P53 In Colorectal Cancer Cells Research Articles

Molecular design, synthesis and anticancer activity of new thiopyrano[2,3-d]thiazoles based on 5-hydroxy-1,4-naphthoquinone (juglone)

A series of 11-substituted 9-hydroxy-3,5,10,11-tetrahydro-2H-benzo[6,7]thiochromeno[2,3-d][1,3]thiazole-2,5,10-triones 3.1–3.13 were synthesized via hetero-Diels-Alder reaction of 5-ene-4-thioxo-2-thiazolidinones and 5-hydroxy-1,4-naphthoquinone (juglone). The structure of newly synthesized compounds was established by means of spectral data and a single-crystal X-ray diffraction analysis. The synthesized compounds were tested on a panel of cell lines representing different types of cancer as well as normal and pseudonormal cells and peripheral human blood lymphocytes. Compound 3.10 was found to be the most active derivative, exhibiting a cytotoxic effect similar to doxorubicin's one (IC50 ranged from 0.6 to 5.98 μM), but less toxic to normal and pseudonormal cells. All synthesized compounds were able to interact with DNA, although their anticancer activity did not correlate with the potency of interaction with DNA. The status of p53 in colorectal cancer cells correlated with the activity of the synthesized derivatives 3.1, 3.7, and 3.10. Compound 3.10 did not have an acute toxic effect on the body of С57BL/6 mice, unlike the well-known anticancer drug doxorubicin, which was used as a positive control. The injection of 3.10 (20 mg/kg) to mice had no effect on the counts of leukocytes, erythrocytes, platelets and hemoglobin level in their blood, in contrast to doxorubicin, which caused anemia and leukopenia, indicating bio-tolerance of 3.10in vivo.

Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile.

TP53 is the most commonly mutated gene in cancer, and gain-of-function mutations have wide-ranging effects. Efforts to reactivate wild-type p53 function and inhibit mutant functions have been complicated by the variety of TP53 mutations. Identified from a screen, the NSC59984 compound has been shown to restore activity to mutant p53 in colorectal cancer cells. Here, we investigated its effects on esophageal adenocarcinoma cells with specific p53 hot-spot mutations. NSC59984 treatment of cells reactivated p53 transcriptional regulation, inducing mitochondrial intrinsic apoptosis. Analysis of its effects on cellular metabolism demonstrated increased utilization of the pentose phosphate pathway and inhibition of glycolysis at the fructose-1,6-bisphosphate to fructose 6-phosphate junction. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; these effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. We found that the effects of NSC59984 were substantially greater in cells harboring the p53 R248W mutation. Overall, these findings demonstrate p53-dependent effects of NSC59984 on cellular metabolism, with increased activity in cells harboring the p53 R248W mutation. This research highlights the importance of defining the mutational status of a particular cancer to create a patient-centric strategy for the treatment of p53-driven cancers.

Mutations in DNA binding domain of p53 impede RSL1D1-p53 interaction to escape from degradation in human colorectal cancer cells

Different from the nucleolus-specific localization in some types of cancer cells, ribosomal L1 domain-containing protein 1 (RSL1D1) distributes throughout the nucleus in human colorectal cancer (CRC) cells. RSL1D1 directly interacts with DNA binding domain (aa 93–292) of wild-type p53 (p53-WT) and thereby recruits p53 to HDM2. The ensuing formation of RSL1D1/HDM2/p53 complex enhances p53 ubiquitination and decreases the protein level of p53 in CRC cells. In this study, we investigated the interaction between RSL1D1 and mutant p53 proteins. We first corroborated that aa 93–224 of p53 is a more precise domain for RSL1D1 binding and mutation in either aa 93–224 or aa 225–292 domain of p53 affects RSL1D1-p53 interaction. R175H mutated p53 does not interact with RSL1D1, whereas R273H mutated p53 still can bind to RSL1D1 but showing a remarkably decreased affinity than p53-WT. Although p53-R273H retains a weakened binding affinity with RSL1D1, it can hardly be recruited to HDM2 by RSL1D1 in HCT116 CRC cells. Accordingly, RSL1D1 loses its capacity to negatively regulate either R175H or R273H p53 mutant via directly interaction in HCT116 cells, thereby facilitating p53 mutants to accumulate and gain oncogenic function. Our findings help explain why mutant p53 proteins are more stable than p53-WT in CRC cells.

Ubiquitin-Like Protein UBD Promotes Cell Proliferation in Colorectal Cancer by Facilitating p53 Degradation.

