TS extracts induce down-regulation of VEGF in HL-60 cells

A number of studies have shown that VEGF is one the most important angiogenic factor closely associated with neovascularization in human tumors. Western blotting and ELISA assay were used to analyze the effects of TS extracts on the expression and release of angiogenic-related protein VEGF in HL-60 cells. As shown in Fig. 4A, treatment of HL-60 cells with TS extracts dose-dependently inhibits the expression of VEGF. In addition, control cells (without treatment) released detectable levels of VEGF into the serum-free culture media at approximately 27 pg/10⁵ cells (Fig. 4B). A concomitant with protein level, TS extracts significantly inhibits VEGF release into culture media in a dose-dependent manner (Fig. 4B).

3.6. Effect of TS extracts on tumor growth in HL-60 xenograft nude mice.

2

Nude mice were used to evaluate the in vivo effect of TS extracts on tumor growth. HL-60 cells were xenograft into nude mice as described in materials and methods. All the animals appeared healthy with no loss of body weight noted during treatment with TS extracts (Fig. 5A). In addition, no signs of toxicity were observed (data not shown) in any of the nude mice. The time course for HL-60 xenograft growth with TS extracts (7.5 and 10.0 mg/kg) or without treatment (control) is shown in Fig. 5B. Evaluation of tumor volume showed significant growth inhibition associated with TS extracts treatment (Fig.

5B). At the end of 21 days, the HL-60 xenograft tumor of each mouse was excised from each sacrificed animal and weighed. Tumor weight in the TS extracts-treated (7.5 and 10.0 mg/kg) mice was inhibited as compared with the control group (Fig. 6A and 6B). In addition, abundant mitosis in nuclei was observed in xenograft tumor section, indicating the proliferating activity, with well differentiation of tumor cells were also noticed (Fig.

7A). While decreased mitotic figures shrunken tumor cells were noted in the 7.5 mg/kg TS extracts treated animals (Fig. 7B), and tumor cells became smaller and shrunken, indicating the regression of tumor cells, in the 10 mg/kg TS extracts treated animals (Fig.

7C). These in vivo data also strongly suggest that TS extracts exerted antitumor activity in HL-60 leukemia xenograft nude mice could be due to the modulation of cell-cycle regulation and/or induction of apoptosis.

3.7. Gallic acid causes G1 arrest and regulates cell-cycle regulatory proteins in HL-60 cells.

Previously we reported that treatment of the HL-60 cells with gallic acid (5-10 g/mL), purified from TS extracts, resulted in sequences of events marked by apoptosis in the HL-60 cells was accompanied by loss of cell viability, ROS generation, internucleosomal DNA fragmentation, cytochrome c release, activation of caspase-3, degradation of

2 present study also showed that TS extracts appreciably inhibits tumor progression through cell-cycle arrest at G1 phase. Therefore, further we intended to investigate the effect of gallic acid (5-10 g/mL), on cell-cycle control in HL-60 cells. The profile of the DNA content in gallic acid-treated HL-60 cells (5 μg/mL for 6-18 h) was obtained using flow cytometric analysis. Fig. 8A showed that gallic acid exposure resulted in progressive and sustained accumulation of cells in G1 phase. Furthermore, the percentage of G1 phase cells increased, while those in the S and G2/M phase decreased after treatment with gallic acid (Fig. 8A). Notably, there was a remarkable accumulation of sub-G1 peak in gallic acid-treated HL-60 cells (5 g/ml for 6-18 h) compared with the unacid-treated group (Fig. 8A). Our findings suggest that gallic acid also promotes cell growth inhibition by inducing G1 phase arrest in human leukemia cells.

