Liqing Du

Expression and function of miRNA in postoperative radiotherapy sensitive and resistant patients of non-small cell lung cancer

PURPOSE To investigate the different miRNA expression profiles of postoperative radiotherapy sensitive and resistant patients of non-small cell lung cancer, explore their potential role and find some radio-sensitivity markers. MATERIALS AND METHODS Thirty non-small cell lung cancer patients who have been treated by postoperative radiotherapy were selected and were divided into radiotherapy sensitive group and resistant group according to overall survival and local or distant recurrence rate. Expression profile of miRNA in these two groups was detected by a microarray assay and the results were validated by quantitative RT-PCR and Northern blot. At the molecular level, the effect of one differently expressed miRNA (miR-126) on the growth and apoptosis of SK-MES-1 cells induced by irradiation was examined. RESULTS Comparing with resistant patients, five miRNAs (miRNA-126, miRNA-let-7a, miRNA-495, miRNA-451 and miRNA-128b) were significantly upregulated and seven miRNAs (miRNA-130a, miRNA-106b, miRNA-19b, miRNA-22, miRNA-15b, miRNA-17-5p and miRNA-21) were greatly downregulated in radiotherapy sensitive group. Overexpression of miRNA-126 inhibited the growth of SK-MES-1 cells and promoted its apoptosis induced by irradiation. The expression level of p-Akt decreased in miRNA-126 overexpression group. After treating with phosphoinositidyl-3 kinase (PI3K) constitutively activator (IGF-1) and inhibitor (LY294002), miRNA-126 overexpression had no significant effects on the apoptosis of SK-MES-1 cells. CONCLUSION We found 12 differently expressed miRNAs in the radiotherapy sensitive and resistant non-small cell lung cancer samples. Moreover, our results showed miRNA-126 promoted non-small cell lung cancer cells apoptosis induced by irradiation through the PI3K-Akt pathway.

Resveratrol inhibits ionising irradiation-induced inflammation in MSCs by activating SIRT1 and limiting NLRP-3 inflammasome activation.

IL-1β, a pro-inflammatory cytokine, has been shown to contribute to radiation injury. Sirt1, an NAD+-dependent class III protein deacetylase, plays an important role in the regulation of the proinflammatory cytokines involved in inflammation-associated diseases. The relationship between Sirt1 and IL-1β, however, has remained elusive. The present study was designed to explore the potential effect of Sirt1 on IL-1β expression induced by radiation and to provide a new target for the development of radiation protection drugs. Our results showed that radiation significantly increased IL-1β mRNA and protein expression and that pretreatment with resveratrol, a Sirt1 activator, inhibited the radiation-induced IL-1β expression in a concentration-dependent manner, whereas the knockdown or inhibition of Sirt1 by nicotinamide significantly enhanced radiation-induced IL-1β expression. This effect can likely be attributed to Sirt1-mediated inhibition of NLRP-3 inflammasome activation because Sirt1 inhibits the transactivation potential of NF-κb by deacetylation, which then suppresses NLRP3 transcription. Taken together, the results demonstrate that Sirt1 exerts anti-inflammatory effects by regulating NLRP3 expression partially through the NF-κb pathway in mesenchymal stem cells. More importantly, our findings suggest that resveratrol is an effective agent in protecting against radiation injury, and we provide a theoretical basis for developing a drug to protect against radiation injury by targeting Sirt1.

Radioprotective and Antioxidant Effect of Resveratrol in Hippocampus by Activating Sirt1

Reactive oxygen species can lead to functional alterations in lipids, proteins, and nucleic acids, and an accumulation of ROS (Reactive oxygen species) is considered to be one factor that contributes to neurodegenerative changes. An increase in ROS production occurs following irradiation. Neuronal tissue is susceptible to oxidative stress because of its high oxygen consumption and modest antioxidant defenses. As a polyphenolic compound, resveratrol is frequently used as an activator of Sirt1 (Sirtuin 1). The present study was designed to explore the radioprotective and antioxidant effect of resveratrol on Sirt1 expression and activity induced by radiation and to provide a new target for the development of radiation protection drugs. Our results demonstrate that resveratrol inhibits apoptosis induced by radiation via the activation of Sirt1. We demonstrated an increase in Sirt1 mRNA that was present on 21 days of resveratrol treatment following irradiation in a concentration-dependent manner. Such mRNA increase was accompanied by an increase of Sirt1 protein and activity. Resveratrol effectively antagonized oxidation induced by irradiation, supporting its cellular ROS-scavenging effect. These results provide evidence that the mitochondrial protection and the antioxidant effect of resveratrol contribute to metabolic activity. These data suggest that Sirt1 may play an important role to protect neurons from oxidative stress.

