Novel therapeutic diiminoquinone exhibits anticancer effects on human colorectal cancer cells in two-dimensional and three-dimensional in vitro models.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer-related mortality. Cancer stem cells (CSCs) in CRC, which are spared by many chemotherapeutics, have tumorigenic capacity and are believed to be the reason behind cancer relapse. So far, there have been no effective drugs to target colon CSCs. Diiminoquinone (DIQ) has shown promising effects on targeting colon cancer. However, there is limited research on the effects of DIQ on eradicating CSCs in CRC. AIM: To investigate the anticancer potential of DIQ on colon CSCs in two-dimensional (2D) and three-dimensional (3D) models using colonospheres and patient-derived organoids. METHODS: Various 2D methods have been used to assess the effect and the mechanism of DIQ on HCT116 and HT29 cell lines including cell proliferation and viability assays, migration and invasion assays, immunofluorescence staining, and flow cytometry. The potency of DIQ was also assessed in 3D culture using the sphere formation assay and colon cancer patient-derived organoid model. RESULTS: Our results showed that DIQ significantly inhibited cell proliferation, migration, and invasion in HCT116 and HT29 cell lines. DIQ treatment induced apoptosis along with an accumulation of HCT116 and HT29 cancer cells in the sub-G1 region and an increase in reactive oxygen species in both CRC cell lines. DIQ reduced sphere-forming and self-renewal ability of colon cancer HCT116 and HT29 stem/progenitor cells at sub-toxic doses of 1 µmol/L. Mechanistically, DIQ targets CSCs by downregulating the main components of stem cell-related -catenin, AKT, and ERK oncogenic signaling pathways. Potently, DIQ displayed a highly significant decrease in both the count and the size of the organoids derived from colon cancer patients as compared to control and 5-fluorouracil conditions. CONCLUSION: This study is the first documentation of the molecular mechanism of the novel anticancer therapeutic DIQ via targeting CSC, a promising compound that needs further investigation.

Thymoquinone Radiosensitizes Human Colorectal Cancer Cells in 2D and 3D Culture Models.

Resistance of cancer cells and normal tissue toxicity of ionizing radiation (IR) are known to limit the success of radiotherapy. There is growing interest in using IR with natural compounds to sensitize cancer cells and spare healthy tissues. Thymoquinone (TQ) was shown to radiosensitize several cancers, yet no studies have investigated its radiosensitizing effects on colorectal cancer (CRC). Here, we combined TQ with IR and determined its effects in two-dimensional (2D) and three-dimensional (3D) culture models derived from HCT116 and HT29 CRC cells, and in patient-derived organoids (PDOs). TQ sensitized CRC cells to IR and reduced cell viability and clonogenic survival and was non-toxic to non-tumorigenic intestinal cells. TQ sensitizing effects were associated with G2/M arrest and DNA damage as well as changes in key signaling molecules involved in this process. Combining a low dose of TQ (3 µM) with IR (2 Gy) inhibited sphere formation by 100% at generation 5 and this was associated with inhibition of stemness and DNA repair. These doses also led to ~1.4- to ~3.4-fold decrease in organoid forming ability of PDOs. Our findings show that combining TQ and IR could be a promising therapeutic strategy for eradicating CRC cells.

Establishment and characterization of prostate organoids from treatment-naïve patients with prostate cancer.

Three-dimensional (3D) organoid culture systems are emerging as potential reliable tools to investigate basic developmental processes of human disease, especially cancer. The present study used established and modified culture conditions to report successful generation and characterization of patient-derived organoids from fresh primary tissue specimens of patients with treatment-naïve prostate cancer (PCa). Fresh tissue specimens were collected, digested enzymatically and the resulting cell suspensions were plated in a 3D environment using Matrigel as an extracellular matrix. Previously established 12-factor medium for organoid culturing was modified to create a minimal 5-factor medium. Organoids and corresponding tissue specimens were characterized using transcriptomic analysis, immunofluorescent analysis, and immunohistochemistry. Furthermore, patient-derived organoids were used to assess the drug response. Treatment-naïve patient-derived PCa organoids were obtained from fresh radical prostatectomy specimens. These PCa organoids mimicked the heterogeneity of corresponding parental tumor tissue. Histopathological analysis demonstrated similar tissue architecture and cellular morphology, as well as consistent immunohistochemical marker expression. Also, the results confirmed the potential of organoids as an in vitro model to assess potential personalized treatment responses as there was a differential drug response between different patient samples. In conclusion, the present study investigated patient-derived organoids from a cohort of treatment-naïve patients. Derived organoids mimicked the histological features and prostate lineage profiles of their corresponding parental tissue and may present a potential model to predict patient-specific treatment response in a pre-clinical setting.

