Pentagamavunone-1 inhibits aggressive breast cancer cell proliferation through mitotic catastrophe and ROS-mediated activities: in vitro and in vivo studies.

Pentagamavunone-1 (PGV-1), an analog of curcumin, has been studied for its cytotoxic effects in 4T1, MCF7, MCF7/HER2, and T47D breast cancer cells. Its antiproliferative effect is partly mediated through G2/M arrest; however, its molecular mechanism during cell cycle progression remains unknown. In this study, we aimed to determine whether PGV-1 has any anticancer effects on highly aggressive breast cancer cells, with a focus on cell cycle regulatory activity, reactive oxygen species (ROS) generation, and their mediated effects on cancer cells. MDA-MB-231 (triple-negative) and HCC1954 (overexpressed HER2) immortalized human breast cancer cells were used in the study. PGV-1 exhibited cytotoxic activity with an irreversible antiproliferative impact on treated cells and had good selectivity when tested in fibroblast cells. Oral PGV-1 administration suppressed tumor growth in a cell-derived xenograft mouse model. PGV-1 induced the phosphorylation of Aurora A kinase and PLK1 in MDA-MB-231 cells, while PLK1 and cyclin B1 phosphorylation were enhanced in the PGV-1-treated HCC1954 cells during prometaphase arrest. Intracellular ROS production was substantially higher upon PGV-1 treatment following mitotic arrest, and this activity caused impairment of mitochondrial respiration, induced senescence, and subsequently triggered early-to-late apoptosis. Collectively, these results suggest that the molecular mechanism of PGV-1 involves the regulation of mitotic kinases to cause cell cycle arrest and the enhancement of ROS production to impair mitochondrial activity and induce cellular senescence. The therapeutic activities demonstrated by PGV-1 in this study show its potential as an appealing candidate for chemotherapy in breast cancer treatment.

Preclinical evaluation of pentagamavunone-1 as monotherapy and combination therapy for pancreatic cancer in multiple xenograft models.

We previously reported that pentagamavunone-1 (PGV-1) effectively inhibited cell proliferation in many types of human tumors, including pancreatic cancer, by inducing M phase (prometaphase) arrest, senescence, and apoptosis with few side effects. However, a detailed evaluation of the effects of PGV-1 on pancreatic cancer cells in an in vivo setting has not yet been conducted. The present study investigated the potential efficacy of PGV-1 as both monotherapy and combination therapy for pancreatic cancer using multiple xenograft mouse assays. A cell-line derived xenograft model (CDX-M) with pancreatic cancer cell line and a patient-derived xenograft mouse model (PDX-M) using resected pancreatic cancer samples without neoadjuvant chemotherapy were established in both heterotopic and orthotopic manners. PGV-1 effectively suppressed tumor formation at the heterotopic and orthotopic sites in CDX-M than in untreated mice. Combination therapy with PGV-1 and gemcitabine more effectively suppressed tumor formation than monotherapy with PGV-1 or gemcitabine when administered after tumor formation. Monotherapy with PGV-1 or gemcitabine less effectively suppressed tumor formation in PDX-M than in CDX-M, whereas combination therapy with PGV-1 and gemcitabine more effectively suppressed tumor formation. PGV-1 as monotherapy and combination therapy with gemcitabine effectively inhibited tumor formation and has potential as an anticancer candidate for pancreatic cancer.

Stabilization of fatty acid synthesis enzyme acetyl-CoA carboxylase 1 suppresses acute myeloid leukemia development.

