Comprehensive and Holistic Analysis of HT-29 Colorectal Cancer Cells and Tumor-Bearing Nude Mouse Model: Interactions Among Fractions Derived From the Chinese Medicine Formula Tian Xian Liquid in Effects on Human Colorectal Carcinoma

Abstract

The Chinese medicine formula Tian Xian Liquid (TXL) has been used clinically for cancer therapy in China for more than 25 years. However, the comprehensive and holistic effects of its bioactive fractions for various antitumor therapeutic effects have not been unraveled. This is the first study to scientifically elucidate the holistic effect of Chinese medicine formula for treating colon cancer, hence allowing a better understanding of the essence of Chinese medicine formula, through the comparison of the actions of TXL and its functional constituent fractions, including ethyl acetate (EA), butanol (BU), and aqueous (WA) fractions. Tissue-specific proliferative/antiproliferative effects of these fractions on human colorectal carcinoma HT-29 cells and splenocytes were studied by using the MTT assay. Their modulations on the expression of markers of antiproliferation, antimetastasis, reversion of multidrug resistance in treated HT-29 cells were examined with real-time polymerase chain reaction and Western blot analysis, and their modulations in a xenografted nude mouse model were examined by Western blot analysis. Results revealed that EA fraction slightly inhibited the proliferation of HT-29 cells, but tissue-specifically exerted the most potent antiproliferative effect on splenocytes. On the contrary, only TXL and BU fraction tissue-specifically contributed to the proliferation of splenocytes, but inhibited the proliferation of HT-29 cells. WA fraction exerted the most potent antiproliferative effect on HT-29 cells and also the strongest inhibitory action on tumor size in the nude mouse model in our previous study. In the HT-29 model, TXL and WA fraction exerted the most pronounced effect on upregulation of p21 mRNA and protein; TXL, and EA and WA fractions exerted the effect on downregulation of G1 phase cell cycle protein, cyclin D1 mRNA and protein; EA and BU fractions exerted the most prominent anti-invasive effect on anti-invasion via downregulation of MMP-1 mRNA; TXL potently reversed most multidrug resistance via downregulation of MDR-1 protein. In conclusion, the comprehensive and holistic effects of TXL were demonstrated with ( a) mutual accentuation and mutual enhancement, ( b) mutual counteraction and mutual suppression, and ( c) mutual antagonism among the 3 constituent fractions. Moreover, the design of the present study may lead to further development of more tissue-specific effective drugs with minimal side effects for clinical use in combating carcinoma.

Keywords: Chinese medicinal formula; Tian Xian Liquid; antitumorigenicity; colon cancer; constituent fractions; holistic.

Figure 1.

Overlaid high-performance liquid chromatography (HPLC) fingerprints of different batches of (A) Tian Xian Liquid (TXL), (B) ethyl acetate fraction of TXL (EA), (C) butanol fraction of TXL (BU), and (D) aqueous fraction of TXL (WA) sample extracts monitored at 275 nm. Four peaks (A-D) were denoted on the chromatograms.

Figure 2.

Proliferative Assay of Tian Xian Liquid (TXL) and its constituent fractions on HT-29 cells at different concentrations after 24 hours of treatment as revealed by the MTT assay. Results are presented in mean ± SEM (n = 3). Student’s t test was performed to compare the antiproliferative effect of each dosage with individual control (%). EA, ethyl acetate fraction of TXL; BU, butanol fraction of TXL; WA, aqueous fraction of TXL. In the TXL-treated cells, corresponding dosage versus control (0%): *P < .05; **P < .01; ***P < .001. In the BU-treated cells, corresponding dosage versus control (0%): ^#P < .05; ^##P < .01; ^###P < .001. In the EA-treated cells, corresponding dosage versus control (0%): ^@P < .05; ^@@P < .01; ^@@@P < .001. In the WA-treated cells, corresponding dosage versus control (0%): ⁺P < .05; ⁺⁺P < .01; ⁺⁺⁺P < .001.

Figure 3.

Effect of Tian Xian Liquid (TXL) and its constituent fractions on proliferation of mouse splenocytes at different concentrations after 24 hours of treatment as revealed by the MTT assay. Results are presented in mean ± SEM (n = 3). Student’s t test was performed to compare the proliferative effect of each dosage with individual control (0%). EA, ethyl acetate fraction of TXL; BU, butanol fraction of TXL; WA, aqueous fraction of TXL. In each type of treated cells, corresponding dosage vs control (0%): *P < .05; **P < .01; ***P < .001.

Figure 4.

mRNA expression of p21 (A), cyclin D1 (B), and MMP-1 (C) in treated HT-29 cells at different time points. The quantification is relative to that of control at 0 hours. The results are expressed as mean fold difference ± standard error of the mean. Comparison with control 0 hours was done by one sample t test. Comparison of treated cells with the control of corresponding time point was done by unpaired t test. CTL, control group. CTL of corresponding time point vs treated cells: *P < .05; **P < .01; ***P < .001.

Figure 5.

Protein expression of p21, cyclin D1, and MMP-1 in treated HT-29 cells at different time points. The results are expressed as mean relative intensity ± standard error of the mean (n = 3). Comparison was done by using unpaired t test. CTL, control group. CTL 0 hours versus treated cells: ^#P < .05; ^##P < .01; ^###P < .001. CTL of corresponding time point versus treated cells: *P < .05; **P < .01; ***P < .001.

Figure 6.

Protein expression of p21, MMP-1, and MDR-1 in tumor excised from nude mouse model (n ≥ 3). The results are expressed as mean relative intensity ± standard error of the mean. Comparison was done by unpaired t test. CTL, control group. CTL versus treated group: *P < .05; **P < .01; ***P < .001.