HCT116 Colorectal Cancer Cells Research Articles

Liensinine sensitizes colorectal cancer cells to oxaliplatin by targeting HIF-1α to inhibit autophagy

BackgroundOxaliplatin is the most common chemotherapeutic agent for patients with colorectal cancer. However, its anti-cancer efficacy is restricted by drug resistance occurring through several mechanisms, including autophagy. Liensinine exerts a considerable anti-tumor effect and can regulate autophagy. Inhibition of autophagy is a strategy to reverse resistance to oxaliplatin. The aim of this study was to check if liensinine can enhance the therapeutic efficacy of oxaliplatin in colorectal cancer and if so, elucidate its mechanism. MethodsTwo colorectal cancer cell lines, HCT116 and LoVo, and one normal intestinal epithelial cell, NCM-460 were used for in vitro experiments. Cell Counting Kit-8 (CCK-8), colony formation, and flow cytometry assays were used to evaluate the cytotoxicity of liensinine and oxaliplatin. Network pharmacology analysis and Human XL Oncology Array were used to screen targets of liensinine. Transfections and autophagy regulators were used to confirm these targets. The relationship between the target and clinical effect of oxaliplatin was analyzed. Patient-derived xenograft (PDX) models were used to validate the effects of liensinine and oxaliplatin. ResultsCCK-8 and colony formation assays both showed that the combination treatment of liensinine and oxaliplatin exerted synergistic effects. Results of the network pharmacology analysis and Human XL Oncology Array suggested that liensinine can inhibit autophagy by targeting HIF-1α/eNOS. HIF-1α was identified as the key factor modulated by liensinine in autophagy and induces resistance to oxaliplatin. HIF-1α levels in tumor cells and prognosis for FOLFOX were negatively correlated in clinical data. The results from three PDX models with different HIF-1α levels showed their association with intrinsic and acquired resistance to oxaliplatin in these models, which could be reversed by liensinine. ConclusionsResearch on the relationship between HIF-1α levels and the clinical effect of oxaliplatin is lacking, and whether liensinine regulates HIF-1α is unknown. Our findings suggest that liensinine overcomes the resistance of colorectal cancer cells to oxaliplatin by suppressing HIF-1α levels to inhibit autophagy. Our findings can contribute to improving prognosis following colorectal cancer therapy.

Knockdown of SETD5 Inhibits Colorectal Cancer Cell Growth and Stemness by Regulating PI3K/AKT/mTOR Pathway.

SET domain-containing 5 (SETD5), a member of protein lysine methyltransferase family, is expressed in multiple cancers, making it potential therapeutic targets. However, the role of SETD5 in colorectal cancer remains largely unknown. The expression of SETD5 in the 30 pairs colorectal cancer tissues samples and cell lines were determined by qRT-PCR. The functions of SETD5 was detected by knocked-down or overexpression in colorectal cancer cell lines SW480 and HCT116 cells. Cell proliferative activity, cell death, and stemness characteristics were assessed. BEZ235, a PI3K/AKT/mTOR pathway inhibitor, was used to perform rescue experiment to analyze whether SETD5 exerted its effects through activating PI3K/AKT/mTOR pathway. SETD5 was substantially upregulated in colorectal cancer, and correlated to metastasis and clinical stage of patients. Knockdown of SETD5 inhibited SW480 and HCT116 cell growth, as evidenced by the inhibition of cell viability and clone-forming. Moreover, Knockdown of SETD5 suppressed the capability of tumor sphere formation of SW480 and HCT116 cells, and reduced the expression of stemness-related proteins Nanog and Sox2. Further western blot analysis revealed that SETD5 knockdown inhibited the phosphorylation of proteins associated with the PI3K/AKT/mTOR pathway. In contrast, overexpression of SETD5 exerted the opposite effects. Mechanistically, by blocking PI3K/AKT/mTOR pathway with BEZ235, the effects of SETD5 overexpression on cell viability and Nanog and Sox2 protein expression were reversed. Our results substantiated that SETD5 functioned as an oncogene by promoting cell growth and stemness in colorectal cancer cells through activating the PI3K/AKT/mTOR signaling pathway.

Targeting Colorectal Cancer: Unravelling the Transcriptomic Impact of Cisplatin and High-THC Cannabis Extract.

Cisplatin and other platinum-derived chemotherapy drugs have been used for the treatment of cancer for a long time and are often combined with other medications. Unfortunately, tumours often develop resistance to cisplatin, forcing scientists to look for alternatives or synergistic combinations with other drugs. In this work, we attempted to find a potential synergistic effect between cisplatin and cannabinoid delta-9-THC, as well as the high-THC Cannabis sativa extract, for the treatment of HT-29, HCT-116, and LS-174T colorectal cancer cell lines. However, we found that combinations of the high-THC cannabis extract with cisplatin worked antagonistically on the tested colorectal cancer cell lines. To elucidate the mechanisms of drug interactions and the distinct impacts of individual treatments, we conducted a comprehensive transcriptomic analysis of affected pathways within the colorectal cancer cell line HT-29. Our primary objective was to gain a deeper understanding of the underlying molecular mechanisms associated with each treatment modality and their potential interactions. Our findings revealed an antagonistic interaction between cisplatin and high-THC cannabis extract, which could be linked to alterations in gene transcription associated with cell death (BCL2, BAD, caspase 10), DNA repair pathways (Rad52), and cancer pathways related to drug resistance.

