Identification of a three-gene prognostic signature for radioresistant esophageal squamous cell carcinoma

BACKGROUND Esophageal squamous cell carcinoma(ESCC)is causing a high mortality rate due to the lack of efficient early prognosis markers and suitable therapeutic regimens.The prognostic role of genes responsible for the acquisition of radioresistance in ESCC has not been fully elucidated.AIM To establish a prognostic model by studying gene expression patterns pertinent to radioresistance in ESCC patients.METHODS Datasets were obtained from the Gene Expression Omnibus and The Cancer Genome Atlas databases.The edgeR,a Bioconductor package,was used to analyze mRNA expression between different groups.We screened genes specifically responsible for radioresistance to estimate overall survival.Pearson correlation analysis was performed to confirm whether the expression of those genes correlated with each other.Genes contributing to radioresistance and overall survival were assessed by the multivariate Cox regression model through the calculation ofβi and risk score using the following formula:∑^(n)_(i=1)βi×PSI.RESULTS We identified three prognostic mRNAs(cathepsin S[CTSS],cluster of differentiation 180[CD180],and SLP adapter and CSK-interacting membrane protein[SCIMP])indicative of radioresistance.The expression of the three identified mRNAs was related to each other(r>0.70 and P<0.05).As to 1-year and 3-year overall survival prediction,the area under the time-dependent receiver operating characteristic curve of the signature consisting of the three mRNAs was 0.716 and 0.841,respectively.When stratifying patients based on the risk score derived from the signature,the high-risk group exhibited a higher death risk and shorter survival time than the low-risk group(P<0.0001).Overall survival of the low-risk patients was significantly better than that of the highrisk patients(P=0.018).CONCLUSION We have developed a novel three-gene prognostic signature consisting of CTSS,CD180,and SCIMO for ESCC,which may facilitate the prediction of early prognosis of this malignancy.

X-Ray Induced Mutation Frequency at the <i>Hypoxanthine Phosphoribosyltransferase</i>Locus in Clinically Relevant Radioresistant Cells

To elucidate the molecular mechanisms underlying cellular radioresistance, clinically relevant radioresistant cell lines were established via long-term exposure to X-rays with stepwise dose escalation. Established cells continue to proliferate despite exposure to 2 Gy X-rays/day for more than 30 days, a standard protocol in cancer radiotherapy. DNA repair fidelity in radioresistant and the parental cells by evaluating the mutation frequency at the hypoxanthine phosphoribosyltransferase (HPRT) locus after exposure to X-rays was determined. Mutation spectrum at the HPRT locus was examined by multiplex polymerase chain reaction. Rejoining kinetics of X-ray-induced DNA double strand breaks (dsbs) was evaluated by the detection of phosphorylated histone H2AX (γH2AX) after X-irradiation. The fold increase in the HPRT mutation frequency due to acute radiation was similar between radioresistant and the parental cell lines. However, fractionated radiation (FR) consisting of 2 Gy X-rays/day increased the mutation frequency at the HPRT locus in parental but not in radioresistant cells. Analysis of the FR-induced mutations at the HPRT locus revealed a high frequency of deletion mutations (>70%) in parental but not in radioresistant cells. As assessed by γH2AX immunostaining, DNA dsbs induced by acute exposure to 10 Gy of X-rays were repaired to the control level within 7 days in radioresistant but not in the parental cells. Moreover, 2 Gy × 5 FR increased the number of γH2AX-positive cells in parental cultures but not in radioresistant cultures. DNA dsbs induced by 2 Gy/day FR are repaired with fidelity in radioresistant but not in parental cells.

The radioresistant and survival mechanisms of Deinococcus radiodurans

Deinococcus radiodurans (D. radiodurans) is distinguished by the most radioresistant organism ever known, and can tolerate extreme environments such as ionizing radiation, ultraviolet radiation, oxidation, and desiccation. D. radiodurans is an important model for studying DNA damage/repair and redox regulation upon high dose ionizing radiation. How D. radiodurans response and repair ROS-induced oxidative damage remains a subject of ongoing investigation. This review provides an overview of the radioresistance characteristics of D. radiodurans. Among them, the DNA damage repair pathway and high-efficiency antioxidant defense system are summarized in detail. Furthermore, a novel model that protects the cell against the ionizing radiation is proposed. This review also discusses the potential application , future challenges and directions in advancing towards D. radiodurans studies.

