According to the results of our literature research; this is the first study to investigate the effect of ASX on human mesothelioma cells, SPC212.
Previous reports deduced that ASX has reduced viability of cancer cells among different types of cancer cells studied so far. In the stufy of Kim et al, the MTT assay was performed for ASX-treated (50 and 100 µM) colon cancer cells to measure cell proliferation (Kim, Kim, & Hong, 2019a). In another study, cytotoxic activity of ASX on A549 and H1703 cells was investigated. ASX reduced cell viability in a concentration and time-dependent manner. In the colony-forming test, ASX was suggested to significantly suppress the ability of A549 and H1703 cells to colonize (Ko vd., 2016). Furthermore, ASX was shown to supress proliferation of LS-180colon cancer cells and decreased the cell viability with dose-dependent manner (Hormozi vd., 2019). ASX exerted an inhibitory effect in non-small cell lung cancer (NSCLC) cell proliferation and viability (Chen vd., 2018). ASX blocked the cell proliferation in the breast cancer cells investigated (McCall vd., 2018). An appropriate concentration of ASX, is effective for preventing the proliferative activity of Caco-2 cells (Kim, Lim, & Kim, 2019b). The common point in ASX toxicity studies is that ASX causes dose and time dependent cytotoxic effects on cell lines. ASX decreased SPC212 cell viability dose- and time-dependently and that IC50 value for 24 h ASX treatment against SPC212 cells were detected as 241.7 µM.
Innate oxidative stress is considered to pathophysiology of cancers because potent factor of angiogenesis. ROS is generated by aerobic cellular metabolism and scavenged by antioxidant mechanisms. ROS production and antioxidant defense capability cause oxidative stress. Oxidative stress results in cell damage (Meng, Ni, Yu, Wang, & Shen, 2017). ASX has antioxidant, anti-inflammatory and immunomodulatory properties. Antioxidants are the focus of researches due to their anticancer properties. Previous studies of ASX reported to reduce oxidative stress and preserve metabolism (Yan vd., 2017). ASX has much more potent antioxidant than other carotenoids such as zeaxanthin, lutein, tunaxanthin, cantaxanthin and b-carotene and a-tochopherol. ASX inhibited stress in the liver by reducting of lipid peroxidation and NK cell activity. Also, ASX showed with an inhibitory effect on hepatic metastases by its antioxidative property (Kurihara, Koda, Asami, Kiso, & Tanaka, 2002). ASX inhibited cytotoxic effects of glutamate in mouse hippocampal HT22 cell. ASX reduced cell viability, glutamate-induced caspase activation and ROS nuclear accumulation (Wen vd., 2015). Oxidative stress is an important factor in diseases such as cancer and causes to mitochondrial dysfunction. Wolf et al. analyzed basal oxidative stress, superoxide measurement and mitochondrial membrane potential. They reported that ASX decreased oxidative stress and protected HeLa cells against oxidative stress (Wolf vd., 2010).
ASX provided decreases in SOD2 level and SOD activity and reduced mitochondrial ROS in gastric epithelial AGS cells (Kim vd., 2019b). In rat glioma cells, ASX showed reactive oxygen species (ROS) and free radical salvaging properties as a quencher of singlet reactive oxygen, nitrogen and two electron oxidants (Afzal vd., 2019). ASX is a potent antioxidant and has terminal carbonyl group which is conjugated to a polyene. It scavenges free radicals. So ASX protects cells from cancer. ASX may result variously from its antioxidant and anti-cancer properties interactions with altering gene expression and cellular signalling cascades (Ambati, Phang, Ravi, & Aswathanarayana, 2014). In our oxidative stress analysis, consistent with literature, ASX also decreased oxidative stress in a dose-dependent way in SPC212 cells
Apoptosis is an organized programmed cell death that occurs in physiological and pathological conditions. Apoptosis can be triggered either by extrinsic pathway and intrinsic pathway. During the apoptosis caspase proteins including caspase-3, caspase-8 and − 9 play critical roles. Caspase-3 is important in this process. Wen et al. examined the activities of caspase proteins in HT22 cells. They demonstrated that glutamate generated the activation of caspase 8/9 in HT22 cells by co- treatment with ASX and ASX prevented activation of caspase-3 (Wen vd., 2015). In vitro studies using cancer cell lines and in vivo tumour models have indicated the anti-proliferative and proapoptotic effects of ASX (Liu vd., 2016; Sowmya, Arathi, Vijay, Baskaran, & Lakshminarayana, 2017; Wu vd., 2016). ASX reduced oxidative stress and cell death in HT22 cells. ASX decreased ROS and thereby aborted intrinsic apoptosis by upregulating antiapoptotic Bcl-2 expression and downregulating proapoptotic Bax expression (Wen vd., 2015). Zhang et al. examined the effect of ASX and other carotenoids at the different dose ranges on K562 cells. They reported that carotenoids (b-carotene, ASX, capsanthin and bixin) decreased the viability of K562 cells and induced cell apoptosis (Zhang, Zhao, Hu, Zhao, & Huang, 2011). Kavitha et al. evaluated the effect of ASX in the hamster buccal pouch (HBP) carcinogenesis model by caspase caspase 3/9 test by enzymatic assay. ASX significantly increased the expression of caspase-9, caspase − 3 and PARP (Kavitha, Kowshik, Kishore, Baba, & Nagini, 2013). Meng et al. reported ASX treatment is not a clear indicator of protective effects on apoptosis for RWPE-1 or PC-3 cells by Annexin V (Meng vd., 2017).
We analysed ASX’s apoptotic effect on SPC212 cells after the cytotoxic and antiproliferative effect analyses. We recognized this finding by Annexin V and caspase 3/7 analysis by flow cytometry. Annexin V test and caspase 3/7 assays exhibited that SPC212 cells, to which ASX was applied, were noticed to be driven to apoptosis different than control cells, which showed relatively higher viability and almost underwent no apoptosis.
In conclusion, we indicated that ASX induced growth inhibition, morphological deformation, oxidative stress and apoptosis in human mesothelioma cells and its IC50 value for SPC212 cells was found to be 241.7 µM for 24 hours. We think that presented results will be an incentive for and provide new insights into future studies since there are no studies about ASX’s effect on lung cancer cells.