| Objective: Qilan preparation, a complex Chinese herbal medicine consisting of ingredients extracted from Astragali Radix(AR; Huang Qi in Chinese; roots of Astragalus membranaceus(Fisch.) Bunge), Gynostemma Pentaphyllum(Thunb.) Makino(G. pentaphyllum; Jiao Gu Lan in Chinese; a perennial liana herb belonging to the Cucurbitaceae), Chuanxiong Rhizoma(CR; Chuan Xiong in Chinese; rhizomes of Ligusticum chuanxiong Hort) and selenium-rich green tea(leaves of Camellia sinensis(L.) Kuntze) and known for ‘fortifying the spleen and boosting qi, quickening the blood and transforming stasis, and resolving toxins and relieving pain’, is used for the prevention and management of oral diseases. The aim of this study was to examine the antitumor effects relative to proliferation, cell cycle distribution and apoptosis of Qilan preparation on Human Tca8113 tongue squamous cell carcinoma(TSCC) cells in vitro and to explore its underlying mechanisms of actions with micro RNA-21 and its target protein PTEN or PDCD4.Methods: Human Tca8113 cells were tested. Cell proliferation, cell cycle distribution and apoptosis were examined using cell counting kit-8(CCK8) and flow cytometry(FCM) after treatment with Qilan preparation for 12 h, 24 h or 48 h. The expression of PTEN and PDCD4 were determined by Western blot. Changes in mi R-21 levels were quantified using Taq Man stem-loop real-time PCR. After mi R-21 was transiently transfected into Tca8113 cells using Lipofectamine®3000, cell proliferation, cell cycle distribution, apoptosis and mi R-21 and PDCD4 expression levels were measured.Result:1. Cell viability, determined by the CCK-8 assay, was significantly reduced at 1.56, 3.12, 6.25, 12.5 and 25 mg/m L Qilan preparation(P<0.05) after treatment for 12, 24 and 48 h compared with the untreated control group in a dose-dependent manner.The IC50 were 8.800 mg/m L, 6.131 mg/m L and 3.628 mg/m L for 12, 24 and 48 h, said that Qilan preparation also exerted an inhibitory effect on Tca8113 cells in a time-dependent manner. 2. When cancer cells were treated with different concentrations of Qilan preparation for 12 h or 24 h, respectively, the number of cells in S-phase steadily increased compared with the untreated control cells(P<0.05), the number of cells in G0/G1-phase significantly decreased(P<0.05), but the percentage in G2/M-phase had non-significantly changed(P>0.05). 3. The amount of apoptotic cells was significantly increased from 15.04±1.67% to 69.74±4.63% when treated with concentrations of the Qilan preparation ranging from 3.12 to 25 mg/m L for 24 h compared with the control group(4.71±1.65%, P<0.05). 4. Qilan preparation treatment exerted a dose-dependent down-regulation of mi R-21 at concentrations ranging from 3.12 to 25mg/m L compared with the control group(P<0.05) on Tca8113 cells. 5. Peak stimulation of PDCD4(2.48±0.12-fold) and PTEN(1.51±0.06-fold) occurred at 6.25mg/m L and 12.5mg/m L, respectively, with no further increases observed at the higher concentrations. There were still significantly increasing PDCD4 and PTEN levels at the higher concentrations compared with the control group(P<0.05). 6. Setting up a series of levels between Lipofectamine®3000 and FAM-NC RNA, FCM tested the number of cells with FAM-green fluorescence and the Median of fluorescence. The results showed that transfection efficiency at(Lipofectamine®3000: FAM-NC RNA = 4.5μL, 60 n M) was the highest. 7. We over-expressed mi R-21 by approximately 14-fold compared to the NC RNA group and 30-fold compared to the NC RNA + Qilan preparation group with a mi R-21 transfection mimic for 24 h before treatment with the Qilan preparation. Based on the results from the earlier experiments, we used 6.25mg/m L as the effective concentration for the reverse test. Determined by the CCK-8 assay and FCM, over-expression of mi R-21 partially reversed the anti-proliferative and pro-apoptosiseffects of the Qilan preparation. Compared with the NC RNA+ Qilan preparation group(55.75±3.63%), the cell viability of mi R-21+Qilan preparation group was increased to 71.08±8.72%, but was still lower than the NC RNA group(97.83±5.63%, P<0.05). The percentage of cells undergoing apoptosis in the mi R-21 + Qilan preparation group decreased to 13.27±0.44% compared with the NC RNA + Qilan preparation group(22.72±1.52%), but was still higher than the NC group(P<0.05). Similar to these results, the expression level of the PDCD4 protein in the mi R-21 + Qilan preparation group was between that of the NC RNA group and the NC RNA + Qilan preparation group(P<0.05). In contrast, over-expression of mi R-21 didn’t changed the S-phase cell cycle arrest due to Qilan preparation treatment(P>0.05).Conclusions:1. Qilan preparation inhibites proliferation and induces apoptosis on Tca8113 cells in vitro; 2. Qilan preparation arrests Tca8113 cell cycle in S-phase in vitro; 3. Qilan preparation down-regulates the expression of mi R-21 and up-regulates the expression of PDCD4 and PTEN in Tca8113 cells in vitro; 4. Qilan prepatration inhibites proliferation and induces apoptosis through micro RNA-21/PDCD4 pathway in Tca8113 cells in vitro; 5. Micro RNA-21/PDCD4 maybe is not the pathway of S-phase cell cycle arrest due to Qilan preparation treatment in Tca8113 cells in vitro. |
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