Anticancer effect of zoledronic acid in endocrine-resistant breast cancer cells via HER-2 signaling.

HER-2 overexpression is a major mechanism involved in endocrine-resistant breast cancer, which has very limited treatment options. Zoledronic acid (ZA) is a drug in the bisphosphonate group used to treat osteoporosis. ZA was reported to exhibit activity in various cancers, with higher efficacy associated with estrogen-deprivation states. ZA inhibits cell proliferation in lung cancer through the epidermal growth factor receptor signaling pathway. Because endocrine-resistant breast cancer cells overexpress HER-2 and grow independently without estrogen, ZA may exert anticancer effects in these cell types. The inhibitory effects and mechanisms of ZA in endocrine-resistant cells through HER-2 signaling were investigated. The efficacy of ZA was higher in the endocrine-resistant breast cancer cells when compared with the wild-type cells. ZA also exhibited a synergistic effect with fulvestrant and may circumvent fulvestrant resistance. ZA decreased phosphorylated ERK (pERK) levels in resistant cell lines and attenuated HER-2 signaling in tamoxifen- and fulvestrant-resistant cells. ZA significantly decreased HER-2 levels and its downstream signaling molecules, including pAKT and pNF-κB in fulvestrant-resistant breast cancer cells. This inhibitory effect may explain the lower IC50 values of ZA in fulvestrant-resistant cells compared with tamoxifen-resistant cells. Moreover, ZA inhibited the migration and invasion in the resistant cell lines, suggesting an ability to inhibit tumor metastasis. The results indicate that ZA has potential for repurposing as an adjuvant treatment for patients with endocrine-resistant breast cancer.

Combining lapatinib and palbociclib inhibits cell proliferation and invasion via AKT signaling pathway in endocrine-resistant breast cancer cells.

Endocrine therapy plays a critical role in patients with hormone receptor-positive breast cancer. Endocrine-resistant breast cancer cells exhibit more HER2 signaling proteins (pAKT and pERK) and mesenchymal biomarkers than wild-type cell lines. In head and neck squamous cell carcinoma, the combination of lapatinib and palbociclib demonstrated synergistic inhibitory effects on cell proliferation and suppressed ERK1/2 phosphorylation. The combination of lapatinib and palbociclib at half-maximal inhibitory concentrations resulted in an increasing cytotoxic effect on cell proliferation. Furthermore, invasion activity was significantly decreased when combining two drugs at nontoxic concentrations more than either single drug alone did. The combination also remarkably suppressed epithelial-mesenchymal transition transcription factors, such as Snail and pAKT, more than monotherapy. Combining drugs, particularly lapatinib and palbociclib for targeting endocrine-resistant breast cancer cells whose tumors overexpressed HER2 after resistance to hormonal therapy, demonstrated better antiproliferative, anti-invasive effects, and suppression of EMT protein and pAKT than a single drug. These results could be from the interruption of the EMT process via the AKT pathway. Thus, this study provides preliminary data for applying this combination to patients with endocrine-resistant breast cancer in further clinical trials.

1'-Acetoxychavicol Acetate from Alpinia galanga Represses Proliferation and Invasion, and Induces Apoptosis via HER2-signaling in Endocrine-Resistant Breast Cancer Cells.