Expression of miRNA-130a in Nonsmall Cell Lung Cancer

MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the posttranscriptional level and are deeply involved in the pathogenesis of several types of cancer. The miRNA-130a has been shown to play a role in antagonizing the inhibitory effects of GAX on endothelial cell proliferation, migration and tube formation, and antagonizing the inhibitory effects of HoxA5 on tube formation in vitro. Here the authors show, for the first time, that miRNA-130a expression is increased in nonsmall cell lung cancer (NSCLC) tissues. Statistical analysis showed that overexpression of miRNA-130a was strongly associated with lymph node metastasis, stage of tumor node metastasis classification and poor prognosis. Moreover, there was a significant difference in miRNA-130a expression levels between smoking and nonsmoking patients. Multivariate Cox regression analysis showed that miRNA-130a was an independent prognostic factor for patients with NSCLC. Together, these data suggest that miRNA-130a may comprise a potential novel prognostic marker for this disease.

Brusatol Enhances the Radiosensitivity of A549 Cells by Promoting ROS Production and Enhancing DNA Damage

NF-E2-related factor 2 (Nrf2) has been identified as a master regulatory factor in the protection of cells from oxidative and electrophilic stress. However, overexpression of Nrf2 in lung cancer may cause chemoresistance, as well as radioresistance. In this study, we examined the relationship between radioresistance and Nrf2 protein levels in H1299, A549, and H460 cells, and finally chose the A549 cell line to continue with due to its strong radioresistance and high Nrf2 protein levels. We found that the Nrf2 inhibitor, brusatol, could prevent the increase and accumulation of Nrf2 after exposure to irradiation. Additionally, following treatment with 80 nM brusatol, A549 cells became sensitive to irradiation, suffering severe DNA damage. Combination treatment with brusatol and ionizing radiation (IR) can distinctly increase the level of reactive oxygen species in A549 cells, causing a 1.8-fold increase compared with the control, and a 1.4-fold increase compared with IR alone. In fact, in the treatment with both brusatol and IR, lung cancer cell proliferation is halted, gradually leading to cell death. Because Nrf2 is closely linked to DNA damage repair, inhibiting the function of Nrf2, as in brusatol treatment, may increase the DNA damage caused by radiotherapy or chemotherapy, possibly enhancing the efficacy of chemotherapeutic drugs. Our study is the first to demonstrate brusatol’s ability to enhance the responsiveness of lung cancer cells to irradiation, and its potential application as a natural sensitizer in radiotherapy.

Mesenchymal stem cells stimulate intestinal stem cells to repair radiation-induced intestinal injury

The loss of stem cells residing in the base of the intestinal crypt has a key role in radiation-induced intestinal injury. In particular, Lgr5+ intestinal stem cells (ISCs) are indispensable for intestinal regeneration following exposure to radiation. Mesenchymal stem cells (MSCs) have previously been shown to improve intestinal epithelial repair in a mouse model of radiation injury, and, therefore, it was hypothesized that this protective effect is related to Lgr5+ ISCs. In this study, it was found that, following exposure to radiation, transplantation of MSCs improved the survival of the mice, ameliorated intestinal injury and increased the number of regenerating crypts. Furthermore, there was a significant increase in Lgr5+ ISCs and their daughter cells, including Ki67+ transient amplifying cells, Vil1+ enterocytes and lysozyme+ Paneth cells, in response to treatment with MSCs. Crypts isolated from mice treated with MSCs formed a higher number of and larger enteroids than those from the PBS group. MSC transplantation also reduced the number of apoptotic cells within the small intestine at 6 h post-radiation. Interestingly, Wnt3a and active β-catenin protein levels were increased in the small intestines of MSC-treated mice. In addition, intravenous delivery of recombinant mouse Wnt3a after radiation reduced damage in the small intestine and was radioprotective, although not to the same degree as MSC treatment. Our results show that MSCs support the growth of endogenous Lgr5+ ISCs, thus promoting repair of the small intestine following exposure to radiation. The molecular mechanism of action mediating this was found to be related to increased activation of the Wnt/β-catenin signaling pathway.

Knockdown of Rad51 expression induces radiation- and chemo-sensitivity in osteosarcoma cells.

Osteosarcoma is the common primary bone malignancy in children and young adults in Eastern countries. Resistance to ionizing radiation (IR) or drugs is an underlying mechanism contributing to the failure of therapy in these patients. Rad51 is the key protein of DNA homologous recombination repair. Although high expression of Rad51 is associated with enhanced resistance to DNA damage induced by chemicals and/or ionizing radiation, the relevance of Rad51 expression in osteosarcoma and its relationship with IR sensitivity and chemo-resistance is not well understood. In this study, we elucidated the possibility of using Rad51 in the treatment of human osteosarcoma in vitro. Changes in chemo- and radiation sensitivity in cultured osteosarcoma cells occurred after suppression of Rad51 expression, using a plasmid vector-mediated short hairpin RNA (shRNA) expression system. The suppression of Rad51 correlated with cell cycle arrest in the G2 phase and inhibited tumor cell proliferation. Our results suggest that Rad51 expression levels might play an important role in radiation- and chemo-sensitivity of human osteosarcoma.

