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Avicularin reversed multidrug-resistance in human gastric cancer through enhancing Bax and BOK expressions
Biomedicine & Pharmacotherapy 103 (2018) 67–74
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Avicularin reversed multidrug-resistance in human gastric cancer through enhancing Bax and BOK expressions Xiang-Feng Guoa, Ji-Peng Liua, Si-Quan Mab, Peng Zhangc, Wen-De Suna,
T
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a
Department of General Surgery, Shanxian Center Hospital, Heze, China Department of General surgery, Heze Second People’s Hospital, Heze, China c Department of General Surgery, Qilu Hospital, Shandong University, Jinan, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: 5-Fu and DDP Gastric cancer Avicularin Apoptosis Bax and BOK
5-Fluorouracil (5-Fu) and cisplatin (DDP) as important therapies in treatment of human gastric cancer have been widely determined. However, the therapeutic effects are usually hampered due to drug resistance or toxicity at high concentrations for application. Avicularin (AL, quercetin-3-α-L-arabinofuranoside), a bio-active flavonol isolated from a number of plants, has been reported to display diverse pharmacological properties. In this study, we explored the hypothesis by which AL reversed 5-Fu or DDP resistance in gastric cancer and the underlying molecular mechanism. Here, in vitro, the drug-resistant cancer cells were incubated to AL or DDP alone or the combination of AL and DDP. Then, MTT, colony formation, Hoechst 33258, flow cytometry and western blot analysis were used to investigate the effects of AL in the regulation of drug-resistance gastric cancer cells. The results indicated that AL treatment markedly re-sensitizes the drug resistant cells (SGC-7901/5-Fu and SGC7901/DDP) to cytotoxicity of 5-Fu or DDP. Molecular mechanism analysis indicated that AL and DDP combination treatment enhanced apoptosis in SGC-7901/DDP cells, accompanied with the up-regulation of cleaved Caspase-3 and PARP, as well as the activation of pro-apoptotic signals, including Bax and BOK. Significantly, down regulation of Bax or BOK expressions using Bax siRNA or BOK siRNA decreased the inhibitory role of DDP in apoptosis of SGC-7901/DDP cells pretreated with AL, demonstrating that AL-reversed DDP resistance was associated with Bax and BOK expression. In vivo, AL and DDP combination significantly reduced gastric tumor growth. Immunohistochemical analysis indicated that co-treatment of AL and DDP significantly induced apoptosis, and reduced tumor cell proliferation in tumor tissue samples. Furthermore, we also found that the Bax, BOK, cleaved Caspase-3 and PARP expression in tumor tissues were highly induced by AL and DDP co-treatment. Together, our findings may provide a novel combination therapeutic strategy in treatment of human gastric cancer.
1. Introduction Gastric cancer is reported as the fourth most commonly diagnosed cancer and is the second most common cause of cancer-associated death in the world [1]. Accordingly, gastric cancer is a heterogeneous disease with various molecular and histological subtypes [2]. Although advances in chemotherapy and surgical approaches, as well as its declining incidence were widely detected, the gastric cancer is still a major global public health problem [3]. Accumulating evidences regarding molecular mechanism of 5-Fu in inhibition of cancer has resulted in the development and progression of therapeutic strategies. Despite these improvements, drug resistance still remains a significant
limitation in the clinical application of 5-Fu [4,5]. In addition, cisplatin (DDP) is a commonly used drug for cancer treatment through crosslinking DNA, resulting in apoptosis of tumor cells [6]. However, resistance to DDP treatment often occurs, which contributes to relapse [7,8]. Thus, it is necessary for the development of more effective therapeutic strategies that could overcome chemoresistance. Many dietary flavonoids exist as glycosides in fruits and vegetables, and they are considered as bioactive food components potentially responsible for various health benefits. Avicularin (AL, Fig. 1A), quercetin-3-α-L-arabinofuranoside, is a glycoside of quercetin, possessing a variety of biological properties, including anti-oxidant, anti-allergic, anti-inflammatory, hepatoprotective, and even anti-tumor activities
Abbreviation: 5-Fu, 5-fluorouracil; AL, avicularin; Bax, B-cell lymphoma 2 associated X; BCA, bicinchoninic acid; BOK, BCL-2-related ovarian killer; DDP, cisplatin; H&E, hematoxylin and eosin; Mcl-1, myeloid cell leukemia 1; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; PARP, poly (ADP-ribose) polymerase; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling ⁎ Corresponding author at: Department of General Surgery, Shanxian Center Hospital, Heze 274300, China. E-mail address: [email protected] (W.-D. Sun). https://doi.org/10.1016/j.biopha.2018.03.110 Received 11 February 2018; Received in revised form 12 March 2018; Accepted 12 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. Avicularin sensitizes SGC-7901/5-Fu and SGC-7901/DDP cells to 5-Fu- and DDP-induced cytotoxicity. (A) The chemical structure of aviculairn (AL). (B) Left, SGC-7901 and SGC-7901/5-Fu cells were treated with 5-Fu at the indicated concentrations for 48 h, followed by MTT analysis. Right, SGC-7901/5-Fu cells were treated with AL for 12 h, and then subjected to the described concentrations of 5-Fu for 36 h, followed by MTT analysis. *P < 0.05, **P < 0.01 and ***P < 0.01 vs Con group without any treatments; +P < 0.05, and ++P < 0.01 vs each corresponding 5-Fu-treated group. (C) Left, SGC-7901 and SGC-7901/DDP cells were treated with DDP at the indicated doses for 48 h, followed by MTT analysis. Right, SGC-7901/DDP cells were treated with AL for 12 h, and then administered with the described concentrations of 5-Fu for 36 h, followed by MTT analysis. *P < 0.05, **P < 0.01 and ***P < 0.01 vs Con group without any treatments; +P < 0.05, and ++ P < 0.01 vs each corresponding DDP-treated group. SGC-7901/5-Fu and SGC-7901/DDP were pre-treated with 10 μM AL for 12 h, followed by 5-Fu (10 μM) or DDP (10 μM) treatment for another 36 h. (D and E) Then, the morphology of cells was captured. (F and G) Colony formation analysis of gastric cancer cells treated as indicated. Data were shown as mean ± S.E.M. And n = 6 in each group. +++P < 0.001 vs 5-Fu or DDP alone group.
cancer that is refractory to standard chemotherapy.
