Human Colorectal Cancer HCT116 Research Articles

Probing the interaction of hesperidin showing antiproliferative activity in colorectal cancer cells and human hemoglobin

Hesperidin, a flavanone glycoside abundant in citrus is known to possess anti-carcinogenic properties. However, its main interaction with cancer cells and blood proteins is not well-studied yet. Here we have explored the interactions of hesperidin with human colorectal cancer cells, HCT116, and human hemoglobin (HHb) with several experimental and theoretical studies. Cellular assays showed that hesperidin interacted with colorectal cancer cells and induced membrane damage, colony formation inhibition, oxidative stress, mitochondrial dysfunction, Bax/Bcl-2, caspase-9, and caspase-3 upregulation, and cytochrome c release determined by cellular, qPCR and ELISA assays. The interaction of the hesperidin with HHb indicated the formation of a static complex mainly with the assistance of hydrogen bonds which lead to partial folding of protein determined by spectroscopy, molecular docking, and molecular dynamic studies. In conclusion, these findings show that hesperidin with potential binding affinity with a plasma protein model can also show promising anticancer activities against colorectal cancer cells.

Novel ST1926 Nanoparticle Drug Formulation Enhances Drug Therapeutic Efficiency in Colorectal Cancer Xenografted Mice.

Cancer is a major public health problem that ranks as the second leading cause of death. Anti-cancer drug development presents with various hurdles faced throughout the process. Nanoparticle (NP) formulations have emerged as a promising strategy for enhancing drug delivery efficiency, improving stability, and reducing drug toxicity. Previous studies have shown that the adamantyl retinoid ST1926 displays potent anti-tumor activities in several types of tumors, particularly in colorectal cancer (CRC). However, phase I clinical trials in cancer patients using ST1926 are halted due to its low bioavailability. In this manuscript, we developed ST1926-NPs using flash nanoprecipitation with polystyrene-b-poly (ethyleneoxide) as an amphiphilic stabilizer and cholesterol as a co-stabilizer. Dynamic light scattering revealed that the resulting ST1926-NPs Contin diameter was 97 nm, with a polydispersity index of 0.206. Using cell viability, cell cycle analysis, and cell death assays, we showed that ST1926-NP exhibited potent anti-tumor activities in human CRC HCT116 cells. In a CRC xenograft model, mice treated with ST1926-NP exhibited significantly lowered tumor volumes compared to controls at low drug concentrations and enhanced the delivery of ST1926 to the tumors. These findings highlight the potential of ST1926-NPs in attenuating CRC tumor growth, facilitating its further development in clinical settings.

Activation of p38 and JNK by ROS Contributes to Deoxybouvardin-Mediated Intrinsic Apoptosis in Oxaliplatin-Sensitive and -Resistant Colorectal Cancer Cells.

Colorectal cancer (CRC) remains a global health burden, accounting for almost a million deaths annually. Deoxybouvardin (DB), a non-ribosomal peptide originally isolated from Bouvardia ternifolia, has been reported to possess antitumor activity; however, the detailed mechanisms underlying this anticancer activity have not been elucidated. We investigated the anticancer activity of the cyclic hexapeptide, DB, in human CRC HCT116 cells. Cell viability, evaluated by MTT assay, revealed that DB suppressed the growth of both oxaliplatin (Ox)-resistant HCT116 cells (HCT116-OxR) and Ox-sensitive cells in a concentration- and time-dependent manner. Increased reactive oxygen species (ROS) generation was observed in DB-treated CRC cells, and it induced cell cycle arrest at the G2/M phase by regulating p21, p27, cyclin B1, and cdc2 levels. In addition, Western blot analysis revealed that DB activated the phosphorylation of JNK and p38 MAPK in CRC. Furthermore, mitochondrial membrane potential (MMP) was dysregulated by DB, resulting in cytochrome c release and activation of caspases. Taken together, DB exhibited anticancer activity against both Ox-sensitive and Ox-resistant CRC cells by targeting JNK and p38 MAPK, increasing cellular ROS levels, and disrupting MMP. Thus, DB is a potential therapeutic agent for the treatment of Ox-resistant CRC.

