Radiation-resistant Cell Line Research Articles

Small molecule inhibitor azd1480 reverses radiotherapy resistance in NSCLC by targeting JAK2/STAT3 pathway

Purpose: To investigate the effect of small molecule inhibitor AZD1480 on radiotherapy resistance in non-small cell lung cancer (NSCLC), and the involvement of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway in the process.
 Methods: Radiation-resistant cell lines A549-20F, A549-30F, A549-40F and H460, H46040F, H460- 40F, and H460-40F were established, and expressions of proteins related to JAK/STAT pathway, mitogen-activated protein (MAPK) pathway and transforming growth factor-β (TGF- β) were assayed. The JAK2V617FH460 overexpression cell line and JAK2H460 cell line were established, and expressions of ATGL and CPT1A were compared. The H460 cells and H460-40F cells were treated with JAK2 small molecule inhibitor AZD1480, and the expressions of ATGL, CPT1A and JAK2/STAT3 pathway-related proteins were compared. The survival and proliferation of cell lines were also compared. 
 Results: The JAK/STAT pathway was significantly enriched, and MAPK and TGF-β were up-regulated. In H460 cells, JAK2/STAT3 route was obvious, suggesting that radiotherapy activated JAK2/STAT3 pathway in NSCLC cells. Significant down-regulations of p- JAK2Y1007, JAK2, p-STAT3s727, STAT3, ATGL and CPT1A proteins expressions were seen in H460 + AZD1480 group, relative to H460 group, but protein levels of p-JAK2Y1007, JAK2, p-STAT3s727, STAT3, ATGL and CPT1A were significantly lower in H460-40F + AZD1480 group than in H460-40F group (p < 0.05). The survival and proliferation rates were significantly lower in A549 + AZD1480 group than in A549 and A549-40F groups (p < 0.05).
 Conclusion: Radiotherapy up-regulates the expressions of ATGL and CPT1A in NSCLC cells by activating the JAK2/STAT3 pathway, while AZD1480, a small molecule inhibitor, reverses the radiation resistance of NSCLC by targeting JAK2/STAT3 pathway and key enzymes of lipid metabolism. Therefore, azd1480 may enhance clinical treatment efficacy in NSCLC patients by reducing radiation resistance.

Detection of MiRNA-126-5p in Non-Small Cell Lung Cancer Cells by Nano-Polymerase Chain Reaction and Its Regulatory Mechanism of Radiation Sensitivity to Cancer Cells

Radiotherapy resistance remains a huge hindrance in treating non-small cell lung carcinoma (NSCLC). Hyperthermia, a treatment method that raises the cell temperature to treat tumors, has been putting in clinical application combined with conventional chemotherapy and radiotherapy to enhance their effectiveness in NSCLC treatment. However, the specific mechanism of this combination therapy has not been extensively researched. In this study, we established a radiation-resistant NSCLC cell line by sequential radiation exposure. It was shown that the combination of hyperthermia and radiotherapy suppressed NSCLC xenograft tumor growth and increased radiation-induced apoptosis. Furthermore, downregulation of miR-126-5p was found in radio-resistant NSCLC cells. The results of bioinformatics analysis based on ENCORI showed that RAD50 is a direct target of miR-126-5p. Overexpressed miR-126-5p increased radiation-induced cell death by suppressing RAD50 expression. Hyperthermia treatment increases miR-126-5p and decreases RAD50, leading to more unrepaired DNA damage and greater cellular death. In conclusion, hyperthermia enhances NSCLC cells’ radio-sensitivity via miR-126-5p/RAD50 axis.

