Asiatic Acid Promotes p 21 WAF 1 / CIP 1 Protein Stability through Attenuation of NDR 1 / 2 Dependent Phosphorylation of p 21 WAF 1 / CIP 1 in HepG 2 Human Hepatoma Cells

More than 60% of currently used anti-tumor drugs are natural origin or derived from natural compounds (Cragg et al., 2005). Plant-derived compounds are important source of these nature derived anti-tumor agents. Nearly 3000 plant species have been reported for the treatment of cancer (Altinbas et al., 2012; Boreddy et al., 2013). Some plant based anticancer agents have been allowed to enter the clinical trial, such as paclitaxel, vincristine, vinblastine, topotecan and irinotecan (Nobili et al., 2009). Asiatic acid, a pentacyclic triterpene found in the tropical medicinal plant Centella asiatica, has antioxidant, antiinflammatory and neuroprotective properties (Huang et al., 2012; Guo et al., 2013). Some studies have reported the anti-tumor effect of asiatic acid. For example, asiatic acid was reported to enhance apoptosis and cell cycle arrest by promoting the activity of extracellular signalregulated kinase and p38 mitogen-activated protein kinase pathways in human breast cancer cells (Hsu et al., 2005). By generation of ROS, asiatic acid could induce apoptosis in human melanoma cells (Park et al., 2005; Xu


Introduction
More than 60% of currently used anti-tumor drugs are natural origin or derived from natural compounds (Cragg et al., 2005).Plant-derived compounds are important source of these nature derived anti-tumor agents.Nearly 3000 plant species have been reported for the treatment of cancer (Altinbas et al., 2012;Boreddy et al., 2013).Some plant based anticancer agents have been allowed to enter the clinical trial, such as paclitaxel, vincristine, vinblastine, topotecan and irinotecan (Nobili et al., 2009).Asiatic acid, a pentacyclic triterpene found in the tropical medicinal plant Centella asiatica, has antioxidant, antiinflammatory and neuroprotective properties (Huang et al., 2012;Guo et al., 2013).Some studies have reported the anti-tumor effect of asiatic acid.For example, asiatic acid was reported to enhance apoptosis and cell cycle arrest by promoting the activity of extracellular signalregulated kinase and p38 mitogen-activated protein kinase pathways in human breast cancer cells (Hsu et al., 2005).By generation of ROS, asiatic acid could induce apoptosis in human melanoma cells (Park et al., 2005 et al., 2012).Asiatic acid could disturb the endoplasmic reticulum and alterations in calcium homeostasis to induce cell death (Gurfinkel et al., 2006).Furthermore, asiatic acid has also been shown to inhibit the angiogenic effects of VEGF (Kavitha et al., 2011).
The cyclin-dependent kinase inhibitor 1A, p21 WAF1/CIP1 , is known as an important inhibitor of cell proliferation by its interactions with complexes of cyclins and cyclindependent kinases (CDK) (Xu et al., 2012;Kan et al., 2013).p21 WAF1/CIP1 is also involved in the regulation of apoptosis (Russo et al., 2013).It has been reported that apoptosis and cell cycle arrest induced by asiatic acid was associated with the increasing levels of p21 WAF1/CIP1 (Hsu et al., 2005), but the underlying molecular mechanism was still unclear.In this study, we investigated the molecular mechanisms that regulate the levels of p21 WAF1/CIP1 by asiatic acid.

Cell culture and treatment
Hepatocellular carcinoma G2 (HepG2) cells and 293T cells were obtained from the Institute of Cell and Biochemistry Research of Chinese Academy of Science.HepG2 cells and 293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37˚C, in a humidified atmosphere of 5% CO 2 , supplemented with penicillin (100 UI/ml) and streptomycin (100 μg/ml).

Viral Production and Infection
Lentiviral shRNA expression plasmids were purchased from Sigma.Two different shRNAs designed against p21 WAF1/CIP1 mRNA, and the sequences are listed as follows: 5'-CCGGAGAGGTTCCTAAGAGTGCTGGCTCGAGC AGCACTCTTAGGAACCTCTTTTTTG-3' (sh-p21-1), 5'-CCGGGAGCGATGGAACTTCGACTTTCTCGAGA AAG-TCGAAGTTCCATCGCTCTTTTTG-3' (sh-2).Stable cell lines were established by the infection of target cells with lentiviruses viruses.Lentiviral vectors coding for shRNA were transfected in 293T cells in combination with packing vectors to obtain viral particles used to infect HepG2 cells.Lentiviruses viruses were harvested at 48 h after the transfection and added to the HepG2 cells with 10 mg/ml polybrene for 24 h infection.Cells were then selected with 3 mg/ml puromycin for 48 h.

Immunoprecipitation
The transfected HepG2 cells were treated with asiatic acid for 12 h.Then the cells were lysed in RIPA buffer.We centrifuged cell lysates at 16000×g, 4˚C for 10 min and collected supernatants.Next, the collected supernatants were incubated with Anti-MYC Agarose for 3 h.The agaroses were washed with RIPA buffer then boiled in 2× loading buffer for 5 min.Finally, the samples were subjected to Western Blot and detected with different antibodies.

Western blot analysis
Cells were lysed for 20 min on ice in RIPA buffer with 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, 10 mM NaF, 2m M Na3VO4, and 5 mg/ml leupeptin.Then cell lysates were centrifuged at 16000×g for 10 min at 4 °C.Protein concentrations were estimated using protein-dye (Bio-Rad,).Samples were separated by SDS-PAGE and then blotted onto a PVDF membrane (Millipore).After blotted with primary antibodies overnight, peroxidase conjugated secondary antibodies were incubated with the membrane.Bound antibodies were detected by Chemiluminescent HRP Substrate (Millipore) and photographed by LAS-3000 luminescent image system (Fujifilm).

In vitro cell proliferation assay
Cell proliferation was examined by using a Cell Counting Kit-8 (Dojindo).Cells were plated and treated with DMSO vehicle control or asiatic acid (10 μM) in 96-well plates at 2,000 per well and cultured in growth medium.Every 24 h, CCK-8 (10 μL) was added to each well containing 100 μL of DMEM medium.Then the plate was incubated for 2 h at 37°C.Absorbance was measured at 450 nm using a microplate reader (Tecan).

Colony formation
HepG2 cells were digested to reconstitute the singlecell suspension and seeded into 6-well plate (200 cells per well).Cells were incubated (37°C, 5% CO 2 ) in DMEM (10% FBS) with DMSO vehicle control or asiatic acid (10 μM).Three weeks later, the colonies were fixed with 10% formalin for 15 min and stained with crystal violet for 20 min then washed with phosphate buffered solution (PBS).Colonies were counted after taking photographs.Colony formation efficiency was calculated as the number of colonies generated divided by total number of input cells.

Statistical analysis
All values are shown as mean ± SD.P values were calculated using student's two-tailed t test; P < 0.05 was considered significant.

Results
Asiatic acid increases the levels of p21 WAF1/CIP1 through enhancing the stability of p21 WAF1/CIP1 protein To confirm whether asiatic acid up-regulates the levels of p21 WAF1/CIP1 , HepG2 human hepatoma cells were treated with asiatic acid for 12 hours (0, 5 and 10 μM).As shown in Figure 1A, the levels of p21 WAF1/ CIP1 protein were up-regulated after the treatment with increasing concentrations of asiatic acid in HepG2 cells.We next examined the expression of p21 WAF1/CIP1 mRNA by quantitative RT-PCR.The results show that there were no significant changes in p21 WAF1/CIP1 mRNA under the treatment with different concentrations of asiatic acid (Figure 1B).P21 WAF1/CIP1 was proved to be degraded through the proteasome pathway (Lee et al., 2007), thus we suppose that the regulation of p21 WAF1/CIP1 protein levels by asiatic acid may be the results of stability regulation.

Knockdown of NDR1/2 kinase inhibits asiatic acidinduced regulation of p21 WAF1/CIP1 stability
In order to further investigate the function of asiatic acid in the regulation of p21 WAF1/CIP1 stability, dual NDR1/ NDR2 knockdown was performed in HepG2 cells before asiatic acid treatment.We found that asiatic acid could not regulate the stability of p21 WAF1/CIP1 protein after the knockdown of NDR1/2 in HepG2 cells (Figure 4A).Furthermore, the levels of MYC-tagged p21 WAF1/ CIP1 (ala-146) protein were also not changed by asiatic acid in MYC-tagged p21 WAF1/CIP1 (ala-146) transfected HepG2 cells (Figure 4B).These results further support a role for asiatic acid in the regulation of the stability of p21 WAF1/CIP1 through inhibiting NDR1/2 kinase dependent phosphorylation of p21 WAF1/CIP1 .

Asiatic acid regulates proliferative activity of HepG2 cells
P21 WAF1/CIP1 acts as a downstream effector of multiple tumor suppressor pathways for promoting anti-proliferative activities (Abbas et al., 2009).We performed proliferation assays (CCK8) to evaluate the cell proliferative activity of HepG2 cells with the treatment of asiatic acid.As shown in Figure 5A, asiatic acid significantly reduced the proliferative activity of HepG2 cells (p< 0.0001).Knockdown of p21 WAF1/CIP1 using Lentiviral-mediated shRNA significantly increased the proliferative activity of HepG2 cells (p< 0.003).Differently, asiatic acid did not significantly inhibit the proliferation of HepG2 cells after stable knockdown of p21 WAF1/CIP1 (p> 0.28) (Figure 5A).Furthermore, colony formation assay was also performed to assess the survival rate of HepG2 cells in the present of asiatic acid.Colony formation of HepG2 cells significantly reduced (p < 0.0002) with the treatment of asiatic acid.In p21 WAF1/CIP1 stable knockdown HepG2 cells, asiatic acid weakly reduced the colony formation (p >0.01).

Discussion
For thousands of years, natural products have been applied to treat and prevent the human cancers (Mondal et al, 2012).Importantly, nearly 100 plant-derived compounds are currently undergoing clinical trials (Harvey, 2008).Meanwhile, the molecular mechanisms and targets of some compounds are still unclear.Asiatic acid, a plant-derived triterpenoid compound, has antitumor effect in some human cancer cell lines (Huang et al., 2012;Guo et al., 2013).Asiatic acid was reported to inhibit cell growth and promote cell apoptosis through mitochondrial death cascade in colon cancer cells (Tang et al., 2007).Asiatic acid could also induce apoptosis in human melanoma cells by generation of ROS (Park et al., 2005;Xu et al., 2012).Down-regulation of the expression and secretion of VEGF by asiatic acid could inhibit the angiogenesis of endothelial cells (Kavitha et al., 2011).Moreover, asiatic acid could inhibit liver fibrosis by blocking TGF-beta/Smad signaling in vivo and in vitro (Tang et al., 2012).
Cyclin-dependent kinase inhibitor p21 WAF1/CIP1 is a potent cyclin-dependent kinase inhibitor.P21 WAF1/CIP1 binds to and inhibits the activity of cyclin-dependent kinases  doi.org/10.7314/APJCP.2014.15.2.963 Asiatic Acid Promotes p21 WAF1/CIP1 Protein Stability in HepG2 Human Hepatoma Cells complexes and thus functions as a regulator of cell cycle progression at G1 (Phalke et al., 2012).Recently, a study has found that apoptosis and cell cycle arrest induced by asiatic acid was associated with the increasing levels of p21 WAF1/CIP1 , but the underlying molecular mechanism was still unclear (Hsu et al., 2005).In the present study, we found that asiatic acid increased the levels of p21 WAF1/CIP1 protein but not the mRNA in HepG2 human hepatoma cells.This implies that asiatic acid may regulate the stability of p21 WAF1/CIP1 .
Several researchers have proved that phosphorylation of p21 WAF1/CIP1 enhanced the degradation of p21 WAF1/CIP1 protein through proteasome pathway.For example, ser-114 phosphorylation of p21 WAF1/CIP1 by glycogen synthase kinase 3β (GSK-3β) promotes its proteasomal degradation after UV Irradiation.Moreover, Human NDR1/NDR2 kinases were reported to phosphorylate p21 WAF1/CIP1 on ser-146 and regulate the stability of p21 WAF1/CIP1 (Cornils et al, 2011).We found that levels of phosphorylated p21 WAF1/ CIP1 protein (ser-146) decreased under the treatment of asiatic acid in HepG2 cells.Differently, phosphorylated p21 WAF1/CIP1 protein (ser-146) accumulated upon the treatment of MG-132 and this effect was not shown in the present of asiatic acid.These results imply that asiatic acid regulates the stability of p21 WAF1/CIP1 by inhibitory phosphorylation of p21 WAF1/CIP1 at ser-146.Our data also showed that phosphorylation of p21 WAF1/CIP1 (ser-114) was not responsible for asiatic acid-induced regulation of p21 WAF1/CIP1 stability by using a specific anti-phosphoserine antibody and immunoprecipitation assays with mutated p21 WAF1/CIP1 (ala-146).
We also found a significant decrease of NDR1/2 kinase expression in HepG2 cells with the treatment of asiatic acid.Dual NDR1/2 siRNA knockdown showed significantly reduced phosphorylation of p21 WAF1/CIP1 (ser-146) and increased levels of p21 WAF1/CIP1 .Further study showed that the levels of mutated p21 WAF1/CIP1 (ala-146) protein were not changed during the treatment of asiatic acid.Moreover, asiatic acid could not regulate the stability of p21 WAF1/CIP1 protein after the knockdown of NDR1/2 in HepG2 cells.These results further support a role for asiatic acid in the regulation of p21 WAF1/CIP1 stability by regulating the phosphorylation of p21 WAF1/CIP1 (ser-146).Importantly, proliferation assay and colony formation assay have shown that asiatic acid significantly inhibited the proliferative activity of HepG2 and stable knockdown of p21 WAF1/CIP1 inhibited this effect.In conclusion, our results reveal that asiatic acid enhances the stability of p21 WAF1/CIP1 via down-regulating the levels of NDR1/2 kinase that phosphorylates p21 WAF1/CIP1 at ser-146 and inhibits the proliferation of HepG2 cells.

Figure 1 .
Figure 1.Effects of Asiatic Acid on the Stability of p21 WAF1/CIP1 in HepG2 Cells.HepG2 cells were treated with asiatic acid for 12 hours with increased concentration.(A) The levels of p21 WAF1/CIP1 and β-actin protein were examined by Western Blot.(B) The levels of p21 WAF1/CIP1 mRNA were examined by real-time PCR

Figure 2 .
Figure 2. Asiatic Acid Inhibits the Phosphorylation of p21 WAF1/CIP1 on Ser-146.(A) HepG2 cells were treated with asiatic acid for 12 hours with increased concentration.The levels of phosphorylated p21 WAF1/CIP1 on ser-146 and β-actin protein were examined by Western Blot.(B) With the treatment of 10 µM MG-132, the levels of phosphorylated p21 WAF1/CIP1 on ser-146 were examined by Western Blot.(C) HepG2 cells were treated with MG-132 (10 µM) in combination with asiatic acid (10 µM), the levels of p21 WAF1/CIP1 were examined by Western Blot.(D) HepG2 cells were transfected with mutated p21 WAF1/CIP1 (ala-146)for 24 hours and were treated with asiatic acid.IP was performed with MYC-beads followed by Western Blot.The intensity of phosphoserine of p21 WAF1/CIP1 was quantified by densitometry and normalized with equal levels MYC-p21 WAF1/CIP1 (ala-146)