Proteins In Breast Cancer Cells Research Articles

Design and synthesis of benzylidenecyclohexenones as TrxR inhibitors displaying high anticancer activity and inducing ROS, apoptosis, and autophagy

Oxidative therapy, a strategy that specifically increases reactive oxygen species (ROS) levels in tumor cells by disrupting the redox homeostasis has gained increasing interest. The antitumor effects of the natural product piperlongumine (PL) appear to result from its ability to increase intracellular ROS levels via inhibition of antioxidative thioredoxin reductase (TrxR). Twenty-seven benzylidenecyclohexenone-based PL analogues (2a-v and 15a-e) were designed, synthesized and evaluated for their pharmacological properties. Most of the compounds exhibited potent antiproliferative activities against five human cancer cell lines, especially against breast tumor cells. One of the most promising analogueues 2c showed 12-fold higher inhibitory activity against the thioredoxin reductase (TrxR) than PL and surpressed the expression of TrxR1 protein in breast cancer cells and inhibited TrxR enzymatic activity. In addition, 2c increased ROS levels and resulted in marked apoptosis by regulating apoptosis-related proteins expressed in the breast cancer cells. Compound 2c also triggered the formation of autophagosomes and autolysosomes by promoting the expression of LC3-II and Beclin-1 and diminishing the expression of LC3-I and p62 proteins. Finally, 2c displayed low acute toxicity and good inhibitory potency to tumors in mice. Overall, 2c is a promising anti-breast cancer candidate warranting further investigation.

SGNLVA-001: A phase I open-label dose escalation and expansion study of SGN-LIV1A administered weekly in breast cancer.

TPS1104 Background: LIV-1 is a highly prevalent transmembrane protein in breast cancer cells. Ladiratuzumab vedotin (LV), SGN-LIV1A, is an investigational antibody-drug conjugate (ADC) that targets LIV-1 via a humanized IgG1 monoclonal antibody conjugated to monomethyl auristatin E (MMAE) by a protease-cleavable linker. LV is internalized when it binds LIV-1 on cell surfaces and MMAE is released, which binds tubulin and induces apoptosis. LV has been shown to be active and tolerable in metastatic breast cancer (mBC) at a recommended dose of 2.5 mg/kg every 21 days (Modi 2017). More frequent, fractionated dosing has improved the activity and/or safety of other ADCs. Thus, this study is actively accruing subjects with metastatic triple negative breast cancer (mTNBC; estrogen receptor (ER)/progesterone receptor (PR)/human epidermal growth factor receptor 2 (HER2) receptor-negative) and endocrine-resistant ER+ or PR+ (hormone receptor [HR+])/HER2-negative mBC to test weekly dosing of LV (Days 1, 8, and 15 of every 3-week cycle). Methods: This study is enrolling up to 82 subjects (42 HR+/HER2-negative and 40 mTNBC) into dose escalation and dose expansion cohorts (NCT01969643). Eligible subjects are females ≥18 years old with pathologically and radiologically confirmed metastatic HR+/HER2-negative or mTNBC with at least 1 measurable lesion per RECIST v1.1. Subjects with HR+/HER2-negative disease must have received no more than 1 prior line of cytotoxic chemotherapy in the locally advanced (LA)/mBC setting, either as single agent or combination therapy. Subjects with mTNBC must have received 1 prior line of cytotoxic chemotherapy in the LA/mBC setting. Progression within 6 months of completion of neoadjuvant or adjuvant therapy is considered an LA/mBC regimen. Subjects must have adequate organ function, ECOG status of ≤1, and no ≥ Grade 2 peripheral neuropathy. Subjects with brain lesions must have received definitive treatment of the lesions. Prior therapy with MMAE-containing agents is not allowed. Dose escalation follows the modified toxicity probability interval method (Ji 2010). Dose expansion cohorts will provide data about activity and tolerability. Tumor assessments will be conducted every 2 cycles per RECIST v1.1 and all subjects will be followed for safety. Pharmacokinetics and markers of pharmacodynamics will be assessed. Primary safety endpoint is the incidence of adverse events and dose-limiting toxicities. Key efficacy endpoints include confirmed overall response rate, duration of response, and progression-free survival. Clinical trial information: NCT01969643 .

The Effects of Houttuynia cordata Thunb and Piper ribesioides Wall Extracts on Breast Carcinoma Cell Proliferation, Migration, Invasion and Apoptosis.

Houttuynia cordata Thunb. (HCT) and Piper ribesioides Wall. (PR) are common herbs that are widely distributed throughout East Asia and possess various biological properties including anti-cancer effects. However, in breast cancer, their mechanisms responsible for anti-carcinogenic effects have not been clarified yet. In this study, the inhibitory effects of HCT and PR ethanolic extracts on breast cancer cell proliferation, migration, invasion and apoptosis were examined. In MCF-7 and MDA-MB-231 cells, HCT and PR extracts at low concentrations can inhibit colony formation and induce G1 cell cycle arrest by downregulating cyclinD1 and CDK4 expression. Additionally, HCT and PR extracts also decreased the migration and invasion of both breast cancer cell lines through inhibition of MMP-2 and MMP-9 secretion. Moreover, the induction of apoptosis was observed in breast cancer cells treated with high concentrations of HCT and PR extracts. Not only stimulated caspases activity, but HCT and PR extracts also upregulated the expression of caspases and pro-apoptotic Bcl-2 family proteins in breast cancer cells. Altogether, these findings provide the rationale to further investigate the potential actions of HCT and PR extracts against breast cancer in vivo.

Nucleolar localization of the Notch4 intracellular domain underpins its regulation of the cellular response to genotoxic stressors

Cell survival is one of the many cellular processes regulated by Notch family of proteins. A comparison of human breast cancer cell lines, which differ in the levels of endogenous Notch4, implicated the protein in regulating susceptibility to apoptosis triggered by genomic damage. In agreement with this observation, increased susceptibility to genotoxic damage was observed following siRNA ablations of Notch4 in two breast cancer cell lines. Further, overexpressing Notch4 intracellular domain (NIC4) tagged to GFP (NIC4-GFP), protected cells from apoptosis triggered by genotoxic drugs. In cells immune-stained for endogenous Notch4, protein was detected in the nucleolus and nucleoplasm, which was also confirmed by the co-localization of NIC4-GFP with RFP-tagged nucleolar proteins in breast cancer cells or the unrelated HEK cell line. Linking functional outcomes to nucleolar localization, NIC4-GFP protection from apoptosis, required the nucleolar proteins Nucleolin and Fibrillarin. Consistently, immunoprecipitation analysis revealed associations between nucleolar proteins—Nucleolin and Nucleophosmin—and Notch4. Microscopy-based biophysical analysis of live cells showed that nucleolar and nucleoplasmic pools of NIC4-GFP are mobile, with some sequestration of nucleolar NIC4-GFP pools. A nucleolar excluded form, NIC4_3RA-GFP, generated by site-directed mutagenesis of the nucleolar localization sequence in NIC4, could not protect from apoptosis triggered by genotoxic stressors. However, transcriptional activity or protection from apoptosis triggered by endoplasmic stress was comparable in cells expressing NIC4_3RA-GFP or NIC4-GFP. Together, the data show that nucleolar localization of NIC4 is critical for the regulation of genomic damage and may be uncoupled from its activities in the nucleoplasm. This study identifies intrinsic features of NIC4 that regulate signaling outcomes activated by the receptor by controlling its spatial localization.

Abstract P2-04-03: The roles of fucosylation in promoting tumorigenic signaling in breast cancer

Abstract Fucosylation is an important form of protein glycosylation involved in the progression of cancer. Aberrant fucose and fucosylated serum protein/oligosaccharide levels have been previously described in breast cancer (BC). Fucosylation has been reported impact proliferation and key signaling pathways (e.g., ERK1/2, p38 MAPK, and E-selectin) in human breast invasive ductal carcinoma. However, precise roles and functions of fucosylated proteins function in BC remain largely unclear. In our preliminary analyses, we performed immunofluorescent lectin staining of BC tissue microarrays and cell lines grown in vitro and found that fucosylation is increased in BC vs. normal breast tissues. To study the functional contributions of fucosylation to BC biology, we either pharmacologically or genetically suppressed fucosylation in BC cell lines. The treated or modified cell lines were characterized for their ability to grow/proliferate in 2D or 3D, as well as on adhesion to extracellular matrix (ECM). We observed that inhibiting fucosylation blunted BC cell proliferation, particularly in 3D, and attenuated adhesive capacity. To determine the mechanism(s) underlying the biological functions of fucosylation in BC cells, we performed parallel fucosylated and phosphoproteomic mass spectrometric analyses on BC cells that were either pharmacologically or genetically inhibited for fucosylation to identify: (i) all fucosylated proteins in BC cells and (ii) signaling pathways that comprise directly fucosylated proteins and that are significantly altered in activity (phosphorylation). All filtered hits were subject to Ingenuity Pathway Analysis. Thirteen signaling pathways were identified in common amongst both pharmacologically or genetically modulated BC groups. These pathways clustered into the following 3 major functional groups: cell-cell/cell-matrix contact, RNA processing, and endosomal/phagosomal dynamics. The most fucosylated proteins included integrins. We validated the direct fucosylation of integrins and further dissected the molecular and functional contributions of fucosylation to integrin signaling in vitro and in vivo in mouse models of breast cancer. Our data identify new roles for fucosylation in regulating key signal transduction pathways in BC. How integrin signaling and BC cell biology is regulated by fucosylation, as well as potential clinical utility of fucosylated integrins for BC, will be discussed. Citation Format: Kara Snyder, Hui Ren, Daniel Lester, Bin Fang, John Koomen, Eric Lau. H. The roles of fucosylation in promoting tumorigenic signaling in breast cancer [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 P2-04-03.

Study on the Antibreast Cancer Mechanism and Bioactive Components of Si-Wu-Tang by Cell Type-Specific Molecular Network.

Si-Wu-Tang (SWT), a traditional Chinese herbal formula, has shown an effect on antibreast cancer. However, the mechanisms and bioactive components of SWT are still unclear. Fortunately, cell type-specific molecular network has provided an effective method. This study integrated the data of formula components, all types of biomolecules in the human body, and nonexpressed protein in breast cancer cells and constructed the breast cancer cell network and the biological network that SWT acted on the breast cancer-related targets by Entity Grammar System (EGS). Biological network showed 59 bioactive components acting on 15 breast cancer-related targets. The antibreast cancer mechanisms were summarized by enrichment analysis: regulation of cell death, response to hormone stimulation, response to organic substance, regulation of phosphorylation of amino acids, regulation of cell proliferation, regulation of signal transmission, and affection of gland development. In addition, we discovered that verbascoside played the role of antibreast cancer by inhibiting cell proliferation, but there was not a report on this effect. The results of CCK8 and western blot were consistent with the antibreast cancer effect of verbascoside based on biological network. Biological network modeling by EGS and network analysis provide an effective way for uncovering the mechanism and identifying the bioactive components of SWT.

Protein‑ and growth‑modulatory effects of carcinoma‑associated fibroblasts on breast cancer cells: Role of interleukin‑6.

Carcinoma-associated fibroblasts (CAFs) secrete factors that increase the expression and/or activities of proteins in breast cancer cells and induce resistance to anti-estrogens, such as fulvestrant. A major factor is interleukin-6 (IL-6). This study demonstrated that, across estrogen receptor (ER) α-positive and -negative cell lines, recombinant human IL-6 (rhIL-6) mimicked most of the CAF-conditioned medium (CM)-induced changes in protein expression patterns; however, in most cases, it failed to recapitulate CAF-CM-triggered alterations in ERK1/2 and AKT activities. The ability of rhIL-6 to induce fulvestrant resistance was dependent upon the culture conditions. In 3D, but not in 2D cultures, rhIL-6 increased the survival of fulvestrant-treated cells, although not to the same extent as observed with CAF-CM. In 2D cultures, rhIL-6 acted in a pro-apoptotic manner and decreased the expression of ATP-binding cassette transporter G2 (ABCG2). The inhibition of the PI3K/AKT pathway had similar effects on apoptosis and ABCG2 expression, linking the failure of rhIL-6 to induce fulvestrant resistance to its inability to activate the PI3K/AKT pathway. In 3D cultures, both CAF-CM and rhIL-6 acted in an anti-apoptotic manner. These activities are likely independent on the PI3K/AKT pathway and ABCG2. Experiments on ERα-negative breast cancer cells revealed a growth-inhibitory effects of both CAF-CM and rhIL-6, which coincided with a reduction in the c-Myc level. These data suggest that IL-6 plays a role in several effects of CAF-CM, including alterations in protein expression patterns, fulvestrant resistance in 3D cultures and growth inhibition. By contrast, IL-6 is unlikely to be responsible for the CAF-CM-induced activation of the PI3K/AKT pathway and fulvestrant resistance in 2D cultures.

Anticancer effects of n-3 EPA and DHA and their endocannabinoid derivatives on breast cancer cell growth and invasion

The anticancer effects of the omega-3 long chain polyunsaturated fatty acids (LCPUFA), EPA and DHA may be due, at least in part, to conversion to their respective endocannabinoid derivatives, eicosapentaenoyl-ethanolamine (EPEA) and docosahexaenoyl-ethanolamine (DHEA). Here, the effects of EPEA and DHEA and their parent compounds, EPA and DHA, on breast cancer (BC) cell function was examined. EPEA and DHEA exhibited greater anti-cancer effects than EPA and DHA in two BC cells (MCF-7 and MDA-MB-231) whilst displaying no effect in non-malignant breast cells (MCF-10a). Both BC lines expressed CB1/2 receptors that were responsible, at least partly, for the observed anti-proliferative effects of the omega-3 endocannabinoids as determined by receptor antagonism studies. Additionally, major signalling mechanisms elicited by these CB ligands included altered phosphorylation of p38-MAPK, JNK, and ERK proteins. Both LCPUFAs and their endocannabinoids attenuated the expression of signal proteins in BC cells, albeit to different extents depending on cell type and lipid effectors. These signal proteins are implicated in apoptosis and attenuation of BC cell migration and invasiveness. Furthermore, only DHA reduced in vitro MDA-MB-231 migration whereas both LCPUFAs and their endocannabinoids significantly inhibited invasiveness. This finding was consistent with reduced integrin β3 expression observed with all treatments and reduced MMP-1 and VEGF with DHA treatment. Attenuation of cell viability, migration and invasion of malignant cells indicates a potential adjunct nutritional therapeutic use of these LCPUFAs and/or their endocannabinoids in treatment of breast cancer.

Hormone-Like Effects of Bisphenol A on p53 and Estrogen Receptor Alpha in Breast Cancer Cells.

Bisphenol A (BPA) is a polymerizing agent commonly found in plastics that has been linked to xenoestrogenic activity. In this study, we analyzed the estrogen-like effects of BPA on the expression of estrogen receptor (ER)α and p53 with hormonal and antihormonal treatments in T-47D and MCF-7 cells. Cells were cultured in medium containing 5% charcoal-stripped fetal bovine serum for 6 days to deplete any endogenous steroids or effectors. The cells were then treated for 24 h with 600 nM BPA, which was determined to be the optimal value by a concentration study of BPA from 1 nM to 2 μM. Extracted cellular proteins were quantified and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)/Western blot analysis. The cell proliferation assays were quantified upon exposure to BPA. Laser confocal microscopy was performed to determine the cytolocalization of p53 and ERα upon treatment with BPA. Western blot analysis revealed that BPA caused an increase in the cellular protein p53 in a concentration-dependent manner. While treatment with BPA did not affect the cytolocalization of p53, an increase in cell proliferation was observed. Our studies provide interesting leads to delineate the possible mechanistic relationship among BPA, ER, and tumor suppressor proteins in breast cancer cells.

3-Chloroplumbagin Induces Cell Death in Breast Cancer Cells Through MAPK-Mediated Mcl-1 Inhibition.

Resistance acquired toward anti-cancer agents is a significant drawback in breast cancer therapy. A key factor contributing to drug resistance is apoptosis suppression associated with the upregulation of anti-apoptotic Bcl-2 family proteins. Specifically, the anti-apoptotic Mcl-1 protein has been shown to play a significant role in drug resistance, making it an important therapeutic target. The present study aimed at determining the antiproliferative activity of 3-chloroplumbagin (ChPL), a naphthoquinone derived from a Dionaea sp., toward breast cancer cells and examining the involvement of Mcl-1 inhibition in ChPL-induced cell death. The results showed that ChPL inhibited breast cancer cell proliferation and induced apoptosis through the intrinsic pathway through down-regulation of anti-apoptotic Bcl-2 family proteins. The induction of apoptosis by ChPL was found to be mediated through MAP kinase signaling inhibition. ChPL inhibited the phosphorylation of MEK and ERK proteins in breast cancer cells, and increased apoptosis induction in cells with reduced ERK expression. Furthermore, ERK silencing decreased the expression of Mcl-1 in ChPL-treated cells. The results of this research indicate that ChPL induces apoptosis in breast cancer cells through MAPK-mediated Mcl-1 inhibition, suggesting further research into its potential in breast cancer treatment.

Abstract 845: c-MET nuclear transportation via membrane-bounded retrograde trafficking in breast cancer cells

Abstract Reactive oxygen species (ROS) can be induced by anti-cancer treatments, such as ionizing irradiation and chemotherapy drugs. Hydrogen peroxide (H2O2) is one of the ROS species highly produced in cancer cells due to rapid proliferation or anti-cancer treatments. ROS serve as important intracellular messengers in cell growth and proliferation signaling pathways. However, ROS also causes cellular stress in cancer cells through oxidizing DNA, protein and lipids. Therefore, balancing ROS stress between signaling and damages at a tolerable level is important for cancer cell survival. c-MET is one of the receptor tyrosine kinases overexpressed in breast cancer. Full-length c-MET is known to be oncogenic for triggering signal transduction cascade from plasma membrane to upregulate cell proliferation and migration in cancer cells. Moreover, c-MET is reported to phosphorylate PARP1 under H2O2 stimulation, and the phosphorylated PARP1 resulting in PARP inhibitor resistance in breast cancer cells. However, it is not yet clear how c-MET travels from cell membrane to the nucleus. Here, we demonstrate that H2O2 induces retrograde transport of c-MET from plasma membrane into the nucleus and that knocking down either COPI or Sec61β attenuates H2O2-induced full-length nuclear c-MET accumulation. However, c-MET kinase inhibitor did not block the nuclear transport of c-MET, suggesting the transportation mechanisms is independent from kinase activity. Our results indicate that membrane-bound retrograde vesicles involve in membrane-to-nucleus transportation of c-MET in breast cancer cells. Moreover, nuclear c-MET interacts with both PARP1 and Ku proteins in breast cancer cells, suggesting the role of full-length nuclear c-MET in mediating repair of ROS-induced DNA damages. Citation Format: Mei-Kuang Chen, Yi Du, Linlin Sun, Jennifer L. Hsu, Yu-Han Wang, Yuan Gao, Jiaxing Huang, Mien-Chie Hung. c-MET nuclear transportation via membrane-bounded retrograde trafficking in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 845.

MiR-202 inhibits cell proliferation, invasion, and migration in breast cancer by targeting ROCK1 gene.

The aim of this study was to research the effect of miR-202 on breast cancer cells proliferation, invasion, and migration. TCGA analysis was used to research miR-202 expression and overall survival in patients with breast cancer. Transfection with miR-202 mimics or cotransfection with miR-202 mimics and ROCK1 expression vector was performed on breast cancer cells. Reverse transcription polymerase chain reaction was used to detect the miR-202 expression of breast cancer tissues and cells. Western blot was conducted to research the expression of ROCK1, E-cadherin, Twist, N-cadherin, and MMP2. Dual luciferase reporter assay was performed to detect the targeted relationship between ROCK1 and miR-202. MTT and transwell assay were used to detect breast cancer cells proliferation, invasion, and migration. Downregulation of miR-202 was positively correlated with poor prognosis of patients withbreast cancer. miR-202 in breast cancer tissues and breast cancer cells was significantly downregulated. Upregulation of miR-202 inhibited the proliferation, migration, and invasion of breast cancer cells. miR-202 promoted E-cadherin expression and inhibited the expression of N-cadherin, Twist, and MMP2. ROCK1 was the target gene of miR-202. miR-202 regulated proliferation, migration, invasion, and expression of related proteins in breast cancer cells by targeting ROCK1 expression. miR-202 inhibited breast cancer cells proliferation, invasion, and migration by targeting the ROCK1 gene.

Upregulation of MIIP regulates human breast cancer proliferation, invasion and migration by mediated by IGFBP2

AimsThe migration and invasion inhibitory protein (MIIP) was initially discovered in a yeast two-hybrid screen for proteins that interact and inhibit the migration and invasion-promoting protein insulin-like growth factor binding protein 2 (IGFBP2). This study aims to evaluate the biological effects of MIIP in breast cancer by targeting IGFBP2. Materials and methodsReverse transcription quantitative real-time polymerase chain reaction and Western blotting were used to evaluate the abnormal expression of MIIP and IGFBP2 in breast cancer tissue or breast cancer cell lines. Transfection assay was used to overexpress MIIP protein in breast cancer cells. MTT assay and colony formation assay were used to detect cell viability of breast cancer cells after MIIP overexpression. Transwell and wound-healing assays were used to detect cell invasion and migration after MIIP overexpression. ResultsMIIP was significantly decreased and IGFBP2 was significantly increased in breast cancer tissues versus para cancerous. Breast cancer tissues of HER2 overexpression and Basal-like were more significant than Luminal A and Luminal B. MIIP was obviously downregulated and IGFBP2 was upregulated in MDA-MB-231, SKBR3 and MCF-7 versus MCF-10A especially in MDA-MB-231. Cell proliferation, cell migration and cell invasion were significantly inhibited after overexpression of MIIP. IGFBP2 was downregulated after overexpression of MIIP. The effects of MIIP on cell proliferation, cell migration and invasion were significantly reversed by IGFBP2. ConclusionThe abnormal expression of MIIP in breast cancer affects the cell biological effects. IGFBP2 was regulated via MIIP which may be associated with these biological effects. These results reveal that MIIP can be a potential target for breast cancer treatment.

H2O2 induces nuclear transport of the receptor tyrosine kinase c-MET in breast cancer cells via a membrane-bound retrograde trafficking mechanism

Reactive oxygen species (ROS) are cellular by-products produced from metabolism and also anticancer agents, such as ionizing irradiation and chemotherapy drugs. The ROS H2O2 has high rates of production in cancer cells because of their rapid proliferation. ROS oxidize DNA, protein, and lipids, causing oxidative stress in cancer cells and making them vulnerable to other stresses. Therefore, cancer cell survival relies on maintaining ROS-induced stress at tolerable levels. Hepatocyte growth factor receptor (c-MET) is a receptor tyrosine kinase overexpressed in malignant cancer types, including breast cancer. Full-length c-MET triggers a signal transduction cascade from the plasma membrane that, through downstream signaling proteins, up-regulates cell proliferation and migration. Recently, c-MET was shown to interact and phosphorylate poly(ADP-ribose) polymerase 1 in the nucleus and to induce poly(ADP-ribose) polymerase inhibitor resistance. However, it remains unclear how c-MET moves from the cell membrane to the nucleus. Here, we demonstrate that H2O2 induces retrograde transport of membrane-associated full-length c-MET into the nucleus of human MCF10A and MCF12A or primary breast cancer cells. We further show that knocking down either coatomer protein complex subunit γ1 (COPG1) or Sec61 translocon β subunit (SEC61β) attenuates the accumulation of full-length nuclear c-MET. However, a c-MET kinase inhibitor did not block nuclear c-MET transport. Moreover, nuclear c-MET interacted with KU proteins in breast cancer cells, suggesting a role of full-length nuclear c-MET in ROS-induced DNA damage repair. We conclude that a membrane-bound retrograde vesicle transport mechanism facilitates membrane-to-nucleus transport of c-MET in breast cancer cells.

Co-Delivery of Eugenol and Dacarbazine by Hyaluronic Acid-Coated Liposomes for Targeted Inhibition of Survivin in Treatment of Resistant Metastatic Melanoma.

