Radiosensitivity Of Breast Cancer Cells Research Articles

MiR-4796 enhances the sensitivity of breast cancer cells to ionising radiation by impairing the DNA repair pathway.

MicroRNAs (miRNAs) are important regulators of DNA damage response (DDR) through post-transcriptional regulation on their target genes, which are implicated in DDR and DNA repair (DR). In this study, we investigated the functional roles and target genes of miR-4796 and miR-1287 in breast cancer cells in response to radiation. The molecular mechanism of miR-4796 in regulating the radiosensitivity of breast cancer cells was also elucidated. Real-time polymerase chain reaction detected miR-4796 and miR-1287 expression; colony formation assay and irradiation therapy tumour xenograft in vivo examined radiosensitising effect; comet assay assessed DNA damage; immunofluorescence imaging determined the formation of γ-H2AX foci; targetscan and RegRNA predicted target mRNAs; luciferase reporter and mutation assays validated target genes; western blotting detected the expression of genes at the protein level; and flow cytometry quantified the activities of nonhomologous end-joining (NHEJ) and homologous recombination (HR). The expressions of miR-4796 and miR-1287 were acutely fluctuated in response to ionising radiation. In the absence of radiation, overexpression of miR-1287 dramatically promoted growth of breast cancer cells in vitro and in vivo, whereas overexpression of miR-4796 did not affect cell growth. When under the treatment with radiation, overexpression of miR-4796 suppressed DR and sensitised cancer cells to radiation both in vitro and in vivo. However, such effect was only observed in cell assays in the overexpressed miR-1287 group, and not confirmed in vivo. We therefore further explored the molecular mechanism of action of miR-4796, and found that miR-4796 targeted multiple components of DDR and DR, including ATM, BRCA1, PARP and RAD51. Moreover, overexpression of miR-4796 inhibited the expression of these DDR components at the protein level. In addition, miR-4796 inhibited HR and NHEJ repair pathways and aggravated radiation-induced DNA damage. The findings here suggest that miR-4796 can enhance radiation-induced cell death by directly targeting multiple DDR components, and repress NHEJ and HR DNA repair pathways. miR-4796 can act as an effective radiation sensitising agent.

CircNCOR1 regulates breast cancer radiotherapy efficacy by regulating CDK2 via hsa-miR-638 binding

BackgroundDespite aggressive local and regional therapy, triple-negative breast cancer (TNBC) is characterized by an increased risk of locoregional recurrence. RNA-sequencing data has identified a large number of circRNAs in primary breast cancers, but the role of specific circRNAs in regulating the radiosensitivity of TNBC is not fully understood. This research aimed to investigate the function of circNCOR1 in the radiosensitivity of TNBC. MethodsCircRNA high-throughput sequencing was conducted on two breast cancer MDA-MB-231 and BT549 cell lines after 6 Gy radiation. The relationship between circNCOR1, hsa-miR-638, and CDK2 was determined by RNA immunoprecipitation (RIP), FISH and luciferase assays. The proliferation and apoptosis of breast cancer cells were measured by CCK8, flow cytometry, colony formation assays, and western blot. ResultsDifferential expression of circRNAs was closely related to the proliferation of breast cancer cells after irradiation. Overexpression of circNCOR1 facilitated the proliferation of MDA-MB-231 and BT549 cells and impaired the radiosensitivity of breast cancer cells. Additionally, circNCOR1 acted as a sponge for hsa-miR-638 to regulate the downstream target protein CDK2. Overexpression of hsa-miR-638 promoted apoptosis of breast cancer cells, while overexpression of CDK2 alleviated apoptosis and increased proliferation and clonogenicity. In vivo, overexpression of circNCOR1 partially reversed radiation-induced loosening of tumor structures and enhanced tumor cell proliferation. ConclusionOur results demonstrated that circNCOR1 bounds to hsa-miR-638 and targets CDK2, thereby regulating the radiosensitivity of TNBC.

Radiation prevents tumor progression by inhibiting the miR‑93‑5p/EphA4/NF‑κB pathway in triple‑negative breast cancer.