Terfenadine, a histamine H1 receptor antagonist, induces apoptosis by suppressing STAT3 signaling in human colorectal cancer HCT116 cells

Terfenadine, a histamine H1 receptor antagonist, induces apoptosis by suppressing STAT3 signaling in human colorectal cancer HCT116 cells

Short Chain Fatty Acid Sodium Butyrate Increases miR-21, miR-143 and miR-145 Expression in Human Colorectal Cancer HCT-116 Cell Line.

Sodium butyrate (NaBu) is a short-chain fatty acid; it is one of the histone deacetylase inhibitors, which can alter both genetic and epigenetic expressions. The present study aimed to elucidate the effect of Na-Bu on the expression of miR-21, miR-143, and miR-145 in human colorectal cancer HCT-116 cell lines. This study was done in Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. HCT-116 cell line was treated with diverse concentrations of NaBu (6.25 mM to 200 mM) at 24, 48, and 72 h. MTT assay was used for assessing the cytotoxicity. Quantitative Real-Time-PCR was performed to investigate the gene expression of miR-21, miR-143, and miR-145. IC50 values were evaluated by MTT assay. IC50 for HCT-116 was 50 mM, 12.5 mM, and 6.25 mM for 24, 48, and 72 h of incubation, respectively. According to the Real-Time-PCR results, 50 mM NaBu after 24 h caused a significant up-regulation in the expression of the miR-21, miR-143, and miR-145 (P<0.05). In 48 h, incubation, 12.5 mM NaBu caused a significant up-regulation in the expression of the miR-21, miR-143, and miR-145 (P<0.05). In treated cells with 6.25 mM NaBu after 72 h of incubation caused a significant up-regulation in the expression of the miR-21, miR-143, and miR-145 compared with untreated cells (P<0.05). The upregulation of miR-21, miR-143, and miR-145 expression are mediated by transcriptional regulation and the activation of this miR promoter is modulated by histone acetylation. The employment of NaBu may represent a promising approach for improving HDACi drug-based therapies for colon cancers.

Lovastatin-Induced Mitochondrial Oxidative Stress Leads to the Release of mtDNA to Promote Apoptosis by Activating cGAS-STING Pathway in Human Colorectal Cancer Cells.

Statins are 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitors widely used in the treatment of hyperlipidemia. The inhibition of HMG-CoA reductase in the mevalonate pathway leads to the suppression of cell proliferation and induction of apoptosis. The cyclic GMP-AMP synthase (cGAS) stimulator of the interferon genes (STING) signaling pathway has been suggested to not only facilitate inflammatory responses and the production of type I interferons (IFN), but also activate other cellular processes, such as apoptosis. It has not been studied, however, whether cGAS-STING activation is involved in the apoptosis induced by statin treatment in human colorectal cancer cells. In this study, we reported that lovastatin impaired mitochondrial function, including the depolarization of mitochondrial membrane potential, reduction of oxygen consumption, mitochondrial DNA (mtDNA) integrity, and mtDNA abundance in human colorectal cancer HCT116 cells. The mitochondrial dysfunction markedly induced ROS production in mitochondria, whereas the defect in mitochondria respiration or depletion of mitochondria eliminated reactive oxygen species (ROS) production. The ROS-induced oxidative DNA damage by lovastatin treatment was attenuated by mitochondrial-targeted antioxidant mitoquinone (mitoQ). Upon DNA damage, mtDNA was released into the cytosol and bound to DNA sensor cGAS, thus activating the cGAS-STING signaling pathway to trigger a type I interferon response. This effect was not activated by nuclear DNA (nuDNA) or mitochondrial RNA, as the depletion of mitochondria compromised this effect, but not the knockdown of retinoic acid-inducible gene-1/melanoma differentiation-associated protein 5 (RIG-I/MDA5) adaptor or mitochondrial antiviral signaling protein (MAVS). Moreover, lovastatin-induced apoptosis was partly dependent on the cGAS-STING signaling pathway in HCT116 cells as the knockdown of cGAS or STING expression rescued cell viability and mitigated apoptosis. Similarly, the knockdown of cGAS or STING also attenuated the antitumor effect of lovastatin in the HCT116 xenograft model in vivo. Our findings suggest that lovastatin-induced apoptosis is at least partly mediated through the cGAS-STING signaling pathway by triggering mtDNA accumulation in the cytosol in human colorectal cancer HCT116 cells.