LncRNA HOTAIR promotes DNA damage repair and radioresistance by targeting ATR in colorectal cancer

Long non-coding RNAs(lncRNAs)have been implicated in cancer progression and drug resistance development.Moreover,there is evidence that lncRNA HOX transcript antisense intergenic RNA(HOTAIR)is involved in colorectal cancer(CRC)progression.The present study aimed to examine the functional role of lncRNA HOTAIR in conferring radiotherapy resistance in CRC cells,as well as the underlying mechanism.The relative expression levels of HOTAIR were examined in 70 pairs of CRC tumor and para-cancerous tissues,as well as in radiosensitive and radioresistant samples.The correlations between HOTAIR expression levels and clinical features of patients with CRC were assessed using the Chi-square test.Functional assays such as cell proliferation,colony formation and apoptosis assays were conducted to determine the radiosensitivity in CRC cells with HOTAIR silencing after treatment with different doses of radiation.RNA pull-down assay andfluorescence in situ hybridization(FISH)were used to determine the interaction between HOTAIR and DNA damage response mediator ataxia-telangiectasia mutated-and Rad3-related(ATR).HOTAIR was significantly upregulated in CRC tumor tissues,especially in radioresistant tumor samples.The elevated expression of HOTAIR was correlated with more advanced histological grades,distance metastasis and the poor prognosis in patients with CRC.Silencing HOTAIR suppressed the proliferation and promoted apoptosis and radiosensitivity in CRC cells.HOTAIR knockdown also inhibited the tumorigenesis of CRC cells and enhanced the sensitivity to radiotherapy in a mouse xenograft model.Moreover,the data showed that HOTAIR could interact with ATR to regulate the DNA damage repair signaling pathway.Silencing HOTAIR impaired the ATR-ATR interacting protein(ATRIP)complex and signaling in cell cycle progression.Collectively,the present results indicate that lncRNA HOTAIR facilitates the DNA damage response pathway and promotes radioresistance in CRC cells by targeting ATR.

Role of glioma stem cells in promoting tumor chemo- and radioresistance: A systematic review of potential targeted treatments

BACKGROUND Gliomas pose a significant challenge to effective treatment despite advancements in chemotherapy and radiotherapy.Glioma stem cells(GSCs),a subset within tumors,contribute to resistance,tumor heterogeneity,and plasticity.Recent studies reveal GSCs’role in therapeutic resistance,driven by DNA repair mechanisms and dynamic transitions between cellular states.Resistance mechanisms can involve different cellular pathways,most of which have been recently reported in the literature.Despite progress,targeted therapeutic approaches lack consensus due to GSCs’high plasticity.AIM To analyze targeted therapies against GSC-mediated resistance to radio-and chemotherapy in gliomas,focusing on underlying mechanisms.METHODS A systematic search was conducted across major medical databases(PubMed,Embase,and Cochrane Library)up to September 30,2023.The search strategy utilized relevant Medical Subject Heading terms and keywords related to including“glioma stem cells”,“radiotherapy”,“chemotherapy”,“resistance”,and“targeted therapies”.Studies included in this review were publications focusing on targeted therapies against the molecular mechanism of GSC-mediated re-sistance to radiotherapy resistance(RTR).RESULTS In a comprehensive review of 66 studies on stem cell therapies for SCI,452 papers were initially identified,with 203 chosen for full-text analysis.Among them,201 were deemed eligible after excluding 168 for various reasons.The temporal breakdown of studies illustrates this trend:2005-2010(33.3%),2011-2015(36.4%),and 2016-2022(30.3%).Key GSC models,particularly U87(33.3%),U251(15.2%),and T98G(15.2%),emerge as significant in research,reflecting their representativeness of glioma characteristics.Pathway analysis indicates a focus on phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin(mTOR)(27.3%)and Notch(12.1%)pathways,suggesting their crucial roles in resistance development.Targeted molecules with mTOR(18.2%),CHK1/2(15.2%),and ATP binding cassette G2(12.1%)as frequent targets underscore their importance in overcoming GSC-mediated resistance.Various therapeutic agents,notably RNA inhibitor/short hairpin RNA(27.3%),inhibitors(e.g.,LY294002,NVP-BEZ235)(24.2%),and monoclonal antibodies(e.g.,cetuximab)(9.1%),demonstrate versatility in targeted therapies.among 20 studies(60.6%),the most common effect on the chemotherapy resistance response is a reduction in temozolomide resistance(51.5%),followed by reductions in carmustine resistance(9.1%)and doxorubicin resistance(3.0%),while resistance to RTR is reduced in 42.4%of studies.CONCLUSION GSCs play a complex role in mediating radioresistance and chemoresistance,emphasizing the necessity for precision therapies that consider the heterogeneity within the GSC population and the dynamic tumor microenvironment to enhance outcomes for glioblastoma patients.

