Trypan Blue Exclusion Assay Research Articles

The Role of Insulin-like Growth Factor Binding Protein (IGFBP)-2 in DNA Repair and Chemoresistance in Breast Cancer Cells.

The role if insulin-like growth factor binding protein-2 (IGFBP-2) in mediating chemoresistance in breast cancer cells has been demonstrated, but the mechanism of action is unclear. This study aimed to further investigate the role of IGFBP-2 in the DNA damage response induced by etoposide in MCF-7, T47D (ER+ve), and MDA-MB-231 (ER-ve) breast cancer cell lines. In the presence or absence of etoposide, IGFBP-2 was silenced using siRNA in the ER-positive cell lines, or exogenous IGFBP-2 was added to the ER-negative MDA-MB-231 cells. Cell number and death were assessed using trypan blue dye exclusion assay, changes in abundance of proteins were monitored using Western blotting of whole cell lysates, and localization and abundance were determined using immunofluorescence and cell fractionation. Results from ER-positive cell lines demonstrated that upon exposure to etoposide, loss of IGFBP-2 enhanced cell death, and this was associated with a reduction in P-DNA-PKcs and an increase in γH2AX. Conversely, with ER-negative cells, the addition of IGFBP-2 in the presence of etoposide resulted in cell survival, an increase in P-DNA-PKcs, and a reduction in γH2AX. In summary, IGFBP-2 is a survival factor for breast cancer cells that is associated with enhancement of the DNA repair mechanism.

Pulsed Electromagnetic Field Enhances Caffeic Acid Phenethyl Ester-induced Death of MCF-7 Breast Cancer Cells.

Caffeic acid phenethyl ester (CAPE) exerts anticancer effects against several cancer types, including breast cancer. Pulsed electromagnetic field (PEMF) improves the efficiency of some chemotherapeutic drugs. In this study, we examined the effects of PEMF stimulation on the anticancer activity of CAPE in MCF-7 breast cancer cells and the underlying signal transduction pathways. MCF-7 cells were seeded and incubated for 24 h. Each of the drugs (5-fluorouracil, paclitaxel, gefitinib, or CAPE) was added to the cells on day 0. Then, cells were immediately stimulated with a 60-min PEMF session thrice a day (with 4-h interval between sessions) for 1-3 days. Cell death and viability were assessed by flow cytometry and trypan blue dye exclusion assay. Molecular mechanisms involved in cell death were confirmed by western blot assay. Compared with treatment with CAPE alone, co-treatment with CAPE and PEMF more strongly reduced the viability of MCF-7 cells, further increased the percentage of the sub-G1 population, poly (ADP-ribose) polymerase (PARP) cleavage, activation of apoptotic caspases, up-regulation of pro-apoptotic proteins, such as Fas cell surface death receptor (FAS) and BCL2 associated X, apoptosis regulator (BAX), and reduced the expression of anti-apoptotic proteins, such as BCL-2 apoptosis regulator (BCL-2), MCL-1 apoptosis regulator, BCL-2 family member (MCL-1), and survivin. PEMF stimulation also increased CAPE-induced phosphorylation of p53, and inhibition of p53 partially restored the PEMF-reduced viability of CAPE-treated MCF-7 cells. PEMF stimulation enhanced CAPE-induced cell death by activating p53, which regulates the expression of apoptosis-related molecules, subsequently activating the caspase-dependent apoptotic pathway in MCF-7 cells, suggesting that PEMF can be utilized as an adjuvant to enhance the effect of CAPE on breast cancer cells.

Effect of dose-rate on nitric oxide-induced apoptosis in A375 human melanoma cells

Purpose: To investigate the effect of dose-rate on nitric oxide (NO)-induced apoptosis in A375 human melanoma cells. Methods: The A375 cells were exposed to NO at a steady-state concentration of 7 μM, similar to the level estimated to occur in vivo in inflamed tissues, and delivered by diffusion through silastic tubing. Trypan blue dye exclusion assay was used to assess cell proliferation and viability. Cell cycle was studied by propidium iodide (PI) staining while Annexin V-FITC and PI assays were used to evaluate apoptosis. Western blotting assay was carried out to determine protein levels of p53, Bax, DR4, DR5, Fas (CD95), Procaspase 8, Procaspase 9 and Procaspase 3. Results: Viability of A375 cells was reduced by 22 % after 24 h leading to extensive apoptosis and cell cycle arrest following exposure to a cumulative dose of 3360 μM/min NO. Treatment with NO stimulated p53 and triggered mitochondrial apoptotic events by inducing conformational changes in Bax. Also, activation of caspase 9 and 3, DR4, DR5, Fas (CD95) and caspase 8 appeared to be mediated concurrently by death receptor processing and downstream caspases. Conclusion: Nitric oxide (NO) induces programmed cell death, thus indicating that it could serve as an effective inhibitor to halt the progression of melanoma or as an enhancer to improve therapeutic strategies for treatment.