Breast cancer (BC) is the most common type of cancer in women. Triple‑negative BC (TNBC) constitutes 10‑15% of all BC cases and is associated with a poor prognosis. It has previously been reported that microRNA (miR)‑93‑5p is dysregulated in plasma exosomes from patients with BC and that miR‑93‑5p improves radiosensitivity in BC cells. The present study identified EphA4 as a potential target gene of miR‑93‑5p and investigated the pathway related to miR‑93‑5p in TNBC. Cell transfection and nude mouse experiments were performed to verify the role of the miR‑93‑5p/EphA4/NF‑κB pathway. Moreover, miR‑93‑5p, EphA4 and NF‑κB were detected in clinical patients. The results revealed that EphA4 and NF‑κB were downregulated in the miR‑93‑5p overexpression group. By contrast, EphA4 and NF‑κB expression levels were not significantly altered in the miR‑93‑5p overexpression + radiation group compared with those in the radiation group. Furthermore, overexpression of miR‑93‑5p with concomitant radiation therapy significantly decreased the growth of TNBC tumors invivo. In conclusion, the present study revealed that miR‑93‑5p targeted EphA4 in TNBC through the NF‑κB pathway. However, radiation therapy prevented tumor progression by inhibiting the miR‑93‑5p/EphA4/NF‑κB pathway. Therefore, it would be interesting to elucidate the role of miR‑93‑5p in clinical research.

Significance of Nuclear Factor-Kappa B (NF-κB) and Survivin in Breast Cancer and Their Association with Radiosensitivity and Prognosis.

Analyze the expression of NF-κB and survivin genes and mRNAs in breast cancer, and evaluate their impact on prognosis. Investigate their association with radiosensitivity in breast cancer. The expression levels of NF-κB and survivin genes in breast cancer were analyzed by bioinformatics, NF-κB and survivin mRNA was verified by RTRCR, and their association with prognosis were assessed. Knockdown of survivin by siRNA was used to analyze its association with radiosensitivity in breast cancer. The gene expression of NFKB1 and BIRC5 are differentially expressed in a variety of tumours and their corresponding normal tissue species. In breast cancer tissues, NFKB1 expression levels were reduced compared to normal tissue, while BIRC5 expression levels were increased (P<0.05). In different molecular subtypes of breast cancer, NFKB1 and BIRC5 were differentially expressed (P<0.05), NFKB1 was highly expressed in the luminal subtype and BIRC5 was highly expressed in the TNBC subtype. In TNBC subtype, NFKB1 expression is higher in IM subtype than other subtypes (P<0.05), and BIRC5 expression is higher in BL-2 than other subtypes (P<0.05). NFKB1 was not associated with tumour size, lymph node stage and distant metastasis (P≥0.05), while BRIC5 was associated with these clinical features (P<0.05). NF-κB and survivin genes were negatively correlated (R = - 0.193, P<0.05). The mRNA levels of NF-κB and survivin are expressed in the same trend in breast cancer patients. NF-κB and survivin were not significantly different in recurrent and non-recurrent patients (P≥0.05). The mRNA levels of the both were not correlated with breast cancer subtypes (P≥0.05). The mRNA expression of NF-κB and survivin correlated with distant metastasis. NF-κB and survivin mRNAs were positively correlated (R=0.903, P<0.05). Gene and mRNA expression of NF-κB and survivin were not associated with patients' survival overall survival (OS) (P≥0.05). Down-regulation of survivin has little effect on the proliferation rate of breast cancer cells (P≥0.05), but increase the apoptosis rate of breast cancer cells (P<0.05).The proliferation rate of cells decreased and the apoptosis rate increased significantly (P<0.05) after the implementation of radiotherapy, and this technique could improve the radiosensitivity of breast cancer cells. NF-κB and survivin interact at the gene and mRNA levels. Regulation of mRNA expression of NF-κB or survivin may help to improve the radiosensitivity of breast cancer cells, more experiments are needed to verify this in the future.

Radiosensitization of Breast Cancer Cells with a 2-Methoxyestradiol Analogue Affects DNA Damage and Repair Signaling In Vitro.

