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Glycyrrhiza glabra L., known as licorice, is one of the most famous herbs in the world. In this study, we investigated the phytochemical and antitumor activities of G. glabra, especially its anti-colorectal cancer activities. G. glabra was extracted with 70% methanol, and the ethyl acetate layer was separated by silica gel, ODS, LH-20 column chromatography, and semi-preparative HPLC to obtain the compounds. The structures were determined by NMR and MS methods. Three new compounds named licopyranol A-C (1-3), and eighteen known compounds (4-21) were isolated. Compounds with an isoprenyl group or dimethylpyran ring showed better antitumor activities. Licopyranol A (1) and glycyrol (5) both inhibited the proliferation, reduced clone formation and promoted apoptosis of RKO cells. The Western blotting assays showed that glycyrol significantly reduced the expression of E-cadherin, ß-catenin, c-Myc, and GSK-3ß proteins in RKO cells, suggesting that glycyrol may inhibit the growth of colorectal cancer RKO cells via the Wnt/ß-catenin signaling pathway.

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Vine pruning residues are by-products of the wine industry that have not received much attention in the past, in spite of being rich in bioactive compounds. In this study, we aimed to test whether an ohmic extract of vine pruning residue (VPE) has anti-colorectal cancer (CRC) properties, and whether responses differ according with cell's mutation profile. VPE decreased human CRC cell proliferation, accompanied by DNA effects and cell cycle modulation. VPE also increased cell sensitivity to the chemotherapeutic drug 5-FU. Our results suggest that tumors harboring BRAF mutations may be more responsive to VPE than KRAS mutated tumors. These effects of the extract were not completely reproduced by the most abundant constituents tested individually at the concentrations present in the effective dose of VPE. Globally, our results indicate that VPE, a polyphenol enriched extract produced by ohmic heating of vine pruning residue, has anti-colorectal cancer potential, including sensitizing to a chemotherapeutical drug, and its use in functional foods or nutraceuticals could be exploited in personalized anti colorectal cancer dietary strategies. Valorization of this lignocellulosic residue should encourage bio-waste recycling, adding value to this agricultural by-product and promoting the sustainable use of natural resources.

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Human colorectal cancer is the third most common cancer disease with a 5­year survival rate of 55% in USA in 2016. The investigation to identify novel biomarker factors with molecular classification may provide notable clinical information to prolong the survival of patients with colorectal cancer. The aryl hydrocarbon receptor (AHR) binds the AHR nuclear translocator in the cytoplasm of various types of cells, including liver cells, and then binds to the xenobiotic responsive element on various genes. AHR was initially discovered via its ligand, the polychlorinated hydrocarbon, 2,3,7,8­tetrachlorodibenzo­p­dioxin (TCDD). The present study was undertaken to determine whether TCDD, an agonist of AHR signaling, impacts the growth of RKO human colorectal cancer cells in vitro. Treatment with TCDD (0.1­100 nM) revealed suppressive effects on colony formation and proliferation of RKO cells, and stimulated death of these cells with subconfluence. These effects of TCDD were abolished by pretreatment with CH223191, an inhibitor of AHR signaling. Western blot analysis demonstrated that TCDD treatment decreased AHR levels and elevated cytochrome P450 family 1 subfamily A member 1 (CYP1A1) levels, indicating a stimulation of AHR signaling. TCDD treatment caused an increase in nuclear factor­κB p65 and ß­catenin levels, although it did not have an effect on Ras levels. Notably, TCDD treatment increased the levels of p53, retinoblastoma, p21 and regucalcin, which are depressors of carcinogenesis. Additionally, action of TCDD on cell proliferation and death were not revealed in regucalcin­overexpressing RKO cells, and regucalcin overexpression depressed AHR signaling associated with CYP1A1 expression. Thus, AHR signaling suppresses the growth of colorectal cancer cells, indicating a role as a significant targeting molecule for colorectal cancer.

