In Vitro and In Vivo Inhibitory Effect of the Combination of Wenxia Changfu Formula(温下肠腑方) with Cisplatin in Non-Small Cell Lung Cancer

Objective： To observe the effect of the combination of Wenxia Changfu Formula （温下肠腑方, WCF） with cisplatin （CDDP） on inhibiting non-small cell lung cancer （NSCLC） in vitro and in vivo and explore its mechanism from its effect on cell cycle. Methods： In vitro, WCF-containing serum was prepared and the rhubarb bl, emodin, and aconitine were detected qualitatively by high-performance liquid chromatogram （HPLC）. A549 cell lines were treated with blank control （dimethyl sulfoxide）, normal serum, normal serum with CDDP （1.25, 2.5, and 5.0 μg/mL, respectively）, WCF-containing serum plus different doses of CDDP （1.25, 2.5, and 5.0 μ g/mL, respectively）. The inhibitory effect was detected by 3-（4,5）-dimethylthiazo（-z- yl）-3,5-diphenylterazolium bromide （MI-I）. The cell cycle was detected by flow cytometry. The protein and mRNA expressions of cyciin D1, proliferating cell nuclear antigen （PCNA）, retinoblastoma （Rb）, and p16 were observed with immunofluorescence and RT-PCR, respectively. In vivo, nude mice xenograft model was established and grouped into the control, CDDP, WCF, and combination groups. The combination＇s inhibition of tumor growth and influence on the weight, spleen, and thymus gland were observed. Results： The inhibitory rate of the combination against A549 cell lines excelled the CDDP alone significantly （P〈0.05）; the combination showed a synergism inhibitory effect （Q=1.19）. Compared with the monotherapy, the combination increased the cell percentage in G0/G1 phase and decreased the cell percentage in S phase significantly （P〈0.05）; the protein and mRNA expressions of cyclin D1, PCNA, and Rb were significantly reduced; the protein and mRNA expressions of p16 were significantly enhanced. Compared with the monotherapy, the combination inhibited the tumor growth significantly in vivo and reduced the weight of tumor （P〈0.05）; compared with the CDDP group, the spleen and thymus gland index of the combination group were enhanced significantly （P〈0.05）. Conclusions： The combination of WCF with CDDP significantly inhibited the A549 cell lines proliferation in vitro and the growth of the tumor in vivo; it inhibited effectively the atrophy of the immune organ caused by chemotherapy. The combination inhibited overproliferation of A549 cell lines by arresting the Go/G1 phase of cell cycle and affecting the protein and mRNA expressions of cell cycle-related proteins, cyclin D1, etc.