Identification of differential gene expression profiles of radioresistant lung cancer cell line established by fractionated ionizing radiation in vitro

Background Radiotherapy plays a critical role in the management of non-small cell lung cancer （NSCLC）. This study was conducted to identify gene expression profiles of acquired radioresistant NSCLC cell line established by fractionated ionizing radiation （FIR） by cDNA microarray. Methods The human lung adenocarcinoma cell line Anip973 was treated with high energy X-ray to receive 60 Gy in 4 Gy fractions. The radiosensitivity of Anip973R and its parental line were measured by clonogenic assay. Gene expression profiles of Anip973R and its parental line were analyzed using cDNA microarray consisting of 21 522 human genes. Identified partly different expressive genes were validated by quantitative reverse transcription-polymerase chain reaction （Q-RT-PCR）. Results Fifty-nine upregulated and 43 downregulated genes were identified to radio-resistant Anip973R. Up-regulated genes were associated with DNA damage repair （DDB2）, extracellular matrix （LOX）, cell adhesion （CDH2）, and apoptosis （CRYAB）. Down-regulated genes were associated with angiogenesis （GBP-1）, immune response （CD83）, and calcium signaling pathway （TNNC1）. Subsequent validation of selected eleven genes （CD24, DDB2, IGFBP3, LOX, CDH2, CRYAB, PROCR, ANXA1 DCN, GBP-1 and CD83） by Q-RT-PCR was consistent with microarray analysis. Conclusions Fractionated ionizing radiation can lead to the development of radiation resistance. Altered gene profiles of radioresistant cell line may provide new insights into mechanisms underlying clinical radioresistance for NSCLC.

TGF-β/Akt/Smad signaling regulates ionizing radiation-induced epithelial-mesenchymal transition in acquired radioresistant lung cancer cells

Objective:To define the properties of lung cancer cells that resisted conventionally fractionated radiation exposure.Methods:Acquired radioresistant lung cancer cell line A549 was constructed by X-ray irradiation with a clinical conventional fraction dose of 2 Gy daily during 30 fractions.Cell morphology,molecular markers,migration capacity and invasion potential were evaluated by the microscope,Western blot,immunofluorescence,wound healing test and transwell chamber assay,respectively.Results:Radioresistant A549 cells shifted from an epithelial to a mesenchymal morphology,termed as epithelial-mesenchymal transition(EMT),and was accompanied by decreased expressions of epithelial markers(F=4.568,P<0.05)and increased expression of mesenchymal markers(F=4.270,P<0.05),greater migratory and invasive capabilities(t=6.386,5.644,P<0.05).The expression of TGF-β,and phosphorylated levels of Akt and Smad3 were also enhanced(F=6.496,4.685,3.370,P<0.05).Furthermore,the EMT phenotype induced by radiation could be reversed through inhibition of TGF-β,Akt or Smad3,indicating a functional relationship be-tween them.Conclusions:EMT mediates acquired radioresistance of lung cancer cells induced by IR with clinical parameters,and the crosstalk mode of TGF-β/Akt/Smad signaling plays a critical regulatory role in this process.

Cholesterol metabolism and tumor radiotherapy

Cholesterol is a lipid that is an essential component of the membrane structure in mammals.Cholesterol homeostasis regulates vital activities of individual cells and governs the overall function of the mammalian body.Cholesterol is mainly obtained through the biosynthesis of endogenous cholesterol and the intake of exogenous cholesterol.Cholesterol metabolism in tumor cells is abnormally active,and cholesterol and its metabolites(precursors and derivatives)play important roles in cancer proliferation,survival,invasion,metastasis,and the resistance to radiation.Preclinical studies have indicated that blocking cholesterol synthesis and uptake can reduce tumor progression and improve the response to anticancer treatment.Therapeutic strategies that target cholesterol synthesis,reduce plasma cholesterol levels,and prevent cholesterol esterification represent promising ways to improve the clinical outcome of cancer patients.

