Metformin suppresses esophageal cancer progression through the radiation­induced cellular senescence of cancer­associated fibroblasts.

Senescent cells are known to secrete proteins, including inflammatory cytokines and damage­associated molecular patterns. This phenomenon is known as the senescence­associated secretory phenotype (SASP). SASP in cancer stromal fibroblasts is involved in cancer growth and progression. Conversely, metformin, an antidiabetic drug, has been reported to inhibit SASP induction by inhibiting the activation of NF­κB, a regulator of SASP. To date, at least to the best of our knowledge, there have been no reports regarding cellular senescence in fibroblasts and tumor progression via the SASP­mediated paracrine pathway. The present study thus aimed to elucidate the induction mechanisms of SASP in radiation­induced fibroblasts and to determine its effects on cancer progression via the paracrine pathway. Furthermore, the present study aimed to determine whether controlling SASP using metformin suppresses cancer progression. A well­differentiated esophageal cancer cell line established by the authors' department and fibroblasts isolated and cultured from the non­cancerous esophageal mucosa of resected esophageal cancer cases were used for the experiments. Fibroblasts were irradiated with 8 Gy radiation, and the changes in the expression of the senescence markers, SA­ß­gal, p21, p16 and NF­κB were evaluated using immunofluorescent staining and western blot analysis in the presence or absence of metformin treatment. The culture supernatants of irradiated fibroblasts treated with metformin and those treated without metformin were collected and added to the cancer cells to evaluate their proliferative, invasive and migratory abilities. Vimentin and E­cadherin expression levels were also evaluated using immunofluorescent staining and western blot analysis. The expression levels of p16, p21 and NF­κB in irradiated fibroblasts were attenuated by treatment with metformin. Supernatants collected from irradiated fibroblasts exhibited the proliferative activity of esophageal cancer cells, and the promotion of migratory and invasion abilities, which may be due to epithelial­mesenchymal transition and changes in cell morphology. These reactions were confirmed to be suppressed by the addition of the supernatant of cultured fibroblasts pre­treated with metformin. On the whole, the present study demonstrates that fibroblasts in the cancer stroma may be involved in tumor progression through cellular senescence.

Meta-stable state photoacid containing ß-estradiol fragment with photomodulated biological activity and anti-cancer stem cells properties.

Photopharmacology is a young and rapidly developing field of research that offers significant potential for new insights into targeted therapy. While it primarily focuses on cancer treatment, it also holds promise for other diseases. The key feature of photopharmacological agents is the presence of a photosensitive and biologically active component in the same molecule. In our current study, we synthesized a spiropyran-based meta-stable state photoacid containing a fragment of ß-estradiol. This compound exhibits negative photochromism and photocontrolled fluorescence under visible-light irradiation due to the initial stabilization of its self-protonated form in solution. We conducted comprehensive biological studies on the HeLa cells model to assess the short- and long-term cytotoxicity of the photoacid, its metabolic effects, its influence on signaling and epithelial-mesenchymal transition super-system pathways, and the proportion of the population enriched with cancer stem cells. Our findings reveal that this derivative demonstrates low cytotoxicity to HeLa cells, yet it is capable of dramatically reducing malignant cells side population enriched in cancer stem cells. Additionally, appropriate structural modification lead to an increase in some other biological effects compared to ß-estradiol. In particular, our substance possesses rare properties of AP-1 suppression and demonstrates some pro-oxidant and metabolic effects, which can be regulated by visible light irradiation. As a result, the new estradiol-based photoacid may be considered a promising multi-acting photopharmacological agent for the next-generation anti-cancer research & development.

Modulation of epithelial-mesenchymal transition by gemcitabine: Targeting ionizing radiation-induced cellular senescence in lung cancer cell.

