| Objective: Tumor is a kind of severe disease threatening the health and lives of human at present. The morbidity and mortality of patients with tumor are higher. Administrating drugs is an important measure in the process of treating tumors. The traditional Chinese drug (TCD) is a precious medicine resource in China. Investigating anti-tumor TCD is an important territory of investigating anti-tumor drugs. Studying the different anti-tumor effect and the related molecular mechanism of different traditional Chinese drug preparations (TCDP) is the important evidence of correctly selecting and using these TCDP clinically. Tumor cell is derived from mutation of normal cell in organism. The immune function of organism, especially the functions of natural killer cell (NK cell) and T cell, play an important role in immunologic surveillance against tumor and preventing the generation and development of tumor. That the tumor cells secrete immunosuppressive substances to inhibit immunofuctions, i.e the tumor-induced immunosuppression, especially to inhibit the immuofunctions of NK cells and T cells, was the important mechanism of making tumor escaping organism immunosurveillance and had important effect on promoting the generation and development of tumor. Thus, the generation and development of tumor is related not only to the immune function of organism, but also to immunosuppressive action induced by tumor cells closely. Up to now, it has been known that there are about twenty kinds of immunosuppressive molecules which could be secreted by tumor cells. Among these, those reported more frequently and having more intensive immunosuppression are transforming growth factorβ1 (TGF-β1), prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF) and interleukin-10 (IL-10). Tumor cells secrete these immunosuppressive molecules to form an extensive immunosuppressive"block hole"region in location of tumor, not only making that the immunocytes there are inhibited severely, but also that once the normal even activated immunocytes enter into there they would become function-inhibited"the silent cell", resulting in that the immunocytes could not attack and eliminate the tumor cells, which should be an important mechanism that the tumor could escape immunosurveillance to generate and to develop. Obviously, it is of vital significance to study the effect of anti-tumor TCDP on the tumor-induced immunosuppression and the related molecular mechanism.Previous studies in our laboratory showed that supernatant from cultured L929 tumor cells had immunosuppression, suggesting there was secretion of immunosuppressive molecules from L929 tumor cells. Artesunate and Oxymatrine are the two kinds of TCDP with some anti-tumor effect. But, so far, there has been no report about the effect of Artesunate and Oxymatrine on the immunosuppression induced by L929 tumor cells and the related molecular mechanism. Therefore, on the basis of previous studies, we study the effect of Artesunate and Oxymatrine on immunosuppression induced by L929 tumor cells and the related molecular mechanism in order to further find out their antitumor mechanism, to guide effectively selecting and rationally using them in clinical and provide the evidences of experiment and theory.Methods: After cultured in complete medium(CM)containing the selected concentration of ART or MOX for 24h and washing off ART or MOX, the L929 tumor cells were re-cultured in CM without ART or MOX for sequential two times of 24h re-culture. The cells and supernatants from the two times of re-cutured L929 tumor cells were harvested respectively. Of them, for the L929 tumor cells treated with ART the cells and supernatant of the first re-culture called A-C1 and A-S1, and the cells and supernatant from the second re-culture called A-C2 and A-S2 respectively; for the L929 tumor cells treated with MOX the cells and supernatant from the first re-culture called O-C1 and O-S1, and the cells and supernatant from the second re-culture called O-C2 and O-S2 respectively. The cells and supernatants harvested from the synchronistically cultured L929 tumor cells in CM without ART or MOX as corresponding control called C-C1, C-S1, C-C2 and C-S2 respectively. Then the effect of different supernatants on seven immunofunctions of murine splenocytes, including NK killing and ConA-induced transformation detected by MTT, and expression levels of CD4+, CD8+, IL-2Rα, CD3ε+ζ+ and CD3ε-ζ+ detected by FCM of direct immunofluorescence were determined. The concentrations of four immunosuppressive molecules, including TGF-β1, PGE2, VEGF and IL-10 in different supernatants were measured by quantitative ELISA. The mRNA expression of the immunosuppressive molecule or its