| Objective To investigate the proliferation-inhibiting and apoptosis-inducing effects of Qi-Boosting Toxin-Resolving Formula (QBTRF) on nasopharyngeal carcinoma cell line HNE1 to explore the possible underlying mechanisms associated with the anti-tumor effects of this formula from these two aspects.Methods These experiments were carried out in vitro.The herbs composing of this formula were made into decoction at first in the whole original formula and several composing fraction way. Then, they were prepared as medicinal serum containing the components of whole original formula (GroupⅠ), the fractions of Qi-boosting (GroupⅡ),toxin-resolving (GroupⅢ),and Yin-enriching (GroupⅣ),and the fraction of components with other therapeutic effects (GroupⅤ).These samples of medicinal serum were cultured together with HNE1 in vitro for several periods of time lasted for 24 h,36 h,48 h or 72 h in a concentration of 15% to investigate their effects on the cells,with MTT assay to determine the proliferation-inhibiting effect, flow cytometry to determine the effects on cell cycle and apoptosis-inducing and immunocytochemistry to determine the effects on proliferating cell nuclear antigen (PCNA) and protein p53 (P53)expression in these cells.Results It was shown that these fractions of medicinal serum all held significant proliferation-inhibiting effects on HNE1 cells when compared before and after the treatment with them. GroupsⅠandⅢshowed their most significant inhibiting activity by the end of culturing period lasted for 48 hours, with the lowest survival rate of cells, while groupsⅡ,ⅣandⅤshowed this kind of effect by the end of culturing period lasted for of 72 hours.Meanwhile, the number of survival cells would decrease further with the concentration of medicinal serum elevated gradually.When compared among the different fractions with their effects on cell cycle and apoptosis-inducing of HNE1 cells,different activities could be seen among them. Firstly, their effects on cell cycle were very significant. Groups I and II showed higher percent of cells at phase G1 and lower percent of cells at phases G2 and S than that of blank controlling group.However, other groups of medicinal serum showed different effects with variation of culturing period in this aspect. When cultured for 24 hours, the percent of cells at phase G1 was higher and this percents of cells at phases G2 and S were lower than that of blank controlling group, while the percents of cells at phases Go to G1 were lower and these percents of cells at phases G2 plus S were higher than that of blank controlling group, when cultured for 24 to 48 hours.Moreover, the number of cells at phase G1 was markedly higher the total number of cells at phases G2 plus S and the number of cells at phase S was also significantly higher than the number of cells at phase G2 within each group,when cultured for 24 to 48 hours.These results suggested that all these groups of medicinal serum could block the cells progressing passing through phases G2/M. Secondly, their effects on apoptosis-inducing were variable.The apoptotic indexes were significantly higher in groupsⅠandⅡthan that of controlling group,while these indexes were lower in groupsⅢ,ⅣandⅤthan that of controlling one.However, apoptotic indexes showed a tendency to elevate with prolonging of the culturing period with medicinal serum among all the groups.As shown in immunocytochemistry for determination of PCNA and P53 expressing, the effects of these groups of medicinal serum were very obvious. When cultured for 24,36 and 48 hours with different groups of medicinal serum respectively, HNE1 cells showed significantly decreased expressing level of activity at various degrees for PCNA among them, with a tendency of further decreasing in the expression level with prolonging of the culturing period and the index of PCNA positive cells being decreased gradually with the concentration of medicinal serum increasing.The expressing level of PCNA was obviously lower in the cells cultured for 36 to 48 hours with the medicinal serum than ones cultured only for 24 hours.In contrast, the expressing level of P53 showed a reversed tendency to that of PCNA when cultured only for 24 hours with medicinal serum.However, the expressing activity of P53 was decreased obviously when cultured for 36 to 48 hours, with more and more cells becoming inactive morphologically with the prolonging of culturing period and mixed with only a few P53 positively stained cells among them.This kind of effect was especially obvious on the cells of groupsⅠandⅢand was not very significant on the cells of groupsⅡ,ⅣandⅤduring the prolonged culturing period lasted for more than 24 hours.Conclusions:Following conclusions could be derived from the results as stated above.1.All fractions of medicinal serum held very significant inhibiting effects on the proliferating activity of HNE1 cells, particularly when cultured with that of fraction I.These effects showed both characters being time-depending and concentration-depending.2.All fractions of medicinal serum could induce HNE1 cells undergoing the process of apoptosis at the concentration of 15%,especially for the composition of original formula. Also, this kind of effect was time-depending.3.All fractions of medicinal serum were able to inhibit the expressing level of PCNA in HNE1 cells during the whole process of culturing and to promote the expressing level of P53 within the first 24 hours,especially for groupⅠandⅡ. Thereafter, this effect would be reversed for P53 activity expressing in the cultured cells.4.QBTRF may bring about its effects on target cells to inhibit their proliferation and to induce apoptosis among them through regulating the expressing activity of associated genes. |
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