PurposeUbiquitin D (UBD) is a member of the ubiquitin-like modifier (UBL) family and is highly expressed in a variety of cancers including colorectal cancer (CRC). However, the mechanisms of its regulatory roles in CRC are largely elusive. In this study, we revealed the effect of UBD on the proliferation of CRC.MethodsThe expression of UBD in clinical tissue samples of CRC and seven CRC cell lines was detected using qRT-PCR, immunohistochemistry (IHC) and Western blotting. CCK-8, colony formation, EdU and flow cytometry assays were used to detect the functional changes of CRC cells transfected with UBD stable expression plasmids in vitro. A xenograft model was constructed to assess the effect of UBD on the growth of CRC cells in vivo. The connection between UBD and p53 was analyzed using Western blotting, immunoprecipitation, proteasome inhibition assay and Cycloheximide (CHX) chase assay.ResultsUBD was overexpressed in CRC tumor tissues compared with nontumor tissues, and its overexpression was positively associated with the tumor size and TNM stage of CRC patients. Functionally, UBD significantly accelerated CRC cell viability and proliferation in vitro and promoted tumorigenesis in vivo. Mechanistically, UBD interacted with p53 in CRC cells, downregulated the expression of p53 by regulating its degradation, shortened the p53 half-life, thereby further affecting the decrease in p21 and the increase in Cyclin D1, Cyclin E, CDK2, CDK4 and CDK6. Moreover, in vivo experiments showed that UBD-induced tumor growth in nude mice was dependent on a decrease in p53.ConclusionsOur study proved that UBD mediates the degradation of p53, thereby facilitating the growth of CRC cells and ultimately promoting the progression of CRC. Therefore, UBD may be a potential therapeutic target and a promising prognostic biomarker for CRC.

Cullin-4B promotes cell proliferation and invasion through inactivation of p53 signaling pathway in colorectal cancer

Cullin 4B (CUL4B) is a member of the Cullin RING E3 ligase family, which is found to be overexpressed in multiple cancers, thus facilitating tumorigenesis and progression. However, the correlation between CUL4B and p53 in colorectal cancer cells (CRC) remains to be further elucidated. In this study, we newly identified that CUL4B functions as a negative regulator of p53, thereby facilitating CRC tumorigenesis and progression. Our data has demonstrated that CUL4B was frequently overexpressed in CRC tissues, and its upregulation was closely correlated with disease progression and poor prognosis. Moreover, CUL4B knockdown suppressed cell proliferation, invasion and epithelial-mesenchymal transition (EMT) of CRC cells. Mechanistically, CUL4B depletion increased the expression of p53 protein and its downstream targets p21, PUMA and MDM2. Furthermore, CUL4B depletion prolonged the half-life of p53 protein, and CUL4B is a binding partner of MDM2. In conclusion, our study shed new lights on the complex regulatory network between CUL4B and p53, and clarifies this CUL4B-p53 axis contributes greatly to CRC tumorigenesis and progression.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

BTG1 might be employed as a biomarker for carcinogenesis and a target for gene therapy in colorectal cancers

Here, BTG1 overexpression inhibited proliferation, induced differentiation, autophagy, and apoptosis in colorectal cancer cells (p2 arrest might be related to Cyclin B1 and Cdc25B hypoexpression in HCT-15 transfectants, while G1 arrest in HCT-116 transfectants overexpressing p21 and p27. BTG1 overexpression decreased the expression of Bcl-2, Bcl-xL, XIAP, Akt1 or survivin and increased the expression of Bax or p53 in colorectal cancer cells. BTG1-induced autophagy was dependent on Beclin-1 expression. BTG1 overexpression might weaken β-catenin pathway in colorectal cancer cells. The chemosensitivity of BTG1 transfectants to paclitaxel, cisplatin, MG132 or SAHA was positively correlated with its apoptotic induction. There was a lower expression level of BTG1 in cancer than matched non-neoplastic mucosa by RT-PCR (p

FBXW7-mutated colorectal cancer cells exhibit aberrant expression of phosphorylated-p53 at Serine-15.

FBXW7 mutations occur in a variety of human cancers including colorectal cancer (CRC). Elucidating its mechanism of action has become crucial for cancer therapy; however, it is also complicated by the fact that FBXW7 can influence many pathways due to its role as an E3-ubiquitin ligase in proteasome degradation. FBXW7 and TP53 are tumour suppressors intensively implicated in colorectal carcinogenesis. Deletion mutations in these two genes in animal models mark the progression from adenoma to carcinoma. Although still largely unknown, the last defense mechanism against CRC at the molecular level could be through a synergistic effect of the two genes. The underlying mechanism requires further investigation. In our laboratory, we have used a phospho-kinase profiler array to illustrate a potential molecular link between FBXW7 and p53 in CRC cells. In vitro and in vivo assessments demonstrated aberrant induction of phosphorylated p53 at Serine 15 [phospho-p53(Ser15)] in human FBXW7-deficient CRC cells as compared to their FBXW7-wild-type counterparts. FBXW7 loss in HCT116 cells promoted resistance to oxaliplatin. Immunoblotting data further confirmed that reduction of phospho-p53(Ser15) may contribute to the decreased efficacy of therapy in FBXW7-mutated CRC cells. The findings may suggest the applicability of phospho-p53(Ser15) as an indicative marker of FBXW7-mutations. Phospho-p53(Ser15) regulation by FBXW7 E3-ligase activity could provide important clues for understanding FBXW7 behavior in tumour progression and grounds for its clinical applicability thereafter.

2-Deoxyglucose Induces Cell Cycle Arrest and Apoptosisin Colorectal Cancer Cells Independent of Its Glycolysis Inhibition

2-Deoxyglucose (2DG) is an anticancer drug with excellent safety profile. Because of its higher dose requirements, its potential is yet to translate into a monotherapy. However, recently, 2DG has been tested as an adjunct in established chemotherapeutic regimens. 2DG enhanced the potency of several chemotherapeutic agents but not all. The rationale selection of known chemotherapeutic agents to use with 2DG is hampered becaue of the lack of complete understanding of mechanism behind 2DG anticancer effects. Although, 2DG is a well-known glycolytic inhibitor, which inhibits the key glycolytic enzyme hexokinase, its anticancer effects cannot be fully explained by this simplistic mechanism alone. In this article, we have shown for the first time that 2DG induced a transient expression of p21 and a continuous expression of p53 in colorectal cancer cells (SW620). The treatment also caused cell cycle arrest at G0/G1 phase and induced apoptosis through the mitochondrial pathway. The effects of 2DG on p21 and p53 protein levels were totally independent of its inhibitory effect on either hexokinase or ATP levels. Results from this study provides key insights into novel molecular mechanisms of 2DG and directs rational selection of other anticancer drugs to combine with 2DG in colorectal cancer treatment.

Abstract 2171: A new small molecule compound restoring p53 pathway induces cell death via active p73 and degradation of mutant p53 in colorectal cancer cells.

Abstract Tumor suppressor p53 is critical for protection against cancer. Over 50% of human cancers harbor mutant p53 which is hyper-stabilized and blocks p53 family member p73/p63 activity. Mutations not only abrogate the p53 tumor suppressor function, but also endow mutant p53 with gain-of-function (GOF) as pro-oncogene which contributes to tumor progression and chemotherapy resistance. Targeting mutant p53 to restore the p53 pathway is a promising strategy for cancer therapy. Small molecule restoring p53 pathway is mostly based on conformational change in mutant p53 or upregulation of wild-type p53. It is barely reported about small molecule compound with ability to both restore p53 pathway and deplete mutant p53 GOF. In this study, we describe a new small molecular compound, NCI-8 that specifically depletes mutant p53 protein and activates p73 to induce p53 responsive transcriptional activity and cell death in mutant p53 colorectal cancer cells. NCI-8 treatment increased p53 responsive bioluminescence and p53 target gene expression such as P21, Puma and DR5 specifically in mutant p53 colorectal cancer cells. Accompanied with p53 pathway restoration, NCI-8 down-regulated mutant p53 in cancer cells with no evident wild-type conformational change in mutant p53. NCI-8- mediated down-regulation of mutant p53 was rescued by MG132, a proteasome inhibitor and nutlin-3, an MDM2 inhibitor. NCI-8 induced ubiquitination of mutant p53. Taken together these results indicate that NCI-8 induces mutant p53 protein degradation via an MDM2-mediated ubiquitin-proteasome pathway. Further, we examined the role of p73 in NCI-8 action to p53 pathway restoration. NCI-8 enhanced p53-responsive bioluminescence with10-fold increase in cells overexpressing p73, but only 2-fold increase in p53-overexpressing cells. NCI-8- induced p53-responsive bioluminescence was significantly reduced by knock-down of p73, but not by knock-down of mutant p53. Knock-down of p73 significantly reduced NCI-8 induction of p53 target genes in mutant p53 cancer cells. These results indicate that p73 is required for NCI-8 to restore the p53 pathway in mutant p53-expressing cancer cells. Importantly, NCI-8 induced cell death in cancer cells, but not in normal cells. No genotoxicity of NCI-8 was detected in cancer and normal cells. Knock-down of p73 reduced cell apoptosis in mutant p53-expressing cancer cells treated with NCI-8, while, overexpression of p73 sensitized these cancer cells to NCI-8 induced cell death. Combination treatments demonstrate that NCI-8 synergizes with CPT11chemotherapy to induce cell death in mutant p53 colorectal cancer cells. These results suggest that p53 pathway restoration and cell death induced by NCI-8 occur via activation of p73 and depletion of mutant p53 GOF. NCI-8 is a promising lead compound specifically targeting mutant p53 for the development of new anti-cancer drugs. Citation Format: Shengliang Zhang, Lanlan Zhou, Bo Hong, Noel Warfel, Antonius Van den heuvel, David Dicker, Levy Kopelovich, Wafik El-Deiry. A new small molecule compound restoring p53 pathway induces cell death via active p73 and degradation of mutant p53 in colorectal cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2171. doi:10.1158/1538-7445.AM2013-2171

