Drug Resistance Of Gastric Cancer Cells Research Articles

Exosomal circ50547 as a potential marker and promotor of gastric cancer progression via miR-217/HNF1B axis

BackgroundExosomes, one of small extracellular vesicles, play a vital role in cell to cell communication and contribute to the advancement of tumors through their cargo molecules. Exosomal circRNAs have emerged as significant players in various types of tumors. Thus, this study aimed to investigate how exosomal circRNAs are involved in the diagnosis and progression of gastric cancer (GC). MethodsSerum exosomes were characterized using transmission electron microscopy, nanoparticle tracking analysis and Western blot. CCK-8, colony formation and transwell assays were conducted to study the function of hsa_circ_0050547 (named as circ50547). qRT-PCR was used to quantify the expression of circ50547 in GC tissues and serum exosomes. Fluorescence in situ hybridization was applied to detect the cellular distribution of circ50547. Stemness and drug-resistance were detected by sphere formation, WB, flow cytometry and half-maximal inhibitory concentration analyses. Bioinformatic analyses, luciferase experiments, qRT-PCR and WB were used to investigate molecular mechanisms. ResultsWe discovered for the first time a new type of GC-derived exosomal circRNA, circ50547. We found that circ50547 is highly expressed in both GC tissues and serum exosomes. Interestingly, we observed that the diagnostic value of exosomal circ50547 is superior to that of serum circ50547. Circ50547 overexpression enhanced the proliferation, migration, invasion, stemness and drug resistance of GC cells, while knockdown of circ50547 showed the opposite effect. Mechanistically, circ50547 acted as a sponge for miR-217 to regulate the expression of HNF1B, which promoted gastric cancer progression. ConclusionExosomal circ50547 may be a promising marker for the diagnosis and prognosis prediction of GC. These findings suggest that it plays an oncogenic role through miR-217/HNF1B signaling pathway in GC.

Inhibition of PI3K/Akt/mTOR Signaling Pathway Suppresses 5-Fluorouracil Resistance in Gastric Cancer.

At present, 5-Fluorouracil (5-FU) is a crucial anti-cancer drug and is widely used for the treatment of various carcinomas, including gastric cancer (GC). The resistance of GC cells to 5-FU is still a matter of great concern. To illustrate the role of PI3K/Akt/mTOR signaling in regulating the cell cycle progression and migration of 5-FU-resistant GC cells. After the establishment of drug-resistant GC cell lines, the effects of 5-FU and/or BEZ235 (the dual inhibitor of PI3K and mTOR) on the activity of parental or drug-resistant GC cells were explored. The viability and localization of GC cells (MKN-45 and MKN-74) and their drug-resistant cells (MKN-45/R and MKN-74/R) were assessed using MTT assays and immunofluorescence staining. The impacts of 5-FU and/or BEZ235 on GC cell cycle progression and cell migration were assessed via flow cytometry analyses and wound healing assays, respectively. GC tissues were collected from patients with GC sensitive or refractory to 5-FU chemotherapy. RT-qPCR and western blot were conducted to measure PI3K, AKT, and mTOR levels in GC cells or tissues. After 5-FU treatment, GC cells displayed 5-FU resistance and the viability of drug-resistant cells (MKN-45/R and MKN-74/R) was higher than that of parental cells (MKN-45 and MKN-74). The IC50 values for MKN-45 and MKN-45/R were 8.93 ug/ml and 140 ug/ml, and the values for MKN-74 and MKN-74/R were 3.93 ug/ml and 114.29 ug/ml. Additionally, the PI3K/Akt/mTOR signaling pathway was activated in drug-resistant GC cells and tumor tissues of patients refractory to 5-FU chemotherapy, as evidenced by high PI3K, Akt, and mTOR levels in MKN-45/R, MKN-74/R, and GC tissues resistant to 5-FU. BEZ235 promoted cell cycle arrest and suppressed the migration of GC cells. Moreover, the combination of BEZ235 and 5-FU led to more effective suppressive influence on cell cycle progression and cell migration relative to the single 5-FU or BEZ235 treatment. Silencing of the PI3K/Akt/mTOR signaling pathway suppressed the 5-FU resistance of GC cells.

