In this study we demonstrated that a natural mixture of wolfberry phytochemicals induced leukemia Jurkat cell apoptosis through elimination of cellular ROS. Previous reports showed that purified resveratrol and quercetin induce Jurkat cell cycle arrest at about 20 to 100 μM [32
]. Compared to those reports, the doses used in the current study were fairly low. Most abundant fractions in our harvested wolfberry extracts were carbohydrates (over 73 %). Working concentration of total polyphenolics in the culture media was at the range of 7.8 – 31.1μg quercetin equivalent/ml (calculated based on total polyphenolics contents in the freeze-dried powder of wolfberry phytochemicals). The rutin doses were at the range of 1.07–4.26 μg/ml.
Wolfberry phytochemicals had inhibitory effects on cancer cell proliferation, including leukemia Jurkat cells (), colon cancer SW480 cells and canine lymphoma F1B cells (data not shown). However, there was no growth inhibition observed in the normal cells treated with same doses of wolfberry phytochemicals (data not shown), suggesting its specificity on anti-cancer cell proliferation. Purified polysaccharides, however, did not inhibit cell proliferation in Jurkat cells in culture (). The results are not consistent with the previous observation in other cancer cells, including colon and prostate cancer cells [12
]. This may be due to difference of cancer cell culture models. The results suggested that other components or synergistic interaction of wolfberry phytochemicals might play roles in inhibition of Jurkat cell proliferation.
Unlike normal cells, most cancer cells grow and proliferate under constitutive oxidative stress condition (e.g. elevated ROS levels), which results in cellular unfolded protein response (UPR) and ER stress [34
]. Phytochemicals, such as polyphenolics, have been demonstrated to induce overloading of ER stress and subsequent apoptosis in cancer cells [33
]. Phytochemical prevention of blood cancer, such as leukemia, has not been well documented [40
]. To our knowledge, this is the first report that phytochemical-eliminated ROS leaded to subsequent apoptosis through down-regulation of ER stress biomarkers in leukemia cells. High antioxidant activity of wolfberry phytochemicals scavenged reactive oxygen species, which resulted in diminishing cellular oxidative status and disrupting of cellular stress homeostasis that cancer cells live on.
A number of publications reported that targeting ER stress could be an attractive strategy for cancer prevention. It has been well documented that overloading ER stress induces cancer cell apoptosis, including leukemia Jurkat cells, though cancer cells naturally live on elevated ER stress. However, a couple of reports addressed that inhibition of ER stress biomarkers causes cancer cell death [41
]. For instance, down-regulation of IRE-1α, PERK, and ATF6α-dependent ER stress signaling by the antibody against the COOH-terminal domain of the GRP78 protein significantly caused inhibition of growth of 1-LN and DU-145 prostate cancer cells [24
]. Under tunicamycin-induced ER stress condition, GRP78 levels were declined by phytochemicals including genistein, epigallocatechin gallate, and salicylic acid [37
]. CHOP has a dual role to protect from and promote cell death [43
]. In this study, wolfberry phytochemicals and/or siRNA knockdown caused down-regulation of ER stress biomarkers, and inhibition of CHOP expression in the progression of Jurkat cell apoptosis, suggesting that precise control of protein levels of ER stress biomarkers is critical to cancer cell survival. Disruption of the balance by either triggering ER stress [15
] or down-regulation of ER stress biomarkers (this study) may lead to leukemia Jurkat cell apoptosis.
β-catenin, a proapoptotic protein, plays a key role in the Wnt signaling pathway. GSK-3β destabilizes β-catenin by phosphorylating it at Ser33, Ser37 and Thr41 [44
]. Mutations in these phosphorylation sites, which result in the stabilization of β-catenin protein levels, have been found in many tumor cell lines [45
]. ER stress-induced dissociation of GRP78 from Wnt leads to downregulation of stabilized β-catenin and consequent cell proliferation [21
]. Wolfberry phytochemicals induced accumulation and stabilization of β-catenin through inactivation of GSK-3β, which in turn leads to activation of cellular Wnt signaling and consequently Jurkat cell apoptosis. On the other hand, GSK-3β is also a critical downstream target of the PI3K/AKT cell survival pathway whose activity can be inhibited by AKT-mediated phosphorylation at Ser9 of GSK-3β [46
Activation and overexpression of NFκB were found in many tumors [30
]. Expression of NFκB, including NFκB p105, p65 and p50, declined dose dependently in wolfberry treated Jurkat cells, indicating that NFκB was one of the molecular targets for induction of apoptosis by wolfberry phytochemicals.
Control of AMPK is important in cancer prevention. AMPK negatively affects mTOR/AKT through phosphorylation of raptor, which leads to inhibition of CDC2 and activation of apoptosis [37
]. Wolfberry phytochemical activation of AMPK might be related to inhibition of CDC2 activity leading to cell cycle arrest in human leukemia Jurkat cells, which was consistent with previous reports in other cancer cell culture models [47
Taken together, wolfberry phytochemicals, a natural mixture of polysaccharides, polyphenolics, and other unknown compounds, dually functioned as an antioxidant, through eliminating cellular ROS, and a modulator of cell survival/death signaling pathways, through targeting ER stress and related pathways in AMPK, β-catenin, NFκB, and PI3K-AKT signaling, which lead to Jurkat cell apoptosis. Targeting multiple signaling pathways by a mixture of wolfberry phytochemicals would be considered as a novel strategy in the development of therapeutic regimens for prevention of cancer, such as leukemia. This may also help overcome the complexity of drug resistance.