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 4068: In vitro masked effect, in vivo anti-tumor activity and toxicology study of KGX101, an interleukin-12 prodrug, in monotherapy or in combination with anti-PD-L1 antibody

Abstract Interleukin-12 (IL-12) has long been limited in its clinical use due to its short half-life (t1/2) and dose-related toxicity, despite its remarkable ability to activate effector functions in T cells and natural killer (NK) cells. To address these challenges, we engineered an IL-12 prodrug called KGX101. In KGX101, the C-terminus of IL-12 is fused with ADCC-impaired IgG1 Fc to extend its half-life. Meanwhile, the N-terminus is concealed with its corresponding receptor soluble domains, IL-12Rβ1 and IL-12Rβ2, using tumor-specific protease cleavable linkers. This innovative design prevents systemic immune system overactivation. Once the linker is cleaved by tumor microenvironment (TME)-specific proteases, KGX101 functions like IL-12, effectively stimulating T cells and NK cells, reshaping the TME, and displaying significant potential in cancer therapy. To confirm KGX101's masking effect, we conducted IFNγ induction efficacy tests using peripheral blood mononuclear cells (PBMCs) from three donors. KGX101 demonstrated a 300 to 2500-fold reduction in activity compared to unmasked IL-12-Fc. Importantly, the masking effect of KGX101 was nearly abolished after protease digestion, affirming its mechanism of action. To further establish KGX101's effectiveness, we conducted in vivo antitumor studies in hPBMC xenograft mouse models using human colorectal cancer HCT116 and PD-L1-overexpressed human malignant melanoma A375. Mice were subcutaneously implanted with a specific mixture of cancer cells and hPBMCs, and then intravenously injected with PBS or KGX101 at various dose levels twice a week for 4-5 cycles. Compared to the PBS control group, KGX101 demonstrated up to a 42% tumor growth inhibition (TGI) in the HCT116 model at 0.18 mg/kg and a 76% TGI in the hPD-L1-A375 model at 0.06 mg/kg. Analysis of tumor-infiltrating lymphocytes (TILs) revealed that KGX101 increased the percentage of T cells in the tumor environment in a dose-dependent manner. We also evaluated the combination of KGX101 with an anti-PD-L1 antibody in an HCT116/hPBMC xenograft mouse model, which showed limited response to anti-PD-L1 monotherapy. The TGI% for KGX101 at 0.06 mg/kg, anti-PD-L1 antibody at 1 mg/kg, and their combination were 36.56%, 6.93%, and 66.22%, respectively, demonstrating a promising antitumor synergy. In terms of pharmacokinetics, a single dose of KGX101 at 0.06 mg/kg and 0.18 mg/kg in the HCT116/hPBMC xenograft mouse model resulted in a serum half-life ranging from 52.75 to 64.87 hours, while the t1/2 in Cynomolgus monkeys was approximately 3 days. In a 4-week repeat dose toxicity study in Cynomolgus monkeys, the highest non-severely toxic dose (HNSTD) of KGX101 was determined to be 0.1 mg/kg. The results from nonclinical toxicology studies support the potential of KGX101 as a well-tolerated therapy for the First-in-Human (FIH) clinical trial. Citation Format: Shumin Yang, Nuo Li, Jing Wang, Li Ma, Yi Zhao, Hui Chen, Haixiang Wu, Weidong Jiang. In vitro masked effect, in vivo anti-tumor activity and toxicology study of KGX101, an interleukin-12 prodrug, in monotherapy or in combination with anti-PD-L1 antibody [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 4068.

