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The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs by various mechanisms and appears to be almost inevitable, even in patients with lung cancer who initially respond well to EGFR-TKIs. Consequently, considerable efforts have been made to develop more effective EGFR-TKIs. Therefore, an understanding of the mechanisms behind TKI resistance is essential for improving EGFR-TKI therapeutic efficacy in non-small cell lung cancer (NSCLC) patients. In this study, we discovered that overexpression of antioxidant-responsive element (ARE)-containing Nrf2 target genes by increased transactivation of Nrf2 occurred because of an acquired Keap1 mutation in the gefitinib-resistant (GR) NSCLC cell line we established. These GR cells also acquired cross-resistance to the irreversible EGFR-TKIs, afatinib and osimertinib, and showed increased viability, invasiveness, proliferation, and tumorigenicity both in vitro and in vivo. These results were confirmed by the fact that inhibition of Nrf2 activity, either by treatment with brusatol or by inducing expression of exogenously introduced wild-type Keap1, suppressed tumor cell proliferation and tumorigenicity in vitro and in vivo. Our data suggest that disruption of the Keap1-Nrf2 pathway is one of the mechanisms by which EGFR-TKI resistance occurs, a fact that must be considered when treating patients with EGFR-TKI.-Park, S.-H., Kim, J. H., Ko, E., Kim, J.-Y., Park, M.-J., Kim, M. J., Seo, H., Li, S., Lee, J.-Y. Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.
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[This corrects the article DOI: 10.1371/journal.pbio.1002152.].
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Epidermal growth factor receptor variant III (EGFRvIII) has been associated with glioma stemness, but the direct molecular mechanism linking the two is largely unknown. Here, we show that EGFRvIII induces the expression and secretion of pigment epithelium-derived factor (PEDF) via activation of signal transducer and activator of transcription 3 (STAT3), thereby promoting self-renewal and tumor progression of glioma stem cells (GSCs). Mechanistically, PEDF sustained GSC self-renewal by Notch1 cleavage, and the generated intracellular domain of Notch1 (NICD) induced the expression of Sox2 through interaction with its promoter region. Furthermore, a subpopulation with high levels of PEDF was capable of infiltration along corpus callosum. Inhibition of PEDF diminished GSC self-renewal and increased survival of orthotopic tumor-bearing mice. Together, these data indicate the novel role of PEDF as a key regulator of GSC and suggest clinical implications.
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Receptores ErbB/metabolismo , Proteínas do Olho/metabolismo , Glioma/etiologia , Células-Tronco Neoplásicas/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Animais , Comunicação Autócrina , Progressão da Doença , Feminino , Glioma/metabolismo , Glioma/mortalidade , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Glioblastoma, the most common primary brain tumor in adults, is an incurable malignancy with poor short-term survival and is typically treated with radiotherapy along with temozolomide. While the development of tumor-treating fields (TTFields), electric fields with alternating low and intermediate intensity has facilitated glioblastoma treatment, clinical outcomes of TTFields are reportedly inconsistent. However, combinatorial administration of chemotherapy with TTFields has proven effective for glioblastoma patients. Sorafenib, an anti-proliferative and apoptogenic agent, is used as first-line treatment for glioblastoma. This study aimed to investigate the effect of sorafenib on TTFields-induced anti-tumor and anti-angiogenesis responses in glioblastoma cells in vitro and in vivo. Sorafenib sensitized glioblastoma cells to TTFields, as evident from significantly decreased post-TTFields cell viability (p < 0.05), and combinatorial treatment with sorafenib and TTFields accelerated apoptosis via reactive oxygen species (ROS) generation, as evident from Poly (ADP-ribose) polymerase (PARP) cleavage. Furthermore, use of sorafenib plus TTFields increased autophagy, as evident from LC3 upregulation and autophagic vacuole formation. Cell cycle markers accumulated, and cells underwent a G2/M arrest, with an increased G0/G1 cell ratio. In addition, the combinatorial treatment significantly inhibited tumor cell motility and invasiveness, and angiogenesis. Our results suggest that combination therapy with sorafenib and TTFields is slightly better than each individual therapy and could potentially be used to treat glioblastoma in clinic, which requires further studies.