Radiation resistance and radiation-related side effects warrant research into alternative strategies in the application of this modality to cancer treatment. Designed in silico to improve the pharmacokinetics and anti-cancer properties of 2-methoxyestradiol, 2-ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16) disrupts microtubule dynamics and induces apoptosis. Here, we investigated whether pre-exposure of breast cancer cells to low-dose ESE-16 would affect radiation-induced deoxyribonucleic acid (DNA) damage and the consequent repair pathways. MCF-7, MDA-MB-231, and BT-20 cells were exposed to sub-lethal doses of ESE-16 for 24 h before 8 Gy radiation. Flow cytometric quantification of Annexin V, clonogenic studies, micronuclei quantification, assessment of histone H2AX phosphorylation and Ku70 expression were performed to assess cell viability, DNA damage, and repair pathways, in both directly irradiated cells and cells treated with conditioned medium. A small increase in apoptosis was observed as an early consequence, with significant repercussions on long-term cell survival. Overall, a greater degree of DNA damage was detected. Moreover, initiation of the DNA-damage repair response was delayed, with a subsequent sustained elevation. Radiation-induced bystander effects induced similar pathways and were initiated via intercellular signaling. These results justify further investigation of ESE-16 as a radiation-sensitizing agent since pre-exposure appears to augment the response of tumor cells to radiation.

Cancer-associated fibroblasts induce growth and radioresistance of breast cancer cells through paracrine IL-6

In breast cancer, the most numerous stromal cells are cancer-associated fibroblasts (CAFs), which are associated with disease progression and chemoresistance. However, few studies have explored the function of CAFs in breast cancer cell radiosensitivity. Here, CAF-derived conditioned media was observed to induce breast cancer cell growth and radioresistance. CAFs secrete interleukin 6 (IL-6) which activates signal transducer and activator of transcription 3 (STAT3) signaling pathway, thus promoting the growth and radioresistance of breast cancer cells. Treatment with an inhibitor of STAT3 or an IL-6 neutralizing antibody blocked the growth and radioresistance induced by CAFs. In in vivo mouse models, tocilizumab (an IL-6 receptor monoclonal antibody) abrogated CAF-induced growth and radioresistance. Moreover, in breast cancer, a poor response to radiotherapy was associated with IL-6 and p-STAT3 expression. These results indicated that IL-6 mediates cross-talk between breast cancer cells and CAFs in the tumor microenvironment. Our results identified the IL-6/STAT3 signaling pathway as an important therapeutic target in breast cancer radiotherapy.

Cancer-Associated Fibroblasts Promote Radioresistance of Breast Cancer Cells via the HGF/c-Met Signaling Pathway

Cancer-associated fibroblasts (CAFs) are an integral part of the tumor microenvironment (TME), which is involved in therapy resistance. This study aimed to investigate the role of CAFs in radiosensitivity of breast cancer cells. The CAFs were isolated from the breast cancer tissues, and the conditioned medium was collected to culture breast cancer cells. Radiation-induced DNA damage was evaluated by immunofluorescence and western blotting. Cytokine array and enzyme-linked immunosorbent assay were performed to analyze the secretion of cytokines and growth factors. An in vitro clonogenic survival assay and in vivo xenograft mouse model were performed to determine the radiosensitivity of breast cancer cells. Finally, the expression of hepatocyte growth factor (HGF) and c-Met in the breast cancer tissues were detected by immunohistochemistry. The CAFs were found to secrete HGF to activate the c-Met signaling pathway, which induced epithelial-to-mesenchymal transition, growth, and radioresistance of breast cancer cells. Furthermore, radiation was observed to enhance HGF secretion by CAFs and increase c-Met expression in breast cancer cells, which led to HGF/c-Met signaling pathway activation. Moreover, radiation-induced tumor necrosis factor α (TNFα) secretion by breast cancer cells promoted CAF proliferation and HGF secretion. Additionally, HGF and c-Met high expression were associated with worse recurrence-free survival in patients with breast cancer who had received radiation therapy. The findings revealed that HGF and TNFα are critical for the crosstalk between breast cancer cells and CAFs in the TME and that the HGF/c-Met signaling pathway is a promising therapeutic target for radiosensitizing breast cancer.

Knockdown of circ-ADAM9 inhibits malignant phenotype and enhances radiosensitivity in breast cancer cells via acting as asponge for miR-383-5p.

