Breast Cancer Cell Lines T47D Research Articles

FGFR2-triggered autophagy and activation of Nrf-2 reduce breast cancer cell response to anti-ER drugs

BackgroundGenetic abnormalities in the FGFR signalling occur in 40% of breast cancer (BCa) patients resistant to anti-ER therapy, which emphasizes the potential of FGFR-targeting strategies. Recent findings indicate that not only mutated FGFR is a driver of tumour progression but co-mutational landscapes and other markers should be also investigated. Autophagy has been recognized as one of the major mechanisms underlying the role of tumour microenvironment in promotion of cancer cell survival, and resistance to anti-ER drugs. The selective autophagy receptor p62/SQSTM1 promotes Nrf-2 activation by Keap1/Nrf-2 complex dissociation. Herein, we have analysed whether the negative effect of FGFR2 on BCa cell response to anti-ER treatment involves the autophagy process and/or p62/Keap1/Nrf-2 axis.MethodsThe activity of autophagy in ER-positive MCF7 and T47D BCa cell lines was determined by analysis of expression level of autophagy markers (p62 and LC3B) and monitoring of autophagosomes’ maturation. Western blot, qPCR and proximity ligation assay were used to determine the Keap1/Nrf-2 interaction and Nrf-2 activation. Analysis of 3D cell growth in Matrigel® was used to assess BCa cell response to applied treatments. In silico gene expression analysis was performed to determine FGFR2/Nrf-2 prognostic value.ResultsWe have found that FGFR2 signalling induced autophagy in AMPKα/ULK1-dependent manner. FGFR2 activity promoted dissociation of Keap1/Nrf-2 complex and activation of Nrf-2. Both, FGFR2-dependent autophagy and activation of Nrf-2 were found to counteract the effect of anti-ER drugs on BCa cell growth. Moreover, in silico analysis showed that high expression of NFE2L2 (gene encoding Nrf-2) combined with high FGFR2 expression was associated with poor relapse-free survival (RFS) of ER+ BCa patients.ConclusionsThis study revealed the unknown role of FGFR2 signalling in activation of autophagy and regulation of the p62/Keap1/Nrf-2 interdependence, which has a negative impact on the response of ER+ BCa cells to anti-ER therapies. The data from in silico analyses suggest that expression of Nrf-2 could act as a marker indicating potential benefits of implementation of anti-FGFR therapy in patients with ER+ BCa, in particular, when used in combination with anti-ER drugs.Graphical

Abstract GS03-10: Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations

Abstract Background: PIK3CA mutations occur in ~40% of HR-positive breast cancers, where alpelisib, an orthosteric PI3Kα inhibitor, is FDA-approved in combination with fulvestrant. Although prior studies have identified potential resistance mechanisms, such as PTEN loss, clinical acquired resistance to orthosteric PI3Kα inhibitors and the role of next-generation allosteric PI3Kα inhibitors remain poorly understood. Methods: To identify on-target and off-target alterations potentially mediating resistance to PI3Kα inhibitors, we used a targeted next-generation sequencing assay (Guardant360; Guardant Health) to analyze ctDNA in serially collected plasma samples from 32 patients with PIK3CA-mutated advanced HR-positive, HER2-negative breast cancer treated with alpelisib and inavolisib. In addition, we performed whole exome sequencing (WES) of 100 tissue samples collected from 8 autopsy series from patients with metastatic, PIK3CA-mutant HR-positive, HER2-negative breast cancer previously treated with PI3Kα inhibitors. Acquired alterations were prioritized through a combination of structural modeling and free-energy perturbation simulation and validated in genomically engineered PIK3CA mutant breast cancer cell lines T47D (PIK3CA H1047R-mutant) or MCF7 (PIK3CA E545K-mutant). Results: We observed that 50% of patients acquire genomic alterations within the PI3K-pathway, including PTEN loss and activating AKT1 mutations. Notably, while secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Ka-inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket including PIK3CA Q859K and PIK3CA W780R. Some mutations had differential effects on PI3Ka-selective vs. pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Ka-inhibitor RLY-2608. Conclusion: In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Ka inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Ka inhibitor. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers. Citation Format: Andreas Varkaris, Ferran Fece de la Cruz, Elizabeth Martin, Bryanna Nordern, Nicholas Chevalier, Ignaty Leshchiner, Anastasia Stavridi, Janice Kim, Alkistis Papatheodoridi, Hakan Gunaydin, Brian Danysh, Laxmi Parida, Ioannis Sanidas, Yongli Ji, Kayao Lau, Gerburg Wulf, Aditya Bardia, Laura Spring, Steven Isakoff, Jochen Lennerz, Levi Pierce, Ermira Pazolli, Gad Getz, Ryan Corcoran, Dejan Juric. Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-10.

Abstract 3334: Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response

Abstract Integrated stress response (ISR) activation has been linked to many human diseases, including cancer and neurological diseases. The ISR controls protein synthesis and proteostasis. ISR main effector ATF4 transcriptional factor regulates the balance between survival and cell death. Under acute ISR, ATF4 promotes cell adaptation and survival. Sustained ISR leads to ATF4-mediated apoptosis. In many cancers, mutations or overexpression of oncogenes, results in enhanced cell proliferation and increased protein synthesis, which activate the ISR. Therefore, protein translation is reduced to maintain proteostasis. In other words, many cancers are primed by the ISR. Therefore, additional push by small molecule ISR-inducers will disrupt the balance and force cancer cells to enter apoptosis. Nelfinavir activates ATF4 through inhibition of eIF2α specific phosphatases CReP and GADD34. PG3 upregulates ATF4 through the HRI/eIF2α/ATF4 pathway. We hypothesize that combined treatment of PG3 and Nelfinavir will sustain upregulation of ATF4, and lead to ATF4-mediated cell apoptosis. Cell viability assays indicated that the combined treatment potently and synergistically inhibited cell viability in colorectal cancer cell lines HT29, SW480 and HCT116 p53−/−, osteosarcoma cell line U2OS and breast cancer cell lines MCF7 and T47D. The combined treatment sustained the induction of ATF4 and enhanced the expression of its proapoptotic target genes, CHOP and PUMA, and cell apoptosis. We confirmed that the enhanced induction of ATF4, CHOP and PUMA is through the ISR because ISRIB (ISR inhibitor) blocked the combined treatment-induced upregulation of ATF4, CHOP and PUMA. Citation Format: Xiaobing Tian, Wafik S. El-Deiry. Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response [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 3334.

Nutraceuticals known to promote hair growth do not interfere with the inhibitory action of tamoxifen in MCF7, T47D and BT483 breast cancer cell lines.

Hair loss/thinning is a common side effect of tamoxifen in estrogen receptor (ER) positive breast cancer therapy. Some nutraceuticals known to promote hair growth are avoided during breast cancer therapy for fear of phytoestrogenic activity. However, not all botanical ingredients have similarities to estrogens, and in fact, no information exists as to the true interaction of these ingredients with tamoxifen. Therefore, this study sought to ascertain the effect of nutraceuticals (+/- estrogen/tamoxifen), on proliferation of breast cancer cells and the relative expression of ERα/β. Kelp, Astaxanthin, Saw Palmetto, Tocotrienols, Maca, Horsetail, Resveratrol, Curcumin and Ashwagandha were assessed on proliferation of MCF7, T47D and BT483 breast cancer cell lines +/- 17β-estradiol and tamoxifen. Each extract was analysed by high performance liquid chromatography (HPLC) prior to use. Cellular ERα and ERβ expression was assessed by qRT-PCR and western blot. Changes in the cellular localisation of ERα:ERβ and their ratio following incubation with the nutraceuticals was confirmed by immunocytochemistry. Estradiol stimulated DNA synthesis in three different breast cancer cell lines: MCF7, T47D and BT483, which was inhibited by tamoxifen; this was mirrored by a specific ERa agonist in T47D and BT483 cells. Overall, nutraceuticals did not interfere with tamoxifen inhibition of estrogen; some even induced further inhibition when combined with tamoxifen. The ERα:ERβ ratio was higher at mRNA and protein level in all cell lines. However, incubation with nutraceuticals induced a shift to higher ERβ expression and a localization of ERs around the nuclear periphery. As ERα is the key driver of estrogen-dependent breast cancer, if nutraceuticals have a higher affinity for ERβ they may offer a protective effect, particularly if they synergize and augment the actions of tamoxifen. Since ERβ is the predominant ER in the hair follicle, further studies confirming whether nutraceuticals can shift the ratio towards ERβ in hair follicle cells would support a role for them in hair growth. Although more research is needed to assess safety and efficacy, this promising data suggests the potential of nutraceuticals as adjuvant therapy for hair loss in breast cancer patients receiving endocrine therapy.

Synthesis, biological evaluation, and stability studies of raloxifene mono- and bis-sulfamates as dual-targeting agents

