Circadian regulation of MGMT expression and promoter methylation underlies daily rhythms in TMZ sensitivity in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. METHODS AND RESULTS: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. CONCLUSION: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity and that scoring MGMT methylation status requires controlling for time of day of biopsy.

The Curcumin Analog PAC Is a Potential Solution for the Treatment of Triple-Negative Breast Cancer by Modulating the Gene Expression of DNA Repair Pathways.

Breast Cancer (BC) is one of the most common and challenging cancers among females worldwide. Conventional treatments for oral cancer rely on the use of radiology and surgery accompanied by chemotherapy. Chemotherapy presents many side effects, and the cells often develop resistance to this chemotherapy. It will be urgent to adopt alternative or complementary treatment strategies that are new and more effective without these negative effects to improve the well-being of patients. A substantial number of epidemiological and experimental studies reported that many compounds are derived from natural products such as curcumin and their analogs, which have a great deal of beneficial anti-BC activity by inducing apoptosis, inhibiting cell proliferation, migration, and metastasis, modulating cancer-related pathways, and sensitizing cells to radiotherapy and chemotherapy. In the present study, we investigated the effect of the curcumin-analog PAC on DNA repair pathways in MCF-7 and MDA-MB-231 human breast-cancer cell lines. These pathways are crucial for genome maintenance and cancer prevention. MCF-7 and MDA-MB-231 cells were exposed to PAC at 10 µM. MTT and LDH assays were conducted to evaluate the effects of PAC on cell proliferation and cytotoxicity. Apoptosis was assessed in breast cancer cell lines using flow cytometry with annexin/Pi assay. The expression of proapoptotic and antiapoptotic genes was determined by RT-PCR to see if PAC is active in programming cell death. Additionally, DNA repair signaling pathways were analyzed by PCR arrays focusing on genes being related and confirmed by quantitative PCR. PAC significantly inhibited breast-cancer cell proliferation in a time-dependent manner, more on MDA-MB-231 triple-negative breast cancer cells. The flow cytometry results showed an increase in apoptotic activity. These data have been established by the gene expression and indicate that PAC-induced apoptosis by an increased Bax and decreased Bcl-2 expression. Moreover, PAC affected multiple genes involved in the DNA repair pathways occurring in both cell lines (MCF-7 and MDA-MB231). In addition, our results suggest that PAC upregulated more than twice 16 genes (ERCC1, ERCC2, PNKP, POLL, MPG, NEIL2, NTHL1, SMUG1, RAD51D, RAD54L, RFC1, TOP3A, XRCC3, XRCC6BP1, FEN1, and TREX1) in MDA-MB-231, 6 genes (ERCC1, LIG1, PNKP, UNG, MPG, and RAD54L) in MCF-7, and 4 genes (ERCC1, PNKP, MPG, and RAD54L) in the two cell lines. In silico analysis of gene-gene interaction shows that there are common genes between MCF-7 and MDA-MB-321 having direct and indirect effects, among them via coexpression, genetic interactions, pathways, predicted and physical interactions, and shared protein domains with predicted associated genes indicating they are more likely to be functionally related. Our data show that PAC increases involvement of multiple genes in a DNA repair pathway, this certainly can open a new perspective in breast-cancer treatment.

Integrative analysis of therapy resistance and transcriptomic profiling data in glioblastoma cells identifies sensitization vulnerabilities for combined modality radiochemotherapy.

BACKGROUND: Inherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance. METHODS: We performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining. RESULTS: Inherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4-two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization. CONCLUSIONS: Here, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.

Banxia xiexin decoction affects drug sensitivity in gastric cancer cells by regulating MGMT expression via IL­6/JAK/STAT3­mediated PD­L1 activity.