The Use of Stem Cell-Derived Organoids in Disease Modeling: An Update.

Organoids represent one of the most important advancements in the field of stem cells during the past decade. They are three-dimensional in vitro culturing models that originate from self-organizing stem cells and can mimic the in vivo structural and functional specificities of body organs. Organoids have been established from multiple adult tissues as well as pluripotent stem cells and have recently become a powerful tool for studying development and diseases in vitro, drug screening, and host-microbe interaction. The use of stem cells-that have self-renewal capacity to proliferate and differentiate into specialized cell types-for organoids culturing represents a major advancement in biomedical research. Indeed, this new technology has a great potential to be used in a multitude of fields, including cancer research, hereditary and infectious diseases. Nevertheless, organoid culturing is still rife with many challenges, not limited to being costly and time consuming, having variable rates of efficiency in generation and maintenance, genetic stability, and clinical applications. In this review, we aim to provide a synopsis of pluripotent stem cell-derived organoids and their use for disease modeling and other clinical applications.

Tideglusib attenuates growth of neuroblastoma cancer stem/progenitor cells in vitro and in vivo by specifically targeting GSK-3ß.

BACKGROUND: Neuroblastoma (NB) is the most frequently diagnosed extracranial solid tumor among the pediatric population. It is an embryonic tumor with high relapse rates pertaining to the presence of dormant slowly dividing cancer stem cells (CSC) within the tumor bulk that are responsible for therapy resistance. Therefore, there is a dire need to develop new therapeutic approaches that specifically target NB CSCs. Glycogen synthase kinase (GSK)-3ß is a serine/threonine kinase that represents a common signaling node at the intersection of many pathways implicated in NB CSCs. GSK-3ß sustains the survival and maintenance of CSCs and renders them insensitive to chemotherapeutic agents and radiation. METHODS: In our study, we aimed at evaluating the potential anti-tumor effect of Tideglusib (TDG), an irreversible GSK-3ß inhibitor drug, on three human NB cell lines, SK-N-SH, SH-SY5Y, and IMR-32. RESULTS: Our results showed that TDG significantly reduced cell proliferation, viability, and migration of the NB cells, in a dose- and time-dependent manner, and also significantly hindered the neurospheres formation eradicating the self-renewal ability of highly resistant CSCs. Besides, TDG potently reduced CD133 cancer stem cell marker expression in both SH-SY5Y cells and G1 spheres. Lastly, TDG inhibited NB tumor growth and progression in vivo. CONCLUSION: Collectively, we concluded that TDG could serve as an effective treatment capable of targeting the NB CSCs and hence overcoming therapy resistance. Yet, future studies are warranted to further investigate its potential role in NB and decipher the subcellular and molecular mechanisms underlying this role.

Genome-wide gene expression analysis of a murine model of prostate cancer progression: Deciphering the roles of IL-6 and p38 MAPK as potential therapeutic targets.

BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths among adult males globally. The poor prognosis of PCa is largely due to late diagnosis of the disease when it has already progressed to an advanced stage marked by androgen-independence, thus necessitating new strategies for early detection and treatment. We construe that these direly needed advances are limited by our poor understanding of early events in the progression of PCa and that would thus represent ideal targets for early intervention. To begin to fill this void, we interrogated molecular "oncophenotypes" that embody the transition of PCa from an androgen-dependent (AD) to-independent (AI) state. METHODS: To accomplish this aim, we used our previously established AD and AI murine PCa cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PCa morphologically and molecularly. We statistically surveyed global gene expressions in these cell lines by microarray analysis. Differential profiles were functionally interrogated by pathways, gene set enrichment and topological gene network analyses. RESULTS: Gene expression analysis of PLum-AD and PLum-AI transcriptomes (n = 3 each), revealed 723 differentially expressed genes (392 upregulated and 331 downregulated) in PLum-AI compared to PLum-AD cells. Gene set analysis demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to tumor aggressiveness including cell migration and invasion facilitated by epithelial-to-mesenchymal transition (EMT). Further analysis demonstrated that the p38 mitogen-activated protein kinase (MAPK) was predicted to be significantly activated in the PLum-AI cells, whereas gene sets previously associated with favorable response to the p38 inhibitor SB203580 were attenuated (i.e., inversely enriched) in the PLum-AI cells, suggesting that these aggressive cells may be therapeutically vulnerable to p38 inhibition. Gene set and gene-network analysis also alluded to activation of other signaling networks particularly those associated with enhanced EMT, inflammation and immune function/response including, but not limited to Tnf, IL-6, Mmp 2, Ctgf, and Ptges. Accordingly, we chose SB203580 and IL-6 to validate their effect on PLum-AD and PLum-AI. Some of the common genes identified in the gene-network analysis were validated at the molecular and functional level. Additionally, the vulnerability to SB203580 and the effect of IL-6 were also validated on the stem/progenitor cell population using the sphere formation assay. CONCLUSIONS: In summary, our study highlights pathways associated with an augmented malignant phenotype in AI cells and presents new high-potential targets to constrain the aggressive malignancy seen in the castration-resistant PCa.

