Inhibition of autophagy by chloroquine potentiates synergistically anti-cancer property of artemisinin by promoting ROS dependent apoptosis

Highlights

• Artemisinin decreased cell viability and colony forming ability of A549 cells but no-toxicity towards normal lung WI38 cells.

• Artemisinin induced autophagy and apoptosis in A549 cells.

• Autophagy inhibition by chloroquine pre-treatment sensitized cell viability of A549 cells towards artemisinin.

• Autophagy inhibition by chloroquine increased AVOs accumulation, decreased MMP and consequently increased ROS.

• Chloroquine pre-treatment also sensitized different cancer cells towards artemisinin.

Abstract

Artemisinin (ART) is a well-known anti-malarial drug, and recently it is shown prospective to selectively kill cancer cells. But low potency makes it inappropriate for use as an anticancer drug. In this study, we modulated the ART-induced autophagy to increase Potency of ART as an anticancer agent. ART reduced the cell viability and colony forming ability of non-small lung carcinoma (A549) cells and it was non-toxic against normal lung (WI38) cells. ART induced autophagy at the early stage of treatment. Pre-treatment with chloroquine (CQ) and followed by ART treatment had synergistic combination index (CI) for cell death. Inhibition of autophagy by CQ pre-treatment led to accumulation of acidic vacuoles (AVOs) which acquainted with unprocessed damage mitochondria that subsequently promoted ROS generation, and resulted releases of Cyt C in cytosol that caused caspase-3 dependent apoptosis cell death in ART-treated A549 cells. Scavenging of ROS by antioxidant N-acetyl-cysteine (NAC) inhibited caspase-3 activity and rescued the cells from apoptosis. Similar effects were observed in other cancer cells SCC25 and MDA-MB-231. The appropriate manipulation of autophagy by using CQ provides a powerful strategy to increase the Potency of selective anticancer property of ART.

Introduction

Artemisinin (ART) is a sesquiterpene lactone isolated from the sweet wormwood Artemisia annua L, usually use as an anti-malarial drug [1]. Recently, ART is also emerging as an anticancer agent and studies show that ART is more cytotoxic to cancer cells than normal cells [2], [3]. It is due to the fact that the metabolic activity and cell divisions in cancer cells are more rapid than normal cells. Therefore, compare to normal cells, cancer cells uptake more iron from environment through transferrin receptor. This iron activates the endoperoxide linkage of ART leading to generation of highly cytotoxic carbon centred radical or reactive oxygen species which is similar of its cytotoxic action against malaria [4], [5]. Therefore, ART not only shows toxicity against cancer cells, but also its toxicity selectivity towards cancer cells makes ART as a promising anticancer agent. Several different molecular mechanisms of anticancer activity of ART have been investigated. Apoptosis has been commonly reported mechanism of its cytotoxic action [6], [7], [8], beside this, induction of autophagy [9], cell cycle arrest [10], ROS generation [7], involvement of iron [3], [10], [11] also been demonstrated well in ART induce cytoxicity in cancer cells. However, the low toxicity and low bio-availability make it unsuitable for use as an anticancer agent. Works are going on to try to increase the potency of artemisinin and make it acceptable as an anticancer drug.

Autophagy is a cellular event, induced by several extracellular stressor or internal needs, where many cytosolic materials like organelles and several proteins are accumulated in a double layer membrane vesicle called autophagosomes then it is fused with lysosomes where internal materials are degraded [12], [13]. The roles of autophagy in cancer cells are not so clear. Some reports which demonstrate that autophagy act as a protective mechanism in 5FU [14], sulforaphane [15], imatinib [16] induce cancer cell death. Whereas under certain circumstances excessive or unquenched autophagy leads to cell death called programme cell death type II (PCDII), this is characteristically differ from apoptosis [17], [18]. So as evidently the autophagy in cancer cells may depend on the kind of tumour, stimuli and also phases of tumorigenesis. If we would try to modulate autophagy induced by anticancer agents in an appropriate manner that may increase its potency against cancer cells.

To increase the potency of anticancer agents by inhibiting autophagy, different autophagy inhibitors have been used [19], [20]. Interestingly, some previous reports suggest that inhibition of autophagy at a late stage can effectively increase cytotoxicity of anticancer agents towards cancer [16], [21], [22]. One of the well known late phase autophagy inhibitor is chloroquine (CQ). For more than six decades CQ has been using to treat several diseases (like, malaria, rheumatoid arthritis, lupus) because of its high effectiveness and well tolerated by human [23]. Recently, it is also found to be effective against cancer [24], [25]. CQ is a weak base that is readily distributed in human body when it is administered. At cellular levels its unprotonated form can diffuse through the plasma membrane, and its protonated forms are trapped within the acidic vacuoles (late endosome and lysosome), causing increased in pH, inactive lysosomal hydrolysates, thus increase the autolysosomes accumulation by inhibiting the autophagy. This function has been found useful in potentiating the killing effect of radiated cancer cells and other chemotherapeutic agents like 5FU [20], AKT inhibitors [26], src kinase inhibitors such as saracatinib [27].

In the present study, we were interested to investigate ART induced autophagy and selective apoptosis in A549 cells. Next we would like to examine whether the clinical potency of ART would be increased when it was treated along with CQ. Results of this report demonstrated that indeed potency of anticancer activity of ART was increased in the presence of CQ which could be a promising strategy for application of ART for treatment of cancer in future.