BACKGROUND: Growing evidence suggests that long non-coding RNAs (lncRNAs) exert pivotal roles in fostering chemoresistance across diverse tumors. Nevertheless, the precise involvement of lncRNAs in modulating chemoresistance within the context of gallbladder cancer (GBC) remains obscure. This study aimed to uncover how lncRNAs regulate chemoresistance in gallbladder cancer, offering potential targets to overcome drug resistance. METHODS: To elucidate the relationship between gemcitabine sensitivity and small nucleolar RNA host gene 1 ( SNHG1 ) expression, we utilized publicly available GBC databases, GBC tissues from Renji Hospital collected between January 2017 and December 2019, as well as GBC cell lines. The assessment of SNHG1, miR-23b-3p, and phosphatase and tensin homolog (PTEN) expression was performed using in situ  hybridization, quantitative real-time polymerase chain reaction, and western blotting. The cell counting kit-8 (CCK-8) assay was used to quantify the cell viability. Furthermore, a GBC xenograft model was employed to evaluate the impact of SNHG1 on the therapeutic efficacy of gemcitabine. Receiver operating characteristic (ROC) curve analyses were executed to assess the specificity and sensitivity of SNHG1. RESULTS: Our analyses revealed an inverse correlation between the lncRNA SNHG1 and gemcitabine resistance across genomics of drug sensitivity in cancer (GDSC) and Gene Expression Omnibus (GEO) datasets, GBC cell lines, and patients. Gain-of-function investigations underscored that SNHG1 heightened the gemcitabine sensitivity of GBC cells in both in vitro and in vivo  settings. Mechanistic explorations illuminated that SNHG1 could activate PTEN -a commonly suppressed tumor suppressor gene in cancers-thereby curbing the development of gemcitabine resistance in GBC cells. Notably, microRNA (miRNA) target prediction algorithms unveiled the presence of miR-23b-3p binding sites within SNHG1 and the 3'-untranslated region (UTR) of PTEN . Moreover, SNHG1 acted as a sponge for miR-23b-3p, competitively binding to the 3'-UTR of PTEN , thereby amplifying PTEN expression and heightening the susceptibility of GBC cells to gemcitabine. CONCLUSION The SNHG1/miR-23b-3p/PTEN axis emerges as a pivotal regulator of gemcitabine sensitivity in GBC cells, holding potential as a promising therapeutic target for managing GBC patients.
Humans ; Deoxycytidine/pharmacology* ; PTEN Phosphohydrolase/genetics* ; Gemcitabine ; RNA, Long Noncoding/metabolism* ; MicroRNAs/genetics* ; Gallbladder Neoplasms/genetics* ; Cell Line, Tumor ; Animals ; Mice ; Drug Resistance, Neoplasm/genetics* ; Mice, Nude ; Antimetabolites, Antineoplastic ; Gene Expression Regulation, Neoplastic

OBJECTIVE To explore downstream regulatory pathway of microRNA-21 (miR-21) in colon cancer cells (RKO) through detecting miR-21 and its target PDCD4, and the influence of miR-21 regulation on the sensitivity of RKO cells to 5-fluorouracil (5-FU). METHODS 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the effect of 5-FU on the viability of RKO cells with knockout of miR-21 or high expression of PDCD4. Real-time was used to determine the expression of PDCD4, ABCC5 and CD44 in RKO cell after knockout of miR-21. RESULTS MTT assay reveals that the IC50 of 5-FU in RKO-WT cells (52.82 ± 0.06 umol/L) was about 67% higher than in miR-21 knockout cells (32.23 ± 0.05 umol/L) (P < 0.05), and the apoptosis ratio elevated after knockout of miR-21. High expression of PDCD4, a target gene of miR-21, can negatively regulate the expression of ABC transporter ABCC5 and the stem cell marker CD44. CONCLUSION MiR-21 can mediate the drug resistance to 5-FU by inhibiting its target PDCD4, which can regulate the expression of ABCC5 and CD44 genes.
ATP Binding Cassette Transporter, Sub-Family G, Member 5 ; ATP-Binding Cassette Transporters ; genetics ; Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis Regulatory Proteins ; physiology ; Cell Line, Tumor ; Colonic Neoplasms ; drug therapy ; pathology ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Humans ; Hyaluronan Receptors ; genetics ; Lipoproteins ; genetics ; MicroRNAs ; physiology ; RNA-Binding Proteins ; physiology

OBJECTIVETo investigate the inhibitory effect of classic demethylating drug 5-aza-2'-deoxycytidine (5-Aza-CdR) on the growth of human lung adenocarcinoma cells in nude mouse xenograft models, and to observe its effect on methylation status and expression of TFPI-2 gene in the nude mouse xenograft tissues.