[9–11]. As reported, AL showed significant ability to attenuate type 2 diabetes process [12]. The biological activities of quercetin and aglycone of avicularin, have been also well explored [13]. However, the biological properties of AL were not fully understood. Here, in our study, we attempted to investigate the role of AL in regulating gastric cancer development. In this study, we explored the effects of AL, combined with 5-Fu or DDP treatment on different regulatory parameters, and the characterization of molecular mechanisms in drug-resistant gastric cancer cell line in vitro and in vivo. Avicularin sensitized drug-resistant SGC-7901 cells to 5-Fu- and DDP-induced cytotoxicity and apoptosis. Co-treatment of AL and DDP significantly increased Caspase-3 and PARP expressions. Meanwhile, pro-apoptotic signals, including Bax and BOK, were sharply expressed in AL and DDP co-treated cells. Of note, knockdown of Bax or BOK proteins eliminated the effects of AL and DDP combination-induced cell death. In vivo, AL and DDP co-treatment reduced the tumor growth without toxicity. The combination regimen held promise as a potential therapeutic strategy for patients with gastric
2. Materials and methods 2.1. Cells and culture The cell lines SGC-7901, and its DDP-resistant cell lines, SGC-7901/ DDP, were purchased from Bioleaf Biotech (Shanghai, China). The 5-Furesistant cell line, SGC-7901/5-Fu, was obtained from Cellbio (Shanghai, China). The gastric cancer cell lines were cultured in RPMI 1640 medium containing penicillin (100 U/mL), streptomycin (100 μg/ mL) and 10% (v/v) fetal bovine serum (Gibco Life Technologies, USA) in a humidified atmosphere of 5% CO2 at 37 °C. Cells were treated with different concentrations of AL, 5-Fu or DDP [14–17]. And the cells were also transfected with Bax si-RNA, BOK siRNA or NC si-RNA (Genescript Co., Ltd, China) using lipofectermin 3000 (Life Technology, USA). The cells were allowed to grow for 24 h for the following experiments. 5-Fu and DDP were purchased from Sigma (USA). 68
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Fig. 2. Avicularin sensitizes SGC-7901/DDP cells to DDP-induced apoptosis. SGC-7901/DDP cells were pre-treated with 10 μM AL for 12 h, followed by 36 h treatment of 10 μM DDP, and the following studies were performed. (A) Hoechst 33258 staining of SGC7901/DDP cells. (B) Flow cytometry analysis of apoptosis. (C) Western blot analysis of cleaved Caspase-9, Caspase-3 and PARP. (D) RT-qPCR analysis of Bax, BOK and Bad. (E) Western blot analysis of Bcl-2, Bax, BOK and Bad. Data were shown as mean ± S.E.M. And n = 6 in each group. +++P < 0.001 vs DDP alone group.
or the two in combination. Cells were then stained with Annexin V-FITC and PI (KeyGen Biotech, China) following the manufacturer’s protocol. The Annexin V-FITC and PI fluorescence was examined with a flow cytometer (BD Accuri™ Flow Cytometer, USA). The cells in early stages of apoptosis were Annexin V positive and PI negative, and the cells in the late stages of apoptosis were both Annexin V and PI positive. The apoptotic cell percentage was analyzed using Flow Jo 7.6 software (TreeStar Inc, USA).
2.2. Colony formation analysis All cells (3 × 103 cells/well) were planted at in 6-well plates. After 14 days, colonies could be observed. The colonies were fixed with 4% paraformaldehyde for 15 min and stained with crystal violet for 15 min at ambient temperature. After washing with PBS, the colonies were viewed and counted under a microscope. Only clearly visible colonies (diameter > 50 μm) were counted. 2.3. Cell viability analysis
2.6. Western blot analysis
MTT solution (300 μL/well, KeyGen Biotech, China) was added to cells after various treatments. Following incubation for another 4 h at 37 °C, the supernatants were removed and DMSO (200 μl, KeyGen Biotech) was added into each well to dissolve the formazan crystals. Then, the 96-well plates were placed in a microplate reader (Bio-Tek, USA) to assess the absorbance at 490 nm.
Cells and tumor tissues were harvested and washed with chilled PBS and harvested in sample buffer (150 mM NaCl, 100 mM NaF, 50 mM Tris-HCl (pH 7.6), 0.5% Nonidet P-40 (NP-40) and 1 mM PMSF). And the tumors were homogenized into 10% (wt/vol) hypotonic buffer (pH 8.0, 1 mM EDTA, 5 μg/ml leupeptin, 25 mM Tris-HCl, 1 mM Pefabloc SC, 5 μg/ml soybean trypsin inhibitor, 50 μg/ml aprotinin, 4 mM benzamidine) to yield a homogenate. Then, the final supernatants from cells and tumors were obtained by centrifugation at 14,000×g for 20 min at 4 °C. Protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, San Diego, CA, USA) with bovine serum albumin as a standard. Sample-loading buffer was added, the mixture was boiled for 5 min. And the total protein extract is used for Western blot analysis. 40 μg of total protein was loaded and proteins were separated using 10% SDS-PAGE and electrophoretically transferred to the polyvinylidene difluoride membranes (Millipore, USA). The membranes were then blocked with 5% skim milk Tris buffered saline with 0.1% Tween 20 (TBST), washed, and then incubated with primary antibodies (1:1000 dilution): anti-Caspase-3 antibody, anti-
2.4. Hoechst 33258 staining After various treatments as described, cells were washed with PBS, fixed with 4% paraformaldehyde and stained with 10 μg/ml Hoechst 33258 in PBS (KeyGen Biotech) for 10 min. The cells were then scanned using inverted fluorescent microscope and te representative images were captured. 2.5. Flow cytometry analysis SGC-7901/DDP cells (2.5 × 105/well) were treated with AL, DDP, 69
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Fig. 3. Avicularin enhances DDP sensitivity dependent on Bax and BOK. (A) SGC-7901/DDP cells were incubated with the indicated concentrations of AL for 24 h, followed by western blot analysis of Bax and BOK. *P < 0.05, **P < 0.01 and ***P < 0.01. (B) Left, SGC-7901/DDP cells were transfected with Bax siRNA for 24 h, followed by AL administration for 12 h, and then the cells were subjected to DDP for another 36 h. Bax expressions were measured using western blot analysis. Right, SGC-7901/DDP cells were transfected with siBax for 24 h, and then were incubated with AL (10 μM) for 12 h, followed by DDP treatment for 36 h. MTT analysis was used for cell viability analysis. (C) Left, SGC-7901/DDP cells were transfected with BOK siRNA for 24 h, followed by AL (10 μM) administration for 12 h, and then the cells were subjected to DDP (10 μM) for another 36 h. Bax expressions were measured by western blot analysis. Right, SGC-7901/DDP cells were transfected with siBax for 24 h, and then were incubated with 10 μM AL for 12 h, followed by DDP treatment for 36 h. MTT analysis was used for cell viability analysis. *P < 0.05, **P < 0.01 and ***P < 0.01 vs Con group; +P < 0.05, and ++P < 0.01 vs DDP-treated group. (D,E) Cells were transfected with Bax or BOK for 24 h, followed by AL treatment for 12 h and subsequent DDP for 36 h. Then, the flow cytometry analysis was used for apoptosis measurement. Data were shown as mean ± S.E.M. And n = 6 in each group. +++P < 0.001 vs DDP alone group; ns: no significant difference.
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Fig. 4. Avicularin sensitizes SGC-7901/DDP cells to DDP treatment in vivo. (A) The representative images of tumor tissues. (B) Tumor volume was measured every two days. (C) Tumor weight was measured. (D) Body weight of mice were recorded. (E) H&E, TUNEL and KI-67 staining of tumor tissue sections. (F) TUNEL and (G) KI-67-positive levels were quantified following IHC analysis. (H,I) Western blot analysis of Bax, BOK, cleaved Caspase-3 and PARP. (J) H&E staining of major organs (liver, heart and kidney) from mice of Con and AL groups. Data were shown as mean ± S.E.M. And n = 8 in each group. ***P < 0.01 vs Con group.
