The In Vitro Reversing Effect Of Arsenic Trioxide On Tumor Immunosuppression From Uterine Cervix Cancer Hela Cells

Objective: Cervix cancer is one of the most common malignancies in women. Traditional therapies of surgery, radiotherapy and chemotherapy can reduce immune function to a certain extent, which is not helpful for further treatment. And therefore auxiliary immunotherapy has gradually been focused. Tumor can escape from immunological surveillance through immunosuppression and this is the important mechanism of the occurrence and development of tumors. Therefore, discovery the effective drugs that can reverse these kind of immunosuppression can open a new approach of anti-tumor drugs. As a broad-spectrum anticancer medicine, As₂O₃ was successfully employed in the treatment of acute promyelocytic leukemia (APL). The inhibition effects of As₂O₃ on the growth of tumer cells were reported, but its down-regulating effects on tumor immunosuppression have not been reported. Accordingly, based on foregone studies, our study planned to change the effects of As₂O₃ on uterine cervix cancer immunosuppression. It is expected to discover the immunological mechanisms of As₂O₃ at point of reversing tumor immunosuppression, to provide experimental basement and theoretic evidences to further study of targeting pathways and sites, exploiting useful, efficient and specially characteristic anti-tumor traditional Chinese medicines that have our own property right, and guiding the proper application for clinical uses.Methods: The suitable inoculated concentration and cultured time of Hela cells were detected by MTT. The maximum concentration which had no change on the proliferation of Hela cells has established by MTT. Re-cultured supernatants of Hela after treated with As₂O₃, followed by being washed completely to remove the As₂O₃, and re-cultured twice with fresh medium, and the corresponding supernatants of Hela treated without As₂O₃ as control were collected. Through MTT, the effects of different supernatants on transformation induced by PHA. And through flow cytometry assay, the effects of different supernatants on PBMC expression of CD3ε+, CD3ε+ζ+, CD4+, IL-2Rα+, CD4+, CD25+, CD3ε-ζ+ and CD16+ were analyzed.Results:1.The inoculated concentration determined by MTT was 2×105/mL, and the cultured time was 48h; The maximum concentration of As₂O₃ non-poisonous was 0.31mg/mL.3.In the normal group (Control) of fresh medium without any supernatants from Hela cells, eight immunofunctions of PBMC (A of transformation induced by PHA, percentage of CD3ε+, CD3ε+ζ+, CD4+, IL-2Rα+, CD4+CD25+, CD3ε-ζ+ and CD16+ were 0.74±0.11, 25.09%±5.84%, 86.07%±16.05%, 40.73%±12.67%, 39.11%±12.83%, 38.53%±16.36%, 83.33%±16.46%, 73.37%±12.75%, respectively. Compared with Control, supernatant of Hela (Control-S1) inhibited all of the eight immunofunctions greatly, which were 0.44±0.11(P<0.001), 54.66%±6.21%(P<0.001), 0.96%±0.34%(P<0.001), 22.2%±4.23%(P<0.001), 20.92%±3.44%(P<0.001), 52.82%±7.28%(P<0.05), 0.74%±0.24%(P<0.05), 2.15%±0.92%(P<0.001), respectively. So their inhibitory rate were 40.7±11.77, 25.09%±5.84%, 86.07%±16.05%, 40.73%±12.67%, 39.11%±12.83%, 38.53%±16.36%, 83.33%±16.46%, 73.37%±12.75%, respectively.4. For Hela after treated with As₂O₃, the inhibition of the first re-cultured supernatant (As-S1) Compared with Control-S1 on transformation induced by PHA, and CD3ε+, CD3ε+ζ+, CD4+, IL-2Rα+, CD4+CD25+, CD3ε-ζ+ , CD16+ expression decreased greatly (P<0.001, P<0.001, P<0.001, P<0.05, P<0.05, P<0.05, P<0.001, P<0.05, respectively). Compared with As-S1, the inhibition of the second re-cultured supernatant (As-S2) on transformation induced by PHA and CD3ε+, CD3ε+ζ+, CD4+, IL-2Rα+ and CD3ε-ζ+ expression increased highly (P<0.05, P<0.05, P<0.001, P<0.05, P<0.05 and P<0.001, respectively) to the level of Control-S2 (All P>0.05), and others had no changes (All P>0.05). Conclusions:1. The inhibitory rate of five immunofunctions of PBMC(transformation induced by PHA, and CD3ε+, CD4+, IL-2Rα+, CD4+CD25+ expressions) after treated by Hela were about 40%; that of T cells CD3ε+ζ+ expression and CD3ε-ζ+ and CD16+ of NK cell were about 70%.2. As₂O₃ down-regulated the inhibition of PBMC immunofunctions caused by Hela cells.3. The Down-regulated effects of As₂O₃ on inhibition of transformation induced by PHA, and the expression of CD3ε+, CD3ε+ζ+, CD4+, IL-2Rα+ and CD3ε-ζ+ from Hela cells can last for 48h; that on the inhibitory of CD16+ expression can last for 96h at least; that on the increased expression of CD4+CD25+ can also last for 96h.4. The down-regulating effects on tumor were one of the anti-tumor mechanisms of As₂O₃.