Metastatic Breast Cancer Cell Line Research Articles

3,6-dichloro-1,2,4,5-Tetrazine Assayed at High Doses in the Metastatic Breast Cancer Cell Line MDA-MB-231 Reduces Cell Numbers and Induces Apoptosis

Background: The lack of treatment options for patients with chemotherapy-resistant cancers is pushing the field toward the development of new therapies. 1,2,4,5-tetrazine derivatives are a class of heterocyclic compounds that exhibit a broad spectrum of antitumor activities. Objective: The purpose of this study was to assess the biological activity of four s-tetrazine derivatives by substitution of two chloride atom of 3,6-dichloro-1,2,4,5-tetrazine with long hydrophobic side chains. Methods: We analyzed the anti-proliferative effects of four s-tetrazine derivatives with MTT assay and their pro-apoptotic effect with AV/ IP flow cytometry analysis and Hoechst 33342 staining. Results: We demonstrated that 3,6-dichloro-1,2,4,5-tetrazine (compound (1)) has a cytotoxic effect and induces apoptosis. Conclusion: 3,6-dichloro-1,2,4,5-tetrazine presents a new cytotoxic drug against metastatic breast cancer cell line MDA-MB-231 in vitro.

53BP1 Accumulation in Circulating Tumor Cells Identifies Chemotherapy-Responsive Metastatic Breast Cancer Patients.

Evidence suggests that the DNA end-binding protein p53-binding protein 1 (53BP1) is down-regulated in subsets of breast cancer. Circulating tumor cells (CTCs) provide accessible “biopsy material” to track cell traits and functions and their alterations during treatment. Here, we prospectively monitored the 53BP1 status in CTCs from 67 metastatic breast cancer (MBC) patients with HER2- CTCs and known hormone receptor (HR) status of the primary tumor and/or metastases before, during, and at the end of chemotherapeutic treatment with Eribulin. Nuclear 53BP1 staining and genomic integrity were evaluated by immunocytochemical and whole-genome-amplification-based polymerase chain reaction (PCR) analysis, respectively. Comparative analysis of CTCs from patients with triple-negative and HR+ tumors revealed elevated 53BP1 levels in CTCs from patients with HR+ metastases, particularly following chemotherapeutic treatment. Differences in nuclear 53BP1 signals did not correlate with genomic integrity in CTCs at baseline or with nuclear γH2AX signals in MBC cell lines, indicating that 53BP1 detected features beyond DNA damage. Kaplan–Meier analysis revealed an increasing association between nuclear 53BP1-positivity and progression-free survival (PFS) during chemotherapy until the final visit. Our data suggest that 53BP1 detection in CTCs could be a useful marker to capture dynamic changes of chemotherapeutic responsiveness in triple-negative and HR+ MBC.

Exosomes Derived from Brain Metastatic Breast Cancer Cells Destroy the Blood-Brain Barrier by Carrying lncRNA GS1-600G8.5.

Brain metastasis is a major cause of death in breast cancer patients. The greatest event for brain metastasis is the breaching of the blood-brain barrier (BBB) by cancer cells. The role of exosomes in cancer metastasis is clear, whereas the role of exosomes in the integrity of the BBB is unknown. Here, we established a highly brain metastatic breast cancer cell line by three cycles of in vivo selection. The effect of exosomes on the BBB was evaluated in vitro by tracking, transepithelial/transendothelial electrical resistance (TEER), and permeability assays. BBB-associated exosomal long noncoding RNA (lncRNA) was selected from the GEO dataset and verified by real-time PCR, TEER, permeability, and Transwell assays. The cells obtained by the in vivo selection showed higher brain metastatic capacity in vivo and higher migration and invasion in vitro compared to the parental cells. Exosomes from the highly brain metastatic cells were internalized by brain microvascular endothelial cells (BMECs), which reduced TEER and increased permeability of BBB. The exosomes derived from the highly metastatic cells promoted invasion of the breast cancer cells in the BBB model. lncRNA GS1-600G8.5 was highly expressed in the highly brain metastatic cells and their exosomes, as compared to the samples with reduced metastatic behavior. Silencing of GS1-600G8.5 significantly abrogated the BBB destructive effect of exosomes. GS1-600G8.5-deficient exosomes failed to promote the infiltration of cancer cells through the BBB. Furthermore, BMECs treated with GS1-600G8.5-deprived exosomes expressed higher tight junction proteins than those treated with the control exosomes. These data suggest the exosomes derived from highly brain metastatic breast cancer cells might destroy the BBB system and promote the passage of cancer cells across the BBB, by transferring lncRNA GS1-600G8.5.

