Effects of ECM protein-coated surfaces on the generation of retinal pigment epithelium cells differentiated from human pluripotent stem cells.

Retinal degeneration diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), initially manifest as dysfunction or death of the retinal pigment epithelium (RPE). Subretinal transplantation of human pluripotent stem cell (hPSC)-derived RPE cells has emerged as a potential therapy for retinal degeneration. However, RPE cells differentiated from hPSCs using current protocols are xeno-containing and are rarely applied in clinical trials. The development of hPSC-derived RPE cell differentiation protocols using xeno-free biomaterials is urgently needed for clinical applications. In this study, two protocols (the activin A and NIC84 protocols) were selected for modification and use in the differentiation of hiPSCs into RPE cells; the chetomin concentration was gradually increased to achieve high differentiation efficiency of RPE cells. The xeno-free extracellular matrix (ECM) proteins, laminin-511, laminin-521 and recombinant vitronectin, were selected as plate-coating substrates, and a Matrigel (xeno-containing ECM)-coated surface was used as a positive control. Healthy, mature hPSC-derived RPE cells were transplanted into 21-day-old Royal College of Surgeons (RCS) rats, a model of retinal degeneration disease. The visual function of RCS rats was evaluated by optomotor response (qOMR) and electroretinography after transplantation of hPSC-derived RPE cells. Our study demonstrated that hPSCs can be efficiently differentiated into RPE cells on LN521-coated dishes using the NIC84 protocol, and that subretinal transplantation of the cell suspensions can delay the progression of vision loss in RCS rats.

Effect of 660-nm LED photobiomodulation on the proliferation and chondrogenesis of meniscus-derived stem cells (MeSCs).

Meniscus-derived stem cells (MeSCs), a unique type of MSC, have outstanding advantages in meniscal cytotherapy and tissue engineering, but the effects and molecular mechanisms of PBM on MeSCs are still unclear. We used 660-nm LED light with different energy densities to irradiate six human MeSC samples and tested their proliferation rate via cell counting, chondrogenic differentiation capacity via the DMMB assay, mitochondrial activity via the MTT assay, and gene expression via qPCR. The proliferation ability, chondrogenic capacity and mitochondrial activity of the 18 J/cm2 group were greater than those of the 4 J/cm2 and control groups. The mRNA expression levels of Akt, PI3K, TGF-ß3, Ki67 and Notch-1 in the 18 J/cm2 group were greater than those in the other groups in most samples. After chondrogenic induction, the expression of Col2A1, Sox9 and Aggrecan in the 18 J/cm2 group was significantly greater than that in the 4 J/cm2 and control groups in most of the samples. The variation in the MTT values and Src, PI3K, Akt, mTOR and GSK3ß levels decreased with time. The results showed that 660-nm LED red light promoted proliferation and chondrogenic differentiation and affected the gene expression of MeSCs, and the effects on gene expression and mitochondrial activity decreased with time.

Nanocomposites of iron oxide, sodium alginate, and eugenol induce apoptosis via PI3K/Akt/mTOR signaling in Hep3 cells and in vivo hepatotoxicity in the zebrafish model.

Hepatic cancer is among the most recurrently detected malignancies worldwide and one of the main contributors to cancer-associated mortality. With few available therapeutic choices, there is an instant necessity to explore suitable options. In this aspect, Nanotechnology has been employed to explore prospective chemotherapeutic approaches, especially for cancer treatment. Nanotechnology is concerned with the biological and physical properties of nanoparticles in the therapeutic use of drugs. In the current work, formulation, and characterization of α-Fe2O3-Sodium Alginate-Eugenol nanocomposites (FSE NCs) using several approaches like SEM and TEM, UV-visible, FTIR, and PL spectroscopy, XRD, EDAX, and DLS studies have been performed. With an average size of 50 nm, the rhombohedral structure of NCs was identified. Further, their anticancer activity against Hep3B liver cancer cell lines has been performed by cell viability, dual staining, DCFH-DA, Annexin-V/-FITC/PI, cell cycle analysis methods, and PI3K/Akt/mTOR signaling proteins were studied to assess the anticancer effects of the NCs in Hep3B cells. Also, anti-cancer activity on animal modeling in-vivo using zebra fishes to hematological parameters, liver enzymes, and histopathology study effectiveness was noticed. Moreover, the NCs reduced the viability, elevated the ROS accumulation, diminished the membrane integrity, reduced the antioxidants, blocked the cell cycle, and triggered the PI3K/Akt/mTOR signaling axis that eventually resulted in cell death. As a result, FSE NCs possess huge potential for use as a possible anticancer candidate.

