Including Cell Viability Assays Research Articles

Cartilage defect repair in a rat model via a nanocomposite hydrogel loaded with melatonin-loaded gelatin nanofibers and menstrual blood stem cells: an in vitro and in vivo study

Cartilage damage caused by injuries or degenerative diseases remains a major challenge in the field of regenerative medicine. In this study, we developed a composite hydrogel system for the delivery of melatonin and menstrual blood stem cells (MenSCs) to treat a rat model of cartilage defect. The composite delivery system was produced by incorporation of melatonin into the gelatin fibers and dispersing these fibers into calcium alginate hydrogels. Various characterization methods including cell viability assay, microstructure studies, degradation rate measurement, drug release, anti-inflammatory assay, and radical scavenging assay were used to characterize the hydrogel system. MenSCs were encapsulated within the nanocomposite hydrogel and implanted into a rat model of full-thickness cartilage defect. A 1.3 mm diameter drilled in the femoral trochlea and used for the in vivo study. Results showed that the healing potential of nanocomposite hydrogels containing melatonin and MenSCs was significantly higher than polymer-only hydrogels. Our study introduces a novel composite hydrogel system, combining melatonin and MenSCs, demonstrating enhanced cartilage repair efficacy, offering a promising avenue for regenerative medicine.Graphical

Euphorbia Pekinensis Rupr. sensitizes colorectal cancer to PD-1 blockade by remodeling the tumor microenvironment and enhancing peripheral immunity

BackgroundImmune checkpoint blockade, such as monoclonal antibodies targeting programmed cell death protein 1 (PD-1), has been a major breakthrough in the treatment of several cancers, but has limited effect in colorectal cancer (CRC), which is a highly prevalent cancer worldwide. Current chemotherapy-based strategies to boost PD-1 response have many limitations. And the role of peripheral immunity in boosting PD-1 response continues to attract attention. Therefore, candidate combinations of PD-1 blockade need to be drugs with multi-targets and multi-modulatory functions. However, it is still unknown whether traditional Chinese medicines with such property can enhance the applicability and efficacy of PD-1 blockade in colorectal cancer. MethodsEuphorbia Pekinensis extract (EP) was prepared and the constituents were analyzed by HPLC. CRC cells were used for in vitro experiments, including cell viability assay, colony formation assay, flow cytometry for 7-AAD staining, western blotting for caspase 3 and caspase 7, HMGB1 and ATP detection. An orthotopic CT26 mouse model was subsequently used to investigate the combination of EP and PD-1 blockade therapy. Tumor volume and tumor weight were assessed, tumor tissues were subjected to histopathological HE staining and TUNEL staining, and tumor-infiltrating immune cells were evaluated by immunofluorescence staining. RNA-sequencing, target prediction and pathway analysis were further employed to explore the mechanism. Molecular docking and cellular thermal shift assay (CETSA) were utilized to verify the direct target of the core component of EP. And, loss-of-function analysis was carried to confirm the upstream-downstream relationship. Flow cytometry was employed to analyze CD8+ T cells in the peripheral blood and spleen. ResultsThe main constituents of EP are diterpenoids and flavonoids. EP dramatically suppresses CRC cell growth and exerts its cytotoxic effect by triggering immunogenic cell death in vitro. Moreover, EP synergizes with PD-1 blockade to inhibit tumorigenesis in tumor-bearing mice. Disruption of ISX nuclear localization by helioscopinolide E is a central mechanism of EP-induced apoptosis in CRC cell. Meanwhile, EP activates immune response by upregulating Phox2b to reshape the immune microenvironment. In addition, EP regulates peripheral immunity by regulating the T cell activation and proliferation, and the ratio of CD8+ T cells in peripheral blood is drastically increased, thereby enhancing the therapeutic efficacy of anti-PD1 immunotherapy. ConclusionEP triggers intra-tumor immunogenic cell death and modulates the immunoregulatory signaling to elicit the tumor immunogenicity. Moreover, EP participates in transcriptional activation of immune response-related pathways. Consequently, multiple stimulating functions of EP on macro- and micro-immune potentiates the anti-tumor effect of PD-1 blockade in CRC.

A novel approach for breast cancer treatment: the multifaceted antitumor effects of rMeV-Hu191.

The therapeutic potential of oncolytic measles virotherapy has been demonstrated across various malignancies. However, the effectiveness against human breast cancer (BC) and the underlying mechanisms of the recombinant measles virus vaccine strain Hu191 (rMeV-Hu191) remain unclear. We utilized a range of methods, including cell viability assay, Western blot, flow cytometry, immunofluorescence, SA-β-gal staining, reverse transcription quantitative real-time PCR, transcriptome sequencing, BC xenograft mouse models, and immunohistochemistry to evaluate the antitumor efficacy of rMeV-Hu191 against BC and elucidate the underlying mechanism. Additionally, we employed transcriptomics and gene set enrichment analysis to analyze the lipid metabolism status of BC cells following rMeV-Hu191 infection. Our study revealed the multifaceted antitumor effects of rMeV-Hu191 against BC. rMeV-Hu191 induced apoptosis, inhibited proliferation, and promoted senescence in BC cells. Furthermore, rMeV-Hu191 was associated with changes in oxidative stress and lipid homeostasis in infected BC cells. In vivo, studies using a BC xenograft mouse model confirmed a significant reduction in tumor growth following local injection of rMeV-Hu191. The findings highlight the potential of rMeV-Hu191 as a promising treatment for BC and provide valuable insights into the mechanisms underlying its oncolytic effect.

