SKBR3 Cell Lines Research Articles

Design synthesis and anti-proliferative activity of some new coumarin substituted hydrazide–hydrazone derivatives

A series of 21 coumarin hydrazide–hydrazone derivatives were designed, synthesized and evaluated potential cytotoxicity effects at 25 μg/mL for 48 h against liver cancer (HepG2) cell line in vitro. Then, seven out of 21 compounds with % cell viability lower than 60% were selected for evaluation of in vitro anti-proliferative activity against liver cancer (HepG2), breast cancer (SKBR-3) and human colon cancer (Caco-2) cell lines. Among the test compounds, 5g, 6d and 6f showed potent activities against both Hep-G2 and SKBR-3 cell lines. More significantly, compound 6d, having a 4-bromophenyl moiety, exhibited best cytotoxic activity against Hep-G2 cell line with IC50 value of 2.84 ± 0.48 μg/mL which is comparable to the standard doxorubicin (IC50 = 2.11 ± 0.13 μg/mL). In addition, compound 6f, having 4-methoxyphenyl moiety, demonstrated the most potent activity (IC50 = 2.34 ± 0.68 μg/mL) against SKBR-3 cell line on comparison with other tested coumarin hydrazide–hydrazone derivatives. Unfortunately, all test compounds, as well as doxorubicin, showed no cytotoxicity toward drug-resistant cell line, Caco-2. Our preliminary results indicated that coumarin hydrazide–hydrazone derivatives could be exploited as leading structures for further anticancer-drug development.

Sodium-Dependent Glucose Transporter 1 (SGLT1) Stabled by HER2 Promotes Breast Cancer Cell Proliferation by Activation of the PI3K/Akt/mTOR Signaling Pathway in HER2+ Breast Cancer.

Aerobic glycolysis is a hallmark of tumor cells. SGLT1 plays a vital role in glucose metabolism. However, whether SGLT1 could promote cell growth and proliferation in breast cancer remains unclear. Here, we investigated the expression of SGLT1 in breast cancer and examined its role in malignant behavior and prognosis. Further, we examined the SGLT1 expression in breast cancer tissues and its relationship with clinicopathologic characteristics. We clarified that SGLT1 was overexpressed in HER2+ breast cancer cell lines and was affected by HER2 status. We further found that SGLT1 affected breast cancer cell proliferation and patient survival by mediating cell survival pathway activation. SGLT1 was overexpressed in HER2+ breast cancers and associated with lymph node metastasis and HER2+ status. Inhibition of HER2 decreased SGLT1 expression, and the extracellular acidification rate was also reduced in the UACC812 and SKBR3 cell lines. These changes could be reversed by proteasome inhibitor treatment. Knockdown of SGLT1 blocked PI3K/Akt/mTOR signaling, thereby inhibiting cell proliferation. Further, we demonstrated that high SGLT1 was significantly correlated with shorter survival in all breast cancer patients and specifically in HER2+ breast cancer patients. Therefore, we conclude that SGLT1 is overexpressed in HER2+ breast cancer, thereby promoting cell proliferation and shortening survival by activating PI3K/Akt/mTOR signaling. This study submits that SGLT1 is promising not only as a novel biomarker of HER2+ breast cancer subtype but also as a potential drug target.

Effects of ICI 182,780, an ERα and ERβ antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells

ABSTRACTObjectiveTo investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability.MethodsCell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy.ResultsRegarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line.ConclusionThe effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors’ involvement in cellular processes of breast cancer resistance.

Evaluation of HER2 expression in urothelial carcinoma cells as a biomarker for circulating tumor cells.