Influence of Oxa-Nano-Liposome on the Drug Resistance of Gastric Cancer Cells Under p53-Mediated Autophagy

The influence of oxaliplatin (Oxa)-nano-liposomes on the drug resistance of gastric cancer cells (GCCs) and the role of p53-mediated autophagy in this process were investigated in this research. Oxa-nano-liposomes were prepared and their quality was evaluated. GCCs treated with Oxa-nano-liposomes were selected and rolled into a negative control (NC) group (cells+ culture medium), a positive control (PC) group (standard Oxa-nano-liposome), and a Oxa-nano-liposome sample group. Cell inhibition rates (IRs) at changeable drug concentrations (DCs) were compared and analyzed. Furthermore, levels of p53 and autophagy-related proteins (ARPs) (such as LC3-II and p62) in the cells were assessed using Western blotting. The results indicated that Oxa-nano-liposomes prepared (Oxa):natural soy phospholipids (NSP):cholesterol:polyethylene glycol (PEG) 2000 = 1:2:1:1 exhibited the best performance. The Oxa-nano-liposome sample group exhibited a higher cell IR to the NC group, showing a great difference (P <0.05). Additionally, the Oxa-nano-liposome sample group demonstrated superior efficacy compared to the PC group. With increasing DC, p53 and LC3-II were upshifted, while p62 was downshifted. In conclusion, Oxa-nano-liposomes effectively inhibited the growth of GCCs, exhibited improved efficacy, and contributed to reducing drug resistance in GCCs towards Oxa-nano-liposomes. Therefore, the Oxa-nano-Liposomes hold significant potential for clinical application. Moreover, p53 regulated the cellular autophagy, enhancing autophagic activity of GCCs.

A transverse colon lipoma complicated with an adenoma is successfully and safely resected en bloc by endoscopic mucosal resection during water immersion

The B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is associated with the progression of gastric cancer (GC). However, its role in drug resistance of gastric cancer stem cell (GCSC) remains unclear. This study aimed to explore the biological function of BMI-1 in GC cells and its role in drug resistance of GCSCs.We assessed BMI-1 expression in the GEPIA database and in our collected samples from patients with GC. We silenced BMI-1 using siRNA to study the cell proliferation and migration of GC cells. We also used Hoechst 33342 staining to verify the effect of adriamycin (ADR) on side population (SP) cells, and measured the effects of BMI-1 on the expression of N-cadherin, E-cadherin, and drug-resistance-related proteins (multidrug resistance mutation 1 and lung resistance-related protein). Finally, we analyzed BMI-1-related proteins uing the STRING and GEPIA databases.BMI-1 mRNA was upregulated in GC tissues and cell lines, especially in MKN-45 and HGC-27 cells. Silencing BMI-1 reduced the proliferation and migration of GC cells. Knocking down BMI-1 significantly decreased epithelial-mesenchymal transition progression, expression levels of drug-resistant proteins, and the number of SP cells in ADR-treated GC cells. Bioinformatics analysis showed that EZH2, CBX8, CBX4, and SUZ12 were positively correlated with BMI-1 in GC tissues.Our study demonstrates that BMI-1 affects the cellular activity, proliferation, migration, and invasion of GC cells. Silencing the BMI-1 gene significantly reduces the number of SP cells and the expression of drug-resistant proteins in ADR-treated GC cells. We speculate that inhibition of BMI-1 increases the drug resistance of GC cells by affecting GCSCs, and that EZH2, CBX8, CBX4, and SUZ12 may participate in BMI-1-induced enhancement of GCSC-like phenotype and viability.

Abstract 1749: The synergic effect of cisplatin and a chemokine receptor antagonist sensitizes drug resistant gastric cancer cells and inhibits tumoroid formation

Abstract Introduction: Gastric cancer (GC) is an important cause of death worldwide. Cisplatin (CDDP) is the most used drug in the chemotherapy of advanced GC. However, CDDP resistance reduces GC survival. The CCR5/CCL5 axis has been associated with GC development, but its role in CDDP resistance in GC has not been elucidated. The aim of this study was to determine the effects of CCR5/CCL5 axis blockade by a chemokine receptor antagonist (CRA), alone and in combination with CDDP, on in vitro and 3D tumorid models from AGS R-CDDP cells. Methodology: AGS R-CDDP cells (CDDP-resistant human gastric adenocarcinoma cells) were previously established using a stepwise drug dosing protocol and characterized. CCL5 was selected through transcriptomic analysis and its expression level was validated by qRT-PCR. Cytotoxicity assays were determined by using MTT. Apoptosis and cell cycle assays were evaluated by flow cytometry. Tumoroid formation was performed from AGS R-CDDP cells in low adhesion plates (NunclonTMSpheraTM). After 10 days in culture, tumoroid >150 µm in diameter were scored and pharmacological stimuli were added on days 14, 17 and 20. CRA was used alone and in combination with CDDP in all assays. Results: CRA/CDDP combination triggered a re-sensitized phenotype in AGS R-CDDP cells, decreasing cell viability but not increasing apoptosis compared to independent treatments. AGS R-CDDP cells treated with CRA/CDDP were mainly arrested in S phase. CCL5 showed a decrease in mRNA levels after the CRA/CDDP combination compared to separate treatments. AGS R-CDDP cells showed a higher potential in the ability to form tumoroids compared to AGS WT cells. Finally, CRA/CDDP combination inhibited tumoroid formation compared to independent treatments. Discussion and Conclusion: CCL5 showed a decrease in mRNA levels after the CRA/CDDP combination, possibly correlating with allosteric inhibition of the CCR5 receptor. CRA/CDDP combination has a synergic effect that sensitizes AGS R-CDDP cells and inhibits tumoroid formation. This combination could be used as a potential coadjuvant in GC therapy allowing to reduce the doses of CDDP and therefore to reduce side effects. Citation Format: Bárbara Mora-Lagos, María Elena Reyes, Lorena Lobos-Gonzalez, Matías del Campo, Carmen Gloria Ili, Kurt Buchegger, Yuselin Mora, Louise Zanella, Ismael Riquelme, Priscilla Brebi. The synergic effect of cisplatin and a chemokine receptor antagonist sensitizes drug resistant gastric cancer cells and inhibits tumoroid formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1749.