METHODSThe nude mouse xenograft model was established by subcutaneous inoculation of human lung adenocarcinoma A549 cells. According to different doses of 5-Aza-CdR, the tumor-bearing nude mice were randomly divided into experimental groups (0.5 mg/kg group, 1 mg/kg group, 2 mg/kg group) and control group (0 mg/kg group). The tumor growth in the nude mice was observed. The methylation status and the expression of TFPI-2 gene mRNA and protein were detected by methylation specific polymerase chain reaction, real-time fluorescent quantitative polymerase chain reaction and Western blot assay.

RESULTSThe nude mice were euthanized at 28 days after intraperitoneal injection of 5-Aza-CdR. The body weight of tumor-bearing nude mice was (27.12 ± 0.38) g in the 0 mg/kg group, (26.80 ± 0.18) g in the 0.5 mg/kg group, (26.67 ± 0.28) g in the 1 mg/kg group, and (26.50 ± 0.26) g in the 2 mg/kg group, showing no significant difference among them (P > 0.05). The volume of xenograft tumors in the 0 mg/kg group was (709.22 ± 2.87)mm³, (400.67 ± 2.68)mm³ in the 0.5 mg/kg group, (285.71 ± 2.91)mm³ in the 1 mg/kg group, and (230.44 ± 3.15)mm³ in the 2 mg/kg group, showing a significant difference (P < 0.05). There were complete methylation of TFPI-2 gene in the 0 mg/kg group, incomplete methylation in the 0.5 and 1 mg/kg groups, and unmethylation in the 2 mg/kg group. The relative mRNA level in the 0, 0.5, 1, 2 mg/kg groups were 1.00 ± 0.00, 1.67 ± 0.07, 3.40 ± 0.24, and 5.55 ± 0.61, respectively (P < 0.05). The relative expression level of TFPI-2 protein in the 0, 0.5, 1, 2 mg/kg groups was 0.18 ± 0.02, 0.36 ± 0.01, 0.64 ± 0.02, and 0.81 ± 0.20, respectively (P < 0.05).

CONCLUSIONS5-Aza-CdR suppresses the tumor growth of human lung adenocarcinoma cells in nude mouse xenograft models, and induces expression of TFPI-2 gene in the xenograft tumor cells. The mechanism might be that 5-Aza-CdR induces re-expression of demethylated TFPI-2 gene by demethylation, and thus inhibits the growth and proliferation of human lung adenocarcinoma cells.

Adenocarcinoma ; drug therapy ; pathology ; Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Proliferation ; drug effects ; DNA Methylation ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic ; Glycoproteins ; genetics ; Heterografts ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Mice ; Mice, Nude ; RNA, Messenger ; metabolism ; Random Allocation

OBJECTIVETo investigate the reversal effects of different concentrations of DNA methylation inhibitor, 5-aza-2-deoxycytidine, on the hypermethylation of maternally expressed gene 3 (MEG3) gene promoter, and then the inhibitory effect of restoration of MEG3 expression on the proliferation of ovarian cancer cells.

METHODSHuman ovarian cancer OVCAR3 cells were treated with various concentration of 5-aza-2-deoxycytidine (0, 1, 5, 10, 20 µmol/L, respectively) for 6 days. Then the methylation status of MEG3 promoter was detected by methylation specific PCR (MSP). The alteration of MEG3 gene expression was detected by RT-PCR. Cell proliferation was determined by MTT assay and EdU incorporation assay.