All three possible sulfamate derivatives of the selective estrogen receptor modulator Raloxifene (bis-sulfamate 7 and two mono-sulfamates 8–9) were synthesized and evaluated as inhibitors of the clinical drug target steroid sulfatase (STS), both in cell-free and in cell-based assays, and also as estrogen receptor (ER) modulators. Bis-sulfamate 7 was the most potent STS inhibitor with an IC50 of 12.2 nM in a whole JEG3 cell-based assay, with the two mono-sulfamates significantly weaker. The estrogen receptor-modulating activities of 7–9 showed generally lower affinities compared to Raloxifene HCl, diethylstilbestrol and other known ligands, with mono-sulfamate 8 being the best ligand (Ki of 1.5 nM) for ERα binding, although 7 had a Ki of 13 nM and both showed desirable antagonist activity. The antiproliferative activities of the sulfamate derivatives against the T-47D breast cancer cell line showed 7 as most potent (GI50 = 7.12 µM), comparable to that of Raloxifene. Compound 7 also showed good antiproliferative potency in the NCI-60 cell line panel with a GI50 of 1.34 µM against MDA-MB-231 breast cancer cells. Stability testing of 7–9 showed that bis-sulfamate 7 hydrolyzed by desulfamoylation at a surprisingly rapid rate, initially leading selectively to 8 and finally to Raloxifene 3 without formation of 9. The mechanisms of these hydrolysis reactions could be extensively rationalized. Conversion of Raloxifene (3) into its bis-sulfamate (7) thus produced a promising drug lead with nanomolar dual activity as an STS inhibitor and ERα antagonist, as a potential candidate for treatment of estrogen-dependent breast cancer.

MicroRNA-190b Targets RFWD3 in Estrogen Receptor-Positive Breast Cancer.

In the year 2020, breast cancer was the most common form of cancer worldwide. Roughly 70% of breast cancers are estrogen receptor-positive (ER+). MicroRNA-190b (miR-190b) has previously been reported to be upregulated in ER+ breast cancers. Previously, we have demonstrated that miR-190b is hypomethylated in ER+ breast cancers, potentially leading to its upregulation. To further study the role of miR-190b in ER+ breast cancer and to identify its clinically relevant targets in breast cancer. Patient cohort and cell line-based RNA-sequencing analysis. The Cancer Genome Atlas was used to obtain gene expression data and clinical information on patients with breast cancer. To identify messenger RNA (mRNA) targets for miR-190b, the ER+ breast cancer cell line T-47D was used to immunoprecipitate biotin-labeled miR-190b followed by RNA sequencing. Western blot was used to confirm miR-190b target. Patient survival based on miR-190b and selected target was studied using the Cancer Genome Atlas. In this study, we confirm that miR-190b is overexpressed in breast cancer via differential expression analysis and show that high expression of miR-190b results in more favorable outcomes in Luminal A patients, hazard ratio (HR) = 0.29, 95% confidence interval [CI] = 0.12-0.71, P = .0063. MicroRNA-190b target analysis identified RING finger and WD repeat domain 3 (RFWD3) as one of miR-190b regulatory targets in ER+ breast cancer. Survival analysis of RFWD3 showed that elevated levels result in poorer overall survival in patients with Luminal A breast cancer (HR = 2.22, 95% CI = 1.33-3.71, P = .002). Gene ontology analysis of our sequencing results indicates that miR-190b may have a role in breast cancer development and/or tumorigenesis and that it may be a suitable tool in characterization between the ER+ subtypes, Luminal A, and Luminal B. We show that miR-190b targets RFWD3 in ER+ breast cancers leading to lower RFWD3 protein expression. Low levels of RFWD3 are associated with better outcomes in patients with Luminal A breast cancer but not in patients with Luminal B breast cancer. These findings provide novel insights into miR-190b role in breast cancer and that its clinical relevance is subtype specific.

Isolation and Cytotoxic Activity Test of Alkaloids from Dichloromethane Fraction of Bark of Tampa Badak (Voacanga foetida (Blume) Rolfe) Against T47D Cell Line

Cancer is a problem in the health sector because this disease has an increasing incidence of morbidity and mortality. While the treatment still leaves some drug side effects, which cause discomfort for cancers sufferers. The arises because most cancer drugs work non-selectively. Because of some of the problems above, it is necessary to encourage the search for alternative drugs treatment of cancer. Voacanga foetida (Blume) Rolfe (Apocynaceae) is a species of plant from the genus Voacanga known as the tampa badak from West Sumatra. This plant is known to be source of indole alkaloids and also bis-indole alkaloids potential as cancer drugs. Therefore, in this study we are interested to explore the potency of this plant as natural product sources with potential cytotoxic activity. The study was started from the isolation process and continued by characterization of the isolated compounds and then testing their cytotoxic activity T47D breast cancer cell line. The isolation process of alkaloid compounds were performed by acid-base extraction, liquid-liquid fractionation, and followed by separation using column chromatography. Then,the potency of cytotoxic activity of the isolates were screened through Brine Shrimp Lethality Test (BSLT) method and evaluated by MTT assay. The structure of three isolated alkaloids (VFB-DB1.1; VFB-DB2.1; and VFB-DB2.2) were charachterized using spectroscopic analyses, including UV and FT-IR. One isolated compound (VFB-DB1.1) was also characterized using 1H NMR spectroscopy. Based on the similarity of 1H NMR spectra of isolated compound when compared with the literature, compound VFB-DB1.1 was identified as voacangine. Then, the BSLT and MTT assay result showed that VFB-DB1.1 exhibited LC50 value of 15.8 µg/mL againt Artemia salina L.and IC50 value of 8.9 µg/mL against T47D breast cancer cells, respectively.

