Part 2:echinacoside Induces Apoptotic Cancer Cell Death By Inhibiting The Nucleotide Pool Sanitizing Enzyme MTh1

echinacoside induces apoptotic cancer cell death by inhibiting the nucleotide pool sanitizing enzyme MTh1

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Cistanche echinacoside has anti-cancer and anti-apoptotic effects

Results

Identification of Echinacoside as an MTh 1 inhibitor

To search for natural compounds capable of inhibiting MTH1, we used the MTH1-catalyzed enzymatic reaction as a high-throughput in the vitro screening assay. Besides oxidized purine nucleotides, MTH1 can also hydrolyze normal deoxyguanosine triphosphate to generate deoxyguanosine monophosphate and pyrophosphate. In the presence of excess inorganic pyrophosphatase, all pyrophosphates are converted into inorganic phosphates, which can be quantified using a malachite green-based absorbance assay,41 thus allowing for indirect measurement of MTH1 activity.32 To test the assay system, we first measured the effect of(S)-crizotinib, a compound with proven potency as an MTH1 inhibitor.33 The result showed that (S)-crizotinib indeed potently inhibitedMTH1 (Figure 1A) with an IC50 of500 nM, which was sevenfold higher than that reported by Huber et al (72 nM).33 This was likely due to the differences in the detection methods or sources of chemicals and enzymes. We adopted a less sensitive chromogenic approach, while Huber et al used a highly sensitive bioluminescent method.33 Keeping this in mind, we screened 12 commercially available natural compounds (Table S1), each of which is a major ingredient of traditional herbs with potential antitumor activity. Echinacoside, a compound purified from the parasitic medicinal plant Cistanche salsa (Figure 1C), significantly inhibited the reaction (Figure 1B), with an IC50 of 7.01±2.13 μM (Figure 1D). Adding 50 times more pyro- phosphatase had no impact on the result while adding five times moreMTH1protein significantly decreased the degree of inhibition, suggesting that Echinacoside specifically inhibited the activity ofMTH1 in the in vitro enzymatic assay.

Echinacoside inhibited cellular MTh 1 to increase intracellular 8-oxog

Next, we asked if Echinacoside can inhibit intracellular MTH1 activity. Inhibition of cellular MTH1 will result in the increase of intracellular 8-oxoG. Avidin has been shown to bind to 8-oxoG with high specificity;42 therefore, we used immunofluorescent staining with Cy3-conjugated avidin to compare intracellular 8-oxoG levels in various cancer cell lines before and after Echinacoside treatment. Human MG-63 osteosarcoma, SK-HEP-1hepatocarcinoma, MCF-7 breast cancer, and SW480 colorectal cancer cells were treated with 0 μM, 15 μM, 30 μM, 60 μM, or 80 μM Echinacoside for 5 hours, 12 hours, or 24 hours. Staining with Cy3-conjugated avidin revealed that treatment with 60 μM Echinacoside for 24 hours clearly and significantly increased the level of cellular 8-oxoG (Cy3-avidin reactive substance) in these cancer cells (Figure 2A and B). Higher concentration (80 μM) of Echinacoside resulted in stronger cellular 8-oxoG staining (Figure 2B), suggesting a dose-response relationship. Similar results were obtained

by immunofluorescent staining with a mouse monoclonal anti-8-oxoG antibody (Figure S1A).43 The concentration of Echinacoside (60 μM) required for a significant increase in cellular 8-oxoG was much higher than the IC50 (7.01 μM) from the in vitro assay. This was likely due to the difference in sensitivity of the two assays. Immunofluorescent staining is far less sensitive than the in vitro enzymatic assay; furthermore, the number of inhibitor molecules that can reach and interact with cellular MTH1 is influenced by complex biological processes; additionally, in the in vitro assay, the less favorite dGTP is used as the substrate, and hence it may be easier (take fewer inhibitors) to inhibit MTH1, resulting in a lower IC50 in the in vitro assay.

The anticancer and antioxidant effects of echinacoside from Cistanche deserticola

Cellular 8-oxoG is generated by ROS, which is antagonized by antioxidants. Thus, an increase in cellular ROS or suppression of the activity of antioxidants would also result in an increase in cellular 8-oxoG level. Echinacoside itself is a potent antioxidant44,45 and thus should enhance rather than suppress the activity of antioxidants. To examine if cellular ROS level was changed by Echinacoside treatment, the same cancer cells were treated with 0 μM, 15 μM, 30 μM, 60 μM, or 80 μM Echinacoside for 5 hours, 12 hours, or 24 hours and then analyzed by flow cytometry after staining with the fluorescent probe 2′,7′-dichlorofluorescein diacetate. The results showed that none of these treatments changed cellular ROS levels (Figure 2C and D). Thus, the increased cellular 8-oxoG level most likely resulted from the inhibition of MTH1 by Echinacoside.