RESULTSAfter treated with 5-aza-2-deoxycytidine, the methylation status of MEG3 in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 0.79 ± 0.00, 0.67 ± 0.00, 0.65 ± 0.03 and 0.61 ± 0.01 folds, respectively (P < 0.05 for all). The relative expressions of MEG3 mRNA in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 2.04 ± 0.16, 2.44 ± 0.17, 3.19 ± 0.34 and 5.34 ± 0.39, respectively (P < 0.05 for all). In contrast to the negative control, the inhibition rates of the OVCAR3 cell growth were increased significantly when treated with 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine in 2, 4 and 6 days. There were (40.78 ± 0.80)%, (35.65 ± 0.33)%, (31.81 ± 0.66)%, (27.33 ± 1.27)% and (17.75 ± 1.85)% of EdU-positive cells in the 0, 1, 5, 10 and 20 µmol/L 5-aza-2-deoxycytidine groups (P < 0.01 for all).

CONCLUSIONSMaternally expressed gene 3 promoter hypermethylation is reversed by 5-aza-2-deoxycytidine in ovarian cancer cells. The downregulation of MEG3 gene might be resulted from the methylation, and the re-expression of MEG3 partly contribute to the growth inhibition of epithelial ovarian cancer cells.

Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DNA Methylation ; Female ; Humans ; Neoplasms, Glandular and Epithelial ; Ovarian Neoplasms ; Promoter Regions, Genetic ; RNA, Messenger

OBJECTIVEThe aim of this study was to explore the effect of SPARC on the anti-cancer effect of gemcitabine and underlying mechanism in pancreatic cancer.

METHODSAfter treating with gemcitabine, the proliferation rate of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells was detected by MTT assay. The cell cycle distribution and cell apoptosis in each group were examined by flow cytometry, and the capability of clone formation was tested by adhesion-dependent clone formation assay. The apoptosis-related proteins were analyzed by Western blot.

RESULTSThe growth of pancreatic cancer cells was inhibited by gemcitabine in a time-dependent and dose-dependent manner. Its IC50 at 24, 48, and 72-h was (40.1 ± 2.5) µmol/L, (15.0 ± 0.5) µmol/L and (6.6 ± 0.1) µmol/L, respectively. The overexpression of SPARC increased the inhibitory effect of gemcitabine on growth of pancreatic cancer MIA PaCa2/SPARC69 cells, presenting a dose- and time- dependent manner. Its IC50 at 24, 48, 72 h was (24.3 ± 1.5) µmol/L, (7.7 ± 0.3) µmol/L and (4.8 ± 0.2) µmol/L, respectively. The clone formation assay showed that before gemcitabine treatment, the clone numbers of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were (2350 ± 125), (2130 ± 120) and (1567 ± 11), respectively. After gemcitabine treatment, the clone numbers of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were ( 1674 ± 79) , (1587 ± 94) and (557 ± 61), respectively. The overexpression of SPARC enhanced the chemosensitivity of MIA PaCa2 cells to gemcitabine chemotherapy. After treating with 10 µmol/L gemcitabine for 48 h, the ratio of G0/G1 cells in MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were (56.0 ± 5.5)%, (55.0 ± 4.5)% and (68.0 ± 7.0)%, respectively. The cells arrested at G0/G1 phase were significantly increased in the MIA PaCa2/SPARC69 cells. The apoptosis rates of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were (22.4 ± 2.5)%, (19.9 ± 2.0)% and (37.7 ± 3.9)%, respectively, indicating that overexpression of SPARC enhanced the gemcitabine-induced apoptosis in MIA PaCa2 cells. The Western blot analysis showed that, compared with MIA PaCa2 and MIA PaCa2/V cells, the expression of caspase-2, -8, -9 and cleaved PARP protein was significantly increased, while the expression of Bcl-2 was not changed significantly in the MIA PaCa2/SPARC69 cells.

CONCLUSIONSPARC can enhance the chemosensitivity of pancreatic cancer cells to gemcitabine via regulating the expression of apoptosis-related proteins.