Cancer cells reprogram lipid metabolism during their malignant progression, but limited information is currently available on the involvement of alterations in fatty acid synthesis in cancer development. We herein demonstrate that acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme for fatty acid synthesis, plays a critical role in regulating the growth and differentiation of leukemia-initiating cells. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPA, a transcription factor regulating myeloid differentiation, for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). We identified ACC1 as a target of the Trib1-COP1 complex and found that an ACC1 mutant resistant to degradation because of the lack of a Trib1-binding site attenuated complex-driven leukemogenesis. Stable ACC1 protein expression suppressed the growth-promoting activity and increased ROS levels with the consumption of NADPH in a primary bone marrow culture, and delayed the onset of AML with increases in mature myeloid cells in mouse models. ACC1 promoted the terminal differentiation of Trib1-COP1-expressing cells and eradicated leukemia-initiating cells in the early phase of leukemic progression. These results indicate that ACC1 is a natural inhibitor of AML development. The upregulated expression of the ACC1 protein has potential as an effective strategy for cancer therapy.

Curcumin Derivatives Verify the Essentiality of ROS Upregulation in Tumor Suppression.

BACKGROUND: Curcumin has been shown to exert pleiotropic biological effects, including anti-tumorigenic activity. We previously showed that curcumin controls reactive oxygen species (ROS) levels through the ROS metabolic enzymes, to prevent tumor cell growth. In this study, we synthesized 39 novel curcumin derivatives and examined their anti-proliferative and anti-tumorigenic properties. METHODS AND RESULTS: Thirty-nine derivatives exhibited anti-proliferative activity toward human cancer cell lines, including CML-derived K562 leukemic cells, in a manner sensitive to an antioxidant, N-acetyl-cysteine (NAC). Some compounds exhibited lower GI50 values than curcumin, some efficiently induced cell senescence, and others markedly increased ROS levels, efficiently induced cell death and suppressed tumor formation in a xenograft mouse model, without any detectable side effects. A clustering analysis of the selected compounds and their measurement variables revealed that anti-tumorigenic activity was most well-correlated with an increase in ROS levels. Pulldown assays and a molecular docking analysis showed that curcumin derivatives competed with co-enzymes to bind to the respective ROS metabolic enzymes and inhibited their enzymatic activities. CONCLUSIONS: The analysis of novel curcumin derivatives established the importance of ROS upregulation in suppression of tumorigenesis, and these compounds are potentially useful for the development of an anti-cancer drug with few side effects.

Pentagamavunon-1 (PGV-1) inhibits ROS metabolic enzymes and suppresses tumor cell growth by inducing M phase (prometaphase) arrest and cell senescence.

We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible growth arrest, and causes apoptosis. In this study, we tested Pentagamavunon-1 (PGV-1), a molecule related to curcumin, for its inhibitory activity on tumor cells in vitro and in vivo. PGV-1 exhibited 60 times lower GI50 compared to that of curcumin in K562 cells, and inhibited the proliferation of cell lines derived from leukemia, breast adenocarcinoma, cervical cancer, uterine cancer, and pancreatic cancer. The inhibition of growth by PGV-1 remained after its removal from the medium, which suggests that PGV-1 irreversibly prevents proliferation. PGV-1 specifically induced prometaphase arrest in the M phase of the cell cycle, and efficiently induced cell senescence and cell death by increasing intracellular ROS levels through inhibition of ROS-metabolic enzymes. In a xenograft mouse model, PGV-1 had marked anti-tumor activity with little side effects by oral administration, whereas curcumin rarely inhibited tumor formation by this administration. Therefore, PGV-1 is a potential therapeutic to induce tumor cell apoptosis with few side effects and low risk of relapse.

Anti-proliferative and Anti-metastatic Potential of Curcumin Analogue, Pentagamavunon-1 (PGV-1), Toward Highly Metastatic Breast Cancer Cells in Correlation with ROS Generation.

