HT-29 Colon Cancer Cells Research Articles

Differentially Methylated Ultra-Conserved Regions Uc160 and Uc283 in Adenomas and Adenocarcinomas Are Associated with Overall Survival of Colorectal Cancer Patients.

Deregulation of the transcribed ultra-conserved regions (T-UCRs) Uc160, Uc283, and Uc346 has been reported in colorectal cancer (CRC) recently. Here, we investigated promoter methylation of these T-UCRs during the adenoma–carcinoma sequence and their clinical significance in CRC patients. Methylation levels were assessed in CRC, adenomas, infiltrated lymph nodes, and metastatic tissue specimens. In situ hybridization was performed in representative tissue specimens. T-UCRs expression levels were also evaluated in HT-29 colon cancer cells before and after the acquired resistance to 5-fluorouracil (5-FU) and oxaliplatin. A gradual increase in T-UCRs methylation levels from hyperplastic polyps to adenomas and to in situ carcinomas (ISC) and a gradual decrease from ISC to infiltrative and metastatic carcinomas was observed (p < 0.001 for Uc160 and Uc283, p = 0.018 for Uc346). Uc160 and Uc283 methylation was associated with the grade of dysplasia in adenoma specimens (p = 0.034 and p = 0.019, respectively). Furthermore, higher Uc160 methylation, mainly in stage III and IV patients, was related to improved overall survival (OS) in univariate (p = 0.009; HR, 0.366) and multivariate analysis (p = 0.005; HR, 0.240). Similarly, higher methylation of Uc283 was associated with longer OS (p = 0.030). Finally, T-UCRs expression was significantly reduced in HT-29 cells after resistance to chemotherapy. This study suggests that promoter methylation of Uc160, Uc283, and Uc346 is altered during CRC development and that Uc160 and Uc283 methylation may have prognostic significance for CRC patients.

A Tryptophan Metabolite, 8-Hydroxyquinaldic Acid, Exerts Antiproliferative and Anti-Migratory Effects on Colorectal Cancer Cells.

8-Hydroxyquinaldic acid, the end-metabolite of tryptophan, is well-known metal chelator; however, its role in humans, especially in cancer promotion and progression, has not been fully revealed. Importantly, 8-hydroxyquinaldic acid is the analog of kynurenic acid with evidenced antiproliferative activity towards various cancer cells. In this study, we revealed that 8-hydroxyquinaldic acid inhibited not only proliferation and mitochondrial activity in colon cancer HT-29 and LS-180 cells, but it also decreased DNA synthesis up to 90.9% for HT-29 cells and 76.1% for LS-180 cells. 8-Hydroxyquinaldic acid induced changes in protein expression of cell cycle regulators (CDK4, CDK6, cyclin D1, cyclin E) and CDKs inhibitors (p21 Waf1/Cip1, p27 Kip1), but the effect was dependent on the tested cell line. Moreover, 8-hydroxyquinaldic acid inhibited migration of colon cancer HT-29 and LS-180 cells and increased the expression of β-catenin and E-cadherin. Importantly, antiproliferative and anti-migratory concentrations of 8-hydroxyquinaldic acid were non-toxic in vitro and in vivo. We reported for the first time antiproliferative and anti-migratory activity of 8-hydroxyquinaldic acid against colon cancer HT-29 and LS-180 cells.

Effect of Hydrogen Sulfide‐Releasing Compounds on Proliferation of Human Colon Cancer HT29 Cells

