Proliferation Of SKBR3 Cells Research Articles

Oxycodone enhances antitumor effect of paclitaxel on human breast cancer SKBR3 cells in vitro

BackgroundThe influences of Oxycodone (OXY) combined with Paclitaxel (PTX) on breast cancer cells are unclear. The present study aimed to examine the effects of OXY combined with PTX on the proliferation, apoptosis, and migration of human breast cancer SKBR3 cells and the underlying mechanism. MethodsThe proliferation, apoptosis and invasion of SKBR3 cells were assessed by CCK-8, colony formation assay, flowcytometric, Transwell assay and scratch assays, respectively. In addition, Western blotting was used to detect the expression of related proteins in these cells. The autophagic bodies were observed under a transmission electron microscope. ResultsOXY (0.25, 0.5 and 1 mM) significantly inhibited the viability, colony-forming, migration, and invasion of SKBR3 cells as compared to the control group. Furthermore, OXY (0.25, 0.5 and 1 mM) markedly induced the apoptosis of SKBR3 cells and the levels of apoptosis-related proteins. In addition, OXY (0.25, 0.5 and 1 mM) and PTX inhibited the proliferation of SKBR3 cells synergistically as compared to PTX group in vitro. Moreover, OXY (0.25, 0.5 and 1 mM) significantly elevated the PTX-induced apoptosis in SKBR3 cells via downregulating the expression of N-cadherin, Becline-1 LC3-Ⅱ, p-Akt and p-mTOR and upregulating E-cadherin expression. Compared with the control group, OXY (1 mM) treatment induced autophagy in SKBR3 cells. ConclusionsThe present study indicates that OXY can enhance the antitumor effect of PTX on breast cancer in vitro. Hence, the combination of OXY with PTX may serve as a potential strategy for the treatment of breast cancer.

Molecular imprinting of miR-559 on a peptide-immobilized poly L-DOPA/silica core-shell and in vitro investigating its effects on HER2-positive breast cancer cells.

In a significant percentage of breast cancers, increased expression of the HER2 receptor is seen and is associated with the spread and worsening of the disease. This research aims to investigate the effect of miR-559 (which targets HER2 mRNA) on SKBR3 breast cancer cells and the possibility of their effective delivery with polymeric nanoparticles and tumor-targeting peptides. L-DOPA monomers were polymerized on the surface of silica nanoparticles in the presence of miR-559 (as a molecular template for molecular imprinting) then an anti-HER2 peptide coupled to the surface of these polymeric nanocomposites (miR-NC-NL2), and the effects of this construct against a HER2-positive breast cancer cells (SKBR3 cells) investigated in vitro conditions. The results showed that miR-NC-NL2 is selective for HER2-positive cells and delivers the miR-559 to them in a targeted manner. miR-NC-NL2 decreased the proliferation of SKBR3 cells and reduced the expression and production of HER2 protein in these cells. Effective and targeted delivery of miR-559 to HER2-positive cancer cells by the miR-NC-NL2 promises the therapeutic potential of this nascent structure based on its inhibitory effect on cancer growth and progression. Of course, animal experiments require a better understanding of this structure's anti-tumor effects.

Exploration of interaction property between nonylphenol and G protein-coupled receptor 30 based on molecular simulation and biological experiments

Nonylphenol (NP), a representative of environmental hormones, can cause extensive biological effects in the human body. In this study, we first analyzed the mutual binding modes of NP and G protein coupled estrogen receptor 30 (GPR30) by molecular simulation. The 3D structure of GPR30 was successfully constructed. We found that the binding sites of NP on GPR30 are similar to that of 17β-Estradiol (E2) on GPR30. The GPR30-E2 bond complex is more stable than GPR30-NP bond complex. Next CCK-8 assay was used to detect the regulatory effect of NP on SKBR-3 cell proliferation. When NP and E2 were used alone, low concentration could promote cell proliferation, while high concentration was the opposite. The presence of E2 can promote the cell proliferation effect of NP, and inhibit the inhibitory intensity. NP could promote both the cell proliferation effect and inhibition intensity of E2. Based on our results, we conclude that the binding modes of NP and GPR30 is similar to that of E2 and GPR30. In biology, NP can play estrogen role by activating GPR30 receptor, but it can also produce cytotoxicity at higher concentration.