Hypoxia-induced Autophagy Contributes to Radioresistance via c-Jun-mediated Beclin1 Expression in Lung Cancer Cells

Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncol- ogy. The hypoxia-inducible factor （HIF）-lα plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by ir- radiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving frac- tion. EGFP-LC3 plasmid was stably transfected into cells to monitor autopbagic processes. Western blotting was used to evaluate the protein expression levels of HIF-lα, c-Jun, phosphorylated c-Jun, Be- clin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phos- phorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Be- clin 1 expression, instead of HIF-1α, could be blocked by SP600125 （a specific inhibitor of c-Jun NH2- terminal kinase）, followed by suppression of autophagy. Under hypoxia, combined treatment of irradia- tion and chloroquine （a potent autophagy inhibitor） significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclinl expression may be considered as a target to reverse the radioresistance in cancer cells.

Targeting fatty acid synthase sensitizes human nasopharyngeal carcinoma cells to radiationvia downregulating frizzled class receptor 10

Objective:Our aim was to test the hypothesis that fatty acid synthase(FASN)expression contributes to radioresistance of nasopharyngeal carcinoma(NPC)cells and that inhibiting FASN enhances radiosensitivity.Methods:Targeting FASN using epigallocatechin gallate(EGCG)or RNA interference in NPC cell lines that overexpress endogenous FASN was performed to determine their effects on cellular response to radiationin vitro using MTT and colony formation assays,andin vivo using xenograft animal models.Western blot,immunohistochemistry,real-time PCR arrays,and real-time RT-PCR were used to determine the relationship between FASN and frizzled class receptor 10(FZD10)expression.FZD10 knockdown and overexpression were used to determine its role in mediating FASN function in cellular response to radiation.Immunohistochemical staining was used to determine FASN and FZD10 expressions in human NPC tissues,followed by analysis of their association with the overall survival of patients.Results:FASN knockdown or inhibition significantly enhanced radiosensitivity of NPC cells,bothin vitro andin vivo.There was a positive association between FASN and FZD10 expression in NPC cell lines grown as monolayers or xenografts,as well as human tissues.FASN knockdown reduced FZD10 expression,and rescue of FZD10 expression abolished FASN knockdown-induced enhancement of radiosensitivity.FASN and FZD10 were both negatively associated with overall survival of NPC patients.Conclusions:FASN contributes to radioresistance,possiblyvia FZD10 in NPC cells.Both FZD10 and FASN expressions were associated with poor outcomes of NPC patients.EGCG may sensitize radioresistance by inhibiting FASN and may possibly be developed as a radiosensitizer for better treatment of NPCs.

CMA down-regulates p53 expression through degradation of HMGB1 protein to inhibit irradiation-triggered apoptosis in hepatocellular carcinoma

AIM To investigate the mechanism of chaperone-mediated autophagy(CMA)-induced resistance to irradiationtriggered apoptosis through regulation of the p53 protein in hepatocellular carcinoma(HCC). METHODS Firstly, we detected expression of lysosome-associated membrane protein 2a(Lamp-2a), which is the key protein of CMA, by western blot in Hep G2 and SMMC7721 cells after irradiation. We further used sh RNA Lamp-2a HCC cells to verify the radioresistance induced by CMA. Next, we detected the HMGB1 and p53 expression after irradiation by western blot, and we further used RNA interference and ethyl pyruvate(EP), as a HMGB1 inhibitor, to observe changes of p53 expression. Finally, an immunoprecipitation assay was conducted to explore the interaction between Lamp-2a and HMGB1, and the data were analyzed. RESULTS We found the expression of Lamp-2a was increased on irradiation while apoptosis decreased in Hep G2 and SMMC7721 cells. The apoptosis was increased markedly in the sh RNA Lamp-2a Hep G2 and SMMC7721 cells as detected by western blot and colony formation assay. Next, we found p53 expression was gradually reduced on irradiation but obviously increased in sh RNA Lamp-2a cells. Furthermore, p53 increased the cell apoptosis on irradiation in Hep3B(p53-/-) cells. Finally, p53 levels were regulated by HMGB1 as measured through RNA interference and the EP treatment. HMGB1 was able to combine with Lamp-2a as seen by immunoprecipitation assay and was degraded via the CMA pathway. The decreased HMGB1 inhibited p53 expression induced by irradiation and further reduced the apoptosis in HCC cells. CONCLUSION CMA pathway activation appears to down-regulate the susceptibility of HCC to irradiation by degrading HMGB1 with further impact on p53 expression. These findings have clinical relevance for radiotherapy of HCC.

