Study On Effect Of Herbal Mixture "Niu Hang Tian Long Yin" On Cell Apoptosis And Mechanism Of Cervical Cancer Cell Line HeLa

Objective:Cervical cancer is the most common gynecologic tumor of genital duct. It threat women's health and life. In general, for invasive carcinoma of uterine cervix, the treatment is surgery and radiotherapy. Because of the limit of surgery and radiotherapy inducing persistent injury to normal organs around the focus and secondary tumor, a new treatment for advanced cervical cancer is needed. In recent years, there is a trend that neoadjuvant chemotherapy and concurrent radiotherapy are regarded as the standard treatment. The goal of the treatment is to reduce tumor bulk and decrease local and distant recurrences. Thus the drugs used as neoajuvant chemotherapy became a hot topic. Combined chemotherapy agents have been studied. The inevitable problem of chemotherapeutic agent's side effect makes us look for new drugs from traditional Chinese medicine. Chinese herbal inducing apoptosis has been reported, but apoptosis is a complex process and limited by many factors. Based on this theory, herbal mixture's mechanism of multi-target is superior to others. "Niu Huang Tian Long Yin" is based on traditional Chinese herbal prescription "Xi Huang Wan" combined with Hirudo, Fructus and so on. To explore the effect and mechanism of "Niu Huang Tian Long Yin" on cervical cancer cell HeLa, serologic pharmacology and a series of typical methods were used. The experiment provided a new thought and method for the study of antitumor.Methods: SD rats were given different dose Chinese herb by gavage. After 3 days, rat's blood was collected asepticly and serum was collected by centrifugation. Medical serum was made into powder through vaccum-freezing process. HeLa cell and human embryo lung cell were cultured in RPMI-1640 contained 10% or 20% FBS in 37.5℃, 5% CO2 HeLa cell were planted on 96-well plate on the same density and were affected by different concentration of contained medical serum for 48 hours, then cell proliferation inhibition was measured by MTT. Cell apoptosis was measured by AO/EB double fluorescent dye staining, flow cytometry, DNA agarose gel electrophoresis and TUNEL. The expression of Bcl-2, c-Myc was measured by flow cytometry.Results: 1 MTT: Cervical cancer cell line HeLa was effected by medical serum for 24 or 48 hours, then cell proliferation inhibition was observed and it was in dose-dependent manner, time-dependent manner (P<0.05). The inhibition rate of HeLa cell was 5.26%, 10.52%, 5.26% and 32.89% respectively in different groups treated for 24 hours. The inhibition rate was 2.38%, 23.80%, 13.10% and 7.14% respectively in different groups treated for 48 hours. The effect of medical serum on human embryo lung cell was different with on HeLa cell. Medical serum of Chinese herbal could promote the proliferation of diploid cell. 2 AO/EB double fluorescent dye staining: There were lots of normal cells but a few of apoptotic cells in control group and high dose group. There were more apoptotic cells in middle dose group and chemotherapeutic agent group and necrosis cell could be observed. 3 Flow cytometry: Apoptotic rate was different between trial groups and control group (P<0.05). Cells in S phase were reduced and in G1 phase were enhanced in trial groups (P<0.05). The expression of Bcl-2 was decreased in middle dose group. On the contrary, c-Myc was incresed. 4 DNA agarose gel electrophoresis: DNA ladder could be found in trial groups. 5 TUNEL: A few of positive cells were observed in control group while a lot of positive cells in trial groups. The positive rate was 11.95%, 29.50%, 17.10%, 37.30% and 4.68% respectively in high dose group, middle dose group, low dose group, chemotherapeutic agent group and control group.Conclusions: 1 Cancer cell line HeLa could be induced apoptosis by Herbal mixture "Niu Huang Tian Long Yin". 2 Apoptosis rate of herbal groups is lower than chemotherapeutic agent group. But medical serum of herbal groups could promote the proliferation of diploid cell. 3 "Niu Huang Tian Long Yin" could reduce the expression of Bcl-2 and enhance t...