Banxia xiexin decoction (BXXX) is a classic preparation used to treat gastrointestinal diseases, and also has certain therapeutic effects on gastrointestinal tumors. BXXX has been reported to regulate the expression of proteins associated with drug resistance and sensitivity in tumors, and thus, the aim of the present study was to investigate the mechanisms of BXXX drug sensitivity in gastric cancer (GC). The expression levels of programmed cell death 1 ligand 1 (PD­L1), 6­O­methylguanine­DNA methyltransferase (MGMT) and STAT3 were immunohistochemically detected in the cancer and adjacent non­cancer tissues of patients with GC, and in vitro experimentation was conducted using drug­resistant and ­sensitive GC cells. The expression levels of PD­L1, MGMT and STAT3 were determined using reverse transcription­quantitative PCR. Different concentrations of BXXX drug serum were used to treat the cells and the cellular inhibition rate was assessed using a Cell Counting Kit­8 assay. Flow cytometry was used to detect apoptosis, and western blot analysis was used to detect the expression levels of IL­6, IFN­Î³, JAK/STAT3 pathway proteins, PD­L1 and MGMT. The association between PD­L1 and MGMT protein expression levels was subsequently assessed via co­immunoprecipitation. Furthermore, in vivo studies were conducted following the establishment of a drug­resistant tumor­bearing mouse model, where GC tumor size was assessed under different treatment conditions, and western blot analysis was used to detect the expression of related pathway proteins. The expression levels of PD­L1, MGMT and STAT3 were significantly increased in GC tissues, GC cells and cisplatin­resistant cells. Furthermore, BXXX inhibited the proliferation of drug­resistant cells and promoted the inhibitory effects of chemotherapeutic drugs on drug­resistant cells. BXXX also inhibited the expression levels of IL­6, IFN­Î³ and JAK/STAT3 pathway proteins, as well as the expression levels of PD­L1 and MGMT. Colivelin, an activator of STAT3, reversed the effects of BXXX on drug­resistant GC cells, and significantly reversed the effect of BXXX on PD­L1 expression. In conclusion, BXXX was found to influence the drug sensitivity of GC cells by regulating the expression of MGMT. This process functions viaPD­L1, which was itself mediated by IL­6/JAK/STAT3 signaling.

Chemical-Genetic Interactions of Bacopa monnieri Constituents in Cells Deficient for the DNA Repair Endonuclease RAD1 Appear Linked to Vacuolar Disruption.

Colorectal cancer is a common cancer worldwide and reduced expression of the DNA repair endonuclease XPF (xeroderma pigmentosum complementation group F) is associated with colorectal cancer. Bacopa monnieri extracts were previously found to exhibit chemical-genetic synthetic lethal effects in a Saccharomyces cerevisiae model of colorectal cancer lacking Rad1p, a structural and functional homologue of human XPF. However, the mechanisms for B. monnieri extracts to limit proliferation and promote an apoptosis-like event in RAD1 deleted yeast was not elucidated. Our current analysis has revealed that B. monnieri extracts have the capacity to promote mutations in rad1∆ cells. In addition, the effects of B. monnieri extracts on rad1∆ yeast is linked to disruption of the vacuole, similar to the mammalian lysosome. The absence of RAD1 in yeast sensitizes cells to the effects of vacuole disruption and the release of proteases. The combined effect of increased DNA mutations and release of vacuolar contents appears to induce an apoptosis-like event that is dependent on the meta-caspase Yca1p. The toxicity of B. monnieri extracts is linked to sterol content, suggesting saponins may be involved in limiting the proliferation of yeast cells. Analysis of major constituents from B. monnieri identified a chemical-genetic interaction between bacopasaponin C and rad1∆ yeast. Bacopasaponin C may have potential as a drug candidate or serve as a model for the development of analogs for the treatment of colorectal cancer.

Vitamin D changes expression of DNA repair genes in the patients with multiple sclerosis.