Fusobacterium nucleatum-induced imbalance in microbiome-derived butyric acid levels promotes the occurrence and development of colorectal cancer.

Colorectal cancer (CRC) ranks among the most prevalent malignant tumors globally. Recent reports suggest that Fusobacterium nucleatum (F. nucleatum) contributes to the initiation, progression, and prognosis of CRC. Butyrate, a short-chain fatty acid derived from the bacterial fermentation of soluble dietary fiber, is known to inhibit various cancers. This study is designed to explore whether F. nucleatum influences the onset and progression of CRC by impacting the intestinal metabolite butyric acid. To investigate the mechanism by which F. nucleatum affects CRC occurrence and development. Alterations in the gut microbiota of BALB/c mice were observed following the oral administration of F. nucleatum. Additionally, DLD-1 and HCT116 cell lines were exposed to sodium butyrate (NaB) and F. nucleatum in vitro to examine the effects on proliferative proteins and mitochondrial function. Our research indicates that the prevalence of F. nucleatum in fecal samples from CRC patients is significantly greater than in healthy counterparts, while the prevalence of butyrate-producing bacteria is notably lower. In mice colonized with F. nucleatum, the population of butyrate-producing bacteria decreased, resulting in altered levels of butyric acid, a key intestinal metabolite of butyrate. Exposure to NaB can impair mitochondrial morphology and diminish mitochondrial membrane potential in DLD-1 and HCT116 CRC cells. Consequently, this leads to modulated production of adenosine triphosphate and reactive oxygen species, thereby inhibiting cancer cell proliferation. Additionally, NaB triggers the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, blocks the cell cycle in HCT116 and DLD-1 cells, and curtails the proliferation of CRC cells. The combined presence of F. nucleatum and NaB attenuated the effects of the latter. By employing small interfering RNA to suppress AMPK, it was demonstrated that AMPK is essential for NaB's inhibition of CRC cell proliferation. F. nucleatum can promote cancer progression through its inhibitory effect on butyric acid, via the AMPK signaling pathway.

DNA-DEPENDENT PROTEIN KINASE INHIBITOR INDUCES APOPTOSIS IN COLON CANCER CELLS

Objectives: Among all types of DNA damage, DNA double-strand breaks (DSBs) are considered the most deleterious form induced by either endogenous factors (oxi-dative damages, mismatches, altered chromatin structures, and missing, or modified nucleotides) or exogenous factors, i.e., ultraviolet (UV) radiation, ionizing radiation (IR), and chemicals or drugs. DNA-dependent protein kinase (DNA-PK) plays a crucial role in repairing DSBs through non-homologous end joining (NHEJ). Cells lacking DNA-PK exhibit heightened sensitivity to IR and various DNA-damaging agents. The inhibition of DNA-PK further intensifies cellular susceptibility to IR and DNA-damaging agents. Several small molecules that inhibit DNA-PK have been developed. This study aimed to evaluate the effect of DNA-PK inhibitor (DNA-PKi) NU7441 on the HCT116 cell line. Methods: DNA-PKi NU7441 was used to assess the effect on anti-proliferation and induction of apoptosis on the HCT116 colorectal cancer cell line. Cells were cultured under standard conditions; crystal violet and apoptosis assay were applied to evaluate cell proliferation and apoptosis. Data were analysed using GraphPad Prism 8.4. Results: DNA-PKi effectively inhibited HCT116 colon cancer cell growth via crystal violet assay (p < 0.01). In addition, DNA-PKi also induced programmed cell death in the HCT116 cell line (p < 0.05). Conclusion: DNA-PKi NU7441 suppressed cell proliferation and induced apoptosis in the HCT116 colon cancer cell line.