Agar-tragacanth/silk fibroin hydrogel containing Zn-based MOF as a novel nanobiocomposite with biological activity

In this study, a novel nanobiocomposite consisting of agar (Ag), tragacanth gum (TG), silk fibroin (SF), and MOF-5 was synthesized and extensively investigated by various analytical techniques and basic biological assays for potential biomedical applications. The performed Trypan blue dye exclusion assay indicated that the proliferation percentage of HEK293T cells was 71.19%, while the proliferation of cancer cells (K-562 and MCF-7) was significantly lower, at 10.74% and 3.33%. Furthermore, the Ag-TG hydrogel/SF/MOF-5 nanobiocomposite exhibited significant antimicrobial activity against both E. coli and S. aureus strains, with growth inhibition rates of 76.08% and 69.19% respectively. Additionally, the hemolytic index of fabricated nanobiocomposite was found approximately 19%. These findings suggest that the nanobiocomposite exhibits significant potential for application in cancer therapy and wound healing.

Cordyceps militaris: A Comprehensive Study on Laboratory Cultivation and Anticancer Potential in Dalton's Ascites Lymphoma Tumor Model.

Cancer, a predominant cause of mortality, poses a formidable challenge in our pursuit of elevating life expectancy. Throughout history, individuals have sought natural remedies with minimal side effects as an appealing substitute for chemotherapeutic drugs. One such remedy is Cordyceps militaris, a renowned medicinal mushroom deeply entrenched in Asian ethnomedicine. Revered for its rejuvenating and curative attributes, it relied upon for ages. The mushroom's soaring demand outpaced natural availability, necessitating controlled laboratory cultivation as the core focus and exploring the potential of methanolic extracts from harvested Cordyceps militaris fruiting bodies against Dalton's Lymphoma Ascites (DLA) cells in vitro, with a specific emphasis on its anticancer traits. For cultivation, we employed a diverse range of rice substrates, among which bora rice showed promising growth of C. militaris fruiting bodies. To assess DLA cell cytotoxicity, several assays, including trypan blue exclusion assay, MTT assay, and LDH assay, were employed at different time points (24-96 h), which provided valuable insights on DLA cell viability and proliferation, shedding light on its therapeutic potential against cancer. Our studies unveiled that methanolic extract prompts apoptosis in DLA cells via AO/EB dual staining, manifesting consistent apoptosis indicators such as membrane blebbing, chromatin condensation, nuclei fragmentation, and cellular shrinkage at 48-96 h of treatment. Furthermore, these striking repercussions of apoptosis were comprehended by an in silico approach having molecular docking simulation against antiapoptotic proteins like BCL-2, BCL-XL, MCL-1, BFL-1 & HSP100. Methanolic C. militaris extracts exhibited cytotoxicity and apoptotic alterations in DLA cells.

Parkin Enhances Gefitinib-induced Anoikis in HeLa Cervical Cancer Cells.

Gefitinib exhibits anticancer activity against cervical cancer cells via anoikis, a type of apoptosis induced by cell detachment from the extracellular matrix. Previous studies have reported that Parkin expression affects the efficacy of anticancer drugs. However, the impact of Parkin expression on the therapeutic effects of gefitinib in human cervical cancer remains unclear. Thus, this study aimed to evaluate whether Parkin over-expression improves the therapeutic effects of gefitinib against HeLa cervical cancer cells. Cell viability and apoptotic death of HeLa cells were measured by trypan blue dye exclusion assay and flow cytometry. Cell detachment, adhesion, spreading, and cell-cell interaction were observed by inverted microscopy. Alteration of adhesion-related molecules was evaluated by confocal microscopy and western blot assay. Parkin expression potentiated gefitinib-induced cell detachment by affecting the organization of the actin cytoskeleton. In addition, Parkin expression induced a further reduction in the reattachment of and interaction between detached cells. The therapeutic efficacy of low-dose gefitinib combined with Parkin expression was equivalent to that of high-dose gefitinib alone. Parkin expression promotes gefitinib-induced anoikis, consequently increasing the efficacy of gefitinib against HeLa human cervical cancer cells. Based on our results, we propose that Parkin can be used to increase the anti-cancer effect of gefitinib on cervical cancer cells.

