Proliferation Of BT-474 Cells Research Articles

Toxicity of new monophenolic synthetic activator of Keap1/Nrf2/ARE redox-sensitive signaling system <i>in vitro</i> and <i>in vivo</i>

One of the promising areas of modern pharmacology is the development of «indirect antioxidants» capable of activating redox-sensitive signaling systems, primarily the Keap1/Nrf2/ARE system. Among its chemical inductors is the hydrophilic monosubstituted monophenol (3’-tert-butyl-4’-hydroxyphenyl)sodium propylthiosulfonate (TS-13) in development. The aim of the study was to investigate TS-13 antiproliferative activity against tumor cell line BT-474 in vitro and acute oral toxicity in mice in vivo. Material and methods. The relationship between TS-13 concentration and proliferative activity of human breast ductal carcinoma cell line BT-474 was determined using the MTT test, the IC<sub>50</sub> was calculated and compared to the previously obtained for MCF-7 line; results were correlated with the functional properties of cells based on gene expression (in silico GSEA). In vivo acute toxicity was studied in 50 female C57Bl/6J mice, who received a TS-13 solution in distilled water at various doses by intragastric gavage. LD<sub>50</sub> obtained experimentally and predicted in silico using the GUSAR web service were compared. Results and discussion. TS-13 inhibited the proliferation of BT-474 cells in a concentration-dependent manner (exponential approximation, IC<sub>50</sub> = 59.5 μM) and was 2.2 times more toxic than for MCF-7 cells. This may be due to functional differences between the BT-474 and MCF-7 lines, as evidenced by the GSEA results. The LD<sub>50</sub> value established in the in vivo experiment was 936 mg/kg body weight, the obtained value satisfactorily corresponds to the predicted in silico (561 mg/kg), although in reality the compound turned out to be somewhat less toxic than could be expected based on its structure. Conclusions. A study of the acute toxicity of the new water-soluble monophenol TC-13 allows the classification of it as slightly toxic (toxicity rating level 4) according to the Hodge – Sterner scale) or as moderately hazardous (hazard class 3) according to GOST 12.1.007-76.

Discovery of potent and selective HER2 PROTAC degrader based Tucatinib with improved efficacy against HER2 positive cancers

HER2 is a validated therapeutic target for HER2 positive breast cancer and gastric cancer. TKIs have significantly improved the prognosis of patients with HER2 positive cancer. However, the pan-HER TKIs always caused gastrointestinal and skin side effects, and acquired drug resistance inevitable compromised their therapeutic efficacy. Herein, we describe the discovery of the first potent and selective HER2 PROTAC degrader based Tucatinib with improved antitumor activity in vitro and in vivo. The preferred selective HER2 PROTAC, CH7C4, efficiently degraded HER2 with DC50 of 69 nM and Dmax of 96%, and inhibited the proliferation of BT-474 cells with IC50 of 0.047 ± 0.006 nM via long lasting HER2 degradation and strong repression of downstream pathway. Moreover, CH7C4 had acceptable pharmacokinetic profiles with a half-life of 5.31 h, and significantly inhibited the growth of BT-474 xenografts in vivo with TGI of 73%. As the first selective HER2 PROTAC degrader with better activity in vitro and in vivo than Tucatinib, CH7C4 provides new insights into the development of new therapeutic strategy for HER2 positive cancer.

CircEPSTI1 Promotes the Proliferation of HER2-Positive Breast Cancer Cells via circEPSTI1/miR-145/ERBB3 Axis.

