Diterpenoid DGA induces apoptosis via endoplasmic reticulum stress caused by changes in glycosphingolipid composition and inhibition of STAT3 in glioma cells

Abstract

Glioma is one of the most common malignant primary brain tumors, with poor prognosis and high recurrence. There are currently few drugs approved for brain tumors; thus, it is necessary to develop new effective drugs. Natural diterpenoids have important biological activities, including antiinflammatory, antioxidative, and antitumor effects. In this study, 7α,14β-dihydroxy-ent-kaur-17-dimethylamino-3,15-dione (DGA), a diterpenoid compound modified from glaucocalyxin A, inhibited the proliferation of many tumor cells, especially glioma. Flow cytometry analysis showed that DGA induced apoptosis in glioma cells. DGA also inhibited xenograft tumors in nude mice. It affected the expression of ceramide synthases (CerS) in glioma cells; CerS1 decreased, and CerS2 and CerS5 increased, resulting in a change in the composition of glycosphingolipids containing varying acyl chain lengths. In glioma cells treated with DGA, the gene transcription of activating transcription factor 4 (ATF4), X-box binding protein-1 (XBP1), and C/EBP-homologous protein (CHOP) in unfolded protein response pathways was upregulated. Meanwhile, the ratio of proapoptotic protein Bcl-2–associated X protein (BAX) to antiapoptotic protein B-cell lymphoma 2 (Bcl-2) also increased. This suggested that an imbalance of glycosphingolipids caused by DGA induced severe endoplasmic reticulum stress and triggered cell apoptosis. Moreover, Western blotting showed DGA inhibited the signal transducers and activators of transcription 3 (STAT3) signaling pathway by reducing the phosphorylation of STAT3 and its upstream kinases, which also promoted the apoptosis of glioma cells. Together, these results explored the anticancer activities of DGA and highlighted it as a potential candidate for treating glioma.

Introduction

Glioma, a primary intracranial tumor originating from glial cells, accounts for 81 % of malignant brain tumors and has a high morbidity and mortality rate [1]. The World Health Organization classifies gliomas into low grade (grade I or II) and high grade (grade III or IV) based on characteristics that reflect aggressiveness and invasiveness; the higher the grade, the worse the clinical outcome [2]. However, there are only a few drugs approved by the US Food and Drug Administration for brain tumors, such as temozolomide, lomustine, and bevacizumab [3]. The current standard of care for newly diagnosed glioma is to perform the largest possible safe surgical resection, followed by radiation therapy with temozolomide [4]. Nevertheless, the overall prognosis of the disease is still poor, with current treatments failing to significantly prolong median overall survival beyond 24 months [5]. Therefore, it is necessary to develop new effective agents against glioma and to study their underlying mechanisms.

The endoplasmic reticulum (ER) is an important organelle in eukaryotic cells where many biological macromolecules such as proteins and sphingolipids are synthesized [6], [7]. ER function can be disrupted by many factors, and unfolded or misfolded proteins accumulate in the ER, a condition known as ER stress [8]. To restore normal function, cells activate a set of tightly controlled regulatory programs, known as the unfolded protein response (UPR) [9]. If ER stress persists or is prolonged, the UPR will activate pathways leading to cell death [8], [10], [11]. Recently, regulating cell proliferation and inducing apoptosis via the UPR pathway have become new therapeutic targets for many cancers [6], [12], [13], [14].

Signal transducers and activators of transcription 3 (STAT3), a key molecule of convergence for numerous oncogenic signaling pathways, is structurally activated in a variety of hematological malignant tumors and solid tumors, such as lymphoma, breast cancer, head and neck tumors, and prostate cancer [15], [16], [17]. STAT3 is activated by phosphorylation and subsequently regulates the expression of many genes involved in cancer cell proliferation and survival, tumor angiogenesis, and metastasis [18]. STAT3 not only acts as a transcriptional inducer, but also affects gene expression through epigenetic modification, induces epithelial-mesenchymal transformation, promotes the tumor microenvironment, promotes the self-renewal and differentiation of tumor stem cells, and helps to establish pre-metastatic niches [19]. It has been confirmed that inhibition of the STAT3 pathway can inhibit the microbiological and invasive potential of some cancers [20].

Terpenoids naturally occur in plants and are believed to have important antiinflammatory, antioxidative, and antitumor effects [21], [22], [23], [24], such as paclitaxel, triptolide, tanshinone, and glaucocalyxin A [25], [26], [27], [28], [29]. Hortelano et al reported a labdane diterpenoid, α-Hispanolol, may exert anti-tumoral effects in vitro and in vivo through the inhibition of cell proliferation and invasion as well as by the induction of apoptosis in human glioblastoma cells [30]. We previously found that 7α,14β-dihydroxy-ent-kaur-17-dimethylamino-3,15-dione (DGA; Fig. 1A), a small diterpenoid derived from the lead natural compound glaucocalyxin A, has antiinflammatory effects on acute lung injury in mice. As a new diterpene compound, DGA has inhibitory effect on the proliferation of many tumor cells in drug screening experiments. Here, DGA inhibited the proliferation of glioma cells in vitro and in vivo. We also demonstrated that DGA affected the expression of different ceramide synthases (CerS) and changed the composition of glycosphingolipids (GSLs) containing different fatty acyl lengths, causing ER stress to induce cell apoptosis. Furthermore, DGA inhibited the STAT3 signaling pathway by decreasing the phosphorylation of STAT3 (Tyr705) and its upstream kinases Janus kinase 2 (JAK2) and Src, which also promoted the apoptosis of tumor cells.