PAICS/DYRK3 Multienzyme Interactions as Coregulators of Purinosome Formation and Metabolism on Radioresistance in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a prevalent type of oral cancer. While therapeutic innovations have made strides, radioresistance persists as a significant hindrance in OSCC treatment. Despite identifying numerous targets that could potentially suppress the oncogenic attributes of OSCC, the exploration of oncogenic protein kinases for cancer therapy remains limited. Consequently, the functions of many kinase proteins in OSCC continue to be largely undetermined. In this research, we aim to disclose protein kinases that target OSCC and elaborate their roles and molecular mechanisms. Through the examination of the kinome library of radiotherapy-resistant/sensitive OSCC cell lines (HN12 and SAS), we identified a key gene, the tyrosine phosphorylation-regulated kinase 3 (DYRK3), a member of the DYRK family. We developed an in vitro cell model, composed of radiation-resistant OSCC, to scrutinize the clinical implications and contributions of DYRK3 and phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS) signaling in OSCC. This investigation involves bioinformatics and human tissue arrays. We seek to comprehend the role of DYRK3 and PAICS signaling in the development of OSCC and its resistance to radiotherapy. Various in vitro assays are utilized to reveal the essential molecular mechanism behind radiotherapy resistance in connection with the DYRK3 and PAICS interaction. In our study, we quantified the concentrations of DYRK3 and PAICS proteins and tracked the expression levels of key pluripotency markers, particularly PPAT. Furthermore, we extended our investigation to include an analysis of Glut-1, a gene recognized for its linkage to radioresistance in oral squamous cell carcinoma (OSCC). Furthermore, we conducted an in vivo study to affirm the impact of DYRK3 and PAICS on tumor growth and radiotherapy resistance, focusing particularly on the role of DYRK3 in the radiotherapy resistance pathway. This focus leads us to identify new therapeutic agents that can combat radiotherapy resistance by inhibiting DYRK3 (GSK-626616). Our in vitro models showed that inhibiting PAICS disrupts purinosome formation and influences the survival rate of radiation-resistant OSCC cell lines. These outcomes underscore the pivotal role of the DYRK3/PAICS axis in directing OSCC radiotherapy resistance pathways and, as a result, influencing OSCC progression or therapy resistance. Our findings also reveal a significant correlation between DYRK3 expression and the PAICS enzyme in OSCC radiotherapy resistance.

Suppresses of LIM kinase 2 promotes radiosensitivity in radioresistant non-small cell lung cancer cells

Radiation resistance has always been one of the main obstacles to tumor radiotherapy. Therefore, understanding the mechanisms underlying radiotherapy resistance is a focus of research. In this study, we induced two radiation-resistant cell lines to mimic the radiation resistance of NSCLC and investigated the mechanisms of radiotherapy resistance. Cell radiosensitivity was analyzed by single-cell gel electrophoresis, colony formation and tumor sphere formation assays. A wound healing assay was used to analyze cell migration. Western blotting and siRNA were used to identify the potential mechanism. In animal model experiments, xenograft tumors were used to verify the difference between radiotherapy-resistant and nonresistant NSCLC models after radiotherapy. Our results showed that NSCLC radiation-resistant cells exhibited more radioresistance and migratory abilities under low-dose irradiation. The expression of LIMK2 and p-CFL1 were upregulated in NSCLC radiation-resistant cells. Knockdown of LIMK2 significantly enhanced the radiosensitivity of NSCLC-resistant cells. In vivo, low-dose radiotherapy suppressed tumor growth, induced apoptosis and upregulated the expression of LIMK2 in xenograft tumors. However, radiotherapy had little effect on the NSCLC radiation resistance model. In conclusion, NSCLC radiation-resistant cells exhibit more radioresistance and migratory ability under low-dose irradiation. Strikingly, knockdown of LIMK2 enhanced the radiosensitivity of NSCLC-resistant cells.

Phase-separated ribosome-nascent chain complexes in genotoxic stress response.

Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered amino-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of Saccharomyces cerevisiae protein complexes according to their amino-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, SGS1 mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together, these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human.

Prognostic evaluation of pancreatic cancer based on the model of chemo-radiotherapy resistance-related genes.

The incidence and mortality of pancreatic cancer are almost the same, and the 5-year survival rate is less than 10%. The high mortality of pancreatic cancer is related to chemo-radiotherapy. The present study aimed to establish a prognostic signature of pancreatic cancer based on chemo-radiotherapy resistant-related genes (CRRGs). In this study, we explored the radiation-resistant and chemotherapy-resistant pancreatic cancer cell lines by colony formation and a subcutaneous tumor model in nude mice. Next, we obtained CRRGs from radiation- and gemcitabine-resistant pancreatic cancer cell lines in the Gene Expression Omnibus (GEO) database. Based on univariate Cox and least absolute shrinkage and selection operator (LASSO) Cox regression analyses, a prognostic model of the pancreatic adenocarcinoma (PAAD) cohort in The Cancer Genome Atlas (TCGA) database (N=177) was established and verified using the GEO cohort data set (N=112). Finally, the functions of candidate target genes were verified by a methyl thiazolyl tetrazolium (MTT) assay, a colony formation assay, and a subcutaneous tumor model in nude mice. Through the in vitro and in vivo experiments, we found that radiotherapy- and chemotherapy-resistant pancreatic cancer cells were cross-resistant to chemotherapy and radiotherapy. We constructed a risk model consisting of nine CRRGs (SNAP25, GPR87, DLL1, LAD1, WASF3, ARHGAP29, ZBED2, GAD1, and JAG1) by using public databases. According to the Kaplan-Meier curve analysis, the survival of the high-risk group was worse than that of the low-risk group. We then used nomograms to predict the 1/3/5-year overall survival (OS) in pancreatic cancer patients. We chose JAG1 as a candidate target since it has been proven to be involved in the stemness maintenance of cancer cells, and found that JAG1 silencing inhibited the proliferation and chemo-radiotherapy tolerance of pancreatic cancer cells. This study established and validated a prognostic signature of pancreatic cancer using nine CRRGs. The in vitro and in vivo experiments showed that JAG1 could promote the proliferation and chemoradiotherapy tolerance of pancreatic cancer cell lines. These findings may offer new insights into the role of CRRGs in pancreatic cancer and provide novel prognostic biomarkers for the treatment of pancreatic cancer.