Metformin inhibits proliferation and promotes apoptosis of HER-2 positive breast cancer cells possibly through the Hippo-YAP pathway

To investigate the effect of metformin on the proliferation and apoptosis of HER-2-positive breast cancer cell line SKBR3 and explore the possible mechanism of its action. SKBR3 cells were treated with different concentrations (20-120 μmol/L) of metformin, and the changes in cell proliferation and colony formation ability were assessed using CCK-8 assay and crystal violet staining, respectively. Flow cytometry was performed to analyze cell apoptosis and cell cycle changes. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect mRNA expressions of YAP, TAZ, EGFR, CTGF, CYR61, E-cadherin, N-cadherin, vimentin and fibronectin in the treated cells, and the protein expressions of YAP and TAZ were detected using Western blotting; immunofluorescence assay was used to observe YAP/TAZ nuclear translocation in the cells. Metformin treatment significantly inhibited the proliferation of SKBR3 cells (P < 0.05) in a concentration- and time-dependent manner. The results of flow cytometry showed that metformin significantly promoted apoptosis and caused cell cycle arrest at G1 phase in SKBR3 cells. Metformin treatment significantly down-regulated the mRNA expressions of YAP, TAZ, EGFR, CTGF and CYR61, N-cadherin, vimentin and fibronectin (P < 0.05) and up-regulated the expression of E-cadherin (P < 0.05); Western blotting results showed that YAP and TAZ protein expressions were significantly down-regulated in the cells after metformin treatment (P < 0.05). Immunofluorescence assay revealed that metformin treatment caused the concentration of YAP and TAZ in the cytoplasm, and significantly reduced their amount in the cell nucleus. Metformin can inhibit proliferation and promote apoptosis and epithelal-mesenchymal transition of HER-2 positive breast cancer cells possibly by that inhibing YAP and TAZ expression and their nuclear localization.

Molecular mechanism study of BPAF-induced proliferation of ERα-negative SKBR-3 human breast cancer cells in vitro/in vivo

Bisphenol AF (BPAF) is a known estrogen disruptor of the ERα pathway. The aim of the present study was to characterize the proliferation effects of BPAF on ERα-negative SKBR-3 breast cancer cells with mechanistic insights. BPAF at low concentrations (0.001–0.1 μM) significantly induced the proliferation of SKBR-3 cells. In a SKBR-3 tumor model in BALB/c nude mice, BPAF at 100 mg/kg body weight/day also significantly promoted the growth of SKBR-3 tumors. Low concentrations of BPAF markedly increased the expression of G protein-coupled estrogen receptor (GPER1), c-Myc, CyclinD1 and c-Fos proteins, and enhanced phosphorylation of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in SKBR-3 cells. Further, BPAF significantly upregulated mRNA levels of related target genes in SKBR-3 cells and SKBR-3 tumor tissues in nude mice. The GPER1 inhibitor G15 and phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin (WM) inhibited phosphorylation of Erk and Akt. The specific signal inhibitors also markedly decreased the expression of target genes and weakened the cell proliferation induced by low-concentration BPAF. The findings showed that GPER1 could independently regulate BPAF-induced proliferation of SKBR-3 cells without requiring ERα. These results provide mechanistic insights into the effects of BPAF regarding ERα-negative human breast cancer development.

MicroRNA-124 suppresses the invasion and proliferation of breast cancer cells by targeting TFAP4.

MicroRNA (miRNA/miR)-124 is widely accepted as the suppressor of different tumors. The present study aimed to improve understanding of the potential role of miR-124 in breast cancer. The gene expression profile change derived from the overexpression of miR-124 was investigated using RNA sequencing and bioinformatics analysis of the breast cancer cell line SKBR3. The results demonstrated that the gene expression profile of SKBR3 cells significantly changed. In addition, the transcription factor activating enhancer-binding protein 4 (TFAP4) gene was identified among the top 10 differentially expressed genes, and was identified as a novel target gene of miR-124 using a dual-luciferase reporter assay. TFAP4 knockdown in notably impaired SKBR3 cell migration and proliferation, which was consistent with decreasing migration and proliferation ability following overexpression of miR-124. Taken together, these results suggest that overexpression of miR-124 can suppress the migration and proliferation of SKBR3 cells by tarsgeting TFAP4. Thus, TFAP4 may act as a novel therapeutic target of breast cancer.