Abstract 3791: MAPK p38 is the link between autophagy and lipid metabolism in colorectal cancer cells

Abstract Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome. Recent progress has demonstrated that autophagy plays a wide variety of physiological and pathophysiological roles, notably in cancer. We have showed previously, that the p38 pathway was involved in Irinotecan sensitivity of colon cancer cells p53WT. p38 being involved in the induction of autophagy we wanted to determine its role in the processes of autophagy in presence and in absence of p53 in colorectal cancer cells. Our results show that the overexpression of the constitutively active p38alpha isoform in HCT116 p53KO cells leads to a massive autophagy, accompanied with a block in G2 / M. The induction of autophagy in this context doesn't leads to the cellular death but rather to survival. The autophagy induction by p38 overexpression in HCT116 was weaker, indicating that the lack of p53 increase p38 role in autophagy induction. Furthermore, we have shown that autophagy induced by p38 comes along with an increase of the lipid metabolism by activating a key enzyme of the synthesis of fatty acids. We demonstrate here, that the role of p38 in the induction of autophagy depends on the presence of p53 and that p38 would be the link between the increase of the lipid metabolism and the induction of autophagy that lead to survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3791. doi:10.1158/1538-7445.AM2011-3791

Activation of p53-Dependent Apoptosis by Acute Ablation of Glycogen Synthase Kinase-3β in Colorectal Cancer Cells

The restoration of checkpoint mechanisms may provide a rational anticancer approach, but the molecular circuitries of how this can be achieved therapeutically are poorly understood. A pivotal signaling network in colorectal cancer cells involves glycogen synthase kinase-3beta (GSK3beta), a multifunctional kinase whose role in tumor cell survival is not defined. We used molecular, genetic, and pharmacologic antagonists of GSK3beta in p53+/+ or p53-/- colorectal cancer cells. We monitored kinase activity in immunoprecipitation, protein expression by immunoblotting, and cell death by multiparametric flow cytometry. A xenograft colorectal cancer model was used to study antitumor activity in vivo. Treatment of p53+/+ colorectal cancer cells with pharmacologic inhibitors of GSK3beta resulted in sustained elevation of p53, with up-regulation of p21(Waf1/Cip1) and loss of survivin levels. Molecular targeting of GSK3beta by overexpression of a GSK3beta dominant-negative mutant, or acute-silencing of GSK3beta by RNA interference, reproduced the induction of transcriptionally active p53 in colorectal cancer cells. This pathway was recapitulated by deregulated Wnt/T-cell factor signaling, with elevation of the tumor suppressor p14ARF, and reduced expression of the p53 antagonist, MDM2. Rather than cell cycle arrest, GSK3beta blockade resulted in p53-dependent apoptosis, which was contributed by acute loss of survivin and inhibition of colorectal cancer growth in mice. Acute ablation of GSK3beta in colorectal cancer cells activates p53-dependent apoptosis and antagonizes tumor growth. This pathway may be exploited for rational treatment of colorectal cancer patients retaining wild-type p53.

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is induced by genistein through the expression of p53 in colorectal cancer cells.

Genistein is an isoflavenoid found in soy that has anti-tumorigenic activities. Treatment of colorectal carcinoma HCT-116 cells with 50 microM genistein results in a 50% reduction in cell proliferation and a 6-fold increase in apoptosis. Genistein induces nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1), a protein with antitumorigenic activities, in a time- and concentration-dependent manner in HCT-116 cells. In addition, p53 and p21 are induced in HCT-116 cells. The induction of p53 (3 hr) precedes the induction of NAG-1 (12 hr), suggesting that genistein-induced NAG-1 expression is mediated by p53. In contrast, NAG-1 is not induced by genistein in the p53-negative colorectal carcinoma cell line HCT-15. Luciferase reporter constructs of the NAG-1 promoter containing 2 p53 sites showed that the p53 sites within the NAG-1 promoter are critical to genistein-induced NAG-1 expression in p53-positive U2OS cells. The expression of p53 was critical for NAG-1 promoter activity since no promoter activity was observed with genistein treatment in HCT-15 cells. However, genistein-induced promoter activity was restored in HCT-15 cells by transfection with wild-type p53. Together our data suggest a relationship between genistein, p53 and NAG-1 forming a novel pathway responsible for the antitumorigenic activity of genistein.

