Colorectal Cancer Cells Research Articles

PD-L1 promotes tumor metastasis by regulating the infiltration of FGFBP2(+)Tm cells in colorectal cancer

PD-L1 promotes tumor metastasis by regulating the infiltration of FGFBP2(+)Tm cells in colorectal cancer

4-(Pyrazolyl)benzenesulfonamide Ureas as Carbonic Anhydrases Inhibitors and Hypoxia-Mediated Chemo-Sensitizing Agents in Colorectal Cancer Cells.

Hypoxia in tumors contributes to chemotherapy resistance, worsened by acidosis driven by carbonic anhydrases (hCA IX and XII). Targeting these enzymes can mitigate acidosis, thus enhancing tumor sensitivity to cytotoxic drugs. Herein, novel 4-(pyrazolyl)benzenesulfonamide ureas (SH7a-t) were developed and evaluated for their inhibitory activity against hCA IX and XII. They showed promising results (hCA IX: KI = 15.9-67.6 nM, hCA XII: KI = 16.7-65.7 nM). Particularly, SH7s demonstrated outstanding activity (KIs = 15.9 nM for hCA IX and 55.2 nM for hCA XII) and minimal off-target kinase inhibition over a panel of 258 kinases. In NCI anticancer screening, SH7s exhibited broad-spectrum activity with an effective growth inhibition full panel GI50 (MG-MID) value of 3.5 μM and a subpanel GI50 (MG-MID) range of 2.4-6.3 μM. Furthermore, SH7s enhanced the efficacy of Taxol and 5-fluorouracil in cotreatment regimens under hypoxic conditions in HCT-116 colorectal cancer cells, indicating its potential as a promising anticancer agent.

Abstract C019: IL27R signaling regulates neutrophils maturation, activation and function in colorectal cancer-liver metastases

Abstract Colorectal cancer (CRC) is the third most common cancer and fourth cause of cancer death worldwide. The liver is the most common organ for CRC metastasis, which is a major cause of death for CRC patients. Therefore, a better understanding of mechanisms facilitating CRC liver metastasis is in urgent need. IL27 is a member of IL6/IL12 cytokines superfamily which have a critical role in tumor microenvironment to affect tumorigenesis and metastasis. IL27 is secreted by activated macrophages and dendritic cells, and IL27R is expressed in various immune cells, mediating IL27-dependent pro-inflammatory or anti-inflammatory immune responses. However, the role of myeloid IL27 signaling in CRC-liver metastasis remains unclear. To study the role of IL27R signaling in myeloid cells, we generated Il27ra fl/fl mice and crossed them with LysM cre deleter to obtain LysM cre+ Il27ra f/f mice. Using MC-38 CRC cells portal vein injection model, we found that myeloid IL27Ra deletion leads to increased CRC-liver metastasis accompanied by increase in neutrophils, particularly Ly6G+Cxcr2-CD101- immature neutrophils, in circulation as well as tumors in LysM cre+ Il27ra f/f mice. Single-cell sequencing of tumor-infiltrating immune cells revealed that lack of IL27Ra in myeloid cells promoted neutrophil recruitment and degranulation as well as reactive oxygen species (ROS) and reactive nitrogen species (RNS) production but inhibited neutrophil maturation and production of pro-inflammatory chemokines and cytokines such as CXCL10, CSF1 and TNF. Furthermore, IL27Ra deficient neutrophils displayed heightened inflammatory response when stimulated with LPS in vitro. Collectively, our work reveals novel role of myeloid IL27Ra signaling that inhibits metastatic liver colonization by promoting neutrophil maturation, restricting neutrophil infiltration and degranulation. Citation Format: Jiani Zhu, Zhengzheng Shi, Ekaterina Koltsova. IL27R signaling regulates neutrophils maturation, activation and function in colorectal cancer-liver metastases [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C019.

