ABSTRACT
Objective To study the enhancement of sensitivity to cisplatin (DDP) mediated by the addition of proteasome inhibitor Oprozomib (OZ) in ovarian cancer cells. Methods SKOV3/DDP and A2780/DDP cells were cultured in vitro, and the logarithmic growth phase cells were used in this study. To study the effect of OZ on drug-resistant cell viability, a series dilutions of OZ (0, 3, 9, 27, 81, 243, 729, 2181, and 6561 nmol/L) was used. The effect of DDP was also investigated with a series titrations (0, 3, 9, 27, 81, 243, 729, 2181 and 6561 nmol/L) in the presence of 50 μl of 500 nmol/L OZ. CCK-8 method was used to detect cell viability; flow cytometry was used to detect the apoptosis rate after treated with DDP+OZ. The experiments were divided into the control group and OZ group to detect the effects of OZ on the activity of proteasome chymotrypsin (CT-L), the synthesis of intracellular glutathione (GSH); the expression of intracellular glutathione synthetase (GSS) was examined using western blotting. The experiments were divided into the control group, DDP group, OZ group, DDP+OZ group, DDP+OZ+GSH group and DDP+OZ+Tempol group to detect the ROS level and apoptosis rate. Results The IC50 of OZ to SKOV3/DDP and A2780/ DDP cells were 140 and 350 nmol/L, respectively; In the presence of OZ, the IC50 of DDP to SKOV3/DDP and A2780/DDP cells were 154 and 232 nmol/L, respectively. The apoptosis rate of ovarian cancer cells increased significantly (P<0.01) after treated with DDP+OZ together. OZ can inhibit the CT-L activity of the proteasome in a dose-dependent manner and can inhibit GSH synthesis and GSS protein expression (P<0.01). The decrease of GSH level leads to the obstruction of ROS clearance, and the ROS level was significantly reduced by the ROS scavenger Tempol (P<0.05, P<0.01). Tempol significantly inhibits the apoptosis induced by DDP+OZ (P<0.01). Conclusion OZ enhanced sensitivity of SKOV3/DDP and A2780/DDP to cis-platinum by inhibiting the generation of GHS which resulted in the accumulation of ROS.
ABSTRACT
BACKGROUND: Cisplatin resistance (DDP-resistance) remains one of the major causes of poor prognosis in females with ovarian cancer. Long non-coding RNAs (lncRNAs) have been shown to participate in the regulation of cellular processes, including chemoresistance. The aim of this study was to explore the role of HOX transcript antisense RNA (HOTAIR) in DDP-resistant ovarian cancer cells. METHODS: DDP-resistant ovarian cancer cell lines (SKOV3/DDP and A2780/DDP) were established. Real-time PCR, western blot, dual-luciferase reporter assay, and flow cytometry were then used to evaluate the effect of HOTAIR/miR-138-5p axis on chemoresistance of DDP-resistant ovarian cancer cells to DDP. RESULTS: We found that HOTAIR was upregulated in DDP-resistant cells, while miR-138-5p was downregulated. Knockdown of HOTAIR increased the expression of miR-138-5p in DDP-resistant cells and miR-138-5p is directly bound to HOTAIR. Upregulation of miR-138-5p induced by HOTAIR siRNA or by its mimics enhanced the chemosensitivity of DDP-resistant cells and decreased the expression of EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) and SIRT1 (sirtuin 1). Furthermore, the HOTAIR silencing-induced chemosensitivity of DDP-resistant cells was weakened by miR-138-5p inhibitor. CONCLUSIONS: These data demonstrate that HOTAIR acts as a sponge of miR-138-5p to prevent its binding to EZH2 and SIRT1, thereby promoting DDP-resistance of ovarian cancer cells. Our work will shed light on the development of therapeutic strategies for ovarian cancer treatment.
