Red Sea Sponge Callyspongia siphonella Extract Induced Growth Inhibition and Apoptosis in Breast MCF-7 and Hepatic HepG-2 Cancer Cell Lines in 2D and 3D Cell Cultures.

Introduction: The increasing incidence of cancer diseases necessitates the urgent exploration of new bioactive compounds. One of the trends in drug discovery is marine sponges which is gaining significant support due to the abundant production of natural pharmaceutical compounds obtained from marine ecosystems. This study evaluates the anticancer properties of an organic extract from the Red Sea sponge Callyspongia siphonella (C. siphonella) on HepG-2 and MCF-7 cancer cell lines. Methods: C. siphonella was collected, freeze-dried, and extracted using a methanol-dichloromethane mixture. The extract was analyzed via Liquid Chromatography-Mass Spectrometry. Cytotoxic effects were assessed through cell viability assays, apoptosis detection, cell cycle analysis, mitochondrial membrane potential assays, scratch-wound healing assays, and 3D cell culture assays. Results: Fifteen compounds were identified in the C. siphonella extract. The extract showed moderate cytotoxicity against MCF-7 and HepG-2 cells, with IC50 values of 35.6 ± 6.9 µg/mL and 64.4 ± 8 µg/mL, respectively, after 48 hours of treatment. It induced cell cycle arrest at the G2/M phase in MCF-7 cells and the S phase in HepG-2 cells. Apoptosis increased significantly in both cell lines, accompanied by reduced mitochondrial membrane potential. The extract inhibited cell migration, with notable reductions after 24 and 48 hours. In 3D cell cultures, the extract had IC50 values of 5.1 ± 2 µg/mL for MCF-7 and 166.4 ± 27 µg/mL for HepG-2 after 7 days of treatment, showing greater potency in MCF-7 spheres compared to HepG-2 spheres. Discussion and Conclusion: The anticancer activity is attributed to the bioactive compounds. The C. siphonella extract's ability to induce apoptosis, disrupt mitochondrial membrane potential, and arrest the cell cycle highlights its potential as a novel anticancer agent. Additional research is required to investigate the underlying mechanism by which this extract functions as a highly effective anticancer agent.

Evaluation of Cytotoxicity of the Methanolic Extract of Red Sea Marine Sponge Xestospongia Testudinaria and Its Related Compounds Against MCF-7 Human Breast Cancer Cells.

Background: Breast cancer is a leading cause of death and one of the most common fatal medical conditions in the world. Chemical compounds of various types have been identified in the Red Sea marine sponge Xestospongia testudinaria, including sterol esters, sterols, indole alkaloids, and brominated polyunsaturated fatty acids. These compounds have demonstrated promising biological features, which in cludes anti-inflammatory, cancer preventive, and antioxidant capacities. Methods: The cytotoxic potential of Xestospongia testudinaria was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and morphological alterations in MCF-7 cell line. Furthermore, the flow cytometry was also utilized to assess apoptosis and identify changes in the cell cycle; besides, cell migration was assessed by scratch wound-healing assay. Results: A significant dose-dependent decrease in the percentage of MCF-7 cell viability was observed with IC50 39.8 ug/mL. Functional studies were performed on MCF-7 to show that Xestospongia testudinaria raises apoptotic cell death and induces growth arrest at the G1/G0 while inhibiting cell migration in scratch assay. Conclusion: These results demonstrated that Xestospongia testudinaria extract has an inhibitory effect on breast cancer cells proliferation, migration and induce apoptosis. Thus, it holds great promise as a potential treatment for breast cancer.

In silico Screening and Validation of Achyranthes aspera as a Potential Inhibitor of BRAF and NRAS in Controlling Thyroid Cancer.