Effects of Vacuolar H+‐ATPase Inhibition and the a4 Isoforms on Secretion and Activation of Cathepsin D in MDA‐MB231 Breast Cancer Cells

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are important for acid‐base balance in the blood and sperm maturation. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. V‐ATPases are hypothesized to create a localized low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells are involved in metastasis by cleaving extracellular matrix proteins and activating other proteases, which facilitates tumor invasion. We are interested in a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor showed a trend of reducing the activity of secreted cathepsin B, but not cathepsin L. However, that trend, was not statistically significant. Cathepsin D is another protease that is secreted by cancer cells including metastatic breast cancer. For this study we looked at the activity of cathepsin D following inhibition of V‐ATPases. Our preliminary data indicates that inhibition of V‐ATPases reduces the activity of secreted cathepsin D. To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for the a4 isoform of the V‐ATPase “a” subunit. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. We hypothesize that the a4 isoform knockout cells will have reduced activation of secreted cathepsin D while not effecting activation of cathepsin L. Separate abstracts submitted by our lab are focused on the a1, a2 and a3 isoform knockouts.Support or Funding InformationAnderson Summer Research Assistantship

Effects of Vacuolar H+‐ATPase Inhibition and the a1 and a3 Isoforms on Secretion and Activation of Cathepsins in MDA‐MB231 Breast Cancer Cells

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation and synaptic signaling. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases create a low pH in a localized environment which promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells cleave extracellular matrix proteins and activate other proteases, facilitating tumor cell invasion. Our lab is investigating a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor reduced the activity of secreted cathepsin B, but not cathepsin L. However, although there was a trend, the difference in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data supports the conclusion that there is a significant difference in the effects of V‐ATPase inhibition on secreted cathepsins. Inhibiting V‐ATPases does not appear to affect the activity of secreted cathepsin L, however it reduces the activity of secreted cathepsins B and D To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for two of the four V‐ATPase “a” subunit isoforms, a1 and a3. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. The a1 isoform is found in intracellular compartments. We hypothesize that the a3 isoform knockout cells will have reduced activation of secreted cathepsins B and D while not effecting activation of cathepsin L. We predict that the a1 isoform knockout will affected the activity of intracellular cathepsins, but not affect the activity of secreted cathepsin B, D or L. Separate abstracts submitted by our lab are focused on the a1 and a3 isoform knockouts.Support or Funding InformationDenison University Anderson AssistantshipDenison University Research Fund

