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Journal of Chinese Integrative Medicine ›› 2011, Vol. 9 ›› Issue (4): 414-422.doi: 10.3736/jcim20110411

• Original Experimental Research • Previous Articles     Next Articles

Inhibitory effects of traditional Chinese herbal medicine Tanreqing injection on proliferation of leukemia cells in vitro and the potential mechanisms

Bo Yang1, Xue-chun Lu1, Feng Zhang2, Yang Liu1, Hong-li Zhu1()   

  1. 1. Department of Geriatric Hematology, Chinese PLA General Hospital, Beijing 100853, China
    2. Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China
  • Received:2010-09-28 Accepted:2010-12-28 Online:2011-04-20 Published:2011-04-15
  • Contact: Zhu Hong-li E-mail:bjzhl202_cn@sina.com

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Objective: The present study investigates the effects of Tanreqing injection, a compound Chinese herbal medicine, on the proliferation of leukemia cells in vitro and discusses the potential mechanisms.
Methods: Tanreqing injection was diluted to a series of concentrations (1∶2, 1∶4, 1∶8, 1∶16, 1∶32, 1∶64, 1∶128, 1∶256 and 1∶512) by volume and then independently applied to treat chronic myeloid leukemia K562 cells and T cell acute lymphocytic leukemia Molt4 cells at the proliferative stage. Cell growth was observed at different time intervals under a microscope. Cell proliferation was determined by cell counting kit-8 assay and the survival curve was delineated. The inhibitory rate and the half inhibitory concentration (IC50) were calculated. Molt4 cells were stained with propidium iodide (PI) and PI/Annexin V and then the cell cycle and apoptosis were analyzed by using flow cytometry. In addition, a real-time quantitative polymerase chain reaction was subjected to detect the expressions of apoptosis-related genes (bcl-2 and caspase-3) after Tanreqing treatment.
Results: Tanreqing injection had inhibitory effects on the proliferation of K562 cells and Molt4 cells. The most toxic concentrations were observed between 1∶2 and 1∶16 where cells were almost necrotic. The inhibitory effect manifested in a concentration- and time-dependent manner. The IC50 of K562 and Molt4 was 1∶333 and 1∶142, respectively. After 1∶32 Tanreqing injection treatment for 72 h, the number of Molt4 cells in the S phase significantly decreased (P<0.05), and the apoptosis rate markedly increased (P<0.05). In addition, increased caspase-3 expression and decreased bcl-2 expression were also observed (P<0.05).
Conclusion: Tanreqing injection can both inhibit the proliferation and promote the apoptosis of leukemia cells in vitro, whereby the potential mechanism seems to be mediated in part by decreasing S phase ratio, down-regulating bcl-2 expression and up-regulating caspase-3 expression.

Key words: Tanreqing injection, leukemia, K562 cells, Molt4 cells, cell proliferation, apoptosis

Figure 1

Growth curve of K562 cells after treatment with Tanreqing injection of different concentrations"

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Figure 2

Growth curve of Molt4 cells after treatment with Tanreqing injection of different concentrations"

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Figure 3

Microscopic observation of K562 cell growth after treatment with Tanreqing injection of different concentrations for 24 h (×100) At the concentrations of 1∶2 (B), 1∶4 (C), 1∶8 (D), and 1∶16 (E), cell death was obvious. At the concentrations of 1∶32 (F) and 1∶64 (G), cell growth was unfavorable. At the concentrations of 1∶128 (H), 1∶256 (I) and 1∶512 (J), cell growth was not significantly different from that in the control group (A)."

Figure 4

Microscopic observation of Molt4 cell growth after treatment with Tanreqing injection of different concentrations for 48 h (×100) At the concentrations of 1∶2 (B), 1∶4 (C), 1∶8 (D), and 1∶16 (E), cell death was obvious. At the concentrations of 1∶32 (F) and 1∶64 (G), cell growth was unfavorable. At the concentrations of 1∶128 (H), 1∶256 (I) and 1∶512 (J), cell growth was not significantly different from that in the control group (A)."

Figure 5

Cell cycle distribution of Molt4 cells treated with Tanreqing injection of different concentrations for 72h A: Control; B: 1∶512; C: 1∶256; D: 1∶128; E: 1∶64; F: 1∶32. “A” in the figures D, E and F represents apoptotic cells. The results show that cells in S phase gradually decreased and apoptotic cells appeared along with gradually increased concentration of Tanreqing injection."

Figure 6

Proportion of Molt4 cells in the S phase after treatment with Tanreqing injection of different concentrations for 72 h *P<0.05, vs control group."

Figure 7

Apoptotic Molt4 cells after treatment with Tanreqing injection of different concentrations for 72 h detected by flow cytometry after PI/Annexin V staining A: Control; B: 1∶512; C: 1∶256; D: 1∶128; E: 1∶64; F: 1∶32. The upper right quadrant represents cells positive for both Annexin V and PI; the lower right quadrant represents cells positive for Annexin V but negative for PI. PI-A: propidium iodide/Annexin V; FITC-A: fluorescein isothiocyanate-Annexin V."

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