Estrogen receptor-positive breast cancer patients have a good prognosis, but 30% of these patients will experience recurrence due to the development of resistance through various signaling pathways. This study aimed to evaluate the mode of anticancer effects of 1'-acetoxychavicol acetate, which is isolated from the rhizomes of Alpinia galanga in estrogen receptor positive (MCF7) human epidermal growth factor receptor 2-overexpressed (MCF7/HER2), and endocrine-resistant breast cancer cells (MCF7/LCC2 and MCF7/LCC9). 1'-Acetoxychavicol acetate showed antiproliferation in a concentration- and time-dependent fashion and had higher potency in human epidermal growth factor receptor 2-overexpressed cell lines. This was associated with down-regulation of human epidermal growth factor receptor 2, pERK1/2, pAKT, estrogen receptor coactivator, cyclin D1, and MYC proto-oncogene while in vivo and significant reduction in the tumor mass of 1'-acetoxychavicol acetate-treated zebrafish-engrafted breast cancer groups. The anti-invasive effects of 1'-acetoxychavicol acetate were confirmed in vitro by the matrigel invasion assay and with down-regulation of C - X-C chemokine receptor type 4, urokinase plasminogen activator, vascular endothelial growth factor, and basic fibroblast growth factor 2 genes. The down-regulation of urokinase plasminogen activator and fibroblast growth factor 2 proteins was also validated by molecular docking analysis. Moreover, 1'-acetoxychavicol acetate-treated cells exhibited lower expression levels of the anti-apoptotic Bcl-2 and Mcl-1 proteins in addition to enhanced stress-activated kinases/c-Jun N-terminal kinase 1/2 and poly-ADP ribose polymerase cleavage, indicating apoptotic cell induction by 1'-acetoxychavicol acetate. Moreover, 1'-acetoxychavicol acetate had higher potency in human epidermal growth factor receptor 2-overexpressed cell lines regarding its inhibition on human epidermal growth factor receptor 2, pAKT, pERK1/2, PSer118, and PSer167-ERα proteins. Our findings suggest 1'-acetoxychavicol acetate mediates its anti-cancer effects via human epidermal growth factor receptor 2 signaling pathway.

NADPH: Quinone oxidoreductase 1 (NQO1) mediated anti-cancer effects of plumbagin in endocrine resistant MCF7 breast cancer cells.

BACKGROUND: PLB is a natural naphthoquinone compound isolated from the roots of Plumbago indica plant. Our previous study reported the inhibitory effect of Plumbagin (PLB) on human endocrine resistant breast cancer cell growth and cell invasion. HYPOTHESIS/PURPOSE: Since PLB is a naphthoquinone compound, it can be reduced by the cytosolic NADPH: quinone oxidoreductase 1 (NQO1) enzyme. NQO1 expression is upregulated in various types of aggressive cancer including breast cancer. This study investigated the impact of NQO1 on anti-cancer effects of PLB in endocrine-resistant breast cancer cells. STUDY DESIGN: This study was an in vitro study using ER-positive cell line (MCF7) and endocrine-resistant cell lines (MCF7/LCC2 and MCF7/LCC9 cells). METHODS: The roles of NQO1 in anti-cancer activity of PLB were investigated by using NQO1 knockdown cells, NQO1 inhibitor and NQO1 overexpressed cells. To study the impact of NQO1 on the effects of PLB on cell viability, apoptosis, invasion and generation of ROS, the following assays were used: MTT assays, annexin V-PE/7-ADD staining flow cytometry, matrigel invasion assays and DCFHDA assays. To study the mechanism of how NQO1 mediated PLB effects in tamoxifen response and apoptosis, we assessed the levels of mRNA expression by using qRT-PCR. RESULTS: 1. In this study, NQO1 was upregulated in endocrine-resistant cells. 2. PLB did not change the expression of NQO1 but it was able to increase NQO1 activity. 3. The inhibitory effects of PLB on cell proliferation, cell invasion and expression of tamoxifen resistant gene were attenuated in NQO1 knockdown cells or in the presence of NQO1 inhibitor. 4. The effects of PLB to induce apoptosis and generate ROS were also decreased when NQO1 activity was inhibited or when the NQO1 expression was reduced. 5. The anti-cancer effects of PLB increased when NQO1 was upregulated. CONCLUSION: The effects of PLB in endocrine-resistant breast cancer cells is dependent on NQO1's activity.

Plumbagin inhibited AKT signaling pathway in HER-2 overexpressed-endocrine resistant breast cancer cells.