Pharmacological Targeting of SERCA in Breast Cancer

About 12% of U.S. woman develop breast cancer at some time during their lifetime. In addition to surgery and radiation therapy, chemotherapy is a key part of treatment following diagnosis. However, current drugs have variable success due to challenging disease subtypes that exhibit drug resistance. As a result, over 40,000 women die every year from intractable metastatic breast cancer. Thus, various new targets against breast cancer cells are currently under study. Aberrant intracellular Ca2+ homeostasis is observed in most cancer cells, hence, proteins that modulate Ca2+ levels may serve as a potential therapeutic target. Thapsigargin (TG) and cyclopiazonic acid (CPA) are agents known to potently inhibit the endoplasmic/sarcoplasmic reticulum (ER/SR) Ca2+‐ATPase (SERCA) and have shown high efficacy for impairing cancer cell growth. However, TG and CPZ are highly cytotoxic for normal cells. In recent years, our lab used spectrometry UV‐visible Ca2+ loading and ATPase activity assays in SR microsomes to identify novel drugs that target SERCA in striated muscle via an inhibitory, calcium‐dependent mechanism. These include drugs of the benzothiazepine (BZT) family (K201, CGP, PH00095) and antiepileptic drugs (sipatrigin, pimozide). The action of these new agents would cause partial SR depletion in cells by affecting Ca2+ sequestration at low cytosolic Ca2+. Interestingly, some of these drugs have been found to be cytoprotective under various stressful conditions. It is possible that these drugs prevent damaging SR Ca2+ overload under pathological conditions, including ischemia, via its action on SERCA. These findings contrast with the cytotoxicity associated with TG and CPZ. We compared the efficacy of these new SERCA inhibitors versus TG/CPZ on cell viability of breast cancer cell lines MCF‐7 and MDA‐MB‐231. MTS assay revealed TG and CPZ were more efficacious than our new compounds to decrease MCF‐7 viability. However, none of the agents tested significantly affected MDA‐MB‐231, a more metastatic breast cancer cell line. When paired with chemotherapeutics, K201, TG and CPZ synergistically reduce MCF‐7 viability. Still, the effects of chemotherapeutics on MDA‐MD‐231 was not significantly changed. Based on our studies, targeting intracellular Ca2+ homeostasis may serve as a potential therapeutic target for some breast cancer cell types but not all. Here, we find that drugs of the BZT family, as well as some antiepileptics, reduce MCF‐7 cell viability and potentially synergize with the action of chemotherapeutics. However, MDA‐MB‐231 remain largely unaffected by these agents. In principle, we thought that MDA‐MB‐231 may not have significant intracellular Ca2+ stores; yet immunoblot analysis reveal comparable levels of SERCA1/2 protein expression in both cell lines. Still, MDA‐MB‐231 seems to preferentially utilize Ca2+ entry from the extracellular space for cell survival. Further studies of plasma membrane Ca2+ transporters are required to elucidate the dependence of intracellular Ca2+ signaling among these breast cancer cell lines.Support or Funding InformationCT Moy Endowed Fund, Eskridge Foundation Grant, SIUSOM ‐ Team Development Grant

Effects of Vacuolar H+‐ATPase Inhibition and the a2 Isoform on Secretion and Activation of Cathepsin B in MDA‐MB231 Breast Cancer Cells

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are important for immune functionality, synaptic signaling, and other processes. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases create a low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells are involved in metastasis. Cathepsins cleave extracellular matrix proteins and activate other proteases, facilitating tumor invasion. We are interested in a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor reduced the activity of secreted cathepsin B, but did not cathepsin L. However, although there was a trend, difference in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data builds on that trend to show a significant difference in the activity of secreted cathepsin B. To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for the V‐ATPase a2 isoform of subunit “a”. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. Additionally, it was found that the a1 and a2 isoforms are not present on the cell surface, thus we hypothesize that the a2 isoform knockout will not affect the activity of secreted cathepsin B, but will affect the activity of cathepsin B in the cell interior. Separate abstracts submitted by our lab are focused on the a1, a3, and a4 isoform knockouts.Support or Funding InformationDenison University Anderson Summer Research Assistantship

Examining the Role of p190RhoGAP on the Organization of the Actin Cytoskeleton in Breast Epithelial Cells

