HCT116 Colorectal Cancer Cells Research Articles

Abstract 120: HIF-1α regulation by Smurf2-mediated protein degradation in a VHL-independent manner

Abstract HIF-1α is the main mediator of hypoxic response in cells and contributes to angiogenesis, metastasis and therapy resistance in cancer. The canonical pathway of HIF-1α degradation in normoxia is through the Von Hippel-Lindau (VHL) E3 ubiquitin ligase. We previously found that knockdown of CDK4 decreased HIF-1α expression in a VHL-independent manner. To explore the mechanism of CDK4-induced HIF-1α stabilization, we performed a proteomic analysis on HIF-1α-interacting proteins with palbociclib treatment, a bioinformatic prediction of HIF-1α-targeting E3 ubiquitin ligases and identified SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) to be involved in a non-canonical HIF-1α regulation mechanism. Overexpression of Smurf2 enhanced the ubiquitination of HIF-1α and reduced expression of HIF-1α in HCT116 colorectal cancer cells. Smurf2 overexpression downregulated HIF-1α target gene SLC2A1 but not HIF1A mRNA level in hypoxia. Knockdown of SMURF2 increased HIF-1α expression in normoxia even when CDK4 was knocked-down. TCGA analysis showed correlation between high SMURF2 mRNA and better prognosis in kidney renal clear cell carcinoma (KIRC). In KIRC, VHL loss is a frequent mutational event that leads to high constitutive expression of HIF-1α and HIF-2α. Thus, we investigated a role for Smurf2 in HIF regulation in this cancer type. Overexpression of Smurf2 suppressed HIF-1α expression in RCC4 VHL-deficient clear cell renal cancer line in normoxia. Knockdown of SMURF2 increased HIF-1α expression in RCC4 cells. Treatment of RCC4 cells with Smurf2 inhibitor Heclin also increased HIF-1α expression in normoxia. HIF-2α expression was reduced upon Smurf2 overexpression in RCC4 cells under normoxia and in SW480 colorectal cancer cells under hypoxia. In summary, Smurf2 mediates VHL-independent HIF-1α degradation, which provides new insights for HIF-1α targeting in cancers especially with VHL deficiency, and targeting of Smurf2 to increase HIF activity may have functional consequences in non-neoplastic disorders. Citation Format: Shuai Zhao, Wafik S. El-Deiry. HIF-1α regulation by Smurf2-mediated protein degradation in a VHL-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 120.

Abstract 3002: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

Abstract Autophagy is a dynamic, multi-step process that cells use to degrade damaged, abnormal, and potentially harmful cellular substances. While autophagy is maintained at a basal level in all cells, it is activated at a higher level in many cancer cells and promotes tumor growth, anti-tumor immune response, and resistance to cancer therapy. As a result, autophagy is increasingly being recognized to have an important role in cancer progression and emerging as a potential target for cancer therapy. We recently discovered that small GTPase Rab27B, a well-known regulator of vesicle trafficking and exosome secretion, also regulates autophagy process. Depletion of Rab27B in colorectal cancer (CRC) cells by CRISPR-Cas9 and siRNA resulted in an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62 indicating a defect in the autophagy flux. To characterize this autophagy defect, we used EGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining and identified that the degradation step of the autophagy flux is inhibited when Rab27B is silenced. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that autophagy defect resulting from Rab27B deletion will cause reduced stress response and tumor growth. Indeed, Rab27B knockout resulted in a 60% reduction in cell viability in response to starvation and a 94% reduction in colony formation in soft agar assay. Rab27B deletion also prevented spheroid formation in vitro. Finally, to analyze the effect of Rab27B deletion in tumor formation in vivo, we performed a xenograft study with wildtype and Rab27B knockout CRC cells HCT116 and observed that Rab27B deletion resulted in a significant reduction of tumor size (p<0.0001). Next, to analyze the therapeutic potential of Rab27B inhibition in CRC, we sought to identify small-molecule inhibitors of Rab27B GTPase activity in vitro through high-throughput drug screening from a panel of ∼2,100 compounds contained within the KU-HTSL chemical library. This screening resulted in the identification of three compounds (BTB00809, BTB03584, and KM01532) that demonstrated Rab27B GTPase activity inhibition. Interestingly, inhibition of Rab27B in CRC cells with the compound km01532 resulted in a similar increase of LC3-II in CRC cells HCT116 and SW480 as seen in the Rab27B knockout. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on investigating the mechanism of how Rab27B functions in the autophagy process, and whether Rab27B can be targeted as a potential therapeutic strategy for CRC using the small molecule compound km01532. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan Dixon. Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3002.

