Beta-Elemene Blocks Epithelial-Mesenchymal Transition in Human Breast Cancer Cell Line MCF-7 through Smad3-Mediated Down-Regulation of Nuclear Transcription Factors

<div><p>Epithelial-mesenchymal transition (EMT) is the first step required for breast cancer to initiate metastasis. However, the potential of drugs to block and reverse the EMT process are not well explored. In the present study, we investigated the inhibitory effect of beta-elemene (ELE), an active component of a natural plant-derived anti-neoplastic agent in an established EMT model mediated by transforming growth factor-beta1 (TGF-β1). We found that ELE (40 µg/ml ) blocked the TGF-β1-induced phenotypic transition in the human breast cancer cell line MCF-7. ELE was able to inhibit TGF-β1-mediated upregulation of mRNA and protein expression of nuclear transcription factors (SNAI1, SNAI2, TWIST and SIP1), potentially through decreasing the expression and phosphorylation of Smad3, a central protein mediating the TGF-β1 signalling pathway. These findings suggest a potential therapeutic benefit of ELE in treating basal-like breast cancer.</p> </div

TGF-β1 induces epithelial-to-mesenchymal changes in MCF-7 cells.

<p>(A) MCF-7 cells treated with 10 ng/ml TGF-β1 for 24 hrs had a spindle-like morphology and lost intercellular junctions. Magnification, 200×. (B) Western immunoblot analysis of expression of EMT-related proteins. Expression levels of E-cadherin and β-catenin (epithelial markers) in TGF-β1-treated MCF-7 cells were markedly decreased, whereas expression levels of N-cadherin and vimentin (mesenchymal markers) were dramatically increased. TGF-β1: transforming growth factor-β1.</p

ELE facilitates TGF-β1-mediated downregulation of Smad3 expression but inhibits increased Smad3 phosphorylation.

<p>(A) RT-PCR analyses showed that TGF-β1 treatment led to downregulation of Smad3 mRNA expression, and ELE enhanced the reduction in Smad3 expression (*: P<0.01, **: P<0.001). (B) Western immunoblot analysis showed that the ratio of p-Smad3/total Smad3 was increased in a time-dependent manner with 10 ng/ml TGF-β1 (*:P<0.01), although expression levels of total Smad3 were slightly decreased. (C) TGF-β1-induced Smad3 phosphorylation was blocked by ELE and total Smad3 was further decreased in MCF-7 cells treated with TGF-β1 and ELE (*: P<0.01, **: P<0.001).</p

ELE blocks TGF-β1-induced EMT in MCF-7 cells.

<p>(A) Representative pictures of MCF-7 cells treated with ELE showed that epithelial morphology of cells was maintained even in the presence of TGF-β1. Magnification, 200×. (B) Western immunoblot analysis showed that expression levels of E-cadherin and β-catenin (epithelial markers) and N-cadherin and vimentin (mesenchymal markers) did not differ in cells treated with TGF-β1 and ELE compared to cells with ELE only.</p

MCF-7 cells acquire migration and invasion abilities in response to TGF-β1 treatment.

<p>(A) Representative pictures of the scratch wound assay. TGF-β1-treated MCF-7 cells migrated faster than controls. The black lines indicated size of the remaining wound. Data from three independent experiments were analyzed and presented as mean ± SD. (P<0.001). (B) Representative pictures in a transwell migration assay (top).The penetrated cells numbers of MCF-7 treated with TGF-β1 for 12 hrs were significantly increased as compared to controls without TGF-β1 (P<0.001). Representative pictures in a transwell invasion assay (bottom). TGF-β1 treatment led to more MCF-7 cells penetrating matrix than controls. (P = 0.002). The results in both transwell migration and invasion assays were obtained from three independent experiments.</p

TGF-β1-mediated upregulation of nuclear transcriptional factors expression was blocked by ELE.

<p>(A) RT-PCR analyses showed that mRNA expression levels of transcriptional factors (SNAI1, SNAI2, TWIST and SIP1) were significantly increased in TGF-β1 treated MCF-7 cells (*: P<0.01, **: P<0.001). (B) Western immunoblot analysis showed that protein expression levels of SNAI1, SNAI2, TWIST and SIP1 were repressed by ELE treatment, especially in SNAI1 and SNAI2.</p

Image_1_Rapid response to fifth-line brigatinib plus entrectinib in an ALK-rearranged lung adenocarcinoma with an acquired ETV6-NTRK3 fusion: a case report.jpeg

The management of non-small cell lung cancer (NSCLC), specifically targeting the anaplastic lymphoma kinase (ALK) with tyrosine kinase inhibitors (TKIs), is challenged by the emergence of therapeutic resistance. Resistance mechanisms to ALK TKIs can be broadly classified into ALK-dependent and ALK-independent pathways. Here, we present a case with lung adenocarcinoma (LUAD) harboring an ALK rearrangement. The patient had developed resistance to sequential ALK TKI therapies, with an acquired ETV6-NTRK3 (E4:N14) fusion as a potential mechanism of ALK-independent resistance to lorlatinib. Subsequently, the patient was treated with the combination of brigatinib plus entrectinib and demonstrated a positive response, achieving an 8-month progression-free survival. Our case provides a potential treatment option for LUAD patients with ALK rearrangements and highlights the utility of next-generation sequencing (NGS) in uncovering genetic alterations that can guide the selection of effective treatment strategies.</p