Circular RNA (circRNA) has been proven to play acritical role in breast cancer progression. Therefore, this study was designed to clarify the role and underlying molecular mechanisms of circ-disintegrin and metalloproteinase9 (circ-ADAM9) in breast cancer. Aquantitative real-time polymerase chain reaction (RT-qPCR) was conducted to assess the expression levels of circ-ADAM9, microRNA-383-5p (miR-383-5p), and profilin2 (PFN2). Cellular growth curves of breast cancer cells were determined by colony-forming assay. Cell viability and apoptosis were measured by MTT and flow cytometry, respectively. The protein expression level was analyzed by western blot. Cell migration and invasion were evaluated by wound healing and Transwell assays. Axenograft experiment was established to clarify the functional role of circ-ADAM9 inhibition in vivo. The interactions among circ-ADAM9, miR-383-5p, and PFN2 were analyzed by dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays. We found that circ-ADAM9 was upregulated in breast cancer tissues and cells compared to controls. Inhibition of circ-ADAM9 expression impaired proliferation, migration, and invasion, but increased radiosensitivity and apoptosis in breast cancer cells; besides, radiotherapy combined with circ-ADAM9 inhibition showed significant inhibitory effects on tumor growth. The functional effects of circ-ADAM9 were related to miR-383-5p, atarget of circ-ADAM9. Overexpression of miR-383-5p-mediated malignant behaviors and radiosensitivity of breast cancer cells were dependent on PFN2. Circ-ADAM9 was found to participate in breast cancer progression through targeting the miR-383-5p/PFN2 axis.

18F-FDG Micro PET/CT imaging to evaluate the effect of BRCA1 knockdown on MDA-MB231 breast cancer cell radiosensitivity

ObjectiveRadioresistance of tumor cells is a major factor associated with failure of radiotherapy (RT). This study aimed to investigate the effect of BRCA1 knockdown on MDA-MB231 breast cancer cell radiosensitivity. Materials and methodsShort hairpin RNA (shRNA) was used to knockdown BRCA1 gene in MDA-MB231 cells. Cell viability and proliferative capacity were assessed by CCK-8 and colony formation assays, respectively. We established xenograft models in nude mice to evaluate tumor volume and tumor weight. The mice were imaged by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) before and after RT to evaluate changes in maximum standardized uptake value (SUVmax) and tumor SUVmax/muscle SUVmax (TMR). Changes in HIF-1α, Glut-1 and Ki-67 were analyzed and the correlation between 18F-FDG uptake and tumor biology was analyzed. ResultsCompared with the control cells, RT significantly reduced cell viability and colony formation capacity in cells with the BRCA1 gene knockdown. In vivo assays showed that there was obvious delay in the tumor growth in the shBRCA1+RT group compared with the control group. 18F-FDG Micro PET/CT indicated a reduction in glucose metabolism in the shBRCA1+RT group, with statistically significant differences in both the SUVmax and TMR. The data showed the expression of HIF-1α, Glut-1 and Ki-67 was downregulated in the shBRCA1+RT group, and both SUVmax and TMR had significant correlation with tumor biology. ConclusionThese results demonstrated that BRCA1 knockdown improves the sensitivity of MDA-MB231 breast cancer cells to RT. In addition, 18F-FDG PET/CT imaging allows non-invasive analysis of tumor biology and assessment of radiosensitivity.

HS-173, a novel PI3K inhibitor enhances radiosensitivity of breast cancer cells

Background: The development of radiosensitizers that modulate activated signaling pathways has enhanced effective cancer treatment via radiation therapy. The phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT) pathway induces cancer progression and radioresistance. Therefore, we investigated if HS-173, a novel PI3K inhibitor, could increase the radiosensitivity in breast cancer cells. Materials and Methods: Breast cancer cell lines (MCF-7, BT-474, and T47D, MDA-MB-231) were exposed to radiation (2–8 Gy). After irradiation, cell viability was assessed using the MTT assay. MDA-MB-231 cells were exposed to radiation (5 Gy) alone and/or in combination with HS-173 (1 μM). After treatment, the levels of PI3K/AKT signaling protein were measured using western blotting. The radiosensitivity of HS-173 was assessed using a clonogenic assay and flow cytometry. Results: We observed that HS-173 decreased the radiation-induced phosphorylation of AKT in MDA-MB-231 cells and increased their radiosensitivity in the clonogenic assay. Upon investigation of the mechanism underlying the enhanced radiosensitivity by HS-173, we observed a significant increase in the IR-induced G2/M cell cycle arrest and apoptosis pathway components, including poly (ADP-ribose) polymerase (PARP-1) and cleaved caspase-3. It can be concluded that HS-173 significantly improved radiosensitivity by inducing apoptosis and G2/M arrest in radio-resistant breast cancer cells. Conclusion: HS-173 may be applied as a radiosensitizer with promising potential in radio-resistant breast cancer treatment.

Circ_0008500 Knockdown Improves Radiosensitivity and Inhibits Tumorigenesis in Breast Cancer Through the miR-758-3p/PFN2 Axis.

Breast cancer is one of the most common malignancies worldwide. Circular RNAs (CircRNAs) were revealed to be implicated in the development of breast cancer. In this research, we aimed to investigate the role and underlying mechanism of circ_0008500 in the development and radiosensitivity of breast cancer. Using real-time quantitative PCR (RT-qPCR) and western blot, we found that hsa_circ_0008500 (circ_0008500) and profilin 2 (PFN2) were increased, while microRNA-758-3p (miR-758-3p) was decreased in breast cancer tissues and cells. Cell viability, the number of colonies, proliferation and apoptosis were detected using CCK-8, colony formation, EdU assays and flow cytometry, respectively. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were devoted to test the interaction between miR-758-3p and circ_0008500 or PFN2. The results showed that circ_0008500 knockdown inhibited cell growth, and facilitated cell apoptosis and radiosensitivity in breast cancer cells in vitro. Moreover, circ_0008500 regulated PFN2 expression by sponging miR-758-3p. Functionally, circ_0008500 knockdown regulated cell behaviors and radiosensitivity by targeting miR-758-3p to downregulate PFN2 expression in vitro. Additionally, in vivo tumor formation assay and immunohistochemistry (IHC) assay demonstrated that circ_0008500 knockdown enhanced the radiosensitivity and repressed tumor growth in vivo. In conclusion, circ_0008500 inhibition promoted the radiosensitivity and restrained the development of breast cancer by downregulating PFN2 expression via targeting miR-758-3p.

RNA Interference Induces BRCA1 Gene Methylation and Increases the Radiosensitivity of Breast Cancer Cells.

Purpose: To investigate the relationship between breast cancer susceptibility gene-1 (BRCA1) gene methylation and the radiosensitivity of breast cancer. Materials and Methods: The authors studied three breast cancer cell lines: MDA-MB-435, MDA-MB-231, and MCF-7 cells. They constructed five short hairpin RNAs (shRNAs) and five small interfering RNAs to target selected promoter loci and initiate sequence-specific methylation in breast cancer cells. Pyrosequencing was used to analyze the state of DNA methylation. Quantitative real-time polymerase chain reaction was used to detect BRCA1 mRNA expression and RNA-directed DNA methylation (RdDM)-related gene expression. Western blotting was performed to analyze BRCA1 protein expression. Colony formation assays and γ-histone H2A foci formation assays were conducted to assess the surviving fraction (SF) and double-strand break (DSB) repair ability of cells after irradiation. Results: The authors constructed five strains of lentivirus vectors and five plasmid vectors targeting BRCA1 promoter region. In MDA-MB-435 cells, lentivirus-mediated RNA interference targeting Site 1 of BRCA1 increased the methylation levels of BRCA1 and reduced BRCA1 mRNA and protein expression. The SF and the ability to repair DNA DSBs were reduced in the combined LV-BRCA1RNAi-Site 1 infection and irradiation group. Conclusions: The authors' findings suggest that the shRNA suppressed the expression levels of the BRCA1 gene and reduced the SF and DNA repair ability of cells after irradiation through RdDM. In summary, the radiosensitivity of breast cancer cells may correlate with BRCA1 methylation. Advances in Knowledge: The authors first utilized a lentivirus-based shRNA-mediated specific-sequence DNA methylation of the BRCA1 gene mediated by RdDM.