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.

Six bufadienolides derivatives are the main active substance against human colorectal cancer HCT116 cells of Huachansu injection

BackgroundHuachansu injection (HCS) is an aqueous extract preparation of Traditional Chinese Medicine (TCM) toad skin (Bufo melanosticus or B. bufo gargarizans). As a national second-class and self-developed TCM preparation, it is widely used to treat human colorectal cancer (CRC), yet its substantial toxic side effects, such as vascular irritation reaction, drug fever, and allergic reactions, pose a considerable challenge during clinical use. In-depth research on the material basis of HCS' traditional efficacy is essential for developing more effective and less toxic medicine. Identifying the main components responsible for the efficacy of HCS is crucial in determining its material basis and explaining any adverse reactions. PurposeThe purpose of this study is to clarify the main active components of HCS as a growth inhibitory agent against CRC HCT116 cells and to further understand the material basis of its anti-CRC properties. MethodsSulforhodamine B (SRB) assay was used to screen the compound with strong inhibitory activity against HCT116 cells from 44 compounds isolated from aqueous extracts of toad skin (B. melanosticus). The screened compounds as standard and MeOH extracts from HCS were determined by HPLC coupled with triple quadrupole mass spectrometry. The partial MeOH extract was subjected to HPLC system using solvents in a gradient of increasing polarity, yielding 12 fractions in total (HCS1 to HCS12). Besides, the screened active compounds were knocked out from HCS to obtain a fraction as a negative control, and the knocked-out components were collected as one fraction using semi-preparative HPLC. Those fractions were submitted to HPLC-MS/MS or HPLC-DAD system to determine the presence or absence of the screened compounds. In addition, cytotoxicity of those prepared fractions against CRC HCT116 cells was evaluated to determine the effect of the screened compounds in the anti-CRC of HCS. ResultsSix compounds, namely arenobufagin, telocinobufagin, gamabufalin, hellebrigenin, 19-hydroxylbufagin and argentinogenin, were screened for their inhibitory activity (with an IC50 value of less than 400 nM), and were present in HCS at a total-mass-percentage of 1.35×10−3 % in HCS MeOH extract. In addition, those 6 compounds were mainly distributed in HCS8, which also showed the strongest inhibitory activity against HCT116 cells. And the results of multicomponent knockout assay indicated the negative control lost its anti-CRC activity significantly, while the activity can be restored after those 6 compounds were added to the negative control. ConclusionsThose six bufadienolides derivatives are responsible for the anti-CRC HCT116 cells activity of HCS, and are the main components of HCS on anti-CRC. Through this research, we can more clearly understand the main anti-CRC active components in HCS, and the work also lays the foundation for the development of more effective and less toxic medicine from HCS.

Anticancer and anti-angiogenic activities of mannooligosaccharides extracted from coconut meal on colorectal carcinoma cells in vitro

Colorectal carcinoma (CRC) is one of the most common malignancies, though there are no effective therapeutic regimens at present. This study aimed to investigate the inhibitory effects of mannooligosaccharides extracted from coconut meal (CMOSs) on the proliferation and migration of human colorectal cancer HCT116 cells in vitro. The results showed that CMOSs exhibited significant inhibitory activity against HCT116 cell proliferation in a concentration-dependent manner with less cytotoxic effects on the Vero normal cells. CMOSs displayed the ability to increase the activation of caspase-8, –9, and –3/7, as well as the generation of reactive oxygen species (ROS). Moreover, CMOSs suppressed HCT116 cell migration in vitro. Interestingly, treatment of human microvascular endothelial cells (HMVECs) with CMOSs resulted in the inhibition of cell proliferation, cell migration, and capillary-like tube formation, suggesting its anti-vascular angiogenesis. In summary, the results of this study indicate that CMOSs could be a valuable therapeutic candidate for CRC treatment.