MACC1 as a Potential Target for the Treatment and Prevention of Breast Cancer

Metastasis associated in colon cancer 1 (MACC1) is an oncogene first identified in colon cancer. MACC1 has been identified in more than 20 different types of solid cancers. It is a key prognostic biomarker in clinical practice and is involved in recurrence, metastasis, and survival in many types of human cancers. MACC1 is significantly associated with the primary tumor, lymph node metastasis, distant metastasis classification, and clinical staging in patients with breast cancer (BC), and MACC1 overexpression is associated with reduced recurrence-free survival (RFS) and worse overall survival (OS) in patients. In addition, MACC1 is involved in BC progression in multiple ways. MACC1 promotes the immune escape of BC cells by affecting the infiltration of immune cells in the tumor microenvironment. Since the FGD5AS1/miR-497/MACC1 axis inhibits the apoptotic pathway in radiation-resistant BC tissues and cell lines, the MACC1 gene may play an important role in BC resistance to radiation. Since MACC1 is involved in numerous biological processes inside and outside BC cells, it is a key player in the tumor microenvironment. Focusing on MACC1, this article briefly discusses its biological effects, emphasizes its molecular mechanisms and pathways of action, and describes its use in the treatment and prevention of breast cancer.

Curcumin Increases Radiosensitivity of Radioresistant Nasopharyngeal Cancer.

To study the effects of curcumin on the proliferation, invasion, apoptosis, and radiosensitivity of the radioresistant nasopharyngeal carcinoma (NPC) C6661-IR strain as well as the potential radiosensitization mechanism. NPC cells were continuously irradiated with different intensities of radiation to induce radiation-resistant cell lines. A plate clone formation assay was used to evaluate the effect of curcumin on the radiosensitivity of NPC cells. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) assay was conducted to detect changes in cell viability. Flow cytometry was employed to analyze apoptosis percentage as well as Transwell® assay and immunofluorescence assay to observe cell invasion. Western blotting was applied to detect the expression levels of Bax, Bcl-2, and pro/cleaved-caspase 3. MiR-205-5p mimics and si-TP53INP1 were synthesized and transfected into C6661-IR cells, and the cells were then incubated with 10 μm/L curcumin. Real-time quantitative reverse transcription PCR (RT-qPCR) was used to measure miR-205-5p levels and western blotting was conducted to detect the expression of TP53INP1. The optimal radiation dose of X-ray was 6 Gy, and this dose was used in all subsequent experiments. Curcumin treatment significantly inhibited the proliferation and invasion of C6661-IR cells, promoted apoptosis and enhanced radiosensitivity. Compared to the 0 Gy+Cur group and the 6 Gy+Cur group, the miR-205-5p levels were higher in the C6661-IR cells of the 0 Gy and 6 Gy groups. Moreover, miR-204-5p was found to directly target TP53INP1. Curcumin downregulated miR-205-5p levels and upregulated TP53INP1 expression (p < 0.05). Thus, modulation of miR-205-5p or TP53INP1 expression attenuates the biological effects of curcumin on C6661-IR cells. Curcumin inhibited the proliferation and invasion of C6661-IR, promoted apoptosis, and enhanced its radiosensitivity to X-rays by mediating miR-205-5p/TP53INP1 expression.

The miR-34a-5p-c-MYC-CHK1/CHK2 Axis Counteracts Cancer Stem Cell-Like Properties and Enhances Radiosensitivity in Hepatocellular Cancer Through Repression of the DNA Damage Response.