In order to examine the molecular mechanism(s) and underlying changes in cell-cycle patterns caused by gallic acid treatment, we investigated the effects of various cyclins and CDKs involved in cell-cycle regulation in HL-60 cells. Cells were treated with gallic acid (5-10 μg/mL) for 0-6 h. Dose-dependent reductions of cyclin D1, CDK4, cyclin E, CDK2, and cyclin A, and induction of p27^KIP expression were observed (Fig. 8B). Notably, gallic acid treatment significantly inhibits the expression of cyclin D1, cyclin E, CDK2 and 4, which are critically required for G1-S transition phase. Therefore, we believed that the gallic acid-induced G1 cell-cycle arrest is mediated by the inhibition of cyclin D1, cyclin E, and CDK2 and 4. However, the experimental treatment did not appear to alter the amount of detectable cyclin B1, CDC2, and p15 protein levels, which was concomitant with TS extracts treatment (Fig. 8B).

4. Discussion

2

Differential regulation of the cell-cycle, and subsequent events leading to apoptotic cell death, account for the anticancer effect of some potential phytochemicals (Sporn and Suh, 2002). Several studies have demonstrated anticancer potential for extracts from a number of herbal medicines or mixtures in vitro or in vivo. Herbal medicine is one of the ancient forms of health care known to humankind and it has been used in most cultures throughout history. Typically, herbal medicines emphasize the use of whole extracts from a plant mix or from complex formulations (Sporn and Nuh, 2002). Our previous study has demonstrated that TS extracts induce apoptotic cell death in cultured human premyelocytic leukemia HL-60 cells (Yang et al., 2006a). The present investigation also a parallel study showing the effect of TS extracts an in vivo human tumor xenograft in nude mice as well as in vitro cell culture models involving HL-60 cells. Summary of our data suggests that TS extracts treatment could be effective in suppressing the proliferation of HL-60 cells as shown by growth inhibition, cell-cycle arrest, and apoptotic induction in vivo and in vitro.

Investigation has shown the nontoxic characteristics of T. sinensis [oral administration of T.

sinensis (1000 mg/kg/day) for 28 days in rats], which increases its potential for application in food and drug products (Liao et al., 2007). Furthermore, in vivo toxicity was also examined superficially from body weight changes and histological studies of vital organs (data not shown). There appeared to be no sign of significant toxicity at TS extracts exposures up to the concentration of 10 mg/kg. This likely indicates that there are no side effects at these doses. Future studies should test whether there is an optimal/effective dose for TS extracts exposure.

Disturbance of the cancer cell-cycle is one of the therapeutic targets in the development of new anticancer drugs. The results of cell-cycle analysis in the present study showed that TS extracts/gallic acid treatment had a profound effect on cell-cycle control, with the premyelocytic leukemia cells accumulating in G1 phase. Progression through the first gap

2 holoenzymes (Takahashi et al., 1999; Youn et al., 2008). The CDK catalytic subunits CDK 4 and CDK 2, and their regulatory subunits, cyclin D1 and cyclin E, are believed to be a crucial event in the regulation of S-phase entry, which appears to define the restriction point in the late G1 phase. Cyclin D expression is frequently deregulated in human neoplasias, and agents that can down-regulate cyclin D expression may be helpful in both their prevention and treatment (Sausville et al., 2000). Further, it has been found that cyclin E, which is one of the key cell-cycle regulators, is over-expressed in primary carcinoma tissue (Wang et al., 1994). Cyclin A is particularly interesting among the clyclin family because it can activate two different types of CDKs and function in both S-phase and mitosis. Cyclin A associated protein kinase activity is critically required for G1 to S-phase transition and further entry into M-S-phase (Johnson and Walker, 1999). The results imply that the expression of cyclin D1, CDK4, cyclin E, Cyclin A, and CDK2 are down-regulated by TS extracts, which corroborates the G1 block in HL-60 cells. It has been shown that impairment of a growth stimulation-signaling pathway induces the expression of CDK inhibitor, which binds to and subsequently inhibits cyclin-CDK activity (Sandal et al., 2002). Our results suggest that inducing p27^KIP expression via treatment with TS extracts/gallic acid may account for a large part of the reduction in CDK activity and, subsequently, block cell-cycle progression. Our study has also demonstrated that there were no significant differences in the expression of cyclin B1 and CDC2 after treatment with the TS extracts and gallic acid. The evidence suggests that the complex formed by the association of cyclin B1 and CDC2 plays a major role at entry into mitosis (Kuo et al., 2006). These results suggest that the observed inhibition of proliferation in HL-60 cells associated with the T. sinensis treatment could be the result of cell-cycle arrest during the G1 phase.

