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Journal of Chinese Integrative Medicine ›› 2011, Vol. 9 ›› Issue (12): 1353-1359.doi: 10.3736/jcim20111212

• Original Experimental Research • Previous Articles     Next Articles

Medicated serum prepared with Chinese herbal medicine Zhizhen Recipe down-regulates activity of nuclear factor-κB and expression of P-glycoprotein in human colorectal cancer multidrug-resistant cell line HCT-8/VCR

Song-bai Cai, Xu Zhang, Zhi-xia Chen, Ling-chun Kong, Song-po Wang()   

  1. Department of Traditional Chinese Medicine, Shanghai First People's Hospital, Shanghai Jiao Tong University, Shanghai 200080, China
  • Received:2011-08-15 Accepted:2011-09-05 Online:2011-12-20 Published:2011-12-15

Objective: To investigate the effects of medicated serum prepared with Chinese herbal medicine Zhizhen Recipe (ZZR) on activity of nuclear factor-κB (NF-κB) and expression and function of P-glycoprotein (P-gp) in human colorectal cancer multidrug-resistant cell line HCT-8/VCR.
Methods: The multidrug resistance of HCT-8/VCR cells was detected by cell counting kit-8 method, and the experimental concentrations of ZZR-medicated serum were determined by the same way. HCT-8 and HCT-8/VCR cells were treated with ZZR-medicated serum of medium dose for 24 h. The activity of NF-κB was determined by enzyme-linked immunosorbent assay. The intracellular distribution of P-gp was detected by laser scanning confocal microscopy, and the mean fluorescence intensity of rhodamine 123 was detected by flow cytometry.
Results: ZZR-medicated sera with volume fraction of 8%, 16% and 32% of medium dose were confirmed as the experimental sera. Compared with the untreated group, NF-κB activities of the ZZR-medicated serum groups (ZZR-medicated serum with volume fraction of 8%, 16% and 32% of medium dose) were obviously down-regulated (P<0.01), which had a negative correlation with the concentrations. After interfering HCT-8/VCR with ZZR-medicated serum of different concentrations for 24 h, P-gp in HCT-8/VCR transmitted gradually from cell membrane to cytoplasm and nuclei. Nuclei became pyknotic and cracking. Compared with the untreated group, the mean fluorescence intensities of ZZR-medicated serum groups declined with concentration gradients (P<0.01). The efflux of intracellular rhodamine 123 decreased, the wave crest shifted to right, and the intracellular fluorescence intensity strengthened (P<0.01).
Conclusion: ZZR-medicated sera of experimental concentrations down-regulate activity of NF-κB and expression and function of P-gp in human colorectal cancer multidrug-resistant cell line HCT-8/VCR and the effect is related to the concentrations.

Key words: colorectal neoplasms, P-glycoprotein, NF-kappa B, compound (traditional Chinese drugs)

Table 1

Sensitivity of HCT-8 and HCT-8/VCR cells to different chemotherapeutic drugs (x±s)"

Drug n IC50 (μg/mL)
HCT-8 HCT-8/VCR Resistance index of HCT-8/VCR (%)
VCR 3 0.14±0.02 9.55±0.23** 70.74
5-FU 3 3.19±0.34 65.71±0.26** 20.60
CDDP 3 0.74±0.04 4.13±0.16** 5.58
EP 3 0.80±0.07 3.79±0.12** 4.74

Table 2

Inhibition rates of HCT-8/VCR cell line treated with different concentrations of medicated serum"

Group n Inhibition rate at different concentrations of medicated serum
2% 4% 8% 16% 32% 64%
Blank serum 3 1.24±0.31 3.56±0.01 2.14±0.02 2.77±0.05 3.43±0.05 3.35±0.08
Low-dose medicated serum 3 0 7.42±0.03 12.91±1.68△△ 11.19±1.15△△ 10.59±2.07△△ 41.10±1.02△△
Medium-dose medicated serum 3 1.61±1.12 11.03±1.31 20.11±1.83△△ 23.70±1.61△△ 24.75±2.86△△ 43.89±3.28△△
High-dose medicated serum 3 1.43±0.07 9.45±0.04 16.24±1.04△△ 21.32±1.59△△ 30.29±3.46△△ 57.71±3.69△△

Table 3

NF-κB activity in HCT-8/VCR cell line treated with medicated serum for 24 h (x±s)"

Group n Absorbance value at 450 nm
HCT-8 3 3.534±0.120*
HCT-8/VCR 3 3.863±0.055
Blank serum 3 3.923±0.125
8% Medicated serum 3 3.457±0.091△△
16% Medicated serum 3 2.946±0.066△△
32% Medicated serum 3 2.868±0.079△△
BAY11-7082 3 2.730±0.042△△

Figure 1

Distribution of P-gp in HCT-8 and HCT-8/VCR cell lines treated with different concentrations of medicated serum (Laser scanning confocal microscopy, ×2 400) A: HCT-8; B: HCT-8/VCR; C: Blank serum; D: 8% Medicated serum; E: 16% Medicated serum; F: 32% Medicated serum; G: BAY11-7082. P-gp: P-glycoprotein."

Table 4

Mean fluorescence intensity of P-gp in cells treated with different concentrations of medicated serum"

Group n Mean flourence intensity
HCT-8 3 33.175±3.735
HCT-8/VCR 3 133.015±6.548
Blank serum 3 134.090±10.400
8% Medicated serum 3 43.148±3.236△△
16% Medicated serum 3 25.840±2.640△△
32% Medicated serum 3 23.448±3.302△△
BAY11-7082 3 8.625±4.406△△

Table 5

Efflux function of P-gp in cells treated with different concentrations of medicated serum"

Group n Mean flourence intensity
HCT-8 3 100.00
HCT-8/VCR 3 24.82±12.07
Blank serum 3 26.31±1.04
8% Medicated serum 3 186.76±40.42△△
16% Medicated serum 3 245.78±10.09△△
32% Medicated serum 3 282.31±77.53△△
BAY11-7082 3 321.69±20.36△△

Figure 2

Efflux function of P-gp in cells treated with different concentrations of medicated serum A: HCT-8; B: HCT-8/VCR; C: Blank serum; D: 8% Medicated serum; E: 16% Medicated serum; F: 32% Medicated serum; G: BAY11-7082. P-gp: P-glycoprotein."

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