Mechanical effects modulate drug resistance in MCF-7-derived organoids: Insights into the wnt/ß-catenin pathway.

Addressing drug resistance poses a significant challenge in cancer treatment, as cancer cells develop diverse mechanisms to evade chemotherapy drugs, leading to treatment failure and disease relapse. Three-dimensional (3D) cell culture has emerged as a valuable model for studying drug resistance, although the underlying mechanisms remain elusive. By obtaining a better understanding of drug resistance within the 3D culture environment, we can develop more effective strategies to overcome it and improve the success of cancer treatments. Notably, the physical structure undergoes notable changes in 3D culture, with mechanical effects believed to play a pivotal role in drug resistance. Hence, our study aimed to explore the influence of mechanical effects on drug resistance by analyzing data related to "drug resistance" and "mechanobiology". Through this analysis, we identified ß-catenin and JNK1 as potential factors, which were further examined in MCF-7 cells cultivated under both 2D and 3D culture conditions. Our findings demonstrate that ß-catenin is activated through canonical and non-canonical pathways and associated with the drug resistance, particularly in organoids obtained under 3D culture.

Comparative evaluation of the therapeutic strategies using a minimal model of luminal-A breast cancer.

Cellular behavior is heavily influenced by cellular interactions, which are often lost in conventional cell culture methods. As a result, in vitro cellular behavior may not accurately reflect in vivo conditions. Three-dimensional (3D) culture, on the other hand, is better suited for studying cellular behavior as it allows for more comprehensive cell communication. In this study, we utilized 3D culture of the MCF-7 cell line to create a minimal model of luminal-A breast cancer and evaluated its histopathological and morphological features using various methods. To determine the optimal therapeutic strategies for eliminating cancer cells, we assessed the effectiveness of diverse therapeutic approaches, including targeting distinct phases of the cell cycle, endocrine therapy, and gene therapy in both 2D and 3D culture systems. Our findings indicate that cells derived from mammospheres respond differently to their parent cells in monolayer culture depending on the therapeutic strategy used. This variability in drug response may be due to the altered microenvironment created by heterogeneous cellular makeup and emerging cellular interactions in the 3D culture. Therefore, it is important to administer a therapeutic approach that can eradicate cells regardless of the microenvironment.

Caspase-9-mediated cleavage of vimentin attenuates the aggressiveness of leukemic NB4 cells.

Vimentin is a main type 3 intermediate filament protein. It seems that abnormal expression of vimentin is contributed to the appearance of the aggressive feature of cancer cells. So that it has been reported that malignancy and epithelial-mesenchymal transition in solid tumors, and poor clinical outcomes in patients with lymphocytic leukemia and acute myelocytic leukemia have been associated with the high expression of vimentin. Vimentin is a non-caspase substrate of caspase-9 although its cleavage by caspase-9 in biological processes has not been reported. In the present study, we sought to understand whether vimentin cleavage mediated by caspase-9 could reverse the malignancy in leukemic cells. Herein, to address the issue, we investigated vimentin changes in differentiation and took advantage of the inducible caspase-9 (iC9)/AP1903 system in human leukemic NB4 cells. Following the transfection and treatment of the cells using the iC9/AP1903 system, vimentin expression, cleavage, and subsequently, the cell invasion and the relevant markers such as CD44 and MMP-9 were evaluated. Our results revealed the downregulation and cleavage of vimentin which attenuates the malignant phenotype of the NB4 cells. Considering the favorable effect of this strategy in keeping down the malignant features of the leukemic cells, the effect of the iC9/AP1903 system in combination with all-trans-retinoic acid (ATRA) treatment was evaluated. The obtained data prove that iC9/AP1903 significantly makes the leukemic cells more sensitive to ATRA.

Synthesis, characterization, and in vitro evaluation of the starch-based α-amylase responsive hydrogels.

Controlled-release drug delivery systems are promising platforms in medicine. Among various types of material in drug delivery, hydrogels are interesting ones. They are water-soluble and tissue compatible polymers with a high capacity to carry and release drugs in a controllable manner. In this study, we introduce the synthesis, characterization, and application of an α-amylase responsive hydrogel in controlled drug delivery. The newly synthesized starch-based hydrogels structurally characterized by means of Fourier-transform infrared spectroscopy and scanning electron microscopy. A proapoptotic drug, doxorubicin, was loaded into the hydrogels and the controlled release of the drug was assessed in the presence of α-amylase and ultimately it was evaluated to controlled-drug release in vitro and subsequently in killing cancer cells. Our results highlight the effectiveness of temporal drug delivery using α-amylase responsive hydrogels in killing cancer cells.

