Ursodeoxycholic acid inhibits the proliferation of colon cancer cells by regulating oxidative stress and cancer stem-like cell growth.
ABSTRACT: The regulation of reactive oxygen species (ROS) exists as a therapeutic target for cancer treatments. Previous studies have shown that ursodeoxycholic acid (UDCA) suppresses the proliferation of colon cancer cells. The aim of this study was to evaluate the effect of UDCA upon the proliferation of colon cancer cells as a direct result of the regulation of ROS.Colon cancer cell lines (HT29 and HCT116) were treated with UDCA. The total number of cells and the number of dead cells were determined using cell counters. A fluorescein isothiocyanate-bromodeoxyuridine flow kit was used to analyze cell cycle variations. Upon exposure to UDCA, the protein levels of p27, p21, CDK2, CDK4 and CDK6 were determined using western blotting, and qRT-PCR was used to determine levels of mRNA. We preformed dichlorofluorescindiacetate (DCF-DA) staining to detect alteration of intracellular ROS using fluorescence activated cell sorting (FACS). Colon cancer stem-like cell lines were generated by tumorsphere culture and treated with UDCA for seven days. The total number of tumorspheres was determined using microscopy.We found that UDCA reduced the total number of colon cancer cells, but did not increase the number of dead cells. UDCA inhibited the G1/S and G2/M transition phases in colon cancer cells. UDCA induced expression of cell cycle inhibitors such as p27 and p21. However, it was determined that UDCA suppressed levels of CDK2, CDK4, and CDK6. UDCA regulated intracellular ROS generation in colon cancer cells, and induced activation of Erk1/2. Finally, UDCA inhibited formation of colon cancer stem-like cells.Our results indicate that UDCA suppresses proliferation through regulation of oxidative stress in colon cancer cells, as well as colon cancer stem-like cells.
Project description:Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G(1) phase. Aberrant expression of CDK4 and CDK6 is a hallmark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM (the calcein acetoxymethyl-ester) is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G(1) phase. The metabolic effects of calcein AM on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phosphate pathway was significantly altered. To elucidate whether these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.
Project description:Background/Aim:LncRNA plays a key role in tumor progression. HAGLR functions as an oncogene in many cancers. However, the molecular mechanism of HAGLR in colon cancer is still unclear. Methods:qRT-PCR was used to measure the expression of HAGLR, miR-185-5p in colon cancer. The expression of CDK4 and CDK6 was detected by Western blot. CCK-8 assay, EdU staining, transwell and Annexin V-FITC/PI assay were used to analyze the effect of HAGLR and miR-185-5p on cell proliferation, invasion, migration and apoptosis. Bioinformatic analysis and luciferase were used to analyze the target genes of HAGLR and miR-185-5p. Nude mice were used to detect mouse tumor changes. Results:Compared with normal colon cancer tissues and cells, the expression of HAGLR was increased in colon cancer tissues and cells. In addition, the expression of HAGLR down-regulation inhibited the growth, migration, and invasion of colon cancer cells. MiR-185-5p was reduced in colon cancer, and CDK4 and CDK6 acted as target genes of miR-185-5p to regulate the progress of colon cancer. And CDK4 and CDK6 were predicted as downstream targets of miR-185-5p. Finally, it was demonstrated that HAGLR regulated tumor progression in vivo. Conclusion:Lnc HAGLR promoted the development of colon cancer by miR-185-5p/CDK4/CDK6 axis, and lnc HAGLR might be potential target for colon cancer.
Project description:BACKGROUND:Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6) are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor. RESULTS:A search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP). An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy. CONCLUSION:These findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with a companion diagnostic biomarker and a putative functional site for kinase-unrelated activities of Cdk4.