While melanoma remains a challenge for oncologists, possibilities are being continuously explored to fight resistant metastatic melanoma more effectively. Eugenol is reported to inhibit survivin protein in breast cancer cells. Survivin is also overexpressed by melanoma cells, and is known to impart resistance to them against chemotherapy-induced apoptosis. To be able to fight resistant melanoma, we formulated hyaluronic acid (HA)-coated liposomes loaded with an effective combination of anti-melanoma agents (Dacarbazine and Eugenol), using a solvent injection method. Quality-by-Design (QbD) was applied to optimize and obtain a final formulation with the desired quality attributes, and within an acceptable size range. The optimized formulation was then subjected to performance analysis in cell lines. Coated-Dacarbazine Eugenol Liposomes were found to possess 95.08% cytotoxicity at a dacarbazine concentration of 0.5 µg/mL, while Dacarbazine Solution showed only 10.20% cytotoxicity at the same concentration. The number of late apoptotic cells was also found to be much higher (45.16% vs. 8.43%). Furthermore, migration assay and proliferation study also revealed significantly higher inhibition of cell migration and proliferation by Coated-Dacarbazine Eugenol Liposomes, signifying its potential against metastasis. Thus, surface-functionalized dacarbazine- and eugenol-loaded liposomes hold great promise against resistant and aggressive metastatic melanoma, with much less unwanted cytotoxicity and reduced doses of the chemotherapeutic agent.

Correlation of OGR1 with proliferation and apoptosis of breast cancer cells.

Effects of ovarian cancer G-protein-coupled receptor 1 (OGR1) protein on proliferation and apoptosis of breast cancer cells, as well as its molecular mechanism were investigated. The MCF-7 cell line highly expressed OGR1 was constructed by transient transfection of eukaryotic expression vector using breast cancer cells. At the same time, cells were transfected with empty vector as controls. The effects of highly expressed OGR1 on cell growth, proliferation, apoptosis and other abilities were identified. In addition, the effects of highly expressed OGR1 on serine-threonine kinase (AKT), p53 and other genes were studied. It was proved in apoptosis experiment that highly expressed OGR1 protein in breast cancer cells could effectively increase the proportion of apoptosis of cells. Cell proliferation experiment revealed that the growth and proliferation abilities of breast cancer cells with highly expressed OGR1 were inhibited to some extent, compared with those of breast cancer cells with low expression of OGR1. Results of western blotting showed that the gene and protein expression levels of p53 in breast cancer cells with highly expressed OGR1 were increased. There was no significant difference in protein expression of AKT between breast cancer cells with low expression of OGR1 and those with highly expressed OGR1. However, the protein content of phosphorylated-AKT (p-AKT) in breast cancer cells with highly expressed OGR1 was lower than that in breast cancer cells with low expression of OGR1. The proliferation and apoptosis of breast cancer cells are influenced by the changes of OGR1 expression, which are correlated with the gene expression levels of AKT and p53 to some extent, but the detailed molecular mechanism requires additional study.

The Expression Level of S100A4 Protein Affects the Migration Activity of Breast Cancer Cells.

Reduced expression of metastatic marker protein S100A4 in triple-negative breast cancer cells MDA-MB-231 leads to a decrease in the migration ability of cells and increases the sensitivity of the modified cells to docetaxel therapy. Cells capable of migration differ from the immotile cells in the content of the S100A4 protein in the cell, and this difference persists after the treatment of cells with the agents that reduce the intracellular level of S100A4. The presence of exogenous S100A4 protein in culture medium reduces the content of this protein in breast cancer cells. The results of the study show that the ability of breast cancer cells to migrate depends on the S100A4 protein concentration in the cell.

MiR-125a-5p inhibits the proliferation and invasion of breast cancer cells and induces apoptosis by targeting GAB2.