1,8-Dihydroxy-3-methoxy-anthraquinone inhibits tumor angiogenesis through HIF-1α downregulation

Photorhabdus luminescens is a gram-negative bioluminescent bacterium known as an intestinal bacterium that coexists in the digestive tract of insect-pathogenic nematodes. As part of our ongoing exploration to identify bioactive compounds from diverse natural resources, the chemical analysis of the cultures of P. luminescens KACC 12254 via LC/MS and TLC-based analyses enabled the isolation and identification of a major fluorescent compound. Its chemical structure was elucidated as 1,8-dihydroxy-3-methoxyanthraquinone (DMA) using HR-ESI-MS and NMR analysis. In this study, we conducted comprehensive investigations utilizing human colorectal cancer HCT116 cells, human umbilical cord vascular endothelial cells (HUVECs), and zebrafish embryos to assess the potential benefits of DMA in suppressing tumor angiogenesis. Our results convincingly demonstrate that DMA effectively suppresses the stability of hypoxia-inducible factor-1α (HIF-1α) protein and its target genes without inducing any cytotoxic effects. Furthermore, DMA demonstrates the ability to inhibit HIF-1α transcriptional activation and mitigate the production of reactive oxygen species (ROS). In our in vitro experiments, DMA exhibits notable inhibitory effects on VEGF-mediated tube formation, migration, and invasion in HUVECs. Additionally, in vivo investigations using zebrafish embryos confirm the antiangiogenic properties of DMA. Notably, DMA does not exhibit any adverse developmental or cardiotoxic effects in the in vivo setting. Moreover, we observe DMA's capability to restrain tumor growth through the downregulation of PI3K/AKT and c-RAF/ERK pathway. Collectively, these compelling findings underscore DMA's potential as a promising therapeutic candidate for targeted intervention against HIF-1α and angiogenesis in cancer treatment.

Interaction mechanism of nordentatin with human α-1 acid glycoprotein and human colorectal cancer HCT-116 cells

Plasma protein binding of therapeutic bioactive materials, especially anticancer compounds can play an important role in their pharmacokinetics and pharmacodynamics. Human α-1 acid glycoprotein (AGP) has shown potential binding affinities to different endogenous and exogenous ligands due to its unique binding site composed of eight anti-parallel β-sheets. Therefore, in this study, the interaction of nordentatin, a member of coumarins with a chemical formula of C19H20O4 with AGP, was measured by multi-spectroscopic and theoretical approaches. Afterward, the anticancer potency of nordentatin against human colorectal cancer HCT-116 cells was assessed. The fluorescence quenching of AGP was observed upon binding of nordentatin through a static quenching mechanism. The spontaneous hydrophobic interaction of nordentatin with AGP had a binding constant value (Kb) of 104 M−1 with a marginal change in the secondary structure of AGP. Cellular assays demonstrated that nordentatin exhibited promising inhibitory effects on the cellular growth of HCT-116 cells through increasing the levels of intracellular ROS and [Ca2+], while it remarkably triggered the MMP reduction. Also, RT-qPCR analysis disclosed that nordentatin stimulated apoptosis by overexpression of P53, Bax/Bcl-2, cytochrome c, caspase-9, and caspase-3 mRNA. Overall, these findings suggested that nordentatin, in addition to potential binding to plasma protein, could be used as an effective preventive or curative plant-based therapy for colorectal cancer.

3β-Hydroxy-12-oleanen-27-oic Acid Exerts an Antiproliferative Effect on Human Colon Carcinoma HCT116 Cells via Targeting FDFT1.

3β-hydroxy-12-oleanen-27-oic acid (ATA), a cytotoxic oleanane triterpenoid with C14-COOH isolated from the rhizome of Astilbe chinensis, has been previously proven to possess antitumor activity and may be a promising antitumor agent. However, its molecular mechanisms of antitumor action were still unclear. This study explored the underlying mechanisms of cytotoxicity and potential target of ATA against human colorectal cancer HCT116 cells via integrative analysis of transcriptomics and network pharmacology in combination with in vitro and in vivo experimental validations. ATA significantly inhibited the proliferation of HCT116 cells in a concentration- and time-dependent manner and induced the cell cycle arrest at the G0/G1 phase, apoptosis, autophagy, and ferroptosis. Transcriptomic analysis manifested that ATA regulated mRNA expression of the genes related to cell proliferation, cell cycle, and cell death in HCT116 cells. The integrated analysis of transcriptomics, network pharmacology, and molecular docking revealed that ATA exerted cytotoxic activity via interactions with FDFT1, PPARA, and PPARG. Furthermore, FDFT1 was verified to be an upstream key target mediating the antiproliferative effect of ATA against HCT116 cells. Of note, ATA remarkably suppressed the growth of HCT116 xenografts in nude mice and displayed an apparent attenuation of FDFT1 in tumor tissues accompanied by the alteration of the biomarkers of autophagy, cell cycle, apoptosis, and ferroptosis. These results demonstrate that ATA exerted in vitro and in vivo antiproliferative effects against HCT116 cells through inducing cell apoptosis, autophagy, and ferroptosis via targeting FDFT1.

ADT-OH synergistically enhanced the antitumor activity of celecoxib in human colorectal cancer cells.

Colorectal cancer is one of the most prevalent cancers in the world, but the research on its prevention, early diagnosis and treatment is still a major challenge in clinical oncology. Thus, there is a pressing requirement to find effective strategies to improve the survival of colon cancer patients. Celecoxib has been accounted to be an effective antitumor drug, but may exhibit significant side effects. In recent studies, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), one of the most commonly used reagents for the synthesis of sustained-release H2 S donors, has also been reported to inhibit cancer progression by affecting processes such as cell cycle, angiogenesis, and apoptosis. Therefore, we evaluated the therapeutic effect of the combination of ADT-OH and celecoxib on colorectal cancer through in vitro and in vivo, hoping to achieve better therapeutic effect and reduce the effect of celecoxib on gastric injury through exogenous administration of H2 S. Our results demonstrated that ADT-OH combined with celecoxib synergistically inhibited the proliferation and migration ability of human colorectal cancer HCT116 cells, altered cell cycle and cytoskeleton, increased intracellular reactive oxygen species (ROS), and promoted cell apoptosis. Noteworthy, in vivo studies also indicated the excellent antitumor therapeutic effect of the combination therapy without apparent toxicity. In general, our results provide a reasonable combination strategy of low-dose ADT-OH and celecoxib in the preclinical application of colorectal cancer.

Synthesis and Cytotoxicity Evaluation of Tyrosine-5 Fluorinated Analogues of RA-VII, An Antitumor Bicyclic Hexapeptide

AbstractRA-VII analogues fluorinated at each ε-position of Tyr-5 were designed. The synthesis of these peptide analogues commenced with the preparation of atropisomeric fluorocycloisodityrosines from protected 3-fluoro-L-tyrosyl-3-boronyl-L-tyrosine mediated by copper(II) acetate and 4-(dimethylamino)pyridine. After N-methylation, the tetrapeptide segment was coupled with the N-terminus of each fluorocycloisodityrosine to afford a hexapeptide. After removal of the C- and N-terminal protecting groups, each peptide was subjected to macrocyclization to produce an analogue. The analogue with a β-oriented fluorine atom was equipotent to RA-VII with regard to cytotoxicity toward human mammary carcinoma MCF-7 cells, and showed 2.1-fold and 1.4-fold lower cytotoxicities than RA-VII toward human promyelocytic leukemia HL-60 and human colorectal cancer HCT-116 cells, respectively, whereas the analogue with an α-oriented fluorine atom showed 7.7-fold, 6.0-fold, and 14-fold lower cytotoxicities than RA-VII toward those cells, respectively.

Anticancer activity and mechanisms of action of Taisui fermentation broth in human colorectal cancer HCT116 cells in vitro and in vivo

Taisui, an unclassified biological object, is considered as a functional food with rich medicinal value in ancient Chinese books. However, there are few studies on Taisui, particularly on its anticancer activity and underlying mechanisms. Herein, we evaluated the anticancer potential of Taisui fermentation broth using HCT116 cells and nude mice xenografts in vitro and in vivo. Taisui fermentation broth inhibited HCT116 cells proliferation by inducing S-phase arrest, apoptosis, and autophagy, by regulating related proteins. Taisui fermentation broth-induced HCT116 cells death was reversed by inhibiting apoptosis and autophagy. Autophagy inhibition suppressed Taisui fermentation broth-induced apoptosis, whereas apoptosis inhibition attenuated Taisui fermentation broth-induced autophagy. Taisui fermentation broth induced apoptosis and autophagy by regulating reactive oxygen species-mediated c-Jun NH2-terminal kinase/p38 signaling in HCT116 cells. Additionally, Taisui fermentation broth inhibited tumor growth in a mouse xenograft model. Taisui fermentation broth may be a promising anticancer agent for the treatment of colorectal cancer.

Abstract 1385: Oncogenic regulation of autophagy and DNA emission

Abstract Background: Cancer cells release higher levels of cell-free DNA (cfDNA) into the circulatory system than normal cells. However, the molecular mechanisms underlying the process of programmed DNA elimination remain poorly understood. Emerging evidence has indicated that oncogenes such as HRAS can be released through extracellular vesicles (EVs). Moreover, studies have shown an active mechanism of DNA emission through autophagy. Autophagy is an evolutionarily conserved process that degrades and recycles cytoplasmic materials such as organelles and proteins in the lysosomes. Interestingly, Microtubule-associated protein 1 light chain-3 (LC3B) is known to degrade nuclear lamina components in HRAS-induced cells, potentially producing a pool of cytoplasmic chromatin. Cytoplasmic chromatin is ultimately degraded in lysosomes or re-routed outside of the cells. The aim of our study was to evaluate the role of autophagy proteins in circulating tumor (ctDNA) and EV-associated DNA emission in cancer cell lines harboring oncogenes (RAS and BRAF) as this aspect of the process is relatively understudied. Methods: We have assessed protein and RNA expression of autophagy genes (LC3B, ATG5 and ATG7) in IEC-18 cells that have been transduced with the HRAS oncogene (RAS3 cells) and human colorectal cancer cell lines SW620 and HCT116 using RT-qPCR and western blot (WB). All conditions were compared to their respective control cells (IEC-18, CCD-841 CoN and CCD18-Co cells. To determine the role of LC3 in nuclear membrane degradation, we employed transmission electron microscopy (TEM). We have further validated the role of autophagy genes in EV-DNA emission by shRNA knockdown using shRNA against highly expressed autophagy genes, such as LC3B in RAS3 cells. EV-DNA was measured using droplet digital PCR (ddPCR). Results: To determine whether autophagy expels oncogenic DNA via EVs in KRAS-mutant CRC cells, we focused on the role of ATG5, LC3B and ATG7, as this aspect of the process is understudied. Our data shows higher LC3B and ATG7 expression in transformed RAS3 cells, compared to control cell lines (IEC18). Moreover, ATG5 is overexpressed in SW620 and HT29 colorectal cancer cells compared to control cell lines. LC3 is known to degrade the nuclear membrane in HRAS-activated cells. Indeed, we observed degradation of the nuclear membrane in RAS3 cells leading to the accumulation of cytosolic DNA shown by TEM. Furthermore, overexpression of LC3B in IEC18 cells using GFP-tagged LC3B resulted in increased EV emission. These findings suggest that the autophagy axis may contribute to oncogenic DNA emission via EVs. Lastly, shRNA knockdown of LC3 resulted in reduction of EV-DNA emission (ddPCR). Impact: EV biogenesis and autophagy pathways play pivotal and interconnected roles during DNA processing and extracellular emission. Therefore, understanding the detailed mechanisms is critical for biomarker discovery and potential therapeutic strategies targeting this process. Citation Format: Thupten Tsering, Kyle Dickinson, Laura Montermini, Janusz Rak, Julia Burnier. Oncogenic regulation of autophagy and DNA emission [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1385.

2,4-Dipropylphloroglucinol inhibits the growth of human lung and colorectal cancer cells through induction of apoptosis

Scientists are finding the most effective chemotherapeutic agents for the treatment of cancer. In the present study, we evaluated the anticancer mechanism of DPPG, a derivative of DAPG (2,4-diacetylphloroglucinol), for the first time. DPPG and DAPG inhibited 83 and 59% of human colorectal cancer HCT116 cell growth at 40.0µg/ml, and 74 and 57% of human lung cancer A549 cell growth at 10.0µg/ml concentrations respectively. Furthermore, DPPG and DAPG inhibited 97 and 73% colony formation of the HCT116 cells at 20.0µg/ml concentration. DPPG and DAPG induced apoptosis in the HCT116 and A549 cells that was confirmed by Hoechst 33342 and FITC-annexin V staining. This result also revealed that ROS generated in both the HCT116 and A549 cells after treatment with DPPG. However, no ROS production was observed in HCT116 and A549 cells after treatment with DAPG. Both DAPG and DPPG significantly increased the CASP3 protein expression that was detected by staining the cells with the super-view 488-CASP3 substrate. Expression of WNT1 gene was eliminated in DPPG and DAPG treated HCT116. Expression of MAPK1 gene was entirely abolished in DPPG treated cells, whereas a significant decrease was observed for DAPG. An intense band of CASP8 gene product was observed agarose gel for DPPG treated HCT116 cells than DAPG. Molecular docking simulation showed the high binding affinities (≥ 6.5kcal/mol) of DPPG and DAPG with target proteins WNT1, MAPK1, CASP8, and CASP3 in HCT116 cells. This manuscript demonstrated that DAPG and DPPG inhibited lung and colorectal cancer cells by inducing apoptosis. DAPG and DPPG inhibited A549 and HCT116 cells growth by inducing apoptosis.

Phloretin induces G2/M arrest and apoptosis by suppressing the β-catenin signaling pathway in colorectal carcinoma cells.

Colorectal carcinoma (CRC) is the third most prevalent cancer, causing a significant mortality worldwide. Present available therapies are surgery, chemotherapy including radiotherapy, and these are known to be associated with heavy side effects. Therefore, nutritional intervention in the form of natural polyphenols has been well recognised to prevent CRC. Phloretin, a known dihydrochalcone is present in apple, pear and strawberry. This has been proven to induce apoptosis in cancer cells and also exhibited anti-inflammatory activity, thus can be explored as a potential anticancer nutraceutical agent. This study demonstrated phloretin's significant in vitro anticancer activity against CRC. Phloretin suppressed cell proliferation, colony forming ability and cellular migration in human colorectal cancer HCT-116 and SW-480 cells. Results also revealed that phloretin generated reactive oxygen species (ROS) which provoked depolarization of mitochondrial membrane potential (MMP) and further contributed to cytotoxicity in colon cancer cells. Phloretin also modulated the cell cycle regulators including cyclins and cyclin-dependent kinases (CDKs) and halted cell cycle at G2/M phase. Moreover, it also induced apoptosis by regulating the expression of Bax and BCl-2. The Wnt/β-catenin signaling is inactivated by phloretin by targeting the downstream oncogenes namely CyclinD1, c-Myc and Survivin which are involved in the proliferation and apoptosis of colon cancer cells. In our study we showed that lithium chloride (LiCl) induced the expression of β-catenin and its target genes and the co-treatment of phloretin circumvent its effect and downregulated the Wnt/β-catenin signaling. In conclusion, our results strongly suggested that phloretin can be utilized as a nutraceutical anticancer agent for combating CRC.