Pterostilbene inhibits hepatocellular carcinoma proliferation and HBV replication by targeting ribonucleotide reductase M2 protein.

Hepatocellular carcinoma (HCC), one of the most deadly diseases all around the world. HBV infection is a causative factor of HCC and closely associated with HCC development. Ribonucleotide reductase (RR) is a key enzyme for cellular DNA synthesis and RR small subunit M2 (RRM2) is highly upregulated in HCC with poor survival rates. We have previously shown that HBV can activate the expression of RRM2 and the activity of RR enzyme for the viral DNA replication in host liver cells. Thus, RRM2 may be an important therapeutic target for HCC and HBV-related HCC. Pterostilbene, a natural plant component, potently inhibited in vitro RR enzyme activity with the IC50 of about 0.62 µM through interacting with RRM2 protein, which was much higher than current RRM2 inhibitory drugs. Pterostilbine inhibited cell proliferation with an MTT IC50 of about 20-40 µM in various HCC cell lines, causing DNA synthesis inhibition, cell cycle arrest at S phase, and accordingly apoptosis. On the other hand, the compound significantly inhibited HBV DNA replication in HBV genome integrated and newly transfected HCC cells, and the EC50 for inhibiting HBV replication was significantly lower than the IC50 for inhibiting HCC proliferation. Notably, pterostilbene possessed a similar inhibitory activity in sorafenib and lamivudine resistant HCC cells. Moreover, the inhibitory effects of pterostilbine against HCC proliferation and HBV replication were significantly reversed by addition of dNTP precursors, suggesting that RR was the intracellular target of the compound. Finally, pterostilbine effectively inhibited HCC xenograft growth with a relatively low toxicity in nude mouse experiments. This study demonstrates that pterostilbene is a novel potent RR inhibitor by targeting RRM2. It can simultaneously inhibit HCC proliferation and HBV replication with a potential new use for treatment of HCC and HBV-related HCC.

MicroRNA-98-5p targeting ribonucleotide reductase small subunit M2 regulating cisplatin resistance in cervical cancer cells / 解剖学报

Objective To investigate the regulation and mechanism of microRNA (miR)-98-5p on cisplatin sensitivity in cisplatin-resistant cervical cancer cells. Methods The cisplatin(DDP) +miR-NC group (transfected miR- NC), DDP + miR-98-5p group (transfected miR-98-5p mimics), DDP + si-NC group (transfected si-NC), DDP + si- ribonucleotide reductase subunit M2 (RRM2) group (transfected si-RRM2), DDP + miR-98-5p + pcDNA group (co- transfected miR-98-5p mimics and pcDNA), DDP + miR-98-5p + pcDNA-RRM2 group (co-transfected miR-98-5p mimics and pcDNA-RRM2) were transfected into HeLa/DDP cells by liposome method. Real-tim PCR, Western blotting, CCK-8, Transwell chamber and dual luciferase reports gene detection assay were used to detect the expression of miR-98-5p, RRM2, cyclin Dl, P21, matrix metalloproteinase (MMP)-2 and MMP-9 in cells, inhibition rate, half inhibitory concentration(IC

Downregulation of RRM2 Attenuates Retroperitoneal Liposarcoma Progression via the Akt/mTOR/4EBP1 Pathway: Clinical, Biological, and Therapeutic Significance.