PUMA mediates the apoptotic response to p53 in colorectal cancer cells.

Although several genes that might mediate p53-induced apoptosis have been proposed, none have previously been shown to play an essential role in this process through a rigorous gene disruption approach. We used a gene-targeting approach to evaluate p53-mediated death in human colorectal cancer cells. Expression of p53 in these cells induces growth arrest through transcriptional activation of the cyclin-dependent kinase inhibitor p21. If p21 is disrupted via gene targeting, the cells die through apoptosis. If the PUMA gene is also disrupted in such cells, apoptosis is prevented. The effects of PUMA on apoptosis were observed after exogenous overexpression of p53 as well as after exposure to hypoxia, a physiologic activator of p53, and DNA damage. The PUMA protein interacts with Bcl-X(L) and promotes mitochondrial translocation and multimerization of Bax. Accordingly, genetic disruption of BAX makes cells resistant to the apoptosis resulting from PUMA expression. These results suggest that the balance between PUMA and p21 is pivotal in determining the responses to p53 activation and provide a model for understanding the basis of p53 mutations in human cancer.

Staining patterns of p53 immunohistochemistry and their biological significance in colorectal cancer.

Immunohistochemistry (IHC) is a cheap and rapid method to detect p53 inactivation but the results are often discordant with gene mutation analysis. This study aimed to investigate whether there is a difference in the immunohistochemical staining patterns of p53-positive cells on comparing tumours with inactivating gene mutations with those without. Tissues of 142 colorectal cancers were investigated for p53 inactivation simultaneously by IHC and gene analysis using SSCP of exons 4-9 and sequencing. In addition, tumours were investigated immunohistochemically for the expression of mdm-2 protein, known to be transcriptionally transactivated by the wild-type (wt) p53 gene. p53-positive cells of tumours without detectable p53 gene mutations were microdissected using a PALM laser microscope system and subjected to p53 sequence analysis. Among the 142 cases of colorectal cancer (male/female=88/54; mean age 66a+/-11 years, range 24-90 years), 74% (n=105) of tumours were positive by p53 IHC and mutations in the p53 gene were found in 51% (73 patients). In 16% (12 patients) with mutations in the p53 gene, IHC for p53 was negative. In tumours with mutations in the p53 gene and positive p53 IHC, staining of all nuclei of the tumour was more frequently (57/61, 93%) found than in tumours without p53 gene mutations, where staining of scattered single cells was predominantly seen (29/44, 66%; p<0.0001). mdm-2 positivity (n=33) showed only staining of scattered single cells, predominantly (24/33, 82%; p<0.0001) in tumours without gene mutations. Single cell microdissection followed by mutation analysis of scattered p53-positive cells revealed no gene mutations. A scattered positive immunohistochemical reactivity of p53 in colorectal cancer cells might therefore represent a functionally active non-mutated p53 gene and should not be considered as a marker of gene mutation and inactivation.

Genetic determinants of p53-induced apoptosis and growth arrest.

Previous studies have suggested that expression of p53 in cancer cells can result in either growth arrest or apoptosis. Accordingly, expression of p53 in a series of colorectal cancer cell lines yielded growth arrest in some lines (A-lines) and apoptosis in others (D-lines). To investigate the basis of this difference, we evaluated the role of p21WAF1/Cip1, a known mediator of p53-induced growth arrest. Inactivation of p21 by homologous recombination converted an A-line to a D-line, suggesting that p21 could protect cells from apoptosis. However, examination of p53-induced p21 expression in naturally occurring D-lines and A-lines demonstrated that the induction of p21 could not account for the differential response to p53. Moreover, when a D-line was fused to an A-line, the resulting hybrid cells underwent apoptosis in response to p53, indicating that the apoptosis pathway was dominant over the growth arrest pathway. Therefore, the apoptotic response to p53 in colorectal cancer cells is modulated by at least two factors: p21-mediated growth arrest that can protect cells from apoptosis in A-cells, and trans-acting factors in D-cells that can overcome this protection, resulting in cell death.