Angiogenesis (i.e. blood vessel formation) and metastasis of tumors are two of the most dangerous aspects of malignant cancer cells. Metastasis requires spatial and temporal coordination of protein complexes known to affect cell adhesiveness and migration/proliferative properties. These protein complexes include E‐cadherin, Rho GTPases (e.g., Rac1 and RhoA), and the actin cytoskeleton. Rho GTPases perform different functions in the cell and have been shown to affect cancer progression, wound healing, and inflammation through regulation of cadherin and actin. For Rho GTPases to function, additional proteins increase or decrease their activity in the cell. These include guanine‐nucleotide‐exchange factors (GEFs) and GTPase‐activating proteins (GAPs). The current study aims to examine p190RhoGAP (GTPase activation protein (GAP) for RhoA) on cell adhesion and migration dynamics of normal Human Mammary Epithelial cells (HMECs) and the metastatic adenocarcinoma breast cancer cell line, MCF‐7. Cells were examined under endogenous conditions, as well as transfected with p190RhoGAP wild‐type and a catalytically inactive p190 RhoGAP‐R1283A mutant that locks RhoA into a constitutively active state. Overexpression of wild‐type and catalytically inactive p190RhoGAP had no major effect on actin stress fiber and cortical actin at cell‐cell contacts. However, HMEC cells overexpressing p190 RhoGAP‐R1283A showed reduced cytosolic RhoA protein, suggesting that loss of RhoA activity affects RhoA protein levels. Although no significant change in wound closure was observed, transwell cell invasion assays show a decreased rate of cell invasion in the presence of the chemo‐attractant, epidermal growth factor (EGF) in MCF‐7 cells. These data indicate a role for p190RhoGAP in the regulation of RhoA during cell migration and invasion. Loss of p190RhoGAP catalytic activity could affect the production of RhoA protein, leading to a decrease in the formation of actin structures (i.e., lamellipodia and filopodia) and a decrease in cell migration and invasion. Future work aims to determine how p190RhoGAP controls spatiotemporal regulation of protein complexes involved in metastatic cancer.Support or Funding InformationSaint Mary's School of Science Research Program and the Robert W. and Beverly J. Summers Scholarship

New BI and TRI-Thiazole copper (II) complexes in the search of new cytotoxic drugs against breast cancer cells

New thiazolyl derivatives (BT and TT) and their copper (II) complexes [Cu2Cl2(BT)2] (Cu-BT) and [Cu4ClO2(TT)2]PF6⋅3.5H2O (Cu-TT) were synthesized and characterized by elemental analysis, 1H NMR and 13C NMR, HRMS, X-ray diffraction, IR and UV–Vis spectroscopies. The crystal structure of Cu-BT shows the formation of a dinuclear complex where each copper(II) center is bonded to two thiazol N atoms, from different BT ligands, one deprotonated amide N atom, an O atom from the ester terminal groups and a chlorine atom. The structure found for Cu-TT is a positively charged tetranuclear moiety containing two deprotonated TT ligands, a chlorine anion, two hydroxide anions acting as bridges between the copper centers and a water molecule. The cytotoxic activity of both copper complexes was evaluated on metastatic breast cancer cell lines, characterized for its rapidly dividing behavior. Both, Cu-BT and Cu-TT, show higher cytotoxic activity against these tumor cells than free BT and TT and also than cisplatin. In addition, we found that both complexes interact with DNA. Consistently, they also show cytotoxicity against a rapidly dividing non-tumor cell line, although with higher IC50, being such interaction and selectivity an indicator of the possible coexistence of more than one mechanism of action.

Abstract P3-10-16: Identification of molecules that enhance the efficacy of eribulin in TNBC and IBC cell lines