Detection of circulating tumor cells (CTC) by techniques based on epithelial cell adhesion molecule (EpCAM) is suboptimal in urothelial carcinoma (UC). As HER2 is thought to be broadly expressed in UC, we explored its utility for CTC detection. HER2 and EpCAM expression was analyzed in 18 UC cell lines (UCCs) by qRT-PCR, western blot and fluorescence-activated cell scanning (FACS) and compared to the strongly HER2-expressing breast cancer cell line SKBR3 and other controls. HER2 expression in UC patient tissues was measured by qRT PCR and correlated with data on survival and risk for metastasis. UCCs with high EpCAM and variable HER2 expression were used for spike-in experiments in the CellSearch system. Twenty-one blood samples from 13 metastatic UC patients were analyzed for HER2-positive CTCs with CellSearch. HER2 mRNA and protein were broadly expressed in UCC, with some heterogeneity, but at least 10-fold lower than in the HER-2+ SKBR3 cells. Variations were unrelated to cellular phenotype or clinicopathological characteristics. EpCAM expression was essentially restricted to UCCs with epitheloid phenotypes. Heterogeneity of EpCAM and HER2 expression was observed also in spike-in experiments. The 7 of 21 blood samples from 6 of 13 patients were enumerated as CTC positive via EpCAM, but only one sample stained weakly positive (1+) for HER2. Detection rate of CTCs by EpCAM in UC is poor, even in metastatic patients. Because of its widespread expression, particularly in patients with high risk of metastasis, detection of HER2 could improve identification of UC CTCs, which is why combined detection using antibodies for EpCAM and HER2 may be beneficial.

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.

Expression of MTERF3 gene in breast carcinoma and the relationship with clinicopathological characteristics.

BackgroundMitochondrial transcription termination factor 3 (MTERF3) is a negative regulator of mitochondrial transcription. It is a modular factor involves in mitochondrial ribosome biogenesis and protein synthesis. However, the association between MTERF3 and breast cancers remains largely unknown. The aim of this study was to investigate the expression of MTERF3 in breast carcinoma and to analyze its clinicopathological significance, and to examine the potential prognostic value of MTERF3 in breast cancer.MethodsThe protein expression levels of MTERF3 in MCF7 (Luminal A), BT-474 (Luminal B), SKBR3 (HER2 overexpression), MDA-MB-468 (Basal like) and MCF10A cell lines were detected by Western blotting. Immunohistochemistry (IHC), Western blotting, and semiquantitative RT-PCR were performed to analyze the protein and mRNA expression levels of MTERF3 in 58 breast cancer tissues and 58 noncancerous breast tissues. The MTERF3 expression data and clinical information from breast cancer patients were downloaded from the TCGA dataset by using the R3.6.1 software. Then the relationship between the expression level of MTERF3 and clinicopathological characteristics and the prognostic value was analyzed. A Cox regression model was performed for the multivariate analysis of the factors that affected the prognosis of breast cancer. The association between the expression levels of MTERF3 and other mitochondrial regulatory genes was analyzed with GEPIA.ResultsMTERF3 is upregulated in breast cancer cell lines compared to noncancerous breast cell line. The IHC results showed that the MTERF3 protein was located in the cytoplasm, and the rate of positive expression in breast cancer tissue was significantly upregulated compared with the adjacent normal tissue. The mRNA and protein expression levels of MTERF3 in breast cancer tissues were significantly higher than that in breast tissues. Moreover, the expression of MTERF3 was significantly correlated with ER status, PR status, breast cancer molecular typing, cancer type, histological diagnosis and primary site (P<0.05). Further analysis showed MTERF3 expression was not related to prognosis. Multivariate Cox regression analysis showed that age, metastasis status and tumor type were independent prognostic factors for breast cancer patients. The expression levels of MTERF3 were positively correlated with the TFAM, TFB1M, TFB2M, MTERF1, TEFM and MFN1 genes but negatively correlated with the MTERF4 and PINK1 genes. In addition, the expression levels of MTERF3 were not correlated with the MTERF2 gene.ConclusionsMTERF3 was significantly upregulated in breast cancer cells and tissues compared with noncancerous cells and tissues. Moreover, the expression level of MTERF3 was correlated with ER status, PR status, breast cancer molecular typing, cancer type, histological diagnosis and primary site. These findings suggested that the upregulation of MTERF3 may be used as a diagnostic and therapeutic target in breast carcinoma.