Ionizing radiation, a standard therapeutic approach for lung cancer, often leads to cellular senescence and the induction of epithelial-mesenchymal transition (EMT), posing significant challenges in treatment efficacy and cancer progression. Overcoming these obstacles is crucial for enhancing therapeutic outcomes in lung cancer management. This study investigates the effects of ionizing radiation and gemcitabine on lung cancer cells, with a focus on induced senescence, EMT, and apoptosis. Human-derived A549, PC-9, and mouse-derived Lewis lung carcinoma cells exposed to 10 Gy X-ray irradiation exhibited senescence, as indicated by morphological changes, ß-galactosidase staining, and cell cycle arrest through the p53-p21 pathway. Ionizing radiation also promoted EMT via TGFß/SMAD signaling, evidenced by increased TGFß1 levels, altered EMT marker expressions, and enhanced cell migration. Gemcitabine, a first-line lung cancer treatment, was shown to enhance apoptosis in senescent cells caused by radiation. It inhibited cell proliferation, induced mitochondrial damage, and triggered caspase-mediated apoptosis, thus mitigating EMT in vitro. Furthermore, in vivo studies using a lung cancer mouse model revealed that gemcitabine, combined with radiation, significantly reduced tumor volume and weight, extended survival, and suppressed malignancy indices in irradiated tumors. Collectively, these findings demonstrate that gemcitabine enhances the therapeutic efficacy against radiation-resistant lung cancer cells, both by inducing apoptosis in senescent cells and inhibiting EMT, offering potential improvements in lung cancer treatment strategies.

Downregulation of SMAD4 protects HaCaT cells against UVB-induced damage and oxidative stress through the activation of EMT.

As a member of the SMAD family, SMAD4 plays a crucial role in several cellular biological processes. However, its function in UVB radiation-induced keratinocyte damage is not yet clarified. Our study aims to provide mechanistic insight for the development of future UVB protective therapies and therapeutics involving SMAD4. HaCaT cells were treated with UVB, and the dose dependence and time dependence of UVB were measured. The cell function of UVB-treated HaCaT cells and the activity of epithelial-mesenchymal transition (EMT) after overexpression or silencing of SMAD4 was observed by flow cytometry, quantitative reverse transcription PCR (qRT-PCR) and Western Blots (WB). We found that a significant decrease in SMAD4 was observed in HaCaT cells induced by UVB. Our data confirm SMAD4 as a direct downstream target of miR-664. The down-regulation of SMAD4 preserved the viability of the UVB-treated HaCaT cells by inhibiting autophagy or apoptosis. Furthermore, the silencing of SMAD4 activated the EMT process in UVB-treated HaCaT cells. Down-regulation of SMAD4 plays a protective role in UVB-treated HaCaT cells via the activation of EMT.

Downregulation of ß-Catenin Contributes to type II Alveolar Epithelial Stem Cell Resistance to Epithelial-Mesenchymal Transition by Lowing Lin28/let-7 Ratios in Fibrosis-Resistant Mice after Thoracic Irradiation.

Transdifferentiation of type II alveolar cells (AECII) is a major cause for radiation-induced lung fibrosis (RILF). Cell differentiation phenotype is determined by Lin28 (undifferentiated marker) and let-7 (differentiated marker) in a see-saw-pattern. Therefore, differentiation phenotype can be extrapolated based on Lin28/let-7 ratio. Lin28 is activated by ß-catenin. To the best of our knowledge this study was the first to use the single primary AECII freshly isolated from irradiated lungs of fibrosis-resistant C3H/HeNHsd strain to further confirm RILF mechanism by comparing its differences in AECII phenotype status/state and cell differentiation regulators to fibrosis-prone C57BL/6j mice. Results showed that radiation pneumonitis and fibrotic lesions were seen in C3H/HeNHsd and C57BL/6j mouse strains, respectively. mRNAs of E-cadherin, EpCAM, HOPX and proSP-C (epithelial phenotype biomarkers) were significantly downregulated in single primary AECII isolated from irradiated lungs of both strains. Unlike C57BL/6j, α-SMA and Vimentin (mesenchymal phenotype biomarkers) were not upregulated in single AECII from irradiated C3H/HeNHsd. Profibrotic molecules, TGF-ß1 mRNA was upregulated and ß-catenin was significantly downregulated in AECII after irradiation (both P < 0.01). In contrast, transcriptions for GSK-3ß, TGF-ß1 and ß-catenin were enhanced in isolated single AECII from irradiated C57BL/6j (P < 0.01-P < 0.001). The Lin28/let-7 ratios were much lower in single primary AECII from C3H/HeNHsd after irradiation vs. C57BL/6j. In conclusion, AECII from irradiated C3H/HeNHsd did not undergo epithelial-mesenchymal transition (EMT) and lower ratios of Lin28/let-7 contributed to AECII relatively higher differentiated status, leading to increased susceptibility to radiation stress and a failure in transdifferentiation in the absence of ß-catenin. Reducing ß-catenin expression and the ratios of Lin28/let-7 may be a promising strategy to prevent radiation fibrosis.

