Novel sesquiterpene lactone analogues as potent anti-breast cancer agents.
ABSTRACT: Triple-negative breast cancer (TNBC) is associated with high grade, metastatic phenotype, younger patient age, and poor prognosis. The discovery of an effective anti-TNBC agent has been a challenge in oncology. In this study, fifty-eight ester derivatives (DETDs) with a novel sesquiterpene dilactone skeleton were organically synthesized from a bioactive natural product deoxyelephantopin (DET). Among them, DETD-35 showed potent antiproliferative activities against a panel of breast cancer cell lines including TNBC cell line MDA-MB-231, without inhibiting normal mammary cells M10. DETD-35 exhibited a better effect than parental DET on inhibiting migration, invasion, and motility of MDA-MB-231 cells in a concentration-dependent manner. Comparative study of DETD-35, DET and chemotherapeutic drug paclitaxel (PTX) showed that PTX mainly caused a typical time-dependent G2/M cell-cycle arrest, while DETD-35 or DET treatment induced cell apoptosis. In vivo efficacy of DETD-35 was evaluated using a lung metastatic MDA-MB-231 xenograft mouse model. DETD-35 significantly suppressed metastatic pulmonary foci information along with the expression level of VEGF and COX-2 in SCID mice. DETD-35 also showed a synergistic antitumor effect with PTX in vitro and in vivo. This study suggests that the novel compound DETD-35 may have a potential to be further developed into a therapeutic or adjuvant agent for chemotherapy against metastatic TNBC.
Project description:A novel plant sesquiterpene lactone derivative, DET derivative (DETD)-35, originating from parental deoxyelephantopin (DET) was previously observed to effectively suppress human triple negative breast cancer (TNBC) MDA-MB-231 cell activity and tumor growth in mice. In this study, the mechanisms underlying the activity of DETD-35 were elucidated. DET and DETD-35 induced reactive oxygen species (ROS) which caused structural damage and dysfunction of mitochondria and increased cytosolic calcium level, subsequently evoking exosome release from the cancer cells. Intriguingly, exosomes induced by both compounds had an atypical function. Cancer cell-derived exosomes commonly show metastatic potential, but upon DET/DETD-35 treatment exosomes showed anti-proliferative activity against MDA-MB-231 cells. Quantitative proteome analysis of TNBC cell-secreted exosomes showed that DET and DETD-35 attenuated the expression of proteins related to cell migration, cell adhesion, and angiogenesis. Furthermore, several exosomal proteins participating in biological mechanisms such as oxidative stress and decrease of transmembrane potential of mitochondria were found deregulated by treatment with either compound. Pretreatment with ROS scavenger, N-acetylcysteine, blockaded DET- or DETD-35-induced oxidative stress and calcium dependent exosome release mechanisms, and also reverted DET- or DETD-35-induced reprogramming exosomal protein expression profiles resulting in attenuation of exosomal toxicity against TNBC cell proliferation. In summary, this study shows that a plant-derived sesquiterpene lactone DET and its analog DETD-35 inhibitory TNBC cell activities through oxidative stress-induced cancer cell releasing exosomes in tandem with alteration of exosomal protein composition and functions. The findings of this study suggest that DETD-35 may be suitable for further development into an anti-TNBC drug.
Project description:Triple negative breast cancer (TNBC) is a highly metastatic cancer among the breast cancer subgroups. A thorny issue for clinical therapy of TNBC is lack of an efficient targeted therapeutic strategy. We previously created a novel sesquiterpene lactone analog (named DETD-35) derived from plant deoxyelephantopin (DET) which exhibits potent effects against human TNBC MDA-MB-231 tumor growth in a xenograft mouse model. Here we studied the mechanisms of both DET and DETD-35 against MDA-MB-231 cells. DETD-35 (3-fold decreased in IC50) exhibited better anti-TNBC cell activity than DET as observed through induction of reactive oxygen species production (within 2 h treatment) and damage to the ER structures, resulting in ER-derived cytoplasmic vacuolation and ubiquitinated protein accumulation in the treated cells. Intriguingly, the effects of both compounds were blockaded by pretreatment with ROS scavengers, N-acetylcysteine and reduced glutathione, and protein synthesis inhibitor, cycloheximide. Further, knockdown of MEK upstream regulator RAF1 and autophagosomal marker LC3, and co-treatment with JNK or ERK1/2 inhibitor resulted in the most significant attenuation of DETD-35-induced morphological and molecular or biochemical changes in cancer cells, while the inhibitory effect of DET was not influenced by MAPK inhibitor treatment. Therefore, DETD-35 exerted both ER stress-mediated paraptosis and apoptosis, which may explain its superior activity to DET against TNBC cells. Although the chemotherapeutic drug paclitaxel induced vacuole-like structures in MDA-MB-231 cells, no paraptotic cell death features were detected. This study provides a strategy for combating TNBC through sesquiterpene lactone analogs by induction of oxidative and ER stresses that cause paraptosis-like cell death.