The important mechanism of endocrine resistance is the crosstalk between estrogen receptor (ER) and HER2 signaling pathways. Aside from ER downregulation, there was an increase in HER2 expression and increased activation of the downstream AKT/ERK pathways in endocrine-resistant cells (MCF-7/LCC2 and MCF-7/LCC9) which is similar to HER2-overexpressed (SKBR3) cells. However, nuclear receptor coactivator 3 (NCOA3), the important ER-coactivator, that upregulated in endocrine-resistant cells did not express in HER2-overexpressed (SKBR3) cells. NCOA3 was able to activate AKT/ERK signalling pathway. Our previous study reported that plumbagin (PLB), a naphthoquinone compound, had a potent cytotoxic activity against endocrine-resistant cells. This study aimed to further investigate the mechanism of anti-cancer effects of PLB on ER and HER-2 signaling. PLB can inhibit estradiol (E2)-induced cell proliferation in MCF-7 wild-type cells but had no effect in the resistant cells. It also inhibited HER2 expression in both endocrine-resistant and HER-2 overexpressed cells. Therefore, the mechanism of PLB may be regulated through HER-2 signaling. PLB inhibited the phosphorylation of AKT (pAKT) and pERK1/2 and induced apoptosis and reduced the expression of anti-apoptotic genes Bcl-2 and pro-caspase 3 and Cleaved Caspase 3 protein in both endocrine-resistant and HER-2 overexpressed cells. However, the inhibitory effect of PLB was more obvious when pre-treated the cells with AKT inhibitor only in HER-2 overexpressed cells. In addition, the inhibitory effect of PLB on pAKT was attenuated when NCOA3 was downregulated. Our finding suggested that the inhibitory effect of PLB on AKT signaling pathways regulated through NCOA3 in HER2-overexpressed endocrine-resistant cells.

Plumbagin inhibits cancer stem-like cells, angiogenesis and suppresses cell proliferation and invasion by targeting Wnt/ß-catenin pathway in endocrine resistant breast cancer.

Fifty percent of advanced stage ER-positive breast cancer patients develop endocrine resistance. Aberrant activation of Wnt/ß-catenin is associated with stem-like phenotypes and epithelial-mesenchymal transition (EMT) process which confers resistance to endocrine therapy. Cancer stem-like cells (CSLCs) can be a vital source of proangiogenic factors including fibroblast growth factor 2 (FGF2) which drives angiogenesis and leads to tumor growth and metastasis. Therefore, targeting Wnt and FGF2 may provide effective treatment for endocrine resistant breast cancer. Our previous in vitro study reported that plumbagin (PLB) was a potent anticancer agent and was able to inhibit EMT in endocrine-resistant cells. This study aimed to further investigate the inhibitory effects of PLB on cancer stem-like phenotypes, tumorigenicity and angiogenesis. The results demonstrated Wnt/ß-catenin signaling was activated and was able to form mammospheres with increased cancer stem cell markers (ALDH1, NANOG, and OCT4) in endocrine-resistant cells. PLB significantly inhibited colony-forming, mammosphere formation and decreased cancer stem cell markers. The inhibitory effects of PLB on cell proliferation and invasion were mediated by Wnt signaling pathway. PLB also significantly reduced Wnt responsive genes and ß-catenin. Moreover, PLB treatment at doses of 2 and 4 mg/kg/day inhibited tumor growth, angiogenesis and metastasis without any adverse effects on body weight and blood coagulation in orthotopic xenograft nude mice. In conclusion, PLB exerted anti-cancer activity and eliminated stem-like properties by attenuating Wnt/ß-catenin signaling and FGF2 expression. These findings suggest that PLB could be a promising agent to treat endocrine resistant breast cancer.

Ethoxy mansonone G as an anticancer agent in estrogen receptor-positive and endocrine-resistant breast cancer.

OBJECTIVES: To study anticancer effects, underlying mechanism and safety of ethoxy mansonone G (EMG) which is the potent derivative of mansonone G (MG) in breast cancer cells. METHODS: Anticancer, antimigration, anti-invasion effects and anchorage-independent growth were investigated by MTT, scratch, matrigel invasion and soft agar assays. Estrogen receptor (ER)-targeted genes and endocrine-resistant genes were assessed by RT-PCR and Western blot. KEY FINDINGS: Ethoxy mansonone G is the most potent MG derivative and has anticancer effects in ER-positive, endocrine-resistant and ER-negative breast cancer cells. Our results demonstrated that EMG can significantly inhibit estrogen-induced cell proliferation and the expression of ER-targeted genes in ER-positive breast cancer cells, suggesting the anti-estrogenic property of EMG which is consisting with the virtual molecular docking that EMG could possibly bind to the ERα. Moreover, EMG has synergistic effect with tamoxifen in endocrine-resistant cells. EMG also inhibited cell proliferation, invasion and anchorage-independent growth by reducing expression of genes involved in endocrine resistance and invasive factors during the metastatic process. CONCLUSION: Ethoxy mansonone G has an anticancer effect in breast cancer cells and is possible to use as a therapeutic agent in patients with breast cancer.

Mechanism of Cepharanthine Cytotoxicity in Human Ovarian Cancer Cells.