Consecutive dosing of UVB irradiation induces loss of ABCB5 expression and activation of EMT and fibrosis proteins in limbal epithelial cells similar to pterygium epithelium.

Pterygium pathogenesis is often attributed to a population of altered limbal stem cells, which initiate corneal invasion and drive the hyperproliferation and fibrosis associated with the disease. These cells are thought to undergo epithelial to mesenchymal transition (EMT) and to contribute to subepithelial stromal fibrosis. In this study, the presence of the novel limbal stem cell marker ABCB5 in clusters of basal epithelial pterygium cells co-expressing with P63α and P40 is reported. ABCB5-positive pterygium cells also express EMT-associated fibrosis markers including vimentin and α-SMA while their ß-catenin expression is reduced. By using a novel in vitro model of two-dose UV-induced EMT activation on limbal epithelial cells, we could observe the dysregulation of EMT-related proteins including an increase of vimentin and α-SMA as well as downregulation of ß-catenin in epithelial cells correlating to downregulation of ABCB5. The sequential irradiation of limbal fibroblasts also induced an increase in vimentin and α-SMA. Taken together, these data demonstrate for the first time the expression of ABCB5 in pterygium stem cell activity and EMT-related events while the involvement of limbal stem cells in pterygium pathogenesis is exhibited via sequential irradiation of limbal epithelial cells. The later in vitro approach can be used to further study the involvement of limbal epithelium UV-induced EMT in pterygium pathogenesis and help identify novel treatments against pterygium growth and recurrence.

Exposure to b-LED Light While Exerting Antimicrobial Activity on Gram-Negative and -Positive Bacteria Promotes Transient EMT-like Changes and Growth Arrest in Keratinocytes.

While blue LED (b-LED) light is increasingly being studied for its cytotoxic activity towards bacteria in therapy of skin-related infections, its effects on eukaryotic cells plasticity are less well characterized. Moreover, since different protocols are often used, comparing the effect of b-LED towards both microorganisms and epithelial surfaces may be difficult. The aim of this study was to analyze, in the same experimental setting, both the bactericidal activity and the effects on human keratinocytes. Exposure to b-LED induced an intense cytocidal activity against Gram-positive (i.e, Staphylococcus aureus) and Gram-negative (i.e., Pseudomonas aeruginosa) bacteria associated with catheter-related infections. Treatment with b-LED of a human keratinocyte cell line induced a transient cell cycle arrest. At the molecular level, exposure to b-LED induced a transient downregulation of Cyclin D1 and an upregulation of p21, but not signs of apoptosis. Interestingly, a transient induction of phosphor-histone γ-H2Ax, which is associated with genotoxic damages, was observed. At the same time, keratinocytes underwent a transient epithelial to mesenchymal transition (EMT)-like phenotype, characterized by E-cadherin downregulation and SNAIL/SLUG induction. As a functional readout of EMT induction, a scratch assay was performed. Surprisingly, b-LED treatment provoked a delay in the scratch closure. In conclusion, we demonstrated that b-LED microbicidal activity is associated with complex responses in keratinocytes that certainly deserve further analysis.

Irradiation Activates MZF1 to Inhibit miR-541-5p Expression and Promote Epithelial-Mesenchymal Transition (EMT) in Radiation-Induced Pulmonary Fibrosis (RIPF) by Upregulating Slug.