Anticancer Activity and GC-MS Profiling of Bioactive Constituents in the Methanolic Extracts of Spatoglossum variabile and Gracilaria corticata

Background: Marine algae stand out as repositories of bioactive metabolites, widely harnessed in treating diverse diseases. Given the often-challenging side effects associated with conventional cancer treatments, researchers are increasingly turning their attention to marine algae as a promising source for discovering novel and potent anticancer compounds. In this study, we meticulously explored the potential anticancer activity of Spatoglossum variabile and Gracilaria corticata sourced from the Indian Ocean. background: Marine algae are rich in bioactive metabolites that are extensively used to treat various diseases. Researchers have considered marine algae for novel and promising anticancer compounds due to the adverse effects of standard cancer treatments Materials and Methods: The secondary metabolites were extracted using methanol solvent according to the standard protocol. The extract was subjected to GC-MS analysis and evaluated for its short- and long-term cytotoxicity and anti-proliferative ability in the HCT116 cell line using MTT, clonogenic, and trypan blue dye exclusion assays, respectively. Results and Discussion: The methanolic extract of Spatoglossum variabile contained 21 unique anticancer compounds, except for the other bioactive compounds. The results indicate that increasing the concentration of algal extracts decreased the cell viability significantly, with an IC50 of 51.49 μg/mL and 37.50 μg /mL in the HCT116 cell line. The results showed a significant reduction in colorectal cancer colony formation and a gradual reduction in cell proliferation. These results underscore the potential for targeted, low-toxicity biomedicines due to their selective cytotoxicity against cancer cells. method: The extract was subjected to GC-MS analysis and evaluated for its short- and long-term cytotoxicity and anti-proliferative ability in HCT116 cell line using MTT, clonogenic, and trypan blue dye exclusion assays, respectively. Conclusion: Our findings show that Spatoglosuum variabile & Gracilaria corticata extracts contain various bioactive compounds, display long-term and short-term cytotoxicity and antiproliferative effects, which indicates that their constituent compounds might be further refined into effective anti-colorectal cancer medications. result: The methanolic extract of Spatoglossum variabile contained 21 unique anti-cancer compounds, in addition to other bioactive compounds. The results indicate that increasing the concentration of algal extracts decreased the cell viability significantly, with an IC50 of 51.49 µg/mL and 37.50 µg /ml in the HCT116 cell line. The results showed a significant reduction in colony formation and a gradual reduction in cell proliferation

Improving the power of drug toxicity measurements by quantitative nuclei imaging

Imaging-based anticancer drug screens are becoming more prevalent due to development of automated fluorescent microscopes and imaging stations, as well as rapid advancements in image processing software. Automated cell imaging provides many benefits such as their ability to provide high-content data, modularity, dynamics recording and the fact that imaging is the most direct way to access cell viability and cell proliferation. However, currently most publicly available large-scale anticancer drugs screens, such as GDSC, CTRP and NCI-60, provide cell viability data measured by assays based on colorimetric or luminometric measurements of NADH or ATP levels. Although such datasets provide valuable data, it is unclear how well drug toxicity measurements can be integrated with imaging data. Here we explored the relations between drug toxicity data obtained by XTT assay, two quantitative nuclei imaging methods and trypan blue dye exclusion assay using a set of four cancer cell lines with different morphologies and 30 drugs with different mechanisms of action. We show that imaging-based approaches provide high accuracy and the differences between results obtained by different methods highly depend on drug mechanism of action. Selecting AUC metrics over IC50 or comparing data where significantly drugs reduced cell numbers noticeably improves consistency between methods. Using automated cell segmentation protocols we analyzed mitochondria activity in more than 11 thousand drug-treated cells and showed that XTT assay produces unreliable data for CDK4/6, Aurora A, VEGFR and PARP inhibitors due induced cell size growth and increase in individual mitochondria activity. We also explored several benefits of image-based analysis such as ability to monitor cell number dynamics, dissect changes in total and individual mitochondria activity from cell proliferation, and ability to identify chromatin remodeling drugs. Finally, we provide a web tool that allows comparing results obtained by different methods.