Radiotherapy has become an increasingly widespread modality for treating hepatocellular cancer (HCC); however, the development of radioresistance significantly limits its effectiveness and invariably leads to tumor recurrence. Cancer stem cell (CSC) theory offers a potential explanation for tumor relapse and radioresistance, but the underlying mechanism remains unknown. Herein we investigate the role of miRNA in molecular regulation of stemness and radioresistance in HCC. Two HCC radiation-resistant cell lines (Huh7-RR and SMMC-7721-RR) were established by selecting the radioresistant subpopulation from HCC cells via clonogenic survival assays. MiRNA Sequencing was used to identify potential radiosensitivity involved miRNA in HCC-RR cells. Xenograft tumor mouse model was established for in vivo study. CSC properties were assessed using sphere formation assay and side population (SP) cells analysis. We found that miR-34a-5p was significantly downregulated in HCC-RR cells. Overexpression of miR-34a-5p counteracts CSC properties and enhances radiosensitivity in HCC. Mechanistic investigation revealed that c-MYC is the direct target of miR-34a-5p. Overexpression of miR-34a-5p reversed c-MYC-induced radioresistance. Moreover, we found that the specific molecular mechanism was that c-MYC activated CHK1 and CHK2, which are two key DNA damage checkpoint kinases, and facilitated the DNA damage response to radiation. Repression of the miR-34a-5p-cMYC-CHK1/CHK2 axis contributes to the acquisition of radioresistance in HCC cells. In summary, the miR-34a-5p-c-MYC-CHK1/CHK2 axis counteracts cancer stem cell-like properties and enhances radiosensitivity in hepatocellular cancer through repression of the DNA damage response.

The role of radiotherapy-related autophagy genes in the prognosis and immune infiltration in lung adenocarcinoma.

BackgroundThere is a close relationship between radiotherapy and autophagy in tumors, but the prognostic role of radiotherapy-related autophagy genes (RRAGs) in lung adenocarcinoma (LUAD) remains unclear.MethodsData used in the current study were extracted from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Weighted gene co-expression network analysis (WGCNA) was executed to recognize module genes associated with radiotherapy. The differentially expressed genes (DEGs) between different radiotherapy response groups were filtered via edgeR package. The differentially expressed radiotherapy-related autophagy genes (DERRAGs) were obtained by overlapping the module genes, DEGs, and autophagy genes (ATGs). Then, prognostic autophagy genes were selected by Cox analyses, and a risk model and nomogram were subsequently built. Gene Set Enrichment Analysis (GSEA) and single-sample Gene Set Enrichment Analysis (ssGSEA) were performed to investigate potential mechanisms through which prognostic autophagy signatures regulate LUAD. Radiotherapy-resistant cell lines (A549IR and PC9IR) were established after exposure to hypo-fractionated irradiation. Ultimately, mRNA expression was validated by quantitative real-time PCR (qRT-PCR), and relative protein levels were measured in different cell lines by western blot.ResultsA total of 11 DERRAGs were identified in LUAD. After Cox analyses, SHC1, NAPSA, and AURKA were filtered as prognostic signatures in LUAD. Then, the risk score model was constructed using the prognostic signatures, which had a good performance in predicting the prognosis, as evidenced by receiver operating characteristics curves. Furthermore, Cox regression analyses demonstrated that risk score was deemed as an independent prognostic factor in LUAD. Moreover, GSEA and ssGSEA results revealed that prognostic RRAGs may regulate LUAD by modulating the immune microenvironment and affecting cell proliferation. The colony formation assay showed that the radiosensitivity of radiation-resistant cell lines was lower than that of primary cells. The western blot assay found that the levels of autophagy were elevated in the radiotherapy-resistant cell lines. Moreover, the expression of DERRAGs (SHC1, AURKA) was higher in the radiotherapy-resistant cells than in primary cells.ConclusionOur study explored the role of RRAGs in the prognosis of LUAD and identified three biomarkers. The findings enhanced the understanding of the relationship between radiotherapy, autophagy, and prognosis in LUAD and provided potential therapeutic targets for LUAD patients.

Identification of Key Genes and FUNCTIONAL Pathway in Radioresistance of Non-Small Cell Lung Cancer.