GTAATGGAATAGCAC-3′ (reverse); BOK: 5′-AGAGGGAGTGATCCTAGGTAGCCA-3′ (forward), 5′-GTTG CAGAGGAATAAGTAC-3′ (reverse); and GAPDH: 5′-TGCGCTGAGGTACTAGCCC (forward), 5′-GGTGAC CTTGGGTCCAATTCGG-3′ (reverse).
Caspase-9 antibody, anti-PARP antibody, anti-Bax antibody, anti-Bcl-2 antibody, anti-Bad antibody, anti-BOK and GAPDH (1:1000 dilution, all from Abcam, USA). Membranes were then incubated with horseradish peroxidase–conjugated secondary antibody (1:5000 dilutions, KeyGen Biotech, China) for 2 h at room temperature. The blots were then incubated with chemiluminescent substrate and exposed to Kodak (Eastman Kodak Company, USA) X-ray film. Every protein expression levels were defined as grey value using ImageJ 1.38 software (National Institutes of Health, USA). GAPDH was used as an internal control.
2.8. Animals and treatments 48, nude male mice (6–8 weeks of age), were purchased from ChinaJapan Union Hospital of Jilin University (Jilin, China). They were fed with free access to pellet food and water in plastic cages at 25 ± 2 °C and kept on a 12 h light/dark cycle. 2 × 106 SGC-7901/DDP cells were subcutaneously injected into the right flank of nude mice. Once the tumor volume reached 50 mm3, treatments were initiated as follows: control (Con) group, PBS (vehicle); AL, aviculairn at 5 mg/kg of body weight once daily, ip; DDP, DDP at 5 mg/kg of body weight every 3 days, ip; AL + DDP, combination of AL (1.25, 2.5 or 5 mg/kg) and DDP [11,12,18–20]. Drugs were administered to mice for 28 days. The body weight of mice was measured daily. The tumor volume was measured daily. The tumor volumes were calculated using the formula V = width2 × length × 0.4. After 28 days, mice were sacrificed, solid tumor, liver, heart and kidney samples were separated. AL (HPLC > 98%) was purchased from Aladdin Chemistry Company (Shanghai,
2.7. RT-qPCR analysis Total RNA was extracted from cells using TRIzol (Invitrogen, USA) following the manufacturer’s instructions. Total RNA was reversetranscribed to cDNA with random primer (Takara Bio, Japan). Gene expression was measured by reverse transcription PCR (RT-PCR) using an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, USA) and SYBR Green PCR Kit (Roche Molecular Bio-chemicals, USA). The Bax, Bad and BOK mRNA expressions were normalized to that of GAPDH Primers (Sangon Biotech, Shanghai, China): Bax: 5′-AGAACCATGGCCAGTTACGG-3′ (forward), 5′-TGAGTAGCT TTCCGCCTGAG-3′ (reverse); Bad: 5′-AAGGTGAAGGTCGGAGTCAAC-3′ (forward), 5′-GCATTGG 71
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3.2. Aviculairn sensitizes SGC-7901/DDP cells to DDP-induced apoptosis
China). All protocols were in accordance with the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of the People’s Republic of China. The animal study was carried out in accordance with the regulations of China-Japan Union Hospital of Jilin University (Jilin, China).
Hoechst 33258 staining and flow cytometry analysis verified that AL pretreatment enhanced DDP-triggered cell apoptosis, which was in a concentration-dependent manner (Fig. 2A and B and Supplementary Fig. S1B). Next, cleaved Caspase-9, Caspase-3 and PARP were abundantly activated in AL and DDP co-treatment group (Fig. 2C). Further, as shown in Supplementary Fig. S1C, we found that AL pretreatment elevated DDP-induced expression of cleaved Caspase-3 and PARP in a dose-dependent manner. RT-qPCR and western blot analysis also suggested that AL and DDP in combination dramatically induced the mRNA and protein expressions of pro-apoptotic signals, including Bax and BOK, while no significant difference was observed in Bad alterations in SGC-7901/DDP cells (Fig. 2D and E). In contrast, Bcl-2 was found to be significantly reduced by AL and DDP in-combinational treatment (Fig. 2E).
2.9. H&E staining Tumor, liver, heart and renal tissue samples isolated from each group of mice as indicated were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm. The histological changes of tissues were assessed by standard hematoxylin and eosin (H&E, Sigma Aldrich, USA) staining. 2.10. TUNEL analysis in tissues The apoptosis of tumor tissues was calculated by using TUNEL assay with the in-situ Apoptosis Detection Kit (KeyGen Biotech) following the manufacturer’s instructions. After deparaffinization and hydration, tumor tissue sections were washed using PBS twice and then incubated with 20 μg/ml proteinase K (Abcam) at 37 °C for 25 min, followed by PBS washes. Then, all sections were incubated with TUNEL mixture. The tumor sections were then counter-stained with DAPI (KeyGen Biotech). Finally, the tissue sections were observed with a confocal microscopy.
3.3. Aviculairn enhances DDP sensitivity by regulation of Bax and BOK pathway The findings above indicated that AL could sensitize the role of DDP in apoptosis induction, which was associated with the elevation of proapoptotic molecules, including Bax and BAK. Therefore, we attempted to explore whether AL-elevated DDP sensitivity was dependent on Bax and BOK. First, we found that AL alone treatment dose-dependently reduced Bax and BOK expressions (Fig. 3A). Next, Bax and BOK expressions were significantly down regulated in SGC-7901/DDP cells using siRNAs. The results of silence were ensured using western blot analysis (Fig. 3B and C). And in the siCon group, AL could elevate the suppressive effects of DDP, while the effect was abolished in Bax siRNA and BOK siRNA groups using MTT analysis. Flow cytometry analysis also suggested that knockdown of Bax or BOK eliminated the inhibitory role of DDP in apoptosis induction in SGC-7901/DDP cells (Fig. 3D and E). Similarly, AL pretreatment dose-dependently enhanced the role of DDP in inducing the protein expressions of Bax and BOK (Supplementary Fig. S1D).
2.11. Immunohistochemical analysis of KI-67 Mouse gastric tumors were sectioned at 4 μm thickness, and stained with KI-67 (Abcam, 1:200) following the manufacturer’s protocols. The tumor sections were analyzed using a microscope. The percentage of KI67-positive cells in each tumor section was quantified by using the HistoQuest software. 2.12. Statistical analysis Data were shown as mean ± S.E.M. Statistical tests were performed using GraphPad Prism Software version 5.0 (GraphPad Sofware Inc., San Diego, CA). For two-group comparisons, a two-tailed unpaired t-test was used. For multiple group comparisons, one-way ANOVA analysis was used. P < 0.05 was considered to be significant.