Enhanced bacterial cancer therapy delivering therapeutic RNA interference of c-Myc

BackgroundBacterial cancer therapy was first trialled in patients at the end of the nineteenth century. More recently, tumour-targeting bacteria have been harnessed to deliver plasmid-expressed therapeutic interfering RNA to a range of solid tumours. A major limitation to clinical translation of this is the short-term nature of RNA interference in vivo due to plasmid instability. To overcome this, we sought to develop tumour-targeting attenuated bacteria that stably express shRNA by virtue of integration of an expression cassette within the bacterial chromosome and demonstrate therapeutic efficacy in vitro and in vivo.ResultsThe attenuated tumour targeting Salmonella typhimurium SL7207 strain was modified to carry chromosomally integrated shRNA expression cassettes at the xylA locus. The colorectal cancer cell lines SW480, HCT116 and breast cancer cell line MCF7 were used to demonstrate the ability of these modified strains to perform intracellular infection and deliver effective RNA and protein knockdown of the target gene c-Myc. In vivo therapeutic efficacy was demonstrated using the Lgr5creERT2Apcflx/flx and BlgCreBrca2flx/flp53flx/flx orthotopic immunocompetent mouse models of colorectal and breast cancer, respectively. In vitro co-cultures of breast and colorectal cancer cell lines with modified SL7207 demonstrated a significant 50–95% (P < 0.01) reduction in RNA and protein expression with SL7207/c-Myc targeted strains. In vivo, following establishment of tumour tissue, a single intra-peritoneal administration of 1 × 106 CFU of SL7207/c-Myc was sufficient to permit tumour colonisation and significantly extend survival with no overt toxicity in control animals.ConclusionsIn summary we have demonstrated that tumour tropic bacteria can be modified to safely deliver therapeutic levels of gene knockdown. This technology has the potential to specifically target primary and secondary solid tumours with personalised therapeutic payloads, providing new multi-cancer detection and treatment options with minimal off-target effects. Further understanding of the tropism mechanisms and impact on host immunity and microbiome is required to progress to clinical translation.

Abstract 1822: Artemisia annua Lpolyphenols augment b-lapachone effects on oxaliplatin-resistant HCT 116 colorectal cancer cells by downregulating TERT, CD44, EGFR and oxaliplatin resistance-related proteins of OXP-R-Hct 116 cells

Abstract We previously demonstrated that β-lapachone (β-Lap) exerts stronger anticancer activity on oxaliplatin-resistant HCT116 colorectal cancer cells (Oxp-R-HCT 116 cells) than parental HCT116 cells (p-HCT 116 cells) by downregulating the oxaliplatin resistance-related upregulated proteins (Oxp-RR-Proteins) proteins in Oxp-R-HCT 116 cells, such as SOD1, MAPK, and nucelophosmin. We also demonstrated polyphenols extracted from Korean Artemisia annua L. (pKAL) exerts anticancer activity on radio-resistant MDA-MB-231 human breast cancer cells by suppressing stem cell phenotype, β-catenin, and MMP-9. In this study, we found that pKAL significantly enhanced the anticancer activity of β-lapachone (β-Lap) on oxaliplatin-resistant (Oxp-R) HCT116 colorectal cancer cells. The aim of this study is to elucidate how pKAL enhance the anticancer effects of β-Lap on Oxp-R-HCT 116 cells. CCK-8 assay, phase-contrast microscopy analysis, and morphological analysis with hematoxylin stain revealed that the anticancer effect of β-Lap is more enhanced by pKAL on Oxp-R-HCT 116 cells over 60 hr. This phenomenon was associated with suppression of oxaliplatin resistance-related upregulated proteins (Oxp-RR-Proteins) (p53, ERK, and β-catenin), and downregulation of cell survival- and stemness-related proteins (TERT, CD44 and EGFR) that were not up-regulated in Oxp-R-HCT 116 cells. Protein-protein interaction network analysis of the 21 proteins that showed large difference in expression between before and after the combined treatment of pKAL and β-Lap in HCT116-OxPt-R cells revealed that the enhanced anticancer effect of β-Lap by pKAL was related to the inhibition of negative regulation of apoptotic process and the induction of DNA damage through TERT, CD44, and EGFR-mediated multiple signaling networks. The detailed western blot analysis for anti-cancer effects of pKAL revealed that pKAL alone also suppressed the expression of TERT, CD44 and EGFR in p-HCT 116 cells as well as Oxp-R-HCT 116 cells while β-Lap alone did not influence the expression of them. In addition, for some of the Oxp-RR-Proteins of Oxp-R-HCT 116 cells (p53, ERK, and β-catenin) were suppressed by pKAL alone, but not others. However, some other Oxp-RR-Proteins of Oxp-R-HCT 116 cells that were not suppressed by pKAL alone were significantly suppressed by the combination treatment of pKAL and β-Lap. These findings suggest that pKAL enhanced the anti-cancer effects β-Lap by suppressing TERT, CD44, and EGFR-mediated multiple signaling networks as well as some of the Oxp-RR-Proteins of Oxp-R-HCT 116 cells. In conclusion, this study suggests that pKAL enhance the anticancer effects of β-Lap on OXP-R-Hct 116 cells by downregulating CD44, EGFR and some of the Oxp-RR-Proteins of Oxp-R-HCT 116 cells. Citation Format: Won Sup Lee, Eun Joo Jung, Hye Jung Kim, Sung Chul Shin, Gon Sup Kim, Jin-Myung Jung, Soon Chan Hong, Choong Won Kim. Artemisia annua Lpolyphenols augment b-lapachone effects on oxaliplatin-resistant HCT 116 colorectal cancer cells by downregulating TERT, CD44, EGFR and oxaliplatin resistance-related proteins of OXP-R-Hct 116 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1822.