Uptake and effect of radioiodine exposure on SKBR-3 cell lines

Radioiodine has been known as an adjuvant therapy for thyroid cancer. Beside for thyroid cell, radioiodine is reported to be taken up by breast cell. It may be proposed as an alternative for breast cancer therapy. Objective: The study aim was to analyze an uptake of radioiodine and cell proliferation rate of breast cancer cell after the exposure of radioiodine. Methods: This study used SKBR-3 cell line as representative of Human Epidermal Growth Factor Receptor (HER2+) subtype. The cells were planted in DMEM medium added 10% fetal bovine serum (FBS), panstrep 1% and amphotericin B. The cells were grown until 80% confluent and then stripped. Subsequently the cells were sub-cultured on plates 12 and 6 wells respectively for measured uptake of radioiodine and cell proliferation rate after the exposure. Radioiodine uptake was calculated by sum efflux of 125 Iodine (125I) and the amount of uptake after the addition of 95% ethanol. Cell proliferation rate was measured by calculated by using clonogenic assay after iodine-131 (131I) exposure. Results: The average of iodine uptake was 194±50 cpm/106 cell. The average cell proliferation after radioiodine exposure was 54%±5%; Conclusions: Radioiodine is taken by SKBR-3 cells and it reduced cell proliferation rate. This finding shows an opportunity for radioiodine as an alternative therapy for breast cancer. Another studies are needed to understand of cell death the mechanism.

Cisplatin-resistant MDA-MB-231 Cell-derived Exosomes Increase the Resistance of Recipient Cells in an Exosomal miR-423-5p-dependent Manner

Chemoresistance blunts the therapeutic effect of cisplatin (DDP) on Triple-Negative Breast Cancer (TNBC). Researchers have not determined to date whether exosomes confer DDP resistance to other breast cancer cells or whether exosomal transfer of miRNAs derived from DDP-resistant TNBC cells confer DDP resistance. The aim of this study was to investigate the role of exosomes in chemoresistance in breast cancer. MDA-MB-231 cells resistant to DDP (231/DDP) were established. Exosomes were isolated from 231/DDP cells (DDP/EXO) and characterized by measuring the levels of protein markers, nanoparticle tracking analysis and transmission electron microscopy. MDA-MB-231, MCF-7 and SKBR-3 cell lines were treated with the isolated DDP/EXOs and cell proliferation and cytotoxicity to DDP were evaluated using MTT assays and apoptosis analyses. Western blotting was used to examine P-glycoprotein (P-gp) expression. Additionally, a microarray was used to analyse microRNA (miRNA) expression profiles in MDA-MB-231 and 231/DDP exosomes. The effects on miRNAs were determined using RT-PCR. Exosomal miR-423-5p was extracted, and differential expression was verified. The MTT cell viability assay, flow cytometry, and Transwell and immunofluorescence assays were performed to determine if differential expression of miR-423-5p sensitized cells to DDP in vitro. Under a transmission electron microscope, the isolated exosomes exhibited a round or oval shape with a diameter ranging between 40 and 100 nm. DDP/EXOs labelled with PKH67 were taken up by MDA-MB-231 cells. After an incubation with DDP/EXOs, the cell lines exhibited a higher IC50 value for cisplatin, P-gp expression, migration and invasion capabilities and a lower apoptosis rate. Furthermore, 60 miRNAs from exosomes derived from 231/DDP cells were significantly up-regulated compared to exosomes from MDA-MB-231 cells. Notably, compared to the corresponding sensitive exosomes, miR-370-3p, miR-423-5p and miR-373 were the most differentially expressed miRNAs in DDP-resistant exosomes. We chose miR-423-5p, and up-regulation and down-regulation of exosomal miR-423-5p expression significantly affected DDP resistance. Exosomes from DDP-resistant TNBC cells (231/DDP) altered the sensitivity of other breast cancer cells to DDP in an exosomal miR-423-5p dependent manner. Our research helps to elucidate the mechanism of DDP resistance in breast cancer.

Trastuzumab in combination with AT-101 induces cytotoxicity and apoptosis in Her2 positive breast cancer cells.

Aim: AT-101 is a polyphenolic compound with potent anti-apoptotic effects in various cancers. In this study, the possible synergistic cytotoxic and apoptotic effect of trastuzumab/AT-101 combination was investigated in HER2-positive breast cancer cell lines. Materials & methods: SKBR-3, MDA-MB-453 and MCF-10A cell lines were treated with a trastuzumab/AT-101 combination. Synergistic cytotoxicity and apoptosis effects were shown and then PI3K and Akt protein levels were studied. Result: The trastuzumab/AT-101 combination induced synergistic cytotoxicity and apoptosis in both breast cancer cells but not in MCF-10A cells. Combination treatment induced cytotoxicity via inhibiting PI3K/AKT but not the MAPK/ERK pathway. Conclusion: The trastuzumab/AT-101 combination may be a good candidate for patients with trastuzumab-resistant Her2-positive breast cancer and inhibition of the PI3K/AKT pathway may be one of the underlying mechanisms.

