HT-29 Human Colon Cancer Cells Research Articles

Comprehensive Analysis of Phycoerythrin 545 Stability and the Apoptotic Impact of Its Degradation Products on HT29 Cells.

This study investigates the properties and potential applications of phycoerythrin 545, a naturally occurring light-harvesting pigment protein from Rhodomonas salina. Phycoerythrin 545, characterized by its bright red color and maximum absorption wavelength at 545 nm, was extracted using freeze-thawing methods, further purified, and analyzed using chromatographic, spectroscopic techniques, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phycoerythrin 545 consists of two subunits, primarily α and β, but lacks the γ subunit, and is stable at 4 °C within a pH range of 3-10. To further characterize it, its susceptibility to degradation by trypsin was assessed. The biological activity of phycoerythrin 545 and its degradation products were investigated in HT29 human colon cancer cells. The results showed that the degradation products, particularly those within 3-10 kDa, significantly decreased the viability of HT29 cells by inducing apoptosis. Mechanistic studies indicated these effects were mediated through the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases and MAPK/c-Jun N-terminal Kinase signaling pathways and involved the regulation of key apoptotic proteins such as p53, Bim, Bad, Bak, and Bax, leading to the activation of the Caspase-3 apoptotic pathway. These findings contribute to understanding the structural and functional properties of phycoerythrin 545, laying a foundation for its exploration in food industry applications and cancer therapy supplementation.

Green synthesis of undoped and Ag-doped NiO nanoparticles: Evaluation and their synergetic effect on antimicrobial, anticancer, and antioxidant activities

In this investigation, we successfully synthesized nickel oxide nanoparticles (NiO NPs) using a combustion method with Acacia nilotica gum as a biofuel, and systematically altered silver (Ag) concentrations within the NiO matrix. Post-calcination at 400 °C, various characterization techniques confirmed the formation, surface morphology, and crystalline characteristics of NiO NPs. The Ag-doped NiO NPs, particularly the NiAg4 sample, demonstrated remarkable antimicrobial efficacy, with superior activity against both fungal and bacterial pathogens. The NiAg4 sample also exhibited potent cytotoxicity against HT-29 human colon cancer cells, achieving an IC50 value of 1.5 mg/mL. Toxicological assessments revealed that both undoped and Ag-doped NiO NPs maintained biocompatibility with human red blood cells, indicating their potential for biomedical applications. Additionally, the NPs showed strong antioxidant activity and protein kinase inhibition, further supporting their therapeutic potential. These findings suggest that Ag-doped NiO NPs synthesized through a green protocol possess enhanced bioactivities, making them promising candidates for future in vivo studies and diverse biomedical applications.

Cytotoxicity assay of Turkish rare endemic Helianthemum germanicopolitanum Bornm. plant extract on HT-29 cell line

Objective: The aim of this study was to investigate the cytotoxic effect of Helianthem germanicopolitanum Bornm., a rare endemic plant in Turkey, against colon cancer. Materials and Methods: After the extraction of Helianthemum germanicopolitanum Bornm. plant, the phytochemical profile of the plant was analyzed by High Performance Liquid Chromatography (HPLC). Cytotoxicity analysis on HT-29 human colon cancer cell line was performed by WST 1 assay. The results were compared with other studies on similar plant species in the literature. Results: H. germanicopolitanum plant contains various flavonoids and these flavonoids have cytotoxic effects on colorectal cancer cells. These cytotoxic effects provide anticarcinogenic effects by activating cell death pathways at certain concentrations. These findings provide an important insight into the therapeutic potential of the plant. Conclusion: Cytotoxicity studies of flavonoids in extracts obtained from extraction procedures on colorectal cancer cell lines show that flavonoids offer anticarcinogenic effects by activating cell death pathways at certain concentrations.

Anti-Metastatic Effects of Standardized Polysaccharide Fraction from Diospyros kaki Leaves via GSK3β/β-Catenin and JNK Inactivation in Human Colon Cancer Cells.