Overexpression of nuclear β-catenin in rectal adenocarcinoma is associated with radioresistance

AIM:To investigate the association between nuclearβ-catenin overexpression in rectal adenocarcinoma and radioresistance.METHODS:A retrospective analysis was conducted.The analysis involved 136 patients with locally advanced rectal adenocarcinoma who underwent shortcourse preoperative radiotherapy and radical resection.The expression ofβ-catenin in both pretreatment biopsy specimens and resected primary tumor tissues was examined by immunohistochemistry.The correlation ofβ-catenin expression with radioresistance was evaluated using the tumor regression grading(TRG)system.The relationship betweenβ-catenin expression and clinicopathological characteristics was also analyzed.Univariate and logistic multivariate regression analyses were adopted to determine the independent factors of radioresistance.RESULTS:Nuclearβ-catenin overexpression was more evident in radioresistant rectal adenocarcinoma than in radiosensitive rectal adenocarcinoma(57.6%vs 16.7%,P<0.001).Nuclearβ-catenin was overexpressed in favor of poor TRG(≤2),whereas membraneβ-catenin was expressed in favor of good TRG(≥3).Nuclearβ-catenin expression in tumor cell differentiation(P=0.018),lymph node metastasis(P=0.022),and TRG(P<0.001)showed significant differences.Univariate analyses demonstrated that radioresistance is associated with nuclearβ-catenin overexpression(P<0.001).In addition,logistic multivariate regression analysis indicated that only three factors,namely,tumor size(P<0.001),tumor cell differentiation(P<0.001),and nuclearβ-catenin overexpression(P<0.001),are associated with radioresistance.By using radioresistance as a prediction target,nuclearβ-catenin-based prediction alone achieved 83%accuracy,65%sensitivity,and88%specificity.CONCLUSION:Nuclearβ-catenin overexpression may be a valuable candidate to predict the response of rectal adenocarcinoma to preoperative radiotherapy.

Mechanisms of radioresistance in hepatocellular carcinoma

Hepatocellular carcinoma(HCC), one of the most common cancers in the world, is characterized by poor prognosis and recurrence after resection. Its prevalence is highest in developing countries, particularly where there is high incidence of hepatitis B virus infection. Several curative treatments are available for early stage HCC; however, these options are not available for advanced disease. New techniques allowing the specific delivery of high-dose radiotherapy enable their use in the treatment of HCC, which has been avoided in the past due to low hepatic tolerance for radiation. This presents a new challenge—the development of resistance to radiotherapy and subsequent disease recurrence. Recently, the mechanisms controlling radioresistance have begun to be elucidated. Understanding the molecular basis of radioresistance is key to developing new strategies with better treatment response and increased patient survival.

AEG-1 expression correlates with CD133 and PPP6c levels in human glioma tissues

Astrocyte elevated gene-1（AEG-1） is associated with tumor genesis and progression in a variety of human cancers.This study aimed to explore the significance of AEG-l in glioma and investigate whether it correlated with radioresistance of glioma cells.Immunohistochemical staining showed that the intensity of AEG-l,CD133 and PPP6 c protein expression in glioma tissues increased significantly,mainly in the cytoplasm.The expression rate of AEG-l,CD133 and PPP6c were 85.9%（67/78）,60.3%（47/78） and 65.8%（51/78）,respectively.AEG-l expression was correlated with age（r=0.227,P=0.045）,clinical stage（r=0.491,P〈0.001） and clinical grade（r=0.450,P〈0.001）.No correlation was found between AEG-l expression and other clinicopathologic parameters（P〉0.05）.The expression of AEG-1 was positively correlated with the expression of CD133（r=0.240,P = 0.035） and PPP6c（r= 0.250,P = 0.027）.In addition,retrieved data on TCGA implied co-occurrence of genomic alterations of AEG-l and PPP6 c in glioblastoma.Our findings indicate that AEG-l is positively correlated with CD133 and AEG-l expression.It may play an important role in the progression of glioma and may serve as potential novel marker of chemoresistance and radioresistance.

