Yu-An Chen, Yi-Ru Lai, Ho Lin, J. Hsieh, Yu-Hsin Lin, Chih-Ho Lai
Background: Prostate cancer (PCa) is one of the most commonly diagnosed cancers in men and usually becomes refractory because of recurrence and metastasis. CD44, a transmembrane glycoprotein, serves as a receptor for hyaluronic acid (HA) and has been found to be abundantly expressed in cancer stem cells (CSCs) that often exhibit a radioresistant phenotype. Cytolethal distending toxin subunit B (CdtB), produced by Campylobacter jejuni, is a genotoxin acts as a type I deoxyribonuclease (DNase I), which is responsible for creating DNA double-strand breaks (DSBs). Nanoparticles loaded with antitumor drugs and specific ligands that recognize cancerous cell receptors are promising methods to overcome the therapeutic challenges. Results: Our results showed that administration of bacterial genotoxin significantly improved the efficacy of radiotherapy in a xenograft mouse model. We further prepared HA-decorated nanoparticles-encapsulated CdtB (HA-CdtB-NPs) and investigated the targeted therapeutic activity in radioresistant PCa cells. The results showed that HA-CdtB-NPs sensitized radioresistant PCa cells by enhancing DSB and causing G2/M cell-cycle arrest, without affecting the normal prostate epithelial cells. Our results demonstrate that HA-CdtB-NPs possess maximum target-specificity and delivery efficiency of CdtB into the nucleus, thereby enhancing the effect of radiation in radioresistant PCa cells. Conclusions: These findings indicate that HA-loaded CdtB nanoparticles exert target-specificity accompanied with radiomimetic activity, which can be developed as an effective agent for overcoming radioresistance in PCa.
{"title":"Novel bacterial genotoxin-loaded nanoparticles for targeting therapy of radioresistant prostate cancer","authors":"Yu-An Chen, Yi-Ru Lai, Ho Lin, J. Hsieh, Yu-Hsin Lin, Chih-Ho Lai","doi":"10.3390/iecc2021-09230","DOIUrl":"https://doi.org/10.3390/iecc2021-09230","url":null,"abstract":"Background: Prostate cancer (PCa) is one of the most commonly diagnosed cancers in men and usually becomes refractory because of recurrence and metastasis. CD44, a transmembrane glycoprotein, serves as a receptor for hyaluronic acid (HA) and has been found to be abundantly expressed in cancer stem cells (CSCs) that often exhibit a radioresistant phenotype. Cytolethal distending toxin subunit B (CdtB), produced by Campylobacter jejuni, is a genotoxin acts as a type I deoxyribonuclease (DNase I), which is responsible for creating DNA double-strand breaks (DSBs). Nanoparticles loaded with antitumor drugs and specific ligands that recognize cancerous cell receptors are promising methods to overcome the therapeutic challenges. \u0000Results: Our results showed that administration of bacterial genotoxin significantly improved the efficacy of radiotherapy in a xenograft mouse model. We further prepared HA-decorated nanoparticles-encapsulated CdtB (HA-CdtB-NPs) and investigated the targeted therapeutic activity in radioresistant PCa cells. The results showed that HA-CdtB-NPs sensitized radioresistant PCa cells by enhancing DSB and causing G2/M cell-cycle arrest, without affecting the normal prostate epithelial cells. Our results demonstrate that HA-CdtB-NPs possess maximum target-specificity and delivery efficiency of CdtB into the nucleus, thereby enhancing the effect of radiation in radioresistant PCa cells. \u0000Conclusions: These findings indicate that HA-loaded CdtB nanoparticles exert target-specificity accompanied with radiomimetic activity, which can be developed as an effective agent for overcoming radioresistance in PCa.","PeriodicalId":20534,"journal":{"name":"Proceedings of The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response","volume":"38 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80770694","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bladder cancer (BC) often requires lifetime monitoring due to its high recurrence rate. Exfoliated bladder cancer cells (EBCCs) may express a series of different biomarkers according to its epithelial-mesenchymal transition (EMT) status, a phenomenon characterized by loss of intercellular adhesion, enhanced cell motility, and cancer invasion. Here, we demonstrated the clinical heterogeneity of EBCCs using an integrated microfluidic assay to separate various EMT subtypes of EBCCs in real-time and under high-throughput based on the principle of inertial focusing. Enriched cells from BC patient-derived urine bladder wash samples were isolated based on cell size and characterized by antibodies targeting EMT biomarkers such as cytokeratin (CK), vimentin (VIM), survivin, and epidermal growth factor receptor (EGFR). This rapid, non-invasive method demonstrates high efficiency of cancer cell recovery under the optimal flow rate and the specific retrieval of various EMT phenotype cell fractions from respective device outlets. The evaluation of clinical samples revealed a vast amount of tumor heterogeneity, reflecting different EMT phenotypes, which can correlate with drug resistance and tumor dormancy. Overall, the separation of heterogeneous clinical samples can better facilitate routine screening procedures and greatly enhance personalized treatment.
