Effects Of Fufang Muji Chongji On Induction Of Apoptosis And Differentiation In Human Tumor Cell Lines

objective:Cancer is the powerful foe of mankind. Now, Chemicals are the capital therapeutics to cure malignancy, but the exploitation cost of chemicals is expensive, it has big adverse reaction and most of it have mutagenesis noxious property. Accordingly, people diversion the vision to the Chinese crude drug, and try to find light adverse reaction and distinct anti-tumour drugs. Chinese crude drugs have many features, for example, they have many targets, component elements and effects. they can impact the tumour genesis and development, at the same time, they can elevate the organism immunity, it is hard for them to form drug resistance. We all know, unbalanced control of proliferation, apoptosis and differentiation are the main characteristics of cancer cells, proliferation, apoptosis and differentiation are regulated by a network of signaling pathways and transcription factors, which are possible targets for a rational tumour therapy. For instance, some anticancer reagents cause cell death through affecting the processes of the cell cycle and some others induce apoptosis, which play an important role in the balance between cell proliferation and cell death. Compounds that induce apoptosis and differentiation are good candidates as cancer chemopreventive and/or chemotherapeutic agents. Increasing attention has been paid to primitive medicinal plants to find new substances with potentially useful biological activities.China is a high-risk region for primary hepatocellular carcinoma. Treatment for primary hepatocellular carcinoma is still difficult, and depends on basic medical research. Recent evidence suggests that apoptosis and differentiation of cells are closely related to the occurrence, progress and metastasis of tumors. Study of the mechanisms of apoptosis anddifferentiation in tumor cells are an important field of tumor therapy and cancer molecular biology.Leukemia, one of the most threatening hematological malignant cancers, has been found to be very sensitive to anticancer reagents which either block the process of the cell cycle or cause apoptosis. This chemotherapy-sensitive property of leukemia entices researchers to look for more specific and potent drugs against it. As potent new drug candidates, natural compounds have been highlighted because of their low degree of cytotoxicity.This paper studied the antitumor activities of Muji Chongji,and its inducing differentiation and apoptosis of HL-60 and HepG2 cells. A mechanism for the Muji Chongji-induction of differentiation and apoptosis were explored preliminarily.Methods:MTT assay was adopted to describe the proliferation of carcinoma cells. Apotosis induced by Muji Chongji was investigated by applying light microscopy, DNA electrophoresis and flow cytometry analysis techniques. Cell cycle was determined with flow cytometry analysis.Immunohistochemistry was performed to determine the protein of Bcl-2 and caspase-3. Cell differentiation was determined by using Giemsa stain. Nitroblue tetrazolium(NBT) reduction, flow cytometry was used to determine the expression changes of CD11b and CD14. Cell differentiation was also determined by using peroxydase staining. The contents of y-glutamyl transpeptidase (γ-GT) and alkaline phosphatase (ALP) in the supernatant were determined by biochemistry techniques .α-fetoprotein (AFP) was detected by radioimmunoassay. Immunohistochemical staining was performed to determine the protein of p21WAF1.Results:(1) The researches on the antitumor activities of Muji Chongji in vitro revealed that the growth of K562, HL-60, Hela, HepG2, Raji, A549 cells was inhibited by Muji Chongji in time-dependent manner. Compared with normal control group and positive control group, Muji Chongji could inhibit the growth of tumor cells obviously(p<0.01).(2) Fufang Muji Chongji treatment led to morphological changes in carcinoma cells HL-60 and HepG2, for example, marginal nuclei,chromatincondensation and nuclear fragmentation. The result of flow cytometry show that apoptotic cells increased after the effect of Muji Chongji on HL-60 and HepG2 cells. Muji Chongji treatment caused a decline in the fraction of S-phase cells and an increase in G0/G1cells. Muji Chongji increased the apoptosis rate of HL-60 and HepG2, and there was a apoptosis peak in DNA tapes. DNA ladders appeared on agarose gel, which demonstrated that Muji Chongji could induce apoptosis. After HL-60 cells were treated with Muji Chongji for different time, The expression levels of Bcl-2 and caspase-3 were detected by immunohistochemistry. The expression of Bcl-2 protein was decreased and caspase-3 protein was increased.(3) The research results mentioned above testify that Muji Chongji has obvious antitumor effect and induction of differentiation of tumor cells. The possible mechanism might relate to modulation of gene expressions associated with proliferation and differentiation, which lead to inhibition of DNA sythesis. Treatment with muji Chongji induced a differentiation in HL-60 cells, NBT deoxidization ability boosted up obviously (p<0.01). After treated with muji Chongji for 48h, the expression of CD11b is increased, but no significant changes of CD14 was observed. Cell differentiation was also determined by using peroxydase staining, the result is hadro-masculine. Our study also showed that muji Chongji induced up-regulation of p21WAF1 protein. (4) Treatment with muji Chongji induced a differentiation in HepG2 cells. First, we can find this effect by morphological findings. The content of AFP in the supernatant was decreased, but ALP was increased. Our study also showed that muji Chongji induced upregulation of p21WAF1 protein, so we presume its molecular mechanisms of action is via accumulation of p21WAF1.Conclusions:(1) Fufang muji Chongji can inhibit the proliferation of many tumour cells, it has extensive killing effect on tumour cells.(2) The anti-tumor mechanism of Fufang muji Chongji is it can induce apoptosis in HepG2 and HL-60 cells, The mechanism of Fufang muji Chongji inducing apoptosis may be related to disturb cell cycle, downregulation expression of Bcl-2 and upregulation expression ofcaspase-3.(3) The anti-tumor mechanism of Fufang muji Chongji is it can induce differentiation in HepG2 and HL-60 cells and there is significant correlation with cell cycle inhibitor p21WAF1.