A polysaccharide fraction from Diospyros kaki (PLE0) leaves was previously reported to possess immunostimulatory, anti-osteoporotic, and TGF-β1-induced epithelial-mesenchymal transition inhibitory activities. Although a few beneficial effects against colon cancer metastasis have been reported, we aimed to investigate the anti-metastatic activity of PLE0 and its underlying molecular mechanisms in HT-29 and HCT-116 human colon cancer cells. We conducted a wound-healing assay, invasion assay, qRT-PCR analysis, western blot analysis, gelatin zymography, luciferase assay, and small interfering RNA gene silencing in colon cancer cells. PLE0 concentration-dependently inhibited metastasis by suppressing cell migration and invasion. The suppression of N-cadherin and vimentin expression as well as upregulation of E-cadherin through the reduction of p-GSK3β and β-catenin levels resulted in the outcome of this effect. PLE0 also suppressed the expression and enzymatic activity of matrix metalloproteinases (MMP)-2 and MMP-9, while simultaneously increasing the protein and mRNA levels of the tissue inhibitor of metalloproteinases (TIMP-1). Furthermore, signaling data disclosed that PLE0 suppressed the transcriptional activity and phosphorylation of p65 (a subunit of NF-κB), as well as the phosphorylation of c-Jun and c-Fos (subunits of AP-1) pathway. PLE0 markedly suppressed JNK phosphorylation, and JNK knockdown significantly restored PLE0-regulated MMP-2/-9 and TIMP-1 expression. Collectively, our data indicate that PLE0 exerts an anti-metastatic effect in human colon cancer cells by inhibiting epithelial-mesenchymal transition and MMP-2/9 via downregulation of GSK3β/β-catenin and JNK signaling.

Histogram-based global thresholding method for image binarization

One of the most fundamental issues in image processing is the thresholding (binarization) method. This method is generally used for segmenting regions with different homogeneity in grayscale images. In other words, it performs clustering based on the intensity levels of pixels in an image histogram. This paper presents a new and effective approach to the global thresholding method of grayscale images. In the proposed method, alpha and beta regions are determined using the mean and standard deviation values of an image histogram. The optimum threshold value is obtained by calculating the average of gray-scale values of the alpha and beta regions. The experiments were carried out on three different image sets to demonstrate the effectiveness of the thresholding method. The result of experimental studies show that the proposed method achieves promising performance compared to many traditional, state-of-the-art thresholding and document binarization methods performed in H-DIBCO'14 (Document Image Binarization Competition), Human HT29 colon-cancer cells (BBBC008) and C. elegans live/dead assay (BBBC010) datasets based on various evaluation criteria.

Colon cancer inhibitory properties of Caulerpa lentillifera polysaccharide and its molecular mechanisms based on three-dimensional cell culture model

Caulerpa lentillifera is rich in polysaccharides, and its polysaccharides show a significant effect in different biological activities including anti-cancer activity. As an edible algae-derived polysaccharide, exploring the role of colon cancer can better develop the application from a dietary therapy perspective. However, more in-depth studies of C. lentillifera polysaccharide on anti-colon cancer activity and mechanism are needed. In this study, we found that Caulerpa lentillifera polysaccharides (CLP) showed potential anti-colon cancer effect on human colon cancer cell HT29 in monolayer (IC50 = 1.954 mg/mL) and spheroid (IC50 = 0.402 mg/mL). Transcriptomics and metabolomics analyses revealed that CLP had an inhibitory effect on HT29 3D spheroid cells by activating aminoacyl-tRNA biosynthesis as well as arginine and proline metabolism pathways. Furthermore, the anti-colon cancer effects of CLP were confirmed through other human colon cancer cell HCT116 and LoVo in monolayer cells (IC50 = 1.890 mg/mL and 1.437 mg/mL, respectively) and 3D spheroid cells (IC50 = 0.344 mg/mL and 0.975 mg/mL, respectively), and three patient-derived organoids with IC50 values of 6.333–8.780 mg/mL. This study provided basic data for the potential application of CLP in adjuvant therapeutic food for colon cancer on multiple levels, while further investigation of detailed mechanism in vivo was still required.

Isoprenoid flavonoids isolated from Sophora davidii and their activities induces apoptosis and autophagy in HT29 cells

Four previously undescribed isoprenoid flavonoids (2–5) were isolated from Sophora davidii, along with five known analogues. The structures of the compounds were established through comprehensive analysis of spectroscopic data, including HRESIMS, 1D and 2D NMR, and absolute configurations determined by theoretical calculations, including ECD and NMR calculation. The cytotoxic effects of the isolated compounds on human HT29 colon cancer cells were evaluated using the MTT assay, compound 1 exhibited cytotoxicity against human HT29 colon cancer cells with an IC50 value of 8.39 ± 0.09 μM. Studies conducted with compound 1 in HT29 cells demonstrated that it may induce apoptosis and autophagy in HT29 by promoting the phosphorylation of P38 MAPK and inhibiting the phosphorylation of Erk MAPK.