Oxidative stress (OS) plays an essential role in demyelination and tissue injury related to pathogenesis of multiple sclerosis (MS). On the other hand, vitamin D (VD) as an antioxidant reduces oxidative stress and has been used as adjuvant therapy in autoimmune diseases. Although VD supplementation is suggested as a protective and immunomodulation factor for MS patients, the molecular mechanisms remain unclear. Given that VD may modulate the immune system of MS patients through the DNA repair pathway, we aimed to evaluate the effects of VD supplementation in DNA repair genes expression including OGG1, MYH, MTH1, and ITPA. Transcript levels were measured using the RT-qPCR method in peripheral blood mononuclear cells (PBMCs) of relapsing-remitting multiple sclerosis (RRMS) patients before and after two months of VD supplementation. Furthermore, in silico analysis and correlation gene expression analysis was performed to find the biological binding sites and the effect of NRF2 on the regulation of DNA repair genes. Our data revealed that in MS patients, 2-month VD treatment significantly altered the expression of MYH, OGG1, MTH1, and NRF2 genes. A significant correlation was observed between DNA repair genes and NRF2 expression, which was confirmed by the presence of antioxidant response element (ARE) binding sites in the promoter of OGG1, MYH, and MTH1 genes. This study demonstrated that the impact of VD on MS patients may be mediated through the improvement of DNA repair system efficiency. This finding brought some new evidence for the involvement of DNA repair genes in the physiopathology of MS patients.

Molecular Characteristics of Thalamic Gliomas in Adults.

The 2016 World Health Organization classification of central nervous system tumor firstly introduces molecular diagnosis to glioma, while the molecular features of adult thalamic gliomas (ATGs) in a relatively large sample have not been reported. We aimed at exploring molecular characteristics in ATGs. The data of 97 and 575 newly diagnosed ATGs and superficial gliomas (SGs) patients were collected, and we performed a comparative analysis of molecular characteristics between them. We analyzed expressions of molecules as follow: H3 K27M, isocitrate dehydrogenase1 (IDH1), Ki-67, O6-Methylguanine-DNA methyltransferase (MGMT) promoter, EGFR, p53, ATRX, GFAP, Oligo2, PTEN, MGMT, and MMP9 by immunohistochemistry. Direct gene sequencing was performed to test the H3 K27M, IDH1, and TERT promoter mutation. The median age at diagnosis of ATGs was 36.0 years, and majority of them were high-grade glioma. We found a significant difference in H3 K27M mutation (P = 0.003), IDH1 mutation (P < 0.001), MGMT promoter methylation (P = 0.005), and Ki67 > 0.1 (P < 0.001) between ATGs and SGs. The statuses of IDH1 (P < 0.001), MGMT promoter (P < 0.001), and Ki67 (P < 0.001) were significantly different between these two groups in lower-grade gliomas. And statuses of IDH1 (P < 0.001), Ki67 (P < 0.001), and EGFR (P = 0.032) were different between these two groups in high-grade gliomas. Only Ki67 > 0.1 was differentially expressed between lower- and high-grade gliomas in ATGs (P = 0.014). The high occurrence of H3 K27M mutation and Ki67 > 0.1, rare occurrence of IDH1 mutation, and MGMT promoter methylation in ATGs suggested that ATGs may be a distinct type of glioma entity.

DNA-editing enzymes as potential treatments for heteroplasmic mtDNA diseases.

Mutations in the mitochondrial genome are the cause of many debilitating neuromuscular disorders. Currently, there is no cure or treatment for these diseases, and symptom management is the only relief doctors can provide. Although supplements and vitamins are commonly used in treatment, they provide little benefit to the patient and are only palliative. This is why gene therapy is a promising research topic to potentially treat and, in theory, even cure diseases caused by mutations in the mitochondrial DNA (mtDNA). Mammalian cells contain approximately a thousand copies of mtDNA, which can lead to a phenomenon called heteroplasmy, where both wild-type and mutant mtDNA molecules co-exist within the cell. Disease only manifests once the per cent of mutant mtDNA reaches a high threshold (usually >80%), which causes mitochondrial dysfunction and reduced ATP production. This is a useful feature to take advantage of for gene therapy applications, as not every mutant copy of mtDNA needs to be eliminated, but only enough to shift the heteroplasmic ratio below the disease threshold. Several DNA-editing enzymes have been used to shift heteroplasmy in cell culture and mice. This review provides an overview of these enzymes and discusses roadblocks of applying these to gene therapy in humans.

Muscle phenotype of AGAT- and GAMT-deficient mice after simvastatin exposure.