Anti-inflammatory and Anti-angiogenic Effects of Bioconverted Picrasma quassioides Extract: A Comparative Study on NF-κB Signaling Pathway Inhibition and Angiogenesis Suppression

Purpose: This study aimed to compare the anti-inflammatory and anti-angiogenic properties of bioconverted Picrasma quassioides extract (B-P.Q) with those of the non-bioconverted extract (P.Q) and peony root bark extract (BOT).Methods: The research utilized several experimental techniques, including cell viability assays on NIH 3T3 fibroblasts, measurement of NF-κB-related signaling protein activation, chorioallantoic membrane (CAM) assays, tube formation assays, and cell migration inhibition assays using human umbilical vein endothelial cells (HUVECs). These methods were employed to evaluate cytotoxicity, anti-inflammatory effects, and anti-angiogenic properties of the extracts.Results: B-P.Q demonstrated no cytotoxicity at the tested concentrations and significantly inhibited the activation of NF-κB-related signaling proteins (p-p38, p-Akt-1, p-SAPK/JNK, p-MEK1/2, and p-IκB) compared to P.Q and BOT. In angiogenesis experiments, B-P.Q showed superior inhibition of new blood vessel formation in CAM assays, effectively suppressed tube formation, and significantly inhibited HUVEC migration compared to controls and P.Q.Conclusion: The study found that bioconverted Picrasma quassioides extract (B-P.Q) has strong anti-inflammatory and anti-angiogenic properties, consistently outperforming non-bioconverted P.Q and BOT in various experimental models. These results suggest that B-P.Q holds significant promise as a therapeutic agent for chronic inflammatory conditions, particularly those involving excessive angiogenesis. The research underscores the effectiveness of bioconversion in enhancing the bioactive properties of natural extracts and opens up new possibilities for developing treatments for inflammatory skin problems.

Progesterone boosts abiraterone-driven target and NK cell therapies against glioblastoma

IntroductionGlioblastoma (GBM) poses a significant challenge in oncology, with median survival times barely extending beyond a year due to resistance to standard therapies like temozolomide (TMZ). This study introduces a novel therapeutic strategy combining progesterone (Prog) and abiraterone (Abi) aimed at enhancing GBM treatment efficacy by modulating the tumor microenvironment and augmenting NK cell-mediated immunity.MethodsWe employed in vitro and in vivo GBM models to assess the effects of Prog and Abi on cell viability, proliferation, apoptosis, and the immune microenvironment. Techniques included cell viability assays, Glo-caspase 3/7 apoptosis assays, RNA-seq and qPCR for gene expression, Seahorse analysis for mitochondrial function, HPLC-MS for metabolomics analysis, and immune analysis by flow cytometry to quantify NK cell infiltration.ResultsProg significantly reduced the IC50 of Abi in TMZ-resistant GBM cell, suggesting the enhanced cytotoxicity. Treatment induced greater apoptosis than either agent alone, suppressed tumor growth, and prolonged survival in mouse models. Notably, there was an increase in CD3−/CD19−/CD56+/NK1.1+ NK cell infiltration in treated tumors, indicating a shift towards an anti-tumor immune microenvironment. The combination therapy also resulted in a reduction of MGMT expression and a suppression of mitochondrial respiration and glycolysis in GBM cells.ConclusionThe combination of Prog and Abi represents a promising therapeutic approach for GBM, showing potential in suppressing tumor growth, extending survival, and modulating the immune microenvironment. These findings warrant further exploration into the clinical applicability of this strategy to improve outcomes for GBM patients.

