Antitumor Effect of Water Decoctions of Taxus Cuspidate on Pancreatic Cancer

Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2014, Article ID 291675, 11 pages http://dx.doi.org/10.1155/2014/291675 Research Article Antitumor Effect of Water Decoctions of Taxus Cuspidate on Pancreatic Cancer Chao Qu1,2 and Zhen Chen1,2 1 2 Department of Integrative Oncology, Fudan University Shanghai Cancer Center, Shanghai 200032, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China Correspondence should be addressed to Zhen Chen; Received 15 December 2013; Accepted 5 January 2014; Published 25 February 2014 Academic Editor: Yoshiharu Motoo Copyright © 2014 C. Qu and Z. Chen. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Taxus cuspidate has been used as a traditional Chinese medicinal herb and considered to affect various physiological functions in the body for thousands of years. As we know that taxol isolated from the Taxus cuspidate has been approved for the treatment of ovarian cancer, it has also shown its antitumor abilities against other kinds of cancers. But the antitumor activity of other components which are free of paclitaxel and hydrophilic paclitaxel derivatives from Taxus cuspidate has not been fully understood. In this study, we investigated the effect of the water decoctions from the leaves of Taxus cuspidate on pancreatic cancer cell proliferation and the potential mechanism(s); though its antitumor activity and mechanism in vitro remain to be elucidated, the water soluble constituents from Taxus cuspidate could be used in clinical for cancer patients. 1. Introduction In traditional medicinal history, herbal medicines, which have been used to treat different medical conditions, is now considered a promising choice for treating cancers [1]. Meanwhile, water decoctions from leaves of Taxus cuspidate have been used for the treatment of human pancreatic cancers for many years in the cancer hospital, Fudan University, Shanghai, China. Recently, much efforts have been put on discovering pharmacological effects of TCM on tumor cells, and some underlying mechanisms have been revealed, such as inhibition of tumor invasion and induction of cell apoptosis [2]. Taxus, a genus of yews, are small coniferous trees or shrubs in the yew family Taxaceae and the natural source of Taxol which is a new antitumor drug, to treat patients with lung, ovarian, breast, head, and neck cancers [3]. Following approval by the US Federal Drug Administration (FDA), paclitaxel (Taxol) has been established as a standard drug for cancer chemotherapy [4, 5], and it has also exhibited its antitumor effect in many clinical and nonclinical trials to treat pancreatic cancer [6, 7]. Inducing a G2/M cell cycle arrest by disrupting disassembly of microtubules is the primary mechanism of Taxol on tumor progression [8]. Following the first observation of the antitumor activities in vivo and in vitro of the water extract [9], researches have showed that aqueous extract of Taxus chinensis could significantly inhibit the proliferation of A549 cells and induce the apoptosis by regulating the expressions of surviving [10]. Polysaccharides contents dissolved in water from Taxus yunnanensis displayed mild cytotoxicity against cancer cells in a concentration-dependent manner [11]. Unlike the clear antitumor mechanism of Taxol, the antitumor mechanism of components which are free of paclitaxel from Taxus cuspidate is still on the way to be uncovered. Here, the water decoction of Taxus cuspidate was prepared, its antiproliferation effect was determined, and the potential mechanisms were explored in human pancreatic cancer. 2. Materials and Methods 2.1. Preparation of Water Decoctions from Taxus Cuspidate. Air-dried leaves of Taxus cuspidate were supplied by the Company of Ningbo Taikang yew biological engineering Co, Ltd. The final decoction of the Taxus cuspidate was prepared at the Department of Pharmacy, Fudan University Shanghai Cancer Center, Shanghai, China, by boiling them with distilled water to the required concentration, the daily 2 Evidence-Based Complementary and Alternative Medicine dosage as described previously [12]. Drug administration was started from 1 week after the injection of tumor cells and continued until the end of the experiment. Body weights were recorded once per week. Mice of the treatment groups were orally administered water decoction (5 g/kg, 10 g/kg, and 20 g/kg) with 0.2 mL each time. Mice of the control group were administered normal saline (10 mice in each group). Treatment point was continued for 4 weeks, and then mice were sacrificed and tumors were removed and weighed. Tissues that would be used for molecular biological analysis were preserved in neutral-buffered formalin at 4∘ C before embedding in paraffin. into pieces with a blade. The residual pieces were grinded into single-cell suspension through a 200 hole mesh screen, the single-cell suspension was centrifuged in 1000 r/min for 5 minutes, and modulated into 106 /mL. Cells were fixed with 1% paraformaldehyde in PBS (phosphate-buffered saline) for 15 minutes and refixed with 70% ethanol. The cells were then treated following the standardized protocol, and cell cycle analyses were done by flow cytometry. 2.2. Cell Lines and Animals. Human pancreatic cancer cell line capan1 was obtained from the American Type Culture Collection and cultured in DMEM supplemented with 10% FBS in a humidified incubator containing 5% CO2 atmosphere at 37∘ C. Female BALB/c-nu/nu nude mice, 4 to 6 weeks sold, were obtained from the Laboratory Animal Center, Shanghai University of Traditional Chinese Medicine, and housed in laminar flow cabinets under specific pathogenfree conditions and provided with food and water ad libitum. 2.3. Mouse Model of Pancreatic Cancer In Vivo. 40 female BALB/c-nu/nu nude mice weighing 18–20 g were used; briefly, 2 × 106 capan1 pancreatic cancer cells per animal in 200 ul of phosphate-buffered saline were subcutaneously injected into the flank of each mouse. The mice are randomly divided into 4 groups, each experimental group contains 10 mice. Length and width of tumors (in millimeters) were measured twice a week with calipers. Tumor volumes were calculated by the formula (𝑎 × 𝑏2 ) × 0.5, where 𝑎 and 𝑏 are the long and short dimensions, respectively. After four-week observation, the mice were sacrificed, tumors were resected, and tumor weights were measured. 2.4. H and E Staining and Immunohistochemistry. For H and E staining, paraffin-embedded sample slides were deparaffinized, hydrated, and then stained with hematoxylin for 1 min. After rinsing, the slides were stained with eosin for 1 min, rinsed, and sealed with cover slips using Permount. Immunohistochemistry (IHC) was performed as described previously [13]. Briefly, specimens (...truncated)