2

Investigations have shown that apoptosis is controlled by both mitochondrial and membrane death receptor pathways. The extrinsic pathway is initiated by the binding of transmembrane death receptors, including Fas, FasL, TNFR1, and TRAIL receptors with cognate extracellular ligands (Reed, 2000). Ligand receptors recruit adaptor proteins such as TRADD and FADD which interact with and trigger the activation of caspase-8.

Activated caspase-8 further cleaves or activates downstream effector caspases, such as caspase-3 (Reed, 2009).The present study indicates that TS extracts-induced apoptosis is associated with up-regulation of Fas and FasL, caspase-8 activation, and down-regulation of Bid in HL-60 cells. Our previous investigation has been demonstrated that treatment of HL-60 cells with TS extracts can induce apoptosis via a mitochondrial pathway that is associated with loss of cell viability, internucleosomal DNA fragmentation, cytochrome c translocation, caspase-3 activation, poly ADP-ribose polymerase (PARP) degradation, and Bcl-2 and Bax dysregulation (Yang et al., 2006a). However, the activation of caspase-9 by TS extracts was still in debate. Caspase-9 is a crucial factor for activation of caspase-3, which cleave several cellular targets including poly ADP ribose polymerase (Reed, 2009).

The current data filled the gape that TS extracts markedly activates caspace-9 from procaspase-9 followed by caspase-3 activation. Analysis of our data suggests that TS extracts-induced apoptosis is controlled by both a mitochondrial and a membrane DR pathway.

Our previous report demonstrated that catalase (H2O2 scavenger) significantly decreases TS extracts-induced cytotoxicity, DNA fragmentation, and ROS production in HL-60 cells (Yang et al., 2006a). The present investigation further confirmed that catalase significantly reduced TS extracts-induced cell-cycle arrest (cyclin D1, CDK4, cyclin E, CDK2, cyclin A, pRb, and p27^KIP) and apoptosis (Fas, FasL, caspase-8, Bid, and caspase-9) in HL-60 cells.

Analysis of our data suggesting TS extracts-induced HL-60 cell-cycle arrest and apoptosis

2 shown that gallic acid-induced intracellular ROS, especially H2O2, play an important role in eliciting an early signal of apoptosis (Sakaguchi et al., 1998; Inoue et al., 2000), and that catalase significantly reduces gallic acid-induced apoptotic cell death (Yang et al., 2006a;

Isuzugawa et al., 2001). In addition, recent studies appear to support the notion that TS extracts may possess protective antioxidant properties (Cho et al., 2003; Hsieh et al., 2004;

Hseu et al., 2008a). Several researchers have shown that antioxidants produce genetic changes that cause apoptosis in cancer cells by mechanisms other than antioxidant effect (Yang et al., 2006b). Thus, TS extracts might serve as a mediator for the reactive oxygen-scavenging system and potentially act as both a pro-oxidant and an antioxidant, depending on the redox state of the biological environment. However, the detailed mechanisms of the chemotherapeutic action of T. sinensis are unknown, and further investigations are needed.