cell-intrinic synthesis rate-limiting enzyme of the immunosuppressive molecule that among the detected four kinds of immunosuppressive molecules secreted by L929 tumor cells its secretion was affected more highly by ART or MOX was detected by RT-PCR usingβ-actin as intrinsic reference. The mRNA expression level was shown by the relative coefficient (RC). The correlations between the congcentrations of immunosuppressive molecules in supernatants and the immunosuppressions of corresponding supernatants were analysed based on professional and statistical analysis to determine the relative molecules that ART and MOX down-regulated the immunosuppression induced by L929 tumor cells. For the immunosuppressive molecule that the inhibitory effect of ART or MOX on its secretion from L929 tumor cells was more higher, using the single-analysis of correlativity the relative correlation between its supernatant' concentration and its mRNA expression or its synthysis rate-limiting enzyme mRNA expression was determined to estimate that the ART or MOX down-regulated the molecule' secretion happened at either gene transcription or post-transcription.Results:1 The effect of supernatants from cultured L929 tumor cells on the immunofunctions of murine splenocytesIn the normal control group replacing supernatant from cultured L929 tumor cells with CM, i.e C-nS group, the detected values of the seven kinds of murine splenocytes' immunofunctions were 65.39±0.87% (NK killing rate), 0.925±0.026 ( A value of ConA-induced transformation),48.24±2.10%(CD4+cells),19.23±0.60% (CD8+ cells),26.50±0.85%(IL-2Rα+cells),21.67±1.29% (CD3ε+ζ+ cells) and 16.33%±0.18% (CD3ε-ζ+ cells). Compared with the C-nS group, the supernatants(C-S1 and C-S2) from synchronistically cultured L929 tumor cells treated without ART or MOX as corresponding control inhibited all of the seven immunofunctions apparently:①in C-S1 group the detected values of the seven kinds of murine splenocytes' immunofunctions respectively were (60.97±3.86)% (NK killing rate ,P<0.01), 0.440±0.038 (A value of ConA-induced transformation, P<0.01), (22.72±2.61)% (CD4+cells, P<0.01), (11.42±1.25)% (CD8+ cells,P<0.01), (10.39±0.49)% (IL-2Rα+cells,P<0.001), (7.89±0.78)% (CD3ε+ζ+cells,P<0.001) and (10.93%±0.16)% (CD3ε-ζ+ cells,P<0.001),and the corresponding inhibitory rate respectively were (8.34±4.55)%, (47.88%±1.89)%, (53.00±3.37)%, (40.71±4.65)%, (59.91%±0.77)%, (63.33±5.77)% and (33.05%±1.52)%;②in C-S2 group the detected values of the seven kinds of murine splenocytes' immunofunctions respectively were (59.59±3.13)% (P<0.01), 0.441±0.047 (P<0.001), (21.89±2.18)%(P<0.01),(12.15±0.65)%(P<0.01),(9.76±0. 94)%(P<0.001), (7.45±0.54)%(P<0.001) and( 10.75%±0.59 ) % ( P<0.001), and the corresponding inhibitory rate respectively were (9.79±3.95)%,(48.00%±2.16)%,(54.69±2.55)%,(36.85±1.41)%,(61.49±0.85)%,(65.63±0.87)%, (33.32%±0.25)%; while the C-S1 group compared with C-S2 groups these values had no changes (P>0.05).2 The effect of supernatant from L929 treated with ART or MOX on the immunofunctions of murine splenocytesFor L929 tumor cells after treated with ART:⑴The first re-culture' supernatant (A-S1) compared with C-S1, the inhibitory effect of A-S1 on the others six immunofunctions except for CD8+ (P>0.05) decreased greatly (P<0.001,P<0.01,P<0.05,P<0.05,P<0.001,P<0.01, respectively);⑵The second re-culture' supernatant (A-S2) compared with A-S1:①The inhibition of CD8+ expression increased greatly(P<0.05);②The inhibitory effect on CD3ε+ζ+ expression increased significantly(P<0.01),but had not reached the level of C-S2 (P<0.01);③The inhibitory effect on the NK killing rate , A of ConA-induced transformation and expression of CD4+,IL-2Rαas well as CD3ε-ζ+ had no changes (All P>0.05).For L929 tumor cells after treated with MOX:①The first re-culture' supernatant (O-S1) compared with C-S1, the inhibitory effect on the others six immunofunctions except for CD8+ (P>0.05) decreased greatly (P<0.05,P<0.01,P<0.05,P<0.05,P<0.001,P<0.01, respectively);②The inhibitory effect on CD3ε+ζ+expression increased markedly(P<0.05),but had not reached the level of C-S2 (P<0.01);③The inhibitory effect on the NK killing rate , A of ConA-induced transformation and the expression of CD4+ ,CD8+,IL-2Rαas well as CD3ε-ζ+ had no changes (All P>0.05).3 The effect of ART and MOX on secretion of four kinds of immunosuppressive molecules from L929 tumor cellsThe L929 tumor cells treated without any TCDP secrete the four kinds of immunosuppressive molecules steadily (C-S1 VS C-S2, all P>0.05), the concentrations of TGF-β1 and PGE2 were more higher(:198.15±3.23)pg/ml and(133.76±5.16)pg/ml respectively, and the concentrations of VEGF and IL-10 were relatively lower: (33.38±0.59)pg/ml and(27.31±0.37)pg/ml respectively. For L929 tumor cells after treated with ART:①The first re-culture' supernatant (A-S1) compared with corresponding C-S1: the concentrations of TGF-β1, PGE2, VEGF and IL-10 in A-S1 decreased greatly:(99.05±8.02)pg/ml( P <0.001, decreased 50.01%), PGE2(66.57±4.71)pg/ml(P <0.001, decreased 50.23%), VEGF(21.03±0.87)pg/ml(P <0.01, decreased 36.99%)and IL-10(22.89±0.57)pg/ml(P <0.01, decreased 16.18%)respectively;②The second re-culture' supernatant (A-S2) compared with A-S1, the concentration of IL-10 increased highly(P<0.05), and the others had no changes(All P>0.05).For L929 tumor cells after treated with MOX:①The first re-culture' supernatant (O-S1) compared with corresponding C-S1: the concentrations of TGF-β1, PGE2,VEGF and IL-10 in O-S1 decreased greatly: TGF-β1(91.35±9.62)pg/ml(decreased 53.89%), PGE2(69.43±2.32)pg/ml(decreased 48.23%), VEGF(21.36±0.75)pg/ml (decreased 36.01%)and IL-10(22.84±0.33)pg/ml(decreased 16.37%)(all P<0.01)respectively;②The second re-culture' supernatant (O-S2) compared with O-S1, the concentrations of the four kinds of immunosuppressive molecules had no changes(all P>0.05).4 The analysis of related molecules to the effect of ART and MOX on the immunosuppression induced by L929 tumor cellsThe professional and statistical analysis demonstrated:①The related molecules to that ART and MOX down-regulated immunosuppression induced by L929 tumor cells on the ConA-induced transformation and the expression of CD4+,IL-2Rαand CD3ε-ζ+of murine splenocytes were TGF-β1 and PGE2.②The related molecules to that ART and MOX down-regulated immunosuppression induced by L929 tumor cells on the NK killing were TGF-β1and PGE2.③The related molecules to that MOX down-regulated immunosuppression induced by L929 tumor cells on the NK killing was TGF-β1.5 The effect of ART and MOX on the expression of TGF-β1 mRNA and COX-2mRNA in L929 tumor cellsIn all the detected four kinds of molecules secreted by L929 tumor cells the secretions of TGF-β1 and PGE2 were affected more higher by ART and MOX. The COX-2 was cell-intrinic synthesis rate-limiting enzyme of PGE2. Detecting the expression of TGF-β1 mRNA and COX-2 mRNA could reveal that the two kinds of TCDP down-regulated the secretions of TGF-β1 and PGE2 happened at either gene transcription or post-transcreption. The results demonstrated①In the L929 tumor cells treated without any TCDP, TGF-β1 mRNA and COX-2 mRNA were expressed highly and steadily (C-C1 VS C-C2 , all P>0.05);②In the L929 tumor cells treated with ART and MOX , the expression of TGF-β1 mRNA and COX-2 mRNA were decreased significantly(A-C1 vs C-C1,O-C1 vs C-C1,A-C2 vs C-C2 and O-C2 vs C-C2: TGF-β1 mRNA,P<0.001; COX-2 mRNA,P<0.005). That ART and MOX down-regulated the secretion of TGF-β1 and PGE2 from L929 tumor cells was positively related to down-regulating the transcription of TGF-β1 mRNA and COX-2 mRNA significantly(after treated with ART : r =1.000, P=0.000; r =0.992, P=0.008; after treated with MOX : r =0.997, P=0.003; r =0.995, P=0.005). Demonstrating that the two kinds of TCDP down-regulated the secretion of TGF-β1 and PGE2 from L929 tumor cells happened at gene transcription.Conclusion:1 The supernatants from cutured L929 tumor cells treated without ART or MOX could inhibit all of the detected seven immunofunctions (ConA-induced transformation, NK killing, and expression of CD4+,CD8+, IL-2Rα, CD3ε+ζ+andCD3ε-ζ+) of mouse splenocytes significantly. The immunosuppressive effect of supernatants from cutured L929 tumor cells treated with ART or MOX on all of the detected seven immunofunctions was reduced markedly.2 The L929 tumor cells treated without ART or MOX could secrete all of the detected four immunosuppressive molecules(TGF-β1,PGE2,VEGF and IL-10)steadily. The secretion of the detected four kinds of immunosuppressive molecules from L929 tumor cells treated with ART or MOX was down-regulated greatly.3 The related immunosuppressive molecules that ART down-regulated the immunosuppressive effect of L929 tumor cells on the ConA-induced transformation, NK killing and expression of CD4+, IL-2Rαand CD3ε-ζ+ of murine splenocytes were TGF-β1and PGE2.4 The related molecules that MOX down-regulated the immunosuppressive effect of L929 tumor cells on expression of CD4+, IL-2Rαand CD3ε-ζ+ of murine splenocytes were TGF-β1 and PGE2. The related molecule that MOX down-regulated the immunosuppressive effect of L929 tumor cells on murine splenocytes'NK killing was TGF-β1.5 That ART and MOX down-regulated the secretion of TGF-β1 and PGE2 from L929 tumor cells was positively related to down-regulating the expression of TGF-β1 mRNA and COX-2 mRNA significantly.
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