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BACKGROUND: Our previous study successfully identified that 3,3'-Dimethylquercetin (DMQ) acted as a potent anticancer agent against human colon cancer cell lines RKO. Thus, this study was conducted to investigate the underlying mechanism by which DMQ displayed inhibitory activity in RKO cells. METHODS: Flow cytometry was used to evaluate the effect of DMQ on the cell cycle arrest, as well as the mitochondrial membrane potential in RKO cells. DAPI staining and DNA fragmentation ladder assays were performed to assess the apoptosis inducing activity of DMQ. Furthermore, western blot analysis was conducted to examine the expression of related proteins responsible for the cell cycle arrest and apoptosis. RESULTS: Treatment with DMQ caused a significant increase in the fraction of G2/M cells, and induced remarkable apoptosis. Furthermore, western blot analysis showed that DMQ arrested cells at G2/M checkpoint by down-regulation of cyclin B1, cdc2 and cdc25c and up-regulation of p21, and induced cell apoptosis via affecting the ratio of Bax/Bcl-2, causing loss of the mitochondrial membrane potential and enhancing the expression of cleaved caspase-9 (C-caspase-9) and cleaved caspase-3 (C-caspase-3). CONCLUSION: These data showed that DMQ could suppress RKO cell growth by arresting RKO cells at G2/M checkpoint and inducing mitochondria-dependent cell apoptosis. Our findings shed light on the potential use of DMQ as a chemotherapeutic agent for CRC.

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OBJECTIVE: To investigate the expression level of IARS2 gene in colon cancer tissues and various cell strains of the cancer; to explore cytologically the effect of IARS2 gene knockdown on proliferation, apoptosis and cell cycle of RKO cells in the cancer. METHODS: Real-time, fluorescence-based quantitative PCR (qPCR) was used to detect the expression of IARS2 gene in human colon cancer and surrounding tissues and in various cell strains of the cancer; the RNA interference target of IARS2 gene was designed and the target was detected by Western blot; the IARS2-siRNA lentiviral vector was established and used to infect the RKO cells of colon cancer; qPCR was employed to determine the effect of gene knockdown; changes of the RKO cells in growth, apoptosis, cell cycle and clone formation were observed after IARS2 gene knockdown. RESULTS: The expression of IARS2 gene was higher in human colon cancer tissues than in surrounding tissues; there was expression of IARS2 gene in colon cancer cells, and the expression level of IARS2 gene mRNA was higher in the RKO cells than in the SW480, HCT116, DLD1, HT-29 and SW620 cells. After infection of the RKO cells with IARS2-siRNA lentivirus, the expression of IARS2 gene was inhibited in the level of mRNA; proliferation rate of the RKO cells was significantly inhibited; the G1 phase arrest of the RKO cells was increased with less RKO cells in S phase; the apoptotic RKO cells increased significantly; and the number of colonies of the RKO cells reduced. CONCLUSION: The expression of IARS2 gene is different in human colon cancer and surrounding tissues; after knockdown of IARS2 gene, proliferation of the RKO cells is inhibited; there are more cells in G phase and fewer cells in S phase; apoptosis of cells is increased; and formation of colonies is reduced. IARS2 gene is probably a cancer-promoting gene.

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Lon is a nuclear-encoded, mitochondrial protease that assists protein folding, degrades oxidized/damaged proteins, and participates in maintaining mtDNA levels. Here we show that Lon is up-regulated in several human cancers and that its silencing in RKO colon cancer cells causes profound alterations of mitochondrial proteome and function, and cell death. We silenced Lon in RKO cells by constitutive or inducible expression of Lon shRNA. Lon-silenced cells displayed altered levels of 39 mitochondrial proteins (26% related to stress response, 14.8% to ribosome assembly, 12.7% to oxidative phosphorylation, 8.5% to Krebs cycle, 6.3% to ß-oxidation, and 14.7% to crista integrity, ketone body catabolism, and mtDNA maintenance), low levels of mtDNA transcripts, and reduced levels of oxidative phosphorylation complexes (with >90% reduction of complex I). Oxygen consumption rate decreased 7.5-fold in basal conditions, and ATP synthesis dropped from 0.25 ± 0.04 to 0.03 ± 0.001 nmol/mg proteins, in the presence of 2-deoxy-d-glucose. Hydrogen peroxide and mitochondrial superoxide anion levels increased by 3- and 1.3-fold, respectively. Mitochondria appeared fragmented, heterogeneous in size and shape, with dilated cristae, vacuoles, and electrondense inclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid, a Lon inhibitor, partially mimics Lon silencing. In summary, Lon is essential for maintaining mitochondrial shape and function, and for survival of RKO cells.

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Radiation therapy for variety of human solid tumors utilizes mechanism of cell death after DNA damage caused by radiation. In response to DNA damage, cytochrome c was released from mitochondria by activation of pro-apoptotic Bcl-2 family proteins, and then elicits massive Ca(2+) release from the ER that lead to cell death. It was also suggested that irradiation may cause the deregulation of Ca(2+) homeostasis and trigger programmed cell death and regulate death specific enzymes. Thus, in this study, we investigated how cellular Ca(2+) metabolism in RKO cells, in comparison to radiation-resistant A549 cells, was altered by gamma (γ)-irradiation. In irradiated RKO cells, Ca(2+) influx via activation of NCX reverse mode was enhanced and a decline of [Ca(2+)]i via forward mode was accelerated. The amount of Ca(2+) released from the ER in RKO cells by the activation of IP3 receptor was also enhanced by irradiation. An increase in [Ca(2+)]i via SOCI was enhanced in irradiated RKO cells, while that in A549 cells was depressed. These results suggest that γ-irradiation elicits enhancement of cellular Ca(2+) metabolism in radiation-sensitive RKO cells yielding programmed cell death.

