Apoptosis Of MKN-45 Cells Research Articles

The Chinese Herbal Formula Weichang'an Reduces the Proliferation of Human Gastric Cancer Cells via Mitochondrial Apoptosis

Abstract Objective The aim of the study was to investigate the effects of Chinese herbal formula Weichang'an (WCA) on the proliferation and mitochondria-mediated apoptosis of human gastric cancer cells. Methods Cell Counting Kit-8 (CCK8) was used to evaluate the antiproliferative activity of WCA on MKN45 cells; Giemsa staining was used to investigate cell colony formation; flow cytometry was used to analyze cell cycle, apoptosis rate, and caspases activation; and Hoechst staining was used to analyze the morphology of cell nuclei. The mitochondrial membrane potential was analyzed by both flow cytometric measurements and fluorescence microscopy. Western blot was used to analyze the protein levels of pro-caspase-3, Bcl-2, Bax, and Bcl-X. MKN45 cells were subcutaneously injected into the right forelimb of 16 nude mice to establish the tumor xenograft model, and a total of 12 nude mouse tumor xenograft models were successfully created. The nude mice were divided into the control group and the WCA-treated group. The two groups received normal saline and WCA treatment at 35.49 g/kg by a single daily oral gavage for 21 days. The body weight and tumor size of the nude mice were measured twice a week. The ultrastructural changes of subcutaneous tumors were observed by a transmission electron microscope. Results WCA can suppress cell proliferation and colony formation. It can also induce changes in the mitochondrial membrane potential and increase the activities of caspases-3, caspases-8, and caspases-9 in MKN45 cells. WCA induced S-phase arrest and apoptosis in MKN45 cells, and decreased the expression levels of antiapoptotic proteins Bcl-2 and Bcl-X in MKN45 cells, while increasing the expression levels of the proapoptotic proteins Bax and pro-caspase-3 compared with the control group. WCA inhibited the growth of a xenografted MKN45 tumor in nude mice, and the protein levels of Bax and pro-caspase-3 were significantly increased, while Bcl-X and Bcl-2 were reduced compared with the control group. The differences are statistically significant (p < 0.05). Conclusion WCA could suppress the proliferation of gastric cancer cells via mitochondrial apoptosis.

Quercetin Promotes Apoptosis of Gastric Cancer Cells through the EGFR-ERK Signaling Pathway

Previous studies have shown that various active components of licorice have anticancer effects. However, few studies have investigated the mechanism of action of licorice in gastric cancer. The effect of active compounds in licorice on the biological activity of gastric cancer cells was investigated in vitro (MKN-45 cells). Network pharmacology and molecular docking were used to predict the potential targets of licorice against gastric cancer and verify the binding stability of target proteins to compounds. In addition, the anticancer effect of licorice was assessed using a mouse model of gastric cancer. The licorice-active component (quercetin) effectively inhibited proliferation, caused cell cycle arrest, and promoted apoptosis in MKN-45 cells, accompanied by increased Cyt-C, decreased BCL-2, and decreased mitochondrial membrane potential and mitochondrial damage. Further research showed that quercetin targeted EGFR, blocked the ERK signaling pathway, and downregulated PTGS2. In the in vivo experiment, quercetin treatment resulted in reduced tumor volume, decreased Ki67 and BCL-2 expression in tumor tissue, increased caspase 3 and BAX levels, and induced mitochondrial damage.

Design, synthesis, and biological evaluation of thiazole/thiadiazole carboxamide scaffold-based derivatives as potential c-Met kinase inhibitors for cancer treatment

As part of our continuous efforts to discover novel c-Met inhibitors as antitumor agents, four series of thiazole/thiadiazole carboxamide-derived analogues were designed, synthesised, and evaluated for the in vitro activity against c-Met and four human cancer cell lines. After five cycles of optimisation on structure–activity relationship, compound 51am was found to be the most promising inhibitor in both biochemical and cellular assays. Moreover, 51am exhibited potency against several c-Met mutants. Mechanistically, 51am not only induced cell cycle arrest and apoptosis in MKN-45 cells but also inhibited c-Met phosphorylation in the cell and cell-free systems. It also exhibited a good pharmacokinetic profile in BALB/c mice. Furthermore, the binding mode of 51am with both c-Met and VEGFR-2 provided novel insights for the discovery of selective c-Met inhibitors. Taken together, these results indicate that 51am could be an antitumor candidate meriting further development.

(-)-Epigallocatechin-3-gallate induced apoptosis by dissociation of c-FLIP/Ku70 complex in gastric cancer cells.

Anti-cancer properties of (-)-epigallocatechin-3-gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE-inhibitory protein (c-FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti-cancer role of EGCG in gastric cancer (GC) cells through dissociation of c-FLIP/Ku70 complex. MKN-45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT-PCR. Protein expression of c-FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c-FLIP/Ku70 complex as well as Ku70 translocation were studied by sub-cellular fractionation and co-immunoprecipitation. EGCG induced apoptosis in MKN-45 cells with substantial up-regulation of P53 and P21, down-regulation of c-Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c-FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c-FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.

CTPS cytoophidia formation affects cell cycle progression and promotes TSN‑induced apoptosis of MKN45 cells.

Cytidine triphosphate synthase (CTPS) forms filamentous structures termed cytoophidia in numerous types of cell. Toosendanin (TSN) is a tetracyclic triterpenoid and induces CTPS to form cytoophidia in MKN45 cells. However, the effects of CTPS cytoophidia on the proliferation and apoptosis of human gastric cancer cells remain poorly understood. In the present study, CTPS-overexpression and R294D-CTPS mutant vectors were generated to assess the effect of CTPS cytoophidia on the proliferation and apoptosis of gastric cancer MKN45 cells. Formation of CTPS cytoophidia significantly inhibited MKN45 cell proliferation (evaluated using EdU incorporation assay), significantly blocked the cell cycle in G1 phase (assessed using flow cytometry) and significantly decreased mRNA and protein expression levels of cyclin D1 (assessed by reverse transcription-quantitative PCR and western blotting, respectively). Furthermore, the number of apoptotic bodies and apoptosis rate were markedly elevated and mitochondrial membrane potential was markedly decreased. Moreover, mRNA and protein expression levels of Bax increased and Bcl-2 decreased markedly in MKN45 cells following transfection with the CTPS-overexpression vector. The proliferation rate increased, percentage of G1/G0-phase cells decreased and apoptosis was attenuated in cells transfected with the R294D-CTPS mutant vector and this mutation did not lead to formation of cytoophidia. The results of the present study suggested that formation of CTPS cytoophidia inhibited proliferation and promoted apoptosis in MKN45 cells. These results may provide insights into the role of CTPS cytoophidia in cancer cell proliferation and apoptosis.

Silencing of circ_0078607 prevents development of gastric cancer and inactivates the ERK1/2/AKT pathway through the miR-188-3p/RAP1B axis.

The aim of this study is to explore the expression and mechanism of circ_0078607 on proliferation and apoptosis of gastric cancer. Real time PCR (RT-PCR) was performed to detect the expression of circ_0078607 in gastric cancer tumor tissues, plasma and cell lines. Cell viability was detected by cell counting Kit-8. Cell proliferation ability was assessed by cell cycle assay. The samples were analyzed by flow cytometry for the detection of apoptosis. Luciferase assay and RNA immunoprecipitation (RIP) were carried out to verify the relationship between circ_0078607 and miR-188-3p, miR-188-3p, and RAP1B. Western blot was employed to detect the protein level of RAP1B, ERK1/2 and AKT. In vivo, the effect of circ_0078607 on gastric cancer tumor growth was detected by lentivirus vector injection. Here, we found the increased level of circ_0078607 in gastric cancer tissues, gastric cancer patients plasma and cell lines. Knockdown of circ_0078607 could prevent proliferation and induce cell apoptosis in MKN-28 cells. Then we verified that circ_0078607 could interact with miR-188-3p by performed luciferase assay and RIP. Furthermore, we observed that RAP1B was a potential target of miR-188-3p. Next, we found that miR-188-3p inhibitor or overexpression of RAP1B could prevent the anti-tumor function of sh-circ_0078607. Silencing of circ_0078607 inhibited ERK1/2/AKT signal pathways via regulating miR-188-3p/RAP1B. In vivo, knockdown of circ_0078607 inhibited tumor growth. Knockdown of circ_0078607 inhibits the proliferation and induces apoptosis of gastric cancer via miR-188-3p/RAP1B signal pathway.

Apoptotic Effects of Anthocyanins from Vitis coignetiae Pulliat Are Enhanced by Augmented Enhancer of the Rudimentary Homolog (ERH) in Human Gastric Carcinoma MKN28 Cells.

Evidence suggests that augmented expression of a certain gene can influence the efficacy of targeted and conventional chemotherapies. Here, we tested whether the high expression of enhancer of the rudimentary homolog (ERH), which serves as a prognostic factor in some cancers, can influence the efficacy of anthocyanins isolated from fruits of Vitis coignetiae Pulliat, Meoru in Korea (AIMs) on human gastric cancer cells. The anticancer efficacy of AIMs was augmented in ERH-transfected MKN28 cells (E-MKN28 cells). Molecularly, ERH augmented AIM-induced caspase-dependent apoptosis by activating caspase-3 and -9. The ERH-augmented apoptotic effect was related to mitochondrial depolarization and inhibition of antiapoptotic proteins, XIAP, and Bcl-2. In addition, reactive oxygen species (ROS) generation was augmented in AIMs-treated E-MKN28 cells compared to AIMs-treated naïve MKN28 cells. In conclusion, ERH augmented AIM-induced caspase-dependent mitochondrial-related apoptosis in MKN28 cells. A decrease in expression of Bcl-2 and subsequent excessive ROS generation would be the mechanism for ERH-augmented mitochondrial-related apoptosis in AIMs-treated MKN28 cells. A decrease in expression of XIAP would be another mechanism for ERH-augmented caspase-dependent apoptosis in AIMs-treated MKN28 cells.

Toosendanin induces apoptosis of MKN‑45 human gastric cancer cells partly through miR‑23a‑3p‑mediated downregulation of BCL2.

Toosendanin (TSN) is a tetracyclic triterpenoid extracted from Melia toosendan Sieb, et Zucc, which primarily grows in specific areas of China. Although toosendanin (TSN) exerts antitumoral effects on various human cancer cells, its influence on gastric cancer (GC) is remains to be elucidated. MicroRNAs (miRNAs/miRs) serve crucial roles in apoptosis and proliferation of cancer cells. miR-23a-3p has been shown to be associated with human GC; however, the specific function of miR-23a-3p in GC remains unclear. Therefore, the present study aimed to elucidate the role of miR-23a-3p in the regulation of GC cell proliferation and apoptosis induced in vitro by TSN treatment. Subsequently, apoptosis-related genes expression levels were quantified by reverse transcription-quantitative PCR and western blot analysis, respectively, and the target relationship between miR-23a-3p and BCL2 was determined by luciferase reporter gene analysis. Additionally, cell proliferation and apoptosis experiments were carried out. The results indicated that TSN inhibited proliferation and induced apoptosis in MKN-45 cells. Moreover, it upregulated the expression of miR-23a-3p. B-cell lymphoma-2 (BCL2) was identified as a potential target gene of miR-23a-3p, which was demonstrated to bind to the 3′-untranslated region of BCL2 mRNA, as detected by the luciferase reporter assay. Further studies revealed that BCL2 expression was downregulated following overexpression of miR-23a-3p. In addition, the overexpression of the miR-23a-3p inhibited proliferation, induced G1 arrest and increased apoptosis in MKN-45 cells. The results of the present study demonstrated that miR-23a-3p inhibited proliferation and induced apoptosis of GC cells, which may be attributable to its direct targeting of BCL2. These results may provide a novel insight into the apoptosis of GC cells, and may lead to investigations into the mechanisms of the effects of TSN.

SiRNA-dependent Gene Silencing of EVI1 Decreased Cell Proliferation in MKN45 Cells

MDS1 and EVI1 complex locus protein EVI1 (<i>MECOM</i>) is an oncogenic transcription factor in many types of cancer. However, the clinical significance of <i>MECOM</i> in esophagogastric cancer has not been well elucidated. The aim of this study was to define the association of amplification of MECOM and esophagogastric cancer patients’ overall survival rate and to investigate the functional role of <i>MECOM</i> in MKN45 cell proliferation and apoptosis. In this study, a total of 3236 esophagus and gastric patients were analyzed from 16 genomic studies using cBioPortal, which provides an open-access resource with multiple cancer genomics data sets. When the patient samples were divided into two subgroups (<i>MECOM</i> amplified group and <i>MECOM</i> non-amplified group), the poor survival rate was significantly associated with <i>MECOM</i> amplification (<i>p</i>= 0.04). To investigate the role of EVI1 on esophagogastric cancer cells, siRNA dependent gene silencing of EVI1 in MKN45 cells was conducted. RT-PCR was used to examine the expression of EVI1 in MKN45 cells. The MTT assay was used to examine cell proliferation. The apoptotic cells were quantified by fluorescence cell counter. Knockdown of EVI1 leads to reduced cell proliferation and induced apoptosis in MKN45 cells. These results indicate that <i>MECOM</i> may be a potential target for esophagus and gastric cancer gene-targeted therapy. Collectively, our result suggested that EVI1 is a probable target gene for esophagogastric cancer cell proliferation.

Effect of trametes robiniophila on the apoptosis of human gastric carcinoma MKN-45 and SGC-7901 cells and the expressions of matrix metalloproteinase 2 and matrix metalloproteinase 9

Objective To analyze the effect of trametes robiniophila on the apoptosis and the expressions of invasion and metastasis related matrix metalloproteinase 2 (MMP-2) and MMP-9 in human gastric carcinoma poorly differentiated cell line MKN-45 and medium differentiated cell line SGC-7901. Methods Human gastric carcinoma cell lines MKN-45 and SGC-7901 incubated with trametes robiniophila at the different concentrations [0 (the negative control group), 5, 10, and 20 mg/ml] for 24 h. When bifluorescently stained with acridine orange-ethidium bromide (AO-EB), morphology was observed by using microscopy. Flow cytometry was used to test the apoptosis ratio after 24, 48, and 72 h. Reverse transcription polymerase chain reaction (RT-PCR) was applied to analyze the expressions of mRNA of MMP-2 and MMP-9 after 24 h, and the absorbance values of the bands after gel electrophoresis were used as their expression levels. Results Under the fluorescence microscope, the cells of the negative control group were green (normal cells), and the membrane was even in size and integrity. The proportion of apoptotic and necrotic cells was increased with the concentration enrichment of trametes robiniophila. The apoptotic cells were yellow, and their cell membrane lost integrity. Apoptotic bodies were found in cancer cells, and cell membrane showed bud projection. The nuclei of the apoptotic cells showed brightly condensed chromatin or fragmented. Chromatin was strongly stained and located in karyotheca. The necrotic cells were dyed red with integrity of size. The apoptotic ratio of MKN-45 cell line induced by trametes robiniophila after 24 h was (6.5±0.8)%, (14.6±1.0)%, (18.0±1.1)%, and (23.1±1.2)%, respectively (F = 333.972, P < 0.01) in negative control group and 5, 10, and 20 mg/ml trametes robiniophila group. After 48 h, the apoptotic ratio was (7.3±1.2)%, (18.3±1.6)%, (24.5±1.3)%, and (27.2±1.7)%, respectively (F = 528.432, P=0.001); after 72 h, the apoptotic ratio was (7.5±0.9)%, (50.2±1.6)%, (58.0±1.9)%, and (69.0±1.4)%, respectively(F = 3 814.238, P < 0.01). After SGC-7901 cell line induced by trametes robiniophila for 24 h, the apoptotic ratio was (12.9±1.0)%, (19.4±1.2)%, (22.0±1.7)%, and (23.0±1.9)%, respectively (F = 120.190, P < 0.01) in negative control group and 5, 10, and 20 mg/ml trametes robiniophila group. For 48 h, the apoptotic ratio was(10.2±1.3)%, (40.9±1.4)%, (51.6±1.9)%, and (66.2±1.9)%, respectively (F = 1 281.342, P < 0.01). For 72 h, the apoptotic ratio was (27.4±1.8)%, (49.7±1.4)%, (65.1±1.4)%, and (69.0±2.0)%, respectively (F = 1 112.767, P < 0.01). The induction of apoptosis showed time and dose dependence (both P < 0.01). There was a trendency that the apoptotic ratio of SGC-7901 cell line was higher than that of the MKN-45 cell line at the same condition. RT-PCR showed that mRNA relative expression level of MMP-2 was 0.64±0.02, 0.49±0.01, 0.36±0.02, and 0.32±0.01, respectively (F = 274.321, P < 0.01) of negative control group and 5, 10, and 20 mg/ml trametes extract group after the effect of trametes extract on gastric cancer MKN-45 cell line. The relative expression level of MMP-9 in MKN-45 cell line was 0.71±0.01, 0.54±0.02, 0.47±0.02, and 0.39±0.02, respectively (F = 203.948, P < 0.01). The expression level of MMP-2 in SGC-7901 cell line was 0.64±0.01, 0.42±0.02, 0.34±0.20, and 0.29±0.01, respectively (F = 305.877, P < 0.01), while the mRNA expression level of MMP-9 was 0.65±0.15, 0.47±0.01, 0.44±0.01, and 0.39±0.02, respectively (F = 265.259, P < 0.01). Compared with the negative control group, the expression levels of MMP-2 and MMP-9 of the two cell lines were both decreased, after incubated with trametes robiniophila at the different concentrations (all P < 0.05), and with the addition of concentration, the expression levels of MMP-2 and MMP-9 were decreased. Conclusion Trametes robiniophila can induce the apoptosis of human gastric carcinoma cell lines MKN-45 and SGC-7901 in vitro and can inhibit the expressions of MMP-2 and MMP-9 and the effect of trametes robiniophila may be related with the differentiation degree. Key words: Stomach neoplasms; Apoptosis; Neoplasm invasiveness; Neoplasm metastasis; Trametes robiniophila

Menadione Induced Apoptosis in MKN45 Cells via Down-regulation of Survivin

Menadione은 종양 억제 물질로 알려진 바 있다. 현재 많은 연구에서 다양한 암세포주에 대하여 Menadione의 잠재적인 항암물질로서의 가능성이 보고되었다. 본 연구에서는 Menadione의 항암효과와 세포사멸작용에 연관된 분자신호를 위암세포주에서 확인하였다. Menadione 처리는 위암세포인 MKN45의 세포생존능을 감소시켰다. 감소된 세포생존능은 Western blotting을 통해 caspase-3 과 caspase-7의 활성화와 PARP가 cleavage 된 것을 확인함으로써 세포사멸작용이 유도되었다는 것을 확인했다. 위세포사멸단백질들의 저해제로 작용하는 survivin의 발현을 menadione이 억제한다는 것을 확인함으로써, 세포사멸과정에 포함된 상위조절인자를 확인했다. 우리는 survivin 발현을 조절하는 전사인자로 알려진 β-catenin 또한 menadione에 의해 하향 조절된다는 것을 확인했다. 이전 연구에서 우리는 menadione이 세포사멸유도를 저해는 XIAP의 발현을 억제한다는 것을 확인했으며, menadione이 AGS세포에서 G2/M 세포주기 정체를 유도한다는 것을 확인하였다. 우리는 또 다른 위암세포주인 MKM45 세포에서 menadione의 이전과 다른 항암 기전을 밝혀냈다. 비록 더 자세한 연구가 필요하겠지만, 이 연구를 통해 증명된 억제기전은 menadione에 의한 항암효과를 이해하는 데 도움이 될 것으로 사료된다.

RETRACTED ARTICLE: Lidocaine inhibits growth, migration and invasion of gastric carcinoma cells by up-regulation of miR-145

BackgroundGastric cancer receives considerable attention not only because it is the most common cancer all through the world, but also because it’s on the top third leading reason for cancer-related death. Lidocaine is a well-documented local anesthetic that has been reported to suppress cancer development. The study explored the effects of lidocaine on the growth, migration and invasion of the gastric carcinoma cell line MKN45 and the mechanism behind.MethodsThe effect of lidocaine on viability, proliferation and apoptosis of MKN45 cells were analyzed by Cell Counting Kit-8 assay, BrdU staining assay and flow cytometry, respectively. Moreover, cell migration and invasion were both examined by Transwell assay. The expression of apoptosis-, migration-, and invasion-related proteins were detected by western blot. The relative expression of miR-145 was determined by qRT-PCR. Moreover, the impact which lidocaine brought on MEK/ERK and NF-κB pathways were examined by western blot.ResultsLidocaine inhibited viability, proliferation, migration, and invasion of MKN45 cells, while enhanced apoptosis. Moreover, miR-145 expression was enhanced by lidocaine; and transfection with miR-145 inhibitor increased cell viability, proliferation, migration, and invasion, but inhibited apoptosis. The up-regulation of miR-145 was partly contributed to the inhibitory effect of lidocaine on gastric cancer cell line MKN45. Finally, lidocaine inactivated MEK/ERK and NF-κB pathways via up-regulation of miR-145.ConclusionsOur results suggested that lidocaine decreased growth, migration and invasion of MKN45 cells via regulating miR-145 expression and further inactivation of MEK/ERK and NF-κB signaling pathways.

Dual inhibitor of PI3K and mTOR (NVP-BEZ235) augments the efficacy of fluorouracil on gastric cancer chemotherapy.

PurposeNVP-BEZ235 is a recently developed dual inhibitor of PI3K and mTOR and shows good inhibitory effects on several types of tumors. However, the efficacy of NVP-BEZ235 on gastric cancer therapy remains unclear. This study aimed to investigate the potential of NVP-BEZ235 as a new agent to enhance chemotherapy for gastric cancer.MethodsHuman gastric cancer MKN-45 cells or nude mice xenografted with MKN-45 cells were treated by NVP-BEZ235 and fluorouracil (5-FU) alone or in combination. The proliferation, invasion, apoptosis, and chemoresistance of gastric cancer cells were examined in vivo and in vitro.ResultsIn vitro, combined treatment with NVP-BEZ235 and 5-FU showed synergistic inhibitory effects on proliferation, migration, and invasion and synergistic stimulating effects on apoptosis of MKN-45 cells. In vivo, NVP-BEZ235 and 5-FU synergistically inhibited the growth and induced apoptosis of MKN-45 xenografts. Mechanistically, NVP-BEZ235 inhibited PI3K/Akt/mTOR signaling; decreased the levels of Bcl-2, MMP9, and VEGF; but increased the levels of Bax and cleaved caspase-3 in MKN-45 xenografts.ConclusionNVP-BEZ235 enhances the antitumor efficacy of 5-FU. Therefore, NVP-BEZ235 is a promising agent to enhance chemotherapy for gastric cancer.

Co-delivery of paclitaxel and tanespimycin in lipid nanoparticles enhanced anti-gastric-tumor effect in vitro and in vivo

Combined administration regimens are commonly used in cancer therapy to reduce cell toxicity and drug resistance. In this study, we use solid lipid nanoparticles (SLNs) as drug carriers and sought to investigate the effect of combined administration of paclitaxel (PTX) and tanespimycin (17-AAG) in gastric cancer. The SLNs loaded with paclitaxel and tanespimycin were prepared using the solvent injection method. The effect of encapsulated SLNs on cell viability and colony formation were measured in three human gastric cell lines. Cell apoptosis assay was carried out in MKN45 cells and xenograft model was used to investigate the effect of encapsulated SLNs in vitro and in vivo. The expression levels of proteins involved in oxidative stress and apoptosis were measured by western blotting analysis. The encapsulated SLNs reduced cell viabilities and colony formation in gastric cell lines. These SLNs could also induce apoptosis in MKN45 cells, inhibit growth of xenograft and influence the protein levels of Hsp90, MnSOD, Cleaved caspase 3 and Cleaved PARP. The effect of encapsulated SLNs exceeded that of single treatment of PTX or 17-AAG. The combination administration of PTX or 17-AAG resulted in a synergetic anti-cancer effect, probably via an increased oxidative stress and apoptosis levels.

Experimental in vivo and in vitro study of inhibitory effect of extract from taraxacum mogonon on human poorly differentiated gastric cancer

Objective To investigate inhibitory effect and mechanism of extract from taraxacum mogonon (ETM) on human poorly differentiated gastric cancer MKN45 cells and their model nude mice tumors. Methods Human poorly differentiated gastric cancer MKN45 cells cultured in vitro were divided into the control group and the experimental group. The experimental group was treated with different concentrations of ETM, while the control group was treated with phosphate buffered saline (PBS). The tumor cell growth was examined by methyl thiazolyl tetrazolium (MTT). Cell apoptosis was detected by flow cytometry (FCM). The expression of Survivin mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and the protein expression of Survivin was detected by Western blot. The nude mouse model with poorly differentiated gastric cancer was established with MKN45 cells. Successful models of nude mice were divided into the experimental group and the control group according to random number table. ETM 0.4 ml (100 mg/ml) was injected into abdominal cavity of the nude mice in the experimental group, while only equal dose of 0.9% NaCl solution was injected in the control group, every 3 days for once, 42 days in total. The weight and volume changes of transplantable tumors were observed. The protein expression of Survivin in transplantable tumors was examined by immunohistochemistry (IHC) method. Results ETM could inhibit the cell proliferation and promote apoptosis of MKN45 cells in a dose-dependent and time-dependent way when its concentration reached 0.2 μmol/L (P < 0.05). The expression of Survivin mRNA and Survivin protein was down regulated in a dose-dependent way in the experimental group. Survivin mRNA expression in the experimental group with 0.1, 0.2, 0.4, 0.8, 1.6 μmol/L of ETM and in the control group was 0.18±0.04, 0.14±0.03, 0.11±0.01, 0.08±0.04, 0.06±0.02 and 0.19±0.03 respectively (F= 132.35, P < 0.05); Survivin protein expression in the experimental group in 0.1, 0.2, 0.4, 0.8, 1.6 μmol/L of ETM, and in the control group was 0.86±0.03, 0.60±0.05, 0.43±0.01, 0.22±0.01, 0.14±0.03 and 0.92±0.06 respectively (F= 76.57, P < 0.05). The difference of experimental group was statistically significant when concentration of ETM reached 0.2 μmol/L or above compared with the control group (P < 0.05). Nude mouse experiment showed that the tumor inhibition rate was (60.3±3.2)% in the experimental group. The volume and weight of transplantable tumor in the experimental group was (326±27) mm3 and (0.31±0.13) g, which was obviously lower than that in the control group [(843±14) mm3 and (0.78±0.25) g]. The difference was statistically significant (t= 3.94, P= 0.043; t= 3.27, P= 0.037). IHC experiment results showed that positive cell count of Survivin protein in the experimental group was obviously less than that in the control group (28±11 vs. 152±20; t= 4.32, P= 0.029). Conclusions ETM has an obvious inhibitory effect on human poorly differentiated gastric cancer MKN45 cell and its nude mouse tomor model. The mechanism may be related with down-regulation of Survivin expression caused by ETM. Key words: Taraxacum; Stomach neoplasms; In vitro; Disease models, animal

Effects of Combination of Anti-CTLA-4 and Anti-PD-1 on Gastric Cancer Cells Proliferation, Apoptosis and Metastasis

Background/Aims: Gastric cancer (GC) is one of the most common and lethal varieties of cancers. Anticancer activities of anti-CTLA-4 and anti-PD-1 antibodies have been explored in different cancers, including GC. The study aimed to explore the role of combination therapy with anti-CTLA-4 and anti-PD-1 antibodies in GC cells, and understand the possible underlying molecular mechanism. Methods: MKN-45 and MGC-803 cells were divided into four groups, namely control, CTLA-4, PD-1, and CTLA-4&PD-1. Cell viability, cell cycle, apoptosis, migration and invasion were measured by MTT, flow cytometry, and transwell assays, respectively. Expression levels of different mRNAs and proteins associated with apoptosis, epithelial mesenchymal transition (EMT), β-catenin, MAPK, and PI3K/AKT pathways were assessed by RT-qPCR and western blot analysis, respectively. The tumor formation in vivo was examined by tumor Xenograft model assay. Results: Combination with anti-CTLA-4 and anti-PD-1 antibodies significantly suppressed cell proliferation, induced apoptosis, as well as inhibited migration, invasion, and EMT in MKN-45 and MGC-803 cells. Western blotting revealed that combination with anti-CTLA-4 and anti-PD-1 antibodies declined the activation of β-catenin, MAPK and PI3K/AKT signal pathways. Moreover, combination of anti-CTLA-4 and anti-PD-1 antibodies inhibited tumor formation in vivo. Furthermore, the mRNA levels of CTLA-4 and PD-1 were significantly decreased in si-CTLA and si-PD-1 transfected cells, and combination with si-CTLA and si-PD-1 also suppressed cell proliferation, migration, invasion, EMT and induced apoptosis in MKN-45 cells. Conclusion: Combination therapy with anti-CTLA-4 and anti-PD-1 antibodies presented the promising outcomes in GC, although further investigations are warranted.

Synthesis and preliminary antiproliferative activity of new pteridin-7(8H)-one derivatives

Pteridines are an important class of heterocyclic compounds with diverse biological activities. Here, we report a series of pteridin-7(8H)-one derivatives and their antiproliferative activities toward MKN-45, MGC-803, EC-109, and H1650. Structure-activity relationship studies showed that compound 12 exerted the most potent antiproliferative activity against MKN-45 and MGC-803 with the IC50 values of 4.32 and 7.01 μM, respectively. Besides, compound 12 induced morphological changes and apoptosis of MKN-45 cells, increased expression of Bax, down-regulated expression of Bcl-2 and caused cleavage of caspase-3/9. Additionally, we first reported the construction of the novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold through the competitive 5-endo cyclization reaction with two C-N bonds and a chiral carbon center established.

Ubiquitination and expression of metastasis associated 1 in gastric cancer MKN-45 cells

Objective To observe the ubiquitination of metastasis associated 1 (MTA1) in gastric cancer cells and its effect on the expression of MTA1. Methods The non-transfected MKN-45 cells [Myc-MTA1 (-), HA-Ub (-)] served as the control group, and three experimental groups of MKN-45 cells were transfected with the myc tag of MTA1 plasmid [Myc-MTA1 (+ ), HA-Ub (-)], HA tagged ubiquitin plasmid [Myc-MTA1 (-), HA-U (+ )], or co-transfected with the two plasmids [Myc-MTA1 (+ ), HA-Ub (+ )]. 2 μg plasmid was transfected into each culture dish, and then the transfected cells were harvested after 24 h culture. MKN-45 cells in the experimental group were treated with proteasome inhibitor MG-132. The expression level of MTA1 was detected by immunoprecipitation (IP) and Western blotting. MTA1 expressing plasmid containing the Myc tag and HAtagged ubiquitin plasmid (HA-Ub) was co-transfected into cells, and the expression of Myc-MTA1 was detected. Flow cytometry was used to detect the apoptosis of MKN-45 cells induced by MG-132. Results (1) With the proteasome inhibitor MG-132 treatment of gastric cancer cell line MKN-45, MTA1 expression levels increased significantly in experimental group, there were (0.78±0.15) μg/ml and (1.37±0.33) μg/ml after 2 h and 6 h, compared with the control group [(0.28±0.09) μg/ml], the difference was statistically significant (t=2.134, 5.562, P<0.05). (2) After 6 h, the degree of ubiquitination in experimental group was (77.28±9.38)%, and the control group was (15.41±2.14)%, the difference was statistically significant (P<0.05). (3) In gastric cancer MKN-45 cells [Myc-MTA1 (+ ), HA-Ub (+ ) group, Myc-MTA1 can be strongly ubiquitin, the degree of ubiquitin was (64.59±7.61)%, compared with the other two groups, the difference was statistically significant (P<0.05). (4) MG-132 treatment of the experimental group and the control group of MKN-45 gastric cancer cells after 24 h of gastric cancer MKN-45 cells transfected with [Myc-MTA1(+ )、HA-Ub(+ )], the apoptosis ratio was (22.90±4.17)%, significantly higher than control group MKN-45 cell apoptosis ratio (8.25±1.04)%. C-poly adenosine diphosphate-ribose polymerase (c-PARP) levels showed that [Myc-MTA1 (+ ), HA-Ub (+ )] the degree of apoptosis was (29.77±5.04)%, significantly higher than the control group MKN-45 cells (13.10±3.28)% (t=1.845, 2.374, P<0.05). Conclusion MTA1 in human gastric cancer cells can be regulated through ubiquitination pathway. Its ubiquitination pathway plays an important role in the development and metastasis of gastric cancer. Key words: Gastric cancer; Metastasis associated 1; Ubiquitination

Lobetyol activate MAPK pathways associated with G1/S cell cycle arrest and apoptosis in MKN45 cells in vitro and in vivo.

The agent lobetyol, which is isolated from Lobelia chinensis, was previously shown to be cytotoxicity againts several cancer cell lines in published report. Today, we perform a study in vitro and in vivo to analyze its anti-carcinoma effect in MKN45 cells and to explore the molecular mechanism. The growth inhibition of lobetyol on MKN45 cells was analyzed with MTT and flow cytometry. Hoechst 33342 staining and TUNEL cover glass staining were used to provide the visual evidence of apoptosis. Western blotting assay was performed to study the activation or blocking of related signaling pathways. Lobetyol induce apoptosis and cell cycle arrest in a time- and dose-dependent manner in MKN45 cells in our study. This process is mediated by the MAPK signaling pathways. This study confirmed the cytotoxicity of lobetyol in MKN45 cells and provided an insight into the molecular mechanism, which demonstrates the potential of lobetyol as an anti-tumor agent.

Black Currant (Ribes nigrum L.) Extract Induces Apoptosis of MKN-45 and TE-1 Cells Through MAPK- and PI3K/Akt-Mediated Mitochondrial Pathways.

Black currant extract (BCE) is rich in polyphenols and can induce apoptosis in various cancer cells, but the molecular mechanism by which BCE induces cancer cell apoptosis has not been reported. The aim of this work was to elucidate the antitumor effect of BCE and the signal transduction pathways involved. MTT test results revealed that the viability of MKN-45 and TE-1 cells treated with BCE gradually decreased in a concentration-dependent manner, with significant effects achieved after 12 h of treatment. MKN-45 and TE-1 cells clearly showed characteristics of apoptosis: shrinkage, cytoplasmic condensation, and formation of cytoplasmic filaments, even partial detachment. In addition, these results showed MKN-45 cells showed a higher level of apoptosis than TE-1 cells when treated with BCE. Western blot assays showed that the Bcl-2/Bax ratio decreased in both MKN-45 and TE-1 cells, indicating that BCE induced apoptosis through the mitochondrial pathway. In addition, BCE-induced apoptosis was mediated by mitochondrial dysfunction involving the PI3K/Akt pathway in both MKN-45 and TE-1 cells. However, BCE-induced cell apoptosis was mediated by the Fas receptor pathway in MKN-45 cells but not in TE-1 cells. BCE-induced apoptosis in MKN-45 cells was associated with the MAP-kinase signaling pathway through the activation of p38 and JNK and the inactivation of Erk1/2. However, it was associated with the MAP-kinase signaling pathway only by means of activation of p38 and JNK in TE-1 cells. These results showed that BCE induces apoptosis of MKN-45 and TE-1 cells through MAPK- and PI3K/Akt-mediated mitochondrial pathways. Thus, BCE may be a promising anticancer candidate.