HT-29 Colorectal Cancer Cells Research Articles

Biocompounds and Bioactivities of Selected Greek Boraginaceae Seeds

The seeds of Boraginaceae species constitute a source of γ-linolenic acid (GLA, 18:3n-6)-rich oils, mainly those of Borago officinalis. However, there are many wild unexplored Boraginaceae taxa that are potentially GLA producers. This work scrutinizes the seeds of several Greek Boraginaceae species for fatty acids (FAs), phenolic compounds, antioxidant activity, and in vitro antiproliferative activity against HT-29 colorectal cancer cells. The FA profiles were checked by GC-FID, and GLA (FA% of total FA) highlights in Symphytum bulbosum (26.2), S. creticum (23.2), and Lithodora zahnii (17.4). The total FA content ranged from 9.2 in Alkanna methanaea to 27.3 g/100 g of seeds in Alkanna corcyrensis. The antioxidant activity of the water/methanol extracts, evaluated by the ABTS and DPPH methods, was in line with other GLA producer seeds. Phenolic acids were analyzed by HPLC-DAD and LC-MS, and rosmarinic acid ranged from 160.2 in S. bulbosum to 479.7 mg/100 g in L. zahnii. The MTT assay showed dose- and time-dependent inhibitory effects of the water/methanol extracts against HT-29 cancer cells, especially those of C. major and L. zahnii (GI50 of 600 and 375 μg/mL for 72 h-exposed cells). This work constitutes the first approach to evaluate the seeds of Boraginaceae taxa from Greece as functional oil providers.

Cytotoxicity of Physalis minima Linn (Solanaceae) fruit against HCT116 and HT29 colorectal cancer cell lines

The pantropical Physalis minima are traditionally used for the prevention and treatment of various illnesses, diseases, and cancers. While most earlier studies on the species have focused on the phytochemistry of the leaf and stem extracts, recent studies have indicated that its fruit may contain bioactive compounds of medical interest. In this study, we investigated the cytotoxicity of extracts from the fruit of P. minima against colorectal cancer cell lines and revealed its phytochemical profile via high-resolution tandem mass spectrometry analysis. Following a 24-h treatment with the fruit extract, cytoplasm shrinkage and nucleus condensation were observed in the colorectal cancer cell lines HCT116 and HT29, indicating the induction of programmed cell death. Phytochemically, 71 putative metabolites were identified. Some of these metabolites have been reported to inhibit cancers to varying degrees, further supporting the correlation of the putative metabolites with the cytotoxicity against colorectal cancer cells demonstrated in this study.

GOLPH3 inhibits erastin-induced ferroptosis in colorectal cancer cells.

Ferroptosis is a novel form of programmed cell death and is considered to be a druggable target for colorectal cancer (CRC) therapy. However, the role of ferroptosis in CRC and its underlying mechanism are not fully understood. In the present study we found that a protein enriched in the Golgi apparatus, Golgi phosphoprotein 3 (GOLPH3), was overexpressed in human CRC tissue and in several CRC cell lines. The expression of GOLPH3 was significantly correlated with the expression of ferroptosis-related genes in CRC. The overexpression of GOLPH3 in Erastin-induced Caco-2 CRC cells reduced ferroptotic phenotypes, whereas the knockdown of GOLPH3 potentiated ferroptosis in HT-29 CRC cells. GOLPH3 induced the expression of prohibitin-1 (PHB1) and prohibitin-2 (PHB2), which also inhibited ferroptosis in Erastin-treated CRC cells. Moreover, GOLPH3 interacted with PHB2 and nuclear factor erythroid 2-related factor 2 (NRF2) in Caco-2 cells. These observations indicate that GOLPH3 is a negative regulator of ferroptosis in CRC cells. GOLPH3 protects these cells from ferroptosis by inducing the expression of PHB1 and PHB2, and by interacting with PHB2 and NRF2.