Regulation of MicroRNA-4697-3p by Parkinson's disease-associated SNP rs329648 and its impact on SNCA112 mRNA.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by a multifaceted genetic foundation. Genome-Wide Association Studies (GWAS) have played a crucial role in pinpointing genetic variants linked to PD susceptibility. Current study aims to delve into the mechanistic aspects through which the PD-associated Single Nucleotide Polymorphism (SNP) rs329648, identified in prior GWAS, influences the pathogenesis of PD. Employing the CRISPR/Cas9-mediated genome editing mechanism, we demonstrated the association of the disease-associated allele of rs329648 with increased expression of miR-4697-3p in differentiated SH-SY5Y cells. We revealed that miR-4697-3p contributes to the formation of high molecular weight complexes of α-Synuclein (α-Syn), indicative of α-Syn aggregate formation, as evidenced by Western blot analysis. Furthermore, our study unveiled that miR-4697-3p elevates SNCA112 mRNA levels. The resultant protein product, α-Syn 112, a variant of α-Syn with 112 amino acids, is recognized for augmenting α-Syn aggregation. Notably, this regulatory effect minimally impacts the levels of full-length SNCA140 mRNA, as evidenced by qRT-PCR. Additionally, we observed a correlation between the disease-associated allele and miR-4697-3p with increased cell death, substantiated by assessments including cell viability assays, alterations in cell morphology, and TUNEL assays. Our research reveals that the disease-associated allele of rs329648 is linked to higher levels of miR-4697-3p. This increase in miR-4697-3p leads to elevated SNCA112 mRNA levels, consequently promoting the formation of α-Syn aggregates. Furthermore, miR-4697-3p appears to play a role in increased cell death, potentially contributing to the pathogenesis of PD.

Abstract 2099: PELP1 inhibition enhances the therapeutic efficacy of topoisomerase inhibitors in triple-negative breast cancer

Abstract BACKGROUND: Triple negative breast cancers (TNBCs) account for 15-24% of breast cancer deaths and are characterized by aggressive clinical course, a worse prognosis, and a higher propensity to metastasize. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1) is an oncogene, its expression is deregulated in TNBC, and its status is a prognostic indicator of poor TNBC survival. Recently, a small molecule inhibitor of PELP1 (SMIP34) which binds to and degrades PELP1 was developed. The Objective of this proposal is to define the mechanism of action of PELP1 inhibitor SMIP34 in TNBC progression and to pave the path for a novel combination therapy. METHODS: We tested the effectiveness of PELP1 inhibitor, SMIP-34, in combination with 140 FDA-approved medications on cell survival of multiple TNBC cell lines. The effect of SMIP34 combination therapy on cell survival was measured using MTT, colony formation, and Annexin V apoptosis assays. Mechanistic studies were done using RT-qPCR, immunoprecipitation, proximity ligation, mass spectrometry, gene reporter, immunofluorescence, and comet assays. Furthermore, the preclinical utility of combination therapy was evaluated using MDA-MB-231 xenografts, patient-derived organoids (PDOs) and explants (PDEs). RESULTS: We performed an in vitro screening of 140 FDA-approved drugs in combination with SMIP34. Results revealed that SMIP34 sensitized TNBC cells to five FDA approved drugs. Of the five drugs, the three drugs Gemcitabine, Valrubicin, and Mitoxantrone, induced DNA damage by inhibiting topoisomerase, a protein that cleaves and reconnects DNA during replication. We validated the ability of SMIP34 to enhance the efficacy of TIs using two additional TNBC cell lines and confirmed their synergistic activity using several in vitro assays including cell viability, colony formation, and apoptosis. In agreement with the role of PELP1 in DNA damage response, Western blotting analyses showed higher levels of γ-H2AX in SMIP34+TI combination therapy when compared to monotherapy. Results from the comet assays further demonstrate increased DNA damage caused by the SMIP-34 + TIs combination therapy. Mechanistic studies using Proximity labelling followed by immunoprecipitation identified topoisomerase 2A and 2B (TOP2A and TOP2B) as potential interactors of PELP1. Furthermore, gene correlation analysis using TCGA datasets revealed that PELP1 expression is positively correlated with TOP2A, and TOP2B in TNBC. SMIP34 plus Mitoxantrone (TI) combination treatment significantly reduced the growth of TNBC PDEXs, and PDOs in vitro, and MDA-MB-231 xenograft tumor growth in vivo, compared to vehicle or monotherapy treatment. CONCLUSION: Together, these results suggest a novel targeted therapy for the treatment of TNBC, involving the combination of topoisomerase inhibitors and the PELP1 inhibitor SMIP34. Citation Format: Khaled Mohamed Nassar, John R. Sanchez, Durga Meenakshi Panneerdoss, Behnam Ebrahimi, Yang Xue, Uday P. Pratap, Salvador C. Alejo, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. PELP1 inhibition enhances the therapeutic efficacy of topoisomerase inhibitors in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2099.

