HT-29 Colorectal Cancer Cells Research Articles

Thymoquinone Radiosensitizes Human Colorectal Cancer Cells in 2D and 3D Culture Models

Simple SummaryRadiotherapy is a standard of care therapy that kills cancer cells but has limited efficacy in targeting the resistant cancer stem cells (CSCs). The use of radiosensitizers in experimental studies and clinical practice is a successful strategy for eradicating CSCs. Here, we investigated the radiosensitizing potential of thymoquinone (TQ), a natural compound with known anti-cancer activity, in 2D and 3D cultures of colorectal cancer, and in patient-derived organoids. We show that TQ sensitized cancer cells and stem/progenitor cells to radiation mainly through the inhibition of cell survival, DNA repair, and stemness in addition to regulating major pathways implicated in this process. Thus, TQ could be used as a sensitizer to effectively target and kill colorectal cancer cells.Resistance of cancer cells and normal tissue toxicity of ionizing radiation (IR) are known to limit the success of radiotherapy. There is growing interest in using IR with natural compounds to sensitize cancer cells and spare healthy tissues. Thymoquinone (TQ) was shown to radiosensitize several cancers, yet no studies have investigated its radiosensitizing effects on colorectal cancer (CRC). Here, we combined TQ with IR and determined its effects in two-dimensional (2D) and three-dimensional (3D) culture models derived from HCT116 and HT29 CRC cells, and in patient-derived organoids (PDOs). TQ sensitized CRC cells to IR and reduced cell viability and clonogenic survival and was non-toxic to non-tumorigenic intestinal cells. TQ sensitizing effects were associated with G2/M arrest and DNA damage as well as changes in key signaling molecules involved in this process. Combining a low dose of TQ (3 µM) with IR (2 Gy) inhibited sphere formation by 100% at generation 5 and this was associated with inhibition of stemness and DNA repair. These doses also led to ~1.4- to ~3.4-fold decrease in organoid forming ability of PDOs. Our findings show that combining TQ and IR could be a promising therapeutic strategy for eradicating CRC cells.

CLEISTANTHIN B SHOWS A POTENT CYTOTOXIC ACTIVITY AGAINST COLORECTAL CANCER CELLS

Objective: The aim was to find out the cleistanthin B sensitive cancer cell type among a panel of cancer cell lines. Methods: The 50% inhibitory concentrations (IC50) of cleistanthin B against different cancer cells were determined by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay. The cell death caused by cleistanthin B in colorectal cancer (CRC) cells was evaluated by acridine orange and ethidium bromide (AO-EB) dual staining. Using short exposure, we generated the 5-fluorouracil</AQ1> and oxaliplatin (5-FU+Ox) surviving cells from the parental HT-29 CRC cell lines. These surviving CRC cells were further treated with cleistanthin B either alone or combined with 5-FU. Annexin V apoptosis assay was used to determine the combined effect of cleistanthin B with 5-FU against HT-29 cells. Results: The IC50 values of cleistanthin B were found to be 3.6±0.55, 5.2±0.51, 8.6±1.02, 10.5±1.50, 18.3±3.71, 25.8±5.50, and 26.7±5.90 μg/mL against HT-29, SW-480, HCT-15, HELA, MDA-MB-231, A549, and DU145, respectively. The IC50 value of cleistanthin B against L132 cells was >100 μg/mL. The cleistanthin B treated HT-29, SW-480, and HCT-15 CRC cells showed apoptotic changes such as chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies in the AO-EB dual staining method. Flow cytometry analysis revealed that cleistanthin B enhances the 5-FU induced apoptosis against 5-FU+Ox surviving HT-29 CRC cells. Conclusion: Cleistanthin B is relatively more potent against CRC cells than other cancer cells, and it induces apoptosis mediated cell death in CRC cells. Cleistanthin B enhances the anticancer activity of 5-FU against HT-29 CRC cells.

Ternary Copper (II) Complex Induced Apoptosis and Cell Cycle Arrest in Colorectal Cancer Cells.

The lack of specificity, severe side effects, and development of drug resistance have largely limited the use of platinum-based compounds in cancer treatment. Therefore, copper complexes have emerged as potential alternatives to platinum-based compounds. Ternary copper (II) complex incorporated with 1-10-phenanthroline and L-tyrosine was investigated for its anti-cancer effects in HT-29 colorectal cancer cells. Cytotoxic effects of ternary copper (II) complex in HT-29 cells was evaluated using MTT assay, Real-Time Cell Analysis (RTCA) and lactate dehydrogenase (LDH) assay. Cell cycle analysis was performed using flow cytometry. Apoptosis induction was studied by Annexin V-FITC/Propidium Iodide (PI) staining and mitochondrial membrane potential analysis (JC-10 staining) using flow cytometry. Intracellular Reactive Oxygen Species (ROS) were detected by DCFH-DA assay. The expression of proteins involved in the apoptotic signalling pathway (p53, caspases, and PARP-1) was evaluated by western blot analysis. Ternary copper (II) complex reduced the cell viability of HT-29 cells in a time- and dose-dependent manner, with IC50 of 2.4 ± 0.4 and 0.8 ± 0.04 μM at 24 and 48 hours, respectively. Cell cycle analysis demonstrated induction of S-phase cell cycle arrest. Morphological evaluation and Annexin V-FITC/PI flow cytometry analysis confirmed induction of apoptosis that was further supported by cleavage and activation of caspase-8, caspase-9, caspase-3, and PARP- 1. Mutant p53 was also downregulated in a dose-dependent manner. No LDH release, mitochondrial membrane potential disruption, and ROS production were observed. Ternary copper (II) complex holds great potential to be developed for colorectal cancer treatment.

Effects of Platostoma palustre ethanolic extracts and commercial herbal tea on the cell viability of colorectal cancer cells

Platostoma palustre jelly is a traditional food. Platostoma palustre has been used as folk medicine and is effective against heat-shock, hypertension and diabetes. Therefore, the aim of this study was to determine the effects of ethanolic extracts and commercial herbal tea of Platostoma palustre in inhibiting colorectal cancer cell viability. The ethanolic extracts of Platostoma palustre by using 90% ethanol for extraction. In this study, 2-fold serial dilution of 100 mg/mL Platostoma palustre extracts were applied. On other hand, the same dilution fold was also performed for 100% commercial herbal tea with Platostoma palustre. Additionally, CT-26 and HT-29 colorectal cancer cell lines were also used in this study. After co-culturing for 24 hours, the cell viability of CT-26 and HT-29 colorectal cancer cell lines were performed by using 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. According to these data, the 1.56-100 mg/mL Platostoma palustre extracts possessed the significant inhibition effects of CT-26 colorectal cancer cell viability. The 3.13-100% commercial herbal tea with Platostoma palustre possessed the significant inhibition effects of CT-26 colorectal cancer cell viability. The 6.25-100 mg/mL Platostoma palustre extracts possessed the significant inhibition effects of HT-29 colorectal cancer cell viability. The 25-100% commercial herbal tea with Platostoma palustre possessed the significant inhibition effects of HT-29 colorectal cancer cell viability. However, the 0.39-3.13 mg/mL Platostoma palustre extracts possessed the significant promoting effects of HT-29 colorectal cancer cell viability. The 0.39-12.5% commercial herbal tea with Platostoma palustre also possessed the significant promoting effects of HT-29 colorectal cancer cell viability. Comparison of CT-26 and HT-29 cell lines was on the cell viability after Platostoma palustre ethanolic extracts and commercial herbal tea treatments, CT-26 cell line was better sensitive than HT-29 cell line on the inhibition of cell viability after treatment of Platostoma palustre ethanolic extracts and the commercial herbal tea. Taken these results together, Platostoma palustre ethanolic extracts and commercial herbal tea may have a potential for inhibiting the growth of colorectal cancer cells.

Cytochalasan Alkaloids as TRAIL Sensitizers from an Endophytic Fungus Chaetomium sp.

Two new cytochalasans with a rare 6/6/5/5/7 pentacyclic ring system, named chaetoconvosins C-D (1: -2: ), together with two known congeners (3: -4: ), were isolated from the fermentation of an endophytic fungus, Chaetomium sp. SG-01, harbored in the fibrous roots of Schisandra glaucescens Diels. Their structures including the absolute configuration were elucidated by extensive spectroscopic (HRESIMS, NMR, and ECD) and X-ray crystallographic analyses. The TRAIL-resistance-overcoming activity of 1: -4: in a TRAIL-resistant HT29 colorectal cancer cell line was evaluated, which revealed that co-treatment of 1: -4: at 50 µM with TRAIL (150 ng/mL) reduced the HT29 cell viability by 19.0%, 24.1%, 17.9%, and 15.5%, respectively, compared to treatment with 1: -4: alone.

Kaempferitrin inhibits colorectal cancer cells by inducing reactive oxygen species and modulating PI3K/AKT signalling pathway

Colorectal cancer is a life-threatening malignancy of high invasive death worldwide, despite improvements in conventional chemotherapy. Hence, there is an urgent need for new and effective anti-colorectal chemotherapeutic agents. Kaempferitrin can inhibit colon cancer cell viability, apoptotic induction, and reactive oxygen species (ROS) generation in a concentration-dependent way. Kaempferitrin had a cytotoxic and anti-proliferative effect on HT-29 colorectal cancer cells at a dosage of 30 µM, regulate apoptotic cell death, cell proliferation, and the cellular antioxidant system. Hyperoxia, cellular stress, ROS, and oxidative damage are the regulators of cancer cells. Increased sensitivity towards oxidative stress is caused by a change in redox state caused by enhanced ROS generation. In HT-29 cells, kaempferitrin also activated caspase-3 and enhanced the ratios of cleaved PARP protein expression, triggering caspase-dependent death. These kaempferitrin-induced processes also correlate with PI3K/AKT, implying downstream targeting of the cancer cell pathway. These findings suggest that kaempferitrin could be a promising therapeutic agent for the treatment of CRC, since it inhibits CRC cell proliferation and induces apoptosis via the PI3K/AKT signalling pathway.

Involvement of TMEM16A/ANO1 upregulation in the oncogenesis of colorectal cancer

The Ca2+-activated Cl− channel ANO1 is widely expressed in epithelial cells, and ANO1 upregulation is implicated in the oncogenesis of many epithelium-originated cancers. However, whether ANO1 plays a causal role in the tumorigenesis of colorectal cancer remains largely unknown. Here, we show that ANO1 channel protein is upregulated in human colorectal cancer tissue samples and its upregulation is correlated with the TNM staging, histological type, pathological differentiation and poor prognosis. Knockdown or pharmacological inhibition of ANO1 suppresses colorectal cancer cell proliferation and induces cell apoptosis. Furthermore, ANO1 knockdown inhibits the growth of subcutaneous xenograft tumors implanted with colorectal cancer HT-29 cells in nude mice. Mechanically, knockdown of endogenous ANO1 inactivates the Wnt/β-catenin signaling through downregulating critical components, such as Frizzled protein 1, β-catenin and upregulating GSK3β. Taken together, our results demonstrate that ANO1 upregulation is involved in the tumorigenesis of colorectal cancer, and inhibition of ANO1 upregulation or inactivating downstream Wnt/β-catenin signaling may have therapeutic potential for colorectal cancer.

Diosmetin Exerts Synergistic Effects in Combination with 5-Fluorouracil in Colorectal Cancer Cells.

5-Fluorouracil (5-FU) is a chemotherapeutic medication commonly used to treat colorectal cancer (CRC); however, the drug-associated adverse effects and toxicity have greatly affected its clinical use. Exploring another therapeutic strategy that lowers the toxicity of 5-FU while having a synergistic effect against CRC is thus a viable option. Diosmetin, a natural flavonoid, has been shown to inhibit the proliferation of many cancer cells, including CRC cells. This study aims to investigate the synergistic effect of diosmetin and 5-FU on HCT116 and HT29 colorectal cancer cells and to explore the apoptotic activity of this combination. The MTT assay was used to assess the viability of cells treated with monotherapy and combination therapy. The combination index (CI) and dose reduction index (DRI) were calculated using the CompuSyn software (version 1.0). The SynergyFinder 2.0 software was used to calculate the synergy score, while the Combenefit software was employed to perform isobologram analysis and synergism determination. The AO/PI double staining technique was used to detect the apoptotic characteristics of cells, whereas the flow cytometry technique was used to investigate the apoptosis induction and cell cycle arrest in cells. The combination of 5-FU and diosmetin showed a synergistic effect in HCT116 cells with a mean CI value of 0.66 ± 0.4, and an additive effect in HT29 cells with a CI value of 1.0 ± 0.2. The DRI of 5-FU in HCT116 cells was three times lower in the combination therapy compared to monotherapy of 5-FU. AO/PI microscopic examination and Annexin V analysis revealed that the combination-treated cells had more apoptotic cells than the monotherapy-treated cells, which was activated mainly through intrinsic apoptosis pathway. HCT116 cell death was confirmed by mitotic arrest in the G2/M phase. Our findings suggest that 5-FU/diosmetin combination exhibits synergistic effect against HCT116 cancer cells, and potentially reduces the unfavorable adverse effect of 5-FU while enhancing the anticancer efficacy by inducing apoptosis and interrupting mitosis. Further research studies are needed to validate the combination’s anti-tumorigenic activities in a xenograft animal model.

Long non-coding RNA AC087388.1 as a novel biomarker in colorectal cancer

BackgroundSeveral investigations have reported diverse roles of long non-coding RNA (lncRNA) in biological processes, tumor development, and progression of colorectal cancer (CRC). In this study, we investigated the lncRNA AC087388.1 tumorigenic role in CRC cells.MethodsThe CRC tissues were collected at the Reza Radiotherapy and Oncology Center, Mashhad, Iran. The human SW-48 and HT-29 CRC cell lines were obtained from the national cell bank of Iran. The cells were cultured according to ATCC (the American Type Culture Collection) recommendations. Quantitative real-time PCR was applied to assess the RNA expression. ShRNA transfection was done to downregulate the target gene. MTT and apoptosis assays were conducted to evaluate cell proliferation and viability, respectively. Colony formation assay, wound healing assay, and invasion assay were applied to determine growth, motility, and invasion of the cells, respectively. ENCORI online tool was used as downstream enrichment analysis.ResultsForty CRC patients were encompassed in this study. The results demonstrated that the lncRNA SLC16A1-AS1, AC087388.1, and ELFN1-AS1 were significantly overexpressed in the CRC tissues in comparison to their normal counterpart margins. All the lncRNAs have shown significant Area Under Curve (AUC) values in the patients. Downregulation of lncRNA AC087388.1 remarkably decreased the cell proliferation and viability of the CRC cells. In addition, the data demonstrated that the downregulation of lncRNA AC087388.1 significantly suppressed cell growth and colony formation capability in the cells. Also, downregulation of lncRNA AC087388.1 attenuated motility and invasion of CRC cells, and significantly decreased the expression of invasion genes. In-silico functional enrichment analysis indicated that the lncRNA AC087388.1 has contributed to crucial signaling pathways in tumorigenesis such as the p53 and Wnt signaling pathways, apoptosis, and cell cycle.ConclusionsAltogether, we showed that lncRNA AC087388.1 has an oncogenic role in tumorigenesis of CRC, and it can be considered as a novel diagnostic and prognostic biomarker in CRC.

Comparison of Colorectal Cancer Stem Cells and Oxaliplatin-Resistant Cells Unveils Functional Similarities.

Colorectal cancer is the second most common cancer in women, the third in men, and an important cause of cancer-related mortality. Recurrence and the development of chemotherapy resistance are major hindrances for patients’ treatment. The presence of cancer stem cells with chemotherapy resistance able to generate proliferating tumor cells contributes to tumor recurrence and resistance. In addition, tumor cells can develop chemoresistance through adaptation mechanisms. In this article, cancer stem cells were isolated from HT29 and SW620 colorectal cancer cell lines. Oxaliplatin resistance was induced by a single drug treatment simulating the usual guidelines of patient treatment. A comparison of these two populations showed similarities since cancer stem cells presented increased oxaliplatin resistance, and resistant cells contained an increased number of cancer stem cells. Cancer stem cells isolated from resistant cells showed increased oxaliplatin resistance. Cell invasion capacity and epithelial-mesenchymal transition were increased both in cancer stem cells and oxaliplatin-resistant cells. mRNA expression analysis showed that both cell types shared a significant proportion of commonly regulated genes. In summary, the data presented indicate that colorectal cancer stem cells and oxaliplatin-resistant cells are highly related cell populations that might have interesting implications in the development of tumor recurrence and resistance to chemotherapy.

Anticancer Effect of Sargassum oligocystom Hydroalcoholic Extract Against SW742, HT-29, WiDr, and CT-26 Colorectal Cancer Cell Lines and Expression of P53 and APC Genes.

Colorectal cancer (CRC) is the third most common cancer in the world, with enhancing morbidity and mortality each year. Due to the drug resistance against CRC, the use of novel compounds besides chemotherapy is required. Natural seafood contains large amounts of biologically active substances with new chemical structures and new medicinal activities. The aim of this study was to evaluate the effects of hydroalcoholic extract of Sargassum oligocystom algae on SW742, HT-29, WiDr, and CT-26 CRC cell lines, and to evaluate the expression of P53 and APC genes using quantitative real-time PCR (RT-qPCR). The cytotoxicity of S. oligocystom hydroalcoholic extract was determined by MTT and trypan blue methods in six different concentrations including 0.1, 0.2, 0.5, 1, 2, and 4mg/mL on various CRC cell lines and a control group. The expression of P53 and APC genes in exposure to 2mg/mL of the extract was also evaluated using RT-qPCR. The LD50 and LD90 of S. oligocystom included 0.5-1 and > 2mg/mL, respectively mostly affecting SW742 and CT-26 cells. In the trypan blue test, 90% viability and death of cells were observed at 0.1 and 4mg/mL of extract, respectively. The 2mg/mL was a safe cytotoxic concentration. A significant viability decrease was observed at concentrations ≥ 1mg/mL (p < 0.001). Sargassum oligocystom extract at 2mg/mL significantly increased the expression of APC ranging 1.98-2.2-fold (p < 0.001) but not P53 gene which ranged 0.5-0.68-fold (p = 0.323) after 24h. These results indicated that the brown algae S. oligocystom extract had significant antitumor effects against the SW742, HT-29, WiDr, and CT-26 CRC cell lines and especially CT-26, suggesting that it may be a potential candidate for further studies and therefore designing drugs of natural anticancer origin. The S. oligocystom had an anticancer effect via an increase in the APC gene expression.

Fabrication of cellulose nanocrystals as potential anticancer drug delivery systems for colorectal cancer treatment

Polysaccharide nanocrystals have great potential to be used as improved drug carriers due to their low cost, high biodegradability, and biocompatibility. This study reports the synthesis of cellulose nanocrystals (CNC) loaded with 5-fluorouracil (CNC/5FU) to evaluate their anticancer activity against colorectal cancer cells. X-ray and Fourier-transform infrared spectroscopy demonstrated that acid hydrolysis successfully degraded the amorphous cellulose to liberate the crystal regions. From transmission electron microscopy, CNC/5FU appeared as rod-like nanocrystals with an average length and width of 69.53 ± 1.14 nm and 8.13 ± 0.72 nm, respectively. The anticancer drug 5FU showed improved thermal stability after being loading onto CNC. From UV–vis spectroscopy data, the drug encapsulation efficiency in CNC/5FU was estimated to be 83.50 ± 1.52%. The drug release of CNC/5FU was higher at pH 7.4 compared to those at pH 4.2 and 1.2. From the cytotoxicity assays, CNC did not affect the viability of CCD112 colon normal cells. On the other hand, CNC/5FU exhibited anticancer effects against HCT116 and HT-29 colorectal cancer cells. The anticancer actions of CNC/5FU against HCT116 cells were then confirmed using an in vitro tumor-on-chip model and clonogenic assay. Mechanistic studies demonstrated that CNC/5FU killed the cancer cells by mainly inducing cell apoptosis and mitochondrial membrane damage. Overall, this study indicated that CNC/5FU could be a potential nanoformulation for improved drug delivery and colorectal cancer treatment.

Porric acid E, a natural compound from Rhytidhysteron sp. BZM-9, suppresses colorectal cancer growth via an autophagy-dependent pathway.

Colorectal cancer (CRC) is one of the major killer diseases worldwide, and more effective therapeutic compounds for CRC treatment are urgently needed. Although bioactive natural products derived from endophytic fungi have been extensively employed as antibiotics and anticancer agents, little is known about the effect of Rhytidhysteron sp. BZM-9 (an endophytic fungus)-derived compounds on CRC. Herein, a natural molecule porric acid E was isolated from Rhytidhysteron sp. BZM-9. Alamar Blue cell viability assay, Western blotting, transmission electron microscopy, flow cytometry analysis, and fluorescence image examination were employed to evaluate the antitumor effects of porric acid E on CRC cell lines. To establish the xenograft tumor model, nude mice received subcutaneous implants consisting of CRC cells on their flanks. Then the mice were treated with porric acid E or vehicle to assess the tumor-killing effects. The results revealed that porric acid E exhibited cytotoxicity by inhibiting proliferation and promoting apoptosis in CRC cells in vitro. Additionally, compared with fluorouracil (5-FU), porric acid E exhibited a more potent inhibitory effect on CRC HT29 cells. Importantly, extensive autophagy induced by porric acid E was detected in CRC cells, whereas inhibition of autophagy could significantly ameliorate porric acid E-mediated cytotoxic effect on CRC cells. Moreover, porric acid E treatment could markedly suppress subcutaneous HT29 xenograft tumor growth in vivo. Bioinformatics prediction indicated that Beclin-1 might be the potential target of porric acid E. These findings might afford a useful and important method for the treatment of CRC through fungal endophyte-derived natural compounds.

Studying the suppressing effect of mesenchymal stem cells derived from amniotic membrane on colorectal cancer

Mesenchymal stem cell therapy is considered as a proper tool for biological activities and treatment of diseases and cancer. Here, the suppressing effect of mesenchymal stem cells derived from the amniotic membrane (AM-MSCs) on colorectal cancer (HT29 cell line) was studied. MSCs were isolated from the human amniotic membrane and identity tests were performed. AM-MSC and HT29 cells were co-cultured and MTT assay was used to determine proper inhibitory concentration. The apoptotic effect of AM-MSC on HT29 cells was studied by Acridine orange staining. Expression of CDK2 protein and the antioxidant effect of AM-MSCs on HT29 was performed by immunocyto chemistry test. The effect of MSC on the HT29 cell line migration was determined by Scratch test. The result showed that AM-MSC had proliferation-inhibitory effects and caused apoptosis of the HT29 cell line. Real Time-PCR assay showed the increase of gene expression of p53, Caspase3 in apoptosis, and p21 in tumor cell line treated with AM-MSC. AM-MSC arrested the cell cycle of tumor cells by reduction of CDK2 protein. The migration of HT29 and ROS expression levels was decreased. Altogether, AM-MSC may play a role in suppressing colorectal cancer HT29 cell line by inhibiting angiogenesis, cell cycle, and induction of apoptosis and has antioxidant properties.

MiR-545 promotes colorectal cancer by inhibiting transferring in the non-normal ferroptosis signaling.

In this study, we examined whether and how miR-545 modulates ferroptosis in colorectal cancer (CRC). HT-29 and HCT-116 human CRC cell viability was examined using a CCK-8 assay and malondialdehyde (MDA) and Fe2+ levels were measured after treatment with the ferroptosis inducers Eradicator of Ras and ST (erastin) and Ras selective lethal 3 (RSL3) with or without miR-545 overexpression or knockdown vectors. Our results demonstrate that miR-545 overexpression inhibited, while miR-545 knockdown further increased, erastin and RSL3-induced upregulation of MDA, reactive oxygen species (ROS), and Fe2+ levels. Similarly, miR-545 overexpression partially reversed, while miR-545 knockdown enhanced, the erastin and RSL3-induced reduction in HT-29 and HCT-116 cell survival rates. Transferrin (TF) was identified as a target gene of miR-545. To determine whether miR-545 suppresses ferroptosis via TF, we overexpressed TF in HT-29 and HCT-116 cells. We found that TF overexpression blocked miR-545-induced changes in ROS, MDA, and Fe2+ levels in HT-29 and HCT-116 cells, thereby inducing CRC cell death. An in vivo assay showed that inhibition of miR-545 decreased tumor growth in nude mice treated with erastin. Together, these findings indicate that miR-545 promotes CRC cell survival by suppressing TF.

CCL20 induces colorectal cancer neoplastic epithelial cell proliferation, migration, and further CCL20 production through autocrine HGF-c-Met and MSP-MSPR signaling pathways.

CCL20-CCR6 interactions promote colorectal cancer through direct effects on neoplastic epithelial cells and through modulating the tumor microenvironment. The mechanism of these effects on neoplastic epithelial cells is poorly understood. This study demonstrates that CCL20 induces secretion of hepatocyte growth factor (HGF) and phosphorylation of HGF’s cognate receptor c-Met in HT29 and HCT116 colorectal cancer cell lines both in concentration- and time-dependent manners. Similar to CCL20, HGF induces migration, autofeedback CCL20 secretion, and ERK1/2 phosphorylation in the colon cancer cells. CCL20-dependent ERK1/2 phosphorylation is blocked by HGF inhibition, and CCL20-dependent migration and CCL20 secretion are blocked by inhibition of HGF or ERK. Interestingly, unlike CCL20, HGF does not induce proliferation of colon cancer cells, and CCL20-dependent cell proliferation is not blocked by direct HGF inhibition. CCL20-dependent proliferation, however, is blocked by the multi-tyrosine kinase inhibitor crizotinib. Exploring this effect, it was found that CCL20 also induces production of MSP and phosphorylation of MSP’s receptor MSPR by the colorectal cancer cells. CCL20-dependent cell proliferation is inhibited by directly blocking MSP-MSPR interactions. Thus, CCL20-mediated migration and CCL20 secretion are regulated through a pathway involving HGF, c-Met, and ERK, while CCL20-mediated proliferation is instead regulated through MSP and its receptor MSPR.

Investigation of the Effects of Apigenin, a Possible Therapeutic Agent, on Cytotoxic and SWH Pathway in Colorectal Cancer (HT29) Cells.

Purpose: Colorectal cancer (CRC) is one of the most common and fatal malignancies in humans, still leading to serious morbidity and mortality. We here aimed to investigate the effects of flavonoid apigenin, which is considered to have anti-tumoral activity on CRC with high epidemiological prevalence, on cell proliferation and cell survivals, and the positive and negative dose-dependent effects of genetic or mutational alterations in SWH pathway components on HT29 CRC cell lines. Methods: Human colon cancer cell lines HT-29 were commercially available. In each flask, 5 groups were formed, each of which consists of 5,000 cells for different dose groups and the cells were plated. After a 24 and 48 h incubation period, cytotoxicity values were measured by MTT assay and gene expression was assessed by real-time polymerase chain reaction (PCR) analysis method. Results: Application of 12.5 and 25 nM of apigenin significantly increased cell death in HT29 cell lines. LATS1, STK3 and TP53 gene expression decreased in the same dose groups compared to control and other groups. Conclusion: It has been concluded that TP53 gene is strongly correlated with LATS1 and STK3 genes among the SWH pathway factors in the progression of CRC and could be used as an important marker for early detection of malignant transmission. In addition, it may be effective in CRC cases especially when 25 nM of apigenin applies for therapeutic purpose.

A novel fluorescent c-met targeted imaging agent for intra-operative colonic tumour mapping: Translation from the laboratory into a clinical trial

BackgroundThe c-Met protein is overexpressed in many gastrointestinal cancers. We explored EMI-137, a novel c-Met targeting fluorescent probe, for application in fluorescence-guided colon surgery, in HT-29 colorectal cancer (CRC) cell line and an in vivo murine model. MethodsHT-29 SiRNA transfection confirmed specificity of EMI-137 for c-Met. A HT-29 CRC xenograft model was developed in BALB/c mice, EMI-137 was injected and biodistribution analysed through in vivo fluorescent imaging. Nine patients, received a single intravenous EMI-137 bolus (0.13 mg/kg), 1–3 h before laparoscopic-assisted colon cancer surgery (NCT03360461). Tumour and LN fluorescence was assessed intraoperatively and correlated with c-Met expression in eight samples by immunohistochemistry. Findingsc-Met expression HT-29 cells was silenced and imaged with EMI-137. Strong EMI-137 uptake in tumour xenografts was observed up to 6 h post-administration. At clinical trial, no serious adverse events related to EMI-137 were reported. Marked background fluorescence was observed in all participants, 4/9 showed increased tumour fluorescence over background; 5/9 had histological LN metastases; no fluorescent LN were detected intraoperatively. All primary tumours (8/8) and malignant LN (15/15) exhibited high c-Met protein expression. InterpretationEMI-137, binds specifically to the human c-Met protein, is safe, and with further refinement, shows potential for application in fluorescence-guided surgery.

Polymeric complex based on poly (styrene-alt-maleic anhydride)- targeted with folic acid for doxorubicin delivery to HT-29 colorectal cancer cells

In this study, a drug-polymer complex for targeted delivery of doxorubicin to cancer cells based on poly (styrene-alt-maleic-anhydride) copolymer was synthesized. The targeted polymer complex has shown a greater effect on cell growth than pure drugs. Targeted polymer complex containing doxorubicin which is targeted by folic acid receptors, was shown to increase the effects of the drug on HT-29 colorectal cancer cells, which have a high concentration of folate receptors on its cell wall. Based on these results, this drug-carrying system may be valuable in reducing the dose of doxorubicin its systemic side effects.

Characterization of Novel α-Mangostin and Paeonol Derivatives With Cancer-Selective Cytotoxicity.

α-Mangostin (aMan) and Paeonol (Pae) have shown anticancer and anti-inflammatory properties. However, these two natural compounds have no clinical value because of their low solubility and low membrane permeability. In this study, we screened chemically synthesized derivatives from these two natural compounds as potential novel chemicals that increase cancer cell cytotoxicity over nontransformed human cells. We found that two derivative compounds, named α-Mangostin-1 (aMan1) and Paeonol-1 (Pae1) more efficiently and more specifically induced cytotoxicity in HCT116, HT29, and SW48 colorectal cancer cell lines than the parental compounds. Both aMan1 and Pae1 arrested HCT116 cells in the G1 phase and HT29 and SW48 cells in the G2-M phase of the cell cycle. Both aMan1 and Pae1 induced apoptosis in human colorectal cancer cells, through a caspase-dependent mechanism. aMan1 and Pae1 induced selective transcriptional responses in colorectal cancer cells involving genes related to metabolic stress and DNA damage response signaling pathways. Finally, experiments on primary colon organoids showed that both derivatives were able to kill cancer-derived organoids without affecting the viability of organoids derived from healthy tissue, where the parental compounds and the currently used chemotherapeutic drug irinotecan failed. In conclusion, our findings expand the knowledge of natural compound derivatives as anticancer agents and open new avenues of research in the derivation of lead compounds aimed at developing novel chemotherapeutic drugs for colorectal cancer treatment that selectively target cancer, but not healthy cells.