Statin-induced myopathy affects more than 10 million people worldwide. But discontinuation of statin treatment increases mortality and cardiovascular events. Recently, L-arginine:glycine amidinotransferase (AGAT) gene was associated with statin-induced myopathy in two populations, but the causal link is still unclear. AGAT is responsible for the synthesis of L-homoarginine (hArg) and guanidinoacetate (GAA). GAA is further methylated to creatine (Cr) by guanidinoacetate methyltransferase (GAMT). In cerebrovascular patients treated with statin, lower hArg and GAA plasma concentrations were found than in non-statin patients, indicating suppressed AGAT expression and/or activity (n = 272, P = 0.033 and P = 0.039, respectively). This observation suggests that statin-induced myopathy may be associated with AGAT expression and/or activity in muscle cells. To address this, we studied simvastatin-induced myopathy in AGAT- and GAMT-deficient mice. We found that simvastatin induced muscle damage and reduced AGAT expression in wildtype mice (myocyte diameter: 34.1 ± 1.3 µm vs 21.5 ± 1.3 µm, P = 0.026; AGAT expression: 1.0 ± 0.3 vs 0.48 ± 0.05, P = 0.017). Increasing AGAT expression levels of transgenic mouse models resulted in rising plasma levels of hArg and GAA (P < 0.01 and P < 0.001, respectively). Simvastatin-induced motor impairment was exacerbated in AGAT-deficient mice compared with AGAT-overexpressing GAMT-/- mice and therefore revealed an effect independent of Cr. But Cr supplementation itself improved muscle strength independent of AGAT expression (normalized grip strength: 55.8 ± 2.9% vs 72.5% ± 3.0%, P < 0.01). Homoarginine supplementation did not affect statin-induced myopathy in AGAT-deficient mice. Our results from clinical and animal studies suggest that AGAT expression/activity and its product Cr influence statin-induced myopathy independent of each other. The interplay between simvastatin treatment, AGAT expression and activity, and Cr seems to be complex. Further clinical pharmacological studies are needed to elucidate the underlying mechanism(s) and to evaluate whether supplementation with Cr, or possibly GAA, in patients under statin medication may reduce the risk of muscular side effects.

Promotion of Cell Death in Cisplatin-Resistant Ovarian Cancer Cells through KDM1B-DCLRE1B Modulation.

Ovarian cancer is the gynecological malignancy with the poorest prognosis, in part due to its high incidence of recurrence. Platinum agents are widely used as a first-line treatment against ovarian cancer. Recurrent tumors, however, frequently demonstrate acquired chemo-resistance to platinum agent toxicity. To improve chemo-sensitivity, combination chemotherapy regimens have been investigated. This study examined anti-tumor effects and molecular mechanisms of cytotoxicity of Oldenlandia diffusa (OD) extracts on ovarian cancer cells, in particular, cells resistant to cisplatin. Six ovarian cancer cells including A2780 and cisplatin-resistant A2780 (A2780cis) as representative cell models were used. OD was extracted with water (WOD) or 50% methanol (MOD). MOD significantly induced cell death in both cisplatin-sensitive cells and cisplatin-resistant cells. The combination treatment of MOD with cisplatin reduced viability in A2780cis cells more effectively than treatment with cisplatin alone. MOD in A2780cis cells resulted in downregulation of the epigenetic modulator KDM1B and the DNA repair gene DCLRE1B. Transcriptional suppression of KDM1B and DCLRE1B induced cisplatin sensitivity. Knockdown of KDM1B led to downregulation of DCLRE1B expression, suggesting that DCLRE1B was a KDM1B downstream target. Taken together, OD extract effectively promoted cell death in cisplatin-resistant ovarian cancer cells under cisplatin treatment through modulating KDM1B and DCLRE1B.

Achievable Central Nervous System Concentrations of the Green Tea Catechin EGCG Induce Stress in Glioblastoma Cells in Vitro.

