Metastasis suppressor protein 1 regulated by PTEN suppresses invasion,migration, and EMT of gastric carcinoma by inactivating PI3K/AKT signaling

Epithelial-mesenchymal transition (EMT) is a crucial event for cancer progression and metastasis. Metastasis suppressor protein 1 (MTSS1) is a metastasis suppressor in several cancers. In this study, we elucidated the potential physiological function of MTSS1 in the invasion and migration of gastric cancer (GC), and its distinct role in EMT and subsequently determined the potential molecular mechanism. We observed that MTSS1 expression was downregulated in GC tissues and several GC cell lines (SGC-7901, MGC-803, MKN-28, MKN-45, and BGC-823). Importantly, forced expression of MTSS1 drastically diminished the cell viability in both SGC-7901 and MKN-45 cells. Moreover, overexpression of MTSS1 attenuated the invasion ability of these two cell lines. In addition to the invasive capability, introduction of MTSS1 led to a loss of migratory potential. Furthermore, augmentation of MTSS1 exhibited the typical EMT phenotype switch, accompanied by enhanced the expression of vimentin and N-cadherin and reduced E-cadherin expression. Interestingly, MTSS1 also repressed transforming growth factor beta 1 (TGF-β1)-induced EMT. Our mechanistic investigations revealed that MTSS1 was positively regulated by the phosphatase and tensin homolog (PTEN), and it functioned as a tumor suppressor, possibly by inactivating the phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene (AKT) pathway in GC cells. Collectively, our data provide insight into an important role for MTSS1 in suppressing tumor cell invasion, migration and EMT, which indicates that MTSS1 may act as a prospective prognostic biological marker and a promising therapeutic target for GC.     

Raddeanin A induces human gastric cancer cells apoptosis and inhibits their invasion in vitro

Raddeanin A is one of the triterpenoid saponins in herbal medicine Anemone raddeana Regel which was reported to suppress the growth of liver and lung cancer cells. However, little was known about its effect on gastric cancer (GC) cells. This study aimed to investigate its inhibitory effect on three kinds of different differentiation stage GC cells (BGC-823, SGC-7901 and MKN-28) in vitro and the possible mechanisms. Proliferation assay and flow cytometry demonstrated Raddeanin A’s dose-dependent inhibitory effect and determined its induction of cells apoptosis, respectively. Transwell assay, wounding heal assay and cell matrix adhesion assay showed that Raddeanin A significantly inhibited the abilities of the invasion, migration and adhesion of the BGC-823 cells. Moreover, quantitative real time PCR and Western blot analysis found that Raddeanin A increased Bax expression while reduced Bcl-2, Bcl-xL and Survivin expressions and significantly activated caspase-3, caspase-8, caspase-9 and poly-ADP ribose polymerase (PARP). Besides, Raddeanin A could also up-regulate the expression of reversion inducing cysteine rich protein with Kazal motifs (RECK), E-cadherin (E-cad) and down-regulate the expression of matrix metalloproteinases-2 (MMP-2), MMP-9, MMP-14 and Rhoc. In conclusion, Raddeanin A inhibits proliferation of human GC cells, induces their apoptosis and inhibits the abilities of invasion, migration and adhesion, exhibiting potential to become antitumor drug.     

Long noncoding RNA NEAT1-modulated miR-506 regulates gastric cancer development through targeting STAT3

Accumulating evidence has indicated that long noncoding RNA NEAT1 exerts critical roles in cancers. So far, the detailed biological role and mechanisms of NEAT1, which are responsible for human gastric cancer (GC), are still largely unknown. Here, we observed that NEAT1 and STAT3 expressions were significantly upregulated in human GC cells including BGC823, SGC-7901, AGS, MGC803, and MKN28 cells compared with normal gastric epithelial cells GES-1, while miR-506 was downregulated. We inhibited NEAT1 and observed that NEAT1 inhibition was able to repress the growth, migration, and invasion of GC cells. Conversely, overexpression of NEAT1 exhibited an increased ability of GC progression in BGC823 and SGC-7901 cells. Bioinformatics analysis, dual luciferase reporter assays, RIP assays, and RNA pull-down tests validated the negative binding correlation between NEAT1 and miR-506. In addition, it was found that miR-506 can modulate the expression of NEAT1 in vitro. STAT3 was predicted as a messenger RNA (mRNA) target of miR-506, and miR-506 mimics can suppress STAT3 mRNA expression. Subsequently, it was observed that downregulation of NEAT1 can restrain GC development by decreasing STAT3, which can be reversed by miR-506 inhibitors. Therefore, it was hypothesized in our study that NEAT1 can be recognized as a competing endogenous RNA to modulate STAT3 by sponging miR-506 in GC. In conclusion, we implied that NEAT1 can serve as an important biomarker in GC diagnosis and treatment.     