Abstract BACKGROUND: Eribulin mesylate, a non-taxane microtubule-dynamics inhibitor, is an effective chemotherapy drug for patients with metastatic breast cancer who have previously received at least two chemotherapeutic regimens. We are seeking to enhance the anti-tumor activity of eribulin in metastatic breast cancer cell lines. The purpose of this study was to identify kinase targets that markedly enhance the efficacy of eribulin using high-throughput nonbiased synthetic lethal RNA interference (RNAi) screening and to validate these targets in combination with eribulin using triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC) cell lines which are heterogeneous diseases and characterized by their high metastatic potential and poor prognosis. METHODS: High-throughput RNAi screening (709-human-kinome library) was performed to identify target kinases. The Bliss scoring method was used to identify potential targets from the screening (using a Bliss score cut-off of 0.15), and protein-protein interactome analysis (STRING V.11) and Ingenuity Pathway Analysis (IPA) were used to identify therapeutic target pathways. To validate the in vitro efficacy of selected targets, we performed CellTiter-Blue or sulforhodamine B cell proliferation and anchorage-independent colony-formation assays in 18 TNBC and 6 IBC cell lines treated with eribulin with or without inhibitors of the discovered targets. RESULTS: In vitro proliferation data demonstrated that single-agent eribulin had a nanomolar range of half-maximal inhibitory concentrations (0.2 - 4.0 nM) in most of the tested cell lines. RNAi screening identified 130 relevant kinase targets. IPA and STRING analysis revealed PI3K/AKT/mTOR, MAPK, JNK, and PAK1 as major canonical pathways that could be targeted to enhance the cytotoxic effect of eribulin. In in vitro cell line validation, PAK1 inhibitor NVS-PAK1-1 and mTOR inhibitor everolimus showed strong combinational anti-tumor effect (combination index values = 0.1 - 0.9), and JNK inhibitor JNK-In-8 showed moderate combinational anti-tumor effect (combination index values = 0.5 - 0.9) in combination with eribulin. Inhibitors of PI3K (copanlisib) and MEK (trametinib) showed combinational anti-tumor effect in some cell lines. CONCLUSION: We identified that PAK1, mTOR, and JNK inhibitors enhanced the anti-proliferation effects of eribulin in TNBC and IBC cell lines. Our data inform new treatment strategies with eribulin for TNBC and IBC that could lead to clinical trials of innovative combination therapy based on eribulin. Citation Format: Jangsoon Lee, Jon A Fuson, Huey Liu, Troy Pearson, Debu Tripathy, Bora Lim, Naoto T Ueno. Identification of molecules that enhance the efficacy of eribulin in TNBC and IBC cell lines [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-16.

Synthesis of Natural (-)-Antrocin and its Enantiomer via Stereoselective Aldol Reaction.

The total synthesis of (−)-antrocin and its enantiomer are presented. Antrocin (−)-1 is an important natural product which acts as an antiproliferative agent in a metastatic breast cancer cell line (IC50: 0.6 μM). The key features of this synthesis are: (a) selective anti-addition of trimethylsilyl cyanide (TMSCN) to α,β-unsaturated ketone; (b) resolution of (±)-7 using chiral auxiliary L-dimethyl tartrate through formation of cyclic ketal diastereomers followed by simple column chromatography separation and acid hydrolysis; (c) substrate-controlled stereoselective aldol condensation of (+)-12 with monomeric formaldehyde and pyridinium chlorochromate (PCC) oxidation for synthesis of essential lactone core in (−)-14; and (d) non-basic Lombardo olefination of the carbonyl at the final step to yield (−)-antrocin. In addition, (+)-9 cyclic ketal diastereomer was converted to (+)-antrocin with similar reaction sequences.

Evaluation of cytotoxic profile of hydroalcoholic extract of fruit rinds of Garcinia pedunculata on human embryonic kidney and breast carcinoma cells

Background: The fruit rinds of Garcinia pedunculata has potential medicinal properties and used in many chronic ailments. It has been demonstrated that cytoprotective effects in various experimental research works. But its cytotoxic effect has not been evaluated. The present study was aimed to screen its relative cytotoxic effect on normal and cancer cell lines.Methods: In the present study, the cytotoxic effect of hydro alcoholic extract of Garcinia pedunculata was evaluated on normal human embryonic kidney (HEK-293) and M.D. Anderson metastatic breast cancer cell lines (MDA-MB 231) using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.Results: Higher dose level of hydro alcoholic extract of Garcinia pedunculata (HAGP) (500 μg/ml) has shown considerable increase (112.503) in the percentage viability of HEK-29 whereas; there is a remarkable decrease in the viable cell population (77.490) in MDA-MB 231.Conclusions: Based on the observed results we could conclude that HAGP has potential cytotoxic effect on the cancer cell line without altering the normal cell growth and proliferation. Thus it has potential to develop as a safer chemotherapeutic agent. Further detailed exploration is required to confirm its therapeutic efficacy in different cancer cell lines.

