Dietary phytochemicals, HDAC inhibition, and DNA damage/repair defects in cancer cells

Genomic instability is a common feature of cancer etiology. This provides an avenue for therapeutic intervention, since cancer cells are more susceptible than normal cells to DNA damaging agents. However, there is growing evidence that the epigenetic mechanisms that impact DNA methylation and histone status also contribute to genomic instability. The DNA damage response, for example, is modulated by the acetylation status of histone and non-histone proteins, and by the opposing activities of histone acetyltransferase and histone deacetylase (HDAC) enzymes. Many HDACs overexpressed in cancer cells have been implicated in protecting such cells from genotoxic insults. Thus, HDAC inhibitors, in addition to unsilencing tumor suppressor genes, also can silence DNA repair pathways, inactivate non-histone proteins that are required for DNA stability, and induce reactive oxygen species and DNA double-strand breaks. This review summarizes how dietary phytochemicals that affect the epigenome also can trigger DNA damage and repair mechanisms. Where such data is available, examples are cited from studies in vitro and in vivo of polyphenols, organosulfur/organoselenium compounds, indoles, sesquiterpene lactones, and miscellaneous agents such as anacardic acid. Finally, by virtue of their genetic and epigenetic mechanisms, cancer chemopreventive agents are being redefined as chemo- or radio-sensitizers. A sustained DNA damage response coupled with insufficient repair may be a pivotal mechanism for apoptosis induction in cancer cells exposed to dietary phytochemicals. Future research, including appropriate clinical investigation, should clarify these emerging concepts in the context of both genetic and epigenetic mechanisms dysregulated in cancer, and the pros and cons of specific dietary intervention strategies.     

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation-resistant prostate cancer cell lines, LNCaP-IRR, PC3-IRR, and Du145-IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP-IRR, PC3-IRR, and Du145-IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras-mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-Akt and Jak-STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2-D DIGE followed by computational image analysis and MS. Twenty-seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.     

Non-targeted radiation effects-an epigenetic connection

Ionizing radiation (IR) is a pivotal diagnostic and treatment modality, yet it is also a potent genotoxic agent that causes genome instability and carcinogenesis. While modern cancer radiation therapy has led to increased patient survival rates, the risk of radiation treatment-related complications is becoming a growing problem. IR-induced genome instability has been well-documented in directly exposed cells and organisms. It has also been observed in distant 'bystander' cells. Enigmatically, increased instability is even observed in progeny of pre-conceptually exposed animals, including humans. The mechanisms by which it arises remain obscure and, recently, they have been proposed to be epigenetic in nature. Three major epigenetic phenomena include DNA methylation, histone modifications and small RNA-mediated silencing. This review focuses on the role of DNA methylation and small RNAs in directly exposed and bystander tissues and in IR-induced transgenerational effects. Here, we present evidence that IR-mediated effects are maintained by epigenetic mechanisms.     

Telomere-Binding Protein TPP1 Modulates Telomere Homeostasis and Confers Radioresistance to Human Colorectal Cancer Cells

Background

Radiotherapy is one of the major therapeutic strategies in cancer treatment. The telomere-binding protein TPP1 is an important component of the shelterin complex at mammalian telomeres. Our previous reports showed that TPP1 expression was elevated in radioresistant cells, but the exact effects and mechanisms of TPP1 on radiosensitivity is unclear.

Principal Findings

In this study, we found that elevated TPP1 expression significantly correlated with radioresistance and longer telomere length in human colorectal cancer cell lines. Moreover, TPP1 overexpression showed lengthened telomere length and a significant decrease of radiosensitivity to X-rays. TPP1 mediated radioresistance was correlated with a decreased apoptosis rate after IR exposure. Furthermore, TPP1 overexpression showed prolonged G2/M arrest mediated by ATM/ATR-Chk1 signal pathway after IR exposure. Moreover, TPP1 overexpression accelerated the repair kinetics of total DNA damage and telomere dysfunction induced by ionizing radiation.

Conclusions

We demonstrated that elevated expressions of TPP1 in human colorectal cancer cells could protect telomere from DNA damage and confer radioresistance. These results suggested that TPP1 may be a potential target in the radiotherapy of colorectal cancer.     