Purpose: Pentagamavunon-1 (PGV-1) is a curcumin analogue that shows cytotoxic activity in various cancer cells. In this study, we evaluated the effect of PGV-1 on a highly metastatic breast cancer cell line, the 4T1 cells, as an anti-metastatic and anti-proliferative agent. Methods: Cell viability was evaluated using MTT assay; while cell cycle profile, apoptosis incidence, and ROS intracellular level were determined by flow cytometry. Cell senescence was observed under senescence-associated-ß-galactosidase (SA-ß-gal) staining assay. The expression of matrixmetalloproteinase-9 (MMP-9) was determined using immunoreaction based-ELISA, while other proteins expression were detected using immunoblotting. Results: Curcumin and PGV-1 showed cytotoxic effects on 4T1 cells with IC50 value of 50 and 4 µM, respectively. The cytotoxic activity of PGV-1 was correlated to the induction of G2/M cell cycle arrest and cell senescence. Furthermore, PGV-1 increased the accumulation of intracellular ROS level. We also revealed that PGV-1 bound to several ROS-metabolizing enzymes, including glyoxalase I (GLO1), peroxiredoxin 1 (PRDX1), N-ribosyldihydronicotinamide: quinone reductase 2 (NQO2), aldo-keto reductase family 1 member c1 (AKR1C1). As an antimetastatic agent, PGV-1 showed less inhibitory effect on cell migration compared to curcumin. However, PGV-1 significantly decreased MMP-9 protein expression in a dose-dependent manner suggesting it still potent to inhibit metastatic cells. Conclusion: Overall, our findings suggest that PGV-1 is potential to be developed as an antiproliferative and anti-metastatic agent.

Curcumin targets multiple enzymes involved in the ROS metabolic pathway to suppress tumor cell growth.

Curcumin has been reported to exhibit anti-tumorigenic activity; however, since its precise actions remain unclear, its effects are considered to be deceptive. In the present study, we confirmed the anti-tumorigenic effects of curcumin on CML-derived leukemic cells in a xenograft model and in vitro culture system. In vitro pull-down and mass analyses revealed a series of enzymes (carbonyl reductase, glutathione-S-transferase, glyoxalase, etc.) that function in a reactive oxygen species (ROS) metabolic pathway as curcumin-binding targets, the expression of which was up-regulated in human leukemia. Curcumin increased ROS levels over the threshold in leukemic cells, and the antioxidant, glutathione (GSH) and overexpression of curcumin-binding enzymes partially mitigated the up-regulation of ROS and growth inhibition caused by curcumin. These results show that curcumin specifically inhibits tumor growth by increasing ROS levels over the threshold through the miscellaneous inhibition of ROS metabolic enzymes. Curcumin has potential in therapy to regulate ROS levels in tumor cells, thereby controlling tumor growth.

Myeloid leukemia factor 1 stabilizes tumor suppressor C/EBPα to prevent Trib1-driven acute myeloid leukemia.

C/EBPα is a key transcription factor regulating myeloid differentiation and leukemogenesis. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPα for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). Here we show that myeloid leukemia factor 1 (MLF1) stabilizes C/EBPα protein levels by inhibiting the ligase activity of the Trib1-COP1 complex. MLF1 directly interacts with COP1 in the nucleus and interferes with the formation of the Trib1-COP1 complex, thereby blocking its ability to polyubiquitinate C/EBPα for degradation. MLF1 overexpression suppressed the Trib1-induced growth advantage in a murine bone marrow (BM) culture and Trib1-induced AML development in BM-transplanted mouse models. MLF1 was expressed in hematopoietic stem cells and myeloid progenitors (common myeloid progenitors and granulocyte-macrophage progenitors) in normal hematopoiesis, which is consistent with the distribution of C/EBPα. An MLF1 deficiency conferred a more immature phenotype on Trib1-induced AML development. A higher expression ratio of Trib1 to MLF1 was a key determinant for AML development in mouse models, which was also confirmed in human patient samples with acute leukemia. These results indicate that MLF1 is a positive regulator that is critical for C/EBPα stability in the early phases of hematopoiesis and leukemogenesis.

Homozygous inactivation of CHEK2 is linked to a familial case of multiple primary lung cancer with accompanying cancers in other organs.