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Radiation therapy for variety of human solid tumors utilizes mechanism of cell death after DNA damage caused by radiation. In response to DNA damage, cytochrome c was released from mitochondria by activation of pro-apoptotic Bcl-2 family proteins, and then elicits massive Ca2+ release from the ER that lead to cell death. It was also suggested that irradiation may cause the deregulation of Ca2+ homeostasis and trigger programmed cell death and regulate death specific enzymes. Thus, in this study, we investigated how cellular Ca2+ metabolism in RKO cells, in comparison to radiation-resistant A549 cells, was altered by gamma (gamma)-irradiation. In irradiated RKO cells, Ca2+ influx via activation of NCX reverse mode was enhanced and a decline of [Ca2+]i via forward mode was accelerated. The amount of Ca2+ released from the ER in RKO cells by the activation of IP3 receptor was also enhanced by irradiation. An increase in [Ca2+]i via SOCI was enhanced in irradiated RKO cells, while that in A549 cells was depressed. These results suggest that gamma-irradiation elicits enhancement of cellular Ca2+ metabolism in radiation-sensitive RKO cells yielding programmed cell death.

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Compostos α-aminocarbonilícos como ácido 5-aminolevulínico (ALA) e aminoacetona (AA) apresentam um grande potencial pró-oxidante, pois sofrem reações de enolização e subseqüente oxidação aeróbica, com a formação de espécies radicalares de oxigênio, íons NH4+ e α-oxoaldeídos potencialmente citotóxicos. A α-aminocetona 1,4-diamino-2-butanona (DAB), um análogo da putrescina, é um agente microbicida de vários parasitas incluindo Trypanosoma cruzi. Acredita-se que o mecanismo de morte desencadeado por DAB nos parasitas seja por meio da inibição competitiva da ornitina descarboxilase (ODC), importante enzima do metabolismo de poliaminas, muito embora tenha sido observado de igual forma danos oxidativos nestes parasitas quando tratados com DAB. O objetivo deste trabalho é esclarecer o mecanismo de oxidação química de DAB e sua ação pró-oxidante à cultura de células de mamíferos (LLC-MK2 e RKO), assim como sua atividade microbicida contra tripomastigotas de Trypanosoma cruzi. Demonstramos aqui que DAB, quimicamente similar ao ALA e AA, sofre reação de oxidação catalisada por íons fosfato, e por íons de metais de transição como Fe(II) e Cu(II), resultando na formação de radicais de oxigênio, H2O2, NH4+, 2-oxo-4-aminobutanal como produto principal da oxidação de DAB e de compostos ciclicos de caracter pirrólico. Danos oxidativos observados em ferritina, apotransferrina e liposomos de cardiolipina e fosfatidilcolina (20:80) contribuem para a nossa hipótese de ação pró-oxidante de DAB. O tratamento de células de mamíferos das linhagens LLC-MK2 (IC50 1,5 mM, tratamento de 24 h) e RKO (IC50 0,3 mM, tratamento de 24 h) com DAB levou à alteração do balanço redox celular, à ativação de resposta antioxidante e ao desencadeamento de morte celular via apoptose e parada de ciclo celular. Em culturas de tripomastigotas de T. cruzi o tratamento com DAB culminou na redução da motilitidade e viabilidade destes parasitas (IC50 0,2 mM, tratamento de 4 h), assim como depleção do...

