HT-29 Colon Cancer Cells Research Articles

Effect of polyamide nanoparticles on apoptosis of colon cancer cells induced by survivin antisense oligonucleotide

In this study, we aimed to observe the effect of polyamidoamine (PAMAM) liposomes on the apoptosis of human colon cancer cells induced by survivin antisense oligonucleotides (ASODNs). PAMAM liposomes and PAMAM were mixed with survivin ASODNs to obtain antisense gene transfection complexes. In addition, the zeta potentials and encapsulation rates of the complexes were measured. The two gene-containing complexes were transfected into HT-29 colon cancer cells to observe changes in cell morphology, detect the inhibitory effect on tumor cells and changes in apoptosis, and observe changes in the cytoskeleton microfilament system using laser confocal microscopy. Caspase-3 activity in the cells was determined using a kinase activity assay, and p38 mitogen-activated protein kinase (p38 MAPK) activity in the cells was measured using immunoprecipitation analysis. The results showed that the zeta potential of the PAMAM liposome-survivin-ASODN complex was higher than that of the PAMAM-survivin-ASODN complex (P < 0.05). There was no significant difference in the gene encapsulation rates between the two complexes (P > 0.05). PAMAM liposomes may efficiently deliver survivin ASODNs to human colon cancer cells, reduce the expression of survivin protein and at the same time induce G2/M phase arrest in cells, and activate caspase-3 by activating p38 MAPK. Cleavage of caspase-3 destroys the structure of the intracellular skeletal microfilament system, finally resulting in apoptosis of colon cancer cells.

Microwave irradiation-based synthesis of anisotropic gold nanoplates using Staphylococcus hominis as reductant and its optimization for therapeutic applications

The pivotal aim of this investigation was to synthesize anisotropic gold nanoplates using Staphylococcus hominis strain MANF2 as reducing agent and optimize statistically its production for therapeutic applications. Initially, gold nanoparticles were synthesized using strain MANF2 via microwave irradiation method which reduced Au3+ ions to Au+ ions, as evident by change in the colour of solution to purple, followed by observation of characteristic surface plasmon resonance peak (λmax) at 530 nm in UV–vis spectrum. Box-Behnken Design-based optimization (N = 17) estimated maximum production of nanoparticles (λmax – 3.18) under the conditions of 1 mM of auric chloride, 1 mL of reducing agent, and irradiation period of 45 s. Data obtained were modelled and trained using multilayer perceptron and Marquardt-Levenberg algorithm of artificial neural networking, respectively which showed the constructed neural network topology “3-8-1” neurons in input, hidden, and output layers, depicting the best fitness function. The optimized nanoparticles were characterized using varied analytical tools viz. UV–vis spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy, Zeta potential analyzer, Dynamic light scattering (DLS), X-ray diffraction (XRD), and Energy-Dispersive X-ray Spectroscopy (EDX). Scanning electron microscopy (SEM) analysis revealed pentagonal and square-shaped gold nanoplates of 100 nm in size with sharp edges. Anti-tubercular activities of gold nanoplates were assessed using luciferase reporter phase (LRP) assay which exhibited relative light unit reduction of 38.2 ± 0.3–92.6 ± 0.4 % at varied concentrations. Anticancer activity of gold nanoplates was demonstrated against HT-29 (colon cancer) cell line using MTT assay. Results showed promising anticancer traits of gold nanoplates with IC50 value of 41.3 μg/mL against HT-29 cells. In conclusion, S. hominis-based synthesized anisotropic/polygonal gold nanoplates can certainly be implied as auspicious anti-tubercular and anticancer agents for designing ideal therapeutic drugs in future.

Naringin Combined with NF-κB Inhibition and Endoplasmic Reticulum Stress Induces Apoptotic Cell Death via Oxidative Stress and the PERK/eIF2α/ATF4/CHOP Axis in HT29 Colon Cancer Cells.