Protein Expression Analysis of an In Vitro Murine Model of Prostate Cancer Progression: Towards Identification of High-Potential Therapeutic Targets.

BACKGROUND: Prostate cancer (PC) is the most frequently diagnosed cancer among men worldwide. The poor prognosis of PC is largely due to late diagnosis of the disease when it has progressed to advanced stages marked by androgen-independence. We interrogated proteomic signatures that embody the transition of PC from an androgen-dependent (AD) to an androgen-independent (AI) state. METHODS: We have previously established AD and AI murine PC cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PC at phenotypic and subcellular levels. We statistically surveyed global protein expression profiles in these cell lines. Differential profiles were functionally interrogated by pathways and protein-protein interaction network analyses. RESULTS: Protein expression pattern analysis revealed a total of 683 proteins, among which 99 were significantly differentially altered in PLum-AI cells as compared to PLum-AD cells (45 increased and 54 decreased). Principal component analysis (PCA) revealed that the two different cell lines clearly separated apart, indicating a significant proteome expression difference between them. Four of the proteins (vimentin, catalase, EpCAM, and caspase 3) that were differentially expressed in PLum-AI cells compared to PLum-AD cells were subjected to biochemical validation by Western blotting. Biological process gene ontology (GO) analysis of the differentially expressed proteins demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to PI3 kinase and androgen receptor pathways. Besides, other relevant biological processes that are enriched in PLum-AI cells included cell adhesion and cell migration processes, cell and DNA damage, apoptosis, and cell cycle regulation. CONCLUSIONS: Our protein expression analysis of a murine in vitro model of PC progression identified differential protein spots that denote this progression and that comprise high-potential targets for early treatment of PC with a personalized patient-specific approach. Efforts are underway to functionally assess the potential roles of these proteins as therapeutic targets for PC progression.

Thymoquinone induces apoptosis and DNA damage in 5-Fluorouracil-resistant colorectal cancer stem/progenitor cells.

The high recurrence rates of colorectal cancer have been associated with a small population of cancer stem cells (CSCs) that are resistant to the standard chemotherapeutic drug, 5-fluorouracil (5FU). Thymoquinone (TQ) has shown promising antitumor properties on numerous cancer systems both in vitro and in vivo; however, its effect on colorectal CSCs is poorly established. Here, we investigated TQ's potential to target CSCs in a three-dimensional (3D) sphere-formation assay enriched for a population of colorectal cancer stem/progenitor cells. Our results showed a significant decrease in self-renewal potential of CSC populations enriched from 5FU-sensitive and resistant HCT116 cells at 10-fold lower concentrations when compared to 2D monolayers. TQ decreased the expression levels of colorectal stem cell markers CD44 and Epithelial Cell Adhesion Molecule EpCAM and proliferation marker Ki67 in colonospheres derived from both cell lines and reduced cellular migration and invasion. Further investigation revealed that TQ treatment led to increased TUNEL positivity and a dramatic increase in the amount of the DNA damage marker gamma H2AX particularly in 5FU-resistant colonospheres, suggesting that the diminished sphere forming ability in TQ-treated colonospheres is due to induction of DNA damage and apoptotic cell death. The intraperitoneal injection of TQ in mice inhibited tumor growth of spheres derived from 5FU-sensitive and 5FU-resistant HCT116 cells. Furthermore, TQ induced apoptosis and inhibited NF-κB and MEK signaling in mouse tumors. Altogether, our findings document TQ's effect on colorectal cancer stem-like cells and provide insights into its underlying mechanism of action.