Amniotic membrane mesenchymal stromal cell-derived secretome in the treatment of acute ischemic stroke: A case report.

BACKGROUND: Stroke is the second and third leading cause of death and disability, respectively. To date, no definitive treatment can repair lost brain function. Recently, various preclinical studies have been reported on mesenchymal stromal cells (MSCs) and their derivatives and their potential as alternative therapies for stroke. CASE SUMMARY: A 45-year-old female suffered an acute stroke, which led to paralysis in the left upper and lower limbs. The amniotic membrane MSC-derived secretome (MSC-secretome) was intravenously transplanted once a week for 4 wk. MSC-secretome-regulated regulatory T cells were investigated for the beneficial effects. The clinical improvement of this patient was accompanied by an increased frequency of regulatory T cells after transplantation. CONCLUSION: Intravenous administration of MSC-secretome can potentially treat patients who suffer from acute ischemic stroke.

Xeno-free culture and proliferation of hPSCs on 2D biomaterials.

Human pluripotent stem cells (human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs)) have unlimited proliferative potential, whereas adult stem cells such as bone marrow-derived stem cells and adipose-derived stem cells have problems with aging. When hPSCs are intended to be cultured on feeder-free or xeno-free conditions without utilizing mouse embryonic fibroblasts or human fibroblasts, they cannot be cultured on conventional tissue culture polystyrene dishes, as adult stem cells can be cultured but should be cultivated on material surfaces grafted or coated with (a) natural or recombinant extracellular matrix (ECM) proteins, (b) ECM protein-derived peptides and specific synthetic polymer surfaces in xeno-free and/or chemically defined conditions. This review describes current developing cell culture biomaterials for the proliferation of hPSCs while maintaining the pluripotency and differentiation potential of the cells into 3 germ layers. Biomaterials for the cultivation of hPSCs without utilizing a feeder layer are essential to decrease the risk of xenogenic molecules, which contributes to the potential clinical usage of hPSCs. ECM proteins such as human recombinant vitronectin, laminin-511 and laminin-521 have been utilized instead of Matrigel for the feeder-free cultivation of hPSCs. The following biomaterials are also discussed for hPSC cultivation: (a) decellularized ECM, (b) peptide-grafted biomaterials derived from ECM proteins, (c) recombinant E-cadherin-coated surface, (d) polysaccharide-immobilized surface, (e) synthetic polymer surfaces with and without bioactive sites, (f) thermoresponsive polymer surfaces with and without bioactive sites, and (g) synthetic microfibrous scaffolds.

Cell-binding peptides on the material surface guide stem cell fate of adhesion, proliferation and differentiation.

Human cells, especially stem cells, need to communicate and interact with extracellular matrix (ECM) proteins, which not only serve as structural components but also guide and support cell fate and properties such as cell adhesion, proliferation, survival and differentiation. The binding of the cells with ECM proteins or ECM-derived peptides via cell adhesion receptors such as integrins activates several signaling pathways that determine the cell fate, morphological change, proliferation and differentiation. The development of synthetic ECM protein-derived peptides that mimic the biological and biochemical functions of natural ECM proteins will benefit academic and clinical application. Peptides derived from or inspired by specific ECM proteins can act as agonists of each ECM protein receptor. Given that most ECM proteins function in cell adhesion via integrin receptors, many peptides have been developed that bind to specific integrin receptors. In this review, we discuss the peptide sequence, immobilization design, reaction method, and functions of several ECM protein-derived peptides. Various peptide sequences derived from mainly ECM proteins, which are used for coating or grafting on dishes, scaffolds, hydrogels, implants or nanofibers, have been developed to improve the adhesion, proliferation or differentiation of stem cells and to culture differentiated cells. This review article will help to inform the optimal choice of ECM protein-derived peptides for the development of scaffolds, implants, hydrogels, nanofibers and 2D cell culture dishes to regulate the proliferation and direct the differentiation of stem cells into specific lineages.