BACKGROUND: Thyroid carcinoma (THCA) is one of the most prevalent endocrine tumors, accounting for 3.4% of all cancers diagnosed annually. Single Nucleotide Polymorphisms (SNPs) are the most prevalent genetic variation associated with thyroid cancer. Understanding thyroid cancer genetics will enhance diagnosis, prognosis, and treatment. METHODS: This TCGA-based study analyzes thyroid cancer-associated highly mutated genes through highly robust in silico techniques. Pathway, gene expression, and survival studies were performed on the top 10 highly mutated genes (BRAF, NRAS, TG, TTN, HRAS, MUC16, ZFHX3, CSMD2, EIFIAX, SPTA1). Novel natural compounds from Achyranthes aspera Linn were discovered to target two highly mutated genes. The natural compounds and synthetic drugs used to treat thyroid cancer were subjected to comparative molecular docking against BRAF and NRAS targets. The ADME characteristics of Achyranthes aspera Linn compounds were also investigated. RESULTS: The gene expression analysis revealed that the expression of ZFHX3, MCU16, EIF1AX, HRAS, and NRAS was up-regulated in tumor cells while BRAF, TTN, TG, CSMD2, and SPTA1 were down-regulated in tumor cells. In addition, the protein-protein interaction network demonstrated that HRAS, BRAF, NRAS, SPTA1, and TG proteins have strong interactions with each other as compared to other genes. The ADMET analysis shows that seven compounds have druglike properties. These compounds were further studied for molecular docking studies. The compounds MPHY012847, IMPHY005295, and IMPHY000939 show higher binding affinity with BRAF than pimasertib. In addition, IMPHY000939, IMPHY000303, IMPHY012847, and IMPHY005295 showed a better binding affinity with NRAS than Guanosine Triphosphate. CONCLUSION: The outcomes of docking experiments conducted on BRAF and NRAS provide insight into natural compounds with pharmacological characteristics. These findings indicate that natural compounds derived from plants as a more promising cancer treatment option. Thus, the results of docking investigations conducted on BRAF and NRAS substantiate the conclusions that the molecule possesses the most suited drug-like qualities. Compared to other compounds, natural compounds are superior, and they are also druggable. This demonstrates that natural plant compounds can be an excellent source of potential anti-cancer agents. The preclinical research will pave the road for a possible anti-cancer agent.

Evaluation of participation and performance indicators in a breast cancer screening program in Saudi Arabia.

OBJECTIVES: To evaluate early performance indicators for breast cancer screening at the King Abdulaziz University Hospital in Saudi Arabia. METHODS: This study retrospectively evaluated data from women who underwent their first breast cancer screening program in Jeddah, Saudi Arabia between 2012 and 2019. Data on screening results were used to estimate performance indicators and generate descriptive statistics. RESULTS: Of the 16000 women invited from 2012 to 2019, a total of 1911 (11.9%) participated. The majority of women (68.8%) were between 40 and 55 years old. Based on the screening process results, 26.6%, 40.1%, 9.7%, 1.3%, 0.7%, and 5.2% of women had BI-RADS scores of R1, R2, R3, R4, R5, and R0 respectively. The remaining 16.3% did not have mammogram records. The recall rate, or the percentage of women who underwent further evaluation, was 19.9%; 18.9% underwent a biopsy procedure. In addition, 1.6% of women had cancer screen-detected, although only 0.7% were diagnosed with breast cancer. CONCLUSION: In light of the low participation and high recall rates, it is essential that the screening program utilizes performance indicators to optimize resource utilization and ensure the quality of the service provided. Additionally, a national framework and standardized performance indicators could mitigate this problem for other cancer screening programs.

Detection of HPV RNA in Extracellular Vesicles from Neuroendocrine Cervical Cancer Cells.

Background: Neuroendocrine carcinoma of the cervix (NECC) is an aggressive and rare type of cervical cancer. The five-year overall survival is low at 30% and there is no standardized therapy based on controlled trials for this type of tumour. Most are locally advanced or metastasized at the time of the diagnosis. Extracellular vesicles (EVs) could be a carrier of viral DNA/RNA, given their vital role in cellular communication. The content of EV derived from NECC cells has not been investigated due to the lack of cell line, and it is not known whether they contain human papillomaviruses (HPV) DNA/RNA or not. Methods: The presence of viral E7 DNA/RNA in EVs purified from a culture of a recently established NECC cell line, GUMC-395, was evaluated by using droplet digital polymerase chain reaction (ddPCR). These EVs were characterized using nanoparticle tracking analysis (NTA) for size distribution, transmission electron microscopy (TEM) for morphology, Western blot for CD63, and bioanalyser for RNA quantity and quality. Results: HPV16 viral-RNA, but not DNA, was detected in EVs from GUMC-395 using ddPCR. NTA identified EVs with a mean diameter of 105.0 nm, TEM confirmed normal morphological shape and size, and Western blot analysis confirmed the presence of EV-associated proteins CD63. The EVs were found to be enriched with small RNAs using a bioanalyser. Conclusions: HPV16 RNA is found in EVs from a neuroendocrine cervical cancer and could be involved in the pathogenesis of the disease and used as a diagnostic biomarker.

Era of Molecular Diagnostics Techniques before and after the COVID-19 Pandemic.