Inhibition of two gastric cancer cell lines induced by fucoxanthin involves downregulation of Mcl-1 and STAT3

Fucoxanthin is a natural carotenoid that had never been previously demonstrated to have anti-tumor effect on human gastric adenocarcinoma SGC-7901 or BGC-823 cells. Here it was found to inhibit proliferation and induce apoptosis through JAK/STAT signal pathway in these cells; the mechanism by which this occurred was investigated. We find that fucoxanthin significantly increased the number of apoptotic cells by propidium iodide (PI) dye staining and flow cytometry. Fucoxanthin (50 or 75 μM) induced SGC-7901 cells cycle arrest at S phase, while BGC-823 cells arrest at G2/M phase. RT-PCR and western blot analysis revealed that the expressions of Mcl-1, STAT3 and p-STAT3 were obviously decreased by fucoxanthin in a dose-dependent manner. Synthetic siRNA targeting Mcl-1 was transfected into cells which had no effect on expressions of STAT3. After pretreatment with AG490 (50 μM) which led to blocking of the JAK/STAT signal pathway, the reductive expressions of Mcl-1, STAT3 and p-STAT3 caused by fucoxanthin were inhibited. This is the first analysis of effects on SGC-7901 and BGC-823 cells by fucoxanthin. Fucoxanthin can induce cell-cycle arrest and apoptosis in these cells. These effects involved downregulation of Mcl-1, STAT3 and p-STAT3. This work is significant for better understanding of mechanisms leading to human gastric adenocarcinoma formation and informing exploitation of anti-tumor marine drug, and for providing Mcl-1 and STAT3 as potential therapeutic targets for gastric adenocarcinoma.     

Retracted: MicroRNA-133a inhibits gastric cancer cells growth,migration, and epithelial-mesenchymal transition process by targeting presenilin 1

Gastric cancer (GC) is one of the most common malignancies and a leading cause of cancer-related death worldwide. Accumulating evidence reported that microRNA (miR)-133a was involved in GC. This study aimed to investigate the function and mechanism of miR-133a in the development and progression of GC. The expression of miR-133a and presenilin 1 (PSEN1) in two GC cell lines, SGC-7901 and BGC-823, were inhibited and overexpressed by transient transfections. Thereafter, cell viability, migration, and apoptosis were measured by trypan blue exclusion assay, transwell migration assay, and flow cytometry assay, respectively. Dual-luciferase reporter assay was conducted to verify whether PSEN1 was a direct target of miR-133a. Furthermore, quantitative real-time polymerase chain reaction and Western blot analysis were mainly performed to assess the expression changes of epithelial-mesenchymal transition (EMT)-associated proteins, apoptosis-related proteins, and Notch pathway proteins. MiR-133a inhibitor significantly increased cell viability and migration, while miR-133a mimic decreased cell viability, migration, and induced apoptosis. miR-133a suppression accelerated transforming growth factor-β1 (TGF-β1)-induce EMT, as evidenced by upregulation of E-cadherin, and downregulation of N-cadherin, vimentin, and Slug. Of contrast, miR-133a overexpression blocked TGF-β1-induce EMT by altering these factors. PSEN1 was a direct target of miR-133a, and suppression of PSEN1 abolished the promoting functions of miR-133 suppression on cell growth and metastasis. Moreover, PSEN1 inhibition decreased Notch 1, Notch 2, and Notch 3 protein expressions. This study demonstrates an antigrowth and antimetastasis role of miR-133a in GC cells. Additionally, miR-133a acts as a tumor suppressor may be via targeting PSEN1.     

