The Experimental Study In The Anticancerous Effect Of Fuganchun 6 On Hepatoma

Objective: At present, it is believed that primarycarcinoma of the liver is one of the most common cancers inChina, and also is the most difficult to conquer. According tostatistics, every year, about 45% of new cases occured all overthe world are in our country. Because of its insidious occuranceand shortage of significant symptom in the initial period, it hascrept into the middle or advanced period when this disease isdiagnosed, and then has a very bad prognosis. In last 20 years,the death rate of primary carcinoma of the liver shows aincreasing tendency. In our country, the 5-years survival rate ofliver cancer is 2.7%, and it has played a secondary role in themalignant tumor that causes sufferers to die. Because a numberof people have been infected with HBV, HCV or other diseases,which result in the liver cancer, so the situation of highincidence of this disease is severe at present.Nowadays, the routine therapies of liver cancer areoperation, radiotherapy and chemotherapy. Up to now, surgicaloperation is regarded as the first choice to treat liver cancer, itseffect is related with the condition of liver function, the wholestatus of sufferer as well as the character of cancer. Therecurrence rate is 61.5% during the period of five years after thetumor mass is excised. Furthermore, only less than 25% of thesufferers could be committed to operation. So operation, byitself, is not enough to eradicate tumor and to prevent therecurrence and the metastasis of tumor, and often, it imposesenormous misery on the sufferer. As regards radiotherapy, thereare a vast of side effects in the wake of it, such as bone marrowdepression, indigestion, immunodepression and weakness ofbody. Chemotherapy is accepted as the common method to treatliver cancer, its effect of selective inhibition on tumor cells isinferior, and the toxicity to other normal tissues is serious. All ofthe three therapies above-mentioned damage the sufferer's bodyto a certain extent. Therefore, the problem of how to improvethe general therapeutic effect on liver cancer has become thefocus in the realm of medicine.With the development of science and technology,Traditional Chinese Medicine (TCM) with the traditionalcharacter of our country shows unparalleled superiority in theliver cancer treatment. In accordance with the TraditionalChinese Medicine theory, the development of carcinoma is theprocess of struggling between the healthy qi and evil qi.Therefore, the most important principle of clinical treatment isto boost the healthy qi and to suppress the evil qi with thepurpose of adjusting the balance of both sides.Fuganchun 6 (FGC-6) is established by Dr Mao Yu Xiangon the basis of his long-term practical experience. Many effectscan be observed in the clinical experiment of FGC-6, such assoothing the patient's suffering, prolonging the life span andimproving living quality.In this research, we treated the hepatoma-22 transplantedmice with the FGC-6, and then observed its effects onimproving the immunity, inducing the apoptosis of tumor cellsand the effect of serum containing FGC-6 on inhibiting theproliferation of H22 cells in vitro. The aim of this research is toexplore the anticancerous mechanism of FGC-6 at cellular andmolecular level, and to povide the laboratory evidence for theclinical appliance.Methods: A total of 50 KM mice, everyone's weight was20±1g, were transplanted with H22 cells in the right armpit. 24hours later, the mice were divided into five groups randomly:⑴normal saline group (NS group): 0.4ml distilled water (DW)was poured into stomach two times per day, 0.2ml sterile normalsaline was injected into abdominal cavity one time per day. ⑵Fluorouracil Ⅰgroup (5-FuⅠgroup): 0.4ml DW was pouredinto stomach two times per day, 0.2ml 20mg/ml 5-Fu wasinjected into abdominal cavity one time per day. ⑶5-FuⅡgroup: 0.4ml DW was poured into stomach two times per day,0.2ml 10mg/ml 5-Fu was injected into abdominal cavity onetime per day. ⑷FGC-6 group: 1.4g/0.4ml FGC-6 was pouredinto stomach two times per day, 0.2ml sterile normal saline wasinjected into abdominal cavity one time per day. ⑸5-Fu+FGC-6 group: 0.7g/0.4ml FGC-6 was poured into stomach twotimes per day, 0.2ml 10mg/ml 5-Fu was injected into abdominalcavity one time per day. Another 10 mice were transplanted withsterile normal saline in the right armpit as the normal controlgroup (N group), they were treated as the mice of NS group. Allmice were treated with different medicine for 10 dayssuccessively. After 24 hours of the last treatment, the weight ofmouse was weighed, the number of leukocyte was counted withcaudal vein blood, the tumor and thymus were taken out andweighed to calculate the inhibition rate of tumor growth and theindex of thymus, the target-cell-killing activity of NK cell wastested by LDH release test, the activity of lymphocyteproliferation in splenocytes was measured by MTT colorimetry,the level of IL-2 produced by splenocytes induced by ConA wasexamined by ELISA. Apoptosis of the tumor cell was observedby DNA ladder analysis, the protein expression level of bcl-2and bax genes were measured by Western blotting to furtherexplore the molecular mechanism of apoptosis.Another 50 mice were divided into 5 groups,the normalcontral group (N group) and 10, 20, 30, 50 times FGC-6 dosegroups. N group: 0.4ml DW was poured into stomach two timesper day; different FGC-6 dose groups: 0.28g/0.4ml, 0.56g/0.4ml,0.84g/0.4ml, 1.4g/0.4ml FGC-6 were poured into stomachrespectively two times per day. Every dose was equivalent to 10,20, 30 or 50 times of one adult person's dose. Three days later,the serum was collected after 1 hour of the last treatment. TheH22 tumor cells were transferred about six to eight generations,the effect of the serum containing FGC-6 on the inhibitionproliferation of the H22 cells was measured by MTT colorimetryand the cell growth curve.Results1 Comparison of the weight of tumor mass and the rate ofinhibition: After H22 cells were transplanted for ten days,tumor mass can be seen in all of the mice of every group.Compared with NS group (2.17±0.74g), the weight of tumormass of 5-FuⅠgroup (1.26±0.33g), FGC-6 group (1.34±0.26g)and 5-Fu+FGC-6 group(1.21±0.26g) were lighter (P﹤0.01),and the rate of inhibition was 42.07%, 38.47% and 44.31%respectively; the weight of tumor mass of 5-Fu+FGC-6 groupwas lighter than 5-FuⅠand FGC-6 groups, but there was nosignificant difference (P﹥0.05); the weight of tumor mass of5-FuⅡgroup(1.73±0.25g) was lighter than that of NS group (P﹤0.05), but was heavier than that of 5-Fu+FGC-6 group (P﹤0.05).2 Comparison of the number of leukocyte: The number ofleukocyte of NS group (6.51±0.86×10~9 /L) was lower thanthat of N group (7.73±1.04×10 ~9/L) (P﹤0.05). Compared withNS group, the number of leukocyte of FGC-6 group (8.31±1.06×10~9/L) increased (P﹤0.01), but that of 5-FuⅠgroup (3.07±0.78×10 ~9/L) and 5-FuⅡgroup (4.38±0.83×10~9/L) decreased(P﹤0.01). The number of leukocyte of 5-Fu+FGC-6 group(6.52±0.83×10~ 9/L) was more than that of 5-FuⅡgroup (P﹤0.01), but there was no significant difference between 5-Fu+FGC-6 and NS groups (P﹥0.05).3 Comparison of the index of thymus: Compared with theindex of thymus of NS group (23.74±3.17), the index ofthymus of N group(29.79±2.11), FGC-6 group(29.06±2.30)and 5-Fu+FGC-6 group(28.72±2.43) were much higher (P﹤0.01), but that of the 5-FuⅠgroup (6.06±1.79) and 5-FuⅡgroup (14.42±1.39) were much lower (P﹤0.01). There was asignificantly difference between 5-Fu+FGC-6 and 5-FuⅡgroups (P﹤0.01).4 Comparison of lymphocyte proliferation in spleen cells:The activity of lymphocyte proliferation in spleen cells wasmeasured by MTT colorimetry. There was no significantdifference between N group (3.30±0.27) and NS group (3.10±0.14). Compared with NS group, the activity of lymphocyteproliferation of FGC-6 (3.84±0.42) and 5-Fu+FGC-6 groups(3.61±0.16) were higher (P﹤0.01), there was a tendency thatthe activity of lymphocyte proliferation of 5-FuⅠ(2.97±0.27)and 5-FuⅡgroups (2.92±0.21) were lower than that of NSgroup, but it was not obvious (P﹥0.05). However, the activityof lymphocyte proliferation of 5-Fu+FGC-6 group markedlyincreased than that of 5-FuⅡgroup (P﹤0.01).5 Comparison of the target-cell-killing activity of NK cell:The activity of NK cell was measured by LDH release test. Thetarget-cell-killing activity of NK cell of NS group was lowerthan that of N group. Compared with NS group, thetarget-cell-killing activity of NK cell of FGC-6 and 5-Fu+FGC-6 groups markedly increased (P﹤0.01); but that of the5-Fu Ⅰgroup obviously decreased (P ﹤0.01). Thetarget-cell-killing activity of NK cell of 5-FuⅡgroup was muchlower than that of 5-Fu+FGC-6 group (P﹤0.01).6 Comparison of the production of IL-2 in murine splenocytesinduced by ConA: The production of IL-2 in murinesplenocytes of NS group was much lower than that of N group.Compared with NS group, the production of IL-2 in murinesplenocytes of FGC-6 and 5-Fu+FGC-6 group was higher (P﹤0.01), but that of the 5-FuⅠgroup was lower (P﹤0.05).7 DNA ladder analysis of tumor mass: The genome DNA oftumor mass was extracted and resolved by agarose gelelectrophoresis. The DNA ladder could be seen in 5-FuⅠ,FGC-6 and 5-Fu+FGC-6 groups. But the DNA of NS groupwas intact and clear, there was no DNA ladder.8 Comparison of the protein expression level of bcl-2 and baxgenes in tumor mass: Western blotting was used to measurethe protein expression level of bcl-2 and bax genes in tumortissue. Comparison with NS group, bcl-2 gene expression waslower in 5-FuⅠ, FGC-6 and 5-Fu+FGC-6 groups(P﹤0.01), itwas contrary that the protein expression level of bax was muchhigher in this three groups(P﹤0.01). Whereas, there wasn'tsignificantly difference between 5-FuⅠ, FGC-6 and 5-Fu+FGC-6 groups(P﹥0.05).9 The inhibition effect of serum containing drug on theproliferation activity of H22 cells in vitro9.1 The result of MTT colorimetry showed that the serum...