Cepharanthine (CEP), a medicinal product derived from Stephania cephalantha Hayata, possesses a potent cytotoxicity against several types of cancers. Recently, we have found that CEP could efficiently inhibit the growth of mutated p53 colon cancer cells, which are often resistant to commonly used chemotherapeutic agents. In this study, we evaluated the cytotoxic effect and the underlying mechanisms of CEP on both chemosensitive CaOV-3 and chemoresistant OVCAR-3 ovarian cancer cell lines. The present study demonstrated that CEP significantly inhibited the growth of CaOV-3 and OVCAR-3 cells in a time- and concentration-dependent manner. CEP arrested CaOV-3 and OVCAR-3 cells in the G1 phase and S phase of cell cycle, respectively. Western blot analysis demonstrated that CEP markedly increased the expression of p21Waf1 protein and decreased the expression of cyclins A and D proteins in both CaOV-3 and OVCAR-3 cells. Additionally, CEP triggered apoptotic cell death in OVCAR-3 cells. Taken together, the above results suggest that CEP is a promising anticancer drug for ovarian cancer.

Bioequivalence and pharmacokinetic/pharmacodynamic correlation of clopidogrel in healthy Thai subjects
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Clopidogrel is an antiplatelet drug, selectively binding to the platelet P2Y12 receptor of adenosine diphosphate. Clopidogrel is a prodrug modified through active metabolite in the liver by two steps of CYP enzyme. The active metabolite is responsible for inhibiting platelet aggregation. OBJECTIVE: The study aimed to assess the bioequivalence of clopidogrel 75 mg generic and reference drugs and to investigate the correlation between pharmacokinetics of active metabolites and its antiplatelet activities. MATERIALS AND METHODS: Determination of clopidogrel, carboxylic acid form, and active metabolite were done by liquid chromatography tandem mass spectrometry, and evaluation of platelet function was also investigated by light transmission aggregometer. 20 subjects were randomized and assigned in a crossover design to take a single 75-mg oral dose of clopidogrel generic and reference drugs in two periods with washout. Pharmacokinetic parameters Cmax, AUC0-t, and AUC0-inf of clopidogrel, carboxylic acid form, and active metabolite were analyzed. RESULTS: Bioequivalence could be shown when testing parameters with ANOVA, as 90% confidence intervals were found to be within the acceptance range of 80 - 125%. For the maximum of platelet aggregation after administration of both products, no significant differences were found. Significant correlation of Cmax of clopidogrel active metabolite and maximum platelet aggregation was found after receiving 0 - 6 hours of both formulations. CONCLUSION: The study found bioequivalence of clopidogrel generic and reference drugs. There were also significant correlations between Cmax of clopidogrel active metabolite and maximum platelet aggregation.
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Salinomycin overcomes acquired tamoxifen resistance through AIB1 and inhibits cancer cell invasion in endocrine resistant breast cancer.

Salinomycin is a monocarboxylic polyether ionophore isolated from Streptomyces albus. It has been widely used as an antibiotic in veterinary medicine in poultry. A recent study demonstrated that salinomycin selectively inhibits human breast cancer stem cells; one possible mechanism of tamoxifen resistance. Our results show that salinomycin is effective in inhibiting MCF-7/LCC2 and MCF-7/LCC9 cell lines which are well-established endocrine resistant cells and has a synergistic effect in combination with tamoxifen using MTT proliferation assay. The inhibitory effect of salinomycin on the reduction of critical ER co-activator; amplified breast 1 (AIB1) mRNA and protein expression is overcoming tamoxifen resistance. Moreover, salinomycin significantly inhibits cell invasion in Matrigel invasion assay. The effect was mediated at least in part by the decrease of matrix metalopeptidase 9 (MMP-9) which is one critical enzyme facilitated in the cell invasion process. In conclusion, salinomycin should be developed as a novel agent used alone or in combination for endocrine-resistant breast cancer.

Plumbagin Enhances Tamoxifen Sensitivity and Inhibits Tumor Invasion in Endocrine Resistant Breast Cancer through EMT Regulation.