Breast cancer is the most common type of cancer worldwide. There are great challenges in the prevention and treatment of breast cancer. In this study, we explored the molecular and biological mechanisms of circular RNA circEPSTI1 (has_circ_0000479) in the regulation of HER2-positive breast cancer cells. The expression of CircEPSTI1, microRNA miR-145, and ERBB3 in HER2-positive breast cancer cells was evaluated by qRT-PCR and western blot assays. Cell proliferation was assessed by CCK-8. Wound-healing and transwell migration assays were performed to evaluate cell migration. A transwell invasion assay was performed to detect cell invasion. The interaction of miR-145, circEPSTI1, and ERBB3 was confirmed bydual-luciferase reporter and RIP assays. CircEPSTI1 was upregulated in the HER2-positive breast cancer tissues and cells. Knockdown of circEPSTI1 inhibited SKBR3 and BT474 cell proliferation, migration, and invasion. Mechanistically, circEPSTI1 directly targeted miR-145, and miR-145 was a downstream mediator of circEPSTI1 in modulating the proliferation, migration, and invasion of SKBR3 and BT474 cells. ERBB3 was identified as a direct and functional target of miR-145 in HER2-positive breast cancer cells. Our findings demonstrate that circEPSTI1, an overexpressed circRNA in HER2-positive breast cancer, promotes the proliferation, migration, and invasion of SKBR3 and BT474 cells through the miR-145/ERBB3 axis.

Abstract 5380: Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death

Abstract Background and goals: ONC201 an experimental anticancer drug, targeting mitochondrial caseinolytic peptidase, ClpP is currently under investigation. Current data on the effect of ONC201 along with observations that ONC201 causes cytotoxic effect, there are data demonstrating that ONC201 is rather cytostatic and does not induce cell death. Recently, we demonstrated ONC201-induced arrest of proliferation and a decrease in the number of mitochondrial nucleoids. Here we extended our study to describe the effect of short-and long-term exposure of BT474 cells to ONC201. Observations: Proliferation and cell cycle arrest. ONC201 in a dose-dependent manner (0-50 µM) inhibited the proliferation of BT474 cells without induction of apoptotic cell death. Cells exposed to 10 µM ONC201 accumulated in G0/G1 phase (49.4 ± 11.5% vs 78.7 ± 5.1%) and declined in the S-phase of the cell cycle (39.2 ± 6.6% vs 9.0 ± 2.1%). In parallel, ONC201 exposure induced a decline in Cyclin E and Cdk2 protein levels. Nucleoids and mtDNA. Exposure of BT474 cells, to 10 µM ONC 201, decreased the number of mitochondrial nucleoids from 249 ± 52 to 155 ± 38 per cell after 24 h. Longer 72 h exposure further reduced the number of nucleoids to 84 ± 36 per cell. ONC201 exposure also induced reduction of mitochondrial size from 2.8 ± 0.5 µm2 to 1.5 ± 0.2 µm2 and depletion of mtDNA. Short-term and long-term consequences of cell exposure to ONC201. Short-term treatment (24 h exposure) demonstrated the reversible effect of ONC201 on the expression of stress proteins (ATF4, CHOP, and GDF-15), cell cycle regulatory proteins (cyclin E, Cdk2), and partial restoration of mitochondrial nucleoids (to 214 ± 46 nucleoids per cell). On the contrary, long-term treatment (72 h) resulted in the irreversible and persistent arrest of proliferation, decline of cell cycle regulatory proteins, and activation of stress proteins. Respiration. ONC201 exposure suppressed State 3, State 4, and uncoupled respiration in digitonin-permeabilized BT474 cells. Complex I activity was decreased from 3.3 ± 0.4 ng-atoms O/min/106 cells to 0.5 ± 0.1 and 0.4 ± 0.3 ng-atoms O/min/106 cells, in cells exposed to ONC201 for 24h and 72h, respectively. Similarly, ONC201 decreased the activity of Complex II from 3.5 ± 0.5 ng-atoms O/min/106 cells to 1.5 ± 0.6 and 0.3 ± 0.1 ng-atoms O/min/106. Conclusions: The consequences of ONC201 treatment were dependent on the duration of exposure to the drug. Short-term (24 h) exposure of BT474 cells resulted in reversible induction of stress proteins (ATF4, CHOP, and GDF-15), which were reversed following 120 h incubation in drug-free media. On the contrary, long-term (72 h) exposure of cells to ONC201 induced irreversible and persistent arrest of proliferation and elevation of expression of stress proteins. Thus, ONC201 can induce prolonged and irreversible elevation of stress proteins and persistent G1/S cell cycle arrest. Citation Format: Artem A. Mishukov, Irina V. Odinokova, Serazhutdin A. Abdullaev, Vitaly K. Zhalimov, Emily M. Fennel, Lucas J. Aponte-Collazo, Paul R. Graves, Lee M. Graves, Ekhson L. Holmuhamedov. Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5380.

AZD4547 and calcitriol synergistically inhibited BT-474 cell proliferation while modified stemness and tumorsphere formation

Fibroblast growth factor receptor (FGFR) overamplification/activation in cancer leads to increased cell proliferation. AZD4547, a FGFR selective inhibitor, hinders breast cancer cells growth. Although luminal B breast tumors may respond to chemotherapy and endocrine therapy, this subtype is associated with poor prognosis, inadequate response and/or acquired drug resistance. Calcitriol, the vitamin D most active metabolite, exerts anti-neoplastic effects and enhances chemotherapeutic drugs activity. In this study, we sought to decrease the concentration of AZD4547 needed to inhibit the luminal-B breast cancer cell line BT-474 proliferation by its combination with calcitriol. Anti-proliferative inhibitory concentrations, combination index and dose-reduction index were analyzed from Sulforhodamine B assays. Western blot and qPCR were used to study FGFR molecular targets. The compound’s ability to inhibit BT-474 cells tumorigenic capacity was assessed by tumorspheres formation. Results: BT-474 cells were dose-dependently growth-inhibited by calcitriol and AZD4547 (IC50 = 2.9 nM and 3.08 μM, respectively). Calcitriol at 1 nM synergistically improved AZD4547 antiproliferative effects, allowing a 2-fold AZD4547 dose-reduction. Mechanistically, AZD4547 downregulated p-FGFR1, p-Akt and tumorsphere formation. Calcitriol also decreased tumorspheres, while induced cell differentiation. Both compounds inhibited MYC and CCND1 expression, as well as ALDH, a stemness marker that positively correlated with FGFR1 and negatively with VDR expression in breast cancer transcriptomic data. In conclusion, the drugs impaired self-aggregation capacity, reduced stemness features, induced cell-differentiation and when combined, synergistically inhibited cell proliferation. Overall, our results suggest that calcitriol, at low pharmacological doses, may be a suitable candidate to synergize AZD4547 effects in luminal B breast tumors, allowing to reduce dose and adverse effects.

Combined Network Pharmacology and Cytology Experiments to Identify Potential Anti-Breast Cancer Targets and Mechanisms of Delphinidin

Delphinidin is a type of anthocyanin monomer with antioxidant, anti-inflammatory, and anti-tumor effects. However, the biological mechanisms underlying its anti-breast cancer activity have not been thoroughly studied. We further studied the effect of delphinidin on breast cancer cells through comprehensive network pharmacology, cellular and molecular experiments. We acquired the know therapeutic targets of delphinidin and obtained differentially expressed genes (DEGs) of breast cancer using RTCGA. We used topological analysis to screen out the 106 core targets of delphinium anti-breast cancer and performed functional analysis. These genes were mainly enriched in the pathways in cancer, Progesterone-mediated oocyte maturation and cell cycle. Then, by taking the intersection of the three analyzed data sets, important core targets (EGFR, TOP2A and PTGS2) were obtained and molecular-docking was performed to validate the result. Additionally, In Vitro experiments, MCF-7 and BT-474 cell proliferation was inhibited in a dose-dependent manner by delphinidin and the expressions of EGFR, TOP2A and PTGS were reduced. Moreover, delphinidin influenced cell cycle, the expressions of cdk1 and cyclin B1 were reduced. Furthermore, delphinidin induced apoptosis by activating the MAPK-Signaling pathway. Collectively, our findings suggested that delphinidin may offer effective approaches in breast cancer prevention and therapy. Supplemental data for this article is available online at http://dx.doi.org/10.1080/01635581.2021.2012582

Abstract 6352: Potential anticancer role of secretion modification region (SMR) peptide reduces mortalin and HER2-overexpressing exosomes in human HER2-positive breast cancer cells

Abstract Background/Significance: Exosomes are 30-100 nm membrane vesicles secreted by many cell types. Tumors secrete large numbers of exosomes that transport oncoproteins and immune suppressive molecules, supporting tumor growth and metastasis. The role of exosomes in intercellular signaling is shown by human epidermal growth factor receptor type 2 (HER2)-expressing breast tumors, where exosomes with HER2 stimulate tumor growth and interfere with activity of the therapeutic antibody Herceptin, which binds to the extracellular domain of HER2 and prolongs the overall survival of HER2-positive breast cancer patients. Exosome release is modulated by growth factors EGF and heregulin, HER2 receptor-activating ligands naturally present in the surrounding tumor microenvironment. The SMRwt peptide reduces secretion of HER2 exosomes; thus, it can potentially be exploited as an anticancer agent. Methods: BT-474 and HCC1569 HER2-positive breast cancer cells overexpressing HER2 and mortalin were used in these experiments. Exosome size and concentration were measured by NanoSight. Purified tumor exosomes were prepared using paramagnetic beads coated with antibodies against tumor specific proteins and confirmed by flow cytometry and electron microscopy. To identify biological functions, we cultured tumor exosomes with breast cancer cells, which increased tumor cell proliferation. To determine SMRwt peptide inhibition effects, we assayed tumor exosome secretion and cell proliferation by NanoSight, Western blot and MTT. Results: We characterized exosomes secreted by mortalin and HER2-overexpressing breast cancer cell lines and analyzed SMRwt peptide's role in reducing tumor exosome release and cell proliferation. Exosome secretion increased in HER2-overexpressing tumor cell lines HCC1596 and BT474. Exosomes from HER2-positive tumor cell-conditioned supernatants stimulated tumor cell growth and tumor exosome release and inhibited Herceptin activity on HCC1569 and BT474 cell proliferation. Addition of SMRwt peptide reduced tumor cell proliferation and tumor exosome release. Conclusions: These data show the role of HER2-positive exosomes in modulating sensitivity to Herceptin and thus, to HER2-driven tumor aggressiveness; and indicate that the SMRwt peptide has potential anticancer functions in HER2-positive HCC1569 and BT474 human breast cancer cells. The data also indicate tumor exosomes affect the proliferation of parental cells. Tumor exosomes derived from BT-474 cells and HCC1569 cells stimulated the proliferation of BT-474 cells and HCC1569 cells, suggesting a biological function for tumor exosomes. Citation Format: Ming Bo Huang. Potential anticancer role of secretion modification region (SMR) peptide reduces mortalin and HER2-overexpressing exosomes in human HER2-positive breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6352.

Upregulation of hsa_circ_0136666 contributes to breast cancer progression by sponging miR-1299 and targeting CDK6.

Circular RNAs (circRNAs) can participate in multiple cancers, including breast cancer. Increasing circRNAs are recognized in various cancers because of the high-throughput sequencing. However, the potential physiological effect of hsa_circ_0136666 in breast cancer progression is unknown. In our study, the biological role of hsa_circ_0136666 in breast cancer development was studied. It was displayed that hsa_circ_0136666 was greatly increased in breast cancer. In addition, overexpression of hsa_circ_0136666 was able to promote Michigan Cancer Foundation-7 (MCF7) and BT474 cell proliferation and triggered cell cycle in G2/M phase. microRNA plays critical role in tumor development and they can act as direct targets of circRNAs. miR-1299 has been implicated as a famous tumor suppressor in many cancers. Here, miR-1299 was predicted as the target of hsa_circ_0136666. Meanwhile, its Upregulation repressed breast cancer proliferation, migration and invasion capacity, which could be reversed by the increase of hsa_circ_0136666. Furthermore, Cyclin-dependent kinase 6 (CDK6) was speculated as the downstream target of miR-1299. In MCF7 and BT474 cells, CDK6 was greatly overexpressed and it was shown that CDK6 contributed a lot to breast cancer progression. Subsequently, it was implied that hsa_circ_0136666 could modulate CDK6 levels positively in vitro. In conclusion, it was revealed that Upregulation of hsa_circ_0136666 promoted breast cancer progression by sponging miR-1299 and targeting CDK6.