B-cell-specific Moloney murine leukemia virus integration site 1 knockdown impairs adriamycin resistance of gastric cancer cells

Background and study aimsThe B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is associated with the progression of gastric cancer (GC). However, its role in drug resistance of gastric cancer stem cell (GCSC) remains unclear. This study aimed to explore the biological function of BMI-1 in GC cells and its role in drug resistance of GCSCs. Patients and methodsWe assessed BMI-1 expression in the GEPIA database and in our collected samples from patients with GC. We silenced BMI-1 using siRNA to study the cell proliferation and migration of GC cells. We also used Hoechst 33342 staining to verify the effect of adriamycin (ADR) on side population (SP) cells, and measured the effects of BMI-1 on the expression of N-cadherin, E-cadherin, and drug-resistance-related proteins (multidrug resistance mutation 1 and lung resistance-related protein). Finally, we analyzed BMI-1-related proteins uing the STRING and GEPIA databases. ResultsBMI-1 mRNA was upregulated in GC tissues and cell lines, especially in MKN-45 and HGC-27 cells. Silencing BMI-1 reduced the proliferation and migration of GC cells. Knocking down BMI-1 significantly decreased epithelial-mesenchymal transition progression, expression levels of drug-resistant proteins, and the number of SP cells in ADR-treated GC cells. Bioinformatics analysis showed that EZH2, CBX8, CBX4, and SUZ12 were positively correlated with BMI-1 in GC tissues. ConclusionOur study demonstrates that BMI-1 affects the cellular activity, proliferation, migration, and invasion of GC cells. Silencing the BMI-1 gene significantly reduces the number of SP cells and the expression of drug-resistant proteins in ADR-treated GC cells. We speculate that inhibition of BMI-1 increases the drug resistance of GC cells by affecting GCSCs, and that EZH2, CBX8, CBX4, and SUZ12 may participate in BMI-1-induced enhancement of GCSC-like phenotype and viability.

RUNX2 Reverses p53-Induced Chemotherapy Resistance in Gastric Cancer.

Gastric cancer is one of the most common malignancies worldwide; however, its overall mortality has not improved significantly over the last decade. Chemoresistance plays a critical role in this issue. This study aimed to clarify the role and mechanism of runt-related transcription factor 2 (RUNX2) in platinum-based chemotherapy resistance. First, a drug-resistant model of gastric cancer cells was established to evaluate the relative expression level of the RUNX2 as a potential biomarker of chemotherapy resistance. Next, exogenous silencing was conducted to study whether RUNX2 could reverse drug resistance and understand the underlying mechanisms. Simultaneously, the correlation between the clinical outcomes of 40 patients after chemotherapy and the RUNX2 expression levels in tumor samples was analyzed. We discovered that RUNX2 was significantly expressed in drug-resistant gastric cancer cells and tissues; it was also reversibly resistant to transformation treatment by exogenous RUNX2 silencing. It is confirmed that RUNX2 negatively regulates the apoptosis pathway of the p53 to reduce the chemotherapeutic effects of gastric cancer. RUNX2 is a possible target for platinum-based chemotherapy resistance.

Thiostrepton confers protection against reactive oxygen species-related apoptosis by restraining FOXM1-triggerred development of gastric cancer.

