Studies On The Anticancer And Reversing Multidrug Resistance Effects Of Abnormal Savda Munziq Total Flavonoids

A large numer of anticancer agents, including alkylating agents, antitumor antibioltics, antimetabolites, and various other agents such as platinum derivatives, procarbazine, and emetine, have been tried, alone and in a variety of combinations and by different routes of administration, to treat carcinoma, but the predictable response rate has always been less than 20%. Additionally, drug toxicity is frequent. Current trials are investigateing the use of plant derivatives , much attention has been paid to Chinese herd, because it has little toxicity, not susceptible to get drug resistance, has multimeans in antitumor.Multidrug resistance (MDR) is the protection of tumor cell population against numerous drugs differing in structure and mechanisms of influence on the cells. MDR is also one of the major causes of failure of chemotherapy of human maligancies. It is related to many active mechanisms. Often several different mechanisms are switched on in the cells, but usually one major mechanism is operating. The active mechanisms read as follows: a) activation of transmembrane proteins effluxing different chemical substances from the cells (P-glycoprotein is the most known efflux pump); b) activation of the enzymes of glutathione detoxification system; alteration of the genes and the proteins involved into the control of apoptosis (especially P53 and Bcl-2); changes of activity and quantity of topoisomerases. The tumor cell MDR and looking for little toxic and more effective chemotherapy drug has long been an interesting subject.Uighur medicine considers that Abnormal Savda is a pathological production resulted from the combustion of different body fluids known as Savda, Sapra, Belghem and Kan. It is main leading cause of cancers, and about 90% of caners attribute to Abnormal Savda in clinical reports. Bahki Yusup and Tursun Tohti Haji, two of the famous Uighur Doctor,according to the traditional therapy, have applied the ASMq-TF for treatment of cancers, which demonstrated remarkable clinical results, there by gaining the honor of"highly skilled doctor"by the local people.To date, the anticancer effects of ASMq-TF have only been little studied and its potential mechanism of action is still unclear, so a possible anticancer effect of Munziq of Abnoraml Savda clearly warrants further investigation.In the present study, we used human clone cells and Caco-2 to examine the anticancer effects of ASMq-TF, and also examine in vivo by used 1,2-dimethylhydrazine (DMH) induced rat colon carcinogenesis.We used Reversal of resistance to 5-Fu in human hepatocarcinoma cell line BEL /Fu toexamine reversal effects of ASMq-TF.Part1 The effect of Abnormal Savda Munziq Total Flavonoids on the proliferation, apop- tosis in Caco-2Colon cancer cells line of Caco-2 was cultured by cell culture technique. The effect of ASMq-TF, on the proliferation of Caco-2 cells after exposure to different concentration and different time was evaluated by MTT. Apoptosis rate was determined by using flow cytometry, DNA fragmentation assay. The study the effects of Abnormal Savda Munziq on the proliferation, apoptosis and correlative gene expression in cancer cells line of Caco-2.The results of MTT assay demonstrated that water soluble of ASMq-TF ethanol extract showed different effects on Caco-2 cell grows at 48h and72h incubation. The IC50, they were IC50 5.99mg/ml(48h)and 3.02mg/ml(72h). DNA fragmentation observed by gel electrophoresis showed that there were no significant DNA ladders in Caco-2 after treatment with different water soluble of ASMq-TF 0.5~5.0mg/ml , and seems to be no detectable difference between control cultures. But ethyl acetate extract induced the typical pattern of DNA ladders in Caco-2 cells at 7.5mg/ml. To study the acting mechanism of the fractions of ASMq-TF ethanol extract, flow cytometry was used to detect the cell cycle distribution of Caco-2 cells after 48h treatment with each fraction, respectively. The percentage of HepG2 cells in phase sub-G1 was most high concentration of 7.5mg/ml.Part2 Effects of Abnormal Savda Munziq Total Flavonoids on DMH induced colon carcinogenesisIn vivo experiment was designed to assess the potential preventive and curative effect of ASMq-TF on 1,2-dimethylhydrazine (DMH) induced rat colon carcinogenesis. Obserb number of aberrant crypt foci (ACF) and aberrant crypt (AC) and crypt multiplicity. The study on the preventive and treatment effects of Abnormal Savda Munziq on colon carcinogenesis.The results showed that all doses (400, 800 and 1600 mg/kg b. w.) of ASMq-TF ethanol extract in the pre-treatment group as well as in the treatment group significantly reduced the number of aberrant crypt foci (ACF), aberrant crypt (AC) and crypt multiplicity (P<0.05), the organ relative weights in pre-treatment and treatment groups were regained by ASMq-TF ethanol extract administration when compared to negative control group (P<0.05). These findings suggested that ASMq-TF ethanol extract had a chemoprotective effects on DMH-induced colon carcinogenesis, by suppressing the development of preneoplastic lesions, and probably exert protection against the initiation and promotion steps of colon carcinogenesis.Part3 Reversal mechanism of resistance to 5-Fu in human hepatocar- cinoma cell line BEL/Fu by Abnormal Savda Munziq Total FlavonoidsA drug-resistant hepatocarcinoma cell line BEL/Fu was used in this study. The aim of this study was to observe the reversal effect of 5-Fu resistance and its potential mechanism in hepatocarcinoma cell line BEL/Fu by Abnormal Savda Munziq.Sentivity to 5-Fu of BEL-/Fu cells was assessed by MTT assay. The reversal action of ASMq-TF, IC50 value and synergic action of vincristine and ASMq-TF were determined by MTT assay. The reversal action of ASMq-TF was also observed by clone forming text. MDR1 expression of Pgp was examined by immunochemical assay and determination of gene expression of MDR1 ,MRP, glutathione S-transferase(GSTpi) and topoisomeraseⅡαof Bel-7402 cell line were investigated using reverse transcription polymerase chain reaction (RT-PCR). Expression of bcl-2 protein and percentage of cell apoptosis was detected by flow cytometry. MDR1 expression of Pgp was examined by immunochemical assay.The reversal effect of ASMq-TF was evaluated using Bel/Fu cell line with innate resistance in this study. The IC50 value of ASMq-TF crude extract was 2000.68μg/ml. ASMq-TF at 390μg/ml concentration was no toxic but had a toxic effect on Bel/Fucell line with 5-FU. The cytotoxicity of 390μg/ml ASMq-TF and 5-FU to Bel/Fu cellline was more remarkable than only with 5-FU (P<0.01). The fold reversal of ASMq-TF combining with 5-FU was 7.8.While ASMq-TF concentrations lower than 390μg/ml had less toxic, it had no synergetic effect with 5-FU. ASMq-TF concentrations higher than 400μg/ml were not suitable due to cytotoxicitiy to tumor cells .The reversal effect of ASMq-TF was confirmed by clone forming text. The inhibited effect of ASMq-TF on Bel/Fu cell line correlated well with ASMq-TF concentration. However, Bel/Fu cell line treated with 5-FU and ASMq-TF showed significantly inhibited effect as comparing with ASMq-TF treatment. Bel/Fu cells treated with 390μg/ml ASMq-TF,The clone forming size is much smaller and the clone forming rate was only 3.5%. At same time, the irregular shaped cells, intracellular toxic granules, very loosely arranged cell and bizarre cells were observed. There was not clone forming and a few of dispersed cells in cells treated with 460μg/ml ASMq-TF .Human Pgp is coded by gene MDR1 and related to MDR of tumor cells. ASMq-TF has been found to downed-regulat MDR1/Pgp gene and proteinexpression. And simultaneously it could up-regulate TopoⅡαmRNA.Taken together, the present study suggested that the apoptosis triggered by ASMq-TF total flavonoids was mediated through DNA fragmentation, cell cycle arrest at sub-G1, up-regulation of p53, p21 and Bax gene expression, down-regulation of Bcl-2 gene expression in concentration- dependent manner. We concluded that the anticancer properties of ASMq-TF was possibly mediated through multiple pathways, suggesting multiple ingredients, rather than single component The acting ingredients in ASMq-TF that exerted the anticancer effect may include total flavonoids which are abundant in ASMq-TF.