Human Malignant Cells Research Articles

Knockdown of NUPR1 inhibits angiogenesis in lung cancer through IRE1/XBP1 and PERK/eIF2α/ATF4 signaling pathways.

The stress response molecule nuclear protein‑1 (NUPR1) is essential for the growth of multiple types of human malignant tumor cells. However, the significance of NUPR1 in lung cancer is still not entirely elucidated. Therefore, this study is aimed to explore the function and underlying mechanisms of NUPR1 in lung cancer. NUPR1 mRNA and protein levels in lung cancer cell lines (A549 or H1299 cells) were silenced through siRNA transfection and western blot observed its successful infection efficiency. Then, using tube formation and wound healing experiments, the effects of si-NUPR1 on angiogenesis and migration of human umbilical vein endothelial cells (HUVEC) were examined, respectively, which indicated inhibitory effects on the angiogenesis and migration of HUVEC. Vascular endothelial growth factor A (VEGFA), a vital molecule in angiogenesis, was detected by PCR and western blot assays, manifesting NUPR1 knockdown represses VEGFA expression. Furthermore, the knockdown of NUPR1 may reduce angiogenesis by lowering VEGFA expression through inositol-requiring enzyme 1 (IRE1)/X box binding protein 1 (XBP1) and protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2 A (eIF2α)/recombinant activating transcription factor 4 (ATF4) signaling pathways in A549 or H1299 cells. In conclusion, these findings demonstrated that NUPR1 knockdown inhibits angiogenesis in A549 and H1299 cells through IRE1/XBP1 and PERK/eIF2α/ATF4 signaling pathways, indicating that NUPR1 could represent a novel lung cancer therapeutic target.

Silver(I) Bromide Phosphines Induce Mitochondrial-Mediated Apoptosis in Malignant Human Colorectal Cells.

Due to its emerging resistance to current therapies, colon cancer remains one of the most difficult types of cancer to treat. Silver, a non-invasive metal, is well-known for its antimicrobial and anti-cancer properties. Two novel silver(I) phosphine complexes, [silver(I) diphenyl-2-pyridylphosphine]Br (1) and [silver(I) is 4-(dimethylamino)phenyldiphenylphosphine]Br (2), were synthesized and characterized by elemental analysis, infrared spectroscopy, and nuclear magnetic resonance (1H, 13C, 31P). To assess the complexes' potentials as antiproliferative agents, experiments were conducted on human colorectal cancer cells (HT-29) in vitro. The evaluation involved the analysis of morphological changes, the performance of an alamarBlue® proliferation assay, and the undertaking of flow cytometric analyses to detect mitochondrial alterations. Complex 1 displayed superior selectivity and significant inhibitory effects on malignant HT-29 cells while exhibiting minimal toxicity towards two non-malignant HEK-293 and MRHF cells. Moreover, after 24 h of treatment, complex 1 (IC50, 7.49 µM) demonstrated higher efficacy in inhibiting cell proliferation compared with complex 2 (IC50, 21.75 µM) and CDDP (IC50, 200.96 µM). Flow cytometric studies indicated that complex 1 induced regulated cell death, likely through mitochondrial-mediated apoptosis. Treatment with complex 1 induced morphological changes indicative of apoptosis, which includes membrane blebbing, PS externalization, increased levels of reactive oxygen species (ROS) and mitochondrial membrane depolarization (ΔΨm). These observations suggest that complex 1 targets the mitochondria and holds promise as a novel metal-based anti-cancer therapeutic for the selective treatment of colorectal cancer.

Effects of microRNA-106b on migration and invasion of human malignant pleural mesothelioma cell NCI-H2452

Objective: To investigate the inhibitory effect of microRNA-106b in the process of migration and invasion of human malignant pleural mesothelioma cell NCI-H2452. Methods: In April 2017, the expression level of miRNA-106b in malignant pleural mesothelioma cells (NCI-H2452, MSTO-211H, NCI-H2052) and normal mesothelial cells MeT-5A was detected and analyzed. Using NCI-H2452 cells as a model, the NCI-H2452 cell model with miRNA-106b overexpression was established by transfecting miRNA-106b mimics. The expression level of miRNA-106b in the cells was detected by real-time fluorescent quantitative PCR. The effect of miRNA-106b on the migration and invasion ability of NCI-H2452 cells was analyzed. The gene expression data of malignant mesothelioma and the downstream target gene data of miRNA-106b in public databases were analyzed to screen the downstream target genes of miRNA-106b in mesothelioma cells that affect cell migration and invasion ability, and to verify the expression of this gene in NCI-H2452 cells with miRNA-106b overexpression. Results: The expression of miRNA-106b in three MPM cells was decreased compared with MeT-5A cells (P<0.001) . The expression level of miRNA-106b was significantly increased after transfection of miRNA-106b mimics (P<0.001) . The scratch migration levels of the experimental group were 28.45%±4.37%, 38.12%±4.82% and 50.06%±8.92% at 24h, 31h and 48h, respectively. Compared with the control group, the migration level decreased by 37.48%±2.65%, 49.21%±3.45% and 68.14%±3.81% (P<0.01) . The number of cell migration and invasion decreased in the experimental group compared with the control group (P<0.001) . Public databases were used to screen and analyze the possibility that TCF21 gene, as a downstream target gene, could affect the migration and invasion ability of MPM cells. The expression level of TCF21 gene was increased after transfection of miRNA-106b mimics in NCI-H2452 cells (P=0.009) . Conclusion: MiRNA-106b can inhibit the migration and invasion of NCI-H2452 cells and increase the expression of TCF21 gene.

