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Modulation of Sp1/RCP, NF-B and p53 DNA binding after IR in human U1-Mel cells. Nuclear extracts from irradiated or nonirradiated confluent U1-Mel cells were examined for activation of transcription factor binding using DNA band shift assays as described in Materials and Methods. A) Time course (in hours) of proteins binding to the Sp1 consensus site before and after 450 cGy; B) effect of various doses of IR (0 –1200 cGy) on proteins binding to the Sp1 consensus site at 2 h after IR; C) time course (in h) of NF-B DNA binding activity after 450 cGy; D) effect of various doses of IR (0 –1200 cGy) on NF-B DNA binding at 12 h postirradiation; E) time course and dose response of nuclear p53 DNA binding activity. Addition of p53 antibody (E) blocked IR-induced DNA binding . Equal amounts of protein were loaded in each lane as determined by Bradford assays. In addition, TF binding to AP-1 and Oct-1 oligos remained unchanged at various times and doses after IR (data not shown), as described previously (5). Gels are representative of experiments performed at least three times. 100 cGy 1.0 Gy.
Source publication
Regulation of transcriptional responses in growth-arrested human cells under conditions that promote potentially lethal damage repair after ionizing radiation (IR) is poorly understood. Sp1/retinoblastoma control protein (RCP) DNA binding increased within 30 min and peaked at 2-4 h after IR (450-600 cGy) in confluent radioresistant human malignant...
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Citations
... The balance between the expression of NF-κB and p53, a guardian of the genome, has been claimed to determine the behavior of cancer cells toward proliferation or apoptosis [23]. Having established the impact of the SP/NK1R axis on the expression levels of NF-κB, we also determined the influence of this pathway on the expression of p53 and one of its downstream targets p21. ...
... The equilibrium between NF-κB and p53 plays a critical role in regulating the proliferation or survival of the cells [23]. While p53 regulates the progression of the cell cycle through modulating the expression of cyclin-dependent kinase (CDK) inhibitors such as p21 [34], the activation of NF-κB is coupled with the transcription of the genes involved in the prevention of apoptosis [35]. ...
Background
Numerous molecules have been introduced to participate in the formation of breast cancer, the most common malignancy in women. Among them, neuropeptide substance P (SP) and its related receptor neurokinin-1 receptor (NK1R) have attracted unprecedented attention in tumorigenesis processes. In this study, we investigated the effect of the SP/NK1R pathway on the induction of oxidative stress in breast cancer and examine the therapeutic potential of NK1R inhibition in this malignancy.
Methods
MCF-7 cells were treated with varying concentrations of SP and aprepitant, an FDA-approved NK1R antagonist, either as a single drug or in a combined modality. Resazurin assay was used to evaluate the anti-cancer ability of aprepitant. The alteration in the intracellular levels of reactive oxygen species (ROS) and gene expression were determined using ROS assay and the qRT-PCR analysis, respectively.
Results
The stimulation of the SP/NK1R axis in the MCF-7 cells was coupled with the accumulation of ROS as well as upregulation of NF-κB and its related pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and IL-6. In contrast, the suppression of NK1R by aprepitant halted the viability of MCF-7 cells, at least partly due to p53-mediated upregulation of p21. Moreover, aprepitant attenuated the oncogenic properties of SP by preventing the oxidative property of this neuropeptide.
Conclusion
Overall, our results suggest that the SP/NK1R pathway might play a critical role in breast cancer pathogenesis, probably through inducing ROS/NF-κB-mediated inflammatory responses. Moreover, it seems that blockage of the axis has promising therapeutic value against breast cancer cells.
Graphical Abstract
Schematic representation proposed for the plausible mechanism by which the stimulation of the SP/NK1R might induce oxidative stress in breast cancer-derived MCF-7 cells. Once SP interacts with NK1R, this signaling axis could disturb the balance between the expression of p53 and NF-κB, an event that leads to the accumulation of ROS within MCF-7 cells. The produced ROS, in turn, elevates the expression of pro-inflammatory cytokines (TNF-α and IL-6) and downregulates the expression of p21. On the other hand, aprepitant, an antagonist of NK1R, could reduce the survival of proliferative capacity of MCF-7 cells by decreasing the intracellular levels of ROS and p53-mediated up-regulation of p21. Along with the effect on p53, aprepitant could also reduce the expression of NF-κB and its related pro-inflammatory cytokines.