Designed peptide-grafted hydrogels for human pluripotent stem cell culture and differentiation.

Human pluripotent stem cells (hPSCs) have the ability to differentiate into cells derived from three germ layers and are an attractive cell source for cell therapy in regenerative medicine. However, hPSCs cannot be cultured on conventional tissue culture flasks but can be cultured on biomaterials with specific hPSC integrin interaction sites. We designed hydrogels conjugated with several designed peptides that had laminin-ß4 active sites, optimal elasticities and different zeta potentials. A higher expansion fold of hPSCs cultured on the hydrogels was found with the increasing zeta potential of the hydrogels conjugated with designed peptides, where positive amino acid (lysine) insertion into the peptides promoted higher zeta potentials of the hydrogels and higher expansion folds of hPSCs when cultured on the hydrogels using xeno-free protocols. The hPSCs cultured on hydrogels conjugated with the optimal peptides showed a higher expansion fold than those on recombinant vitronectin-coated plates, which are the gold standard of hPSC cultivation dishes. The hPSCs could differentiate into specific cell lineages, such as mesenchymal stem cells (MSCs) and MSC-derived osteoblasts, even after being cultivated on hydrogels conjugated with optimal peptides for long periods of time, such as 10 passages.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 transfection of guide RNA targeting on MMP9 as anti-cancer therapy in human cutaneous squamous cell carcinoma cell line A431.

Introduction: Cutaneous squamous cell carcinoma (SCC) is the second most common form of skin malignancy, representing around 20% of all skin cancers. It is the main cause of death due to non-melanoma skin cancer every year. Metastatic cutaneous SCC is associated with poor prognosis in patients and warrants a more effective and specific approach such as disruption of genes associated with cancer metastasis. Material and methods: Matrix metalloproteinases (MMPs) are enzymes involved in cancer progression and are regarded as major oncotargets. Among others, MMP9 plays critical roles in tumour progression, angiogenesis, and invasion of cutaneous SCC. We aimed to determine whether the MMP9 gene is a suitable gene target for anti-cancer therapy for cutaneous SCC. We performed clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 transfection of guide RNA (gRNA) targeting the MMP9 gene into human cutaneous SCC cell line A431. Results: Following CRISPR transfection treatment, the viability (p < 0.01) and migratory activities (p < 0.0001) of in vitro cutaneous SCC cells were found to be reduced significantly. The use of quantitative polymerase chain reaction (qPCR) also revealed downregulation of the mRNA expression levels of cancer-promoting genes TGF-ß, FGF, PI3K, VEGF-A, and vimentin. Direct inhibition of the MMP9 gene was shown to decrease survivability and metastasis of cutaneous SCC cell line A431. Conclusions: Our findings provided direct evidence that MMP9 is important in the viability, proliferation, and metastasis of cutaneous SCC cells. It serves as a positive foundation for future CRISPR-based targeted anti-cancer therapies in treating skin cancer and other forms of malignancies that involve MMPs as the key determinants.

Structural, Optical, Antibacterial, and Anticancer Properties of Cerium Oxide Nanoparticles Prepared by Green Synthesis Using Morinda citrifolia Leaves Extract.