Abstract 6067: Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines

Abstract Background: Our overarching goal is to therapeutically sensitize human colorectal cancer stem cells to differentiation-inducing effects of retinoid agents. Tumorigenesis in colorectal cancer (CRC) results from the acquisition of mutations in a colonic stem cell which transforms it into a cancer stem cell (CSC). There are no curative treatments for advanced CRC due to the resistance of CSCs to chemotherapy and/or radiation. All-trans retinoic acid (ATRA) is known to be a greatly effective treatment for acute promyelocytic leukemia (APL) which is caused by translocation in the retinoic acid receptor alpha (RARA) gene. We conjecture that the response of retinoic acid (RA) agents may have effects on solid tumors depending on their pathway genotype. Results from our bioinformatics analysis on mutations and overexpression of RA signaling component genes in CRC showed that the majority of RA pathway genes are overexpressed in CRC. Therefore, we hypothesize that the ability of retinoid and retinoid metabolism blocking agents to induce differentiating effects in CSCs depends on the RA pathway genotype expressed in CRC cells. Methods: The ability of RA agents (ATRA, 13-cis retinoic acid, 9-cis retinoic acid) and RA metabolism blocking agents (Liarozole and Talarozole) to inhibit proliferation and induce differentiation on CRC cell lines with different mutations in RA signaling were evaluated. Additionally, Nanostring Profiling was used to measure the effects of the agents on mRNA expression of CSC markers, indicators of cell differentiation, and RA receptors. Results: Our results show that the RA pathway genotype affects the response of RA agents in CRC cell lines. Both HCT116 and SW480 CRC cell lines, which are both mutant in RARA and RARA and RXRG respectively displayed resistance to ATRA. HT29 CRC cells express wild- type RA receptors and are sensitive to ATRA. All three cell lines showed similar dose responses to the CYP26A1 inhibitor Liarozole. This indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism increases intracellular RA levels in a similar manner on all 3 cell lines. Nanostring Profiling shows that ATRA treatment of HT29 cells induces: an increase in the expression of RARA, CYP26A1 (ATRA metabolizing enzyme) and KRT20 (differentiation marker); and a decrease in the expression of CSC markers LGR5 and ALDH1A1. This indicates an active RA signaling pathway, induced differentiation, and a decrease in stem cells. Discussion: Overall, our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Therefore, further investigation on how to therapeutically sensitize human colonic CSCs to the differentiation-inducing effects of retinoid agents should provide insight into designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6067.

Abstract 5949: Aspirins metabolites inhibit human colon cancer cell growth: Potential role in colorectal cancer prevention

Abstract Aspirin’s role in colorectal cancer (CRC) prevention is evidential from several epidemiological data. Numerous theories and targets have been proposed, a consensus has not been reached regarding its mechanism of action. One of the unexplored area in aspirin’s CRC prevention is the role of Dihydroxy benzoic acids (DHBAs), which are generated from aspirin’s metabolism by gut microflora and cytochrome P450 (CYP450). Out of the screened DHBAs, 2,3-DHBA and 2,5-DHBA showed concentration dependent inhibition of CDK1, CDK2, CDK4 and CDK6 enzyme activity in-vitro. We demonstrated that 2,3-DHBA and 2,5-DHBA inhibits CDK-1 enzyme activity beginning at 500 µM, while CDK2 and CDK4 activity was inhibited only at higher concentrations (>750 µM). 2,3-DHBA inhibited CDK6 enzyme activity from 250 µM, while 2,5-DHBA inhibited its activity >750 µM. Using colony formation assays, we further demonstrated the role of 2,3-DHBA and 2,5-DHBA in cancer cell cycle inhibition in 3 different cancer cell lines. Out of the 2 DHBAs, 2,5-DHBA was highly effective in inhibiting clonal formation in HCT-116 and HT-29 CRC cell lines (250-500 µM), and in the MDA-MB-231 breast cancer cell line (~100 µM); whereas both aspirin and salicylic acid failed to inhibit all four CDKs and colony formation. Based on the present results, it is suggested that 2,3-DHBA and 2,5-DHBA may contribute to the chemopreventive properties of aspirin, possibly through the inhibition of CDKs. The present data and the proposed mechanisms should open new areas for future investigations. Citation Format: Poojitha Tummala, Ranjini Sankaranarayanan, Rakesh Dachineni, Jayarama Gunaje Bhat. Aspirins metabolites inhibit human colon cancer cell growth: Potential role in colorectal cancer prevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5949.