... The activation of NF-κB could occur in response to DNA damaging agents and provoke multiple radioresistance signals, which attenuate the lethal effects of radiation. Inhibition of NF-κB was proved to be an effective strategy to enhance tumor radiosensitivity [27,28]. It can be inferred that TMEM59L might enhance radiosensitivity by inhibiting PI3K/AKT and NF-κB activation. ...
Radiotherapy is one of the cornerstone of the glioblastoma treatment paradigm. However, the resistance of tumor cells to radiation results in poor survival. The mechanism of radioresistance has not been fully elucidated. This study aimed to screen the differential expressed genes related with radiosensitivity. The differentially expressed genes were screened based on RNA sequencing in 15 pairs of primary and recurrent glioblastoma that have undergone radiotherapy. Candidate genes were validated in 226 primary and 134 recurrent glioblastoma (GBM) obtained from the Chinese Glioma Genome Atlas (CGGA) database. RNA and protein expression were verified by Quantitative Real-time PCR (qPCR) and western blot in irradiated GBM cell lines. The candidate gene was investigated to explore the relationship between mRNA levels and clinical characteristics in the CGGA and The Cancer Genome Atlas dataset. Kaplan–Meier survival analysis and Cox regression analysis were used for survival analysis. Gene ontology and KEGG pathway analysis were used for bioinformatics analysis. Four genes (TMEM59L, Gelsolin, ZBTB7A and ATX) were screened. TMEM59L expression was significantly elevated in recurrent glioblastoma and lower in normal brain tissue. We selected TMEM59L as the target gene for further study. The increasing of TMEM59L expression induced by radiation was confirmed by mRNA and western blot in irradiated GBM cell. Further investigation revealed that high expression of TMEM59L was enriched in IDH mutant and MGMT methylated gliomas and associated with a better prognosis. Gene ontology and KEGG pathway analysis revealed that TMEM59L was closely related to the DNA damage repair and oxidative stress respond process. We speculated that the high expression of TMEM59L might enhance radiotherapy sensitivity by increasing ROS-induced DNA damage and inhibiting DNA damage repair process.
... Moreover, inhibition of NF-κB was proved to be an effective strategy to enhance tumor radiosensitivity. 26, 27 Our result showed that TMEM59L expression was negatively related to these pathways. It can be inferred that TMEM59L might affect radiosensitivity by affecting PI3K/Akt and NF-κB inhibition. ...
Background:
Radiotherapy (RT) is one of the cornerstone of the glioblastoma (GBM) treatment paradigm. RT can significantly improve the prognosis of GBM; however, the resistance of tumor cells to radiation results in poor survival. The mechanism of radioresistance has not been fully elucidated.
Methods：
The differentially expressed genes were screened based on RNA sequencing dataset of 226 primary glioblastomas and 134 recurrent glioblastomas that have undergone radiotherapy, combined with 15 pairs of primary and recurrent GBM obtained from the Chinese Glioma Genome Atlas. Real-time quantitative PCR was performed in three irradiated GBM cell lines（U87, LN229 and U251）to verify the result. The selected gene was investigated to the relationship between mRNA levels and clinical characteristics in the CGGA and TCGA dataset. Kaplan-Meier survival analysis and Cox regression analysis were used for survival analysis. Gene ontology (GO) and KEGG pathway analysis was used for bioinformatics analysis.
Results:
TMEM59L expression was significantly elevated in recurrent GBM who have received radiotherapy. The result was validated in three irradiated GBM cells. The high-expression of TMEM59L was enriched in IDH mutant and MGMT methylated gliomas and associated with a better prognosis. Gene ontology and KEGG pathway analysis demonstrated that TMEM59L was closely associated with cell proliferation, apoptosis, drug response, and DNA damage.
Conclusion：
TMEM59L expression was increased after irradiation and resulted in longer survival. We speculated that the high expression of TMEM59L might affect radiotherapy sensitivity by contributing to reactive oxygen species and DNA damage repair.
... Expression. In addition to the NF-κB axis, which is considered a crucial aspect in cancer cell apoptosis, the p53 and its proapoptotic target gene expression could induce apoptotic cell death in several human cancer cells [36][37][38]. Toward this end, we further evaluated the relative mRNA expression of p53 and downstream proapoptotic target genes such as Bax to investigate the SP/NK1R signaling activity in the SW480 cell line. As presented in Figure 4, our findings demonstrated that the mRNA expression of p53 and Bax was not significantly different among all groups compared to control. ...
... Our results demonstrated that the treated group with AP (10 μM) decreases both expression of NF-κB p65 protein and mRNA expression of NF-κB antiapoptotic target genes such as survivin and Bcl-2 (Figures 3(a) and 3(b)). To further investigate apoptotic mechanisms by AP, recent evidence has been found that inducing apoptosis by AP is associated with increased p53 in several tumor cells [16,35,36]. In support of this idea, it is well found that the activation of PI3K/Akt is correlated with p53 pathways [16]. ...
Background:
Colorectal cancer (CRC) is recognized as one of the most common malignancies with a high mortality rate worldwide, supporting the necessity for an effective novel antitumor drug to improve current therapy's effectiveness. Substance P (SP) is the essential member of the tachykinins (TKs) family, which binds to the specific receptors, known as neurokinin-1 receptor (NK1R), exerting its multiple influences such as tumor cell proliferation, angiogenesis, and metastasis. Aprepitant, as a specific NK1R antagonist, is suggested as a novel antitumor agent, promoting apoptotic processes in tumor cells; however, the exact antitumor mechanism of aprepitant on molecular signaling in CRC is not entirely known.
Method:
The resazurin assay was conducted to assess the cytotoxic effects of aprepitant on the viability of the CRC cell line (SW480). The level of reactive oxygen species (ROS) was measured after 24-hour treatment with SP and aprepitant. PI/annexin V-FITC staining was conducted to assess apoptosis. Also, the expression of NF-κB antiapoptotic target genes and proapoptotic p53 target genes was measured by real-time- (RT-) PCR assay. Western blotting assay was performed to determine the expression of PI3k/AKT/NF-κB proteins.
Results:
We found that aprepitant stimulates apoptotic cell death and attenuates the PI3K/Akt pathway and its downstream proapoptotic target gene, including NF-κB in SW480 cells. Also, the obtained results from the quantitative RT-PCR assay showed that aprepitant could decrease the level of mRNA of NF-κB antiapoptotic target genes.
Conclusion:
Towards this end, this study suggests that SP/NK1R system plays a vital role in the development of CRC, and pharmaceutical targeting of NK1R using aprepitant might be a promising treatment against CRC.
... Moreover, NF-kB transcriptionally activates several genes associated with angiogenesis and metastasis, such as Matrix Metalloproteinase-9 (MMP-9), tissue Plasminogen Activator (tPA), ICAM-1, and Vimentin (30,98,121,129). More importantly, also, Cyclooxygenase-2 (COX-2) is regulated by NF-kB (130). ...
Currently, radiation therapy is one of the standard therapies for cancer treatment. Since the first applications, the field of radiotherapy has constantly improved, both in imaging technologies and from a dose-painting point of view. Despite this, the mechanisms of resistance are still a great problem to overcome. Therefore, a more detailed understanding of these molecular mechanisms will allow researchers to develop new therapeutic strategies to eradicate cancer effectively. This review focuses on different transcription factors activated in response to radiotherapy and, unfortunately, involved in cancer cells’ survival. In particular, ionizing radiations trigger the activation of the immune modulators STAT3 and NF-κB, which contribute to the development of radiation resistance through the up-regulation of anti-apoptotic genes, the promotion of proliferation, the alteration of the cell cycle, and the induction of genes responsible for the Epithelial to Mesenchymal Transition (EMT). Moreover, the ROS-dependent damaging effects of radiation therapy are hampered by the induction of antioxidant enzymes by NF-κB, NRF2, and HIF-1. This protective process results in a reduced effectiveness of the treatment, whose mechanism of action relies mainly on the generation of free oxygen radicals. Furthermore, the previously mentioned transcription factors are also involved in the maintenance of stemness in Cancer Stem Cells (CSCs), a subset of tumor cells that are intrinsically resistant to anti-cancer therapies. Therefore, combining standard treatments with new therapeutic strategies targeted against these transcription factors may be a promising opportunity to avoid resistance and thus tumor relapse.
... Although the suppression of the NF-κB pathway appears to be closely associated with the induction of apoptosis, the p53 overexpression also represents another attractive strategy for improving the sensitivity of cancer cells to apoptosis [40]. ...
... Our results revealed that administration of NK1R antagonist had no significant enhancing effect on transcriptional activity of pro-apoptotic target genes of p53. Results are obtained as the mean ± SD of two independent experiments (p < 0.05) Moreover, several studies have shown that the execution of apoptotic cell death is potentially dependent on a balance between the activity of p53 and NF-κB axis [40,55]. Contrary to results mentioned above, our data demonstrated that aprepitant (15 µM) did not alter the protein expression level of p53, as well as the mRNA level of pro-apoptotic p53 target genes such as p21and Bax. ...
One of the most prevalent malignancies is esophageal squamous cell carcinoma (ESCC), which is associated with high morbidity and mortality. Substance P (SP), as one of the peptides released from sensory nerves, causes the enhancement of cellular excitability through the activation of the neurokinin-1 (NK1) receptor in several human tumor cells. Aprepitant, a specific, potent, and long-acting NK1 receptor antagonist, is considered as a novel agent to inhibit proliferation and induce apoptosis in malignant cells. Since the antitumor mechanism of aprepitant in ESCC is not completely understood, we conducted this study and found that aprepitant induced growth inhibition of KYSE-30 cells and arrested cells in the G2/M phase of the cell cycle. Aprepitant also caused apoptotic cell death and inhibited activation of the PI3K/Akt axis and its downstream effectors, including NF-κB in KYSE-30 cells. Besides, quantitative real-time (qRT)-PCR analysis showed a significant down-regulation of NF-κB target genes in KYSE-30 cells, indicating a probable NF-κB-dependent mechanism involved in aprepitant cytotoxicity. Thus, the present study recommends that SP/NK1R system might, therefore, be considered as an emerging and promising therapeutic strategy against ESCC.
... The observed alterations in the expression of these genes may be through the down-regulation of NF-ĸB caused by TLR4 inhibition. The protective effect of NF-ĸB against apoptosis has been studied by a myriad of researches [47] and its inhibition has been reported to modulate different members of the BCL2 family to induce apoptosis [48]. Moreover, TLR4 also induces TNF-α transcription which leads to higher NF-кB activation [18] and subsequently resistance to apoptosis through transcription of BCLxL and BCL2 [49,50]. ...
Background:
Despite all advances in the treatment of ovarian cancer (OC), it remains the most lethal gynecological malignancy worldwide. There are growing amounts of evidence indicating the role of inflammation in initiating chemoresistance. Therefore, Toll-like receptor 4 (TLR4), a mediator of inflammation in cancer cells, may be a proper anticancer target.
Methods:
The effects of TLR4 activation by LPS was studied using MTT, colony formation, staining, scratch, and qRT-PCR assays as the first step. Then the same assays, in addition to anoikis resistance, cell cycle and annexin V/PI apoptosis tests, were used to investigate whether the inhibition of TLR4 using a small molecule inhibitor, TAK-242, could suppress the proliferation of various OC cell lines: A2780CP, 2008C13, SKOV3, and A2780S.
Results:
The activation of TLR4 using LPS showed enhanced proliferation and invasion in the TLR4-expressing cell line (SKOV3). Next, treatment with the inhibitor revealed that TAK-242 suppressed the inflammatory condition of ovarian cancer cells, as evident by the down-regulation of IL-6 gene expression. We also found that TAK-242 halted cancer cell proliferation by inducing cell cycle arrest and apoptosis through the modulation of genes involved in these processes. Given the fact that the overexpression of TLR4 contributes to drug resistance, it was tempting to investigate the effect of TAK-242 in a combined-modality strategy. Interestingly, we found enhanced cytotoxicity when TAK-242 was used in combination with doxorubicin.
Conclusion:
TAK-242 serves as an appealing therapeutic strategy in the TLR4-expressing OC cells, either in the context of monotherapy or in combination with a chemotherapeutic drug.
... In contrast to the activation of p53 that is associated with the induction of cell cycle arrest and tumor cells apoptosis, downregulation of NF-кB induces cancer cell death via the suppression of deathrepressor genes. 46 We found that while the expressions of BAD and FAS were increased, TAK-242 decreased the mRNA expression levels of the antiapoptotic target genes of NF-кB. Consistently, there have been recent studies indicating that TAK-242 is able to block UV-induced photocarcinogenesis in keratinocytes and mice through the suppression of NF-κB. ...