Developing a circularly permuted variant of Renilla luciferase as a bioluminescent sensor for measuring Caspase-9 activity in the cell-free and cell-based systems.

Biosensors and whole cell biosensors consisting of biological molecules and living cells can sense a special stimulus on a living system and convert it to a measurable signal. A major group of them are the bioluminescent sensors derived from luciferases. This type of biosensors has a broad application in molecular biology and imaging systems. In this project, a luciferase-based biosensor for detecting and measuring caspase-9 activity is designed and constructed using the circular permutation strategy. The spectroscopic method results reveal changes in the biosensor structure. Additionally, its activity is examined in a cell-free coupled assay system. Afterward, the biosensor is utilized for measuring the cellular caspase-9 activity upon apoptosis induction in a cancer cell line. In following the gene of biosensor is sub-cloned into a eukaryotic vector and transfected to HEK293T cell line and then its activity is measured upon apoptosis induction in the presence and absence of a caspase-9 inhibitor. The obtained results show that the designed biosensor detects the caspase-9 activity in the cell-free and cell-based systems.

Delay in apoptosome formation attenuates apoptosis in mouse embryonic stem cell differentiation.

Differentiation is an inseparable process of development in multicellular organisms. Mouse embryonic stem cells (mESCs) represent a valuable research tool to conduct in vitro studies of cell differentiation. Apoptosis as a well known cell death mechanism shows some common features with cell differentiation, which has caused a number of ambiguities in the field. The research question here is how cells could differentiate these two processes from each other. We have investigated the role of the mitochondrial apoptotic pathway and cell energy level during differentiation of mESCs into the cardiomyocytes and their apoptosis. p53 expression, cytochrome c release, apoptosome formation, and caspase-3/7 activation are observed upon induction of both apoptosis and differentiation. However, remarkable differences are detected in time of cytochrome c appearance, apoptosome formation, and caspase activity upon induction of both processes. In apoptosis, apoptosome formation and caspase activity were observed rapidly following the cytochrome c release. Unlike apoptosis, the release of cytochrome c upon differentiation took more time, and the maximum caspase activity was also postponed for 24 h. This delay suggests that there is a regulatory mechanism during differentiation of mESCs into cardiomyocytes. The highest ATP content of cells was observed immediately after cytochrome c release 6 h after apoptosis induction and then decreased, but it was gradually increased up to 48 h after differentiation. These observations suggest that a delay in the release of cytochrome c or delay in ATP increase attenuate apoptosome formation, and caspase activation thereby discriminates apoptosis from differentiation in mESCs.

Caspase-9 suppresses metastatic behavior of MDA-MB-231 cells in an adaptive organoid model.

Caspase-9, a cysteine-aspartate protease traditionally associated with intrinsic apoptosis, has recently emerged as having non-apoptotic roles, including influencing cell migration-an aspect that has received limited attention in existing studies. In our investigation, we aimed to explore the impact of caspase-9 on the migration and invasion behaviors of MDA-MB-231, a triple-negative breast cancer (TNBC) cell line known for its metastatic properties. We established a stable cell line expressing an inducible caspase-9 (iC9) in MDA-MB-231 and assessed their metastatic behavior using both monolayer and the 3D organotypic model in co-culture with human Foreskin fibroblasts (HFF). Our findings revealed that caspase-9 had an inhibitory effect on migration and invasion in both models. In monolayer culture, caspase-9 effectively suppressed the migration and invasion of MDA-MB-231 cells, comparable to the anti-metastatic agent panitumumab (Pan). Notably, the combination of caspase-9 and Pan exhibited a significant additional effect in reducing metastatic behavior. Interestingly, caspase-9 demonstrated superior efficacy compared to Pan in the organotypic model. Molecular analysis showed down regulation of epithelial-mesenchymal transition and migratory markers, in caspase-9 activated cells. Additionally, flow cytometry analysis indicated a cell cycle arrest. Moreover, pre-treatment with activated caspase-9 sensitized cells to the chemotherapy of doxorubicin, thereby enhancing its effectiveness. In conclusion, the anti-metastatic potential of caspase-9 presents avenues for the development of novel therapeutic approaches for TNBC/metastatic breast cancer. Although more studies need to figure out the exact involving mechanisms behind this behavior.

Porous scaffolds with the structure of an interpenetrating polymer network made by gelatin methacrylated nanoparticle-stabilized high internal phase emulsion polymerization targeted for tissue engineering.

The interlacing of biopolymers and synthetic polymers is a promising strategy to fabricate hydrogel-based tissue scaffolds to biomimic a natural extracellular matrix for cell growth. Herein, open-cellular macroporous 3D scaffolds with a semi-interpenetrating network were fabricated through high internal phase emulsion templating. The scaffolds are prepared by (I) the curing of PEG diacrylate (PEGDAC) and gelatin methacrylate (GelMA) in the continuous aquatic phase of a coconut oil-in-water emulsion stabilized by GelMA nanoparticles, and (II) the removal of the internal phase. The effect of the main contributing parameters such as pH, GelMA content, and GelMA/PEGDAC weight ratio on the emulsion features was investigated systematically. Due to the isoelectric point of GelMA at around pH 6, the GelMA particle (aggregation) size decreased at both sides of pH from 1000 to 100-140 nm because of the increased number of positive and negative charges on GelMA. These GelMA nanoparticles were able to produce stable emulsions with narrowly distributed small emulsion droplets. Moreover, the stability and emulsion droplet size were enhanced and increased, respectively, with GelMA content increasing and GelMA/PEGDAC weight ratio decreasing. These trends lie in the prevented coalescence phenomenon caused by the improved viscosity and likely partially formed network by GelMA chains in the continuous phase. Hence, the following formulation was selected for scaffold preparation: φ oil = 74%, pH = 12, GeMA = 4 wt%, and GelMA/PEGDAC = 10/8. Then, PCL in different contents was infiltrated into the scaffold to balance hydrophilicity and hydrophobicity. The cell culture assay proved that the scaffold with a pore size of 60-180 µm and containing 51.2 wt% GelMA, 10.3 wt% PEG, and PCL 27.2 wt% provided a suitable microenvironment for mouse fibroblast cell (L929) adhesion, growth, and spreading. These results show that this strategy suggests promising culture for tissue engineering applications.

The effect of caspase-9 in the differentiation of SH-SY5Y cells.

Neuroblastoma is the most common solid malignant tumor in infants and young children. Its origin is the incompletely committed precursor cells from the autonomic nervous system. Neuroblastoma cells are multipotent cells with a high potency of differentiation into the neural cell types. Neural differentiation leads to the treatment of neuroblastoma by halting the cell and tumor growth and consequently its expansion. Caspases are a family of proteins involved in apoptosis and differentiation. The present study aimed to investigate the potential role of caspase-9 activation on the differentiation of the human neuroblastoma SH-SY5Y cells. Here we investigated the caspase-9 and 3/7 activity during 1,25-dihydroxycholecalciferol (D3)-mediated differentiation of SH-SY5Y cells and took advantage of the inducible caspase-9 system in putting out the differentiation of the neuroblastoma cells. D3-induced differentiation of the cells could lead to activation of caspase-9 and caspase-3/7, astrocyte-like morphology, and increased expression of Glial fibrillary acidic protein (GFAP). By using the inducible caspase-9 system, we showed differentiation of SH-SY5Y cells to astrocyte-like morphology and increased level of GFAP expression. Furthered studies using a specific caspase-9 inhibitor showed inhibition of differentiation mediated by D3 or caspase-9 to astrocyte-like cells. These results show the potency of caspase-9 to direct differentiation of the human neuroblastoma SH-SY5Y cells into cells showing an astrocyte-like morphology.

Cancer Therapy and Imaging Through Functionalized Carbon Nanotubes Decorated with Magnetite and Gold Nanoparticles as a Multimodal Tool.

Pharmacotherapy and imaging are two critical facets of cancer therapy. Carbon nanotubes and their modified species such as magnetic or gold nanoparticle conjugated ones they have been introduced as good candidates for both purposes. Gold nanoparticles enhance effects of X-rays during radiotherapy. Nanomaterial-mediated radiofrequency (RF) hyperthermia refers to using RF to heat tumors treated with nanomaterials for cancer therapy. The combination of hyperthermia and radiotherapy, synergistically, causes a significant reduction in X-ray doses. The present study was conducted to investigate the ability and efficiency of the multi-walled carbon nanotubes functionalized with magnetic Fe3O4 and gold nanoparticles (mf-MWCNT/AuNPs) for imaging and cancer therapy. The mf-MWCNT/AuNPs were utilized for imaging approaches such as ultrasounds, CT scan, and MRI. They were also examined in thermotherapy and radiotherapy. The MCF-7 cell line was used as an in vitro model to study thermotherapy and radiotherapy. The mf-MWCNT/AuNPs are beneficial as a contrast agent in imaging by ultrasounds, CT scan, and MRI. They are also radio waves and X-rays absorbent and enhance the efficiency of thermotherapy and radiotherapy in the elimination of cancer cells. The valuable properties of mf-MWCNT/AuNPs in radio- and thermotherapies and imaging strategies make them a good candidate as a multimodal tool in cancer therapy. Graphical Abstract The mf-MWCNT/AuNPs are beneficial as a contrast agent in imaging by US (ultrasounds), CT scan, and MRI. They are also radio waves and X-rays absorbent and enhance the efficiency of thermotherapy and radiotherapy in the elimination of cancer cells. The valuable properties of the mf-MWCNT/AuNPs in radio- and thermotherapies and imaging strategies make them a good candidate as a multimodal tool in cancer therapy.

The non-apoptotic role of caspase-9 promotes differentiation in leukemic cells.

Caspase family contains cysteine proteases involving in the key cellular processes, such as apoptosis, inflammation, and autophagy. There is a growing body of evidence that caspase family also plays a role in cellular differentiation. Evidence suggests that caspase-9 is among the most important members with non-apoptotic roles in the execution of differentiation. Since drug-induced differentiation in some types of cancer cells is a promising treatment, we have investigated caspase-9 activity during differentiation of a cancer cell; leukemia. We demonstrate that caspase-9 has increased activity during differentiation and also the inhibition of caspase-9 will prevent the granulocytic differentiation of leukemic cells. In addition, we studied the differentiation induction mediated by caspase-9 using an inducible variant of caspase-9. Results indicate the caspase-9 mediated differentiation accompanied by a reduction in the expression of CD33 and an increase in CD15. Notably, all of the events occur when cell viability remains constant. Owing to the evidence, caspase-9 activity is considered as a central factor in the execution of differentiation in leukemic cells.

Carbon nanotube-delivered iC9 suicide gene therapy for killing breast cancer cells in vitro.

AIM: To induce a safe death to MCF-7 human breast cancer cell line through gene therapy based on iC9 suicide gene. MATERIALS & METHODS: To induce apoptosis to MCF-7 cell line, iC9 gene was transfected using pyridine-functionalized multi-walled carbon nanotubes. Then, to enhance chemotherapy, iC9 suicide gene therapy was performed alongside. RESULTS: The results show that the MCF-7 cells were efficiently eliminated in a high percentage by this approach. Furthermore, the suicide gene by itself/in combination with the chemotherapeutic drugs managed to pass the cell cycle arrests. CONCLUSION: We introduced an in vitro treatment approach based on suicide gene therapy and the first step was taken toward the enhancement of chemotherapy, although more investigation is required.

Diethyl [(3-phenoxy-2-oxo-4-phenyl-azetidin-1-yl)-phenyl-methyl]-phosphonate as a potent anticancer agent in chemo-differentiation therapy of acute promyelocytic leukemia.

Organophosphonates are a group of chemical agents which have high bioactivity. In the present study, we aimed to investigate the anticancer activity of the synthesized ß-lactam derivatives of α-amino phosphonates on solid tumor and human leukemic cell lines. The results show that one of these compounds, Diethyl [(3-phenoxy-2-oxo-4-phenyl-azetidine-1-yl)-phenyl-methyl]-phosphonate, is a potent anticancer agent which especially shows anti-leukemic activity. Flow cytometry study showed that this chemical agent causes the G1 phase cell cycle arrest and consequently apoptosis in NB4 cell line as an acute promyelocytic leukemia model. In fact, this agent induces cell differentiation and apoptosis, at low and high concentrations, respectively. Its combination with All-Trans Retinoic Acid shows a higher percentage of cells in the terminal differentiation stage. This evidence suggested that diethyl phosphonate might be a proper candidate for chemo-differentiation therapy in acute promyelocytic leukemia and even in other types of acute myeloid leukemia.