Progestins and breast cancer hallmarks: The role of the ERK1/2 and JNK pathways in estrogen receptor positive breast cancer cells

Progestins used in hormonal contraceptives and menopausal hormone therapy (MHT) have been linked to increased breast cancer risk. Whether the association holds for all progestins is unclear and the underlying mechanisms remain poorly understood. We directly compared the effects of four progestins (medroxyprogesterone acetate (MPA), norethisterone acetate (NET-A), levonorgestrel (LNG) and drospirenone (DRSP)) to each other and the natural progestogen progesterone (P4) on selected cancer hallmarks. To provide mechanistic insight into these effects, we assessed the role of the progesterone receptor (PR), and the extracellular signal-related kinase (ERK1/2) and c-Jun N terminal (JNK) signaling pathways. We showed that the increased proliferation of the luminal T47D breast cancer cell line by P4 and all progestins, albeit to different extents, was inhibited by PR knockdown and inhibition of both the ERK1/2 and JNK pathways. While knockdown of the PR also blocked the upregulation of MKI67 and CCND1 mRNA expression by selected progestogens, only a role for the ERK1/2 pathway could be established in these effects. Similarly, only a role for the ERK1/2 pathway could be confirmed for progestogen-induced colony formation, whereas both the ERK1/2 and JNK pathways were required for cell migration in response to the three older progestins implicated in the etiology of breast cancer, MPA, NET-A and LNG. Together our results show that all the progestins elicit their effects on cell proliferation via a mechanism requiring the PR, ERK1/2 and JNK pathways. While the ERK1/2 and JNK pathways are also required for increased cell migration by the older progestins, only a role for the ERK1/2 pathway could be established in their effects on colony formation. Notably, the cytoplasmic PR was not needed for activation of the ERK1/2 pathway by the progestogens. Given that DRSP showed significantly lower proliferation than MPA and NET-A, and that it had no effect on breast cancer cell migration and colony formation, hormonal formulations containing the newer generation progestin DRSP may provide a better benefit/risk profile towards breast cancer than those containing the older generation progestins.

RAD50 Deficient in a Breast Cancer Model Predicts Sensitivity to PARP Inhibitors.

Breast and ovarian tumors with pathogenic variants in BRCA1 or BRCA2 genes are more sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi) treatment than wild-type tumors. Pathogenic variants in non-BRCA1/2 homologous recombination repair genes (HRR) also concede sensitivity to PARPi treatment. RAD50 participates in the Mre11-Rad50-Nbn (MRN) complex of the HRR pathway and plays an important role in DNA repair. The objective of this study is to evaluate whether RAD50 protein deficiency modulates the PARPi response in breast cancer cell lines. T47D breast cancer cell line was modified using small interfering RNA and CRISPR/Cas9 technology, to knockout the RAD50 gene. PARPi response (niraparib, olaparib and rucaparib alone or in combination with carboplatin), in T47D and T47D-edited clones, was evaluated by cell viability, cell cycle, apoptosis and protein expression analyses. Treatment with niraparib and carboplatin exerted a synergistic effect on T47D-RAD50 deficient cells and an antagonistic effect on T47D cells parental. Cell cycle analysis demonstrated an increase in the G2/M population in cells treated with niraparib or rucaparib alone or in combination with carboplatin. T47D-RAD50 deficient cells treated with rucaparib and carboplatin exhibited twofold levels in late apoptosis, also showing differences in PARP activation. All T47D RAD50 deficient clones treated with niraparib or rucaparib combined with carboplatin, or rucaparib alone showed increased levels of H2AX phosphorylation. T47D RAD50 deficient cells treated with PARP inhibitors alone or in combination with carboplatin showed cell cycle arrest in the G2/M phase, leading to death by apoptosis. Thus, RAD50 deficiency may be a good biomarker for predicting PARPi response.

Abstract LB_A24: Baicalein suppresses tumorigenesis of the ER𝛼 positive of breast cancer cells by targeting multiple signaling pathways