Combining RANK/RANKL and ERBB-2 targeting as a novel strategy in ERBB-2-positive breast carcinomas

BackgroundERBB-2 is overexpressed in about 20% of breast cancers (BCs), indicating poor prognosis. The receptor activator of nuclear factor-κB (RANK) pathway is implicated in ERBB-2 (+) BC. The purpose of this study was to elucidate the underlying molecular mechanism of this interaction and the beneficial impact of dual targeting of RANK and ERBB-2 pathways.MethodsWe used SKBR3, MCF7, MDA-MB-453, and BT-474 human BC cell lines. We examined RANK and RANKL expression using RT-PCR, Western blot, and immunofluorescence. The evaluation of RANK expression in a cohort of BC patients was performed using immunohistochemistry. The interaction between RANK and ERBB family members was detected using proximity ligation assay (PLA), which enables the visualization of interacting proteins. We used inhibitors of both pathways [trastuzumab (T), pertuzumab (P), denosumab (D)]. NF-κB pathway activation was studied using Western blot. Cell growth and viability was evaluated using XTT, flow cytometry, and clonogenic assay. For cell migration evaluation, scratch assay was performed. Data were analyzed by one-way ANOVA.ResultsCell lines express RANK and RANKL. RANK immunostaining was also detected in human BC tissue samples. RANK receptor dimerizes with ERBB family members. RANK/ERBB-2 dimer number seems to be associated with ERBB-2 expression (SKBR3, 5.4; BT-474, 8.2; MCF7, 0.7; MDA-MB-453, 0.3). RANK/ERBB-2 dimers were decreased in the presence of the inhibitors D, T, and P, while they were increased after RANKL (R) treatment in SKBR3 (m, 5.4; D, 1.2; T, 1.9; DT, 0.6; TP, 1; DTP, 0.4; R, 11.8) and BT-474 (m, 8.2; D, 3.1; T, 4.3; DT, 0.7; TP, 3.4; DTP, 3.2; R, 11.6). Combination targeting of SKBR3 further decreased NF-κB pathway activation compared to single targeting. In SKBR3, RANKL and ERBB-2 blockage resulted in reduced cell proliferation, increased apoptosis, and lower metastatic potential compared to mock cells (m) and reversed values in RANKL presence. The combination treatment of SKBR3 with D, T, and P had an advantage in functional traits compared to single targeting. Denosumab suppressed NF-κB signaling and diminished proliferation rate in MDA-MB-453 cells. MCF7 did not correspond to inhibitors.ConclusionsThe results indicate a novel physical and molecular association between ERBB-2 and RANK pathways that affects ERBB-2 (+) BC growth. We also present data suggesting that the combination of anti-ERBB-2 agents and RANKL inhibitors have a potential direct anti-tumor effect and should be further tested in certain BC patients.

IN-VITRO AND IN-VIVO STUDIES OF THE POSSIBLE CHEMOPREVENTIVE FUNCTIONS OF SOYBEAN ISOFLAVONE AND FLAVONOIDS ON BREAST CANCER

Objectives: In this study, we assess the possible influence of soybean isoflavone (genistein) and other flavonoids (quercetin and catechin) on breast cancer chemoprevention. We design in-vitro and in-vivo experiments to analyze the effect of genistein, quercetin and catechin on cell proliferation, cell migration, and angiogenesis of breast cancer cells. Methods: In cell proliferation experiment, MCF-7 cells, SKBR-3 cells, and HUVEC cells were treated with genistein and other flavonoids (catechin and/or quercetin) for 48[Formula: see text]h to assess the influence on cell growth of normal and breast cancer cells. In cell motility test, we analyze the effect of isoflavone and flavonoids on migration ability of MCF-7 cells by 16[Formula: see text]h and SKBR-3 cells by 24[Formula: see text]h in two different concentrations (1.25[Formula: see text][Formula: see text]g/ml and 2.5[Formula: see text][Formula: see text]g/ml). In the in-vivo experiment, SKBR-3 cells mixed with PBS and catechin, respectively, were injected subcutaneously into nude mice, then we investigated the effect of catechin on cell growth by observing subcutaneous tumor size changes after 15 days. Results: The results suggest that genistein and quercetin can significantly inhibit proliferation of breast cancer cells, and their inhibitory effects are independent of estrogen receptor. In cell motility tests, all of the three phytochemicals were effective in the inhibition of cell migration on two breast cancer cell lines, except for quercetin on cell migration of SKBR-3 cell line. In the in-vitro experiment, catechin showed stimulatory effect on cell proliferation of HUVEC cell line, which may consider positive effect on angiogenesis, rather than inhibitory effect. However, in the in-vivo experiment, it showed no significant change in tumor size between the groups of with and without catechin treatment. Conclusions: According to our study, the results suggest that isoflavone and flavonoids tend to inhibit cell growth and metastasis of breast cancer cells. Our in-vivo experiment does not reach a significant result, and it may be due to lower catechin concentration. Under in-vivo environment, we should also consider the possible metabolic forms of catechin that cause different result from the in-vitro study.

Synthesis and cytotoxicity evaluation of glycosidic derivatives of lawsone against breast cancer cell lines

Breast cancer is the most incident and mortal cancer type in women, with an estimated 2 million new cases expected by 2020 worldwide, with 600,000 deaths. As not all breast cancer types respond to the anti-hormonal therapy, the development of new antineoplastic drugs is necessary. Lawsone (2-hydroxy-1,4-naphtoquinone) is a natural bioactive naphtoquinone displaying a range of activities, with dozens of derivatives described in the literature, including some glycosides possessing antitumor activity. Here, a series of glycosides of lawsone are reported for the first time and all compounds displayed good activity against the SKBR-3 cell line, with IC50 below 10 µM. The most promising derivative was the glycosyl triazole derived from peracetylated d-glucose (11), which showed better cytotoxicity against SKBR-3 (IC50 = 0.78 µM), being the most selective toward this tumoral cell (SI > 20). All compounds described in this work were more active than lawsone, indicating the importance of the carbohydrate and glycosyl triazole moiety for activity.

Mechanistic insight into anti-cancer activity of plumbagin in endocrine resistant and HER2-overexpressed breast cancer cells

Abstract Background Plumbagin (PLB) is a naphthoquinone compound isolated from the root of Plumbago indica. In our previous study reported that PLB was able to inhibit cell growth in estrogen receptor (ER)-positive and endocrine resistant cells in vitro. The overexpression of human epidermal growth factor receptor 2 (HER2) and nuclear receptor coactivator 3 (NCOA3) was reported in endocrine resistant cells. However, the molecular mechanism underlying of inhibitory effect of PLB remains to be elucidated. We further investigated the mechanisms of PLB on cell apoptosis and HER2 signaling in endocrine resistant and HER2-overexpressing breast cancer. Methods The experiments were performed on MCF-7/LCC9 (endocrine resistant) and SKBR3 (HER2-overexpressing) cell lines. Basal levels of HER2 downstream signaling and NCOA3 in cancer cells used this study were determined by quantitative PCR (qPCR) and western blot. Functional and molecular studies of PLB included cell proliferation and apoptosis were evaluated by colony-forming assay, flow cytometry, qPCR and western blot analyses. Results PLB significantly inhibited the proliferation of MCF-7/LCC9 and SKBR3 cells in a concentration-dependent manner. PLB dramatically induced apoptosis in both cell lines and reduced apoptosis-related gene expression. NCOA3 mRNA and AKT phosphorylation significantly reduced in MCF-7/LCC9 cells after treatment with PLB. PLB also decreased phosphorylation of SKBR3 cells. Conclusions These findings demonstrated the NCOA3 and AKT signaling might be involved in anti-cancer mechanism of PLB in endocrine resistant HER2-overexpressed breast cancer, supporting further investigations on the therapeutic potential of PLB in animal model and/or phase I clinical trials. Legal entity responsible for the study The authors. Funding Ratchadaphiseksomphot Endowment Fund (RA62/034 to W.K.) and Special Task Force for Activating Research (STAR) Ratchadaphiseksomphot Endownment Fund to Overcoming Cancer Drug Resistance Research Group. Disclosure All authors have declared no conflicts of interest.

Targeting the TIGIT-PVR immune checkpoint axis as novel therapeutic option in breast cancer

ABSTRACTImmune checkpoints are intensively investigated as targets in cancer therapy. T-cell immunoreceptor with immunoglobulin (Ig) and ITIM domains (TIGIT) and its ligand poliovirus receptor (PVR) are recently emerging as novel promising targets in immunotherapy. Here, we show that high expression of PVR represents an independent prognostic marker being associated with poor outcome for breast cancer patients. Furthermore, PVR mRNA, as well as protein expression, is associated with more aggressive breast cancer subtypes such as HER2 positive and triple-negative breast cancer. In vitro, blocking TIGIT or PVR resulted in enhanced immune cell-mediated lysis of breast cancer cell lines SKBR-3, MDA-MB-231, MDA-MB-468, and BT549 and additionally increased the cytotoxic effects of a bispecific T cell engager BiTE® antibody construct targeting EGFR. Taken together, our data identify the immune checkpoint factor PVR as a novel prognostic marker in breast cancer and indicate that blocking the TIGIT-PVR axis might represent a novel therapeutic option for the treatment of breast cancer patients.

The Effect of Dracocephalum kotschyi Alcoholic Extract on the BCL2 and BAX Expression in SKBR3 Cell Line

The Effect of Dracocephalum kotschyi Alcoholic Extract on the BCL2 and BAX Expression in SKBR3 Cell Line

Comparative anti-proliferative effects of potential HER2 inhibitors on a panel of breast cancer cell lines

Breast cancer is one of the most lethal types of cancer in women worldwide. The human epidermal growth factor receptor 2 (HER2) is considered as a validated target in breast cancer therapy. Previously, we have used quantitative structure activity relationship QSAR equations and their associated pharmacophore models to screen for new promising HER2 structurally diverse inhibitory leads which were tested against HER2-overexpressing SKOV3 ovarian cancer cell line. In this study, we sought to explore the effect of most active ligands against different normal and breast cancer cell lines that represent different breast cancer subtypes with distinguished expression levels in HER2 and HER1. We have tested the promising compounds against SKBR3, MDA-MB-231, MCF7, human fibroblast, and MCF10 cell lines. To understand the inhibitory effects of the active ligands against HER2 over expressed breast cancer cell lines, all inhibitors and the control compound, lapatinib, were docked into the active site of HER2 enzyme performed using Ligand Fit docking engine and PMF scoring function. Five ligands exhibited promising results with relatively low IC50 values on cells that amplify HER2 and high IC50 on those that do not express such a receptor. The most potent compound (compound 13) showed an IC50 of 0.046µM. To test their toxicity against normal cells, the active compounds were tested against both normal fibroblast and normal breast cancer cell MCF-10 and relatively high IC50 values were scored. The IC50 values on HER2 over-expressed breast cancer and normal fibroblast cells provided a promising safety index. Docking results showed the highest similarity in the binding site between the most active ligand and the lapatinib. Our pharmacophore model resulted in a high potent ligand that shows high potency against HER2 positive breast cancer and relatively low toxicity towards the normal human cells.

Phospholipase-Cγ1 Signaling Protein Down-Regulation by Oligoclonal-VHHs based Immuno-Liposome: A Potent Metastasis Deterrent in HER2 Positive Breast Cancer Cells.

Objective The purpose of this study was to develop multivalent antibody constructs via grafting anti-HER2 antibodies, including Herceptin and oligoclonal-variable domain of heavy chain antibodies (VHHs), onto liposome membranes to enhance antibody activity and compare their effect on phospholipase C (PLC) signaling pathway with control. Materials and Methods In this experimental study, SKBR3 and BT-474 cell lines as HER2 positive and MCF10A cell line as normal cell were screened with anti-HER2 antibodies, including constructs of multivalent liposomal antibody developed with Herceptin and anti-HER2 oligoclonal-VHHs. To confirm the accuracy of the study, immunofluorescent assay, migration assay and immuno-liposome binding ability to HER2 were evaluated. Finally, the antibodies effect on PLCγ1 protein level was measured by an immunoassay method (ELISA). Results In the present study, by using multivalent form of antibodies, we were able to significantly inhibit the PLCγ1 protein level. Interestingly, the results of migration assay, used for study the motility of different types of cell, shows correspondingly decreased number of immigrated cells in SKBR3 and BT-474 cell lines. Since MCF10A cells show no overexpression of HER2, as expected, the result did not show any change in PLCγ1 level. Moreover, immunofluorescent assay has confirmed high expression of HER2 in SKBR3 and BT-474 cell lines and low HER2 expression on MCF10A cell line. High binding of immuno-liposome to SKBR3 and BT-474 cells and low binding to MCF10A confirmed that in this study anti-HER2 antibodies have conserved binding ability to HER2 even after conjugation with liposome. ConclusionPLCγ1 protein levels did indeed decrease after treatment with immuno-liposome form of compounds in both two tested cell lines, verifying the inhibition ability of them. Moreover, an elevated antibody activity is associated with liposomes conjugation suggesting that immuno-liposome may be a potential target for enhancing the antibody activity.

Multicomponent Conjugates of Anticancer Drugs and Monoclonal Antibody with PAMAM Dendrimers to Increase Efficacy of HER-2 Positive Breast Cancer Therapy

PurposeConjugation of nanocarriers with antibodies that bind to specific membrane receptors that are overexpressed in cancer cells enables targeted delivery. In the present study, we developed and synthesised two PAMAM dendrimer-trastuzumab conjugates that carried docetaxel or paclitaxel, specifically targeted to cells which overexpressed HER-2.MethodsThe 1H NMR, 13C NMR, FTIR and RP-HPLC were used to analyse the characteristics of the products and assess their purity. The toxicity of PAMAM-trastuzumab, PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined using MTT assay and compared with free trastuzumab, docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. The cellular uptake and internal localisation were studied using flow cytometry and confocal microscopy, respectively.ResultsThe PAMAM-drug-trastuzumab conjugates in particular showed extremely high toxicity toward the HER-2-positive SKBR-3 cells and very low toxicity towards to HER-2-negative MCF-7 cells. As expected, the HER-2-positive SKBR-3 cell line accumulated trastuzumab from both conjugates rapidly; but surprisingly, although a large amount of PAMAM-ptx-trastuzumab conjugate was observed in the HER-2-negative MCF-7 cells. Confocal microscopy confirmed the intracellular localisation of analysed compounds. The key result of fluorescent imaging was the identification of strong selective binding of the PAMAM-doc-trastuzumab conjugate with HER-2-positive SKBR-3 cells only.ConclusionsOur results confirm the high selectivity of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates for HER-2-positive cells, and demonstrate the utility of trastuzumab as a targeting agent. Therefore, the analysed conjugates present an promising approach for the improvement of efficacy of targeted delivery of anticancer drugs such as docetaxel or paclitaxel.

Mathematical modeling of drug-induced receptor internalization in the HER2-positive SKBR3 breast cancer cell-line

About 20% of breast cancer tumors over-express the HER2 receptor. Trastuzumab, an approved drug to treat this type of breast cancer, is a monoclonal antibody directly binding at the HER2 receptor and ultimately inhibiting cancer cell growth. The goal of our study was to understand the early impact of trastuzumab on HER2 internalization and recycling in the HER2-overexpressing breast cancer cell line SKBR3. To this end, fluorescence microscopy, monitoring the amount of HER2 expression in the plasma membrane, was combined with mathematical modeling to derive the flux of HER2 receptors from and to the membrane. We constructed a dynamic multi-compartment model based on ordinary differential equations. To account for cancer cell heterogeneity, a first, dynamic model was expanded to a second model including two distinct cell phenotypes, with implications for different conformational states of HER2, i.e. monomeric or homodimeric. Our mathematical model shows that the hypothesis of fast constitutive HER2 recycling back to the plasma membrane does not match the experimental data. It conclusively describes the experimental observation that trastuzumab induces sustained receptor internalization in cells with membrane ruffles. It is also concluded that for rare, non-ruffled (flat) cells, HER2 internalization occurs three orders of magnitude slower than for the bulk, ruffled cell population.

Magnetic Nanoparticles (MNPs) Influence on SK-BR3 Breast Cancer Cell Line - in vitro Study

Nanotechnologies involving the use of magnetic nanoparticles made of nickel, iron or cobalt are nowadays developing in anti-tumor therapy and diagnostic. The most important potential of MNPs is for drug delivery, meaning they are functionalized by binding to chemotherapeutic agents, nucleic acids, antibodies, etc. The present study is questioning the possibility of oleic acid-coated iron MNPs to induce cell death per se, selectively in tumor cells (breast cancer adenocarcinoma SK-BR3 cell line) without previous coupling with effector substances.