Anti-proliferative and Anti-migratory Activities of Diphyllin on Human Colorectal Cancer Cells

Published on:June 2022 Journal of Young Pharmacists, 2022; 14(2):198-202 Original Article | doi:10.5530/jyp.2022.14.37 Authors: Sagar Puli, Ramasamy Raveendran* Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Dhanvantari Nagar, Puducherry, INDIA. Abstract: Background: Colorectal cancer (CRC) is the third leading cause of death, probably because of its invasive and metastatic potential. Agents that can mitigate the growth and prevent migration may benefit CRC. Diphyllin and its analogues belong to glycosylated compounds previously reported for their in vitro cytotoxicity against many viruses, candida, and human malignant cells. This study aims to evaluate the anti-proliferative and antimigratory activities of diphyllin on human CRC cells. Methods: The MTT assay was performed to determine the 50% inhibitory concentrations (IC50) in HT-29, SW-480, and HCT-15 CRC cells. The acridine orange and ethidium bromide (AO/EB) dual staining was used to ascertain the type of cell death. HT-29 cells were subjected to short-term 5-fluorouracil+oxaliplatin (5-FU+Ox) to eliminate the most sensitive cells. The remaining surviving cells were further treated with diphyllin alone or combined with 5-FU. The combination effect of diphyllin with 5-FU in 5-FU+Ox surviving HT-29 cells was determined by flow cytometry-based Annexin V apoptosis assay. The wound-healing assay was used to detect the anti-migratory activity of diphyllin on 5-FU+Ox surviving HT-29 CRC cells. Results: The IC50 values of diphyllin were found to be 2.9±0.38, 1.3±0.28, and 3.9±0.65 μg/mL against HT-29, SW-480, and HCT-15, respectively. Microscopic examination manifested apoptotic changes like chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies in diphyllin treated CRC cells. Flow cytometry analysis revealed that diphyllin enhances the apoptosis in 5-FU+Ox surviving HT-29 cells. Wound-healing assay displayed the inhibitory activity of diphyllin in 5-FU+Ox surviving HT-29 cells. Conclusion: Diphyllin shows anti-proliferative activity by inducing apoptosis in HT-29, SW-480, HCT-15 cells and 5-FU+Ox surviving HT-29 cells and impairs their migration. Key words: Anticancer, Apoptosis, Cytotoxicity, Flow cytometry, Woundhealing.

CREB1 contributes colorectal cancer cell plasticity by regulating lncRNA CCAT1 and NF-κB pathways.

The CREB1 gene encodes an exceptionally pleiotropic transcription factor that frequently dysregulated in human cancers. CREB1 can regulate tumor cell status of proliferation and/or migration; however, the molecular basis for this switch involvement in cell plasticity has not fully been understood yet. Here, we first show that knocking out CREB1 triggers a remarkable effect of epithelial-mesenchymal transition (EMT) and leads to the occurrence of inhibited proliferation and enhanced motility in HCT116 colorectal cancer cells. By monitoring 45 cellular signaling pathway activities, we find that multiple growth-related pathways decline significantly while inflammatory pathways including NF-κB are largely upregulated in comparing between the CREB1 wild-type and knocked out cells. Mechanistically, cells with CREB1 knocked out show downregulation of MYC as a result of impaired CREB1-dependent transcription of the oncogenic lncRNA CCAT1. Interestingly, the unbalanced competition between the coactivator CBP/p300 for CREB1 and p65 leads to the activation of the NF-κB pathway in cells with CREB1 disrupted, which induces an obvious EMT phenotype of the cancer cells. Taken together, these studies identify previously unknown mechanisms of CREB1 in CRC cell plasticity via regulating lncRNA CCAT1 and NF-κB pathways, providing a critical insight into a combined strategy for CREB1-targeted tumor therapies.

The Synergistic Cytotoxic Effects of GW5074 and Sorafenib by Impacting Mitochondrial Functions in Human Colorectal Cancer Cell Lines.

Colorectal cancer (CRC) ranks third in the United States for incidence or mortality. Surgical resection is the primary treatment for patients at an early stage, while patients with advanced and metastatic CRC receive combined treatment with chemotherapy, radiotherapy, or targeted therapy. C-RAF plays a key role in maintaining clonogenic and tumorigenic capacity in CRC cells and it might be a potential therapeutic target for CRC. Sorafenib is a popular oral multi-kinase inhibitor, including a B-RAF inhibitor that targets the RAF-MEK-ERK pathway. Sorafenib, as a single agent, has tumor-suppressing efficacy, but its clinical application is limited due to many complex drug resistance mechanisms and side effects. GW5074 is one of the C-RAF inhibitors and has the potential to enhance the efficacy of existing cancer chemotherapies. In this study, we investigated whether the combination of sorafenib with GW5074 could reduce the dosage of sorafenib and enhance its tumor-suppressive effect in two CRC cell lines, HCT116 and LoVo cells. Our findings demonstrate that GW5074 can potentiate the cytotoxicity of sorafenib and dramatically reduce the half-maximal inhibitory concentration (IC50) dose of sorafenib from 17 and 31 µM to 0.14 and 0.01 µM in HCT116 and LoVo cells, respectively. GW5074, similar to sorafenib, suppressed the cellular proliferation and induced cellular apoptosis and cytosolic ROS, but had no further enhancement on the above-mentioned effects when combined with sorafenib. The synergistic effects of GW5074 and sorafenib were mainly found in mitochondrial functions, including ROS generation, membrane potential disruption, and fission–fusion dynamics, which were examined by using the flow cytometry analysis. In summary, the C-RAF inhibitor GW5074 might potentiate the cytotoxicity of the B-RAF inhibitor sorafenib mediated through mitochondrial dysfunctions, suggesting that GW5074 potentially serves as a sensitizer for sorafenib application to reduce the risk of drug resistance of CRC treatment. Our findings also provide novel insights on using C-RAF inhibitors combined with sorafenib, the current CRC therapeutic drug choice, in CRC treatment.