Abstract The anti-tumor properties of baicalein, a flavonoid compound, have been extensively documented. However, the specific effects of baicalein on ER𝛼 positive breast cancer cell lines, namely T47-D and MCF-7, are less well-known. In this study, we conducted comprehensive in vitro experiments to elucidate the comparative impacts of baicalein on these two cell lines. Our results demonstrate that baicalein exerts robust anti-cancer effects, inducing morphological changes, reducing colony formation, and inhibiting cell proliferation in both T47-D and MCF-7 cells. These effects were more pronounced at higher concentrations. Additionally, baicalein treatment impeded the migratory capabilities of both cell lines, as shown by wound healing assays. Furthermore, our data show that baicalein triggers apoptosis, leading to an increase in early apoptotic, late apoptotic, and dead cells. This apoptotic process appears to be caspase 3/7-dependent. Interestingly, the pro-apoptotic effects were more potent in T47-D cells, accompanied by significant upregulation of pro-apoptotic genes and downregulation of anti-apoptotic markers. Baicalein treatment also induced oxidative stress and DNA damage, evidenced by an increase in reactive oxygen species (ROS) and phosphorylation of ATM and H2A.X. Importantly, baicalein inhibited the PI3K signaling pathway, a key mediator of cancer progression. Genome-wide transcriptomic analysis revealed significant changes in gene expression patterns, with baicalein treatment causing both upregulation and downregulation of several genes. Pathway analysis revealed that cell cycle and ferroptosis pathways were activated in both cell lines upon baicalein treatment. In conclusion, our findings highlight the potential of baicalein as a therapeutic agent against breast cancer, while also revealing nuanced differences in response between the T47-D and MCF-7 breast cancer cell lines. Citation Format: Besa Xhabija, Aidan J. McLaughlin, Ibiere Lovelyn Epelle, Tristan H. Nielson, Elisabeta Ujeniuc, Mariam I. Duhaini. Baicalein suppresses tumorigenesis of the ER𝛼 positive of breast cancer cells by targeting multiple signaling pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A24.

Investigating the Properties and Cytotoxicity of Cisplatin-Loaded Nano-Polybutylcyanoacrylate on Breast Cancer Cells

Background: This study aimed to develop a novel drug formulation using polybutylcyanoacrylate (PBCA) nanoparticles to deliver cisplatin, a commonly used chemotherapeutic agent for breast cancer treatment.Materials and Methods: PBCA nanoparticles were synthesized using a mini-emulsion polymerization method, and the resulting NPs were comprehensively characterized for their physical properties, such as size, size distribution, zeta potential, drug loading, and encapsulation efficiency. In addition, the cytotoxicity of the NPs was assessed, along with their ability to release the entrapped drugs over time. Results: The results showed that the PBCA nanoparticles had a mean size of 457 ± 7.4 nm, a size distribution of 0.253±0.011 and a negative zeta potential of -12.3 ± 1.3 mV. The drug encapsulation efficiency and loading capacity of cisplatin-PBCA were found to be 45.6 ± 2.7% and 3.5 ±0.8%, respectively; The release of the drug from the PBCA was estimated to be approximately 12.2±1.1% after 45 hours. The cytotoxic effects of the nanoparticle formulation were significantly enhanced compared to the free drug. The cytotoxicity of cisplatin-PBCA was evaluated in the T-47D breast cancer cell line, showing promising results as a potential drug formulation for breast cancer therapy. Conclusions: These findings suggest that cisplatin-PBCA may offer advantages over traditional cisplatin formulations, potentially improving the efficacy and reducing the toxicity of breast cancer treatment.

Anti-breast cancer potential of a new xanthine derivative: In silico, antiproliferative, selectivity, VEGFR-2 inhibition, apoptosis induction and migration inhibition studies

BackgroundThe overexpression of VEGFR-2 receptors in breast cancer provides a valuable approach to anticancer strategies. Targeting VEGFR-2, a new semisynthetic compound (T-1-MCPAB) has been designed. MethodsComputational methods (ADMET, toxicity, DFT, Molecular Docking, Molecular Dynamics Simulations, MM-GBSA, PLIP, and PCAT) were conducted. In addition to the semi-synthesis, in vitro studies (anti-VEGFR-2, anti-proliferative, flow cytometry, and wound scratch assay) were employed. ResultsADME and toxicity profiles of T-1-MCPAB studies indicated its overall drug-likeness showing results much better than Sorafenib. Then, T-1-MCPAB’s exact 3D structure, stability, and reactivity were evoked by the DFT calculations. Molecular docking, molecular dynamics simulations, MM-GPSA, PLIP, and PCAT studies denoted the correct binding and inhibiting potential of T-1-MCPAB, towards VEGFR-2 protein. After the semisynthesis, T-1-MCPAB inhibited VEGFR-2 with an IC50 of 0.135 µM, which was comparable to sorafenib's IC50 of 0.0591 µM. T-1-MCPAB also showed a notable performance against MCF7 and T47D breast cancer cell lines with IC50 values of 30.95 µM and 63.64 µM, respectively, and had high selectivity index values of 3.7 and 1.8, respectively. Furthermore, T-1-MCPAB influenced early and late apoptosis and significantly decreased the potential of MCF7 cells to heal and migrate. ConclusionT-1-MCPAB is a promising VEGFR-2 inhibitor with potential for breast cancer treatment. Further chemical and biological studies are needed to explore its potential as a therapeutic agent.

Development and characterisation of a novel inhibitory anti-GH monoclonal antibody.

Excess growth hormone (GH) has been implicated in multiple cancer types and there is increasing interest in the development of therapeutic inhibitors targeting GH-GH receptor (GHR) signalling. Here we describe a panel of anti-GH monoclonal antibodies (mAbs) generated using a hybridoma approach and identify two novel inhibitory mAbs (1-8-2 and 1-46-3) that neutralised GH signalling. mAbs 1-8-2 and 1-46-3 exhibited strong inhibitory activity against GH-dependent cell growth in a Ba/F3-GHR cell viability assay, with EC50 values of 1.00 ± 0.27 and 0.5 ± 0.1 µg/mL, respectively. Cross-reactivity with the human placental hormones, placental lactogen (PL) and placental GH, was observed by ELISA, but neither antibody cross-reacted with mouse GH or human prolactin (PRL). mAb 1-8-2 had a binding affinity for GH of KD 0.62 ± 0.5 nM, while mAb 1-46-3 had a KD of 2.68 ± 0.53 nM, as determined by bio-layer interferometry. mAb 1-46-3 inhibited GH-dependent signal transduction in T-47D and LNCaP cancer cell lines and reduced GH-dependent cell growth and migration in the breast cancer cell line T-47D. mAb 1-46-3 inhibited T-47D cell viability more effectively than the GHR antagonist B2036. In conclusion, we describe two novel inhibitory anti-GH mAbs and provide in vitro evidence supporting development of these entities as anti-cancer therapeutics.

SAT428 The Effects Of Diphenyl Phthalate (DP) On Estrogen Receptor Alpha (ERα;) And Tumor Suppressor Gene (BRCA-1) In Breast Cancer Cells

Abstract Disclosure: K. Whittaker: None. E. Hallman: None. A. Zanib: None. S. Pfiffner: None. R. Yaldo: None. S. Dinda: None. Phthalates are a group of compounds that have been linked to xenoestrogenic activity which can lead to the proliferation of breast cancer cells. Specifically, Diphenyl Phthalate (DP) is a compound found in cosmetics, toys, plastic containers, and medical equipment. Previous studies have linked phthalates as endocrine-disrupting compounds (EDC) which may have an effect on diseases such as breast cancer. Due to the endocrine-disrupting actions of phthalates, it is critical to further investigate DP’s potential impact on breast cancer cells. This study investigates the estrogen-like effects of DP, both alone and in combination with hormones and antihormones on T-47D and MCF-7 breast cancer cell lines. The breast cancer cells were cultured in a medium that contained 5% charcoal-stripped fetal bovine serum (FBS) to ensure depletion of any endogenous steroids or growth factors. The cells were treated with varying concentrations of DP (25-250uM) for 24 hours. Through western blot analysis, alterations in the expression of ERα; related to the varying concentrations of DP were observed. A concentration-dependent decrease of ERα; expression levels was observed in both cell lines when compared to the control. A concentration-dependent increase of BRCA1 was seen in both cells. The optimal concentration of DP was revealed to be 200uM in T-47D breast cancer cells as it exhibited the most estrogen-like effect. The optimal concentration (200uM) was then used alone and in combination with hormones and antihormones for further investigation of DP on T47-D cells. Treatment with DP, E2 and the combination of DP and E2 for 24 hours showed a significant down-regulation of ERα in T-47D cells when compared to the control. These effects were sensitive to the presence of ICI showing significant down-regulation of ERα (approximately 80%) in T-47D cells. Image cytometric analysis with propidium iodide staining was utilized to examine the effects of DP on cellular viability in T-47D cells. Cells were treated for 6 days with DP concentrations ranging from 25-250 µM, and all concentrations displayed an increase in cell proliferation compared to the control in T47D cells. DP alone and in combination with E2 showed an increase in cell proliferation that was reversed when treated in combination with anti-hormones. RT-qPCR studies in T-47D cells revealed a transcriptional expression of ESR1 and BRCA-1 mRNA levels that correlate with the translational data from western blot analyses. Confocal microscopy, cell viability, and RT-qPCR analyses are in progress as well as evaluation on the effects of DP with hormones and antihormones on MCF-7 cells. Our studies offer an intriguing direction to explore the molecular mechanisms of DP as an EDC on the steroid receptors and tumor suppressor gene in breast cancer cells. Presentation: Saturday, June 17, 2023

Ultra Sensitive Breast Cancer Cell Lines Detection Using Dual Nanocavities Engraved Junctionless FET.

This article reports breast cancer cell lines (Hs578T, MDA-MB-231, MCF-7, and T47D) and healthy breast cells (MCF-10A) detection based on the modulation of its electrical properties by deploying dual nanocavity engraved junctionless FET. The device has a dual gate to enhance gate control and has two nanocavities etched under both gates for breast cancer cell lines immobilization. As the cancer cells are immobilized in the engraved nanocavities, which were earlier filled with air, the dielectric constant of the nanocavities changes. This results in the modulation of the device's electrical parameters. This electrical parameters modulation is then calibrated to detect the breast cancer cell lines. The reported device demonstrates a higher sensitivity toward the detection of breast cancer cells. The JLFET device optimization is done for improving the performance by optimizing the nanocavity thickness and the SiO2 oxide length. The variation in the dielectric property of cell lines plays a key role in the detection technique of the reported biosensor. The sensitivity of the JLFET biosensor is analyzed in terms of ∆VTH, ∆ION, ∆gm , and ∆SS . The reported biosensor shows the maximum sensitivity for T47D ( κ = 32 ) breast cancer cell line with ∆VTH = 0.800 V, ∆ION = 0.165 mA/μm, ∆gm = 0.296 mA/V-μm , and ∆SS = 5.41 mV/decade. Moreover, the effect of variation in the occupancy of the cavity by the immobilized cell lines has also been studied and analyzed. With increased cavity occupancy the variation in the device performance parameter enhances Further, the sensitivity of the proposed biosensor is compared with the existing biosensors and it is reported to be highly sensitive as compared to the existing biosensors. Hence, the device can be utilized for array based screening of cell lines of breast cancer and diagnosis with the benefit of easier fabrication and cost effectiveness of the device.

Radiosensitizing Effects of CDK4/6 Inhibitors in Hormone Receptor-Positive and HER2-Negative Breast Cancer are through Downregulating DNA Repair Mechanism and NF-κB-Signaling Pathway

CDK4/6 inhibitors combined with endocrine therapy are demonstrated to prolong survival for hormone receptor (HR)-positive and HER2-negative advanced breast cancer. We sought to assess whether cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors can enhance the radiosensitivity and their underlying mechanisms in this subtype of breast cancer through both in vitro and in vivo approaches. We used MCF-7 and T-47D (HR-positive and HER2-negative) breast cancer cell lines, and different doses of CDK4/6 inhibitors (ribociclib and palbociclib) and radiotherapy (RT) to assess the biological functions and mechanisms through in vitro study. The radiation enhancing effect was assessed by clonogenic assay, numbers of the DNA damage response-related molecules, γH2AX and 53BP1, were assessed by immunofluorescence. The p-ERK, c-Myc, and DNA-double strand break (DNA-DSB)-related molecules, p-DNA-PKcs, and p-ATM, were assessed by western blotting. We used NF-κB p65 Transcription Factor Assay Kit to evaluate the transcriptional activity of NF-κB. We evaluated the antitumor effect of combined RT (2 Gy every other day for total 6 Gy) and ribociclib through the MCF-7 orthotopic xenograft model. The synergistic effects of combining RT with different concentrations of ribociclib and palbociclib pretreatment were demonstrated by colonogenic assay. We revealed that CDK4/6 inhibitors synergistically increased the numbers of RT-induced γH2AX and 53BP1, downregulated the expression of p-DNA-PKcs and p-ATM activated by RT, and diminished the expression of RT-triggering p-ERK expression, and NF-κB activation and its down-streaming gene, c-Myc. When compared with control, ribociclib, and RT group, combined ribociclib and RT significantly shrunk the tumor growth of MCF-7 cell xenograft tumors, and downregulated the immunohistochemical expression of p-ERK, p-NF-κB p65, and c-Myc than control group. Our results demonstrated that combining CDK4/6 inhibitors enhanced the radiosensitivity in HR-positive and HER2-negative breast cancer cells, and conceivably radioenhancing mechanisms of CDK4/6 inhibitors are at least through lessening DNA-DSB repairing processes and weakening the activation of ERK signaling and NF-κB signaling by RT.

Autoantibody Diversity Is Augmented in Women with Breast Cancer and Is Related to the Stage of the Disease.

Breast cancer (BC) is the most frequent malignant neoplasia and leading cause of cancer mortality for women. A timely diagnosis of BC is crucial to ensure the best chances of survival. Among the various screening tools for BC, antibodies directed towards self-antigens or tumor-associated antigens (autoantibodies) have emerged as an alternative to image-based screening modalities. However, little attention has been paid to the global diversity of autoantibodies. This work aimed to analyze the diversity of autoantibodies reactive to antigens expressed by the BC cell line T47D in the sera of Mexican women with BC, benign breast pathology (BBP), or without breast pathology (WBP). We found that the diversity of antibodies in the sera was higher in the BC and BBP groups than in the WBP group. Likewise, the diversity changed with the progression of BC. Our results show and measure the complexity of the antibody response in breast health and disease.

Efficacy of Different Solvents in the Extraction of Bioactive Compounds and Anticancer Activities of Thyme (Thymus vulgaris L.) Leaves and Twigs

Thyme contains phytochemicals that exhibit cytotoxic and cytogenic activities. Differ solvents have different abilities in dissolving plant bioactive compounds. The objective of this study is to determine the anticancer properties of thyme leaves and twigs including profile of bioactive compounds, cytotoxicity, ability of apoptosis induction and caspase-3 activation of different solvent extracts on T47D breast cancer cell line. An experimental study was conducted in eight groups. T47D breast cancer cell line was treated with thyme extracts as treatment group, doxorubicin as positive control, while untreated T47D cells were used as negative control. Thyme was extracted by maceration using 6 different solvents: methanol, ethanol, chloroform, ethyl acetate, dichloromethane, and n-hexane. Bioactive compounds of all extracts were analyzed by GC-MS. Cytotoxicity was investigated through MTT assay on T47D breast cancer cell line. The percentage of apoptosis and caspase-3 were detected by flow cytometry using Annexin V-PI and BD Cytofix/Cytoperm, respectively. Thymol as the main bioactive compound in thyme was found in all extracts. MTT results revealed that six solvents extracts had moderate to weak cytotoxicity with dichloromethane had the lowest IC50 values at 120.23 µg/ml. All thyme extracts promoted apoptosis in T47D cells with apoptosis percentage of methanol, chloroform, ethyl acetate, ethanol, dichlorometane, n-Hexane and doxorubicin were 82.67; 80.98; 73.13; 72.28; 60.62; 27.74 and 98.43%, respectively. Among all extracts, methanol extract showed the highest apoptosis and caspase-3 activation percentage. We conclude that all thyme extracts had anticancer properties in T47D breast cancer cell line although efficacy of each solvent extract was differed.

Preparation, Characterization and Cytotoxic Studies of Cisplatin-containing Nanoliposomes on Breast Cancer Cell Lines

Objective: Today, cancer is one the most important challenges in modern medicine. Breast carcinoma is one type of cancer that is treated with cisplatin, a chemotherapy drug. By using liposomal nanocarriers, this study seeks to increase the therapeutic efficiency of cisplatin. Method: The zeta potential, particle size, and drug-release characteristics of nanoliposomal cisplatin were evaluated after it had been synthesized using the reverse phase evaporation technique. The cytotoxicity rate of nanoliposomal cisplatin was then assessed using the T-47D breast cancer cell line. Results: This study’s liposomal nanoparticles (NPs) had a zeta potential of -24.9 mV and a particle size of 342.3 nm. 3.51% and 79.6%, respectively, were found to be the drug loading level and amount of encapsulated drug. A significant improvement over the free drug was seen in this nanoliposome’s cytotoxic effect on the T-47D breast cancer cell line (P<0.05). Conclusion: According to what we have discovered, cisplatin liposomal nanocarriers may prove to be a cutting-edge chemotherapy treatment for breast cancer.

Cryptobrachytone C from Cryptocarya pulchrinervia (Kosterm) Leaves on Proliferation, Apoptosis, Migration and Clonogenicity of MCF-7 and T47D Cell Lines.

Cryptocarya pulchrinervia is an Indonesian indigenous plant that grows in Sumatra, Kalimantan and Papua. One of the new compounds extracted from this plant was cryptobrachytone C, which was known to be cytotoxic against cancer cells of Murine leukemia P388 with IC50 10.52 μM. In this study, the cytotoxicity and anticancer properties of cryptobrachytone C on proliferation, apoptosis, migration and clone formation of MCF-7 and T47D breast cancer cell lines were examined, which had not previously been done before. The cytotoxicity of the compound was measured using an MTT (3- (4,5-dimethylthiazol-2- yl) -2,5-di-phenyl-tetrazolium bromide) assay. The cell proliferation was measured using a BrdU assay, and the cell apoptosis was measured using annexin-V FITC, while the cell migration was measured using a transwell filter. The cytotoxic test result demonstrated that cryptobrachytone C was cytotoxic against MCF-7 cells with IC50 12.94 ± 0.32 μM but not against T47D cells with IC50 65.33 ± 2.33 μM nor against normal MRC-5 cells with IC50 122.57 ± 19.84 μM. The cell proliferation assay showed that cryptobrachytone C at IC50 concentration had antiproliferative properties against MCF-7 cancer cell lines (p < 0.05) but did not significantly reduce T47D cell proliferation (p < 0.07). Although the results of the cell apoptosis test showed that cryptobrachytone C could induce the apoptosis of the MCF-7 and T47D cells, it was insignificant (p > 0.05). The cell migration test showed that cryptobrachytone at IC50 concentrations could inhibit the migration of the MCF-7 and T47D cells. The clonogenic test showed that cryptobrachytone C at IC50 concentration can induce the inhibition of the formation of MCF-7 and T47D cell colonies. The cryptobrachytone C anti-cancer character was more signi icant on the MCF-7 cell line compared to the T47D. This study showed that cryptobrachytone C was cytotoxic and had potential as an anti-cancer compound against MCF-7 and T47D breast cancer cell lines.