Wild-type HCT116 Research Articles

Ceragenin CSA-13 induces cell cycle arrest and antiproliferative effects in wild-type and p53 null mutant HCT116 colon cancer cells

Antimicrobial peptides of the cathelicidin family play a central role in the host defense system. Our group has reported previously that cathelicidin-related or cathelicidin-modified antimicrobial peptides, such as FF/CAP-18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1 and colon cancer-derived cell line HCT116. Ceragenin CSA-13, which mimics the hydrophobic and cationic morphology of cathelicidin-related peptides, was developed to reduce synthetic costs and resolve stability issues in the presence of proteases. In this study, we evaluated the antiproliferative effect of CSA-13 on HCT116 cells. We evaluated the effects of CSA-13 in HCT116 cells by measuring cell growth, detecting apoptosis, analyzing the cell cycle, and examining mitochondrial membrane depolarization. Treatment with CSA-13 suppressed HCT116 cell proliferation in a dose-dependent manner, increasing the incidence of apoptosis detected by the binding of Annexin V. Furthermore, cell cycle analysis showed that the cell cycle of CSA-13-treated wild-type and p53 null mutant HCT116 cells was arrested at the G1/S phase, indicating that CSA-13 affects the cell cycle by a p53-independent pathway. Our study showed that CSA-13 exerts an antiproliferative effect in cancer cells similar to that of FF/CAP-18, suggesting that membrane-permeabilizing capability is the common underlying mechanism for anticancer and antimicrobial effects of CSA-13 and anitimicrobial peptides.

Role of p53, Bax, p21, and DNA-PKcs in radiation sensitivity of HCT-116 cells and xenografts

Molecular factors that dictate tumor response to ionizing radiation in rectal cancer are not well described. We investigated the contribution of p53, p21, Bax, and DNA-PKcs in response to ionizing radiation in an isogeneic colorectal cancer system in vitro and in vivo. HCT-116 DNA-PKcs(-/-) cells and xenografts were radiosensitive compared with wild-type (WT) HCT-116 cells. HCT-116 p53(-/-) cells and tumor xenografts displayed a radioresistant phenotype. Separately, p21 or Bax deficiency was associated with a radiosensitive phenotype in vitro and in vivo. In vivo, Bax deficiency led to increased tumor necrosis and decreased microvessel density. In vitro, HCT-116 Bax(-/-) cells had decreased levels of vascular endothelial growth factor. HCT-116 WT cells had a more radioresistant phenotype after pancaspase inhibition, but pancaspase inhibition did not alter radiosensitivity in HCT-116 Bax(-/-) cells subjected to ionizing radiation. There was no difference in cell growth in HCT-116 WT cells subjected to transient apoptosis-inducing factor (AIF) inhibition; however, HCT-116 Bax(-/-) cells treated with AIF siRNA followed by ionizing radiation had a significant survival advantage compared with control-treated cells, implicating AIF in the radiosensitivity of Bax(-/-) cells. These data might be used along with other markers to predict response to radiation in patients with rectal cancer.

Dual Regulatory Roles of Human AP-Endonuclease (APE1/Ref-1) in CDKN1A/p21 Expression

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein involved in repair of oxidative DNA damage as well as in transcriptional regulation, is often overexpressed in tumor cells. APE1 was earlier shown to stimulate p53’s DNA binding and its transactivation function in the expression of cyclin-dependent kinase inhibitor p21 (CDKN1A) gene. Here, we show APE1’s stable binding to p53 cis elements which are required for p53-mediated activation of p21 in p53-expressing wild type HCT116 cells. However, surprisingly, we observed APE1-dependent repression of p21 in isogenic p53-null HCT116 cells. Ectopic expression of p53 in the p53-null cells abrogated this repression suggesting that APE1’s negative regulatory role in p21 expression is dependent on the p53 status. We then identified APE1’s another binding site in p21’s proximal promoter region containing cis elements for AP4, a repressor of p21. Interestingly, APE1 and AP4 showed mutual dependence for p21 repression. Moreover, ectopic p53 in p53-null cells inhibited AP4’s association with APE1, their binding to the promoter and p21 repression. These results together establish APE1’s role as a co-activator or co-repressor of p21 gene, dependent on p53 status. It is thus likely that APE1 overexpression and inactivation of p53, often observed in tumor cells, promote tumor cell proliferation by constitutively downregulating p21 expression.

