Effects Of Compound Zhebei Granule And Active Ingredients On Expression Of Surface Antigen In KG-1a Cells And Regulation Of Signal Pathway

1 ObjectiveFormer basic studies found that Compound Zhe Bei Granule (CZBG) could reverse the multiple drug resistance of tumor, by means of regulating the relative proteins and enzymes expressing and inducing the cell apoptosis. Clinical trials also confirmed that CZBG combined with chemotherapy achieved higher remission rate of refractory acute leukemia. Based on preliminary studies, we explore the mechanism of reversing drug resistance activity of CZBG and its effective ingredients through cell and animal experiments.2 Methods2.1Cell experimentThe experiments included:①Culturing of KG-1a cell, identifying its specific surface antigens. ②Through MTT method, detecting the specific surface antigens, cell apoptosis and signal pathway proteins (PETN, AKT, ERK1/2) of KG-1a cell after receiving tetramethylpyrazine (TMP), peimine (PM) and tetrandrine (TET) treatments.③By Western-blot method, detecting the PTEN, Caspase9 and NF-κB expressing after TMP and TET treatments. ④Testing the AKT, Caspasae9, ERK, mTOR, PTEN and NF-κB expressions of KG-1a intervened by TMP, PM and TET through real-time PCR. 2.2Animal experimentThe experiments included:①Observing the effects of CZBG with doxorubicine on specific surface antigens of KG-la xenograft tumor tissues by flow cytometry. ②etecting the effects of CZBG with doxorubicine on mTOR, PTEN, NF-κB and PI3K expressions of KG-la xenograft tumor tissues by immunohistochemical method.3 Results3.1 Cell experimentKG-la cell had LSC characteristics for high expressions of CD34、CD33、 CD123、CD96, while low expression of CD117. Compared to control group, the results of drug intervention groups at 48hrs were as follows, ①TMP, PM and TET inhibited the cell proliferation remarkably, and the IC50 were 103.43μg/mL、 163.55μg/mL、8.69μg/mL, respectively. ②TMP had no effects on early, later and total apoptosis rate of KG-la cell; PM increased the later apoptosis rate; TET increased both early and later apoptosis rate. ③TMP reduced the expressing rates of CD34+CD38-, CD34+CD33+, CD34+CD123+and CD33+CD123+, as well as the fluorescence intensity of CD7, CD56 and CD44; PM had negative influence on expressing rates of CD34+CD123+, CD33+CD123+ and CD34+CD96+; TET reduced the expressing rates of CD34+CD38-, CD34+CD96+, CD7 and CD56, and the fluorescence intensity of CD7、CD56. ④TMP had no effects either on the expressing rate of ERK1/2, PTEN and AKT, or on the fluorescence intensity of PTEN, but reduced the fluorescence intensity of ERK1/2、AKT; PM was negative in affecting the expressing rate of PTEN, AKT and ERK1/2, and the fluorescence intensity of PTEN and AKT; TET improved the expression of PTEN.⑤Both TMP and PM had no effects on genetic expressing rates of AKT, Caspasae9, ERK, mTOR, PTEN and NF-κB; TET influenced the genetic expression of mTOR、 NF-κB.3.2Animal experimentThe results are as follows:①The results of specific surface antigens of KG-1a cell:compared to normal saline group, the expressing rates of CD34+CD38-, CD34+CD38-CD123+, CD34+CD38-CD96+ and CD34+CD38-CD33+ in CZBG groups reduced; compared to doxorubicine group, the expressing rates of CD34+CD38- and CD34+CD38-CD96+ in medium and high doses CZBG groups, and ones of CD34+CD38-CD123+ and CD34+CD38-CD33+ in high dose group decreased. ②The results of specific signal pathway proteins:compared to normal saline group, the optical densities of mTOR and NF-κB in each CZBG groups decreased; compared to doxorubicine group, mTOR in high dose CZBG group, NF-κB in medium and high dose CZBG groups performed lower optical densities.4 ConclusionsThe results are as follows:①Human acute medullary leukemia clone KG-1a cell has LSC like features. ②TMP, PM and TET can inhibit the cell proliferation and improve cell apoptosis. ③ TMP and TET can reduce the expressions of specific surface antigens in KG-1a cell, and regulate the AKT & ERK1/2 pathways at protein level. ④ CZBG combined with chemotherapy can inhibit the expressions of surface antigens in KG-1a xenograft tumor tissues, regulate AKT pathway at protein level as well.