Gastric cancer is a leading cause of tumor-associated death worldwide. Metastasis and chemoresistance are crucial barriers for gastric cancer treatment. The Forkhead Box M1 (FOXM1) transcription factor has been reported as a promising treatment target for various types of tumors, but its effects on gastric cancer progression are not fully understood. In the present study, we found that FOXM1 expression levels were significantly up-regulated in human gastric cancer cell lines and tissues, and its expression was much higher in patients with metastasis. We then found that suppressing FOXM1 with its inhibitor thiostrepton (THIO) significantly reduced the proliferation of gastric cancer cells, while induced G0/G1 and apoptosis. Moreover, reactive oxygen species (ROS) production, mitochondrial impair and autophagy were remarkably provoked in gastric cancer cells treated with THIO, which were required for the regulation of apoptotic cell death. Furthermore, THIO exposure considerably suppressed the migration, invasion and angiogenesis in gastric cancer cells. The inhibitory effects of THIO on tumor growth and metastasis were confirmed in an established gastric cancer xenograft mouse model without detectable toxicity. Intriguingly, our in vitro studies showed that the anti-cancer effects of THIO on gastric cancer were almost abolished upon FOXM1 over-expression, indicating the necessity of FOXM1 suppression in THIO-inhibited tumor growth. In addition, higher FOXM1 expression was detected in gastric cancer cells with chemoresistance. Both in vitro and in vivo studies illustrated that THIO strongly promoted the drug-resistant gastric cancer cells to chemotherapies, proved by the considerably decreased cell proliferation and epithelial-mesenchymal transition (EMT) process. Together, these findings revealed that FOXM1 was a promising therapeutic target for gastric cancer treatment, and THIO exerted potential as an therapeutic agent for the disease.

Low SP1 SUMOylation-dependent SNHG17 upregulation promotes drug resistance of gastric cancer through impairing hsa-miR-23b-3p-induced Notch2 inhibition.

Specificity protein 1 (SP1), a transcription factor mediated by SUMOylation modifiers, is upregulated in gastric cancer (GC) and shares negative correlation with patient prognosis. Here, we paid main attention to the role of SP1 SUMOylation in the drug resistance of GC cells and the possible long non-coding RNA (lncRNA) SNHG17/microRNA-23b-3p (miR-23b-3p)/Notch2 network engaged in this process. Tumor tissues and non-tumor tissues were isolated from GC patients who received treatment with capecitabine and cisplatin (DDP). Co-immunoprecipitation was utilized to detect the SUMOylation level of SP1. Using gain- and loss-of-function approaches, we assessed the impacts of SNHG17/miR-23b-3p/Notch2 on sensitivity of DDP-resistant GC cells in vitro and in vivo. A series of assays such as luciferase activity detection and RNA pull-down were conducted for mechanistic exploration. SP1 expression was increased due to low SP1 SUMOylation level in the recurrent GC tissues. This increase led to upregulated SNHG17 expression and SP1 binding sites existed in the SNHG17 promoter. In addition, SNHG17 could bind to miR-23b-3p while miR-23b-3p targeted Notch2. Loss of SNHG17 reduced the resistance of DDP-resistant GC cells to DDP, which was achieved through miR-23b-3p-dependent Notch2 inhibition. Finally, SP1 silencing attenuated the resistance of GC to DDP in mice. Low SP1 SUMOylation induces SNHG17 upregulation and blocks miR-23b-3p-induced Notch2 inhibition, contributing to the resistance of GC to DDP. This study may aid in the development of therapeutic targets overcoming the chemoresistance of GC.

Study on the Mechanism of Action of STAT3 in the Drug Resistance of Gastric Cancer Cells.

Gastric cancer is the most common digestive tract malignancy in China and has a poor prognosis, with a 5-year overall survival rate of only 35.1%. Because its early symptoms are not obvious and early diagnosis is complicated, there is an urgent need to find biological targets for diagnosis and treatment. This research detected the expression of STAT3 in gastric cancer tissues and adjacent tissues by Western blot and immunohistochemical experiments and conducted corresponding basic experiments to explore the role of STAT3 in inhibiting the proliferation of cisplatin-resistant gastric cancer cells and promoting their apoptosis, including the construction of cisplatin-resistant gastric cancer cell line, the knock-out STAT3 in drug-resistant gastric cancer cells by CRISPR-Cas9, and the comparison of the proliferation and apoptosis of drug-resistant cells and drug-resistant cells STAT3(-). The mechanism provides a possible intervention target for clinically improving the prognosis of patients with cisplatin-resistant gastric cancer.

The E3 ubiquitin ligase, FBXW5, promotes the migration and invasion of gastric cancer through the dysregulation of the Hippo pathway

F-box and WD repeat domain-containing 5 (FBXW5), with WD40 repeats, can bind to the PPxY sequence of the large tumor suppressor kinases 1/2 (LATS1/2) kinase domain, resulting in ubiquitination. Ubiquitination and the subsequent degradation of LATS1/2 abrogate the Hippo pathway and worsen gastric cancer (GC). However, the effects and molecular mechanisms of FBXW5 in GC remain unexplored. To elucidate the clinical significance of FBXW5, immunohistochemistry was conducted to reveal the positive correlation between FBXW5 expression and lymph node metastasis (p < 0.001) and TNM stage (training cohort: p = 0.018; validation cohort: p = 0.001). Further, patients with high FBXW5 expression were found to have poor prognosis (training cohort: log-rank p = 0.020; validation cohort: log-rank p = 0.025). Cell experiments revealed the promoting effects of FBXW5 on the proliferation, invasion, metastasis, and chemoresistance of GC cells. Blocking LATS1-YAP1 leads to the loss of FBXW5-mediated regulation of the Hippo pathway and partial functions. Further, co-immunoprecipitation and in vivo ubiquitination assays revealed the interaction between FBXW5 and LATS1, which promoted the ubiquitination and degradation of LATS1. Based on mouse xenograft assays, FBXW5 silencing attenuated the growth of subcutaneous tumor xenografts. Altogether, FBXW5 was found to inactivate the Hippo signaling pathway by enhancing LATS1 ubiquitination and degradation, which promoted the invasion, metastasis, and drug resistance of GC cells.

Intracellular MUC20 variant 2 maintains mitochondrial calcium homeostasis and enhances drug resistance in gastric cancer.

Signet ring cell carcinoma (SRCC) is a particular histologic variant of gastric cancer (GC). However, the critical factor related to the aggressive characteristics of SRCC has not been determined. We collected surgically resected tissues from 360 GC patients in the Kumamoto University cohort and generated survival curves via the Kaplan-Meier method. In vitro, we identified the specific transcript variant of MUC20 in SRCC cells by direct sequencing and investigated the role of MUC20 in GC progression using GC cells with MUC20 silencing and forced expression. In vivo, we examined chemoresistance using MUC20 variant 2 (MUC20v2)-overexpressing non-SRCC cells to construct a xenograft mouse model. We analyzed a comprehensive GC cell line database to identify the specifically expressed genes in gastric SRCC. We focused on MUC20 and investigated its role in GC progression. Survival analysis revealed that GC patients with high MUC20 expression exhibited a poor prognosis and that MUC20 expression was significantly correlated with SRCC histological type. Moreover, we found that gastric SRCC cells specifically expressed MUC20v2, which was dominantly expressed in the cytoplasm. Silencing MUC20v2 caused cell death with characteristic morphological changes in gastric SRCC cells. To further determine the types of cell death, we examined apoptosis, pyroptosis and ferroptosis by detecting cleaved PARP, gasdermin E-N-terminal (GSDME-N), and lipid reactive oxygen species (ROS) levels, respectively. We found that apoptosis and pyroptosis occurred in MUC20-silenced gastric SRCC cells. In addition, MUC20v2-overexpressing GC cells exhibited chemoresistance to cisplatin (CDDP) and paclitaxel (PTX). RNA sequencing revealed that the pathways involved in intracellular calcium regulation were significantly upregulated in MUC20v2-overexpressing GC cells. Notably, forced expression of MUC20v2 in the cytoplasm of GC cells led to the maintenance of mitochondrial calcium homeostasis and mitochondrial membrane potential (MMP), which promoted cell survival and chemoresistance by suppressing apoptosis and pyroptosis. Finally, we investigated the significance of MUC20v2 in a xenograft model treated with CDDP and showed that MUC20v2 overexpression caused chemoresistance by inhibiting cell death. These findings highlight the novel functions of MUC20v2, which may confer cell survival and drug resistance in GC cells. MUC20v2 protects GC cells from apoptosis and pyroptosis by maintaining mitochondrial calcium levels and mitochondrial membrane potential and subsequently induces drug resistance.

LRP5 Promotes Gastric Cancer via Activating Canonical Wnt/β-Catenin and Glycolysis Pathways

The overactivation of canonical Wnt/β-catenin pathway is one of the main cascades for the initiation, progression, and recurrence of most human malignancies. As an indispensable coreceptor for the signaling transduction of the canonical Wnt/β-catenin pathway, LRP5 is up-regulated and exerts a carcinogenic role in most types of cancer. However, its expression level and role in gastric cancer (GC) has not been clearly elucidated. The current work showed that LRP5 was overexpressed in GC tissues and the expression of LRP5 was positively associated with the advanced clinical stages and poor prognosis. Ectopic expression of LRP5 enhanced the proliferation, invasiveness, and drug resistance of GC cells invitro, and accelerated the tumor growth in nude mice, through activating the canonical Wnt/β-catenin signaling pathway and up-regulating aerobic glycolysis, thus increasing the energy supply for GC cells. Additionally, the expression of LRP5 and glycolysis-related genes showed an obviously positive correlation in GC tissues. By contrast, the exact opposite results were observed when the endogenous LRP5 was silenced in GC cells. Collectively, these results not only reveal the carcinogenic role of LRP5 during GC development through activating the canonical Wnt/β-catenin and glycolysis pathways, but also provide a valuable candidate for the diagnosis and treatment of human GC.

Betulinic acid targets drug-resistant human gastric cancer cells by inducing autophagic cell death, suppresses cell migration and invasion, and modulates the ERK/MEK signaling pathway.

The main purpose of this study was to examine the anticancer effects of betulinic acid - a plant triterpene, against gastric cancer, along with demonstrating its underlying mechanism. The MTT assay and clonogenic assays were executed to assess cellular viability in control and betulinic acid treated cells. Transmission electron microscopy and western blotting were implemented to study autophagy stimulation by betulinic acid. The ERK/MEK signaling pathway was monitored by western blotting. Migration and invasion of SGC-7901 cells was investigated via transwell chamber assay. Results of this investigation indicated that betulinic acid induced remarkable cytotoxicity against gastric cancer SGC-7901 cells, in contrast to normal gastric GES-1 cells. The cytotoxicity of betulinic acid was observed due to its autophagy stimulation tendency in target cells. Autophagic cell death was supported by the data attained from western blotting showing enhanced LC3-II, and lowered LC3-I and p62 expressions. Moreover, betulinic acid was observed to block the ERK/MEK signaling pathway in SGC-7901 cells, which was associated with declined levels of expressions of the phosphorylated ERK and MEK proteins. Finally, the transwell chamber assay revealed a potential lowering of migration and invasion by betulinic acid in the SGC-7901 cells. In conclusion, these results demonstrated that betulinic acid exhibited significant anti-gastric cancer effects mediated via autophagy induction, blocking of ERK/MEK signaling and suppression of migration and invasion. Therefore, betulinic acid may prove as a lead molecule in gastric cancer management and research.

Banxia xiexin decoction affects drug sensitivity ingastric cancer cells by regulating MGMT expression via IL‑6/JAK/STAT3‑mediated PD‑L1 activity.

Banxia xiexin decoction (BXXX) is a classic preparation used to treat gastrointestinal diseases, and also has certain therapeutic effects on gastrointestinal tumors. BXXX has been reported to regulate the expression of proteins associated with drug resistance and sensitivity in tumors, and thus, the aim of the present study was to investigate the mechanisms of BXXX drug sensitivity in gastric cancer (GC). The expression levels of programmed cell death 1 ligand 1 (PD-L1), 6-O-methylguanine-DNA methyltransferase (MGMT) and STAT3 were immunohistochemically detected in the cancer and adjacent non-cancer tissues of patients with GC, and in vitro experimentation was conducted using drug-resistant and -sensitive GC cells. The expression levels of PD-L1, MGMT and STAT3 were determined using reverse transcription-quantitative PCR. Different concentrations of BXXX drug serum were used to treat the cells and the cellular inhibition rate was assessed using a Cell Counting Kit-8 assay. Flow cytometry was used to detect apoptosis, and western blot analysis was used to detect the expression levels of IL-6, IFN-γ, JAK/STAT3 pathway proteins, PD-L1 and MGMT. The association between PD-L1 and MGMT protein expression levels was subsequently assessed via co-immunoprecipitation. Furthermore, in vivo studies were conducted following the establishment of a drug-resistant tumor-bearing mouse model, where GC tumor size was assessed under different treatment conditions, and western blot analysis was used to detect the expression of related pathway proteins. The expression levels of PD-L1, MGMT and STAT3 were significantly increased in GC tissues, GC cells and cisplatin-resistant cells. Furthermore, BXXX inhibited the proliferation of drug-resistant cells and promoted the inhibitory effects of chemotherapeutic drugs on drug-resistant cells. BXXX also inhibited the expression levels of IL-6, IFN-γ and JAK/STAT3 pathway proteins, as well as the expression levels of PD-L1 and MGMT. Colivelin, an activator of STAT3, reversed the effects of BXXX on drug-resistant GC cells, and significantly reversed the effect of BXXX on PD-L1 expression. In conclusion, BXXX was found to influence the drug sensitivity of GC cells by regulating the expression of MGMT. This process functions viaPD-L1, which was itself mediated by IL-6/JAK/STAT3 signaling.

The CAGE-MiR-181b-5p-S1PR1 Axis Regulates Anticancer Drug Resistance and Autophagy in Gastric Cancer Cells.

Cancer-associated gene (CAGE), a cancer/testis antigen, has been known to promote anticancer drug resistance. Since the underlying mechanisms of CAGE-promoted anticancer drug resistance are poorly understood, we established Anticancer drug-resistant gastric cancer cells (AGSR) to better elucidate possible mechanisms. AGSR showed an increased expression level of CAGE and autophagic flux compared with anticancer drug-sensitive parental gastric cancer cells (AGS cells). AGSR cells showed higher invasion potential, growth rate, tumor spheroid formation, and angiogenic potential than AGS cells. CAGE exerted effects on the response to anticancer drugs and autophagic flux. CAGE was shown to bind to Beclin1, a mediator of autophagy. Overexpression of CAGE increased autophagic flux and invasion potential but inhibited the cleavage of PARP in response to anticancer drugs in CAGE CRISPR–Cas9 cell lines. TargetScan analysis was utilized to predict the binding of miR-302b-5p to the promoter sequences of CAGE, and the results show that miR-302b-5p directly regulated CAGE expression as illustrated by luciferase activity. MiR-302b-5p regulated autophagic flux and the response to anticancer drugs. CAGE was shown to bind the promoter sequences of miR-302b-5p. The culture medium of AGSR cells increased CAGE expression and autophagic flux in AGS cells. ImmunoEM showed CAGE was present in the exosomes of AGSR cells; exosomes of AGSR cells and human recombinant CAGE protein increased CAGE expression, autophagic flux, and resistance to anticancer drugs in AGS cells. MicroRNA array revealed miR-181b-5p as a potential negative regulator of CAGE. MiR-181b-5p inhibitor increased the expression of CAGE and autophagic flux in addition to preventing anticancer drugs from cleaving poly(ADP-ribose) polymerase (PARP) in AGS cells. TargetScan analysis predicted sphingosine 1-phosphate receptor 1 (SIPR1) as a potential target for miR-181b-5p. CAGE showed binding to the promoter sequences of S1PR1. The downregulation or inhibition of S1PR1 led to decreased autophagic flux but enhanced the sensitivity to anticancer drugs in AGSR cells. This study presents a novel role of the CAGE–miR-181b-5p–S1PR1 axis in anticancer drug resistance and autophagy.

Discovery of sertraline and its derivatives able to combat drug-resistant gastric cancer cell via inducing apoptosis

Resistance phenomena during chemotherapy of tumor has been severely hampering the applications of chemotherapeutics. Due to advantage of drug repurposing, discovery of new chemosensitizers based on approved drugs is an effect strategy to find new candidates. Herein, we found antidepressant drug – sertraline, could sensitize drug-resistant gastric cancer cell (SGC-7901/DDP) with the IC50 value of 18.73 μM. To understand the structure–activity relationship and improve the activity, 30 derivatives were synthesized and evaluated. The IC50 value of the best compound was improved to 5.2 μM. Moreover, we found apoptosis induction and cell cycle arrest was the reason for the cell death of the drug-resistant cells after treatment of sertraline and derivatives, and PI3K/Akt/mTOR pathway was involved.

Long noncoding RNA (lncRNA) EIF3J-DT induces chemoresistance of gastric cancer via autophagy activation