Antitumorigenic Effect of Combination Treatment with BRAF Inhibitor and Cisplatin in Colorectal Cancer In Vitro and In Vivo

AbstractIn colorectal cancer (CRC), BRAF inhibitor (BRAFi) monotherapy appears ineffective, while cisplatin treatment is associated with adverse effects, drug resistance, and reduced efficacy. Herein, a combinatorial approach is being explored to increase the likelihood of effectively killing colorectal cancer cells. The combined effect of BRAFi (PLX4720, Vemurafenib, Dabrafenib, Encorafenib) and cisplatin treatment is examined in BRAFV600E‐mutated (RKO, HT29, Colo‐205) and BRAFwt (Caco‐2) cell lines, as well as in mouse xenografts of RKO cells. Following cisplatin‐only treatment, all cell lines show accumulation within subG1 (apoptotic cells) and G2/M phases, as well as phosphorylation of ERK1/2 and H2AX. Following BRAFi‐only treatment, BRAFV600E‐mutated cells show accumulation within G0/G1 phase, reduced distribution in the S and G2/M phases, inhibition of ERK1/2 phosphorylation, and increased phosphorylation of H2AX. Combined BRAFi and cisplatin treatment synergistically decrease RKO cells viability, reduce phosphorylation of ERK1/2, and increase phosphorylation of H2AX. Importantly, in mouse xenografts of RKO cells, combined PLX4720 and cisplatin treatment show superior therapeutic potential than each monotherapy (P < 0.001). Taken together, in in vitro and in vivo preclinical models, BRAFi and cisplatin combined treatment has shown an improved antitumor effect, rendering it a potential anticancer treatment strategy for BRAF‐mutant colon cancer patients.

High mobility group A1 (HMGA1) promotes the tumorigenesis of colorectal cancer by increasing lipid synthesis.

Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to meet the high energy and biosynthetic demands required for their proliferation. High mobility group A1 (HMGA1) is a structural transcription factor and frequently overexpressed in human colorectal cancer (CRC). Here, we show that HMGA1 promotes CRC progression by driving lipid synthesis in a AOM/DSS-induced CRC mouse model. Using conditional knockout (Hmga1△IEC) and knock-in (Hmga1IEC-OE/+) mouse models, we demonstrate that HMGA1 enhances CRC cell proliferation and accelerates tumor development by upregulating fatty acid synthase (FASN). Mechanistically, HMGA1 increases the transcriptional activity of sterol regulatory element-binding protein 1 (SREBP1) on the FASN promoter, leading to increased lipid accumulation in intestinal epithelial cells. Moreover, a high-fat diet exacerbates CRC progression in Hmga1△IEC mice, while pharmacological inhibition of FASN by orlistat reduces tumor growth in Hmga1IEC-OE/+ mice. Our findings suggest that targeting lipid metabolism could offer a promising therapeutic strategy for CRC.

Impact and mechanism study of dioscin on biological characteristics of colorectal cancer cells

Impact and mechanism study of dioscin on biological characteristics of colorectal cancer cells

Abstract B022: Characterizing invasiveness in CCCR cells: Role of miR-100, miR-125b and EVs