Subject(s)
Humans , Female , Ovarian Neoplasms/genetics , Cisplatin/pharmacology , Drug Resistance, Neoplasm/genetics , RNA, Long Noncoding/genetics , Gene Expression Regulation, Neoplastic/drug effects , Up-Regulation , Apoptosis/drug effects , MicroRNAs/antagonists & inhibitors , Cell Line, Tumor , Gene Knockout Techniques/methods , Sirtuin 1/antagonists & inhibitors , Real-Time Polymerase Chain Reaction , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitorsABSTRACT
In previous studies, we found interferon-a (IFN-a) could reduce protein levels of p11, 5-hydroxytryptamine receptor 1b(5-HT1b) and 5-hydroxytryptamine receptor 4 (5-HT4), but does not influence their messenger RNA levels in SH-sy5ycells. Thus, we investigated the post-transcriptional modulation of these molecules by IFN-a. SH-sy5y cells were treatedwith IFN-a, NH4Cl or MG132 alone or in combination, and then the protein levels of p11, 5-HT1b and 5-HT4 wereanalyzed by western blots. The regulatory effects of p11 on 5-HT1b and 5-HT4 were also determined in p11 knock-downcells. NH4Cl but not MG132 could reverse the protein level of p11 in IFN-a-treated SH-sy5y cells. MG132 could recoverthe protein levels of 5-HT1b and 5-HT4 in p11 knock-down cells. The down-regulation effects of IFN-a on p11, 5-HT1band 5-HT4 were associated with the lysosome and ubiquitin–proteasome-mediated pathways. p11 was identified as a potentregulator to modulate the ubiquitination of 5-HT1b and 5-HT4. Therefore, it could be potential target therapies in IFN-ainduced depression.
ABSTRACT
@# Objective: To investigate the relationship between long non-coding RNA (lncRNA) HIT and cisplatin (DDP) resistance in osteosarcoma cells and the mechanism related to epithelial-mesenchymal transition (EMT). Methods: 42 pairs of osteosarcoma tissues and corresponding para-cancerous tissues (more than 5 cm away from the edge of cancer tissues) were collected at the Department of Orthopedics, Tianjin Hospital during June 2017 to June 2018. Quantitative Real-time PCR (qRT-PCR) was used to detect the mRNAexpression of HIT and EMT related markers (Snail and E-cadherin) in the collected tissues. The DDP-resistant osteosarcoma U2OS cell line was constructed and human adrenal 293T cell line was used as control. Two sets of siRNA vectors targeting HIT loaded on lentivirus were transfected into cells with DDP-resistance as the interference group A and group B. Meanwhile, the U2OS cell line was transfected with HIT full-length vector and blank vector respectively, as over-expression group and blank group. The DDP 50% inhibitory concentration (IC50) was detected by MTT assay. qRT-PCR was used to detect the mRNA expressions of HIT, Snail and E-cadherin. Western blotting was used to detect the protein expressions of Snail and E-cadherin. RNA binding protein immunoprecipitation (RNAIP) assay was used to clarify the combination of HIT and Snail protein in the U2OS and 293T cells. Results: The mRNAexpressions of HIT and Snail in osteosarcoma tissues were significantly higher than those in para-cancerous tissues, while the mRNA expression of Ecadherin was significantly lower than that in the paracancerous tissues. The mRNA expression of HIT and E-cadherin in osteosarcoma tissues was negatively correlated (all P<0.01). The DDP IC50 in the DDP-resistance group was significantly higher than that in the control group, interference group A and B, and the DDP IC50 in over-expression group was significantly higher than that in blank group (all P<0.01). The expression of HIT in resistance group was significantly higher than that in the control group, and the HIT expressions in interference group A and B were significantly lower than that in DDP-resistance and control group; moreover, the expression of HIT in over-expression group was significantly higher than that in blank group (all P<0.05 or P<0.01). The mRNAexpression of Snail in DDPresistance group was significantly higher than that in the control group and interference group A and B, while the mRNA expression of E-cadherin in DDP-resistance group was significantly lower than that in the control group and interference group A and B; and the mRNA expression of E-cadherin in over-expression group was significantly lower than that in blank group. The protein expression of Snail in the DDP-resistance group was significantly higher than that in the control group and interference group A and B,while E-cadherin protein expression was significantly lower; and protein expression of Snail in over-expression group was significantly higher than that in blank group (all P<0.05 or P<0.01). The expression of HIT in the U20S and 293T cells treated by anti-Snail antibody induced by immunomagnetic beads was significantly higher than that in the cells treated by IgG antibody (P<0.01). Conclusion: HIT can promote EMT and cisplatin-resistance in osteosarcoma cells through up-regulation of Snail protein and inhibition of E-cadherin transcription activity.