Currently, new advancements in the area of nanotechnology opened up new prospects in the field of medicine that could provide us with a solution for numerous medical complications. Although a several varieties of nanoparticles is being explored to be used as nanomedicines, cerium oxide nanoparticles (CeO2 NPs) are the most attractive due to their biocompatibility and their switchable oxidation state (+3 and +4) or in other words the ability to act as prooxidant and antioxidant depending on the pH condition. Green synthesis of nanoparticles is preferred to make it more economical, eco-friendly, and less toxic. The aim of our study here is to formulate the CeO2 NPs (CeO2 NPs) using Morinda citrifolia (Noni) leaf extract and study its optical, structural, antibacterial, and anticancer abilities. Their optical and structural characterization was accomplished by employing X-ray diffractography (XRD), TEM, EDAX, FTIR, UV-vis, and photoluminescence assays. Our CeO2 NPs expressed strong antibacterial effects against Gram-positive S. aureus and S. pneumonia in addition to Gram-negative E. coli and K. pneumonia when compared with amoxicillin. The anticancer properties of the green synthesized CeO2 NPs against human acute lymphoblastic leukemia (ALL) MOLT-4 cells were further explored by the meticulous study of their ability to diminish cancer cell viability (cytotoxicity), accelerate apoptosis, escalate intracellular reactive oxygen species (ROS) accumulation, decline the mitochondria membrane potential (MMP) level, modify the cell adhesion, and shoot up the activation of proapoptotic markers, caspase-3, -8, and -9, in the tumor cells. Altogether, the outcomes demonstrated that our green synthesized CeO2 NPs are an excellent candidate for alternative cancer therapy.

Synthesis of Zinc Oxide (ZnO)-Titanium Dioxide (TiO2)-Chitosan-Farnesol Nanocomposites and Assessment of Their Anticancer Potential in Human Leukemic MOLT-4 Cell Line.

Leukemia is the most prevalent cancer in children and one of the most common and deadly cancers that affect adults. Several metal oxide nanoparticles, biopolymers, and phytochemicals have been discovered to target cancer cells selectively while inflicting low to no damage to healthy cells. Among the existing nanoparticle synthesis methodologies, biologically synthesized nanoparticles using phytochemicals have emerged as a straightforward, economical, and environmentally sound strategy. The synergistic antitumor potential of ZnO-TiO2-chitosan-farnesol nanocomposites (NCs) against leukemia MOLT-4 cells was investigated in the current study. After synthesizing the NCs, characterization of the same was carried out using XRD, DLS, FESEM, TEM, PL, EDX, and FTIR spectroscopy. To analyze its anticancer activity, MOLT-4 cells were cultured and treated at diverse dosages of NCs. The cell viability upon treatment was examined by MTT assay. The morphological and nuclear modifications were observed by dual staining. ROS and MMP levels were observed by DCFH-DA staining and Rh-123 dye, respectively. Furthermore, the caspase 3, 8, and 9 levels were examined by performing ELISA. The XRD patterns exhibited a hexagonal structure of the NCs. In the DLS spectrum, the hydrodynamic diameter of the NCs was observed to be 126.2 nm. The electrostatic interface between the ZnO-TiO2-chitosan-farnesol NCs was confirmed by the FTIR spectra. A significant loss of cell viability in a dosage-dependent trend confirmed the cytotoxic effect of the NCs. An elevated ROS level and MMP depletion suggested apoptosis-associated cell death via the intrinsic pathway, which was confirmed by elevated expressions of caspase 3, 8, and 9 markers. Thus, the results showed that the synthesized NCs demonstrated a remarkable anticancer potential against leukemic cells and can be potentially valuable in cancer treatments. The findings from this study conclude that this is a new approach for modifying the physicochemical characteristics of ZnO-TiO2-chitosan-farnesol composites to increase their properties and synergistically exhibit anticancer properties in human leukemic cancer cells.

Synthesis, Characterization, and Antiproliferative Effect of CuO-TiO2-Chitosan-Amygdalin Nanocomposites in Human Leukemic MOLT4 Cells.

