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Background and purpose:Trastuzumab has a relatively low incidence of drug resistance,which can be used as an adjuvant treatment to improve clinical efficacy.It has been used to treat breast cancer in the past,but its application in other cancers has been less studied.This study aimed to explore the effects of trastuzumab assisted modified DOF fortnightly regimen on serum tumor markers and survival rate in cisplatin-resistant gastric cancer patients,in order to provide more references for the selection of clinical treatment methods for cisplatin-resistant gastric cancer.Methods:Eighty patients with cisplatin-resistant gastric cancer treated in Harison International Peace Hospital from January 2017 to January 2019 were selected as the study objects,and they were divided into observation group and control group according to random number table method.All of them received improved DOF fortnightly treatment,and trastuzumab adjuvant treatment was added to the observation group on this basis.The serum tumor markers[serum carcinoembryonic antigen(CEA),carbohydrate antigen 19-9(CA19-9),CA72-4],serum neovascular markers[vascular endothelial growth factor(VEGF),pigment epithelial derived factor(PEDF),angiopoietin-2(Ang-2)],biochemical indicators[N-terminal pro B type natriuretic peptide(NT proBNP),aspartate transaminase(AST),blood urea nitrogen(BUN),alanine aminotransferase(ALT)],adverse reactions and survival rate were compared between two groups.This study was approved by the Ethics Committee of Harison International Peace Hospital(number:20160511).Results:After treatment,CEA,CA19-9 and CA72-4 in both groups decreased,and CEA,CA19-9 and CA72-4 levels were lower in the observation group than in the control group with statistical significance(P＜0.01).After treatment,VEGF,PEDF and Ang-2 in two groups decreased,and the difference was statistically significant(P＜0.01).The levels of VEGF,PEDF and Ang-2 were compared between the two groups before and after treatment,and there was no significant difference(P＞0.05).The levels of NT-proBNP,AST,BUN and ALT were compared between the two groups before and after treatment,and there was no statistically significant difference(P＞0.05).The number of patients with fatigue,gastrointestinal reaction and myelosuppression and the total incidence of adverse reactions were compared between the two groups,and there was no statistically significant difference(P＞0.05).At 5 years after treatment,11 cases(27.5%)survived and 29 cases(72.5%)died in the observation group.There were 3 cases(7.5%)of survival and 37 cases(92.5%)of death in the control group.The median survival was 2 years(95%CI:1.8-2.2)in the observation group and 1 year(95%CI:0.6-1.4)in the control group.The survival rate of 1-5 years was higher in the observation group than in the control group.The difference was statistically significant(log-rank χ2 = 13.853,P = 0.001).Conclusion:In the clinical treatment of cisplatin-resistant gastric cancer,trastuzumab assisted modified DOF fortnightly regimen suggests that it can reduce the expression levels of serum tumor markers,improve the 5-year survival rate of patients,and has certain drug safety.
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Objective:To investigate the effect of circ_DCAF6 on the proliferation,migration and apoptosis of colorectal cancer cells resistant to cisplatin(DDP)and its possible mechanism.Methods:SW480 and DDP-resistant cells SW480/DDP were cultured in vitro.The expression of circ_DCAF6 and miR-485-3p in the cells was detected by RT-qPCR,and the expression of FOXK1 protein was detected by Western blotting.After si-NC,si-circ_DCAF6,miR-NC,or miR-485-3p was transfected into SW480/DDP cells,or si-circ_DCAF6 and anti-miR-485-3p were co-trans-fected into SW480/DDP cells,respectively,CCK-8 method was used to detect the effect of DDP at different concentrations(0,0.156,0.625,2.5,10,40,160 p g/mL)on the 24 h survival rate of trans-fected cells,and the half inhibitory concentration(IC50 value)was calculated;Cell migration and apoptosis were detected and the regulatory relationship between circ_DCAF6,miR-485-3p and FOXK1 was verified.Results:Compared with SW480 cells,circ_DCAF6 and FOXK1 protein ex-pressions were increased in SW480/DDP cells,while miR-485-3p expression was decreased(P＜0.05).Silencing circ_DCAF6 or overexpression of miR-485-3p inhibited the proliferation and mi-gration of SW480/DDP cells and the expression of FOXK1 protein,increased the sensitivity of SW480/DDP cells to DDP,and promoted cell apoptosis(P＜0.05).Knockdown of miR-485-3p atten-uated the effects of circ_DCAF6 silencing on the malignant phenotype of SW480/DDP cells.circ_DCAF6 could target miR-485-3p;FOXK1 was the target gene of miR-485-3p.Conclusion:Silencing circ_DCAF6 could inhibit the proliferation and migration of SW480/DDP cells and promote cell apoptosis.Its mechanism of action is related to the regulation of miR-485-3p/FOXK1 axis.
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OBJECTIVE To explore the mechanism of Compound lizard powder reducing cisplatin resistance in gastric cancer by regulating glycolytic activity based on phosphoinositide 3-kinase（PI3K）/protein kinase B（Akt） signaling pathway. METHODS Human gastric cancer MKN45 and MKN45/DDP （cisplatin-resistant） cells were cultured in vitro and intervened with different mass concentrations of cisplatin （0.1， 0.2， 0.4， 0.8， 1.6， 3.2 μg/mL） to detect the survival rate， half inhibitory concentration （IC50） and drug resistance index. MKN45/DDP cells were inoculated subcutaneously in the right anterior axilla of nude mice to prepare a transplanted tumor model of gastric cancer. After successful modeling， they were randomly divided into model group， cisplatin group （0.002 g/kg）， Compound lizard powder group （2.8 g/kg） and combination group （the same dose as each single drug group）， with 8 nude mice in each group. Each administration group was given relevant solution， twice a week （cisplatin， i.p.） or twice a day （Compound lizard powder， i. g.）， for 4 consecutive weeks. During the experiment， the body weight of nude mice was monitored， and tumor volume and inhibitory rate of tumor were calculated. The levels of inflammatory factors （tumor necrosis factor- α， interleukin-6） in tumor tissue， the mRNA and protein expressions of multidrug resistance-associated protein 1 （MRP1）， P-glycoprotein （P-gp）， glucose transporter-1 （GLUT1） and lactate dehydrogenase A （LDHA）， as well as the protein expressions of PI3K， phosphorylated PI3K （p-PI3K）， Akt， phosphorylated Akt （p-Akt）， hexokinase-2 （HK2） and pyruvate kinase M2 （PKM2） were all detected. RESULTS With the intervention of different concentrations of cisplatin， the survival rate of MKN45/DDP-resistant cells was significantly higher than that of MKN45 parent cells （P＜0.05）. IC50 value of MKN45/DDP and MKN45 cells were（1.052 0±0.221 9） and （0.372 1±0.238 0）μg/mL， and the drug resistant index was 2.827. Compared with the model group， cisplatin group， Compound lizard powder group and combination group all had certain inhibitory effects on the tumor growth in nude mice； the inhibitory rates of tumor increased significantly （P＜0.05）； the levels of inflammatory factors， the mRNA and protein expressions of MRP1， P-gp， GLUT1 and LDHA （except for cisplatin group）， the phosphorylation levels of PI3K and Akt protein （except for cisplatin group） as well as the protein expressions of HK2 and PKM2 were decreased significantly， while the combination group was significantly better than the cisplatin group （P＜0.05）. CONCLUSIONS Compound lizard powder may inhibit tumor growth in transplanted tumor model nude mice with gastric cancer-resistant cells by reducing the secretion of tumor-related inflammatory factors， inhibiting the expression of glycolysis， drug resistance-related proteins and genes， inhibiting the activation of the PI3K/Akt signaling pathway， thus having a certain effect of enhancing cisplatin efficacy and reversing drug resistance.
الملخص
ObjectiveTo explore the mechanism of Buzhong Yiqitang-containing serum in alleviating the cisplatin resistance in human non-small cell lung cancer （A549/DDP） cells via regulating the nuclear factor E2-related factor 2 （Nrf2）/reactive oxygen species （ROS） signaling pathway. MethodThe serum containing Buzhong Yiqitang was prepared and A549/DDP cells were cultured and randomly grouped： blank （10% blank serum）， cisplatin （10% blank serum+20 mg·L-1 cisplatin）， Buzhong Yiqitang （10% Buzhong Yiqitang-containing serum+20 mg·L-1 cisplatin）， ML385 （10% blank serum+5 μmol·L-1 ML385+20 mg·L-1 cisplatin）， Buzhong Yiqitang+ML385 （10% Buzhong Yiqitang-containing serum+5 μmol·L-1 ML385+20 mg·L-1 cisplatin）， tertiary butylhydroquinone （TBHQ） （10% blank serum+5 μmol·L-1 TBHQ+20 mg·L-1 cisplatin）， and Buzhong Yiqitang+TBHQ （10% Buzhong Yiqitang-containing serum+5 μmol·L-1 TBHQ+20 mg·L-1 cisplatin）. The median inhibitory concentration （IC50） of cisplatin in each group was determined by the cell counting kit-8 （CCK-8） method and the resistance index （RI） was calculated. The apoptosis rate was detected by flow cytometry. The ROS content of each group was determined with the DCFH-DA fluorescence probe. Western blot was employed to determine the protein levels of Nrf2， cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3）， cytochrome C （Cyt C）， and B-cell lymphoma-2 （Bcl-2）. ResultCompared with those in the cisplatin group， the IC50 and RI of A549/DDP cells to cisplatin in Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 groups decreased （P˂0.05）. Compared with the blank group， the cisplatin， Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 groups showed increased apoptosis rate of A549/DDP cells （P˂0.05）. Compared with the blank group， cisplatin promoted the expression of Nrf2 （P˂0.05）. Compared with the cisplatin group， Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 inhibited the expression of Nrf2 （P<0.05）， elevated the ROS level （P˂0.05）， up-regulated the protein levels of cleaved Caspase-3 and Cyt C， and down-regulated the protein level of Bcl-2 （P<0.05）， which were the most significant in the Buzhong Yiqitang+ML385 group. Compared with the cisplatin group， the TBHQ group showed increased IC50 and RI of cisplatin （P<0.05）， decreased apoptosis rate of A549/DDP cells （P<0.05）， up-regulated protein levels of Nrf2 and Bcl-2 （P<0.05）， lowered level of ROS （P˂0.05）， and down-regulated protein levels of cleaved Caspase-3 and Cyt C （P<0.05）. Compared with the TBHQ group， Buzhong Yiqitang+TBHQ decreased the IC50 and RI of cisplatin in A549/DDP cells （P<0.05）， increased the apoptosis rate （P<0.05）， down-regulated the protein levels of Nrf2 and Bcl-2 （P<0.05）， increased ROS （P˂0.05）， and up-regulated the protein levels of cleaved Caspase-3 and Cyt C （P<0.05）. ConclusionBuzhong Yiqitang induced apoptosis by inhibiting Nrf2/ROS pathway to alleviate cisplatin resistance in A549/DDP cells.
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Objective @#To investigate the reversal effect and mechanism of cinobufagin (CBG) on cisplatin resist- ance in human ovarian cancer cells . @*Methods @#A2780 cell line and its cisplatin-resistant cell line A2780/DDP are common ovarian cancer cells in clinic , so these two cell lines were selected as the research objects . The cell viabil- ity was detected by cell Counting Kit-8 (CCK-8) assay , and the cell proliferation ability was detected by plate clo- ning and 5-ethynyl-2 ′-deoxyuridine (EdU) assay. Hoechst staining was used to observe cell apoptosis . Cell scratch test and Transwell test were used to evaluate cell migration and invasion ability. Western blot and quantitative reverse transcription PCR (RT-qPCR) were used to detect the protein and mRNA expressions of phosphatidylinosi- tol 3-kinase/protein kinase ( PI3K/AKT) signaling pathway and epithelial-mesenchymal transition ( EMT) . @*Results@#Compared with A2780 cells , the drug resistance indexes of A2780/DDP cells were 5 . 636 , 5 . 864 , 5 . 695 , respectively. After treatment of A2780/DDP cells with CBG (2 , 4 , 6 mg/ml) , the reversal resistance indexes were 1 . 617 , 2. 570 , 3 . 461 , respectively. CBG treatment significantly increased the level of apoptosis and inhibi- ted the proliferation , migration and invasion of the cells in a concentration-dependent manner (P < 0. 05) . Western blot results showed that compared with A2780 cells , the relative ratio of P-PI3K/PI3K and P-AKT/AKT protein levels , as well as the protein expression of N-cadherin , Vimentin , and Snail were higher in the control group (A2780/DDP) cells , while the protein expression of E-cadherin was lower ( t P-PI3K/PI3K = 8 . 115 , t P-AKT/AKT = 17. 62 , t N-cadherin = 6. 126 , t Vimentin = 4. 001 , t Snail = 17. 333 , t E-cadherin = 4. 620 , P < 0. 01) ; As the dose of CBG increased , the protein expression levels of P-PI3K , P-AKT , N-cadherin , Vimentin , and Snail in drug-resistant cells de- creased , while the protein expression level of E-cadherin increased ( FP-PI3K = 268. 5 , FP-AKT = 190. 5 , FN-cadherin = 24. 02 , F Vimentin = 57 . 65 , FSnail = 87 . 24 , FE-cadherin = 135 . 8 , P < 0. 05) . qRT-PCR results showed that with the in- crease of CBG concentration , the mRNA expression levels of PI3K , AKT , N-cadherin , Vimentin and Snail de- creased , while the mRNA expression level of E-cadherin gradually increased ( FPI3K = 101 . 1 , FAKT = 558. 3 , FN-cadherin = 86. 97 , F Vimentin = 105 . 9 , FSnail = 85 . 71 , FE-cadherin = 80. 96 , P < 0. 01) .@*Conclusion @#CBG can reverse cisplatin resistance of ovarian cancer A2780/DDP cell line , and its mechanism may be related to the regulation of PI3K/AKT signaling pathway and inhibition of EMT by CBG.
الملخص
OBJECTIVE@#To investigate the role of myosin heavy chain 9 (MYH9) in regulation of cell proliferation, apoptosis, and cisplatin sensitivity of non-small cell lung cancer (NSCLC).@*METHODS@#Six NSCLC cell lines (A549, H1299, H1975, SPCA1, H322, and H460) and a normal bronchial epithelial cell line (16HBE) were examined for MYH9 expression using Western blotting. Immunohistochemical staining was used to detect MYH9 expression in a tissue microarray containing 49 NSCLC and 43 adjacent tissue specimens. MYH9 knockout cell models were established in H1299 and H1975 cells using CRISPR/Cas9 technology, and the changes in cell proliferation cell were assessed using cell counting kit-8 (CCK8) and clone formation assays; Western blotting and flow cytometry were used to detect apoptosis of the cell models, and cisplatin sensitivity of the cells was evaluated using IC50 assay. The growth of tumor xenografts derived from NSCLC with or without MYH9 knockout was observed in nude mice.@*RESULTS@#MYH9 expression was significantly upregulated in NSCLC (P < 0.001), and the patients with high MYH9 expression had a significantly shorter survival time (P=0.023). In cultured NSCLC cells, MYH9 knockout obviously inhibited cell proliferation (P < 0.001), promoted cell apoptosis (P < 0.05), and increased their chemosensitivity of cisplatin. In the tumor-bearing mouse models, the NSCLC cells with MYH9 knockout showed a significantly lower growth rate (P < 0.05). Western blotting showed that MYH9 knockout inactivated the AKT/c- Myc axis (P < 0.05) to inhibit the expression of BCL2- like protein 1 (P < 0.05), promoted the expression of BH3- interacting domain death agonist and the apoptosis regulator BAX (P < 0.05), and activated apoptosis-related proteins caspase-3 and caspase-9 (P < 0.05).@*CONCLUSION@#High expression of MYH9 contributes to NSCLC progression by inhibiting cell apoptosis via activating the AKT/c-Myc axis.