Abstract 4715: Azathioprine and Z36 induced cell death in human colorectal cancer HCT-116 and HCT-15 cells

Abstract Rationale and Objectives: Cancer accounts for a huge global health burden and is the third highest cause of death across the globe. Cancer cells are known to have higher basal reactive oxygen species (ROS) levels, owing to their higher metabolism and proliferation. Higher basal ROS in cancer cells made them susceptible to exogenous ROS stimuli that lead to cell death. Therefore, in the present study, we have analyzed the effects of Azathioprine, a glutathione (GSH) inhibitor and an intracellular ROS producer. The effect of Azathioprine was analyzed alone or in combination with a BCL-2 family protein inhibitor Z36, on human colorectal cancer cell lines HCT-116 and HCT-15. Methods: The effects of the drugs have been investigated using various methods including cell viability assay, DNA fragmentation assay, and Annexin V-FITC/PI apoptosis assay. In addition, morphological changes evident in cell death were detected by Giemsa staining. Combination dosage for drug synergistic effects was computed through the use of CompuSyn software. Western blot was performed to detect changes in the expression of cell death-related proteins following drug treatments. Results and conclusion: Azathioprine at a dose of 5 µM has been demonstrated to induce cell death in both HCT116 and HCT15 cells in a concentration and time-dependent manner. On the contrary, Z36, a novel BCLXL inhibitor, has demonstrated similar cell viability results at an even lower dose of 2.5 µM. When both drugs were combined at lower doses, no significant effects were observed on the cell viability of either cell line, demonstrating that the combined treatment has no synergistic or additive effects. This suggests that both drugs do not augment each other and might induce cell death via upregulation of ROS using a similar mechanism. Citation Format: Yousuf Tahir Ali, Mahwish Fatima, Muhammad Areeb Ahmed, Hasan Salman Siddiqi, Azhar Hussain, Mati Ur Rehman. Azathioprine and Z36 induced cell death in human colorectal cancer HCT-116 and HCT-15 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4715.

Noxa inhibits oncogenesis through ZNF519 in gastric cancer and is suppressed by hsa-miR-200b-3p

While Phorbol-12-myristate-13-acetate-induced protein 1 (Noxa/PMAIP1) assumes a pivotal role in numerous tumors, its clinical implications and underlying mechanisms of gastric cancer (GC) are yet enigmatic. In this investigation, our primary objective was to scrutinize the clinical relevance and potential mechanisms of Noxa in gastric cancer. Immunohistochemical analysis was conducted on tissue microarrays comprising samples from a meticulously characterized cohort of 84 gastric cancer patients, accompanied by follow-up data, to assess the expression of Noxa. Additionally, Noxa expression levels in gastric cancer clinical samples and cell lines were measured through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. The effect of Noxa expression on the prognosis of patients with gastric cancer was evaluated using Kaplan–Meier survival. Further insight into the role of Noxa in driving gastric cancer progression was gained through an array of experimental techniques, including cell viability assays (CCK8), plate cloning assays, transwell assays, scratch assays, and real-time cell analysis (RTCA). Potential upstream microRNAs (miRNAs) that might modulate Noxa were identified through rigorous bioinformatics analysis, substantiated by luciferase reporter assays and Western blot experiments. Additionally, we utilized RNA sequencing, qRT-PCR, and Western blot to identify proteins binding to Noxa and potential downstream target. Finally, we utilized BALB/c nude mice to explore the role of Noxa in vivo. Our investigation unveiled a marked downregulation of Noxa expression in gastric cancer and underscored its significance as a pivotal prognostic factor influencing overall survival (OS). Noxa overexpression exerted a substantial inhibitory effect on the proliferation, migration and invasion of GC cells. Bioinformatic analysis and dual luciferase reporter assays unveiled the capacity of hsa-miR-200b-3p to interact with the 3′-UTR of Noxa mRNA, thereby orchestrating a downregulation of Noxa expression in vitro, consequently promoting tumor progression in GC. Our transcriptome analysis, coupled with mechanistic validation, elucidated a role for Noxa in modulating the expression of ZNF519 in the Mitophagy-animal pathway. The depletion of ZNF519 effectively reversed the oncogenic attributes induced by Noxa. Upregulation of Noxa expression suppressed the tumorigenesis of GC in vivo. The current investigation sheds light on the pivotal role of the hsa-miR-200b-3p/Noxa/ZNF519 axis in elucidating the pathogenesis of gastric cancer, offering a promising avenue for targeted therapeutic interventions in the management of this challenging malignancy.

Anti-proliferative, Pro-apoptotic, and Chemosensitizing Potential of 3-Acetyl-11-keto-β-boswellic Acid (AKBA) Against Prostate Cancer Cells.

Prostate cancer incidences are rising worldwide at an alarming rate. Drug resistance and relapse are two major challenges in the treatment of prostate cancer. Therefore, new multimodal, safe, and effective therapeutic agents are urgently required which could effectively mitigate the menace of tumor recurrence and chemo-resistance. Plant-derived products are increasingly being utilized due to their antioxidant, antibacterial, and anti-tumor potential. In the current study, 3-acetyl-11-keto-β-boswellic acid, a triterpenoid isolated from plant Boswellia, was utilized to ascertain its chemotherapeutic potential against human prostate cancer cells. Various in vitro assays including cell viability, nuclear staining, mitochondria potential, reactive oxygen species (ROS) generation, and quantification of apoptosis, were performed for the evaluation of the cytotoxic potential of AKBA. We observed that AKBA (10-50µM) dose-dependently suppressed cell proliferation and caused programmed cell death in PC3 cells via both intrinsic and extrinsic pathway. Intriguingly, AKBA was also found to chemosensitize PC3 cells in synergistic combination with doxorubicin. To the best of our knowledge, this is the first study to document the synergistic chemosensitizing impact of AKBA when combined with doxorubicin in prostate cancer cells.This showcases the potential of AKBA in combinatorial therapy or adjuvant therapy for the management of prostate cancer. In sum, our results suggested that AKBA is a promising drug-like molecule against prostate cancer. Our investigation introduces a novel perspective, elucidating a previously unexplored dimension, and uncovering a compelling chemosensitizing phenomenon along with a strong synergistic effect arising from the concurrent application of these two agents.