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Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/terapia , Terapia por Estimulação Elétrica/métodos , Glioblastoma/terapia , Sorafenibe/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Autofagia , Neoplasias Encefálicas/tratamento farmacológico , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Terapia Combinada/métodos , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sorafenibe/administração & dosagemRESUMO
Radiation therapy (RT) provides therapeutic benefits for patients with glioblastoma (GBM), but inevitably induces poorly understood global changes in GBM and its microenvironment (TME) that promote radio-resistance and recurrence. Through a cell surface marker screen, we identified that CD142 (tissue factor or F3) is robustly induced in the senescence-associated ß-galactosidase (SA-ßGal)-positive GBM cells after irradiation. F3 promotes clonal expansion of irradiated SA-ßGal+ GBM cells and orchestrates oncogenic TME remodeling by activating both tumor-autonomous signaling and extrinsic coagulation pathways. Intratumoral F3 signaling induces a mesenchymal-like cell state transition and elevated chemokine secretion. Simultaneously, F3-mediated focal hypercoagulation states lead to activation of tumor-associated macrophages (TAMs) and extracellular matrix (ECM) remodeling. A newly developed F3-targeting agent potently inhibits the aforementioned oncogenic events and impedes tumor relapse in vivo. These findings support F3 as a critical regulator for therapeutic resistance and oncogenic senescence in GBM, opening potential therapeutic avenues.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/radioterapia , Tromboplastina , Linhagem Celular Tumoral , Recidiva Local de Neoplasia , Transdução de Sinais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/metabolismo , Microambiente TumoralRESUMO
Liver cancer is a common malignancy worldwide, with a poor prognosis and a high recurrence rate despite the available treatment methodologies. Tumor-treating fields (TTFields) have shown good preclinical and clinical results for improving the prognosis of patients with glioblastoma and malignant pleural mesothelioma. However, there is minimal evidence for the effect of TTFields on other cancer types. Thus, the present study aimed to investigate the therapeutic efficacy of TTFields in an in vitro model, and to further elucidate the underlying mechanisms. In the present study, two hepatocellular carcinoma (HCC) cell lines (Hep3B and HepG2) were treated with TTFields (intensity, 1.0 V/cm; frequency, 150 kHz) in order to determine the potential antitumor effects of this approach. TTFields significantly inhibited the proliferation and viability of HCC cell lines, as measured using Trypan blue and MTT assays, as well as colony formation in three-dimensional cultures. The TTFields also significantly inhibited the migration and invasion of HCC cells in Transwell chamber and wound-healing assays. Moreover, TTFields enhanced the production of reactive oxygen species in the cells and increased the proportion of apoptotic cells, as evidenced by increased caspase-3 activity, as well as PARP cleavage in western blotting experiments. All of these effects were increased following the application of TTFields in combination with the multi-kinase inhibitor sorafenib, which demonstrated a synergistic effect. Thus, to the best of our knowledge, these results demonstrate for the first time the potential of TTFields in improving the sensitivity of HCC cells to sorafenib, which may lay the foundation for future clinical trials for this combination treatment strategy.
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Background: Gold nanoparticles (GNP, AuNPs) have received much attention as a tool to improve the therapeutic index of radiation therapy. This study aimed to evaluate the normal in vitro toxicity of AuNPs at kilovoltage energies on hepatocytes to provide scientific support for using AuNPs with radiotherapy. Methods: Using the same treatment protocol applied to tumor cell lines, hepatocytes were exposed to AuNPs and/or radiation at various time points. Results: The combination of X-ray irradiation and AuNPs did not have any significant effect on cell survival and apoptosis in normal hepatocytes. Furthermore, the combination treatment resulted in no or little change in the level of gamma-H2A histone family member X (γ-H2AX), a marker for DNA double-strand breaks (DSB), nor on the proportion of cells in the G2/M phase. Additionally, interleukin-8 (IL-8) secretion was measured using an enzyme-linked immunosorbent assay (ELISA) to assess its role in tumor progression and angiogenesis. The combination of irradiation and AuNP treatment revealed no significant reduction in hepatocyte viability, proliferation, or secretory capacity compared to cells receiving either treatment alone. According to this study, AuNPs in combination with radiation do have potentially in the treatment of hepatocellular carcinoma (HCC) with no critical cytotoxicity on normal tissue. Conclusions: Therefore, it is postulated that radiation and AuNPs are an effective combination therapy against HCC with no little cytotoxic effects on normal tissue, a hypothesis which warrants further investigation in in vivo, as well as in in vitro.