3.4. Aviculairn sensitizes SGC-7901/DDP cells to DDP treatment in vivo The findings above elucidated that AL combined with DDP could dramatically induced cytotoxicity in DDP-resistant SGC-7901 cells. Here, we confirmed the effects of AL and DDP combination in vivo. DDP or AL alone showed no inhibitory role in DDP-resistant tumor growth, whereas the two in combination markedly reduced the tumor growth (Fig. 4A–C). Significantly, we found that AL-enhanced anti-cancer role of CCP in vivo was in a dose-dependent manner, as evidenced by the reduced tumor volume and tumor weight (Supplementary Fig. S2A and B). DDP-alone treatment resulted in the decrease of body weight; however, AL and DDP in combination exhibited less effect on the body weight change. And AL single treatment showed no side-effects on the body weight of mice, compared to the Con group (Fig. 4D). H&E staining indicated that tumor cell density was significantly reduced by AL and DDP in combination. Consistently, AL and DDP co-treatment dramatically enhanced TUNEL-positive levels in tumor tissue sections, while KI-67 levels were significantly reduced by AL and DDP combination (Fig. 4E–G). Western blot analysis further confirmed that AL and DDP double treatment induced apoptosis, supported by the improved Bax, BOK, cleaved Caspase-3 and PARP expressions (Fig. 4H and I). Of note, AL dose-dependently promoted the effects of DDP on the induction of Bax, BOK, cleaved Caspase-3 and PARP (Supplementary Fig. S2C and D). Finally, H&E staining indicated that there were no significant histological alterations observed in major organs treated with or without AL, indicating the safe use of AL (Fig. 4J).
3. Results 3.1. Aviculairn sensitizes SGC-7901/5-Fu and SGC-7901/DDP cells to 5Fu- and DDP-induced cytotoxicity Fig. 1B and C indicated that compared to SGC-7901 cells, SGC7901/5-Fu and SGC-7901/DDP cells were resistant to 5-Fu or DDP treatment. AL (10 μM) showed no potential side-effects on the cell viability of SGC-7901/5-Fu and SGC-7901/DDP. Of note, the SGC7901/5-Fu and SGC-7901/DDP cells were pretreated with AL for 12 h, followed by the indicated doses of AL for another 36 h, AL significantly sensitized SGC-7901/5-Fu and SGC-7901/DDP cells to 5-Fu or DDPinduced cytotoxicity. The morphology of drug resistant gastric cancer cells exhibited that AL pretreatment enhanced 5-Fu or DDP-induced toxicity in vitro (Fig. 1D and E). Cell proliferation was significantly reduced by AL and 5-Fu or DDP combination detected by colony formation analysis (Fig. 1F and G). In addition, we found that AL could dose-dependently sensitize SGC-7901/DDP cells to DDP-induced reduction of colony formation (Supplementary Fig. S1A). Together, the findings above illustrated that AL dramatically improved 5-Fu or DDP sensitivity in gastric cancer cells. 72
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4. Discussion
cells. As previously reported, BOK is interacted with Mcl-1, an antiapoptotic protein. BOK-induced apoptosis could be suppressed by Mcl-1 [48,49]. Therefore, if AL and DDP combination-induced apoptosis is also linked to anti-apoptotic molecules suppression, further study is still necessary to comprehensively reveal the underlying mechanism. In conclusion, it could be concluded that AL synergized the cytotoxicity of 5-Fu or DDP in gastric cancer cells through inducing apoptosis in vitro and in vivo with few side effects. Additionally, increase of Bax and BOK expressions by AL was, at least partly, a major mechanism for sensitizing drug-resistant gastric cancer cells. The results supplied a rationale for effective combination treatment strategies for patients with drug-resistant tumors.
Gastric cancer is the second leading cause of cancer mortality worldwide [1,21]. And application of chemotherapeutic agents singly or prescription combination was still used as the mainstream treatment [22]. Presently, 5-Fu and DDP are still important chemotherapeutic drugs applied for the treatment of human gastric cancer. However, intrinsic or acquired resistance weakens the benefit of these approaches [6–8]. Thus, it is essential to find effective methods to sensitize tumor cells to chemotherapies. Currently, a variety of natural compounds have been proposed to overcome the drug resistance through synergistic effects [23,24]. In our present study, a novel mechanism in overcoming the drug resistance was explained. Flavonoids have been extensively suggested to have a variety of biological activities, such as anti-oxidant, anti-inflammatory, and anti-tumor actions [25,26]. Avicularin is a plant flavonoid and a quercetin glycoside [9–11]. Here, we illustrated that AL could elevate the sensitivity to 5-Fu or DDP in 5-Fu or DDP resistant gastric cancer cells through inducing apoptosis dependent on Bax and BOK. Apoptosis as a major molecular mechanism by which cell death was induced by chemotherapy drugs was widely determined [27]. Thus, defects in apoptosis could enhance the cancer cell survival and also confer resistance to chemotherapy [28]. Apoptosis is regulated following the activation of caspases, which is a group of aspartate-specific cysteine proteases that could catalyze the addition or removal of specific cysteine or aspartic acid residues from targeting substrates, subsequently activating or suppressing the action of the target substrate [29,30]. Caspase-9 is well known as an initiator of intrinsic apoptosis [31]. Caspase-3 is a major executioner in apoptosis, which is tightly implicated in chemoresistance of certain type of malignancies, such as colon and breast cancer [32]. Loss of Caspase-3 is frequently observed in a number of solid tumors and is related to poor survival of patients [33,34]. Caspase-3 activation promotes its down-streaming signal of PARP, subsequently contributing to cell death [35]. In our study, we found that AL and DDP in combination significantly induced apoptosis, along with the increased Caspase-9, Caspase-3 and PARP cleavage. In addition, Bax is well known as a central cell death regulator. Bax is a major pro-apoptotic member of the B-cell lymphoma 2 (Bcl2) family proteins, regulating apoptosis in both normal and cancer cells [36]. Recently, increase in Bax expressions is linked to a good response to chemotherapies in leukemia [37,38]. In contrast, decrease of Bax levels is tightly associated with a poor prognosis in colon cancer [39]. Moreover, the reduction of Bax plays an essential role in tumorigenesis [40]. There are studies indicated that reduction of Bax was participated in acquisition of resistance to 5-Fu and DDP in cancer [41,42]. And increasing evidences have also suggested that Bax could be considered as a promising target for cancer treatment [43]. However, Bcl-2 is an essential anti-apoptotic signal, contributing to tumor growth [44,45]. In our study, AL could improve Bax expressions, while reduce Bcl-2 levels in DDP-resistant gastric cancer cells. The elevation of Bax leads to apoptosis induced by DDP. Of note, under the condition of Bax suppression, the effects of DDP and AL combination on apoptosis induction were eliminated, contributing to cell survival. The findings indicated that Bax could be served as a therapy target in the combinational treatment for tumor. BOK is also an essential pro-apoptotic signal that facilitate the disruption and release of Cyto c, an apoptogenic factor from the intermembrane space of mitochondria, Caspase-3 activation, and executing the final steps of programmed cell death [46,47]. BOK is frequently eliminated in cancers. In our study, we found that AL combined with DDP treatment significantly increased BOK expressions in DDP-resistant gastric cancer cells. Intriguingly, BOK knockdown significantly abolished AL and DDP combination-induced apoptosis. The elevated cell viability was observed in BOK knockdown cells with AL and DDP combinational treatment. The results indicated that targeting BOK might be also a molecular target of AL in DDP-resistant gastric cancer