Gene Expression Profiles Predict Sensitivity of Prostate Cancer to Radiotherapy

Ionizing radiation (IR) is the most common treatment used to control localized primary prostate cancer (PC). However, for a significant number of patients, radiotherapy fails to adequately control the tumor. Thus, a main clinical problem today is the lack of a specific marker that may be used to predict the treatment outcome and to identify prostate cancer patients who are unlikely to respond to radiation therapy. In this study, we used human PC xenografts with predetermined radioresistant/sensitive phenotypes, and gene expression microarrays, correlated their specific transcripttional profiles with response to radiation. Employing unsupervised two-way hierarchical clustering, we identified four gene clusters displaying different expression patterns. Two clusters showed higher expression levels in the resistant xenografts and the other two clusters showed higher expression levels in the sensitive xenografts. Expression levels of 113 genes differed by at least 3 fold between sensitive and resistant xenografts. These genes represent members of several cellular pathways, some of which are known to be associated with response to radiation. All or several of these genes could serve as predictive tools to determine at biopsy the expected response of a particular tumor to radiotherapy. Indeed, the profiles we identified enabled us to predict the degree of radiosensitivity of a panel of established PC cell lines. Importantly, irradiation of the PC xenografts did not induce any significant changes in gene expression, regardless of their susceptibility phenotype. These data strongly support the first of two models: a: a random effect of irradiation on a homogeneous population of cells, rather than b: of a tumor comprised of a mixture of radioresistant and radiosensitive cell subpopulations. Our findings imply that each of the radio-phenotypes represents different intrinsic characteristics that affect the ability of a tumor to survive radiotherapy.

Constitutive Overexpression of Bcl-2, Survivin and ER Stress Chaperone GRP-78 Confers Intrinsic Radioresistance in Human Hepatocellular Carcinoma Cells: Insight into the Mechanistic Pathways Involved

We present evidence here that abundantly expressed b-catenin-triggered NF-kB-dependent upregulation of inducible nitric oxide synthase(iNOS) found in hepatoma Mahlavu cells (RT-resistant variant designated as RR-Mal), but not in Hep 3B cells (RT-sensitive variant designated as RS-3B) is a key element contribrting to the radioresisitance through the activation of two prominent radioprotective pathways. First, high iNOS expression found in RR-Mal, but not in RS-3B cells was found to perturb calcium homeostasis that triggered ER stress response leading to the overproduction of ER chaperone GRP-78 via robust generation of cleaved ATF-6a (50 kDa) subunits and their nuclear translocation. Meanwhile, both abundantly expressed NF-κB and COX-2 found in RR-Mal cells could also provoke an increased production of PGE2 resulting in robust production of Bcl-2. Interestingly, when RR-Mal cells were treated with PDTC (a NF-κB inhibitor) or celecoxib (a COX-2 inhibitor), a concentration-dependent downregulation of Bcl-2 could be demonstrated implying that Bcl-2 overexpression was indeed mediated through NF-κB/Cox-2/PGE2 pathway. Importantly, we also unveiled that siRNA-mediated silencing of survivin in RR-Mal cells could result in a concomitant downregulation of GRP-78 due to a severe inhibition of ATF-6a (50 kDa) expression. Taken together, our data demonstrate that constitutively overexpressed b-catenin/NF-κB/iNOS and NF-κB/COX-2/PGE2 pathways that overproducing GRP-78, survivin and Bcl-2 expressions are responsible for radioresistance acquisition in RR-Mal cells. Thus, both pathways could be served as potential targets for overcoming radioresistance.

3-59 Mechanism on X-ray Irradiation Suppressing Radioresistance in Non-small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer, which is the leading cause of death in lung cancer patient. Routine treatment of NSCLC cannot effectively change the survival rate of patients, one important reason is the increased radioresistance of tumor cells after conventional radiotherapy.

Radioresistance of MnSOD and its relation with Bcl-2 and p53 mRNA expression

The molecular mechanisms of radioresistance of manganese ssuperoxide dismutase (MnSOD) was investigated in thisstudy. We assayed cell necrocis and apoptosis induced by ionizing radiation. Furthermore, the possible roles of oncogenes bc1-2and p53 mRNA expression in MnSOD gene-transfected cells were for investigated. me results showed that 10 Gy X-ray could induce the expression of bc1-2 mRNA and suppress p53 mRNA expression in sense MnSOD transfected cells, whereas Bc1-2 mRNAexpression did not change and p53 mRNA expression increased in antisense MnSOD transfected cells. It is suggested that Bc1-2 isrequired for the radioresistance of MnSOD and the status of p53 play a role in radiation-induced cell death.