Differential biological activity of two extracts of ripe fruits of Carica candamarcensis Hook on human neutrophils

We evaluated the effect of the total macerate (TM) and seed oil (SO) of mature Carica candamarcensisfruits, on the release of Matrix metalloproteinase 9 (MMP9) and the phosphorylation of MAPK in neutrophils. The antioxidant capacity of these extracts was evaluated by ABTS assay. Neutrophils stimulated with different dilutions of TM or SO were analyzed for cytotoxicity, MMP9 release, and MAPK phosphorylation, using trypan blue exclusion assays, zymography, and immunoblotting, respectively. Both extracts show antioxidant activity, being higher in TM; none presented cytotoxic effect. The 5% and 2.5% dilutions of TM significantly reduced MMP9 release, and all decreased MAPK phosphorylation. SO significantly increased the release of MMP9 and MAPK phosphorylation, the effect being greater when they were prestimulated with lipopolysaccharide.TM may have anti-inflammatory potential, while SO could have a priming effect that needs to be confirmed.

Comparative Efficacy of the Dimethyl Sulfoxide, Glycerol and Methanol on the Post-Thaw Cell Viability of HeLa Cells

Cryoprotectants are used to protect cells during freezing. The concentration, type, and freeze-thaw conditions of these substances vary depending on the type of cell to be used. It is very important to determine the appropriate cryopreservation method for the particular cell. This study aims to provide insights into the optimal cryopreservation method for HeLa cells by comparing the performance of different cryoprotectants and evaluating their effectiveness under various freezing and storage conditions. Cell suspensions were frozen with a freezing media composed of cryoprotectant + fetal bovine serum + medium at a ratio of 5:10:85 (v:v:v) and stored under the following conditions: 3 months (-20 °C), 1 month (-80 °C), and 6 months (-80 °C). Cell viability and recovery rates were analyzed immediately post-thaw and after 48 h using the trypan blue dye exclusion assay. In 3 months (-20 °C), viability and recovery rates were higher in the methanol group. Glycerol showed better performance in 1 month (-80 °C). DMSO was the most efficient in 6 months (-80 °C). Methanol failed at -80 °C storage temperature. This study demonstrates the effect of these cryoprotectants in HeLa cells on cell viability and cell recovery rates immediately after thawing and after 48 hours of cultivation.

Characterization of a hemolytic and antibiotic-resistant Pseudomonas aeruginosa strain S3 pathogenic to fish isolated from Mahananda River in India.

Virulent strain Pseudomonas aeruginosa isolated from Mahananda River exhibited the highest hemolytic activity and virulence factors and was pathogenic to fish as clinical signs of hemorrhagic spots, loss of scales, and fin erosions were found. S3 was cytotoxic to the human liver cell line (WRL-68) in the trypan blue dye exclusion assay. Genotype characterization using whole genome analysis showed that S3 was similar to P. aeruginosa PAO1. The draft genome sequence had an estimated length of 62,69,783 bp, a GC content of 66.3%, and contained 5916 coding sequences. Eight genes across the genome were predicted to be related to hemolysin action. Antibiotic resistance genes such as class C and class D beta-lactamases, fosA, APH, and catB were detected, along with the strong presence of multiple efflux system genes. This study shows that river water is contaminated by pathogenic P. aeruginosa harboring an array of virulence and antibiotic resistance genes which warrants periodic monitoring to prevent disease outbreaks.

Hesperidin Enhanced the Antimigratory Activity and Senescence-Mediated G2/M Arrest Effect of PGV-1 Against T47D Luminal Breast Cancer Cells

Luminal breast cancer cells exhibit proliferative and metastatic characteristics. Exploration of the effective treatment with minimum side effects is necessary. This study aimed to confirm the combination treatment of a potent anticancer candidate PGV-1 and hesperidin on luminal breast cancer cells, T47D, covering its cytotoxic and anti-migratory activities. PGV-1 showed much stronger cytotoxicity with an IC50 value of 2 μM than that of hesperidin (200 μM) as evaluated by trypan blue exclusion assay, but the combination of the two compounds exhibited a synergistic effect. Propidium iodide (PI) staining with flow cytometry proved that the combination treatment increased the cell population in the G2/M phase. Additionally, the combination treatment increased senescent cells as shown in senescence associated β-galactosidase (SA-β-gal) assay which might correlate to its antiproliferative properties. In addition, the scratch wound healing assay showed that the combination also inhibited cell migration significantly. Molecular docking of the two compounds demonstrated potential interaction with their protein targets in cell cycle machinery, i.e. KIF1, CDK1, TOP2A, CA12, ESR1, FN1, and TYMS. Altogether, these findings strengthen the evidence of anti-cancer properties enhancement of PGV-1 in luminal breast cancer through combining the compound with hesperidin.

Abstract 4633: Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is always diagnosed as stage IV with a 5 year survival rate of less than 3% due to its aggressive metastatic phenotype. ATC lacks differentiation and loses its thyroid-like functions making it a difficult cancer to treat due to its lack of targetable genetic lesions and unresponsiveness to conventional treatments. Therefore, we investigated genes that had aberrant expression in ATC in relation to differentiation. Using NCBI Gene expression omnibus (GSE33630, GSE85457), which provides publicly available transcriptomic datasets, and bioinformatics software, GEO2R, we found that Homeobox D4 (HOXD4) has 12.5FC higher expression in ATC vs. normal tissue (p=0.000552) and 4.36FC higher in ATC vs. papillary thyroid cancer (PTC) in human patients (p=6.69e-08). HOXD4 is a transcription factor that plays a crucial role in cell differentiation and has been associated with a worse prognosis in cancers such as ovarian serous carcinoma. As loss of differentiation is essential to ATC’s refractory nature and therapeutic resistance, we are exploring modulation of differentiation by all-trans retinoic acid (ATRA). ATRA was used to induce differentiation in ATC cell line T238 therefore abrogating the aggressive phenotype of this cancer. T238 treated with ATRA had a 26% decrease in proliferation at 24 hours, 46% at 48 hours, and 87% at 72 hours measured using a trypan blue exclusion assay. T238 treated with ATRA also had a 44% decrease in migratory capacity at 24 hours and 24% at 48 hours measured by a cell scratch wound assay. Immortalized normal thyroid cell line NTHY-ori-3-1 did not have a significant decrease in either proliferation or migratory capacity when treated with ATRA. T238 cells treated with ATRA for 24 hours had a 30% decrease in HOXD4 protein levels. Lastly, immunohistochemistry staining of HOXD4 in human patient tissue samples confirmed the over-expression of HOXD4 in ATC (19.5% staining) vs. normal tissue (2.5%) and PTC (7.5%). Overall, we hypothesize that the aberrant expression of HOXD4 in ATC plays a crucial role in the aggressive metastatic phenotype and therefore differentiation agents such as ATRA can target this biomarker to modulate the ATC phenotype. Therefore, HOXD4 can be used as a potential prognostic indicator for early detection of ATC. Future studies will involve CRISPRi based knockdown of HOXD4 in vitro and validation of these results in animal in vivo models. Citation Format: Kaci Kopec, Tara Jarboe, Nicole R. DeSouza, Humayun K. Islam, Jan Geliebter, Raj K. Tiwari. Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid 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 4633.

Abstract 5982: Cannabinoid derived product is a potential novel therapeutic for papillary thyroid carcinoma

Abstract Papillary thyroid carcinoma (PTC) is the most common thyroid cancer that usually affects women ages 20-50, presenting as an asymptomatic neck mass. Total or partial thyroidectomy shows an excellent prognosis; however, investigation into non-invasive therapeutics with minimal adverse effects is ongoing. In addition to driver mutations such as BRAF, RAS, and RET-PTC, p53 mutations have also been identified. The loss of function of p53 is linked to cancer progression from PTC to poorly differentiated thyroid cancer and possibly anaplastic thyroid cancer. The disruption of the Wnt cell pathway is documented to be related to malignant cell transformation in thyroid cancer, leading to remarkable upregulation of c-myc, resulting in enhancement of tumor growth and drug resistance. Previous studies revealed that upregulation of BCL-2 occurs at later stages of tumorigenesis and is involved in chemotherapy resistance in some forms of cancer. In PTC, the downregulation of BCL-2 is linked to its favorable prognosis. Cannabinoid Derived Products (CDPs) have shown effectiveness in inhibiting different types of cancer, with research dating back to the 1970s. Ongoing in-vitro and in-vivo studies show that CDPs can effectively modulate tumor growth. However, the apoptotic properties of CDPs are largely dependent on the cancer type and drug dose/concentration. Our investigation focuses on CDPs and its potential anti-cancer effects by modulation of gene expression. We investigated its effects on gene expression of p53, c-Myc, and BCL-2 in K1 cell line which consists of PTC cells obtained from metastasis of well-differentiated PTC. K1 PTC cells (1 x106 cells/mL) were cultured with CDPs and incubated at 37°C under 5% CO2 for 24 and 48 hours. Cells were harvested, and cell viability was determined via trypan blue exclusion assay. Using qRT-PCR, we determined the effects of CDPs on the expression of TP53, c-Myc, and BCL-2. Results show that the CDPs decreased the viability (p< 0.001) of K1 PTC cells in a dose and time-dependent manner. Within 24 hours, the cannabinoid-containing product increased the gene expression of TP53 (p < 0.01) and decreased the gene expression of BCL-2 (p< 0.01) and c-Myc (p< 0.05) in K1 PTC cells. The results suggest that CDPs interact as a potential regulator in cancer with the upregulation of p53 and downregulation of BCL-2 and c-Myc. Further in-vitro and in-vivo studies are needed to understand the mechanism and therapeutic potential of cannabinoid-containing products in papillary thyroid cancer. Citation Format: Carolina Taico, Ibrahim Musa, Fariba Ardalani, Anish Maskey, Nan Yang, Joseph Breslin, Raj Tiwari, Jan Geliebter, Xiu Min Li. Cannabinoid derived product is a potential novel therapeutic for papillary thyroid carcinoma [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 5982.

Abstract 1458: Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents a very aggressive type of pancreatic cancer (9% survival rate at 5 years). PDX1 is an important transcription factor for embryonic development of the pancreas, endocrine lineage differentiation and the maintenance of mature beta cells. Notably, in PDAC patients, the expression of PDX1 is downregulated in tumor cells compared to adjacent non-tumoral tissue. Interestingly, increasing tumor PDX1 expression has been shown to enhance the overall survival rate in PDAC patients. Therefore, our aim was to analyze the role of PDX1 on tumor aggressiveness of PDAC cells. To induce PDX1 expression, PANC1 cells were treated with BRD7552 (a PDX1 inducer) for 9 days. Overexpression of PDX1 was confirmed by Western Blot analysis and no cytotoxic effects were observed by MTT or Trypan Blue exclusion assays on treated cells compared to control. Wound healing and transwell migration assays showed a significant reduction in the migration rate compared to the control group. A higher proportion of cells in G1 phase was observed in treated cells compared to control by propidium iodide staining followed by flow cytometry assay. Furthermore, the cell confluence assay showed a significant reduction in proliferation rate in treated cells compared to control, but no differences were observed on the proportion of KI67+ and PH3+ cells by immunostaining. To assess the in ovo effects of BRD7552, treated PANC1 cells were implanted onto the chorioallantoic membrane of chick embryos and tumor growth was measured at different stages observing a significant reduction in tumor growth in PANC1 cells compared to control between days 3 and 8 post implantation. No significant differences in morphology or color were observed between treated and control implanted cells. Hematoxylin-eosin staining showed reduced invasion of treated cells into the chorioallantoic membrane compared to control. In conclusion, the overexpression of PDX1 affects cell cycle, reduces proliferation rate and inhibits migratory potential in vitro. Moreover, in an in ovo model this overexpression diminished tumor growth and invasion in PANC1 cells. In conclusion, the overexpression of PDX1 induced by BRD7552 drives pancreatic ductal adenocarcinoma to a less aggressive tumoral phenotype. Citation Format: Jimena Mosna, Federico Garde, Marcelo Stinson, Candela Pastore, Pablo Sanchis, Abel Carcagno. Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness [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 1458.

Abstract 6643: Hyperthermic induction of immunotherapeutic response in in vitro melanoma model

Abstract Immunotherapy has revolutionized the treatment of advanced melanoma, yet challenges persist with non-responders, development of resistance, and dosage concerns. Our strategy to enhance checkpoint inhibitor efficacy is to combine it with hyperthermia. Moderate hyperthermia, 39-45°C, is an interesting anti-neoplastic therapy which induces production of tumor associated antigens rendering tumor sites more amenable to immunotherapy. To investigate temperature-induced alterations on carcinogenic phenotypes in vitro, B16-F10 melanoma cells were grown at 37°C under normal culture conditions, or incubated at 41°C for 2 hours for moderate, febrile hyperthermic induction. Hyperthermia-induced changes were analyzed by trypan blue exclusion assay, scratch wound assay, collection of conditioned media for cytokine array, and protein isolation for Western Blot. Conditioned media from the hyperthermia-treated group showed an inflammatory shift in cytokine and chemokine expression, with increased TNFa and decreased IL-4 expression. Prior studies found the hyperthermia-induced apoptotic effect is directly correlated to endogenous TNFa levels, elucidating a potential molecular mechanism. Treatment for two hours at 41°C decreased proliferation of B16-F10 cells by 62% after 48 hours and 94% after 72 hours. Additionally, hyperthermia decreased cellular migration by 70% after 24 hours. Further evaluation of cellular interactions in important pathways responsible for these hyperthermia-induced changes were investigated via Western blot. Expression of constitutively active MAPK signaling cascade effectors pERK and ERK was decreased by 86% and 50%, respectively, helping to regulate pro-tumorigenic proliferative signaling. Some cancer cells have been demonstrated to resist hyperthermia via mechanisms to inhibit induction of caspase-3, however, in our model caspase-3 expression increased by 31% following 41°C treatment, thus allowing for induction of apoptosis via caspase-3 in this model. Cellular stress may induce these cell death pathways, as well as heat shock responses. Following febrile hyperthermic induction, hsp70 expression increased by 188% compared to cells cultured at 37°C. Hsp70 has a well-established role in supporting tumor-specific immune responses. Induction of hyperthermia in combination with anti-PDL1, anti-PD-1, or IL-15 immunotherapy is currently being evaluated in a C57BL/6 mouse model by our group. These studies aim to assess if hyperthermia augments response to immunotherapies in vivo. Our current works suggests that moderate, non-ablative hyperthermia helps initiate anti-tumor immune responses, assisting in the induction of anti-tumorigenic cell programs and, potentially, the production of additional tumor associated antigens, thereby making it an interesting candidate for combination with immune checkpoint inhibition and other immunotherapeutic approaches. Citation Format: Tara Jarboe, Danielle Quaranto, Nicole R. DeSouza, Kaci Kopec, Jan Geliebter, Raj K. Tiwari, Mark D. Hurwitz. Hyperthermic induction of immunotherapeutic response in in vitro melanoma model [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 6643.

Abstract 7196: Evaluation of nab-sirolimus in combination with fulvestrant or PI3K pathway inhibitors to overcome resistance in breast cancer cell lines

Abstract Background: Endocrine therapy (ET) is the standard of care for hormone receptor positive (HR+) breast cancer (BC), but ET resistance develops in most patients (pts). One resistance mechanism involves cross talk between the PI3K-AKT-mTOR and estrogen receptor (ER) signaling pathways, resulting in increased ER signaling and tumor proliferation. This is mitigated by mTOR inhibition as demonstrated by improved outcomes with exemestane plus the mTOR inhibitor (mTORi) everolimus in pts with HR+ advanced BC. While PI3K inhibitors (PI3Ki [e.g. alpelisib]) are efficacious in pts with HR+, PIK3CA-mutated BC, resistance can occur due to acquired genomic alterations in the PI3K-AKT-mTOR pathway. Combination therapy that targets a critical downstream node, mTOR, could delay or overcome acquired resistance due to activation of upstream components of the PI3K-AKT-mTOR pathway. nab-Sirolimus is an intravenously administered, albumin-bound nanoparticle form of the mTORi sirolimus, with significantly greater tumor accumulation, mTOR target suppression, and improved antitumor effects than oral mTORis in nonclinical tumor models. We evaluated the antiproliferative and cytotoxic effects of nab-sirolimus in combination with ET or PI3Kis in BC cell lines. Methods: HR+, PI3K-mutated BC cell lines (MCF7 or MDA-MB-361) were incubated for 5 days with increasing concentrations of nab-sirolimus and the selective ER degrader fulvestrant. MDA-MB-361 cells or HR-negative (HR−), PI3K-mutated BC cells, MDA-MD-453, were incubated for 5 days with increasing concentrations of nab-sirolimus and PI3K-AKT-mTOR pathway inhibitors gedatolisib or alpelisib. Antiproliferative/cytotoxic effects of single-agent and combination treatment were assessed via an automated trypan blue exclusion assay. Results: Addition of 20 or 80 nM nab-sirolimus to fulvestrant (12.5-500 nM) markedly decreased cell viability and nearly doubled cell death in MCF7 and MDA-MB-361 cells. Antiproliferative effects of low-dose gedatolisib (2.5-10 nM) were enhanced by 61-71% when nab-sirolimus (20 or 80 nM) was added to MDA-MB-453 cells. While low doses (2.5, 5, and 10 nM) of gedatolisib alone led to limited cytotoxicity (4.2-9.6%), up to 48.9% of cell death occurred when 20 or 80 nM nab-sirolimus was added. Results were similar with alpelisib (1 uM) plus nab-sirolimus (20 or 80 nM). Western blot showed increased p4EBP1 in response to fulvestrant or PI3Ki treatment, which was reversed with the addition of nab-sirolimus. Conclusions: nab-Sirolimus enhanced the cytotoxic effects of fulvestrant in HR+ BC cells and of PI3Kis in HR+ and HR− BC cells. Addition of nab-sirolimus to ET or other PI3K-AKT-mTOR pathway inhibitors may overcome mechanisms of resistance; further exploration is warranted to elucidate clinical relevance. Citation Format: Khine Nyein Myint, Sean Wallace, Shihe Hou, Maria Zalath, Brian McMorran, Andrew Kwon, Igor Vivanco. Evaluation of nab-sirolimus in combination with fulvestrant or PI3K pathway inhibitors to overcome resistance in breast cancer cell lines [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 7196.

Brucine Entrapped Titanium Oxide Nanoparticle for Anticancer Treatment: An In Vitro Study.

Backgroundand Objective. The public's health has been seriously threatened by cervical cancer during recent times. In terms of newly diagnosed cases worldwide, it ranks as the ninth most prevalent malignancy. Multiple investigations have proven that nanoparticles can effectively combat cancer due to their small dimensions and extensive surface area. In the meantime, bioactive compounds which are biocompatible are being loaded onto nanoparticles to promote cancer therapy. The current study investigates the anticancerous potential of Brucine-entrapped titanium oxide nanoparticles (TiO2 NPs) in cervical cancer cell line (HeLa). Materials and Methods. The physiochemical, structural, and morphological aspects of Brucine-entrapped TiO2 NPs were evaluated by UV-visible spectrophotometer, Fourier transform-infrared spectroscopy (FT-IR), dynamic light scattering (DLS), scanning electron microscopy (SEM), and energy dispersive X-ray (EDAX). The cytotoxic effect against the HeLa cell line was assessed using a tetrazolium-based colorimetric assay (MTT), a trypan blue exclusion (TBE) assay, phase contrast microscopic analysis, and a fluorescence assay including ROS and DAPI staining. Furthermore, estimation of antioxidant markers includes catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD). Results. The UV spectrum at 266 nm revealed the formation of TiO NPs. The FT-IR peaks confirmed the effective entrapment of brucine with TiO2 NPs. The average size (100.0 nm) of Brucine-entrapped TiO2 NPs was revealed in DLS analysis. The micrograph of the SEM revealed the formation of ellipsoidal to tetragonal-shaped NPs. The Ti, O, and C signals were observed in EDAX. In MTT assay, Brucine-entrapped TiO2 NPs showed inhibition of cell proliferation in a dose-wise manner and IC50 was noticed at the concentration of 30 µg/mL. The percentage of viable cells gradually reduced in the trypan blue exclusion assay. The phase contrast microscopic analysis of Brucine-entrapped TiO2 NP-treated cells showed cell shrinkage, cell wall deterioration, and cell blebbing. The intracellular ROS level was increased in a dose-wise manner when compared to control cells in ROS staining. The condensed nuclei and apoptotic cells were increased in treated cells, as noted in DAPI staining. In treated cells, the antioxidant markers such as CAT, GSH, and SOD levels were substantially lower compared to the control cells. Conclusion. The synthesized Brucine entrapped TiO2 NPs exhibited remarkable anticancer activity against the HeLa cell line.

In vitro cytotoxic and inhibitory effect of tramadol, flunitrazepam and levonogestrel on neutrophils and myeloperoxidase

The abuse of flunitrazepam, tramadol and levonorgestrel among adolescents is a major problem, with adverse effects on the immune system. Neutrophils serve as the host primary defense and employ the role of an essential enzyme, myeloperoxidase. This study investigated the in vitro effect of flunitrazepam, tramadol and levonorgestrel on the activity of the neutrophil. Neutrophils were isolated from blood samples of volunteers and seeded in culture plates, and then treated with flunitrazepam, tramadol and levonorgestrel. Trypan blue exclusion assay was used to assess the cytotoxic effect of flunitrazepam, (15, 150, 1500, 15,000, 150,000 ng/mL), tramadol (300, 3000, 30,000 µg/ml) and levonorgestrel (18.5, 185 and 1850 ng/mL) on the neutrophils, and the effect flunitrazepam (150,000 ng/mL), tramadol (30,000 µg/ml) and levonorgestrel (1850 ng/mL) on myeloperoxidase activity was assessed. The viability of neutrophils treated with flunitrazepam, (15,000, 150,000 ng/mL), tramadol (300, 3000, 30,000 µg/ml) and levonorgestrel (18.5, 185 and 1850 ng/mL) decreased significantly (p < 0.05) compared with control. Myeloperoxidase activity in neutrophils treated with flunitrazepam (150,000 ng/mL), tramadol (30,000 µg/ml) and levonorgestrel (1850 ng/mL) decreased significantly (p < 0.05) compared with background and positive control. This study revealed that flunitrazepam, tramadol and levonorgestrel altered the activity of myeloperoxidase and phagocytic activities of the neutrophil.

Effects of different cryopreservation parameters on the differences between trypan blue and fluorescent SYTO 13/GelRed assays

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how (i) the absence or presence of different cryoprotectants, (ii) different cell–cryoprotectant incubation conditions, and (iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays.