Repression of PDK1- and LncRNA HOTAIR-Mediated EZH2 Gene Expression Contributes to the Enhancement of Atractylenolide 1 and Erlotinib in the Inhibition of Human Lung Cancer Cells

Abstract

Background/Aims: We previously showed that the major bioactive compound of Atractylodes macrocephula Koidz atractylenolide 1 (ATL-1) inhibited human lung cancer cell growth by suppressing the gene expression of 3-Phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1). However, the potentially associated molecules and downstream effectors of PDK1 underlying this inhibition, particularly the mechanism for enhancing the anti-tumor effects of epidermal growth factor receptor-tyrosine-kinase inhibitors (EGFR-TKIs), remain unknown. Methods: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Western blot analyses were performed to examine the protein expressions of PDK1 and of zeste homolog 2 (EZH2). The levels of long non-coding RNA (lncRNA) and HOX transcript antisense RNA (HOTAIR) were examined via qRT-PCR. RNA-binding protein immunoprecipitation assays were used to analyze HOTAIR interaction with EZH2. The promoter activity of the EZH2 gene was determined using Secrete-Pair Dual Luminescence Assay Kit. Exogenous expressions of PDK1, HOTAIR, and EZH2 were conducted via transient transfection assays. A xenografted tumor model was used to further evaluate the effect of ATL-1 in the presence or absence of erlotinib in vivo. Results: We showed that the combination of ATL-1 and EGFR-TKI erlotinib further inhibited growth and induced cell arrest of the human lung cancer cells, determined by both MTT and flow cytometry assays. ATL-1 inhibited the protein expression and the promoter activity of EZH2, which was reversed in cells with PDK1 overexpression. Interestingly, ATL-1 inhibited the expression levels of HOTAIR. While silencing HOTAIR inhibited the expressions of PDK1 and EZH2, overexpression of HOTAIR reduced the ATL-1-reduced PDK1 and EZH2 protein expressions and EZH2 promoter activity. In addition, ATL-1 reduced the HOTAIR binding to the EZH2 protein. Moreover, we found that exogenously expressed EZH2 antagonized the effect of ATL-1 on cell growth inhibition. Consistent with the in vitro results, ATL-1 inhibited tumor growth and the expression levels of HOTAIR, protein expressions of EZH2 and PDK1 in vivo. Importantly, there was synergy of the combination of ATL-1 and erlotinib in this process. Conclusion: Here, we provide the first evidence that ATL-1 inhibits lung cancer cell growth through inhibiting not only the PDK1 but also the lncRNA HOTAIR, which results in the reduction of one downstream effector EZH2 expression. The novel interplay between the HOTAIR and EZH2, as well as repressions of the PDK1 and HOTAIR coordinate the overall effects of ATL-1. Importantly, the combination of ATL-1 and EGFR-TKI erlotinib exhibits synergy. Thus, targeting the PDK1- and HOTAIR-mediated downstream molecule EZH2 by the combination of ATL-1 and erlotinib potentially facilitates the development of an additional novel strategy to combat lung cancer.

The Epigenome in Multiple Myeloma: Impact on Tumor Cell Plasticity and Drug Response

“…HOTAIR is well-known to influence chromatin compactness by serving as a molecular scaffold for PRC2, thereby recruiting and affecting PRC2 occupancy on genes genome-wide (95, 96). Importantly, HOTAIR overexpression has been reported in several solid tumors and hematological malignancies, including AML and diffuse large B cell lymphoma, and has been positively correlated with initiation, progression, drug resistance, and poor prognosis (95–99). In MM, however, no aberrant expression of HOTAIR in MM cells so far has been reported (100).…”

Atractylenolide I Induces Apoptosis and Suppresses Glycolysis by Blocking the JAK2/STAT3 Signaling Pathway in Colorectal Cancer Cells

“…Pharmacokinetic studies in rats have demonstrated that, after oral administration, AT-I is readily absorbed and persists for extended periods of time, suggesting good oral bioavailability (Wang et al, 2008). AT-I has a wide range of biological and pharmacological properties, including neuroprotective, antiallergic, anti-inflammatory, and anti-cancer activities (Wang et al, 2009;Lim et al, 2012;More and Choi, 2017;Xiao et al, 2018). Previous studies have shown that AT-I is beneficial in treating various cancers through the regulation of different molecules and pathways.…”

Transfer of metastatic traits via miR‐200c in extracellular vesicles derived from colorectal cancer stem cells is inhibited by atractylenolide I

“…Herein, we demonstrated the utility of ATL‐1 for this purpose. Research has shown the unique therapeutic potential of ATLs against cancers such as melanoma, 39 lung cancer, 40 and ovarian cancer 41 . In particular, Zhang et al revealed that ATL‐II, another member of the ATL family with similar structural characteristics as ATL‐1, improved the chemotherapeutic efficacy against CRC by suppressing the viability and proliferation of CRC cells.…”