Colon Cancer Cell Line HT-29 Research Articles

In vitro cytotoxic potential of Gymnema sylvestre fractions on HT-29 cell line

The HT-29 colon cancer cell line was used in the current work to investigate the anti-cancer efficacy of Gymnema sylvestre extracts. The viability, apoptosis, flow cytometry, and mode of action of the active fraction were also evaluated. The active fractions of G. sylvestre caused cell cycle arrest at the S phase and G2/M phase, and also caused programmed cell death. The alcoholic fractions showed anti-cancer effect in HT-29 cancer cells. By applying methanolic fraction of Gymnema sylvestre to the HT-29 cells, IC50 was observed at 140.39 μg/mL, whereas for chloroform fraction of Gymnema sylvestre showed IC50 at 154.44 μg/mL. The herbal extract for colon cancer treatment may be developed with the help of researchers due to G. sylvestre’s anti-cancer properties.

CRISPR Cas9-mediated ablation of pyruvate carboxylase gene in colon cancer cell line HT-29 inhibits growth and migration, induces apoptosis and increases sensitivity to 5-fluorouracil and glutaminase inhibitor.

Pyruvate carboxylase (PC) is an important anaplerotic enzyme that replenishes the tricarboxylic acid cycle (TCA) intermediates. It prevents the collapse of the TCA cycle upon its intermediates are removed during high anabolic demand. We have recently shown that overexpression of PC protein was associated with staging, metastasis and poor survival of colorectal cancer patients. Herein, we generated the PC knockout (PC KO) colon cancer cell lines, HT-29, by CRISPR-Cas9 technique, as a model to understand the role of this enzyme in colorectal cancer. The PC KO HT-29 cell lines had no detectable PC protein and did not show abnormal cellular or nuclear structures. However, PC KO HT-29 cells showed a 50-60% reduction in their growth rate and a 60-70% reduction in migration. The deficient growth phenotype of PC KO HT-29 cells was associated with apoptotic induction with no apparent cell cycle disruption following five days of growth. Down-regulation of key lipogenic enzymes, including acetyl-CoA carboxylase-1 and fatty acid synthase, was also associated with growth inhibition, suggesting that the de novo lipogenesis is impaired. Furthermore, PC KO HT-29 cells were 50% and 60% more sensitive to 5-fluorouracil and glutaminase inhibitor, CB-839, at their IC50 concentrations, respectively, following 48h exposure. The increased cytotoxicity of CB-839 to PC KO HT-29 cells was associated with increased poly (ADP-ribose) polymerase cleavage. However, this was not observed with PC KO cells exposed to 5-fluorouracil, suggesting that PC KO HT-29 cells were prone to CB-839-induced apoptosis. Collectively, these findings indicate that ablation of PC expression in HT-29 cells disrupts the metabolic homeostasis of cells and inhibits proliferation and migration, accompanied by apoptotic induction. This study highlights the crucial role of PC in supporting the survival of HT-29 cells during exposure to chemotherapeutic drugs.

HSD3B1 Expression Is Upregulated by Interleukin 4 in HT-29 Colon Cancer Cells via Multiple Signaling Pathways.

3β-Hydroxysteroid dehydrogenase/isomerase is essential for the synthesis of active steroid hormones. Interleukin 4 (IL4) induces the expression of HSD3B1 in various human cancer cell lines. Here, we demonstrated that administration of IL4 to an HT-29 colon cancer cell line induced high expression of HSD3B1 at the mRNA and protein levels. In the HT-29 cells, IL4 stimulated the activity of signal transducer and activator of transcription 6 (STAT6) and promoted its binding to the STAT6-binding site in the HSD3B1 promoter. The STAT6 inhibitor significantly suppressed HSD3B1 induction by IL4 in a dose-dependent manner. Moreover, inhibition of the PI3-kinase/AKT pathway strongly suppressed the IL4-induced HSD3B1 expression. Glycogen synthase kinase 3 (GSK3), a downstream target of AKT, had a stimulatory effect on the IL4-induced HSD3B1 expression. However, IL4 stimulated the phosphorylation of AKT, which inhibited the GSK3 activity at the early stage. Hence, GSK3 potentiated the HSD3B1 levels at the late stage of the IL4 stimulation. Additionally, inhibitors of mitogen-activated protein kinases (MAPKs), ERK1/2 and p38, but not of JNK, partly reduced the HSD3B1 expression following the IL4 stimulation. We further demonstrated that IL4 potently promoted steroid synthesis. Our results indicate that IL4 induces HSD3B1 expression via multiple signaling pathways in HT-29 cells and may play a role in the regulation of steroid synthesis.

Downregulation of MACC1 facilitates the reversal effect of verapamil on the chemoresistance to active metabolite of irinotecan in human colon cancer cells

The aim of this study is to investigate the reversal effect of verapamil (VER) on chemoresistance to irinotecan (CPT-11) in human colon cancer cells and relevant mechanisms. Cell counting kit-8 (CCK-8) test and colony-forming unit (CFU) experiment results show that VER strengthens the sensitivity of human colon cancer cell line HT29 to CPT-11 but has a small effect on SW480 cells. High-throughput transcriptome sequencing, RT-PCR, and Western blot results show that the inhibition of metastasis-associated in colon cancer-1 (MACC1) expression by VER is the key factor for reversal effect on chemoresistance to CPT-11. Transfection experiments further show that VER can reverse the resistance of human colon cancer cells to SN-38, the active metabolite of CPT-11, when MACC1 is overexpressed. The nude mouse transplantation tumor experiment provides an in vivo proof that VER can strengthen sensitivity to CPT-11 in drug-resistant human colon cancer cells, and the effect might be related to the inhibited expression of MACC1. In summary, VER might strengthen the reversal effect of VER on chemoresistance to CPT-11 in human colon cancer cells and facilitate the apoptosis of human colon cancer cells by downregulating MACC1 expression.

Fermentation Extract of Naringenin Increases the Expression of Estrogenic Receptor β and Modulates Genes Related to the p53 Signalling Pathway, miR-200c and miR-141 in Human Colon Cancer Cells Exposed to BPA.

The estrogenic receptor beta (ERβ) protects against carcinogenesis by stimulating apoptosis. Bisphenol A (BPA) is related to promoting cancer, and naringenin has chemoprotective activities both can bind to ERβ. Naringenin in the colon is metabolized by the microbiota. Cancer involves genetic and epigenetic mechanisms, including miRNAs. The objective of the present study was to evaluate the co-exposure effect of colonic in vitro fermented extract of naringenin (FEN) and BPA, to elucidate molecular effects in HT-29 colon cancer cell line. For this, we quantified genes related to the p53 signaling pathway as well as ERβ, miR-200c, and miR-141. As an important result, naringenin (IC50 250 µM) and FEN (IC50 37%) promoted intrinsic pathways of apoptosis through phosphatase and tensin homolog (PTEN) (+2.70, +1.72-fold, respectively) and CASP9 (+3.99, +2.03-fold, respectively) expression. BPA decreased the expression of PTEN (−3.46-fold) gene regulated by miR-200. We suggest that once co-exposed, cells undergo a greater stress forcing them to mediate other extrinsic apoptosis mechanisms associated with death domain FASL. In turn, these findings are related to the increase of ERβ (5.3-fold with naringenin and 13.67-fold with FEN) gene expression, important in the inhibition of carcinogenic development.

Anticancer Activity of Calcium Channel Blockers in Colon Cancer Cell Culture

Objective: Repurposing non-cancer drugs for cancer treatment has many advantages. We can access to a new cancer drug easily, quickly and cheaply. In addition, we generally know safety prifile of repurped drugs. There are few studies in the literature investige the anticancer effects of Calcium channel blockers (CCBs). We planned to investigate the anticancer effects of CCBs on colon cancer cell line.
 Material and Method: We adminestered different doses of T-type CCB NNC-55-0396 and L-type CCB amlodipine on colon cancer cell line HT-29. MTT analysis was performed at 48 hours to measure cell viability. The dose-response curve was constructed using GraphPad Prism.8 programme.
 Results: Amlodipine caused more than 90% cytotoxicity at all concentrations of 500, 250, 100, 50, 10 µg/ml in MTT analysis at 48 hours. Similarly, NNC-55-0396 caused more than 90% cytotoxicity at all 80, 40, 20, 10.5 µM concentrations.
 Discussion: In our study, NNC-55-0396 and amlodipine molecules showed severe cytotoxicity on HT-29 cells. There are publications indicating that it may have other anticancer effects other than cytotoxicity. They are promising molecules as anti-cancer drugs. They should be investigated in clinical studies alone or in combination with other cancer drugs.

Utilizing TPGS for Optimizing Quercetin Nanoemulsion for Colon Cancer Cells Inhibition

Background: Colorectal cancer is one of the most challenging cancers to treat. Exploring novel therapeutic strategies is necessary to overcome drug resistance and improve patient outcomes. Quercetin (QR) is a polyphenolic lipophilic compound that was chosen due to its colorectal anticancer activity. Nanoparticles could improve cancer therapy via tumor targeting by utilizing D-tocopheryl polyethylene glycol succinate (vitamin-E TPGS) as a surfactant in a nanoemulsion preparation, which is considered an efficient drug delivery system for enhancing lipophilic antineoplastic agents. Thus, this study aims to develop and optimize QR-loaded nanoemulsions (NE) using TPGS as a surfactant to enhance the QR antitumor activity. Method: The NE was prepared using a self-assembly technique using the chosen oils according to QR maximum solubility and TPGS as a surfactant. The prepared QR-NE was evaluated according to its particle morphology and pH. QR entrapment efficiency and QR in vitro drug release rate were determined from the selected QR-NE then we measured the QR-NE stability. The anticancer activity of the best-selected formula was studied on HT-29 and HCT-116 cell lines. Results: Oleic acid was chosen to prepare QR-NE as it has the best QR solubility. The prepared NE, which had particles size < 200 nm, maximum entrapment efficiency > 80%, and pH 3.688 + 0.102 was selected as the optimal formula. It was a physically stable formula. The prepared QR-NE enhanced the QR release rate (84.52 ± 0.71%) compared to the free drug. QR-NPs significantly improved the cellular killing efficiency in HCT-116 and HT-29 colon cancer cell lines (lower IC50, two folds more than free drug). Conclusion: The prepared QR-NE could be a promising stable formula for improving QR release rate and anticancer activity.

Simultaneous enrichment and sequential separation of O-linked glycopeptides and phosphopeptides with immobilized titanium (IV) ion affinity chromatography materials

Protein phosphorylation and O-linked glycosylation are two critical posttranslational modifications (PTMs) and they both target identical amino acid residues, generating “crosstalk”. Analysis of the crosstalk between these PTMs is crucial for deciphering their biological functions. Although several strategies have been established to enrich N-linked glycopeptides and phosphopeptides simultaneously, they are not appropriate for O-linked glycopeptides and phosphopeptides. In this study, we established a method for the simultaneous enrichment and sequential elution of O-linked glycopeptides and phosphopeptides into two fractions using commercially available immobilized titanium (IV) ion affinity chromatography (Ti4+-IMAC) materials. The established method exhibited high selectivity, repeatability, and recovery of the targeted peptides. Particularly, the overlap between the O-linked glycopeptide and phosphopeptide fractions was very low (∼3%). The application of this method to cell lysates of the colon cancer HT29 cell line resulted in a comparable number of enriched phosphopeptides as the coeluted method. However, the number of identified O-linked glycopeptides with our established method was 9.7-fold higher than that with the coelution method. Our study demonstrated that the established strategy was beneficial for the simultaneous analysis of O-linked glycopeptides and phosphopeptides, which might facilitate the study of the biological function of PTM crosstalk.

Inhibition of Aryl Hydrocarbon Receptor (AhR) Expression Disrupts Cell Proliferation and Alters Energy Metabolism and Fatty Acid Synthesis in Colon Cancer Cells.

Simple SummaryCancer cells undergo metabolic modifications in order to meet their high energetic demand. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor primarily known as a xenobiotic sensor. However, this receptor seems to have a wide range of physiological roles in many processes including cell proliferation, migration or control of immune responses. AhR is often overexpressed in tumor cells of various tissue origin, and several studies have indicated that AhR may also contribute to regulation of cellular metabolism, including synthesis of fatty acids (FA), one of the major steps in metabolic transition. Potential links between the AhR and the control of tumor cell proliferation and metabolism thus deserve more attention.The aryl hydrocarbon receptor (AhR) plays a wide range of physiological roles in cellular processes such as proliferation, migration or control of immune responses. Several studies have also indicated that AhR might contribute to the regulation of energy balance or cellular metabolism. We observed that the AhR is upregulated in tumor epithelial cells derived from colon cancer patients. Using wild-type and the corresponding AhR knockout (AhR KO) variants of human colon cancer cell lines HCT116 and HT-29, we analyzed possible role(s) of the AhR in cell proliferation and metabolism, with a focus on regulation of the synthesis of fatty acids (FAs). We observed a decreased proliferation rate in the AhR KO cells, which was accompanied with altered cell cycle progression, as well as a decreased ATP production. We also found reduced mRNA levels of key enzymes of the FA biosynthetic pathway in AhR KO colon cancer cells, in particular of stearoyl-CoA desaturase 1 (SCD1). The loss of AhR was also associated with reduced expression and/or activity of components of the PI3K/Akt pathway, which controls lipid metabolism, and other lipogenic transcriptional regulators, such as sterol regulatory element binding transcription factor 1 (SREBP1). Together, our data indicate that disruption of AhR activity in colon tumor cells may, likely in a cell-specific manner, limit their proliferation, which could be linked with a suppressive effect on their endogenous FA metabolism. More attention should be paid to potential mechanistic links between overexpressed AhR and colon tumor cell metabolism.

Phytochemical Screening, Antioxidant, Antidiabetic and Anticancer Activities of Elaeocarpus variabilis Fruit

This study focused on the determination of secondary metabolites to an evaluation of the biological activity of Elaeocarpus variabilis Zmarzty fruit extract, an endemic medicinal plant. Their total Phenolic, tannin, flavonoids, alkaloids and saponins contents were quantified and their antioxidant activity, determined by DPPH, total antioxidant, ABTS, FRAP and Fe2+ chelating activity. The ethyl acetate extract exhibited the highest radical scavenging activity. The most relevant contents in total phenolics (Gallic Acid Equivalent mg/g), tannin (Gallic Acid Equivalent mg/g), total flavonoids (Rutin equivalent mg/g), total alkaloids (Quinine equivalent mg/g), and total saponin (Diosgenin equivalent mg/g) were observed. Significant correlations were established between antioxidant activity and secondary metabolites. Though the in vitro anti-diabetic activity was carried out using -amylase and -glucosidase inhibition, this indicates that the absorption and digestion of carbohydrates would probably reduce the blood glucose levels and hence help the management of type-2 diabetes. The ethyl acetate extract exhibited the potential of anticancer activity on human HT-29 colon cancer cell line IC50 values (30.0 ± 1.0) the valuable activity that compared to doxorubicin (17.0 ± 0.5). While this is the first study concerning extract from E. variabilis, our findings highlighted the richness’ in flavonoids of those extracts and their amazing potential as a source of antioxidant, anti-inflammatory, anti-diabetic, and anticancer agents.

Identification of a novel interplaying loop of PPARγ and respective lncRNAs are involved in colorectal cancer progress

Long noncoding RNAs (lncRNAs) as regulatory molecules play important roles in early treatment and diagnosis of cancers. Considering the role of PPARγ in colorectal cancer (CRC) as a tumor suppressor, the GEO database was used to identify candidate genes that affect the activation of PPARγ protein in CRC cell lines. Then were selected 5 genes containing PPARγ response element (PPRE) in up to 4000 bp upstream and were affected by PPARγ protein activation in HT-29 colon cancer cell line using UCSC database. Expression meta-analysis was applied to map the expression network between candidate genes and all known lncRNAs through expression correlation and lncRNAs that correlated with a greater number of candidate genes (R > 0.5, P.value < 0.001). Moreover, were selected 3 lncRNAs as lncRNAs affected by PPARγ protein activation. Next, the expression levels of candidate genes and lncRNAs were evaluated using RT-qPCR in HT-29 cell line. Results showed a significant increase (FDR <0.05) in the expression level of 5 candidate genes and lncRNAs LINC01133, MBNL1-AS, LOC100288911 after treatment with pioglitazone as PPARγ ligand compared to the untreated group in HT-29 cells. Although additional tests are needed to confirm bioinformatics predictions, it can be concluded that increased expression of PPARγ may increase genes and lncRNAs expression. In summary, this study could be suggested identifying lncRNAs affected by PPARγ activation could be a new strategy in understanding the function and activity of PPARγ in colon cancer.

Diindolylmethane Derivatives: New Selective Blockers for T-Type Calcium Channels.

The natural product indole-3-carbinol (I3C) and its major digestive product 3,3′-diindolylmethane (DIM) have shown clinical promise in multiple forms of cancer including breast cancer. In this study, we explored the calcium channel activity of DIM, its synthetic derivative 3,3′-Diindolylmethanone (DIM-one) and related I3C and DIM-one analogs. For the first time, DIM, DIM-one and analog IX were identified as selective blockers for T-type CaV3.3 (IC50s DIM 2.09 µM; DIM-one 9.07 µM) while compound IX inhibited both CaV3.2 (6.68 µM) and CaV3.3 (IC50 = 3.05 µM) using a FLIPR cell-based assay to measure inhibition of T-type calcium channel window current. Further characterization of DIM by electrophysiology revealed it inhibited inward Ca2+ current through CaV3.1 (IC50 = 8.32 µM) and CaV3.3 (IC50 = 9.63 µM), while IX partially blocked CaV3.2 and CaV3.3 inward Ca2+ current. In contrast, DIM-one preferentially blocked CaV3.1 inward Ca2+ current (IC50 = 1.53 µM). The anti-proliferative activities of these compounds revealed that oxidation of the methylene group of DIM shifted the selectivity of DIMs from breast cancer cell line MCF-7 to colon cancer cell line HT-29.

Expression of Long Nonencoding Ribonucleic Acid SNHG20 in Colon Cancer Tissue in Its Influences on Chemotherapeutic Sensitivity of Colon Cancer Cells.

Noncoding RNA (ncRNA) is a kind of RNA that plays a key role in a variety of biological processes, illnesses, and tumours despite the fact that it cannot be translated into proteins. The HT29 colon cancer cell line was utilized to create a 5-FU drug-resistant cell strain (control group), a lentivirus SNHG20 carrier (OE-SNHG20 group), and an SNHG20 shRNA carrier (SNHG20 shRNA carrier group) (SE-SNHG20 group). To determine the expression of cell SNHG20, a real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was utilized, and cholecystokinin-octapeptide (CCK-8) was used to detect the difference in 5-FU inhibitory concentration 50. The goal of the study was to see how variations in long nonencoding ribonucleic acid (lncRNA) SNHG20 expression affect colon cancer cell 5-fluorouracil (5-FU) chemotherapeutic sensitivity by collecting colon cancer and normal para cancer tissues and analysing the differences in SNHG20 expression. The ability of cell cladogenesis was tested using platform cladogenesis. Cell apoptosis was detected using flow cytometry. Western blots revealed the presence of protein phosphatidylinositol kinase (PI3K), protein kinase B (AKT), caspase-3, e-cadherin, and matrix metalloproteinase 9 (MMP-9) enzymes. The findings revealed that SNHG20 expression was considerably upregulated (P < 0.05) in colon cancer tissue and 5-FU drug-resistant colon cancer cells. Cell 5-FU IC50, cell cladogenesis, cell survival rate, and MMP-9, P-PI3K, and P-AKT expression were all significantly improved. Cell apoptosis and expressions of E-cadherin and caspase-3, on the other hand, were considerably decreased (P < 0.05). Cell 5-FU IC50, cell cladogenesis, cell survival rate, and the expressions of MMP-9, P-PI3K, and P-AKT were all significantly lower in the SE-SNHG20 group, although cell apoptosis and the expressions of E-cadherin and caspase-3 were significantly higher (P < 0.05). The results revealed that lncRNA SNHG20 could inhibit the chemotherapeutic sensitivity of colon cancer cells to 5-FU by regulating PI3K/AKT pathways. The inhibition of lncRNA SNHG20 expression could promote the apoptosis and proliferation of 5-FU-resistant colon cancer cells.

Targeting Colorectal Cancer Cells with Niosomes Systems Loaded with Two Anticancer Drugs Models; Comparative In Vitro and Anticancer Studies.

Colorectal cancer (CRC) is considered one of the most commonly diagnosed malignant diseases. Recently, there has been an increased focus on using nanotechnology to resolve most of the limitations in conventional chemotherapy. Niosomes have great advantages that overcome the drawbacks associated with other lipid drug delivery systems. They are simple, cheap, and highly stable nanocarriers. This study investigated the effectiveness of using niosomes with their amphiphilic characteristics in the incorporation of both hydrophilic and hydrophobic anticancer drugs for CRC treatment. Methods: Drug-free niosomes were formulated using a response surface D-optimal factorial design to study the cholesterol molar ratio, surfactant molar ratio and surfactant type effect on the particle size and Z-potential of the prepared niosomes. After numerical and statistical optimization, an optimized formulation having a particle size of 194.4 ± 15.5 nm and a Z-potential of 31.8 ± 1.9 mV was selected to be loaded with Oxaliplatin and Paclitaxel separately in different concentrations. The formulations with the highest entrapment efficiency (EE%) were evaluated for their drug release using the dialysis bag method, in vitro antitumor activity on HT-29 colon cancer cell line and apoptosis activity. Results: Niosomes prepared using d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) at a molar ratio 4, cholesterol (2 molar ratio) and loaded with 1 molar ratio of either Oxaliplatin or Paclitaxel provided nanosized vesicles (278.5 ± 19.7 and 251.6 ± 18.1 nm) with a Z-potential value (32.7 ± 1.01 and 31.69 ± 0.98 mV) with the highest EE% (90.57 ± 2.05 and 93.51 ± 2.97) for Oxaliplatin and Paclitaxel, respectively. These formulations demonstrated up to 48 h drug release and increased the in vitro cytotoxicity and apoptosis efficiency of both drugs up to twice as much as free drugs. Conclusion: These findings suggest that different formulation composition parameters can be adjusted to obtain nanosized niosomal vesicles with an accepted Z-potential. These niosomes could be loaded with either hydrophilic drugs such as Oxaliplatin or hydrophobic drugs such as Paclitaxel. Drug-loaded niosomes, as a unique nanomicellar system, could enhance the cellular uptake of both drugs, resulting in enhanced cytotoxic and apoptosis effects against HT-29 colon cancer cells. Oxaliplatin–niosomes and Paclitaxel–niosomes can be considered promising alternative drug delivery systems with enhanced bioavailability of these two anticancer drugs for colorectal cancer treatment.

Investigation of the potential antitumor activity of PLK1 inhibitor SBE13 in colon cancer cell line HT29

Background: High levels of Polo-like kinase 1 (PLK1), which have been found to be abnormally expressed in many tumor types, are known to contribute to tumorigenesis and poor prognosis. Therefore, specific targeting of PLK1 is an important strategy for cancer therapy. In this study, it was aimed to investigate the cytotoxic effect of SBE13, one of the PLK1 inhibitors, against HT29 colon adenocarcinoma cells and its apoptotic potential.&#x0D; Methods: The cytotoxic effect of SBE13 on HT29 was determined by XTT colorimetric assay. Flow cytometry was also used to determine apoptosis. &#x0D; Results: SBE13 showed a dose-dependent cytotoxic effect in HT29 cells and its IC50 value was calculated as 11.79 µM for 48 h. Moreover, the Annexin V binding assay revealed that SBE13 treatment significantly increased the apoptosis in HT29 cells.&#x0D; Conclusion: Generally, SBE13 exerts a cytotoxic effect promoted by apoptosis in colon cancer cells HT29. Although the anticancer efficacy of SBE13 in colon cancer is promising, this potential effect should be reinforced by further studies.

Cytotoxic effect of partially purified conotoxin from Conus virgo on Human Colon Cancer cell line HT-29

The present study focused on partial purification of conotoxin from the crude venom of Conus virgo and tested for its anticancer potential against human colon cancer cell line HT-29. The screening of crude venom was carried out using human cancer cell lines such as MCF-7 and HT-29 by MTT method. The result showed IC50 for HT-29 at 40 μg/ml for 24 h. incubation whereas MCF-7 cell line failed to show the decreased cell viability (71% cell viability at a maximum concentration of 140 μg/ml). The conotoxin was purified using DEAE-cellulose column chromatography; purified fraction-1 exhibited significantly decreased cell viability at 20, 40, 60, 80, 100, 120 and 140 ng/ml concentrations in HT-29cells and IC50 was found at 120 ng/ml for 24h. incubation. In Cytomorphological observation, distinct cellular changes such as cell shrinkage, loss of adherence and loss of colony formation were observed. The nuclear morphological damages were observed in propidium iodide staining. DNA fragmentation was observed in the treatment of fraction-1 on HT-29 cells when compared to control cells using agarose gel electrophoresis. Based on these findings, the fraction 1 conotoxin induced the nuclear damage and caused cell death in HT-29 cells, so it could be a good candidate for colon cancer chemotherapy.

5-Keto-D-Fructose, a Natural Diketone and Potential Sugar Substitute, Significantly Reduces the Viability of Prokaryotic and Eukaryotic Cells.

5-Keto-D-fructose (5-KF) is a natural diketone occurring in micromolar concentrations in honey, white wine, and vinegar. The oxidation of D-fructose to 5-KF is catalyzed by the membrane-bound fructose dehydrogenase complex found in several acetic acid bacteria. Since 5-KF has a sweetening power comparable to fructose and is presumably calorie-free, there is great interest in making the diketone commercially available as a new sugar substitute. Based on a genetically modified variant of the acetic acid bacterium Gluconobacter oxydans 621H, an efficient process for the microbial production of 5-KF was recently developed. However, data on the toxicology of the compound are completely lacking to date. Therefore, this study aimed to investigate the effect of 5-KF on the viability of prokaryotic and eukaryotic cells. It was found that the compound significantly inhibited the growth of the gram-positive and gram-negative model organisms Bacillus subtilis and Escherichia coli in a concentration-dependent manner. Furthermore, cell viability assays confirmed severe cytotoxicity of 5-KF toward the colon cancer cell line HT-29. Since these effects already occurred at concentrations of 5 mM, the use of 5-KF in the food sector should be avoided. The studies performed revealed that in the presence of amines, 5-KF promoted a strong Maillard reaction. The inherent reactivity of 5-KF as well as the Maillard products formed could be the trigger for the observed inhibition of prokaryotic and eukaryotic cells.

Abstract 5803: Exploring the anti-cancer activities of 5-Fluorouracil, metformin, and calcitriol single, dual, and triple therapies against colon cancer cells in vitro: The role of PI3K/Akt/mTOR pathway

Abstract Background: Chemoresistance to 5-Fluorouracil (5-FU) is a common clinical problem during the treatment of colorectal cancer (CRC) and its pathogenesis includes hyperactivation of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) oncogenic pathway that induces aerobic glycolysis. Although both calcitriol and metformin ameliorated the tumoricidal actions of 5-FU, there is no report on the potential anti-cancer effects of 5-FU, calcitriol, and metformin triple therapy. Objectives: This study aimed to measure the chemotherapeutic effects of 5-FU, calcitriol, and/or metformin single, dual, and triple treatments against CRC in vitro. Materials and methods: The HT29, SW480 and SW620 colon cancer cell lines were treated for 12 hours with 5-FU (50 µM), calcitriol (25 µM), and/or metformin (39.8 mM) single/dual/triple protocols followed by measuring cell cycle and cell apoptosis by flow cytometer. We also measured the gene and protein expression of cell cycle and apoptosis regulatory molecules alongside the PI3K/Akt/PTEN/mTOR network. Results: All therapies induced HT29 arrest at the S-phase of cell cycle, with calcitriol/metformin and 5-FU/calcitriol/metformin regimens most prominent (1.7-fold for both) relative to untreated cells. Alternatively, all therapies equally induced G1-phase arrest in the SW480 cells (~1.5-fold), whereas metformin-alone showed maximal numbers of SW620 cells in the G0/G1-phase (1.6-fold). 5-FU/metformin co-therapy also showed the highest numbers of apoptotic HT29 (11%) and SW480 (13%) cells, whilst 5-FU/calcitriol/metformin displayed the lowest percentage (81%) of viable SW620 cells. Triple therapy also revealed maximal downregulations of markers of cell cycle induction (CCND1/CCND3), cell survival (BCL2) and the PI3K/Akt/mTOR pathway, which coincided with highest expression of PTEN, cell cycle inhibitors (p21/p27), and pro-apoptotic (BAX/Cytochrome-C/Caspase-3) molecules in all three cell lines. Conclusions: The anti-cancer effects of metformin monotherapy were greater than calcitriol, whilst the 5-FU/metformin approach was better relative to the other dual therapies. However, the triple therapy protocol revealed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhanced regulation of the PI3K/PTEN/Akt/mTOR pathway. Citation Format: Mohammed A. Baghdadi, Jawwad Ahmad, Shakir Idris, Mai Alhadrami, Riyad Almaimani, Mohammed Aslam, Bassem Refaat. Exploring the anti-cancer activities of 5-Fluorouracil, metformin, and calcitriol single, dual, and triple therapies against colon cancer cells in vitro: The role of PI3K/Akt/mTOR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5803.

Anticancer, antioxidant, and antimicrobial properties of solvent extract of Lobophora variegata through in vitro and in silico studies with major phytoconstituents

Seaweeds possess a wide range of bioactive compounds which have been play a vital role in production of novel pharmaceutical and nutraceutical products resources. The present study employed in silico molecular docking to characterize the biomedical potential of brown seaweed Lobophora variegata through antibacterial, antibiofilm, antioxidant and anticancer activities. The antibacterial activity of L. variegata extracts was analyzed against Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Pseudomonas aeruginosa strains producing biofilms. The antioxidant activity was assessed using (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), superoxide, and hydroxyl radical scavenging assays. Further, the anticancer, antibacterial, and antioxidant activities of the bioactive compounds were evaluated through molecular docking using GLIDE ligand docking module and prime MM-GBSA module in the Schrodinger software. The phytochemical analysis of the methanolic extract of L. variegata found more phycocompounds which was confirmed by FT-IR and GC-MS. The significant level of phenol and flavonoid were expressed in methanolic extract of L. variegata. In addition, the potent antioxidant activity of the methanolic extract was efficiently expressed during the free radical scavenging assays of ABTS, hydroxyl, and superoxide denoted as 34.94, 26.64, and 29.86 mg/g, respectively. The antibacterial and biofilm activities of the methanolic extract of L. variegata had significant results. Moreover, the methanolic extract exhibited cytotoxicity against human colon cancer HT-29 cell lines at an inhibitory concentration is 193.65 μg/mL. Through the computational analysis, glyceryl monostearate, 2-palmitoylglycerol, phthalic acid, 2-chloropropyl ethyl ester, phthalic acid, 5-methylhex-2-yl ethyl ester, and l-(+)-ascorbic acid 2,6-dihexadecanoate results as top ranked molecules and shown to be a promising compounds, which were evaluated for anticancer, antioxidant, and antibacterial properties against multi-target proteins. Based on the experimental results, L. variegata could be a valuable marine source for developing novel pharmaceutical applications.

SK1 Inhibitor RB005 Induces Apoptosis in Colorectal Cancer Cells through SK1 Inhibition Dependent and Independent Pathway.

Colorectal cancer (CRC) is the fourth leading cause of cancer- related death globally, with a high incidence rate in economically fast-growing countries. Sphingosine- 1-phosphate (S1P) is a bioactive lipid mediator that plays critical roles in cancer cell proliferation, migration, and angiogenesis converted by the isoforms of sphingosine kinase (SK1 and SK2). SK1 is highly expressed in colorectal cancer; its inhibitors suppress the formation of S1P and increase ceramide levels having a pro-apoptotic function. RB005 is a selective SK1 inhibitor and a structural analog of PP2A activator FTY720. The purpose of this study is to test whether RB005, an SK1 inhibitor, can be used as an anticancer agent by inhibiting the growth of colon cancer cells. We performed MTT and colony-forming assay using colon cancer cell lines HT29 and HCT116 cells to examine the cell toxicity effect of RB005. To determine whether apoptosis of RB005 in colon cancer cell line is due to SK1 inhibition or other mechanisms due to its structural similarity with FTY720, we conducted LC/MS, siRNA knockdown, and PP2A activity experiments. RB005 notably inhibited CRC cell growth and proliferation compared to PF543 and ABC294640 by inducing the mitochondria-mediated intrinsic apoptotic pathway. Apoptotic cell death is caused by increased mitochondrial permeability Initiated by the activation of pro-apoptotic protein BAX, increased ceramides, and activation of PP2A. Also, RB005 treatment in HT29 cells did not change the expression level of SK1, but strikingly decreased SK1 activity and S1P levels. All these events of cell death and apoptosis were less effective when SK1 was knocked down by siRNA. This result indicates that RB005 shows the in-vitro anti-CRC effect by inhibiting SK1 activity and PP2A activation, increasing proapoptotic ceramide levels following the activation of the intrinsic apoptotic pathway.