Project description:There is no effective clinical therapy for triple-negative breast cancers (TNBCs), which have high low-density lipoprotein (LDL) requirements and express relatively high levels of LDL receptors (LDLRs) on their membranes. In our previous study, a novel lipid emulsion based on a paclitaxel-cholesterol complex (PTX-CH Emul) was developed, which exhibited improved safety and efficacy for the treatment of TNBC. To date, however, the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul have not been investigated. In order to offer powerful proof for the therapeutic effects of PTX-CH Emul, we systematically studied the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul and made a comparative evaluation of antineoplastic effects on TNBC (MDA-MB-231) and non-TNBC (MCF7) cell lines through in vitro and in vivo experiments. The in vitro antineoplastic effects and in vivo tumor-targeting efficiency of PTX-CH Emul were significantly more enhanced in MDA-MB-231-based models than those in MCF7-based models, which was associated with the more abundant expression profile of LDLR in MDA-MB-231 cells. The results of the cellular uptake mechanism indicated that PTX-CH Emul was internalized into breast cancer cells through the LDLR-mediated internalization pathway via clathrin-coated pits, localized in lysosomes, and then released into the cytoplasm, which was consistent with the internalization pathway and intracellular trafficking of native LDL. The findings of this paper further confirm the therapeutic potential of PTX-CH Emul in clinical applications involving TNBC therapy.
Project description:BACKGROUND:Triple negative breast cancer (TNBC) is an aggressive malignancy with poor prognosis, in part because of the current lack of any approved molecularly targeted therapy. We evaluated various combinations of three different drugs: nintedanib, an antiangiogenic TKI targeting VEGF receptors, paclitaxel (PTX), or a PD-L1 antibody, using models of orthotopic primary or advanced metastatic TNBC involving a metastatic variant of the MDA-MB-231 human cell line (called LM2-4) in SCID mice and two mouse lines (EMT-6 and a drug-resistant variant, EMT-6/CDDP) in immunocompetent mice. These drugs were selected based on the following: PTX is approved for TNBC; nintedanib combined with docetaxel has shown phase III clinical trial success, albeit in NSCLC; VEGF can act as local immunosuppressive factor; and PD-L1 antibody plus taxane therapy was recently reported to have encouraging phase III trial benefit in TNBC. METHODS:Statistical analyses were performed with ANOVA followed by Tukey's Multiple Comparison Test or with Kruskal-Wallis test followed by Dunn's Multiple Comparison Test. Survival curves were analyzed using a Log-rank (Mantel Cox) test. Differences were considered statistically significant when p values were?<?0.05. RESULTS:Toxicity analyses showed that nintedanib is well tolerated when administered 5-days ON 2-days OFF; PTX toxicity differed in mice, varied with cell lines used and may have influenced median survival in the metastatic EMT6/CDDP model; while toxicity of PD-L1 therapy depended on the cell lines and treatment settings tested. In the LM2-4 system, combining nintedanib with PTX enhanced overall antitumor efficacy in both primary and metastatic treatment settings. In immunocompetent mice, combining nintedanib or PTX with the PD-L1 antibody improved overall antitumor efficacy. Using the advanced metastatic EMT-6/CDDP model, optimal efficacy results were obtained using the triple combination. CONCLUSIONS:These results suggest circumstances where nintedanib plus PTX may be potentially effective in treating TNBC, and nintedanib with PTX may improve PD-L1 therapy of metastatic TNBC.