NPM1 promotes cell proliferation by targeting PRDX6 in colorectal cancer

Colorectal cancer is a malignant tumor that begins in the colorectal mucosal epithelium. NPM1 is a nucleolar phosphoprotein that has been linked to tumor progression in humans. NPM1 is significantly overexpressed in a variety of tumors, including colorectal cancer, but its role and mechanism in colorectal cancer remain unknown. Therefore, the purpose of this study was to discover the role of NPM1 in promoting colorectal cancer proliferation via PRDX6 and its molecular mechanism. NPM1 knockdown or overexpression inhibited or promoted the proliferation and cell cycle progression of HCT-116 and HT-29 colorectal cancer cells, respectively, according to our findings. Furthermore, NPM1 knockdown or overexpression increased or decreased intracellular ROS levels. Animal experiments revealed that NPM1 knockdown or overexpression inhibited or promoted the growth of colorectal cancer cells transplanted subcutaneously. NPM1 knockdown or overexpression reduced or increased PRDX6 expression and related enzyme activities, respectively, according to our findings. NPM1 formed a complex with CBX3 as evidenced by immunoprecipitation, and the double luciferase reporter gene assay confirmed that the CBX3-NPM1 complex promoted PRDX6 transcription. Our data support the role of NPM1 in promoting the proliferation of colorectal cancer, which may be accomplished by CBX3 promoting the expression of the antioxidant protein PRDX6 and thus inhibiting intracellular ROS levels. NPM1 and PRDX6 are potential colorectal cancer therapeutic targets.

Anticancer Activity of Cannabidiol (CBD) in Human Colorectal Cancer Cells: A Mechanistic Study

ObjectivesCannabidiol (CBD) is a major non-psychoactive bioactive component of the phytocannabinoids abundant in cannabis. Its potential use for treatment of colorectal cancer has been proposed by several studies. This study investigated the anti-cancer activity of CBD and the mechanism of that activity in human colorectal cancer cells. MethodsHuman colorectal cancer cell lines (SW620, SW480, HCT116, and Caco-2) and a human normal colon cell line (CCD18CO) were maintained in DMEM supplemented with 10% FBS and antibiotics. Cell viability was measured by MTT assay. Cell cycle phase was measured by flow cytometry. Apoptosis was analyzed by measuring caspase 3/7 enzyme activity. Western blotting was performed to compare expression of proteins regulating cell cycle arrest, apoptosis, and endoplasmic reticulum (ER) stress. Selective inhibitors for cannabinoid receptors were used to test the relevance of each receptor for CBD-mediated cell viability repression and apoptosis induction. Different types of CBD derivatives were tested for effects on cell viability. Data were analyzed using one-way analysis of variance (ANOVA), or two-way ANOVA followed by Bonferroni post-hoc tests (Dunnett T3 tests for unequal variance) for intergroup comparisons. ResultsCBD repressed viability of SW620, SW480, HCT116, and Caco-2 human colorectal cancer cells, with respective IC50 values of 5.4, 10.4, 10.8, and 20 μM for 24-hour treatment and 4.7, 5.8, 5.7, and 13.8 μM for 48-hour treatment. Moreover, CBD treatment led to G1-phase cell cycle arrest and an increased sub-G1 population with downregulation of cyclin D1, cyclin D3, CDK2, CDK4, and CDK6. It also increased activity of caspase 3/7, production of cleaved PARP, and expression of ER stress proteins (BiP, IRE1α, p-eIF2, ATF3 and ATF4). Repression of cell viability and induction of apoptotic cell death occurred through a mechanism dependent on cannabinoid receptor type 2 (CB2). Suppression of cell viability was also observed in cells treated with other of non-psychoactive cannabinoid derivatives (cannabidivarin, cannabigerol, cannabicyclol, cannabigerovarin). ConclusionsOur data indicate that CBD and its derivatives could be promising agents for the prevention of human colorectal cancer. Funding SourcesNo funding source.