Objective: To investigate whether miR-125a-5p can inhibit the proliferation and invasion of breast cancer cells and induce apoptosis by targeting GAB2. Methods: qRT-PCR was used to detect the expression of miR-125a-5p in normal mammary epithelial cells and breast cancer cell lines; The miR-125a-5p overexpression plasmid was transiently transfected into MDA-MB-157 cells, and the proliferation, invasion and apoptosis of breast cancer cells were detected by CCK8 kit, Transwell chamber and flow cytometry, respectively; Gene silencing was used to knock down GAB2 gene in MDA-MB-157 cells, and the changes of proliferation, invasion, apoptosis and apoptosis-related proteins in breast cancer cells were detected by CCK8 kit, Transwell chamber, flow cytometry and western blot, respectively; The direct interaction between miR-125a-5p and GAB2 was detected by dual-luciferase reporter assay. The miR-125a-5p overexpression plasmid was transiently transfected into MDA-MB-157 cells, and the expression levels of GAB2 and apoptosis-related proteins were detected by western blot. Results: The expression of miR-125a-5p in breast cancer cell lines, MDA-MB-157 cells, MDA-MB-361 cells and MDA-MB-415 cells, was significantly lower than that in normal breast epithelial cells, MCF-10A cells; The proliferation and invasion ability of MDA-MB-157 cells transfected with miR-125a-5p were significantly inhibited, and the apoptosis rate was significantly increased; Since GAB2 knocked down, the proliferation and invasion ability of MDA-MB-157 cells were significantly inhibited, while the apoptosis rate was significantly increased, the Bax protein expression was significantly down-regulated, and the Bcl-2 protein expression was significantly up-regulated; The dual-luciferase reporter assay demonstrated that miR-125a-5p can specifically target GAB2. Transfected with miR-125a-5p, the GAB2 protein expression and Bax protein expression were significantly down-regulated, but the Bcl-2 protein expression was significantly up-regulated. Conclusion: miR-125a-5p inhibits the proliferation and invasion of breast cancer cells and induces their apoptosis by negatively regulating GAB2.

Microparticle Delivery of Protein Markers for Single-Cell Western Blotting from Microwells.

Immunoblotting confers protein identification specificity beyond that of immunoassays by prepending protein electrophoresis (sizing) to immunoprobing. To accurately size protein targets, sample analysis includes concurrent analysis of protein markers with known molecular masses. To incorporate protein markers in single-cell western blotting, microwells are used to isolate individual cells and protein marker-coated microparticles. A magnetic field directs protein-coated microparticles to >75% of microwells, so as to 1) deliver a quantum of protein marker to each cell-laden microwell and 2) synchronize protein marker solubilization with cell lysis. Nickel-coated microparticles are designed, fabricated, and characterized, each conjugated with a mixture of histidine-tagged proteins (42.3-100 kDa). Imidazole in the cell lysis buffer solubilizes protein markers during a 30 s cell lysis step, with an observed protein marker release half-life of 4.46 s. Across hundreds of individual microwells and different microdevices, robust log-linear regression fits (R2 > 0.97) of protein molecular mass and electrophoretic mobility are observed. The protein marker and microparticle system is applied to determine the molecular masses of five endogenous proteins in breast cancer cells (GAPDH, β-TUB, CK8, STAT3, ER-α), with <20% mass error. Microparticle-delivered protein standards underpin robust, reproducible electrophoretic cytometry that complements single-cell genomics and transcriptomics.

Antitumor effects of berberine against EGFR, ERK1/2, P38 and AKT in MDA-MB231 and MCF-7 breast cancer cells using molecular modelling and in vitro study

Berberine is an alkaloid plant-based DNA intercalator that affects gene regulation, particularly expression of oncogenic and tumor suppressor proteins. The effects of berberine on different signaling proteins remains to be elucidated. The present study aimed to identify the effects of berberine against key oncogenic proteins in breast cancer cells. Molecular docking and molecular dynamics simulations were used for EGFR, p38, ERK1/2, and AKT. The effects of berberine and lapatinib on MAPK and PI3K pathways in MDA-MB231 and MCF-7 cells were evaluated using immunoflorescence assays, and the amounts of phosphorylated kinases were compared to total kinases after treating with different concentrations of berberine. Simulations showed berberine accurately interacted with EGFR, AKT, P38, and ERK1/2 active sites in silico (scores = -7.57 to -7.92 Kcal/mol) and decreased the levels of active forms of corresponding enzymes in both cell lines; however, berberine binding to p38 showed less stability. Cytotoxicity analysis indicated that MDA-MB231 cells were resistant to berberine compared to MCF-7 cells [72 h IC50 = 50 versus 15 μM, respectively). Also, lapatinib strongly activated AKT but suppressed EGFR in MDA-MB231 cells. The activity of EGFR, AKT, P38, and ERK1/2 were affected by berberine; however, berberine dramatically reduced EGFR and AKT phosphorylation. By way of its multikinase inhibitory effects, berberine might be a useful replacement for lapatinib, an EGFR inhibitor which can cause acquired drug resistance in patients.