HeLa细胞中LRP16影响电离辐射诱导的NF-κB核转位

前期研究显示抑制LRP16的表达可以明显增加肿瘤细胞对辐射诱导凋亡的敏感性,但具体机制尚不清楚．大量研究表明,NF-κB信号通路在肿瘤产生辐射抵抗中起着重要的作用. 为研究LRP16影响肿瘤细胞对辐射敏感性的可能机制,首先通过免疫 荧光技术检测电离辐射刺激后不同时间点NF-κB的核转位情况;然后分别过表达和抑制LRP16的表达,采用Western印迹方法检测NF-κB在核蛋白与浆蛋白中的表达情况、 IκB-α总体蛋白水平及磷酸化水平．结果发现,电离辐射后1 h,可见NF-κB明显入核;过表达LRP16可以促进NF-κB入核、提高IκB-α的磷酸化水平、促进IκB-α 的降解;反之,抑制LRP16的表达可以抑制NF-κB入核、降低IκB-α的磷酸化水平、 阻碍IκB-α的降解．上述研究结果表明,在HeLa细胞中LRP16可以影响电离辐射诱导的NF-κB核转位,该研究为LRP16参与肿瘤细胞产生辐射抵抗现象提供一种可能的机制．     

Epigenetic modifications in radiation-induced non-targeted effects and their clinical significance

BackgroundIonizing radiation (IR) plays an important role in the diagnosis and treatment of cancer. Besides the targeted effects, the non-targeted effects, which cause damage to non-irradiated cells and genomic instability in normal tissues, also play a role in the side effects of radiotherapy and have been shown to involve both alterations in DNA sequence and regulation of epigenetic modifications.Scope of reviewWe summarize the recent findings regarding epigenetic modifications that are involved in radiation-induced non-targeted effects as well as their clinical significance in radiotherapy and radioprotection.Major conclusionsEpigenetic modifications play an important role in both the realization and modulation of radiobiological effects. However, the molecular mechanisms underlying non-targeted effects still need to be clarified.General significanceA better understanding of the epigenetic mechanisms related to radiation-induced non-targeted effects will guide both individualized clinical radiotherapy and individualized precise radioprotection.     

Low-dose ionizing radiation: induction of differential intracellular signalling possibly affecting intercellular communication

Given the complexity of the carcinogenic process and the lack of any mechanistic understanding of how ionizing radiation at low-level exposures affects the multistage, multimechanism processes of carcinogenesis, it is imperative that concepts and paradigms be reexamined when extrapolating from high dose to low dose. Any health effect directly linked to low-dose radiation exposure must have molecular/biochemical and biological bases. On the other hand, demonstrating some molecular/biochemical or cellular effect, using surrogate systems for the human being, does not necessarily imply a corresponding health effect. Given the general acceptance of an extrapolated LNT model, our current understanding of carcinogenesis cries out for a resolution of a real problem. How can a low-level acute, or even a chronic, exposure of ionizing radiation bring about all the different mechanisms (mutagenic, cytotoxic, and epigenetic) and genotypic/phenotypic changes needed to convert normal cells to an invasive, malignant cell, given all the protective, repair, and suppressive systems known to exist in the human body? Until recently, the prevailing paradigm that ionizing radiation brings about cancer primarily by DNA damage and its conversion to gene and chromosomal mutations, drove our interpretation of radiation carcinogenesis. Today, our knowledge includes the facts both that epigenetic events play a major role in carcinogenesis and that low-dose radiation can also induce epigenetic events in and between cells in tissues. This challenges any simple extrapolation of the LNT model. Although a recent delineation of “hallmarks” of the cancer process has helped to focus on how ionizing radiation might contribute to the induction of cancers, several other hallmarks, previously ignored—namely, the stem cells in tissues as targets for carcinogenesis and the role of cell–cell communication processes in modulating the radiation effects on the target cell—must be considered, particularly for the adaptive response, bystander effects, and genomic instability phenomena.     

Differentiation-induced radioresistance in muscle cells

DNA damage induces cell cycle arrest and DNA repair or apoptosis in proliferating cells. Terminally differentiated cells are permanently withdrawn from the cell cycle and partly resistant to apoptosis. To investigate the effects of genotoxic agents in postmitotic cells, we compared DNA damage-activated responses in mouse and human proliferating myoblasts and their differentiated counterparts, the myotubes. DNA double-strand breaks caused by ionizing radiation (IR) induced rapid activating autophosphorylation of ataxia-teleangiectasia-mutated (ATM), phosphorylation of histone H2AX, recruitment of repair-associated proteins MRE11 and Nbs1, and activation of Chk2 in both myoblasts and myotubes. However, IR-activated, ATM-mediated phosphorylation of p53 at serine 15 (human) or 18 (mouse) [Ser15(h)/18(m)], and apoptosis occurred in myoblasts but was impaired in myotubes. This phosphorylation could be enforced in myotubes by the anthracycline derivative doxorubicin, leading to selective activation of proapoptotic genes. Unexpectedly, the abundance of autophosphorylated ATM was indistinguishable after exposure of myotubes to IR (10 Gy) or doxorubicin (1 microM/24 h) despite efficient phosphorylation of p53 Ser15(h)/18(m), and apoptosis occurred only in response to doxorubicin. These results suggest that radioresistance in myotubes might reflect a differentiation-associated, pathway-selective blockade of DNA damage signaling downstream of ATM. This mechanism appears to preserve IR-induced activation of the ATM-H2AX-MRE11/Rad50/Nbs1 lesion processing and repair pathway yet restrain ATM-p53-mediated apoptosis, thereby contributing to life-long maintenance of differentiated muscle tissues.     

Ataxia telangiectasia and Rad3-related overexpressing cancer cells induce prolonged G₂ arrest and develop resistance to ionizing radiation

We investigated whether ataxia telangiectasia and rad3-related (ATR) kinases regulate prolongation of ionizing radiation (IR) induced-G? arrest and radioresistance in ataxia telangiectasia mutated-intact cancer cells. ATR overexpressing cancer cells showed prolonged-G? arrest after IR exposure and were significantly resistant to DNA damaging stresses. The phosphorylation of p-Ser1?-p53, p-Ser3??-Chk1, and p-Tyr1?-Cdk1 phosphorylation was increased until 36 h after IR exposure in ATR-overexpressing cells, whereas p-Ser1?-histone H3 decreased. ATR-overexpressing cells also showed rapid attenuation of increased γ-H2AX foci after IR exposure compared with control cells. In contrast, ATR knockdown cells had limited clearance of γ-H2AX foci after IR exposure. In conclusion, ATR overexpression seems to primarily induce prolonged G? arrest after IR exposure, which increases IR resistance by enhancing DNA damage repair. These results may provide useful clues for understanding the function of ATR in controlling IR-induced G? arrest and radiation response.     

The roles of polyphenols in cancer chemoprevention

Oxidative stress imposed by reactive oxygen species (ROS) plays a crucial role in the pathophysiology associated with neoplasia, atherosclerosis, and neurodegenerative diseases. The ROS-induced development of cancer involves malignant transformation due to altered gene expression through epigenetic mechanisms as well as DNA mutations. Considerable attention has been focused on identifying naturally occurring antioxidative phenolic phytochemicals that are able to decrease ROS levels, but the efficacies of antioxidant therapies have been equivocal at best. Several studies have shown that some antioxidants exhibit prooxidant activity under certain conditions and potential carcinogenicity under others, and that dietary supplementation with large amounts of a single antioxidant may be deleterious to human health. This article reviews the intracellular signaling pathways that respond to oxidative stress and how they are modulated by naturally occurring polyphenols. The possible toxicity and carcinogenicity of polyphenols is also discussed.     

NOD2 agonist murabutide alleviates radiation-induced injury through DNA damage response pathway mediated by ATR

Injury-induced by ionizing radiation (IR) severely reduces the quality of life of victims. The development of radiation protectors is regarded as one of the most resultful strategies to alleviate damages caused by IR exposure. In the present study, we investigated the radioprotective effects of the agonist of nucleotide-binding-oligomerization-domain-containing proteins 2 called murabutide (MBD) and clarified the potential mechanisms. Our results showed that the pretreatment with MBD effectively protected cultured cells and mice against IR-induced toxicity and the pretreatment with MBD in vitro and in vitro also inhibited apoptosis caused by IR exposure. The downregulation of γ-H2AX and the upregulation of ATR signaling pathways by MBD treatment indicated that the radioprotective effects of MBD were due to the stimulation of DNA damage response (DDR) pathway to repair DNA double-strand breaks caused by IR exposure. As the radioprotective effects of MBD were diminished by the ATR selective inhibitor rather than the ATM inhibitor, ATR pathway was confirmed to be a more crucial checkpoint pathway in mediating the stimulation of DDR pathway by MBD. Taken together, our data provide a novel and effective protector to relieve the injury induced by IR exposure.     

Functional validation of miRNAs targeting genes of DNA double-strand break repair to radiosensitize non-small lung cancer cells

DNA-Double strand breaks (DSBs) generated by radiation therapy represent the most efficient lesions to kill tumor cells, however, the inherent DSB repair efficiency of tumor cells can cause cellular radioresistance and impact on therapeutic outcome. Genes of DSB repair represent a target for cancer therapy since their down-regulation can impair the repair process making the cells more sensitive to radiation. In this study, we analyzed the combination of ionizing radiation (IR) along with microRNA-mediated targeting of genes involved in DSB repair to sensitize human non-small cell lung cancer (NSCLC) cells. MicroRNAs are natural occurring modulators of gene expression and therefore represent an attractive strategy to affect the expression of DSB repair genes. As possible IR-sensitizing targets genes we selected genes of homologous recombination (HR) and non-homologous end joining (NHEJ) pathway (i.e. RAD51, BRCA2, PRKDC, XRCC5, LIG1). We examined these genes to determine whether they may be real targets of selected miRNAs by functional and biological validation. The in vivo effectiveness of miRNA treatments has been examined in cells over-expressing miRNAs and treated with IR. Taken together, our results show that hsa-miR-96-5p and hsa-miR-874-3p can directly regulate the expression of target genes. When these miRNAs are combined with IR can decrease the survival of NSCLC cells to a higher extent than that exerted by radiation alone, and similarly to radiation combined with specific chemical inhibitors of HR and NHEJ repair pathway.     