Biopolymer‐based nanoparticles as promising candidate for inducing apoptosis in liver cancer cells

AbstractLiver cancer is one of the most common causes of death all around the globe. On the other hand, conventional modalities, such as chemotherapy and surgery, have not been effectively suppressed cancer incidence due to some deficiencies. As a result, a novel folic acid (FA)‐decorated poly lactic‐co‐glycolic acid (PLGA)‐Alginate (Alg) nanocarrier was efficiently developed for active doxorubicin (Dox) delivery to human HepG2 liver cancer cells. The in vitro assays, including cell viability assay (MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) test), apoptosis, cell cycle arrest, and cellular uptake, were applied to evaluate the cancer cell growth suppression and apoptosis‐inducing ability of the nanocarrier. More than 85% cytotoxicity was attained after 24 h of treatment with 400‐nM concentration of FA‐PLGA‐Dox‐Alg. More than 21% and 61% apoptosis were observed after 24 h of treatment with 100‐nM concentrations of FA‐PLGA‐Dox‐Alg in apoptosis and cell cycle arrest assays. The cellular uptake of the FA‐PLGA‐Alg nanocarrier was about 30% higher than that of the PLGA‐Alg carrier which indicated the cancer cell targeting ability of the FA‐PLGA‐Alg sample. These results have indicated the potential capability of FA‐PLGA‐Dox‐Alg in the inhibition of liver cancer cells.

Scoparone attenuates glioma progression and improves the toxicity of temozolomide by suppressing RhoA/ROCK1 signaling.

Glioma, a type of malignant brain tumor, has become a challenging health issue globally in recent years. In this study, we investigated the potential therapeutic role of scoparone in glioma and the underlying mechanism. Initially, transcriptome sequencing was conducted to identify genes that exhibited differential expression in glioma cells treated with scoparone compared to untreated cells. Subsequently, the impact of scoparone on the proliferation, migration, and invasion of glioma cells was assessed in vitro using a range of assays including cell viability, colony formation, wound healing, and transwell assays. Moreover, the apoptotic effects of scoparone on glioma cells were evaluated through flow cytometry and western blot analysis. Furthermore, we established a glioma xenograft mouse model to assess the in vivo antitumor activity of scoparone. Lastly, by integrating transcriptome analysis, we endeavored to unravel the molecular mechanisms underlying the observed antitumor effects of scoparone by examining the expression levels of RhoA/ROCK1 signaling pathway components using western blot analysis and qRT-PCR. Our transcriptome sequencing results revealed that scoparone significantly downregulated RhoA/ROCK1 signaling in glioma cells. Furthermore, scoparone treatment inhibited glioma cell proliferation, migration, and invasion, and promoted cell apoptosis in vitro. Moreover, scoparone reduced tumor growth and prolonged survival in a glioma xenograft mouse model, and improved the toxicity of temozolomide. Finally, our results showed that the antitumor effects of scoparone were mediated by the suppression of RhoA/ROCK1 signaling. Scoparone could be a promising therapeutic agent for glioma by suppressing RhoA/ROCK1 signaling. These findings pave the way for future research endeavors aimed at the development and optimization of scoparone-based therapeutic strategies.