Currently, combination therapy is considered the most effective solution for a selective chemotherapeutic effect in the treatment of colon cancer. This study investigated the death of both colon cancer HT29 cells and healthy vascular smooth muscle TG-Ha-VSMC cells (VSMCs) induced by naringin combined with endoplasmic reticulum (ER) stress and NF-κB inhibition. Naringin combined with tunicamycin and BAY 11-7082 suppressed the proliferation of HT29 cells in a dose-dependent manner and induced particularly apoptotic death without significantly affecting healthy VSMCs according to Annexin V/PI staining and AO/EB staining analyses. Insufficient antioxidant defense and heat shock response as well as excessive ROS generation were observed in HT29 cells following combination therapy. Quantitative real-time PCR and western blot analysis demonstrated that drug combination-induced mitochondrial apoptosis was activated through the ROS-mediated PERK/eIF2α/ATF4/CHOP pathway. Additionally, naringin combination significantly reduced the sXBP expression induced by tunicamycin+BAY 11-7082 in a dose-dependent manner. In conclusion, this study found that naringin combined with tunicamycin+BAY 11-7082 efficiently induced apoptotic cell death in HT29 colon cancer cells via oxidative stress and the PERK/eIF2α/ATF4/CHOP pathway, suggesting that naringin combined with tunicamycin plus BAY 11-7082 could be a new combination therapy strategy for effective colon cancer treatment with minimal side effects on healthy cells.

The Effects of Doxorubicin, Ethanol Extract and Flavonoid-rich Fraction of Euphorbia Splendida Mobayen on the PARP Level, and APC Gene Expression in HT-29 Human Colon Cancer Cell Line

Background: Natural products derived from various sources are being used to develop chemotherapeutic drugs. Euphorbiaceae is widely used to treat different types of cancers. Colorectal cancer (CRC) is the second and third cause of cancer in women and men, respectively. CRC is strongly associated with the deregulation of the Adenomatous polyposis coli (APC) gene and Poly [ADP-ribose] polymerase (PPAR) protein. Objectives: The current study aimed to examine the effect of doxorubicin, ethanol extract, and the flavonoid-rich fraction of Euphorbia Splendida Mobayen on colon cancer HT-29 cell line death, APC gene expression, and PPAR concentration. Methods: Following treatment of cells by Euphorbia ethanol extract, Euphorbia flavonoid-rich fraction, and doxorubicin, cell viability assay was used to investigate the viability status of the HT-29 cell line. Total RNA was isolated from the cell line and converted into cDNA. The expression level of the APC gene was determined by quantitative real-time PCR. Poly (ADP-ribose) polymerase (PPAR) protein was detected by the ELIZA method. Results: We found that Euphorbia ethanol extract, Euphorbia flavonoid-rich fraction, and doxorubicin can stimulate dose-dependent cell death in the HT-29 cell line, increase ACP gene expression (P = 0.001, P = 0.041, P = 0.019), and decrease PARP level (P = 0.001, P = 0.001, P = 0.001, respectively). Conclusions: The findings indicated that doxorubicin, ethanol extract, and the flavonoid-rich fraction of Euphorbia Splendida Mobayen had cytotoxic effects on human colon cancer HT-29 cell line by possibly stimulating apoptosis.

Investigating an in silico approach for prioritizing antidepressant drug prescription based on drug-induced expression profiles and predicted gene expression.

In clinical practice, an antidepressant prescription is a trial and error approach, which is time consuming and discomforting for patients. This study investigated an in silico approach for ranking antidepressants based on their hypothetical likelihood of efficacy. We predicted the transcriptomic profile of citalopram remitters by performing an in silico transcriptomic-wide association study on STAR*D GWAS data (N = 1163). The transcriptional profile of remitters was compared with 21 antidepressant-induced gene expression profiles in five human cell lines available in the connectivity-map database. Spearman correlation, Pearson correlation, and the Kolmogorov-Smirnov test were used to determine the similarity between antidepressant-induced profiles and remitter profiles, subsequently calculating the average rank of antidepressants across the three methods and a p value for each rank by using a permutation procedure. The drugs with the top ranks were those having a high positive correlation with the expression profiles of remitters and that may have higher chances of efficacy in the tested patients. In MCF7 (breast cancer cell line), escitalopram had the highest average rank, with an average rank higher than expected by chance (p = 0.0014). In A375 (human melanoma) and PC3 (prostate cancer) cell lines, escitalopram and citalopram emerged as the second-highest ranked antidepressants, respectively (p = 0.0310 and 0.0276, respectively). In HA1E (kidney) and HT29 (colon cancer) cell types, citalopram and escitalopram did not fall among top antidepressants. The correlation between citalopram remitters' and (es)citalopram-induced expression profiles in three cell lines suggests that our approach may be useful and with future improvements, it can be applicable at the individual level to tailor treatment prescription.