Formulation, development and evaluation of hyaluronic acid-conjugated liposomal nanoparticles loaded with regorafenib and curcumin and their in vitro evaluation on colorectal cancer cell lines

Colorectal cancer is one of the major causes of global cancer, with chemotherapy and radiation therapy being effective but limited due to low specificity. Regorafenib, a multikinase inhibitor, provides hope to patients with metastatic colorectal cancer and was approved by the FDA in 2012. However, due to resistance issues and adverse events, its efficacy is compromised, necessitating further refinement. Meanwhile, curcumin, a compound of turmeric, exhibits anticancer effects through antioxidant and anti-inflammatory actions, induction of the apoptosis, arrest of cell cycle, inhibition of angiogenesis, and modulation of signaling pathways. Unfortunately, its clinical utility is limited by its poor bioavailability, pointing towards innovative drug delivery strategies for enhanced efficacy in colorectal cancer treatment.Hyaluronic acid (HA)-decorated liposomes (LIPO) have been developed to target colorectal cells through an overexpressed CD44 receptor, increasing antitumor and antimetastasis efficacy. This study investigates the possibility of loading curcumin (CUR) or regorafenib (REGO) into a liposomal formulation for passive and HA-actively targeted treatment, evaluating its critical quality attributes (CQA) (size, zeta potential, polydispersity index) and cytotoxic activity in the HT29 colorectal cancer cell line. The average particle size of the plain liposomes and those decorated with HA was 144.00 ± 0.78 nm and 140.77 ± 1.64 nm, respectively. In contrast, curcumin-loaded plain liposomes and HA-decorated liposomes had 140 ± 2.46 nm and 164.53 ± 15.13 nm, respectively. The prepared liposomes had a spherical shape with a narrow size distribution and an acceptable zeta potential of less than −30 mV. The encapsulation efficiency was 99.2 % ± 0.3 and 99.9 ± 0.2 % for HA-decorated and bare regorafenib loaded. The % EE was 98.9 ± 0.2 % and 97.5 ± 0.2 % for bare liposomal nanoparticles loaded with curcumin and coated with curcumin. The IC50 of free REGO, CUR, REGO-LIPO, CUR-LIPO, REGO-LIPO-HA and CUR-LIPO-HA were 20.17 ± 0.78, 64.4 ± 0.33, 224.8 ± 0.06, 49.66 ± 0.22, 73.66 ± 0.6, and 27.86 ± 0.49 µM, respectively. The MTT assay in HT29 cells showed significant cytotoxic activity of the HA-decorated liposomal formulation compared to the base uncoated formulation, indicating that hyaluronic acid-targeted liposomes loaded with regorafenib or curcumin could be a promising targeted formulation against colorectal cancer cells.

Momordica cochinchinensis (Gac) Aril Suppresses Proliferation and Induces Apoptosis of Colorectal Cancer Cells.

Gac aril contains high level of carotenoids. This carotenoid possesses several pharmacological properties including antioxidant, anti-inflammatory, and anti-tumor activities. To investigate the anti-cancer activity of Gac aril extract on human colorectal cancer cells and its related mechanisms. Colorectal cancer cell lines HCT116 and HT29 were treated with Gac aril extract and its effects on cytotoxicity and anti-proliferation were analyzed using the MTT/MTS and colony formation assay, respectively. Then, further related mechanisms responsible for anti-proliferation were investigated by cell death detection ELISA and Flow cytometry. The results showed that treated cells became rounded up and there was a loss of contact with neighboring cells, leading to a reduction of cell viability. The cytotoxic effects were evaluated IC50 for HCT116 and HT29 cells were 2.16 mg/mL and 1.29 mg/mL, respectively but it not toxic to normal HEK293 at the same dose. Moreover, Gac aril extract significantly inhibits proliferative ability with increasing concentrations having a greater effect. Subsequently, the cellular mechanism responsible for suppressive proliferation was validated. It shows apoptosis induction and arrest of cell cycle. Our findings demonstrated that Gac aril extract can induce apoptosis and arrest of cell cycle at S and G2/M phases in both HCT116 and HT29 colorectal cancer cells.

Neutrophil-to-lymphocyte ratio in colorectal tissue affects prognosis in patients with colorectal cancer

The objective of this investigation was to analyse the correlation between the neutrophil-to-lymphocyte ratio (NLR) status in the immune microenvironment (IME) and the prognostic outcomes of patients who have undergone radical surgery for colorectal cancer (CRC). In light of the continued prevalence of CRC in China, this study utilised Kaplan-Meier and Cox regression analyses to assess the prognostic relevance of NLR status in IME among patients with CRC. Furthermore, cellular experiments, such as cell scratching, were conducted to elucidate the underlying mechanisms of NLR's impact on CRC. The NLR status in IME has been found to have a significant impact on the prognosis of patients with CRC. Patients who exhibit elevated intratumoural and extratumoural NLR are associated with a poor prognosis. Experimental evidence indicates that tumour-associated neutrophil (TAN) augments the migratory, invasive, and proliferative potential of HT-29, HCT-116 and LOVO colorectal cancer cells, while concurrently reducing their sensitivity to oxaliplatin. Conversely, lymphocytes have demonstrated cytotoxic effects on HT-29 cells. The NLR status in IME may serve as a prognostic biomarker for resectable CRC.