Angiogenesis is tightly regulated by an intricate balance between stimulators and inhibitors. Among them, VEGF, a soluble angiogenic factor produced by many tumors as well as normal cell lines, plays a key role in regulating normal and pathologic angiogenesis (Tonini et al., 2003; Hseu et al., 2011b). A previous report clearly evidenced that increased angiogenesis in bone marrow region from patients with acute myeloid leukemia (Hussong et al., 2000). These observations also suggest that the increased anagiogeneis is critically mediated by VEGF expression, which play crucial role for the further onset of tumor progression. Therefore, the therapeutic strategies have been developed for acute myeloid leukemia also targets anti-angiogenic processes, with promising results, because of the critical dependence of tumor growth and metastasis on angiogenesis. It is noteworthy that TS extracts significantly down-regulates both VEGF expression and release in HL-60 cells. A similar pattern of results were found in our previous study that TS extracts potentially inhibits VEGF-induced angiogenesis in

2

vascular endothelial cells (Hseu et al., 2011a). Taken together, the inhibitory effect of TS extracts on VEGF activity or angiogenesis in lekemia or endothelial cells are strong evidence for development of anti-cancer/anti-angiogenic drug from this vital source.

Furthermore, tumor inhibition was observed after treatment with TS extracts in the nude mice xenograft model in this study. Both incidence and mean tumor volume and weight were significantly reduced by TS extracts. Experiments using animals and circulating blasts from leukemia patients have yielded evidence that apoptosis also occurs in response to chemotherapy in vivo. Human acute-leukemia cell lines (HL-60 cells) have proven particularly informative in study of chemotherapy-associated apoptotic proteolytic events (Hseu et al., 2004). Moreover, in this study the in vivo toxicity of TS extracts was also examined superficially from body weight change and histological study of vital organs (data not shown), with no apparent signs of significant negative effects at exposures of 7.5 and 10 mg/kg. Analysis of our data suggests that TS extracts exert anti-proliferative action and growth inhibition on HL-60 cells in vitro or in vivo.

Natural products, including plants, provide rich resources for anticancer drug discovery.

In our previous study, a number of compounds, including gallic acid, methyl gallate, ethyl gallate, kaempferol, kaempferol-3-O-β-D-glucoside, quercetin, quercitrin, quercetin-3-O-β-D-glucoside, and rutin, was isolated from the leaves of T. sinensis; identity of the compounds was determined by HPLC and based on the analogous information reported in the literature (Yang et al., 2006a; Hsu et al., 2003). The total phenolic content of the TS extract was estimated to be 130 ± 26 mg of gallic acid equivalent/g of plant extracts (Yang et al., 2006a). The yield of gallic acid, the natural phenolic component purified from TS extracts, was about 6%. Although it remains unclear which of the components of T.

sinensis are active compounds, gallic acid has received increased attention recently because of some interesting new findings regarding its biological activities (Chen et al.,

2

2009). Gallic acid is widely distributed in various plants and fruits, such as gallnuts, sumac, oak bark, green tea, apple peels, grapes, strawberries, pineapples, bananas, lemons, and in red and white wine (You et al., 2010). Even though the therapeutic utility of gallic acid in this regard is unknown, its common occurrence in fruits and food as well as its small molecular weight (170 Da) might be an advantage in terms of safety and dosing design.

Studies have demonstrated that gallic acid selectively induces cancer cell death by apoptosis; however, gallic acid shows no cytotoxicity against normal cells (Yang et al., 2006a). Other workers have shown that gallic acid causes inactivating phosphorylation of CDC25A/CDC25C-CDC2, leading to cell-cycle arrest, and apoptosis induction in human prostate carcinoma DU 145 cells (Agarwal et al., 2007). Raina et al (2008) revealed that gallic acid treatment remarkably decreased human prostate cancer cell xenografted tumor incidence in mice. Therefore, gallic acid may be a useful phytochemical for cancer chemoprevention (Surh, 2003). These results corroborate other studies which have implicated that gallic acid is the main constituent responsible for the antiproliferative activity (Chen et al., 2009). Moreover, in future we have planned to investigate antitumor effect of other bioactive compounds isolated from the aqueous leaf extracts of T. sinensis.

The results obtained in vitro and in vivo in this study imply that T. sinensis could act as a chemopreventive agent with respect to inhibition of the growth of human leukemia HL-60 cells through the induction of cell-cycle arrest and apoptosis. We also believed that the antitumor activity of T. sinensis may be the abundance of gallic acid. These data provide an important step that might help model the effects of T. sinensis for potential future studies with animal models and human patients, and thereby facilitate the development of nutraceutical products using this agent.