Effects of Viable and Heat-Inactivated Bifidobacterium longum D42 on Proliferation and Apoptosis of HT-29 Human Colon Cancer Cells.

Bifidobacterium longum is a common probiotic; both viable and heat-inactivated Bifidobacterium longum have many probiotic effects, such as anticancer effects. But some mechanisms of anticancer effects are still unclear, especially for heat-inactivated probiotics. In this study, we analyzed the effects of viable and heat-inactivated Bifidobacterium longum D42 on human colon cancer cells (HT-29). Cell proliferation, membrane permeability and apoptosis were detected by using the CCK-8 method, LDH method and Annexin V-FITC/PI kits. The ROS level and mitochondrial membrane potential were examined using the fluorescent probes DCFH-DA and JC-1. Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot were used to detect the expression of mitochondrial apoptosis pathway genes and proteins. The results showed that viable and heat-inactivated Bifidobacterium longum D42 at concentrations of 1 × 106 CFU/mL significantly inhibited the proliferation of and increased the level of LDH release of HT-29 colon cancer cells. We found that they could increase the apoptosis rate of HT-29 cells. Moreover, they could also induce apoptosis by inducing cells to produce ROS and destroying the mitochondrial membrane potential of cells. Further studies found that they could increase the mRNA transcription and protein expression levels of the Caspase-3, Caspase-9 and Bax genes in cells, and reduce the mRNA transcription and protein expression levels of the Bcl-2 gene. In summary, our findings revealed that viable and heat-inactivated Bifidobacterium longum D42 have inhibitory effects on proliferation and promote the apoptosis of human colon cancer cells, and also have certain adjuvant drug therapeutic effects and have potential application value in the adjuvant treatment of colon cancer.

Diterpenoids from Torreya grandis and their cytotoxic activities

Eight previously undescribed diterpenoids, along with eleven previously reported analogues, were obtained from the supercritical CO2 extracts of Torreya grandis aril. The structures of these compounds were elucidated based on HRESIMS, NMR, ECD, and single-crystal X-ray diffraction data. In the MTT assay, compound 18 exhibited significant inhibitory effects on two human colon cancer cell lines, HT-29 and HCT 116 cells, with IC50 values of 7.37 μM and 6.55 μM, respectively. It was found that compound 18 induced apoptosis and significantly inhibited the migration of HCT 116 colon cancer cells in a concentration-dependent manner.

Fucoidan from brown seaweed Tubinaria decurrens: Structure and structure - anticancer activity relationship

The aims of this study are to determine the structure of a fucoidan from brown seaweed Turbinaria decurrens, to investigate its anticancer activity and structure–activity relationship. SEC-MALLS, IR, ESI-MS and NMR spectra analysis indicated that dominant structure of the fucoidan, with a Mw 122.6 KDa, has a backbone of (1 → 3)– and (1 → 4)–α–L–Fucp residues, branched at C–4, sulfate groups are attached at C–2, C–3 and C–4; branches are (1 → 4)–β–D–Galp residues and sulfated at C–2. The fucoidan was hydrolyzed by HCl aqueous solution to obtain hydrolyzed fucoidans. It is assumed that native and hydrolyzed fucoidans have a rod-like conformation in solution with cross–sectional radius of gyration (Rgc) ranged from 0.53 to 1.52 nm as estimated from SAXS measurements. The fucoidans show great anticancer activity against HT29 human colon cancer cell line with IC50 ranging from 5.41 ± 0.36 to 73.52 ± 2.54 μg/mL. Anticancer activity of the fucoidan could be significantly improved by lowering molecular weight, furthermore, fucoidan required small molecular weight, small molecular weight distribution and rod–like structure with a short branch length for high anticancer activity.

Impact of Butein on Apoptosis, Migration, Invasion, and Epithelial-Mesenchymal Transitions of HT-29 Human Colon Cancer Cells

Impact of Butein on Apoptosis, Migration, Invasion, and Epithelial-Mesenchymal Transitions of HT-29 Human Colon Cancer Cells

The mechanism of anticancer effects of some pyrrolopyrimidine derivatives on HT-29 human colon cancer cells

In the present work, the mechanism of anticancer activity of some pyrrolopyrimidine derivatives was evaluated. Compounds 5 and 8 exhibiting significant antiproliferative activity against HT-29 cells with IC50 values of 4.17 μM and 2.96, arrested the cells at the G2/M phase and significantly induced apoptosis. The apoptotic potential of the compounds has been verified via ELISA assay, which resulted in increased BAX, PUMA, BIM, and cleaved caspase 3 expression and decreased BCL-XL and MCL-1 protein levels in HT-29 cells. Moreover, the immunofluorescence technique showing that compounds 5 and 8-treatment reduced Ki67 immunolocalization and increased the caspase 3 and p53 immunolocalization confirmed the apoptotic activity. While treatment of HT-29 cells to compounds 5 and 8 inhibited Akt and ERK1/2, there are no alterations in JNK and p38 signaling pathways. According to molecular docking results, compounds 5 and 8 occupied the active site of Akt kinase and showed important hydrogen bonding interactions with key amino acids. Also, siRNA-mediated depletion of BIM, PUMA, and BAX/BAK expression decreased apoptotic response in HT-29 cells upon exposure to compound 5 and compound 8. Compounds 5 and 8 trigger the activation of mitochondrial apoptosis in HT-29 cells. Additionally, we found that proapoptotic BH3-only proteins BIM and PUMA are required for the full engagement of mitochondrial apoptosis signaling. However, p53 was dispensable for compound 5- or compound 8-induced apoptosis in HT-29 cells.

Cytotoxic activity of cordycepin produced by marine-derived fungus Emericella sp. against HT29 human colon cancer cell lines.

Colorectal cancer accounted for the third most common cancer in the world. The search for new drug candidates that can be used for colorectal cancer treatment from marine-derived fungi, Emericella sp. The present study was performed to isolate the cytotoxic compound from Emericella sp. The isolation method was carried out by using a combination of chromatographic techniques to afford compound 1. The cytotoxic activity and the exosome production property were determined by using proliferation and luciferase assay against HT29 CD63 Nluc cells, respectively. The chemical structure of compound 1 was identified as cordycepin based on spectroscopy methods such as mass spectrometry and nuclear magnetic resonance (1D and 2D NMR) analyses and comparison with authentic spectral data. The biological activity assay showed that cordycepin exhibited cytotoxic activity with an IC50 value of 92.05µM through proliferation assay, and also inhibited the exosome production by luciferase assay with an IC50 value of 86.47µM. Cordycepin was isolated from culture broth Emericella sp., exhibiting moderate cytotoxic activity and inhibitory activity of exosome production. Thus, cordycepin is a potential compound to be investigated further for its exosome production inhibition activity for further use as an anticancer lead compound.

Evaluation of dimedone-derived compounds as inhibitors against human colon cancer: Insights from 2D-QSAR, ADMET prediction, Osiris, Molinspiration, and molecular modeling

For this research, we performed a 2D-QSAR analysis on a set of 34 molecules derived from dimedone with inhibitory activity against human colon cancer (HT-29). The investigation incorporated principal component analysis (PCA), multiple linear regression (MLR), multiple non-linear regression (MNLR), and artificial neural network (ANN). The evaluations of the QSAR models demonstrated high predictive power (R2MLR = 0.884; R2CV (MLR) = 0.86), (R2MNLR = 0.810; R2CV (MNLR) = 0.879), and (R2ANN = 0.9; R2CV (ANN) = 0.89). The reliability of the models was validated through internal, external, Y-randomization, and validation domain applicability assessments. Utilizing the QSAR model predictions, we designed four new molecular structures that exhibited superior inhibitory activity against the HT-29 human colon cancer cell line compared to the 34 previously tested compounds. Subsequently, we examined the ADMET predictions, Molinspiration, and Osiris properties of the four compounds. The results revealed excellent ADMET predictions and Molinspiration for all four compounds, while only one designed compound fulfilled all Osiris properties. Molecular docking was employed to investigate the bindings between the newly designed molecule C and the c-Met protein. The findings indicated that the newly designed compound exhibited high stability in c-Met. Finally, MD simulations were employed to assess the stability and binding modes of compound C. Based on the MD results, compound C shows promise as a potential c-Met agonist candidate. Overall, our results suggest that this investigated compound has the potential to serve as a novel inhibitor against human colon cancer.

Anti-proliferative activity of chitosan-coated oxypeucedanin nano-chitosomes (COPD-NCs) against human HT-29 colon cancer cells: in vitro study.

Oxypeucedanin (OPD) as a powerful anti-proliferative agent found in the Angelicae dahuricae has been used to suppress cancer cell growth. However, the hydrophobic chemical structure has limited its solubility and bio-accessibility. This is the first time OPD is encapsulated into a nano-liposomal structure and coated with poly-cationic chitosan polymer as the oxypeucedanin drug delivery system to evaluate its antioxidant and anti-colon cancer potential. The chitosan-coated oxypeucedanin nano-chitosomes (COPD-NCs) were synthesized utilizing the thin-layer hydration method and characterized by FESEM, DLS, FTIR, and zeta potential analysis. The anti-cancer potential of COPD-NC was analyzed by measuring the cell survival rate (MTT assay) and studying the cellular death type (AO/PI staining) following the increased treatment concentrations of COPD-NC on the HT-29 colon cancer cell line. Moreover, the COPD-NCs' apoptotic activity was verified by analyzing Cas-3 and Cas-9 gene expression profiles. Finally, the COPD-NCs' antioxidant activity was evaluated by applying ABTS, DPPH, and FRAP antioxidant assays. The 258.26-nm COPD-NCs significantly inhibited the HT-29 colon cancer cells compared with the normal fibroblast HFF cells. The up-regulated Cas-3 and Cas-9 gene expression exhibited the COPD-NCs' apoptotic activity. Also, the COPD-NCs' apoptotic activity was verified by detecting the increased apoptotic bodies following the AO/PI fluorescent staining in the increased exposure doses of COPD-NCs. Ultimately, the COPD-NCs meaningfully inhibited the ABTS-DPPH radicals and exhibited an appropriate FRAP-reductive potential. The designed nanostructure for COPD-NCs significantly improved its antioxidant potential and selective cytotoxicity on human HT-29 human cancer cells, which makes them a safe selective natural drug delivery system. Therefore, the COPD-NCs can selectively induce apoptotic death in human HT-29 cancer cells and have the potential to be studied as an anti-colon cancer compound. However, further cancer and normal cell lines are required to verify their selective cytotoxicity.

Cytotoxic activities of alkaloid constituents from the climbing stems and rhizomes of Sinomenium acutum against cancer stem cells.

From the methanolic extract of the climbing stems and rhizomes of Sinomenium acutum, two new aporphine analogues, acutumalkaloids I and II, were isolated together with fifteen known compounds including lysicamine. The chemical structures of the isolated new compounds were elucidated based on chemical/physicochemical evidence such as NMR and MS spectra. For acutumalkaloids I and II, the absolute configurations were established by comparison of experimental and predicted electronic circular dichroism (ECD) data. We compared anti-proliferative activities of isolated compounds with reported naturally occurring Wnt/β-catenin pathway inhibitor, nuciferine. Among the isolated compounds, we found lysicamine have anti-proliferative activity against both of HT-29 human colon cancer cell line and its cancer stem cells (CSCs). The IC50 values of lysicamine against non-CSCs and its CSCs were lower than that of nuciferine. In addition, the results of western blotting analysis suggested that lysicamine inhibited the expression of Wnt/β-catenin pathway target protein such as survivin. These results suggested that lysicamine show cytotoxic activity via inhibition of Wnt/β-catenin pathway.

Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer

PurposeFluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer.MethodsEpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed.ResultsAc2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers.ConclusionOur encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer.

Anticancer Activity of Mineral-Supplemented Organically Cultivated Carrot on HT-29 Cells and Its Anti-Inflammatory Effect on Mice Splenocytes

Carrot (Daucus carota) is one of the world’s most significant root vegetables, with various bioactivities. This study aimed to investigate the anticancer activity and anti-inflammatory effects of natural dream cultivation carrot (NC). Natural dream cultivation is a cultivation method based on organic farming incorporating minerals. An MTT assay was used to evaluate the inhibitory rate of carrot samples on HT-29 human colon cancer cells, and qPCR was used to assess the mRNA expression of the cell cycle and apoptosis-related genes in the cancer cells. The nitrite oxide (NO) concentration was determined using the Griess method. The levels of inflammatory cytokines in LPS-induced mouse splenocytes were determined using an enzyme-linked immunosorbent assay, and the activity of NK cells was determined using LDH analysis. The results revealed that NC effectively inhibited cancer cell growth rate. Moreover, NC upregulated the mRNA expression of cell-cycle-arrest-related genes (p53 and p21) and apoptosis-related genes (Bim, Bad, Bax, Bak, caspase-9, and caspase-3) in cancer cells while downregulating the expression of anti-apoptotic genes, Bcl-2 and Bcl-xL. NC inhibited NO production and the release of inflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-γ, and IL-12) in LPS-induced mouse splenocytes. NC also demonstrated the ability to stimulate NK cell activation. This study explored the potential mechanisms underlying carrots’ anticancer and anti-inflammatory properties by investigating their inhibitory effects on cancer cells and regulating the inflammatory response. The innovative mineral-supplemented organic cultivation method, as explored in this study, opens new avenues for harnessing the potential of carrots as a functional food source with promising applications in cancer and inflammation management. This research not only provides insights into the bioactive potential of carrots but also contributes to the future development of novel dietary interventions and therapeutics.

Development of 1,3,6-Tribenzoylated Glucose as an Antiausterity Agent Targeting Tumor Microenvironment.

One aspect of cancer-specific environments, nutrient starvation, is a factor in cancer cell resistance to treatment with chemotherapeutic agents and development of malignancy. Our newly synthesized novel glucose derivative β-1,3,6-O-tribenzoyl-D-glucose (3) showed preferential cytotoxicity against PANC-1 human pancreatic cancer cells as well as HT-29 human colon cancer cells depending on low nutritional environment. The amount of ester functionalization in 3 is important. None of the mono- and tetrabenzoylated D-glucose analog showed cytotoxicity, and dibenzoylated D-glucoses showed only limited cytotoxicity. Fluorescence imaging with double staining of Hoechst 33342 and propidium iodide clearly showed that 3 actually causes cell death in a nutrient deprived medium. We thus demonstrate that an inexpensive natural product, D-glucose, is a unique template for attachment of acyl moieties to target tolerance to nutrient starvation. We expect these compounds will lead to additional compounds to treat refractory cancers by diversification of chemically modified glucose.

GlyH-101 and CaCCinh-A01 Inhibited HT-29 Proliferation by Activating the Mitochondrial Apoptosis Pathway and Arresting the Cell Cycle.

GlyH-101 and CaCCinh-A01 are effective blockers of cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated chloride channels (CaCCs), respectively. Available evidence suggests that GlyH-101 and CaCCinh-A01 can suppress cell proliferation, block invasion and metastasis, and cause several cancer cell types to undergo apoptosis, demonstrating their anti-tumor properties. The aim of this study was to investigate the effect of GlyH-101 and CaCCinh-A01 on HT-29 cell activity and to suggest the possible molecular mechanisms by which inhibitors of CFTR and CaCCs inhibit HT-29 cell activity. Human colon HT-29 cancer cells were treated with GlyH-101 or CaCCinh-A01 or GlyH-101 plus CaCCinh-A01 complex. Cell viability was determined by MTT assay, the apoptosis and cell cycle were determined by flow cytometry, and reactive oxygen species (ROS) leves were determined by 2',7'-Dichlorodihydrofluorescein diacetate staining. The expression of proteins related to apoptosis and cell cycle regulation was measured by western blotting. The proliferative ability of HT-29 cells was dose- and time-dependently reduced by GlyH-101 and CaCCinh-A01. Treatment with GlyH-101 and CaCCinh-A01 resulted in cell necrosis and apoptosis, up-regulated ROS levels, activated the mitochondrial apoptosis pathway, prompted arrest of the cell cycle in S phase, and increased the levels of proteins related to the cell cycle. Additionally, the combination of these two inhibitors had a stronger regulatory effect on HT-29 cell proliferation than either GlyH-101 or CaCCinh-A01 treated alone. GlyH-101 and CaCCinh-A01 inhibited cell proliferation through cell cycle arrest and mitochondrial-related pathways in vitro. The combination of these inhibitors could further enhance their anti-proliferative effects. Our findings propose new lead compounds with anti-colon cancer activity, and also provide new evidence for the effectiveness of chloride channels-targeted therapy in anticancer therapy.