PurposeFor better understanding of radiotherapy resistance and its potential mechanism.MethodsWe established radioresistance cell lines of non-small cell lung cancer (NSCLC) followed by microarray analysis. 529 differentially expressed genes (DEGs) were then screened between radiation resistant cell lines compared with the sensitive cell lines. The biological functions and enrichment pathways of the above DEGs were identified using Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analyses. Gene Set Enrichment Analysis (GSEA) revealed that the radiation resistance group had the most gene sets enriched in altered immune response, such as TNF signaling pathway, when compared to the radiation sensitive group. Protein-protein interaction (PPI) network was carried out through the STRING database, and then five hub genes (CXCL10, IFIH1, DDX58, CXCL11, RSAD2) were screened by Cytoscape software. RT-PCR confirmed the expression of the above hub genes. ChIP-X Enrichment Analysis showed that STAT1 might be the transcription factor of the above hub genes. Considering that PD-L1 could be activated by STAT1 in a variety of tumors and ultimately lead to immune exhaustion, RT-PCR and Western blot verified the expression level of PD-L1.ResultsFive hub genes (CXCL10, IFIH1, DDX58, CXCL11, RSAD2) were screened and verified to be highly expressed in radioresistance group, STAT1 might be the transcription factor of the above hub genes. Our study found that the expression level of PD-L1 was increased after radiotherapy resistance.ConclusionAlthough immune system activation occurs followed by radiation resistance, we hypothesized that the upregulation of PD-L1 expression caused by STAT1 activation might be one of the mechanisms of radiotherapy resistance.

LINC01578 affects the radiation resistance of lung cancer cells through regulating microRNA-216b-5p/TBL1XR1 axis

ABSTRACT Radiation resistance largely limits the survival of patients with non-small-cell lung cancer (NSCLC). To understand the mechanism underlying radiation resistance, we explored the influence of LINC01578 in radiation-resistant NSCLC cells. LINC01578, miR-216b-5p and Transducin (beta)-like 1 X-linked receptor 1 (TBL1XR1) expression was evaluated in patients with NSCLC, and their correlation with patients’ prognosis was examined. Radiation-resistant NSCLC cell line (A549-RR) was induced and treated with oligonucleotide or plasmid transfection, and cell biological functions were captured. The interplay between LINC01578, miR-216b-5p and TBL1XR1 was clarified. NSCLC patients showed high LINC01578 and TBL1XR1 expression, and low miR-216b-5p expression, which was correlated to shorter patients’ prognosis, respectively. LINC01578 or TBL1XR1 deficiency or miR-216b-5p elevation suppressed the functional activities of A549-RR cells. LINC01578 suppression elevated miR-216b-5p expression, consequently leading to the down-regulation of TBL1XR1. miR-216b-5p silencing or TBL1XR1 overexpression compromised LINC01578 knockdown’s effects on radiation resistance of A549-RR cells. In brief, LINC01578 suppresses miR-216b-5p and enhances TBL1XR1 expression, thus to promote biological functions of radiation-resistant NSCLC cells.

Studies on Screening of MicroRNA Related to Irradiation Response and the Regulation Mechanism of miRNA-96-5p on Irradiation Resistance in Rectal Cancer Cells

<h3>Purpose/Objective(s)</h3> To screen microRNAs that are positively correlated with radiation resistance in rectal cancer, and to investigate their regulatory mechanism. <h3>Materials/Methods</h3> Fresh rectal cancer tissues were obtained intraoperatively and divided into two equal parts, one of which was pre-stored in a refrigerator at -150℃, and the other was constructed with Patients Derived Xenograft (PDX) models for rectal cancer and irradiated to transplantation tumor screen out radiation-sensitive tumor tissues and radiation-resistant tumor tissues. According to the screening results, the pre-stored samples were screened for differential microRNAs in radiation reactivity, and the microRNAs that were positively correlated with irradiation resistance were obtained. On the base of screening results, the pre-stored samples were screened for differential microRNAs in irradiation reactivity by miRNAs microarrays, and the microRNAs that were positively correlated with irradiation resistance were obtained. Biological prediction software was used to predict the target genes regulated by microRNA, and the prediction results were verified in radiation-resistant rectal cancer cell lines. Lentivirus plasmid transfection technique was used to construct rectal cancer cell lines with down-regulated target microRNA expression, in order to further verify the effects of the changes in the expression of the microRNA on the radiation reactivity, proliferation, invasion and migration of rectal cancer cells. The target genes regulated by microRNAs were screened and verified, and the expression of the key genes in the signal transduction pathways involved in the regulation were detected. <h3>Results</h3> The results of microRNA screening and validation showed that there were 14 microRNAs with fold change > 1.5. Among them, miRNA-552-3p, miRNA-96-5p, miRNA-182-5p, and miRNA-183-5p were up-regulated in irradiation-resistant tumor tissues. The enrichment signaling pathway analysis showed that the Wnt signal transduction pathway was highly active in irradiation-resistant tumor tissues. The clone formation experiment under radiation interference showed that HR-8348 was the most radiation-resistant cell line among the five cell lines, and the expression level of miRNA-96-5p was positively correlated with the radiation-resistant ability of rectal cancer cell lines. The results of prediction and validation showed that the expression of GPC3 gene was directly regulated by miRNA-96-5p, and the down-regulated expression of miRNA-96-5p inhibited the activity of Wnt signal transduction pathway. <h3>Conclusion</h3> The high expression of miRNA-96-5p enhanced the radiation resistance of rectal cancer cells, and its effect may be related to the down-regulation of GPC3 expression and the abnormal activation of Wnt signal transduction pathway.