Effects of gamma irradiation on cell cycle, apoptosis and telomerase activity in p53 wild-type and deficient HCT116 colon cancer cell lines

Radiotherapy serves as adjunctive treatment to chemotherapy and surgical resection of colorectal cancer. However, the cellular response to irradiation varies depending on the expression of tumor suppressor p53, which plays a significant role in the regulation of cell cycle arrest, apoptosis and telomerase activity in various cancers. The present study aimed to investigate cell cycle arrest, apoptosis and telomerase activity with respect to p53 expression in p53 wild-type (+/+) and deficient (−/−) HCT116 colon cancer cell lines following 5 Gy γ-irradiation. Cell cycle arrest and apoptosis were evaluated using flow cytometry. The telomerase activity was measured using a TRAP (telomerase repeat amplification protocol) assay. Following treatment with irradiation, G1/S cell cycle arrest occurred in the p53+/+ cells, whereas the p53−/− cells accumulated in the G2 phase. No differences were observed in the apoptotic ratios between the two cell lines following irradiation. Decreased telomerase activity was observed in the p53+/+ cells, whereas telomerase activity was increased in the p53−/− cells. The results showed that while telomerase activity and G1 cell cycle arrest were regulated depending on the p53 status, G2 arrest and the apoptotic response were promoted via a p53-independent pathway.

Abstract 2636: The H1047R point mutation in PI3KCA changes morphology in human colon cancer cells.

Abstract INTRODUCTION: The PI3K signaling pathway involves a wide range of cell signaling phenomena, including cell proliferation, differentiation, angiogenesis, survival, adhesion, cytoskeletal rearrangement, invadopodia formation and motility. Mutations in the catalytic subunit p110α (PIK3CA) of Class 1A phosphatidyinositol 3-kinase (PI3K) occur in up to 1/3 of human colorectal cancers (CRC). The kinase domain mutation H1047R is the most frequent cancer-specific mutation in p110α and results in a gain of function. In vivo studies have demonstrated that cell lines bearing the p110α mutation are more metastatic than the cells with wild type p110α. We have previously shown that inhibition of PI3K-p110α significantly delayed or abrogated the migration of HCT116 parental cell line, which is a CRC cell line that is heterozygous for the PIK3CA mutation and thus contains both the H1047R mutation in p110α and a wild type allele of PIK3CA. The aim of our study was to investigate the mechanism of how the H1047R mutation of PI3K results in the increased cell motility in HCT116 cell line. We hypothesize that the rearrangement of the cytoskeleton may be responsible for the changes in cell morphology and cell motility in the presence of the H1047R mutation of PI3K. METHODS: HCT116 cells engineered to contain either the H1047R mutant (MUT) or wild type (WT) PI3KCA allele were used in this study. Cell morphology was observed by microscopic approaches and F-actin, the major component of cytoskeleton, was stained with Alexa Fluor® 488 Phalloidin. Effect of PI3K-p110α inhibition on cell morphology was tested at a series of time points with 1.0 μM of A66 and PIK75, which are specific inhibitors of p110α. RESULTS: The morphological appearance of MUT cells was considerably different as compared to WT cells. The WT HCT116 cells normally show a rounded and more clumped morphology. However, the MUT HCT116 cells become elongated and actin filaments appear to align along the length of the cell. The cells also adopt less clumped and more fibroblastic morphology. Furthermore, when the MUT cells were treated with the p110α specific inhibitors, the cell morphology then again resembled the WT cells. The morphological changes in MUT HCT116 cells possibly contribute to enhanced migratory capacity. CONCLUSION: The morphologic appearance of MUT PIK3CA bearing colon cancer cells is markedly different from WT cells. The inhibition of mutant PIK3CA function results in morphologic changes that resemble the WT allele bearing cells. Thus, the interaction of PI3K and the cell cytoskeleton appears to be an important aspect in regulating cell motility and subsequently, metastases. The PI3K pathway remains a desirable target in CRC therapeutics. Citation Format: Guanghua Wan, Essence Hand, Judy Lin Cannon, Ashwani Rajput. The H1047R point mutation in PI3KCA changes morphology in human colon 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 2636. doi:10.1158/1538-7445.AM2013-2636

