title: In vitro and in vivo anticancer effects of Lithospermum erythrorhizon extract on B16F10 murine melanoma.
abstract: BACKGROUND
  Lithospermum erythrorhizon has long been used in traditional Asian medicine for the treatment of diseases including skin cancer. In this study, hexane extract from the roots of Lithospermum erythrorhizon (LEH) was chemically characterized and its anticancer activity was tested against the most aggressive form of skin cancer.
  
  
  METHODS
  The in vitro anticancer studies viz. cell growth, cell cycle and apoptosis, and the expression of tumor regulating proteins were analyzed against B16F10 melanoma cells. In addition, C57BL/6 mice models were used to evaluate the in vivo anticancer potential of LEH. Mice were intraperitoneally injected with LEH at doses of 0.1 and 10mg/kg every 3 days. The tumor inhibition ratio was determined after 21 days of treatment and the histopathological analyses of the tumor tissues were compared. Further, LEH was purified and its active compounds were structurally elucidated and identified by NMR spectra and quantified by HPLC analyses.
  
  
  RESULTS
  LEH effectively inhibits the growth of melanoma cells with an IC(50) of 2.73μg/ml. Cell cycle analysis revealed that LEH increased the percentage of cells in sub-G1 phase by dose dependent manner. LEH exhibited down regulation of anti-apoptotic Bcl-2 family proteins and up regulation of apoptotic Bax protein expression. Importantly, LEH induced cleavage of poly (ADP-ribose) polymerase (PARP) and activated the caspase cascade (caspase 3) with this cleavage mediating the apoptosis of B16F10 cells. LEH treatment at a dose of 10mg/kg for 21 days in experimental mice implanted with tumors resulted in significant reduction of the tumor growth (43%) and weight (36%). Histopathology analysis of LEH treated tumor tissues showed evidence of increased necrotic cells in a concentration dependent manner. Meanwhile, five naphthoquinone compounds [Shikonin (1); Deoxyshikonin (2); β-Hydroxyisovalerylshikonin (3); Acetylshikonin (4) and Isobutyrylshikonin (5)] were purified from LEH and responsible for its anticancer activity.
  
  
  CONCLUSIONS
  LEH induced apoptosis in B16F10 cells by activation of caspase 3 and inducing sub-G1 cell cycle arrest. LEH exhibited both in vitro and in vivo anticancer activity. Shikonin derivatives in the LEH are responsible for the anticancer activity.
authors.affiliation: Department of Horticultural Bioscience, Pusan National University, Miryang, Republic of Korea.
authors.forename: Seetharaman
authors.initials: S
authors.lastname: Rajasekar
authors.forename: Da Jung
authors.initials: DJ
authors.lastname: Park
authors.forename: Cheol
authors.initials: C
authors.lastname: Park
authors.forename: Sejin
authors.initials: S
authors.lastname: Park
authors.forename: Young Hoon
authors.initials: YH
authors.lastname: Park
authors.forename: Sun Tae
authors.initials: ST
authors.lastname: Kim
authors.forename: Yung Hyun
authors.initials: YH
authors.lastname: Choi
authors.forename: Young Whan
authors.initials: YW
authors.lastname: Choi
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D005260:Female; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D029684:Lithospermum; D008546:Melanoma, Experimental / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D009285:Naphthoquinones / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use*; D008517:Phytotherapy; D010936:Plant Extracts / Q000494:pharmacology / Q000627:therapeutic use*; D018517:Plant Roots; D047368:Tumor Burden / Q000187:drug effects
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D009285:Naphthoquinones; D010936:Plant Extracts; C016101:shikonin; D020169:Caspases