In clinical practice, there are a number of cancer patients with clear family histories, but the patients lack mutations in known familial cancer syndrome genes. Recent advances in genomic technologies have enhanced the possibility of identifying causative genes in such cases. Two siblings, an elder sister and a younger brother, were found to have multiple primary lung cancers at the age of 60. The former subsequently developed breast cancer and had a history of uterine myoma. The latter had initially developed prostate cancer at the age of 59 and had a history of colon cancer. Single-nucleotide polymorphism (SNP) genotyping revealed that ∼10% of the genomes were homozygous in both patients. Exome sequencing revealed nonsynonymous mutations in five genes in the runs of homozygosity: CHEK2, FCGRT, INPP5J, MYO18B, and SFI1. Evolutionary conservation of primary protein structures suggested the functional importance of the CHEK2 mutation, p.R474C. This mutation altered the tertiary structure of CHK2 by disrupting the salt bridge between p.R474 and p.E394. No such structural changes were observed with the other mutated genes. Subsequent cell-based transfection analysis revealed that CHK2 p.R474C was unstable and scarcely activated. We concluded that the homozygous CHEK2 variant was contributory in this case of familial cancer. Although homozygous inactivation of CHEK2 in mice led to cancers in multiple organs, accumulation of additional human cases is needed to establish its pathogenic role in humans.

COP1 targets C/EBPα for degradation and induces acute myeloid leukemia via Trib1.

The ubiquitin ligase constitutively photomorphogenic 1 (COP1) is involved in many biological responses in mammalian cells, but its role in tumorigenesis remains unclear. Here we show that COP1 is a ubiquitin ligase for the tumor suppressor CCAAT/enhancer-binding protein (C/EBPα) and promotes its degradation in vivo, thereby blocking myeloid differentiation of hematopoietic cells for tumorigenesis. In this process, mammalian homolog of Tribbles, Trib1, which contains a COP1-binding motif, is essential for down-regulation of C/EBPα expression. Murine bone marrow transplantation experiments showed that coexpression of COP1 accelerates development of acute myeloid leukemia induced by Trib1, which pathologically resembles that of p42C/EBPα-deficient mice. Interestingly, coexpression of ligase activity-deficient COP1 mutant abrogated Trib1-induced leukemogenesis. These results indicate that COP1 and Trib1 act as an oncoprotein complex functioning upstream of C/EBPα, and its ligase activity is crucial for leukemogenesis.

The COP1 E3-ligase interacts with FIP200, a key regulator of mammalian autophagy.

BACKGROUND: The ubiquitin ligase COP1, COnstitutively Photomorphogenic 1, functions in many biological responses in mammalian cells, but its downstream pathway remains unclear. RESULTS: Here, we identified FIP200, a key regulator of mammalian autophagy, as a novel COP1-interacting protein by yeast two-hybrid screening. The interaction was confirmed by a GST-pulldown assay. Split-GFP analysis revealed that interaction between COP1 and FIP200 predominantly occurred in the cytoplasm and was enhanced in cells treated with UV irradiation. Different forms of FIP200 protein were expressed in cultured mammalian cells, and ectopic expression of COP1 reduced one of such forms. CONCLUSIONS: These data suggest that COP1 modulates FIP200-associated activities, which may contribute to a variety of cellular functions that COP1 is involved in.

CSN5 specifically interacts with CDK2 and controls senescence in a cytoplasmic cyclin E-mediated manner.

The fifth component (CSN5) of the mammalian COP9 signalosome complex plays an essential role in cell proliferation and senescence, but its molecular mediator remains to be determined. Here, we searched for interactors among various cell cycle regulators, and found that CSN5, but not the CSN holo-complex, bound to CDK2 in vivo and in vitro. Depletion of CSN5 enhanced phosphorylation of CDK2 by Akt, resulting in cytoplasmic accumulation of CDK2 together with cyclin E in a leptomycin B-resistant manner, and impaired phosphorylation of the retinoblastoma protein. Additional knockdown of CDK2, which reduced the expression of cyclin E to the normal level, did not restore cell proliferation, but significantly suppressed senescence in CSN5-depleted cells. Enforced expression of cytoplasmic cyclin E induced premature senescence in immortalized cell lines. These results show that CSN5 functions through CDK2 to control premature senescence in a novel way, depending on cyclin E in the cytoplasm.