Increasing pieces of evidence indicate that inflammatory processes facilitate tumorigenesis; tumor cells simulate the mechanisms by which innate immune cells produce pro‐inflammatory cytokines to exploit them for their own survival and proliferation. Toll‐like receptor 4 (TLR4), which serves as one of the most well‐known receptors on the surface of the immune cells, is often expressed ectopically in the tumor cells resulting in tumor progression, invasion, and chemoresistance. In this study, we examined the anticancer effects of TAK‐242, a small molecule inhibitor of TLR4, on different breast cancer cell lines: MCF7, SKBR3, MDA‐MB‐231, and BT‐474. Our results showed that the TLR4 inhibition, as revealed by the downregulation of TLR4 downstream genes, exerted desirable cytotoxicity on the TLR4‐expressing cells, at least partly, through the downregulation of EGFR and c‐Myc genes. TAK‐242 also inhibited the proliferation of anoikis‐resistant cells and suppressed the clonal growth of the indicated cells. The results of this study propose a mechanistic pathway by which the inhibition of TLR4 using TAK‐242 could augment apoptotic cell death through the alteration of both nuclear factor‐кB‐ and p53‐related apoptosis genes in breast cancer cells, especially cells with overexpression of TLR4. Taken together, this study supports the idea that the activation of inflammatory pathways may have a crucial role in breast cancer progression and the inhibition of TLR4 using TAK‐242, either as a single agent or in combination, seems to be a novel promising strategy that could be clinically available in foreseeable future. In this study, we found that the inhibition of toll‐like receptor 4 using TAK‐242 results in the inhibition of proliferation and induction of apoptosis in the toll‐like receptor 4 (TLR4)‐expressing breast cancer cell lines.
... [7][8][9] Sp1 is also an important mediator of radiation-associated gene expression in human head and neck squamous cell carcinoma. 21 Several studies have reported that radiation can phosphorylate Sp1, which increases its transcriptional activity, 22 while Sp1 DNAbinding activity is increased in a transient and reversible manner in response to RT. 23 Evidence above further confirmed that inhibiting the expression of Sp1 may be a novel strategy for reducing radioresistance. ...
Background/aims
Radioresistance remains a significant obstacle in the therapy of cervical cancer, and the mechanism of it is still unclear. We aimed to investigate the role of specificity protein 1 (Sp1) in radioresistance of cervical cancer.
Methods
Sp1 was examined immunohistochemically on tissues from 36 human cervical cancer patients. We used RT-qPCR and Western blot to examine the expression of Sp1 in irradiated cervical cancer cell lines SiHa and HeLa. The role of Sp1 in radioresistance of cervical cancer cells was assessed by colony-formation assay and cell cycle analysis. Dual-luciferase reporter assay was performed to detect the downstream of Sp1.
Results
High Sp1 expression was positively correlated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis, and lymphovascular space invasion (LVSI) of cervical cancer. The expression of Sp1 was dose-dependently increased in irradiated cervical cancer cell lines at both mRNA and protein levels. Colony-formation assay showed that alteration of Sp1 expression affected the survival of cervical cancer cells with radiotherapy (RT) treatment. Knockdown of Sp1 significantly strengthened the cellular response to radiation by inducing G2/M arrest in cervical cancer cells. Overexpression of Sp1 significantly decreased G2/M arrest in cervical cancer cells, which was related to upregulation of CDK1 expression. Dual-luciferase reporter assay showed the direct effect of Sp1 on the transcriptional activation of CDK1.
Conclusion
Sp1 may contribute to radioresistance through inhibiting G2/M phase arrest by targeting CDK1, and be considered as a potential therapeutic target to promote the effect of RT for patients with cervical cancer.
... miR-205 contains three putative Sp1 transcription factor-binding sites in its promoter region, which in response to radiation, increased the transcription of miR-205 and promoted radioresistance in ESCC (Figure 2). These results demonstrated that Sp1 could be phosphorylated by high dose radiation that in turn elevated its transcriptional activity [51,52]. ...
The five-year survival rate of esophageal cancer patients is less than 20%. This may be due to increased resistance (acquired or intrinsic) of tumor cells to chemo/radiotherapies, often caused by aberrant cell cycle, deregulated apoptosis, increases in growth factor signaling pathways, and/or changes in the proteome network. In addition, deregulation in non-coding RNA-mediated signaling pathways may contribute to resistance to therapies. At the molecular level, these resistance factors have now been linked to various microRNA (miRNAs), which have recently been shown to control cell development, differentiation and neoplasia. The increased stability and dysregulated expression of miRNAs have been associated with increased resistance to various therapies in several cancers, including esophageal cancer. Therefore, miRNAs represent the next generation of molecules with tremendous potential as biomarkers and therapeutic targets. Yet, a detailed studies on miRNA-based therapeutic intervention is still in its infancy. Hence, in this review, we have summarized the current status of microRNAs in dictating the resistance/sensitivity of tumor cells against chemotherapy and radiotherapy. In addition, we have discussed various strategies to increase radiosensitivity, including targeted therapy, and the use of miRNAs as radiosensitive/radioresistance biomarkers for esophageal cancer in the clinical setting. Abstract The five-year survival rate of esophageal cancer patients is less than 20%. This may be due to