MiR-143-3p Increases the Radiosensitivity of Breast Cancer Cells Through FGF1.

Breast cancer is a common malignant tumor in women. At present, the main treatment for breast cancer is radiotherapy. Due to the difference in radiosensitivity between individuals or tumor cells, the effect of radiotherapy is not good. Therefore, in radiotherapy, how to use various auxiliary means to reduce the radiation resistance of tumor, Therefore, it has become an important research topic to improve the radiosensitivity of the tumor. Fibroblast growth factor-1 (FGF1) plays an important role in tumor migration. Therefore, the study of miR-143-3p increasing the radiosensitivity of breast cancer cells through FGF1 is proposed in this paper. In this study, a control group experiment was set up to study. During the experiment, the relative expression of miR-143-3p was detected by fluorescent quantitative PCR of miRNA, and the cell irradiation experiment was created to analyze the radiosensitivity of breast cancer cells by comparing their survival fraction. The results of this study showed that when the radiation dose was 0, the survival scores of the three groups were all 1. The survival fraction of the experimental group decreased from 0.26 ± 0.045 to 0.068 ± 0.008 when the dose was added to 4Gy. The survival fraction of the experimental group was always greater than that of the two control groups. The results of this study show that miR-143-3p can increase the radiosensitivity of breast cancer cells through FGF1.

KLF5-trancripted miR-125b-5p is involved in enhancing the radio-sensitivity of breast cancer cells by targeting BRCA1

KLF5-trancripted miR-125b-5p is involved in enhancing the radio-sensitivity of breast cancer cells by targeting BRCA1

Lipid Droplet Biosynthesis Impairment through DGAT2 Inhibition Sensitizes MCF7 Breast Cancer Cells to Radiation.

Breast cancer is the most frequent cancer in women worldwide and late diagnosis often adversely affects the prognosis of the disease. Radiotherapy is commonly used to treat breast cancer, reducing the risk of recurrence after surgery. However, the eradication of radioresistant cancer cells, including cancer stem cells, remains the main challenge of radiotherapy. Recently, lipid droplets (LDs) have been proposed as functional markers of cancer stem cells, also being involved in increased cell tumorigenicity. LD biogenesis is a multistep process requiring various enzymes, including Diacylglycerol acyltransferase 2 (DGAT2). In this context, we evaluated the effect of PF-06424439, a selective DGAT2 inhibitor, on MCF7 breast cancer cells exposed to X-rays. Our results demonstrated that 72 h of PF-06424439 treatment reduced LD content and inhibited cell migration, without affecting cell proliferation. Interestingly, PF-06424439 pre-treatment followed by radiation was able to enhance radiosensitivity of MCF7 cells. In addition, the combined treatment negatively interfered with lipid metabolism-related genes, as well as with EMT gene expression, and modulated the expression of typical markers associated with the CSC-like phenotype. These findings suggest that PF-06424439 pre-treatment coupled to X-ray exposure might potentiate breast cancer cell radiosensitivity and potentially improve the radiotherapy effectiveness.

Retraction.

Retraction: "Inhibition of Notch signaling pathway enhanced the radiosensitivity of breast cancer cells," by Jian-Heng Peng, Xiao-Lin Wang, Liang Ran, Jun-Long Song, Zhi-Ting Zhang, Xin Liu, Hong-Yuan Li, J Cell Biochem. 2018; 8398-8409: The above article, published online on 15 June 2018 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/jcb.27036) has been retracted by agreement between the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. A detailed investigation revealed that several image elements of the experimental data were published elsewhere in a different scientific context. Thus, the editors consider the conclusions of this article to be invalid.

A radiosensitizer, gallotannin-rich extract from Bouea macrophylla seeds, inhibits radiation-induced epithelial-mesenchymal transition in breast cancer cells

BackgroundRadioresistance can pose a significant obstacle to the effective treatment of breast cancers. Epithelial–mesenchymal transition (EMT) is a critical step in the acquisition of stem cell traits and radioresistance. Here, we investigated whether Maprang seed extract (MPSE), a gallotannin-rich extract of seed from Bouea macrophylla Griffith, could inhibit the radiation-induced EMT process and enhance the radiosensitivity of breast cancer cells.MethodsBreast cancer cells were pre-treated with MPSE before irradiation (IR), the radiosensitizing activity of MPSE was assessed using the colony formation assay. Radiation-induced EMT and stemness phenotype were identified using breast cancer stem cells (CSCs) marker (CD24−/low/CD44+) and mammosphere formation assay. Cell motility was determined via the wound healing assay and transwell migration. Radiation-induced cell death was assessed via the apoptosis assay and SA-β-galactosidase staining for cellular senescence. CSCs- and EMT-related genes were confirmed by real-time PCR (qPCR) and Western blotting.ResultsPre-treated with MPSE before irradiation could reduce the clonogenic activity and enhance radiosensitivity of breast cancer cell lines with sensitization enhancement ratios (SERs) of 2.33 and 1.35 for MCF7 and MDA-MB231cells, respectively. Pretreatment of breast cancer cells followed by IR resulted in an increased level of DNA damage maker (γ-H2A histone family member) and enhanced radiation-induced cell death. Irradiation induced EMT process, which displayed a significant EMT phenotype with a down-regulated epithelial marker E-cadherin and up-regulated mesenchymal marker vimentin in comparison with untreated breast cancer cells. Notably, we observed that pretreatment with MPSE attenuated the radiation-induced EMT process and decrease some stemness-like properties characterized by mammosphere formation and the CSC marker. Furthermore, pretreatment with MPSE attenuated the radiation-induced activation of the pro-survival pathway by decrease the expression of phosphorylation of ERK and AKT and sensitized breast cancer cells to radiation.ConclusionMPSE enhanced the radiosensitivity of breast cancer cells by enhancing IR-induced DNA damage and cell death, and attenuating the IR-induced EMT process and stemness phenotype via targeting survival pathways PI3K/AKT and MAPK in irradiated breast cancer cells. Our findings describe a novel strategy for increasing the efficacy of radiotherapy for breast cancer patients using a safer and low-cost natural product, MPSE.

Investigation of miR-93-5p and its effect on the radiosensitivity of breast cancer

ABSTRACT Accumulating evidence suggests that intrinsic resistance to radiotherapy reduces the survival of patients with cancer. The present study investigated whether miR-93-5p affects proliferation, migration, apoptosis, and radiosensitivity of breast cancer (BC) cells. MDA-MB-468, MCF-7, and MDA-MB-231 BC cells were incubated with hsa-miR-93-5p mimics, hsa-miR-93-5p inhibitor, and negative control RNA with or without exposure to ionizing radiation to determine cell proliferation, migration, and apoptosis using the Cell Counting Kit-8 assay, wound healing assay and apoptotic assay, respectively. Overexpression of miR-93-5p inhibited the migratory abilities (P = 0.001) and decreased the cell proliferation (P = 0.049) of MCF-7 cells. In MCF-7 cells, a significant increase in apoptosis was detected after treatment with miR-93-5p compared with the negative control (P = 0.001) and miR-93-5p inhibitor (P = 0.004). In MDA-MB-468 cells, the proportion of apoptotic cells increased following exposure to ionizing radiation (P = 0.001). The percentage of apoptotic MDA-MB-231 cells in the miR-93-5p group was significantly increase compared with that determined in the negative control (P = 0.044) and hsa-miR-93-5p inhibitor (P = 0.046) groups. In conclusion, our findings showed that miR-93-5p reduces BC cell proliferation and migratory capacity, and increases the ratio of apoptotic cells. Overexpression of miR-93-5p could increase radiosensitivity in BC cells by increasing apoptosis. This evidence provides new insight into the treatment of BC and identifies miR-93-5p as a potential therapeutic target.

ALG3 contributes to stemness and radioresistance through regulating glycosylation of TGF-\u03b2 receptor II in breast cancer

BackgroundRadiotherapy is a conventional and effective local treatment for breast cancer. However, residual or recurrent tumors appears frequently because of radioresistance. Novel predictive marker and the potential therapeutic targets of breast cancer radioresistance needs to be investigated.MethodsIn this study, we screened all 10 asparagine-linked glycosylation (ALG) members in breast cancer patients’ samples by RT-PCR. Cell viability after irradiation (IR) was determined by CCK-8 assay and flow cytometry. The radiosensitivity of cell lines with different ALG3 expression was determined with the colony formation assay by fitting the multi-target single hit model to the surviving fractions. Cancer stem-like traits were assessed by RT-PCR, Western blot, and flow cytometry. The mechanisms of ALG3 influencing radiosensitivity was detected by Western blot and immunoprecipitation. And the effect of ALG3 on tumor growth after IR was verified in an orthotopic xenograft tumor models. The association of ALG3 with prognosis of breast cancer patients was confirmed by immunohistochemistry.ResultsALG3 was the most significantly overexpressing gene among ALG family in radioresistant breast cancer tissue. Overexpression of ALG3 predicted poor clinicopathological characteristics and overall survival (OS), and early local recurrence-free survival (LRFS) in breast cancer patients. Upregulating ALG3 enhanced radioresistance and cancer stemness in vitro and in vivo. Conversely, silencing ALG3 increased the radiosensitivity and repressed cancer stemness in vitro, and more importantly inhibition of ALG3 effectively increased the radiosensitivity of breast cancer cells in vivo. Mechanistically, our results further revealed ALG3 promoted radioresistance and cancer stemness by inducing glycosylation of TGF-β receptor II (TGFBR2). Importantly, both attenuation of glycosylation using tunicamycin and inhibition of TGFBR2 using LY2109761 differentially abrogated the stimulatory effect of ALG3 overexpression on cancer stemness and radioresistance. Finally, our findings showed that radiation played an important role in preventing early recurrence in breast cancer patients with low ALG3 levels, but it had limited efficacy in ALG3-overexpressing breast cancer patients.ConclusionOur results suggest that ALG3 may serve as a potential radiosensitive marker, and an effective target to decrease radioresistance by regulating glycosylation of TGFBR2 in breast cancer. For patients with low ALG3 levels, radiation remains an effective mainstay therapy to prevent early recurrence in breast cancer.

PLK1 Inhibition Sensitizes Breast Cancer Cells to Radiation via Suppressing Autophagy.

Polo-like kinase 1 (PLK1) is a protein kinase that is overexpressed in breast cancer and may represent an attractive target for breast cancer treatment. However, few studies have investigated the relationship between PLK1 and radiosensitivity in breast cancer. Here, we attempted to explore whether PLK1 inhibition could sensitize breast cancer cells to radiation. Breast cancer cells were treated with PLK1 small interference RNA or the PLK1-inhibitor, GSK461364. Cell proliferation was assessed using a colony formation assay. Cell cycle analyses were performed by flow cytometry. DNA damage, autophagy, and reactive oxygen species induced by ionizing radiation were detected by immunofluorescence, Western blot, and flow cytometry, respectively. Microtubule-associated protein 1 light chain 3 alpha (LC3) puncta were detected using an immunofluorescence assay. A clonogenic survival assay was used to determine the effect of PLK1 inhibition on cell radiosensitivity. A xenograft mouse model of breast cancer cells was used to investigate the potential synergistic effects of PLK1 inhibition and irradiation invivo. Finally, the expression of PLK1 and LC3 in the breast cancer tissues was evaluated by immunohistochemistry. PLK1 inhibition significantly suppressed the proliferation and increased the radiosensitivity of breast cancer cells. Pharmacologic inhibition of PLK1 by the selective inhibitor, GSK461364, enhanced the radiosensitivity of breast cancer cells invivo (n = 4, P = .002). Mechanistically, PLK1 inhibition led to the downregulation of radiation-induced reactive oxygen species and autophagy, thereby increasing the radiosensitivity of breast cancer cells. Additionally, we detected a positive correlation between the expression of PLK1 and LC3 in human breast cancer samples (n = 102, R = 0.486, P = .005). Our findings indicate that PLK1 inhibition enhances the radiosensitivity of breast cancer cells in a manner associated with the suppression of radiation-induced autophagy. The inhibition of PLK1 represents a promising strategy for radiosensitizing breast cancer.