Evaluation of the Impact of Imprinted Polymer Particles on Morphology and Motility of Breast Cancer Cells by Using Digital Holographic Cytometry

Breast cancer is the second most common cancer type worldwide and breast cancer metastasis accounts for the majority of breast cancer-related deaths. Tumour cells produce increased levels of sialic acid (SA) that terminates the monosaccharide on glycan chains of the glycosylated proteins. SA can contribute to cellular recognition, cancer invasiveness and increase the metastatic potential of cancer cells. SA-templated molecularly imprinted polymers (MIPs) have been proposed as promising reporters for specific targeting of cancer cells when deployed in nanoparticle format. The sialic acid-molecularly imprinted polymers (SA-MIPs), which use SA for the generation of binding sites through which the nanoparticles can target and stain breast cancer cells, opens new strategies for efficient diagnostic tools. This study aims at monitoring the effects of SA-MIPs on morphology and motility of the epithelial type MCF-7 and the highly metastatic MDAMB231 breast cancer cell lines, using digital holographic cytometry (DHC). DHC is a label-free technique that is used in cell morphology studies of e.g., cell volume, area and thickness as well as in motility studies. Here, we show that MCF-7 cells move slower than MDAMB231 cells. We also show that SA-MIPs have an effect on cell morphology, motility and viability of both cell lines. In conclusion, by using DH microscopy, we could detect SA-MIPs impact on different breast cancer cells regarding morphology and motility.

Mechanisms of Tumor-Lymphatic Interactions in Invasive Breast and Prostate Carcinoma.

During the last few years, diverse studies have shown that tumors can actively interact with the lymphatic system and promote metastases development. In order to examine the molecular mechanisms involved in this interaction, we co-cultured tumor and lymphatic endothelial cells (LEC) and subsequently analyzed the molecular alterations of LECs. Therefore, LECs were co-cultivated with either a highly or weakly metastatic breast cancer cell line using contact (mixture) and non-contact (transwell) co-cultures. mRNA profiles from LECs were subsequently analyzed for genes specifically induced by highly metastatic tumor cells (“metastatic specific”). Among the up-regulated “metastatic specific” genes, we found candidates involved in cell cycle, cell adhesion and motility (BST2, E-selectin, and HMMR), cytokines (CCL7, CXCL6, CXCL1, and CSF2) and factors of the complement system (C1R, C3, and CFB). Among the down-regulated genes, we detected the hyaluronan receptor STAB2, angiogenic factor apelin receptor (APLNR), and the glycosylation enzyme MAN1A1. In an additional prostate cancer co-culture model, we could confirm a “metastatic specific” upregulation of E-selectin and CCL7 in LECs after interaction with the prostate cancer cell lines LNCAP (highly metastatic) and DU145 (weakly metastatic). These data allowed us to identify a set of genes regulated in LECs during in vitro communication with cancer cells, which might subsequently facilitate lymphatic metastasis.

Investigation of the effects of rapamycin on the mTOR pathway and apoptosis in metastatic and non-metastatic human breast cancer cell lines.

The aim of this study was to analyze the effects of rapamycin treatment on apoptosis via mTOR pathway in metastatic and non-metastatic human breast cancer cell lines by immunohistochemical and TUNEL analysis. MCF-7 and MDA-MB 231 cell lines were incubated under standard conditions forming Rapamycin and control groups. In immunohistochemical evaluation; mTOR pathway was evaluated with anti-IGF1, anti-PI3K, anti-pAKT1/2/3, anti-mTORC1, anti-mTORC2 and anti-ERK1 antibodies. The effect of apoptosis was also confirmed by TUNEL method. In this study, activation of PI3K/AKT/mTOR and related molecular pathways in the MDA-MB 231 and MCF-7 breast cancer cell line was evaluated and it was observed that these pathways could play a key role in cancer development. Increased apoptotic cells were observed in mTORC1 inhibition by Rapamycin administration. Targeting the mTOR pathway in breast cancer treatment may be a treatment option. In addition, the demonstration and confirmation of increased apoptosis in Rapamycin treated groups suggested that Rapamycin, an inhibitor of mTOR, is promising in the treatment of breast cancer (Tab. 2, Fig. 3, Ref. 66).

Chemosensory bitter taste receptors T2R4 and T2R14 activation attenuates proliferation and migration of breast cancer cells.

The emerging significance of the bitter taste receptors (T2Rs) role in the extraoral tissues alludes to their potential role in many pathophysiological conditions. The dysregulation of T2R expression and function in disease conditions has now been demonstrated in airways diseases, neurological disorders, and in some cancers. However, the role of T2Rs in the pathophysiology of breast cancer is unexplored thus far. Previously, we demonstrated differential expression of the 25 T2Rs in breast cancer (BC) cells. Based on our previous findings we selected two T2Rs, T2R4 and T2R14 for this work. The objective of the current study is to investigate the expression of T2R4 and T2R14 in BC clinical samples and to examine their physiological role using highly metastatic BC and non-cancerous cell lines. Using approaches, which involve receptor knockdown, pharmacological activation and biochemical assays we report that (i) T2R4 and T2R14 expression patterns are dissimilar, with decreased levels of T2R4 and increased levels of T2R14 in BC clinical samples compared to non-cancerous controls. (ii) Activation of T2Rs with their respective agonist elicited physiological responses in metastatic breast cancer cells, and no responses were seen in non-tumorigenic breast epithelial cells. (iii) Agonist activation of T2Rs (irrespective of T2R subtype) induced anti-proliferative, pro-apoptotic, and anti-migratory responses in highly metastatic breast cancer cells. Taken together, our findings demonstrate that the chemosensory T2R signaling network is involved in evoking physiological responses in the metastatic breast cancer cell line.

Paclitaxel May Inhibit Epithelial-Mesenchymal Transition Properties of Triple-negative Breast Cancer Cell Line via Altering the Expression of EMT-promoting and –inhibiting MicroRNAs

Background: Abnormal expressions of microRNAs are related to various cancers such as breast cancer for which paclitaxel is widely used as a chemotherapeutic agent. We aimed to investigate the effect of paclitaxel treatment on the expression level of miR-199a-5p and miR-10b, involved in epithelial-mesenchymal transition (EMT) process in breast cancer cell lines. Methods: Human breast cancer cell lines BT-474, SKBR-3, MDA-MB-231, and MCF-7 were cultured and MTT assay was used to determine IC50 of paclitaxel. RNA was extracted, cDNA was synthesized, and the expression level of miRNAs and genes was quantitatively determined using real-time PCR. Results: After treatment with paclitaxel, the expression level of miR-199a-5p significantly decreased in MCF-7 and SKBR-3 cell lines, while it increased in MDA-MB-231 and BT-474. The expression level of miR-10b was also significantly reduced in MCF-7, MDA-MB-231, and SKBR-3 and increased in BT-474 cell lines following treatment with paclitaxel. Our results further indicated that paclitaxel reduced the expression level of vimentin and MMP-9 in MDA-MB-231 cell line. Conclusion: Our findings revealed the increased expression of EMT-inhibitor miR-199a-5p and the decreased expression of metastamir miR-10b after treatment of MDA-MB-231 metastatic breast cancer cell line. Reduced expressions of vimentin and MMP-9 were also observed, corroborating the inhibition of metastasis markers in this type of breast cancer. The therapeutic effect of paclitaxel may in part be due to the change in the balance of EMT-promoting and EMT-inhibiting miRNAs.

SNX27-retromer assembly recycles MT1-MMP to invadopodia and promotes breast cancer metastasis.

A variety of metastatic cancer cells use actin-rich membrane protrusions, known as invadopodia, for efficient ECM degradation, which involves trafficking of proteases from intracellular compartments to these structures. Here, we demonstrate that in the metastatic breast cancer cell line MDA-MB-231, retromer regulates the matrix invasion activity by recycling matrix metalloprotease, MT1-MMP. We further found that MT2-MMP, another abundantly expressed metalloprotease, is also invadopodia associated. MT1- and MT2-MMP showed a high degree of colocalization but were located on the distinct endosomal domains. Retromer and its associated sorting nexin, SNX27, phenocopied each other in matrix degradation via selectively recycling MT1-MMP but not MT2-MMP. ITC-based studies revealed that both SNX27 and retromer could directly interact with MT1-MMP. Analysis from a publicly available database showed SNX27 to be overexpressed or frequently altered in the patients having invasive breast cancer. In xenograft-based studies, SNX27-depleted cell lines showed prolonged survival of SCID mice, suggesting a possible implication for overexpression of the sorting nexin in tumor samples.