Conflict of interest statement

2

The authors have no conflict of interest to declare.

Acknowledgments

This work was supported by grants NSC-97-2320-B-039-042, NSC-99-2320-B-039-035-MY3, CMU95-037, and CMU95-333 from the National Science Council and China Medical University of Taiwan.

References

Agarwal, C., Veluri, R., Kaur, M., Chou, S.C., Thompson, J.A., Agarwal, R., 2007.

Fractionation of high molecular weight tannins in grape seed extract and identification of procyanidin B2-3,3'-di-O-gallate as a major active constituent causing growth inhibition and apoptotic death of DU145 human prostate carcinoma cells. Carcinogenesis. 28, 1478-1484.

Ahmad, N., Adham, V.M., Afaq, F., Feyes, D.K., Mukhtar, H., 2001. Resveratrol causes WAF-1/p21-imediated G1-phase arrest of cell cycle and induction of apoptosis in human epridermoid carcinoma A431 cells. Clin Cancer Res. 7, 1466-1473.

Bak, M.J., Jeong, J.H., Kang, H.S., Jin, K.S., Ok, S., Jeong, W.S., 2009. Cedrela sinensis leaves suppress oxidative stress and expression of iNOS and COX-2 via MAPK signaling pathways in RAW264.7 cells. J Food Sci Nutr. 14, 269-276.

Chang, H.C., Hung, W.C., Huang, M.S., Hsu, H.K., 2002. Extract from the leaves of Toona sinensis Roemor exerts potent antiproliferative effect on human lung cancer cells. Am J Chin Med. 30, 307-314.

Chang, H.L., Hsu, H.K., Su, J.H., Wang, P.H., Chung, Y.F., Chia, Y.C., Tsai, L.Y., Wu, Y.C., Yuan, S.S.F., 2006. The fractioned Toona sinensis leaf extract induced apoptosis of human ovarian cancer cells and inhibits tumor growth in a murine xenograft model.

2

Gynecol Oncol. 102, 309-314.

Chen, H.M., Wu, Y.C., Chia, Y.C., Chang, F.R., Hsu, H.K., Hsieh, Y.C., Chen, C.C., Yuan, S.S., 2009. Gallic acid, a major compound of Toona sinensis leaf extracts, contains a ROS-mediated anti-cancer activity in human prostate cancer cells. Cancer Lett.

286, 161-171.

Chia, Y.C., Rajbanshi, R., Calhoun, C., Chiu, R.H., 2010. Anti-neoplastic effect of gallic acid, a major component of Toona sinensis leaf extract, on oral squmous carcinoma cells. Molecules. 15, 8377-8389.

Cho, E.J., Yokozawa, T., Rhyu, D.Y., Kim, H.Y., Shibahara, N., Park, J.C., 2003. The inhibitory effects of 12 medicinal plants and their component compounds on lipid peroxidation. Am J Chin Med. 31, 907-917.

Collins, A., Yuan, L., Kiefer, T.L., Cheng, Q., Lai, L., Hill, S.M., 2003. Overexpression of the MT1 melatonin receptor in MCF-7 human breast cancer cells inhibits mammary tumor formation in nude mice. Cancer Lett. 189, 49-57.

Edmonds, J.M., Staniforth, M., 1998. Toona sinensis: Meliaceae. Curtis’s Bot Mag. 15, 186-193.

Eskes, R., Desagher, S., Antonsson, B., Martinou, J.C., 2000. Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol. 20, 929-935.

Hseu, Y.C., Chang, W.H., Chen, C.S., Liao, J.W., Huang, C.J., Lu, F.J., Chia, Y.C., Hsu, H.K., Wu, J.J., Yang, H.L., 2008a. Antioxidant activities of Toona Sinensis leaves extracts using different antioxidant models. Food Chem Toxicol. 46, 106-114.