DNA/BSA binding studies and in vitro anticancer and antibacterial studies of isoelectronic Cu(I)- and Ag(I)-pyridinyl Schiff base complexes incorporating triphenylphosphine as co-ligands

Four mononuclear Ag(I) and Cu(I) Schiff base complexes of the type [M(L)(PPh3)2]X [where M = Ag+ or Cu+, L = (E)-N-(2-fluorophenyl)-1-(pyridin-2-yl)methanimine (fpm) or (E)-1-(pyridin-2-yl)-N-(p-tolyl)methanimine (ptm) and X = NO3–] have been synthesized and characterized by various spectroscopic and analytical techniques. The analysis shows that the complexes are four-coordinates formed via the nitrogen atoms of a bidentate fpm or ptm ligands and two phosphorus atoms from the triphenylphosphine co-ligands. Two of the complexes were structurally characterized by single crystal X-ray diffraction revealing a distorted trigonal pyramidal geometry around the Ag(I) or Cu(I) center. The binding affinity of the complexes with calf thymus-DNA (CT-DNA) and bovine serum albumin (BSA) was determined using UV–visible spectroscopy. The interaction of the complexes with CT-DNA revealed the strong stacking interaction between the aromatic chromophore of the complexes and the base pairs of the CT-DNA. The study of the complexes with BSA showed moderate affinity to BSA, suggesting that complexes 1–4 can be delivered and dispositioned in vivo. Invitro anticancer activity of the synthesized complexes was evaluated against non-cancerous human embryonic kidney 293 (HEK293), cervical cancer cell (HELA), breast cancer cell (MDA-MB231) and malignant human melanoma cell (A375). High antiproliferative effects of the complexes on HELA (with EC50 values between 1.82±1.23 and 10.66±2.87 μM better than cisplatin with EC50 > 50 μM) and MDA-MB231(≈2 to 7-fold effective than cisplatin) were recorded. Further, the invitro antibacterial activity of the synthesized compounds was evaluated against S. aureus, E. coli, S. typhi and P. aeruginosa using the disc diffusion method from which the Cu(I) complexes showed better inhibitory effects than the Ag(I) complexes.

Iron oxide nanoparticles induce ferroptosis via the autophagic pathway by synergistic bundling with paclitaxel.

In recent years, inhibiting tumor cell activity by triggering cell ferroptosis has become a research hotspot. The development of generic targeted nanotherapeutics might bring new ideas for non‑invasive applications. Currently, the potential mechanism underlying the universal application of paclitaxel (PTX)‑loaded iron oxide nanoparticles (IONP@PTX) to different types of tumors is unclear. The present study aimed to prepare IONP@PTX for targeted cancer therapy and further explore the potential mechanisms underlying the inhibitory effects of this material on the NCI‑H446 human small cell lung cancer and brain M059K malignant glioblastoma cell lines. First, a CCK‑8 assay was performed to determine cell viability, and then the combination index for evaluating drug combination interaction effect was evaluated. Intracellular reactive oxygen species (ROS) and lipid peroxidation levels were monitored using a DCFH‑DA fluorescent probe and a C11‑BODIPY™ fluorescent probe, respectively. Furthermore, western blotting assay was performed to determine the expression of autophagy‑ and iron death‑related proteins. The experimental results showed that, compared with either IONP monotherapy, PTX monotherapy, or IONP + PTX, IONP@PTX exerted a synergistic effect on the viability of both cell types, with significantly increased total iron ion concentration, ROS levels and lipid peroxidation levels. IONP@PTX significantly increased the expression of autophagy‑related proteins Beclin 1 and histone deacetylase 6 (HDAC6) in both cell lines (P<0.05), increased the expression of light chain 3 (LC3)‑II/I in NCI‑H446 cells (P<0.05) and decreased that of sequestosome1 (p62) in M059K cells (P<0.05). Moreover, the addition of rapamycin enhanced the IONP@PTX‑induced the upregulation of Beclin 1, LC3‑II/I and HDAC6 and the downregulation of mTORC1 protein in both cell lines (P<0.05). Moreover, rapamycin enhanced the IONP@PTX‑induced downregulation of p62 protein in NCI‑H446 cells (P<0.05), suggesting that IONP@PTX induces ferroptosis, most likely through autophagy. Collectively, the present findings show that IONP works synergistically with PTX to induce ferroptosis via the autophagic pathway.

Progress in construction of mouse models to investigate the pathogenesis and immune therapy of human hematological malignancy.

Hematological malignancy is a disease arisen by complicate reasons that seriously endangers human health. The research on its pathogenesis and therapies depends on the usage of animal models. Conventional animal model cannot faithfully mirror some characteristics of human features due to the evolutionary divergence, whereas the mouse models hosting human hematological malignancy are more and more applied in basic as well as translational investigations in recent years. According to the construction methods, they can be divided into different types (e.g. cell-derived xenograft (CDX) and patient-derived xenograft model (PDX) model) that have diverse characteristics and application values. In addition, a variety of strategies have been developed to improve human hematological malignant cell engraftment and differentiation in vivo. Moreover, the humanized mouse model with both functional human immune system and autologous human hematological malignancy provides a unique tool for the evaluation of the efficacy of novel immunotherapeutic drugs/approaches. Herein, we first review the evolution of the mouse model of human hematological malignancy; Then, we analyze the characteristics of different types of models and summarize the ways to improve the models; Finally, the way and value of humanized mouse model of human immune system in the immunotherapy of human hematological malignancy are discussed.

T-cell dysfunction by pseudohypoxia and autocrine purinergic signaling in chronic lymphocytic leukemia

AbstractAcquired T-cell dysfunction is common in chronic B-cell malignancies. Given the strong connection between T-cell metabolism and function, we investigated metabolic alterations as the basis of T-cell dysfunction induced by malignant cells. Using B-cell malignant cell lines and human peripheral blood mononuclear cells, we first established a model that recapitulates major aspects of cancer-induced T-cell dysfunction. Cell lines derived from chronic lymphocytic leukemia (CLL) (PGA-1, CII, and Mec-1), but not from other B-cell malignancies, altered the T-cell metabolome by generating a pseudohypoxic state. T cells were retained in aerobic glycolysis and were not able to switch to oxidative phosphorylation (OXPHOS). Moreover, T cells produced immunosuppressive adenosine that negatively affected function by dampening the activation, which could be restored by the blocking of adenosine receptors. Subsequently, we uncovered a similar hypoxic-like signature in autologous T cells from primary CLL samples. Pseudohypoxia was reversible upon depletion of CLL cells ex vivo and, importantly, after the in vivo reduction of the leukemic burden with combination therapy (venetoclax and obinutuzumab), restoring T-cell function. In conclusion, we uncovered a pseudohypoxic program connected with T-cell dysfunction in CLL. Modulation of hypoxia and the purinergic pathway might contribute to therapeutic restoration of T-cell function.

Raf/MEK/ERK Signaling Pathway Is Involved in the Inhibition of Glioma Cell Proliferation and Invasion in the Ketogenic Microenvironment.

A high-fat, low-carbohydrate ketogenic diet has been used to treat malignant glioma, in which the Raf/MEK/ERK signaling pathway is overactivated. However, whether the Raf/MEK/ERK signaling pathway is involved in the therapeutic effect of ketone bodies remains unknown. In this study, we investigated the effects of a major ketone body, 3-hydroxybutyric acid (3-HBA), on the proliferation and metastasis of malignant glioblastoma cells and the underlying mechanism. Two human malignant glioblastoma cell lines (U87 and U251) were treated with different concentrations of 3-HBA with or without the Raf inhibitor PAF C-16 for 24 h. Cell proliferation, cell cycle, cell invasion, and phospholipase D1 (PLD1) activity were determined. Protein and gene expression levels of Raf/MEK/ERK signaling pathway members were examined. 3-HBA significantly decreased cell proliferation, invasion, and intracellular PLD1 activity in both U87 and U251 glioblastoma cell lines. 3-HBA treatment significantly increased the proportion of cells in the G1 phase and decreased the proportion of cells in S phase in U87 cells. In the U251 line, the proportion of treated cells in S phase was increased and proportion of cells in G2 was decreased. 3-HBA treatment also significantly decreased the protein expression levels of Raf, MEK, p-MEK, ERK, p-ERK, and PLD1 while increasing p53 expression; an effect that was similar to treatment with the Raf inhibitor. Co-treatment of 3-HBA with the Raf inhibitor further enhanced the effects of the 3-HBA in both cell lines. We confirmed that a ketogenic microenvironment can inhibit glioma cell proliferation and invasion by downregulating the expression of PLD1 through the Raf/MEK/ERK signaling pathway.

Green supported silver nanoparticles over modified reduced graphene oxide: Investigation of its antioxidant and anti-ovarian cancer effects

Abstract A green biosynthesis of silver nanoparticles (AgNPs) decorated Mentha longifolia root extract-functionalized graphene oxide (GO) nanohybrid material has been described. Initially, the Mentha longifolia root was coated on GO’s surface. The phytochemicals of the plant acted as reducing agent for reduction of silver ions and GO to form the rGO-Mentha/Ag nanocomposite. The nanocomposite was characterized using FE-SEM, EDX, FT-IR, TEM, elemental mapping, and XRD analysis. The cells treated with rGO-Mentha/Ag nanocomposite were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay for 48 h about the cytotoxicity and anti-human ovarian cancer properties on normal (HUVEC) and human ovarian cancer cell lines, i.e., SKOV3 and A2780. The IC50 of rGO-Mentha/Ag nanocomposite were 181.2 and 196.4 µg/mL against SKOV3 and A2780cell lines, respectively. The viability of malignant human ovarian cell line reduced dose-dependently in the presence of rGO-Mentha/Ag nanocomposite. After clinical study, rGO-Mentha/Ag nanocomposite can be introduced as a novel composite in the treatment of human ovarian cancer.

Antioxidant Property and Inhibition of Tyrosinase and Melanin Synthesis of the Korean Fir (Abies koreana Wilson) Needle Extracts

Korean fir (Abies koreana Wilson) is traditionally used in folk medicine for its antibacterial, memory-enhancing, and anti-inflammatory properties. In this study, we evaluated the antioxidant and skin-whitening effects of the methanol and ethanol extracts of Korean fir needles. The extracts were tested for their antioxidant capacity using various assays, including radical scavenging (1,1-diphenyl-2-picrylhydrazyl, O2-, H2O2 and NOž), SOD-like, ferrous ion chelating, and reducing power assays. The total phenolic and flavonoid contents were determined by the Folin-Ciocalteu method. The non-toxic doses of the extracts were determined by MTT assay using human malignant melanoma SK mel-100 cells, and the tyrosinase activity and melanin contents were measured using an enzyme-substrate assay. The results showed that the antioxidant activity of the Korean fir needle extracts increased in a dose-dependent manner, as confirmed by their radical scavenging activities in the 2,2-diphenyl-1-1-picrylhydrazyl and 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) assays. The Korean fir needle extract significantly reduced tyrosinase activity and melanin content in a dose-dependent manner (p &lt; 0.01), suggesting its potential use as a skin-whitening agent. The methanol extracts of the Korean fir needles exhibited significantly higher phenolic (8306 mg gallic acid equivalents/100 g) content, with higher superoxide (IC50 = 4.22 mg/mL) and nitric oxide (IC50 = 1.50 mg/mL) radical scavenging activities and inhibition of tyrosinase and melanin synthesis than those of ethanol extracts (p &lt; 0.05). Overall, our results demonstrate the potential of Korean fir (Abies koreana Wilson) needles as a source of tyrosinase inhibitors and antioxidants for inhibiting melanin biosynthesis, which could have applications in the cosmetic and pharmaceutical industries.

In vitro effects of undifferentiated callus extracts from Plantago major L, Rhodiola rosea L and Silybum marianum L in normal and malignant human skin cells

Background and objectivesThe occurrence of non-melanoma and melanoma skin cancers is currently increasing rapidly with one in every three cancers diagnosed as a skin cancer. A useful strategy to control the progression of skin cancer could be the use of plant flavonoids that suppress pro-inflammatory cytokines involved in tumor initiation and progression. In this study, the anti-inflammatory and antioxidant activity of undifferentiated callus extracts from Plantago major L, Silybum marianum L and Rhodiola rosea L was investigated both in normal and malignant skin cells. MethodsAntioxidant activity of the extracts was analyzed by using the Trolox Equivalent Antioxidant Capacity (TEAC) assay. High-Performance Thin-Layer Chromatography (HPTLC) was performed to demonstrate the phytochemical profile, and the total flavonoid content was analyzed with an aluminum chloride colorimetric method. The anti-inflammatory effect was investigated by cell treatments using the plant extracts. Thereafter, the possible suppression of induced IL-6 response was measured from the cultured skin cancer cell lines A2058 and A431, and normal primary keratinocytes with Enzyme-Linked Immunosorbent Assay (ELISA). ResultsThe HPTLC analysis assessed that the extracts contained a complex phytochemical profile that was rich in phenolic and flavonoid compounds. Dose response assays showed that concentrations between 15 and 125 μg/mL of all three plant extracts could be used to investigate an effect on the IL-6 production. The S. marianum extract had the most pronounced anti-inflammatory effect, which significantly inhibited induced IL-6 production in both normal keratinocytes and skin cells derived from epidermal carcinoma. The extract from S. marianum also had the highest flavonoid content and showed the highest antioxidant activity of the three extracts tested. ConclusionAll in all, we have confirmed that undifferentiated callus extracts of S. marianum possess properties such as antioxidant and anti-inflammatory activities in both normal and malignant keratinocytes, and thus could be a promising agent controlling the pro-inflammatory IL-6 production.

Innovative retargeted oncolytic herpesvirus against nectin4-positive cancers.

Nectin4 is a recently discovered tumor associated antigen expressed in cancers that constitute relevant unmet clinical needs, including the undruggable triple negative breast cancer, pancreatic ductal carcinoma, bladder/urothelial cancer, cervical cancer, lung carcinoma and melanoma. So far, only one nectin4-specific drug-Enfortumab Vedotin-has been approved and the clinical trials that test novel therapeutics are only five. Here we engineered R-421, an innovative retargeted onco-immunotherapeutic herpesvirus highly specific for nectin4 and unable to infect through the natural herpes receptors, nectin1 or herpesvirus entry mediator. In vitro, R-421 infected and killed human nectin4-positive malignant cells and spared normal cells, e.g., human fibroblasts. Importantly from a safety viewpoint, R-421 failed to infect malignant cells that do not harbor nectin4 gene amplification/overexpression, whose expression level was moderate-to-low. In essence, there was a net threshold value below which cells were spared from infection, irrespective of whether they were malignant or normal; the only cells that R-421 targeted were the malignant overexpressing ones. In vivo, R-421 decreased or abolished the growth of murine tumors made transgenic for human nectin4 and conferred sensitivity to immune checkpoint inhibitors in combination therapies. Its efficacy was augmented by the cyclophosphamide immunomodulator and decreased by depletion of CD8-positive lymphocytes, arguing that it was in part T cell-mediated. R-421 elicited in-situ vaccination that protected from distant challenge tumors. This study provides proof-of-principle specificity and efficacy data justifying nectin4-retargeted onco-immunotherapeutic herpesvirus as an innovative approach against a number of difficult-to-drug clinical indications.

Novel Strategy to Regulate PD-L1 Expression and Enhance Anti-Tumor Effect

Abstract The programmed death 1/PD ligand 1(PD-1/PD-L1 axis is a major player in the tumor immune evasion strategy. We show that the immunomodulatory small tellurium molecules, AS101 [ammonium trichloro(dioxoethylene-o, o′)tellurate] and SAS [octa-O-bis-(R,R)-tartarate ditellurane], suppress PD-L1 expression on a variety of human and mouse malignant cells by inhibition of the α4β1 VLA-4 (very late antigen-4) integrin activity. As a consequence, pAkt and its downstream effector pNFκB are inhibited. VLA-4/pAkt/pNFκB signaling blockade was found crucial for decreasing PD-L1 expression. Moreover, the tellurium compounds had physiological effects. SAS augmented killing of human and mouse malignant cells by stimulated syngeneic splenocytes or CD8+ T cells. Furthermore, SAS prevented the development of chemoresistance in malignant cells. IL-10 production by tumor cells, the inhibition of which by AS101 we showed suppressed chemoresistance, is shown here to be closely associated with inhibition of VLA-4 and PD-L1 expression suggesting that AS101 and SAS compounds may, at least partially, induce chemosensitivity by virtue of inhibition of the VLA-4/IL-10/PD-L1 pathway. AS101 or SAS treatment of B16 melanoma-bearing mice decreased tumor cell PD-L1 expression, led to increased CD8+ T-cell infiltration into the tumors and tumor shrinkage. Combined treatment with αPD-1 antibody and either of the tellurium compounds significantly increased antitumor effects. These results collectively suggest that VLA-4 integrin signaling serves as a critical component of cancer evasion and is a potential target for cancer treatment. Furthermore, AS101 or SAS may be suitable for effective combinatorial cancer immunotherapy with αPD-1.

Anti-cancer activity, and molecular docking of novel hybrid heterocyclic steroids revealed promising anti-hepatocellular carcinoma agent: Implication of cyclin dependent kinase-2 pathway.