Inhibition of PI3K/AKT molecular pathway mediated by membrane estrogen receptor GPER accounts for cryptotanshinone induced antiproliferative effect on breast cancer SKBR-3 cells

BackgroundBreast cancer is the most frequently diagnosed malignancy among women and the second leading cause of cancer death worldwide. Among which nuclear estrogen receptor (nER) negative breast cancer is always with much poor prognosis. Recently, membrane G protein coupled estrogen receptor (GPER), a newly recognized estrogen receptor has been documented to take essential part in the development and treatment of breast cancer. The present study was designed to investigate the anti nER negative breast cancer effect of cryptotanshinone (CPT), an important active compound of traditional Chinese medicine Danshen and its possible molecular pathway.MethodsThe following in vitro tests were performed in nER negative but GPER positive breast cancer SKBR-3 cells. The effect of CPT on cell proliferation rate and cell cycle distribution was evaluated by MTT cell viability test and flow cytometry assay respectively. The role of PI3K/AKT pathway and the mediated function of GPER were tested by western blot and immunofluorescence. Technique of gene silence and the specific GPER agonist G-1 and antagonist G-15 were employed in the experiments to further verify the function of GPER in mediating the anticancer role of CPT.ResultsThe results showed that proliferation of SKBR-3 cells could be blocked by CPT in a time and dose dependent manner. CPT could also exert antiproliferative activities by arresting cell cycle progression in G1 phase and down regulating the expression level of cyclin A, cyclin B, cyclin D and cyclin-dependent kinase 2 (CDK2). The antiproliferative effect of CPT was further enhanced by G-1 and attenuated by G-15. Results of western blot and immunofluorescence showed that expression of PI3K and p-AKT could be downregulated by CPT and such effects were mediated by GPER which were further demonstrated by gene silence test.ConclusionThe current study showed that the antiproliferative action of CPT on SKBR-3 cells was realized by inhibition of GPER mediated PI3K/AKT pathway. These findings provide further validation of GPER serving as useful therapeutic target.

Biological function of long noncoding RNA snaR in HER2-positive breast cancer cells.

Long noncoding RNA, snaR (small NF90-associated RNA), has been reported to be upregulated in various cancer cell lines. We evaluated the additional role of snaR in HER2-positive breast cancer cell lines. We explored changes of expression of snaR among the selected long noncoding RNAs which have a potential in cancer proliferation or progression. The proliferation, migration, and invasion of HER2-positive breast cancer cells (SK-BR3) were evaluated by snaR with RNA interruption in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, wound-healing assay, and Transwell assay. The expression of snaR was remarkably upregulated in SK-BR3 cell lines together with ANRIL, while the SFMBT2 was downregulated in SK-BR3 cell lines. Although Nespas, 7SK, PSF inhibiting RNA, mascRNA, Hoxa11as, NRON, AK023948, MER11C, p53 mRNA, CAR Intergenic 10, HUC 1 and 2, ZFAS1, SCA8, and SNHG5 were also upregulated and UCA1 was downregulated, the differences were not dominent. Based on the expression result, we explored the functional role of snaR in HER2-positive breast cancer. Downregulation of snaR with small interfering RNA was identified to significanlty inhibit migration as well as proliferation of SK-BR3 cells. In this study, snaR was identified as upregulated and to play a role in cancer progression of HER2-positive breast cancer cells. These results suggest snaR as a potential biomarker for HER2-positive breast cancer.

Cytotoxic Effect of Emodin on Growth of SKBR3 Breast Cancer Cells

Background: The poor prognosis of breast cancer is due to its resistance to the conventional treatments. Therefore, researchers are studying about herbs which have anticancer effects. Emodin is a hydroxy-anthraquinone that is found in many medicinal plants and has biological and anticancer effects. According to previous studies, it inhibited the growth of cancer cells by apoptosis. Objectives: In this study, we aimed to determine the effect(s) of emodin on growth and proliferation of SKBR3 cancer cells. Methods: SKBR3 cells were cultivated for 24 hours. Then different concentrations of emodin (0, 10, 25 and 50 µM) were added to the test wells and incubated for 24, 48 and 72 hours. Cell viability was examined by MTT assay after 24, 48 and 72 hours. Apoptosis was determined in cells treated with emodin (0, 10, 25 and 50 µM) using flow cytometric assay. Alterations in expression of apoptotic-related genes (Caspase 3, 8, 9, Bcl2 and Bax) were determined by real time PCR. Caspase 3 activity was measured using a colorimetric assay. Results: Emodin had inhibitory effects on the proliferation of SKBR3 cells with IC50 value of 25 µM. Emodin induced apoptosis and increased the mRNA expression of Caspase 3, 8, 9 and Bax and decreased the mRNA expression of Bcl2 in SKBR3 cells. It also increased the activity of Caspase 3. Conclusions: Emodin had an inhibitory effect on the growth of SKBR3 cell line in a dose and time dependent manner. This study indicated that emodin induces apoptosis in SKBR3 cells through the alterations of the expression of apoptosis-related genes and increases the activity of Caspase 3.