Understanding miRNAs regulatory roles in epithelial-mesenchymal transition (EMT) would help establish new avenues for further uncovering the mechanisms underlying radiation-induced pulmonary fibrosis (RIPF) and identifying preventative and therapeutic targets. Here, we demonstrated that miR-541-5p repression by Myeloid Zinc Finger 1 (MZF1) promotes radiation-induced EMT and RIPF. Irradiation could decrease miR-541-5p expression in vitro and in vivo and inversely correlated to RIPF development. Ectopic miR-541-5p expression suppressed radiation-induced-EMT in vitro and in vivo. Knockdown of Slug, the functional target of miR-541-5p, inhibited EMT induction by irradiation. The upregulation of transcription factor MZF1 upon irradiation inhibited the expression of endogenous miR-541-5p and its primary precursor (pri-miR-541-5p), which regulated the effect of the Slug on the EMT process. Our finding showed that ectopic miR-541-5p expression mitigated RIPF in mice by targeting Slug. Thus, irradiation activates MZF1 to downregulate miR-541-5p in alveolar epithelial cells, promoting EMT and contributing to RIPF by targeting Slug. Our observation provides further understanding of the development of RIPF and determines potential preventative and therapeutic targets.

Detection of cancer stem cells by EMT-specific biomarker-based peptide ligands.

The occurrence of epithelial-mesenchymal transition (EMT) within tumors, which enables invasion and metastasis, is linked to cancer stem cells (CSCs) with drug and radiation resistance. We used two specific peptides, F7 and SP peptides, to detect EMT derived cells or CSCs. Human tongue squamous carcinoma cell line-SAS transfected with reporter genes was generated and followed by spheroid culture. A small molecule inhibitor-Unc0642 and low-dose ionizing radiation (IR) were used for induction of EMT. Confocal microscopic imaging and fluorescence-activated cell sorting analysis were performed to evaluate the binding ability and specificity of peptides. A SAS xenograft mouse model with EMT induction was established for assessing the binding affinity of peptides. The results showed that F7 and SP peptides not only specifically penetrated into cytoplasm of SAS cells but also bound to EMT derived cells and CSCs with high nucleolin and vimentin expression. In addition, the expression of CSC marker and the binding of peptides were increased in tumors isolated from Unc0642/IR-treated groups. Our study demonstrates the potential of these peptides for detecting EMT derived cells or CSCs and might provide an alternative isolation method for these subpopulations within the tumor in the future.

Not Only Hypoxia- but Radiation-Induced Epithelial-Mesenchymal Transition Is Modulated by Hypoxia-Inducible Factor 1 in A549 Lung Cancer Cells.

Hypoxia leads to post-treatment metastasis and recurrences of cancer via the epithelial-mesenchymal transition (EMT). Radiotherapy itself may also contribute to the acquisition of EMT phenotypes. Despite extensive studies on the EMT driven by either hypoxia or radiation stimuli, the molecular mechanisms characterizing these EMT events remain unclear. Thus, we aimed to evaluate the differences in the molecular pathways between hypoxia-induced EMT (Hypo-EMT) and radiation-induced EMT (R-EMT). Further, we investigated the therapeutic effects of HIF-1α inhibitor (LW6) on Hypo-EMT and R-EMT cells. A549 cells, lung adenocarcinoma cell line, acquired enhanced wound-healing activity under both hypoxia and irradiation. Localization of E-cadherin was altered from the cell membrane to the cytoplasm in both hypoxia and irradiated conditions. Of note, the expression levels of vimentin, one of the major EMT markers, was enhanced in irradiated cells, while it decreased under hypoxia condition. Importantly, LW6 significantly blocked EMT-related malignant phenotypes in both Hypo-EMT cells and R-EMT cells with concomitant re-location of E-cadherin onto the cell membrane. Moreover, LW6 deflected stress responsive signalling, JNK, activated sustainably under hypoxic condition, and the blockage of JNK impaired EMT phenotypes. Together, this work demonstrated the molecular events underlying Hypo-EMT and R-EMT, and highlighted HIF-1α as a therapeutic target not only in Hypo- EMT, but also in R-EMT.

Role of Mitochondria in Radiation Responses: Epigenetic, Metabolic, and Signaling Impacts.

Until recently, radiation effects have been considered to be mainly due to nuclear DNA damage and their management by repair mechanisms. However, molecular biology studies reveal that the outcomes of exposures to ionizing radiation (IR) highly depend on activation and regulation through other molecular components of organelles that determine cell survival and proliferation capacities. As typical epigenetic-regulated organelles and central power stations of cells, mitochondria play an important pivotal role in those responses. They direct cellular metabolism, energy supply and homeostasis as well as radiation-induced signaling, cell death, and immunological responses. This review is focused on how energy, dose and quality of IR affect mitochondria-dependent epigenetic and functional control at the cellular and tissue level. Low-dose radiation effects on mitochondria appear to be associated with epigenetic and non-targeted effects involved in genomic instability and adaptive responses, whereas high-dose radiation effects (>1 Gy) concern therapeutic effects of radiation and long-term outcomes involving mitochondria-mediated innate and adaptive immune responses. Both effects depend on radiation quality. For example, the increased efficacy of high linear energy transfer particle radiotherapy, e.g., C-ion radiotherapy, relies on the reduction of anastasis, enhanced mitochondria-mediated apoptosis and immunogenic (antitumor) responses.

PPM1A as a key target of the application of Jiawei­Maxing­Shigan decoction for the attenuation of radiation­induced epithelial­mesenchymal transition in type II alveolar epithelial cells.

Radiation­induced lung tissue injury is an important reason for the limited application of radiotherapy on thoracic malignancies. Previously, we reported that administration of Jiawei­Maxing­Shigan decoction (JMSD) attenuated the radiation­induced epithelial­mesenchymal transition (EMT) in alveolar epithelial cells (AECs) via TGF­ß/Smad signaling. The present study aimed to examine the role of protein phosphatase Mg2+/Mn2+­dependent 1A (PPM1A) in the anti­EMT activity of JMSD on AECs. The components in the aqueous extract of JMSD were identified by high­performance liquid chromatography coupled with electrospray mass spectrometry. Primary rat type II AECs were treated with radiation (60Co γ­ray at 8 Gy) and JMSD­medicated serum. PPM1A was overexpressed and knocked down in the AECs via lentivirus transduction and the effects of JMSD administration on the key proteins related to TGF­ß1/Smad signaling were measured by western blotting. It was found that radiation decreased the PPM1A expression in the AECs and JMSD­medicated serum upregulated the PPM1A expressions in the radiation­induced AECs. PPM1A overexpression increased the E­cadherin level but decreased the phosphorylated (p­)Smad2/3, vimentin and α­smooth muscle actin (α­SMA) levels in the AECs. By contrast, the PPM1A knockdown decreased the E­cadherin level and increased the p­Smad2/3, vimentin and α­SMA levels in the AECs and these effects could be blocked by SB431542 (TGF­ß1/Smad signaling inhibitor). JMSD administration increased the E­cadherin level and decreased the p­Smad2/3, vimentin and α­SMA levels in the AECs; however, these effects could be blocked by siPPM1A­2. In conclusion, PPM1A is a key target of JMSD administration for the attenuation of the radiation­induced EMT in primary type II AECs via the TGF­ß1/Smad pathway.

NK Cells Lose Their Cytotoxicity Function against Cancer Stem Cell-Rich Radiotherapy-Resistant Breast Cancer Cell Populations.

Cancer stem cells (CSCs) can be induced from differentiated cancer cells in the tumor microenvironment or in response to treatments and exhibit chemo- and radioresistance, leading to tumor recurrence and metastasis. We previously reported that triple negative breast cancer (TNBC) cells with acquired radioresistance exhibited more aggressive features due to an increased CSC population. Therefore, here, we isolated CSCs from radiotherapy-resistant (RT-R)-TNBC cells and investigated the effects of these CSCs on tumor progression and NK cell-mediated cytotoxicity. Compared to MDA-MB-231 and RT-R-MDA-MB-231 cells, CD24-/low/CD44+ cells isolated from RT-R-MDA-MB-231 cells showed increased proliferation, migration and invasion abilities, and induced expression of tumor progression-related molecules. Moreover, similar to MDA-MB-231 cells, CD24-/low/CD44+ cells recruited NK cells but suppressed NK cell cytotoxicity by regulating ligands for NK cell activation. In an in vivo model, CD24-/low/CD44+ cell-injected mice showed enhanced tumor progression and lung metastasis via upregulation of tumor progression-related molecules and altered host immune responses. Specifically, NK cells were recruited into the peritumoral area tumor but lost their cytotoxicity due to the altered expression of activating and inhibitory ligands on tumors. These results suggest that CSCs may cause tumor evasion of immune cells, resulting in tumor progression.