Radiation-induced cytochrome c release and the neuroprotective effects of the pan-caspase inhibitor z-VAD-fmk in the hypoglossal nucleus.

Numerous studies have demonstrated that neuronal cell death occurs via extrinsic (death receptors) and intrinsic (mitochondria) pathways. Radiation induces caspase activation fundamentally via the mitochondrial pathway. To investigate the role of caspase, a cell permeable pan-caspase inhibitor, z-VAD-fmk [N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone], was used to investigate the effects of caspase blockade in vivo following irradiation. Adult male Sprague-Dawley rats (weight, 250–300 g) underwent irradiation at room temperature with a 4-Gy dose of radiation. Since z-VAD-fmk does not penetrate the blood-brain barrier, it was applied intracerebroventricularly via a bolus injection (0.2 μg/h for 1 h). Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) demonstrated that z-VAD-fmk reduced the numbers of TUNEL-positive cells within the hypoglossal nucleus, suggesting that intervention in the caspase cascade following radiation may have therapeutic applications. The caspase inhibitor z-VAD-fmk reduced the expression and activation of caspase-3, caspase-8 and caspase-9 in the irradiated rats, indicating that caspase may be a potential therapeutic target in the treatment of brain radiation injury. Treatment with z-VAD-fmk also reduced the appearance of cytochrome c within the cytosolic fraction following radiation. The hypoglossal nucleus may be used as a model of radiation-induced injury in the central nervous system, providing visual information and displaying apoptotic nuclear morphology.

Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells

BackgroundMethotrexate is an inhibitor of folic acid metabolism. Homologous recombination is one of the most important ways to repair double-stranded breaks in DNA and influence the radio- and chemosensitivity of tumor cells. But the relationship between methotrexate and homologous recombination repair has not been elucidated.MethodsInduction of double-strand breaks by methotrexate in HOS cells is assessed by the neutral comet assay. Inhibition of subnuclear repair foci by methotrexate is measured by immunofluorescence. Western blot and quantitative real-time PCR are conducted to detect whether methotrexate affects the expression level of genes involved in homologous recombination. In addition, we used a pCMV3xnls-I-SceI construct to determine whether methotrexate directly inhibits the process of homologous recombinational repair in cells, and the sensitivity to methotrexate in the Ku80-deficient cells is detected using clonogenic survival assays.ResultsThe result showed that methotrexate can regulate the repair of DNA double-strand breaks after radiation exposure, and methotrexate inhibition caused the complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigation revealed that methotrexate led to a significant reduction in the transcription of RAD51 genes. Treatment with methotrexate resulted in a decreased ability to perform homology-directed repair of I-SceI-induced chromosome breaks. In addition, enhancement of cell death was observed in Ku mutant cells compared to wild-type cells.ConclusionsThese results demonstrate that methotrexate can affect homologous recombination repair of DNA double-strand breaks by controlling the expression of homologous recombination-related genes and suppressing the proper assembly of homologous recombination–directed subnuclear foci.

Tat-SmacN7 induces radiosensitization in cancer cells through the activation of caspases and induction of apoptosis

A major concern in cancer therapy is resistance of tumors such as human non-small cell lung cancer and esophageal cancer to radiotherapy. Intrinsic radioresistance of these cancer cells limits therapeutic efficiency. Here, we determined in two cancer cell lines the potential radiosensitizing activity of Tat-SmacN7, a small molecule compound, which mimics the activity of Smac, a mitochondrial protein released during apoptosis. We found that Tat-SmacN7 can enter the cells and promote RNA expression and the activity of caspase-3, -8 and -9 and sensitized the cancer cells to radiation with a sensitization enhancement ratio (SER) of 1.5-1.6. Tat-SmacN7 radiosensitization was mediated by both extrinsic and intrinsic apoptosis pathways through activation of caspases. Consistently, blockage of caspase activation, through treatment with a caspase inhibitor, z-VAD-fmk, inhibited apoptosis and abrogated Tat-SmacN7 radiosensitization. Our study demonstrates that Tat-SmacN7 also has radiosensitization effects in vivo, so it could be further developed as a novel class of radiosensitizers for the treatment of radioresistant human non-small cell lung cancer and esophageal cancer.