Antimetabolites, Antineoplastic ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Caspase 2 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cell Cycle ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cysteine Endopeptidases ; metabolism ; Deoxycytidine ; administration & dosage ; analogs & derivatives ; pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; Humans ; Osteonectin ; metabolism ; Pancreatic Neoplasms ; metabolism ; pathology ; Poly(ADP-ribose) Polymerases ; metabolism ; Time Factors

OBJECTIVETo investigate the effects of E2F-1 gene silencing on multidrug resistance of human gastric cancer SGC7901/DDP cells and its possible mechanisms.

METHODSGastric cancer SGC7901/DDP cells were seeded in 6 well plates and divided into three groups: the experimental group, blank control and the negative control groups. For the experimental group, the SGC7901/DDP cells were transfected with recombinant lentivirus vector (Lv-shRNA-E2F-1), while the negative control with an control lentiviral vector (Lv-shRNA-NC) and the blank control with no treatment. The E2F-1 protein level was analyzed by Western blot. MTT assay was used to detect the half maximal inhibitory concentration (IC50) of three chemotherapy drugs including adriamycin, 5-fluorouracil (5-Fu) and cisplatine (DDP) of the three cell groups. Flow cytometry (FCM) was used to detect the pump-out rate of adriamycin and apoptosis rate of the three cell groups. Semi-quantitative RT-PCR and Western blot were also used to detect the protein and mRNA levels of multidrug resistance-associated genes (MDR1, MRP) and apoptosis-related genes (c-Myc, Skp2, cyclinD1).

RESULTSThe expression of E2F-1 protein in the experimental group was significantly lower than that in the negative control and blank control groups (0.794 ± 0.033 vs. 1.487 ± 0.082 vs. 1.511 ± 0.084, P < 0.01). The IC50 of the three chemotherapy drugs (adriamycin, 5-Fu and cisplatine) in the experimental group was significantly lower than that of the negative control and blank control groups, respectively (P < 0.01). Compared with the negative control and blank control groups, the pump-out rate of adriamycin of the experimental group was significantly declined [(0.16 ± 0.01)% vs. (0.37 ± 0.01)% vs. (0.35 ± 0.02)%, P < 0.01]. However, the apoptosis rate of the experimental group was significantly higher than that of the negative control and blank control groups [(33.82 ± 1.26)% vs. (17.34 ± 0.81)% vs. (13.16 ± 1.06)%, P < 0.01]. The results of RT-PCR and Western blot assays showed that mRNA and protein expressions of five genes (MDR1, MRP, CyclinD1, c-Myc, Skp2) in the experimental group were significantly lower than that in the negative control and blank control groups, respectively (P < 0.01).

CONCLUSIONSE2F-1 gene silencing enhances the chemosensitivity of gastric cancer SGC7901/DDP cells to the chemotherapeutic drugs, directly or indirectly downregulated the expression of MDR1 and MRP, and finally reverses the multidrug resistance of the gastric cancer cells. The mechanism may be associated with the suppression of cyclinD1, c-Myc and Skp2.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Antibiotics, Antineoplastic ; pharmacology ; Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Cyclin D1 ; genetics ; metabolism ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; E2F1 Transcription Factor ; genetics ; metabolism ; Fluorouracil ; pharmacology ; Gene Silencing ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; S-Phase Kinase-Associated Proteins ; genetics ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Transfection

Based on the representative articles in recent years, the different mechanisms of decitabine on immune regulation in patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT) are summarized. Decitabine improves the expression of WT1 gene to stimulate specific cytotoxic T cells which can enhance graft versus leukemia effect (GVL) and improve the expression of FOXP3 gene to stimulate regulatory T cells so as to inhibit the acute graft versus host disease (GVHD). Through the above-mentimed mechanisms, decitabine can improve both therapeutic effect and quality of life in the patients with AML after allogeneic HSCT.
Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Graft vs Leukemia Effect ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia, Myeloid, Acute ; immunology ; therapy ; Quality of Life ; T-Lymphocytes, Cytotoxic ; T-Lymphocytes, Regulatory ; Transplantation, Homologous