ABSTRACT Chemotherapy is currently the main treatment for unresectable or advanced postoperative gastric cancers. However, its efficacy is negatively affected by the occurrence of chemoresistance, which severely affects patient prognosis. Recently, dysregulation in autophagy has been suggested as a potential mechanism for chemoresistence, and long noncoding RNA (lncRNA) also shows its regulatory role in cancer drug resistance. Using RNA sequencing, we found that lncRNA EIF3J-DT was highly expressed in drug-resistant gastric cancer cells. In-vitro and in-vivo experiments showed that EIF3J-DT activated autophagy and induced drug resistance in gastric cancer cells by targeting ATG14. Bioinformatics and experimental results showed that EIF3J-DT regulated the expression of ATG14 through direct binding to enhance stabilization of ATG14 mRNA and via blocking the degradation of ATG14 mRNA through competitively binding with microRNA (miRNA) MIR188-3p. Therefore, EIF3J-DT increased the expression of ATG14, contributing to activation of autophagy and chemoresistance. Furthermore, it was confirmed that EIF3J-DT and ATG14 were highly expressed in gastric cancer patients resistant to chemotherapy, and this was closely associated with patient prognosis. In conclusion, EIF3J-DT is involved in the regulation of autophagy and chemoresistance in gastric cancer cells by targeting ATG14. It may be a suitable new target for enhancing chemosensitivity and improving prognosis. Abbreviations: 3-MA: 3-methyladenine; 5-Fu: 5-fluorouracil; ATG: autophagy related; C-CASP3: cleaved caspase 3; C-CASP7: cleaved caspase 7; C-PARP: cleaved PARP; CQ: chloroquine; CR: complete response; DIG: digoxigenin; ESR1: estrogen receptor 1; FBS: fetal bovine serum; FISH: fluorescence in situ hybridization; IHC: immunohistochemistry; ISH: in situ hybridization; lncRNA: long noncoding RNA; miRNA: microRNA; MUT: mutant; NC: negative control; OXA: oxaliplatin; PBS: phosphate-buffered saline; PD: progressive disease; PFA: paraformaldehyde; PR: partial response; qPCR: quantitative polymerase chain reaction; RAPA: rapamycin; SD: stable disease; TEM: transmission electron microscopy; WT: wild type.

PD-1/PD-L1 enhanced cisplatin resistance in gastric cancer through PI3K/AKT mediated P-gp expression

BackgroundProgrammed cell death receptor 1 (PD-1) is an immunosuppressive molecule expressed on T cells, and its ligand (PD-L1) which expressed on tumor cells play pivotal roles in regulating host immune responses. However, little is known whether PD-1/PD-L1 axis could directly activates intracellular oncogenic signaling pathways in tumor cells, leading to tumor resistance. MethodsIn the present study, the expression of PD-1 and PD-L1 in the tissues of gastric cancer was detected by western blot and immunofluorescence. The effect of PD-L1-Fc and cisplatin on resistant gastric cancer cells was examined by MTT assay and Flow Cytometry. In addition. The effect of PD-L1-Fc on the expression of P-gp in gastric cancer cells and resistant gastric cancer cells was detected by quantitative real-time reverse-transcription PCR (qRT-PCR) and western blot. The molecular mechanisms of the regulation of cisplatin and PD-L1-Fc treatment were evaluated by western blot. ResultsWe found that the level of PD-1 was significantly increased in human gastric cancer tissues and drug-resistant gastric cancer cells and P-gp was the same result. The PD-L1 could reduce the level of cell damage caused by cisplatin. In addition, we found PD-L1 can also up-regulate the expression of P-gp. Mechanistically, PD-L1 activated the PI3K/AKT signaling pathway in which PI3K/AKT pathway inhibition attenuated the upregulation of P-gp. ConclusionPD-1/PD-L1 enhanced cisplatin resistance in gastric cancer through PI3K/AKT mediated P-gp expression.

JP3 enhances the toxicity of cisplatin on drug-resistant gastric cancer cells while reducing the damage to normal cells

Background: Cisplatin (DDP) is a highly effective chemotherapeutic agent to most solid tumors including gastric cancer (GC), however, its clinical value is limited due to severe toxic side effects and secondary drug resistance. JP3, a JWA protein based MMP2-targeted polypeptide, known to inhibit the growth of GC in vivo. However, the bidirectional effects of JP3 in DDP-resistant GC and normal cells have not been demonstrated. The present study aims to investigate the actions of JP3 on protecting normal cells from the toxicity of DDP while enhancing its anti-tumor effects on GC cells.Methods: Routine laboratory experimental methods including CCK-8 assay, Western blotting, Hoechst staining, immunofluorescence (IF) and qRT-PCR were used in mechanism investigation; protein docking analysis and coimmunoprecipitation (Co-IP) were used for prediction and confirmation of interactions between JP3 and CK2. Mouse xenograft model was used for screening the treatment of JP3 plus DDP on GC growth.Results: DDP showed similar toxicities to normal cells and DDP-resistant GC cells; JP3 competitively inhibited the binding of XRCC1 to CK2, reduced the DNA repair and anti-apoptosis capacity of DDP-resistant GC cells in combination with DDP treatment; meanwhile, JP3 protected normal cells from DDP-induced oxidative stress and DNA damage through ERK/Nrf2 signaling. JP3 combined with DDP showed similar bidirectional effects in vivo.Conclusions: JP3 enhanced the inhibitory effects of DDP on tumor growth while reduced toxic side effects of DDP on normal cells. The results of this study provide a new insight for the treatment of drug-resistant GC.