Bioactive compounds and bioactivity of the heads of wild artichokes

Wild artichokes (Cyanara algarbiensis, C. baetica, C. cardunculus, and C. humilis) heads are globally consumed, although some taxa remain understudied, constituting natural reservoirs of bioactive phytochemicals. This study was focused on four Cyanara taxa collected in diverse locations in the SE of the Iberian Peninsula in comparison with cultivated C. scolymus. Analyses of vitamin C was effected by HPLC-DAD, and the methanol:water (80:20, v/v) extracts were analyzed for: i) phenolic compounds by HPLC-DAD and LC-MS; ii) phenolic acids and flavonoid using the Folin-Ciocalteu and aluminum chloride methods; iii) the antioxidant activity through the DPPH and ABTS•+ methods; iv) the in vitro antiproliferative activity against HT-29 cells using the MTT assay; and v) caspase-3 activity, which was performed to verify induced apoptosis. Vitamin C highlighted in C. humilis (59.9 mg/100 g fw), and total phenolics (by chromatography quantitation) reached 645.9 mg/100 g fw in C. baetica. The antioxidant activity stood out in C. baetica, with 0.50 (DPPH) and 0.52 (ABTS•+) mmol TE/100 g fw. Chlorogenic acid, apigenin-7-O-glucoronide, and apigenin-7-O-rutinoside highlighted in C. baetica (159.5, 274.5, and 50.3 mg/100 g fw); and quercetin-3-O-glucoside and 1,5-di-O-caffeoylquinic acid stood out in C. algarbiensis (61.5 and 93.5 mg/100 g fw). Artichoke extracts inhibited HT-29 colorectal cancer cells proliferation, particularly the ones from C. algarbiensis and C. baetica (GI50 of 975 and 650 μg/mL for 72-h exposed cells). This work constitutes a whole approach to the knowledge of phytochemicals and bioactivities of wild artichoke taxa to discern their use as functional foods.

Specific photodamage on HT-29 cancer cells leads to endolysosomal failure and autophagy blockage by cathepsin depletion

Endolysosomes perform a wide range of cellular functions, including nutrient sensing, macromolecule digestion and recycling, as well as plasma membrane repair. Because of their high activity in cancerous cells, endolysosomes are attractive targets for the development of novel cancer treatments. Light-activated compounds termed photosensitizers (PS) can catalyze the oxidation of specific biomolecules and intracellular organelles. To selectively damage endosomes and lysosomes, HT-29 colorectal cancer cells were incubated with nanomolar concentrations of meso-tetraphenylporphine disulfonate (TPPS2a), an amphiphilic PS taken up via endocytosis and activated by green light (522 nm, 2.1 J.cm−1). Several cellular responses were characterized by a combination of immunofluorescence and immunoblotting assays. We showed that TPPS2a photosensitization blocked autophagic flux without extensive endolysosomal membrane rupture. Nevertheless, there was a severe functional failure of endolysosomes due to a decrease in CTSD (cathepsin D, 55%) and CTSB (cathepsin B, 52%) maturation. PSAP (prosaposin) processing (into saposins) was also considerably impaired, a fact that could be detrimental to glycosphingolipid homeostasis. Therefore, photosensitization of HT-29 cells previously incubated with a low concentration of TPPS2a promotes endolysosomal dysfunction, an effect that can be used to improve cancer therapies.

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.

Ultrasound-assisted extraction of quinoa seed malt extract for the determination of nutritional content, antioxidant, and anticancer activity against HT-29 colorectal cancer cell lines

Ultrasound-assisted extraction of quinoa seed malt extract for the determination of nutritional content, antioxidant, and anticancer activity against HT-29 colorectal cancer cell lines

In Vitro Assessment of the Anti-Proliferative and Anti-Viability Effects of Salivary Gland Extracts from Hyalomma ticks (Acari: Ixodidae) on Human Colorectal Cancer Cells.