Project description:Mammalian cell division is thought to be driven by sequential activation of several Cyclin-dependent kinases (Cdk), mainly Cdk4, Cdk6, Cdk2 and Cdk1. Since mice lacking Cdk4, Cdk6 or Cdk2 are viable, it has been proposed that they play compensatory roles. We report here that mice lacking Cdk4 and Cdk2 complete embryonic development to die shortly thereafter presumably due to heart failure. However, conditional ablation of Cdk2 in adult mice lacking Cdk4 does not result in obvious abnormalities. Moreover, these double mutant mice recover normally after partial hepatectomy. In culture, Cdk4(-/-);Cdk2(-/-) embryonic fibroblasts become immortal, display robust pRb phosphorylation and have normal S phase kinetics. These observations indicate that Cdk4 and Cdk2 are dispensable for the mammalian cell cycle and for adult homeostasis.
Project description:Cell cycle progression, including genome duplication, is orchestrated by cyclin-dependent kinases (CDKs). CDK activation depends on phosphorylation of their T-loop by a CDK-activating kinase (CAK). In animals, the only known CAK for CDK2 and CDK1 is cyclin H-CDK7, which is constitutively active. Therefore, the critical activation step is dephosphorylation of inhibitory sites by Cdc25 phosphatases rather than unrestricted T-loop phosphorylation. Homologous CDK4 and CDK6 bound to cyclins D are master integrators of mitogenic/oncogenic signaling cascades by initiating the inactivation of the central oncosuppressor pRb and cell cycle commitment at the restriction point. Unlike the situation in CDK1 and CDK2 cyclin complexes, and in contrast to the weak but constitutive T177 phosphorylation of CDK6, we have identified the T-loop phosphorylation at T172 as the highly regulated step determining CDK4 activity. Whether both CDK4 and CDK6 phosphorylations are catalyzed by CDK7 remains unclear. To answer this question, we took a chemical-genetics approach by using analogue-sensitive CDK7(as/as) mutant HCT116 cells, in which CDK7 can be specifically inhibited by bulky adenine analogs. Intriguingly, CDK7 inhibition prevented activating phosphorylations of CDK4/6, but for CDK4 this was at least partly dependent on its binding to p21 (cip1) . In response to CDK7 inhibition, p21-binding to CDK4 increased concomitantly with disappearance of the most abundant phosphorylation of p21, which we localized at S130 and found to be catalyzed by both CDK4 and CDK2. The S130A mutation of p21 prevented the activating CDK4 phosphorylation, and inhibition of CDK4/6 and CDK2 impaired phosphorylations of both p21 and p21-bound CDK4. Therefore, specific CDK7 inhibition revealed the following: a crucial but partly indirect CDK7 involvement in phosphorylation/activation of CDK4 and CDK6; existence of CDK4-activating kinase(s) other than CDK7; and novel CDK7-dependent positive feedbacks mediated by p21 phosphorylation by CDK4 and CDK2 to sustain CDK4 activation, pRb inactivation, and restriction point passage.
Project description:Cyclin D-dependent kinases (CDK4 and CDK6) are positive regulators of cell cycle entry and they are overactive in the majority of human cancers. However, it is currently not completely understood by which cellular mechanisms CDK4/6 promote tumorigenesis, largely due to the limited number of identified substrates. Here we performed a systematic screen for substrates of cyclin D1-CDK4 and cyclin D3-CDK6. We identified the Forkhead Box M1 (FOXM1) transcription factor as a common critical phosphorylation target. CDK4/6 stabilize and activate FOXM1, thereby maintain expression of G1/S phase genes, suppress the levels of reactive oxygen species (ROS), and protect cancer cells from senescence. Melanoma cells, unlike melanocytes, are highly reliant on CDK4/6-mediated senescence suppression, which makes them particularly susceptible to CDK4/6 inhibition.
