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Journal of Hematology & Oncology May 2018Chemoresistance to temozolomide (TMZ) is a major challenge in the treatment of glioblastoma (GBM). We previously found that miR-519a functions as a tumor suppressor in...
BACKGROUND
Chemoresistance to temozolomide (TMZ) is a major challenge in the treatment of glioblastoma (GBM). We previously found that miR-519a functions as a tumor suppressor in glioma by targeting the signal transducer and activator of transcription 3 (STAT3)-mediated autophagy oncogenic pathway. Here, we investigated the effects of miR-519a on TMZ chemosensitivity and autophagy in GBM cells. Furthermore, the underlying molecular mechanisms and signaling pathways were explored.
METHODS
In the present study, two stable TMZ-resistant GBM cell lines were successfully generated by exposure of parental cells to a gradually increasing TMZ concentration. After transfecting U87-MG/TMZ and U87-MG cells with miR-519a mimic or inhibitor, a series of biochemical assays such as MTT, apoptosis, and colony formation were performed to determine the chemosensitive response to TMZ. The autophagy levels in GBM cells were detected by transmission electron microscopy, LC3B protein immunofluorescence, and Western blotting analysis. Stable knockdown and overexpression of miR-519a in GBM cells were established using lentivirus. A xenograft nude mouse model and in situ brain model were used to examine the in vivo effects of miR-519a. Tumor tissue samples were collected from 48 patients with GBM and were used to assess the relationship between miR-519a and STAT3 expression.
RESULTS
TMZ treatment significantly upregulated miR-519a in U87-MG cells but not in U87-MG/TMZ cells. Moreover, the expression of miR-519a and baseline autophagy levels was lower in U87-MG/TMZ cells as compared to U87-MG cells. miR-519a dramatically enhanced TMZ-induced autophagy and apoptotic cell death in U87-MG/TMZ cells, while inhibition of miR-519a promoted TMZ resistance and reduced TMZ-induced autophagy in U87-MG cells. Furthermore, miR-519a induced autophagy through modification of STAT3 expression. The in vivo results showed that miR-519a can enhance apoptosis and sensitized GBM to TMZ treatment by promoting autophagy and targeting the STAT3/Bcl-2/Beclin-1 pathway. In human GBM tissues, we found an inverse correlation between miR-519a and STAT3 expression.
CONCLUSIONS
Our results suggested that miR-519a increased the sensitivity of GBM cells to TMZ therapy. The positive effects of miR-519a may be mediated through autophagy. In addition, miR-519a overexpression can induce autophagy by inhibiting STAT3/Bcl-2 pathway. Therefore, a combination of miR-519a and TMZ may represent an effective therapeutic strategy in GBM.
Topics: Animals; Apoptosis; Autophagy; Cell Line, Tumor; Drug Resistance, Neoplasm; Glioblastoma; Humans; Mice; MicroRNAs; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Signal Transduction; Temozolomide; Transfection; Xenograft Model Antitumor Assays
PubMed: 29843746
DOI: 10.1186/s13045-018-0618-0 -
Journal of Experimental & Clinical... Nov 2021Resistance to oxaliplatin is a major obstacle for the management of locally advanced and metastatic colon cancer (CC). Although long noncoding RNAs (lncRNAs) play key...
BACKGROUND
Resistance to oxaliplatin is a major obstacle for the management of locally advanced and metastatic colon cancer (CC). Although long noncoding RNAs (lncRNAs) play key roles in CC, the relationships between lncRNAs and resistance to oxaliplatin have been poorly understood yet.
METHODS
Chemo-sensitive and chemo-resistant organoids were established from colon cancer tissues of the oxaliplatin-sensitive or -resistant patients. Analysis of the patient cohort indicated that lnc-RP11-536 K7.3 had a potential oncogenic role in CC. Further, a series of functional in vitro and in vivo experiments were conducted to assess the effects of lnc-RP11-536 K7.3 on CC proliferation, glycolysis, and angiogenesis. RNA pull-down assay, luciferase reporter and fluorescent in situ hybridization assays were used to confirm the interactions between lnc-RP11-536 K7.3, SOX2 and their downstream target HIF-1α.