Effect of Fu-Zheng-Xiao-Liu Granules on Expression of Human Epidermal Growth Factor Receptor 2 (HER-2) and Proliferation and Apoptosis of Breast Cancer Cell Line SKBR-3.

BackgroundPrevious research showed that granulized Fu-Zheng-Xiao-Liu has a significant effect on breast cancer. However, it remains unclear whether HER-2 plays a role in this anti-cancer effect.Material/MethodsSerum of male SD rats administered Fu-Zheng-Xiao-Liu granules (SF) was prepared and used to treat HER-2 positive breast cancer cell line SKBR-3. PBS and herceptin were used as negative and positive controls, respectively. MTT was used to detect the proliferation of SKBR-3 cells. Flow cytometry was used to measure the apoptosis of SKBR-3 cells. Western blot and immunofluorescence were used to measure the expression change of HER-2.ResultsSerum of male SD rats administered Fu-Zheng-Xiao-Liu granules had significantly reduced HER-2 expression at both mRNA level and protein level, significantly inhibited proliferation of SKBR-3 cells, and significantly increased apoptosis of SKBR-3 cells, compared to that of the blank control group or serum control group.ConclusionsFu-Zheng-Xiao-Liu granules affect proliferation and apoptosis through inhibition of HER-2 transcription and translation, providing an experimental basis for further study of the mechanism by which Fu-Zheng-Xiao-Liu granules affect breast cancer.

Molecular mechanisms of resistance to phosphatidyl inositol 3-kinase inhibitors in triple-negative breast cancer cells

To explore the molecular mechanisms of resistance to phosphatidyl inositol 3-kinase (PI3K) inhibitors in triple-negative breast cancer (TNBC) cells. HCC70 cells (TNBC) were transfected with siFZD7, siWANT5B or siGSK3 using lipofectamine 2000 transfection reagent. The expression levels of key proteins of WNT/β-catenin and PI3K/AKT/mTOR pathways were determined by Western blot analysis. After HCC70, MCF-7 (ER-positive) and SK-BR3 (HER2-positive) cells were treated with PI3K/AKT/mTOR inhibitors, the inhibition rates of cell proliferation were measured by MTT assay, and half maximal inhibitory concentrations (IC50) were calculated. The altered activities of WNT/β-catenin and PI3K/AKT/mTOR proteins were detected by Western blot and luciferase report gene assay, respectively. The nuclear translocation of β-catenin protein was examined by immunofluorescence assay. Xenograft nude mouse model was used to evaluate the tumorigenicity of breast cancer cells treated with BKM120 in vivo. The expression levels of p-LRP6, p-4EBP1 and β-catenin proteins in the tumor tissues were determined by immunohistochemical staining. The expression levels of FZD7, WANT5B and GSK3 proteins were significantly reduced in the HCC70 cells transfected with the target siRNAs. Meanwhile, the activity of WNT/β-catenin was enhanced and PI3K/AKT/mTOR pathway was inhibited. PI3K/AKT/mTOR inhibitors suppressed MCF-7 and SK-BR3 cell proliferation. The IC50 of GDC-094, BKM120, XL147, perifosine, everolimus, and BEZ235 in MCF-7 cells were 0.46 mmol/L, 1.44 mmol/L, 4.34 mmol/L, 11.35 μmol/L, 53.71 μmol/L and 12.87 μmol/L respectively, and 0.63 mmol/L, 0.58 mmol/L, 3.74 mmol/L, 13.22 μmol/L, 60.00 μmol/L and 11.38 μmol/L in the SK-BR3 cells, respectively. The results of luciferase report gene assay showed that the luciferase activities in HCC70, MCF-7 and SK-BR3 cells treated with BKM120 were 1.75±0.05, 1.13±0.02 and 0.43±0.01, respectively. The luciferase activities in HCC70 and SK-BR3 cells were significantly different from that of the control cells (1.00±0.02, P<0.05). The immunohistochemical analysis showed that BKM120 inhibited mTOR activity, and the enhanced WNT/β-catenin activity reversed the phenotype of inhibitory mTOR induced by BKM120. BKM120 suppressed the tumorigenic ability of MCF-7 and SK-BR3 cells in vivo, but had no effect on cultured HCC70 cells. The immunohistochemical analysis showed nuclear translocation of β-catenin protein and increased expression level of p-LRP-6 protein in transplanted tumor tissues from HCC70 cells treated with BKM120, increased the level of p-LRP-6 protein, and no changes of p-4EBP1 protein expression. However, no nuclear translocation of β-catenin protein and no decrease of p-LRP6 and p-4EBP1 protein levels in the transplanted tumor tissue of MCF-7 cells after treatment with BKM120. The triple-negative breast cancer HCC70 cells have drugs-resistance to PI3K inhibitors. The WNT/β-catenin signaling pathway may regulate the PI3K/AKT/mTOR pathway, therefore, inducing the drug-resistance of TNBC cells to PI3K inhibitors.