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OBJECTIVE Despite the status of cisplatin (DDP) as a classical chemotherapeutic agent in the treatment of cancer, the development of multidrug resistance often leads to a failure of DDP therapy.Traditional Chinese medicine(TCM)as adjuvant chemotherapy of cancer drugs in China has been widely used in cancer treatment.ZuoJin WAN (ZJW),a TCM formula,was proved reversing drug resistance in gastric cancer,but its exact mechanism was still unclear. METHODS CCK-8 assay was used to detect the cell viability. The levels of proteins and mRNA were evaluated using Western blot and q-PCR. Mitochondrial membrane potential was measured by fl ow cytometry. Depolymerisa-tion of F-actin and translocation of G-actin(gamma-actin)from the cytoplasm to the mitochondria was detected using an immuno fl uorescence assay. RESULTS phosphorylated coflin-1 (p-coflin-1) was overexpressed in the DDP-resistant human gastric cancer cell lines SGC7901/DDP and BGC823/DDP, relative to the respective parent cell lines(SGC7901 and BGC823),and DDP induced the dephosphory-lation of p-coflin-1 in both parent lines but not in the DDP-resistant lines. However, ZJW could induce the dephosphorylation of pcoflin-1 and promote coflin-1 translocation from the cytoplasm into the mito-chondria in both SGC7901/DDP and BGC823/DDP cells. This mitochondrial translocation of coflin-1 was found to induce the conversion of flamentous actin to globular-actin, activate mitochondrial dam-age and calcium overloading, and induce the mitochondrial apoptosis pathway. These effects of ZJW on DDP-resistant human gastric cancer cell lines could be reversed via transfection with coflin-1-specifc siRNA,or treatment with a PP1 and PP2A inhibitor.CONCLUSION ZJW can be used as an inhibitor of chemoresistance in gastric cancer, which may partly be due to dephosphorylation of p-coflin-1 via the activation of PP1 and PP2A.
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AIM:To investigate the effect of Shenci capsule combined with cisplatin ( DDP) in reversing DDP resistance by PI3K/AKT/mTOR signaling pathway in nude mice bearing A 549/DDP tumor.METHODS:The patient-de-rived lung adenocarcinoma A 549/DDP cell xenograft model was established .The tumor-bearing nude mice were randomly divided into control group , Shenci capsule group , DDP group and Shenci capsule combined with DDP group .The mice in control group was treated with normal saline , while the mice in other groups were treated with different drugs for 21 d.After treatment, the mice were killed and lung cancer tissues were collected .Flow cytometry was used to analyze the cell cycle and apoptosis.FQ-PCR was used to determined the mRNA levels of PTEN , P-glycoprotein, PI3K, AKT and mTOR in A549/DDP lung tumor .RESULTS:Compared with control group , the cell proliferation in all the drug treatment groups was inhibited .Compared with other drug treatment groups , Shenci capsule combined with DDP blocked the cell cycle of A 549/DDP cells at G2/M phase, promoted the apoptosis rate , increased the mRNA expression of PTEN and inhibited the mRNA expression of P-glycoprotein, PI3K, AKT and mTOR.CONCLUSION:Shenci capsule increases the sensitivity of A 549/DDP resistant cells in nude mice to DDP by blocking PI 3K/AKT/mTOR signaling pathway , increasing the expression of PTEN or inhibiting P-glycoprotein-mediated resistance pathway .