Both natural and synthetic compounds that release hydrogen sulfide (H2S) such as diallyl trisulfide and naproxen‐H2S hybrids growth and proliferation of human colon HT29 cells (Lai et al. J. Cell. Mol. Med 19: 474, 2014; Kodela et al. Drug Des. Dev. Ther. 9:4873, 2015). Aims of the present study were: (a) to investigate the pharmacological action of L‐cysteine (substrate for the biosynthesis of H2S) and NaHS (H2S‐releasing compound) on proliferation of HT29 cells at different time‐points, and (b) to study the role of enzymes (cystathionine β‐synthase, CBS and cystathionine γ‐lyase, CSE) and K+‐ATP channels on the response elicited by H2S‐producing compounds in HT29 cells. The colon adenoma HT29 cells were treated with different concentrations of L‐cysteine (1 μM – 1 mM) and NaHS (1 μM – 1 mM) for 24 and 48 hours, in the absence and presence of inhibitors of CBS and CSE, amino‐oxyacetic acid (AOA, 1 mM), an inhibitor of K+‐ATP channels, glibenclamide (GLB, 10 μM). After incubation, the anti‐proliferative response on cells caused by various agents was determined by MTT Assay. At equimolar concentrations (10 μM and 1 mM), both L‐cysteine and NaHS caused concentration‐dependent inhibitions of cell proliferation after incubation for 24 h, but increased cell growth at these same concentrations after 48 h of incubation. For instance, at 10 μM, both L‐cysteine and NaHS increased cell proliferation by 37% and 51% after 48 h of incubation. Pretreatment of cells with AOA (1 mM) had no significant (P&gt;0.05) effect on the inhibitory response caused by L‐cysteine and NaHS after 24 h incubation. Interestingly, AOA blocked and even reversed the increase in proliferation caused by L‐cysteine (10 μM and 1 mM) and NaHS (10 μM and 1 mM) after 48 h incubation. After 24 h, both L‐cysteine and NaHS caused additional inhibitory effects on cell proliferation in HT 29 cells pretreated with GLB (10 μM). GLB blocked and even reversed the increase in proliferation caused by L‐cysteine and NaHS in HT29 cells after 48 h incubation. We conclude that H2S‐releasing compounds can elicit a time‐dependent inhibitory or excitatory action on the proliferation of HT29 cells. The inhibitory/excitatory effects caused by these compounds depends, at least in part, on endogenous biosynthesis of new gas and the activity of K+‐ATP channels in HT29 cells.Support or Funding InformationThis research is supported by Tittle III under the Award Number P031B090216;Title III, Part B, Historically Black Graduate Institutions (HBGI) (CFDA No. 84.031B).

Cyanidin Chloride Induces Apoptosis by Inhibiting NF-κB Signaling through Activation of Nrf2 in Colorectal Cancer Cells.

Colorectal cancer (CRC) is the third most common cancer worldwide and a leading cause of cancer-related deaths in developed countries. Anthocyanins are a class of flavonoids, widely distributed in food, exhibiting important biological effects. Cyanidin chloride (CyCl) is the common type of anthocyanin with antioxidative and anti-inflammatory potential. The present study aimed to investigate the molecular mechanisms underlying the chemotherapeutic effects of CyCl in colorectal cancer cells. We found that CyCl treatment induced apoptosis as well as a significant inhibition of cellular proliferation and colony formation in three colon cancer HCT116, HT29, and SW620 cells. In addition, CyCl suppressed nuclear factor-kappa B (NF-κB) signaling and induced the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in tumor necrosis factor-alpha (TNF-α)-stimulated colon cancer cells. Nrf2 and NF-κB are two key transcription factors regulating antioxidative responses and cellular proliferation, respectively. In this study, knockdown of Nrf2 by small interfering RNA (siRNA) transfection inhibited the effect of CyCl on NF-κB signaling and apoptosis, suggesting that there is functional crosstalk between Nrf2 and NF-κB. Our findings demonstrate the important role of Nrf2 in inducing apoptosis through the involvement of NF-κB signaling in colorectal cancer cells, suggesting that CyCl may be used as a potential therapeutic agent for CRC.

A comparative study to elucidate the biological activities of crude extracts from rice bran and wheat bran in cell line models.

The present study investigated the nutritional composition of bran from rice (RB) and wheat (WB) and compared the natural virtues of crude extracts based on phenolic composition, antidiabetic and anticancer activities. The profiling of phenolic-rich ethyl acetate extracts (RBE and WBE) confirms that RBE is rich in catechol (0.122mg/g dw), p-coumaric acid (0.159mg/g dw), kaempferol (0.374mg/g dw) and apigenin (0.399mg/g dw); and WBE is affluent with catechol (0.144mg/g dw), ferulic acid (0.160mg/g dw), caffeic acid (0.083mg/g dw) and ellagic acid (0.074mg/g dw). RBE exhibited better antioxidant activity, inhibited the activity of α-amylase (IC50-353.41µg/mL) and α-glucosidase (IC50-314.22µg/mL), hindered glycation process (IC50-451.11µg/mL), and enhanced glucose uptake in L6 muscle cells (20.4%) indicating its potential in diabetic management. RBE was toxic to HT29 colon cancer cells and decreased cell membrane integrity. RBE and WBE arrested cell-cycle transition in HT29 cells from G0 to G1 and G2 to M phase respectively and induced apoptosis (27.15% and 5.9%, respectively for RBE and WBE) suggesting anticancer activities of the extract. The study indicates that bran from rice and wheat are a potential source of dietary fibre and phytochemicals with antidiabetic and anticancer properties for developing value-added products with nutraceutical benefits.

Changes in extracellular matrix in different stages of colorectal cancer and their effects on proliferation of cancer cells.

BACKGROUNDThe extracellular matrix is the main component of the tumor microenvironment. Extracellular matrix remodels with the oncogenesis and development of tumors. Previous studies usually focused on the changes of proteins in normal colorectal tissues and colorectal cancers. Little is known about the changes in the extracellular matrix in different stages of colorectal cancer and the effects of these changes on the development of this cancer.AIMTo test the changes of type I collagen, type IV collagen, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-3 (TIMP-3) in different stages of colorectal cancer and the effects of these changes on the proliferation of cancer cells.METHODSThe extracellular matrix from various stages of colorectal cancer and normal colon tissue was obtained by using acellular technology. We used proteomics to detect the differential expression of proteins between normal colon tissues and colorectal cancer tissues, and then we used Western blot to observe their expression in each stage of colorectal cancer and in normal colon tissue. By co-culturing the extracellular matrix and HT29 colon cancer cells in vivo and in vitro, we tested the cancer cell proliferation rate in vitro by methyl thiazolyl tetrazolium (MTT) assay and in vivo by measuring the tumor volume.RESULTSThe expression of type I collagen and MMP-2 increased with increased tumor stage. The expression of MMP-9 was higher in colorectal cancer tissues and was highest in stage III cancer. The expression of type IV collagen and TIMP-3 decreased with increased tumor stage. The proliferation rate of cancer cells in the extracellular matrix of colorectal cancer was higher than that in the extracellular matrix of the normal colon.CONCLUSIONThese data suggest that the extracellular matrix structure and composition become disorganized during the development of tumors, which is more conducive for the growth of cancer cells.

Anticancer Activities of Meroterpenoids Isolated from the Brown Alga Cystoseira usneoides against the Human Colon Cancer Cells HT-29.

Colorectal cancer (CRC) is one of the most common types of cancers and a leading cause of cancer death worldwide. The current treatment for CRC mainly involves surgery, radiotherapy, and chemotherapy. However, due to the side effects and the emergence of drug resistance, the search for new anticancer agents, pharmacologically safe and effective, is needed. In the present study, we have investigated the anticancer effects of eight algal meroterpenoids (AMTs, 1-8) isolated from the brown seaweed Cystoseira usneoides and their underlying mechanisms of action using HT-29, a highly metastatic human colon cancer cell line. All the tested meroterpenoids inhibited the growth of HT-29 malignant cells and were less toxic towards non-cancer colon cells, with the AMTs 1 and 5 exhibiting selectivity indexes of 5.26 and 5.23, respectively. Treatment of HT-29 cells with the AMTs 1, 2, 3, 4, 5, and 7 induced cell cycle arrest in G2/M phase and, in some instances, apoptosis (compounds 2, 3, and 5). Compounds 1-8 also exhibited significant inhibitory effects on the migration and/or invasion of colon cancer cells. Mechanistic analysis demonstrated that the AMTs 1, 2, 5, 6, 7, and 8 reduced phosphorylation levels of extracellular signal-regulated kinase (ERK) and the AMTs 2, 3, 4, 5, 7, and 8 decreased phosphorylation of c-JUN N-terminal kinase (JNK). Moreover, the AMTs 1, 2, 3, 4, 7, and 8 inhibited phosphorylation levels of protein kinase B (AKT) in colon carcinoma cells. These results provide new insights into the mechanisms and functions of the meroterpenoids of C. usneoides, which exhibit an anticancer effect on HT-29 colon cancer cells by inducing cell cycle arrest and apoptosis via the downregulation of ERK/JNK/AKT signaling pathways.

A melt-electrowritten filter for capture and culture of circulating colon cancer cells.

Metastasis is the major cause of death in cancer patients accounting for about 90% of the mortality. The detection and analysis of the hallmark of metastasis, circulating tumor cells (CTCs), have significant impact in cancer biology and clinical practice. However, the scarcity of CTCs in blood, particularly in that of colorectal cancer patients, is a serious bottleneck in the development of CTC-based precision medicine. Herein, the melt electrowriting (MEW) technology was used for reproductive fabrication of a biocompatible antibody-presenting polycaprolactone filter with tailored porous structure. It is demonstrated, for the first time, that such filter can be used not only to catch cancer cells spiked in whole blood but also to culture the cancer cells directly on site. Specifically, HT29 colon cancer cells can be captured with an efficiency of 85%, and when spiked into 4 mL of whole blood, 47% were captured on one Ø12mm filter. Furthermore, repeated capture and culture experiments have shown that as few as 20 HT29 colon cancer cells spiked into 4 mL of whole blood can be captured on the filter and within 2 weeks be expanded on site to become tumor bodies that are visible to the untrained eye. This filter allows for downstream analysis, such as flow cytometry, immunocytochemistry, Western blotting, and rt-qPCR. This technology represents a simple and cost-effective platform that potentially enables fast and efficient culture of rare CTCs from patients’ blood. This provides non-invasive alternatives for solid biopsy tumor materials for treatment screening, with great potential to realize precision medicine for cancer treatment.

Abstract B104: Immunomodulatory effect of 15-hydroxy prostaglandin dehydrogenase in colon cancer

Abstract Cyclooxygenase-2, the key player in the biosynthesis of prostaglandin-E2 (PGE2) is often upregulated in colon cancer. In addition, TNF-α, the prime regulator of COX-2 and mPGES-1, also contributes to increased synthesis of PGE2. However, 15-hydroxy prostaglandin dehydrogenase, a tumor suppressor playing major role in PGE2 catabolism, is often downregulated in colon cancer. Interestingly, colon cancer patients expressing high levels of cysteinyl leukotriene receptor-2 (CysLT2R) show good prognosis and therefore, we investigated a potential link between CysLT2R-signaling and 15-PGDH in metastatic colon cancer. Elevation of 15-PGDH expression by LTC4, a CysLT2R ligand, exhibited antitumor activity in colon cancer cells with significant phosphorylation of β-catenin with concurrent activation of CASPASE-3 and dramatic downregulation of TNF-α as well as NFκβ1 mRNA expression in HT-29 and Caco-2 colon cancer cells. The interaction between programmed death ligand 1 (PD-L1) and programmed death receptor 1 (PD-1) of cancer cells and T cells, respectively, leads to T-cell inactivation and tumor escape. We observed that recombinant TNF-α stimulated colon cancer cells expressed high PD-L1 compared to the unstimulated counterparts, which was downregulated by LTC4 treatment. Protumorigenic role and association of tumor-associated macrophage (TAMs) with PD-L1 was also evident with increase in CD-47 mRNA expression. Human (SW480, SW620) and mouse (MC-38) colon cancer cells exposed to conditioned media of chemically differentiated THP-1 and RAW 264.7 cells, respectively, showed abundant expression of PD-L1 in both mRNA and protein level along with CD-47 in mRNA level, which was inhibited by LTC4 stimulation via activation of CysLT2R in a 15-PGDH dependent manner. Hence restoration of 15-PGDH expression through CysLT2R-signaling promotes immunomodulatory effect, indicating a promising therapeutic alternative for metastatic colon cancer with high TAM infiltration. Citation Format: Shakti R. Satapathy, Anita Sjolander. Immunomodulatory effect of 15-hydroxy prostaglandin dehydrogenase in colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B104.

Amino Alcohol Acrylonitriles as Activators of the Aryl Hydrocarbon Receptor Pathway: An Unexpected MTT Phenotypic Screening Outcome.

Lead (Z)-N-(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide, 1 showed MCF-7 GI50 =30 nM and 400-fold selective c.f. MCF10A (normal breast tissue). Acetamide moiety modification (13 a-g) to introduce additional hydrophobicity was favoured with MCF-7 breast cancer cell activity enhanced at 1.3 nM. Other analogues were potent against the HT29 colon cancer cell line at 23 nM. Textbook SAR data was observed in the MCF-7 cell line, in an MTT assay, via the ortho (17 a), meta (17 b) and para (13 f). The amino alcohol -OH moiety was pivotal, but no stereochemical preference noted. But, these data did not fit our homology modelling expectations. Aberrant MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) screening results and metabolic interference confirmed by sulforhodamine B (SRB) screening. Interfering analogues resulted in 120 and 80-fold CYP1A1 and CYP1A2 amplification, with no upregulation of SULT1A1. This is consistent with activation of the AhR pathway. Piperidine per-deuteration reduced metabolic inactivation. 3-OH / 4-OH piperidine analogues showed differential MTT and SRB activity supporting MTT assay metabolic inactivation. Data supports piperidine 3-OH, but not the 4-OH, as a CYP substrate. This family of β-amino alcohol substituted 3,4-dichlorophenylacetonitriles show broad activity modulated via the AhR pathway. By SRB analysis the most potent analogue was 23 b, (Z)-3-(4-(3-(4-phenylpiperidin-1-yl)-2-hydroxypropoxy)phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile.

Quantum Dot Labelling of Tepary Bean (Phaseolus acutifolius) Lectins by Microfluidics.

Lectins are bioactive proteins with the ability to recognize cell membrane carbohydrates in a specific way. Diverse plant lectins have shown diagnostic and therapeutic potential against cancer, and their cytotoxicity against transformed cells is mediated through the induction of apoptosis. Previous works have determined the cytotoxic activity of a Tepary bean (Phaseolus acutifolius) lectin fraction (TBLF) and its anti-tumorigenic effect on colon cancer. In this work, lectins from the TBLF were additionally purified by ionic-exchange chromatography. Two peaks with agglutination activity were obtained: one of them was named TBL-IE2 and showed a single protein band in two-dimensional electrophoresis; this one was thus selected for coupling to quantum dot (QD) nanoparticles by microfluidics (TBL-IE2-QD). The microfluidic method led to low sample usage, and resulted in homogeneous complexes, whose visualization was achieved using multiphoton and transmission electron microscopy. The average particle size (380 nm) and the average zeta potential (−18.51 mV) were determined. The cytotoxicity of the TBL-IE2 and TBL-IE2-QD was assayed on HT-29 colon cancer cells, showing no differences between them (p ≤ 0.05), where the LC50 values were 1.0 × 10−3 and 1.7 × 10−3 mg/mL, respectively. The microfluidic technique allowed control of the coupling between the QD and the protein, substantially improving the labelling process, providing a rapid and efficient method that enabled the traceability of lectins. Future studies will focus on the potential use of the QD-labelled lectin to recognize tumor tissues.

Antiproliferative activity of cycloalkanecarboxamide derivatives possessing sulfonate or sulfamate moiety.

A series of cycloalkanecarboxamide-containing sulfonate and sulfamate derivatives were prepared, and their antiproliferative activity was tested against NCI-60 cancer cell lines panel. Compound 1f possessing cyclohexyl and p-(tert-butyl)benzenesulfonate moieties was the most active among all the target compounds. It exerted broad-spectrum anticancer activity against all the nine cancer types involved in the NCI-60 panel. Additionally, compound 1g containing cyclohexyl and p-fluorobenzenesulfonate moieties was the most potent against HT29 colon cancer cell line (IC50=4.73µM) with selectivity index more than 4.23 towards HT29 than normal fibroblasts. It exerts its antiproliferative activity against HT29 through the induction of apoptosis (increasing caspase 3/7 activity) but not necrosis. Structure-activity relationship studies are presented in detail.

High Levels of Apoptosis Are Induced in the Human Colon Cancer HT-29 Cell Line by Co-Administration of Sulforaphane and a Peptide Nucleic Acid Targeting miR-15b-5p.

Sulforaphane (SFN) is one of most important dietary constituents of broccoli (Brassica oleracea) and other cruciferous vegetables, which have been reported to exhibit health benefits, including prevention and therapy of cancer, such as colorectal carcinoma (CRC). The objective of this study was to determine whether the anticancer effect of SFN on colon cancer HT-29 cell line could be improved by the combined treatment with molecules inhibiting microRNAs (miRNAs) involved in CRC. As miRNA inhibiting molecules we focused on peptide-nucleic acids (PNAs). As miRNA to be targeted, miR-15b-5p was selected on the basis of several information present in the literature and confirming that miR-15b-5p is overexpressed in colon cancer patients, and that its targeting decreases cell migration and metastasis in colorectal cancer. In this article, we described for the first time the efficacy of targeting miR-15b-5p by using a PNA against miR-15b-5p (R8-PNA-a15b), functionalized with an octoarginine peptide (R8) for maximizing cellular uptake. The miR-15b-5p downregulation in the colon cancer HT-29 cell line was associated with inhibition of in vitro cell growth and activation of the proapoptotic pathway, demonstrated by a sharp increase of late apoptotic cells in HT-29-treated cell populations. A second conclusion of this study is that the R8-PNA-a15b might be proposed in "combo-therapy" associated with SFN. To our knowledge, no report is available in the literature on a combination between SFN and miRNA-targeting molecules. Our data demonstrate that this combined treatment leads to a very high proportion of apoptotic HT-29 cells (over 85%), a value higher than the sum of the values of apoptotic cells obtained after singularly administered regents (either SFN or R8-PNA-a15b).

Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis

BackgroundP53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells. PurposeTo explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation. Study design and methodsDifferent mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53. ResultsThe triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation. ConclusionOur study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.

Novel nano-vehicle for delivery and efficiency of anticancer auraptene against colon cancer cells

The aim of this study is to devise, prepare and characterize nano encapsulated auraptene (AUR) and evaluate cytotoxic and apoptotic effects on HT-29 colon cancer cells. Herein, AUR nano formulations were prepared by triblock (PCL-PEG-PCL) and pentablock (PLA-PCL-PEG-PCL-PLA) biodegradable copolymers in order to increase AUR bioavailability as an anticancer agent. The preparation of nano particles (NPs) was done with rotor stator homogenization (RSH) and Ultrasonic homogenization (USH) methods. The physicochemical characteristics of prepared nanoparticles (NPs) were studied using HNMR, FTIR, GPC, DLS and SEM techniques. The smaller hydrodynamic size (110 nm) and polydispersity index (PDI: 0.288) as well as higher cellular uptake (89%) were observed in PB NPs rather than TB NPs. The highest cytotoxic and apoptotic effects were observed in AUR loaded PB NPs compared to AUR loaded TB NPs and free AUR obtained by MTT assay, cell cycle arrest, Annexin V-FITC, DAPI staining and RT-PCR techniques. Real time PCR results indicated that Bax /Bcl2 expression ratio as an apoptosis predicting criterion, in free AUR, AUR loaded TB and AUR loaded PB have increased 6, 9 and 13 times, respectively (p value < 0.05). In conclusion, using biodegradable nano-vehicles for sustained delivery of natural anti-cancer compounds may open new perspectives for treatment of cancer patients.

Effect of Lactobacillus acidophilus CICC 6074 S-Layer Protein on Colon Cancer HT-29 Cell Proliferation and Apoptosis.

The objective of this research was to investigate the effects of Lactobacillus acidophilus CICC 6074 S-layer protein on the viability, adhesion, cell cycle, and apoptosis of human colon cancer HT-29 cells and to explore their molecular mechanism of tumor suppression. The S-layer protein at doses of 0, 25, 50, and 100 mg/L significantly suppressed the proliferation of HT-29 cells. The S-layer protein exerts its cytotoxic activities against colon cancer HT-29 cells by arresting the cell cycle in the G1 phase through upregulating the expression of p53, p21, and p16 and downregulating the expression of CDK1 (cyclin-dependent kinases) and cyclin B. Morphological changes were further observed by transmission electron microscopy, and the cells treated with the S-layer protein showed obvious characteristic changes of apoptosis including chromatin condensation, nuclear fragmentation, vacuoles, and so on. Furthermore, our mechanism studies indicated that the S-layer protein may induce HT-29 cell apoptosis through the death receptor apoptotic pathway and mitochondrial pathway and impede cell invasion by inhibiting the synthesis of the PI3K/AKT pathway and FasL. These results demonstrated that the L. acidophilus CICC 6074 S-layer protein may be a potential anticarcinogenic agent.

Apoptosis Induction with Enhancement of BAX/BCL2 Gene Expression Ratio via Combination Therapy in HT29 Colon Cancer Cells

Background and Aim: Combination therapy is one of the new strategies that minimize resistance to chemotherapy and reduces drug toxicity. Here, we investigated the effect of combination therapy with 5-Fluorouracil and Gamma Tocopherol on cell survival and BAX/BCL2 gene expression ratio in HT29 colon cancer cells. Methods: The proliferation of cancer cells was determined via colony formation assay. BAX/BCL2 ratio was evaluated after incubation with concentrations of 5-Fluorouracil and Gamma Tocopherol via real-time-PCR. Results: The average number of colonies in the cells treated with 5-Fluorouracil, Gamma Tocopherol and their combination of them was 63±4, 78±3, and 28±2, respectively which significantly decreased in the combination group. In contrast with the control group, the BAX/BCL2 ratio remarkably increased when the cell underwent combinational treatment (p<0.05). Conclusion: 5-Fluorouracil and Gamma Tocopherol reduced HT 29 cell proliferation. Our results suggest that combination therapy with 5- Fluorouracil and Gamma Tocopherol can be considered as a strategy for induction of apoptosis via increasing the BAX/BCL2 ratio. *Corresponding Author: Nadereh Rashtchizadeh; Email: rashtchizadeh@rocketmail.com Please cite this article as: Bazzaz R, Yaghmaei P, Dastmalchi S, Rashtchizadeh N. Apoptosis Induction with Enhancement of BAX/BCL2 Gene Expression Ratio via Combination Therapy in HT29 Colon Cancer Cells. Arch Med Lab Sci. 2020;6:1-7 (e21). https://doi.org/10.22037/amls.v6.33489

Tributyltin(IV) ferulate, a novel synthetic ferulic acid derivative, induces autophagic cell death in colon cancer cells: From chemical synthesis to biochemical effects

Ferulic acid (FA) is a natural phenolic phytochemical that has low toxicity and exhibits therapeutic effects against various diseases, behaving as an antioxidant. FA also displays modest antitumor properties that have been reported at relatively high concentrations. With the aim of improving the anti-tumor efficacy of FA, we synthesized the novel compound tributyltin(IV) ferulate (TBT-F). The coordination environment at the tin center was investigated spectroscopically. Following synthesis, chemical characterization and computational analysis, we evaluated TBT-F effects in colon cancer cells. The results showed that TBT-F, at nanomolar range concentrations, was capable of reducing the viability of HCT116, HT-29 and Caco-2 colon cancer cells. On the other hand, FA was completely inefficacious at the same treatment conditions. Cell viability reduction induced by TBT-F was associated with G2/M cell cycle arrest, increase in membrane permeabilization and appearance of typical morphological signs. TBT-F-induced cell death seemed not to involve apoptotic or necroptotic markers whereas autophagic vacuoles appearance and increase in LC3-II and p62 autophagic proteins were observed after treatment with the compound. The autophagy inhibitor bafylomicin A1 markedly prevented the effect of TBT-F on colon cancer cells, thus indicating that autophagy is triggered as a cell death process.Taken together, our results strongly suggest that the novel ferulic derivative TBT-F is a promising therapeutic agent for colon cancer since it is capable of triggering autophagic (type-II) cell death that may be important in case of resistance to classic apoptosis.

β-Lactams with antiproliferative and antiapoptotic activity in breast and chemoresistant colon cancer cells

A series of novel 1,4-diaryl-2-azetidinone analogues of combretastatin A-4 (CA-4) have been designed, synthesised and evaluated in vitro for antiproliferative activity, antiapoptotic activity and inhibition of tubulin polymerisation. Glucuronidation of CA-4 by uridine 5-diphosphoglucuronosyl transferase enzymes (UGTs) has been identified as a mechanism of resistance in cancer cells. Potential sites of ring B glucuronate conjugation are removed by replacing the B ring meta-hydroxy substituent of selected series of β-lactams with alternative substituents e.g. F, Cl, Br, I, CH3. The 3-phenyl-β-lactam 11 and 3-hydroxy-β-lactam 46 demonstrate improved activity over CA-4 in CA-4 resistant HT-29 colon cancer cells (IC50 = 9 nM and 3 nM respectively compared with IC50 = 4.16 μM for CA-4), while retaining potency in MCF-7 breast cancer cells (IC50 = 17 nM and 22 nM respectively compared with IC50 = for 4 nM for CA-4). Compound 46 binds at the colchicine site of tubulin, and strongly inhibits tubulin assembly at micromolar concentrations comparable to CA-4. In addition, compound 46 induced mitotic arrest at low concentration in both cell lines MCF-7 and HT-29 together with downregulation of expression of antiapoptotic proteins Mcl-1, Bcl-2 and survivin in MCF-7 cells. These novel antiproliferative and antiapoptotic β-lactams are potentially useful scaffolds in the development of tubulin-targeting agents for the treatment of breast cancers and chemoresistant colon cancers.

The Prevention and Inhibition Effect of Anthocyanins on Colorectal Cancer.

Anthocyanins are a type of flavonoids that are natural water soluble glycosidic pigments with efficacious anti-cancer effects, which have good biological activity against many cancers including colorectal cancer (CRC). However, the exact molecular mechanism used by anthocyanins against cancer is unclear; it is also unclear what a reasonable dosage might be for their use against colorectal cancer. Western blotting, immunohistochemistry, MTT assay, xenograft model, and hematoxylin-eosin (HE) staining were used to perform the experiments. Compared with the control group, anthocyanins could significantly inhibit the cell viability and proliferation and promote the apoptosis of human colon cancer HT29 cells. Furthermore, anthocyanins reduced tumor weight and volume in a colon tumor mouse model and downregulated the expression of PI3K protein, inhibited AKT expression and phosphorylation, decreased the Bcl-2 and Bax ratio and reduced survivin protein expression in the tumor tissue. Anthocyanins promoted apoptosis of CRC cells and inhibited colon cancer growth of xenografted tumors. Mechanistically, anthocyanins enhanced the Bcl-2/Bax and caspase-dependent apoptotic pathways through targeting the PI3K/AKT/survivin pathway, resulting in impairment of growth of CRC.