RESULTS
In this study, we identified a novel lncRNA, lnc-RP11-536 K7.3, was associated with resistance to oxaliplatin and predicted a poor survival. Knockout of lnc-RP11-536 K7.3 inhibited the proliferation, glycolysis, and angiogenesis, whereas enhanced chemosensitivity in chemo-resistant organoids and CC cells both in vitro and in vivo. Furthermore, we found that lnc-RP11-536 K7.3 recruited SOX2 to transcriptionally activate USP7 mRNA expression. The accumulative USP7 resulted in deubiquitylation and stabilization of HIF-1α, thereby facilitating resistance to oxaliplatin.
CONCLUSION
In conclusion, our findings indicated that lnc-RP11-536 K7.3 could promote proliferation, glycolysis, angiogenesis, and chemo-resistance in CC by SOX2/USP7/HIF-1α signaling axis. This revealed a new insight into how lncRNA could regulate chemosensitivity and provide a potential therapeutic target for reversing resistance to oxaliplatin in the management of CC.
Topics: Antineoplastic Agents; Carcinogenesis; Colorectal Neoplasms; Drug Resistance, Neoplasm; Female; Humans; Male; Organoids; Oxaliplatin; RNA, Long Noncoding; Signal Transduction
PubMed: 34740372
DOI: 10.1186/s13046-021-02143-x -
Molecular Cancer Sep 2022Chemoresistance is a major factor contributing to the poor prognosis of patients with pancreatic cancer, and cancer stemness is one of the most crucial factors...
BACKGROUND
Chemoresistance is a major factor contributing to the poor prognosis of patients with pancreatic cancer, and cancer stemness is one of the most crucial factors associated with chemoresistance and a very promising direction for cancer treatment. However, the exact molecular mechanisms of cancer stemness have not been completely elucidated.
METHODS
mA-RNA immunoprecipitation and sequencing were used to screen mA-related mRNAs and lncRNAs. qRT-PCR and FISH were utilized to analyse DDIT4-AS1 expression. Spheroid formation, colony formation, Western blot and flow cytometry assays were performed to analyse the cancer stemness and chemosensitivity of PDAC cells. Xenograft experiments were conducted to analyse the tumour formation ratio and growth in vivo. RNA sequencing, Western blot and bioinformatics analyses were used to identify the downstream pathway of DDIT4-AS1. IP, RIP and RNA pulldown assays were performed to test the interaction between DDIT4-AS1, DDIT4 and UPF1. Patient-derived xenograft (PDX) mouse models were generated to evaluate chemosensitivities to GEM.
RESULTS
DDIT4-AS1 was identified as one of the downstream targets of ALKBH5, and recruitment of HuR onto mA-modified sites is essential for DDIT4-AS1 stabilization. DDIT4-AS1 was upregulated in PDAC and positively correlated with a poor prognosis. DDIT4-AS1 silencing inhibited stemness and enhanced chemosensitivity to GEM (Gemcitabine). Mechanistically, DDIT4-AS1 promoted the phosphorylation of UPF1 by preventing the binding of SMG5 and PP2A to UPF1, which decreased the stability of the DDIT4 mRNA and activated the mTOR pathway. Furthermore, suppression of DDIT4-AS1 in a PDX-derived model enhanced the antitumour effects of GEM on PDAC.
CONCLUSIONS
The ALKBH5-mediated mA modification led to DDIT4-AS1 overexpression in PDAC, and DDIT-AS1 increased cancer stemness and suppressed chemosensitivity to GEM by destabilizing DDIT4 and activating the mTOR pathway. Approaches targeting DDIT4-AS1 and its pathway may be an effective strategy for the treatment of chemoresistance in PDAC.
Topics: AlkB Homolog 5, RNA Demethylase; Animals; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Mice; Pancreatic Neoplasms; RNA Helicases; RNA, Antisense; RNA, Long Noncoding; TOR Serine-Threonine Kinases; Trans-Activators; Transcription Factors; Up-Regulation
PubMed: 36056355
DOI: 10.1186/s12943-022-01647-0 -
Journal of Experimental & Clinical... Apr 2021Cancer stem cell (CSC)-related chemoresistance leads to poor outcome of the patients with colorectal cancer (CRC). In this study, we identified the...
BACKGROUND
Cancer stem cell (CSC)-related chemoresistance leads to poor outcome of the patients with colorectal cancer (CRC). In this study, we identified the chemoresistance-relevant molecules and decipher the involved mechanisms to provide potential therapeutic target for CRC. We focused on Sec62, a novel target with significantly increased expression in chemoresistant CRC tissues, and further investigated its role in the progression of CRC.
METHODS
Through analyzing the differentially-expressed genes between chemoresistant and chemosensitive CRCs, we selected Sec62 as a novel chemoresistance-related target in CRC. The expression and clinical significance of Sec62 were determined by immunoblotting and immunohistochemistry in tissues and cell lines of CRC. The roles of Sec62 in drug resistance, stemness and tumorigenesis were evaluated in vitro and in vivo using functional experiments. GST pull-down, western blot, coimmunoprecipitation and Me-RIP assays were performed to further explore the downstream molecular mechanisms.
RESULTS
Sec62 upregulation was associated with the chemoresistance of CRC and poor outcome of CRC patients. Depletion of Sec62 sensitized CRC cells to chemotherapeutic drugs. Sec62 promoted the stemness of CRC cells through activating Wnt/β-catenin signaling. Mechanistically, Sec62 bound to β-catenin and inhibited the degradation of β-catenin. Sec62 competitively disrupted the interaction between β-catenin and APC to inhibit the β-catenin destruction complex assembly. Moreover, Sec62 expression was upregulated by the mA-mediated stabilization of Sec62 mRNA.
CONCLUSIONS
Sec62 upregulated by the METTL3-mediated mA modification promotes the stemness and chemoresistance of CRC by binding to β-catenin and enhancing Wnt signalling. Thus, mA modification-Sec62-β-catenin molecular axis might act as therapeutic targets in improving treatment of CRC.
Topics: Animals; Cell Line, Tumor; Colorectal Neoplasms; Drug Resistance, Neoplasm; Female; Fluorouracil; HEK293 Cells; Humans; Membrane Transport Proteins; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Oxaliplatin; Wnt Signaling Pathway; Xenograft Model Antitumor Assays; beta Catenin; Mice
PubMed: 33858476
DOI: 10.1186/s13046-021-01934-6 -
Journal of Experimental & Clinical... Aug 2022Tyrosine kinase inhibitors (TKIs) such as sunitinib are multitarget antiangiogenic agents in clear cell renal cell carcinoma (ccRCC). They are widely used in the...
BACKGROUND
Tyrosine kinase inhibitors (TKIs) such as sunitinib are multitarget antiangiogenic agents in clear cell renal cell carcinoma (ccRCC). They are widely used in the treatment of advanced/metastatic renal cancer. However, resistance to TKIs is common in the clinic, particularly after long-term treatment. YTHDC1 is the main nuclear reader protein that binds with mA to regulate the splicing, export and stability of mRNA. However, the specific role and corresponding mechanism of YTHDC1 in renal cancer cells are still unclear.
METHODS
The Cancer Genome Atlas (TCGA) dataset was used to study the expression of YTHDC1 in ccRCC. Cell counting kit-8 (CCK-8), wound healing, Transwell and xenograft assays were applied to explore the biological function of YTHDC1 in ccRCC. Western blot, quantitative real time PCR (RT‒qPCR), RNA immunoprecipitation PCR (RIP-qPCR), methylated RIP-qPCR (MeRIP-qPCR) and RNA sequencing (RNA-seq) analyses were applied to study the YY1/HDAC2/YTHDC1/ANXA1 axis in renal cancer cells. The CCK-8 assay and xenograft assay were used to study the role of YTHDC1 in determining the sensitivity of ccRCC to sunitinib.