Despite the growth of molecular diagnosis from the era of Hippocrates, the emergence of COVID-19 is still remarkable. The previously used molecular techniques were not rapid enough to screen a vast population at home, in offices, and in hospitals. Additionally, these techniques were only available in advanced clinical laboratories.The pandemic outbreak enhanced the urgency of researchers and research and development companies to invent more rapid, robust, and portable devices and instruments to screen a vast community in a cost-effective and short time. There has been noteworthy progress in molecular diagnosing tools before and after the pandemic. This review focuses on the advancements in molecular diagnostic techniques before and after the emergence of COVID-19 and how the pandemic accelerated the implantation of molecular diagnostic techniques in most clinical laboratories towardbecoming routine tests.

The Therapeutic Potential of Milk Extracellular Vesicles on Colorectal Cancer.

Colorectal cancer remains one of the leading prevalent cancers in the world and is the fourth most common cause of death from cancer. Unfortunately, the currently utilized chemotherapies fail in selectively targeting cancer cells and cause harm to healthy cells, which results in profound side effects. Researchers are focused on developing anti-cancer targeted medications, which is essential to making them safer, more effective, and more selective and to maximizing their therapeutic benefits. Milk-derived extracellular vesicles (EVs) from camels and cows have attracted much attention as a natural substitute product that effectively suppresses a wide range of tumor cells. This review sheds light on the biogenesis, methods of isolation, characterization, and molecular composition of milk EVs as well as the therapeutic potentials of milk EVs on colorectal cancer.

Conditionally Reprogrammed Cells and Robotic High-Throughput Screening for Precision Cancer Therapy.

Cancer is a devastating disease that takes the lives of millions of people globally every year. Precision cancer therapy is based on a patient's tumor histopathology, expression analyses, and/or tumor RNA or DNA analysis. Only 2%-20% of patients with solid tumors benefit from genomics-based precision oncology. Therefore, functional diagnostics and patient-derived cancer models are needed for precision cancer therapy. In this review, we will summarize the potential use of conditional cell reprogramming (CR) and robotic high-throughput screening in precision cancer medicine. Briefly, the CR method includes the co-culturing of irradiated Swiss-3T3-J2 mouse fibroblast cells alongside digested primary non-pathogenic or pathogenic cells with the existence of Rho-associated serine-threonine protein kinase inhibitor called Y-27632, creating an exterior culture environment, allowing the cells to have the ability to gain partial properties of stem cells. On the other hand, quantitative high-throughput screening (qHTS) assays screen thousands of compounds that use cells in a short period of time. The combination of both technologies has the potential to become a driving force for precision cancer therapy.

Anti-Proliferative, Cytotoxic and Antioxidant Properties of the Methanolic Extracts of Five Saudi Arabian Flora with Folkloric Medicinal Use: Aizoon canariense, Citrullus colocynthis, Maerua crassifolia, Rhazya stricta and Tribulus macropterus.

Saudi Arabian flora have a history of use as folklore remedies, although such properties have yet to be explored rigorously, and the safety of such remedies should be assessed. This study determined the anti-proliferative, cytotoxic, and antioxidant properties of extracts of the following five plants indigenous to Saudi Arabia: Aizoon canariense, Citrullus colocynthis, Maerua crassifolia, Rhazya stricta, and Tribulus macropterus. The aerial parts of the five plants were collected from various locations of the western and northern regions of Saudi Arabia and used to prepare methanolic extracts. Three approaches were used to determine the proliferation and cytotoxicity effects using HaCaT cells: MTT, FACS, and confocal microscopy. Meanwhile, two approaches were used to study the antioxidant potential: DPPH (acellular) and RosGlo (cellular, using HaCaT cells). C. colocynthis possessed anti-proliferative activity against HaCaT cells, showing a significant decrease in cell proliferation from 24 h onwards, while R. stricta showed significant inhibition of cell growth at 120 and 168 h. The IC50 values were determined for both plant extracts for C. colocynthis, with 17.32 and 16.91 µg/mL after five and seven days of treatment, respectively, and for R. stricta, with 175 and 105.3 µg/mL after five and seven days of treatment. R. stricta and M. crassifolia exhibited the highest capacities for scavenging the DPPH radical with IC50 values of 335 and 448 µg/mL, respectively. The subsequent ROS-Glo H2O2 assay confirmed these findings. The R. stricta and M. crassifolia extracts showed potent antioxidant activity in both acellular and cellular models. The C. colocynthis extract also demonstrated significant anti-proliferation and cytotoxic activity, as did the R. stricta extract. These properties support their usage in folk medicine and also indicate a further potential for development for holistic medicinal use or as sources of new active compounds.