The synthetic retinoid ST1926 attenuates prostate cancer growth and potentially targets prostate cancer stem-like cells.

Retinoids are vitamin A derivatives that regulate crucial biological processes such as cellular proliferation, apoptosis, and differentiation. The use of natural retinoids in cancer therapy is limited due to their toxicity and the acquired resistance by cancer cells. Therefore, synthetic retinoids were developed, such as the atypical adamantyl retinoid ST1926 that provides enhanced bioavailability and reduced toxicity. We have assessed the in vitro and in vivo antitumor properties and mechanism of action of ST1926 in targeting cancer stem-like cells population of human prostate cancer (PCa) cell lines, DU145 and PC3, and mouse PCa cell lines, PLum-AD and PLum-AI. We demonstrated that ST1926 substantially reduced proliferation of PCa cells and induced cell cycle arrest, p53-independent apoptosis, and early DNA damage. It also decreased migration and invasion of PCa cells and significantly reduced prostate spheres formation ability in vitro denoting sufficient eradication of the self-renewal ability of the highly androgen-resistant cancer stem cells. Importantly, ST1926 potently inhibited PCa tumor growth and progression in vivo. Our results highlight the potential of ST1926 in PCa therapy and warrant its clinical development.

Novel 2-(5-Imino-5H-isoquinolones[3,4-b]quinoxalin-7-ylmethyl)-benzonitrile (DIQ3) and Other Related Derivatives Targeting Colon Cancer Cells: Syntheses and in Vitro Models.

Chemotherapy has been shown to be effective in reducing the progression and development of cancer in metastatic patients. However, drug selectivity is still a major issue for most chemotherapeutics. In this study, we synthesized four novel heterocyclic compounds having similarity in structure with quinone systems whereby nitrogen atoms replace the oxygen atoms. The anticancer activity of these compounds (DIQ3-6) was tested against HCT116 human colon cancer cells. We showed that all four heterocycles caused significant reduction in colon cancer cell viability at doses as low as 4 µM, a concentration that was not cytotoxic to normal human FHs74Int intestinal cell lines. Interestingly, these heterocycles inhibited colon sphere formation in 3D cultures at first generation (G1), mainly because of inhibition of proliferation as evidenced by Ki67 staining. Thus, DIQ3 causes sufficient eradication of the self-renewal ability of the highly resistant cancer stem cells. This study represents the first documentation of the activity of these novel heterocyclic compounds, particularly compound DIQ3, and their potential therapeutic use in targeting colon cancer self-renewal capacity. Our findings provide the basis for proposing these nontoxic and stable compounds for additional testing against cancer.

Sphere-Formation Assay: Three-Dimensional in vitro Culturing of Prostate Cancer Stem/Progenitor Sphere-Forming Cells.

Cancer Stem Cells (CSCs) are a sub-population of cells, identified in most tumors, responsible for the initiation, recurrence, metastatic potential, and resistance of different malignancies. In prostate cancer (PCa), CSCs were identified and thought to be responsible for the generation of the lethal subtype, commonly known as Castration-Resistant Prostate Cancer (CRPC). In vitro models to investigate the properties of CSCs in PCa are highly required. Sphere-formation assay is an in vitro method commonly used to identify CSCs and study their properties. Here, we report the detailed methodology on how to generate and propagate spheres from PCa cell lines and from murine prostate tissue. This model is based on the ability of stem cells to grow in non-adherent serum-free gel matrix. We also describe how to use these spheres in histological and immuno-fluorescent staining assays to assess the differentiation potential of the CSCs. Our results show the sphere-formation Assay (SFA) as a reliable in vitro assay to assess the presence and self-renewal ability of CSCs in different PCa models. This platform presents a useful tool to evaluate the effect of conventional or novel agents on the initiation and self-renewing properties of different tumors. The effects can be directly evaluated through assessment of the sphere-forming efficiency (SFE) over five generations or other downstream assays such as immuno-histochemical analysis of the generated spheres.

Primary versus castration-resistant prostate cancer: modeling through novel murine prostate cancer cell lines.