Natural Compound Shikonin Induces Apoptosis and Attenuates Epithelial to Mesenchymal Transition in Radiation-Resistant Human Colon Cancer Cells.

Radiation resistance represents an imperative obstacle in the treatment of patients with colorectal cancer, which remains difficult to overcome. Here, we explored the anti-proliferative and migration-inhibiting properties of the natural product shikonin on a radiation-resistant human colon carcinoma cell line (SNU-C5RR). Shikonin reduced the viability of these cells in a dose-dependent manner; 38 μM of shikonin was determined as the half-maximal inhibitory concentration. Shikonin induced apoptotic cell death, as demonstrated by increased apoptotic body formation and the number of TUNEL-positive cells. Moreover, shikonin enhanced mitochondrial membrane depolarization and Bax expression and also decreased Bcl-2 expression with translocation of cytochrome c from mitochondria into the cytosol. In addition, shikonin activated mitogen-activated protein kinases, and their specific inhibitors reduced the cytotoxic effects of shikonin. Additionally, shikonin decreased the migration of SNU-C5RR cells via the upregulation of E-cadherin and downregulation of N-cadherin. Taken together, these results suggest that shikonin induces mitochondria-mediated apoptosis and attenuates epithelial-mesenchymal transition in SNU-C5RR cells.

A proteomic investigation of isogenic radiation resistant prostate cancer cell lines.

To model the problem of radiation resistance in prostate cancer, cell lines mimicking a clinical course of conventionally fractionated or hypofractionated radiotherapy have been generated. Proteomic analysis of radiation resistant and radiosensitive DU145 prostate cancer cells detected 4410 proteins. Over 400 proteins were differentially expressed across both radiation resistant cell lines and pathway analysis revealed enrichment in epithelial to mesenchymal transition, glycolysis and hypoxia. From the radiation resistant protein candidates, the cell surface protein CD44 was identified in the glycolysis and epithelial to mesenchymal transition pathways and may serve as a potential therapeutic target.

MiRNA‐31 increases radiosensitivity through targeting STK40 in colorectal cancer cells

ObjectiveTo propose and verify that miRNA‐31 increases the radiosensitivity of colorectal cancer and explore its potential mechanism.MethodA bioinformatics analysis was performed to confirm that the expression of miRNA‐31 was higher in colorectal cancer than in normal colorectal tissue. The expression of miRNA‐31 was detected to verify the change in its expression in a radiotherapy‐resistant cell line. Methylation was detected to explore the cause of the decrease in miRNA‐31 expression. Overexpression or inhibition of miRNA‐31 further confirmed the change in its expression in colorectal cancer cell lines. Bioinformatics methods were used to screen the downstream target genes and for experimental verification. A luciferase assay was performed to determine the miRNA‐31 binding site in STK40. Overexpression or inhibition of STK40 in colorectal cancer cell lines further confirmed the change in STK40 expression in vitro.ResultsThe bioinformatics results showed higher expression of miRNA‐31 in tumors than in normal tissue, and miRNA‐31 mainly participated in the pathway related to cell replication. Next, we observed the same phenomenon: miRNA‐31 was expressed at higher levels in colorectal tumors than in normal colorectal tissue and its expression decreased in radiation‐resistant cell lines after radiation, implying that miRNA‐31 increased the radiosensitivity of colorectal cancer cell lines. No significant change in upstream methylation was observed. miRNA‐31 regulated the radiosensitivity of colorectal cancer cell lines by inhibiting STK40. Notably, miRNA‐31 played a role by binding to the 3′ untranslated region of SK40. STK40 negatively regulated the radiosensitivity of colorectal cancer cells.ConclusionsmiRNA‐31 increases the radiosensitivity of colorectal cancer cells by targeting STK40; miRNA‐31 and STK40 are expected to become potential biomarkers for increasing the sensitivity of tumor radiotherapy in clinical treatment.

