antibodies ZL and [Pt(en)]2ZL (en = ethylenediamine and ZL = 1-hydroxy-3(1H-imidazol-1-yl)ethane-1,1-diylbisphosphonic acid) were prepared in our lab [6]. Their structures were identified by IR, 1H/13C NMR, and ESI-MS. Their purities were over 99.5 % determined by HPLC. They were both dissolved in phosphate-buffered saline (PBS) to give the stock solution of 4 × 103 μM and stored at −4 °C. Cisplatin was purchased from Shandong Boyuan Pharmaceutical Co., Ltd. The reagents 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), bicinchoninic acid (BCA) protein assay kit, bovine serum albumin (BSA), and crystal violet were purchased from Solarbio Science and Technology (Shanghai, China). Fluorescein isothiocyanate (FITC)-Annexin V/propidium iodide (PI) apoptosis assay kit was purchased from Nanjing KeyGEN Biotech (Jiangsu, China). Hoechst 33342, RNase A, senescence-associated β-galactosidase (SA-β-gal) staining
Tumor Biol. Fig. 1 Chemical structure of [Pt(en)]2ZL
kit, Triton X-100, electrochemiluminescence (ECL), αtubulin antibody, FITC-conjugated anti-mouse secondary antibody, hematoxylin and eosin staining kit, PI dye, and onestep TdT-mediated dUTP nick-end labeling (TUNEL) apoptosis assay kit were purchased from Beyotime (Shanghai, China). Monoclonal antibodies specific to p21 and caspase-3 were purchased from Santa Cruz Biotechnology (Berkeley, CA, USA), while monoclonal antibodies specific to p53, survivin, PARP, cyclin D1, cyclin E, and CDK2 were purchased from Cell Signal Technologies (Beverly, MA, USA). β-Tubulin antibody was purchased from Abcam Trading Co., Ltd (Shanghai, China). The secondary antibodies to antimouse and anti-rabbit immunoglobulin G (IgG)-conjugated horseradish peroxidase (HRP) were purchased from Bioworld Technology (Minneapolis, MN, USA). Cell culture Human gastric cancer cell lines SGC7901 and MGC803, hepatoma cell line BEL7404, esophageal cancer cell line EC109, breast adenocarcinoma cell line MCF-7, ovary cancer cell line SKOV3, and human normal gastric mucosal epithelial cell line GES-1 were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained in HG-Dulbecco modified Eagle’s medium (Biological Industries, Kibbutz Beit Haemek, Israel), supplemented with 10 % (v/v) fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated in a 37 °C incubator (Thermo Electron Corporation, USA) with a humidified atmosphere containing 5 % CO2. Cell viability assay The MTT method was applied to investigate the sensitivity of various human cancer cells to [Pt(en)]2ZL as previously reported [23]. Briefly, cells (5 × 104 cells/mL) were seeded in 96-well plates with 100-μL culture medium per well. After an overnight incubation, the medium was removed and replaced by various concentrations of the drug. The plates were incubated at 37 °C in 5 % CO2 for 48 h. Before next processing, the cells were observed under the microscope and photographed by a Panasonic Lumix DMC-FH2. Then, 20 μL MTT solution (5 mg/mL) was added to each well. After plates were cultured at 37 °C for 4 h, the supernatants were removed and 150 μL
DMSO was added to each well for dissolving the formazan crystal. The absorbance was measured at the wavelength of 490 nm using a microplate reader (BioTek Instruments, Inc. Vermont, USA). All experiments were performed at least three times. The half maximal inhibitory concentration (IC50) values (concentration of drug that inhibits cell growth by 50 %) were determined from the dose–response curve according to the inhibition rate for each concentration. Colony formation assay SGC7901 cells were seeded in 6-well plates at a density of 1 × 103 cells/mL per well and incubated at 37 °C overnight to allow cell attachment. Then, the cells were treated with 0, 1, 2, and 4 μM of [Pt(en)]2ZL, respectively. After 7-day exposure to the drug, cells were