RESULTS
Our results demonstrated that YTHDC1 is downregulated in ccRCC tissues compared with normal tissues. Low expression of YTHDC1 is associated with a poor prognosis in patients with ccRCC. Subsequently, we showed that YTHDC1 inhibits the progression of renal cancer cells via downregulation of the ANXA1/MAPK pathways. Moreover, we also showed that the YTHDC1/ANXA1 axis modulates the sensitivity of tyrosine kinase inhibitors. We then revealed that HDAC2 inhibitors resensitize ccRCC to tyrosine kinase inhibitors through the YY1/HDAC2 complex. We have identified a novel YY1/HDAC2/YTHDC1/ANXA1 axis modulating the progression and chemosensitivity of ccRCC.
CONCLUSION
We identified a novel YY1/HDAC2/YTHDC1/ANXA1 axis modulating the progression and chemosensitivity of ccRCC.
Topics: Annexin A1; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Histone Deacetylase 2; Humans; Kidney Neoplasms; MAP Kinase Signaling System; Nerve Tissue Proteins; Protein Kinase Inhibitors; RNA Splicing Factors; Sunitinib; YY1 Transcription Factor
PubMed: 35974388
DOI: 10.1186/s13046-022-02460-9 -
Journal of Experimental & Clinical... Aug 2023Gemcitabine resistance has brought great challenges to the treatment of pancreatic cancer. The N6-methyladenosine (m6A) mutation has been shown to have a significant...
BACKGROUND
Gemcitabine resistance has brought great challenges to the treatment of pancreatic cancer. The N6-methyladenosine (m6A) mutation has been shown to have a significant regulatory role in chemosensitivity; however, it is not apparent whether gemcitabine resistance can be regulated by fat mass and obesity-associated protein (FTO).
METHODS
Cells with established gemcitabine resistance and tissues from pancreatic cancer patients were used to evaluate FTO expression. The biological mechanisms of the effects of FTO on gemcitabine resistant cells were investigated using CCK-8, colony formation assay, flow cytometry, and inhibitory concentration 50. Immunoprecipitation/mass spectrometry, MeRIP-seq, RNA sequencing and RIP assays, RNA stability, luciferase reporter, and RNA pull down assays were employed to examine the mechanism of FTO affecting gemcitabine resistant pancreatic cancer cells.
RESULTS
The results revealed that FTO was substantially expressed in cells and tissues that were resistant to gemcitabine. Functionally, the gemcitabine resistance of pancreatic cancer could be enhanced by FTO, while its depletion inhibited the growth of gemcitabine resistant tumor cells in vivo. Immunoprecipitation/mass spectrometry showed that the FTO protein can be bound to USP7 and deubiquitinated by USP7, leading to the upregulation of FTO. At the same time, FTO knockdown significantly decreased the expression level of NEDD4 in an m6A-dependent manner. RNA pull down and RNA immunoprecipitation verified YTHDF2 as the reader of NEDD4, which promoted the chemoresistance of gemcitabine resistant cells. FTO knockdown markedly increased the PTEN expression level in an NEDD4-dependent manner and influenced the chemosensitivity to gemcitabine through the PI3K/AKT pathway in pancreatic cancer cells.
CONCLUSION
In conclusion, we found that gemcitabine resistance in pancreatic cancer can be influenced by FTO that demethylates NEDD4 RNA in a m6A-dependent manner, which then influences the PTEN expression level and thereby affects the PI3K/AKT pathway. We also identified that the FTO level can be upregulated by USP7.