BACKGROUND: Retroperitoneal liposarcoma (RLPS) is a rare tumor with high recurrence rate. Ribonucleotide reductase small subunit M2 (RRM2) protein is essential for DNA synthesis and replication. Our previous study has demonstrated that RRM2 downregulation inhibited the proliferation of RLPS cells, but further association between RRM2 and RLPS and relevant mechanisms remains to be explored. METHODS: RRM2 expression was evaluated in RLPS tumor tissues and cell lines by using real-time PCR and immunohistochemical analysis. The effect of RRM2 downregulation on cell proliferation, apoptosis, cell cycle, cell migration and invasion was tested by lentivirus. The effect of RRM2 inhibition on tumor growth in vivo was assessed by using patient-derived tumor xenograft (PDX) of RLPS and RRM2 inhibitor. The underlying mechanisms of RRM2 in RLPS were explored by protein microarray and Western blotting. RESULTS: The results showed that RRM2 mRNA expression was higher in RLPS tissues than in normal fatty tissues (P<0.001). RRM2 expression was higher in the dedifferentiated, myxoid/round cell, and pleomorphic subtypes (P=0.027), and it was also higher in the high-grade RLPS tissues compared to that in the low-grade RLPS tissues (P=0.004). There was no correlation between RRM2 expression and overall survival (OS) or disease-free survival (DFS) in this group of RLPS patients (P>0.05). RRM2 downregulation inhibited cell proliferation, promoted cell apoptosis, facilitated cell cycle from G1 phase to S phase and inhibited cell migration and invasion. Inhibition of RRM2 suppressed tumor growth in NOD/SCID mice. Protein microarray and Western blot verification showed that activity of Akt/mammalian target of rapamycin/eukaryotic translation initiation factor 4E binding protein 1 (Akt/mTOR/4EBP1) pathway was downregulated along with RRM2 downregulation. CONCLUSION: RRM2 was overexpressed in RLPS tissues, and downregulation of RRM2 could inhibit RLPS progression. In addition, suppression of RRM2 is expected to be a promising treatment for RLPS patients.

Ribonucleotide reductase subunit M2B deficiency leads to mitochondrial permeability transition pore opening and is associated with aggressive clinicopathologic manifestations of breast cancer.

Ribonucleotide reductase small subunit M2B (RRM2B) plays an essential role in maintaining mitochondrial homeostasis. Mitochondrial permeability transition pore (MPTP) is a key regulator of mitochondrial homeostasis. MPTP contributes to cell death and is crucial in cancer progression. RRM2B's relation to MPTP is not well known, and the role of RRM2B in cancer progression is controversial. Here, our aim was to study the role of RRM2B in regulating MPTP and the association between RRM2B and clinicopathological manifestations in breast cancer. Analysis of Rrm2b-/- mice cells found changes consistent with MPTP opening, including mitochondrial swelling and upregulation of cyclophilin D (CypD), a protein that activates MPTP opening. Silencing of RRM2B gene expression in MCF7 and KB cell lines led to MPTP opening. Accordingly, dysfunctional oxidative phosphorylation and elevated superoxide levels were also detected in RRM2B-silenced MCF7 and KB cell lines, which was consistent with the findings by gene set enrichment analysis of 159 breast cancer cases that genes involving respiratory electron transport were enriched in high-RRM2B breast cancer, and genes involving biologic oxidation were enriched in low-RRM2B breast cancers. A metabolomic study revealed that spermine levels in RRM2B-silenced MCF7 and KB cells were only 5% and 8% of control levels, respectively. Addition of exogenous spermine to RRM2B-silenced MCF7 and KB cells was able to reverse the MPTP opening induced by RRM2B deficiency. These results suggest that RRM2B may induce MPTP opening through reducing spermine levels. Immunohistochemical analysis of 148 breast cancer cases showed that RRM2B and CypD protein levels were inversely correlated in breast cancer specimens (P<0.05), so were their associated clinicopathologic parameters that high-level RRM2B expression was associated with better clinicopathological features. We conclude that RRM2B deficiency leads to MPTP opening mediated by spermine. Coupling of low RRM2B and high CypD expression is associated with aggressive manifestations of breast cancer.

CREB1 directly activates the transcription of ribonucleotide reductase small subunit M2 and promotes the aggressiveness of human colorectal cancer.