Retracted: Suppression of BMX-ARHGAP fusion gene inhibits epithelial-mesenchymal transition in gastric cancer cells via RhoA-mediated blockade of JAK/STAT axis

Gastric cancer (GC) is one of the main causes of cancer-related mortality worldwide. Epithelial-mesenchymal transition (EMT) is an important biological process involving the process by which malignant tumor cells obtain the ability of migration, invasion, resistance of apoptosis, and degradation in the extracellular matrix. The current study aimed at investigating whether bone marrow X kinase Rho GTPase activating protein 12 (BMX-ARHGAP) fusion gene affects GC. First, short hairpin RNA (shRNA) against BMX-ARHGAP or BMX-ARHGAP were introduced to treat SGC-7901 cells with the highest BMX-ARHGAP among the five GC cell lines (SGC-7901, MKN-45, NCI-N87, SNU-5, and AGS). Next, cell vitality, drug resistance, migration, and invasion of SGC-7901 cells, activities of Rho and JAK/STAT axis, as well as EMT and lymph node metastasis (LNM) were evaluated. The survival rate of the mice was then determined through the transfection of the specific pathogen-free NOD-SCID mice with treated SGC-7901 cells. The results showed that BMX-ARHGAP expression was associated with the infiltration degree of GC tumor and poor prognosis for patients with GC. BMX-ARHGAP silencing was found to play an inhibitory role in the Rho and JAK/STAT axis to reduce cell vitality, drug resistance, migration and invasion, reverse EMT process, as well as inhibit LNM. BMX-ARHGAP overexpression was observed to have induced effects on GC cells as opposed to those inhibited by BMX-ARHGAP silencing. The survival rate of mice was increased after transfection with silenced BMX-ARHGAP. These findings provided evidence that the suppression of BMX-ARHGAP resulted in the inhibition of RhoA to restraint the development of GC cells by blocking the JAK/STAT axis.     

转录因子SOX2 对胃癌干细胞自我更新、增殖和迁移的影响

目的:检测SOX2在胃癌组织中的表达,探讨SOX2对胃癌干细胞自我更新、增殖和转移能力的影响。方法:采用免疫组化检测SOX2在胃癌及癌旁组织中的表达情况。通过肿瘤球形成实验富集、分离胃癌干细胞。构建SOX2过表达慢病毒并感染胃癌干细胞中,通过实时定量PCR和western bolt检测感染慢病毒后胃癌干细胞中SOX2表达情况。分别利用肿瘤球形成实验检测SOX2对胃癌肿瘤干细胞自我更新能力的影响,CCK-8实验检测SOX2对胃癌干细胞增殖能力的影响,流式细胞术分析SOX2对胃癌干细胞的细胞周期的影响,Transwell实验检测SOX2对胃癌干细胞转移能力的影响。结果:SOX2在胃癌组织中表达显著低于癌旁组织。肿瘤球形成实验能够有效富集胃癌细胞SGC-7901和BGC-823的干细胞。慢病毒载体感染能够显著增强SOX2在胃癌干细胞中的表达。过表达SOX2能够抑制胃癌干细胞的自我更新、增殖和侵袭能力。结论:SOX2在胃癌中发挥抑癌基因的功能,其机制可能通过抑制肿瘤干细胞的自我更新、增殖和侵袭转移能力而抑制胃癌的发生发展。     

白藜芦醇通过Pi3K/AKT 通路抑制胃癌SGC-7901 细胞增殖和迁移

目的:验证白藜芦醇是否可以抑制胃癌SGC-7901细胞增殖和迁移及其信号通路。方法:用不同浓度白藜芦醇干预SGC-7901细胞,再用LY-294002和IGF-1分别用来抑制和激活Pi3K/AKT通路。MTT法测细胞增殖,划痕试验和Transwell试验测细胞迁移,Western blot检测细胞迁移相关蛋白(MMP-2、MMP-9)、细胞迁移相关蛋白(P21、P27)、以及AKT、p-AKT的表达情况;结果:相比于对照组,白藜芦醇组胃癌细胞增殖和迁移减弱(P=0.001),p-AKT表达减少(P0.001);LY-294002可以抑制p-AKT的表达(P=0.004),和白藜芦醇一样可以抑制胃癌细胞的增殖和迁移;IGF-1可以显著增加p-AKT的表达(P0.001),可以逆转白藜芦醇对胃癌细胞增殖和迁移的抑制作用。结论:白藜芦醇通过抑制Pi3K/AKT信号通路抑制胃癌细胞增殖和迁移。     

Downregulation of microRNA-92b-3p suppresses proliferation,migration, and invasion of gastric cancer SGC-7901 cells by targeting Homeobox D10

To investigate the effect and mechanism of microRNA-92b-3p (miR-92b-3p) targeting Homeobox D10 (HOXD10) on proliferation, migration, and invasion of gastric cancer, we detected t he expression of miR-92b-3p and HOXD10 in SGC-7901 cells. The effects of miR-92b-3p or HOXD10 on proliferation, migration, invasion, and matrix metalloproteinase (MMP)-2/9 expression in SGC-7901 cells were measured by the Cell Counting Kit-8 assay, Transwell assay, and Western blot, respectively. The results showed that miR-92b-3p expression was increased, and HOXD10 expression was decreased in SGC-7901 cells, compared with human normal gastric epithelial cells GES-1. Functional experiments demonstrated that cell proliferation, migration, invasion, and expression of MMP-2/9 in SGC-7901 cells were significantly inhibited by miR-92b-3p silencing and HOXD10 overexpression. Moreover, HOXD10 was a potential target gene of miR-92b-3p as evidenced by the TargetScan software and double luciferase reporter assay. In the rescue experiment, knockdown of HOXD10, accompanied by higher expression of MMP-2/9, could significantly eliminate the inhibitory effects of miR-92b-3p silencing on cell proliferation, migration, and invasion. In conclusion, miR-92b-3p is highly expressed in gastric cancer SGC-7901 cells, and interfering with its expression might inhibit SGC-7901 cell proliferation, migration, and invasion via downregulating MMP-2/9 expression and targeting HOXD10.     

RASAL1 influences the proliferation and invasion of gastric cancer cells by regulating the RAS/ERK signaling pathway

The aim of this study was to investigate the biological characteristics of the RASAL1 gene in a well-differentiated gastric cancer cell line MKN-28 and a poorly differentiated gastric cancer cell line BGC-823 cells, using RNA interference and gene transfection technology, respectively. MKN-28 cells were transfected with the shRNA of RASAL1 and BGC-823 cells were transfected with the pcDNA 3.1 plasmid vector containing RASAL1. RT-PCR and western blotting were then used to detect the expression of RASAL1 mRNA and protein. The activities of RAS and extracellular signal-regulated kinase 1/2 were analyzed by the pull-down method and western blotting. The proliferate capacity, apoptosis rate, invasive and migratory potentials of MKN-28 or BGC-823 cells were also measured by Cell Counting Kit-8 cell proliferation assay, propidium iodide/Annexin V staining coupled with flow cytometry, and transwell chamber assays, respectively. Measurement of RASAL1 mRNA and protein expression in two cells revealed successful transfection of the shRNA of RASAL1 and RASAL1-pcDNA3.1 plasmid into these two cells. Moreover, decreased expression of RASAL1 in MKN-28 cells resulted in increased expression of RAS-GTP and p-ERK1/2. Interestingly, decreased expression of RASAL1 inhibited apoptosis and facilitated cell proliferation, invasion and migration. The increased expression of RASAL1 in BGC-823 cells caused declined expression of RAS-GTP and p-ERK1/2, as well as promoted apoptosis and restrained cell proliferation, invasion and migration. The down-regulation of RASAL1 promoted the proliferation, invasion and migration of gastric cancer MKN-28 cells, and up-regulation of RASAL1 inhibited the proliferation, invasion and migration of BGC-823 gastric cancer cells by regulating the RAS/ERK signaling pathway. Thus, our results suggest that RASAL1 may play an important role as a tumor suppressor gene in gastric cancer.     