Downregulation of serine/arginine-rich splicing factor 3 induces G1 cell cycle arrest and apoptosis in colon cancer cells

Serine/arginine-rich splicing factor 3 (SRSF3) likely has wide-ranging roles in gene expression and facilitation of tumor cell growth. SRSF3 knockdown induced G1 arrest and apoptosis in colon cancer cells (HCT116) in association with altered expression of 833 genes. Pathway analysis revealed 'G1/S Checkpoint Regulation' as the most highly enriched category in the affected genes. SRSF3 knockdown did not induce p53 or stimulate phosphorylation of p53 or histone H2A.X in wild-type HCT116 cells. Furthermore, the knockdown induced G1 arrest in p53-null HCT116 cells, suggesting that p53-dependent DNA damage responses did not mediate the G1 arrest. Real-time reverse transcription-polymerase chain reaction and western blotting confirmed that SRSF3 knockdown reduced mRNA and protein levels of cyclins (D1, D3 and E1), E2F1 and E2F7. The decreased expression of cyclin D and E2F1 likely impaired the G1-to-S-phase progression. Consequently, retinoblastoma protein remained hypophosphorylated in SRSF3 knockdown cells. The knockdown also induced apoptosis in association with reduction of BCL2 protein levels. We also found that SRSF3 knockdown facilitated skipping of 81 5'-nucleotides (27 amino acids) from exon 8 of homeodomain-interacting protein kinase-2 (HIPK2) and produced a HIPK2 Δe8 isoform. Full-length HIPK2 (HIPK2 FL) is constantly degraded through association with an E3 ubiquitin ligase (Siah-1), whereas HIPK2 Δe8, lacking the 27 amino acids, lost Siah-1-binding ability and became resistant to proteasome digestion. Interestingly, selective knockdown of HIPK2 FL induced apoptosis in various colon cancer cells expressing wild-type or mutated p53. Thus, these findings disclose an important role of SRSF3 in the regulation of the G1-to-S-phase progression and alternative splicing of HIPK2 in tumor growth.

Curcumin Enhances the Effect of Chemotherapy against Colorectal Cancer Cells by Inhibition of NF-κB and Src Protein Kinase Signaling Pathways

ObjectiveDevelopment of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Herein, we examined the effectiveness of a combination treatment regimen of 5-fluorouracil (5-FU) and curcumin in colorectal cancer (CRC) cells.MethodsWild type HCT116 cells and HCT116+ch3 cells (complemented with chromosome 3) were treated with curcumin and 5-FU in a time- and dose-dependent manner and evaluated by cell proliferation assays, DAPI staining, transmission electron microscopy, cell cycle analysis and immunoblotting for key signaling proteins.ResultsThe individual IC50 of curcumin and 5-FU were approximately 20 µM and 5 µM in HCT116 cells and 5 µM and 1 µM in HCT116+ch3 cells, respectively (p<0.05). Pretreatment with curcumin significantly reduced survival in both cells; HCT116+ch3 cells were considerably more sensitive to treatment with curcumin and/or 5-FU than wild-type HCT116 cells. The IC50 values for combination treatment were approximately 5 µM and 1 µM in HCT116 and 5 µM and 0.1 µM in HCT116+ch3, respectively (p<0.05). Curcumin induced apoptosis in both cells by inducing mitochondrial degeneration and cytochrome c release. Cell cycle analysis revealed that the anti-proliferative effect of curcumin and/or 5-FU was preceded by accumulation of CRC cells in the S cell cycle phase and induction of apoptosis. Curcumin potentiated 5-FU-induced expression or cleavage of pro-apoptotic proteins (caspase-8, -9, -3, PARP and Bax), and down-regulated anti-apoptotic (Bcl-xL) and proliferative (cyclin D1) proteins. Although 5-FU activated NF-κB/PI-3K/Src pathway in CRC cells, this was down-regulated by curcumin treatment through inhibition of IκBα kinase activation and IκBα phosphorylation.ConclusionsCombining curcumin with conventional chemotherapeutic agents such as 5-FU could provide more effective treatment strategies against chemoresistant colon cancer cells. The mechanisms involved may be mediated via NF-κB/PI-3K/Src pathways and NF-κB regulated gene products.

