SiRNA Knocking Down in HepG2 Cells Identifies PFKFB4 and HNF4α as Key Genes Important for Cancer Cell Survival.

Ovarian cancer is one of the leading causes of cancer death for women throughout the Western world. Kaempferol, a natural flavonoid, has shown promise in the chemoprevention of ovarian cancer. A common concern about using dietary supplements for chemoprevention is their bioavailability. Nanoparticles have shown promise in increasing the bioavailability of some chemicals. Here we developed five different types of nanoparticles incorporating kaempferol and tested their efficacy in the inhibition of viability of cancerous and normal ovarian cells. We found that positively charged nanoparticle formulations did not lead to a significant reduction in cancer cell viability, whereas nonionic polymeric nanoparticles resulted in enhanced reduction of cancer cell viability. Among the nonionic polymeric nanoparticles, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nanoparticles incorporating kaempferol led to significant reduction in cell viability of both cancerous and normal cells. Poly(DL-lactic acid-co-glycolic acid) (PLGA) nanoparticles incorporating kaempferol resulted in enhanced reduction of cancer cell viability together with no significant reduction in cell viability of normal cells compared with kaempferol alone. Therefore, both PEO-PPO-PEO and PLGA nanoparticle formulations were effective in reducing cancer cell viability, while PLGA nanoparticles incorporating kaempferol had selective toxicity against cancer cells and normal cells. A PLGA nanoparticle formulation could be advantageous in the prevention and treatment of ovarian cancers. On the other hand, PEO-PPO-PEO nanoparticles incorporating kaempferol were more effective inhibitors of cancer cells, but they also significantly reduced the viability of normal cells. PEO-PPO-PEO nanoparticles incorporating kaempferol may be suitable as a cancer-targeting strategy, which could limit the effects of the nanoparticles on normal cells while retaining their potency against cancer cells. We have identified two nanoparticle formulations incorporating kaempferol that may lead to breakthroughs in cancer treatment. Both PEO-PPO-PEO and PLGA nanoparticle formulations had superior effects compared with kaempferol alone in reducing cancer cell viability.

Insulinoma-associated 1 (INSM1) is neuroendocrine (NE) transcription factor which temporal and spatial expression pattern is restricted to regions undergoing NE differentiation during embryogenesis. INSM1 is however re-expressed at high levels in small-cell lung cancer (SCLC) together with an array of other NE markers. The NE signature of SCLC is a central diagnostic tool for the disease but recent data pinpoints that this distinct signature might play a role in SCLC pathogenesis. The functional role of INSM1 in SCLC was here investigated by shRNA-mediated silencing of INSM1 in a panel of SCLC cell lines. Upon silencing of INSM1, significant reduction in cell viability was observed as measured by MTT assay. This reduction in cell viability was concomitant with decreased cell proliferation and increased apoptosis as measured by BrdU incorporation and cleaved caspase 3/7 levels, respectively. Accordingly with phenotypic growth changes it was demonstrated that INSM1 silencing caused reduced phosphorylation of members of the growth- and survival-promoting PI3K/Akt, MAPK and JAK-STAT signaling pathways and reduced levels of the inhibitors of apoptosis proteins (IAPs) members c-IAP1, XIAP and survivin. Importantly, it was shown that INSM1 is an upstream regulator of signatures previously shown to play a role in SCLC tumorigenesis, including achate-scute homolog 1 (ASCL1) and Sonic Hedgehog (SHh) pathway. Upon silencing of INSM1, a decrease in mRNA and protein levels of ASCL1 and downstream targets aldehyde dehydrogenase 1 (ALDH1) and delta-like ligand 3 (DLL3) was observed. Furthermore, mRNA levels of the SHh positive mediators Gli2, Gli3 and Smoothened was downregulated while the SHh inhibitor Patched was upregulated upon INSM1 silencing. Futhermore, overexpression of the Notch1 intracellular domain in SCLC cells resulted in a significant reduction in INSM1 expression and cell viability. This suggest that a master inhibitor of the NE signature, Notch1 signaling, could be an inhibitor of INSM1-driven tumorigenesis. In conclusion, INSM1 is a potential key driver of growth- and survival-promoting pathways in SCLC and warrants further investigation of INSM1 as a therapeutic target. Citation Format: Camilla L. Christensen, Takeshi Shimamura, Esra A. Akbay, Signe R. Michaelsen, Hans S. Poulsen, Kwok-kin Wong. Insulinoma-associated 1 is a key transcriptional regulator of growth and survival-promoting pathways in small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2013-3126

Cerebral vascular deposition of the amyloid-β (Aβ) peptide, a condition known as cerebral amyloid angiopathy (CAA), is associated with intracerebral hemorrhaging and contributes to disease progression in Alzheimer's disease (AD) and vascular cognitive impairment and dementia (VCID). Familial mutations at positions 22 and 23 within the Aβ peptide lead to early onset and severe CAA pathology. Here, we evaluate the effects of fibrillar Aβ peptides on the viability of primary-cultured human cerebral smooth muscle (HCSM) cells, which are the major site of amyloid deposition in cerebral blood vessel walls. Comparisons are made of the familial E22Q (Dutch) mutant of Aβ40 with wild-type Aβ40 and Aβ42. In agreement with previous studies, we find that there is a significant reduction in cell viability when Aβ40-Dutch or Aβ42-WT peptides are added to HCSM cell cultures as monomeric Aβ, whereas Aβ40-WT is relatively nontoxic. The binding of Aβ fibrils derived from sporadic CAA or familial Dutch-type CAA brain tissue to the membrane surface of HCSM cells does not result in a significant loss of cell viability. In contrast, when Aβ40-WT monomers and sporadic CAA fibrils are coincubated in HCSM cell cultures, there is a significant reduction in HCSM cell viability that is accompanied by an increase in cell surface fibril formation. Lastly, intrathecal administration of Aβ40-Dutch fibrillar seeds promotes fibrillar amyloid accumulation in the smooth muscle of meningeal vessels in the rTg-D transgenic rat model of CAA. Together, the present findings suggest that fibrillar Aβ seeds propagate the expansion of new amyloid fibrils on cerebral vascular smooth muscle, leading to membrane disruption and cell death.

Children with Group 3 medulloblastoma (G3 MB) have a very poor prognosis, and many do not survive beyond 5 years after diagnosis. A factor that may contribute to this is the lack of available targeted therapy. Expression of protein lin‐28 homolog B (LIN28B), a regulator of developmental timing, is upregulated in several cancers, including G3 MB, and is associated with worse survival in this disease. Here, we investigate the role of the LIN28B pathway in G3 MB and demonstrate that the LIN28B–lethal‐7 (let‐7; a microRNA that is a tumor suppressor)–lymphokine‐activated killer T‐cell‐originated protein kinase (PBK; also known as PDZ‐binding kinase) axis promotes G3 MB proliferation. LIN28B knockdown in G3‐MB‐patient‐derived cell lines leads to a significant reduction in cell viability and proliferation in vitro and in prolonged survival of mice with orthotopic tumors. The LIN28 inhibitor N‐methyl‐N‐[3‐(3‐methyl‐1,2,4‐triazolo[4,3‐b]pyridazin‐6‐yl)phenyl]acetamide (1632) significantly reduces G3 MB cell growth and demonstrates efficacy in reducing tumor growth in mouse xenograft models. Inhibiting PBK using HI‐TOPK‐032 also results in a significant reduction in G3 MB cell viability and proliferation. Together, these results highlight a critical role for the LIN28B–let‐7–PBK pathway in G3 MB and provide preliminary preclinical results for drugs targeting this pathway.

Objectives Targeted inhibitors of the PI3 kinase (PI3K) pathway have shown promising but incomplete antitumor activity in preclinical chordoma models. The aim of this study is to advance methodology for a high-throughput drug screen using chordoma models to identify new combination therapies for chordoma. Study Design Present work is an in vitro study. Setting The study conducted at an academic research laboratory. Materials and Methods An in vitro study on automated high-throughput screening of chordoma cells was performed using a library of 1,406 drugs as both mono- and combination therapies with PI3K inhibitors. Combination indices were determined for dual therapies and synergistic outliers were identified as potential therapeutic agents. T (brachyury) siRNA knockdown in combination with PI3K pathway inhibition was also assessed. Results Fifty-nine combination therapies were identified as having potential therapeutic efficacy. Effective combinations included PI3K inhibitors with GSK1838705A (ALK/IGF-1R inhibitor), LY2874455 (VEGFR/FGFR inhibitor), El1 (selective Ezh2 inhibitor), and (-)-p-bromotetramisole oxalate (alkaline phosphatase inhibitor). The top ranking targets identified included ALK, PDGFR, VEGFR, aurora kinase, and BCL-2. T (brachyury) inhibition produced significant reduction in cell viability and growth; however PI3K inhibition in combination with T (brachyury) knockdown did not result in further reduction in growth and viability in vitro. Conclusion High throughput with in vitro combination screening is feasible with chordoma cells and allows for rapid identification of synergistic dual-therapies. Potential combination therapies and targetable pathways were identified. T (brachyury) knockdown produced significant reduction in cell viability, but did not show additional benefit with PI3K pathway inhibition in this model. Further in vitro and in vivo validation of these therapeutic combinations is warranted.

Costus speciosus is a medicinal plant commonly known as wild ginger distributed in South and Southeast Asian countries. Leaves of this plant are used for ayurvedic treatment regimes in malignancies and mental illness. Rhizome extract from the plant is used to treat malignancies, pneumonia, urinary disorders, jaundice, rheumatism, and diabetes. The goal of this study was to investigate the effects of methanol extract of leaves of C. speciosus on the growth of human hepatocellular carcinoma (HepG2) cells and understand possible mechanisms of its action. Viability of HepG2 cells were measured by MTS assay after 24 h and 48 h treatment with extracts of 1, 10, 50, 100, and 200 μg/mL concentrations. Cell cycle analysis and apoptosis were evaluated by flow cytometry and caspase-3 induction. HepG2 cells treated with 100 μg/mL methanol leaf extract for 24 h displayed a significant reduction in cell viability (P ≤ 0.05). The methanol extract perturbed cell cycle progression, modulated cell cycle and regulated, signal molecules were involved in induction of apoptosis in HepG2 cells. Our findings indicate that phytochemicals of leaves of C. speciosus shows potential for natural therapeutic product development for hepatocellular carcinoma. This is the first report to demonstrate in vitro anticancer activity of leaf extract of C. speciosus in relation to liver cancer.

Hepatology Basic Science

Hepatocellular carcinoma (HCC) has attracted global and local interest, with alarming current and forecasted statistics. This is mainly due to its late-stage diagnosis, high recurrence rate and potential resistance to conventional therapies. Thus, there is an emerging need to explore new molecular targets and novel treatment strategies to effectively combat HCC. In this study, we analyzed four GEO datasets (GSE112790, GSE62232, GSE60502 and GSE84402) to identify upregulated differentially expressed genes (DEGs) associated with HCC. Enrichment analysis and protein‒protein interaction (PPI) network construction were performed on the overlapping upregulated DEGs to predict and prioritize potential therapeutic targets. Six hub genes (RRM2, TOP2A, CCNB1, CDK1, BIRC5 and PBK) were identified, with RRM2 emerging as the top candidate. The role of RRM2 in HCC was then validated through a literature review, in silico analysis and CRISPR/Cas9-mediated gene disruption, followed by in vitro functional assays in HepG2 cells. RRM2 knockdown resulted in significant reductions in cell viability, proliferation inhibition, migration impairment, G2/M phase cell cycle arrest and apoptosis. Our findings emphasize RRM2 as a critical player in HCC progression. Targeting RRM2 with CRISPR/Cas9 effectively reversed several hallmarks of HCC, offering new opportunities for the development of precise HCC therapeutic options.Graphical

We investigated the possible anticancer mechanisms of Pteris vittata [PV] n-hexane extract on MCF-7 [breast cancer cell line]. Cultured cell lines were treated with various concentrations of this extract ± Baf-A1 [autophagic inhibitor]. Cells’ viability, apoptotic markers [caspase-7, Bax, and Bcl-2], autophagic markers [light chain 3 [LC-3] and P62/SQSTM1]], and the tumor suppressor P53 and its mRNA were checked by their corresponding methods. Treated cell lines showed significant concentration and time-dependent reductions in cell viability in response to PV-n-hexane extract and also exhibited a concomitant induction of apoptosis [increased chromatin condensation, nuclear fragmentation, and pro-apoptotic Bax, and cleaved caspase-7 levels while decreased Bcl-2 levels] and autophagy [increased autophagosomes vacuoles, and LC3B II levels while decreased P62/SQSTM1 levels]. Moreover, PV-n-hexane extract-treated cells showed significant increases in the P53 and its mRNA levels. The addition of Baf-A1 reversed the PV-n-hexane extract autophagic effects and increased apoptotic cell percentage with a much increase in the cleaved caspase-7 and P53 protein and its mRNA levels. We concluded that the PV-n-hexane extract exhibits cytotoxic effects on the MCF-7 cell line with significant reductions in cell viability and concomitant autophagy and apoptosis induction. Inhibition of autophagy in the PV-treated MCF-7 cells enhances apoptosis via a p35-dependent pathway.

Purpose: The aim of this study was to access the safety profiles of 2 fusion proteins with anti-vascular endothelial growth factor action (ziv-aflibercept and aflibercept) on retinal pigment epithelium cells and Muller-Glia cells in culture by assessing cell viability post drug exposure. Methods: Primary human retinal pigment epithelium cells (pRPE) and Muller-Glia cells (Mio-M1) were exposed to the clinical standardized concentrations of ziv-aflibercept (25 mg/mL) and aflibercept (40 mg/mL). Progressively higher concentrations of NaCl (300, 500, 1,000, 1,500, 2,000, 5,000, and 10,000 mosm/kg) were also applied to cells to assess the possibility of potentiating hyperosmotic cytotoxity effect. The study was applied to measure pRPE and Mio-M1 viability by a tetrazolium dye-reduction assay (XTT). Results: Cell viability of both pRPE and Mio-M1 presented no significant changes after exposure of ziv-aflibercept and aflibercept. Progressive NaCl concentrations did not significantly alter cell viability. The exposure to the negative control of 75 µL/mL of dimethyl sulfoxide showed significant reduction in cell viability. Conclusions: At clinical doses, neither ziv-aflibercept nor aflibercept caused any significant reduction in cell viability in vitro. Furthermore, injection solutions of NaCl with higher osmolality caused no significant reduction in cell viability.

Laser-induced photothermal therapy using gold nanoparticles (AuNPs) has emerged as a promising approach to cancer therapy. However, optimizing various laser parameters is critical for enhancing the photothermal conversion efficacy of plasmonic nanomaterials. In this regard, the present study investigates the photothermal effects of dodecanethiol-stabilized hydrophobic ultrasmall spherical AuNPs (TEM size 2.2 ± 1.1 nm), induced by a 343 nm wavelength ultrafast femtosecond-pulse laser with a low intensity (0.1 W/cm2) for 5 and 10 min, on the cell morphology and viability of human hepatocellular carcinoma (HepG2) cells treated in vitro. The optical microscopy images showed considerable alteration in the overall morphology of the cells treated with AuNPs and irradiated with laser light. Infrared thermometer measurements showed that the temperature of the cell medium treated with AuNPs and exposed to the laser increased steadily from 22 °C to 46 °C and 48.5 °C after 5 and 10 min, respectively. The WST-1 assay results showed a significant reduction in cell viability, demonstrating a synergistic therapeutic effect of the femtosecond laser and AuNPs on HepG2 cells. The obtained results pave the way to design a less expensive, effective, and minimally invasive photothermal approach to treat cancers with reduced side effects.

CRLF2 Rearrangement Status in Ph-like ALL Predicts Intrinsic Glucocorticoid Resistance In Vitro that is Reversible with Targeted MAPK and PI3K Pathway Inhibition

This study introduces advanced nanoparticle-based drug delivery systems (NDDS) designed for targeted colorectal cancer treatment. We developed and characterized three distinct formulations: Bevacizumab-loaded chitosan nanoparticles (BEV-CHI-NP), polymeric micelles (BEV-PM), and BEV-conjugated exosomes enriched with AS1411 and N1-methyladenosine (AP-BEV + M1A-EXO). Each formulation exhibited optimized physicochemical properties, with particle sizes between 150 and 250nm and surface charges ranging from + 14.4 to + 43mV, ensuring stability and targeted delivery. The AP-BEV + M1A-EXO formulation demonstrated targeted delivery to VEGF, a protein commonly overexpressed in colorectal cancer cells, as indicated by localized staining. This suggests a more precise delivery of the therapeutic agent to VEGF-enriched regions. In contrast, the BEV-CHI-NP formulation exhibited a broader pattern of tumor suppression, evidenced by reduced overall staining intensity. The BEV-PM group showed moderate effects, with a relatively uniform protein expression across tumor tissues. In vivo studies indicated that the AP-BEV + M1A-EXO formulation achieved a notable reduction in tumor volume (~ 65.4%) and decreased levels of tumor biomarkers, including CEA and CA 19-9, compared to conventional BEV-API treatment. In vitro experiments using human colon tumor organoids (HCTOs) further supported these findings, showing a significant reduction in cell viability following exposure to AP-BEV + M1A-EXO. These results suggest that combining aptamer specificity with exosome-based delivery systems could enhance the precision and effectiveness of colorectal cancer therapies, representing a potential advancement in treatment strategies. In vivo experiments further revealed that the AP-BEV + M1A-EXO formulation outperformed conventional BEV-API treatment, achieving a four-fold increase in tumor suppression. This formulation resulted in a 65.4% reduction in tumor volume and a significant decrease in tumor biomarkers, including CEA and CA 19-9. In vitro studies also demonstrated a significant reduction in cell viability in human colon tumor organoids exposed to AP-BEV + M1A-EXO. These findings highlight the potential of combining aptamer specificity with exosome-based delivery systems to enhance the precision and efficacy of colorectal cancer therapies, marking a promising step forward in cancer treatment innovation.

IntroductionJunctional Adhesion Molecule-A (JAM-A) is a transmembrane protein with important physiological functions in regulating cell-cell adhesion. JAM-A levels have been reported to regulate HER2 expression (Brennan et al, Oncogene 2013;32(22):2799–804), thus we hypothesised that JAM-A also regulates expression of HER3 in breast cancer cells. HER3 is the most potent binding partner of HER2 in activating tumour growth signalling, and accumulating evidence suggests that HER3 plays an important role in resistance to anti-HER2 therapies. Furthermore HER3 is frequently overexpressed in HER2-negative breast cancers, and, along with other HER family members, may drive HER2-independent tumourigenic mechanisms.Material and methodsUsing in vitro assays with a panel of breast cell lines, we are investigating whether JAM-A knockdown or over-expression influences expression or activity of FOXA1 and HER3 at mRNA and protein level, and DNA-protein binding assay to show FOXA1/HER3 promoter gene interaction.Results and discussionsResults from our study showed that stable overexpression of JAM-A in MCF7 breast cancer cells (MCF7-JAM) increased both mRNA and protein expression of HER3. Correspondingly, transient gene silencing of JAM-A reduced the mRNA and protein expression of HER3 in MCF7 and SKBR3 cells. In MCF7 cells, JAM-A gene silencing phenocopied that of HER3 gene silencing by reducing protein expression of the HER downstream effectors phospho-AKT and phospho-ERK, in parallel with significant reductions in cell viability (measured by Alamar Blue assay). To begin exploring the mechanism whereby JAM-A regulates HER3 expression, we focused on the HER3 transcription factor FOXA1 following a bioinformatic search. JAM-A gene silencing reduced FOXA1 expression in all cell lines tested; while JAM-A overexpression had the opposite effect in increasing FOXA1 expression. FOXA1 gene silencing was sufficient to reduce HER3 expression in the same cells. Furthermore FOXA1 in nuclear extracts of breast cancer cells bound specifically to an oligonucleotide sequence corresponding to the HER3 gene promoter, and gene silencing of JAM-A reduced FOXA1 binding activity to this sequence. Taken together, our data provide novel evidence of a direct relationship between levels of JAM-A, FOXA1 and HER3 in breast cancer cells.ConclusionIn conclusion, we suggest that JAM-A merits investigation as a novel target to inhibit HER3-dependent tumourigenic signalling in breast cancer.

Many epithelial cancers have been shown to overexpress the enzyme cyclooxygenase-2 (COX-2), an enzyme responsible for metabolizing anandamide (AEA) to prostamides. AEA has demonstrated cytotoxicity in COX-2 overexpressing cancers via its metabolism to novel J-series prostamides, namely 15d-PMJ2. Fatty acid amide hydrolase (FAAH) degrades AEA into arachidonic acid and ethanolamine (EA), limiting the cytotoxic capability of AEA. Cell lines with high FAAH expression have demonstrated resistance to AEA. By understanding the metabolic characteristics of FAAH, we can design AEA analogs which circumvent FAAH breakdown. To examine the effects of altering polarity, steric bulk, and functional groups on AEA-mediated cytotoxicity, we investigated known AEA derivatives which possess these properties. Arvanil, Arachidonoyl Diethanolamine (ADA), Arachidonoyl Serinol (AS), and R1-methanandamide (m-AEA) add steric bulk to the molecule via aromatic rings, extra EA arms and additional alcohol/methyl functional groups respectively. Arachidonoyl glycine (NAGly) substitutes the terminal EA alcohol with a carboxylic acid increasing polarity. Arachidonoyl-2’-chloroethylamine (AC) substitutes the terminal EA alcohol with a highly soluble chlorine. Furthermore, it is known that Arvanil, ADA, AS, and m-AEA resist degradation of the molecule by FAAH and NAGly is a known substrate of COX-2. Therefore our goal was to determine which structural modifications improve AEA-mediated cytotoxicity. To determine this, JWF2 tumorigenic keratinocytes were exposed to differing concentrations of the AEA analogs for 24 hours and cell viability was measured by conducting MTS assays. Arvanil demonstrated a 90% reduction in cell viability, NAGly demonstrated a 70% reduction in cell viability, and m-AEA demonstrated a 100% reduction in cell viability at 20 µM, the optimal dosage of AEA. Due to the fact that ADA, AS, and AC did not show a significant reduction in cell viability these molecules were not further examined. MTS assays were conducted in other epithelial cancer cell lines with varying expressions of COX-2 and FAAH. Each cell line was exposed to varying concentrations of either NAGly, Arvanil, or both for 24 hours. NAGly demonstrated a 40% reduction in cell viability in HT-29 colon cancer cells (low COX-2, high FAAH). Arvanil demonstrated a 60% reduction in A431 tumorigenic keratinocytes. NAGly demonstrated a 60% reduction in cell viability and Arvanil demonstrated a 100% reduction in cell viability in patient-derived primary melanoma. These findings suggest that modulation and substitute to the core structure of AEA will result in decreased susceptibility to FAAH degradation and enhanced antineoplastic activity. Citation Format: Andrew Morris, Eman Soliman, Rukiyah Van Dross, Colin Burns. Structural modification of the chemotherapeutic anandamide: Designing anti-cancer agents and investigating their COX-2 metabolic products [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2193. doi:10.1158/1538-7445.AM2017-2193