Sprouty-2对胃癌细胞上皮间质转化及侵袭转移的影响

目的:研究Sprouty2(SPRY2)基因在胃癌肿瘤细胞上皮间质转化(EMT)和侵袭转移的影响。方法:体外培养人胃癌细胞(BGC-823),采用慢病毒介导的sh RNA沉默SPRY2基因,并用实时定量PCR与Western blot检测其SPRY2、E-钙黏蛋白(E-cadherin)、波形蛋白(vimentin)的表达,采用细胞划痕实验、Transwell实验检测SPRY2基因沉默后的胃癌细胞侵袭转移能力变化。结果:在慢病毒介导sh RNA沉默SPRY2基因的人胃癌BGC-823细胞中,SPRY2的m RNA和蛋白表达明显降低(P0.05),SPRY2沉默后人胃癌细胞E-cadherin的蛋白表达增多(P0.05),vimentin的蛋白表达减少(P0.05)。此外,SPRY2沉默后,胃癌细胞迁移能力和侵袭能力明显减弱(P值均P0.05)。结论:Sprouty-2基因通过调节E-cadherin与vimentin的表达参与胃癌细胞的上皮-间质转化,进而促进胃癌细胞的迁移与侵袭。     

RSPO2 enhances cell invasion and migration via the WNT/β-catenin pathway in human gastric cancer

R-spondins comprise a group of secreted WNT agonists. R-spondin2 (RSPO2) plays a crucial role in the activation of the WNT/β-catenin pathway and oncogenesis, though its specific role in human gastric cancer (GC) remains unclear. In the current study, RSPO2 expression levels were upregulated in cancer specimens and cell lines (AGS and BGC-823). Inhibition of RSPO2 expression levels had distinct effects on cell invasion, migration, and epithelial-mesenchymal transition (EMT) in AGS and BGC-823 cells in vitro. Furthermore, RSPO2 positively correlated with leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), the receptor of RSPO2. Silencing RSPO2 reduced the expression of LGR5 and WNT/β-catenin effector molecule β-catenin together with downstream targets TCF-4 and Cyclin-D1. These observations demonstrate that upregulation of RSPO2 in GC specimens and cell lines is closely related to tumor invasion and migration and that RSPO2 promotes EMT in gastric cancer cells by activating WNT/β-catenin signaling.     

LINC00665 induces gastric cancer progression through activating Wnt signaling pathway

Long noncoding RNAs (lncRNAs) are recently recognized as noteworthy regulators of different tumors, counting gastric cancer (GC). Lately, long intergenic noncoding RNA (LINC) 00665 has been verified to display significant parts in several cancers. Be that as it may, its role and mechanism in GC movement still stay uninvestigated. As of now, we observed LINC00665 was obviously GC cells (MKN28, BGC-823, SGC7-901, AGS, HGC-27) in comparison to GES-1 cells, which was identified as human normal gastric epithelial cells. Then, LINC00665 was obviously downregulated in GC cells including AGS and BGC-823 cells. Loss of LINC00665 greatly repressed AGS and BGC-823 cell survival and cell expansion. Moreover, GC cell apoptosis was significantly induced by the loss of LINC00665. For another, we found that the GC cell cycle was also captured in G1 and G2 phases. The experiments on cell migration and invasion indicated that knockdown of LINC00665 restrained GC cell migration and invasion. Modifications in Wnt signaling are closely associated with the development of cancers. Here, we found that Wnt signaling was significantly inactivated by the silence of LINC00665 in GC cells. β-catenin and cyclinD1 were restrained whereas GSK-3β was induced by the inhibition of LINC00665 in GC cells. Furthermore, we confirmed the impact of LINC00665 in vivo using xenograft models. Taken these together, we indicated that LINC00665 could function as a novel biomarker in GC progression.     