P21WAF1/CIP1 deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells

p21WAF1/CIP1 is a critical regulator of cell cycle progression. However, the role of p21 in mitochondrial function remains poorly understood. In this study, we examined the effect of p21 deficiency on mitochondrial function in HCT116 human colon cancer cells. We found that there was a significant increase in the mitochondrial mass of p21−/− HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53−/− cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1α and TFAM and AMPK activity were also elevated in p21−/− cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1α axis. However, the increase in mitochondrial biogenesis in p21−/− cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21−/− cells exhibited significant proliferation impairment in galactose medium, suggesting that p21 deficiency induces a defect in the mitochondrial respiratory chain in HCT116 cells. Taken together, our results suggest that the loss of p21 results in an aberrant increase in the mitochondrial mass and in mitochondrial dysfunction in HCT116 cells, indicating that p21 is required to maintain proper mitochondrial mass and respiratory function.

NDV-induced apoptosis in absence of Bax; evidence of involvement of apoptotic proteins upstream of mitochondria

BackgroundRecently it was shown that following infection of HeLa cells with Newcastle disease virus (NDV), the matrix (M) protein binds to Bax and subsequently the intrinsic pathway of apoptosis is activated. Moreover, there was very little alteration on mRNA and protein levels of Bax and Bcl-2 after infection with NDV.FindingIn order to further investigate the role of members of the Bcl-2 family, Bax-knockout and wild-type HCT116 cells were infected with NDV strain AF2240. Although both cells underwent apoptosis through the activation of the intrinsic pathway and the release of cytochrome c from mitochondria, the percentage of dead Bax-knockout cells was significantly lower than wt cells (more than 10% at 48 h post-infection). In a parallel experiment, the effect of NDV on HT29 cells, that are originally Bcl-2-free, was studied. Apoptosis in HT29 cells was associated with Bax redistribution from cytoplasm to mitochondria, similar to that of HeLa and wt HCT116 cells.ConclusionAlthough the presence of Bax during NDV-induced apoptosis contributes to a faster cell death, it was concluded that other apoptotic protein(s) upstream of mitochondria are also involved since cancer cells die whether in the presence or absence of Bax. Therefore, the classic Bax/Bcl-2 ratio may not be a major determinant in NDV-induced apoptosis.

Apoptotic-like death occurs through a caspase-independent route in colon carcinoma cells undergoing mitotic catastrophe

We have examined the relationship between chemotherapy-induced mitotic catastrophe and cell death by apoptosis in both wild-type and p53(−/−) HCT116 human colon carcinoma cells treated with nanomolar concentrations of paclitaxel (PTX), a drug that acts on tubulin altering the normal development of mitosis. After treatment, HCT116 cells entered mitosis regardless of the presence of functional p53, which resulted in changes in the distribution of cells in the different phases of the cell cycle, and in cell death. In the presence of PTX, the percentage of polyploid cells observed was higher in p53-deficient cells, indicating that mitotic slippage was favored compared to wild-type cells, with the presence of large multinucleate cells. PTX caused mitotic catastrophe and about 50–60% cells that were entering an aberrant mitosis died through an apoptotic-like pathway characterized by the presence of phosphatidylserine in the outer cell membrane, which occurred in the absence of significant activation of caspases. Lack of p53 facilitated endoreduplication and polyploidy in PTX-treated cells, but cells were still killed with similar efficacy through the same apoptotic-like mechanism in the absence of caspase activity.

