• Journal of Microbiology and Biotechnology
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• v.33 no.9
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• pp.1206-1212
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• 2023

The Wnt/β-catenin pathway plays essential roles in regulating various cellular behaviors, including proliferation, survival, and differentiation [1-3]. The intracellular β-catenin level, which is regulated by a proteasomal degradation pathway, is critical to Wnt/β-catenin pathway control [4]. Normally, casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), which form a complex with the scaffolding protein Axin and the tumor suppressor protein adenomatous polyposis coli (APC), phosphorylate β-catenin at Ser45, Thr41, Ser37, and Ser33 [5, 6]. Phosphorylated β-catenin is ubiquitinated by the β-transducin repeat-containing protein (β-TrCP), an F-box E3 ubiquitin ligase complex, and ubiquitinated β-catenin is degraded via a proteasome pathway [7, 8]. Colorectal cancer is a significant cause of cancer-related deaths worldwide. Abnormal up-regulation of the Wnt/β-catenin pathway is a major pathological event in intestinal epithelial cells during human colorectal cancer oncogenesis [9]. Genetic mutations in the APC gene are observed in familial adenomatous polyposis coli (FAP) and sporadic colorectal cancers [10]. In addition, mutations in the N-terminal phosphorylation motif of the β-catenin gene were found in patients with colorectal cancer [11]. These mutations cause β-catenin to accumulate in the nucleus, where it forms complexes with transcription factors of the T-cell factor/lymphocyte enhancer factor (TCF/LEF) family to stimulate the expression of β-catenin responsive genes, such as c-Myc and cyclin D1, which leads to colorectal tumorigenesis [12-14]. Therefore, downregulating β-catenin response transcription (CRT) is a potential strategy for preventing and treating colorectal cancer. Plant cytokinins are N⁶-substituted purine derivatives; they promote cell division in plants and regulate developmental pathways. Natural cytokinins are classified as isoprenoid (isopentenyladenine, zeatin, and dihydrozeatin), aromatic (benzyladenine, topolin, and methoxytopolin), or furfural (kinetin and kinetin riboside), depending on their structure [15, 16]. Kinetin riboside was identified in coconut water and is a naturally produced cytokinin that induces apoptosis and exhibits antiproliferative activity in several human cancer cell lines [17]. However, little attention has been paid to kinetin riboside's mode of action. In this study, we show that kinetin riboside exerts its cytotoxic activity against colon cancer cells by suppressing the Wnt/β-catenin pathway and promoting intracellular β-catenin degradation.

• Korean Journal of Medicinal Crop Science
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• v.25 no.5
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• pp.328-334
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• 2017

Background: In this study, we evaluated the anti-cancer activity and potential molecular mechanism of 70% ethanol extracts of the root of Aralia cordata var. continentalis (Kitagawa) Y. C. Chu (RAc-E70) against human colorectal cancer cells. Methods and Results: RAc-E70 suppressed the proliferation of the human colorectal cancer cell lines, HCT116 and SW480. Although RAc-E70 reduction cyclin D1 expression at the protein and mRNA levels, RAc-E70-induced reduction in cyclin D1 protein level occurred more dramatically than that of cyclin D1 mRNA. The RAc-E70-induced downregulation of cyclin D1 expression was attenuated in the presence of MG132. Additionally, RAc-E70 reduced HA-cyclin D1 levels in HCT116 cells transfected with HA-tagged wild type-cyclin D1 expression vector. RAc-E70-mediated cyclin D1 degradation was blocked in the presence of LiCl, a $GSK3{\beta}$ inhibitorbut, but not PD98059, an ERK1/2 inhibitor and SB203580, a p38 inhibitor. Furthermore, RAc-E70 phosphorylated cyclin D1 at threonine-286 (T286), and LiCl-induced $GSK3{\beta}$ inhibition reduced the RAc-E70-mediated phosphorylation of cyclin D1 at T286. Conclusions: Our results suggested that RAc-E70 may downregulate cyclin D1 expression as a potential anti-cancer target through $GSK3{\beta}$-dependent cyclin D1 degradation. Based on these findings, RAc-E70 maybe a potential candidate for the development of chemopreventive or therapeutic agents for human colorectal cancer.

• Molecules and Cells
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• v.38 no.6
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• pp.562-572
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• 2015

We previously reported the role of histone deacetylase 3 (HDAC3) in response to anti-cancer drugs. The decreased expression of HDAC3 in anti-cancer drug-resistant cancer cell line is responsible for the resistance to anti-cancer drugs. In this study, we investigated molecular mechanisms associated with regulation of HDAC3 expression. MG132, an inhibitor of proteasomal degradation, induced the expression of HDAC3 in various anti-cancer drug-resistant cancer cell lines. Ubiquitination of HDAC3 was observed in various anti-cancer drug-resistant cancer cell lines. HDAC3 showed an interaction with SIAH2, an ubiquitin E3 ligase, that has increased expression in various anti-cancer drug-resistant cancer cell lines. miRNA array analysis showed the decreased expression of miR-335 in these cells. Targetscan analysis predicted the binding of miR-335 to the 3'-UTR of SIAH2. miR-335-mediated increased sensitivity to anti-cancer drugs was associated with its effect on HDAC3 and SIAH2 expression. miR-335 exerted apoptotic effects and inhibited ubiquitination of HDAC3 in anti-cancer drug-resistant cancer cell lines. miR-335 negatively regulated the invasion, migration, and growth rate of cancer cells. The mouse xenograft model showed that miR-335 negatively regulated the tumorigenic potential of cancer cells. The down-regulation of SIAH2 conferred sensitivity to anti-cancer drugs. The results of the study indicated that the miR-335/SIAH2/HDAC3 axis regulates the response to anti-cancer drugs.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2699-2703
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• 2012

The functional significance of the proteasome activator $REG{\gamma}$ in the regulation of cell proliferation and apoptosis has been recognized. However, pathological contributions to tumor development remain to be elucidated. Both oncogenic proteins and tumor suppressors are targeted by $REG{\gamma}$ for proteasomal degradation. It has been proposed that the role of the $REG{\gamma}$ in the pathogenesis of cancer is cell- and context-specific. In this study, we aimed to explore the potential involvement of $REG{\gamma}$ in laryngeal carcinomas, comparing protein expression in tumor and adjacent tissues by immunohistochemical staining and Western blot analysis. We also characterized the correlation between the expression of $REG{\gamma}$ and the previously identified substrates p53 and p21. We showed that $REG{\gamma}$ was abnormally highly expressed in cancer tissues. Statistical analysis revealed that there was a positive relationship between the level of $REG{\gamma}$ and the expression of p53 and p21. Our study suggests that $REG{\gamma}$ overexpression can facilitate the growth of laryngeal cancer cells.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.2
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• pp.963-967
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• 2014

Previous studies have suggested anti-tumor effects of asiatic acid in some human cancer cell lines. This agent is reported to increase the levels of $p21^{WAF1/CIP1}$ in human breast cancer cell lines. However, the molecular mechanisms have not been established. Here we report that asiatic acid up-regulates $p21^{WAF1/CIP1}$ protein expression but not the level of $p21^{WAF1/CIP1}$ mRNA in HepG2 human hepatoma cells. Furthermore, we found that the asiatic acid induced increase of $p21^{WAF1/CIP1}$ protein was associated with decreased phosphorylation (ser-146) of $p21^{WAF1/CIP1}$. Knockdown of NDR1/2 kinase, which directly phosphorylates $p21^{WAF1/CIP1}$ protein at ser-146 and enhances its proteasomal degradation, increased the levels of $p21^{WAF1/CIP1}$ protein and eliminated the regulation of $p21^{WAF1/CIP1}$ stability by asiatic acid. At the same time, the expression of NDR1/2 kinase decreased during treatment with asiatic acid in HepG2 cells. Moreover, asiatic acid inhibited the proliferation of HepG2 cells, this being attenuated by knockdown of $p21^{WAF1/CIP1}$. In conclusion, we propose that asiatic acid inhibits the expression NDR1/2 kinase and promotes the stability of $p21^{WAF1/CIP1}$ protein through attenuating NDR1/2 dependent phosphorylation of $p21^{WAF1/CIP1}$ in HepG2 cells.

• BMB Reports
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• v.44 no.1
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• pp.40-45
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• 2011

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects cells and tissues from extracellular damage by eliminating superoxide anion radicals produced during metabolism. Two different forms of EC-SOD exist, and their different enzyme activities are a result of different disulfide bond patterns. Although only two folding variants have been discovered so far, five folding variants are theoretically possible. Therefore, we constructed five different mutant EC-SOD expression vectors by substituting cysteine residues with serine residues and evaluated their expression levels and enzyme activities. The mutant EC-SODs were expressed at lower levels than that of wild-type EC-SOD, and all of the mutants exhibited inhibited extracellular secretion, except for C195S ECSOD. Finally, we demonstrated that co-expression of wild-type EC-SOD and any one of the mutant EC-SODs resulted in reduced secretion of wild-type EC-SOD. We speculate that mutant EC-SOD causes malfunctions in systems such as antioxidant systems and sensitizes tissues to ROS-mediated diseases.

• Molecules and Cells
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• v.42 no.11
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• pp.773-782
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• 2019

Cellular senescence is an irreversible form of cell cycle arrest. Senescent cells have a unique gene expression profile that is frequently accompanied by senescence-associated heterochromatic foci (SAHFs). Protein kinase CK2 (CK2) downregulation can induce trimethylation of histone H3 Lys 9 (H3K9me3) and SAHFs formation by activating SUV39h1. Here, we present evidence that the PI3K-AKT-mTOR-reactive oxygen species-p53 pathway is necessary for CK2 downregulation-mediated H3K9me3 and SAHFs formation. CK2 downregulation promotes SUV39h1 stability by inhibiting its proteasomal degradation in a p53-dependent manner. Moreover, the dephosphorylation status of Ser 392 on p53, a possible CK2 target site, enhances the nuclear import and subsequent stabilization of SUV39h1 by inhibiting the interactions between p53, MDM2, and SUV39h1. Furthermore, $p21^{Cip1/WAF1}$ is required for CK2 downregulation-mediated H3K9me3, and dephosphorylation of Ser 392 on p53 is important for efficient transcription of $p21^{Cip1/WAF}$. Taken together, these results suggest that CK2 downregulation induces dephosphorylation of Ser 392 on p53, which subsequently increases the stability of SUV39h1 and the expression of $p21^{Cip1/WAF1}$, leading to H3K9me3 and SAHFs formation.

• Molecules and Cells
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• v.44 no.10
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• pp.710-722
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• 2021

Hypoxia, or low oxygen tension, is a hallmark of the tumor microenvironment. The hypoxia-inducible factor-1α (HIF-1α) subunit plays a critical role in the adaptive cellular response of hypoxic tumor cells to low oxygen tension by activating gene-expression programs that control cancer cell metabolism, angiogenesis, and therapy resistance. Phosphorylation is involved in the stabilization and regulation of HIF-1α transcriptional activity. HIF-1α is activated by several factors, including the mitogen-activated protein kinase (MAPK) superfamily. MAPK phosphatase 3 (MKP-3) is a cytoplasmic dual-specificity phosphatase specific for extracellular signal-regulated kinase 1/2 (Erk1/2). Recent evidence indicates that hypoxia increases the endogenous levels of both MKP-3 mRNA and protein. However, its role in the response of cells to hypoxia is poorly understood. Herein, we demonstrated that small-interfering RNA (siRNA)-mediated knockdown of MKP-3 enhanced HIF-1α (not HIF-2α) levels. Conversely, MKP-3 overexpression suppressed HIF-1α (not HIF-2α) levels, as well as the expression levels of hypoxia-responsive genes (LDHA, CA9, GLUT-1, and VEGF), in hypoxic colon cancer cells. These findings indicated that MKP-3, induced by HIF-1α in hypoxia, negatively regulates HIF-1α protein levels and hypoxia-responsive genes. However, we also found that long-term hypoxia (>12 h) induced proteasomal degradation of MKP-3 in a lactic acid-dependent manner. Taken together, MKP-3 expression is modulated by the hypoxic conditions prevailing in colon cancer, and plays a role in cellular adaptation to tumor hypoxia and tumor progression. Thus, MKP-3 may serve as a potential therapeutic target for colon cancer treatment.

• Biomolecules & Therapeutics
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• v.27 no.2
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• pp.231-239
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• 2019

Suppressor of Variegation 3-9 Homolog 2 (SUV39H2) methylates the lysine 9 residue of histone H3 and induces heterochromatin formation, resulting in transcriptional repression or silencing of target genes. SUV39H1 and SUV39H2 have a role in embryonic development, and SUV39H1 was shown to suppress cell cycle progression associated with Rb. However, the function of human SUV39H2 has not been extensively studied. We observed that forced expression of SUV39H2 decreased cell proliferation by inducing $G_1$ cell cycle arrest. In addition, SUV39H2 was degraded through the ubiquitin-proteasomal pathway. Using yeast two-hybrid screening to address the degradation mechanism and function of SUV39H2, we identified translationally controlled tumor protein (TCTP) as an SUV39H2-interacting molecule. Mapping of the interacting regions indicated that the N-terminal 60 amino acids (aa) of full-length SUV39H2 and the C-terminus of TCTP (120-172 aa) were critical for binding. The interaction of SUV39H2 and TCTP was further confirmed by co-immunoprecipitation and immunofluorescence staining for colocalization. Moreover, depletion of TCTP by RNAi led to up-regulation of SUV39H2 protein, while TCTP overexpression reduced SUV39H2 protein level. The half-life of SUV39H2 protein was significantly extended upon TCTP depletion. These results clearly indicate that TCTP negatively regulates the expression of SUV39H2 post-translationally. Furthermore, SUV39H2 induced apoptotic cell death in TCTP-knockdown cells. Taken together, we identified SUV39H2, as a novel target protein of TCTP and demonstrated that SUV39H2 regulates cell proliferation of lung cancer cells.

• Molecules and Cells
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• v.44 no.2
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• pp.101-115
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• 2021

The INO80 chromatin remodeling complex has roles in many essential cellular processes, including DNA replication. However, the mechanisms that regulate INO80 in these processes remain largely unknown. We previously reported that the stability of Ino80, the catalytic ATPase subunit of INO80, is regulated by the ubiquitin proteasome system and that BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, stabilizes Ino80 via deubiquitination and promotes replication fork progression. However, the E3 ubiquitin ligase that targets Ino80 for proteasomal degradation was unknown. Here, we identified the C-terminus of Hsp70-interacting protein (CHIP), the E3 ubiquitin ligase that functions in cooperation with Hsp70, as an Ino80-interacting protein. CHIP polyubiquitinates Ino80 in a manner dependent on Hsp70. Contrary to our expectation that CHIP degrades Ino80, CHIP instead stabilizes Ino80 by extending its half-life. The data suggest that CHIP stabilizes Ino80 by inhibiting degradative ubiquitination. We also show that CHIP works together with BAP1 to enhance the stabilization of Ino80, leading to its chromatin binding. Interestingly, both depletion and overexpression of CHIP compromise replication fork progression with little effect on fork stalling, as similarly observed for BAP1 and Ino80, indicating that an optimal cellular level of Ino80 is important for replication fork speed but not for replication stress suppression. This work therefore idenitifes CHIP as an E3 ubiquitin ligase that stabilizes Ino80 via nondegradative ubiquitination and suggests that CHIP and BAP1 act in concert to regulate Ino80 ubiquitination to fine-tune its stability for efficient DNA replication.