The main aim of this study was to synthesize copper oxide- (CuO-) titanium oxide- (TiO2-) chitosan-amygdalin nanocomposites (CTCANc) and to characterize them physically and biologically (antimicrobial and anticancer activity using MOLT4 blood cancer cell line) to endorse their useful applications as potential drug candidates in anticancer avenues. CuO-TiO2-chitosan-amygdalin nanocomposites were synthesized according to standard, reported methods. Physical characterization of the nanocomposites was performed using methods like X-ray diffractometer (XRD), and morphological and ultrastructural analysis of nanocomposites were done using electron microscope scanning and transmission. FTIR was recorded using a Perkin-Elmer spectrometer, and photoluminescence (PL) spectra were done using the spectrometer. Further, antibacterial activities were assessed using standard bacterial cultures. To demonstrate the nanocomposite's anticancer effects, MTT assay, morphological analysis, apoptosis studies using acridine orange/ethidium bromide (AO/EtBr) dual staining, reactive oxygen species (ROS) analysis, and levels of antioxidant enzymes were analyzed using the MOLT4 blood cancer cell line. Synthesized nanocomposites were characterized using XRD and showed various peaks, respectively, for CuO-TiO2, amygdalin, and chitosan. MTT assay indicated an IC50 value of 38.41 µg/ml concentration of CTCANc. Hence, 30 and 40 µg/ml were used for the subsequent experiments. Morphological analysis, staining for apoptosis using AO/EtBr, mitochondrial membrane potential (MMP or ΔΨm) analysis, ROS analysis, and determination of the SOD, CAT, MDA, and GSH levels were performed. Observations like a significant loss of morphology, induction of apoptosis, elevated ROS, and decreased MMP were significant in 30 and 40 µg/ml nanocomposite-treated cells when compared to control cells. The bimetallic nanocomposites exhibited typical nanocomposites characteristics and significant antibacterial and anticancer effects. The study results endorse the antibacterial, anticancer activity of CuO-TiO2-chitosan-amygdalin nanocomposites and strongly suggest that further in-depth research using CuO-TiO2-chitosan-amygdalin nanocomposites could reveal their efficacy in the clinical scenario.

CuO-TiO2-Chitosan-Berbamine Nanocomposites Induce Apoptosis through the Mitochondrial Pathway with the Expression of P53, BAX, and BCL-2 in the Human K562 Cancer Cell Line.

In this study, cells from human Chronic Myelogenous Leukemia (K562) were cultivated with CuO-TiO2-Chitosan-Berbamine nanocomposites. We examined nanocomposites using XRD, DLS, FESEM, TEM, PL, EDAX, and FTIR spectroscopy, as well as MTT for cytotoxicity, and AO/EtBr for apoptotic morphology assessment. The rate of apoptosis and cell cycle arrests was determined using flow cytometry. Flow cytometry was also employed to identify pro- and antiapoptotic proteins such as Bcl2, Bad, Bax, P53, and Cyt C. The FTIR spectrum revealed that the CuO-TiO2-Chitosan-Berbamine nanocomposites were electrostatically interlocked. The nanocomposites' XRD signals revealed a hexagonal shape. In the DLS spectrum, nanocomposites were found to have a hydrodynamic diameter. As a result of their cytotoxic action, nanocomposites displayed concentration-dependent cytotoxicity. The nanocomposites, like Doxorubicin, caused cell cycle phase arrest in K562 cells. After treatment with IC50 concentrations of CuO-TiO2-Chitosan-Berbamine nanocomposites and Doxorubicin, a substantial percentage of cells were in G2/M stage arrest. Caspase-3, -7, -8, -9, Bax, Bad, Cyt C, and P53 expression were considerably enhanced in K562 cells, whereas Bcl2 expression was decreased, indicating that these cells may have therapeutic potential against human blood cancer/leukemia-derived disorders. As a result, the nanocomposites demonstrated outstanding anticancer potential against leukemic cells. CuO-TiO2-Chitosan-Berbamine, according to our findings.

In silico Prediction of Deleterious Single Nucleotide Polymorphism in S100A4 Metastatic Gene: Potential Early Diagnostic Marker.

S100A4 protein overexpression has been reported in different types of cancer and plays a key role by interacting with the tumor suppressor protein Tp53. Single nucleotide polymorphisms (SNP) in S100A4 could directly influence the biomolecular interaction with the tumor suppressor protein Tp53 due to their aberrant conformations. Hence, the study was designed to predict the deleterious SNP and its effect on the S100A4 protein structure and function. Twenty-one SNP data sets were screened for nonsynonymous mutations and subsequently subjected to deleterious mutation prediction using different computational tools. The screened deleterious mutations were analyzed for their changes in functionality and their interaction with the tumor suppressor protein Tp53 by protein-protein docking analysis. The structural effects were studied using the 3DMissense mutation tool to estimate the solvation energy and torsion angle of the screened mutations on the predicted structures. In our study, 21 deleterious nonsynonymous mutations were screened, including F72V, E74G, L5P, D25E, N65S, A28V, A8D, S20L, L58P, and K26N were found to be remarkably conserved by exhibiting the interaction either with the EF-hand 1 or EF-hand 2 domain. The solvation and torsion values significantly deviated for the mutant-type structures with S20L, N65S, and F72L mutations and showed a marked reduction in their binding affinity with the Tp53 protein. Hence, these deleterious mutations might serve as prospective targets for diagnosing and developing personalized treatments for cancer and other related diseases.