echinacoside caused extensive DNA damages specifically in cancer cells Incorporation of 8-oxoG into DNA will stimulate BER and MMR, which generate single-strand DNA breaks (SSB) and gaps.16,17 Normally, these breaks, and gaps are sealed by repair mechanisms involving gap filling and ligation by DNA polymerases and ligases.46,47 However, rapid increases in cellular SSB can saturate cellular repair capacity, leading to the formation of numerous double-strand DNA breaks (DSB)18 and DDR signaling. To examine if the Echinacoside-induced increase in cellular 8-oxoG caused DNA damages, we examined one of the early markers of DNA damage, 53BP1, which binds to sites ofDSB as the earliest cellular response to DSB.48 The MG-63, SK-HEP-1, MCF-7, and SW480 cancer cell lines and the noncancer cell lines human normal liver L-O2,49 human embryonic kidney HEK 293, and mouse fibroblast NIH/3T3 were treated with 0 μM, 15 μM, 30 μM, 60 μM, or 80 μM Echinacoside for 5 hours, 12 hours, or 24 hours. Fluorescent immunostaining showed that treatment with 60 μM Echinacoside for 24 hours specifically increased the number of cells with five or more strongly stained nuclear 53BP1 foci in all cancer but not in any of the noncancer cell lines (Figure 2E and F). A significant increase in the number of 53BP1+ cells was seen as early as 5 hours after initiation of Echinacoside treatment (Figure 2F). Higher concentration (80 μM) of Echinacoside or longer treatment time (12 hours and 24 hours) resulted in larger increases in both the number of53BP1+ cells (Figure 2F) and the number of cellular 53BP1 foci. Together, these results suggest that the inhibition of MTH1 by Echinacoside caused 8-oxoG accumulation and extensive DNA damages in cancer but not in noncancer cells.

echinacoside suppressed cancer cell proliferation

In cycling cells, unrepaired DNA strand breaks will cause a collapse of DNA replication forks, which will then lead to blockage of cell proliferation by induction of cell cycle arrest and apoptosis.50 To check the effects of Echinacoside on cell growth and proliferation, we first analyzed the growth of Echinacoside-treated MG-63, SK-HEP-1, MCF-7, and SW480 cancer cells by a colony formation assay. The results showed that the cancer cells treated with 60 μM or 80 μM Echinacoside formed much fewer colonies, and the colonies that formed were much smaller (Figure 3A and B), indicating greatly suppressed growth potential. Next, we analyzed the proliferation of these cancer cells, together with the L-O2, HEK 293, and NIH/3T3 noncancer cells by an MTT assay. The results showed that the Echinacoside dose-dependently inhibited the proliferation of cancer but not the noncancer cells (Figure 3C). Time-course study of cancer cells treated with 60 μM Echinacoside showed that after 5 hours, there was no significant difference between nontreatment and treatment groups, but after 12 hours, cancer cell proliferation was significantly inhibited by 60 μM Echinacoside (Figure 3D).

stages of the cell cycle by flow cytometry revealed that Echinacoside treatment dose-dependently reduced the percentage of cells in both S and G2/M phases, while the percentage of cells in the G1 phase increased (Figure 4C and D). After 80 μM Echinacoside treatment of MG-63 cells for 24 hours, cells in the G2/M phase decreased from 15% to nearly zero, and cells in the S phase decreased from 37% to 17%, while cells in the G0/1 phase increased from 43% to 80% (Figure 4E). These results indicate that treatment with Echinacoside induced cell cycle arrest and blocked the cancer cells in the G1 phase.

Echinacoside induced apoptosis in cancer cells

Consistent with observations on cell proliferation, Western blot analysis showed that treatment with 60 μM and 80 μM Echinacoside for 24 hours increased the levels of active caspase-3 and cleaved poly (ADP-ribose) polymerase proteins in the cancer cells (Figure 5A and B), suggesting induction of caspase-dependent apoptosis. We then examined several cellular markers of apoptosis. First, the nuclear morphology of Echinacoside-treated cancer cells was revealed by staining with the DNA dye DAPI, which showed that 24 hours treatment with 60 μM and 80 μM Echinacoside increased the number of hallmarks of apoptosis, including pyknosis and condensed chromatin (brighter nuclei) (Figure 5C). Second, the DNA extracted from similarly treated cancer cells was analyzed by electrophoresis on agarose gel, which revealed a typical apoptotic ladder pattern (Figure S1B). Finally, cellular active caspase-3 was detected by immunofluorescent staining, which revealed strong activated caspase-3 signals in Echinacoside-treated cancer cells (Figure S1C).

Next, the percentage of apoptotic cells was measured by Annexin V-FITC and PI double staining and flow cytometry. Analysis of cells treated with 0 μM, 15 μM, 30 μM, 60 μM, 80 μM, or 160 μM Echinacoside for 2 hours, 5 hours, 12 hours, or 24 hours showed that 60 μM or higher concentrations of Echinacoside induced significant apoptosis in the MG-63, SK-HEP-1, MCF-7, and SW480 cancer cells (Figure 6A), but not in the noncancer L-O2, HEK 293, and NIH/3T3 cells (Figure S2). After treatment of MG-63 cells with 60 μM, 80 μM, or 160 μM Echinacoside for 24 hours, the percentage of apoptotic cells increased from 8.89% to 33.72%, 39.01%, and 48.12%, respectively (Figure 6A). The time-course study showed that after 5 hours, there was no significant difference between nontreatment and treatment groups, but significant apoptosis was seen after treatment with 60 μM or higher concentrations of Echinacoside for 12 hours (Figures 6B and S3).

Finally, measurement of mitochondrial membrane potential by the JC-1 fluorescent dye clearly showed a prominent loss of mitochondrial membrane potential after treatment with 60 μM Echinacoside for 12 hours (Figure 6C), but not after 5 hours, indicating the activation of the intrinsic apoptosis pathway following the DNA damages caused by elevated 8-oxodG.

Phenylethanoid Glycosides in cistanche (2)

The anti-cancer and anti-inflammatory effects of cistanche echinacoside

Discussion

Echinacoside is a natural compound isolated from the medicinal plants Cistanche and echinacea.51,52 It has been shown to possess versatile health promotional and disease preventive properties, including neural protection, hepatoprotection, anti-inflammation, antifatigue, antisenescence, antidiabetes, and antitumor activities.53–58 The best known and accepted bioactivity of Echinacoside is its antioxidative and ROS- scavenging action;44,45 however, it has also been shown to cause oxidative DNA damages in cancer cells, with the underlying mechanisms remaining unclear.58 In the present study, using a high-throughput in a vitro screening assay, we found that Echinacoside effectively inhibited the MTH1-catalyzed enzymatic reaction. Increasing the amount of the MTH1 enzyme decreased the degree of inhibition while increasing the amount of the inorganic pyrophosphatase did not affect the inhibition, indicating that Echinacoside specifically inhibited the activity of MTH1 in the in vitro assay. Treatment of different human cancer cell lines with Echinacoside caused significant elevation of the cellular 8-oxoG level without changing the cellular ROS level. Given that Echinacoside itself is a potent antioxidant, these results suggested that the increased intracellular 8-oxoG level was likely resulted from the inhibition of cellular MTH1 by Echinacoside.

Treatment with Echinacoside caused extensive DNA damages and significant upregulation of the G1/S-CDK blocker p21, which were followed by marked apoptotic cell death and suppression of cell proliferation specifically in cancer but not in the noncancer cells. Furthermore, a prominent loss of mitochondrial membrane potential after

Echinacoside treatment indicated activation of the intrinsic apoptosis pathway. Echinacoside-induced DNA damages and upregulation of p21 were observed within 5 hours of treatment, while cancer cell apoptosis, disruption of mitochondrial membrane potential, and growth inhibition were observed 12 hours after initiation of treatment. These data support that cancer cell apoptosis and growth inhibition were the results of the extensive DNA damages caused by the inhibition of MTH1. Recent studies have shown that a reduction in the size of the cellular dNTP pool could also induce DNA replication stress and DNA damages.59,60 However, 200% increase in 8-oxoG level in the Echinacoside-treated cancer cells argues against the possibility of a reduced dNTP pool size, and Bcl2, a protein that reduces dNTP pool size,60 was significantly decreased in Echinacoside-treated SW480 cancer cells;58 moreover, our data clearly showed that Echinacoside directly inhibited MTH1, which could at least be partially responsible for the DNA damages and cellular effects caused by Echinacoside.

The dNTP pool is a critical target of ROS, and oxidized dNTPs are important sources of oxidative DNA damages.3,10 Repair-associated DNA SSBs and DSBs can lead to cellular senescence and apoptosis, which are implicated in aging and a high degree of intratumor heterogeneity and high mutation rates in cancer cells. In contrast, inhibition ofMTH1 targets a phenotype that distinguishes most cancer cells from normal cells and hence represents a novel anticancer strategy that is not limited by genetic adaptations. Interestingly, small molecule antagonists of the antiapoptosis protein Bcl266,67 and agonists of the proapoptosis Bax68 have been developed and were demonstrated to be promising novel anticancer agents. Given their complementary mechanisms of action, combining MTH1 inhibitors and apoptosis-promoting chemicals would create an exciting new generation of anticancer drugs.

For the first time, we demonstrated a new function for Echinacoside as an anticancer natural compound. In the in vitro assay, Echinacoside inhibited MTH1 with an IC50 of7.01 μM. This IC50 value is higher than that of the MTH1 inhibitors reported so far.32,33,69 Using (S)-crizotinib as a positive control, we showed that our assay is sevenfold less sensitive than that used by Huber et al.33 Thus, the actual IC50 of Echinacoside is likely to be lower. Nevertheless, to develop it as a therapeutic agent, the efficacy of the natural Echinacoside molecule will probably need to be improved. As a natural product that has been used as a herbal medicine for a long time, Echinacoside could serve as a good chemical scaffold for developing efficient and probably safe MTH1 inhibitors.37,70 Given that natural products have been a rich source of novel chemical scaffolds for rational structure-based drug design,70 approaches similar to what we used here,

together with the vast interests in natural products for drug discovery, will be useful in finding target-based, effective, and safe new drugs.

echinacoside in cistanche (2)