Antitumor Cytokine DR5-B-Conjugated Polymeric Poly(N-vinylpyrrolidone) Nanoparticles with Enhanced Cytotoxicity in Human Colon Carcinoma 3D Cell Spheroids

Self-assembled nanoparticles based on amphiphilic poly(N-vinylpyrrolidone) (Amph-PVP) were proposed earlier as a new drug delivery system. In the current work, we study the antitumor activity of Amph-PVP-based self-assembled polymeric micelles covalently conjugated with the antitumor receptor-specific TRAIL variant DR5-B (P-DR5-B). The Amph-PVP polymer was synthesized by the earlier developed one-step technique (Kulikov et al., Polym. Sci. Ser. D, 2017). To stabilize Amph-PVP associates, the hydrophobic core was loaded with the model substance prothionamide. For the covalent conjugation with DR5-B, the hydrophilic ends of polymeric chains were modified with maleimide, and a DR5-B N-terminal amino acid residue valine was mutated to cysteine (DR5-B/V114C). DR5-B/V114C was conjugated to the surface of polymeric micelles by the selective covalent interaction of N-terminal cysteine residue with maleimide on Amph-PVP. The cytotoxicity of DR5-B-conjugated Amph-PVP polymeric nanoparticles was investigated in 3D multicellular tumor spheroids (MCTS) of human colon carcinoma HCT116 and HT29 cells, generated by the RGD-induced self-assembly technique (Akasov et al., Int. J. Pharm., 2016). In DR5-B-sensitive HCT116 MCTS, the P-DR5-B activity slightly increased compared to that of DR5-B. However, in DR5-B-resistant HT29 MCTS, P-DR5-B significantly surpassed DR5-B in the antitumor activity. Thus, the conjugation of DR5-B with the Amph-PVP nanoparticles enhanced its tumor-cell killing capacity. In the current study, we obtain a new nano-scaled delivery system based on Amph-PVP self-aggregates coated with covalently conjugated antitumor DR5-specific cytokine DR5-B. P-DR5-B overcomes DR5-B-resistance of the human colon carcinoma MCTS in vitro. This makes Amph-PVP polymeric nanoparticles a prospective and versatile nano-scaled delivery system for the targeted proteins.

Topoisomerase poisoning by the flavonoid nevadensin triggers DNA damage and apoptosis in human colon carcinoma HT29 cells

Nevadensin, an abundant polyphenol of basil, is reported to reduce alkenylbenzene DNA adduct formation. Furthermore, it has a wide spectrum of further pharmacological properties. The presented study focuses the impact of nevadensin on topoisomerases (TOPO) in vitro. Considering the DNA-intercalating properties of flavonoids, first, minor groove binding properties (IC50 = 31.63 µM), as well as DNA intercalation (IC50 = 296.91 µM) of nevadensin, was found. To determine potential in vitro effects on TOPO I and TOPO IIα, the relaxation and decatenation assay was performed in a concentration range of 1–500 µM nevadensin. A partial inhibition was detected for TOPO I at concentrations ≥ 100 µM, whereas TOPO IIα activity is only inhibited at concentrations ≥ 250 µM. To clarify the mode of action, the isolating in vivo complex of enzyme assay was carried out using human colon carcinoma HT29 cells. After 1 h of incubation, the amount of TOPO I linked to DNA was significantly increased by nevadensin (500 µM), why nevadensin was characterized as TOPO I poison. However, no effects on TOPO IIα were detected in the cellular test system. As a subsequent cellular response to TOPO I poisoning, a highly significant increase of DNA damage after 2 h and a decrease of cell viability after 48 h at the same concentration range were found. Furthermore, after 24 h of incubation a G2/M arrest was observed at concentrations ≥ 100 µM by flow cytometry. The analysis of cell death revealed that nevadensin induces the intrinsic apoptotic pathway via activation of caspase-9 and caspase-3. The results suggest that cell cycle disruption and apoptotic events play key roles in the cellular response to TOPO I poisoning caused by nevadensin in HT29 cells.

Insight into the Web of Stress Responses Triggered at Gene Expression Level by Porphyrin-PDT in HT29 Human Colon Carcinoma Cells

Photodynamic therapy (PDT), a highly targeted therapy with acceptable side effects, has emerged as a promising therapeutic option in oncologic pathology. One of the issues that needs to be addressed is related to the complex network of cellular responses developed by tumor cells in response to PDT. In this context, this study aims to characterize in vitro the stressors and the corresponding cellular responses triggered by PDT in the human colon carcinoma HT29 cell line, using a new asymmetric porphyrin derivative (P2.2) as a photosensitizer. Besides investigating the ability of P2.2-PDT to reduce the number of viable tumor cells at various P2.2 concentrations and fluences of the activating light, we assessed, using qRT-PCR, the expression levels of 84 genes critically involved in the stress response of PDT-treated cells. Results showed a fluence-dependent decrease of viable tumor cells at 24 h post-PDT, with few cells that seem to escape from PDT. We highlighted following P2.2-PDT the concomitant activation of particular cellular responses to oxidative stress, hypoxia, DNA damage and unfolded protein responses and inflammation. A web of inter-connected stressors was induced by P2.2-PDT, which underlies cell death but also elicits protective mechanisms that may delay tumor cell death or even defend these cells against the deleterious effects of PDT.

Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells

Several species of mycoplasmas, including Mycoplasma fermentans, are associated with certain human cancers. We previously isolated and characterized in our laboratory a strain of human mycoplasma M. fermentans subtype incognitus (MF-I1) able to induce lymphoma in a Severe Combined Immuno-Deficient (SCID) mouse model, and we demonstrated that its chaperone protein, DnaK, binds and reduces functions of human poly-ADP ribose polymerase-1 (PARP1) and ubiquitin carboxyl-terminal hydrolase protein-10 (USP10), which are required for efficient DNA repair and proper p53 activities, respectively. We also showed that other bacteria associated with human cancers (including Mycoplasmapneumoniae, Helicobacterpylori, Fusobacteriumnucleatum, Chlamydiathrachomatis, and Chlamydia pneumoniae) have closely related DnaK proteins, indicating a potential common mechanism of cellular transformation. Here, we quantify dnaK mRNA copy number by RT-qPCR analysis in different cellular compartments following intracellular MF-I1 infection of HCT116 human colon carcinoma cells. DnaK protein expression in infected cells was also detected and quantified by Western blot. The amount of viable intracellular mycoplasma reached a steady state after an initial phase of growth and was mostly localized in the cytoplasm of the invaded cells, while we detected a logarithmically increased number of viable extracellular bacteria. Our data indicate that, after invasion, MF-I1 is able to establish a chronic intracellular infection. Extracellular replication was more efficient while MF-I1 cultured in cell-free axenic medium showed a markedly reduced growth rate. We also identified modifications of important regulatory regions and heterogeneous lengths of dnaK mRNA transcripts isolated from intracellular and extracellular MF-I1. Both characteristics were less evident in dnaK mRNA transcripts isolated from MF-I1 grown in cell-free axenic media. Taken together, our data indicate that MF-I1, after establishing a chronic infection in eukaryotic cells, accumulates different forms of dnaK with efficient RNA turnover.

Cytotoxic and apoptotic activity of Canavalia seed extract in HT-29 human colon carcinoma cells

Introduction and Aim: Concanavalin the lectin from Canavalia spp. share high structural similarity among the same taxon and exhibit wide range of biological functions. The antiproliferative activity of lectins ConA (C. ensiformis), ConC (C. cathartica), ConG (C. gladiata) and ConM (C. rosea) were studied in human colon carcinoma. Materials and Methods: Human colon carcinoma HT-29 cells and human normal kidney HEK293T cells were examined. The cytotoxicity was evaluated by MTT assay. Cell apoptosis was analyzed by AO-EB (acridine orange-ethidium bromide dual staining) method. Cell cycle was investigated by flow cytometry (RNase-propidium iodide-based flow cytometry). The expression of cell signaling genes was analyzed by qRT-PCR. Results: All concanavalins significantly inhibited the proliferation of HT-29 cells in dose-dependent manner (IC50 values were ConG-10.45, ConA-14.86, ConC-33.34 and ConM-48.98 µg/mL respectively). ConA and ConG induced apoptotic morphology in HT-29 cells without affecting HEK293T cells. Similarly, both lectins increased the sub-G0/G1 proportions in HT-29 cells dose-dependently. ConG showed down-regulation of AKT1, ERK1/2 and mutant p53 in HT-29 cells. Conclusion: Concanavalin extract is shown to possess cytotoxic effect through inducing apoptosis, especially causing cell cycle arrest at G0/G1 phase in HT-29 cells.

The Inhibition of Proliferation and Invasion of Human Colon Carcinoma Cell Line (Caco-2 cells) by Cell-free Supernatants from Lactobacillus Rhamnosus and Lactobacillus Acidophilus

Background: The epidemiological studies indicated that colorectal cancer is one of the most common types of cancer in the world and is considered a leading cause of cancer-related death. The present study aimed to investigate the inhibitory effect of lactobacillus acidophilus supernatant (LAS) and lactobacillus rhamnosus supernatant (LRS) on the growth and invasiveness of the human colon carcinoma cell line (Caco2) in-vitro. Methods: In this experimental study, the anti-proliferative activity and anti-invasion potential of LAS and LRS were determined by MTT and transwell chambers assays, respectively. The expression of mitochondrial membrane potential-9 (MMP-9) and matrix metalloproteinase-12 (MMP12) genes were analyzed by real-time PCR.Results: The results indicated that supernatants of these two lactobacilli had cytotoxic effects on Caco-2 cells at a concentration of 25% v/v and higher. Thus, the minimum concentrations (25% V/V) of supernatants were chosen for further experiments. LAS and LRS could significantly suppress the invasiveness of Caco-2 cells. Also, the expression of MMP12 was significantly increased in Caco-2 cells when treated with LAS, whereas LRS had no significant effect on the invasive capacity and the gene expression levels of MMP12. The expression of MMP-9 was statistically decreased in Caco2 cells treated with LAS and LRS (P<0.00001).Conclusion: In general, it was shown that LAS and LRS exert anti-cancer activity against the growth, invasion, and metastasis of Caco2 cells in-vitro. It seems that these two bacteria could be used as prophylactic and therapeutic agents for the prevention and treatment of colorectal cancer.