Topics: Humans; Gemcitabine; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Ubiquitin-Specific Peptidase 7; RNA Stability; Pancreatic Neoplasms; PTEN Phosphohydrolase; Alpha-Ketoglutarate-Dependent Dioxygenase FTO
PubMed: 37605223
DOI: 10.1186/s13046-023-02792-0 -
Micromachines Jul 2016With a mortality rate over 580,000 per year, cancer is still one of the leading causes of death worldwide. However, the emerging field of microfluidics can potentially... (Review)
Review
With a mortality rate over 580,000 per year, cancer is still one of the leading causes of death worldwide. However, the emerging field of microfluidics can potentially shed light on this puzzling disease. Unique characteristics of microfluidic chips (also known as micro-total analysis system) make them excellent candidates for biological applications. The ex vivo approach of tumor-on-a-chip is becoming an indispensable part of personalized medicine and can replace in vivo animal testing as well as conventional in vitro methods. In tumor-on-a-chip, the complex three-dimensional (3D) nature of malignant tumor is co-cultured on a microfluidic chip and high throughput screening tools to evaluate the efficacy of anticancer drugs are integrated on the same chip. In this article, we critically review the cutting edge advances in this field and mainly categorize each tumor-on-a-chip work based on its primary organ. Specifically, design, fabrication and characterization of tumor microenvironment; cell culture technique; transferring mechanism of cultured cells into the microchip; concentration gradient generators for drug delivery; in vitro screening assays of drug efficacy; and pros and cons of each microfluidic platform used in the recent literature will be discussed separately for the tumor of following organs: (1) Lung; (2) Bone marrow; (3) Brain; (4) Breast; (5) Urinary system (kidney, bladder and prostate); (6) Intestine; and (7) Liver. By comparing these microchips, we intend to demonstrate the unique design considerations of each tumor-on-a-chip based on primary organ, e.g., how microfluidic platform of lung-tumor-on-a-chip may differ from liver-tumor-on-a-chip. In addition, the importance of heart⁻liver⁻intestine co-culture with microvasculature in tumor-on-a-chip devices for in vitro chemosensitivity assay will be discussed. Such system would be able to completely evaluate the absorption, distribution, metabolism, excretion and toxicity (ADMET) of anticancer drugs and more realistically recapitulate tumor in vivo-like microenvironment.
PubMed: 30404302
DOI: 10.3390/mi7080130 -
World Journal of Gastroenterology Nov 2014The evaluation of therapeutic efficacy is necessary to predict the outcome of patients with metastatic colorectal cancer (CRC). In these patients, there is a critical... (Review)
Review
The evaluation of therapeutic efficacy is necessary to predict the outcome of patients with metastatic colorectal cancer (CRC). In these patients, there is a critical need for predictive chemosensitivity assays and biomarkers to optimize efficacy and minimize toxicity. The introduction of targeted agents has improved the progression-free survival and overall survival of patients with metastatic disease. However, approximately 50% of patients do not show a positive response to chemotherapy and the selection of patients likely to respond to a specific regimen remains challenging. Cell culture-based chemosensitivity tests use autologous viable tumor cells to evaluate susceptibility to specific agents in vitro and predict their direct effects. Adenosine triphosphate-based assays and methyl thiazolyl-diphenyl-tetrazolium bromide-based assays are used widely as sensitivity tests because of their short assay period, technical simplicity, and the requirement of small amount of specimen. Among protein- and gene-based chemosensitivity assays, assessment of KRAS mutation status predicts the response to epidermal growth factor receptor-targeted therapy in CRC patients. The validation of predictive and prognostic markers enables the selection of therapeutic regimens with optimal efficacy and minimal toxicity for each patient, which has been termed personalized treatment. This review summarizes currently available predictive and prognostic chemosensitivity tests for metastatic CRC.