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Avicularin reversed multidrug-resistance in human gastric cancer through enhancing Bax and BOK expressions Xiang-Feng Guoa, Ji-Peng Liua, Si-Quan Mab, Peng Zhangc, Wen-De Suna,
T
⁎
a
Department of General Surgery, Shanxian Center Hospital, Heze, China Department of General surgery, Heze Second People’s Hospital, Heze, China c Department of General Surgery, Qilu Hospital, Shandong University, Jinan, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: 5-Fu and DDP Gastric cancer Avicularin Apoptosis Bax and BOK
5-Fluorouracil (5-Fu) and cisplatin (DDP) as important therapies in treatment of human gastric cancer have been widely determined. However, the therapeutic effects are usually hampered due to drug resistance or toxicity at high concentrations for application. Avicularin (AL, quercetin-3-α-L-arabinofuranoside), a bio-active flavonol isolated from a number of plants, has been reported to display diverse pharmacological properties. In this study, we explored the hypothesis by which AL reversed 5-Fu or DDP resistance in gastric cancer and the underlying molecular mechanism. Here, in vitro, the drug-resistant cancer cells were incubated to AL or DDP alone or the combination of AL and DDP. Then, MTT, colony formation, Hoechst 33258, flow cytometry and western blot analysis were used to investigate the effects of AL in the regulation of drug-resistance gastric cancer cells. The results indicated that AL treatment markedly re-sensitizes the drug resistant cells (SGC-7901/5-Fu and SGC7901/DDP) to cytotoxicity of 5-Fu or DDP. Molecular mechanism analysis indicated that AL and DDP combination treatment enhanced apoptosis in SGC-7901/DDP cells, accompanied with the up-regulation of cleaved Caspase-3 and PARP, as well as the activation of pro-apoptotic signals, including Bax and BOK. Significantly, down regulation of Bax or BOK expressions using Bax siRNA or BOK siRNA decreased the inhibitory role of DDP in apoptosis of SGC-7901/DDP cells pretreated with AL, demonstrating that AL-reversed DDP resistance was associated with Bax and BOK expression. In vivo, AL and DDP combination significantly reduced gastric tumor growth. Immunohistochemical analysis indicated that co-treatment of AL and DDP significantly induced apoptosis, and reduced tumor cell proliferation in tumor tissue samples. Furthermore, we also found that the Bax, BOK, cleaved Caspase-3 and PARP expression in tumor tissues were highly induced by AL and DDP co-treatment. Together, our findings may provide a novel combination therapeutic strategy in treatment of human gastric cancer.
1. Introduction Gastric cancer is reported as the fourth most commonly diagnosed cancer and is the second most common cause of cancer-associated death in the world [1]. Accordingly, gastric cancer is a heterogeneous disease with various molecular and histological subtypes [2]. Although advances in chemotherapy and surgical approaches, as well as its declining incidence were widely detected, the gastric cancer is still a major global public health problem [3]. Accumulating evidences regarding molecular mechanism of 5-Fu in inhibition of cancer has resulted in the development and progression of therapeutic strategies. Despite these improvements, drug resistance still remains a significant
limitation in the clinical application of 5-Fu [4,5]. In addition, cisplatin (DDP) is a commonly used drug for cancer treatment through crosslinking DNA, resulting in apoptosis of tumor cells [6]. However, resistance to DDP treatment often occurs, which contributes to relapse [7,8]. Thus, it is necessary for the development of more effective therapeutic strategies that could overcome chemoresistance. Many dietary flavonoids exist as glycosides in fruits and vegetables, and they are considered as bioactive food components potentially responsible for various health benefits. Avicularin (AL, Fig. 1A), quercetin-3-α-L-arabinofuranoside, is a glycoside of quercetin, possessing a variety of biological properties, including anti-oxidant, anti-allergic, anti-inflammatory, hepatoprotective, and even anti-tumor activities
Abbreviation: 5-Fu, 5-fluorouracil; AL, avicularin; Bax, B-cell lymphoma 2 associated X; BCA, bicinchoninic acid; BOK, BCL-2-related ovarian killer; DDP, cisplatin; H&E, hematoxylin and eosin; Mcl-1, myeloid cell leukemia 1; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; PARP, poly (ADP-ribose) polymerase; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling ⁎ Corresponding author at: Department of General Surgery, Shanxian Center Hospital, Heze 274300, China. E-mail address: [email protected] (W.-D. Sun). https://doi.org/10.1016/j.biopha.2018.03.110 Received 11 February 2018; Received in revised form 12 March 2018; Accepted 12 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. Avicularin sensitizes SGC-7901/5-Fu and SGC-7901/DDP cells to 5-Fu- and DDP-induced cytotoxicity. (A) The chemical structure of aviculairn (AL). (B) Left, SGC-7901 and SGC-7901/5-Fu cells were treated with 5-Fu at the indicated concentrations for 48 h, followed by MTT analysis. Right, SGC-7901/5-Fu cells were treated with AL for 12 h, and then subjected to the described concentrations of 5-Fu for 36 h, followed by MTT analysis. *P < 0.05, **P < 0.01 and ***P < 0.01 vs Con group without any treatments; +P < 0.05, and ++P < 0.01 vs each corresponding 5-Fu-treated group. (C) Left, SGC-7901 and SGC-7901/DDP cells were treated with DDP at the indicated doses for 48 h, followed by MTT analysis. Right, SGC-7901/DDP cells were treated with AL for 12 h, and then administered with the described concentrations of 5-Fu for 36 h, followed by MTT analysis. *P < 0.05, **P < 0.01 and ***P < 0.01 vs Con group without any treatments; +P < 0.05, and ++ P < 0.01 vs each corresponding DDP-treated group. SGC-7901/5-Fu and SGC-7901/DDP were pre-treated with 10 μM AL for 12 h, followed by 5-Fu (10 μM) or DDP (10 μM) treatment for another 36 h. (D and E) Then, the morphology of cells was captured. (F and G) Colony formation analysis of gastric cancer cells treated as indicated. Data were shown as mean ± S.E.M. And n = 6 in each group. +++P < 0.001 vs 5-Fu or DDP alone group.
cancer that is refractory to standard chemotherapy.