High-throughput screening identifies candidate drugs for the treatment of recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a benign neoplasm of the larynx caused mainly by human papillomavirus type 6 or 11 and its standard treatment involves repeated surgical debulking of the laryngeal tumors. However, significant morbidity and occasional mortality due to multiple recurrences occur. Conditional reprogramming (CR) was used to establish a HPV-6 positive culture from an RRP patient, named GUMC-403. High-throughput screening was performed at the National Center for Advanced Technology (NCATS) to identify potential drugs to treat this rare but morbid disease. GUMC-403 cells were screened against the NPC library of >2800 approved drugs and the MIPE library of >1900 investigational drugs to identify new uses for FDA-approved drugs or drugs that have undergone significant research and development. From the two libraries, we identified a total of 13 drugs that induced significant cytotoxicity in RRP cells at IC50 values that were clinically achievable. We validated the efficacy of the drugs in vitro using CR 2D and 3D models and further refined our list of drugs to panobinostat, dinaciclib and forskolin as potential therapies for RRP patients.

Extracellular vesicles from triple-negative breast cancer cells promote proliferation and drug resistance in non-tumorigenic breast cells.

PURPOSE: Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype, is genetically heterogeneous which challenges the identification of clinically effective molecular makers. Extracellular vesicles (EVs) are key players in the intercellular signaling communication and have been shown to be involved in tumorigenesis. The main goal of this study was to evaluate the role and mechanisms of EVs derived from TNBC cells in modulating proliferation and cytotoxicity to chemotherapeutic agents in non-tumorigenic breast cells (MCF10A). METHODS: EVs were isolated from TNBC cell lines and characterized by nanoparticle tracking analysis, Western blot, and transmission electron microscopy. MCF10A cells were treated with the isolated EVs and evaluated for cell proliferation and cytotoxicity to Docetaxel and Doxorubicin by the MTT and MTS assays, respectively. Gene and miRNA expression profiling was performed in the treated cells to determine expression changes that may be caused by EVs treatment. RESULTS: MCF10A cells treated with HCC1806-EVs (MCF10A/HCC1806-EVs) showed a significant increase in cell proliferation and resistance to the therapeutic agents tested. No significant effects were observed in the MCF10A cells treated with EVs derived from MDA-MB-231 cells. Gene and miRNA expression profiling revealed 138 genes and 70 miRNAs significantly differentially expressed among the MCF10A/HCC1806-EVs and the untreated MCF10A cells, affecting mostly the PI3K/AKT, MAPK, and HIF1A pathways. CONCLUSION: EVs isolated from the HCC1806 TNBC cells are capable of inducing proliferation and drug resistance on the non-tumorigenic MCF10A breast cells, potentially mediated by changes in genes and miRNAs expression associated with cell proliferation, apoptosis, invasion, and migration.

Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis.

BACKGROUND: Skin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium. METHODS: Conditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium. RESULTS: Conditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers. CONCLUSIONS: Our results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation.

HPV positive neuroendocrine cervical cancer cells are dependent on Myc but not E6/E7 viral oncogenes.

Using conditional cell reprogramming, we generated a stable cell culture of an extremely rare and aggressive neuroendocrine cervical cancer. The cultured cells contained HPV-16, formed colonies in soft agar and rapidly produced tumors in immunodeficient mice. The HPV-16 genome was integrated adjacent to the Myc gene, both of which were amplified 40-fold. Analysis of RNA transcripts detected fusion of the HPV/Myc genes, arising from apparent microhomologous recombination. Spectral karyotyping (SKY) and fluorescent-in-situ hybridization (FISH) demonstrated coordinate localization and translocation of the amplified Myc and HPV genes on chromosomes 8 and 21. Similar to the primary tumor, tumor cell cultures expressed very high levels of the Myc protein and, in contrast to all other HPV-positive cervical cancer cell lines, they harbored a gain-of-function mutation in p53 (R273C). Unexpectedly, viral oncogene knockdown had no effect on the growth of the cells, but it did inhibit the proliferation of a conventional HPV-16 positive cervical cancer cell line. Knockdown of Myc, but not the mutant p53, significantly inhibited tumor cell proliferation. On the basis of these data, we propose that the primary driver of transformation in this aggressive cervical cancer is not HPV oncogene expression but rather the overexpression of Myc.

Complete genome sequence of canine papillomavirus virus type 12.

Papillomaviruses, of the family Papillomaviridae, are epitheliotropic, nonenveloped, circular, double-stranded DNA viruses that contribute to benign and malignant tumors in humans and animals. We report here the whole-genome sequence of canine papillomavirus type 12, found at a pigmented plaque located on the skin of a mixed-breed bloodhound.