Suppression of HCT116 Human Colon Cancer Cell Motility by Polymethoxyflavones is Associated with Inhibition of Wnt/β-Catenin Signaling

Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). In this study, we examined the effect of polymethoxyflavones present in citrus peels on Wnt/β-catenin signaling in the HCT116 CRC cell line. We found that 5,7,3′,4′-tetra-methoxyflavone (TMF) and 7,8,3′,4′-TMF inhibited the expression of target genes of Wnt/β-catenin signaling and the transcriptional activities of β-catenin/Tcf and suppressed the motility of HCT116 cells. Because the binding of β-catenin to Tcf-4 was disrupted by 5,7,3′,4′-TMF and 7,8,3′,4′- TMF, we suggest that they are inhibitors of the Wnt/β-catenin signaling and may have potential applications in CRC prevention.

Ficus dubia Latex Extract Induces Cell Cycle Arrest and Apoptosis by Regulating the NF-κB Pathway in Inflammatory Human Colorectal Cancer Cell Lines.

Simple SummaryIn this study, the anti-tumor activity of Ficus dubia latex extract (FDLE) against HCT-116 and HT-29 human colorectal cancer cell lines in normal and inflammatory conditions was investigated. The results indicated that an inflammation-activated NF-κB increased proliferation-related proteins and decreased apoptosis-related proteins, leading to hyperproliferation of colorectal cancer cell lines. FDLE exhibited remarkable antiproliferative activity in both conditions, however, more effective anti-proliferation was observed due to its more effective regulation of NF-κB inactivation and some proteins related to cell cycle progression and apoptosis induction in inflammatory condition. Our finding may promote the antiproliferative study of FDLE in inflammation-induced colorectal cancer progression.Colorectal cancer is one of the most diagnosed cancers that is associated with inflammation. Ficus dubia latex is recognized as a remedy with various therapeutic effects in traditional medicine, including anti-inflammatory and antioxidant activity. The present study aims to compare the anti-tumor activity of Ficus dubia latex extract (FDLE) against HCT-116 and HT-29 human colorectal cancer cell lines in normal and inflammatory condition and explore its mechanism of action. FDLE exhibited remarkable antiproliferative activity against HCT-116 and HT-29 colorectal cancer cell lines in both conditions using MTT and colony formation assays and more effective anti-proliferation was observed in inflammatory condition. Mechanistically, FDLE induced cell cycle arrest at G0/G1 phase by down-regulating NF-κB, cyclin D1, CDK4 and up-regulatingp21 in both cell in normal condition. In inflammatory condition, FDLE not only exhibited stronger induction of cell cycle arrest in both cells by down-regulating NF-κB, cyclin D1, CDK4 and down-regulating p21, but also selectively induced apoptosis in HCT-116 cells by down-regulating NF-κB and Bcl-xl and up-regulating Bid, Bak, cleaved caspase-7 and caspase-3 through stronger ability to regulate these proteins. Our results demonstrated that the phytochemical agent in the latex of Ficus dubia could potential be used for treatment and prevention of human colorectal cancer, especially in inflammation-induced hyperproliferation progression.

Transcriptome Profiling of HCT-116 Colorectal Cancer Cells with RNA Sequencing Reveals Novel Targets for Polyphenol Nano Curcumin.

Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. The gemini nanoparticle formulation of polyphenolic curcumin significantly inhibits the viability of cancer cells. However, the molecular mechanisms and pathways underlying its toxicity in colon cancer are unclear. Here, we aimed to uncover the possible novel targets of gemini curcumin (Gemini-Cur) on colorectal cancer and related cellular pathways. After confirming the cytotoxic effect of Gemini-Cur by MTT and apoptotic assays, RNA sequencing was employed to identify differentially expressed genes (DEGs) in HCT-116 cells. On a total of 3892 DEGs (padj < 0.01), 442 genes showed a log2 FC >|2| (including 244 upregulated and 198 downregulated). Gene ontology (GO) enrichment analysis was performed. Protein–protein interaction (PPI) and gene-pathway networks were constructed by using STRING and Cytoscape. The pathway analysis showed that Gemini-Cur predominantly modulates pathways related to the cell cycle. The gene network analysis revealed five central genes, namely GADD45G, ATF3, BUB1B, CCNA2 and CDK1. Real-time PCR and Western blotting analysis confirmed the significant modulation of these genes in Gemini-Cur-treated compared to non-treated cells. In conclusion, RNA sequencing revealed novel potential targets of curcumin on cancer cells. Further studies are required to elucidate the molecular mechanism of action of Gemini-Cur regarding the modulation of the expression of hub genes.

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.

Faecalibacterium prausnitzii Ameliorates Colorectal Tumorigenesis and Suppresses Proliferation of HCT116 Colorectal Cancer Cells.