Caspases interplay with kinases and phosphatases to determine cell fate.

Cellular differentiation is one of the critical processes in the life of multicellular organisms. In this phenomenon, a non-specialized cell is converted to a specialized one with its own specific function and morphology. One of the requirements for specialization is silencing of the pathways involved in cell proliferation in parallel with turning on the molecular mechanisms involved in differentiation. Similar to other biological phenomena, the change in cellular state from the proliferative to the differentiated needs molecular switches to persuade the change in response to the internal or external inducers. The quiddity of these molecular switches has not been identified, yet. However, there exists a growing body of evidence showing that the same agents involved in apoptosis have a broad contribution to differentiation progression. To our knowledge, this evidence is still ambiguous because it has raised fundamental questions that require more proof to be answered. The most important questions are: How can two totally different cellular fates act through a similar pathway? What is the separating edge? What forces a cell to choose one of them (death or differentiation)? To address these issues, we will concentrate on three groups of molecules; caspases as the key players of apoptosis, protein kinases, and phosphatases as the major regulators of many cellular and biochemical processes. The evidence reveals a triangle of caspases, kinases, and phosphatases in which their communication leads to the fine-tuning of caspases and consequently they determine cell fate.

Cancer stem cells, cancer-initiating cells and methods for their detection.

The cancer stem cell (CSC) hypothesis considers CSCs as the main culprits of tumor initiation, propagation, metastasis and therapy failure. CSCs represent a minority subpopulation of cells within a tumor. Their detection, characterization and monitoring are crucial steps toward a better understanding of the biological roles of these special cells in the development and propagation of tumors which, in turn, improves clinical reasoning and treatment options. Nowadays, in vitro and in vivo assays are available that address the self-renewal and differentiation potential of CSCs, and advanced in vivo molecular imaging technology facilitates the detection and provides an unprecedented in vivo observation platform to study the behavior of CSCs in their natural environment. Here, we provide a brief overview of CSCs and describe modern cellular models and labeling techniques to study and trace CSCs.

Acquired expression of osteopontin selectively promotes enrichment of leukemia stem cells through AKT/mTOR/PTEN/ß-catenin pathways in AML cells.

AIMS: Acute myeloid leukemia (AML) initiation and progression have been attributed to subpopulations of self-renewing leukemia stem cells (LSCs), which contribute to progression, recurrence and therapeutic resistance in leukemia. Osteopontin (OPN) plays an important role in promoting survival and drug resistance in LSCs. The aim of this study was to explore OPN roles in modulating curcumin-mediated LSC enrichment and survival in AML cell lines and primary CD34+/CD38- bone-marrow-derived AML cells. MATERIALS AND METHODS: The growth inhibitory effects of curcumin (CUR) were evaluated by MTT assay in U937 and CD34+ KG-1 AML cell lines as well as primary CD34+/CD38- bone-marrow derived AML cells isolated by MACS technique. The proportion of LSC markers (CD34, CD38 and CD123) were evaluated by flow cytometry. The expression levels of OPN, AKT, mTOR, PTEN, ß-catenin and NF-κB were investigated by qRT-PCR. Short interfering RNA (siRNA) against OPN was used in AML cells incubated with or without CUR. KEY FINDINGS: Proportions of CD34+/CD38-/CD123+ and CD34+/CD38+/CD123+ LSCs compartment co-expressing an increased level of OPN could be enriched in AML cell lines and in patient's primary cells by CUR treatment. The expression levels of AKT, mTOR, PTEN, and ß-catenin and NF-κB1, were also significantly up-regulated concurrently with OPN in the enriched CD34+ AML cells. SIGNIFICANCE: The increased in CUR-mediated OPN level is involved in a complex interplay of various signaling pathways resulting in cytoprotection and enrichment of CD34+ LSC compartment in CUR-treated AML cells. AKT/mTOR/PTEN/ß-catenin/NF-kB signaling pathways may play roles in modulating OPN-mediated LSC cell survival and enrichment.