Abstract 4715: Azathioprine and Z36 induced cell death in human colorectal cancer HCT-116 and HCT-15 cells

Abstract Rationale and Objectives: Cancer accounts for a huge global health burden and is the third highest cause of death across the globe. Cancer cells are known to have higher basal reactive oxygen species (ROS) levels, owing to their higher metabolism and proliferation. Higher basal ROS in cancer cells made them susceptible to exogenous ROS stimuli that lead to cell death. Therefore, in the present study, we have analyzed the effects of Azathioprine, a glutathione (GSH) inhibitor and an intracellular ROS producer. The effect of Azathioprine was analyzed alone or in combination with a BCL-2 family protein inhibitor Z36, on human colorectal cancer cell lines HCT-116 and HCT-15. Methods: The effects of the drugs have been investigated using various methods including cell viability assay, DNA fragmentation assay, and Annexin V-FITC/PI apoptosis assay. In addition, morphological changes evident in cell death were detected by Giemsa staining. Combination dosage for drug synergistic effects was computed through the use of CompuSyn software. Western blot was performed to detect changes in the expression of cell death-related proteins following drug treatments. Results and conclusion: Azathioprine at a dose of 5 µM has been demonstrated to induce cell death in both HCT116 and HCT15 cells in a concentration and time-dependent manner. On the contrary, Z36, a novel BCLXL inhibitor, has demonstrated similar cell viability results at an even lower dose of 2.5 µM. When both drugs were combined at lower doses, no significant effects were observed on the cell viability of either cell line, demonstrating that the combined treatment has no synergistic or additive effects. This suggests that both drugs do not augment each other and might induce cell death via upregulation of ROS using a similar mechanism. Citation Format: Yousuf Tahir Ali, Mahwish Fatima, Muhammad Areeb Ahmed, Hasan Salman Siddiqi, Azhar Hussain, Mati Ur Rehman. Azathioprine and Z36 induced cell death in human colorectal cancer HCT-116 and HCT-15 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4715.

Abstract 6005: Generation of new oncology cell models through long-term acclimation under hypoxic and hyperbaric culture conditions

Abstract Traditional cancer cell lines used as surrogate models in vitro and in vivo have made significant contributions to cancer research and drug discovery. While cell culture and animal studies are critical steps in determining the efficacy, pharmacodynamics, and mechanism of action of novel anti-cancer drugs, it is well-known that cells grown in 2D culture systems have modified growth characteristics and different responses to chemotherapeutic drugs compared to tumors in vivo as a result of differing structural and environmental conditions that fail to replicate in vivo tumor microenvironments. AVATAR bioreactors (Xcell Biosciences, San Francisco, CA) were invented to address these limitations by providing discrete control over oxygen and hyperbaric pressure levels to better mimic the in vivo environment and allow cultured cells to be grown in conditions which mimic the human body. To better serve preclinical research needs, we generated new tumor cell models by long-term acclimating (LTA) existing cancer cells in the AVATAR incubator system using 1% O2 and 2 PSI conditions similar to the conditions expected in the tumor microenvironment (TME). Six different cancer cell lines of diverse origins including pancreas (MIA PaCa-2), breast (BT-474), colon (HCT116), prostate (LNCaP, PC-3) and melanoma (A-375) were adapted for 2-3 months with phenotypic changes recorded. Key marker changes were commonly observed in LTA cells compared to parental lines. For example, in HCT116 colorectal cancer cells, TME culture conditions induced a higher expression level of E-cadherin in both protein and mRNA levels (2.2-fold change), indicating a mesenchymal-epithelial transition of the cell after adaptation. This coincides with the observation that LTA adapted HCT116 cell bodies had a more angular cell morphology as compared to parental cells grown under conventional normoxic culture conditions. In-depth gene-expression analysis using a Nanostring tumor signaling pathway panel identified several common and tissue-specific gene and pathway changes. These included VEGFA, EGF receptor signaling pathway (EGFR, E-Cadherin, GRB7, c-Fos), TGF pathway (IGFBP3, TGFB1, TGFB2, SMAD7), and extracellular matrix signaling (LAMB3, ITGA5, ITGB3, ITGB8, PDGFA, CCND2). Current studies are focusing on drug response testing in 2D and 3D cultures based on above-mentioned gene and signaling pathway changes. Cell proliferation and migration capacity of xenograft LTA cells will be evaluated for the purposes of creating novel murine models for drug screening applications. Citation Format: Yewei Xing, Abriel Czachorowski, James Lim, Scott Wise. Generation of new oncology cell models through long-term acclimation under hypoxic and hyperbaric culture conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6005.