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Drug repositioning is an alternative process for drug development in cancer. Specifically, it is a strategy for the discovery of new antitumor drugs by screening previously approved clinical drugs. On the basis of this strategy, aripiprazole, an antipsychotic drug, was found to have anticancer activity. In this study, we investigated the radiosensitizing effects of aripiprazole on head and neck cancer cells at sublethal doses of ionizing radiation (IR) in vitro and in vivo. Treatment with aripiprazole suppressed the growth of head and neck cancer cells in a concentration-dependent manner, as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Intriguingly, aripiprazole significantly enhanced the sensitivity of these cells to the IC50 dose of IR. The combination of aripiprazole with IR synergistically increased annexin and propidium iodide double-positive and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cell populations, and induced cleaved poly(ADP-ribose) polymerase and caspase-3 expression, indicating the induction of apoptosis in these cells. Aripiprazole and IR-induced apoptosis were accompanied by an increase in reactive oxygen species and was almost completely suppressed by the addition of the antioxidant, N-acetylcysteine. Finally, aripiprazole greatly sensitized xenograft tumors to IR at doses that did not affect tumor growth. Taken together, these results suggest that aripiprazole could be considered a potent radiosensitizer for head and neck cancer.
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Neoplasias de Cabeça e Pescoço , Aripiprazol/farmacologia , Aripiprazol/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Marcação In Situ das Extremidades Cortadas , Radiação Ionizante , Espécies Reativas de Oxigênio/metabolismoRESUMO
The primary motivation of this investigative study is trying to find an alternative treatment that can be used to slow down or treat glioblastoma due to the witnessed toxic side effects of the current drugs coupled with limited effectiveness in overall treatment. Consequently, a Chinese plant extract emodin proves to play a critical role in this investigative study since results from the Western blot and the other accompanying assays for anti-cancer effects indicate that it cannot work a lot to suppress cell migration and possible invasion, but rather emodin can be combined with radiation to give desired outcomes. Our result shows that the kind of radiation which acts well with emodin is neutron radiation rather than gamma radiation. Emodin significantly enhanced the radiosensitivity of LN18 and LN428 cells to γ-rays through MTT assay and cell counting. Accordingly, exposure to neutron radiation in the presence of emodin induced apoptotic cell death and autophagic cell death to a significantly higher extent, and suppressed cell migration and invasiveness more robustly. These effects are presumably due to the ability of emodin to amplify the effective dose from neutron radiation more efficiently. Thus, the study below is one such trial towards new interventional discovery and development in relation to glioblastoma treatment.
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Emodina , Emodina/farmacologia , Emodina/uso terapêuticoRESUMO
Glioblastoma is a deadly cancer tumor in the brain and has a survival rate of about 15 months. Despite the high mortality rate, temozolomide has proven to increase the survival rate of patients when combined with radiotherapy. However, its effects may be limited because some patients develop therapeutic resistance. Curcumin has proven to be a cancer treatment due to its broad anticancer spectrum, high efficiency and low toxic level. Additionally, curcumin significantly enhanced radiation efficacy under high and low Linear Energy Transfer (LET) radiation conditions in vitro. In combination with radiation, curcumin increased the cell population in the sub-G1 phase and the reactive oxygen species (ROS) level, ultimately increasing GBM cellular apoptosis. The radiosensitizing effects of curcumin are much higher in neutron (high LET)-irradiated cell lines than in γ (low LET)-irradiated cell lines. Curcumin plus neutron combination significantly inhibited cell invasion compared with that of single treatment or curcumin combined γ-ray treatment. Curcumin enhances the radiosensitivity of Glioblastoma (GBM), suggesting it may have clinical utility in combination cancer treatment with neutron high-LET radiation.
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Curcumina , Glioblastoma , Apoptose , Linhagem Celular Tumoral , Curcumina/farmacologia , Glioblastoma/patologia , Humanos , Transferência Linear de Energia , Tolerância a RadiaçãoRESUMO
Lung cancer is one of the most common reasons for cancer-induced mortality across the globe, despite major advancements in the treatment strategies including radiotherapy and chemotherapy. Existing reports suggest that CXCR4 is frequently expressed by malignant tumor and is imperative for vascularization, tumor growth, cell migration, and metastasis pertaining to poor prognosis. In this study, we infer that CXCR4 confers resistance to ionizing radiation (IR) in nonsmall cell lung cancer (NSCLC) cells. Further, on the basis of colony forming ability, one finds that drug-resistant A549/GR cells with improved CXCR4 expression exhibited more resistance to IR than A549 cells evidenced along with a reduction in the formation of γ-H2AX foci after IR. Transfection of shRNA against CXCR4 or treatment of pharmacological inhibitor (AMD3100) both led to sensitization of A549/GR cells towards IR. Conversely, the overexpression of CXCR4 in A549 and H460 cell lines was found to improve clonogenic survival, and reduce the formation of γ-H2AX foci after IR. CXCR4 expression was further correlated with STAT3 activation, and suppression of STAT3 activity with siSTAT3 or a specific inhibitor (WP1066) significantly stymied the colony-forming ability and increased γ-H2AX foci formation in A549/GR cells, indicating that CXCR4-mediated STAT3 signaling plays an important role for IR resistance in NSCLC cells. Finally, CXCR4/STAT3 signaling was mediated with the upregulation of Slug and downregulation of the same with siRNA, which heightened IR sensitivity in NSCLC cells. Our data collectively suggests that CXCR4/STAT3/Slug axis is paramount for IR resistance of NSCLC cells, and can be regarded as a therapeutic target to enhance the IR sensitivity of this devastating cancer.
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Receptores CXCR4/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Prognóstico , TransfecçãoRESUMO
PARK7 is involved in many key cellular processes, including cell proliferation, transcriptional regulation, cellular differentiation, oxidative stress protection, and mitochondrial function maintenance. Deregulation of PARK7 has been implicated in the pathogenesis of various human diseases, including cancer. Here, we aimed to clarify the effect of PARK7 on stemness and radioresistance of glioblastoma stem cells (GSCs). Serum differentiation and magnetic cell sorting of GSCs revealed that PARK7 was preferentially expressed in GSCs rather than differentiated GSCs. Immunohistochemical staining showed enhanced expression of PARK7 in glioma tissues compared to that in normal brain tissues. shRNA-mediated knockdown of PARK7 inhibited the self-renewal activity of GSCs in vitro, as evidenced by the results of neurosphere formation, limiting dilution, and soft-agar clonogenic assays. In addition, PARK7 knockdown suppressed GSC invasion and enhanced GSC sensitivity to ionizing radiation (IR). PARK7 knockdown suppressed expression of GSC signatures including nestin, epidermal growth factor receptor variant III (EGFRvIII), SOX2, NOTCH1, and OCT4. Contrarily, overexpression of PARK7 in CD133- non-GSCs increased self-renewal activities, migration, and IR resistance, and rescued the reduction of GSC factors under shPARK7-transfected and serum-differentiation conditions. Intriguingly, PARK7 acted as a co-chaperone of HSP90 by binding to it, protecting EGFRvIII from proteasomal degradation. Knockdown of PARK7 increased the production of reactive oxygen species, inducing partial apoptosis and enhancing IR sensitivity in GSCs. Finally, PARK7 knockdown increased mouse survival and IR sensitivity in vivo. Based on these data, we propose that PARK7 plays a pivotal role in the maintenance of stemness and therapeutic resistance in GSCs.
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Receptores ErbB/metabolismo , Glioblastoma/enzimologia , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/enzimologia , Proteína Desglicase DJ-1/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/genética , Glioblastoma/patologia , Humanos , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia , Proteína Desglicase DJ-1/genéticaRESUMO
Glioblastoma multiforme (GBM) or glioblastoma is the most deadly malignant brain tumor in adults. GBM is difficult to treat mainly due to the presence of glioblastoma stem cells (GSCs). Epidermal growth factor receptor variant III (EGFRvIII) has been linked to stemness and malignancy of GSCs; however, the regulatory mechanism of EGFRvIII is largely unknown. Here, we demonstrated that Anoctamin-1 (ANO1), a Ca2+-activated Cl- channel, interacts with EGFRvIII, increases its protein stability, and supports the maintenance of stemness and tumor progression in GSCs. Specifically, shRNA-mediated knockdown and pharmacological inhibition of ANO1 suppressed the self-renewal, invasion activities, and expression of EGFRvIII and related stem cell factors, including NOTCH1, nestin, and SOX2 in GSCs. Conversely, ANO1 overexpression enhanced the above phenomena. Mechanistically, ANO1 protected EGFRvIII from proteasomal degradation by directly binding to it. ANO1 knockdown significantly increased survival in mice and strongly suppressed local invasion of GSCs in an in vivo intracranial mouse model. Collectively, these results suggest that ANO1 plays a crucial role in the maintenance of stemness and invasiveness of GSCs by regulating the expression of EGFRvIII and related signaling molecules, and can be considered a promising therapeutic target for GBM treatment.
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Anoctamina-1/genética , Receptores ErbB/genética , Glioblastoma/genética , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Nestina/genética , Estabilidade Proteica , RNA Interferente Pequeno/genética , Receptor Notch1/genética , Fatores de Transcrição SOXB1/genéticaRESUMO
A newly diagnosed or recurrent Glioblastoma multiforme (GBM) can be treated with Tumor-treating fields (TTFields), an emerging type of alternative electric field-based therapy using low-intensity electric fields. TTFields have a penchant to arrest mitosis, eventually leading to apoptosis. Therefore, it is regarded as a potential anticancer therapy. However, in this study, we confirmed the combined efficacy of sorafenib and TTFields to improve the treatment efficiency of malignant GBM. Experimentation revealed the ability of sorafenib to decrease the signal transducer and activator of transcription 3 (STAT3) and this inhibition increased the sensitivity of TTFields in preventing tumor expansion. It was found that both combinatorial as well as monotherapy aimed to inhibit or reduce the level of STAT3, but the extent was different and based upon the reaction conditions. This drug is also capable of arresting multiple kinase pathways along with STAT3-related proteins (Mcl-1 and Survivin). STAT3 silencing can also be accomplished by RNA interference and can increase the TTFields-sensitizing effect of sorafenib. If the effects are reversed and gene regulating STAT3 is expressed more, it annihilates the effects of treatment. Moreover, sorafenib plus TTFields significantly inhibited xenograft tumor growth and combinatorial treatment reduced STAT3 expression more effectively in vivo. These in vitro and in vivo results indicate that sorafenib tends to sensitize GBM cells to TTFields-induced apoptosis by inhibiting STAT3.
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Carbon ion radiotherapy (CIRT) is more effective than conventional photon beam radiotherapy in treating osteosarcoma (OSA); however, the outcomes of CIRT alone are still unsatisfactory. In this study, we aimed to investigate whether miR-29b acts as a radiosensitizer for CIRT. The OSA cell lines U2OS and KHOS were treated with carbon ion beam alone, γ-ray irradiation alone, or in combination with an miR-29b mimic. OSA cell death as well as invasive and migratory abilities were analyzed through viability, colony formation, Transwell, and apoptosis assays. miR-29 expression was downregulated in OSA tissues compared to that in normal tissues and was associated with metastasis and relapse in patients with OSA. Further, miR-29b was found to directly target the transcription factor Sp1 and suppress the activation of the phosphatase and tensin homolog (PTEN)-AKT pathway. Conversely, Sp1 was found to attenuate the inhibitory effects of miR-29b in OSA cells. When used in combination with miR-29b mimic, carbon ion beam markedly inhibited invasion, migration, and proliferation of OSA cells and promoted apoptosis by inhibiting AKT phosphorylation in a Sp1/PTEN-mediated manner. Taken together, miR-29b mimic improved the radiosensitivity of OSA cells via the PTEN-AKT-Sp1 signaling pathway, presenting a novel strategy for the development of carbon ion beam combination therapy.