GABPA protects against gastric cancer deterioration via negatively regulating GPX1

BackgroundTo explore the anti-cancer role of GABPA in the progression of gastric cancer (GC), and the underlying mechanism.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was conducted to detect the expression pattern of GABPA in 45 pairs of GC and non-tumoral tissues. The relationship between GABPA expression and clinic pathological indicators of GC patients was analyzed. In AGS and SGC-7901 cells overexpressing GABPA, their migratory ability was determined by trans well and wound healing assay. The interaction between GABPA and its downstream target GPX1 was explored by dual-luciferase reporter assay, and their synergistical regulation on GC cell migration was finally elucidated.ResultsGABPA was downregulated in GC tissues in comparison to normal ones. Low level of GABPA predicted high incidences of lymphatic and distant metastasis in GC. Overexpression of GABPA blocked AGS and SGC-7901 cells to migrate. GABPA could target GPX1 via the predicted binding site. GPX1 was upregulated in clinical samples of GC, and negatively correlated to GABPA level. The anticancer effect of GABPA on GC relied on the involvement of GPX1.ConclusionsGABPA is downregulated in GC samples, which can be utilized to predict GC metastasis. Serving as a tumor suppressor, GABPA blocks GC cells to migrate by targeting GPX1.     

G蛋白偶联胆汁酸受体1促进胃癌细胞的增殖、迁移和侵袭的实验研究

目的:探讨G蛋白偶联胆汁酸受体1(G-protein coupled bile acid receptor 1,GPBAR1/TGR5)对胃癌细胞增殖、迁移和侵袭的影响。方法:免疫组织化学染色方法(Immunohistochemistry,IHC)检测胃癌及癌旁组织芯片中TGR5表达情况;qRT-PCR及Western blot检测胃癌细胞系中TGR5表达水平;小干扰RNA处理AGS、MKN-45胃癌细胞后构建TGR5敲减细胞系,慢病毒载体转染胃癌SGC-7901细胞构建TGR5过表达细胞系;CCK-8实验、平板克隆形成实验、裸鼠皮下移植瘤实验检测TGR5对细胞增殖的影响;流式细胞仪检测TGR5对细胞周期及凋亡的影响;Tanswell实验检测TGR5对胃癌细胞迁移及侵袭的影响;Western blot检测上皮间充质转化(Epithelial-mesenchymal transition,EMT)相关分子β-连环蛋白(β-catenin)、锌脂蛋白转录因子(Snail)、E盒结合锌指蛋白(Zinc finger E-box binding homeobox 1,ZEB)1在AGS、MKN-45及SGC-7901胃癌细胞中的表达。结果:TGR5在胃癌及癌旁组织中均有表达,胃癌组织TGR5高表达率(41.0%)显著高于癌旁组织(9.5%),伴肠化生癌旁组织TGR5高表达率(50%)显著高于不伴肠化生的癌旁组织(0%),胃癌组织TGR5表达与肿瘤大小相关。TGR5在正常人胃上皮永生化细胞株GES-1及各胃癌细胞系中均有表达。TGR5表达敲低的AGS和MKN-45细胞增殖能力减弱、凋亡率显著升高、侵袭和迁移能力显著降低。过表达TGR5的SGC-7901细胞增殖能力增强、克隆形成能力提高、凋亡率明显减低、侵袭和迁移能力显著升高。此外,TGR5过表达显著上调了间质细胞标志物β-catenin、Snail、ZEB1的表达水平。结论:TGR5能够增强胃癌细胞增殖及迁移能力,并抑制细胞凋亡。TGR5可能通过EMT途径介导胃癌细胞转移。     

CDX2基因高表达对胃癌细胞生物学行为的影响

目的探讨尾侧型同源转录因子-2(CDX2)基因过表达对胃癌BGC-823细胞增殖、迁移、凋亡等生物学特征的影响。方法采用脂质体转染法建立CDX2基因过表达的胃癌BGC-823稳定细胞株,分别采用RT-PCR、Western blotting和免疫细胞化学等方法检测转染重组表达载体pEGFP-C1/CDX2后,BGC-823细胞中CDX2基因及其蛋白的表达。MTT法检测CDX2基因过表达对细胞的增殖能力的影响;划痕实验检测CDX2过表达对细胞迁移能力的影响;流式细胞术检测CDX2过表达对细胞的凋亡的影响;应用基因芯片技术检测转染前后相关基因的差异表达。结果 RT-PCR及Western blotting检测结果显示,与对照组相比,转染pEGFP-C1/CDX2后,BGC-823细胞中CDX2基因和蛋白均呈高表达;CDX2过表达能明显降低转然组BGC-823细胞增殖能力和迁移能力;但对细胞凋亡影响不明显;基因芯片结果提示CDX2基因高表达能影响某些基因的表达。结论 CDX2过表达能明显抑制胃癌细胞增殖、降低迁移能力,提示CDX2在胃癌中可能发挥抑癌基因的作用。     

Casticin Potentiates TRAIL-Induced Apoptosis of Gastric Cancer Cells through Endoplasmic Reticulum Stress

Background

Casticin is one of the main active components obtained from Fructus Viticis and has been reported to exert anti-carcinogenic activity on a variety of cancer cells but the precise mechanism underlying this activity remains unclear.

Materials and Methods

Apoptotic activities of casticin (1.0 µmol/l) and TRAIL (25, 50 ng/ml) alone or in combination in the gastric cancer cell lines BGC-823, SGC-7901 and MGC-803 were detected by the use of a cell apoptosis ELISA detection kit, flow cytometry (FCM) with propidium iodide (PI) staining and activities of caspase-3, -8 and -9 by ELISA and cleavage of polyADP-ribose polymerase (PARP) protein using western blot analysis. Death receptors (DR) expression levels were evaluated using FCM analysis and western blotting. 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was used as a probe to measure the increase in reactive oxygen species (ROS) levels in cells. Multiple interventions, such as siRNA transfection and pharmacological inhibitors were used to explore the mechanisms of these actions.

Results

Subtoxic concentrations of casticin significantly potentiated TRAIL-induced cytotoxicity and apoptosis in BGC-823, SGC-7901 and MGC-803 cells. Casticin dramatically upregulated DR5 receptor expression but had no effects on DR4 or decoy receptors. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by the co-application of TRAIL and casticin. Gene silencing of the CCAAT/enhancer binding protein homologous protein (CHOP) and pretreatment with salubrinal, an endoplasmic reticulum (ER) stress inhibitor, attenuated casticin-induced DR5 receptor expression, and apoptosis and ROS production. Casticin downregulated the expression levels of the cell survival proteins cFLIP, Bcl-2, XIAP, and survivin. In addition, casticin also induced the expressions of DR5 protein in other gastric cancer cells (SGC-7901 and MGC-803).

Conclusion/Significance

Casticin enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-ER stress-CHOP pathway.     

Hsa-miR-34a-5p reverses multidrug resistance in gastric cancer cells by targeting the 3′-UTR of SIRT1 and inhibiting its expression

Multidrug resistance (MDR) is a major obstacle to chemotherapy, which leads to ineffective chemotherapy, an important treatment strategy for gastric cancer (GC). The abnormality of microRNAs (miRNAs) is critical to the occurrence and progression of MDR in various tumors. In this study, hsa-miR-34a-5p was found to be decreased in multidrug resistant GC cells SGC-7901/5-Fluorouracil (SGC-7901/5-Fu) compared to the parental SGC-7901 cells. Overexpression of hsa-miR-34a-5p in SGC-7901/5-Fu cells promoted apoptosis and decreased migration and invasiveness after chemotherapy. In addition, overexpression of hsa-miR-34a-5p suppressed the growth of drug-resistant tumor in vivo. The mechanism of the effects of hsa-miR-34a-5p could include the regulation of the expression of Sirtuin 1 (SIRT1), P-glycoprotein (P-gp) or Multidrug resistance-related protein 1 (MRP1) through direct binding to the 3′-untranslated region (UTR) of SIRT1. Functional gain-and-loss experiments indicated that hsa-miR-34a-5p enhances the chemotherapy sensitivity of MDR GC cells by inhibiting SIRT1, P-gp and MRP1. In conclusion, hsa-miR-34a-5p can reverse the MDR of GC cells by inhibiting the expression of SIRT1, P-gp or MRP1.