الموضوعات
Animals , Humans , Mice , Apoptosis , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Proliferation , Cisplatin/pharmacology , Cytoskeletal Proteins/metabolism , Lung Neoplasms/metabolism , Mice, Nude , Myosin Heavy Chains/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transductionالملخص
Aim To investigate the effect of silencing circRNA ciRS-7 on cisplatin resistance of gastric cancer cells and its related mechanism. Methods Cisplatin-resistant gastric cancer cells HGC-27/DDP were constructed, ciRS-7 was silenced and HGC-27/DDP cells were treated with cisplatin. Real-time quantitative PCR (qRT-PCR) was used to detect the expression levels of ciRS-7 and microrNA-944 (miR-944). Cell counting kit-8 (CCK-8) was used to detect cell survival rate. Clone formation assay was used to detect the number of clones. Western blot was used to detect the protein expression levels of CyclinD1, proliferating cell nuclear antigen (PCNA), B-lymphocytoma-2-associated X protein (Bax), B-lymphocytoma-2 (Bcl-2) and cleaved aspartate-specific cysteine proteinase 3 (cleaved-caspase-3). Flow cytometry was used to detect cell apoptosis rate. In situ end labeling (TUNEL) was used to detect apoptosis index. Dual luciferase assay was used to verify the targeting relationship between ciRS-7 and miR-944. Results CiRS-7 was highly expressed in cisplatin-resistant gastric cancer tissues and cells, while miR-944 was low expressed in cisplatin-resistant gastric cancer tissues and cells. The survival rate, clonal formation number, protein expression levels of CyclinD1, PCNA and Bcl-2 in cisplatin-treated HGC-27/DDP cells were significantly reduced by silencing ciRS-7, while ciRS-7 expression level, apoptosis rate, apoptosis index, Bax, cleaved-caspase-3 protein expression levels of cisplatin-treated HGC-27/DDP cells were significantly raised. CiRS-7 targetedly inhibited the expression of miR-944. Conclusions Silencing ciRS-7 can reverse cisplatin resistance of gastric cancer cells, and the mechanism may be related to the promotion of miR-944 expression by silencing ciRS-7.
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Objective:To investigate the effects of serum exosomes of patients with gastric cancer on cisplatin resistance, clonal formation, migration, invasion and apoptosis of the AGS gastric cancer cells, and the corresponding molecular mechanisms.Methods:The experimental study was conducted. The exosomes of patients with gastric cancer was separated from their serum, and the expression of lncRNA HOTTIP was analyzed using the quantitative real time polymerase chain reaction (qRT-PCR). Normal gastric epithelial cell line GES1, gastric cancer cell line AGS and human embryonic kidney cell 293T were cultured in vitro. AGS cells were incubated with exosomes (Exo),with phos-phate buffered saline (PBS) treatment as control, and transfected with si-NC or si-HOTTIP-3, named as Exo group, PBS group, si-NC+Exo group, and si-HOTTIP-3+Exo group. The AGS cells were trans-fected with si-NC, si-HOTTIP-1, si-HOTTIP-2, si-HOTTIP-3, oe-HOTTIP, vector, oe-HOTTIP+miR-138-5p mimic, oe-HOTTIP+mimic NC, miR-138-5p inhibitor, inhibitor NC, miR-138-5p inhibitor+si-TJP1 and miR-138-5p inhibitor+si-NC. They were recorded as si-NC group, si-HOTTIP-1 group, si-HOTTIP-2 group, si-HOTTIP-3 group, oe-HOTTIP group, vector group, oe-HOTTIP+miR-138-5p mimic group, oe-HOTTIP+mimic NC group, miR-138-5p inhibitor group, inhibitor NC group, miR-138-5p inhibitor+si-TJP1 group and miR-138-5p inhibitor+si-NC group. The 293T cells transfected with mimic NC+HOTTIP wt, miR-138-5p mimic+HOTTIP wt, mimic NC+HOTTIP mut, miR-138-5p mimic+HOTTIP mut, mimic NC+TJP1 3'UTR wt, miR-138-5p mimic+TJP1 3'UTR wt, mimic NC+TJP1 3'UTR mut, miR-138-5p mimic+TJP1 3'UTR mut were recorded as the mimic NC+HOTTIP wt group, miR-138-5p mimic+HOTTIP wt group, mimic NC+HOTTIP mut group, miR-138-5p mimic+HOTTIP mut group, mimic NC+TJP1 3'UTR wt group, miR-138-5p mimic+TJP1 3'UTR wt group, mimic NC+TJP1 3'UTR mut group, miR-138-5p mimic+TJP1 3'UTR mut group. The cell counting kit-8 (CCK8) was used to analyze the cisplatin sensitivity of gastric cancer cells. The colony formation experiment was used to analyze the colony formation of gastric cancer cells. The Transwell experiment was used to analyzed cell migration and invasion of gastric cancer cells. The flow cytometry experiment was used to analyze cell apoptosis of gastric cancer cells. The Western bolt assay was used to analyze the expression of exosome marker proteins, including the CD63 and CD81, and the protein of TJP1, the drug-resistance related proteins, including the P-gp and MCL-1. The dual-luciferase assay was used to analyze the targeted relationships among lncRNA HOTTIP, miR-138-5p and TJP1. Observation indicators: (1) expression of lncRNA HOTTIP; (2) resistance of gastric cancer cells to cisplatin regulated by exosome-mediated lncRNA HOTTIP; (3) regulation of cisplatin resistance in gastric cancer cells mediated by miR-138-5p through lncRNA HOTTIP overexpression; (4) targeting of TJP1 gene 3′-untranslated region (UTR) by miR-138-5p; (5) regulation of cisplatin resistance in gastric cancer cells by TJP1 through miR-138-5p inhibition. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the t test. The one-way ANOVA was used for comparison for multiple groups and the Tukey′s test was used for further pairwise compari-son. Count data were described as absolute numbers, and the chi-square test was used for comparison. Correlation analysis was conducted using the Pearson′s test. Results:(1) Expression of lncRNA HOTTIP. The expression of lncRNA HOTTIP in the serum exosome of patients with gastric cancer was higher than that in healthy volunteers, showing a significant difference ( P<0.05). Results of transmi-ssion electron microscopy examination showed that the serum exosomes were circular or oval in shape. Results of Western bolt assay showed the expression of marker proteins of CD63 and CD81 in serum exosomes. (2) Resistance of gastric cancer cells to cisplatin regulated by exosome-mediated lncRNA HOTTIP. Compared with the PBS group, the biochemical half maximal inhibitory concentra-tion (IC50), the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the Exo group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). Compared with the si-NC+Exo group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the si-HOTTIP-3+Exo group decreased, while the cell apoptosis rate increased, showing significant differences between them ( P<0.05). (3) Regulation of cisplatin resistance in gastric cancer cells mediated by miR-138-5p through lncRNA HOTTIP overexpression. Compared with the vector group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the oe-HOTTIP group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). Compared with the oe-HOTTIP+mimic NC group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the oe-HOTTIP+miR-138-5p mimic group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). (4) Targeting of TJP1 gene 3′-UTR by miR-138-5p. Results of dual-luciferase assay showed that the luciferase activity in 293T cells treatment with mimics of control+vectors of wild type of TJP1 gene 3′-UTR and 293T cells treatment with mimics of miR-138-5p+vectors of wild type of TJP1 gene 3′-UTR was 1.00±0.09 and 0.21±0.03, respectively, showing a significant difference between them ( t=15.02, P<0.05). (5) Regulation of cisplatin resistance in gastric cancer cells by TJP1 through miR-138-5p inhibition. Compared with the inhibitor group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the miR-138-5p inhibitor group increased, while the cell apoptosis rate decreased, showing significant differences between them ( P<0.05). Compared with the miR-138-5p inhibitor+si-NC group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P-gp protein, expression of MCL-1 protein in the miR-138-5p inhibitor+si-TJP1 group decreased, while the cell apoptosis rate increased, showing significant differences between them ( P<0.05). Conclusion:Serum exosomes-mediated lncRNA HOTTIP can promote cisplatin resistance, clonal formation, migration, invasion and apoptosis of gastric cancer cells and inhibit cell apoptosis of gastric cancer cells through regulating the expression of miR-138-5p/TJP1.
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@#Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck. In recent years, the incidence rate has been increasing. Mitochondria are dynamic organelles involved in various cell behaviors in eukaryotic cells. Mitochondrial dysfunction is closely related to tumor development. As a switch that determines cancer cell death, targeting mitochondria has become the focus of OSCC treatment. This article reviews the relationship between mitochondria and tumorigenesis and development, OSCC treatment, and cisplatin resistant OSCC. Current studies have found that mitochondrial dysfunction promotes cell carcinogenesis, and the mitochondrial morphology and function of cancer cells are significantly changed. The increase of mitochondrial fission improves the invasiveness of cancer cells, and mitophagy dysfunction can induce cancer cell apoptosis. The emergence of drugs and the development of nanotechnology in targeted drug delivery systems have opened up new methods for targeting mitochondria to treat OSCC, reducing the side effects of systemic medication. The cisplatin resistance of OSCC is generated through the mitochondrial pathway, and the mitochondrial function and mutation mechanism of mitochondrial DNA are clarified in order to provide new ideas for targeting mitochondria to treat cisplatin resistant OSCC.
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ObjectiveTo investigate the inhibitory effect of Astragalus polysaccharide （APS） on epithelial-mesenchymal transition （EMT） induced by transforming growth factor-β1 （TGF-β1） in cisplatin （DDP）-resistant lung adenocarcinoma cell line A549/DDP cells transplanted into nude mice and the molecular mechanism in improving DDP resistance. MethodBALB/c nude mice were randomly divided into a blank group， a model group， a DDP group， and a combination group （APS combined with DDP）. A549/DDP cells were infected with TGF-β1-overexpressed lentiviral vector and the negative control. The infected cells were inoculated subcutaneously in nude mice. The A549/DDP cells with TGF-β1 gene overexpression were inoculated into all groups except the control group with negative TGF-β1 gene overexpression. The drug intervention was performed eight days after cell inoculation. The mice in the combination group received intragastric administration of APS （0.3 g·kg-1·d-1） and intraperitoneal injection of cisplatin （0.003 5 g·kg-1）， and those in the cisplatin group received intraperitoneal injection of cisplatin （0.003 5 g·kg-1）. After 32 days of cell inoculation， the nude mice were killed and the tumor tissues and lungs were collected. The tumor weight was recorded and the inhibition rate was calculated. The number of metastatic nodules of the lung tumor on the whole slide was counted under the microscope. Immunohistochemistry， Western blot， and real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） were used to detect the protein and gene expression of EMT molecular markers α-catenin and N-cadherin， and tumor drug resistance markers human lung resistance protein （LRP）， multidrug resistance-associated protein （MRP）， and P-glycoprotein （P-gp） in the transplanted tumor. ResultCompared with the blank group， the model group showed increased tumor weight and metastatic nodules of the lung tumor （P<0.05）， decreased protein and mRNA expression of α-catenin （P<0.05）， and elevated protein and mRNA expression of N-cadherin， LRP， MRP， and P-gp （P<0.05）. Compared with the model group and the cisplatin group， the combination group showed reduced tumor weight and metastatic nodules of the lung tumor （P<0.05）， increased protein and mRNA expression of α-catenin （P<0.05）， and decreased protein and mRNA expression of N-cadherin， LRP， MRP， and P-gp （P<0.05）. ConclusionAPS can inhibit the growth and metastasis of the transplanted tumor of lung adenocarcinoma and improve cisplatin resistance， which may be related to the inhibition of EMT of tumor cells.
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Objective:To explore the effects of miR-485-5p on cisplatin-resistant ovarian cancer cells and it’s mechanism.Methods:RT-qPCR was used to detect the expression of miR-485-5p in human normal ovarian epithelial cell line (IOSE-80) and ovarian cancer cell lines (A2780,SKOV3,OVCAR3,OVCA433) . Cisplatin (DDP) -resistant ovarian cancer cells were constructed and the expression of miR-485-5p and EGFR was detected. CCK8 assay was used to detect cell proliferation ability in each group. Cell apoptosis was measured by flow cytometry.Results:Compared with IOSE-80 cell, miR-485-5p expression was decreased in each ovarian cancer cell lines, while the expression of EGFR was increased (all P<0.05) .The expression of miR-485-5p in SKOV3/DDP cell lines was further decreased while EGFR expression was further increased than that in SKOV3 cells ( P<0.05) . Transfection of miR-485-5p mimic into SKOV3/DDP cells could inhibit cell proliferation and induce apoptosis, but the results were reversed when cells were transfected with miR-485-5p inhibitor (all P<0.05) . The proliferation was increased while apoptosis was decreased in EGFR transfected SKOV3/DDP cells, but this effects was partially offseted by miR-485-5pmimic. Conclusion:miR-485-5p participates in the regulation of cisplatin resistance of ovarian cancer cells, and overexpression of miR-485-5p can promote the chemosensitivity of ovarian cancer, which may be achieved through negative regulation of EGFR.
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Objective To explore the related genes that play a key regulatory role in cisplatin resistance in lung adenocarcinoma. Methods Bioinformatics methods were used to download the differentially-expressed genes between cisplatin sensitive group and drug resistant group in patients with lung adenocarcinoma in TCGA database and GDSC database. GO function analysis and KEGG pathway enrichment analysis were carried out to analyze the differentially-expressed genes. The protein-protein interaction network was constructed and hierarchical cluster analysis was carried out to screen the key genes. The key genes were verified at the cell level by real-time fluorescence quantitative PCR and ELISA. Then the expression of the selected key gene in A549/DDP cells was silenced by siRNA and its sensitivity to cisplatin was detected. Results We screened out 178 differentially-expressed genes. After cluster analysis, CXCL9, CXCL10, NKX2-1 and SFTPA1 were regarded as the key genes of cisplatin resistance in lung adenocarcinoma. CXCL10 was temporarily selected for subsequent verification and function experiment. The mRNA expression of CXCL10 in A549/DDP cells was significantly higher than that in A549 cells (P < 0.001), and the expression of CXCL10 protein in the supernatant of A549/DDP cells was higher than that in A549 cells, which were consistent with the prediction of bioinformatics. The sensitivity of A549/DDP cells to DDP increased after silencing CXCL10 expression. Conclusions CXCL10 is a key gene to regulate cisplatin resistance in lung adenocarcinoma. Downregulating the expression of CXCL10 can become a potential target for reversing cisplatin resistance in lung adenocarcinoma.
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@#Objective To reveal the potential mechanism of cisplatin resistance in non-small cell lung cancer A549 cells by comparing the expression profiles of wild-type A549 cells and cisplatin-resistant A549 cells (A549/DPP) through whole transcriptome sequencing analysis. Methods The cisplatin resistant A549 (A549/DDP) cell line was first established. Then, the whole-transcriptome analysis was conducted both on A549 and A549/DDP cells. Next, the differentially expressed RNAs of lncRNA-seq, circRNA-seq, and miRNA-seq data were identified, respectively, followed by functional enrichment analysis. Finally, a comprehensive analysis based on the whole transcriptome data was performed and the construction of the ceRNA network was carried out. Results A total of 4 517 lncRNA, 123 circRNA, and 145 miRNA were differentially expressed in A549/DDP cells compared with the A549 cell line. These different RNAs were significantly enriched in cancer-related pathways. The ceRNA network contained 12 miRNAs, 4 circRNAs, 23 lncRNAs, and 9 mRNA nodes, of which hsa-miR-125a-5p and hsa-miR-125b-5p were important miRNAs based on the topological analysis. Conclusion Tumor necrosis factor signaling pathway and p53 signaling pathway are involved in A549/DPP resistance. Hsa-miR-125a-5p and hsa-miR-125b-5p may be potential targets for reversing cisplatin resistance.
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AIM: To study the effects and mechanism of down-regulating lncRNA LINC00176 on cisplatin resistance and autophagy in lung cancer A549/DDP cells. METHODS: The qRT-PCR method was used to determine the expression changes of LINC00176 in normal bronchial epithelial 16HBE cells and lung cancer A549, A549/DDP, NCI-H1299 and SK-MES-1 cells. A549/DDP cells were divided into Control group, si-NC group, and si-LINC00176 group, si-LINC00176+ Anti-miR-NC group, and si-LINC00176+ Anti-miR-138-5p group. MTT experiment detected the half inhibitory concentration (IC
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Actin-like 6A (ACTL6A), also known as BAF53A, is an SWI / SNF subunit of chromatin-remodeling factors and plays an important role in regulating stem cell function. Recent studies found that ACTL6A was involved in tumor occurrence and development. However, the mechanism of ACTL6A in cisplatin resistance is still unclear. This study investigated the biological function and molecular mechanism of ACTL6A in maintaining cancer stem cell function and cisplatin resistance. First, analysis from TCGA, GEO, and GEPIA databases showed that ACTL6A expression levels in lung adenocarcinoma (LUAD) tissues and cisplatin resistant cells were dramatically higher than that in adjacent normal tissues and cisplatin sensitive cells (P < 0. 05), and ACTL6A high expression was positively associated with a poor prognosis of LUAD. Knockdown of ACTL6A enhanced cisplatin sensitivity (P < 0. 05), reduced tumor sphere (P<0. 05), inhibited cell migration (P<0. 05), and promoted cell apoptosis (P<0. 05) in A549 cells. Western blotting showed that knockdown of ACTL6A increased the protein expression of E-cadherin, and decreased the protein expression of N-cadherin, vimentin, and twist. Moreover, knockdown of ACTL6A inhibited the expression of cancer stem cell markers, including ALDH3A1, ALDH4A1, SOX2, OCT4, and Nanog. Subsequently, Hippo / YAP signaling-related proteins were analyzed by Western blotting. The results showed the expression of beta-TRCP and YAP was decreased in A549 cell with knockdown of ACTL6A. However, phosphorylation levels at S127 and S397 of YAP were increased and inhibited translocation of YAP into the nucleus for regulating related gene expression. In summary, ACTL6A maintained the stemness of lung cancer stem cells and promoted cisplatin resistance in A549 cells by inhibiting activation of the Hippo signaling pathway.
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Objective: To investigate the effect of Buzhong Yiqi Tang on cisplatin resistance tumor cells A549/DDP in nude mice with lung adenocarcinoma, and determine the optimal concentration. Method: The 36 BALB/C nude mice were randomly divided into no-load group, model group, cisplatin group, low-dose Buzhong Yiqi Tang group (1.46 g · kg-1), medium-dose Buzhong Yiqi Tang group (2.92 g · kg-1) and high-dose Buzhong Yiqi Tang group (5.84 g · kg-1). The no-load group was inoculated with A549/DDP cells transfected by uninfected lentiviral transforming growth factor-β1(TGF-β1), and the other groups were inoculated with TGF-β1 over-expression A549/DDP cells. After the intervention, the nude mice were put to death. The tumors of mice were weighed, and the inhibition rate was calculated, liver, spleen and lung metastases of tumors were counted under microscope, and pathological observation was made for lung histomorphological changes. Immunohistochemistry, Western blot and Real-time polymerase chain reaction(Real-time PCR) were used to detect the expressions of lung resistance-related protein (LRP), multidrug resistance-related protein (MRP), P-glycoprotein (P-gp) in transplantation tumor. Result: Compared with the model group, the weight of tumors and the number of pulmonary metastatic nodules decreased in the Buzhong Yiqi Tang intervention group. With the increase of the dosage of Buzhong Yiqi Tang, the weight of tumors gradually decreased, while the inhibition rate increased. The effect of the high-dose group was the most significant (PPPConclusion: Buzhong Yiqi Tang can inhibit the growth and metastasis of transplanted tumors, alleviate lung injury and improve the drug resistance of A549/DDP to cisplatin, with a certain dose-effect relationship. High-dose concentration (5.84 g ·kg-1) was the best concentration in nude mice.
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Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a human oncoprotein that is overexpressed in multiple kinds of cancers including non-small cell lung cancer (NSCLC). CIP2A plays an 'oncogenic nexus' to participate in the tumorigenesis and chemoresistance in several cancer types. AKT and mTORC1 overactivation are detected in NSCLC and many other cancers. Previous studies found that the CIP2A/AKT/mTOR pathway controls cell growth, apoptosis, autophagy process. Polyphyllin I (PPI) and polyphyllin VII (PPVII) are natural components extracted from Paris polyphylla that display anti-cancer properties. In the present study, we investigated whether PPI and PPVII can be used in the cisplatin (DDP)-resistant human NSCLC cell line A549/DDP. Results demonstrated that PPI and PPVII treatment significantly suppressed A549/DDP cell proliferation, migration, invasion and EMT, induced apoptosis and autophagy. Further examination of the mechanism revealed that the PPI and PPVII significantly upregulated the p53, induced caspase-dependent apoptosis and suppressed the CIP2A/AKT/mTOR pathway. The activation of autophagy was mediated through PPI and PPVII induced inhibition of mTOR. We propose that PPI and PPVII might be developed as candidate drugs for DDP-resistant NSCLC.
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Objective Gastric cancer is the most common cancer in the world. In China, Patients with gastric cancer are mostly treated with platinum-based chemotherapy. Programmed cell death 4 (PDCD4) was found as an important proapoptosis recently, the aim of the present study was to investigate the role of PDCD4 reversed the apoptosis induced by cisplatin in gastric cancer cell. The study will provide the target marker for treatment and diagnosis of cisplatin resistance in gastric cancer.Methods Stable transfection with pCMV-PDCD4 vector into human cisplatin resistance gastric cancer cell line-SGC7901/DDP; the cells were divided into control group, over-expression group, control with cisplatin group, over-expression with cisplatin group for following experiments. Hoechst dying with immunofluorescence and flow cytometry were used to measure the cell apoptosis in vitro; Real-time PCR was used to detect the mRNA expression levels of PDCD4, and the protein levels of PDCD4, pAK, pGSK3β, BCL-2 and Bak were detected by Western blot. The cells were divided into vector group, PDCD4 group, PDCD4 with activator group for detect the level of PARP(C) by Western blot.Results Compared with control group, the Results of real-time PCR and western blot were showed the level of PDCD4 was augmented in over-expression group (also in the over-expression with cisplatin group), which was indicated stable transfection with PDCD4 was successful. Immunofluorescence (with hoechst dying) and flow cytometry demonstrated that PDCD4 facilitated cell apoptosis exposed to cisplatin. PDCD4 overexpression attenuated the protein levels of pAkt, pGSK3β and BCL-2, but increased the protein levels of BAK. Furthermore, incubation with SC-79 (the activator of Akt) reversed cell apoptosis induced by PDCD4.Conclusion Overexpression of PDCD4 promotes the apoptosis induced by cisplatin through pAKT/pGSK3β pathway, which is favorable to reverse cisplatin resistance in gastric cancer.
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@#Objective: To study the regulatory effects and possible mechanism of long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) on chemotherapy sensitivity to cisplatin (DDP) of colorectal cancer (CRC)．Methods: A total of 112 pairs of matched cancer and adjacent non-cancerous tissues were obtained from the CRC patients who underwent surgical resection in the First Affiliated Hospital of Xinxiang Medical University betweenApril 2006 and March 2011.All specimens were confirmed by pathological examinations. Tumor tissues and corresponding adjacent non-cancerous tissues from 30 cisplatin-sensitive CRC patients and 30 cisplatin-resistant patients were selected. Human CRC cell lines (HT29, SW480, HCT116, RKO and LoVo) and normal colonic epithelial cell line NCM460 were also collected for this study; and DDP-resistant RKO/DDP and LoVo/DDP cell lines were constructed. siPVT1, siNC, LV-PVT1 and LV-NC were transfected into LoVo and RKO cells or LoVo/DDP and RKO/DDP cells using lipofectamineTM2000. The expression of lncRNA PVT1 in CRC tissues and cells was tested by Real-time qPCR. CCK-8 assay, flow cytometry and WB were performed to test the effect of PTV1 knockout or enforcement on cell proliferation, apoptosis and expressions of apoptosis-related proteins, respectively. The CRC subcutaneous transplanted xenograft model was established on athymic nude mice to study the effect of PVT1 over-expression on tumor growth and DDP resistance. Results: PVT1 was highly expressed in the cancer tissues and CRC cells, and its expression was positively associated with cisplatin resistance of CRC. After knockdown of PVT1, the proliferation of cisplatinresistant CRC cells was significantly suppressed, while the apoptosis was significantly enhanced (P<0.05 or P<0.01); Mechanically, the levels of drug resistance-associated molecules, including MDR1 and MRP1, as well as the expression of anti-apoptotic Bcl-2 were significantly downregulated whereas the levels of pro-apoptotic Bax and cleaved caspase-3 were increased in PVT1-silenced DDP-resistant CRC cells. Over-expression of PVT1 reversely increased proliferation and decreased apoptosis of CRC cells (P<0.05 or P<0.01). In addition, PVT1 over-expression in CRC cells significantly promoted DDP-resistance in vivo (P<0.05). Conclusion: Collectively, knockdown of PVT1 expression can significantly suppress cell proliferation and promote apoptosis of DDP-resistant CRC cells. Overexpression of PVT1 can significantly promote the growth of CRC cells in vitro and transplanted xenograft in vivo. PVT1 regulates endogenous apoptosis pathways and further promotes the sensitivity of CRC cells to cisplatin chemotherapy via inhibiting the expressions of MDR1 and MRP1.
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Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.