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Glioblastomas (GBMs) are characterized by four subtypes, proneural (PN), neural, classical, and mesenchymal (MES) GBMs, and they all have distinct activated signaling pathways. Among the subtypes, PN and MES GBMs show mutually exclusive genetic signatures, and the MES phenotype is, in general, believed to be associated with more aggressive features of GBM: tumor recurrence and drug resistance. Therefore, targeting MES GBMs would improve the overall prognosis of patients with fatal tumors. In this study, we propose peroxisome proliferator-activated receptor gamma (PPARγ) as a potential diagnostic and prognostic biomarker as well as therapeutic target for MES GBM; we used multiple approaches to assess PPARγ, including biostatistics analysis and assessment of preclinical studies. First, we found that PPARγ was exclusively expressed in MES glioblastoma stem cells (GSCs), and ligand activation of endogenous PPARγ suppressed cell growth and stemness in MES GSCs. Further in vivo studies involving orthotopic and heterotopic xenograft mouse models confirmed the therapeutic efficacy of targeting PPARγ; compared to control mice, those that received ligand treatment exhibited longer survival as well as decreased tumor burden. Mechanistically, PPARγ activation suppressed proneural-mesenchymal transition (PMT) by inhibiting the STAT3 signaling pathway. Biostatistical analysis using The Cancer Genomics Atlas (TCGA, n = 206) and REMBRANDT (n = 329) revealed that PPARγ upregulation is linked to poor overall survival and disease-free survival of GBM patients. Analysis was performed on prospective (n = 2) and retrospective (n = 6) GBM patient tissues, and we finally confirmed that PPARγ expression was distinctly upregulated in MES GBM. Collectively, this study provides insight into PPARγ as a potential therapeutic target for patients with MES GBM.
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Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Glioblastoma/metabolismo , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/diagnóstico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Camundongos , PPAR gama/genética , Prognóstico , RNA Interferente Pequeno/genética , Transdução de Sinais , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor-treating fields (TTFs) - a type of electromagnetic field-based therapy using low-intensity electrical fields - has recently been characterized as a potential anticancer therapy for glioblastoma multiforme (GBM). However, the molecular mechanisms involved remain poorly understood. Our results show that the activation of autophagy contributes to the TTF-induced anti-GBM activity in vitro or in vivo and GBM patient stem cells or primary in vivo culture systems. TTF-treatment upregulated several autophagy-related genes (~2-fold) and induced cytomorphological changes. TTF-induced autophagy in GBM was associated with decreased Akt2 expression, not Akt1 or Akt3, via the mTOR/p70S6K pathway. An Affymetrix GeneChip miRNA 4.0 Array analysis revealed that TTFs altered the expression of many microRNAs (miRNAs). TTF-induced autophagy upregulated miR-29b, which subsequently suppressed the Akt signaling pathway. A luciferase reporter assay confirmed that TTFs induced miR-29b to target Akt2, negatively affecting Akt2 expression thereby triggering autophagy. TTF-induced autophagy suppressed tumor growth in GBM mouse models subjected to TTFs as determined by positron emission tomography and computed tomography (PET-CT). GBM patient stem cells and a primary in vivo culture system with high Akt2 levels also showed TTF-induced inhibition. Taken together, our results identified autophagy as a critical cell death pathway triggered by TTFs in GBM and indicate that TTF is a potential treatment option for GBM.
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Autofagia/efeitos da radiação , Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , MicroRNAs/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Campos Eletromagnéticos , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite the importance of radiation therapy, there are few radiation-related markers available for use in clinical practice. A larger catalog of such biomarkers is required to help clinicians decide when radiotherapy should be replaced with a patient-specific treatment. Arachidonate 15-lipoxygenase (15-LOX-1) enzyme is involved in polyunsaturated fatty acid metabolism. When colorectal cancer (CRC) cells were exposed to radiation, 15-LOX-1 was upregulated. To verify whether 15-LOX-1 protects against or induces DNA damage, we irradiated sh15-LOX-1 stable cells. We found that low 15-LOX-1 is correlated with radioresistance in CRC cells. These data suggest that the presence of 15-LOX-1 can be used as a marker for radiation-induced DNA damage. Consistent with this observation, gene-set-enrichment analysis based on microarray experiments showed that UV_RESPONSE was decreased in sh15-LOX-1 cells compared to shCon cells. Moreover, we discovered that the expression of the histone H2A variant macroH2A2 was sevenfold lower in sh15-LOX-1 cells. Overall, our findings present mechanistic evidence that macroH2A2 is transcriptionally regulated by 15-LOX-1 and suppresses the DNA damage response in irradiated cells by delaying H2AX activation.
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Breast cancer stem cells (BCSCs) are a small subpopulation of breast cancer cells that have been proposed to be a primary cause of failure of therapies, including ionizing radiation (IR). Their embryonic stem-like signature is associated with poor clinical outcome. In the present study, the function of octamer-binding transcription factor 4 (Oct4), an embryonic stem cell factor, in the resistance of BCSCs to IR was investigated. Mammosphere cells exhibited increased expression of stemness-associated genes, including Oct4 and sexdetermining region Ybox 2 (Sox2), and were more resistant to IR compared with serum-cultured monolayer cells. IRresistant MCF7 cells also exhibited significantly increased expression of Oct4. To investigate the possible involvement of Oct4 in IR resistance of breast cancer cells, cells were transfected with Oct4. Ectopic expression of Oct4 increased the clonogenic survival of MCF7 cells following IR, which was reversed by treatment with small interfering RNA (siRNA) targeting Oct4. Oct4 expression decreased phosphorylated histone H2AX (γ-H2AX) focus formation and suppressed IRinduced premature senescence in these cells. Mammosphere, IRresistant and Oct4overexpressing MCF7 cells exhibited enhanced phosphorylation of signal transducer and activation of transcription 3 (STAT3) (Tyr705) and inhibitor of nuclear factor κB (NFκB), and blockade of these pathways with siRNA against STAT3 and/or specific inhibitors of STAT3 and NFκB significantly increased IRinduced senescence. Secretome analysis revealed that Oct4 upregulated interleukin 24 (IL24) expression through STAT3 and NFκB signaling, and siRNA against IL24 increased IRinduced senescence, whereas recombinant human IL24 suppressed it. The results of the present study indicated that Oct4 confers IR resistance on breast cancer cells by suppressing IRinduced premature senescence through STAT3- and NFκB-mediated IL24 production.
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Neoplasias da Mama/radioterapia , Interleucinas/genética , Fator 3 de Transcrição de Octâmero/genética , Tolerância a Radiação/genética , Fator de Transcrição STAT3/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Senescência Celular/genética , Senescência Celular/efeitos da radiação , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Técnicas de Inativação de Genes , Humanos , Células MCF-7 , NF-kappa B/genética , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/efeitos da radiação , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Fatores de Transcrição SOXB1/genética , Transdução de Sinais/genéticaRESUMO
Cancer stem cells, a small subpopulation of cells with stem cell-like characteristics found within most solid tumors, are widely reported to be responsible for the malignancy of aggressive cancer cells, and targeting these cells presents a sound therapeutic strategy for reducing the risk of tumor relapse. In the present study, we examined the effects of an extract of Saccharina japonica (ESJ) on glioblastoma stem cells (GSCs). Saccharina japonica is a member of the Phaeophyceae (brown algae) family, which displays biological activities, including antitumor effects. ESJ inhibited the sphere-forming ability of GSCs in vitro as evidenced by neurosphere formation and limiting dilution assays. Treatment with ESJ partially induced apoptosis, reduced cell invasiveness, and sensitized GSCs to ionizing radiation. In addition, ESJ inhibited the maintenance of stemness in GSCs by suppressing the expression of epidermal growth factor receptor (EGFR)/EGFR variant III (EGFRvIII) and Notch intracellular domain. Intriguingly, the observed ESJ-induced suppression also appeared to induce the proteasomal degradation of EGFR/EGFRvIII. Our results indicate that ESJ could be considered a potent therapeutic adjuvant that targets GSCs.