Cell lines representing the progression of prostate cancer (PC) from an androgen-dependent to an androgen-independent state are scarce. In this study, we used previously characterized prostate luminal epithelial cell line (Plum), under androgen influence, to establish cellular models of PC progression. Cells derived from orthotopic tumors have been isolated to develop an androgen-dependent (PLum-AD) versus an androgen-independent (PLum-AI) model. Upon immunofluorescent, qRT-PCR and Western blot analyses, PLum-AD cells mostly expressed prostate epithelial markers while PLum-AI cells expressed mesenchymal cell markers. Interestingly, both cell lines maintained a population of stem/progenitor cells. Furthermore, our data suggest that both cell lines are tumorigenic; PLum-AD resulted in an adenocarcinoma whereas PLum-AI resulted in a sarcomatoid carcinoma when transplanted subcutaneously in NOD-SCID mice. Finally, gene expression profiles showed enrichment in functions involved in cell migration, apoptosis, as well as neoplasm invasiveness and metastasis in PLum-AI cells. In conclusion, these data suggest that the newly isolated cell lines represent a new in vitro model of androgen-dependent and -independent PC.

Metformin and Ara-a Effectively Suppress Brain Cancer by Targeting Cancer Stem/Progenitor Cells.

BACKGROUND: Gliomas and neuroblastomas pose a great health burden worldwide with a poor and moderate prognosis, respectively. Many studies have tried to find effective treatments for these primary malignant brain tumors. Of interest, the AMP-activated protein kinase (AMPK) pathway was found to be associated with tumorigenesis and tumor survival, leading to many studies on AMPK drugs, especially Metformin, and their potential role as anti-cancer treatments. Cancer stem cells (CSCs) are a small population of slowly-dividing, treatment-resistant, undifferentiated cancer cells that are being discovered in a multitude of cancers. They are thought to be responsible for replenishing the tumor with highly proliferative cells and increasing the risk of recurrence. METHODS: Metformin and 9-ß-d-Arabinofuranosyl Adenine (Ara-a) were used to study the role of the AMPK pathway in vitro on U251 (glioblastoma) and SH-SY5Y (neuroblastoma) cell lines. RESULTS: We found that both drugs are able to decrease the survival of U251 and SH-SY5Y cell lines in a 2D as well as a 3D culture model. Metformin and Ara-a significantly decreased the invasive ability of these cancer cell lines. Treatment with these drugs decreased the sphere-forming units (SFU) of U251 cells, with Ara-a being more efficient, signifying the extinction of the CSC population. However, if treatment is withdrawn before all SFUs are extinguished, the CSCs regain some of their sphere-forming capabilities in the case of Metformin but not Ara-a treatment. CONCLUSION: Metformin and Ara-a have proved to be effective in the treatment of glioblastomas and neuroblastomas, in vitro, by targeting their cancer stem/progenitor cell population, which prevents recurrence.

Berberis libanotica Ehrenb extract shows anti-neoplastic effects on prostate cancer stem/progenitor cells.

Cancer stem cells (CSCs), including those of advanced prostate cancer, are a suggested reason for tumor resistance toward conventional tumor therapy. Therefore, new therapeutic agents are urgently needed for targeting CSCs. Despite the minimal understanding of their modes of action, natural products and herbal therapies have been commonly used in the prevention and treatment of many cancers. Berberis libanotica Ehrenb (BLE) is a plant rich in alkaloids which may possess anti-cancer activity and a high potential for eliminating CSCs. We tested the effect of BLE on prostate cancer cells and our data indicated that this extract induced significant reduction in cell viability and inhibited the proliferation of human prostate cancer cell lines (DU145, PC3 and 22Rv1) in a dose- and time-dependent manner. BLE extract induced a perturbation of the cell cycle, leading to a G0-G1 arrest. Furthermore, we noted 50% cell death, characterized by the production of high levels of reactive oxidative species (ROS). Inhibition of cellular migration and invasion was also achieved upon treatment with BLE extract, suggesting a role in inhibiting metastasis. Interestingly, BLE extract had a major effect on CSCs. Cells were grown in a 3D sphere-formation assay to enrich for a population of cancer stem/progenitor cells. Our results showed a significant reduction in sphere formation ability. Three rounds of treatment with BLE extract were sufficient to eradicate the self-renewal ability of highly resistant CSCs. In conclusion, our results suggest a high therapeutic potential of BLE extract in targeting prostate cancer and its CSCs.