Topics: Adenocarcinoma; Antineoplastic Agents; Biomarkers, Tumor; Colorectal Neoplasms; Drug Screening Assays, Antitumor; Genetic Predisposition to Disease; Genetic Testing; Humans; Molecular Targeted Therapy; Phenotype; Precision Medicine; Predictive Value of Tests
PubMed: 25469008
DOI: 10.3748/wjg.v20.i44.16398 -
Molecular Oncology Oct 2022To investigate the effect of Apatinib (an inhibitor targeting VEGFR-2) enhances chemosensitivity of ABT-199 on diffuse large B-cell lymphoma (DLBCL). Viability assay and...
To investigate the effect of Apatinib (an inhibitor targeting VEGFR-2) enhances chemosensitivity of ABT-199 on diffuse large B-cell lymphoma (DLBCL). Viability assay and flow cytometric assay for determining apoptosis, cell cycle, mitochondrial membrane potential, reactive oxygen species and immunoblotting were used to explore the combination effect in DLBCL cell lines, DLBCL patient samples, and DLBCL mouse models. RNA sequencing assay helped identify mechanisms of ABT-199 plus Apatinib. The results show that ABT-199 combined with Apatinib inhibited cell proliferation, reduced colony-forming capacity, and induced apoptosis and cell cycle arrest in DLBCL cells. Mechanistically, the combination therapy inhibited tumour cell growth and promoted tumour cell death by regulating EDN1 and MAPK-related pathways and activating the intrinsic apoptotic pathway. The effect of the combination therapy was also validated in primary DLBCL blasts and xenograft mouse models. Our findings indicate that Apatinib enhances the chemosensitivity of ABT-199 and might serve as a promising therapeutic strategy for DLBCL.
Topics: Humans; Mice; Animals; Vascular Endothelial Growth Factor Receptor-2; Reactive Oxygen Species; Xenograft Model Antitumor Assays; Cell Line, Tumor; Lymphoma, Large B-Cell, Diffuse; Apoptosis; Cell Proliferation
PubMed: 36053810
DOI: 10.1002/1878-0261.13309 -
Brain and Behavior Dec 2022We attempted to investigate influence of microRNA-433-3p on malignant progression of glioma and identify its molecular mechanism, thus laying groundwork for glioma...
OBJECTIVE
We attempted to investigate influence of microRNA-433-3p on malignant progression of glioma and identify its molecular mechanism, thus laying groundwork for glioma management.
METHODS
Expression data along with clinical data of glioma were accessed from the TCGA database for differential and survival analyses to look for the target differentially expressed genes. Quantitative reverse transcriptase PCR (qRT-PCR) and western blot were utilized to assess NR5A2 mRNA and protein expression in different glioma cell lines, respectively. MTT, Transwell assay, and flow cytometry were carried out to assay the impact of NR5A2 on behaviors of glioma cells in vitro. Bioinformatics analysis was used to identify the upstream microRNA of NR5A2 in glioma, while dual-luciferase and western blot assays were used to detect binding of microRNA and NR5A2. Chemosensitivity of glioma cells was evaluated by cisplatin cytotoxicity test.
RESULTS
NR5A2 was upregulated in both glioma tissues and cell lines. Dual-luciferase assay result showed binding site of microRNA-433-3p on NR5A2 mRNA 3'UTR, and microRNA-433-3p reduced NR5A2 expression. Cell assays revealed that silencing NR5A2 could hamper proliferation, invasion, and migration and enhance chemosensitivity to cisplatin while promoting glioma cell apoptosis and blocking glioma cells in G0/G1 phase. Rescue experiments also indicated that microRNA-433-3p suppressed glioma malignant progression via inhibiting NR5A2.
CONCLUSION
MicroRNA-433-3p which is significantly poorly expressed in glioma targets NR5A2 to suppress glioma malignant progression and enhance chemosensitivity to cisplatin.
Topics: Humans; Cisplatin; Gene Expression Regulation, Neoplastic; Cell Line, Tumor; Glioma; MicroRNAs; Apoptosis; RNA, Messenger; Cell Proliferation; Receptors, Cytoplasmic and Nuclear
PubMed: 36303447
DOI: 10.1002/brb3.2632