Faecalibacterium prausnitzii is one of the most abundant commensals of gut microbiota that is not commonly administered as a probiotic supplement. Being one of the gut’s major butyrate-producing bacteria, its clinical significance and uses are on the rise and it has been shown to have anti-inflammatory and gut microbiota-modulating properties in the treatment of inflammatory bowel illness, Crohn’s disease, and colorectal cancer. Colorectal cancer (CRC) is a silent killer disease that has become one of the leading causes of cancer-related death worldwide. This study aimed to evaluate the anti-tumorigenic and antiproliferative role of F. prausnitzii as well as to study its effects on the diversity of gut microbiota in rats. Findings showed that F. prausnitzii probiotic significantly reduced the colonic aberrant crypt foci frequency and formation in Azoxymethane (AOM)-induced CRC in rats. In addition, the administration of F. prausnitzii lowered the lipid peroxidation levels in the colon tissues. For in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, the cell-free supernatant of F. prausnitzii suppressed the growth of HCT116 colorectal cancer cells in a time/dose-dependent manner. 16S rRNA gene sequencing using rat stool samples showed that the administration of F. prausnitzii modulated the gut microbiota of the rats and enhanced its diversity. Hence, these findings suggest that F. prausnitzii as a probiotic supplement can be used in CRC prevention and management; however, more studies are warranted to understand its cellular and molecular mechanisms of action.

Photoaffinity Labeling-Based Chemoproteomic Strategy Reveals RBBP4 as a Cellular Target of Protopanaxadiol against Colorectal Cancer Cells.

Protopanaxadiol (PPD), a main ginseng metabolite, exerts powerful anticancer effects against multiple types of cancer; however, its cellular targets remain elusive. Here, we synthesized a cell-permeable PPD probe via introducing a bifunctional alkyne-containing diazirine photo-crosslinker and performed a photoaffinity labeling-based chemoproteomic study. We identified retinoblastoma binding protein 4 (RBBP4), a chromatin remodeling factor, as an essential cellular target of PPD in HCT116 colorectal cancer cells. PPD significantly decreased RBBP4-dependent trimethylation at lysine 27 of histone H3 (H3K27me3), a crucial epigenetic marker that correlates with histologic signs of colorectal cancer aggressiveness, and PPD inhibition of proliferation and migration of HCT116 cells was antagonized by RBBP4 RNA silencing. Collectively, our study highlights a previously undisclosed anti-colorectal cancer cellular target of the ginseng metabolite and advances the fundamental understanding of RBBP4 functions via a chemical biology strategy.

LINC00586 Represses ASXL1 Expression Thus Inducing Epithelial-To-Mesenchymal Transition of Colorectal Cancer Cells Through LSD1-Mediated H3K4me2 Demethylation.

Colorectal cancer (CRC) is a major public health problem on a global scale by virtue of its relatively high incidence. The transition of tumor cells from an epithelial to a mesenchymal-like phenotype, so-called epithelial-to-mesenchymal transition (EMT), is a key hallmark of human cancer metastasis, including CRC. Understanding the signaling events that initiate this phenotypic switch may provide opportunities to limit the metastasis of CRC. In this study, we aim to identify long non-coding RNA (lncRNA) mediated epigenetic regulation under the context of CRC. 54 paired samples of tumor tissues and surrounding non-tumor tissues were collected from CRC patients. Cultured human CRC cells HCT116 and LoVo were assayed for their viability and migration using CCK-8 tests and transwell migration assays. The expression of EMT-specific markers (E-cadherin, N-cadherin and vimentin) was analyzed biochemically by RT-qPCR and immunoblot analyses. Interaction among LINC00586, LSD1, and ASXL1 was determined by RNA immunoprecipitation and chromatin immunoprecipitation. In vivo analysis of LINC00586 was performed in nude mice xenografted with HCT116 cells. LINC00586 was overexpressed in CRC tissues and associated with patient survival. LINC00586 knockdown repressed HCT116 and LoVo cell viability, migration, their phenotypic switch from epithelial to a mesenchymal, and tumorigenesis in vivo. We demonstrated LINC00586 recruited the LSD1 into the ASXL1 promoter region and epigenetically silenced the ASXL1 expression. An ASXL1 gene resisting to LINC00586 attack was demonstrated in cultured HCT116 and LoVo cells and mouse xenograft models of human CRC. Overall, discovery of the LINC00586/LSD1/ASXL1 axis partially explains epigenetic mechanism regulating EMT in CRC, providing a therapeutic target to limit CRC metastasis.