To identify new steroidal agents with potential biological activities, we synthesized hybrid steroids containing thiazole, pyrazole, isoxazole, thiophene or phthalazine moiety. Epi-androsterone 1 reacted with phenylthiosemicarbazide to afford the corresponding androstane-4-phenyl-3-thiosemicarbazone derivative 2. The latter product was used in the synthesis of a series of annulated steroid derivatives. Also, Epi-androsterone 1 reacted with the thienopyridazine derivative 16 to afford the thieno[3,4-d]pyridazino-N-ylidenoandrostane derivative 17. Compound 17 reacted readily with electron-poor olefins to yield the corresponding phthalazine steroid derivatives. Detailed experimental and spectroscopic evidences for the structures of the newly synthesized compounds are explained. Compounds 3, 7, 8a, 12a, 14, 17 and 21a, were investigated individually as anticancer agents on different panel of human malignant cell lines. Moreover, a computer modelling investigation was performed to speculate the macromolecular targets for the most promising candidate. The results revealed a concentration-dependent reduction in the number of viable cells in all cancer cell lines. Most notably, compound 7 was the most effective compound against all tested cancer cell lines, especially against HepG2 cell line; therefore, the mode of action of this compound against HCC was investigated. Compound 7 was able to induce cell cycle arrest, and DNA fragmentation in HepG2 cells. Moreover, compound 7 induced apoptosis via upregulating the expression of caspase-3, -8, -9, P53, Bax and inhibiting the expression of BCL2, and CDK2 genes. Our results highlighted compound 7 as a promising anti-hepatocellular carcinoma agent, with theoretical, and practical potential binding affinity with CDK2; therefore, more investigations are required to elucidate its chemotherapeutic value as anti-HCC agent.

Parasporin-4, a novel apoptosis inducer of breast cancer cells produced by Bacillus thuringiensis

Parasporin (PS) proteins have cytocidal activity preferential for various human malignant cells. The purpose of this investigation was to see if the PS separated from B. thuringiensis strain E8 isolate had any particular cytotoxicity against breast cancer. The extracted spores-crystal proteins were solubilized and digested with proteinase K. Cytotoxicity effects were analysed by MTT assay. Caspases activities were measured using ELISA. SDS-PAGE analysis was performed for determination of molecular weight of Cry protein. Identification of extracted proteins function was evaluated by MALDI-TOF MS analysis. Breast cancer cells line (MCF-7) was highly susceptible to 1mg/mL PS and showed apoptosis characteristics, but it has no effects on the normal cells (HEK293). Apoptosis evaluation showed that caspases 1, 3, 9 and BAX were remarkably up-regulated in cancer cells, indicating the intrinsic pathway activation in these cells. PS Size was determined using SDS-PAGE in E8 isolate as 34kDa and a 25kDa digested peptide was identified as PS4. The function of PS4 was reported as an ABC-transporter by spectrometry. The data of the present study show that PS4 is a selective cytotoxic protein against breast cancer and a molecule with a lot of potentials for next researches.

Chaotic fractals: Why chaos is the dynamic of carcinogenesis

We can see the fractals in our environment every day (trees, snowflakes, broccoli, etc.). Even the shapes of the DNA helix and anatomical structures are fractal, for example, the branching of blood vessels, bronchi, and cell membranes [1]. Unlike euclidean geometry, fractal geometry reveals how an object with irregularities in many dimensions can be identified by examining how the number of features in one dimension relates to the number of similarly shaped features in other dimensions [2]. Mandelbrot used fractal geometry to describe such irregular shapes and demonstrated that this geometry was an appropriate mathematical language for describing chaotic systems [1]. In fractal geometry, the fractal dimension (FD) is a statistical quantity that gives an indication of how completely a fractal appears to fill space, as one zooms down to finer and finer scales. The FD provides a measure of the complexity of a structure. Increased FD is an indicator of chaos [3]. A complex adaptive system (CAS) is a type of system that is composed of many interacting components, called agents, which can adapt and change their behavior based on their interactions with the environment and with other agents. CAS are characterized by their ability to self-organize and evolve over time, often resulting in emergent properties and behaviors that cannot be predicted from the properties of the individual agents alone. Examples of CAS include ecosystems, economies, social networks, and the human brain. It is also worth noting that a CAS can have both chaotic and regular behavior depending on the circumstances and the complexity of the system. Stem cells can also be considered CAS because they possess many of the characteristics that define CAS. Stem cells have the ability to self-renew, differentiate into multiple cell types, and respond to signals from their environment [4]. Some studies suggest an important role of the feedback loop between cancer cells and the microenvironment. Also, putting cells into an “inappropriate” microenvironmental context can otherwise trigger pathological issues, and even neoplastic transformation [5]. Cancer has previously been demonstrated to be a chaotic behavior of the stem cell [6]. The FD of chromatin has been demonstrated to increase during carcinogenesis and tumor growth in diffuse large B-cell lymphoma, chronic lymphocytic leukemia, oropharyngeal carcinoma, and hepatocarcinoma compared to equivalent normal tissue. A research study of over 3000 cancer specimens revealed the prevalence of fractal chromatin structure in neoplasias, as well as the importance of this arrangement in the creation of chromosomal abnormalities [7]. Fractal analysis of the cell surface is a rather sensitive method that has been recently introduced to characterize cell progression toward cancer. Analysis of FD of cell surface imaged with atomic force microscopy (AFM) showed strong segregation between normal and malignant human cervical epithelial cells [8]. There is a growing literature that shows FD to be a useful measure of the pathologies of the tumoral vascular architecture, and tumor/parenchymal border [3, 9]. Entropy-based fractal image modeling has been used to increase the diagnostic accuracy of mammographic detection of breast tumors. Mammographic density correlates with the FD of breast cancer and eventually with a higher growth rate. Breast masses with higher FD show increased aggressiveness and poor prognosis [5]. The FD of the lung tumor on contrast-enhanced computed tomography (CT) images demonstrated that the use of FD as a predictive indicator of therapeutic response or progression is warranted [10]. Similarly, the FD of liver mass in contrast-enhanced CT is a useful prognostic biomarker for hepatocellular carcinoma patients treated with sunitinib [11]. The FD analysis of thyroid ultrasound images is used for the prediction and early detection of thyroid malignancy [12]. Moreover, fractal geometry is applied in many radiographic analyses. At least, the application of chaos theory opens up the opportunity for novel dynamic access to carcinogenesis. Of course, the application of chaos theory cannot solve all problems, but such an interdisciplinary approach may increase the understanding of carcinogenesis. Mesut Tez: Writing—review and editing (equal). None. The author declares no conflict of interest. Not applicable. Not applicable. If no data are available, there is no data for this review.