The role of the repair of double-stranded DNA breaks in the radioresistance of yeast cells

The role of DNA double-strand break (DSB) repair in radioresistance of Saccharomyces cerevisiae G1 cells is discussed. The contribution of rapid and slow DNA DSB repair to radioresistance of diploid yeast has been estimated. The contribution of the DNA DSB repair involving no homologous chromosome interaction is shown to be insignificant in comparison with the recombinational repair. The rapid DNA DSB repair efficiency calculation method based on the proposed yeast radiation inactivation model is given. The calculations are in a satisfactory agreement with the experimental data. Possible mechanisms of radiation induction of lethal sectoring in yeast are discussed. This phenomenon is supposed to be due to the DNA DSB processing during vegetative division of irradiated cells. A general scheme of radiation inactivation of yeast cells is proposed.     

Mechanisms of radioresistance and the underlying signaling pathways in colorectal cancer cells

Radiotherapy is one of the most common modalities for the treatment of a wide range of tumors, including colorectal cancer (CRC); however, radioresistance of cancer cells remains a major limitation for this treatment. Following radiotherapy, the activities of various cellular mechanisms and cell signaling pathways are altered, resulting in the development of radioresistance, which leads to therapeutic failure and poor prognosis in patients with cancer. Furthermore, even though several inhibitors have been developed to target tumor resistance, these molecules can induce side effects in nontumor cells due to low specificity and efficiency. However, the role of these mechanisms in CRC has not been extensively studied. This review discusses recent studies regarding the relationship between radioresistance and the alterations in a series of cellular mechanisms and cell signaling pathways that lead to therapeutic failure and tumor recurrence. Our review also presents recent advances in the in vitro/in vivo study models aimed at investigating the radioresistance mechanism in CRC. Furthermore, it provides a relevant biochemical basis in theory, which can be useful to improve radiotherapy sensitivity and prolong patient survival.     

A cancer chemopreventive agent silibinin, targets mitogenic and survival signaling in prostate cancer

There are many epigenetic variables that affect the biological responses of autocrine, paracrine and endocrine regulatory molecules, which determine the growth and development of different cancers including prostate cancer (PCA). One of the focuses of the current cancer chemoprevention studies is the search for non-toxic chemopreventive agents that inhibit mitogenic and cell survival signaling in cancer cells. In general, advanced stage cancer cells harbor many constitutively active mitogenic signaling and anti-apoptotic mechanisms, which make them less dependent on external growth factors as well as resistant to chemotherapeutic agents. In this regard, silibinin (a naturally occurring flavanone) has shown the pleiotropic anticancer effects in different cancer cells. Our extensive studies with PCA have shown that inhibition of mitogenic and cell survival signaling, such as epidermal growth factor receptor, insulin-like growth factor receptor type I and nuclear factor kappa B signaling are the most likely molecular targets of silibinin's efficacy in PCA. We have observed that silibinin inhibits prostate tumor growth in animal models without any apparent signs of toxicity. At the same time, silibinin is also physiologically available in different organs of the body including plasma and prostate, which is generally required for the pharmacological dosing and translational mechanistic studies of the compound. There are substantial amount of data to support the inhibitory effect of silibinin on mitogenic and cell survival signaling in PCA, which are reviewed in the present communication.     

Dysregulation of IRP1-Mediated Iron Metabolism Causes Gamma Ray-specific Radioresistance in Leukemia Cells

Iron is required for nearly all organisms, playing important roles in oxygen transport and many enzymatic reactions. Excess iron, however, can be cytotoxic. Emerging evidence suggests that radioresistance can be achieved in lower organisms by the protection of proteins, but not DNA, immediately following ionizing radiation (IR) exposure, allowing for improved DNA repair. One potential mechanism for protein protection is controlling and limiting the amount of free iron in cells, as has been demonstrated in the extremophile Deinococcus Radiodurans, reducing the potential for oxidative damage to proteins during exposure to IR. We found that iron regulatory protein 1 (IRP1) expression was markedly reduced in human myeloid leukemia HL60 cells resistant to low linear energy transfer (LET) gamma rays, but not to high LET alpha particles. Stable knockdown of IRP1 by short-hairpin RNA (shRNA) interference in radiosensitive parental cells led to radioresistance to low LET IR, reduced intracellular Fenton chemistry, reduced protein oxidation, and more rapid DNA double-strand break (DSB) repair. The mechanism of radioresistance appeared to be related to attenuated free radical-mediated cell death. Control of intracellular iron by IRPs may be a novel radioresistance mechanism in mammalian cells.