MA Chun-Hong, SUN Wen-Sheng, CAO Ying-Lin, ZHANG Li-Ning and SONG Jing 世界华人消化杂志 1998 0 0 2
关键词：tumor necrosis factor/pharmacology; interleukin/pharmacology; liver neoplasms/drug therapy; drug synergism 期刊 sjhrxhzz 0 Original Articles fur -->
Abstract
AIM Toobserve the effect of recombinant tumor necrosis factor α (rHTNFα) and mutantinterleukin-2 (IL-2) on the hepatocarcinoma cell line in vitro.
METHODS The hepatocarcinoma cellline (H7402) was used as a cell culture system in vitro. H7402 was cultured with differentconcentrations of rHTNFα and/or mutatant IL-2. Thecytotoxity was measured by short-term MTT method.
RESULTS The inhibitory effect onH7402 became stronger with the increase of the concentration of rHTNFα (r=0.909，P<0.05). Mutatant IL-2 had no direct inhibitory effect, but couldenhance the effect of rHTNFα in inhibiting H7402 cellline. The concentration of both rHTNFα and mutantIL-2 affected this enhancement: 2.5×103 U/ml of mutant IL-2 could increasecytotxity of rHTNFα (1000 U/ml) to H7402 effectively.When rHTNFα reached 5×104 U/ml, theenhancement appeared only when the concentration of mutant IL-2 was at 2×104 U/ml. If the dose of rHTNFα was at 8×104 U/ml, the inhibitory effect did not rise significantly until mutant IL-2 was at 4×104 U/ml.
CONCLUSION rHTNFα combined with mutatant IL-2 in low concentration can yield goodinhibitory effect on hepatocarcinoma.INTRODUCTION
Studies of effect of tumor necrosis factor α(TNFα) on hepatocarcinoma cells has rarely been reported, althougheffects of TNFα on other carcinoma cells have beenmeasured by many authors. In order to study the effect of TNFα on hepatocarcinoma cells and provide a certain theoretical evidencefor the clinical use of TNFα, we measured the deathrate of hepatocarcinoma cell line H7402 after culturing with recombinant human tumornecrosis factor α(rHTNFα) and/or mutant interleukin-2 (IL-2).
MATERIALS AND METHODS
MATERIALS
Recombinant human tumor necrosis factor α (1×104 U/ml)was provided by the Chinese Academy of Military Medicine. Cys-125 of mutantinterleukin-2(Quancheng Biological Company, Jinan, Shandong) was mutated into Ser-125.Human hepatocarcinoma cell line H7402, provided by the Department of Immunology, ShandongMedical Institute, was cultured on 1640 medium with 10% fetal calf serum (Sigma) at 37℃under 5% CO2 . MTT〔3-(4,5)〕 dimethythiazol-zyls-2,5-diphenytetrazolium bromide (Sigma) was used to measure the death rate of H7402.
METHODS
rHTNFα and mutant IL-2 were diluted at 1×103 /ml,1×104 U/ml, 5×104 U/ml, 8×104 U/ml and 2.5×103 U/ml,5×103 U/ml, 1×104 U/ml, 2×104 U/ml, 4×104 U/mlrespectively. H7402 was suspended at a concentration of 5×105 cells/ml, thenseeded at 96 plates (100?μl per cole). Twenty-fourhours after incubation at 37℃, rHTNFα and/or mutant IL-2 were added (1640 medium as control) into H7402.Ten hours later, assay was made by short-term MTT method[1] . Results wereexpressed as cytotoxic index (CI).
RESULTS
Results of MTT method indicated (Table 1) that: ① rHTNFαcould kill H7402 directly. The linear regression showed ahigh positive correlation between the concentration and the cytotoxity of rHTNFα(r=0.909, P<0.05). ②Mutant IL-2 had no direct inhibitory effect on H7402 andthere was no correlation between its concentration and its CI. ③Mutant IL-2 could enhance the effect of rHTNFα on inhibiting H7402 cell line. Table 1 Inhibitory effect of rHTNFα andmutant IL-2 on H7402 IL-2
(U/ml) | | rHTNFα(U/ml) | | 0 | 103 | 104 | 5×104 | 8×104 | | 0 | A | 0.25±0.02 | 0.235±0.03 | 0.19±0.01 | 0.11±0.30 | 0.095±0.02 | | | CI | | 0.06 | 0.24 | 0.56 | 0.71 | | 2.5×103 | A | 0.285±0.03 | 0.22±0.01 | 0.155±0.01 | 0.125±0.03 | 0.175±0.03 | | | CI | | 0.12b | 0.38 | 0.50 | 0.47 | | 5×103 | A | 0.28±0.04 | 0.23±0.03 | 0.145±0.01 | 0.11±0.01 | 0.135±0.02 | | | CI | | 0.08 | 0.22 | 0.56 | 0.59 | | 1×104 | A | 0.32±0.04 | 0.295±0.02 | 0.14±0.01 | 0.15±0.01 | 0.23±0.01 | | | CI | | | 0.20 | 0.44 | 0.08 | | 2×104 | A | 0.335±0.04 | 0.285±0.04 | 0.235±0.02 | 0.05±0.01 | 0.26±0.03 | | | CI | | | 0.06 | 0.80b | 0.24 | | 4×104 | A | 0.40±0.05 | 0.30±0.04 | 0.205±0.03 | 0.15±0.01 | 0.03±0.01 | | | CI | | | 0.18 | 0.40 | 0.88b | b P<0.01.
More analysesindicated that the concentrations of both rHTNFα and mutant IL-2 affected the enhancement effect of mutantIL-2 on rHTNFα:2500?U/ml of mutant IL-2 could increase cytotoxity of rHTNFα(1×103 U/ml) effectively to H7402 (P<0.01), no further increaseoccurred with the rising of concentration of rHTNFα. When rHTNFα reached 5×104 U/ml, the enhancement appeared only whenthe concentration of mutant IL-2 was at 2×104 U/ml (P<0.01). If the dose of rHTNFα was increased to 8×104 U/ml, its inhibitoryeffect did not rise significantly until the dose of mutant IL-2 reached 4×104 U/ml.In a word, rHTNFα combinedwith mutant IL-2 in low concentration can yield inhibiting effect on hepatocarcinoma aswell as in high concentration, however, when the concentration of any one of rHTNFα and mutant IL-2 was high andanother was low, there was no enhancement of cytotoxity.
DISCUSSION
TNFα has been usedwidely in biotherapy of cancer, for it can inhibit or kill many kinds of carcinoma cellsefficiently. However, TNFα has different effects on various carcinoma cells[2] . The present study showed that rHTNFαhad strong cytotoxity on hepatocarcinomacells and there was a positive correlation with its concentration. Since TNFα has severe side-effect in highconcentration, it is valuable to use TNFα with other cytokines in order to improve its inhibitory effect andreduce its side effects. IL-2 is one of the cytokines which can increase the cytotoxity ofTNFα. Mutant IL-2 ismore stable and has better activity because of its point mutation at coden 125 (Cys-125 toSer-125)[3] . Our results showed thatmutant IL-2 had no direct inhibitory effect, but could enhance the effect of rHTNFα on inhibiting H7402 cell line.The concentration of both rHTNFα and mutant IL-2 affected this enhancement: small dose of mutantIL-2 could raise the cytotoxity of small dose of rHTNFα by about two times, this enhancement did not change withthe rising of concentration of IL-2. If the dose of rHTNFα was increased, that of IL-2 must be also increased inorder to streng-then the inhibitory effect of rHTNFα. After all, it was supposed that rHTNFα combined with mutant IL-2 in lowconcentration was a better choice than combined in high concentration when hepaticcarcinoma was concerned. This paper provided a valuable theoretical evidence forconcomitant administration of two cytokines clinically.
REFERENCES
1 Yang GZ. Outlineand technique of immuno-biological engineering. 1st., Beijing: High Educational PublishingHouse, 1993:208-209
2 Ruggeiero V,Lantham K, Baglioni C. Cytostatic and cytotoxic activity of tumor necrosis factor on humancancer cells. J Immunol, 1987;138(8):2711-2717
3 Alice W, Lu SD,David MF. Site-specfic mutagenesis of the human interleukin-2 gene: structure functionanalysis of the Cysteine residues. Science, 1984 Jun 29;244(4656):1431-1433
(MA Chun-Hong, SUN Wen-Sheng, CAO Ying-Lin, ZHANG Li-Ning and SONG Jing)
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