Depletion of CSN5 inhibits Ras-mediated tumorigenesis by inducing premature senescence in p53-null cells.

The mammalian COP9 signalosome (CSN) complex is involved in cell transformation, but its molecular mechanism remains undetermined. Here we show that disruption of the fifth component (CSN5) prevented the formation of tumors by p53-null cells transformed with an active form of Ras in subcutaneously injected mice. Depletion of CSN5 suppressed cell proliferation, and induced premature senescence characterized by upregulation of senescence-associated-ß-galactosidase activity and increased expression of CDK inhibitors. CSN5-depleted cells exhibited enhanced activation of the PI3 kinase-Akt pathway, and chemical inhibition of this pathway reduced the level of senescence. Thus, CSN5 is suggested to be a novel target in cancer therapy and for drugs against tumor cells harboring mutated p53.

CSN5/Jab1 controls multiple events in the mammalian cell cycle.

The COP9 signalosome (CSN) complex is critical for mammalian cell proliferation and survival, but it is not known how the CSN affects the cell cycle. In this study, MEFs lacking CSN5/Jab1 were generated using a CRE-flox system. MEFs ceased to proliferate upon elimination of CSN5/Jab1. Rescue experiments indicated that the JAMM domain of CSN5/Jab1 was essential. CSN5/Jab1-elimination enhanced the neddylation of cullins 1 and 4 and altered the expression of many factors including cyclin E and p53. CSN5/Jab1-elimination inhibited progression of the cell cycle at multiple points, seemed to initiate p53-independent senescence and increased the ploidy of cells. Thus, CSN5/Jab1 controls different events of the cell cycle, preventing senescence and endocycle as well as the proper progression of the somatic cell cycle.

New twist in the regulation of cyclin D1.

Among the cell cycle-related mammalian cyclins, cyclin D1 is more closely connected with cell proliferation in response to extracellular signals than the cell cycle clock itself. Because both its mRNA and protein are labile, the intracellular abundance of cyclin D1 is thought to be largely regulated at the level of transcription. However, recent findings suggest that, in certain cell types, cyclin D1 is post-translationally regulated, and a disturbance of this regulatory mechanism induces aberrant entry into the cell cycle and proliferation, sometimes leading to diseases such as cancer. In this review, we summarize recent findings and discuss the physiological role and cellular function of the novel mechanism of regulation of cyclin D1 in terms of the control of cell proliferation.

Mammalian COP9 signalosome.

The COP9 signalosome (CSN) complex is highly conserved from yeast to human. Although the plant CSN was first identified as a negative regulator of photomorphogenesis, the mammalian CSN is linked to different biological responses such as checkpoint control, signal transduction, development and the cell cycle. Frequent over-expression of the CSN subunit in a variety of human cancers suggests its involvement in cell transformation and tumorigenesis. The best-known biochemical function associated with the CSN is the control of protein stability via the ubiquitin-proteasome system through regulation of cullin-RING-E3 ubiquitin ligase activity by deneddylation, by controlling the activity of COP1 E3 ligase, or by counteracting ubiquitin-mediated degradation through a CSN-associated deubiquitinating enzyme. In addition to affecting the stability of transcription factors, the CSN may regulate gene transcription by directly associating with chromatin. This review summarizes recent findings and discusses the physiological role and the cellular function of the mammalian CSN in terms of the regulation of cell proliferation.

Isolation and characterization of cytoplasmic cyclin D1 mutants.