Radiation-Induced Overexpression of TGFß and PODXL Contributes to Colorectal Cancer Cell Radioresistance through Enhanced Motility.

The primary cause of colorectal cancer (CRC) recurrence is increased distant metastasis after radiotherapy, so there is a need for targeted therapeutic approaches to reduce the metastatic-relapse risk. Dysregulation of the cell-surface glycoprotein podocalyxin-like protein (PODXL) plays an important role in promoting cancer-cell motility and is associated with poor prognoses for many malignancy types. We found that CRC cells exposed to radiation demonstrated increased TGFß and PODXL expressions, resulting in increased migration and invasiveness due to increased extracellular matrix deposition. In addition, both TGFß and PODXL were highly expressed in tissue samples from radiotherapy-treated CRC patients compared to those from patients without this treatment. However, it is unclear whether TGFß and PODXL interactions are involved in cancer-progression resistance after radiation exposure in CRC. Here, using CRC cells, we showed that silencing PODXL blocked radiation-induced cell migration and invasiveness. Cell treatment with galunisertib (a TGFß-pathway inhibitor) also led to reduced viability and migration, suggesting that its clinical use may enhance the cytotoxic effects of radiation and lead to the effective inhibition of CRC progression. Overall, the results demonstrate that downregulation of TGFß and its-mediated PODXL may provide potential therapeutic targets for patients with radiotherapy-resistant CRC.

A Wnt-mediated phenotype switch along the epithelial-mesenchymal axis defines resistance and invasion downstream of ionising radiation in oral squamous cell carcinoma.

BACKGROUND: Dynamic transitions of tumour cells along the epithelial-mesenchymal axis are important in tumorigenesis, metastasis and therapy resistance. METHODS: In this study, we have used cell lines, 3D spheroids and tumour samples in a variety of cell biological and transcriptome analyses to highlight the cellular and molecular dynamics of OSCC response to ionising radiation. RESULTS: Our study demonstrates a prominent hybrid epithelial-mesenchymal state in oral squamous cell carcinoma cells and tumour samples. We have further identified a key role for levels of E-cadherin in stratifying the hybrid cells to compartments with varying levels of radiation response and radiation-induced epithelial-mesenchymal transition. The response to radiation further entailed the generation of a new cell population with low expression levels of E-cadherin, and positive for Vimentin (ECADLow/Neg-VIMPos), a phenotypic signature that showed an enhanced capacity for radiation resistance and invasion. At the molecular level, transcriptome analysis of spheroids in response to radiation showed an initial burst of misregulation within the first 30 min that further declined, although still highlighting key alterations in gene signatures. Among others, pathway analysis showed an over-representation for the Wnt signalling pathway that was further confirmed to be functionally involved in the generation of ECADLow/Neg-VIMPos population, acting upstream of radiation resistance and tumour cell invasion. CONCLUSION: This study highlights the functional significance and complexity of tumour cell remodelling in response to ionising radiation with links to resistance and invasive capacity. An area of less focus in conventional radiotherapy, with the potential to improve treatment outcomes and relapse-free survival.

Down-regulation of autophagy-associated protein increased acquired radio-resistance bladder cancer cells sensitivity to taxol.

BACKGROUND: As a bladder-preserving therapy, radiation therapy (RT) has been widely used in the treatment of bladder cancer (BCa) and made great progress in the past few decades. However, some BCa patients have low RT responsiveness and local recurrence rate after RT could reach 50%. Acquired radio-resistance (ARR) is one of the important reasons for the failure of RT. Unfortunately, these ARR cells also lack sensitivity to chemotherapy and cause tumor recurrence and metastasis. PURPOSE: To build ARR-phenotype BCa cell model, discuss the possible molecular mechanism of ARR and find effective target molecules to overcome ARR. MATERIALS AND METHODS: Five thousand six hundred and thirty-seven cells were subjected 30 times to 2 Gy of γ-rays and the surviving cells were called 5637R. Colony formation and MTT assay were applied to evaluate cells sensitivity to ionizing radiation (IR) and anti-neoplastic agents, respectively. Cells abilities of migration and invasion were determined using transwell method. Quantitative real-time polymerase chain reaction (RT-qPCR) and western blot (WB) were respectively utilized to compare the difference of gene and protein expression between 5637 and 5637R cells. Molecule inhibitors and small interfering RNA (siRNA) systems were employed to decrease the expression of target proteins, respectively. RESULTS: BCa cells survived from fractionated irradiation (FI) exhibited tolerance to both IR and chemotherapy drugs. These ARR cells (5637R) had elevated migration and invasion abilities, accompanied by increased expression of epithelial mesenchymal transition (EMT)-related transcription factors (ZEB1/Snail/Twist). Moreover, 5637R cells showed enhanced cancer stem cell (CSC)-like characteristics with activated KMT1A-GATA3-STAT3 circuit, a newly reported self-renewal pathway of human bladder cancer stem cell (BCSC). Combined with Kaplan-Meier's analysis, we speculated that GATA3/MMP9/STAT3 could be an effective molecular panel predicting poor prognosis of BCa. In order to enhance the sensitivity of resistant cells to radiation, we introduced ERK inhibitor (FR 180204) and STAT3 inhibitor (S3I-201). However, both of them could not enhance ARR cells response to IR. On the other hand, siRNAs were respectively implemented to inhibit the expression of endogenous Beclin1 and Atg5, two important autophagy-related genes, in BCa cells, which significantly increased 5637R cells death upon taxol exposing. Similarly, chloroquine (CQ), a classic autophagy inhibitor, enhanced the cytotoxicity of taxol only on 5637R cells. CONCLUSIONS: Long-term FI treatment is an effective method to establish the ARR-phenotype BCa cell model, by enriching BCSCs and enhancing cells migration and invasion. Both inhibiting the expression of autophagy-related proteins and using autophagy inhibitor can increase the sensitivity of ARR cells to taxol, suggesting that autophagy may play an important role in ARR cells chemical tolerance.

Radiation exposure triggers the malignancy of non­small cell lung cancer cells through the activation of visfatin/Snail signaling.

It is estimated that one­half of patients with non­small cell lung cancer (NSCLC) undergo radiotherapy worldwide. However, the outcome of radiotherapy alone is not always satisfactory. The aim of the present study was to evaluate the effects of radiotherapy on the malignancy of NSCLC cells. It was demonstrated that radiation therapy could increase the migration and invasion of NSCLC cells in vitro. Moreover, the upregulation of visfatin, a 52­kDa adipokine, mediated radiation­induced cell motility. A neutralizing antibody specific for visfatin blocked radiation­induced cell migration. Radiation and visfatin induced the expression of Snail, a key molecule that regulates epithelial to mesenchymal transition in NSCLC cells. Furthermore, visfatin positively regulated the mRNA stability of Snail in NSCLC cells, but had no effect on its protein degradation. This may be explained by visfatin­mediated downregulation of microRNA (miR)­34a, which was shown to bind the 3' untranslated region of Snail mRNA to promote its decay. Collectively, these findings suggested that radiation could induce cell motility in NSCLC cells through visfatin/Snail signaling.

Keap1-Nrf2 Pathway Regulates ALDH and Contributes to Radioresistance in Breast Cancer Stem Cells.