α-Aminocarbonyl componds such as 5-aminolevunilic acid (ALA) and aminoacetone (AA) have been shown to exhibit pro-oxidant properties. These compounds undergo phosphate-catalyzed enolization in physiological pH and subsequent aerobic oxidation, yielding reactive oxygen species, NH4+ ions and an α-oxoaldehyde highly cytotoxic. The α-aminoketone 1,4-diamino-2-butanone (DAB) is a putrescine analogue and a microbicidal agent to various parasites including Trypanosoma cruzi. The mechanism of DAB toxicity to these parasites is attributed to DAB competitive inhibition of ornithine decarboxylase (ODC), a key enzyme on polyamine biosynthesis, although it has also been shown DAB isto implicated in oxidative damage to these parasites. Our aim is to clarify the mechanism of DAB aerobic oxidation and of its putative pro-oxidant activity to mammalian cell cultures (LLC-MK2 and RKO cell linages) and to Trypanosoma cruzi trypomastigotes. Here we show that, similar to ALA and AA, DAB undergoes aerobic oxidation in presence of phosphate ions and of transition metal ions such as Fe(II) and Cu(II), yielding oxygen radicals, H2O2, NH4+ and 2-oxo-4-aminobutanal accompanied by its condensation cyclic products displaying pyrrolic characteristics. Oxidative alterations to ferritin, apotransferrin and liposomes of cardiolipin and phosphatidylcholine (20:80) were observed under DAB treatment strongly supporting our hypothesis of DAB pro-oxidative activity. DAB treatment of mammalian cultured cells LLC-MK2 (IC50 1.5 mM, 24 h incubation) and RKO (IC50 0.3 mM, 24 h incubation) resulted in redox imbalance, induction of antioxidant response, activation of apoptosis pathway and cell cycle arrest. DAB is shown here to trigger Trypanosoma cruzi trypomastigotes decreased parasite motility and viability (IC50 0.2 mM, 4 h incubation), as well as redox thiol imbalance parallel to increase TcSOD activity. In addition, DAB efficiently hampered host cell (LLC-MK2) invasion by trypomastigotes...

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PURPOSE: To reveal the interaction between beta-Lapachone (beta-lap) and ionizing radiation in causing cell death in RKO human colon adenocarcinoma cells, and to elucidate the potential usefulness of combined beta-lap treatment and radiotherapy for cancer treatment. MATERIALS AND METHODS: The cytotoxicities of various treatments were determined in vitro using clonogenic and apoptotic cell death. The changes in cell cycle distribution were studied using flow cytometry and an in vitro kinase assay. The tumor growth was studied using RKO tumors grown s.c. in the hind leg BALB/c- nuslc nude mice. RESULTS: beta-Lap caused clonogenic cell death and rapid apoptosis in RKO cells in vitro, in a dose dependent manner. The repair of sublethal radiation damage was almost completely inhibited when cells were maintained in beta-lap during the interval between the two-dose irradiation. Flow cytometry study demonstrated that beta-lap induced apoptosis, independent of the cell cycle phase, and completely prohibited the induction of radiation-induced G2 arrest in irradiated cells. The prohibition of radiation-induced G2 arrest is unclear, but may be related to the profound suppression of the p53, p21 and cyclin B1-Cdc2 kinase activities observed in cells treated with beta-lap. The combination of beta-lap and radiation markedly enhanced the radiation-induced growth suppression of tumors. CONCLUSION: beta-Lap is cytotoxic against RKO cells, both in vitro and in vivo, and also sensitized cells to ionizing radiation by inhibiting sublethal radiation damage repair. beta-lap is potentially useful as a potent anti-cancer chemotherapy drug and potent radiosensitizer against cancer cells.

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PURPOSE: To reveal the interaction between beta-Lapachone (beta-lap) and ionizing radiation in causing cell death in RKO human colon adenocarcinoma cells, and to elucidate the potential usefulness of combined beta-lap treatment and radiotherapy for cancer treatment. MATERIALS AND METHODS: The cytotoxicities of various treatments were determined in vitro using clonogenic and apoptotic cell death. The changes in cell cycle distribution were studied using flow cytometry and an in vitro kinase assay. The tumor growth was studied using RKO tumors grown s.c. in the hind leg BALB/c- nuslc nude mice. RESULTS: beta-lap caused clonogenic cell death and rapid apoptosis in RKO cells in vitro, in a dose dependent manner. The repair of sublethal radiation damage was almost completely inhibited when cells were maintained in beta-lap during the interval between the two-dose irradiation. Flow cytometry study demonstrated that beta-lap induced apoptosis, independent of the cell cycle phase, and completely prohibited the induction of radiation- induced G2 arrest in irradiated cells. The prohibition of radiation-induced G2 arrest is unclear, but may be related to the profound suppression of the p53, p21 and cyclin B1-Cdc2 kinase activities observed in cells treated with beta-lap. The combination of beta-lap and radiation markedly enhanced the radiation-induced growth suppression of tumors. CONCLUSION: beta-lap is cytotoxic against RKO cells, both in vitro and in vivo, and also sensitized cells to ionizing radiation by inhibiting sublethal radiation damage repair. beta-lap is potentially useful as a potent anti-cancer chemotherapy drug and potent radiosensitizer against caner cells.

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