Bufalin Induces Apoptosis of Human Osteosarcoma U-2 OS Cells through Endoplasmic Reticulum Stress, Caspase- and Mitochondria-Dependent Signaling Pathways

Abstract

Bone cancer is one of the cancer-related diseases, and there are increased numbers of patients with bone cancer worldwide. Therefore the efficacy of treatment of bone cancer is considered extremely vital. Bufalin has been showed to have biological activities including anticancer activities in vitro and in vivo. However, the exact associated mechanisms for bufalin induced apoptosis in human bone cancer cells are still unclear. In the present study, we investigated the effect of bufalin on the cytotoxic effects in U-2 OS human osteosarcoma cells. For examining apoptotic cell deaths, we used flow cytometry assay, Annexin V/PI double staining, and TUNNEL assay. Reactive oxygen species (ROS), Ca2+, mitochondrial membrane potential (ΔΨm), and caspase-8, -9 and -3 activities were measured by flow cytometry assay. Furthermore, western blotting and a confocal laser microscopy examination were used for measuring the alterations of apoptotic associated protein expression and translocation, respectively. The results indicated that bufalin induced cell morphological changes, decreased the viable cell number, induced apoptotic cell death, and increased the apoptotic cell number, and affected apoptotic associated protein expression in U-2 OS cells. Bufalin increased apoptotic proteins such as Bak, and decreased anti-apoptotic proteins such as Bcl-2 and Bcl-x in U-2 OS cells. Furthermore, bufalin increased the protein levels of cytochrome c (Cyto c), AIF (Apoptosis inducing factor) and Endo G (Endonuclease G) in cytoplasm that were also confirmed by confocal microscopy examination. Based on those findings, bufalin induced apoptotic cell death in U-2 OS cells may be via endoplasmic reticulum (ER) stress, caspase-, and mitochondria-dependent pathways; thus, we may suggest that bufalin could be used as an anti-cancer agent for the treatment of osteosarcoma in the future, and further in vivo studies are needed.

Bufalin Enhances Immune Responses in Leukemic Mice Through Enhancing Phagocytosis of MacrophageIn Vivo

“…Bufalin has biological activities including inducing cancer cell apoptosis (11)(12)(13)(14)(15)(16)(17) and DNA damage in NCI-H460 human lung cancer cells (18). Recently, we also found that bufalin significantly inhibits cell adhesion, migration and invasion of gefitinib-resistant NCI-H460 cells (19) and induced apoptosis of U-2 OS human osteosarcoma cells (20) and SCC 4 human tongue cancer cells (21).…”

Chemical evaluation and cytotoxic mechanism investigation of Clinacanthus nutans extract in lymphoma SUP‐T1 cells

“…Caspase are hydrolysis enzymes which play an important role in apoptotic cell death. Many studies shown that induction of caspase protein could trigger apoptosis in cancer cells . Therefore, it is estimated that some key biomarkers like caspase‐3, ‐7, and ‐8 can prove if MHA could induce caspase‐dependent cell death.…”

In situ phase transition of microemulsions for parenteral injection yielding lyotropic liquid crystalline carriers of the antitumor drug bufalin

“…It is a bufanolide steroid compound. The broad spectrum of anticancer activities of BUF has received considerable attention [26][27][28][29][30][31][32].…”

“…The mechanisms of bufalin-induced cell death comprise its (i) influence on the expression of apoptosis-related genes, (ii) inhibition of tumor cell proliferation, (iii) generation of reactive oxygen species, (iv) endoplasmic reticulum stress, (v) JNK (c-Jun N-terminal kinase) activation, (vi) protein kinase modulating effect, (vii) caspase-and mitochondria-dependent signaling pathways, and (viii) cell cycle arrest via the c-Myc/NF-kappa B pathway [26][27][28][29][30][31][32][33][34][35][36][37]. Despite that BUF is considered as a promising antitumor agent, its poor water solubility, high cardiac toxicity, short circulation half-life and narrow therapeutic window have limited the clinical application of the drug [26].…”