The polyphenolic compounds present in green tea are preventative against cancer in several animal tumor models. However, direct cytotoxic effects on cancer cells have also been reported. In order to determine whether drinking of green tea has chemopreventive or cytotoxic effects on brain cancer cells, we investigated the effect of the major green tea polyphenol EGCG as a pure substance and as tea extract dietary supplement on primary human glioblastoma cell cultures at the CNS-achievable concentration of 100 nM reported in the literature. We compared this with the effect of the cytotoxic concentration of 500 µM determined to be specific for the investigated primary glioblastoma cultures. After treatment with 500 µM EGCG, strong induction of autophagy and apoptosis was observed. Under treatment with 100 nM EGCG, glioblastoma cells proliferated over the entire observation period of 6 days without any detectable signs of cell death. Only within the first 12 h of treatment was increased accumulation of autophagic vacuoles and increased reactive oxygen species production as a stress response demonstrated. Mild forms of stress, such as treatment with 100 nM EGCG, activate different endogenous repair mechanisms to protect cells. Our data imply that drinking of green tea may have chemopreventive effects, but no direct cytotoxic properties.

Radiosensitivity enhancement of human thyroid carcinoma cells by the inhibitors of histone deacetylase sodium butyrate and valproic acid.

Radiotherapy is one of the leading treatments for clinical cancer therapy. External beam radiotherapy has been proposed as an adjuvant treatment for patients bearing differentiated thyroid cancer refractory to conventional therapy. Our purpose was to study the combined effect of HDAC inhibitors (HDACi) and ionizing irradiation in thyroid cancer cell lines (Nthy-ori 3-1, WRO, TPC-1 and 8505c). HDACi radiosensitized thyroid cancer cells as evidenced by the reduction of survival fraction, whereas they had no effect in the normal cells. HDACi enhanced radiation-induced cell death in WRO cells. Gamma-H2AX foci number increased and persisted long after ionizing exposure in the HDACi-treated cells (WRO and TPC-1). Moreover, the expression of the repair-related gene Ku80 was differentially modulated only in the cancer cells, by the compounds at the protein and/or mRNA levels. We present in vitro evidence that HDACi can enhance the radiosensitivity of human thyroid cancer cells.

Effects of electromagnetic field, cisplatin and morphine on cytotoxicity and expression levels of DNA repair genes.

Morphine (Mor) is widely used as an analgesic drug in cancers and in combination with chemotherapy is known to have DNA damaging effects on non-targeted cell. This study surveyed the effect of Mor in combination with 50-Hz electromagnetic field (EMF) and co-treatment of cisplatin in combination with Mor and EMF on the expression of genes involved in DNA repair pathways. MCF-7 and SH-SY5Y cells were treated with 5.0 µM Mor and then exposed to 50-Hz 0.50 mT EMF in the intermittent pattern of 15 min field-on/15 min field-off. Gene expression, cisplatin and bleomycin cytotoxicity were measured using real-time PCR and MTT assay. Mor treated cells showed significant down-regulation of the examined genes, while in "Mor + EMF" treatments the genes were not significantly changed. IC50 of cisplatin was significantly elevated in both cell lines when co-treated with "Mor + EMF" compared with Mor treated cells. Non-homologous end joining (NHEJ) related genes were significantly decreased in co-treatment of cisplatin and "Mor + EMF" which led to bleomycin higher cytotoxicity in SH-SY5Y not in MCF-7. Our data is promising for providing a cell line-specific sensitization by combination of cisplatin and "Mor + EMF" treatment with local administration of double strand breaking agents.

Interrogation of ethnomedicinal plants for synthetic lethality effects in combination with deficiency in the DNA repair endonuclease RAD1 using a yeast cell-based assay.

ETHNOPHARMACOLOGICAL RELEVANCE: Plant materials used in this study were selected based on the ethnobotanical literature. Plants have either been utilized by Thai practitioners as alternative treatments for cancer or identified to exhibit anti-cancer properties. AIM OF THE STUDY: To screen ethnomedicinal plants using a yeast cell-based assay for synthetic lethal interactions with cells deleted for RAD1, the yeast homologue of human ERCC4 (XPF) MATERIALS AND METHODS: Ethanolic extracts from thirty-two species of medicinal plants utilized in Thai traditional medicine were screened for synthetic lethal/sick interactions using a yeast cell-based assay. Cell growth was compared between the parental strain and rad1∆ yeast following exposure to select for specific toxicity of plant extracts. Candidate extracts were further examined for the mode of action using genetic and biochemical approaches. RESULTS: Screening a library of ethanolic extracts from medicinal plants identified Bacopa monnieri and Colubrina asiatica as having synthetic lethal effects in the rad1∆ cells but not the parental strain. Synthetic lethal effects for B. monneiri extracts were more apparent and this plant was examined further. Genetic analysis indicates that pro-oxidant activities and defective excision repair pathways do not significantly contribute to enhanced sensitivity to B. monneiri extracts. Exposure to B. monneiri extracts resulted in nuclear fragmentation and elevated levels of ethidium bromide staining in rad1∆ yeast suggesting promotion of an apoptosis-like event. Growth inhibition also observed in the human Caco-2 cell line suggesting the effects of B. monnieri extracts on both yeast and human cells may be similar. CONCLUSIONS: B. monneiri extracts may have utility in treatment of colorectal cancers that exhibit deficiency in ERCC4 (XPF).