Project description:This study was designed to purify molecules possess anti-cancer cell activity from the fruit body of Ganoderma leucocontextum. Bio-activity-guided purification and chromatographic separation of Ganoderma leucocontextum extract led to the enrichment of bioactive fractions and isolation of a single compound. The purified compound was identified as Ganoderiol F, which induced cancer cell death. In the in vivo experiments, we founded ethanol extract and ethyl acetate fraction inhibited tumor growth in the mice injected with 4T1 cells. We found that Ganoderiol F-mediated suppression of breast cancer cell viability occurred through cell cycle arrest. Ganoderiol F down-regulated expression of cyclin D, CDK4, CDK6, cyclin E and CDK2 and inhibited cell cycle progression arresting the cells in G1 phase. In addition, Ganoderiol F up-regulated pro-apoptotic Foxo3, down-regulated anti-apoptotic c-Myc, Bcl-2 and Bcl-w leading to apoptosis in human breast cancer cells MDA-MB-231. These results showed that c-Myc, cyclin D-CDK4/CDK6 and cyclin E-CDK2 are the central components of Ganoderiol F regulation of cell cycle progression. Hence Ganoderiol F may serve as a potential CDK4/CDK6 inhibitor for breast cancer therapy. Abbreviations: GLE: Ganoderma leucocontextum ethanol extract; GLEA: Ganoderma leucocontextum ethyl acetate fraction; GLPE: Ganoderma leucocontextum petroleum ether fraction; RP-HPLC: reversed-phase high-performance liquid chromatograph; DMEM: Dulbecco's modified Eagle's medium; FBS: fetal bovine serum; PAGE: polyacrylamide gel electrophoresis.
Project description:Diabetes (T1DM and T2DM) is characterized by a deficit in ?-cell mass. A broader understanding of human ?-cell replication mechanism is thus important to increase ?-cell proliferation for future therapeutic interventions. Here, we show that p27 (Kip1), a CDK inhibitor, is expressed abundantly in isolated adult human islets and interacts with various positive cell cycle regulatory proteins including D-type cyclins (D1, D2 and D3) and their kinase partners, CDK4 and CDK6. Also, we see interaction of cyclin E and its kinase partner, CDK2, with p27 suggesting a critical role of p27 as a negative cell cycle regulator in human islets. Our data demonstrate interaction of p27 with GSK-3 in ?-cells and show, employing rodent ?-cells (INS-1), isolated human islets and purified ?-cells derived from human islets, that siRNA-mediated depletion of GSK-3 or p27 or 1-AKP / BIO - mediated GSK-3 inhibition results in increased ?-cell proliferation. We also see reduction of p27 levels following GSK-3 inactivation or depletion. Our data show that serum induction of quiescent INS-1 cells leads to sequential phosphorylation of p27 on its S10 and T187 residues with faster kinetics for S10 corresponding with the decreased levels of p27. Altogether our findings indicate that p27 levels in ?-cells are stabilized by GSK-3 and thus p27 down regulation following GSK-3 depletion / inactivation plays a critical role in promoting ?-cell replication.
Project description:8-Hydroxyquinaldic acid, the end-metabolite of tryptophan, is well-known metal chelator; however, its role in humans, especially in cancer promotion and progression, has not been fully revealed. Importantly, 8-hydroxyquinaldic acid is the analog of kynurenic acid with evidenced antiproliferative activity towards various cancer cells. In this study, we revealed that 8-hydroxyquinaldic acid inhibited not only proliferation and mitochondrial activity in colon cancer HT-29 and LS-180 cells, but it also decreased DNA synthesis up to 90.9% for HT-29 cells and 76.1% for LS-180 cells. 8-Hydroxyquinaldic acid induced changes in protein expression of cell cycle regulators (CDK4, CDK6, cyclin D1, cyclin E) and CDKs inhibitors (p21 Waf1/Cip1, p27 Kip1), but the effect was dependent on the tested cell line. Moreover, 8-hydroxyquinaldic acid inhibited migration of colon cancer HT-29 and LS-180 cells and increased the expression of ?-catenin and E-cadherin. Importantly, antiproliferative and anti-migratory concentrations of 8-hydroxyquinaldic acid were non-toxic in vitro and in vivo. We reported for the first time antiproliferative and anti-migratory activity of 8-hydroxyquinaldic acid against colon cancer HT-29 and LS-180 cells.