This study was aimed to investigate the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA) on DLC-1 gene transcription regulation and molecular biological behaviours in the human multiple myeloma RPMI-8226 cells. The cells were treated respectively with 5-Aza-CdR and TSA alone, or the both combination; the cell proliferation and apoptosis, DLC-1 expression, the protein expression of Ras homolog family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) were examined by CCK-8 method, RT-PCR and ELISA, respectively. The results showed that the 5-Aza-CdR and TSA had cell growth inhibitory and apoptosis-inducing effects in dose-dependent manner (P < 0.05). Compared with a single drug (5-Aza-CdR or TSA alone), the effects were significantly enhanced after treatment with the combination of 5-Aza-CdR and TSA (P < 0.05). DLC-1 was weakly expressed in the control group; the treatment with 5-Aza-CdR alone enhanced its re-expression dose-dependently (P < 0.05). Compared with 5-Aza-CdR alone, 5-Aza-CdR plus TSA enhanced DLC-1 re-expression significantly.Compared with the control, 5-Aza-CdR and TSA significantly decreased RhoA and Rac1 protein expression (P < 0.05). It is concluded that 5-Aza-CdR and TSA can effectively reverse DLC-1 expression of RPMI-8226 cells; TSA has a synergistic effect on its re-expression. 5-Aza-CdR and TSA have significant cell growth inhibitory and apoptosis-inducing effects on RPMI-8226 cells. These effects may be related to the inhibition of Rho/Rho kinase signalling pathway.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Azacitidine ; administration & dosage ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; GTPase-Activating Proteins ; metabolism ; Gene Expression ; drug effects ; Humans ; Hydroxamic Acids ; administration & dosage ; pharmacology ; Multiple Myeloma ; genetics ; pathology ; Tumor Suppressor Proteins ; metabolism

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.
Adenine Nucleotides/*pharmacology ; Antimetabolites, Antineoplastic/*pharmacology ; Apoptosis/*drug effects ; Arabinonucleosides/*pharmacology ; Caspase 3/metabolism ; Caspase 7/metabolism ; Cell Line, Tumor ; Cell Proliferation/drug effects ; G2 Phase Cell Cycle Checkpoints/drug effects ; Gene Knockdown Techniques ; Humans ; Leupeptins/pharmacology ; Lung Neoplasms/metabolism/pathology ; M Phase Cell Cycle Checkpoints/drug effects ; Mesothelioma/metabolism/pathology ; Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors/genetics/metabolism ; RNA Interference ; RNA, Messenger/metabolism ; RNA, Small Interfering/metabolism ; Stilbenes/*pharmacology ; bcl-X Protein/antagonists & inhibitors/*genetics/*metabolism

BACKGROUNDCancer testis antigens (CTAs) are a novel group of tumor associated antigens. Demethylating agent decitabine was reported to be able to up-regulate CTAs through its hypomethylation mechanism, thus enhance the immunogenicity of leukemia cells. However, few researches have ever focused on the questions that whether this immunostimulatory effect of decitabine could induce autologous CTA specific cytotoxic T lymphocytes (CTLs) in vivo, and if so, whether this effect contributes to disease control. In this study, we aimed to show that decitabine could induce specific autologous CTLs against some mouse CTAs in leukemia cells in vitro and in vivo.

METHODSSeveral mouse CTAs were screened by RT-PCR. CTL specific to one of the CTAs named P1A was detected and sorted by P1A specific dimer by flow cytometry. The activity of specific CTLs was measured by real time RT-PCR.

RESULTSWe firstly screened expression of some CTAs in mouse leukemia cells before and after decitabine treatment and found that decitabine treatment did up-regulate expression of many CTAs. Then we measured the CTLs' activity specific to a mouse CTA P1A in vivo and showed that this activity increased after decitabine treatment. Finally, we sorted these in vivo induced P1A specific CTLs by flow cytometry and demonstrated their cytotoxicity against decitabine treated leukemia cells.

CONCLUSIONSOur study showed the autologous immune response induced by decitabine in vivo. And more importantly, we firstly proved that this response may contribute to disease control. We believe that this immunostimulatory effect is another anti-cancer mechanism of decitabine, and this special effect would inspire new applications of decitabine in the field of leukemia treatment in the future.

Animals ; Antigens, Neoplasm ; metabolism ; Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Flow Cytometry ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes, Cytotoxic ; drug effects ; metabolism