Genetic assessment and folate receptor autoantibodies in infantile-onset cerebral folate deficiency (CFD) syndrome.

INTRODUCTION: Cerebral folate deficiency (CFD) syndromes are defined as neuro-psychiatric conditions with low CSF folate and attributed to different causes such as autoantibodies against the folate receptor-alpha (FR) protein that can block folate transport across the choroid plexus, FOLR1 gene mutations or mitochondrial disorders. High-dose folinic acid treatment restores many neurologic deficits. STUDY AIMS AND METHODS: Among 36 patients from 33 families the infantile-onset CFD syndrome was diagnosed based on typical clinical features and low CSF folate. All parents were healthy. Three families had 2 affected siblings, while parents from 4 families were first cousins. We analysed serum FR autoantibodies and the FOLR1 and FOLR2 genes. Among three consanguineous families homozygosity mapping attempted to identify a monogenetic cause. Whole exome sequencing (WES) was performed in the fourth consanguineous family, where two siblings also suffered from polyneuropathy as an atypical finding. RESULTS: Boys (72%) outnumbered girls (28%). Most patients (89%) had serum FR autoantibodies fluctuating over 5-6 weeks. Two children had a genetic FOLR1 variant without pathological significance. Homozygosity mapping failed to detect a single autosomal recessive gene. WES revealed an autosomal recessive polynucleotide kinase 3´phosphatase (PNKP) gene abnormality in the siblings with polyneuropathy. DISCUSSION: Infantile-onset CFD was characterized by serum FR autoantibodies as its predominant pathology whereas pathogenic FOLR1 gene mutations were absent. Homozygosity mapping excluded autosomal recessive inheritance of any single responsible gene. WES in one consanguineous family identified a PNKP gene abnormality that explained the polyneuropathy and also its contribution to the infantile CFD syndrome because the PNKP gene plays a dual role in both neurodevelopment and immune-regulatory function. Further research for candidate genes predisposing to FRα-autoimmunity is suggested to include X-chromosomal and non-coding DNA regions.

Traditional Chinese Medicine Curcumin Sensitizes Human Colon Cancer to Radiation by Altering the Expression of DNA Repair-related Genes.

BACKGROUND/AIM: The aim of the present study was to investigate the radio-sensitizing efficacy of curcumin, a traditional Chinese medicine (TCM) on colon cancer cells in vitro and in vivo. MATERIALS AND METHODS: Human colon cancer HT-29 cells were treated with curcumin (2.5 µM), irradiation (10 Gy) and the combination of irradiation and curcumin. Cell proliferation was assessed using the MTT assay. Apoptotic cells were detected by Annexin V-PE/7-AAD analysis. PCR was performed to determine differential-expression profiling of 95 DNA-repair genes in irradiated cells and cells treated with both irradiation and curcumin. Differentially-expressed genes were confirmed by Western blotting. In vivo radio-sensitizing efficacy of curcumin was assessed in a xenograft mouse model of HT-29 colon cancer. Curcumin was administrated daily by intraperitoneal injection at 20 mg/kg/dose. Mice received irradiation (10 Gy) twice weekly. Apoptosis of the cancer cells following treatment was determined by TUNEL staining. RESULTS: Irradiation induced proliferation inhibition and apoptosis of HT-29 cells in vitro. Concurrent curcumin treatment sensitized the HT-29 tumor to irradiation (p<0.01). DNA repair-related genes CCNH and XRCC5 were upregulated and LIG4 and PNKP downregulated by the combination of curcumin and irradiation compared with irradiation alone (p<0.05). Combined treatment of curcumin and irradiation resulted in a significantly greater tumor-growth inhibition and apoptosis compared to irradiation treatment alone (p<0.01). CONCLUSION: Curcumin sensitizes human colon cancer in vitro and in vivo to radiation. Downregulation of LIG4 and PNKP and upregulation of XRCC5 and CCNH DNA-repair-related genes were involved in the radio-sensitizing efficacy of curcumin in colon cancer.

Tanshinone IIA induces apoptosis via inhibition of Wnt/ß­catenin/MGMT signaling in AtT­20 cells.

A strategy to suppress the expression of the DNA repair enzyme O6­methylguanine­DNA methyltransferase (MGMT) by inhibition of Wnt/ß­catenin signaling may be useful as a novel treatment for pituitary adenoma. Previous studies have reported that Tanshinone IIA (TSA), a major quinone compound isolated from Salvia miltiorrhiza, had antitumor effects. However, whether TSA has antitumor effects against pituitary adenoma and whether the mechanisms are associated with the Wnt/ß­catenin/MGMT pathway remains to be clarified. In the present study, TSA treatment caused apoptosis in AtT­20 cells in a concentration­dependent manner, as demonstrated by cell viability reduction, phophatidylserine externalization detected by Annexin V staining and mitochondrial membrane potential disruption detected by JC­1 staining, which were associated with activation of caspase­3 and DNA fragmentation detected by TUNEL in AtT­20 cells. T­cell factor (TCF)­lymphoid­enhancing factor (LEF) reporter activity was determined by dual luciferase reporter assay and the interaction between ß­catenin and TCF­4 were detected using a co­immunoprecipitation kit. The results indicated TSA treatment increased ß­catenin phosphorylation, inhibited ß­catenin nuclear translocation, reduced ß­catenin/TCF­4 complex formation and TCF­LEF luciferase reporter activity, and subsequently reduced the expression of cyclin D1 and MGMT. Notably, overexpression of MGMT in ß­catenin knock down AtT­20 cells abrogated the TSA­mediated effects in AtT­20 cells. In conclusion, TSA induced apoptosis via inhibition of Wnt/ß­catenin­dependent MGMT expression, which may provide novel insights into the understanding of the mechanism of the antitumor effects of Salvia miltiorrhiza.

Olea europaea Leaf Extract Improves the Efficacy of Temozolomide Therapy by Inducing MGMT Methylation and Reducing P53 Expression in Glioblastoma.

Unmethylated O6-methylguanine-DNA-methyltransferase (MGMT) promoter leads to Temozolomide (TMZ) resistance in most of the glioblastoma multiforme (GBM) patients. We previously investigated the synergistic effect of Olea europaea leaf extract (OLE) on TMZ cytotoxicity through modulating microRNA expression. To date, knowledge about the effect of OLE on MGMT methylation is insufficient. The aim of the current study was to evaluate the potential modulating effect of OLE on the TMZ response of GBM tumors through MGMT methylation. Exposure to 1 mg/mL OLE caused a significant induction of CpG island methylation in the MGMT gene using Methyl quantitative PCR assay (P < 0.001). In WST-1 analysis, the use of 350 µM TMZ plus 1 mg/mL OLE significantly increased the TMZ response of MGMT unmethylated cells (P = 0.003). Using the comet assay, the impact of 1 mg/mL OLE plus 350 µM TMZ on the formation of DNA strand breaks was significantly higher than that of 450 µM TMZ alone (P < 0.001) and Western blot analysis revealed that, when cells are treated with 1-mg/mL OLE, the total p53 protein levels tended to decrease. The results presented in this study uniquely demonstrated that OLE synergistically increased the TMZ response of GBM tumors by regulating MGMT gene methylation and p53 expression. However, further studies to validate our findings are required.

Low-intensity red and infrared lasers affect mRNA expression of DNA nucleotide excision repair in skin and muscle tissue.

Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols.

Lipoic acid inhibits the DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) and triggers its depletion in colorectal cancer cells with concomitant autophagy induction.

Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy.