Artesunate induces mitochondria-mediated apoptosis of human retinoblastoma cells by upregulating Kruppel-like factor 6

Ying Yang,Xiaobing Qian,Kin Chiu,Yihui Wu,Jing Zhuang,Jin Qiu,Jieting Zeng,Haoting Chen,Qianying Gao,Nandan Wu

doi:10.1038/s41419-019-2084-1

Abstract

Retinoblastoma (RB) is the most common primary intraocular malignancy in children. Intravitreal chemotherapy achieves favorable clinical outcomes in controlling RB vitreous seeds, which are a common reason for treatment failure. Thus, a novel, effective and safe intravitreal chemotherapeutic drug is urgently required. The malaria drug artesunate (ART) recently demonstrated remarkable anticancer effects with mild side effects. The purpose of this study is to investigate the anti-RB efficacy, the underlying mechanism and the intraocular safety of ART. Herein, we verified that ART inhibits RB cell viability and induces cell apoptosis in a dose- and time-dependent manner. Microarray analysis revealed that Kruppel-like factor 6 (KLF6) was upregulated after ART treatment, and this was further confirmed by real-time PCR and western blot assays. Silencing of KLF6 expression significantly reversed ART-induced RB cell growth inhibition and apoptosis. Furthermore, ART activated mitochondria-mediated apoptosis of RB cells, while silencing KLF6 expression significantly inhibited this effect. In murine xenotransplantation models of RB, we further confirmed that ART inhibits RB tumor growth, induces tumor cell apoptosis and upregulates KLF6 expression. In addition, KLF6 silencing attenuates ART-mediated inhibition of tumor growth in vivo. Furthermore, we proved that intravitreal injection of ART in Sprague-Dawley (SD) rats is safe, with no obvious retinal function damage or structural disorders observed by electrophysiology (ERG), fundal photographs, fundus fluorescein angiography (FFA) or optical coherence tomography (OCT) examinations. Collectively, our study revealed that ART induces mitochondrial apoptosis of RB cells via upregulating KLF6, and our results may extend the application of ART to the clinic as an effective and safe intravitreal chemotherapeutic drug to treat RB, especially RB with vitreous seeds.

Highlights

Retinoblastoma (RB) is the most common childhood cancer of the eye; it arises from the retina and causes serious damage to vision, even endangering lives[1]
Using real-time PCR and western blot assays for further verification, we found that Kruppel-like factor 6 (KLF6), which is known as a tumor suppressor that induces apoptosis in various cancer cells[23,24], was upregulated in both RNA and protein expression after ART treatment (Fig. 2c–e)
We revealed a novel anti-RB mechanism of ART: ART treatment upregulates KLF6 expression, which causes mitochondrial dysfunction, increases the Bcl-2-associated X protein (Bax)/Bcl-2 ratio, promotes the release of cyt-c, and stimulates the cleavage of caspase-9 and caspase-3, resulting in cell apoptosis (Supplementary Fig. S6)

Summary

Introduction

Retinoblastoma (RB) is the most common childhood cancer of the eye; it arises from the retina and causes serious damage to vision, even endangering lives[1]. The current standard treatment for RB includes. Yang et al Cell Death and Disease (2019)10:862 safe intravitreal chemotherapeutic drug is urgently required for the treatment of RB, especially with vitreous seeds. Accumulating evidence has demonstrated that ART effectively inhibits the growth of various cancer cells, including leukemia, renal cell carcinoma, esophageal cancer, ovarian cancer, and RB15–19. In addition to cell and animal experiments, the antitumor effect and safety of ART has already been verified in patients. Zhang et al.[20] reported that the combination of ART with vinorelbine and cisplatin can elevate outcomes in advanced non-small-cell lung cancer patients without additional side effects. Many studies have explored the possible antitumor mechanisms of ART, such as cell cycle arrest, induction of cell apoptosis, regulation of tumorrelated gene expression, and inhibition of angiogensis[21,22]. The underlying molecular mechanism of ART action on RB cells remains unclear

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