Project description:Successful adhesion of circulating tumor cells (CTCs) to microvascular endothelium of distant metastatic tissue is the key starting step of metastatic cascade that could be effectively chemoprevented as we demonstrated previously. Here, we hypothesize that the hetero-adhesion may produce secretory biomarkers that may be important for both premetastatic diagnosis and chemoprevention. We show that co-incubation of triple-negative breast cancer (TNBC) cell line MDA-MB-231 with human pulmonary microvascular endothelial monolayers (HPMEC) secretes Cyr61 (CCN1), primarily from MDA-MB-231. However, addition of metapristone (RU486 metabolite) to the co-incubation system inhibits Cyr61 secretion probably via the Cyr61/integrin ?v?1 signaling pathway without significant cytotoxicity on both MDA-MB-231 and HPMEC. Transfection of MDA-MB-231 with Cyr61-related recombinant plasmid or siRNA enhances or reduces Cyr61 expression, accordingly. The transfection significantly changes hetero-adhesion and migration of MDA-MB-231, and the changed bioactivities by overexpressed CYR61 could be antagonized by metapristone in vitro. Moreover, the circulating MDA-MB-231 develops lung metastasis in mice, which could be effectively prevented by oral metapristone without significant toxicity. The present study, for the first time, demonstrates that co-incubation of MDA-MB-231 with HPMEC secrets CYR61 probably via the CYR61/integrin ?v?1 signaling pathway to promote adhesion-invasion of TNBC (early metastatic step). Metapristone, by interfering the adhesion-invasion process, prevents metastasis from happening.
Project description:<h4>Purpose</h4>Triple-negative breast cancer (TNBC) has aggressive characteristics and fewer treatment options than other subtypes. The purpose of this study was to explore prognostic biomarkers for TNBC that can be easily detected from the blood samples.<h4>Methods</h4>MDA-MB-231 and MDA-MB-231BR, a brain metastatic variant of the human TNBC cell line MDA-MB-231, were used as less and more aggressive models of TNBC, respectively. The extent to which the candidate gene/protein identified by RNA sequencing correlated well with aggressiveness of TNBC and how much protein was detected from the blood of tumor-bearing mice were evaluated.<h4>Results</h4>Both the in vitro proliferation and in vivo tumor growth of MDA-MB-231BR were more rapid than those of MDA-MB-231. RNA sequencing identified ESM1 as a gene that was expressed significantly more in MDA-MB-231BR than in MDA-MB-231, and qRT-PCR confirmed a significantly higher expression of ESM1 in MDA-MB-231BR xenograft in vivo. Furthermore, Kaplan-Meier analysis of relapse-free survival demonstrated that TNBC patients with high ESM1 expression had clearly worse relapse-free survival than those with low ESM1 expression, which was consistent with our preclinical findings. Endocan, a protein of ESM1 gene product, was successfully detected in both conditioned medium from MDA-MB-231BR and plasma samples from mice bearing MDA-MB-231BR xenograft, which showed a significantly distinct pattern from less aggressive MDA-MB-231. Moreover, bisulfite sequence analysis revealed that overexpression of ESM1 in MDA-MB-231BR might be attributed to DNA demethylation in an upstream region of the ESM1 gene.<h4>Conclusion</h4>This study indicates that endocan could be used as a blood-based prognostic biomarker in TNBC patients.