[9–11]. As reported, AL showed significant ability to attenuate type 2 diabetes process [12]. The biological activities of quercetin and aglycone of avicularin, have been also well explored [13]. However, the biological properties of AL were not fully understood. Here, in our study, we attempted to investigate the role of AL in regulating gastric cancer development. In this study, we explored the effects of AL, combined with 5-Fu or DDP treatment on different regulatory parameters, and the characterization of molecular mechanisms in drug-resistant gastric cancer cell line in vitro and in vivo. Avicularin sensitized drug-resistant SGC-7901 cells to 5-Fu- and DDP-induced cytotoxicity and apoptosis. Co-treatment of AL and DDP significantly increased Caspase-3 and PARP expressions. Meanwhile, pro-apoptotic signals, including Bax and BOK, were sharply expressed in AL and DDP co-treated cells. Of note, knockdown of Bax or BOK proteins eliminated the effects of AL and DDP combination-induced cell death. In vivo, AL and DDP co-treatment reduced the tumor growth without toxicity. The combination regimen held promise as a potential therapeutic strategy for patients with gastric
2. Materials and methods 2.1. Cells and culture The cell lines SGC-7901, and its DDP-resistant cell lines, SGC-7901/ DDP, were purchased from Bioleaf Biotech (Shanghai, China). The 5-Furesistant cell line, SGC-7901/5-Fu, was obtained from Cellbio (Shanghai, China). The gastric cancer cell lines were cultured in RPMI 1640 medium containing penicillin (100 U/mL), streptomycin (100 μg/ mL) and 10% (v/v) fetal bovine serum (Gibco Life Technologies, USA) in a humidified atmosphere of 5% CO2 at 37 °C. Cells were treated with different concentrations of AL, 5-Fu or DDP [14–17]. And the cells were also transfected with Bax si-RNA, BOK siRNA or NC si-RNA (Genescript Co., Ltd, China) using lipofectermin 3000 (Life Technology, USA). The cells were allowed to grow for 24 h for the following experiments. 5-Fu and DDP were purchased from Sigma (USA). 68
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Fig. 2. Avicularin sensitizes SGC-7901/DDP cells to DDP-induced apoptosis. SGC-7901/DDP cells were pre-treated with 10 μM AL for 12 h, followed by 36 h treatment of 10 μM DDP, and the following studies were performed. (A) Hoechst 33258 staining of SGC7901/DDP cells. (B) Flow cytometry analysis of apoptosis. (C) Western blot analysis of cleaved Caspase-9, Caspase-3 and PARP. (D) RT-qPCR analysis of Bax, BOK and Bad. (E) Western blot analysis of Bcl-2, Bax, BOK and Bad. Data were shown as mean ± S.E.M. And n = 6 in each group. +++P < 0.001 vs DDP alone group.
or the two in combination. Cells were then stained with Annexin V-FITC and PI (KeyGen Biotech, China) following the manufacturer’s protocol. The Annexin V-FITC and PI fluorescence was examined with a flow cytometer (BD Accuri™ Flow Cytometer, USA). The cells in early stages of apoptosis were Annexin V positive and PI negative, and the cells in the late stages of apoptosis were both Annexin V and PI positive. The apoptotic cell percentage was analyzed using Flow Jo 7.6 software (TreeStar Inc, USA).
2.2. Colony formation analysis All cells (3 × 103 cells/well) were planted at in 6-well plates. After 14 days, colonies could be observed. The colonies were fixed with 4% paraformaldehyde for 15 min and stained with crystal violet for 15 min at ambient temperature. After washing with PBS, the colonies were viewed and counted under a microscope. Only clearly visible colonies (diameter > 50 μm) were counted. 2.3. Cell viability analysis
2.6. Western blot analysis
MTT solution (300 μL/well, KeyGen Biotech, China) was added to cells after various treatments. Following incubation for another 4 h at 37 °C, the supernatants were removed and DMSO (200 μl, KeyGen Biotech) was added into each well to dissolve the formazan crystals. Then, the 96-well plates were placed in a microplate reader (Bio-Tek, USA) to assess the absorbance at 490 nm.
Cells and tumor tissues were harvested and washed with chilled PBS and harvested in sample buffer (150 mM NaCl, 100 mM NaF, 50 mM Tris-HCl (pH 7.6), 0.5% Nonidet P-40 (NP-40) and 1 mM PMSF). And the tumors were homogenized into 10% (wt/vol) hypotonic buffer (pH 8.0, 1 mM EDTA, 5 μg/ml leupeptin, 25 mM Tris-HCl, 1 mM Pefabloc SC, 5 μg/ml soybean trypsin inhibitor, 50 μg/ml aprotinin, 4 mM benzamidine) to yield a homogenate. Then, the final supernatants from cells and tumors were obtained by centrifugation at 14,000×g for 20 min at 4 °C. Protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, San Diego, CA, USA) with bovine serum albumin as a standard. Sample-loading buffer was added, the mixture was boiled for 5 min. And the total protein extract is used for Western blot analysis. 40 μg of total protein was loaded and proteins were separated using 10% SDS-PAGE and electrophoretically transferred to the polyvinylidene difluoride membranes (Millipore, USA). The membranes were then blocked with 5% skim milk Tris buffered saline with 0.1% Tween 20 (TBST), washed, and then incubated with primary antibodies (1:1000 dilution): anti-Caspase-3 antibody, anti-
2.4. Hoechst 33258 staining After various treatments as described, cells were washed with PBS, fixed with 4% paraformaldehyde and stained with 10 μg/ml Hoechst 33258 in PBS (KeyGen Biotech) for 10 min. The cells were then scanned using inverted fluorescent microscope and te representative images were captured. 2.5. Flow cytometry analysis SGC-7901/DDP cells (2.5 × 105/well) were treated with AL, DDP, 69
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Fig. 3. Avicularin enhances DDP sensitivity dependent on Bax and BOK. (A) SGC-7901/DDP cells were incubated with the indicated concentrations of AL for 24 h, followed by western blot analysis of Bax and BOK. *P < 0.05, **P < 0.01 and ***P < 0.01. (B) Left, SGC-7901/DDP cells were transfected with Bax siRNA for 24 h, followed by AL administration for 12 h, and then the cells were subjected to DDP for another 36 h. Bax expressions were measured using western blot analysis. Right, SGC-7901/DDP cells were transfected with siBax for 24 h, and then were incubated with AL (10 μM) for 12 h, followed by DDP treatment for 36 h. MTT analysis was used for cell viability analysis. (C) Left, SGC-7901/DDP cells were transfected with BOK siRNA for 24 h, followed by AL (10 μM) administration for 12 h, and then the cells were subjected to DDP (10 μM) for another 36 h. Bax expressions were measured by western blot analysis. Right, SGC-7901/DDP cells were transfected with siBax for 24 h, and then were incubated with 10 μM AL for 12 h, followed by DDP treatment for 36 h. MTT analysis was used for cell viability analysis. *P < 0.05, **P < 0.01 and ***P < 0.01 vs Con group; +P < 0.05, and ++P < 0.01 vs DDP-treated group. (D,E) Cells were transfected with Bax or BOK for 24 h, followed by AL treatment for 12 h and subsequent DDP for 36 h. Then, the flow cytometry analysis was used for apoptosis measurement. Data were shown as mean ± S.E.M. And n = 6 in each group. +++P < 0.001 vs DDP alone group; ns: no significant difference.
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Fig. 4. Avicularin sensitizes SGC-7901/DDP cells to DDP treatment in vivo. (A) The representative images of tumor tissues. (B) Tumor volume was measured every two days. (C) Tumor weight was measured. (D) Body weight of mice were recorded. (E) H&E, TUNEL and KI-67 staining of tumor tissue sections. (F) TUNEL and (G) KI-67-positive levels were quantified following IHC analysis. (H,I) Western blot analysis of Bax, BOK, cleaved Caspase-3 and PARP. (J) H&E staining of major organs (liver, heart and kidney) from mice of Con and AL groups. Data were shown as mean ± S.E.M. And n = 8 in each group. ***P < 0.01 vs Con group.