Tamoxifen is widely used as the first line drug for estrogen receptor-positive subtype which is expressed in 70% of overall breast cancer patients. However, approximately 50% of these patients develop acquired resistance after 5 years of treatment, which is characterized by tumor recurrence and metastasis. The epithelial mesenchymal transition (EMT) is an important process in breast cancer invasion. Fundamentally, targeting the EMT represents a crucial therapeutic strategy for preventing or treating breast cancer metastasis. Plumbagin (PLB) is a natural naphthoquinone with significant anticancer effects against several types of tumor cells including breast cancer. In this study, we investigated the effect of PLB on human endocrine-resistant breast cancer cell growth, invasion and the possible mechanisms underlying such actions. PLB exhibited potent cytotoxic activity at a micromolar concentration against endocrine-resistant breast cancer cells. Interestingly, a fixed low concentration of PLB and tamoxifen combination resulted in an increase in growth inhibition in endocrine-resistant cells. In addition, PLB also significantly suppressed mesenchymal biomarker expressions that govern the EMT process, resulting in attenuated metastatic capabilities. In conclusion, PLB should be developed as a pharmacological agent for the use as a single treatment or in combination for endocrine-resistant breast cancer. Copyright © 2016 John Wiley & Sons, Ltd.

Induction of HEXIM1 activities by HMBA derivative 4a1: Functional consequences and mechanism.

We have been studying the role of Hexamethylene bisacetamide (HMBA) Induced Protein 1 (HEXIM1) as a tumor suppressor whose expression is decreased in tamoxifen resistant and metastatic breast cancer. HMBA was considered the most potent and specific inducer for HMBA inducible protein 1 (HEXIM1) prior to our studies. Moreover, the ability of HMBA to induce differentiation is advantageous for its therapeutic use when compared to cytotoxic agents. However, HMBA induced HEXIM1 expression required at mM concentrations and induced dose limiting toxicity, thrombocytopenia. Thus we structurally optimized HMBA and identified a more potent inducer of HEXIM1 expression, 4a1. The studies reported herein tested the ability of 4a1 to induce HEXIM1 activities using a combination of biochemical, cell phenotypic, and in vivo assays. 4a1 induced breast cell differentiation, including the stem cell fraction in triple negative breast cancer cells. Clinically relevant HEXIM1 activities that are also induced by 4a1 include enhancement of the inhibitory effects of tamoxifen and inhibition of breast tumor metastasis. We also provide mechanistic basis for the phenotypic effects of 4a1. Our results support the potential of an unsymmetrical HMBA derivative, such as 4a1, as lead compound for further drug development.

Lead optimization of HMBA to develop potent HEXIM1 inducers.

The potency of a series of Hexamethylene bis-acetamide (HMBA) derivatives inducing Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) was determined in LNCaP prostate cancer cells. Several compounds with unsymmetrical structures showed significantly improved activity. Distinct from HMBA, these analogs have increased hydrophobicity and can improve the short half-life of HMBA, which is one of the factors that have limited the application of HMBA in clinics. The unsymmetrical scaffolds of the new analogs provide the basis for further lead optimization of the compounds using combinatorial chemistry strategy.

Determination of hexamethylene bisacetamide, an antineoplastic compound, in mouse and human plasma by LC-MS/MS.

Hexamethylene bisacetamide (HMBA) is a polar compound which has recently been discovered to have antineoplastic activity by up-regulating the expression of an endogenous antiproliferative breast cancer protein, HEXIM1 (hexamethylene bisacetamide inducible protein 1) in vivo. HMBA has been shown in the past to induce terminal differentiation in multiple leukemia types at a concentration of 2-5mM, but its phase I and II clinical trials were largely unsuccessful due to serious side effects (notably, thrombocytopenia) with dose escalation. In this work, a sensitive and simple LC-MS/MS method for direct determination of HMBA in mouse and human plasma is described. Plasma samples were prepared by deproteinization with acetonitrile. Separation was achieved on a Waters Atlantis(®) T3 (2.1 mm × 50 mm, 3 µm) column with retention times of 2.2 and 3.7 min for HMBA and 7MBA (internal standard), respectively. The quantitation was carried out by tandem mass spectrometry using positive MRM mode. The linear range of the method was 0.500-100 ng/mL in both mouse and human plasma with injection volume of 5 µL. This method has been validated in accordance with the US Food and Drug Administration (FDA) guidelines for bioanalytical method development and applied to the determination of HMBA concentrations in FVB mice over time after a single dose of HMBA in saline (0.9% NaCl) at 10mg/kg.