Abstract PO-044: Exploiting a radiotherapy induced immunologic double bind in advanced prostate cancer

Abstract An evolutionary double bind occurs in cancer when resistance to a commonly used therapy increases the sensitivity to a second therapy. Typically, the second therapy is most effective in the recurrent setting and less effective when given up front. There is a tremendous need for novel second-line therapies able to exploit these new sensitivities. Prostate cancer remains one of the most treatable cancers, however, advanced disease is typically metastatic and currently incurable. Radiation therapy (RT) is often used in the resistant setting. Given locally, RT induces double-strand DNA breaks in tumor cells. This results in cellular stress responses that either trigger apoptosis and cell death, or DNA repair and cell survival. Radiosensitivity is related to the DNA repair capabilities of a tumor. Activation of DNA damage response pathways, which are mediated by ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR), induces changes in surviving cancer cells. Most notably, it has been shown that RT, as well as other DNA damaging agents, can increase natural killer cell (NK) ligand expression on tumor cells. Here we utilize an isogenic cell line model of radioresistant prostate cancer. Our pre-clinical model shows that alterations in DNA repair pathways required to maintain resistance to RT correlate with changes in NK-cell ligands, including: major histocompatibility complex class I chain-related protein A/B (MICA/B), Nectin-2 (CD112), and poliovirus receptor (PVR). This results in a 2-fold increase in sensitivity to NK-cell mediated killing in the radiation resistant cell line. An “evolutionary game assay” was performed by preparing mixtures of parental and radiation-resistant cell lines at 4 different ratios: 100:0, 90:10, 10:90 and 0:100. This was repeated for six treatment scenarios: untreated, 6Gy radiation, 1:1 NK cells, 5:1 NK cells, 6Gy with 1:1 NK cells, and 6Gy with 5:1 NK cells. Comparison of monotypic growth rates to coculture mixtures of parental and radiation-resistant cell lines enable quantification of competitive interactions through the use of evolutionary math models. This assay quantifies effective fitness (growth rate) to experimentally determine the frequency-dependent gradient of selection. Preliminary results indicate the emergence of three qualitatively unique evolutionary scenarios: 1) dominance of resistance (radiation therapy), 2) dominance of sensitive (5:1 NK), and 3) coexistence (untreated; 1:1 NK). These results suggest the existence of an evolutionary double-bind when NK-cell administration follows radiation. This was experimentally validated and confirmed by repeating the evolutionary assay with sequential radiation and NK therapy. Importantly, there is a minimum threshold of NK cells required to achieve the double-bind. Direct measurement of the fitness landscape underlying radiation and NK treatment enabled the discovery and validation of an evolutionary strategy to re-sensitize tumors after radiation treatment. Citation Format: Kimberly Luddy, Jeffrey West, Mark Robertson-Tessi, Alexander Anderson, Laure Marignol, Robert Gatenby, Cliona O'Farrelly. Exploiting a radiotherapy induced immunologic double bind in advanced prostate cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-044.

MiR-34a reverses radiation resistance on ECA-109 cells by inhibiting PI3K/AKT/mTOR signal pathway through downregulating the expression of SIRT1

Background Radiotherapy is an effective treatment for esophageal squamous cell carcinoma (ESCC). However, many ESCC patients relapsed after receiving radiotherapy due to the inherent resistance. The function of miR-34a and SIRT1, as well as the correlation between miR-34a and SIRT1 has been widely claimed in multiple types of malignant tumors. This study aimed to investigate the effects of miR-34a on radiation resistance against ESCC and the underlying mechanism. Methods In this study, CCK8, flow cytometry, wounding healing assays, and cell clone formation assay were used to determine the in vitro anti-tumor effects of radiation on radiation-resistant ESCC cell line (rECA-109). The luciferase activity and Western Blot assays were used to investigate the relationship among miR-34a, SIRT1, and the anti-radiation resistant effects. The xenograft experiments were used to verify the important function of miR-34a and SIRT1 in radiation resistance against ESCC. The apoptosis state of tumor tissues was evaluated by TUNEL assay. Results The introduction of miR-34a significantly induced the cell death and apoptosis of rECA-109 and inhibit the migration of rECA-109 treated by radiation. The anti-tumor effect was accompanied by the downregulation of SIRT1 and the inhibition of PI3K/AKT/mTOR signal pathway. The radiation resistance on rECA-109 cells was reversed by silencing SIRT1, accompanied by the PI3K/AKT/mTOR signal pathway inhibited. In vivo experiments revealed that the radiation resistance on ESCC was reversed by the introduction of miR-34a, the effect of which was promoted by the activation of SIRT1. Conclusion Our results showed that miR-34a could reverse the radiation resistance on rECA-109 cells by downregulating the expression of SIRT1through inhibiting the PI3K-AKT-mTOR signal pathway.