fixed with 95 % ethanol for 30 min and stained with 1 % crystal violet. Subsequently, cell colonies (≥50 cells) were counted under inverted light microscopy [24]. Images of representative colonies were photographed by a Panasonic Lumix DMC-FH2. The colony formation rate was calculated according the formula of (colony counts)/(cells inoculated) × 100 %. SA-β-gal staining SGC7901 cells were seeded in 6-well plates at a density of 1 × 103 cells/mL per well and incubated at 37 °C overnight to allow cell attachment. Then, the cells were treated with 0, 1, 2, and 4 μM of [Pt(en)]2ZL. After 7-day exposure to the drug, cells were stained using a senescence kit according to the manufacturer’s instructions. Briefly, cells were fixed with formalin for 15 min at room temperature, washed with PBS thrice, and incubated at 37 °C with SA-β-gal working solution overnight [25]. Cells were considered positive when the cytoplasm was stained with SA-β-gal. Then, the samples were counted and imaged using a Panasonic Lumix DMC-FH2. The experiment was repeated three times and the results were analyzed statistically. Immunofluorescence staining For determining the effect of [Pt(en)]2ZL on microtubule polymerization, immunofluorescence staining was performed. After 48 h exposure to the drug, the cells were washed twice with PBS, fixed with 4 % paraformaldehyde for 30 min, then washed twice with PBS, and permeabilized with 0.5 % Triton
Tumor Biol.
X-100 in PBS for 5 min at 4 °C. Then, the cells were blocked with 5 % BSA in PBS for 1 h. The primary α-tubulin antibody was diluted (1:500) with 1 % BSA in PBS and incubated at 4 °C overnight. The cells were washed twice with PBS for 5 min to remove unbound primary antibody, and then, cells were incubated with FITC-conjugated anti-mouse secondary antibody (1:500) for 2 h at 37 °C. The cells were washed twice with PBS for 5 min to remove unbound secondary antibody. Subsequently, nucleus was stained with PI and then immunofluorescence was detected using a fluorescence microscope (BX51TRF, Olympus) [26]. Cell cycle analysis SGC7901 cells were seeded at a density of 2 × 105 cells/mL per well in 6-well plates and incubated at 37 °C overnight to allow cell attachment. After incubation with 0, 10, 20, and 30 μM of [Pt(en)]2ZL for 24 h, respectively, cells were harvested, washed with ice-cold PBS, and fixed with 70 % ethanol at 4 °C overnight. The fixed cells were washed and resuspended in 200 μL PBS containing 50 μg/mL PI and 50 μg/mL RNase A. Then, the samples were incubated at 37 °C for 30 min in the dark and analyzed for DNA content by FCM (FACS Calibur, Becton Dickinson, USA), and the populations of G0/G1, S, and G2/M phases were quantified using FlowJo software as previously described. TUNEL assay In order to further confirm the anti-cancer effect of [Pt(en)]2ZL, apoptotic SGC7901 cells were examined by the TUNEL assay. This method was performed to label 3′-end of fragmented DNA, thus allowing the identification of apoptotic cells. The cells cultured in 24-well plates were treated with DNase I and [Pt(en)]2ZL (30 μM), fixed with 4 % paraformaldehyde for 30 min, rinsed with PBS thrice, and then permeabilized by 0.1 % Triton X-100 for 2 min on ice followed by TUNEL staining for 1 h at 37 °C. The FITC-labeled TUNELpositive cells were imaged under a fluorescence microscopy (BX51TRF, Olympus). The cells with green fluorescence were defined as apoptotic cells. Hoechst 33342-PI staining assay Cells (2 × 105 cells/mL) were seeded on glass slides placed in 6-well plates for 24 h. After growing to approximately 40– 50 % confluence, the cells were treated with the indicated concentration of [Pt(en)]2ZL for 48 h, respectively. The cells were washed with PBS thrice, followed by fixation with 4 % paraformaldehyde for 15 min, and then washed thrice with PBS. Hoechst 33342 was subsequently added at a final concentration of 10 μg/mL in the dark for 30 min, and then, PI was added at a final concentration of 10 μg/mL in the dark for