Differential Regulation of NK Cell Receptors in Acute Lymphoblastic Leukemia.

Cancer immunotherapies are preferred over conventional treatments which are highly cytotoxic to normal cells. Focus has been on T cells but natural killer (NK) cells have equal potential. Concepts in cancer control and influence of sex require further investigation to improve successful mobilization of immune cells in cancer patients. Acute lymphoblastic leukemia (ALL) is a hematological malignancy mainly of B cell (B-ALL) and T cell (T-ALL) subtypes. Influence of ALL on NK cell is still unclear. Targeted next-generation sequencing was conducted on 62 activating/inhibitory receptors, ligands, effector, and exhaustion molecules on T-ALL (6 males) and normal controls (NC) (4 males and 4 females). Quantitative PCR (q-PCR) further investigated copy number variation (CNV), methylation index (MI), and mRNA expression of significant genes in T-ALL (14 males), NC (12 males and 12 females), and B-ALL samples (N = 12 males and 12 females). Bioinformatics revealed unique variants particularly rs2253849 (T>C) in KLRC1 and rs1141715 (A>G) in KLRC2 only among T-ALL (allele frequency 0.8-1.0). Gene amplification was highest in female B-ALL compared to male B-ALL (KLRC2, KLRC4, and NCR3, p < 0.05) and lowest in male T-ALL cumulating in deletion of KLRD1 and CD69. MI was higher in male ALL of both subtypes compared to normal (KIR2DL1-2 and 4 and KIR2DS2 and 4, p < 0.05) as well as to female B-ALL (KIR3DL2 and KIR2DS2, p < 0.05). mRNA expressions were low. Thus, ALL subtypes potentially regulated NK cell suppression by different mechanisms which should be considered in future immunotherapies for ALL.

Rescue of photoreceptor with human mesenchyme stem cell and human mesenchyme stem cell expressing erythropoietin in total degeneration of retina animal model.

PURPOSE: This study aimed to investigate the efficacy of human-derived umbilical cord mesenchymal stem cells (HDUMSC) and human-derived umbilical cord mesenchymal stem cells expressing erythropoietin (HDUMSC-EPO) to rescue total degenerated retina in a rat model. METHODS: The study included four treatment groups, namely negative control using normal saline (HBSS) injection, positive control using sodium iodide 60 mg/kg (SI), SI treated with HDUMSC, and SI treated with HDUMSC-EPO given via subretinal and intravenous routes, to test the efficacy of retinal regeneration following SI-induced retinal degeneration. Retinal function in both phases was tested via electroretinography (ERG) and histological staining examining the outer nuclear layer (ONL). RESULTS: There was a statistically significant result (P < 0.05) in the SI treated with HDUMSC-EPO only when comparing day 11 (mean = 23.6 µv), day 18 (mean = 25.2 µv), day 26 (mean = 26.3 µv), and day 32 (mean = 28.2 µv) to the b-wave ERG on day 4 rescue injection day (mean = 12.5 µv). The SI treated with HDUMSC-EPO showed significant improvement in b-wave ERG readings in the Sprague-Dawley (SD) rat but did not restore baseline readings prior to degeneration (day 0). Both treated groups' ONL thicknesses did not show significant changes compared to the negative control group (HBSS) following rescue therapy. CONCLUSION: Total retinal degeneration following intravenous SI injection was observed at 60 mg/kg. SI treated with HDUMSC and HDUMSC-EPO showed no regenerative potential compared to baseline in SI-induced total retina degeneration on ERG or histology, whereas SI treated with HDUMSC-EPO group showed a substantial increase in b-wave ERG amplitude over time.

Camptothecin Encapsulated in ß-Cyclodextrin-EDTA-Fe3O4 Nanoparticles Induce Metabolic Reprogramming Repair in HT29 Cancer Cells through Epigenetic Modulation: A Bioinformatics Approach.

Cancer progresses through a distinctive reprogramming of metabolic pathways directed by genetic and epigenetic modifications. The hardwired changes induced by genetic mutations are resilient, while epigenetic modifications are softwired and more vulnerable to therapeutic intervention. Colon cancer is no different. This gives us the need to explore the mechanism as an attractive therapeutic target to combat colon cancer cells. We have previously established the enhanced therapeutic efficacy of a newly formulated camptothecin encapsulated in ß-cyclodextrin-EDTA-Fe3O4 nanoparticles (CPT-CEF) in colon cancer cells. We furthered this study by carrying out RNA sequencing (RNA-seq) to underscore specific regulatory signatures in the CPT-CEF treated versus untreated HT29 cells. In the study, we identified 95 upregulated and 146 downregulated genes spanning cellular components and molecular and metabolic functions. We carried out extensive bioinformatics analysis to harness genes potentially involved in epigenetic modulation as either the cause or effect of metabolic rewiring exerted by CPT-CEF. Significant downregulation of 13 genes involved in the epigenetic modulation and 40 genes from core metabolism was identified. Three genes, namely, DNMT-1, POLE3, and PKM-2, were identified as the regulatory overlap between epigenetic drivers and metabolic reprogramming in HT29 cells. Based on our results, we propose a possible mechanism that intercepts the two functional axes, namely epigenetic control, and metabolic modulation via CPT-CEF in colon cancer cells, which could skew cancer-induced metabolic deregulation towards metabolic repair. Thus, the study provides avenues for further validation of transcriptomic changes affected by these deregulated genes at epigenetic level, and ultimately may be harnessed as targets for regenerating normal metabolism in colon cancer with better treatment potential, thereby providing new avenues for colon cancer therapy.

Treatment of HT29 Human Colorectal Cancer Cell Line with Nanocarrier-Encapsulated Camptothecin Reveals Histone Modifier Genes in the Wnt Signaling Pathway as Important Molecular Cues for Colon Cancer Targeting.

Cancer cells are able to proliferate in an unregulated manner. There are several mechanisms involved that propel such neoplastic transformations. One of these processes involves bypassing cell death through changes in gene expression and, consequently, cell growth. This involves a complex epigenetic interaction within the cell, which drives it towards oncogenic transformations. These epigenetic events augment cellular growth by potentially altering chromatin structures and influencing key gene expressions. Therapeutic mechanisms have been developed to combat this by taking advantage of the underlying oncogenic mechanisms through chemical modulation. Camptothecin (CPT) is an example of this type of drug. It is a selective topoisomerase I inhibitor that is effective against many cancers, such as colorectal cancer. Previously, we successfully formulated a magnetic nanocarrier-conjugated CPT with ß-cyclodextrin and iron NPs (Fe3O4) cross-linked using EDTA (CPT-CEF). Compared to CPT alone, it boasts higher efficacy due to its selective targeting and increased solubility. In this study, we treated HT29 colon cancer cells with CPT-CEF and attempted to investigate the cytotoxic effects of the formulation through an epigenetic perspective. By using RNA-Seq, several differentially expressed genes were obtained (p < 0.05). Enrichr was then used for the over-representation analysis, and the genes were compared to the epigenetic roadmap and histone modification database. The results showed that the DEGs had a high correlation with epigenetic modifications involving histone H3 acetylation. Furthermore, a subset of these genes was shown to be associated with the Wnt/ß-catenin signaling pathway, which is highly upregulated in a large number of cancer cells. These genes could be investigated as downstream therapeutic targets against the uncontrolled proliferation of cancer cells. Further interaction analysis of the identified genes with the key genes of the Wnt/ß-catenin signaling pathway in colorectal cancer identified the direct interactors and a few transcription regulators. Further analysis in cBioPortal confirmed their genetic alterations and their distribution across patient samples. Thus, the findings of this study reveal that colorectal cancer could be reversed by treatment with the CPT-CEF nanoparticle-conjugated nanocarrier through an epigenetic mechanism.