GTAATGGAATAGCAC-3′ (reverse); BOK: 5′-AGAGGGAGTGATCCTAGGTAGCCA-3′ (forward), 5′-GTTG CAGAGGAATAAGTAC-3′ (reverse); and GAPDH: 5′-TGCGCTGAGGTACTAGCCC (forward), 5′-GGTGAC CTTGGGTCCAATTCGG-3′ (reverse).
Caspase-9 antibody, anti-PARP antibody, anti-Bax antibody, anti-Bcl-2 antibody, anti-Bad antibody, anti-BOK and GAPDH (1:1000 dilution, all from Abcam, USA). Membranes were then incubated with horseradish peroxidase–conjugated secondary antibody (1:5000 dilutions, KeyGen Biotech, China) for 2 h at room temperature. The blots were then incubated with chemiluminescent substrate and exposed to Kodak (Eastman Kodak Company, USA) X-ray film. Every protein expression levels were defined as grey value using ImageJ 1.38 software (National Institutes of Health, USA). GAPDH was used as an internal control.
2.8. Animals and treatments 48, nude male mice (6–8 weeks of age), were purchased from ChinaJapan Union Hospital of Jilin University (Jilin, China). They were fed with free access to pellet food and water in plastic cages at 25 ± 2 °C and kept on a 12 h light/dark cycle. 2 × 106 SGC-7901/DDP cells were subcutaneously injected into the right flank of nude mice. Once the tumor volume reached 50 mm3, treatments were initiated as follows: control (Con) group, PBS (vehicle); AL, aviculairn at 5 mg/kg of body weight once daily, ip; DDP, DDP at 5 mg/kg of body weight every 3 days, ip; AL + DDP, combination of AL (1.25, 2.5 or 5 mg/kg) and DDP [11,12,18–20]. Drugs were administered to mice for 28 days. The body weight of mice was measured daily. The tumor volume was measured daily. The tumor volumes were calculated using the formula V = width2 × length × 0.4. After 28 days, mice were sacrificed, solid tumor, liver, heart and kidney samples were separated. AL (HPLC > 98%) was purchased from Aladdin Chemistry Company (Shanghai,
2.7. RT-qPCR analysis Total RNA was extracted from cells using TRIzol (Invitrogen, USA) following the manufacturer’s instructions. Total RNA was reversetranscribed to cDNA with random primer (Takara Bio, Japan). Gene expression was measured by reverse transcription PCR (RT-PCR) using an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, USA) and SYBR Green PCR Kit (Roche Molecular Bio-chemicals, USA). The Bax, Bad and BOK mRNA expressions were normalized to that of GAPDH Primers (Sangon Biotech, Shanghai, China): Bax: 5′-AGAACCATGGCCAGTTACGG-3′ (forward), 5′-TGAGTAGCT TTCCGCCTGAG-3′ (reverse); Bad: 5′-AAGGTGAAGGTCGGAGTCAAC-3′ (forward), 5′-GCATTGG 71
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3.2. Aviculairn sensitizes SGC-7901/DDP cells to DDP-induced apoptosis
China). All protocols were in accordance with the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of the People’s Republic of China. The animal study was carried out in accordance with the regulations of China-Japan Union Hospital of Jilin University (Jilin, China).
Hoechst 33258 staining and flow cytometry analysis verified that AL pretreatment enhanced DDP-triggered cell apoptosis, which was in a concentration-dependent manner (Fig. 2A and B and Supplementary Fig. S1B). Next, cleaved Caspase-9, Caspase-3 and PARP were abundantly activated in AL and DDP co-treatment group (Fig. 2C). Further, as shown in Supplementary Fig. S1C, we found that AL pretreatment elevated DDP-induced expression of cleaved Caspase-3 and PARP in a dose-dependent manner. RT-qPCR and western blot analysis also suggested that AL and DDP in combination dramatically induced the mRNA and protein expressions of pro-apoptotic signals, including Bax and BOK, while no significant difference was observed in Bad alterations in SGC-7901/DDP cells (Fig. 2D and E). In contrast, Bcl-2 was found to be significantly reduced by AL and DDP in-combinational treatment (Fig. 2E).
2.9. H&E staining Tumor, liver, heart and renal tissue samples isolated from each group of mice as indicated were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm. The histological changes of tissues were assessed by standard hematoxylin and eosin (H&E, Sigma Aldrich, USA) staining. 2.10. TUNEL analysis in tissues The apoptosis of tumor tissues was calculated by using TUNEL assay with the in-situ Apoptosis Detection Kit (KeyGen Biotech) following the manufacturer’s instructions. After deparaffinization and hydration, tumor tissue sections were washed using PBS twice and then incubated with 20 μg/ml proteinase K (Abcam) at 37 °C for 25 min, followed by PBS washes. Then, all sections were incubated with TUNEL mixture. The tumor sections were then counter-stained with DAPI (KeyGen Biotech). Finally, the tissue sections were observed with a confocal microscopy.
3.3. Aviculairn enhances DDP sensitivity by regulation of Bax and BOK pathway The findings above indicated that AL could sensitize the role of DDP in apoptosis induction, which was associated with the elevation of proapoptotic molecules, including Bax and BAK. Therefore, we attempted to explore whether AL-elevated DDP sensitivity was dependent on Bax and BOK. First, we found that AL alone treatment dose-dependently reduced Bax and BOK expressions (Fig. 3A). Next, Bax and BOK expressions were significantly down regulated in SGC-7901/DDP cells using siRNAs. The results of silence were ensured using western blot analysis (Fig. 3B and C). And in the siCon group, AL could elevate the suppressive effects of DDP, while the effect was abolished in Bax siRNA and BOK siRNA groups using MTT analysis. Flow cytometry analysis also suggested that knockdown of Bax or BOK eliminated the inhibitory role of DDP in apoptosis induction in SGC-7901/DDP cells (Fig. 3D and E). Similarly, AL pretreatment dose-dependently enhanced the role of DDP in inducing the protein expressions of Bax and BOK (Supplementary Fig. S1D).
2.11. Immunohistochemical analysis of KI-67 Mouse gastric tumors were sectioned at 4 μm thickness, and stained with KI-67 (Abcam, 1:200) following the manufacturer’s protocols. The tumor sections were analyzed using a microscope. The percentage of KI67-positive cells in each tumor section was quantified by using the HistoQuest software. 2.12. Statistical analysis Data were shown as mean ± S.E.M. Statistical tests were performed using GraphPad Prism Software version 5.0 (GraphPad Sofware Inc., San Diego, CA). For two-group comparisons, a two-tailed unpaired t-test was used. For multiple group comparisons, one-way ANOVA analysis was used. P < 0.05 was considered to be significant.