MicroRNA-101 inhibits cell proliferation and induces apoptosis by targeting EYA1 in breast cancer.

MicroRNAs (miRNAs or miRs) regulate gene expression by negatively modulating the stability or translational efficiency of their target genes by targeting the 3'-untranslated region(3'-UTR). Aberrant miRNA expression has been reported in various types of cancer; miRNAs can function as either oncogenes or tumor suppressor genes in cancer. In this study, we examined the expression level of miR‑101 in breast cancer tissues and cell lines by RT-qPCR, and found that miR‑101 expression was downregulated in breast cancer tissues and cell lines; indeed, in 6of the 28tissue samples, miR‑101 could not be detected. Furthermore, miR‑101, when transfected into SKBR3cells, inhibited cell proliferation and promoted apoptosis, while miR‑101 inhibitor had the opposite effect. A dual-luciferase reporter assay revealed that miR‑101 targeted the 3'-UTR of eyes absent homolog1(Drosophila)(EYA1). Western blot analysis demonstrated a significantly decreased protein level of EYA1 in the SKBR3 cells transfected with miR‑101 mimic, whereas transfection with miR‑101 inhibitor led to an increased level of EYA1. Moreover, an increased expression of EYA1 was also found in breast cancer tissues and cell lines. The silencing of EYA1 using siRNA targeting EYA1(EYA1‑siRNA) significantly inhibited SKBR3 cell proliferation and promoted apoptosis, and also suppressed the increased proliferation induced by transfection with miR‑101 inhibitor. The protein expression levels of Notch signaling components (jagged1, Hes1 and Hey1) were significantly decreased by transfection with miR‑101 mimic and EYA1-siRNA, and were increased by transfection with miR‑101 inhibitor. Furthermore, the elevated protein expression levels of jagged1, Hes1 and Hey1 induced by transfection with miR‑101 inhibitor in the SKBR3 cells were significantly decreased by transfection with EYA1-siRNA. Taken together, these results suggest that miR‑101 is down-regulated in breast cancer, and can inhibit cell proliferation and promote apoptosis by targeting EYA1 through the Notch signaling pathway.

Downregulation of LncRNA GAS5 causes trastuzumab resistance in breast cancer.

Therapeutic resistance to trastuzumab caused by dysregulation of long noncoding RNAs (lncRNAs) is a major obstacle to clinical management of HER2-positive breast cancer. To investigate which lncRNAs contribute to trastuzumab resistance, we screened a microarray of lncRNAs involved in the malignant phenotype of trastuzumab-resistant SKBR-3/Tr cells. Expression of the lncRNA GAS5 was decreased in SKBR-3/Tr cells and in breast cancer tissue from trastuzumab-treated patients. Inhibition of GAS5 promoted SKBR-3 cell proliferation, and GAS5 knockdown partially reversed lapatinib-induced inhibition of SKBR-3/Tr cell proliferation. GAS5 suppresses cancer proliferation by acting as a molecular sponge for miR-21, leading to the de-repression of phosphatase and tensin homologs (PTEN), the endogenous target of miR-21. Moreover, mTOR activation associated with reduced GAS5 expression was required to suppress PTEN. This work identifies GAS5 as a novel prognostic marker and candidate drug target for HER2-positive breast cancer.

Abstract P5-04-15: Inhibitory effects of calcitriol and the vitamin D analogue paricalcitol in combination with chemotherapy on the growth of HER2 overexpressing breast cancer cells

Abstract Introduction: Despite recent advances in therapy, additional options for improving the outcomes of patients with HER2-overexpressing breast cancer (BC) are needed. The role of vitamin D in relation to BC outcomes is controversial. Both calcitriol and synthetic vitamin D analogues have been shown to increase sensitivity to chemotherapy in BC cell lines. In this study, we aimed to determine the effect of combined treatment with either calcitriol or the vitamin D analogue paricalcitol plus doxorubicin or paclitaxel in HER2+ BC cells. Methods: Cell characterization was performed using western blot for estrogen receptor (ER) α, progesterone receptor (PR) A-B, HER2 receptor and vitamin D receptor (VDR) A-B. Flow cytometry was used to determine the quantitative expression of HER2 in an established breast cancer cell line known to express VDR (SKBR3 [ERα-, PR A-B -, HER2+]). The effects of calcitriol and paricalcitol, alone or in combination with doxorubicin or paclitaxel, were evaluated using a Sulforhodamine B growth assay. We calculated IC20 inhibitory concentrations by non-linear regression analysis using sigmoidal fitting of dose-response curves. Results are presented as the mean cell proliferation percentage (%) ± standard deviation. Statistical analyses were carried out using one-way ANOVA and the Holm-Sidak method. Results: Calcitriol, paricalcitol, doxorubicin and paclitaxel inhibited cell proliferation in a dose dependent manner. Calcitriol 100nM in combination with doxorubicin 0.01 μM inhibited the proliferation of SKBR3 cells to 30.9% ± 24.1, compared to 74.6% ± 6.6 with calcitriol alone (p = 0.003) and 86.6% ± 4.1 with doxorubicin alone (p &amp;lt; 0.001). Similarly, the combination of calcitriol 100nM plus paclitaxel 0.001 μM inhibited the proliferation of SKBR3 cells to 11.6% ± 10.4, compared to 77.5% ± 6.1 with calcitriol alone (p &amp;lt; 0.001) and 50.7% ± 8.4 with paclitaxel alone (p &amp;lt; 0.001). Paricalcitol 25nM in combination with doxorubicin 0.01 μM inhibited the proliferation of SKBR3 cells to 49.1% ± 25.2, compared to 72.3 ± 13.2 with paricalcitol alone (p = 0.21) and with doxorubicin alone (p = 0.05). Paricalcitol 10nM in combination with paclitaxel 0.001μM inhibited the proliferation of SKBR3 cells to 26.1% ± 23.1, compared to 72.3% ± 13.2 with paricalcitol alone (p = 0.05) and 43.2% ± 12.1 with paclitaxel alone (p =0.285). Conclusions: Simultaneous treatment of SKBR3 cells with calcitriol plus either doxorubicin or paclitaxel showed a cooperative growth-inhibiting effect when compared with each cytotoxic drug alone. The combination of paricalcitol with each of the cytotoxic agents also showed a trend towards a higher growth-inhibiting effect, but failed to reach statistical significance when compared with each drug alone. Although these results point towards the presence of a cooperative antiproliferative effect of Vitamin D compounds on HER2+ BC cells when combined with chemotherapy, the concentrations needed to reach such an effect were high, which could potentially limit their use in clinical practice. Citation Format: Mateos-Soria AS, Garcia-Becerra RA, Diaz-Nieto L, Ventura-Gallegos JL, Jacobo-Herrera N, Soto-Perez-de-Celis E, Zentella-Dehesa A, Chávarri-Guera Y. Inhibitory effects of calcitriol and the vitamin D analogue paricalcitol in combination with chemotherapy on the growth of HER2 overexpressing breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-15.

ANTI-TUMOR EFFECTS OF EPHEDRINE AND ANISODAMINE ON SKBR3 HUMAN BREAST CANCER CELL LINE

Background: To investigate the effects of ephedrine and anisodamine on the proliferation of human breast cancer.&#x0D; Materials and Methods: SKBR3 cell was treated with or without ephedrine and / or anisodamine, respectively. The trypan blue exclusion assay was&#x0D; used to determine cell numbers. Flow cytometry was used to assess cell cycle distribution and apoptosis. The concentration of cAMP and cyclin D1&#x0D; was analyzed by enzyme-linked immunosorbent assay. Western blot was used to measure PKA.&#x0D; Results: Ephedrine and anisodamine inhibited cell proliferation and arrested SKBR3 cells at G0/G1 phases. Ephedrine and anisodamine increased the&#x0D; level of CD1 in SKBR3 cells. Furthermore, significant change in intracellular cAMP concentration was found in SKBR3 cells treated with ephedrine&#x0D; and anisodamine. The phosphorylation of PKA substrate was not activated after 48 hours of treatment with ephedrine and anisodamine.&#x0D; Conclusion: Ephedrine and anisodamine inhibit the proliferation of SKBR3 cells via a significantly change of intracellular cAMP concentration.