Differential effect of polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles on BT-474 human breast cancer cell viability

Polyvinylpyrrolidone superparamagnetic iron oxide nanoparticles (PVP-SPIONs) have unique properties. Due to these characteristics, PVP-SPIONs have been used in several medical applications such as magnetic resonance imaging (MRI) contrast agent or drug delivery system. However, a more comprehensive understanding of the environmental safety of PVP-SPIONs is vital for consumption of these nanomaterials. In this study, we describe the effects of PVP-SPIONs on cell viability of the BT-474 human breast cancer cells. Cell viability of the BT-474 cells treated with PVP-SPIONs (10–800 μg/ml) was assessed by MTT assay. MRC-5 cell line was used as a control. Quantitative real-time PCR was performed to investigate the mRNA expression levels of apoptotic (caspase 3) and anti-apoptotic (BCL2) genes Confluent BT-474 monolayers exposed to PVP-SPIONs showed biphasic effects on cell proliferation. PVP-SPIONs at 10–100 μg /ml promote proliferation of BT-474 cells but not the MRC-5 cells. At higher dosage, PVP-SPIONs have toxicity on BT-474 cells. The results of real-time PCR was in line with MTT assay. The increase of cell proliferation at low PVP-SPIONs concentrations is different from what would be expected for these nanoparticles. Our results suggest that more attentions are needed to ensure the safer use of SPION in nanomedicine.

Inhibition of long non-coding RNA ROR reverses resistance to Tamoxifen by inducing autophagy in breast cancer.

This study explored the mechanism underlying long non-coding RNA ROR regulating autophagy on Tamoxifen resistance in breast cancer. Cancer tissues and adjacent normal tissues were collected from 74 breast cancer patients. Human breast cancer BT474 cells were assigned into blank, phosphate buffered saline, Tamoxifen, negative control + Tamoxifen, siROR + Tamoxifen, 3-methyladenine + Tamoxifen, and siROR + 3-methyladenine + TA groups. The expression of long non-coding RNA ROR and expressions of multi-drug resistance-associated P-glycoprotein and glutathione S-transferase-π messenger RNA were detected using quantitative real-time polymerase chain reaction. The expressions of light chain 3, Beclin 1, multi-drug resistance-associated P-glycoprotein, and glutathione S-transferase-π protein were determined using western blotting. Cell proliferation, invasion, and migration abilities were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Transwell assay, and scratch test, respectively. The long non-coding RNA ROR expression was higher in the breast cancer tissues than that in the adjacent normal tissues. Compared with the blank group, light chain 3 and Beclin 1 expressions were increased in the siROR + Tamoxifen group but decreased in the 3-methyladenine + Tamoxifen group; these data indicated that downregulated long non-coding RNA ROR promoted autophagy. In comparison with the blank group, multi-drug resistance-associated P-glycoprotein and glutathione S-transferase-π messenger RNA and protein expressions were reduced in the siROR + Tamoxifen group but elevated in the 3-methyladenine + Tamoxifen group, suggesting that downregulated long non-coding RNA ROR suppressed the drug resistance to Tamoxifen and the inhibition of autophagy reversed the effect of long non-coding RNA ROR on drug resistance. Compared with the Tamoxifen, negative control, and siROR + 3-methyladenine + Tamoxifen groups, the cell proliferation, invasion, and migration in the siROR + Tamoxifen group were much decreased; these results implied that downregulated long non-coding RNA ROR suppressed BT474 cell proliferation, invasion, and migration and reversed the effect of Tamoxifen on the BT474 cells. These results indicate that inhibition of long non-coding RNA ROR reverses resistance to Tamoxifen by inducing autophagy in breast cancer.

Quercetin induces caspase-dependent extrinsic apoptosis through inhibition of signal transducer and activator of transcription 3 signaling in HER2-overexpressing BT-474 breast cancer cells.

Flavonoids are assumed to exert beneficial effects in different types of cancers at high concentrations. Yet, their molecular mechanisms of action remain unknown. The present study aimed to examine the effect of quercetin on proliferation and apoptosis in HER2-expressing breast cancer cells. The anti-proliferative effects of quercetin were examined by proliferation, MTT and clonogenic survival assays. The effect of quercetin on expression of apoptotic molecules was determined by western blotting. Luciferase reporter assay was performed to measure signal transducer and activator of transcription 3 (STAT3) transcriptional activity. ELISA assay was performed to measure intracellular MMP-9 levels. Immunocytochemistry was performed to evaluate the nuclear STAT3 level. The results revealed that quercetin inhibited the proliferation of BT-474 cells in a dose- and time-dependent manner. Quercetin also inhibited clonogenic survival (anchorage-dependent and -independent) of BT-474 cells in a dose-dependent manner. These growth inhibitions were accompanied with an increase in sub-G0/G1 apoptotic populations. Quercetin induced caspase-dependent extrinsic apoptosis upregulating the levels of cleaved caspase-8 and cleaved caspase-3, and inducing the cleavage of poly(ADP-ribose) polymerase (PARP). In contrast, quercetin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease the mitochondrial membrane potential and did not affect the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX). Quercetin reduced the expression of phospho-JAK1 and phospho-STAT3 and decreased STAT3-dependent luciferase reporter gene activity in the BT-474 cells. Quercetin inhibited MMP-9 secretion and decreased the nuclear translocation of STAT3. Our study indicates that quercetin induces apoptosis at concentrations >20 µM through inhibition of STAT3 signaling and could serve as a useful compound to prevent or treat HER2-overexpressing breast cancer.

Induction of caspase-dependent extrinsic apoptosis by apigenin through inhibition of signal transducer and activator of transcription 3 (STAT3) signalling in HER2-overexpressing BT-474 breast cancer cells

Phytoestrogen intake is known to be beneficial to decrease breast cancer incidence and progression. But its molecular mechanisms of action are still unknown. The present study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of BT-474 cells in a dose- and time-dependent manner. Apigenin also inhibited clonogenic survival (anchorage-dependent and -independent) of BT-474 cells in a dose-dependent manner. These growth inhibitions were accompanied with an increase in sub-G0/G1 apoptotic populations. Apigenin-induced extrinsic a caspase-dependent apoptosis up-regulating the levels of cleaved caspase-8 and cleaved caspase-3, and inducing the cleavage of poly (ADP-ribose) polymerase (PARP). Whereas, apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential without affecting the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX). Apigenin reduced the expression of phospho-JAK1, phospho-JAK2 and phospho-STAT3 and decreased signal transducer and activator of transcription 3 (STAT3) dependent luciferase reporter gene activity in BT-474 cells. Apigenin inhibited CoCl2-induced VEGF secretion and decreased the nuclear translocation of STAT3. Our study indicates that apigenin induces apoptosis through inhibition of STAT3 signalling and could serve as a useful compound to prevent or treat HER2-overexpressing breast cancer.

The effect of HDAC inhibitor SNDX-275 on inhibiting breast cancer BT474 cell proliferation

Objective To explore the effect and molecular mechanism of HDAC inhibitor SNDX-275 inhibiting cell proliferation in ErbB2-overexpressing breast cancer BT474 cells. Methods Breast cancer BT474 cells were treated with HDAC inhibitor SNDX-275, setting as test group, and the cell line treated with phosphate buffered saline (PBS) as control. The concentration of SNDX-275 were 0, 0.5, 1.0, 2.0, 3.0, 4.0 μmol/L respectively. Cell proliferation was analyzed by MTS assay and colony formation assay, the expressions of ErbB2, ErbB3, p-Akt were analyzed by Western blotting, and the expressions of miR-125a, miR-125b were analyzed by RT-PCR. After transfecting miRNA125 inhibitor into BT474 cells, the inhibition rate of SNDX-275 was tested by MTS assay . Results MTS result showed that SNDX-275 inhibited cell proliferation in BT474 cells in a dose-dependent manner. The inhibition rate of 4.0 μmol/L SNDX-275 was about (68.00±4.45)%. Clone assay indicated SNDX-275 could inhibit the proliferation of BT474 cells. Western blotting result indicated that SNDX-275 significantly inhibited the protein expressions of ErbB2, ErbB3 and p-Akt, RT-PCR result illustrated 2 μmol/L SNDX-275 could increase the expressions of miR-125a and miR-125b about 3.22±1.17, 5.42±0.38 times compared with the PBS control respectively, the difference has a statistical significance (t=4.338, P=0.049; t=21.805, P=0.002). MTS result indicated that compared with the PBS control, the inhibition rate of SNDX-275 group was (56.97±3.56)%, while the inhibition rate of SNDX-275 and miRNA125 inhibitor group was (10.67±2.21)%, with a statistical significance(t=-10.993, P=0.008). Conclusion SNDX-275 could inhibit cell proliferation of ErbB2-overexpressing breast cancer BT474 cells, by inhibiting ErbB2-ErbB3-Akt signal pathway through up-regulating miR-125a and miR-125b. Key words: Breast neoplasms; Cell proliferation; MicroRNAs

Hyaluronidase-incorporated hyaluronic acid–tyramine hydrogels for the sustained release of trastuzumab

We developed an injectable hydrogel system for the sustained release of protein drugs that incorporated both protein drugs and hyaluronidase. Trastuzumab and hyaluronidase were incorporated in hydrogels composed of hyaluronic acid–tyramine (HA–Tyr) conjugates through the enzymatic crosslinking utilizing hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). Through electrostatic interactions with the HA, trastuzumab was retained in the hydrogel to minimize its burst release. Hyaluronidase was incorporated in the hydrogel to release trastuzumab from the hydrogels. The hydrogels were degraded and showed sustained release of trastuzumab in phosphate buffer over four weeks in vitro. Both the rates of drug release and gel degradation were controlled by the concentration of hyaluronidase. Trastuzumab released from the hydrogels inhibited the proliferation of BT-474 cells in vitro. In an animal model, the single subcutaneous injection of a mixture solution of HA–Tyr conjugates, H2O2, HRP, trastuzumab and hyaluronidase inhibited tumor growth significantly, whereas injection of trastuzumab alone at the same dose failed to do so. Compared to trastuzumab alone, the hyaluronidase-incorporated HA–Tyr hydrogels improved the pharmacokinetic profile of trastuzumab in the plasma of mice. Furthermore, they were fully degraded over two weeks, and the formation of fibrous capsules was not observed in mice.

Modular anti‐EGFR and anti‐Her2 targeting of SK‐BR‐3 and BT474 breast cancer cell lines in the presence of ErbB receptor‐specific growth factors

Over the last decade, a number of monoclonal antibodies and small molecule inhibitors emerged as potent therapeutic agents in the treatment of Her2/neu overexpressing breast cancer. Numerous patients, however, do not adequately respond to anti-epidermal growth factor receptor (EGFR)/Her2 receptor targeting. Receptor- and, in turn, growth-stimulating effects, which potentially hamper antiproliferative cell treatment, have barely been investigated. BT474 and SK-BR-3 breast cancer cell lines were treated with Trastuzumab, Pertuzumab, and Lapatinib alone using different combinations and concentrations. Moreover, epidermal growth factor (EGF) or heregulin (HRG) was added to reveal potential growth factor-mediated compensatory effects. Receptor and intracellular signaling were analyzed as a function of cell treatment. Read-out parameters were cell proliferation and apoptosis. BT474 cells were efficiently driven into quiescence by Trastuzumab, but not by Pertuzumab treatment. Simultaneous EGF or HRG administration, however, restored the BT474 cell proliferation capacity. In contrast, neither therapeutic antibody treatment caused a profound inhibition of SK-BR-3 cell-cycle progress. Lapatinib turned out to be the most potent cell-cycle inhibitor in both cell lines even though its impact was significantly abrogated in the presence of EGF and HRG. The compensatory effect of EGF on Lapatinib-induced cell-cycle inhibition was reversed by Trastuzumab as well as by Pertuzumab treatment. Most importantly, HRG-caused compensation of Lapatinib-induced cell-cycle exit was reversed by Pertuzumab but not by Trastuzumab. Apparently, multiple anti-EGFR/Her2 targeting by using Trastuzumab, Pertuzumab, and Lapatinib more efficiently affects receptor function (interaction and activation) and consequently enhances their antiproliferative capacity. Growth inhibition by anticancer drugs targeted to Her/ErbB receptors, however, can be significantly undermined in the presence of EGF and in particular by HRG treatment, which suggests that specific therapeutic growth factor sequestration might further enhance anti-EGFR/Her2 targeting.

Abstract 5362: The role of adam10 in trastuzumab resistance of her2 positive breast cancer cell lines

Abstract Members of a disintegrin and metalloproteases (ADAMs) are responsible for shedding of a variety of ErbB ligands. One of them, ADAM10, has been shown to cleave several pro-ligands, including betacellulin and HB-EGF. ADAM10 further may play a role in trastuzumab resistance since it is also responsible for HER2 shedding, thereby removing the binding site for trastuzumab. It was reported that ADAM10 inhibitor INCB 7839 could reduce HER2 cleavage in patients with HER2 positive breast cancer. However, a direct correlation of trastuzumab treatment and ADAM10 and the precise role of ADAM10 in trastuzumab resistance have not been elucidated. The aim of this project is therefore to investigate the role of ADAM10 in trastuzumab treatment and resistance in HER2 positive breast cancer cell lines. Using western blot, we showed that one hour of trastuzumab treatment leads to an upregulation of ADAM10 in conditioned medium and cell lysates of BT474 cells, which was correlated with an elevated level of betacellulin. Additionally, the use of a specific ADAM10 inhibitor in turn leads to downregulation of betacellulin levels in culture medium. Whereas one hour of trastuzumab treatment could not decrease pHER2, its combination with ADAM10 inhibitor reduced pHER2 level. To further investigate the role of ADAM10 in relation to trastuzumab treatment, we used cell viability studies to assess the effect of a specific ADAM10 inhibitor with or without trastuzumab treatment. Although monotherapy with ADAM10 inhibitor only had minimal influence, combining it with trastuzumab significantly enhanced trastuzumab in reducing cell proliferation of BT474 cells. Interestingly, the addition of ADAM10 inhibitor to trastuzumab exerts an even greater inhibition of cell proliferation in the BT474 trastuzumab resistant cell line. Since the N-domain of ADAM10 was shown to be cleaved by ADAM9 in other cell lines, we also assessed ADAM9 level in response to trastuzumab treatment. In BT474 lysates, ADAM9 level was increased after one hour of treatment, correlating with increased ADAM10 level in the medium. ADAM9 level was also found to be increased in the trastuzumab-resistant HER2 positive cell line in comparison to the sensitive cell line. Thus, ADAM10 as well as ADAM9 may play a key role in trastuzumab resistance in HER2 positive breast cancer cells. Our results suggest that the increased level of ADAM10 results in HER ligand sheddase which in turn may lead to the activation of HER receptors and the maintenance of pHER2. We demonstrated that ADAM10 inhibitor in combination with trastuzumab is able to decrease pHER2 and potentiate the trastuzumab effect in HER2 positive breast cancer cells. The role of ADAM9 in its regulation of ADAM10 in relation to trastuzumab treatment is currently under investigation. We recommend that inhibition of ADAM10 should be considered as a potential strategy to improve trastuzumab response in HER2 positive breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5362. doi:10.1158/1538-7445.AM2011-5362

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