The saliva and salivary glands of ticks possess a wide range of immuno-pharmacologically active molecules that effectively modulate the activity of enzymes, antibodies, and amines that have a role in different biological processes. Derived components from saliva and salivary glands of hard ticks Ixodidae have been characterized as potential natural sources for discovering promising anti-cancer drug candidates. The anti-cancer activity of salivary gland extracts (SGEs) from Hyalomma anatolicum, Hyalomma dromedarii, Hyalomma marginatum, and Hyalomma schulzei was assessed. MTT assays and flow cytometry were done on the HT-29 colorectal cancer cell line to evaluate the anti-viability and proliferative inhibition. Based on the MTT assay results, the SGEs from Hy. dromedarii had the highest and lowest substantial anti-viability effects on the HT-29 cancer cell and human foreskin fibroblast (HFF) normal cell, respectively. The cytometric assessment revealed a significant increase in the apoptosis and necrosis ratio of the HT-29 cancer cells after treatment with Hy. dromedarii SGEs. The results demonstrated that Hy. dromedarii SGEs have significant anti-proliferative, anti-viability, and apoptotic potential. The result of this study suggests that Hy. dromedarii SGEs is an appropriate candidate for further investigations to identify and purify the mechanisms and molecules involved in the anti-cancer activity of the SGEs.

Sulforaphane Inhibits IL-1β-Induced IL-6 by Suppressing ROS Production, AP-1, and STAT3 in Colorectal Cancer HT-29 Cells.

Colorectal cancer (CRC) stands as a major cause of cancer-related mortality globally, accounting for approximately 881,000 deaths each year. Traditional approaches such as chemotherapy and surgery have been the primary treatment modalities, yet the outcomes for patients with metastatic CRC are often unsatisfactory. Recent research has focused on targeting the pathways involved in oxidative stress, inflammation, and metastasis to enhance the survival of CRC patients. Within this context, sulforaphane (SFN), a notable phytochemical found predominantly in cruciferous vegetables, has been recognized as a potential anticancer agent. However, the specific mechanisms through which SFN may exert its chemopreventive effects in CRC remain unclear. This study explores the impact of SFN on IL-1β-induced IL-6 activation and MAPK and AP-1 signaling in HT-29 cells. Our findings reveal that SFN treatment not only diminishes IL-1β-stimulated IL-6 expression but also reduces oxidative stress by curtailing reactive oxygen species (ROS) production. Furthermore, it hinders the proliferation and invasiveness of HT-29 cells through the modulation of MAPK/AP-1 and STAT3 signaling pathways. These results indicate that SFN mitigates IL-1β-induced IL-6 expression in CRC cells by attenuating ROS production and disrupting MAPK/AP-1 signaling. This suggests that SFN holds significant potential as a chemotherapeutic agent for both treating and preventing CRC.

Abstract 3031: Overcoming drug resistance in cancer: A paradigm shift with FoxM1 targeting

Abstract In cancer treatment, overcoming drug resistance remains a formidable challenge. The oncogenic transcription factor FoxM1 is widely recognized for its crucial role in promoting drug resistance to various chemotherapeutic and target therapies. Its involvement is attributed to pivotal pathways, encompassing cell cycle progression, angiogenesis, and metastasis. Despite its prominent role in tumor drug resistance, achieving modulation of FoxM1 using small molecule therapeutics has proven elusive. 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent, is known to induce a significant upregulation of FoxM1. In this study, we demonstrate that our novel compound A3 leads to a robust dose-dependent reduction of FoxM1 driven by 5-FU in HT-29 colorectal cancer cells. Notably, exposure of 5-FU-treated HT-29 cells to A3 was shown to promote apoptosis via pro-apoptotic factors, including PUMA. The mechanism of action was further elucidated through a comprehensive analysis of cell signaling molecules. This study presents an innovative approach using small molecule modulators to control FoxM1 levels, providing profound insights for potential therapeutic interventions in cancer treatment. Citation Format: Martin Pettersson, Su Jung Bae, Kyu-kwang Cho, Hye-yeoung Yun, Hee-Sung Park. Overcoming drug resistance in cancer: A paradigm shift with FoxM1 targeting [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 3031.

Abstract 6015: Effect of antineoplastic drugs on cell-to-cell communication in the progression of colorectal cancer

Abstract Background: In the past few years, there has been notable progress in colorectal cancer research, resulting in novel development and delivery of drugs specifically designed for individual patient requirements. However, one of the major roadblocks in designing and treating colorectal cancer is the drug resistance found in advanced stages. Thus, there is a profound need to establish new methods to overcome this barrier. Previous work illustrated that cell-to-cell communications are dysfunctional for multitude of reasons in colorectal cancer cells. Restoration of cell-to-cell communication is a mean to deliver antineoplastic drugs via bystanding effect. Prior research has demonstrated that restoring gap junctions of cell-to-cell communication in breast cancer cells enhances the efficacy of cisplatin, a drug previously deemed resistant. Hence, this study seeks to assess the potency and efficacy of antineoplastic agents known to show resistance in colorectal cancer, with the intention to investigate how gap junctions can increase the efficacy of antineoplastic agents via restoration of cell-to-cell communication. Method: We assessed cell viability utilizing the RealTime Glo MT Cell Viability assay (Promega, Inc.). Four specific colorectal cancer cell lines, Caco2, HT-29, SW620, and SW480, were used. Four antineoplastic agents, doxorubicin, cediranib, nilotinib, and erlotinib, were selected for the study and subsequently in the combination study of gap junction enhancer. The lethal dose at 50% (LD50) was calculated for each antineoplastic agent in each cell line. Results: The results show significant variation of LD50. HT-29 cells dosed with doxorubicin showed LD50 of 8.9 µM, 1.6 µM, and 0.5 µM at 24 hours, 36 hours, and 48 hours of exposure, respectively. HT-29 cells dosed with erlotinib showed LD50 of 15.8 µM and 8.9 µM at 48 hours and 60 hours of exposure, respectively. SW620 dosed with doxorubicin showed LD50 is 0.5 µM at 36 hours of exposure. SW620 dosed with erlotinib showed LD50 of 15.8 µM and 0.5 µM at 48 hours and 60 hours of exposure, respectively. Caco2 cells dosed with cediranib showed LD50 of 2.8 µM and 1.6 µM at 36 hours and 48 hours of exposure, respectively. Conclusion: The findings from this study revealed varying LD50 values for these cell lines when treated with the four distinct drugs. This enabled us to determine a foundational effect of these drugs on these cell lines, which had not been previously identified. Discovering this foundational effect will allow us to investigate the synergy effect when combined with gap junction enhancer by setting a baseline effect using these results. Citation Format: Tomas Lugo, Annelise Nguyen. Effect of antineoplastic drugs on cell-to-cell communication in the progression of colorectal cancer [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 6015.

Abstract 5452: AXIN2 differentially modulates stem cell subpopulations providing a potential therapeutic opportunity for targeting colorectal cancer stem cells

Abstract Constitutively activated WNT-signaling promotes stem cell (SC) overpopulation which drives most colorectal cancer (CRC) development. It is not known, however, how WNT-signaling activity regulates different SC sub-populations. We previously reported that SC population size decreases as AXIN2 is knocked down, and increases when AXIN2 is overexpressed. Changes in cell cycle and apoptotic rate were also observed. Here, we present our recent results on changes between different SC subpopulations (ALDH+ vs. LGR5+) that occur due to AXIN2 modulation. We hypothesize that modulation of AXIN2 promotes differential expansion between different CRC SC subpopulations (ALDH+ and LGR5+). We observed that AXIN2 not only increases LGR5+ SC population size, but also expands the ALDH+ SC population to a greater extent as shown by an increased ALDH+-to-LGR5+ SCs ratio. This indicates that AXIN2, via WNT signaling, regulates SC population size, particularly ALDH+ SCs. Since the ALDH SC marker is also a key component in the retinoic acid (RA) signaling pathway, we surmised that increased AXIN2 would not only lead to elevated ALDH levels, but also upregulate the RA-signaling pathway components in CRC cells. To this end, we conducted NanoString mRNA profiling to study the effects of AXIN2 knockdown and overexpression in HT29 and SW480 CRC cells. The results showed that increased AXIN2 significantly up-regulated several key components of the RA-signaling pathway. Taken together, our results indicate that AXIN2 drives a shift in SC subpopulation sizes favoring the ALDH+ SCs. It also promotes the RA-signaling component expression - a pathway known to regulate SC differentiation. As a potential treatment strategy for CRC, therapeutically inducing high levels of AXIN2 could shift the balance between SC populations towards ALDH+ SCs, which may provide a way to target cancer SCs with retinoids to induce their differentiation. Citation Format: Chi Zhang, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. AXIN2 differentially modulates stem cell subpopulations providing a potential therapeutic opportunity for targeting colorectal cancer stem 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 5452.

Abstract 6773: Tissue engineered colorectal cancer microspheres for drug screening

Abstract Three-dimensional (3D) disease models have garnered widespread interest for use in later stages of the drug discovery process, such as preclinical efficacy and toxicology studies, due to their pathophysiologically relevant properties. However, there is a need and opportunity for 3D cancer models to be used earlier in the drug screening process. To meet this need, the 3D models must strike a balance between throughput, which includes scalability and uniformity, and physiological relevance, such as the ability to modulate key attribute of the tumor microenvironment. Here we report the creation of 3D colorectal cancer (CRC) tissue models, referred to as VivoSpheres, and demonstrated their relevance to cancer drug screening. The VivoSphere production platform couples tissue engineering toolkits with microfluidics, enabling the scalable production of engineered cancer microspheres. The model supports the long-term maintenance of the cancer cell phenotype. In a preliminary study, we were able to generate more physiologically relevant drug responses. We formed CRC VivoSpheres by encapsulating HT-29 CRC cells within poly(ethylene glycol)-fibrinogen hydrogel microspheres using our previously developed microfluidic platform. CRC VivoSpheres were rapidly produced with high cell densities (20 × 106 cells/ml) and high uniformity on day 0 with a coefficient of variation (COV) < 7%. This high uniformity was maintained for 15 days (COV ≤ 10%), which is critical for long-term dose studies. The cells maintained high viability and showed high proliferative capability with a significant increase in colony size and expression of Ki67 up to day 29. The encapsulated cells maintained the CRC phenotype over time with the expression of CD44 (cancer stem cell marker) and CK20 (CRC marker). After establishing shipping conditions that maintained cell viability for remote use, the HT-29 VivoSpheres were shipped to the oncology team at Southern Research for drug testing. The CRC VivoSpheres were treated with DMSO, GANT61, and SRI-38832, the latter two of which are GLI1 inhibitors. Phase contrast images and western blot were used to assess the response of CRC VivoSpheres to the treatments. Oncogenic GLI1 transcription activity and NBS1 overexpression have been found to contribute to chemotherapeutic resistance, negating the anti-tumor effects of 5-fluorouracil. While 2D cultured HT-29s responded to treatment with GANT61, HT-29 VivoSpheres continued to express NBS1 following GANT1 treatment, but downregulated NBS1 in response to the GLI1 inhibitor SRI-38832, which is the same response Southern Research has seen in in vivo tumor models. In conclusion, we have developed tissue-engineered 3D CRC models that hold promise for use in drug screening. These models have demonstrated an initial capability to reproduce the CRC phenotype and mimic in vivo drug response. Citation Format: Yuan Tian, Mohammadjafar Hashemi, Benjamin Anbiah, Ruowen Zhang, Iman Hassani, Rebecca Boohaker, Elizabeth Lipke. Tissue engineered colorectal cancer microspheres for drug screening [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 6773.

Diterpenoids from Scutellaria barbata D. Don Exert the Antiproliferative Effect on Colorectal Cancer Cells Via Hedgehog Signaling Pathway

Background: Scutellaria barbata D. Don, also known as “Banzhilian” in China, the diterpenes of Banzhilian (Bzl-D) is a commonly used herbal medicine for treating various tumors including colorectal cancer (CRC). Objectives: This study aimed to examine the function of Bzl-D in inhibiting CRC development and explore its potential targets and mechanisms involving the Hedgehog (HH) pathway. Materials and methods: To determine the major components in Bzl-D, we performed ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS) analysis. The proliferation, migration, invasion, and apoptosis abilities of the CRC HT-29 cell line were examined, and the related protein expression was tested. Tumorigenesis after treatment was detected by in vivo experiments. Results: Nineteen diterpenoids were detected, Bzl-D significantly inhibited the cell proliferation and metastasis and induced the apoptosis of HT-29 cells through binding to Sonic HH (SHH) and down-regulating the key proteins (SHH, patched, smoothened, and glioma-associated oncogene) of the HH pathway, thus. Of note, Bzl-D remarkably suppressed the growth of HT-29 xenografts in nude mice. Conclusion: This research demonstrates that Bzl-D is a promising anti-rectal cancer drug, providing a new entry point for targeted therapy of CRC.

Phenolic Profiles and Antitumor Activity against Colorectal Cancer Cells of Seeds from Selected Ribes Taxa

Seeds from several Ribes taxa were surveyed for phenolic compounds and in vitro antiproliferative activity against HT-29 colorectal cancer cells. Total phenolic compounds were analyzed through the Folin–Ciocalteu procedure, while LC coupled to a single mass spectrometer (MS) Orbitrap using an electrospray interface (ESI) was performed to determine the phenolic profiles. Antitumor effects were established using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Total phenolics ranged from 11.4 in R. alpinum to 94.8 mg of caffeic acid equivalents (CAE)/g in R. nigrum ‘Koksa’. Concerning phenolic compounds, four were hydroxylated benzoic acids, four cinnamic acid derivatives, eight flavonoids, and nine flavonoid glycosides. The growth inhibition against HT-29 cancer cells was exercised much better by R. nigrum ‘Koksa’ and Ribes ‘Erkeeni’ (GI50 37 and 42 µg/mL). All Ribes extracts, except for R. nigrum ‘Hara katarlik’, showed higher activity than R. rubrum (GI50 at 72 h: 99 µg/mL). Interestingly, the extract from Ribes ‘Erkeeni’, which exhibited high bioactivity, contains all detected phenolic compounds, unlike R. nigrum ‘Koksa’, which lacks only populnin. Therefore, the high bioactivity found for such extracts could be due to a synergy of all detected compounds. This work constitutes a comprehensive action for expanding knowledge on the phenolic profiles and antitumor activity of GLA-rich Ribes seeds.

Broccoli extracellular vesicles enhance the therapeutic effects and restore the chemosensitivity of 5-fluorouracil on colon cancer

Broccoli contains an amount of biologically active substances, which bring beneficial effects on human health. Plant extracellular vesicles have been shown to be novel key factors in cancer diagnosis and tumor therapy. To date, the challenge of overcoming chemoresistance to 5-fluorouracil (5-FU) to facilitate the clinical management of colorectal cancer (CRC) has not been successful. Nevertheless, the functions of broccoli extracellular vesicles (BEVs) in the progression of CRC and 5-FU resistance are predominantly unclear. Herein, we showed that BEVs isolated from broccoli juice were effectively taken up by colorectal cancer HT-29 cells. The co-administration of BEVs and 5-FU significantly inhibited the proliferation and migration of colorectal cancer HT-29 cells, effectively blocking cell cycle progression. Furthermore, the co-administration of BEVs and 5-FU induced apoptosis by stimulating ROS production and disrupting mitochondrial function. Importantly, we found that BEVs reversed 5-FU resistance in HT-29 cells by suppressing the abnormal activation of the PI3K/Akt/mTOR signaling pathway. Collectively, our findings represent a novel strategy for utilizing BEVs to improve the efficacy of colorectal cancer treatment and enhance 5-FU chemosensitivity.

Detailed survey of an in vitro intestinal epithelium model by single-cell transcriptomics

The co-culture of two adult human colorectal cancer cell lines, Caco-2 and HT29, on Transwell is commonly used as an invitro gut mimic, yet the translatability of insights from such a system to adult human physiological contexts is not fully characterized. Here, we used single-cell RNA sequencing on the co-culture to obtain a detailed survey of cell type heterogeneity in the system and conducted a holistic comparison with human physiology. We identified the intestinal stem cell-, transit amplifying-, enterocyte-, goblet cell-, and enteroendocrine-like cells in the system. In general, the co-culture was fetal intestine-like, with less variety of gene expression compared to the adult human gut. Transporters for major types of nutrients were found in the majority of the enterocytes-like cells in the system. TLR 4 was not expressed in the sample, indicating that the co-culture model is incapable of mimicking the innate immune aspect of the human epithelium.