Apigenin induces caspase-dependent apoptosis by inhibiting signal transducer and activator of transcription 3 signaling in HER2-overexpressing SKBR3 breast cancer cells.

Phytoestrogens have been demonstrated to inhibit tumor induction; however, their molecular mechanisms of action have remained elusive. The present study aimed to investigate the effects of a phytoestrogen, apigenin, on proliferation and apoptosis of the human epidermal growth factor receptor 2 (HER2)-expressing breast cancer cell line SKBR3. Proliferation assay, MTT assay, fluorescence-activated cell sorting analysis, western blot analysis, immunocytochemistry, reverse transcription-polymerase chain reaction and ELISA assay were used in the present study. The results of the present study indicated that apigenin inhibited the proliferation of SKBR3 cells in a dose-and time-dependent manner. This inhibition of growth was accompanied by an increase in the sub-G0/G1 apoptotic population. Furthermore, apigenin enhanced the expression levels of cleaved caspase-8 and -3, and induced the cleavage of poly(adenosine diphosphate ribose) polymerase in SKBR3 cells, confirming that apigenin promotes apoptosis via a caspase-dependent pathway. Apigenin additionally reduced the expression of phosphorylated (p)-janus kinase 2 and p-signal transducer and activator of transcription 3 (STAT3), inhibited CoCl2-induced vascular endothelial growth factor (VEGF) secretion and decreased the nuclear localization of STAT3. The STAT3 inhibitor S31-201 decreased the cellular proliferation rate and reduced the expression of p-STAT3 and VEGF. Therefore, these results suggested that apigenin induced apoptosis via the inhibition of STAT3 signaling in SKBR3 cells. In conclusion, the results of the present study indicated that apigenin may be a potentially useful compound for the prevention or treatment of HER2-overexpressing breast cancer.

Silibinin induces apoptosis and inhibits proliferation of estrogen receptor (ER)-negative breast carcinoma cells through suppression of nuclear factor kappa B activation.

Silibinin is a traditionally well-known drug for its hepatoprotective efficacy against various types of liver afflictions. In addition, it has recently been considered broadly as a potential chemopreventive agent against many types of cancers. The current study was designed to evaluate the restrictive effects of pharmacological doses of silibinin on SKBR3, an ErbB2-overexpressed and ER-negative human breast carcinoma cell line. Effect of silibinin on metabolic activity and proliferation of human breast carcinoma (SKBR3) cell line were evaluated by MTT and BrdU assays respectively. Furthermore, the proapoptotic effect of silibinin was investigated using flow cytometry. The NF-κB phosphorylation assay was also used to assess the effect of silibinin on NF-κB activation. The alkalizing effect of silibinin on SKBR3 cell line was evaluated by measuring pH of media of the silibinin-treated cells compared to control. Our results indicate that silibinin inhibited metabolic activity and cell proliferation of SKBR3 cells in a dose-dependent manner. Moreover, silibinin significantly induced apoptosis in SKBR3 cells. On the other hand, silibinin significantly inhibited activation of NF-κB which is known to be highly active in this cell line. Alkalizing effect of silibinin was also observed. The results obtained here indicate that silibinin may be an efficacious therapeutic agent against ER-negative breast carcinomas with high inhibitory effect on NF-κB.

Icariin and icaritin stimulate the proliferation of SKBr3 cells through the GPER1-mediated modulation of the EGFR-MAPK signaling pathway

Icariin (ICA) and icaritin (ICT), with a similar structure to genistein, are the important bioactive components of the genus Epimedium, and regulate many cellular processes. In the present study, using the estrogen receptor (ER)-negative breast cancer cell line, SKBr3, as a model, we examined the hypothesis that ICA and ICT at low concentrations stimulate SKBr3 cell proliferation in vitro through the functional membrane, G protein‑coupled estrogen receptor 1 (GPER1), mediated by the epithelial growth factor receptor (EGFR)‑mitogen-activated protein kinase (MAPK) signaling pathway. MTT assay revealed that ICA and ICT at doses of 1 nM to 1 µM markedly stimulated SKBr3 cell proliferation in a dose-dependent manner. The ICA- and ICT-stimulated cell growth was completely suppressed by the GPER1 antagonist, G-15, indicating that the ICA‑ and ICT-stimulated cell proliferation was mediated by GPER1 activation. Semi-quantitative RT-PCR analysis revealed that treatment with ICA and ICT enhanced the transcription of c-fos, a proliferation-related early gene. The ICA- and ICT-stimulated mRNA expression was markedly attenuated by G-15, AG-1478 (an EGFR antagonist) or PD98059 (a MAPK inhibitor). Our data also demonstrated that ICA and ICT increased the phosphorylation of ERK1/2. The ICA- and ICT-stimulated ERK1/2 phosphorylation was blocked by pre-treatment of the cells with G-15 and AG-1478 or PD 98059. Flow cytometric analysis confirmed that the ICA- and ICT-stimulated SKBr3 cell proliferation involved the GPER1-mediated modulation of the EGFR‑MAPK signaling pathway. To the best of our knowledge, our current findings demonstrate for the first time that ICA and ICT promote the progression of ER-negative breast cancer through the activation of membrane GPER1.

In vitro Study of Nucleostemin as a Potential Therapeutic Target in Human Breast Carcinoma SKBR-3 Cells

Although nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis and expression has been observed in some types of human cancer and stem cells, the molecular mechanisms involved in mediation of cell proliferation and cell cycling remains largely elusive. The aim of the present study was to evaluate NS as a potential target for gene therapy of human breast carcinoma by investigating NS gene expression and its effects on SKBR-3 cell proliferation and apoptosis. NS mRNA and protein were both found to be highly expressed in all detected cancer cell lines. The apoptotic rate of the pcDNA3.1-NS-Silencer group (12.1-15.4 ± 3.8%) was significantly higher than those of pcDNA3.1-NS (7.2-12.0 ± 1.7%) and non-transfection groups (4.1-6.5 ± 1.8%, P<0.01). MTT assays showed the knockdown of NS expression reduced the proliferation rate of SKBR-3 cells significantly. Matrigel invasion and wound healing assays indicated that the number of invading cells was significantly decreased in the pcDNA3.1-NS-siRNA group (P<0.01), but there were no significant difference between non-transfected and over-expression groups (P>0.05). Moreover, RNAi-mediated NS down- regulation induced SKBR-3 cell G1 phase arrest, inhibited cell proliferation, and promoted p53 pathway-mediated cell apoptosis in SKBR-3 cells. NS might thus be an important regulator in the G2/M check point of cell cycle, blocking SKBR-3 cell progression through the G1/S phase. On the whole, these results suggest NS might be a tumor suppressor and important therapeutic target in human cancers.

Altered features and increased chemosensitivity of human breast cancer cells mediated by adipose tissue-derived mesenchymal stromal cells

BackgroundMesenchymal stromal cells (MSCs) represent heterogeneous cell population suitable for cell therapies in regenerative medicine. MSCs can also substantially affect tumor biology due to their ability to be recruited to the tumor stroma and interact with malignant cells via direct contacts and paracrine signaling. The aim of our study was to characterize molecular changes dictated by adipose tissue-derived mesenchymal stromal cells (AT-MSCs) and the effects on drug responses in human breast cancer cells SKBR3.MethodsThe tumor cells were either directly cocultured with AT-MSCs or exposed to MSCs-conditioned medium (MSC-CM). Changes in cell biology were evaluated by kinetic live cell imaging, fluorescent microscopy, scratch wound assay, expression analysis, cytokine secretion profiling, ATP-based viability and apoptosis assays. The efficiency of cytotoxic treatment in the presence of AT-MSCs or MSCs-CM was analyzed.ResultsThe AT-MSCs altered tumor cell morphology, induced epithelial-to-mesenchymal transition, increased mammosphere formation, cell confluence and migration of SKBR3. These features were attributed to molecular changes induced by MSCs-secreted cytokines and chemokines in breast cancer cells. AT-MSCs significantly inhibited the proliferation of SKBR3 cells in direct cocultures which was shown to be dependent on the SDF-1α/CXCR4 signaling axis. MSC-CM-exposed SKBR3 or SKBR3 in direct coculture with AT-MSCs exhibited increased chemosensitivity and induction of apoptosis in response to doxorubicin and 5-fluorouracil.ConclusionsOur work further highlights the multi-level nature of tumor-stromal cell interplay and demonstrates the capability of AT-MSCs and MSC-secreted factors to alter the anti-tumor drug responses.