15 min. After washed with PBS, the slides were analyzed by fluorescence microscopy (BX51TRF, Olympus). Apoptotic cells were identified on the basis of morphological changes in the cell nuclear assembly, such as chromatin condensation, fragment staining with Hoechst 33342, and necrosis or late apoptosis cells staining with PI [27]. Annexin V-FITC/PI staining assay Cell apoptosis was further measured using the Annexin VFITC/PI apoptosis detection kit. Briefly, cells were seeded into 6-well plates at a density of 2 × 105 cells/mL per well. After exposure to the indicated concentration of [Pt(en)]2ZL for 48 h, the cells were harvested, washed with PBS, and then resuspended in 500-μL binding buffer. Finally, the cells were stained with 5 μL of Annexin V and 5 μL of PI for 15 min at room temperature in the dark. All samples were then analyzed using the flow cytometer [28]. Western blot analysis The cells after reaching confluence were incubated in media containing various concentrations of [Pt(en)]2ZL. After incubation for 48 h, cells were washed with ice-cold PBS for 30 min and collected. Then, the cells were extracted and prepared in RIPA buffer (Beyotime, Shanghai, China) with 1 % PMSF (Beyotime, Shanghai, China) by centrifuging at 15,000 rpm for 7 min at 4 °C. The protein contents were determined using a BCA protein assay kit. The protein samples (50–100 μg) were boiled with gel-loading buffer for 5 min at 100 °C and transferred to polyvinyl difluoride (PVDF) membranes (Merck Millipore Corporation, Billerica, MA, USA). The membranes were blocked with a solution containing 5 % (w/v) fat-free milk, incubated at room temperature for 1.5 h, and then incubated at 4 °C overnight with each of following antibodies: anti-p21 antibody (1:500), anti-p53 antibody (1:1000), anti-caspase-3 antibody (1:1000), anti-PARP antibody (1:1000), anti-Bcl-2 antibody (1:1000), anti-Bax antibody (1:1000), anti-survivin antibody (1:1000), anti-cyclin D1 antibody (1:1000), anti-cyclin E antibody (1:1000), anti-CDK2 antibody (1:1000), and antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000). After washing the membrane with Tris-buffered saline and 0.1% Tween 20 (TBST) (20 mM Tri-Cl, pH 7.5; 150 mM NaCl; 0.1% Tween) thrice (10 min each), the membranes were subsequently incubated with goat anti-mouse or anti-rabbit IgGHRP secondary antibody (1:10,000) at 37 °C for 2 h. Finally, the blots were washed with TBST followed by washing twice with PBS. ECL (Beyotime, Shanghai, China) was added to the membranes, which was developed and fixed for 3 min. The protein bands were revealed by enhanced chemiluminescence substrate and exposed on Kodak radiographic film. To assess protein loading, GAPDH was selected as the internal reference [28].
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Statistical analysis The data was expressed as mean ± standard deviation (SD) of three independent experiments. Continuous variables were analyzed using Student’s t test. A value of p < 0.05 was considered statistically significant, and if p < 0.01, it was noted. All analyses were performed using SPSS 17.0 statistical package.
Results
[Pt(en)]2ZL inhibits the colony formation of SGC7901 cells Colony formation assay revealed that [Pt(en)]2ZL elicited anti-proliferative effects on SGC7901 cells in a dosedependent manner. After culture with [Pt(en)]2ZL for 7 days, the number of SGC7901 cell colonies was fewer and their size was smaller compared to the control group (Fig. 3c). At the drug concentration of 4 μM, the colony formation rate of SGC7901 cells was only 13.07 %, which decreased significantly compared with the untreated well (Fig. 3d).
In vitro anti-cancer activity of [Pt(en)]2ZL The inhibition effect of [Pt(en)]2ZL on cellular viability was examined using the mitochondrial MTT reduction assay. After exposure to the drug for 48 h, [Pt(en)]2ZL showed considerable cytotoxic effect against cancer cell lines SKOV3, BEL7404, EC109, MGC803, MCF-7, and SGC7901, and the IC50 values were 193.54 ± 5.74, 69.89 ± 4.19, 44.47 ± 5.77, 38.52 ± 3.00, 56.10 ± 6.26, and 25.15 ± 0.75 μM, respectively (Fig. 2a). Morphologic changes of these cancer cell lines also showed time- and concentration-dependent effects on the cell viability induced by the new complex 1 (Fig. S1). Since the gastric cancer cell lines SGC7901 displayed the highest sensitivity to [Pt(en)]2ZL, it was chosen for further biological investigation. In addition, the IC50 value of [Pt(en)]2ZL against the normal human gastric mucosal epithelial cell line GES-1 at 48 h was 79.16 ± 6.13 μM (Fig. 2b), which indicated that [Pt(en)]2ZL can be used as a potential anti-cancer drug with good selectivity for inhibiting human gastric carcinoma cells rather than normal gastric cells. Morphologic changes in SGC7901 cells induced by [Pt(en)]2ZL were similar with our previous study [6]. Specifically, cell shrinkage, rounding up, cell member bleeding, and decrease of cell density were observed under microscope (Figs. 3a and S1). Moreover, [Pt(en)]2ZL inhibited the cell viability of SGC7901 in a dose- and time-dependent manner (Fig. 3b). The IC50 value of [Pt(en)]2ZL against SGC7901 at 24 h was 70.69 ± 1.08 μM, which was larger than twofold of that at 48 h. Fig. 2 Cytotoxicity of [Pt(en)]2ZL against various tumor cells. a Sensitivity of different human cancer cell lines to [Pt(en)]2ZL treated for 48 h. b Selectivity of [Pt(en)]2ZL in inhibiting human normal gastric cells (GES-1) and gastric cancer cells (SGC7901)
[Pt(en)]2ZL induces senescence-like growth arrest of SGC7901 cells To investigate the SA-β-gal activity and senescence-like growth arrest induced by [Pt(en)]2ZL in SGC7901 cells, SA-β-gal staining assay was performed. [Pt(en)]2ZL increased the number of SA-β-gal-positive cells in a dose-dependent manner after treated for 7 days, while only a small number of SA-β-gal-positive cells were detected in the untreated SGC7901 cells (Fig. 4a). In comparison with the untreated cells, enlarged and flattened morphology was clearly observed in the SGC7901 cells treated by [Pt(en)]2ZL. In particular, the percentage of SA-β-gal-positive SGC7901 cells was 47.83 % with the treatment of 4 μM [Pt(en)]2ZL, which was significantly increased compared to the SA-β-gal-positive rate of 2.08 % without treatment of [Pt(en)]2ZL (Fig. 4b). [Pt(en)]2ZL facilitates microtubule polymerization in SGC7901 cells To gain insight into the mechanism of mitosis of [Pt(en)]2ZL, its effect on the microtubule polymerization was also examined by immunofluorescence assay. As shown in Fig. 4c, [Pt(en)]2ZL can induce the microtubule assembly in a dose-dependent manner. [Pt(en)]2ZL altered the microtubule network with rare characteristic spindle poles and formation of thick microtubule bundles. Compared to the treated groups, the untreated cells
Tumor Biol.
Fig. 3 Inhibition effect of [Pt(en)]2ZL on the growth of human gastric cancer cells SGC7901. a Morphological changes of SGC7901 induced by [Pt(en)]2ZL at the indicated concentrations for 48 h (magnification ×200). b Quantitative analysis of the cell viability of SGC7901 treated by
[Pt(en)]2ZL at different concentrations for 24 and 48 h. c Cell colonies of SGC7901 affected by [Pt(en)]2ZL at the indicated concentration for 7 days. d Quantitative analysis of the SGC7901 cell colonies after incubated with [Pt(en)]2ZL for 7 days (p < 0.05)
exhibited a normal network of unaligned microtubule. In general, [Pt(en)]2ZL enhanced microtubule polymerization and induced the bundling of microtubule, which further demonstrated the anti-proliferative activity of [Pt(en)]2ZL.
untreated SGC7901 cells were homogenously stained with a faint blue fluorescence and maintained regular form. After treatment with [Pt(en)]2ZL for 48 h, there was an increase in the intensity of blue fluorescence indicating nuclear shrinkage or condensation, which is one sign of the early apoptosis, and pink fluorescence emitted indicating the occurrence of late apoptosis. Moreover, the SGC7901 cells stained by single Hoechst 33342 exhibited the formation of typical apoptotic makers such as crescents and apoptotic bodies, which were stained with intense and bright blue (Fig. S2). This further demonstrated that [Pt(en)]2ZL can significantly induce the apoptosis of SGC7901 cells. TUNEL assay with fluorescent microscopy was carried out to determine the effect of [Pt(en)]2ZL on the DNA fragmentation of SGC7901 cells. According to the theory of apoptosis detection by TUNEL assay, TUNELpositive cells with green fluorescence indicated typical apoptosis. After treated by 30 μM [Pt(en)]2ZL, a significant increase in the number of TUNEL-positive cells was observed. Cells treated with DNase I were used as the positive control (Fig. 5b). To quantify the apoptotic death of SGC7901 cells induced by [Pt(en)]2ZL, Annexin V-FITC and PI staining were carried out for FCM analysis. The early apoptosis cells were determined by Annexin V-FITC-positive and PI-negative cells (Annexin V-FITC+/PI−), while the late apoptosis cells were determined by Annexin V-FITC+/PI+. As shown in Fig. 5c, d, SGC7901 cells underwent early and late apoptosis in a dose-dependent manner after treated by the indicated concentration of [Pt(en)]2ZL for 48 h. After treatment with 30 μM [Pt(en)]2ZL for 48 h, the early and late apoptosis rates
[Pt(en)]2ZL induces G1/S phase arrest in SGC7901 cells To examine whether [Pt(en)]2ZL-induced growth inhibition was associated with the regulation of cell cycle, the cell cycle distribution in the presence of [Pt(en)]2ZL was analyzed by flow cytometry. Compared to the control group, the SGC7901 cells treated by [Pt(en)]2ZL exhibited a longer G1/S phase with a concomitantly shorter G2/M phase in the cycle distribution in a dose-dependent manner (Fig. 4d). In particular, treatment of cells with 30 μM [Pt(en)]2ZL resulted in a substantial accumulation of cells in the G0/G1 phase (from 25.92 to 41.03 %) and a similar accumulation in the S phase fraction (from 52.76 to 58.96 %). These results suggested that [Pt(en)]2ZL inhibited SGC7901 cell proliferation through arrest of cell cycle progression at the G1/S phase. [Pt(en)]2ZL induces apoptosis in SGC7901 cells To further examine whether [Pt(en)]2ZL restrained cell viability through the induction of apoptosis, the SGC7901 cells were stained with Hoechst 33342, PI, and TUNEL-FITC. As shown in Figs. 5a and S2, the cells underwent remarkable nuclear morphological changes after treatment with [Pt(en)]2ZL, indicating the occurrence of apoptosis as previous reports [20]. The
Tumor Biol.
Fig. 4 [Pt(en)]2ZL induced growth arrest of SGC7901 cells by inhibiting cell division progress. a [Pt(en)]2ZL treatment increased SA-β-galpositive cells in SGC7901 in a dose-dependent manner after culture for 7 days (magnification ×200). b Quantitative analysis of the SA-β-galpositive cells in SGC7901 affected by [Pt(en)]2ZL in a dose-dependent manner (p < 0.05). c The effect of [Pt(en)] 2 ZL on microtubule morphology of SGC7901 cells observed by immunofluorescence
microscopy. Untreated cells exhibited a normal network of unaligned microtubule; [Pt(en)]2ZL altered the microtubule network with rare characteristic spindle poles and formation of thick microtubule bundles (magnification ×200). d After exposure to the indicated concentration of [Pt(en)]2ZL for 24 h, SGC7901 cells exhibit longer G0/G1 and S phases with a concomitantly shorter G2/M phase in a dose-dependent manner
increased to 20.5 and 25.3 % in SGC7901 cells, respectively. Conversely, the percentage of viable cells reduced to 50.6 % (Fig. 5c). These results further indicated that [Pt(en)]2ZL can stimulate the progress of apoptosis in SGC7901 cells.
dose-dependent manner. [Pt(en)]2ZL also upregulated the expressions of cyclin E and CDK2, although it showed no remarkable difference between different dosages of [Pt(en)]2ZL-treated groups. Consistently, these results suggested that [Pt(en)]2ZL can induce the cell cycle arrest at the G1/S phase.
[Pt(en)]2ZL regulates the expressions of cell cycle regulatory proteins in SGC7901 cells To further determine the effect of [Pt(en)]2ZL on the regulation of cell cycle, several cell cycle regulatory proteins were evaluated using Western blot analysis. SGC7901 cells were treated with the indicated concentrations of [Pt(en)]2ZL for 48 h. As illustrated in Fig. 6, [Pt(en)]2ZL upregulated the expression of p21 and p53, while it downregulated the protein level of cyclin D1 in a
[Pt(en)]2ZL regulates the expressions of apoptosis-related proteins in SGC7901 cells In order to investigate the mechanism how [Pt(en)]2ZL induced apoptosis of SGC7901 cells, changes in the level of apoptosis-related proteins were examined by Western blot analysis (Fig. 7). The above study showed that [Pt(en)]2ZL upregulated the expressions of p21 and p53 after 48 h
Tumor Biol.
Fig. 5 [Pt(en)]2ZL-induced apoptosis of SGC7901 cells. a Apoptosis assayed by Hoechst 33342-PI staining (magnification ×200). b Apoptosis assayed by TUNEL-FITC staining (magnification ×200). c Statistical analysis on the percentage of cells in the Annexin V/PI staining (p < 0.05). d Apoptosis of SGC7901 cells evaluated by Annexin V/PI
flow cytometric analysis, with the viable cells (Q4, Annexin V−, and PI−), early apoptosis cells (Q3, Annexin V+, and PI−), late apoptosis cells (Q2, Annexin V+, and PI+), and necrosis cells (Q1, Annexin V−, and PI+) shown in each quadrant
treatment in a dose-dependent manner. To further discover the mechanism involved in propagating and executing apoptosis of SGC7901 cells, the effect of [Pt(en)]2ZL on the Bcl-2 family proteins was also examined. After 48 h treatment with the indicated concentration of [Pt(en)]2ZL, the expression of Bcl2 and the ratio of Bcl-2/Bax were downregulated (Fig. S3). On the other hand, the expression of Bax was promoted, which is known to be a heterodimer with Bcl-2. These alterations of Bcl-2 family proteins caused by [Pt(en)]2ZL would result in an obvious shift from survival to apoptosis of SGC7901 cells.
Since p53, p21, and Bcl-2 family proteins were significantly altered after treatment with [Pt(en)]2ZL for 48 h, their downstream proteins were focused for further study. As expected, [Pt(en)]2ZL suppressed the expression of caspase-3 (procaspase 3) and survivin. Furthermore, the PARP expression was also reduced and the cleaved PARP expression was elevated, which occurs at the downstream of caspase-3 cleavage (Fig. 7a). Therefore, [Pt(en)]2ZL induced apoptosis of the SGC7901 cancer cells mainly through the mitochondriadependent apoptotic pathway.
Tumor Biol.
Fig. 6 [Pt(en)]2ZL changed the expression of cell cycle-related proteins in SGC7901 cells. a Changes of the expression of upstream proteins associated with the cell cycle regulation in SGC7901 cells induced by
[Pt(en)]2ZL at the indicated concentration. b Quantitative analysis of the relative expression level of each cell cycle-related protein affected by [Pt(en)]2ZL at the indicated concentration (p < 0.05)
Discussion
Mitotic slippage usually is accompanied by cell cycle arrest and apoptosis [29]. Cells undergoing mitotic slippage may eventually die at the G1 phase, and they may also lead to a condition known as senescence during this cellular process [30]. It is widely recognized that senescent cells have many special features, such as limit of proliferative capacity, changes of cell shape, and increase of SA-β-gal activity [31]. Similar to the previous reports, [Pt(en)] 2 ZL induced senescence-like growth arrest in SGC7901 cells in response to DNA damage via SA-β-gal staining test (Fig. 4a). More precisely, senescent cell will lose the reactivity to mitogen and the ability to synthesize DNA in the maintenance of metabolic activity, and cell cycle was arrested at the G1 phase [8, 32]. In addition, cell senescence can accompany with abnormal microtubule assembly [29]. Microtubules are crucial components of mitotic spindle in the process of mitosis, which polymerize or depolymerize in a dynamic balance during cell cycle [33]. In the present study, the immunofluorescence analysis using the antibodies specific to α-tubulin proved that [Pt(en)]2ZL treatment induced abnormal microtubule network arrangement and organization in the cytoplasm of SGC7901 cells (Fig. 4c). This result suggests that the disorder of mitosis may induce the inhibition of proliferation and cell cycle arrest in SGC7901 cells [34]. In addition, these experimental
The present study evaluated the anti-tumor effect of a novel dinuclear platinum complex, [Pt(en)]2ZL. It was demonstrated that [Pt(en)]2ZL showed significant inhibitory effect on the growth of various cancer cell lines, especially on SGC7901 cells. [Pt(en)]2ZL can significantly inhibit the proliferation and growth of SGC7901 cells in the time- and dosedependent manners. The ability of [Pt(en)]2ZL to inhibit 50 % growth of SGC7901 cells is as low as 25.15 ± 0.75 μM after 48 h of treatment (Fig. 2a). Besides, its inhibitory effect is selective in terms of its higher IC50 value for inhibiting the human normal gastric cell lines GES-1 (Fig. 2b). Cell colony formation assay was then performed to see the anti-proliferative effect of [Pt(en)]2ZL with low concentrations on long-term survival of SGC7901 cells. Through this assay, one can observe that the anti-proliferative effect on the single cell with the complex [Pt(en)]2ZL treatment has no interference of various cell-cell interaction effects, such as paracrine effect. Notably, low concentrations (<5 μM) of [Pt(en)]2ZL strongly suppressed the ability of SGC7901 cells to form cell colonies in a dose-dependent manner, which clearly demonstrated that [Pt(en)]2ZL was a potent antiproliferative compound (Fig. 3c). Fig. 7 [Pt(en)]2ZL changed the expression of apoptotic proteins in SGC7901 cells. a Changes of the expression of upstream proteins associated with the apoptosis of SGC7901 cells induced by [Pt(en)]2ZL at the indicated concentration. b Quantitative analysis of the relative expression level of each apoptotic protein affected by [Pt(en)]2ZL at the indicated concentration (p < 0.05)
Tumor Biol.
phenomena implied that [Pt(en)]2ZL may be a novel potential microtubule-polymerizing agent [29, 32]. The cell cycle is a sequential event, which is divided into G0/G1, S, and M phases [17]. DNA is synthesized during the G1/S phase. Many anti-cancer compounds have been reported to arrest the cell cycle at different cell cycle phase and then induce apoptotic cell death [21, 35]. [Pt(en)]2ZL induced cell cycle arrest at G1/S phase after 24-h treatment as measured by flow cytometry (Fig. 4d). The results of cell cycle arrest from this study are similar to that observed in one previous report about liriodenine treatment in human colon cancer cells, resulting from changes in the expressions of p53-p21-cyclin D branch [36]. Our present study indicates that [Pt(en)]2ZL enhanced p21 protein expression in a p53-dependent manner, leading to a downregulation of the G1-S checkpoint cyclin D. For instance, the relationship between the level of cyclin ECDK2 complex and the transition from G1 to S is still unclear, and the inhibition effect of [Pt(en)]2ZL on the formation of this complex may contribute to the G1/S phase arrest, which was also observed in the research of Vijay R et al. about the anticancer effect of another dinuclear platinum compound, BBR3610-DACH, on colorectal cell HCT116 [37]. These studies also provided the first indication that [Pt(en)]2ZL can inhibit cancer cell growth possibly through interference with mitosis. In addition, it was observed that [Pt(en)]2ZL can significantly induce the senescence and microtubule polymerization of SGC7901 cells. These phenomena may be related with the cell cycle arrest and the changes in its checkpoints [6, 38]. When drugs induce DNA damage, the cell cycle arrest will allow stress-triggered mechanisms to repair it. Once this procedure Fig. 8 Proposed mechanism for [Pt(en)]2ZL induced the antiproliferative and cytotoxic activity through cell cycle arrest and apoptosis in SGC7901 cells
fails, cells will enter into the apoptotic process [32, 39]. In this case, the experimental data showed that [Pt(en)]2ZL could remarkably induce apoptosis of SGC7901 cells. The fluorescence staining and the flow cytometry of Annexin V-FITC/PI staining further demonstrated that [Pt(en)]2ZL can induce the apoptosis of SGC7901 in a dose-dependent manner (Fig. 5). Changes in the treated cells were similar to those described by previous researches, which can be seen via the formation of several apoptotic bodies [40]. Moreover, induction of apoptosis by [Pt(en)]2ZL through the intrinsic pathway was also seen in the activation of mitochondria-mediated apoptosis in this case. Once this pathway has been activated, several factors will be involved in the alteration of downstream apoptotic signaling [41]. It is well known that mitochondria play a critical role in the regulation of apoptosis. In particular, the increased ratio of pro-apoptotic protein Bax/anti-apoptotic protein Bcl-2, followed by the activation of tumor suppressor gene p53, can lead to the collapse of mitochondrial transmembrane potential, which implies the dysfunction of the mitochondria and triggers the cell death program. Additionally, survivin also participates in this mitochondrial apoptotic pathway. In this study, it was demonstrated that the treatment of SGC7901 cells with [Pt(en)]2ZL can result in the increase of p53 and Bax but decrease of Bcl-2. These results suggested that [Pt(en)]2ZL may induce apoptosis through the mitochondrion-related mechanisms [42]. So far, there are abundant examples reported in the literature that caspase family exerts pivotal effects on the mitochondrial-mediated apoptotic pathway [43]. In order to understand the cause of the anti-tumor effect of [Pt(en)]2ZL on SGC7901 cells, the level
Tumor Biol.
of caspase-3 was examined and then found that [Pt(en)]2ZL could notably increase caspase-3 activity but decrease procaspase 3. Besides, it has been reported that nitrogenbisphosphonates can induce apoptosis via inducing the expression of cleaved PARP [44, 45]. Likewise, in our study, the decreased PARP levels and increased cleaved-PARP levels in SGC7901 cells were observed after 48-h treatment with [Pt(en)]2ZL, which occurred at the downstream of caspase-3 cleavage. All these results provided the evidence that [Pt(en)]2ZL exerted its apoptotic activity via altering the caspase-3 and PARP activity in the mitochondrial pathway. There are several reports suggesting that the crucial effector survivin is a multifunctional protein that regulates mitotic progression, enhances proliferation, inhibits apoptosis, and promotes angiogenesis [46]. The overexpression of survivin in tumors might play a critical role in tumorigenesis and metastasis. Recently, Hsiao C et al. found that the expression of survivin significantly downregulated, which was accompanied with the activation of other apoptotic proteins [21]. Furthermore, survivin inhibits apoptosis primarily through targeting the terminal effector caspases in the apoptotic pathway [47]. Our findings illustrated that the apoptosis of gastric cancer cell SGC7901 induced by [Pt(en)]2ZL was associated with the downregulation of the expression of survivin. In conclusion, our studies demonstrated that [Pt(en)]2ZL possessed an anti-proliferative and cytotoxic activity through arrest of cell cycle and induction of apoptosis in human gastric cancer cell line SGC7901 (Fig. 8). The arrest of G1/S phase was found to be mediated by the alteration in p53-p21-cyclin D branch. Moreover, the apoptotic mechanism may be related with the activation of caspase-3 and the cleavage of PARP. Taken together, the novel compound [Pt(en)]2ZL is a promising anti-cancer drug for treating human gastric cancer.
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19. Acknowledgments The authors are grateful to the financial support from National Natural Science Foundation of China (21371082 and 21501074), Natural Science Foundation of Jiangsu Province (BK20141102 and BK20151118), and Key Medical Talent Project of Jiangsu Province (RC2011097).
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