Abstract 1338: Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells

Abstract Human leukocyte antigen-G (HLA-G) suppresses lymphoid and myeloid immune systems by binding ILT2 and ILT4 respectively and activating ITIM signaling. HLA-G is abundantly expressed in placental cytotrophoblasts to block fetal rejection. HLA-G is not expressed in most normal cells but is derepressed in in about 50% of malignancies. We engineered ILT2 and ILT4 to produce activating signals as Chimeric ILT Receptors or CIR. Preliminary results indicated robust killing of Acute Myeloid Leukemia (AML) cells by Natural Killer (NK) cells expressing either ILT2 or ILT4 CIRs. Here, we evaluated the effect of alteration of activating domains in the CIR constructs to produce signals that mimic the effect of IL-18 signaling in CIR-NK cells to enhance cytotoxicity, NK cell growth and persistence. Further, HLA-G expression and CIR-NK cell targeting of primary human cell types were rigorously screened. Activated NK cells derived from peripheral blood (2 to 5 donors) were transduced with γ-retroviruses directing expression of CIR proteins (CIR.X.Y or CIR.Y.X) where X stimulates cytotoxicity through CD3ζ, DAP10 or DAP12, and Y directs coactivation through 4-1BB, fusion of the MyD88 death domain or recruitment of endogenous MyD88 with receptor-derived TIR domains and soluble IL-15. CIR-NK cells were cocultured with GFPffluc-expressing AML and solid tumor target cell lines or normal primary human cells. NK cell proliferation and cytotoxicity of CIR-NK cells was monitored by Incucyte microscopy and supernatants were analyzed for cytokine release by ELISA. At low E:T (1:20 - 1:40) in 7-day cocultures, mock-transduced NK cells displayed innate killing activity against HLA-G+ Molm13 and Kasumi1 AML cells that was augmented by CIR constructs containing direct fusions of MyD88 and DAP10 or DAP12 (mock = 2.06E7 ± 4.53E6 vs. CIR-ILT4.MyD88.DAP10 = 1.03E7 ± 5.46E6) with 7-fold elevation of IFN-γ production. CIR constructs that recruited endogenous MyD88 signaling through the TLR2 TIR domain (4-1BB.DAP10.TLR2) also exhibited enhanced anti-HLA-G activity (9.13E6 ± 5.33E6). Against HLA-G+ HT-1376 bladder cancer cells (E:T = 1:10-1:20), CIR-NK cells also displayed enhanced tumor cell killing with enhanced NK cell growth. Conversely, CIR-enhanced killing was not observed against HLA-G− HCT-116 colorectal cancer cells. The persistence of CIR-NK cell potency after 4 weeks of expansion varied markedly with different activation domains employed. When screened against primary human cells (cardiomyocytes, hepatocytes, hepatic endothelial cells, corneal, colon and lung epithelial cells), no CIR-specific targeting was observed at high E:T ratios (1:1 and 1:5). In conclusion, CIR-NK cells exhibit potent and specific anti-tumor activity against HLA-G+ solid and leukemia cells. Alteration of CIR-NK cell intracellular signaling can optimize the potency and persistence of NK cell anti-tumor activity. Citation Format: MyLinh Duong, Jihyun Park, Raphel Ognar, Simon Wain-Hobson, Henri Bayle. Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1338.

Abstract 5881: Upregulation of TET2 expression by DNA methyltransferase (DNMT) inhibitors is associated with resistance in DNMT1 knockout colorectal cancer cells

Abstract Ten-eleven-translocation 2 (TET2) is a member of the TET family proteins (TET1-3) that convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and causes site-specific DNA demethylation. The effects of DNMT1 on TET2 phenotype following DNMT inhibitor treatment is unclear in human cancer. In colorectal cancer HCT116 cells with DNMT1 gene intact (DNMT1+/+) and deletion (DNMT1−/−) status, we found that TET2 expression was robustly increased in DNMT1−/− cells after treatment with 0.5 µM and 5 µM decitabine for 72 h, 96 h, and 120 h. The augmentation in TET2 expression was accompanied by a dose-dependent re-expression of p16ink4A in DNMT1−/− cells. Particularly, TET2 upregulation was associated with resistance to DNMT inhibitors, showing a lack of induction of DNA damage, cell-cycle arrest and apoptosis after drug exposure in DNMT1−/− compared to DNMT1+/+ cells. The treatment with 4’-thio-5-aza-2’-deoxycytidine (aza-T-dCyd) only up-regulated TET2 and re-expressed p16ink4A at the lower dose of 0.5 µM, which was consistent with our previous findings that high dose of aza-T-dCyd at 5 µM predominantly eliminates the cancer cells with severe DNA damage by undergoing apoptosis and cell death (Scientific Reports, 13:5964, 2023). Moreover, both decitabine and aza-T-dCyd had minimal effects on TET2 upregulation and p16ink4A re-expression in cells with intact DNMT1 gene. The data suggest that the absence of DNMT1 gene made the cells prone to TET2 upregulation upon DNMT inhibitor challenges. Future study is needed to investigate the interactive relationship between DNMT1 and TET2 as well as demethylation, and its impact on cancer treatment with DNMT inhibitors. Citation Format: Angelo B. Laranjeira, Dat Nguyen, Lorraine C. Pelosof, James H. Doroshow, Sherry X. Yang. Upregulation of TET2 expression by DNA methyltransferase (DNMT) inhibitors is associated with resistance in DNMT1 knockout colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5881.

Abstract 4635: RNF208: A novel diagnostic and prognostic biomarker for colorectal cancer and key regulator of malignant behavior

Abstract Background: RNF208, a member of the RING finger protein family, has been implicated in ubiquitin-mediated degradation of Vimentin, regulating invasion in breast cancer, and serves as a prognostic factor in uveal melanoma. However, its diagnostic and prognostic value in colorectal cancer (CRC), as well as its role in regulating malignant behavior in CRC, remains unclear. This study aims to reveal the potential of RNF208 as a diagnostic and prognostic marker for CRC and its function in regulating malignant behavior in CRC through bioinformatics analysis and experimental validation. Methods: In this study, based on the ExoRbase2.0 human plasma exosome database (CRC=35; Healthy=118) and TCGA-COAD dataset (CRC=478; Healthy=41), we conducted bioinformatics analysis of the transcriptomic differences in plasma exosomes between CRC patients and normal individuals using ExoRbase2.0. Survival data from the TCGA-COAD dataset were integrated to select key molecules for CRC diagnosis and prognosis. Plasma samples from 39 CRC patients and 16 normal individuals were collected from hospital, and exosomes were isolated and purified using a reagent kit. qPCR was employed to validate the levels of RNF208 in plasma exosomes, confirming the results of bioinformatics analysis. Biological behavior was assessed through CCK-8 proliferation assays, flow cytometry experiments, and Transwell migration assays to investigate the relationship between RNF208 and malignant behavior in the HCT116 colorectal cancer cell line. Results: Bioinformatics analysis revealed a significant correlation between elevated RNF208 expression in plasma exosomes and the incidence of CRC, as well as poor prognosis. The qPCR results from clinical samples further confirmed this correlation, and ROC curves demonstrated high sensitivity and accuracy, suggesting RNF208's potential as a diagnostic and independent prognostic factor for CRC. CCK-8 experiments showed that RNF208 knockdown suppressed proliferation activity in HCT116 cells, flow cytometry indicated that RNF208 knockdown promoted apoptosis in HCT116 cells, and Transwell migration assays demonstrated that RNF208 knockdown inhibited the migration of HCT116 cells. Conclusion: RNF208 serves as a novel diagnostic and prognostic biomarker for colorectal cancer, promoting the proliferation and migration of colorectal cancer cells and contributing to the further deterioration of the disease. Citation Format: Fangyue Guo, Shanfeng Zhang, Pei Li. RNF208: A novel diagnostic and prognostic biomarker for colorectal cancer and key regulator of malignant behavior [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4635.

O209: Stress-induced extracellular vesicles promote EGFR-ERK dependent growth and cetuximab resistance in colorectal cancer

Abstract Introduction Extracellular vesicles (EVs) are nanoized vesicles, which deliver bioactive cargoes to target cells. Our lab has shown that metabolic stress leads to changes in the trafficking and also the cargo and functions of EVs secreted from colorectal cancer (CRC) cells: a so-called “EV switch”. Proteomic analysis revealed that switched EVs carry increased levels of the EGFR-ligand Amphiregulin (AREG). The aim of this work was to investigate the pro-tumorigenic properties of switched EVs in vitro and in vivo. Methods Size exclusion chromatography was used to isolate EVs from HCT116 and Caco-2 CRC cells grown under glutamine-replete and -depleted conditions, and their effect on recipient cell growth measured using the IncuCyte Live Cell Imager. Co-incubation assays with an AREG-neutralising antibody were undertaken to test the role of switched EVs. The in vivo role of switched EVs were investigated using a novel chick embryo model. In addition, we tested whether the growth inhibitory effects of cetuximab could be reversed by co-treatment with switched EVs. Results Switched EVs from HCT116 and Caco-2 cells contain increased levels of AREG and promote EGFR-ERK dependent growth in recipient cells in vitro and in vivo. These effects were inhibited by co-treatment with an AREG neutralising antibody. Caco-2 (KRAS-wild type), but not HCT116 (KRAS-mutant), cell growth was inhibited by cetuximab, which was partially reversed by co-treatment with switched EVs. AREG neutralisation reversed this EV-induced cetuximab rescue. Conclusion Switched EVs mediate CRC cell growth and cetuximab resistance, and could serve as novel biomarkers to predict treatment response.

Targeted inhibition of colorectal cancer proliferation: Thedual-modulatory role of 2,4-DTBP on anti-apoptotic Bcl-2 and Survivin proteins.

The anti-apoptotic proteins, Bcl-2 and Survivin, are consistently overexpressed in numerous human malignancies, notably in colorectal cancer. 2,4-Di-tert-butylphenol (2,4-DTBP) is a naturally occurring phenolic compound known for its diverse biological activities, including anti-cancer properties. The mechanism behind 2,4-DTBP-induced inhibition of cell proliferation and apoptosis in human colorectal cancer cells, specifically regarding Bcl-2 and Survivin, remains to be elucidated. In this study, we employed both in silico and invitro methodologies to underpin this interaction at the molecular level. Molecular docking demonstrated a substantial binding affinity of 2,4-DTBP towards Bcl-2 (ΔG = -9.8 kcal/mol) and Survivin (ΔG = -5.6 kcal/mol), suggesting a potential inhibitory effect. Further, molecular dynamic simulations complemented by MM-GBSA calculations confirmed the significant binding of 2,4-DTBP with Bcl-2 (dGbind = -54.85 ± 6.79 kcal/mol) and Survivin (dGbind = -32.36 ± 1.29 kcal/mol). Invitro assays using HCT116 colorectal cancer cells revealed that 2,4-DTBP inhibited proliferation and promoted apoptosis in both a dose- and time-dependent manner. Fluorescence imaging and scanning electron microscopy illustrated the classical features associated with apoptosis upon 2,4-DTBP exposure. Cell cycle analysis through flow cytometry highlighted a G1 phase arrest and apoptosis assay demonstrated increased apoptotic cell population. Notably, western blotting results indicated a decreased expression of Bcl-2 and Survivin post-treatment. Considering the cytoprotective roles of Bcl-2 and Survivin through the inhibition of mitochondrial dysfunction, our findings of disrupted mitochondrial bioenergetics, characterized by reduced ATP production and oxygen consumption, further accentuate the functional impairment of these proteins. Overall, the integration of in silico and invitro data suggests that 2,4-DTBP holds promise as a therapeutic agent targeting Bcl-2 and Survivin in colorectal cancer.

Rosemary essential oil potentiates the antitumour activity of 5-fluorouracil in human colorectal carcinoma cells.

Improving response rates in colorectal cancer (CRC) is an urgent clinical need. This study aimed to explore the synergistic action of Lebanese rosemary essential oil (REO) and 5-fluorouracil (5-FU) in HCT116 CRC cells. We tested the cell viability of monotherapy and combination therapy. The combination index was calculated using CompuSyn software to evaluate drug-drug interactions and the level of synergistic cytotoxicity. We also evaluated cell migration and cytopathology. Furthermore, cell apoptosis-related proteins (i.e. Bax and Bcl-2) were measured by Western blot analysis. The REO/5-FU combination synergistically reduced cell viability, effectively decreased cell migration, and increased the Bax/Bcl-2 ratio in HCT116 cells. This triggered a proapoptotic morphology and initiated an apoptotic cascade in HCT116 cells, as indicated by a higher Bax/Bcl-2 ratio. Our results provide evidence of the REO/5-FU combination as a better approach to improve 5-FU anticancer efficacy and allow the use of lower 5-FU doses due to the adjuvant effect of REO.

Novel Stable Protease Inhibitor from Phoenix dactylifera(L.) Flowers with Antimicrobial and Antitumoral Activities.

A novel protease inhibitor isolated from date palm Phoenix dactylifera(L.) flowers (PIDF) was purified and characterized. A heat and acidic treatment step followed by ethanol precipitation and reverse-phase high-performance chromatography was applied to purify this natural protease inhibitor to homogeneity with a single band of about 19 kDa. The stability study depicted that PIDF was fully stable at 40 °C and retained 65% of its initial activity after heating at 50 °C for 24 h. Its thermal stability at 70 °C was markedly enhanced by adding calcium, bovine serum albumin, and sorbitol as well as by metal divalent cations, especially Mg2+ and Hg2+. This protease inhibitor showed high inhibitory activity against therapeutic proteases, including pepsin, trypsin, chymotrypsin, and collagenase, and acted as a potent inhibitor of some commercial microbial proteases from Aspergillus oryzae, Bacillus. sp, and Bacillus licheniformis. Moreover, a potent antibacterial spectrum against Gram (+) and Gram (-) bacterial strains and an efficient antifungal effect were observed. Its cytotoxicity toward human colorectal cancer cell LoVo and HCT-116 lines suggested that PIDF could serve as a new therapeutic target inhibiting human colorectal cancer.

Abstract 1229 ''Targeting Notch, IL-1 and Leptin crosstalk may have promising approach in HCT-15 colorectal cancer cells''

Abstract 1229 ''Targeting Notch, IL-1 and Leptin crosstalk may have promising approach in HCT-15 colorectal cancer cells''

Peptide mimetic NC114 induces growth arrest by preventing PKCδ activation and FOXM1 nuclear translocation in colorectal cancer cells.

The peptide mimetic, NC114, is a promising anticancer compound that specifically kills colorectal cancer cells without affecting normal colon epithelial cells. In our previous study, we observed that NC114 inhibited the Wnt/β-catenin pathway, with significant downregulation of both Ser 675-phosphorylated β-catenin and its target genes, cyclin D1 and survivin. However, the molecular mechanism responsible for its cytotoxic effect has not yet been fully characterized. In the present study, we demonstrated that NC114 prevented cell cycle progression from S to G2/M phase by downregulating cell cycle-related gene expression, and also induced growth arrest in SW480 and HCT-116 colorectal cancer cells. A novel covariation network analysis combined with transcriptome analysis revealed a series of signaling cascades affected by NC114 treatment, and identified protein kinase C-δ (PKCδ) and forkhead box protein M1 (FOXM1) as important regulatory factors for NC114-induced growth arrest. NC114 treatment inhibits the activation of PKCδ and its kinase activity, which suppresses MEK/ERK signaling. Attenuated MEK/ERK signaling then results in a reduction in FOXM1 phosphorylation and subsequent nuclear translocation of FOXM1 and β-catenin. Consequently, formation of a T-cell factor-4 (TCF4)/β-catenin transcription complex in the nucleus is inhibited and transcription of its target genes, such as cell cycle-related genes, is downregulated. The efficacy of NC114 on tumor growth was confirmed in a xenograft model. Collectively, elucidation of the mechanism by which NC114 induces growth arrest in colorectal cancer cells should provide a novel therapeutic strategy for colorectal cancer treatment.

Modulation of intestinal metabolites by calorie restriction and its association with gut microbiota in a xenograft model of colorectal cancer

BackgroundColorectal cancer (CRC) is a common malignant tumor, and its occurrence and development are closely related to dysbiosis of gut microbes. Previously, we found calorie restriction altered the composition of the microbial community in a colorectal cancer mouse model and inhibited in vivo growth of CRC cells. Here, we aim to further investigate alteration in the intestinal metabolites and explore the interplay between gut microbiota and intestinal metabolites upon calorie restriction.MethodsHuman colorectal cancer HCT116 cells were used to establish a colorectal cancer xenograft mouse model. The changes of intestinal metabolites in the ad libitum group and calorie restriction group were investigated through untargeted metabolomics analysis. The integrative analysis of gut microbiota and metabolites to elucidate the associations between gut microbiota and intestinal metabolites.ResultsCompared with the mice in the ad libitum group, mice upon calorie restriction exhibited downregulation of Isoleucyl-Valine, and upregulation of D-Proline, 1-Palmitoylphosphatidylcholine, and 4-Trimethylammoniobutanoic acid. Additionally, an integrative analysis of gut microbiota and metabolites revealed that Lactobacillus, Parabacteroides and rC4-4 genus were upregulated in the calorie restriction group and positively correlated with D-Proline, 4-Trimethylammoniobutanoic acid or 1-Palmitoylphosphatidylcholine, while negatively correlated with Isoleucyl-Valine. In contrast, the Nitrospirae and Deferribacteres phylum exhibited opposite trends.ConclusionCalorie restriction affects the abundance of gut microbes such as Nitrospirae phylum and Lactobacillus genus in mouse model of colorectal cancer, leading to changes in the metabolites such as D-Proline、Isoleucyl-Valine, which contributes to the suppression of in vivo growth of CRC by calorie restriction.

Anticancer Effect by Combined Treatment of Artemisia annua L. Polyphenols and Docetaxel in DU145 Prostate Cancer Cells and HCT116 Colorectal Cancer Cells.

Docetaxel (DTX), a semi-synthetic analogue of paclitaxel (taxol), is known to exert potent anticancer activity in various cancer cells by suppressing normal microtubule dynamics. In this study, we examined how the anticancer effect of DTX is regulated by polyphenols extracted from Korean Artemisia annua L. (pKAL) in DU145 prostate cancer cells (mutant p53) and HCT116 colorectal cancer cells (wild-type p53). Here, we show that the anticancer effect of DTX was enhanced more significantly by pKAL in HCT116 cells than in DU145 cells via phase-contrast microscopy, CCK-8 assay, Western blot, and flow cytometric analysis of annexin V/propidium iodide-stained cells. Notably, mutant p53 was slightly downregulated by single treatment of pKAL or DTX in DU145 cells, whereas wild-type p53 was significantly upregulated by pKAL or DTX in HCT116 cells. Moreover, the enhanced anticancer effect of DTX by pKAL in HCT116 cells was significantly associated with the suppression of DTX-induced p53 upregulation, increase of DTX-induced phospho-p38, and decrease of DTX-regulated cyclin A, cyclin B1, AKT, caspase-8, PARP1, GM130, NF-κB p65, and LDHA, leading to the increased apoptotic cell death and plasma membrane permeability. Our results suggest that pKAL could effectively improve the anticancer effect of DTX-containing chemotherapy used to treat various cancers expressing wild-type p53.