In vitro and in vivo effects of crude extract of Acanthus ebracteatus Vahl. on human cervical carcinoma : role of P53 and VEGF

Abstract

The aim of this study was to examine the effects of aqueous crude extract of Acanthus ebracteatus Vahl. (AE) on tumor growth and tumor angiogenesis in human cervical cancer cells contained human papillomavirus (HPV)-16 DNA - implanted nude mice model. In vitro the growth inhibitory effect of AE was studied by using four different cell types including CaSki (HPV-16 positive), HeLa (HPV-18 positive) cells, Hepatocellular carcinoma cells, HepG2, and Human dermal fibroblast cells, HDFs. The cell growth-inhibitory activity and IC50 of AE were determined at different incubation time 24-, 48-, and 72-hours by using Trypan blue exclusion method and MTT assay. In vivo study Female BALB/c nude mice (weighing 20-25 g, age 5-6 weeks) were used for establishment of HPV16-positive cervical cancer mice model. CaSki cells, 1×107 cells/MEM 200µl were injected subcutaneously in the middle dorsum of each animal (HPV group). One week after, mice were fed orally with AE 300 or 3,000 mg/kg BW/day for 14 and 28 days (HPV-AE groups), distilled water were used as vehicle control. After 14- and 28-day of treatments, the tumor microvasculature and capillary vascularity (CV) were determined by using laser scanning confocal microscopic system. Tumor tissue sample of each mouse was collected for immunohistochemical examination of biomarkers, vascular endothelial growth factor (VEGF) and p53 proteins. Under in vitro study, the 3-hr incubation of AE (10-3-104 µg/ml) in three types of cancer cells, CaSki, HeLa and HepG2, and in one normal cell, HDFs, did not show the significant cell toxicity effects. However, AE exhibited the weak anti-proliferative effects on CaSki, Hela and HepG2 at 48-hr exposure time. In addition, AE could inhibit the growth of CaSki cells, significantly when compared to normal cells, HDFs (p<0.05). In vivo study, the increasing in tumor volume were observed during day 14th - 28th, and high dose treatment of AE (3,000 mg/ kg BW) could inhibit the increase of tumor volume (P<0.001). A large number of microvascular network around tumor area was observed in HPV-Veh groups on day 21st and 35th after cancer-cell inoculation. Tumor capillary vascularity (CV) in HPV-Veh was significantly increased when compared to Con-Veh (P<0.001). High dose treatment of AE significantly attenuated the increase of tumor angiogenesis on both 14- and 28-days of treatments (P<0.001). Tumor VEGF expression was significantly increased in HPV-Veh group, and % of VEGF expression in HPV-3,000AE both 14- and 28-days were significantly less than HPV-Veh and HPV-300AE groups. The elevation of p53 expression in HPV-AE treated groups was observed as AE-dose dependent manner (P < 0.001). Our novel findings demonstrated that aqueous crude extract of Acanthus ebracteatus Vahl. could inhibit tumor growth and tumor angiogenesis in cervical cancer mice model. Therefore, it indicated that Acanthus ebracteatus Vahl. might be developed as a new therapeutic substance for cervical cancer treatment in the near future.