Abstract 3783: Role of microRNA 28-3p in prostate cancer progression

Abstract Prostate cancer (PCa) is a leading cause of cancer incidence and mortality among men. Prostate cancer is driven by androgens acting via androgen receptor (AR). Therefore, this cancer is treated with androgen deprivation therapy (ADT) initially. However, the cancer rapidly evolves to be resistant to ADT and progresses to a more invasive stage referred to as ‘Castration Resistant prostate cancer’ (CRPC). CRPC is treated with second generation of AR pathway inhibitors such as Enzalutamide and Abiraterone. These treatments are effective initially. However, drug resistance develops in a significant fraction of patients and the cancer undergoes ‘neuroendocrine trans-differentiation (NED) to a lethal variant called ‘neuroendocrine prostate cancer’ (NEPC). As a result of NED, PCa cells express neuronal markers such as synaptophysin, chromogranin A and enolase. The molecular basis of NED is not yet fully defined. With a focus on identifying the role of microRNAs (miRNAs) in NEPC, we performed small RNA sequencing in CRPC tumors with and without NED. This sequencing identified miR-28-3p to be significantly downregulated in CRPC-NE samples as compared to CRPC-adenocarcinomas (CRPC-Adeno). In view of this, we examined the role of this miRNA in NEPC. Analyses of TCGA data of prostate adenocarcinomas suggest that miR 28-3p has an oncogenic role in primary prostate tumors. However, its expression declines in tumors with increasing Gleason score. We profiled the expression of miR-28-3p in patient-derived xenograft models and clinical CRPC-NE and CRPC-Adeno tissues by real time PCR. Our profiling showed a trend towards downregulation with increased NED. Basal expression of miR 28-3p in various prostate cancer cell lines showed an elevated level in androgen dependent cell lines whereas significantly less expression in NEPC cell lines, LASCPC-01 and NCI-H660. In this study, we generated PC3 cell line that stably expresses miR-28 for understanding its functional role in regulating PCa. Preliminary functional assays including cell viability assay, cell cycle studies and invasion and migration assay suggest a tumor suppressor role of miR-28-3p in advanced prostate cancer. In conclusion, our data shows that miR 28-3p potentially has a biphasic role in PCa, where it is oncogenic in early stages and acts as a tumor suppressor in late stage PCa. Citation Format: Amritha Sreekumar, Sharanjot Saini. Role of microRNA 28-3p in prostate cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3783.

RAW 264.7 Macrophage Cell Line: In Vitro Model for the Evaluation of the Immunomodulatory Activity of Zingiberaceae

The immune response plays an essential role in the body's defense against infection. Macrophages are promising targets against which to screen agents that modulate immune responses. In vitro analysis, primarily using cell lines, is preferred to screen bioactivity initially. RAW 264.7 cells comprise a model macrophage cell line close to primary murine macrophages. Indonesian people often use the Zingiberaceae family or “empon-empon" in the favorable treatment of several diseases, including immune disorders. This review highlights several studies of Zingiberaceae using RAW 264.7 cells, focusing on the observed immunomodulatory activity and assay methods. The research on RAW 264.7 macrophage cell line in Zingiberaceae was gathered using Scopus, PubMed, ScienceDirect, and Google Scholar from 2012-2021. Based on the reviews, the Zingiberaceae family has immunomodulatory effects by increasing or decreasing inflammatory mediators such as NO, IL-6, TNF-α, IL-1β, IL-10, and phagocytosis activity in RAW 264.7 cells. Several methods can assess the activation of RAW 264.7 cells, including cell viability assays using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT); measuring nitric oxide production (Griess reaction), cytokine production (enzyme-linked immunosorbent assay [ELISA]), and phagocytosis (neutral red uptake assay); and detecting DNA (real-time quantitative polymerase chain reaction [qPCR]). The information provided in this review presents avenues for future investigations of the immunomodulatory activity of Zingiberaceae.

Targeted PEGylated Chitosan Nano-complex for Delivery of Sodium Butyrate to Prostate Cancer: An In Vitro Study.

Introduction: Cancer remains a challenging issue against human health throughout the world; As a result, introducing novel approaches would be beneficial for cancer treatment. In this research, sodium butyrate (Sb) is one of the effective anti-cancer therapeutics (also a potent survival factor for normal cells) that was used for prostate cancer suppression in the platform of modified chitosan (CS) nano-complex (polyethylene glycol (PEG)-folic acid (FA)-Sb-CS). Methods: Different analytical devices including Fourier transform infrared, dynamic light scattering, high-performance liquid chromatography, scanning electron microscopy, and transmission electron microscopy were applied for the characterization of synthetics. On the other hand, biomedical tests including cell viability assay, molecular and functional assay of apoptosis/autophagy pathways, and cell cycle arrest analysis were potentially implemented on human PC3 (folate receptor-negative prostate cancer) and DU145 (folate receptor-positive prostate cancer) and HFF-1 normal cell lines. Results: The quality of the syntheses was effectively verified, and the size range from 140 to 170 nm was determined for the PEG-CS-FA-Sb sample. Also, 75 ± 5% of drug entrapment efficiency with controlled drug release manner (Sb release of 54.21% and 74.04% for pHs 7.4 and 5.0) were determined for nano-complex. Based on MTT results, PEG-CS-FA-Sb has indicated 72.07% and 33.53% cell viability after 24 h of treatment with 9 mM on PC3 and DU145 cell lines, respectively, which is desirable anti-cancer performance. The apoptotic and autophagy genes overexpression was 15-fold (caspase9), 2.5-fold (BAX), 11-fold (ATG5), 2-fold (BECLIN1), and 3-fold (mTORC1) genes in DU145 cancer cells. More than 50% of cell cycle arrest and 45.05% of apoptosis were obtained for DU145 cancer cells after treatment with nano-complex. Conclusion: Hence, the synthesized Sb-loaded nano-complex could specifically suppress prostate cancer cell growth and induce apoptosis and autophagy in the molecular and cellular phases.

Anti-cancer effects of ginsenoside CK on acute myeloid leukemia in vitro and in vivo

ObjectivesAcute myeloid leukemia (AML) is a malignant disease characterized by clonal proliferation of myeloid cells, and its treatment continues to be a challenge due to high morbidity and mortality. Ginsenoside compound K, a major active metabolite of the protopanaxadiol-type ginsenosides, exhibits biological activities in various cancer cells and animal models. Here, we investigated the role of CK in anticancer potential in AML both in vitro and in vivo. Materials and methodsTo investigate the inhibitory effects of CK in AML cells, in vitro experiments, including cell viability assays, colony forming assays, and cell cycle and apoptosis assays were performed. AML animal experiment was established and quantitative analysis of lung tumor growth nodules and spleen weight and H&E staining were carried out to further determine the effects of CK on AML. In addition, the potential key genes induced and influenced by CK during treatment was identification by RNA-seq and qRT-PCR. ResultsCK suppressed AML cell activity and induced apoptosis and G1 cell cycle arrest based on the experiment results. Moreover, significantly down-regulated expression genes of BCL2, KIT, DNMT3A, MYC and CSF-1 and up-regulated expression gene of TET2 in CK treatment AML cells were discovered. ConclusionOur results demonstrated that CK could be used as an anti-AML drug with significant therapeutic efficacy and good biosafety.

β-Arrestin2-biased Drd2 agonist UNC9995 alleviates astrocyte inflammatory injury via interaction between β-arrestin2 and STAT3 in mouse model of depression

BackgroundMajor depressive disorder (MDD) is a prevalent and devastating psychiatric illness. Unfortunately, the current therapeutic practice, generally depending on the serotonergic system for drug treatment is unsatisfactory and shows intractable side effects. Multiple evidence suggests that dopamine (DA) and dopaminergic signals associated with neuroinflammation are highly involved in the pathophysiology of depression as well as in the mechanism of antidepressant drugs, which is still in the early stage of study and well worthy of investigation.MethodsWe established two chronic stress models, including chronic unpredictable mild stress (CUMS), and chronic social defeat stress (CSDS), to complementarily recapitulate depression-like behaviors. Then, hippocampal tissues were used to detect inflammation-related molecules and signaling pathways. Pathological changes in depressive mouse hippocampal astrocytes were examined by RNA sequencing. After confirming the dopamine receptor 2 (Drd2)/β-arrestin2 signaling changes in the depressive mice brain, we then established the depressive mouse model using the β-arrestin2 knockout mice or administrating the β-arrestin2-biased Drd2 agonist to investigate the roles. Label-free mass spectrometry was used to identify the β-arrestin2-binding proteins as the underlying mechanisms. We modeled neuroinflammation with interleukin-6 (IL-6) and corticosterone treatment and characterized astrocytes using multiple methods including cell viability assay, flow cytometry, and confocal immunofluorescence.ResultsDrd2-biased β-arrestin2 pathway is significantly changed in the progression of depression, and genetic deletion of β-arrestin2 aggravates neuroinflammation and depressive-like phenotypes. Mechanistically, astrocytic β-arrestin2 retains STAT3 in the cytoplasm by structural combination with STAT3, therefore, inhibiting the JAK–STAT3 pathway-mediated inflammatory activation. Furtherly, pharmacological activation of Drd2/β-arrestin2 pathway by UNC9995 abolishes the inflammation-induced loss of astrocytes and ameliorates depressive-like behaviors in mouse model for depression.ConclusionsDrd2/β-arrestin2 pathway is a potential therapeutic target for depression and β-arrestin2-biased Drd2 agonist UNC9995 is identified as a potential anti-depressant strategy for preventing astrocytic dysfunctions and relieving neuropathological manifestations in mouse model for depression, which provides insights for the therapy of depression.

GABRP promotes CD44s-mediated gemcitabine resistance in pancreatic cancer.

BackgroundPancreatic ductal adenocarcinoma (PDAC) has the worst five-year overall survival rate among all cancer types. Acquired chemoresistance is considered one of the main reasons for this dismal prognosis, and the mechanism of chemoresistance is unknown.MethodsWe previously identified a subpopulation of chemoresistant CD44high-expressing PDAC cells. Subsequently, we selected the candidate gene, gamma-aminobutyric acid receptor subunit Pi (GABRP), from three Gene Expression Omnibus datasets as the potential CD44 downstream target mediating the gemcitabine resistance. Loss and gain of function such as stable knockdown of CD44 by small hairpin (sh) RNA-mediated silencing technique and overexpression (O/E) of CD44s had been studied for comparing the gemcitabine resistance among CD44high-expressing cells, shCD44 cells, CD44low-expressing cells and O/E CD44s expressing cells. Functional assays including cell viability, colony formation, invasion, quantitative PCR and western blotting techniques were performed to validate the roles of CD44 and GABRP playing in mediating the gemcitabine resistance in pancreatic cancer cells.ResultsCD44s depletion significantly reduced gemcitabine resistance in shCD44 single clone cells compared to CD44high-expressing cells. Knockdown of CD44 cells formed less colonies, became less invasive and remarkably decreased the mRNA level of GABRP. While overexpression of CD44s had the opposite effect on gemcitabine resistance, colony formation and invasive property. Of note, long term gemcitabine resistant pancreatic cancer cells detected increased expression of CD44 and GABRP. Clinically, GABRP expression was significantly upregulated in the tissues of patients with pancreatic cancer compared to the normal samples, and the overall survival rate of patients with low GABRP expression was longer. CD44 and GABRP co-expression was positively correlated in 178 pancreatic cancer patients.ConclusionOur findings suggest that GABRP may serve as a CD44s downstream target to diminish gemcitabine resistance in pancreatic cancer, and both CD44s and GABRP molecules have the potential to become prognostic biomarkers for PDAC patients with gemcitabine resistance.

Human tau fibrillization and neurotoxicity in the presence of magnesium oxide nanoparticle fabricated through laser ablation method

In this study, the acceleratory effect of magnesium oxide nanoparticles (MgO NPs) on the amyloid fibrillization of human tau protein, a major protein involved in the onset of Alzheimer’s disease (AD) was investigated. The MgO NPs were fabricated through laser ablation synthesis in solution (LASiS), well-characterized, and explored further for tau aggregation and relevant neurotoxicity by different assays. The results showed that the MgO NPs have a size of around 30 nm, a hydrodynamic radius of 57.09 nm, and a zeta potential of −18.06 mV. The data from ThT and ANS fluorescence-based assays along with circular dichroism (CD) spectroscopy clearly indicated that MgO NPs could significantly promote tau fibrillization, concentration-dependently. Considering the acceleratory effect of MgO NPs against tau fibrillization, cellular assays including cell viability, reactive oxygen species (ROS), and caspase-3 assays indicated that the neurotoxicity of tau amyloid fibrils formed with MgO NPs was higher than that of tau samples aged alone against N2a neuron-like cells. Therefore, it was concluded that the interaction of MgO NPs with tau can lead to acceleration of tau aggregation and underlying neurotoxicity. This study, then can provide useful information about the direct effect of MgO NPs against memory proteins and subsequent adverse effects.

Small Molecule Anticancer Compound Modulates Cell Cycle DNA Damage Response Pathway and Inhibit Tumorigenesis in Triple Negative Breast Cancer

ObjectiveTo investigate the mechanism of action and therapeutic benefits of NSC001, a small molecule anticancer compound, in the treatment of triple negative breast cancer (TNBC), a clinical subtype of breast cancer that is more aggressive, has poor prognosis. In TNBC, patients harbor tumors that lack expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Her2).HypothesisWe hypothesize that NSC001 will have a beneficial therapeutic effect on TNBC cells through the regulation of specific tumor associated signaling pathways in vitro.Material and methodsWe screened 1360 compounds from the National Cancer Institute (NCI) Diversity and Mechanistic Set library using the cell counting kit‐8 (CCK‐8) reagent from Dojindo Molecular Technologies, Inc. The screening was performed by applying 10µM of each candidate drug upon plates harboring the aggressive TNBC cell line, MDA‐MB‐231. Treatment time for all screening was fixed at 24 hours. The efficacy and potential mechanism of action of one of the potent drug candidates identified (NSC001) was further investigated on three TNBC cell lines (MDA‐MB‐231, MDA231‐LM2‐4175, and MDA‐MB‐468) using mass spectrometry and proteomic analysis. Afterwards, the results from the proteomic analysis were validated using molecular methods including cell viability assays, apoptosis assays, cell cycle assays, and western blotting.ResultsWe identified eight compounds that reduced the viability of MDA‐MB‐231 cells by up to 80% following a 24 hour treatment time point. Next, our in vitrodata show that NSC001 reduces the cell viability of TNBC cells in a dose‐and‐time dependent manner. Cell death data show that NSC001 induces potent apoptosis in a dose dependent manner in TNBC cells. Also, the results from colony formation assay show that 100nM [NSC001] is sufficient to inhibit the proliferative capacity of TNBC cells. Mass spectrometry and proteomic data reveals that NSC001 activates the cell cycle G2/M DNA damage checkpoint regulation in all the three TNBC cell lines used. Furthermore, we confirmed this finding in our cell cycle assay where we observed a clear G2/M cell cycle phase arrest. The arrest was potently induced at a dose significantly lower than the IC50 of NSC001. Lastly, we validated our proteomic results using western blotting, which confirmed that this low dose of NSC001 induces expression of p21, a cell cycle checkpoint regulator and tumor suppressor protein which is known to trigger cell cycle arrest in most cancer cell lines.ConclusionOverall, our data suggest that NSC001 may be a potent small molecule anticancer compound which should be further investigated for its potential to be used clinically in the treatment of TNBC patients.