As the small subunit of Ribonucleotide reductase (RR), RRM2 displays a very important role in various critical cellular processes such as cell proliferation, DNA repair, and senescence, etc. Importantly, RRM2 functions like a tumor driver in most types of cancer but little is known about the regulatory mechanism of RRM2 in cancer development. In this study, we found that the cAMP responsive element binding protein 1 (CREB1) acted as a transcription factor of RRM2 gene in human colorectal cancer (CRC). CREB1 directly bound to the promoter of RRM2 gene and induced its transcriptional activation. Knockdown of CREB1 decreased the expression of RRM2 at both mRNA and protein levels. Moreover, knockdown of RRM2 attenuated CREB1-induced aggressive phenotypes of CRC cells in vitro and in vivo. Analysis of the data from TCGA database and clinical CRC specimens with immunohistochemical staining also demonstrated a strong correlation between the co-expression of CREB1 and RRM2. Decreased disease survivals were observed in CRC patients with high expression levels of CREB1 or RRM2. Our results indicate CREB1 as a critical transcription factor of RRM2 which promotes tumor aggressiveness, and imply a significant correlation between CREB1 and RRM2 in CRC specimens. These may provide the possibility that CREB1 and RRM2 could be used as biomarkers or targets for CRC diagnosis and treatment.

ATR-CHK1-E2F3 signaling transactivates human ribonucleotide reductase small subunit M2 for DNA repair induced by the chemical carcinogen MNNG.

BACKGROUND: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent and an environmental carcinogen, causes DNA lesions and even carcinomas. DNA damage responses induced by MNNG activate various DNA repair genes and related signaling pathways. The present study aimed to investigate the regulatory mechanisms of human RR small subunit M2 (hRRM2) in response to MNNG. RESULTS: In this study, we demonstrated that the RRM2 gene was transactivated by MNNG exposure more strongly than the other small subunit, p53R2. The upregulated RRM2 translocated to the nucleus for DNA repair. Further study showed that E2F3 transactivated RRM2 expression by directly binding to its promoter after MNNG exposure. The transactivation was enhanced by the upregulation of NFY, which bound to the RRM2 promoter adjacent to the E2F3 binding site and interacted with E2F3. In response to MNNG treatment, E2F3 accumulated mainly through its phosphorylation at S124 and was dependent on ATR-CHK1 signaling. In comparison, p53R2 played a relatively weaker role in the MNNG-induced DNA damage response, and its transcription was regulated by the ATR-CHK2-E2F1/p53 pathway. CONCLUSIONS: We suggest that MNNG-stimulated ATR/CHK1 signaling stabilizes E2F3 by S124 phosphorylation, and then E2F3 together with NFY co-transactivate RRM2 expression for DNA repair. GENERAL SIGNIFICANCE: We propose a new mechanism for RRM2 regulation to maintain genome stability in response to environmental chemical carcinogens.

Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein.

Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment.

E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2.

As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease.

Correlation between RRM2 Expression Level of Cervical Cancer Cell Line Siha and C33a and Cell Sensi-tivity to Gemcitabine / 中国药房

OBJECTIVE:To study the correlation between RRM2 expression of cervical cancer cell line C33a and Siha and cell sensitivity to gemcitabine(Gem). METHODS:C33a and Siha were treated with 0.1-3.2 μmol/L and 0.5-512 μmol/L of gemcitabine respectively for 72 h;cell viability was measured by MTT assay to calculate the value of IC50. The expression of RRM2 was mea-sured by Western blot and RT-PCR. Siha cell was treated with gradient concentration and large dose of Gem to establish Siha/Gem drug-resistant cell line. RNA interference technology knockdown the expression of RRM2 in Siha/Gem cell,and IC50 of Gem to cell was determined before and after knockdown. RESULTS:The IC50 values of Gem to Siha and C33a were 16.8 μmol/L and 0.63μmol/L. Compared with C33a cells,the expression of RRM2 in Siha cell was higher. Compared with Siha cells,Siha/Gem drug-re-sistant cell(drug resistant index of 16.26)showed higher RRM2 expression. Siha/Gem drug-resistant cell knockdown RRM2,IC50 of Gem to it was decreased,and inverse drug resistant times was 4.24. CONCLUSIONS:There is an negative correlation between RRM2 expression and Gem sensitivity in cervical cancer cell lines. The knockdown of RRM2 in Siha/Gem increases the sensitivity to Gem.

The conserved Lys-95 charged residue cluster is critical for the homodimerization and enzyme activity of human ribonucleotide reductase small subunit M2.

Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G1/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells.