Xanthohumol regulates miR-4749-5p-inhibited RFC2 signaling in enhancing temozolomide cytotoxicity to glioblastoma.

AIMS: Xanthohumol (XN), a natural prenylated flavonoid isolated from Humulus lupulus L. (hops), possess the therapeutic effects in glioblastoma multiforme (GBM), which is a grade IV aggressive glioma in adults. However, low bioavailability and extractive yield limit the clinical applications of XN. To comprehensively investigate XN-mediated gene networks in inducing cell death is helpful for drug development and cancer research. Therefore, we aim to identify the detailed molecular mechanisms of XN's effects on exhibiting cytotoxicity for GBM therapy. METHODS AND KEY FINDINGS: XN significantly induced GBM cell death and enhanced temozolomide (TMZ) cytotoxicity, a first-line therapeutic drug of GBM. XN-mediated transcriptome profiles and canonical pathways were identified. DNA repair signaling, a well-established mechanism against TMZ cytotoxicity, was significantly correlated with XN-downregulated genes. Replication factor C subunit 2 (RFC2), a DNA repair-related gene, was obviously downregulated in XN-treated cells. Higher RFC2 levels which occupied poor patient survival were also observed in high grade GBM patients and tumors. Inhibition of RFC2 reduced cell viability, induced cell apoptosis, and enhanced both XN and TMZ cytotoxicity. By intersecting array data, bioinformatic prediction, and in vitro experiments, microRNA (miR)-4749-5p, a XN-upregulated microRNA, was identified to target to RFC2 3'UTR and inhibited RFC2 expression. A negative correlation existed between miR-4749-5p and RFC2 in GBM patients. Overexpression of miR-4749-5p significantly promoted XN- and TMZ-mediated cytotoxicity, and reduced RFC2 levels. SIGNIFICANCE: Consequently, we suggest that miR-4749-5p targeting RFC2 signaling participates in XN-enhanced TMZ cytotoxicity of GBM. Our findings provide new potential therapeutic directions for future GBM therapy.

FoxM1-mediated RFC5 expression promotes temozolomide resistance.

Although methylguanine-DNA-methyltransferase (MGMT) plays an important role in resistance to temozolomide (TMZ) in glioma, 40% of gliomas with MGMT inactivation are still resistant to TMZ. The underlying mechanism is not clear. Here, we report that forkhead box M1 (FoxM1) transcriptionally activates the expression of DNA repair gene replication factor C5 (RFC5) to promote TMZ resistance in glioma cells independent of MGMT activation. We showed that RFC5 expression is positively correlated with FoxM1 expression in human glioma cells and FoxM1 is able to transcriptionally activate RFC expression by interaction with the RFC5 promoter. Furthermore, knockdown of FoxM1 or RFC5 partially re-sensitizes glioma cells to TMZ. Consistently, thiostrepton, a FoxM1 inhibitor, in combination with TMZ significantly inhibits proliferation and promotes apoptosis in glioma cells. Taken together, these findings suggest that the FoxM1-RFC5 axis may mediate TMZ resistance and thiostrepton may serve as a potential therapeutic agent against TMZ resistance in glioma cells.

DNA methylation and expression of the folate transporter genes in colorectal cancer.

Folate has a central role in the cell metabolism. This study aims to explore the DNA methylation pattern of the folate transporter genes FOLR1, PCFT, and RFC1 as well as the corresponding protein expressions in colorectal cancer (CRC) tissue and adjacent non-cancerous mucosa (ANCM). Our results showed statistically significant differences in the DNA-methylated fraction of all three genes at several gene regions; we identified three differentially methylated CpG sites in the FOLR1 gene, five CpG sites in the PCFT gene, and six CpG sites in the RFC1 gene. There was a pronounced expression of the FRα and RFC proteins in both the CRC and ANCM tissues, though the expression was attenuated in cancer compared to the paired ANCM tissues. The PCFT protein was undetectable or expressed at a very low level in both tissue types. Higher methylated fractions of the CpG sites 3-5 in the RFC1 gene were associated with a lower protein expression, suggestive of epigenetic regulation by DNA methylation of the RFC1 gene in the colorectal cancer. Our results did not show any association between the RFC and FRα protein expression and tumor stage, TNM classification, or tumor location. In conclusion, this is the first study to simultaneously evaluate both DNA methylation and protein expression of all three folate transporter genes, FOLR1, PCFT, and RFC1, in colorectal cancer. The results encourage further investigation into the possible prognostic implications of folate transporter expression and DNA methylation.

Overexpression of RFC3 is correlated with ovarian tumor development and poor prognosis.

Replication factor C3 (RFC3) is an oncogene that can potentially predict prognosis in a variety of human cancers. RFC3 expression in ovarian carcinoma has not yet been determined. In this study, we evaluated the messenger RNA (mRNA) and protein expression levels of RFC3 in normal ovarian and ovarian carcinoma tissues using reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blots (WB). Results showed that higher RFC3 mRNA and protein levels were detected in ovarian carcinoma tissues by RT-PCR and WB. High RFC3 expression was defined as positive staining in >70 % of each tumor cell. High RFC3 expression was detected in 28.1, 17.6, 11.1, and 5.0 % of invasive carcinomas, borderline tumors, cystadenomas, and in normal ovary cells, respectively. Overexpression of RFC3 was associated with later pN status (p = 0.001), pM status (p = 0.001), and advanced International Federation of Gynecology and Obstetrics (FIGO) stage (p = 0.012) in ovarian carcinomas. Univariate survival analyses showed that RFC3 overexpression was also associated with shortened patient survival (mean 7.7 months in tumors with RFC3 overexpression vs 92.9 months in tumors with normal RFC3 levels; p < 0.001). In multivariate analyses, RFC3 protein levels were a significant prognostic factor for ovarian carcinoma (p < 0.001). In conclusion, our findings suggest that RFC3 protein is an important and independent biomarker with prognostic implications for patients with ovarian carcinoma.

