Plumbagin induces apoptotic and autophagic cell death through inhibition of the PI3K/Akt/mTOR pathway in human non-small cell lung cancer cells
Introduction
Apoptosis (also known as type I cell death) and autophagy (also known as type II cell death) are two primary morphologically distinctive modes of programmed cell death. Apoptosis is the best described mechanism of cell death and is morphologically characterized by membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and formation of apoptotic bodies [1]. The classic autophagic pathway acts downstream of the mammalian target of rapamycin (mTOR) kinase [2], [3], [4]. Active mTOR kinase in the mTOR complex 1 (mTORC1) phosphorylates and inhibits Unc-51-like kinase 1 (ULK1), a key proautophagy adapter involved in nucleation of the autophagophore membrane. mTORC1 is a well-known negative regulator of autophagy. Microtubule-associated protein 1A/1B-light chain 3 (LC3), the mammalian homolog of the yeast protein Atg8 (Aut7/Apg8p), is important for transport and maturation of autophagosomes. Newly synthesized free cytosolic LC3/Atg8 is immediately cleaved at its C-terminus by Atg4 to form the cytosolic LC3-I [5]. Upon induction of autophagy, LC3-I is converted into LC3-II after conjugation with phosphatidylethanolamine requiring Atg3 and Atg7 [5]. LC3-II is then incorporated to the growing autophagosome structure that, upon maturation, fuses with the lysosome compartment, leading to the degradation of autophagosomal contents [2], [3], [4]. Currently, there is no cancer therapeutic approach that specifically targets the apoptotic and/or autophagic pathway [2], [3], [4], [6].
Lung cancer is the leading cause of cancer-related death in humans worldwide [7]. There are two major types of lung cancer: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). NSCLC accounts for 70–85% of all cases of lung cancers. There is an increase in the incidence of lung cancer with an estimation of 228,190 new cases of lung cancer in the US, accounting for 14% of cancer diagnosis [7]. Although current therapeutic strategies for the treatment of NSCLC have made some advancement by the platinum-based standard chemotherapy, the average 5-year survival rate is about 17% which has not been significantly improved over the last 40 years [7], [8]. Failure of chemotherapy in NSCLC is mainly due to multidrug resistance and dose-limiting adverse reactions. Both apoptosis and autophagic cell death are found to be suppressed in NSCLC. This highlights the urgent need for safe and efficacious new therapeutics for NSCLC.
Plumbagin (PLB; 5-hydroxy-2-methyl-1,4-naphthoquinone, see Fig. S1) is a natural naphthoquinone isolated from the roots of the medicinal herb Plumbago zeylanica L. (also known as Chitrak). PL has also been found in Juglans regia (English walnut), Juglans cinerea (butternut and white walnut) and Juglans nigra (black walnut). PLB is also isolated from isolated from Chinese medicinal plants such as the Plumbaginaceae, Droseraceae and Ebenaceae. PLB has been reported to have various pharmacological activities including anti-inflammatory, anticancer, antibacterial and antifungal activities in vitro and in vivo [9]. Previous studies have showed that PLB exhibited anti-proliferative effect in various cancer cell lines via intracellular reactive oxygen species (ROS) generation, apoptosis induction and cell cycle arrest [10], [11]; but the underlying mechanism is not fully understood. PLB induced cell apoptosis via modulation of cellular redox status and generation of ROS in human prostate cancer cells [12], tongue carcinoma cells [13], and leukemia cells [14], [15]. Initial studies have showed that PLB induced the apoptosis of lung cancer cells by inhibiting the activation of NF-κB [16], [17] or up-regulating p53 expression via c-JNK phosphorylation [18]. In MCF-7 cells, PI5K-1B plays a crucial role in ROS generation induced by PLB [19]. PLB inhibited invasion and migration of breast and gastric cancer cells through down-regulation of the expression of chemokine receptor CXCR4 [20]. PLB also inhibited osteoclastogenesis and reduced human breast cancer-induced osteolytic bone metastasis in mice through suppression of RANKL signaling [21], [22]. In addition, PLB promoted autophagy of human breast cancer cells via the inhibition of Akt/mTOR pathway [23]. PLB inhibited tumor angiogenesis and tumor growth in human colon carcinoma and prostate cancer xenograft mouse models [24]. PLB plumbagin blocked the Ras/Rac/cofilin and Ras/MEK signaling pathways mediated by vascular endothelial growth factor receptor 2 in human umbilical vein endothelial cells [24]. In order to further explore the anticancer effect of PLB in NSCLC, we examined the pro-apoptotic and pro-autophagic effects of PLB in A549 and H23 cells and the underlying mechanisms.
Section snippets
Chemicals and reagents
PLB (Mr: 188 Dal) was obtained from Sigma–Aldrich Inc. (St. Louis, MO) with a purity of more than 98%. 4′,6-Diamidino-2-phenylindole (DAPI) and 5-(and 6)-chloromethyl-2′,7′-dichlorodihy-drofluorescein diacetate (CM-H2DCFDA) were bought from Invitrogen (Carlsbad, CA). RPMI-1640 medium was obtained from Coring Cellgro Inc. (Herndon, VA). PLB, N-acetylcysteine (NAC), vitamin C, reduced glutathione (GSH), 5,6-dimethylxanthenone 4-acetic acid (DMXAA), Dulbecco’s phosphate buffered saline (PBS) and
PLB inhibits the proliferation of A549 and H23 cells
To investigate the potential growth inhibition of PLB in NSCLC cells, we first examined the effect of PLB on cell proliferation in A549 and H23 cells using the WST-1 assay. The concentration-dependent inhibitory effects of PLB on the growth of A549 and H23 cells are showed in Fig. S2A. When A549 cells were treated with PLB at 0.5, 5, 25, 100 and 200 μM for 24 h, the percentage of cellular viability over the control cells (100%) was 86.9%, 73.0%, 37.0%, 29.9%, and 21.4%, respectively. The
Discussion
Cancer cells evade programmed cell death to support malignant growth [40], [41]. Therefore, understanding the mechanisms of programmed cell death and designing therapeutic approaches to trigger cell death in cancer cells are critical for effectively treating the disease. The treatment of NSCLC remains a major challenge because of poor efficacy and severe toxicities of standard and new chemotherapy. There is an increased interest in seeking new therapies for lung cancers from natural compounds.
Conflict of Interest
All authors have no conflicts of interest.
Acknowledgements
The authors appreciate the financial support from the Startup Fund of the College of Pharmacy, University of South Florida, Tampa, FL 33612. Drs. Yan-Cong Li, Minghua Li, and Zhi-Wei Zhou are holders of postdoctoral scholarships from College of Pharmacy, University of South Florida, Tampa, FL 33612.
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2022, Seminars in Cancer BiologyCitation Excerpt :Because of the current attention of abiding conventional therapeutics and introducing novel pharmacophores from nature to provoke cancer theragnosis [45,46] the below section focuses on the autophagic cell death mediated by the natural bioactive compounds that are indispensable comrades for cancer therapeutics. Plumbagin, a naphthoquinone derivative isolated from Plumbago zeylanica apart from inducing apoptosis, exhibits autophagic cell death by inhibition of PI3K/AKT/mTOR signaling which is further augmented by two synthetic mitogen activated protein kinase inhibitors, SB202190 and SB203580 [47]. Similarly, a bisindolic alkaloid, voacamine enhances chemosensitization towards doxorubicin-induced apoptosis independent cell death by provoking autophagy [3].
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