Two distinct subpopulations of marginal zone B cells exhibit differential antibody-producing capacities and radioresistance

Marginal zone(MZ)B cells,which are splenic innate-like B cells that rapidly secrete antibodies(Abs)against blood-borne pathogens,are composed of heterogeneous subpopulations.Here,we showed that MZ B cells can be divided into two distinct subpopulations according to their CD80 expression levels.CD80^(high)MZ B cells exhibited greater Ab-producing,proliferative,and IL-10-secreting capacities than did CD80^(low)MZ B cells.Notably,CD80^(high)MZ B cells survived 2-Gy whole-body irradiation,whereas CD80^(low)MZ B cells were depleted by irradiation and then repleted with one month after irradiation.Depletion of CD80^(low)MZ B cells led to accelerated development of type II collagen(CII)-induced arthritis upon immunization with bovine CII.CD80^(high)MZ B cells exhibited higher expression of genes involved in proliferation,plasma cell differentiation,and the antioxidant response.CD80^(high)MZ B cells expressed more autoreactive B cell receptors(BCRs)that recognized double-stranded DNA or CII,expressed more immunoglobulin heavy chain sequences with shorter complementarity-determining region 3 sequences,and included more clonotypes with no N-nucleotides or with B-1a BCR sequences than CD80^(low)MZ B cells.Adoptive transfer experiments showed that CD21^(+)CD23^(+)transitional 2 MZ precursors preferentially generated CD80^(low)MZ B cells and that a proportion of CD80^(low)MZ B cells were converted into CD80^(high)MZ B cells;in contrast,CD80^(high)MZ B cells stably remained CD80^(high)MZ B cells.In summary,MZ B cells can be divided into two subpopulations according to their CD80 expression levels,Ab-producing capacity,radioresistance,and autoreactivity,and these findings may suggest a hierarchical composition of MZ B cells with differential stability and BCR specificity.

Regulator of G protein signaling 20 contributes to radioresistance of non-small cell lung cancer cells by suppressing pyroptosis

Objective:To investigate the potential role of the regulator of G protein signaling 20(RGS20)in radioresistance of non-small cell lung cancer(NSCLC).Methods:A total of 35 lung adenocarcinoma(LUAD)patients from The Cancer Genome Atlas(TCGA),who un-derwent radiotherapy,were enrolled and divided into radiosensitive(n=16)and radioresistant(n=19)groups based on clinical prognosis.The expression and prognosis of RGS20 were analyzed by Gene Expression Profiling Interactive Analysis(GEPIA)database.A radioresistant cell line(A549R)was constructed by irradiating A549 cells with 6 Gy X-rays for 10 fractions.Cell survival was measured by colony formation assay.The regulatory effect of RGS20 on pyroptosis were verified by LDH release and Western blot assay,and the underlying mech-anism was investigated by transfecting RGS20 siRNA and applying a GSDMD inhibitor.Results:A total of 2,181 differentially expressed genes(DEGs)were identified by analyzing the data of radio-sensitive and radioresistant individuals from the TCGA-LUAD dataset.These DEGs were enriched in G alpha(z)signalling events analyzed by Reactome database.RGS20 exhibited significant upregulation among the DEGs,and its higher expression predicted poor prognosis in LUAD patients.In vitro,the expression of RGS20 protein was increased by irradiation in A549 cells,whereas it remained at much high levels in A549R cells regardless of irradiation.After irradiation,the expressions of pyroptosis-related proteins were significantly increased in A549 cells(P<0.05),with no significant changes were observed in A549R cells.Treatment with LDC7559 significantly reduced LDH release(P<0.01)and improved the survival rate of irradiated A549 cells(P<0.01).Furthermore,knockdown of RGS20 gene in A549R cells significantly increased LDH release(P<0.001)and enhanced radiosensitivity(P<0.01),while LDC7559 administration reversed LDH release(P<0.01)and radiation-induced cell death increased by siRGS20(P<0.05).Meantime,the increased expression level of GSDMD-NT was observed in A549 and A549R cells transfected with siRGS20(P<0.05).Conclusion:RGS20 contributes to the radioresistance of NSCLC cells,which might be a potential target for NSCLC radiotherapy.