3.4. Aviculairn sensitizes SGC-7901/DDP cells to DDP treatment in vivo The findings above elucidated that AL combined with DDP could dramatically induced cytotoxicity in DDP-resistant SGC-7901 cells. Here, we confirmed the effects of AL and DDP combination in vivo. DDP or AL alone showed no inhibitory role in DDP-resistant tumor growth, whereas the two in combination markedly reduced the tumor growth (Fig. 4A–C). Significantly, we found that AL-enhanced anti-cancer role of CCP in vivo was in a dose-dependent manner, as evidenced by the reduced tumor volume and tumor weight (Supplementary Fig. S2A and B). DDP-alone treatment resulted in the decrease of body weight; however, AL and DDP in combination exhibited less effect on the body weight change. And AL single treatment showed no side-effects on the body weight of mice, compared to the Con group (Fig. 4D). H&E staining indicated that tumor cell density was significantly reduced by AL and DDP in combination. Consistently, AL and DDP co-treatment dramatically enhanced TUNEL-positive levels in tumor tissue sections, while KI-67 levels were significantly reduced by AL and DDP combination (Fig. 4E–G). Western blot analysis further confirmed that AL and DDP double treatment induced apoptosis, supported by the improved Bax, BOK, cleaved Caspase-3 and PARP expressions (Fig. 4H and I). Of note, AL dose-dependently promoted the effects of DDP on the induction of Bax, BOK, cleaved Caspase-3 and PARP (Supplementary Fig. S2C and D). Finally, H&E staining indicated that there were no significant histological alterations observed in major organs treated with or without AL, indicating the safe use of AL (Fig. 4J).
3. Results 3.1. Aviculairn sensitizes SGC-7901/5-Fu and SGC-7901/DDP cells to 5Fu- and DDP-induced cytotoxicity Fig. 1B and C indicated that compared to SGC-7901 cells, SGC7901/5-Fu and SGC-7901/DDP cells were resistant to 5-Fu or DDP treatment. AL (10 μM) showed no potential side-effects on the cell viability of SGC-7901/5-Fu and SGC-7901/DDP. Of note, the SGC7901/5-Fu and SGC-7901/DDP cells were pretreated with AL for 12 h, followed by the indicated doses of AL for another 36 h, AL significantly sensitized SGC-7901/5-Fu and SGC-7901/DDP cells to 5-Fu or DDPinduced cytotoxicity. The morphology of drug resistant gastric cancer cells exhibited that AL pretreatment enhanced 5-Fu or DDP-induced toxicity in vitro (Fig. 1D and E). Cell proliferation was significantly reduced by AL and 5-Fu or DDP combination detected by colony formation analysis (Fig. 1F and G). In addition, we found that AL could dose-dependently sensitize SGC-7901/DDP cells to DDP-induced reduction of colony formation (Supplementary Fig. S1A). Together, the findings above illustrated that AL dramatically improved 5-Fu or DDP sensitivity in gastric cancer cells. 72
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4. Discussion
cells. As previously reported, BOK is interacted with Mcl-1, an antiapoptotic protein. BOK-induced apoptosis could be suppressed by Mcl-1 [48,49]. Therefore, if AL and DDP combination-induced apoptosis is also linked to anti-apoptotic molecules suppression, further study is still necessary to comprehensively reveal the underlying mechanism. In conclusion, it could be concluded that AL synergized the cytotoxicity of 5-Fu or DDP in gastric cancer cells through inducing apoptosis in vitro and in vivo with few side effects. Additionally, increase of Bax and BOK expressions by AL was, at least partly, a major mechanism for sensitizing drug-resistant gastric cancer cells. The results supplied a rationale for effective combination treatment strategies for patients with drug-resistant tumors.
Gastric cancer is the second leading cause of cancer mortality worldwide [1,21]. And application of chemotherapeutic agents singly or prescription combination was still used as the mainstream treatment [22]. Presently, 5-Fu and DDP are still important chemotherapeutic drugs applied for the treatment of human gastric cancer. However, intrinsic or acquired resistance weakens the benefit of these approaches [6–8]. Thus, it is essential to find effective methods to sensitize tumor cells to chemotherapies. Currently, a variety of natural compounds have been proposed to overcome the drug resistance through synergistic effects [23,24]. In our present study, a novel mechanism in overcoming the drug resistance was explained. Flavonoids have been extensively suggested to have a variety of biological activities, such as anti-oxidant, anti-inflammatory, and anti-tumor actions [25,26]. Avicularin is a plant flavonoid and a quercetin glycoside [9–11]. Here, we illustrated that AL could elevate the sensitivity to 5-Fu or DDP in 5-Fu or DDP resistant gastric cancer cells through inducing apoptosis dependent on Bax and BOK. Apoptosis as a major molecular mechanism by which cell death was induced by chemotherapy drugs was widely determined [27]. Thus, defects in apoptosis could enhance the cancer cell survival and also confer resistance to chemotherapy [28]. Apoptosis is regulated following the activation of caspases, which is a group of aspartate-specific cysteine proteases that could catalyze the addition or removal of specific cysteine or aspartic acid residues from targeting substrates, subsequently activating or suppressing the action of the target substrate [29,30]. Caspase-9 is well known as an initiator of intrinsic apoptosis [31]. Caspase-3 is a major executioner in apoptosis, which is tightly implicated in chemoresistance of certain type of malignancies, such as colon and breast cancer [32]. Loss of Caspase-3 is frequently observed in a number of solid tumors and is related to poor survival of patients [33,34]. Caspase-3 activation promotes its down-streaming signal of PARP, subsequently contributing to cell death [35]. In our study, we found that AL and DDP in combination significantly induced apoptosis, along with the increased Caspase-9, Caspase-3 and PARP cleavage. In addition, Bax is well known as a central cell death regulator. Bax is a major pro-apoptotic member of the B-cell lymphoma 2 (Bcl2) family proteins, regulating apoptosis in both normal and cancer cells [36]. Recently, increase in Bax expressions is linked to a good response to chemotherapies in leukemia [37,38]. In contrast, decrease of Bax levels is tightly associated with a poor prognosis in colon cancer [39]. Moreover, the reduction of Bax plays an essential role in tumorigenesis [40]. There are studies indicated that reduction of Bax was participated in acquisition of resistance to 5-Fu and DDP in cancer [41,42]. And increasing evidences have also suggested that Bax could be considered as a promising target for cancer treatment [43]. However, Bcl-2 is an essential anti-apoptotic signal, contributing to tumor growth [44,45]. In our study, AL could improve Bax expressions, while reduce Bcl-2 levels in DDP-resistant gastric cancer cells. The elevation of Bax leads to apoptosis induced by DDP. Of note, under the condition of Bax suppression, the effects of DDP and AL combination on apoptosis induction were eliminated, contributing to cell survival. The findings indicated that Bax could be served as a therapy target in the combinational treatment for tumor. BOK is also an essential pro-apoptotic signal that facilitate the disruption and release of Cyto c, an apoptogenic factor from the intermembrane space of mitochondria, Caspase-3 activation, and executing the final steps of programmed cell death [46,47]. BOK is frequently eliminated in cancers. In our study, we found that AL combined with DDP treatment significantly increased BOK expressions in DDP-resistant gastric cancer cells. Intriguingly, BOK knockdown significantly abolished AL and DDP combination-induced apoptosis. The elevated cell viability was observed in BOK knockdown cells with AL and DDP combinational treatment. The results indicated that targeting BOK might be also a molecular target of AL in DDP-resistant gastric cancer
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