Assessment of anti-cancer effects of koenimbine on colon cancer cells.

Recent studies have highlighted the role of natural elements in reduction of cancer cell growth and apoptosis. Koenimbine, a natural product isolated from Murraya koenigii (L) Spreng is a substance with cytotoxic effects on cancer cells. The effects of koenimbine on HT-29 and SW48 colon cancer cells were evaluated by MTT and Annexin V assays. Expression levels of Wnt/β-catenin pathway genes were quantified by real time PCR. The IC50 values of koenimbine in HT-29 and SW48 was calculated to be 50μg/ml based on the results of MTT assay. This value was 75μg/ml in IEC-18 cells which were used as normal control. Annexin V assays revealed induction of cell apoptosis and necrosis in HT-29 and SW48 cells but not IEG18 cells by koenimbine. Koenimbin treatment resulted in significant down-regulation of CYCLD1 expression in SW48 cell line, but up-regulation of this gene in HT29 cell line. Expression of TBLR1, DKK1, GSK3B and β-catenin was significantly decreased after koenimbin treatment in HT-19 cell line. Moreover, expression of DKK1 and GSK3B was significantly decreased after koenimbin treatment in SW-40 cell line. TCF4 expression was not detected in any of cell lines either before or after treatment with koenimbin. The current in vitro study showed the cytotoxic effects of koenimbin on two colon cancer cell lines and the effects of this substance on expression of selected genes from Wnt-β catenin pathway. Future in vivo studies are needed before suggestion of this substance as an anti-cancer drug.

Characterizing therapeutic signatures of transcription factors in cancer by incorporating profiles in compound treated cells.

Cancers are promoted by abnormal alterations in biological processes, such as cell cycle and apoptosis. An immediate reason for those aberrant processes is the deregulation of their involved transcription factors (TFs). Thus, the deregulated TFs in cancer have been experimented as successful therapeutic targets, such as RARA and RUNX1. This therapeutic strategy can be accelerated by characterizing new potential TF targets. Two kinds of therapeutic signatures of TFs in A375 (skin) and HT29 (colon) cancer cells were characterized by analyzing TF activities under effective and ineffective compounds to cancer. First, the therapeutic TFs (TTs) were identified as the TFs that are significantly activated or repressed under effective compared to ineffective compounds. Second, the therapeutically correlated TF pairs (TCPs) were determined as the TF pairs whose activity correlations show substantial discrepancy between the effective and ineffective compounds. It was facilitated by incorporating (i)compound-induced gene expressions (LINCS), (ii) compound-induced cell viabilities (GDSC) and (iii) TF-target interactions (TRUST2). As a result, among 627 TFs, the 35 TTs (such as MYCN and TP53) and the 214 TCPs (such as FOXO3 and POU2F2 pair) were identified. The TTs and the proteins on the paths between TCPs were compared with the known therapeutic targets, tumor suppressors, oncogenes and CRISPR-Cas9 knockout screening, which yielded significant consequences. We expect that the results provide good candidates for therapeutic TF targets in cancer. The data and Python implementations are available at https://github.com/jmjung83/TT_and_TCP. Supplementary data are available at Bioinformatics online.

Different Polarity Extracts of Polygonum minus towards Cytotoxic Activities against Colon Cancer Cell Lines (HT-29, HCT-116, CT-26)

Aims: To investigate the cytotoxic properties of different polarity solvents of Polygonum minus extracts towards colon cancer cell lines, HT-29, HCT-116 and CT-26.
 Study Design: Experimental study.
 Place and Duration of Study: Central Laboratory, Tissue Culture Laboratory, Universiti Sultan Zainal Abidin, Terengganu between September 2019 until December 2019.
 Methodology: The different polarity solvents of P. minus extracts had been led to tetrazolium salt reduction (MTT) assay and an inhibition concentration of 50 (IC50) value for their cytotoxic potential against colon cancer cells. Then, cell morphology observation and fluorescence double staining of treatment cells were determined using a light inverted microscope and acridine orange/propidium iodide staining.
 Results: The results indicated that an extraction yields aligned from 0.01% for acetone and ethyl acetate to 0.45% for aqueous solution with decreasing order of aqueous solution > 70% aqueous ethanol > 50% aqueous ethanol > methanol > ethanol > acetone and ethyl acetate. Meanwhile, the ethyl acetate extract showed a higher cytotoxic effect at IC50 values of 7.00 ± 0.06 µg/mL and 7.00 ± 0.30 µg/mL towards the HCT-116 and CT-26 cells; and 50% aqueous ethanol towards HT-29 cells (24.00 ± 0.01 µg/mL). The different solvent extracts of P. minus induced cytotoxic effects on the treated cell lines by altering their normal cell morphology and cell membrane integrity (except for acetone extract).
 Conclusion: Therefore, the use of different polarity solvent extracts of P. minus as an anti-cancer agent is promising more on ethyl acetate and warrants further investigation.

Composition of polysaccharides and radiosensitizing activity of native and sulfated laminarans from the Tаuуа basicrassa Kloczc. et Krupn

Polysaccharide fractions of alginate, laminarans and fucoidans were obtained from the brown alga Tauya basicrassa. Yields of alginate and laminarans were large (19.7 % and 5.62 %, respectively), whereas the content of fucoidans (0.52 %) was not significant. Alginate and laminarans had typical structures for those substances. Fucoidans were low- and medium-sulfated heterogeneous polysaccharides. The fucoidan fraction 1TbF1 was sulfated fucogalactan containing a backbone from 1,6-linked residues of β-d-galactopyranose with branches at C3 and C4, terminal fucose and galactose residues and fragments from 1,3-; 1,4-; and 1,2-fucose residues. Sulfate groups were found at positions 2 and 4 of fucose, and positions 2, 3 and 4 of galactose residues. Laminaran 2TbL was subjected to a sulfation to obtain the derivative 2TbLS with partial sulfation (46 %) at C2, C4 and C6. It was shown that 2TbL and 2TbLS inhibited colony formation of sensitize-tested colon cancer cells HT-29 and HCT-116 to X-ray radiation.

A Natural Flavone Tricin from Grains Can Alleviate Tumor Growth and Lung Metastasis in Colorectal Tumor Mice.

Tricin, a flavone isolated from rice bran, has been shown to be chemopreventive in a colorectal cancer (CRC) mouse model. This study aimed to illustrate the inhibitory activities of tricin in colon cancer cells and in a metastatic CRC mouse model. BALB/c mice injected with mouse Colon26-Luc cells into the rectum wall were treated with tricin (37.5 mg/kg) daily for 18 days. Orthotopic colon tumor growth and metastasis to lungs were assessed by in vivo bioluminescence imaging. Results showed that tricin suppressed Colon-Luc cells motility and downregulated phosphorylated Akt, Erk1/2 and NF-κB expressions of human colon cancer HT-29 cells. While tricin treatment suppressed tumor growth and lung metastasis as well as altered the populations of myeloid-derived suppressor cells and regulatory T cells in spleens. In summary, the tumor microenvironment modulatory and anti-metastatic effects of tricin in colon cancer mouse model were shown for the first time, suggesting the potential development of tricin-containing food supplements for CRC patients.

A novel circular RNA hsa_circ_0060927 may serve as a potential diagnostic biomarker for human colorectal cancer.

CYP24A1 plays a role in strictly regulated vitamin D metabolism pathway and has been nominated as a prognostic biomarker for colorectal cancer (CRC). Increasing evidence suggests that circular RNAs (circRNAs) are involved in cancer initiation and progression; however, their diagnostic values in cancers as the new potential biomarkers are still not fully understood. In the present study, by using quantitative real-time polymerase chain reaction (qRT-PCR), we demonstrated that CYP24A1 and hsa_circ_0060927 were only transcribed in 33 and 25 out of 83 CRC tissues, respectively. In these samples, we demonstrated that CYP24A1 was up-regulated in both linear and circular forms (P<0.05). Furthermore, we showed that CYP24A1and hsa_circ_0060927 transcription were induced after vitamin D treatment in HCT-116 and HT-29 colon cancer cell lines. Importantly, analysis of the ROC curve in expressed samples indicated that CYP24A1 and hsa_circ_0060927 could serve as the potential diagnostic markers of CRC, but due to the fact that these transcripts are not expressed in many samples, the diagnostic role of these transcripts could be rejected. Taken together, for the first time, the results of our study illustrated the expression levels hsa_circ_0060927 in CRC; however, further studies are required to validate the present results.

Abstract 227: Maltose enhanced the growth of bladder and colon cancer cells unlike some other disaccharides: Cellobiose, isomaltose, lactose, and sucrose

Abstract The addiction of most cancer cells to the metabolism of glucose is well known. The metabolism by cancer cells of other saccharides is less well characterized. We have studied the impact on growth of human bladder and colon cancer cells of mono- and disaccharides. The standard RPMI 1640 medium contains 2 mg glucose per ml. Substitution for glucose by other monosaccharides (fructose, galactose and mannose) resulted in similar growth in some cell lines but greatly diminished growth in others. For the Caco-2 and HT29 colon cancer cells, co-incubation with 2-deoxyglucose was more inhibitory for growth with fructose than with glucose as substrate. There was a similar situation with some bladder cancer cell lines (5637, HT1197 and RT4) whereas with other bladder cancer cells (HT1376, T24 and UM-UC-3) 2-deoxyglucose caused greater inhibition with glucose. In those cell lines in which alkaline phosphatase activity could be induced after incubation with butyrate (Caco-2, HT29, 5637, HT1197 and HT1376), induction by butyrate was observed with any of the monosaccharides. Similarly with the disaccharides lactose, maltose and trehalose there was induction of alkaline phosphatase by butyrate. In the course of this work it was apparent that maltose could enhance growth to an extent that was sometimes not significantly different to that seen with glucose and was not seen with cellobiose, isomaltose, lactose or sucrose. HT29 cells were the only cell line to have a substantial increased growth with trehalose. The increased growth with maltose was inhibited by 2-deoxyglucose. In conclusion, maltose increased growth of bladder and colon cancer cells without a period of adaptation. Citation Format: Michael A. Lea, Charles desBordes. Maltose enhanced the growth of bladder and colon cancer cells unlike some other disaccharides: Cellobiose, isomaltose, lactose, and sucrose [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 227.

Abstract 4380: Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer

Abstract Background: The estrogen receptor beta (ERβ) is the predominant estrogen receptor in the normal colon mucosa, and its expression is downregulated in colorectal cancer (CRC). On the other hand, ER alpha (ERα) is very little expressed in the normal colon tissue. An increased ERα/β ratio has been shown in colon carcinomas. However, the role of ERα in CRC development and progression needs further evaluation. Aim: To investigate the role of ERα expression in CRC prognosis. Methods: A tumor tissue microarray of primary CRCs from 351 patients was stained for the expression of ERα. The nuclear staining intensity was evaluated using the Immuno-Reactive Score (IRS). Cox regression analysis and Kaplan-Meier curves were used to evaluate overall (OS) and disease-free survival (DFS). Colon cancer (CC) cells lines, SW-480, HCT-116 and HT-29 were used for in-vitro investigations. Two CC mouse models were used to investigate the expression of ERα. The zebrafish xenograft model was used to study CC cell metastasis. Untreated and ERα specific agonist (PPT, 40 nM) treated colon cancer cells were injected into the zebrafish perivitelline space. After 48 h the tale was checked for the presence of the metastasized cells. Results: ERα nuclear expression was negative in 232 and positive in 119 CRC patients, with a significantly higher expression in cancer tissue compared to normal tissue. Patients with positive ERα expression had higher TNM stage and more lymph node and distant metastasis. Positive ERα expression was independently associated with worse OS (HR=0.42; 95% CI: 0.26-0.51) and DFS (HR=0.32; 95% CI: 0.18-0.56), after adjustment for age and TNM stage. Moreover, patients with positive ERα expression had higher frequency of activated K-RAS mutation and higher expression levels of cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear β-catenin, both with potent pro-tumorigenic properties. In the colon tissue from the colitis-associated colon cancer (CAC) mouse model with the Cysltr1 gene disruption, ERα expression was significantly decreased compared to wild-type (WT) mice, while in the colon of ApcMin/+ mice the ERα expression was significantly increased. In-vitro, PPT treatment significantly increased cell migration and wound closure compared to untreated cells. The zebrafish xenograft model showed that fish injected with PPT stimulated CC cells had significantly more tail metastasis compared with the control group (80% versus 67% respectively). Conclusion: Positive ERα expression correlates with poor prognosis and metastasis in colorectal cancer. Our results suggest that ERα-selective antagonists might be beneficial to combine therapeutic opportunities for improving the prognosis of CRC patients. Citation Format: Geriolda Topi, Shakti R Satapathy, Roy Roy Ehrnström, Marie-Louise Lydrup, Anita Sjölander. Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4380.

A Scaffold-Free 3-D Co-Culture Mimics the Major Features of the Reverse Warburg Effect In Vitro.

Most tumors consume large amounts of glucose. Concepts to explain the mechanisms that mediate the achievement of this metabolic need have proposed a switch of the tumor mass to aerobic glycolysis. Depending on whether primarily tumor or stroma cells undergo such a commutation, the terms ‘Warburg effect’ or ‘reverse Warburg effect’ were coined to describe the underlying biological phenomena. However, current in vitro systems relying on 2-D culture, single cell-type spheroids, or basal-membrane extract (BME/Matrigel)-containing 3-D structures do not thoroughly reflect these processes. Here, we aimed to establish a BME/Matrigel-free 3-D microarray cancer model to recapitulate the metabolic interplay between cancer and stromal cells that allows mechanistic analyses and drug testing. Human HT-29 colon cancer and CCD-1137Sk fibroblast cells were used in mono- and co-cultures as 2-D monolayers, spheroids, and in a cell-chip format. Metabolic patterns were studied with immunofluorescence and confocal microscopy. In chip-based co-cultures, HT-29 cells showed facilitated 3-D growth and increased levels of hexokinase-2, TP53-induced glycolysis and apoptosis regulator (TIGAR), lactate dehydrogenase, and: translocase of outer mitochondrial membrane 20 (TOMM20), when compared with HT-29 mono-cultures. Fibroblasts co-cultured with HT-29 cells expressed higher levels of mono-carboxylate transporter 4, hexokinase-2, microtubule-associated proteins 1A/1B light chain 3, and ubiquitin-binding protein p62 than in fibroblast mono-cultures, in both 2-D cultures and chips. Tetramethylrhodamin-methylester (TMRM) live-cell imaging of chip co-cultures revealed a higher mitochondrial potential in cancer cells than in fibroblasts. The findings demonstrate a crosstalk between cancer cells and fibroblasts that affects cellular growth and metabolism. Chip-based 3-D co-cultures of cancer cells and fibroblasts mimicked features of the reverse Warburg effect.

Abstract 3755: PDGF-induced tumor cell growth despite VEGF/EGF inhibition and absence of PDGF receptor expression in colon cancer

Abstract Anti-Epithelial/Vascular Endothelial Growth Factor (EGF/VEGF)-targeted therapy plays important roles in advanced stage colorectal cancer (CRC) patients but fails to show clinical efficacy in specific subgroups which lack of clear underlying mechanisms. In vitro studies indicated that cross-talk or alternative receptor/ligand activities can bypass anti-growth factor signaling arrest and therefore may explain failure of monoclonal antibody (mAb) targeted therapy. In this study we analyzed effects of Platelet Derived Growth Factor (PDGF)-mediated bypassed signaling in CRC. We first investigated human UICC (Union for International Cancer Control ) stage I-IV tumors (n=46) from our Center and found increased PDGFRβ and VEGFR1/2 gene expression (p&amp;lt;0.01/p&amp;lt;0.001). Interestingly, further analysis on human HT29 colon cancer in vitro showed upregulated VEGFR1/2 expression upon VEGF stimulation. This was even more pronounced upon stimulation with PDGF. Inhibition of VEGFR2 and EGFR resulted in decreased HT29 cell proliferation, while additional treatment with VEGF and particularly PDGF attenuated the inhibiting effect and resulted in altered downstream signaling events. Treatment with the tyrosine kinase inhibitor Regorafenib alone, that blocks VEGFR/EGFR+PDGFR, prevented PDGF and VEGF-induced proliferation. This effect was not observed for either VEGFR2 mAb treatment with Ramucirumab or EGFR mAb therapy with Cetuximab. To conclude, PDGF-induced human colon cancer proliferation in the absence of PDGFR and simultaneously inhibited VEGFR2 and EGFR expression in HT29 colon cancer cells suggests bypassed alternative PDGF receptor partners for ligand binding on the tumor cell surface and subsequent intracellular signaling. This may explain failure of anti-VEGF/EGF-targeted monoclonal antibody therapies in CRC patient subgroups and underlines the necessity to effectively inhibit PDGF-mediated signaling events beside VEGF/EGF blockade in patients with increased PDGF expression within their tumors. Citation Format: Romana Moench, Martin Gasser, Karol Nawalaniec, Tanja Grimmig, Minghua Cao, Yueming Luo, Li-Li Hsiao, Ana Maria Waaga-Gasser. PDGF-induced tumor cell growth despite VEGF/EGF inhibition and absence of PDGF receptor expression in colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3755.

Anti-cancer effects of Porphyra haitanensis polysaccharides on human colon cancer cells via cell cycle arrest and apoptosis without causing adverse effects in vitro.

In this study, the anticancer effects of Porphyra haitanensis polysaccharides (PHPs) on human colon cancer cells and non-cancerous cells were evaluated. PHP was extracted by an ultrasonic/microwave-assisted method, and three fractions of polysaccharides (PHP-F1, PHP-F2 and PHP-F3) were obtained through a DEAE-52 cellulose ion-exchange column. The results of the cytotoxicity test showed that all of the PHP fractions had inhibitory effects on the growth of colon cancer cells HT-29, LoVo and SW-480, but no toxic effects on the normal human cells HaCaT. The fractions PHP-F2 and PHP-F3 had the most significant cytotoxicity on HT-29 cells. Studies on intracellular reactive oxygen species (ROS) levels, cell apoptosis, the apoptosis index (using Hoechst 33342 staining) and analysis of cell cycle arrest using flow cytometry revealed that the fractions PHP-F2 and PHP-F3 could apparently induce oxidative stress and apoptosis in HT-29 cells and cause cell G0-G1 phase arrest. These findings suggest that polysaccharides from P. haitanensis have anticancer effects on human colon cancer cells and therefore might be regarded as new candidates for the prevention and treatment of colon cancers.

Novel long noncoding RNAs upregulation may have synergistic effects on theCYP24A1 and PFDN4 biomarker role in human colorectal cancer.

Long noncoding RNAs (lncRNAs) are emerging as the master regulators of tumor initiation, proliferation, and metastasis; however, their diagnostic value as potential biomarkers should be clarified. Vitamin D influences the expression of several genes in various pathways, including theCYP24A1 gene in the vitamin D metabolism pathway. In the present research, we surveyed the expression levels and clinical significance of novel lncRNAs related to CYP24A1 and PFDN4 genes in colorectal cancer (CRC) using real-time polymerase chain reaction. Furthermore, we assessed the expression of these genes after vitamin D treatment in HCT-116 and HT-29 colon cancer cell lines. Our results indicated that the transcription of CYP24A1, PFDN4, and nearby lncRNAs was affected by vitamin D treatment in HCT-116 and HT-29 cell lines. Moreover, CYP24A1, PFDN4, lnc-CYP24A1-3:1, and lnc-TSHZ2-19:1 were upregulated and had the potential to distinguish colorectal cancer tissues from the adjacent tissues by the large area under the receiver operating characteristic curve (0.94, 0.66, 0.70, and 0.60, respectively). lnc-TSHZ2-19:1 expression level significantly correlated with gender (p = .03). In conclusion, CYP24A1, PFDN4, lnc-CYP24A1-3:1, and lnc-TSHZ2-19:1 can be used as potential diagnostic biomarkers in the specific and sensitive assessment of CRC. Besides this, vitamin D treatment may modulate the expression of these genes in a cell-specific manner.

Designing a pH-responsive drug delivery system for the release of black-carrot anthocyanins loaded in halloysite nanotubes for cancer treatment

Anthocyanins extracted from black-carrot were encapsulated into halloysite nanotubes (HNT) as a potential carrier for the first time. Anthocyanins were loaded into HNT under vacuum conditions. TEM images indicated the successful entrance of anthocyanins into the lumen of HNT. Anthocyanins loading efficiency was determined to be 4% using thermogravimetric analysis (TGA). XRD patterns showed that all reflections shifted to higher values for HNT loaded with anthocyanins compared to pure HNT which indicates reduced d-value of the crystalline forms. In vitro release of anthocyanins was studied under gastrointestinal conditions using the pH-differential method. The drug release from HNT was modeled and based on R2 values, the Korsmeyer-Peppas model was introduced as the best model predicting the release behavior. Cytotoxic effects of the samples against MCF-7 human breast cancer and HT-29 colon cancer cell lines were evaluated using SRB assay. The cell viability was reduced by 2 folds against anthocyanins-loaded HNT compared to pure anthocyanins in both cell lines. Anthocyanins-loaded HNT is suggested as a promising pH-responsive drug delivery system for the treatment of cancer.

Almorexant, an Orexin Antagonist with Anti-Tumoral Properties in Human Colon Cancer

Colorectal cancer, which is the third most common cancer, is the main cause of digestive cancer death. Previous studies have demonstrated that orexins, hypothalamic neuropeptides involved in sleep and food intake regulations, have anti-tumoral properties in digestive cancers. In the present work, we investigated the anti-tumoral role of an orexin antagonist, almorexant, in colon cancer. The anti-tumoral role of almorexant has been determined by in vitro and in vivo studies using HT-29 colon cancer cell line, which expressed endogenous orexin receptor 1 subtype (OX1R). Our in vitro study indicated that almorexant was able to reduce HT-29 cell viability by induction of mitochondrial apoptosis involving the tyrosine phosphatase SHP2 and the p38 signaling pathways. In contrast, no effect was observed in the colon cancer cell line HCT-116, which does not express OX1R, demonstrating that the anti-tumoral effect of almorexant was mediated by OX1R. When HT-29 cells were xenografted in nude mice, the administration of almorexant strongly reduced the tumor development with a potency similar to orexin. Our study supports that almorexant, a small molecule analog of orexin peptide, could represent a putative candidate in the treatment of colorectal cancer.

The Effect of Novel 7-methyl-5-phenyl-pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazine Sulfonamide Derivatives on Apoptosis and Autophagy in DLD-1 and HT-29 Colon Cancer Cells.

The discovery of cytotoxic drugs is focused on designing a compound structure that directly affects cancer cells without an impact on normal cells. The mechanism of anticancer activity is mainly related with activation of apoptosis. However, recent scientific reports show that autophagy also plays a crucial role in cancer cell progression. Thus, the objective of this study was to synthesize 7-methyl-5-phenyl-pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazine utilizing nucleophilic substitution reaction at the position N1. The biological activity of tested compounds was assessed in DLD-1 and HT-29 cell lines. The induction of apoptosis was confirmed by Annexin V binding assay and acridine orange/ethidium bromide staining. The loss of mitochondrial membrane potential and caspase-8 activity was estimated using cytometer flow analysis. The concentration of p53, LC3A, LC3B and beclin-1 was measured using the ELISA technique. Our study revealed that anticancer activity of 7-methyl-5-phenyl-pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazine derivatives is related with initiation of apoptosis occur on the intrinsic pathway with mitochondrial membrane decrease and extrinsic with increase of activity of caspase-8. Moreover, a decrease in beclin-1, LC3A, and LC3B were observed in two cell lines after treatment with novel compounds. This study showed that novel 7-methyl-5-phenyl-pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazine derivatives might be a potential strategy in colon cancer treatment.