{"title":"Heterogeneity of biomarker expression in clinical urine biopsies","authors":"Yatian Fu, B. L. Khoo","doi":"10.3390/iecc2021-09221","DOIUrl":"https://doi.org/10.3390/iecc2021-09221","url":null,"abstract":"Bladder cancer (BC) often requires lifetime monitoring due to its high recurrence rate. Exfoliated bladder cancer cells (EBCCs) may express a series of different biomarkers according to its epithelial-mesenchymal transition (EMT) status, a phenomenon characterized by loss of intercellular adhesion, enhanced cell motility, and cancer invasion. Here, we demonstrated the clinical heterogeneity of EBCCs using an integrated microfluidic assay to separate various EMT subtypes of EBCCs in real-time and under high-throughput based on the principle of inertial focusing. Enriched cells from BC patient-derived urine bladder wash samples were isolated based on cell size and characterized by antibodies targeting EMT biomarkers such as cytokeratin (CK), vimentin (VIM), survivin, and epidermal growth factor receptor (EGFR). This rapid, non-invasive method demonstrates high efficiency of cancer cell recovery under the optimal flow rate and the specific retrieval of various EMT phenotype cell fractions from respective device outlets. The evaluation of clinical samples revealed a vast amount of tumor heterogeneity, reflecting different EMT phenotypes, which can correlate with drug resistance and tumor dormancy. Overall, the separation of heterogeneous clinical samples can better facilitate routine screening procedures and greatly enhance personalized treatment.","PeriodicalId":20534,"journal":{"name":"Proceedings of The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75295820","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
I. Tabet, Esin Orhan, C. Velázquez, Lise Fenou, C. Theillet
In Triple Negative Breast Cancer (TNBC), chemotherapy is the only systemic treatment and sustained remissions are rare. We propose to widen therapeutic options. About 30% TNBC tumors are BRCA1 deficient, presentoing defective DNA repair and increased sensitivity to genotoxic drugs. We hypothesized that BRCA-deficient TNBC are highly sensitive to replication stress inducing drugs, thus, opening new therapeutic perspectives. Our preliminary results shown that BRCA1-deficient TNBC cell lines and a CRISPR/Cas9 BRCA1 KO isogenic model display increased sensitivity to gemcitabine. Cell cycle distribution of gemcitabine treated BRCA1-deficient cells were characterized by an elevated Sub-G1 fraction caused by increased numbers of cells in replication catastrophe. This was illustrated by 80% of BRCA1-deficient cells showing persistent (48-72h post treatment) gH2AX staining in absence of RPA32 co-staining, whereas in the isogenic BRCA1 WT model gH2AX and RPA32 positive cell numbers started decreasing at 24h. Interestingly, we noted that in addition to replication catastrophe, BRCA-deficient cells treated with gemcitabine underwent aberrant mitosis as shown by a clear increase of micro-nuclei. Interestingly, in vivo experiments appear to reproduce in vitro data. Indeed, a BRCA hyper methylated TNBC PDX, showed a higher sensitivity to gemcitabine than the BRCA1 WT. In conclusion, our data suggest that BRCA-deficient tumors are more sensitive to the replication poison Gemcitabine. Furthermore, this sensitivity seems to be mediated by an accentuated replicative stress response that is not well managed. Upon gemcitabine treatment, the cells undergo important DNA damage that leads to stalled replication forks, and DNA breakage. In the absence of BRCA1, the HR pathway is compromised, which leads to fork collapse and accumulation of single stranded DNA, therefore exhausting the pool of RPA within the cell and inducing Replicative catastrophe. In addition to deficient replication gemcitabine treated BRCA-deficient, but not BRCA-proficient cells, are subjected to mitotic catastrophe.
{"title":"Exploring sensitivity to replicative stress in BRCA deficient Triple Negative Breast Cancer","authors":"I. Tabet, Esin Orhan, C. Velázquez, Lise Fenou, C. Theillet","doi":"10.3390/iecc2021-09226","DOIUrl":"https://doi.org/10.3390/iecc2021-09226","url":null,"abstract":"In Triple Negative Breast Cancer (TNBC), chemotherapy is the only systemic treatment and sustained remissions are rare. We propose to widen therapeutic options. About 30% TNBC tumors are BRCA1 deficient, presentoing defective DNA repair and increased sensitivity to genotoxic drugs. We hypothesized that BRCA-deficient TNBC are highly sensitive to replication stress inducing drugs, thus, opening new therapeutic perspectives. Our preliminary results shown that BRCA1-deficient TNBC cell lines and a CRISPR/Cas9 BRCA1 KO isogenic model display increased sensitivity to gemcitabine. Cell cycle distribution of gemcitabine treated BRCA1-deficient cells were characterized by an elevated Sub-G1 fraction caused by increased numbers of cells in replication catastrophe. This was illustrated by 80% of BRCA1-deficient cells showing persistent (48-72h post treatment) gH2AX staining in absence of RPA32 co-staining, whereas in the isogenic BRCA1 WT model gH2AX and RPA32 positive cell numbers started decreasing at 24h. Interestingly, we noted that in addition to replication catastrophe, BRCA-deficient cells treated with gemcitabine underwent aberrant mitosis as shown by a clear increase of micro-nuclei. \u0000Interestingly, in vivo experiments appear to reproduce in vitro data. Indeed, a BRCA hyper methylated TNBC PDX, showed a higher sensitivity to gemcitabine than the BRCA1 WT. In conclusion, our data suggest that BRCA-deficient tumors are more sensitive to the replication poison Gemcitabine. Furthermore, this sensitivity seems to be mediated by an accentuated replicative stress response that is not well managed. Upon gemcitabine treatment, the cells undergo important DNA damage that leads to stalled replication forks, and DNA breakage. In the absence of BRCA1, the HR pathway is compromised, which leads to fork collapse and accumulation of single stranded DNA, therefore exhausting the pool of RPA within the cell and inducing Replicative catastrophe. In addition to deficient replication gemcitabine treated BRCA-deficient, but not BRCA-proficient cells, are subjected to mitotic catastrophe.","PeriodicalId":20534,"journal":{"name":"Proceedings of The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82388979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
H. Gad, Z. Zhenjun, Carlos Benítez-Buelga, K. Sanjiv, Huang Xiangwei, He Kang, Feng Mingxuan, Zhao Zhicong, U. W. Berglund, Xia Qiang, T. Helleday
While the repair of DNA double-strand breaks is known to be confined to different phases of the cell cycle and differentially activated at telomeres, less is known of compartmentalisation of base excision repair (BER). Here, we report that Endonuclease VIII like protein 3 (NEIL3) relocates to telomeres following oxidative DNA damage specifically during mitosis and recruits the APE1 to damaged telomeres. Using META-FISH, we demonstrate that NEIL3, but not NEIL1 or NEIL2, is required to initiate base excision repair at oxidised telomeres in mitotic cells, a process dependent on APE1 and Polβ. Repetitive exposure of oxidizing damage in NEIL3 depleted cells induced chromatin bridges and damaged telomeres. Interestingly, we identify that NEIL3 is elevated in Hepatocellular carcinoma (HCC), the most common type of a liver cancer, which correlates with poor survival. We demonstrate that HCC cell lines (6/6) depend on NEIL3 catalytic activity for survival and prevention of senescence, which is not the case for non-transformed cells where NEIL3 is dispensable. In conclusion, we demonstrate a novel function for NEIL3 in repair of oxidative DNA damage at telomeres in mitosis which is important to prevent senescence of HCC. Furthermore, these data suggest NEIL3 could be a target for therapeutic intervention of HCC.
{"title":"NEIL3-mediated mitotic base excision repair of oxidative lesions at telomeres prevents senescence in hepatocellular carcinoma","authors":"H. Gad, Z. Zhenjun, Carlos Benítez-Buelga, K. Sanjiv, Huang Xiangwei, He Kang, Feng Mingxuan, Zhao Zhicong, U. W. Berglund, Xia Qiang, T. Helleday","doi":"10.3390/iecc2021-09227","DOIUrl":"https://doi.org/10.3390/iecc2021-09227","url":null,"abstract":"While the repair of DNA double-strand breaks is known to be confined to different phases of the cell cycle and differentially activated at telomeres, less is known of compartmentalisation of base excision repair (BER). Here, we report that Endonuclease VIII like protein 3 (NEIL3) relocates to telomeres following oxidative DNA damage specifically during mitosis and recruits the APE1 to damaged telomeres. Using META-FISH, we demonstrate that NEIL3, but not NEIL1 or NEIL2, is required to initiate base excision repair at oxidised telomeres in mitotic cells, a process dependent on APE1 and Polβ. Repetitive exposure of oxidizing damage in NEIL3 depleted cells induced chromatin bridges and damaged telomeres. Interestingly, we identify that NEIL3 is elevated in Hepatocellular carcinoma (HCC), the most common type of a liver cancer, which correlates with poor survival. We demonstrate that HCC cell lines (6/6) depend on NEIL3 catalytic activity for survival and prevention of senescence, which is not the case for non-transformed cells where NEIL3 is dispensable. In conclusion, we demonstrate a novel function for NEIL3 in repair of oxidative DNA damage at telomeres in mitosis which is important to prevent senescence of HCC. Furthermore, these data suggest NEIL3 could be a target for therapeutic intervention of HCC.","PeriodicalId":20534,"journal":{"name":"Proceedings of The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87256008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Wojciech Żwierełło, Marta Skórka-Majewicz, Justyna Antoniewicz, Konrad Grzeszczak, Karolina Rogulska, Agnieszka Maruszewska
IntroductionFluorine (F) is an element that belongs to the group of halogens. Small amounts of fluoride are necessary for the proper development of bones and teeth. However, increased intake of fluorine and continuous exposure has negative effects on the human organism. Some recent works have shown that fluoride affects many metabolic pathways that can theoretically be involved in the development of invasive potential in many types of cancers, including brain neoplasms. In light of recent studies, the influence of fluoride on the invasiveness of cancer cells seems highly probable but is practically unexplored. Methods„Wound healing” assayAfter 72 hours or three months of passaging in appropriate NaF concentrations (0.1-10 µM), U-87MG cells were grown in 6-well plates (controls + NaF concentrations indicated). After reaching confluence (~ 80%), the cell layers were scratched with 200 µl pipette tips and washed with PBS to remove cell debris. Fresh medium without serum was added to each well and the wound closure was visualized at 0, 3, 6, 12, and 24 hours using a microscope.Cell migration testAfter 72 hours or three months of passage in appropriate NaF concentrations (0.1-10 µM), a total of 1 × 105 U-87MG cells (controls + 0.1-10 µM concentration of NaF, in serum-free EMEM containing 1% serum albumin bovine species ) were inoculated in the upper chamber of a 24-well Transwell system with a pore size of 8.0 µm. EMEM containing 10% FBS was added to the lower chamber. After incubation, non-migrating and non-invasive cells on the upper surface were removed with a cotton swab and cells on the lower surface were fixed with 4% paraformaldehyde and stained with Giemsa. Photographs were taken and cells were counted under the microscope. ResultsOur observations showed that both in the case of short-term and long-term culture in the presence of sodium fluoride, the mobility of glioblastoma cells significantly increased. Importantly, the effect was visible at the lowest concentration (0.1 µM ) and increased at higher concentrations (1-10 µM) of NaF. ConclusionsThe results of these studies can shed new light on the therapeutic approach in people with brain tumors and draw attention to environmental factors such as fluoride, which may already hamper the treatment of patients at low doses. Considering the numerous processes taking place in the brain under the influence of fluoride, it seems extremely important to investigate the influence of this environmental toxin on the progression and development of brain tumors.
{"title":"Determination of the effect of selected low fluoride concentrations on migratory abilities of human glioma U-87MG cell line.","authors":"Wojciech Żwierełło, Marta Skórka-Majewicz, Justyna Antoniewicz, Konrad Grzeszczak, Karolina Rogulska, Agnieszka Maruszewska","doi":"10.3390/iecc2021-09225","DOIUrl":"https://doi.org/10.3390/iecc2021-09225","url":null,"abstract":"IntroductionFluorine (F) is an element that belongs to the group of halogens. Small amounts of fluoride are necessary for the proper development of bones and teeth. However, increased intake of fluorine and continuous exposure has negative effects on the human organism. Some recent works have shown that fluoride affects many metabolic pathways that can theoretically be involved in the development of invasive potential in many types of cancers, including brain neoplasms. In light of recent studies, the influence of fluoride on the invasiveness of cancer cells seems highly probable but is practically unexplored. \u0000Methods„Wound healing” assayAfter 72 hours or three months of passaging in appropriate NaF concentrations (0.1-10 µM), U-87MG cells were grown in 6-well plates (controls + NaF concentrations indicated). After reaching confluence (~ 80%), the cell layers were scratched with 200 µl pipette tips and washed with PBS to remove cell debris. Fresh medium without serum was added to each well and the wound closure was visualized at 0, 3, 6, 12, and 24 hours using a microscope.Cell migration testAfter 72 hours or three months of passage in appropriate NaF concentrations (0.1-10 µM), a total of 1 × 105 U-87MG cells (controls + 0.1-10 µM concentration of NaF, in serum-free EMEM containing 1% serum albumin bovine species ) were inoculated in the upper chamber of a 24-well Transwell system with a pore size of 8.0 µm. EMEM containing 10% FBS was added to the lower chamber. After incubation, non-migrating and non-invasive cells on the upper surface were removed with a cotton swab and cells on the lower surface were fixed with 4% paraformaldehyde and stained with Giemsa. Photographs were taken and cells were counted under the microscope. \u0000ResultsOur observations showed that both in the case of short-term and long-term culture in the presence of sodium fluoride, the mobility of glioblastoma cells significantly increased. Importantly, the effect was visible at the lowest concentration (0.1 µM ) and increased at higher concentrations (1-10 µM) of NaF. \u0000ConclusionsThe results of these studies can shed new light on the therapeutic approach in people with brain tumors and draw attention to environmental factors such as fluoride, which may already hamper the treatment of patients at low doses. Considering the numerous processes taking place in the brain under the influence of fluoride, it seems extremely important to investigate the influence of this environmental toxin on the progression and development of brain tumors.","PeriodicalId":20534,"journal":{"name":"Proceedings of The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response","volume":"523 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86584810","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
R. Carriero, Lucia Maita, S. Bione, G. Biamonti, A. Montecucco
Rapidly dividing cancer cells show elevated levels of DNA double-strand breaks (DSBs) resulting from replication stress and linked to genome instability. To verify the hypothesis that a low level of endogenous replicative DNA damage may impact gene expression programs and cell biology features relevant to cancer progression, we used DNA ligase I (LigI) defective 46BR.1G1 fibroblasts, deriving from a patient who died at 19 for lymphoma, and the 7A3 cell clone, obtained from 46BR.1G1 by stably expressing ectopic wild-type LigI. LigI deficiency impairs maturation of newly synthesized DNA and increases the number of DSBs and γH2AX foci, features associated with genome instability commonly found also in pre-neoplastic lesions. In order to decipher the strategy used to cope with replicative DNA damage, we have compared gene expression profiles in 46BR.1G1 and 7A3 cells. Among the differentially expressed genes, we identified a group of long noncoding RNAs (lncRNAs) which show significant transcriptional alteration in 46BR.1G1 cells, and appear to be relevant for cancer progression. An interesting up-regulated lncRNA in 46BR.1G1 cells is LINP1 (lncRNA in nonhomologous end joining (NHEJ) pathway 1) which has been shown to be involved in DNA repair. We have observed that LINP1 up-regulation contributes to proliferation and survival of 46BR.1G1 that could account for genome instability. Moreover, we observed that LINP1 is upregulated at later times in control human fibroblasts exposed to exogenous sources of DNA damage. Our observations support the notion that LINP1 lncRNA targeting could reduce the DNA repair efficacy of tumour cells. * These authors contributed equally to this work.
{"title":"LINP1 lncRNA expression profile is modulated in response to DNA damage","authors":"R. Carriero, Lucia Maita, S. Bione, G. Biamonti, A. Montecucco","doi":"10.3390/iecc2021-09218","DOIUrl":"https://doi.org/10.3390/iecc2021-09218","url":null,"abstract":"Rapidly dividing cancer cells show elevated levels of DNA double-strand breaks (DSBs) resulting from replication stress and linked to genome instability. To verify the hypothesis that a low level of endogenous replicative DNA damage may impact gene expression programs and cell biology features relevant to cancer progression, we used DNA ligase I (LigI) defective 46BR.1G1 fibroblasts, deriving from a patient who died at 19 for lymphoma, and the 7A3 cell clone, obtained from 46BR.1G1 by stably expressing ectopic wild-type LigI. LigI deficiency impairs maturation of newly synthesized DNA and increases the number of DSBs and γH2AX foci, features associated with genome instability commonly found also in pre-neoplastic lesions. In order to decipher the strategy used to cope with replicative DNA damage, we have compared gene expression profiles in 46BR.1G1 and 7A3 cells. Among the differentially expressed genes, we identified a group of long noncoding RNAs (lncRNAs) which show significant transcriptional alteration in 46BR.1G1 cells, and appear to be relevant for cancer progression. An interesting up-regulated lncRNA in 46BR.1G1 cells is LINP1 (lncRNA in nonhomologous end joining (NHEJ) pathway 1) which has been shown to be involved in DNA repair. We have observed that LINP1 up-regulation contributes to proliferation and survival of 46BR.1G1 that could account for genome instability. Moreover, we observed that LINP1 is upregulated at later times in control human fibroblasts exposed to exogenous sources of DNA damage. Our observations support the notion that LINP1 lncRNA targeting could reduce the DNA repair efficacy of tumour cells. \u0000 * These authors contributed equally to this work.","PeriodicalId":20534,"journal":{"name":"Proceedings of The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91298994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Magdalena Nowak, Marta Skórka-Majewicz, Wojciech Żwierełło
IntroductionFluorine compounds are common environmental pollutants and may excessively penetrate the human body, especially the brain (fluoride penetrates the blood-brain barrier). Some of the latest studies have shown that fluoride may interfere with some of the metabolic pathways involved in the development of invasive potential in many types of cancer (eg Wnt/catenin or NF-κB). One of the stages of tumor invasion is the degradation of the extracellular matrix by metalloproteinases (MMP-2 and MMP-9), which allows the migration and metastasis of cancer cells. Taking into account the above facts, we decided to check whether low concentrations of fluoride affect the expression level of genes encoding MMP-2, MMP-9, and their TIMP-2 and TIMP-3 inhibitors in human glioblastoma cells. MethodsU-87MG human glioblastoma cells were cultured with EMEM medium (10% FBS, 2 mM glutamine, 1% NEAA), 1 mM sodium pyruvate, 100 IU / ml penicillin, 10 μg / ml streptomycin) under optimal conditions (at 37 ° C, in an atmosphere of 5% CO2, with 95% humidity). Cells were treated with sodium fluoride (NaF; 1-5 µM) for 24, 48 and 72 hours.The analysis of the expression level of the MMP-2, MMP-9, Timp-2, and Timp-3 genes was carried out by RT-PCR. Results The results indicate that NaF (0.1-5 µM) can disrupt the expression of MMP-2, MMP-9, Timp-2, and Timp-3. In the case of MMP-2, there was an approx. 2-fold increase in expression in 48h (5 µM NaF) and about 2.5-fold increase in expression in 72h (0.1-5 µM NaF). For MMP-9, an approximately 3-fold increase in expression was observed in 24h (0.1 µM NaF) and 48h (5 µM NaF). Both Timp-2 and Timp-3 showed a significant increase in expression observed at all time points especially at the highest concentration of NaF (5 µM). ConclusionsThe obtained results may suggest that even low concentrations of fluorine compounds may have an undesirable influence promoting the invasive potential of human glioblastoma cells. AcknowledgmentsThe project was implemented with the use of funds for science granted by the Pomeranian Medical University in Szczecin.
{"title":"Changes in gene expression of metalloproteinases-2 and -9 and their inhibitors TIMP2 and TIMP3 in human glioma cells exposed to low levels of fluoride.","authors":"Magdalena Nowak, Marta Skórka-Majewicz, Wojciech Żwierełło","doi":"10.3390/iecc2021-09229","DOIUrl":"https://doi.org/10.3390/iecc2021-09229","url":null,"abstract":"IntroductionFluorine compounds are common environmental pollutants and may excessively penetrate the human body, especially the brain (fluoride penetrates the blood-brain barrier). Some of the latest studies have shown that fluoride may interfere with some of the metabolic pathways involved in the development of invasive potential in many types of cancer (eg Wnt/catenin or NF-κB). One of the stages of tumor invasion is the degradation of the extracellular matrix by metalloproteinases (MMP-2 and MMP-9), which allows the migration and metastasis of cancer cells. Taking into account the above facts, we decided to check whether low concentrations of fluoride affect the expression level of genes encoding MMP-2, MMP-9, and their TIMP-2 and TIMP-3 inhibitors in human glioblastoma cells. \u0000MethodsU-87MG human glioblastoma cells were cultured with EMEM medium (10% FBS, 2 mM glutamine, 1% NEAA), 1 mM sodium pyruvate, 100 IU / ml penicillin, 10 μg / ml streptomycin) under optimal conditions (at 37 ° C, in an atmosphere of 5% CO2, with 95% humidity). Cells were treated with sodium fluoride (NaF; 1-5 µM) for 24, 48 and 72 hours.The analysis of the expression level of the MMP-2, MMP-9, Timp-2, and Timp-3 genes was carried out by RT-PCR. \u0000Results The results indicate that NaF (0.1-5 µM) can disrupt the expression of MMP-2, MMP-9, Timp-2, and Timp-3. In the case of MMP-2, there was an approx. 2-fold increase in expression in 48h (5 µM NaF) and about 2.5-fold increase in expression in 72h (0.1-5 µM NaF). For MMP-9, an approximately 3-fold increase in expression was observed in 24h (0.1 µM NaF) and 48h (5 µM NaF). Both Timp-2 and Timp-3 showed a significant increase in expression observed at all time points especially at the highest concentration of NaF (5 µM). \u0000ConclusionsThe obtained results may suggest that even low concentrations of fluorine compounds may have an undesirable influence promoting the invasive potential of human glioblastoma cells. \u0000AcknowledgmentsThe project was implemented with the use of funds for science granted by the Pomeranian Medical University in Szczecin.","PeriodicalId":20534,"journal":{"name":"Proceedings of The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response","volume":"61 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78552860","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Hausmann, Charlotte Neitzel, H. Hahn, Ruth Winter, I. Falková, D. Heermann, Goetz Pilarczyk, G. Hildenbrand, M. Falk
Application of ionizing radiation has an increasing impact on bio-medical research, and cancer diagnosis and treatment. Nevertheless, there are a lot of open questions concerning the understanding of radiation DNA damaging mechanisms and repair processes within the light of radio-sensitivity and thus, individualized medical applications. The three-dimensional architecture of genomes on the micro-, meso- and nano-scale acts in combination with epigenetic modifications as an important player of gene regulation and, consequently, fundamental biological processes such as DNA damage response and repair. So far only little is known about the impact of chromatin architecture on DNA double strand break (DSB) repair pathway selection and progression at individual damage sites. How does a cell nucleus manage DSBs and re-organize the chromatin towards functionally intact repair units? Is there a radiosensitivity-related difference in this reaction? We present investigations of spatial and topological parameters of chromatin and repair foci during a time period of repair to glimpse key aspects related to these questions. Nano-probing of radiation-induced chromatin damage sites and the recruited DNA repair proteins in combination with super-resolution Single Molecule Localization Microscopy (SMLM) are powerful methods for geometric and topological analyses of these structures in single cells and single DSB sites and, thus, to study mechanisms of their formation and repair pathway regulation. We used variable tools for such investigations based on image-free high-precision SMLM, nano-scaled molecule distribution analyses, appropriate metrics following Ripley´s distance frequencies and cluster formation analyses, as well as topological quantifications employing persistence homology. Comparing the topology of repair foci by persistence homology suggests general similarities in repair cluster formation, indicating a well-defined non-random, molecule topology at given time points during repair. However, at the same time, the data reveal a specific nano-architecture of DNA damage foci depending on the chromatin domain and cell type. Showing how chromatin architecture around complex damage sites and repair focus nano-architecture may contribute to ongoing repair process, our studies contribute to the molecular understanding of cellular radiation response and its regulation in cancer and non-cancer cells at sub-light microscopic chromatin levels.
{"title":"Space and time in the universe of the cell nucleus after ionizing radiation attacks: a comparison of cancer and non-cancer cell response","authors":"M. Hausmann, Charlotte Neitzel, H. Hahn, Ruth Winter, I. Falková, D. Heermann, Goetz Pilarczyk, G. Hildenbrand, M. Falk","doi":"10.3390/iecc2021-09219","DOIUrl":"https://doi.org/10.3390/iecc2021-09219","url":null,"abstract":"Application of ionizing radiation has an increasing impact on bio-medical research, and cancer diagnosis and treatment. Nevertheless, there are a lot of open questions concerning the understanding of radiation DNA damaging mechanisms and repair processes within the light of radio-sensitivity and thus, individualized medical applications. The three-dimensional architecture of genomes on the micro-, meso- and nano-scale acts in combination with epigenetic modifications as an important player of gene regulation and, consequently, fundamental biological processes such as DNA damage response and repair. So far only little is known about the impact of chromatin architecture on DNA double strand break (DSB) repair pathway selection and progression at individual damage sites. How does a cell nucleus manage DSBs and re-organize the chromatin towards functionally intact repair units? Is there a radiosensitivity-related difference in this reaction? We present investigations of spatial and topological parameters of chromatin and repair foci during a time period of repair to glimpse key aspects related to these questions. Nano-probing of radiation-induced chromatin damage sites and the recruited DNA repair proteins in combination with super-resolution Single Molecule Localization Microscopy (SMLM) are powerful methods for geometric and topological analyses of these structures in single cells and single DSB sites and, thus, to study mechanisms of their formation and repair pathway regulation. We used variable tools for such investigations based on image-free high-precision SMLM, nano-scaled molecule distribution analyses, appropriate metrics following Ripley´s distance frequencies and cluster formation analyses, as well as topological quantifications employing persistence homology. Comparing the topology of repair foci by persistence homology suggests general similarities in repair cluster formation, indicating a well-defined non-random, molecule topology at given time points during repair. However, at the same time, the data reveal a specific nano-architecture of DNA damage foci depending on the chromatin domain and cell type. Showing how chromatin architecture around complex damage sites and repair focus nano-architecture may contribute to ongoing repair process, our studies contribute to the molecular understanding of cellular radiation response and its regulation in cancer and non-cancer cells at sub-light microscopic chromatin levels.","PeriodicalId":20534,"journal":{"name":"Proceedings of The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78077198","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Radium-223 dichloride (Ra-223, Xofigo®) is the first approved α-particle-emitting radionuclide for the treatment of symptomatic bone metastases in patients with castration-resistant prostate cancer (CRPC) with no known evidence of visceral metastases. Ra-223 is a calcium-mimetic that preferentially binds with the bone mineral hydroxyapatite at areas of high bone turnover, such as bone metastases. This highly localized radiotherapy causes a high frequency of unrepairable double-stranded DNA breaks (DSBs), resulting in a potent antitumor effect on bone metastases. Recent evidence has suggested that patients with mutations in the DNA damage response pathway (DDR), may have differential outcomes to Ra-223 treatment (1). DDR comprises a dynamic network of signalling pathways for the maintenance of genomic integrity. Ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) are critical proteins which orchestrate the DDR and their activation is dependent on the type of DNA lesion. ATM is the primary responder to DSBs whilst ATR is activated by a range of lesions including single strand DNA structures at resected ends of DBSs and after replication fork stalling. In this study, we evaluated the impact of combining DDR kinase inhibitors with Ra-223 to investigate whether a greater radiosensitisation response occurs in comparison to standard X-rays in PC3 and DU145 human prostate cell lines and normal prostate epithelial RWPE-1 cells. Cell assays including clonogenic survival, DNA damage assays and flow cytometry were used to assess the effect of DDR kinase inhibitors in combination with ionising radiation. Cells were pre-treated with DDR inhibitors one-hour before exposure to 2Gy X-rays or an equivalent dose of 0.25Gy Ra-223. Our data show that, in all prostate models, DDR kinase inhibitors in combination with Ra-223 significantly enhanced radiosensitivity (p<0.005) response in comparison to combined treatment with X-rays. Furthermore, a greater quantity of residual DSBs at 24 hours post combination treatment was observed after Ra-223 exposure in comparison to X-ray exposure (p<0.001). Promisingly, this combined treatment had minimal effect on RWPE-1 normal cells. Our findings strongly support the combination of DNA damage induction by Ra-223 with DDR kinase inhibitors as a novel potential treatment option for mCRPC patients in order to improve clinical outcome. References: Velho PI, Qazi F, Hassan S, et al. Efficacy of Radium-223 in Bone-metastatic Castration-resistant Prostate Cancer with and Without Homologous Repair Gene Defects. European Urology. 2019; 76: 170-176.
{"title":"Synergistic activity of DNA damage response kinase inhibitors in combination with the targeted alpha therapy radium-223 dichloride for metastatic castration-resistant prostate cancer","authors":"V. Dunne, T. Wright, F. Liberal, K. Prise","doi":"10.3390/iecc2021-09191","DOIUrl":"https://doi.org/10.3390/iecc2021-09191","url":null,"abstract":"Radium-223 dichloride (Ra-223, Xofigo®) is the first approved α-particle-emitting radionuclide for the treatment of symptomatic bone metastases in patients with castration-resistant prostate cancer (CRPC) with no known evidence of visceral metastases. Ra-223 is a calcium-mimetic that preferentially binds with the bone mineral hydroxyapatite at areas of high bone turnover, such as bone metastases. This highly localized radiotherapy causes a high frequency of unrepairable double-stranded DNA breaks (DSBs), resulting in a potent antitumor effect on bone metastases. Recent evidence has suggested that patients with mutations in the DNA damage response pathway (DDR), may have differential outcomes to Ra-223 treatment (1). \u0000DDR comprises a dynamic network of signalling pathways for the maintenance of genomic integrity. Ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) are critical proteins which orchestrate the DDR and their activation is dependent on the type of DNA lesion. ATM is the primary responder to DSBs whilst ATR is activated by a range of lesions including single strand DNA structures at resected ends of DBSs and after replication fork stalling. \u0000In this study, we evaluated the impact of combining DDR kinase inhibitors with Ra-223 to investigate whether a greater radiosensitisation response occurs in comparison to standard X-rays in PC3 and DU145 human prostate cell lines and normal prostate epithelial RWPE-1 cells. \u0000Cell assays including clonogenic survival, DNA damage assays and flow cytometry were used to assess the effect of DDR kinase inhibitors in combination with ionising radiation. Cells were pre-treated with DDR inhibitors one-hour before exposure to 2Gy X-rays or an equivalent dose of 0.25Gy Ra-223. \u0000Our data show that, in all prostate models, DDR kinase inhibitors in combination with Ra-223 significantly enhanced radiosensitivity (p<0.005) response in comparison to combined treatment with X-rays. Furthermore, a greater quantity of residual DSBs at 24 hours post combination treatment was observed after Ra-223 exposure in comparison to X-ray exposure (p<0.001). Promisingly, this combined treatment had minimal effect on RWPE-1 normal cells. \u0000Our findings strongly support the combination of DNA damage induction by Ra-223 with DDR kinase inhibitors as a novel potential treatment option for mCRPC patients in order to improve clinical outcome. \u0000References: \u0000 \u0000Velho PI, Qazi F, Hassan S, et al. Efficacy of Radium-223 in Bone-metastatic Castration-resistant Prostate Cancer with and Without Homologous Repair Gene Defects. European Urology. 2019; 76: 170-176.","PeriodicalId":20534,"journal":{"name":"Proceedings of The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response","volume":"28 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76487370","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hannah Smith, A. Mukhopadhyay, Y. Drew, E. Willmore, N. Curtin
Background: PARP inhibitors (PARPi) exploit defects in homologous recombination repair (HRR) to selectively kill tumour cells. Continuous, PARP inhibition is required for cytotoxicity. PARPis rucaparib, olaparib and niraparib have been approved for use in ovarian cancer on continuous schedules. Previous studies demonstrate prolonged PARP inhibition by rucaparb1. Aim: To determine if persistent PARP inhibition is a class effect. Methods: IGROV-1 (human ovarian cancer) cells were treated with 1µM of rucaparib, olaparib, niraparib, talazoparib or pamiparib for 1h before drug was washed off and replaced with fresh media for 0, 1, 24, 48 or 72h prior to harvesting. Cellular PARP activity was measured using a GCLP-validated assay2 in comparison with untreated controls and where 1µM inhibitor was added to the reaction. Results: Rucaparib, olaparib, niraparib, talazoparib and pamiparib each inhibited PARP activity in permeabilised cells >99% when 1µM was present during the reaction. After 2 h in drug-free medium rucaparib-induced PARP inhibition was maintained at 92.3 ± 4.3% but was much less with talazoparib (58.6 ±5.0%), pamiparib (56.0 ± 4.5%) olaparib (48.3 ± 19.8%) and niraparib (37.3 ± 11.6%). PARP inhibition in rucaparib-treated cells was maintained for 72h in drug-free medium (77.7 ± 12.3%). This sustained PARP inhibition was not observed with the other PARPis. PARP inhibition was only 12.3 ± 5.2% and 12.5 ± 4.9% 72h after talazoparib and pamiparib, respectively, and undetectable with olaparib and niraparib. Conclusion: Rucaparib is unique in its ability to cause persistent PARP inhibition and it is not a class effect. These data have clinical implications for the different uses of PARPi, as a single agent use to exploit HRR defects versus chemo- and radiosensitisation. 1 Murray, J.; et al. BJC, 110, 1977-1984 (2014) 2 Plummer ER, et al Clin Cancer Res. 11 3402-3409 (2005)
{"title":"Differences in durability of PARP inhibition by PARP inhibitors in ovarian cancer cells","authors":"Hannah Smith, A. Mukhopadhyay, Y. Drew, E. Willmore, N. Curtin","doi":"10.3390/iecc2021-09194","DOIUrl":"https://doi.org/10.3390/iecc2021-09194","url":null,"abstract":"Background: \u0000PARP inhibitors (PARPi) exploit defects in homologous recombination repair (HRR) to selectively kill tumour cells. Continuous, PARP inhibition is required for cytotoxicity. PARPis rucaparib, olaparib and niraparib have been approved for use in ovarian cancer on continuous schedules. Previous studies demonstrate prolonged PARP inhibition by rucaparb1. \u0000Aim: \u0000To determine if persistent PARP inhibition is a class effect. \u0000Methods: \u0000IGROV-1 (human ovarian cancer) cells were treated with 1µM of rucaparib, olaparib, niraparib, talazoparib or pamiparib for 1h before drug was washed off and replaced with fresh media for 0, 1, 24, 48 or 72h prior to harvesting. Cellular PARP activity was measured using a GCLP-validated assay2 in comparison with untreated controls and where 1µM inhibitor was added to the reaction. \u0000Results: \u0000Rucaparib, olaparib, niraparib, talazoparib and pamiparib each inhibited PARP activity in permeabilised cells >99% when 1µM was present during the reaction. After 2 h in drug-free medium rucaparib-induced PARP inhibition was maintained at 92.3 ± 4.3% but was much less with talazoparib (58.6 ±5.0%), pamiparib (56.0 ± 4.5%) olaparib (48.3 ± 19.8%) and niraparib (37.3 ± 11.6%). PARP inhibition in rucaparib-treated cells was maintained for 72h in drug-free medium (77.7 ± 12.3%). This sustained PARP inhibition was not observed with the other PARPis. PARP inhibition was only 12.3 ± 5.2% and 12.5 ± 4.9% 72h after talazoparib and pamiparib, respectively, and undetectable with olaparib and niraparib. \u0000Conclusion: \u0000Rucaparib is unique in its ability to cause persistent PARP inhibition and it is not a class effect. These data have clinical implications for the different uses of PARPi, as a single agent use to exploit HRR defects versus chemo- and radiosensitisation. \u00001 Murray, J.; et al. BJC, 110, 1977-1984 (2014) \u00002 Plummer ER, et al Clin Cancer Res. 11 3402-3409 (2005)","PeriodicalId":20534,"journal":{"name":"Proceedings of The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response","volume":"69 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85534240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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