Post-transcriptional regulation of S-phase genes in the dinoflagellate, Karenia brevis.

Karenia brevis is a toxic dinoflagellate responsible for red tides in the Gulf of Mexico. The molecular mechanisms controlling its cell cycle are important to bloom formation because blooms develop through vegetative cell division. This study identifies a suite of conserved S-phase genes in K. brevis-proliferating cell nuclear antigen (PCNA), ribonucleotide reductase 2, replication factor C, and replication protein A-and characterizes their expression at the mRNA and protein level over the cell cycle. In higher eukaryotes, the expression of these genes is controlled by transcription, activated at S-phase entry by the E2F transcription factor, which ensures their timely availability for DNA synthesis. In the dinoflagellate, these transcripts possess a 5'-transspliced leader sequence, which suggests they may be under post-transcriptional control as demonstrated in trypanosomes. Using quantitative polymerase chain reaction (qPCR), we confirmed that their transcript levels are unchanged over the cell cycle. However, their proteins are maximally expressed during S-phase. This suggests their cell-cycle-dependent expression may be achieved at the level of translation and/or stability. Proliferating cell nuclear antigen further undergoes an increase in size of ∼9 kDa that dominates during S-phase. This coincides with a change in its distribution, with prominent staining of chromatin-bound PCNA occurring during S-phase. We hypothesize that the change in the observed size of PCNA is due to post-translational modification. Together, these studies demonstrate post-transcriptional regulation of S-phase genes in K. brevis. Differential expression of these S-phase proteins may be useful in the development of biomarkers to assess bloom growth status in the field.

Mutational and expressional analysis of RFC3, a clamp loader in DNA replication, in gastric and colorectal cancers.

Parts of gastric (GC) and colorectal cancers (CRC) exhibit microsatellite instability (MSI) that causes frameshift mutations and contributes to cancer development. DNA replication and repair play crucial roles in maintenance of genome stability, and their alterations contribute to cancer development. In this study, we analyzed mutation of RFC1 and RFC3, clamp loaders in DNA replication, in GC and CRC with MSI. We analyzed mononucleotide repeats in RFC1 and RFC3 in 29 GC with high MSI (MSI-H), 20 GC with low MSI (MSI-L), 45 GC with stable MSI (MSS), 35 CRC with MSI-H, 20 CRC with MSI-L, and 45 CRC with MSS by single-strand conformation polymorphism. We also analyzed RFC3 expression in the GC and CRC. We found RFC3 frameshift mutations in 7 GC (24.1%) and 9 CRC with MSI-H (25.7%) but not in cancers with MSI-L or MSS. The mutations consisted of 14 c.244delA, one 243_244delAA, and one c.244dupA, which would result in premature stops of RFC3 amino acid synthesis. Loss of RFC3 expression was observed in 51% of the GC and 65% of the CRC, but all of the cancers with RFC3 frameshift mutations were weak or negative. Our data indicate RFC3 mutation and loss of RFC3 expression occur in large fractions of GC and CRC and suggest that these alterations may contribute to the cancer pathogenesis by deregulating DNA repair and replication.

Dioxin mediates downregulation of the reduced folate carrier transport activity via the arylhydrocarbon receptor signalling pathway.

Dioxins such as 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) are common environmental contaminants known to regulate several genes via activation of the transcription factor aryl hydrocarbon receptor (AhR) associated with the development of numerous adverse biological effects. However, comparatively little is known about the molecular mechanisms by which dioxins display their toxic effects in vertebrates. The 5' untranslated region of the hepatocellular Reduced folate carrier (Rfc1; Slc19a1) exhibits AhR binding sites termed dioxin responsive elements (DRE) that have as yet only been found in the promoter region of prototypical TCDD target genes. Rfc1 mediated transport of reduced folates and antifolate drugs such as methotrexate (MTX) plays an essential role in physiological folate homeostasis and MTX cancer chemotherapy. In order to determine whether this carrier represents a target gene of dioxins we have investigated the influence of TCDD on functional Rfc1 activity in rat liver. Pre-treatment of rats with TCDD significantly diminished hepatocellular Rfc1 uptake activity in a time- and dose-dependent manner. In further mechanistic studies we demonstrated that this reduction was due to TCDD-dependent activation of the AhR signalling pathway. We additionally showed that binding of the activated receptor to DRE motifs in the Rfc1 promoter resulted in downregulation of Rfc1 gene expression and reduced carrier protein levels. As downregulation of pivotal Rfc1 activity results in functional folate deficiency associated with an elevated risk of cardiovascular diseases or carcinogenesis, our results indicate that deregulation of this essential transport pathway represents a novel regulatory mechanism how dioxins display their toxic effects through the Ah receptor.