Anti-proliferative effect of an analogue of the LL-37 peptide in the colon cancer derived cell line HCT116 p53+/+ and p53−/−

Antimicrobial peptides of the cathelicidin family are found in many mammalian species, and are focused on various effects other than antimicrobial action. In this study, we evaluated the anti-proliferative effect of an analogue peptide, FF/CAP18, derived from an endogenous cathelicidin family member against the colon cancer cell line HCT116. FF/CAP18 significantly decreased the proliferation of HCT116 cells in a dose-dependent fashion. Furthermore, the treatment of HCT116 with FF/CAP18 caused loss of mitochondrial membrane potential, and resulted in the immunoreactivity to the single-strand DNA antibody, suggesting the early stage of apoptosis. Interestingly, the anti-proliferative effect of FF/CAP18 was constant regardless of the genotype of p53 (wild-type and p53 mutant type HCT116 cells). Therefore, the signaling pathway of p53 is not involved in the growth suppression effect of the cathelicidin analogue peptide. These results indicate that the treatment of certain types of cancer cells with FF/CAP18 may increase the sensitivity of the chemotherapeutic reagents, which might relate to the reduction of the side effects.

Human cytomegalovirus infection is sensitive to the host cell DNA methylation state and alters global DNA methylation capacity

Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that infects and establishes latency in the majority of the human population and may cause fatal infections in immunocompromised patients. Recent data implies a close interaction between HCMV encoded proteins and cellular epigenetic mechanisms such as histone acetylation and deacetylation. In this study, we investigated the interactions between HCMV infection and the DNA methylation machinery in different host cells using several approaches. We found that colon cancer cell line HCT-116 lacking the DNMT1 and DNMT3b methyltransferases was susceptible to HCMV-AD169 infection, while wild-type cells were non-susceptible. Treatment of wild-type HCT-116 cells with 5-azacytidine rendered them susceptible to infection. Further investigation of HCMV infected MRC-5 fibroblasts demonstrated significant global hypomethylation, a phenomenon that was virus strain-specific and associated with the re-localization of DNMT1 and DNMT3b from the nucleus to the cytoplasm. The cytoplasmic accumulation of DNMT1 was also evident in in vitro infected macrophages and in epithelial cells in tissue samples from patients with inflammatory bowel disease and concomitant HCMV infection. Foscavir treatment of virus infected fibroblasts did not affect the majority of the virus induced nuclear exclusion of DNMT1, which suggest that it is dependent on viral IE gene products. In conclusion, HCMV infection results in profound effects on the host cell DNA methylation machinery and is associated with inflammation in vivo. Our results improve the understanding of cytomegalovirus pathogenesis and open the search for new antiviral therapy targets. These findings may also contribute to the further understanding of mechanisms involved in DNA methylation abnormalities in physiological and pathological conditions.

2′‐Hydroxyflavanone induces apoptosis through Egr‐1 involving expression of Bax, p21, and NAG‐1 in colon cancer cells

Natural flavanones exhibit cancer preventive and/or therapeutic effects. The objective of this study was to investigate the molecular mechanism underlying the action of the antitumor activity of hydroxyflavanone using the HCT116 colon cancer cell line. We investigated the effect of hydroxyflavanones on antitumor activity. We found that 2'-hydroxyflavanone (2'-HF) potently inhibited the clonogenicity of HCT116 cells. 2'-HF triggered apoptosis in both wild-type and p53-null HCT116 cells, as revealed by DNA fragmentation and caspase activation. 2'-HF upregulated nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) expression through induction of Egr-1. Silencing of NAG-1 or Egr-1 using small interfering RNA (siRNA) could attenuate 2'-HF-induced apoptosis. Egr-1 also upregulated the proapoptotic gene Bax and the cell cycle inhibitor p21. Dietary 2'-HF may possess antitumor activity against human colon cancer.

Abstract 2943: KPT-SINE (Selective Inhibitors of Nuclear Export) induce apoptosis in colon cancer cells in-vitro and in-vivo through nuclear localization of Tumor Suppressor Proteins (TSPs)

Abstract Chromosome Region Maintenance 1/Exportin-1 (CRM1) is a key nuclear export protein whose inhibition leads to the nuclear accumulation of TSPs and negative regulators of cell proliferation. Through CRM1 inhibition, nuclear localization of these proteins restores cell cycle checkpoints and genome surveying functions culminating in apoptosis of tumor cells. Conversely, CRM1 inhibition of normal cells induces reversible cell cycle arrest. We are developing novel small molecules, KPT-SINE, which irreversibly bind and inhibit CRM1 nuclear export killing a broad range of tumor cell lines and xenografts. We used human colon cancer cells HCT116 to track the molecular and cellular events that follow KPT-SINE treatment and ultimately lead to cancer cell death. Methods: Using our proprietary in silico hierarchical structure-based discovery platform, we designed and tested novel SINE in a cell-based microscopy assay to confirm CRM1-mediated nuclear export inhibition. Cytotoxic IC50s of KPT-SINE were determined on a panel of cancer cell lines. KPT-SINE – KPT-185, -251 and -276 were selected for additional testing based on their potency and pharmacokinetics. These include; 1. Nuclear localization of TSPs, 2. Effects on cell cycle and viability, 3. Effects on TSP mRNA and protein expression. Additionally, we transiently transfected mutant CRM1-C528S into cells and treated with KPT-SINE to show binding to Cysteine 528. We tested the effects of KPT-276 in an in vivo HCT116 xenograft model. Results: KPT-276, a potent inhibitor in the cell-based microscopy assay (EC50 = 130 nM), was a robust inhibitor of HCT116 proliferation (IC50 = 400 nM). KPT-SINE inhibition of CRM1 started within 30 minutes and reached a maximum in 8 hrs. Additionally, KPT-SINE effects were blocked by transient transfection of CRM1-C528S, confirming KPT-SINE - Cysteine 528 interaction. Washout experiments demonstrated that 4 hrs of KPT-SINE incubation sustained 24 hrs of CRM1 inhibition. We also identified nuclear accumulation of p53, FOXO3a, pRb, APC, IαB, p27, PTEN, and p21, which was followed by cell cycle arrest and cell death. Although KPT-276 treatment had less effect on cell cycle and cytotoxicity assays of p53null HCT116 cells, the p53 wildtype and null HCT116 cells showed similar kill curves after 8 days of treatment. Mouse xenografts treated with 75 mg/kg QDX5 of KPT-276 each week for 4 weeks inhibited tumor growth by more than 70%. Further analysis of the tumor molecular markers from KPT-276 treated animals will be reported. Conclusions: In this study we described the correlative effects of KPT-SINE from the accumulation of nuclear TSPs through to cancer cell death in mouse models. These results suggest that KPT-SINE display potent in vivo efficacy in the xenograft model of human colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2943. doi:1538-7445.AM2012-2943

Abstract 1628: Tumor suppressor gene demethylation and reactivation in human colon cancer cells by black raspberry anthocyanins

Abstract Our laboratory has shown that the anthocyanins (ACs) in black raspberries (BRBs) are responsible for much of their chemopreventive effects in the rat esophagus. The present study was undertaken to determine the effects of BRB ACs on human colon cancer cells in vitro. Three days of AC treatment at 5 and 25 μg/ml medium significantly decreased cell proliferation and increased apoptosis in HCT116, Caco2, and SW480 human colon cancer cell lines. The ACs also reduced the activities and protein expression levels of DNA methyltransferases 1 and 3 (DNMT1, DNMT3) in all three cell lines. Promoter methylation of p16 (CDKN2A) and the Wnt pathway inhibitors, Sfrp2, Sfrp5 and Wif1, was decreased by AC treatment resulting in increased mRNA expression levels of all three Wnt inhibitors. ACs did not alter global methylation LINE-1. DNMT1 and DNMT3B knockout (KO) HCT116 cells were generated using siRNA to determine the role of these methyltransferases in AC-induced demethylation. In DNMT1 KO cells, the promoter methylation of Sfrp5 was reduced when compared with wild type HCT116 cells. AC treatment further reduced promoter methylation of Sfrp5 in DNMT1 KO cells suggesting that the ACs targeted protein(s) other than DNMT1 to regulate methylation. Promoter methylation of Sfrp5 was decreased to almost zero by treatment of DNMT3B KO cells with ACs. Interestingly, promoter methylation of p16 was reduced in both DNMT1 and DNMT3B KO cells, and AC treatment further reduced p16 methylation in both lines. In conclusion, these results suggest that ACs demethylated and reactivated tumor suppressor genes through regulation of DNMT1 and DNMT3B. This research was supported by NCI grant CA148818. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1628. doi:1538-7445.AM2012-1628

Abstract 4155: Comprehensive glucan profile exploration of mutated K-ras knockdown in colorectal cancer

Abstract (Background) In colorectal cancer (CRC), K-ras mutation is found in nearly 40% of the patients, and it is of prognostic significance (Onozato W et al, J Surg Oncol, 2010). Moreover, its knockdown in CRC cell lines with mutated K-ras gene results in robust reduction of ability of cell proliferation and anchorage independent growth (Shirasawa S, Science, 1993), both of which reflect metastatic ability. On the other hand, robust alterations of glycan structures have been reported in CRC promotion steps, accompanied by remarkable phenotypic changes, however there is no report that mentions the relationship of K-ras mutation and glycan change. (Materials and Methods) We examined glycan change of CRC cell lines (DLD1 and HCT116) somatically knocked out for K-ras gene by lectin array including 41 lectins to elucidate whether K-ras mutation-induced glycan changes play a critical role in CRC promotion. (Result) (1) In DLD1, we compared DLD1 wild type genotype cells (DLD1, DKS-5) with those that were knocked out for mutated K-ras gene (DKO-3, DKS-8), and the signals of MAL, MPA, UEA-I, and TJA-II were remarkably decreased in the knockdown cells. (2) In HCT116, we compared HCT116 wild type genotypic cells (HCT116, Hk2-10) with those that were knocked out for the mutated K-ras gene (Hke-3), and both MAL and MPA were remarkably declined. (3) In both cell lines, expression changes of glycans which can bind with MAL and MPA were consistent with the results of the lectin blotting, and both lectin signals were confirmed to be commonly altered by removing the mutated K-ras gene. (Conclusion) Mutated K-ras may be involved in critical phenotype change of CRC, accompanied by abnormal sialic acid recognition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4155. doi:1538-7445.AM2012-4155

Abstract 4664: Co-treatment of cancer cells with DNA damaging drugs and quercetin suppresses cell growth independent of p21 and Bax induction

Abstract Quercetin, one of the most abundant polyphenols, has been evaluated for its potential cancer preventive functions and for its anticancer activity in vitro and in vivo. Its interactions with chemotherapeutics, however, have not been established. Some reports indicate enhancement of drug activity by quercetin but others suggest precautions in co-administering antioxidants such as quercetin and chemotherapeutic drugs. Therefore, further investigations were needed to determine the conditions under which the co-treatment of cancer cells with drugs and quercetin could be clinically beneficial. This study was done to examine how quercetin modulates responses to the chemotherapeutic drugs, 5FU, camptothecin, and VP16, in colon and prostate cancer cells, and to determine the biochemical processes involved in the interactions. Survival of HCT116 colorectal cancer cells (p53 wild-type &amp; null) was determined by assay of clonogenicity of cells exposed to selected concentrations of 5-FU, camptothecin, or VP16 in the presence or absence of quercetin. For cell cycle and biochemical experiments, cells were exposed for 24 hours to 10 µM 5FU, 1 µM camptothecin, or 10 µM VP-16 with or without 50 µM quercetin. The effects of the drugs on the cell cycle were measured by flow cytometry. The induction of p53 and its transcriptional targets, p21 and Bax, as well as the levels of cell cycle regulators, cyclin B1 and survivin, were determined by immunoblotting. Additionally, cell migration assays were used to evaluate the effect of combination treatments on the motility of RKO colorectal and PPC1 prostate cancer cells. Quercetin synergistically inhibited the clonogenicity of the wild-type HCT116 cells, but also inhibited effects on the cell cycle of all the drugs tested. In contrast, for p53-null cells, the combination of low concentrations of 5-FU with up to 6 µM quercetin promoted clonogenic survival. Exposure of wild-type cells to 50 µM quercetin reduced drug-induced up-regulation of p53, p21, and Bax. Combinations of quercetin and the drugs also reduced the levels of cyclin B1 and survivin. RKO and PPC1 cells exposed to the drugs had reduced migratory capacity. Although quercetin alone reduced their migratory capacity, combination treatments did not further reduce cell migration. In summary, quercetin in combination with 5-FU reduced the survival of p53-proficient HCT116 colorectal cancer cells independently of p21 and Bax, but combination of this flavonoid with low concentrations of 5-FU provided a survival advantage for p53-null cells. Further investigations are needed to determine the mechanisms and circumstances under which the combinations of bioactive dietary compounds and chemotherapeutic drugs are beneficial. Supported by grants from NCI/NIH (2U54-CA118948-06, and SC2CA138178). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4664. doi:1538-7445.AM2012-4664

Abstract 564: Bim-independent apoptosis by benzyl isothiocyanate in human breast cancer cells is mediated by PUMA

Abstract Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, is a promising agent for chemoprevention of breast cancer as evidenced by efficacy against xenografted breast cancer cells (e.g., MDA-MB-231 cells) and in a transgenic mouse model (MMTV-neu). We have shown previously that mammary cancer chemoprevention with dietary BITC administration in the MMTV-neu mice is associated with apoptosis induction. In cellular models of human breast cancer (MCF-7 and MDA-MB-231 cells), the BITC-induced apoptosis is initiated by reactive oxygen species (ROS) resulting from inhibition of complex III of the mitochondrial respiratory chain. The present study builds upon these observations to provide insights into the molecular circuitry of BITC-induced apoptosis downstream of ROS production. Because BITC-induced apoptosis in different cell types is associated with activation of c-Jun N-terminal kinase (JNK), we initially focused on Bim as this protein is a downstream mediator of JNK-regulated apoptosis. Bim protein expression (extra-long form) was increased by about 2-3-fold upon 24-hour treatment of MDA-MB-231 cells with pharmacologically relevant concentrations of BITC (2.5 and 5 μM BITC). However, RNA interference of Bim had no effect on BITC-induced apoptosis as judged by analysis of cytoplasmic histone-associated DNA fragment release into the cytosol. Moreover, the BITC-treated MCF-7 cells exhibited a marked decrease in Bim (extra-long form) protein level. BITC treatment resulted in induction of PUMA in both MCF-7 and MDA-MB-231 cells, but as expected, this response was more pronounced in the MCF-7 cells which express wild-type p53. The BITC-induced apoptosis was partially but statistically significantly attenuated by RNA interference of PUMA in MCF-7 cells. Consistent with these observations, genetic depletion of PUMA in HCT-116 cells conferred partial but significant protection against BITC-induced apoptosis. Attenuation of BITC-induced apoptosis in PUMA knockout cells was accompanied by enhanced G2/M phase cell cycle arrest compared with isogenic wild-type HCT-116 cells. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with PUMA induction in the tumor as evidenced by immunohistochemistry and western blotting. In conclusion, these results indicate that Bim-independent apoptosis by BITC is partly mediated by PUMA. This investigation was supported by the US PHS grant CA129347 awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 564. doi:1538-7445.AM2012-564

Regulation of Tumor Suppressor p53 and HCT116 Cell Physiology by Histone Demethylase JMJD2D/KDM4D

JMJD2D, also known as KDM4D, is a histone demethylase that removes methyl moieties from lysine 9 on histone 3 and from lysine 26 on histone 1.4. Here, we demonstrate that JMJD2D forms a complex with the p53 tumor suppressor in vivo and interacts with the DNA binding domain of p53 in vitro. A luciferase reporter plasmid driven by the promoter of p21, a cell cycle inhibitor and prominent target gene of p53, was synergistically activated by p53 and JMJD2D, which was dependent on JMJD2D catalytic activity. Likewise, overexpression of JMJD2D induced p21 expression in U2OS osteosarcoma cells in the absence and presence of adriamycin, an agent that induces DNA damage. Furthermore, downregulation of JMJD2D inhibited cell proliferation in wild-type and even more so in p53−/− HCT116 colon cancer cells, suggesting that JMJD2D is a pro-proliferative molecule. JMJD2D depletion also induced more strongly apoptosis in p53−/− compared to wild-type HCT116 cells. Collectively, our results demonstrate that JMJD2D can stimulate cell proliferation and survival, suggesting that its inhibition may be helpful in the fight against cancer. Furthermore, our data imply that activation of p53 may represent a mechanism by which the pro-oncogenic functions of JMJD2D become dampened.

Critical Role of p53 Upregulated Modulator of Apoptosis in Benzyl Isothiocyanate-Induced Apoptotic Cell Death

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.