Harmaline exerts potentially anti-cancer effects on U-87 human malignant glioblastoma cells in vitro

Harmaline is a β-carboline alkaloid that can be extracted from the seeds of Peganum harmala. Harmaline has been shown to exhibit a potent cytotoxic effect against tumor cells. In this study, the anti-glioblastoma activity of harmaline was investigated in vitro. Cell viability, apoptosis, and cell cycle arrest were assessed in U-87 cells treated with harmaline at different doses. Reactive oxygen species (ROS) generation and the mRNA expression of apoptosis-associated genes were assessed. The anti-metastatic effect of harmaline on U-87 cells was evaluated by gelatin zymography assay where matrix metalloproteinase [MMP]-2/-9 enzymatic activity was measured, and the scratch assay was used to assess migratory responses. Flow cytometry demonstrated that harmaline could suppress the proliferation and induce sub-G1 cell cycle arrest and apoptotic cell death in glioblastoma cells. Harmaline treatment was also associated with an upregulation of the cell cycle-related genes, p21 and p53, and pro-apoptotic Bax, as well as the induction of ROS. The zymography assay indicated that the essential steps of metastasis were potently suppressed by harmaline through inhibiting the expression of MMP-2 and - 9. In addition, the migration of U-87 cells was significantly reduced after harmaline treatment. Our data suggest a basis for further research of harmaline which has potential cytotoxic activities in glioblastoma cells; inducing cell cycle arrest and apoptosis, repression of migration, possibly invasion, and metastasis.

Sodium butyrate supresses malignant human mast cell proliferation, downregulates expression of KIT and promotes differentiation.

Sodium butyrate (NaBu) is a class I histone deacetylase inhibitor (HDACi) that can impede the proliferation of transformed cells. Although some HDACi downregulate the expression of the stem cell factor receptor (KIT/CD117), the effect of NaBu on KIT expression and human mast cell proliferation requires further elucidation. In this study, we examined the effects of NaBu on three transformed human mast cell lines, HMC-1.1, HMC-1.2 and LAD2. NaBu (100 µM) inhibited the proliferation and metabolic activity of all three cell lines without significantly affecting their viability, suggesting that although the cells had ceased to divide, they were not yet undergoing apoptosis. Cell cycle analysis using the cell-permeant dye, propidium iodide, indicated that NaBu significantly blocked the cell cycle progression of HMC-1.1 and HMC-1.2 from G1 to G2/M phases. Furthermore, NaBu downregulated the expression of C-KIT mRNA and KIT protein expression in all three cell lines, but this effect was most significant in the HMC-1.1 and HMC-1.2, both of which harbour activating mutations in KIT, which proliferate more rapidly than LAD2. These data support earlier observations showing that human mast cell lines are sensitive to histone deacetylase inhibition. However, our data presents the novel observation that inhibition of cell proliferation by NaBu was not associated with a loss in cell viability but rather an arrest of the cell cycle. Higher concentrations of NaBu led to modest increases in histamine content, tryptase expression, and granularity. In conclusion, NaBu treatment of human mast cell lines led to a modest enhancement of the hallmarks of mature mast cells.

