MCF-7 Cell Death Research Articles

Benzimidazole based hybrids as privileged candidates for topoII inhibition: Design, synthesis and molecular modeling studies

Two series of new benzimidazole derivatives were designed and synthesized. The antiproliferative activity of the new derivatives was screened against five human cancer cell lines namely; HepG-2, MCF-7, PC-3, HCT-116 and Hela. Relative to Doxorubicin (IC50 = 6.72 µM), compounds 18 and 19 showed broad spectrum anticancer effect against all the tested cancer cell lines, with IC50 range of 5.16–20.35 µM, however they were safe to WI-38 normal cells with IC50 of 32.61 µM and 50.62 µM, respectively. Inspection of human TopoII inhibitory activity disclosed that compound 18 was the most potent (IC50 = 6.9 µM) compared to Staurosporine (IC50 = 4.64 µM). Further mechanistic studies implied that compound 18 triggered the apoptotic death of MCF-7 cells via decreasing Bcl-2, activating caspases 8 and 9, and increasing the accumulation of ROS. In addition, it caused cell cycle arrest at the G1-phase and lowered the binding activity of transcription factors’ protein to DNA. The biological results were assisted by molecular modeling studies. The physicochemical parameters of compounds 18 and 19 conferred convenient drug-like properties.

Effect of aza-BODIPY-photodynamic therapy on the expression of carcinoma-associated genes and cell death mode

BackgroundBreast cancer is the most common cancer affecting women worldwide.Photodynamic therapy(PDT) has now proven to be a promising form of cancer therapy due to its targeted and low cytotoxicity to healthy cells and tissues.PDT is a technique used to create cell death localized by light after application of a light-sensitive agent.Aza-BODIPY is a promising photosensitizer for use in PDT. Our results showed that aza-BODIPY-PDT induced apoptosis, probably through p53 and caspase3 in MCF-7 cells. Future studies should delineate the molecular mechanisms underlying aza-BODIPY-PDT-induced cell death for a better understanding of the signaling pathways modulated by the therapy so that this novel technology could be implemented in the clinic for treating breast cancer. AimIn this study,we aimed to determine the change in the expression levels of 88 carcinoma-associated genes induced by aza-BODIPY-PDT were analyzed so as to understand the specific pathways that are modulated by aza-BODIPY-PDT. Material methodIn this study,the molecular basis of the anti-cancer activity of aza-BODIPY-PDT was investigated.Induction of apoptosis and necrosis in MCF-7 breast cancer cells after treatment with aza- BODIPY derivative with phthalonitrile substituents (aza-BODIPY) followed by light exposure was evaluated by Annexin V 7- Aminoactinomycin D (7-AAD) flow cytometry. ResultsAza-BODIPY-PDT induced cell death in MCF-7 cells treated with aza-BODIPY-PDT; flow cytometry revealed that 28 % of the cells died by apoptosis. Seven of the 88 carcinoma-associated genes that were assayed were differentially expressed —EGF, LEF1, WNT1, TCF7, and TGFBR2 were downregulated, and CASP3 and TP53 were upregulated — in cells subjected to aza-BODIPY-PDT.This made us think that the aza-BODIPY-PDT induced caspase 3 and p53-mediated apoptosis in MCF7 cells. ConclusionIn our study,it was determined that the application of aza-BODIPY-PDT to MCF7 cells had a negative effect on cell connectivity and cell cycle.The fact that the same effect was not observed in control cells and MCF7 cells in the dark field of aza-BODIPY indicates that aza-BODIPY has a strong phodynamic anticancer effect.

Bioactives of noni plant (Morinda citrifolia L.) as anti-cancer herbals: A scoping review

In Indonesia, the noni plant (Morinda citrifolia L.) has gained popularity as an alternative treatment for various diseases. Cancer is the second cause of death worldwide, with chemotherapy as the primary treatment. There is growing interest in exploring phytochemicals found in noni plants such as anthraquinones, chitosan, phenols, and flavonoids. These antioxidant compounds hold potential as alternative treatments for cancer. This study explores the active compounds in noni plants that exhibit anti-cancer properties. This study employed a scoping review approach. Previous original articles were retrieved from Scopus, Science Direct, ClinicalKey, Springer Link, and Google Scholar with keywords in Morinda citrifolia L., component, bioactive, antioxidant, anti-inflammatory, and anti-cancer. We included 34 articles that met the inclusion criteria. The findings of this study highlight the anti-cancer activity of several phytochemicals found in noni plants. Damnacanthal has been shown to reduce the number of MCF-7 cells. Nordamnacanthal inhibited the development of the H400 cell cycle by increasing the activity of Cytochrome C, Caspase-9, and Caspase 3/7 and induced the death of MDA-MB231 and MCF-7 cells. Chitosan nanoparticles can inhibit the growth of A549 cells. Scopoletin can inhibit cancer cell proliferation by inducing cells in the G0 / G1 and S phases. Epicatechin can inhibit the growth of PC-9 cells. Damnachanthal, nordamnacanthal, chitosan nanoparticles, scopoletin, and epicatechin are active compounds presented in noni plants that can act as anti-cancer.

Marine Natural Compound (Neviotin A) Displays Anticancer Efficacy by Triggering Transcriptomic Alterations and Cell Death in MCF-7 Cells.

We investigated the anticancer mechanism of a chloroform extract of marine sponge (Haliclona fascigera) (sample C) in human breast adenocarcinoma (MCF-7) cells. Viability analysis using MTT and neutral red uptake (NRU) assays showed that sample C exposure decreased the proliferation of cells. Flow cytometric data exhibited reactive oxygen species (ROS), nitric oxide (NO), dysfunction of mitochondrial potential, and apoptosis in sample C-treated MCF-7 cells. A qPCR array of sample C-treated MCF-7 cells showed crosstalk between different pathways of apoptosis, especially BIRC5, BCL2L2, and TNFRSF1A genes. Immunofluorescence analysis affirmed the localization of p53, bax, bcl2, MAPKPK2, PARP-1, and caspase-3 proteins in exposed cells. Bioassay-guided fractionation of sample C revealed Neviotin A as the most active compound triggering maximum cell death in MCF-7, indicating its pharmacological potency for the development of a drug for the treatment of human breast cancer.

Design, synthesis, in vitro anticancer, molecular docking and SAR studies of new series of pyrrolo[2,3-d]pyrimidine derivatives

The current study involves the design and synthesis of a newly synthesized pyrrolo[2,3-d]pyrimidine derivatives to contain chlorine atoms in positions 4 and 6 and trichloromethyl group in position 2 using microwave technique as a new and robust approach for preparation of this type of pyrrolo[2,3-d]pyrimidine derivatives. The chemical structure of the synthesized pyrrolo[2,3-d]pyrimidine derivatives 3–19 was well-characterized using spectral and elemental analyses as well as single-crystal X-ray diffraction. All compounds were tested in vitro against seven selected human cancer cell lines, namely, MCF7, A549, HCT116, PC3, HePG2, PACA2 and BJ1 using MTT assay. It was found that compounds 14a, 16b and 18b were the most active toward MCF7 with IC50 (1.7, 5.7, and 3.4 μg/ml, respectively) relative to doxorubicin (Dox.) (26.1 μg/ml). Additionally, compound 17 exerted promising cytotoxic effects against HePG2 and PACA2 with IC50 (8.7 and 6.4 μg/ml, respectively) relative to Dox. (21.6 and 28.3 μg/ml, respectively). The molecular docking study confirmed our ELISA result which showed the promising binding affinities of compounds 14a and 17 against Bcl2 anti-apoptotic protein. At the gene expression level, P53, BAX, DR4 and DR5 were up-regulated, while Bcl2, Il-8, and CDK4 were down-regulated in 14a, 14b and 18b treated MCF7 cells. At the protein level, compound 14b increased the activity of Caspase 8 and BAX (18.263 and 14.25 pg/ml) relative to Dox. (3.99 and 4.92 pg/ml, respectively), while the activity of Bcl2 was greatly decreased in 14a treated MCF7 (2.4 pg/ml) compared with Dox. (14.37 pg/ml). Compounds 14a and 14b caused cell cycle arrest at the G1/S phase in MCF7. Compounds 16b and 18b induced the apoptotic death of MCF7 cells. In addition, the percentage of fragmented DNA was increased significantly in 14a treated MCF7 cells.

The effect of hesperetin on estrogen receptor gene expression and its relationship with the downstream pathways of estrogen receptor alpha.

Estrogen receptor (ER) is a transcription factor that affects the expression of some genes involved in the progression and development of breast cancer (BC). Hesperetin (Hst) is a flavonoid that inhibits the proliferation of BC cells. In this study, we investigated the effect of Hst on the cell viability of MCF-7 cells and the gene expression of the ERα, ERβ, IL-6, Ps2, and Cyclin D1. In this study, cell viability was determined by MTT assay. The cells were seeded in RPMI-1640 medium and then exposed to different concentrations of Hst (0, 25, 50, 100, 200, and 400 µM) for 24h, and IC50 was calculated. Real-time PCR was used to assess the expression of ERα, ERβ, pS2, Cyclin D1, and IL-6 mRNA. MCF-7 cells were seeded in RPMI-1640 medium and then exposed to different concentrations of Hst (0, 25, 50, 100, and 200 µM) for 24h. Real-time PCR was carried out using a Step One Real-Time PCR System (ABI, USA) and Amplicon SYBR Green reagents. The MTT assay revealed increased cytotoxicity with higher concentrations of Hst, and the IC50 was calculated at 200 µM. Real-time PCR analysis following treatment with Hst showed a significant increase in ERα gene expression at 25 µM of Hst and a decrease in expression at 50, 100, and 200 µM of Hst (p < 0.0001). ERβ gene expression significantly decreased across all concentrations of Hst (p < 0.0001), while IL-6 gene expression decreased significantly in all concentrations (p < 0.0001). pS2 gene expression increased significantly with all concentrations of Hst (p < 0.0001), while Cyclin D1 gene expression did not significantly decrease upon Hst exposure (p > 0.05). The results of our study demonstrate that Hst has the ability to induce cell death in MCF-7 cells. Furthermore, it was observed that Hst reduces the expression of the ER gene and enhances its activity, which can affect the downstream pathways of the ER.

Viability of HepG2 and MCF-7 cells is not correlated with mitochondrial bioenergetics

Alterations in metabolism are a hallmark of cancer. It is unclear if oxidative phosphorylation (OXPHOS) is necessary for tumour cell survival. In this study, we investigated the effects of severe hypoxia, site-specific inhibition of respiratory chain (RC) components, and uncouplers on necrotic and apoptotic markers in 2D-cultured HepG2 and MCF-7 tumour cells. Comparable respiratory complex activities were observed in both cell lines. However, HepG2 cells exhibited significantly higher oxygen consumption rates (OCR) and respiratory capacity than MCF-7 cells. Significant non-mitochondrial OCR was observed in MCF-7 cells, which was insensitive to acute combined inhibition of complexes I and III. Pre-treatment of either cell line with RC inhibitors for 24–72 h resulted in the complete abolition of respective complex activities and OCRs. This was accompanied by a time-dependent decrease in citrate synthase activity, suggesting mitophagy. High-content automated microscopy recordings revealed that the viability of HepG2 cells was mostly unaffected by any pharmacological treatment or severe hypoxia. In contrast, the viability of MCF-7 cells was strongly affected by inhibition of complex IV (CIV) or complex V (CV), severe hypoxia, and uncoupling. However, it was only moderately affected by inhibition of complexes I, II, and III. Cell death in MCF-7 cells induced by inhibition of complexes II, III, and IV was partially abrogated by aspartate. These findings indicate that OXPHOS activity and viability are not correlated in these cell lines, suggesting that the connection between OXPHOS and cancer cell survival is dependent on the specific cell type and conditions.

In situ forming alginate/gelatin hybrid hydrogels containing doxorubicin loaded chitosan/AuNPs nanogels for the local therapy of breast cancer

In this study, pH-sensitive in situ gelling hydrogels based on oxidized alginate and gelatin-containing doxorubicin (DOX) loaded chitosan/gold nanoparticles (CS/AuNPs) nanogels were fabricated via Schiff-base bond formation. The obtained CS/AuNPs nanogels indicated a size distribution of about 209 nm with a zeta potential of +19.2 mV and an encapsulation efficiency of around 72.6 % for DOX. The study of the rheological properties of hydrogels showed that the value of G' is higher than G″ for all hydrogels, which confirms the elastic behavior of hydrogels in the applied frequency range. The rheological and texture analysis demonstrated the higher mechanical properties of hydrogels containing β-GP and CS/AuNPs nanogels. The release profile of DOX after 48 h indicates the 99 % and 73 % release amount at pH = 5.8 and pH = 7.4, respectively. MTT cytotoxicity study showed that the prepared hydrogels are cytocompatible on MCF-7 cells. By the Live/Dead assay, it was demonstrated that the cultured cells on DOX-free hydrogels were almost alive in the presence of CS/AuNPs nanogels. However, the hydrogel-containing drug and free DOX in the same concentration caused high death of MCF-7 cells as expected, which showed the potential of the developed hydrogels for application in the local treatment of breast cancer.

Novel Thiazolidine-2,4-dione-trimethoxybenzene-thiazole Hybrids as Human Topoisomerases Inhibitors.

Cancer is a complex and heterogeneous disease and is still one of the leading causes of morbidity and mortality worldwide, mostly as the population ages. Despite the encouraging advances made over the years in chemotherapy, the development of new compounds for cancer treatments is an urgent priority. In recent years, the design and chemical synthesis of several innovative hybrid molecules, which bring different pharmacophores on the same scaffold, have attracted the interest of many researchers. Following this strategy, we designed and synthetized a series of new hybrid compounds that contain three pharmacophores, namely trimethoxybenzene, thiazolidinedione and thiazole, and tested their anticancer properties on two breast cancer (MCF-7 and MDA-MB-231) cell lines and one melanoma (A2058) cell line. The most active compounds were particularly effective against the MCF-7 cells and did not affect the viability of the normal MCF-10A cells. Docking simulations indicated the human Topoisomerases I and II (hTopos I and II) as possible targets of these compounds, the inhibitory activity of which was demonstrated by the mean of direct enzymatic assays. Particularly, compound 7e was proved to inhibit both the hTopo I and II, whereas compounds 7c,d blocked only the hTopo II. Finally, compound 7e was responsible for MCF-7 cell death by apoptosis. The reported results are promising for the further design and synthesis of other analogues potentially active as anticancer tools.

Biological activities and ecological aspects of Limonium pruinosum (L.) collected from Wadi Hof Eastern Desert, Egypt, as a promising attempt for potential medical applications

Very few researchers have focused on the biological efficacy of Limonium plants. In this concern, no investigations were commenced to delve into the in vitro and ex vivo biological actions of Limonium pruinosum in Egypt. Therefore, this work aims to assess for the first time the antimicrobial, antioxidant, and antitumor activities of Limonium pruinosum extract in addition to studying its ability to suppress the transcription of cell cycle–stimulating genes. L. pruinosum ethyl acetate extract exhibits considerable antibacterial and antibiofilm activity versus E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Results revealed that L. pruinosum exerts antioxidant effectiveness concerning DPPH, nitric oxide (NO), and hydroxyl radical (OH) scavenging ability with an IC50 (35.88 ± 2.2, 51.31 ± 1.06, and 65.87 ± 1.19 μg/mL) respectively. The results proved the effectiveness of L. pruinosum in closing wounds in gastric epithelial cells (GES-1) by (79.9343 ± 1.98%) compared with control (68.3637 ± 2.32%) in 48 h. Additionally, L. pruinosum had anticancer activity contrary to breast cancer MCF-7 and liver cancer HepG-2 cell lines with IC50 values of 96.73 ± 2.18 and 81.81 ± 0.99 μg/mL, respectively, while it had no cytotoxic activity against (Wi-38) normal cells. Also, L. pruinosum extract provoked considerable early- and late-apoptotic cell populations and was effective in inducing cell death of MCF-7. Our findings evoked that L. pruinosum has promising antibacterial, antioxidant, and wound healing activities and a good breast tumor suppressor arresting the cell cycle-stimulating genes, which may be an auspicious approach for the treatment of breast cancer.

Synergistic cytotoxic effects of an extremely low-frequency electromagnetic field with doxorubicin on MCF-7 cell line

Breast cancer is one of the leading causes of cancer deaths in women worldwide. Magnetic fields have shown anti-tumor effects in vitro and in vivo as a non-invasive therapy method that can affect cellular metabolism remotely. Doxorubicin (DOX) is one of the most commonly used drugs for treating breast cancer patients. It can be assumed that combining chemotherapy and magnetotherapy is one of the most effective treatments for breast cancer. This study aimed to investigate the potential cytotoxic effect of DOX at low concentrations in combination with extremely low-frequency electromagnetic fields (ELF–EMF; 50 Hz; 20 mT). The breast cancer cell line MCF-7 was examined for oxidative stress, cell cycle, and apoptosis. MCF-7 cells were treated with various concentrations of DOX as an apoptosis-inducing agent and ELF–EMF. Cytotoxicity was examined using the MTT colorimetric assay at 12, 24, and 48 h. Consequently, concentration- and time-dependent cytotoxicity was observed in MCF-7 cells for DOX within 24 h. The MTT assay results used showed that a 2 μM concentration of DOX reduced cell viability to 50% compared with control, and as well, the combination of ELF–EMF and DOX reduced cell viability to 50% compared with control at > 0.25 μM doses for 24 h. In MCF-7 cells, combining 0.25 μM DOX with ELF–EMF resulted in increased ROS levels and DOX-induced apoptosis. Flow cytometry analysis, on the other hand, revealed enhanced arrest of MCF-7 cells in the G0-G1 phase of the cell cycle, as well as inducing apoptotic cell death in MCF-7 cells, implying that the synergistic effects of 0.25 μM DOX and ELF–EMF may represent a novel and effective agent against breast cancer.

Radiosensitivity Enhancement using Triptorelin Conjugated Bismuth Sulfide Nanoparticles (Bi2S3@BSA) in Radiotherapy for Breast Cancer Cells

The aim of this study was to assess the radiosensitivity of bismuth sulfide nanoparticles conjugated with a synthetic agonist analog of gonadotropin-releasing hormones in targeted radiotherapy for breast cancer. After synthesis and characterization of nanoparticles, cytotoxicity of nanoparticles was measured by MTT assay, and the survival fraction was determined by colony formation assay. Finally, flow cytometry was performed to identify the mechanism of radiosensitization. Characterization results determined the spherical shape of Bi2S3@BSA with an average size of 8.649 ± 1.6 nm, and Fourier transform infrared confirmed the successful binding of triptorelin to the surface of the nanoparticles. MTT test results show that the Bi2S3@BSA–triptorelin did not cause any toxicity ( P &lt; 0.05 ) even up to 75 μg/ml. At all doses of ionizing radiation, colony formation assays showed that the nontoxic concentration of Bi2S3@BSA–triptorelin significantly increased cell death in MCF-7 cells compared to Bi2S3@BSA ( P &lt; 0.05 ). The apoptosis test also confirmed colony formation assay results at all doses and introduced apoptosis as a mechanism of radiosensitivity produced by nanoparticles. Certainly, targeted bismuth sulfide nanoparticles can be a good candidate for increasing radiosensitivity against tumor cells.

Cannabis sativa L. modulates altered metabolic pathways involved in key metabolisms in human breast cancer (MCF-7) cells: A metabolomics study

The present study investigated the ability of Cannabis sativa leaves infusion (CSI) to modulate major metabolisms implicated in cancer cells survival, as well as to induce cell death in human breast cancer (MCF-7) cells. MCF-7 cell lines were treated with CSI for 48 h, doxorubicin served as the standard anticancer drug, while untreated MCF-7 cells served as the control. CSI caused 21.2% inhibition of cell growth at the highest dose. Liquid chromatography–mass spectroscopy (LC-MS) profiling of the control cells revealed the presence of carbohydrate, vitamins, oxidative, lipids, nucleotides, and amino acids metabolites. Treatment with CSI caused a 91% depletion of these metabolites, while concomitantly generating selenomethionine, l-cystine, deoxyadenosine triphosphate, cyclic AMP, selenocystathionine, inosine triphosphate, adenosine phosphosulfate, 5'-methylthioadenosine, uric acid, malonic semialdehyde, 2-methylguanosine, ganglioside GD2 and malonic acid. Metabolomics analysis via pathway enrichment of the metabolites revealed the activation of key metabolic pathways relevant to glucose, lipid, amino acid, vitamin, and nucleotide metabolisms. CSI caused a total inactivation of glucose, vitamin, and nucleotide metabolisms, while inactivating key lipid and amino acid metabolic pathways linked to cancer cell survival. Flow cytometry analysis revealed an induction of apoptosis and necrosis in MCF-7 cells treated with CSI. High-performance liquid chromatography (HPLC) analysis of CSI revealed the presence of cannabidiol, rutin, cinnamic acid, and ferulic. These results portray the antiproliferative potentials of CSI as an alternative therapy for the treatment and management of breast cancer as depicted by its modulation of glucose, lipid, amino acid, vitamin, and nucleotide metabolisms, while concomitantly inducing cell death in MCF-7 cells.

Chidamide enhances cytotoxicity of doxorubicin by promoting autophagy and apoptosis in breast cancer

BackgroundBreast cancer (BC) is a prevalent disease that harms women's health, and in-depth investigations of the pathogenesis, treatment, and prevention of BC are the focus of many research programs. Chidamide (CHI) is a histone deacetylase suppressor that depresses histone deacetylase functions, thereby influencing cell growth through an epigenetic mechanism. However, CHI effects upon BC are largely unknown. Present research aimed to confirm the possibility of using CHI combined with chemotherapy drug doxorubicin (DOX) to prevent chemotherapeutic BC resistance in vivo and in vitro.MethodsIn this study, CCK8 (a plate colony formation assay) was applied to detect cell proliferation. Flow cytometry detection showed the apoptotic cell death of both T47D and MCF-7 cells. Nude mouse xenografts were used to detect tumor growth and pulmonary metastasis. High-throughput sequencing was used to detect expression of different genes.ResultsOur data showed that CHI treatment reduced BC cell proliferation, tumor growth, and cell invasion. CHI treatments stimulated BC cell apoptosis by promoting ULK2-mediated autophagy and increasing MCF-7 cell sensitivity to DOX, resulting in decreased tumor growth.ConclusionCollectively, our results illustrated that CHI enhanced DOX cytotoxicity by promoting apoptosis and autophagy in BC cells, which advised that CHI could be a candidate drug for BC patient treatments.

Artemisia sieberi Besser essential oil inhibits the growth and migration of breast cancer cells via induction of S-phase arrest, caspase-independent cell death and downregulation of ERK

Ethnopharmacological relevanceArtemisia sieberi Besser is a medicinal herb that has been traditionally used across the Middle East for the treatment of cancer. Further pharmacological studies on its extracts revealed that they possess cytotoxic activity against certain cancer cells, however, there were no studies conducted on the anticancer potential of Artemisia sieberi essential oil (ASEO). Aim of the studyTo evaluate the anticancer potential of ASEO, elucidate the oil's mode of action for the first time and investigate its chemical composition. Materials and methodsArtemisia sieberi was collected from Hail, Saudi Arabia, and its essential oil was obtained via hydrodistillation. The oil's activity against HCT116, HepG2, A549 and MCF-7 cells was assessed using SRB assay, while its anti-metastatic potential was assessed via a migration assay. Cell-cycle analysis and apoptosis assay were conducted via flow cytometry, while protein expression levels were investigated using Western blotting. The oil's chemical constituents were identified using GCMS. ResultsASEO exerted its highest cytotoxic activity against MCF-7 with an IC50 value of 38.7 μg/ml. Further studies showed that the oil inhibited MCF-7 cells' migration, induced S-phase arrest and apoptosis. Western blot analysis showed no change in the expression level of caspase-3 after treatment, indicating the induction of caspase-independent apoptosis-like cell death in MCF-7. Treatment of MCF-7 with the oil resulted in downregulation of the protein expression levels of total ERK and its downstream target, LC3, indicating that any potential activation of the ERK signalling pathway during the cancer cells' growth would be inhibited. Finally, GCMS analysis identified the oil's major components as cis-crysanthenyl acetate (48.56%), davanone (10.28%), 1,8-cineole (6.81%) and caryophyllene diepoxide (5.34%), whereby it is suggested that these compounds might be responsible for the oil's bioactivity. ConclusionASEO possessed in vitro anticancer activity and modulated the ERK signalling pathway. This is the first study to explore the anticancer potential of ASEO in detail and reflects the significance of investigating essential oils from medicinal plants that have been traditionally used against cancer. This work might pave the way for further in vivo studies that could result in developing the oil into a natural effective anticancer treatment.

Abstract 5418: AI-driven image analysis enables simplified, label-free cytotoxicity screening

Abstract The increasing use of precious, patient-derived cells has driven the need for non-perturbing and label-free cell measurements, particularly in the oncology field. To address this we developed the Incucyte® AI Cell Health Analysis Software Module, which uses two pre-trained deep neural networks to perform automated, unbiased analysis of Phase contrast images to segment individual cells and perform label-free Live/Dead cell classification. The neural networks which perform cell instance segmentation and infer cell viability were trained on a wide diversity of cell types with varied morphologies, ensuring that the analysis is applicable across a variety of adherent and suspension tumor cell types. Here, we demonstrate the application of this analysis across diverse and commonly used biological models of breast cancer, glioblastoma, and B-cell lymphoma. In each case, cells were treated with chemotherapeutic compounds or biosimilar antibodies and Phase contrast images were acquired at regular intervals over 3 - 4 days using the Incucyte® Live-Cell Analysis System. Using the Incucyte® AI Cell Health Analysis cells were accurately segmented and the percentage of dead cells were quantified over time without the requirement for a fluorescent reporter or other exogenous label, and with limited user input. Four breast cancer cell lines were treated with a panel of chemotherapeutics designed to target specific expression patterns. AI Cell Health analysis showed that Estrogen receptor (ER) inhibitor Tamoxifen selectively induced &amp;gt;60% cell death only in ER positive cell lines BT474 and MCF7; dual epidermal growth factor receptor (EGFR/HER2) inhibitor Lapatinib induced cell death in AU565, BT474 and MCF7 which express these surface markers. In contrast, Lapatinib and Tamoxifen induced morphological change - but minimal cell death - in triple negative MDA-MB-231 cells. Three glioblastoma cell lines A172, U87 and T98G were treated with a larger panel of chemotherapeutic compounds and for four of the active compounds, efficacy was also determined. Cisplatin, doxorubicin, vinblastine and taxol induced concentration-dependent cell death in A172 and T98G cells; U87 cells displayed resistance to each of these compounds with a maximal 46.5% cell death induced by doxorubicin. Ramos B-cell lymphoma cells were exposed to increasing concentrations of monoclonal antibody Rituximab and the biosimilar Truxima®. The antibodies induced specific cell death via the surface marker CD20 in a time and concentration-dependent manner with similar efficacy (IC50 Rituximab 94.7 ng/mL; Truxima® 110.3 ng/mL), while antibody control IgG1 remained non-perturbing to cells. These results demonstrate that the Incucyte® AI Cell Health Analysis is applicable to a broad range of cancer types cultured in 2D monolayer. This unbiased method enables accurate, label-free quantification of cytotoxic effects induced by clinically relevant therapeutics. Citation Format: Gillian Lovell, Daniel A. Porto, Jasmine Trigg, Nevine Holtz, Nicola Bevan, Timothy Dale, Daniel Appledorn. AI-driven image analysis enables simplified, label-free cytotoxicity screening. [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 5418.

Free Fatty Acids from Cow Urine DMSO Fraction Induce Cell Death in Breast Cancer Cells without Affecting Normal GMSCs.

The objective of this study was to explore the biological relevance of free fatty acids derived from cow urine DMSO fraction (CUDF) by employing in vitro and in silico approaches. Metabolic heterogeneity at the intra- and intercellular levels contributes to the metabolic plasticity of cancer cells during drug-induced response. Free fatty acid (FFA) availability at intra- and intercellular levels is related to tumor heterogeneity at interpatient and xeno-heterogeneity levels. We collected fresh urine from healthy cows and subjected it to fractionation in DMSO using drying, vortexing, and centrifugation. Finally, the sterile filtrate of cow urine DMSO fraction (CUDF) was evaluated for antiproliferative and proapoptotic effects in MCF-7 and ZR-75-1 breast cancer cells using routine cell-based assays. Intracellular metabolites were studied with the help of a novel in-house vertical tube gel electrophoresis (VTGE) method to reveal the nature of CUDF components in MCF-7 cells. Identified intracellular FFAs were studied for their molecular interactions with targeted receptor histone deacetylase (HDAC) using molecular docking and molecular dynamics (MD) simulations. CUDF showed a significant reduction in cell viability and cell death in MCF-7 and ZR-75-1 breast cancer cells. Interestingly, FFAs tetracosanedioic acid, 13Z-docosenoic acid (erucic acid), nervonic acid, 3-hydroxy-tetradecanoic acid, and 3-hydroxcapric acid were found inside the treated MCF-7 cancer cells. These FFAs, including tetracosanedioic acid, indicated a specific affinity to HDAC at their inhibitory sites, similar to trichostatin A, a known inhibitor. This study reports on FFAs derived from CUDF as potential antiproliferative and pro-cell death agents against breast cancer cells. MD simulations hinted at tetracosanedioic acid and other FFAs as inhibitors of HDAC that could explain the observed effects of FFAs in cancer cells.

Momordica cochinchinensis Suppresses the Human Breast Cancer Cells Growth and Migration by Inhibiting Mevalonate Pathway

Background: Momordica cochinchinensis (MC) is shown with high antioxidation and is used as herbal medicine in South-East countries. Objectives: This study aims to determine the four parts (aril, fruit, leaf, and seed) of MC extract against human breast cancer cells with underlying mechanisms of actions. Materials and Methods: The effects of MC extract were examined by sulforhodamine B, colony formation, wound healing, real time polymerase chain reaction (RT-PCR), and Western blotting method. Results: Four parts of MC extracts significantly increased MCF-7 cells death, interestingly, seed extract had the greatest activity with IC50 values of 412.3 ± 49.8 µg/mL for 24 h and 113.3 ± 6.4 µg/mL for 48 h. MC extracts potentiated the anticancer drug effect, doxorubicin, with lower IC50 values when compared with MC treatment alone. Moreover, MC extracts decreased colony formation and accompanied by inhibited cell migration. Furthermore, Seed extract inhibited migration by reducing matrix metalloproteinases (MMP) 2, MMP 9, and vascular endothelial growth factor A (VEGFA) inhibition. The molecular analyses revealed that seed extract inhibited cell viability by decreasing cyclin D1 consistent with activating p21, cytochrome c, and caspase-3 protein levels along with reduction of mevalonate (MVA) pathway, Rac1, and RhoA levels, it appeared to inhibit growth and migration. Conclusion: Taken together, MC extract induces MCF-7 cell death and decreases cell migration relatively via attenuation of MVA pathway.

The effect of acute acid exposure on immunomodulatory protein secretion, cell survival, and cell cycle progression in tumour cell lines.

Cancer develops when multiple systems fail to suppress uncontrolled cell proliferation. Breast cancers and oesophageal squamous cell carcinoma (OSCC) are common cancers prone to genetic instability. They typically occur in acidic microenvironments which impacts on cell proliferation, apoptosis, and their influence on surrounding cells to support tumour growth and immune evasion. This study aimed to evaluate the impact of the acidic tumour microenvironment on the production of pro-tumorigenic and immunomodulatory factors in cancer cell lines. Multiple factors that may mediate immune evasion were secreted including IL-6, IL-8, G-CSF, IP-10, GDF-15, Lipocalin-2, sICAM-1, and myoglobin. Others, such as VEGF, FGF, and EGF that are essential for tumour cell survival were also detected. Treatment with moderate acidity did not significantly affect secretion of most proteins, whereas very low pH did. Distinct differences in apoptosis were noted between the cell lines, with WHCO6 being better adapted to survive at moderate acid levels. Conditioned medium from acid-treated cells stimulated increased cell viability and proliferation in WHCO6, but increased cell death in MCF-7. This study highlights the importance of acidic tumour microenvironment in controlling apoptosis, cell proliferation, and immune evasion which may be different at different anatomical sites. Immunomodulatory molecules and growth factors provide therapeutic targets to improve the prognosis of individuals with cancer.

Fabrication of biocompatible magnetic maltose/MIL-88 metal–organic frameworks decorated with folic acid-chitosan for targeted and pH-responsive controlled release of doxorubicin

Recently, metal–organic frameworks (MOFs) have attracted tremendous attention as promising porous drug delivery systems for cancer treatment. In this work, for the first time, a novel magnetic maltose disaccharide molecule modified with MIL-88 metal–organic framework (Fe3O4@C@MIL-88) was prepared, and then this targeted system was used for the delivery of the doxorubicin (DOX) drug. Eventually, Fe3O4@C@MIL-88-DOX were successfully decorated with folic acid conjugated chitosan (Fe3O4@C@MIL-88-DOX-FC) as a new targeted and controlled release drug system for treatment of MCF-7 breast cancer. The encapsulation efficiency of the DOX in the Fe3O4@C@MIL-88 was obtained at ∼83.6%. The in vitro drug release profiles showed a pH-responsive controlled release of DOX in acidic pH confirming the performance of the systems in the cancerous environment. The DOX release mechanism from systems at pH 5 also showed that the kinetic data well fitted to the Korsmeyer-Peppas and Fickian diffusion. Furthermore, in vitro cytotoxicity and DAPI staining study clearly illustrated that the synthesized Fe3O4@C@MIL-88 system had low cytotoxicity and good biocompatibility against MCF-7 cancer cells and MCF-10A normal cells. Whereas, Fe3O4@C@MIL-88-DOX and Fe3O4@C@MIL-88-DOX-FC exhibited good antitumor activity as a result of targeted delivery of DOX, which indicated the MCF-7 cell death with apoptotic effects. Based on these findings, the resulting carriers could be used as promising targeted drug delivery systems for cancer therapy.