Influence of High‐Polyphenol Sorghum on Benzo[a]pyrene‐Metabolizing Enzymes

Colorectal cancer ranks among the top five most common types of cancer in the United States. Lifestyle, diet, and environmental factors are correlated to the increased risk of colon cancer. One of those environmental factors is the polycyclic aromatic hydrocarbon (PAHs) Benzo[a]pyrene (BaP), formed by the incomplete combustion of organic material. Human exposure to BaP occurs through air pollution, tobacco smoke, engine exhaust, and the consumption of BaP‐exposed foods. BaP crosses cellular membranes, and it becomes metabolically activated by Phase I enzymes, primarily cytochrome P450s (CYPs) and Epoxide Hydrolase, and subsequently has the potential to bind to DNA. The binding of BaP to DNA may result in adverse genotypic alterations, which may result in tumorigenesis. On the other hand, cellular Phase II enzymes play a role in the inactivation of BaP and its metabolites, which can facilitate the excretion of these molecules. Polyphenols, a group of plant micronutrients known for their antioxidant and anti‐inflammatory properties, have been suggested to contribute to the prevention of colon cancer. Extracts from two types of Sorghum bicolor were provided by the USDA ARS Kansas. Preliminary studies in our lab suggest that polyphenol‐rich varietals of the grain Sorghumaffect pathways in carcinogenesis. Specifically, mRNA expression of UDP‐glucuronosyl transferase (UGT) 1A1, a Phase II PAH‐bioinactivating enzyme, is significantly increased in high‐polyphenol sorghum treated HCT116 cells. Therefore, we propose to investigate the impact of high‐polyphenol sorghum on BaP‐exposed colon cancer cells, assessing the expression and activity of Phase I and Phase II enzymes in particular. Our hypothesis is that compared to controls, high polyphenol sorghum extracts will decrease expression and activity of Phase I, and increase expression and activity of Phase II enzymes in BaP‐treated colon cancer cells.Human colorectal cancer HCT116 cells were incubated with either 0, 1, 2, or 5 µM BaP alone, Sorghum extracts alone, or in the presence of high‐polyphenol sorghum and Benzo[a] pyrene for up to 48 hours. RNA was extracted and reverse‐transcribed to cDNA. mRNA expression of carcinogen activating and inactivating enzymes was measured using qPCR. BaP alone increased mRNA expression of CYP1A1 as expected. Our high polyphenol Sorghum extract dramatically decreased mRNA expression of the Phase I enzyme CYP1B1, but also moderately increased mRNA expression of Epoxide Hydrolase. We plan to complete assessment of the expression and catalytic activity of Phase I and Phase II enzymes in the presence of high‐polyphenol sorghum and Benzo[a] pyrene. Our initial results suggest that high‐polyphenol Sorghum may be able to shift the balance of Phase I and Phase II enzymes in BaP‐exposed cells, and thus provide an avenue of a dietary approach to support chemoprevention.

O-17 - Preclinical assessment of nanoparticles conjugated with 64Cu-DOTA-PEG-BBN targeting gastrin-releasing peptide receptors

1508 Objectives: Gold andiron oxide based nanoparticleshave emerged as innovative tools for cancer imaging and therapy, as benefit of their size, biocompatibility and pharmacokinetics. Their surface functionalization with high number of recognizable biomolecules (gastrin-releasing peptides, GRP, namely bombesin, BBN, bombesin-like short chain peptide, BBN(7-14), and Neuromedin B, NMB) forms high-specific targeted systems, suitable for multimodal PET-MRI or PET-CT imaging aimed for early detection of tumours and/or therapy monitoring and follow-up. The aim of this study was to investigate preclinically and comparatively evaluate the potential as imaging agents of activated N-Hydroxysuccinimide gold nanoparticles, coated with polyethylene glycol (PEG) and conjugated to 64Cu-DOTA-BBN/BBN(7-14)/NMB and iron oxide nanoparticles coated with PEG and conjugated to 68Ga-DOTA-BBN/BBN(7-14)/NMB, respectively. Methods: The morphology and size distribution of gold/iron oxide nanoparticles were examined by TEM/SEM coupled with energy dispersion spectroscopy (EDS) techniques and, after functionalization, by UV-Vis spectroscopy and DLS. The gold/iron oxide NPs were fconjugated to PEG(4)-BBN(7-14), PEG(4)-BBN, and PEG(4)-NeuromedinB, previously radiolabelled with Cu-64 using DOTA as chelating agent. The radiolabelled nanoconjugates were incubated with neuroendocrine colorectal cancer cells HT-29 and HCT116, and prostate cancer cells DU-145, all over-expressing GRPRs. We investigated the specific binding of Bombesin (BBN), Bombesin-like peptide sequence BBN(7-14) and Neuromedin B (NMB) to GRP receptors in the presence of cold competitor antagonist ligands. In-vivo PET imaging was performed on high-resolution micro-PET-CT system, on tumor bearing nude mice (Nu/J) inoculated with HT-29, HCT116 and DU-145 tumors, respectively. Results: In vitro binding kinetics assessment showed an increased retention of over 30% of radioisotope inside tumor cells in the case of AuNP-PEG(4)-64Cu-DOTA-BBN(7-14), compared to 64Cu-DOTA-PEG(4)-BBN(7-14) alone. Similarly, IONP-PEG(4)-64Cu-DOTA-NMB was better uptaken and internalized, compared to 64Cu-DOTA-PEG(4)-NMB alone. These results are attributed to the increased concentration and multiple binding sites of BBN-like peptides to GRPRs allowed by their exposure on the surface of NPs. Subsequently, higher internalization rate is promoted, by receptor-mediated endocytosis. Biodistribution/imaging studies, and the results show high and stable tumor uptake of the radiolabelled nanoconjugates, fast blood-clearance and allowed for determination of key PKs parameters, path of accumulation and excretion rate. Conclusions: The combination of size-controlled distribution of nanoparticles, functionalization with high-specific targeting peptides and Cu-64 radioisotope labelling resulted in highly promising imaging/therapeutic scaffolds, based on gold/iron oxide NPs coated with PEG(4) and bearing specific targeting 64Cu-DOTA-BBN analogs. Their preliminary in vitro and in vivo evaluation shows good results regarding uptake, retention, stability and PKs parameters. Further investigations are to be performed to determine the detailed dosimetry and therapeutic efficacy to extend the potential use to targeted radionuclide therapy, by use of highly cytotoxic potency of Auger-electrons emitted by Cu-64, inside the tumour, and specifically affecting the tumour cell metabolism. Research Support: This work was supported by a grant of the Romanian Ministry of Research and Innovation, UEFISCDI project number 64PCCDI/2018.

Butyrate Drives Metabolic Rewiring and Epigenetic Reprogramming in Human Colon Cancer Cells.

Butyrate (B) is a short-chain fatty acid produced by dietary fiber, known to inhibit histone deacetylases (HDACs) and possess cancer-preventive/anticancer effects. However, the role of B in metabolic rewiring, epigenomic reprogramming, transcriptomic network, NRF2 signaling, and eliciting cancer-preventive effects in colorectal cancer (CRC) HCT116 cell remains unclear. Sodium butyrate (NaB) dose-dependently inhibits the growth of CRC HCT116 cells. NaB inhibits NRF2/NRF2-target genes and blocks NRF2-ARE signaling. NaB increases NRF2 negative regulator KEAP1 expression through inhibiting its promoter methylation. Associative analysis of DEGs (differentially expressed genes) from RNA-seq and DMRs (differentially methylated regions) from CpG methyl-seq identified the tumor suppressor gene ABCA1 and tumor promote gene EGR3 are correlated with their promoters' CpG methylation indicating NaB regulates cancer markers through modulating their promoter methylation. NaB activated the mitochondrial tricarboxylic acid (TCA) cycle while inhibited the methionine metabolism which are both tightly coupled to the epigenetic machinery. NaB regulates the epigenetic enzymes/genes including DNMT1, HAT1, KDM1A, KDM1B, and TET1. Altogether, B's regulation of metabolites coupled to the epigenetic enzymes illustrates the potential underlying biological connectivity between metabolomics and epigenomics. B regulates KEAP1/NRF2 signaling, drives metabolic rewiring, CpG methylomic, and transcriptomic reprogramming contributing to the overall cancer-prevention/anticancer effect in the CRC cell model.

Molecular Hydrogen Inhibits Colorectal Cancer Growth via the AKT/SCD1 Signaling Pathway.

Objective Molecular hydrogen (H2) has been considered a potential therapeutic target in many cancers. Therefore, we sought to assess the potential effect of H2 on colorectal cancer (CRC) in this study. Methods The effect of H2 on the proliferation and apoptosis of RKO, SW480, and HCT116 CRC cell lines was assayed by CCK-8, colony formation, and flow cytometry assays. The effect of H2 on tumor growth was observed in xenograft implantation models (inhalation of 67% hydrogen two hours per day). Western blot and immunohistochemistry analyses were performed to examine the expression of p-PI3K, PI3K, AKT, pAKT, and SCD1 in CRC cell lines and xenograft mouse models. The expression of SCD1 in 491 formalin-fixed, paraffin-embedded CRC specimens was investigated with immunochemistry. The relationship between SCD1 status and clinicopathological characteristics and outcomes was determined. Results Hydrogen treatment suppressed the proliferation of CRC cell lines independent of apoptosis, and the cell lines showed different responses to different doses of H2. Hydrogen also elicited a potent antitumor effect to reduce CRC tumor volume and weight in vivo. Western blot and IHC staining demonstrated that H2 inhibits CRC cell proliferation by decreasing pAKT/SCD1 levels, and the inhibition of cell proliferation induced by H2 was reversed by the AKT activator SC79. IHC showed that SCD1 expression was significantly higher in CRC tissues than in normal epithelial tissues (70.3% vs. 29.7%, p = 0.02) and was correlated with a more advanced TNM stage (III vs. I + II; 75.9% vs. 66.3%, p = 0.02), lymph node metastasis (with vs. without; 75.9% vs. 66.3%, p = 0.02), and patients without a family history of CRC (78.7% vs. 62.1%, p = 0.047). Conclusion This study demonstrates that high concentrations of H2 exert an inhibitory effect on CRC by inhibiting the pAKT/SCD1 pathway. Further studies are warranted for clinical evaluation of H2 as SCD1 inhibitor to target CRC.