Abstract Introduction: Extracellular vesicles have been found to be important regulators of intercellular communication between cancer cells. EV cargo varies greatly between different cell types, the composition of which provides important insights into the role of donor and recipient cells. Cetuximab Resistant Colorectal Cancer (CCCR) cells upregulate miR-100 and miR-125b and select them as cargo for their respective secreted EVs. These miRNAs are responsible for altered gene expression in the CCCR cells and can also be functionally transferred as part of EVs between donor and recipient cells. Bioinformatic analysis identified Cingulin (CGN) mRNA as one of the possible targets of miR-100 and miR-125b. The action of miRNAs leads to degradation of CGN mRNA ultimately decreasing cellular concentration of the functional protein. Absence of CGN has been associated with increased invasiveness and metastatic potential in the cancer cells. We carried out immunofluorescent imaging of CCCR cells which revealed downregulation of CGN in CCCR cells compared to the parental Cetuximab sensitive Colorectal Cancer (CC) cells. Similar effects were observed in recipient cells that obtained the miRNAs as part of EVs originating from CCCR donor cells. This project helps elucidate the roles of miR-100 and miR-125b in CCCR cells and how EVs facilitate their functional transfer between nearby cancer cells. Methods: CCCR cell lines were derived from parental CC cells in the lab of Dr. Robert Coffey by iterative selection of cetuximab resistance after growth in 3D. CRISPR/Cas9 technology was used to knockout miR-100 and miR-125b in CCCR cells. Transwell co-culture experiments were carried out to analyze EV transfer of miRNAs. Transfer was monitored by immunofluorescence using antibodies against CGN. Cells were stained and imaged as Z stacks under the 63x Immersion setting. ImageJ was used to analyze the stacks and CGN concentrations between the cells. For quantification of immunofluorescence, additional Z stack images were obtained from an inverted microscope and fluorescence digital camera. Images were analyzed using Fiji software to obtain specific CGN concentrations between the cells. Results and Discussion: Immunofluorescent imaging revealed statistically significant downregulation of CGN in CCCR cells. The protein was also downregulated in the recipient knockout cells as part of the Transwell co-culture experiment with CCCR donor cells. CGN is a tight junction protein and its absence confers increased invasiveness in 3D growth with enhanced metastasis. Decreased CGN concentration in the miR-100 and miR-125b enriched EV recipient knockout cells is indicative of functional transfer of the two miRNAs between cancer cells. These distinctive cellular dynamics could be harnessed to develop potential cancer therapies that provide a better prognosis and limit the metastatic potential of targeted cancer cells. Citation Format: Muhammad Shameer, Hannah M Nelson, Shimian Qu, Liyu Huang, Kevin C Corn, Sydney N Chapman, Nicole L Luthcke, Sara A Schuster, Tellie D Stamaris, Lauren A Turnbull, Lucas L Guy, Xiao Liu, Danielle L Michell, Elizabeth M Semler, Kasey C Vickers, Qi Liu, Jeffrey L Franklin, Alissa M Weaver, Marjan Rafat, Robert J Coffey, James G Patton. Characterizing invasiveness in CCCR cells: Role of miR-100, miR-125b and EVs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B022.

Exploring the Molecular Basis of Anticancer Activity in Various Moss Species Against Colorectal Cancer Cells

Background: Mushrooms are shown to protect against the side effects of cancer. Therefore, mushrooms with proven anticancer properties and active ingredients are fascinating in the search for new cancer drugs. Objective: In this study, the effects of extracts from Hericium coralloides (M1), Lactarius deliciosus (M2), Lepista nuda (M3), Pleurotus ostreatus (M4), and Suillus collitinus (M5) together on HCT116 were investigated. Mesenchymal stem cells (MSCs) were used to study the effect on healthy cells. Methods: MTT was used to determine cell viability. Dose-response curves were generated, the IC50 values of the compounds were calculated, and the effect of the extracts was compared using it. The FTIR was used to analyze the quantitative changes of the cellular components. Results and Discussion: The evaluation of the IC50 values of all fungal species showed that they reduced the cell viability of HCT116 cells. In contrast, no significant reduction in cell viability was observed in MSCs. Changes in the ratio of cell membrane lipids, proteins, and cell nucleic acids between control and fungal-treated HCT116 cells were detected by FTIR. Many of the chemotherapeutic agents are of plant origin, and many resources should still be explored to inhibit the side effects of cancer therapy. Conclusion: The data obtained through this experiment will serve as a reference for studies to be a new source of anticancer drugs in modern pharmacology.

Abstract B020: The Ratio of Alternatively Spliced KRAS Isoforms Plays a Role in The Tumorigenesis of Colorectal Cancer

Abstract One of the most frequently mutated oncogenes, KRAS, has two alternatively spliced isoforms, KRAS4A(4A) and KRAS4B(4B). The isoforms differ by the inclusion or exclusion of the fourth exon, resulting in 4A or 4B respectively. They undergo different post-transcriptional modifications, indicating the non-redundant nature of the two isoforms. Studies also show spatiotemporal regulation of 4A in the gastrointestinal tract of mice during development. Although 4B is considered the major isoform, the ratio of 4A to 4B is cell-type specific. We show 4A is more abundant in colorectal cancer (CRC) cells like CaCO2 and HCT116, compared to other cancer cell lines like HeLa cells and MDA-MB-468 cells. Differential splicing analysis on existing data from flow-sorted mouse colon-crypt cells (stem-like vs. differentiated) and murine intestinal organoids of colon cancer (WT vs. tumor) shows that while differentiated colon cells and WT organoids have decreased levels of 4A, stem-like cells and tumor cells show increased levels of 4A. This supports the stem-cell theory of cancer, indicating that the dysregulation of KRAS isoforms impacts colon cell differentiation in colon crypts and could lead to CRC. Interestingly, we also found that the 3’ splice site(3’SS) of exon 4A is highly conserved. RBM39, a 3’SS recognition homolog, and SRSF1 have also been shown to play a role in KRAS splicing. We hypothesize that RBM39 regulates KRAS splicing by binding the 3’SS, and SRSF1 may play a role either by binding the pre-mRNA directly or through its interaction with RBM39. Therefore, we propose a new model for alternative splicing of KRAS. The abundance of KRAS splice variants may determine the fate of colon cell differentiation and drive CRC tumorigenesis. Citation Format: Harika Reddy Pulipelli, Dr. Klemens J. Hertel. The Ratio of Alternatively Spliced KRAS Isoforms Plays a Role in The Tumorigenesis of Colorectal Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B020.

Development of an optimized protocol for generating knockout cancer cell lines using the CRISPR/Cas9 system, with emphasis on transient transfection.

The clustered regularly interspaced short palindromic repeats (CRISPR) system offers cost-effectiveness, high efficiency, precision, and ease of use compared to traditional gene editing techniques. In this study, we employed findings from prestigious investigations to develop an optimized approach for generating knockout cancer cell lines using a transient transfection method. This protocol introduces a distinctive approach that follows rigorous guidelines for designing gRNA to reduce off-target effects, a major challenge in CRISPR applications. Our step-by-step instructions allow researchers, particularly those with limited laboratory equipment and funding, as well as those undertaking CRISPR projects for the first time, to generate knockout cell lines using CRISPR technology in just ten weeks. This protocol covers all needs for enhancing various yields, such as transfection efficiency, and includes leveraging robust bioinformatics tools, conducting essential assays, isolating monoclonal cells via limiting dilution, validating knockout cells, and providing comprehensive troubleshooting recommendations. Using this method, we successfully created several new generations of colorectal cancer cell lines with monoallelic and biallelic knockouts of the epithelial cell adhesion molecule (EpCAM) gene. Our method, optimized for a wide spectrum of cancer cell lines, makes CRISPR more accessible for applications in personalized and precision medicine. It expands opportunities for novel investigations into cancer mechanisms and paves the way for potential therapeutic interventions.

Discovery of Novel 5-Cyano-3-phenylindole-Based LSD1/HDAC Dual Inhibitors for Colorectal Cancer Treatment.

The dual inhibition of histone lysine-specific demethylase 1 (LSD1) and histone deacetylase (HDAC) has emerged as a promising strategy for cancer therapy. In this study, we report the discovery of novel 5-cyano-3-phenylindole-based LSD1/HDAC dual inhibitors, evaluated through both in vitro and in vivo assays. Among these inhibitors, compound 20c was identified as particularly potent, exhibiting high inhibitory activity against LSD1 (IC50 = 39.0 nM) and HDAC1/2/3/6/8 (IC50 = 1.4, 1.0, 1.3, 2.9, and 16.0 nM, respectively). Compound 20c effectively modulated the expression of biomarkers associated with LSD1 and HDAC inhibition and demonstrated superior antiproliferative activity compared to SAHA and 4SC-202 across multiple colorectal cancer cell lines. Following pharmacokinetic studies, 20c was further assessed in HCT-116 and HT-29 xenograft mouse models. It demonstrated significantly enhanced antitumor efficacy compared to SAHA, without causing observable toxicity. These findings highlight the potential of LSD1/HDAC dual inhibitors for the treatment of malignant cancers.

Enhancing the anticancer effect of metformin through nanoencapsulation: Apoptotic induction, inflammatory reduction, and suppression of cell migration in colorectal cancer cells.

Colorectal cancer (CRC) continues to be a significant health challenge, necessitating the development of efficient therapeutic strategies. Drug repurposing, which involves the use of existing medications for new purposes, presents a promising opportunity. Metformin, a widely used antidiabetic drug, has demonstrated potential anticancer effects. To enhance its efficacy, we formulated nano-metformin, metformin encapsulated within pectin nanoparticles. Our study aimed to evaluate the superiority of nano-metformin over free metformin in treating CRC. The cytotoxicity of both metformin and nano-metformin on Caco-2 CRC cells was assessed using the MTT assay, revealing a significant dose-dependent inhibition of cell growth using nano-metformin. The anti-inflammatory potential was evaluated by measuring the levels of nitric oxide and the pro-inflammatory cytokines IL-2 and IL-6 following lipopolysaccharide (LPS) induction, and the results revealed that treating LPS-induced cells with nano-metformin significantly reduced the production of these inflammatory mediators. To elucidate the mechanism of cell death, we employed an acridine orange/ethidium bromide staining assay, which revealed the enhancement of apoptotic cell death following treatment with nano-metformin. Additionally, we examined the expression of key apoptotic regulators using real-time qPCR. Nano-metformin, in particular, significantly downregulated the expression of the antiapoptotic markers Bcl-2 and Survivin while upregulating the proapoptotic caspases 3, 7, and 9. The comet assay revealed significant DNA damage induced by treatment with the nano-metformin compared with that in the free form. Moreover, nano-metformin significantly reduced the migration ability of cells. In conclusion, our work revealed the superior efficacy of our formulated nanoform over free metformin, highlighting its potential as a promising therapeutic agent for CRC treatment.

Abstract A011: Identifying surface biomarkers from colorectal cancer-derived extracellular vesicles using a bead-based approach

Abstract Colorectal cancer (CRC) accounts for the second most common cause of cancer-related mortality globally. The search for prognostic biomarkers subserving to detected cancer at an early stage, to improve tumor classification and to stratify therapeutic patient management groups is actively ongoing. Surgical resections and chemotherapeutics remain a mainstay in the cure and control of solid tumors. Despite their usefulness in the clinical scenario, shortcomings are well recognized, leading researchers to investigate for a circulating source of tumor-derived materials in biofluids, including mostly circulating tumor DNA (ctDNA), circulating tumor cells (CTCs) and extracellular vesicles (EVs). Characterization of EVs surface markers and assessment of their molecular cargo, can lead to the identification of early metastatic colorectal cancer signatures. Cell surface markers of three CRC cell lines (HCT116, DLD1 and LOVO) were characterized using flow cytometry. EVs were isolated from 48-hour serum-deprived cell lines using the polymer pull down method (20% Polyethelene glycol (PEG)). The concentration and particle size distribution of isolated EVs was checked by the NanoSight NS300. Avidin-coated Polystyrene beads were conjugated with CD9 biotin antibodies; and were used to immunoprecipitated (IP) CD9+ EVs. Following a 2-hour incubation and a series of washing steps, the PE-labelled antibodies of interest (CD9, CD63, EpCAM, Vim, CD44, CD133 and CD29) were added and read using xMAP technology. To increase the number of surface markers that can be read in tandem, IP was multiplexed using EpCAM, CD9 and CD63. This was followed by the isolation of EVs from plasma of metastatic CRC patients (N=3) using size exclusion chromatography (Izon qEV1 columns-35nm in conjunction with the automatic fraction collector) and polymer pull down. The concentration and size were assessed by Nanosight NS300 and surface markers of tumor-derived EVs were assessed using the immunoprecipitation technique on the Luminex system. Comparison of signals between EVs isolated from cell lines and matched originator cells showed that cell surface signatures in EVs match closely with their originator cells. The mean fluorescence intensities quantified cell surface marker expression in the different exosome-bead complexes. Interestingly, one of the metastatic cancer patients with invasive colorectal adenocarcinoma and lung metastasis showed EpCAM and Vimentin positivity suggesting epithelial-mesenchymal plasticity. These results show the importance of multiplexing both at the level of immunoprecipitation and at end-measurement, given the dynamics of cell surface marker changes during disease progression. The use of the mesenchymal marker; Vimentin in a panel for cell surface markers stems from studies on biotinylated cell surface markers on various cancer cell lines and identification of Vimentin in such models. Interestingly we find Vimentin on EVs surface in this study. Citation Format: Jessica Debattista, Laura Grech, Christian Scerri, Malcolm Buhagiar, Godfrey Grech. Identifying surface biomarkers from colorectal cancer-derived extracellular vesicles using a bead-based approach [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A011.

Fatty acid dehydratase HACD3 poses protein kinase activity and promotes the malignant progression of colorectal cancer.

HACD3 is a member belonging to the very long-chain fatty acid dehydratase family. However, little is known about HACD3's function besides its weak dehydratase activity. The current study aims to investigate the potential role of HACD3 in Colorectal Cancer (CRC). The study evaluates HACD3 expression in CRC patient samples. Moreover, the study investigates in vitro cell proliferation, colony formation, invasion, migration, and cell cycle progression on HACD3 knockdown and overexpressing CRC cells, along with in vivo tumorigenesis within NSG mice, Hacd3-/-; ApcMin/+ mice and Hacd3ΔIEC; ApcMin/+mice. The study identifies the molecular target(s) of HACD3 via phosphoproteomics, followed by biological assay verification. The study identifies that HACD3 is highly expressed in CRC tissues and promotes the malignant progression of cancer cells. Additionally, HACD3 interacts with CDK2, leading to CDK2 T160 phosphorylation via a domain between amino acids 298-324 of HACD3. Thus, fatty acid dehydratase HACD3 possesses protein kinase activity and stimulates tumorigenesis, partly by activating the CDK2 pathway. Therefore, inhibiting HACD3 could facilitate CRC treatment.

Fe3O4 nanoparticles containing gambogic acid inhibit metastasis in colorectal cancer via the RORB/EMILIN1 axis

ABSTRACT This research aims to study the effect of magnetic nanoparticles of Fe3O4 (MNP Fe3O4) containing gambogic acid (GA-MNP Fe3O4) on colorectal cancer (CRC). MNP Fe3O4 enhanced the antitumor effect of GA by inhibiting the malignant behavior of CRC cells. RORB was a target of GA, and GA activated RORB expression to inhibit metastasis of CRC. Knockdown of RORB impaired the effect of GA-MNP Fe3O4 on CRC metastasis. EMILIN1 was a target of RORB, and RORB promoted transcription of EMILIN1. Overexpression of EMILIN1 reversed the effect of knockdown of RORB on GA-MNP Fe3O4 and inhibited metastasis in CRC. These findings revealed that MNP Fe3O4 enhanced the antitumor effect of GA and activated RORB to promote EMILIN1 transcription and inhibit CRC metastasis.

Green Hydrothermal Synthesis of Gallic Acid Carbon Dots: Characterization and Cytotoxic Effects on Colorectal Cancer Cell Line.

Colorectal cancer remains a leading cause of cancer-related deaths worldwide, necessitating the development of novel therapeutic approaches. Carbon dots (CDs) have emerged as promising nanoparticles for biomedical applications due to their unique properties. Gallic acid (GA), a known anticancer agent, is effective against various tumor types. This study explores the potential of gallic acid-derived carbon dots (GA-CDs) as an innovative anticancer agent against HCT-116 colorectal cancer cells, focusing on apoptosis signaling pathways. GA-CDs were synthesized using a one-pot hydrothermal method. Characterization was conducted using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, and ultraviolet-visible (UV-Vis) absorption spectroscopy. The cytotoxicity of GA and GA-CDs on HCT-116 cells was evaluated using the MTT assay at various concentrations over 24 and 48 hours. Cellular uptake was assessed via fluorescence microscopy, and apoptosis was analyzed using acridine orange/propidium iodide (AO/PI) staining. Total RNA extraction followed by complementary DNA (cDNA) synthesis via RT-PCR was performed, and real time-PCR (Q-PCR) was conducted to examine the expression of apoptosis-related genes Caspase-3, Bax, and Bcl-2. Characterization confirmed the successful synthesis of spherical GA-CDs. GA-CDs exhibited dose- and time-dependent cytotoxicity, with IC50 values of 88.55 µg/mL for GA-CDs and 192.2 µg/mL for GA after 24 hours. Fluorescence microscopy confirmed the efficient uptake of GA-CDs by HCT-116 cells. AO/PI staining showed a significant increase in apoptotic cell numbers after treatment with GA-CDs. Q-PCR analysis revealed overexpression of Caspase-3 and Bax genes in GA-CD-treated cells, though no significant changes were observed in the expression of Bcl-2 or the Bax/Bcl-2 ratio. GA-CDs demonstrated potent anticancer properties by inducing apoptosis and reducing cell viability in HCT-116 cells. These findings suggest the potential of GA-CDs as a novel therapeutic agent for colorectal cancer treatment, warranting further investigation into their mechanism of action and in vivo efficacy.

Camptothecin and Its Derivatives from Traditional Chinese Medicine in Combination with Anticancer Therapy Regimens: A Systematic Review and Meta-Analysis

Background/Objectives: Camptothecin (CPT) and its derivatives, irinotecan and topotecan, are integral components of cancer chemotherapy, often used in combination therapies. This meta-analysis evaluates the efficacy of CPT-based combination treatments in cancer patients. Methods: We systematically searched the literature database using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses checklist for articles published between 2000 and 2022. Published studies were retrieved through an electronic search on the Web of Science, PubMed, and Google Scholar databases. A total of 138 studies were downloaded and examined, and 71 eligible studies were selected for meta-analysis after excluding studies that did not meet the inclusion criteria. Results: Ultimately, a total of 71 studies were included in the analysis, comprising non-small cell lung cancer (NSCLC), colorectal cancer (COLRC), oesophageal/gastric cancer (O/GC), and small cell lung cancer (SCLC). For NSCLC, the objective response rate (RR) was 31.8% (95% CI: 27.3–37.1%, p = 0.025), with irinotecan plus cisplatin showing significantly higher efficacy compared to other irinotecan-based combinations. In COLRC, irinotecan and 5-fluorouracil/leucovorin plus bevacizumab demonstrated superior efficacy with a RR of 44% (95% CI: 34–58, p < 0.001) and minimal haematological toxicity. In O/GC, irinotecan-based combinations showed an average RR of 43% (95% CI: 27–70, p < 0.001) and average overall survival (OS) and progression-free survival (PFS) rates of 10.2 and 5.5 months, respectively. For SCLC, irinotecan-based combinations had a higher control response than topotecan-based ones, while the latter exhibited higher rates of stable and progressive disease. The overall RR for SCLC was 45% (95% CI: 34.3–60.2, p < 0.001). Conclusions: The existing evidence underscored the potential of CPT-based combination therapy in various cancers. Among the various combinations discussed in this analysis, irinotecan plus cisplatin demonstrated the highest objective RR in 12 trials focused on NSCLC. This study provides valuable insights into potential treatment strategies for various types of cancer, emphasising the importance of personalised and tailored approaches to maximise efficacy and minimise adverse effects.

METTL3‑mediated N6‑methyladenosine modification of MMP9 mRNA promotes colorectal cancer proliferation and migration.

N6‑methyladenosine (m6A) is the predominant chemical modification of eukaryotic mRNA, dynamically mediated by the RNA methyltransferase, methyltransferase-like 3 (METTL3). m6A modification plays a critical role in cancer progression through post‑transcriptional regulation in various types of cancer. However, the role of METTL3 and its associated m6A modification in colorectal tumorigenesis remains to be fully elucidated. In the present study, it was demonstrated that METTL3 expression and the m6A levels were both upregulated in colorectal cancer (CRC) and positively associated with clinical progression, based on the bioinformatics analysis of cancer databases. Furthermore, knockdown and overexpression of METTL3 notably affected CRC cell viability, apoptosis and migration in vitro. Similarly, xenograft animal models confirmed that METTL3 promoted CRC tumorigenicity in vivo. Mechanistically, it was revealed that the m6A modification of matrix metallopeptidase 9 (MMP9) mRNA mediated by METTL3 promoted its expression in CRC by decreasing its degradation. Collectively, the findings of the present study suggested that the METTL3/MMP9 axis could serve as a novel promising therapeutic candidate for CRC.

Enrichment of cancer stem cell subpopulation alters the glycogene expression profile of colorectal cancer cells.

Colorectal cancer (CRC) has a high mortality rate, resulting from the processes of metastasis and disease recurrence. Cancer stem cells (CSCs) are believed to be crucial for both processes, as they ensure the maintenance of the tumor bulk, in addition to being intrinsically resistant to conventional therapies. Thus, the present study aimed to investigate glycobiomarkers in colorectal cancer stem cell subpopulations. For this purpose, a sphere formation assay was standardized for CACO-2 and HT-29 cell lines, which were monitored through gene expression analysis of five known CSC markers (CD24, CD44, ALDH1, LGR5, and PROM1). Compared to the parental condition (2D), a reduction in CD24 expression was seen in CACO-2, while in HT-29 an increase in the expression levels of ALDH1, LGR5, and PROM1 was observed. Regarding glycogenes, eight of them (ST3GAL1, OGT, OGA, MGAT5, GFAT1, GFAT2, B4GALT1 e B3GNT2) have had their expression monitored. An increase in B3GNT2, OGT, and OGA was observed in the HT-29 sphere condition. On the other hand, no change in the glycogenes expression was observed in CACO-2. In silico correlation analyses (CSCs markers versus glycogenes) using TCGA data from colon and rectum carcinoma samples showed a weak positive correlation between LGR5 vs OGA expression regardless of the sample location. In addition, an increase in the expression of LGR5, OGA, and OGT as well as a decrease in the expression of ALDH1 were observed in colon carcinoma samples when compared to the adjacent normal tissue. Interestingly, greater OGA expression resulted in both lower overall survival of colon carcinoma patients and lower disease-free survival of rectum carcinoma patients. Therefore, our data indicates that OGA expression correlates with CSC markers and directly impacts the survival of colorectal carcinoma patients.

High expression of ADAR mediated by OGT promotes chemoresistance in colorectal cancer through the A-to-I editing pathway.

Colorectal cancer (CRC) is a malignant tumor with poor prognosis and adverse therapeutic effect. The study aims to elucidate the contribution of OGT-mediated glycosylation of ADAR to chemoresistance in CRC through its role and regulatory mechanisms. Variations in OGT expression levels and their impact on CRC cell chemoresistance were investigated using gain-of-function and loss-of-function assays. Through a series of molecular biology experiments, we confirmed that ADAR is the downstream target of OGT regulation, emphasizing the role of OGT-mediated glycosylation in stabilizing ADAR. Furthermore, RNA immunoprecipitation (RIP) assays were conducted to examine the effects of ADAR-mediated A-to-I editing on the mRNA stability and translation of genes associated with DNA damage repair. Elevated OGT expression was found to enhance CRC's malignancy and resistance to chemotherapy. OGT's influence leads to the glycosylation of ADAR, thereby increasing its protein levels. ADAR, through its role in A-to-I editing, modulates the mRNA editing of genes implicated in DNA damage repair. This regulation enhances the expression of these genes, improves DNA repair capabilities, and ultimately, fosters chemoresistance in CRC cells. In conclusion, ADAR promotes PARP1 expression under the positive regulation of OGT-mediated O-glycosylation modification to enhance drug resistance in COAD cells. It provides the research basis for overcoming the drug resistance of CRC.