Project description:Triple negative breast cancer (TNBC) is a difficult to treat disease due to the absence of the three unique receptors estrogen, progesterone and herceptin-2 (HER-2). To improve the current therapy and overcome the resistance of TNBC, there is unmet need to develop an effective targeted therapy. In this regard, one of the logical and economical approaches is to develop a tumor hypoxia-targeting drug formulation platform for selective delivery of payload to the drug-resistant and invasive cell population of TNBC tumors. Toward this, we developed a Carbonic Anhydrase IX (CA IX) receptor targeting human serum albumin (HSA) carriers to deliver the potent anticancer drug, Paclitaxel (PTX). We used Acetazolamide (ATZ), a small molecule ligand of CA IX to selectively deliver HSA-PTX in TNBC cells. A novel method of synthesis involving copper free 'click' chemistry (Dibenzocyclooctyl, DBCO) moiety with an azide-labeled reaction partner, known as Strain-Promoted Alkyne Azide Cycloaddition (SPAAC) along with a desolvation method for PTX loading were used in the present study to arrive at the CA IX selective nano-carriers, HSA-PTX-ATZ. The anticancer effect of HSA-PTX-ATZ is higher compared to HSA, PTX and non-targeted HSA-PTX in MDA-MB-231 and MDA-MB-468 cells. The cell killing effect is associated with induction of early and late phases of apoptosis. Overall, our proof-of-concept study shows a promising avenue for hypoxia-targeted drug delivery that can be adapted to several types of cancers.
Project description:Triple-negative breast cancer (TNBC), an aggressive, metastatic and recurrent breast cancer (BC) subtype, currently suffers from a lack of adequately described spontaneously metastatic preclinical models that faithfully reproduce the clinical scenario. We describe two preclinical spontaneously metastatic TNBC orthotopic murine models for the development of advanced therapeutics: an immunodeficient human MDA-MB-231-Luc model and an immunocompetent mouse 4T1 model. Furthermore, we provide a broad range of multifactorial analysis for both models that could provide relevant information for the development of new therapies and diagnostic tools. Our comparisons uncovered differential growth rates, stromal arrangements and metabolic profiles in primary tumors, and the presence of cancer-associated adipocyte infiltration in the MDA-MB-231-Luc model. Histopathological studies highlighted the more rapid metastatic spread to the lungs in the 4T1 model following a lymphatic route, while we observed both homogeneous (MDA-MB-231-Luc) and heterogeneous (4T1) metastatic spread to axillary lymph nodes. We encountered unique metabolomic signatures in each model, including crucial amino acids and cell membrane components. Hematological analysis demonstrated severe leukemoid and lymphoid reactions in the 4T1 model with the partial reestablishment of immune responses in the immunocompromised MDA-MB-231-Luc model. Additionally, we discovered ?-immunoglobulinemia and increased basal levels of G-CSF correlating with a metastatic switch, with G-CSF also promoting extramedullary hematopoiesis (both models) and causing hepatosplenomegaly (4T1 model). Overall, we believe that the characterization of these preclinical models will foster the development of advanced therapeutic strategies for TNBC treatment, especially for the treatment of patients presenting both, primary tumors and metastatic spread.
Project description:Triple negative breast cancer (TNBC) as a metastatic disease is currently incurable. Reliable and reproducible methods for testing drugs against metastasis are not available. Stromal cells may play a critical role in tumor progression and metastasis. In this study, we determined that fibroblasts and macrophages secreted IL-8 upon induction by tumor cell-conditioned media (TCM) from MDA-MB-231 cancer cells. Our data showed that the proliferation of MDA-MB-231 cells co-cultured with fibroblasts or macrophages was enhanced compared to the monoculture. Furthermore, TNBC cell migration, a key step in tumor metastasis, was promoted by conditioned media (CM) from TCM-induced fibroblasts or macrophages. Knockdown of the IL-8 receptor CXCR2 by CRISPR-Cas9 reduces MDA-MB-231 cell proliferation and migration compared to wild type. In a mouse xenograft tumor model, the growth of MDA-MB-231-CXCR2-/- tumor was significantly decreased compared to the growth of tumors from wild-type cells. In addition, the incidence of thoracic metastasis of MDA-MB-231-CXCR2-/- tumors was reduced compared to wild type. We found that the auto- and paracrine loop exists between TNBC cells and stroma, which results in enhanced IL-8 secretion from the stromal components. Significantly, inhibition of the IL-8 signaling pathway by reparixin, an inhibitor of the IL-8 receptor, CXCR1/2, reduced MDA-MB-231 tumor growth and metastasis. Taken together, these findings implicate IL-8 signaling as a critical event in TNBC tumor growth and metastasis via crosstalk with stromal components.