Restored microRNA-519a enhances the radiosensitivity of non-small cell lung cancer via suppressing EphA2.

Accumulating evidence has demonstrated that microRNA-519a (miR-519a) acts as the tumor suppressor in various cancers, but little is known regarding its intrinsic regulatory mechanisms in non-small cell lung cancer (NSCLC). Here, we aimed to investigate the role of miR-519a-targeted ephrinA2 receptor (EphA2) in radiosensitivity of NSCLC. MiR-519a and EphA2 expression in NSCLC and paracancerous tissues were detected using RT-qPCR and western blot analysis. A549 cell line was cultured and radiation-resistant cell line A549R was constructed using fractionated X-ray irradiation of these cells at 60 Gy. Colony formation ability and radioresistance of parent strain A549 and resistant strain A549R were detected with restored miR-519a and depleted EphA2. MTT assay was used to measure cell proliferation, flow cytometry was performed for determination of cell cycle distribution and apoptosis. The migration and invasion abilities were assessed by Transwell assay. The target relationship between miR-519a and EphA2 was verified. Results suggested that miR-519a was downregulated and EphA2 was upregulated in NSCLC tissues and cells, and miR-519a targeted EphA2. MiR-519a expression declined, while EphA2 expression elevated in A549R cells versus A549 cells. Upregulated miR-519a and downregulated EphA2 suppressed D0, Dq, survival fraction (SF2) and N-value, arrested cells at G0/G1 phase, advanced the apoptosis and attenuated migration, proliferation, and invasion of A549 and A549R cells. Overexpression of EphA2 reversed the promotion of upregulated miR-519a on radiosensitivity of NSCLC cells. Our results revealed that miR-519a enhances radiosensitivity of NSCLC by inhibiting EphA2 expression. Moreover, miR-519a serves as a target for NSCLC treatment.

MicroRNA-195-3p inhibits cyclin dependent kinase 1 to induce radiosensitivity in nasopharyngeal carcinoma

ABSTRACT MicroRNAs (miRNAs) are revealed to participate in the progression of multiple malignancies, including nasopharyngeal carcinoma (NPC). This work is intended to decipher the function of microRNA-195-3p (miR-195-3p) in regulating the radiosensitivity of NPC cells and its mechanism. MiR-195-3p and cyclin-dependent kinase 1 (CDK1) expressions were detected in NPC tissues and cells using qRT-PCR and Western blot, respectively. Moreover, radiation-resistant cell lines were induced by continuous irradiation with different doses. Furthermore, the CCK-8 experiment, colony formation assay and flow cytometry were utilized to examine the growth, apoptosis and cell cycle of radioresistant cells. Bioinformatics prediction and dual-luciferase reporter gene assay were applied to prove the targeting relationship between miR-195-3p and CDK1 mRNA 3ʹUTR. The data showed that miR-195-3p was remarkably down-modulated in NPC tissues and was associated with increased tumor grade, lymph node metastasis and clinical stage of the patients. MiR-195-3p expression was significantly down-modulated in radiation-resistant NPC tissues and NPC cell lines relative to radiation-sensitive NPC tissues and human nasopharyngeal epithelial cells, while CDK1 expression was notably up-modulated. MiR-195-3p overexpression inhibited the growth of NPC cells, decreased radioresistance, promoted apoptosis, and impeded the cell cycle progression. CDK1 was a target gene of miR-195-3p, and CDK1 overexpression counteracted the effects of miR-195-3p on NPC cell growth, apoptosis, cell cycle progression and radiosensitivity. In summary, miR-195-3p improves the radiosensitivity of NPC cells by targeting and regulating CDK1.