Preliminary Study On Recombinant Ganoderma Lucidum Immunoregulatory Protein For The Anti-tumor Activity Of H22 Hepatocellular Carcinoma

Malignant tumors is the second human killer in all of life threat to human health and disease,after cardiovascular disease. Hepato- cellular carcinoma is the fifth in common tumors and the third in tumor-related death rate.Each year one million people die of HCC in the world.China is a dominant country of hepatocellular carcinoma and has the highest incidence of hepatocellular carcinoma.There are many risk factors to lead to HCC, hepatitis B virus (HBV),hepatitis C virus (HCV) infection and alcohol are the top three.Because HBV infection,aflatoxin and environmental pollution problems did not control effectively,incidence of hepatocellular carcinoma is still upward tendency which is a serious threat to the health of the people in China.Hepatocellular carcinoma is insidious and difficult to diagnosis.Because of the characteristics of fast progress and age at onset of small,most of the patients have lost the opportunity to surgical resection or can not tolerate surgery.It will recurrence even if surgical resection,so non-surgical treatment methods in the treatment of primary hepatocellular carcinoma occupies an important position. China began the screening research of anti-cancer drugs since the 20th century 50's,samples were extracted from natural resources at first,with the development of science , people extracted the active ingredients from natural plant,identified the relationship between chemical structure and pharmacological , transformed chemical synthesis or modified its structure or structure-oriented reform to enhance the biological activity to reduce side effects and enhance the practical value.Recombinant Ganoderma lucidium immunoregulatory protein is a number of fungal immunomodulatory proteins family and extracted from Ganoderma lucidum mycelium and then recombined.Previous studies showed that, rLZ-8 can inhibit the systemic allergic reactions in mice , stimulate human peripheral blood lymphocyte proliferation and IL-2, TNF-αand INF-γcytokine production such as the immunological activity, but the anti-tumor activity of hepatocellular carcinoma has not been reported.To investigate the anti-tumor activity of rLZ-8 on mouse H22 hepatocellular carcinoma in vitro and in vivo.rLZ-8 was used to inhibite H22 hepatocellular carcinoma in vitro,the TC0 ,IC50 and TC90 were 0.5μg·ml-1,5μg·ml-1 and 12μg·ml-1 , which were detected by CPE and MTT assay respectively.rLZ-8 drug group was significant deviation compared with H22 normal control group by optical density at 570nm,it showed that rLZ-8 had powerful lethal effect to H22 cells in vitro.In PI simple staining flow cytometry experiment,the apoptosis rates of low- ,moderate- ,and high-dose rLZ-8 were 9.23 %,27.51 %,38.60 % ,while H22 normal control group was 4.06 %.The apoptosis rates increased with the dose and showed a dose-dependent relation- ship.We used Annexin V-FITC/PI double staining flow cytometry to detect the effect of rLZ-8 anti-tumor activity of H22 hepatocellular carcinoma,the apoptosis rates of low, moderate,and highdose rLZ- 8 were 18.9 %,22.3 %,34.0 % ,which were highter than H22 normal control group.In the detection of NK and LAK cell activity , the concentration of rLZ-8 increased from 1.25 ng·ml-1 to 20 ng·ml-1, NK cell killing activity increased from 26.9 % to 57.7 % while LAK cell killing activity increased from 18.7 % to 56.2 %.To establish an experimental vivo model of mouse ascites tumor and solid tumor by H22 hepatocellular carcinoma,we observed the therapeutical effect of ascites tumor and solid tumor model ,after the treatment with different concentrations of rLZ-8.Weighed daily and observed the changes of body weight in mouse that ascites tumor was treated 10 days by rLZ-8.We used rLZ-8 to treat solid tumor for 10 days,after the next day, all mouse were killed by cervical dislocation, taked out of tumors and weighted, calculated inhibition rate.The solid tumor inhibition rates of low- ,moderate- ,and high-dose rLZ-8 were 16.7 %,30.0 %,42. 5 %.It was significant deviation compared with model group(P <0.05).The ascites tumor weights of low- ,moderate- ,and high-dose rLZ-8 were 38.69 g±0.03 g,36.7 g±0.02 g,32.6 g±0.03 g,which were highter than model group,it was significant deviation compared with model group(P <0.05).The results showed that, rLZ-8 had a good therapeutic effect on ascites tumor and solid tumorl which were infected by H22 hepatoma carcinoma and can inhibit tumor hyperplasia effectively.Recombinant Ganoderma Lucidum Immunoregulatory Protein (rLZ-8) has effect on anti-tumor,the mechanism is related to inhibit tumor cell proliferation and induce apoptosis of tumor cells.