Enhancement of PDT-cytotoxicity via ROS induced by indomethacin in metastatic breast cancer

Breast cancer is one of the most common types of cancers prevalent in women. Several types of breast cancers can easily metastasize to bone and cause disease complications such as hypercalcemia and pathologic fracture, thus compromising the quality of life of people affected by it. Bisphosphonate drugs are often used for the treatment of bone metastasis to suppress osteoclastic bone resorption. However, bisphosphonate has adverse effects on the gastrointestinal tract and kidneys and also induces osteonecrosis of the jaw. Photodynamic therapy (PDT) is an alternative cancer treatment approach with minimal invasiveness. It is a combination treatment that uses photosensitizers, which accumulate in tumor cells, followed by laser irradiation. We previously reported that the cellular incorporation of 5-aminolevulinic acid (5-ALA), which was a precursor of protoporphyrin IX (PpIX), was regulated by reactive oxygen species derived from mitochondria (mitROS). In this study, we investigated the incorporation of 5-ALA, accumulation of PpIX, and subsequent effects on cell viability after laser irradiation of two different breast cancer cell lines with different metastaticites. The highly metastatic breast cancer cell line 4T1E/M3 showed a significant increase in ROS production after treatment with indomethacin (IND). In addition, IND treatment enhanced the cellular uptake of 5-ALA via PEPT1 upregulation in 4T1E/M3, but not in the non-metastatic cell line. Overall, metastatic breast cancer is likely to be sensitive to ROS and activate signaling pathways associated with 5-ALA transportation, suggesting that ALA-PDT could be an effective treatment with low invasiveness for metastatic breast cancer.

Docosahexaenoic acid suppresses migration of triple-negative breast cancer cell through targeting metastasis-related genes and microRNA under normoxic and hypoxic conditions.

There is insufficient evidence with respect to the effect of the standard anticancer therapeutic agents as well as common dietary supplements on the expression of such genes and microRNAs (miRNAs). Therefore, this study was aimed to study the effect of applying linoleic acid (LA) and docosahexaenoic acid (DHA) fatty acids alone or combined with Taxol on the expression of the matrix metalloproteinase (MMP)-9, MMP-2, vimentin, and talin2 genes, tumor-suppressor miR-194 and, onco-miR-106b in triple-negative breast cancer cell line, known as MDA-MB-231. MDA-MB-231 as metastatic breast cancer cell line was cultured and treated using 0.3 μM Taxol, 100 μM DHA, and 50 μM LA for 24 hours, alone or combined with Taxol under the normoxic and hypoxic conditions. Cells were harvested, after RNA extraction and complementary DNA synthesis, analysis of the expression levels of the studied genes and miRNAs was done through the use of the quantitative real-time polymerase chain reaction (qRT-PCR). Wound healing assay and Western blot analysis were also performed for confirmation. The results of qRT-PCR showed that treating the MDA-MB-231 cells with DHA caused an increase in the miR-194 expression and a decrease in the miR-106b expression, leading to the downregulation of the MMP-2 and MMP-9, and vimentin, and upregulation of the talin2 under the normoxic and hypoxic conditions. The results of the wound healing scratch assay revealed that the administration of the DHA and the DHA-Taxol combination caused the repression of cell migration in comparison with the control groups under the normoxic and hypoxic conditions. The results of the Western blot analysis demonstrated that DHA and the DHA-Taxol combination caused an increase in the expression of the talin2 protein rather than the control cells under both normoxic and hypoxic conditions. This study showed that DHA has significant antimetastatic effects against the triple-negative breast cancer cells. DHA could serve as a promising supplementation for suppressing the breast cancer cell migration, especially under the hypoxic condition.