Hseu, Y.C., Chen, S.C., Chen, H.C., Liao, J.W., Yang, H.L., 2008b. Antrodia camphorata inhibits proliferation of human breast cancer cells in vitro and in vivo. Food Chem Toxicol. 46, 2680-2688.

2 endothelial growth factor (VEGF)-induced angiogenesis in vascular endothelial cells. J Ethnopharmacol. 134, 111-121.

Hseu, Y.C., Chen, S.C., Tsai, P.C., Chen, C.S., Lu, F.J., Yang, H.L., 2007. Inhibition of cyclooxygenase-2 and induction of apoptosis in estrogen-nonresponsive breast cancer cells by Antrodia camphorata. Food Chem Toxicol. 45, 1107-1115.

Hseu, Y.C., Wu, C.R., Chang, H.W., Kumar, K.J.S., Lin, M.K., Chen, C.S., Cho, H.J., Huang, C.Y., Huang, C.Y., Lee H.Z., Hsieh, W.T., Chung, J.G., Wang, H.M., Yang, H.L., 2011b. Inhibitory effects of Physalis angulata on tumor metastasis and angiogenesis. J Ethnopharmacol. 135, 762-771.

Hseu, Y.C., Yang, H.L., Lai, Y.C., Lin, J.G., Chen, G.W., Chang, Y.H., 2004. Induction of apoptosis by Antrodia camphorata in human premyelocytic leukemia HL-60 cells.

Nutr Cancer. 48, 189-197.

Hsieh, T.J., Liu, T.Z., Chia, Y.C., Chern, C.L., Lu, F.J., Chuang, M.C., Mau, S.Y., Chen, S.H., Syu, Y.H., Chen, C.H., 2004. Protective effect of methyl gallate from Toona sinensis (Meliaceae) against hydrogen peroxide-induced oxidative stress and DNA damage in MDCK cells. Food Chem Toxicol. 42, 843-850.

Hsu, H.K., Yang, Y.C., Hwang. J.H., Hong, S.J., 2003. Effects of Toona sinensis leaf extract on lipolysis in differentiated 3T3-L1 adipocytes. Kaohsiung J Med Sci. 19.

3855-3890.

Hussong, J.W., Rodgers, G.M., Shami, P.J., 2000. Evidence increased aniogenesis in patients with acute myeloid leukemia. Blood. 95, 309-313.

Inoue, M., Sakaguchi, N., Isuzugawa, K., Tani, H., Ogihara, Y., 2000. Role of reactive oxygen species in gallic acid-induced apoptosis. Biol Pharm Bull. 23, 1153-1157.

2 sensitivity to the apoptosis inducer gallic acid. Biol Pharm Bull. 24, 1022-1026.

Jiang, S.H., Wang, C.L., Chen, Z.Q., Chen, M.H., Wang, Y.R., Liu, C.J., Zhou, Q.L., Li, Z.J., 2009. Antioxidant properties of the extract and subtractions from old leaves of Toona sinensis Roem (Meliaceae). J Food Biochem. 33, 425-441.

Johnson, D. G., Walker, C.L., 1999. Cyclins and cell cycle checkpoints. Ann Rev Pharmacol Toxicol. 39, 295-312.

Kim, J.J., Kim, H.P., Jung, C.H., Hong, M.H., Hong, M.C., Bae, H.S., Lee, S.D., Park, S.Y., Park, J.H., Ko, S.G., 2006. Inhibition of cell cycle progression via p27^kip1 upregulation and apoptosis induction by an ethanol extract of Rhus verniciflua stokes in AGS gastric cancer cells. Int J Mol Med. 18, 201-208.

Kuo, P.L., Hsu, Y.L., Cho, C.Y., 2006. Plumbagin induced G2-M arrest and autophagy by

Kuo, P.L., Hsu, Y.L., Cho, C.Y., 2006. Plumbagin induced G2-M arrest and autophagy by