To elucidate the mechanism governing the subcellular distribution of cyclin D1 protein, we randomly mutagenized human cyclin D1 cDNA and isolated mutants that encode the protein predominantly located in the cytoplasm. Experiments with Leptomycin B suggested a defect in transportation from the cytoplasm to the nucleus rather than enhanced nuclear exportation. Sequencing revealed that the mutations responsible for the cytoplasmic localization of cyclin D1 resided in the vicinity of the cyclin box, which affected interaction with a catalytic partner, Cdk4. We propose that interaction between cyclin D1 and Cdk4 triggers the mechanism controlling the nuclear transportation of this kinase complex.

Stable form of JAB1 enhances proliferation and maintenance of hematopoietic progenitors.

Overexpression of JAB1 is observed in a variety of human cancers, but how JAB1 is involved in tumor development remained to be investigated. Here we analyzed mice with modified Jab1 expression. Mice ectopically expressing a more stable form of JAB1 protein under the control of a constitutive promoter were rescued from the embryonic lethality caused by the Jab1(-/-) allele and developed a myeloproliferative disorder in a gene dosage-dependent manner. Hematopoietic cells from the bone marrow of Jab1 transgenic mice had a significantly larger stem cell population and exhibited higher and transplantable proliferative potential. In contrast, Jab1(+/-) mice, which express approximately 70% as much JAB1 protein as their wild-type littermates, showed inefficient hematopoiesis. Expression of the tumor suppressor p16(INK4a) was inversely correlated with that of JAB1, and the oncoprotein SMYD3, a newly identified JAB1 interactor, suppressed transcription of p16 in cooperation with JAB1. Thus, the expression and function of JAB1 are critical for the proliferation and maintenance of hematopoietic progenitors.

Small mitochondrial ARF (smARF) is located in both the nucleus and cytoplasm, induces cell death, and activates p53 in mouse fibroblasts.

The ARF transcript produces two proteins, the full-length ARF, p19(ARF), and a short mitochondrial version, smARF. To explore the functional difference between the two, we generated GFP-fused expression vectors for each protein and introduced them into NIH3T3 murine fibroblasts, which sustains a global deletion in the INK4a locus but contains a functional p53 gene. GFP-p19ARF was located within the nucleolus as previously reported, whereas GFP-smARF was detected mainly in the nucleoplasm. GFP-smARF induced cell death although to a slightly lesser extent than p19ARF. GFP-smARF stabilized p53 thereby inducing expression of the target genes, MDM2 and p21. We suggest that smARF has functions other than mitochondria-mediated autophagy, and induces p53 expression and cell death via a novel mechanism.

The myeloid leukemia factor interacts with COP9 signalosome subunit 3 in Drosophila melanogaster.

The human myeloid leukemia factor 1 (hMLF1) gene was first identified as an NPM-hMLF1 fusion gene produced by chromosomal translocation. In Drosophila, dMLF has been identified as a protein homologous to hMLF1 and hMLF2, which interacts with various factors involved in transcriptional regulation. However, the precise cellular function of dMLF remains unclear. To generate further insights, we first examined the behavior of dMLF protein using an antibody specific to dMLF. Immunostaining analyses showed that dMLF localizes in the nucleus in early embryos and cultured cells. Ectopic expression of dMLF in the developing eye imaginal disc using eyeless-GAL4 driver resulted in a small-eye phenotype and co-expression of cyclin E rescued the small-eye phenotype, suggesting the involvement of dMLF in cell-cycle regulation. We therefore analyzed the molecular mechanism of interactions between dMLF and a dMLF-interacting protein, dCSN3, a subunit of the COP9 signalosome, which regulates multiple signaling and cell-cycle pathways. Biochemical and genetic analyses revealed that dMLF interacts with dCSN3 in vivo and glutathione S-transferase pull-down assays revealed that the PCI domain of the dCSN3 protein is sufficient for this to occur, possibly functioning as a structural scaffold for assembly of the COP9 signalosome complex. From these data we propose the possibility that dMLF plays a negative role in assembly of the COP9 signalosome complex.