Tumor recurrence after radiotherapy due to the presence of breast cancer stem cells (BCSCs) is a clinical challenge, and the mechanism remains unclear. Low levels of ROS and enhanced antioxidant defenses are shown to contribute to increasing radioresistance. However, the role of Nrf2-Keap1-Bach1 signaling in the radioresistance of BCSCs remains elusive. Fractionated radiation increased the percentage of the ALDH-expressing subpopulation and their sphere formation ability, promoted mesenchymal-to-epithelial transition and enhanced radioresistance in BCSCs. Radiation activated Nrf2 via Keap1 silencing and enhanced the tumor-initiating capability of BCSCs. Furthermore, knockdown of Nrf2 suppressed ALDH+ population and stem cell markers, reduced radioresistance by decreasing clonogenicity and blocked the tumorigenic ability in immunocompromised mice. An underlying mechanism of Keap1 silencing could be via miR200a, as we observed a significant increase in its expression, and the promoter methylation of Keap1 or GSK-3ß did not change. Our data demonstrate that ALDH+ BCSC population contributes to breast tumor radioresistance via the Nrf2-Keap1 pathway, and targeting this cell population with miR200a could be beneficial but warrants detailed studies. Our results support the notion that Nrf2-Keap1 signaling controls mesenchymal-epithelial plasticity, regulates tumor-initiating ability and promotes the radioresistance of BCSCs.

Radiation-induced H3K9 methylation on E-cadherin promoter mediated by ROS/Snail axis : Role of G9a signaling during lung epithelial-mesenchymal transition.

Lung cancer patients who have undergone radiotherapy developed severe complications such as pneumonitis and fibrosis. Upon irradiation, epithelial cells acquire mesenchymal phenotype via a process called epithelial to mesenchymal transition (EMT), which plays a vital role in organ fibrosis. Several mechanisms have been studied on EMT, however, the correlation between radiation-induced EMT and epigenetic changes are not well known. In the present study, we investigated the role of histone methyltransferase G9a on radiation-induced EMT signaling. There was an increase in total global histone methylation level in irradiated epithelial cells. Western blot analysis on irradiated cells showed an increased expression of H3K9me2/3. The pre-treatment of G9a inhibitor enhanced E-cadherin expression and decreased the mesenchymal markers like N-cadherin, vimentin in the radiated group. Surprisingly, radiation-induced ROS generation and pERK1/2 levels were also inhibited by G9a inhibitor BIX01294, which is showing its antioxidant potential. The ChIP-qPCR analysis on the E-cadherin promoter suggested that G9a and Snail might have formed complex to enrich suppressive marker H3K9me2/3. On the whole, our present study suggested that 1] ROS could modify H3K9 methylation via G9a and promote radiation-induced lung EMT in Beas2B and A549 cells 2] E-cadherin promoter enrichment with heterochromatin mark H3K9me2 expression upon irradiation could be modified by regulating G9a methyltransferase.

MiRNA-155-5p inhibits epithelium-to-mesenchymal transition (EMT) by targeting GSK-3ß during radiation-induced pulmonary fibrosis.

Radiation-induced pulmonary fibrosis (RIPF) is a major lung complication in using radiotherapy to treat thoracic diseases. MicroRNAs (miRNAs) are reported to be the therapeutic targets for many diseases. However, the miRNAs involved in the pathogenesis of RIPF are rarely studied as potential therapeutic targets. Alveolar epithelial cells participate in RIPF formation by undergoing epithelial-mesenchymal transition (EMT). Here we demonstrated the critical role of miR-155-5p in radiation-induced EMT and RIPF. Using the previously established EMT cell model, we found that miR-155-5p was significantly down-regulated through high-throughput sequencing. Irradiation could decrease the expression of miR-155-5p in intro and in vivo, and it was inversely correlated to RIPF formation. Ectopic miR-155-5p expression inhibited radiation-induced-EMT in vitro and in vivo. Knockdown of glycogen synthase kinase-3ß (GSK-3ß), the functional target of miR-155-5p, reversed the induction of EMT and enhanced the phosphorylation of p65, a subunit of NF-κB, which were mediated by the down-regulation of miR-155-5p. Moreover, our finding demonstrated that ectopic miR-155-5p expression alleviated RIPF in mice by the GSK-3ß/NF-κB pathway. Thus, radiation downregulates miR-155-5p in alveolar epithelial cells that induces EMT, which contributes to RIPF using GSK-3ß/NF-κB pathway. Our observation provides further understanding on the regulation of RIPF and identifies potential therapeutic targets.