Abstract P4-08-13: The Use of Reverse Phase Protein Arrays (RPPA), Principal Component Analysis (PCA), and CScape Pathway Mapping to Identify New Tumor Vulnerability and Actionable Targets in Human Malignant Triple-Negative Breast cancer cell lines

Abstract Breast cancer is the major leading cause of cancer-related fatalities nationally and globally. There will be 287,850 new breast cancer cases and 43,250 breast cancer related deaths in the United States in 2022. The relapsed, treatment-resistant, undruggable, and incurable breast cancers are often associated with EGFR/HER2/K-RAS/SIAH pathway activation. The EGFR/HER2/K-RAS/SIAH pathway is a major tumor-driver whose hyperactivation is associated malignant tumor growth, multidrug-resistant phenotypes, early tumor relapse, and systematic metastasis. Seven-In-Absentia (SINA) homologues (SIAH) are extraordinarily evolutionarily-conserved E3 ubiquitin ligases that play a critical gatekeeper role downstream of the EGFR/HER2/K-RAS pathway. SIAH is a major tumor vulnerability that is ideally positioned to become an attractive target for innovative targeted therapy development against metastatic breast cancer (MBC). Prior studies have shown that tumor growth was abolished in malignant tumor cell lines such as MDA-MB-231, MDA-MB-468, MiaPaCa, A459, and HeLa following SIAH inhibition in xenograft models; however, the underpinning molecular mechanisms that give rise to this striking anti-EGFR/K-RAS and anticancer phenotype remain unclear. Specific objectives: to delineate the molecular mechanism(s) of why anti-SIAH2PD targeted therapy is so effective in impeding and eradicating the stage IV and aggressive tumors, we conducted reverse phase protein array (RPPA)-based kinomic analysis to delineate how major cancer signaling pathways and EGFR/HER2/K-RAS/SIAH-dependent signaling networks are rewired and remodeled in response to anti-SIAH2 targeted therapy. Brief statement of methods: About 300 proteins/phosphoproteins were quantititatively measured by the RPPA platform to identify new tumor vulnerabilities and actionable targets, compensatory signaling network activation/inhibition in response to anti-SIAH targeted therapies in five highly malignant cancer cell lines. Doxycycline (DOX)-inducible Tet-ON MDA-MB-231, MDA-MB-468, MiaPaCa, HeLa and A459 cell lines were amplified from single cell and DOX-induced SIAH2PD expression was confirmed. Each of the cell lines was then subjected to one of four experimental conditions: Tet-ON control cells without DOX induction (group A), Tet-ON control cells with DOX induction (group B), Tet-ON-SIAH2PD cancer cells without DOX-induction (no SIAH2PD inhibitor) (group C), Tet-ON-SIAH2PD cancer cells with DOX-induction (SIAH2PD inhibitor) (group D). Reverse Phase Protein Array (RPPA) in conjunction with Principal Component Analysis (PCA) was conducted to quantify fold-changes of proteins/phosphoproteins in response to SIAH inhibition. The ratios of D/C/B/A, D/C, D/B, C/A, and B/A were calculated using GAPDH normalized data. Summary of results: Supported by statistical analyses, we identified 6 unique phospho-proteins that were either up- or down-regulated in response to SIAHLoss-of-function. Many have known roles in controlling and regulating cell growth, cell death, NFκB signaling, stress response, DNA damage, and cell attachment pathways, supporting a tumor eradication phenotype in the absence of SIAH function in these cancer cell lines. Conclusion: Cancer landscape (CScape) functional protein pathway mapping has categorized the synergistic feedforward, feedback, and compensatory signaling pathway activation/inactivation in response to SIAH blockade in these EGFR/HER2/K-RAS-driven malignant human cell lines. Further validation analysis will be conducted to gain better insight into the global cancer pathway alterations to reveal the molecular mechanism(s) of why SIAH inhibition works so effectively to shut down malignant tumor growth in the preclinical models. Citation Format: Andrew P. Howell, Julia Wulfkuhle, Rosa I. Gallagher, Emanuel F. Petricoin, Amy H. Tang. The Use of Reverse Phase Protein Arrays (RPPA), Principal Component Analysis (PCA), and CScape Pathway Mapping to Identify New Tumor Vulnerability and Actionable Targets in Human Malignant Triple-Negative Breast cancer cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-13.

In Silico, In Vitro, and Ex Vivo Biological Activity of Some Novel Mebeverine Precursors

In silico analysis aimed at predicting the pharmacodynamic profile of the compounds was performed. Based on these predictions, ex vivo bioelectrical activity (BEA) and immunohistochemical effects of the compounds were established. A thorough biological evaluation of the compounds was conducted assessing their in vitro antimicrobial and cytotoxic activity. All the newly synthesized compounds exerted drug-like properties, whereby 3-methyl-1-phenylbutan-2-amine 3 showed a significant change in BEA due to Ca2+ channel regulation, Ca2+ influx modulation, and a subsequent change in smooth muscle cell response. The immunohistochemical studies showed a good correlation with the obtained data on the BEA, defining amine 3 as a leader structure. No cytotoxicity to human malignant leukemic cell lines (LAMA-84, K-562) was observed for all tested compounds. Based on the experimental results, we outlined 3-methyl-1-phenylbutan-2-amine 3 as a potential effective choice for orally active long-term therapy of IBS.