| Cancer is the second leading cause of death worldwide. Cancer continues to represent the largest cause of mortality in the world and claims over 6 million lives each year. China is confronted with an increasing incidence of cancer and cancer deaths annually. Mortality that results from the common forms of cancer will be unacceptably high in the 21th century. Despite many therapeutic advances in the understanding of the processes in carcinogenesis, overall mortality statistics are unlikely to change until there is a reorientation of the concepts for the use of natural products as new chemopreventive agents.Natural products include thousands of compounds that exist in fruits, vegetables, plants, and herbs, and several clinical anticancer drugs, have been derived from natural products. Thus, development of compounds with anticancer effects from natural products has currently become a very important topic. Natural compounds isolated from medicinal plants, as rich sources of novel anticancer drugs, have been of increasing interest since then. Cancer prevention and treatment using traditional Chinese medicines have also attracted increasing interest, and the development of pharmacology and molecular biology make it possible to screen effective extracts with anticancer activity from the Traditional Chinese medicinal herbs and many relevant prescriptions.Traditional Uighur Medicine, the main part of Traditional Chinese Me-dicine, has been used for pharmaceutical and dietary therapy for several millennia. Traditional Uighur medicine has its own traditional theory of prevention and treatment of cancer with prescriptions containing Abnormal Savda Munziq (ASMq).(Jighur 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 Doctors, have applied the ASMq for treatment of cancers according to the traditional therapy, which demonstrated remarkable clinical results, thereby gaining the honor of "highly skilled doctor" by the local people.To date, the anticancer effects of ASMq 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 hepatoma cells (HepG2) to examine the anticancer effects of ASMq aqueous extract, ethanol extract, active fractions and total flavonoids of ASMq to demonstrate and elucidate the mechanisms responsible for regulation of cell grows, apoptosis and apoptosis related gene expression.Part 1Preparation and identification of Abnormal Savda MunziqextractionsThe therapeutic utilization of plants is part of universal human culture, and products derived from plants have been used frequently by human for the treatment of diseases or to protect against them. Presently, phytotherapy is considered an alternative to mitigate side effects due theindiscriminate use of synthetic drugs. In the last few years, several herbal products capable of anticancer effects in various test system have been identified. Although the mechanism of the protective effect is unclear, the fact that the consumption of some Chinese herbal preparations lowers the incidence of carcinogenesis at a wide vareity of sites is broadly supported. The Si Jun Zi Tang (Chinese herbal preparation) and paclitaxel (derived from the pacific yew tree) are examples of successful anticancer agents. Because of rapid development and employment of modern analytical equipment and technology, natural active components with anticancer activity have been studied and identified from Chinese medicinal plants. Cancer prevention and treatment using traditional Chinese medicines have attracted increasing interest and lay the foundation for developing new anticancer medicines.In this study, phytochemistry techniques were applied to prepare the extractions of ASMq, which were used for the anticancer study of mechanism of action.1. Preparation of ASMq aqueous extract: ASMq is composed of 10 kinds of herbal medicine. High quality herbs (1.42kg) were selected by a specialist, mixed according to the relevant recipe and prepared by a professional pharmacist. Briefly, herbs were minced and seeped in boiling water in the proportion of 1:10 (w/v) for 3 hours. This was repeated two additional times for 3 hours of boiling. The resulting crude extract was filtered and vacuum dried to powder (495.Og).2. Preparation of ASMq ethanol extract: High quality herbs (7.81kg) were selected by a specialist, mixed according to the relevant recipe and prepared by a professional pharmacist. Briefly, herbs were minced and extracted in 95% ethanol in the proportion of 1:6 (w/v) for 4 hours. This was repeated three additional times for 4 hours ofboiling. The resulting crude extract was filtered and removed the petroleum aether extract, and vacuum dried to powder (935.0g).3. Physical chemistry methods were used to identify the main chemicals in the aqueous extract of ASMq. The results demonstrated that it includes alkaloids, flavonoids, terpenes, coumarins, polysaccharides, etc.4. Aqueous extract of ASMq (475.Og) was extracted by petroleum aether, ethyl acetate and n-bothanol successively. This experiment resulted in petroleum aether extract (1.2g), ethyl acetate extract (8.6g), n-bothanol extract (35. lg) and water-soluble remains (425.Og).5. Ethanol extract of ASMq (467.5g) was extracted by ethyl acetate and n-bothanol successively. This experiment resulted in ethyl acetate extract (52.5g), n-bothanol extract (158.3g), and water-soluble remains (254.8g). The ethyl acetate extract was found to include rich of flavonoids.6. Total alkaloids, total flavonoids and total polysaccharides of ASMq were isolated by corresponding phytochemistry techniques.Part 2The effect of Abnormal Savda Munziq Aqueous and Ethanolextract on the proliferation, apoptosis and correlative geneexpression in human hepatoma (HepG2) cellsIn the present study, we used human hepatoma cells (HepG2) to examine the anticancer effects of ASMq aqueous extract to demonstrate and elucidate the mechanisms responsible for anti-proliferative and induction of apoptosis.Aqueous extract and ethanol extract of ASMq was tested on HepG2 proliferation (MTT assay), cell viability (neutral red assay), lactatedehydrogenase (LDH) release over 48 or 72 hours, respectively. Our results showed that ASMq aqueous and ethanol extract possess a significant (p<0.05) time- and concentration-dependent inhibition of HepG2 proliferation and viability, with increased cytoplasmic leakage.In order to clarify in greater detail the anti-cancer mechanisms of ASMq aqueous and ethanol extract, we also observed their inhibitory effects on cellular protein, DNA and RNA synthesis. Our experimental data suggested that, ASMq aqueous extract obviously decreased the intracellular level of DNA and protein synthesis at 24 h and 48 h incubation, respectively. Concentration-dependent manner was discovered at 24h incubation, while time-dependent manner was detected at low concentrations of ASMq (0.5, 1.0, and 2.5 mg/ml). As a matter of fact, it will be hypothesized from the data that the cellular DNA and protein synthesis inhibition effect seems to be accelerated by increasing concentration of ASMq and increasing exposure time to ASMq. All these results seem to reflect that one of the mechanisms for the anticancer action of ASMq is the inhibitory effect on cellular DNA and protein synthesis.In additionally, the genotoxicity of ASMq aqueous and ethanol extract were investigated using the DNA fragmentation assay by agarose gel electrophoresis. No DNA fragments were detected when analyzing control cultures and ASMq treatment cultures after 24 h incubation. However, DNA fragments (DNA ladders) showing varying sizes between 250 and 1000 base pairs were clearly visible after agarose gel electrophoresis. Significant DNA fragmentation was observed in HepG2 cells after 48h incubation with ASMq aqueous and ethanol extract in a concentration dependent manner.Apoptosis is now known as an important type of cell death in response to cytotoxic treatment. The administration of many natural compounds with anticancer effect has been shown to be capable of inducing theapoptotic death of cancer cells. To study the acting mechanism of ASMq aqueous and ethanol extract, flow cytometry was used to analyze the cell cycle distribution of HepG2 cells after 24 h and 48h treatment with different concentration (0.5-7.5mg/ml) of ASMq aqueous or ethanol extract. The results demonstrated that the ASMq aqueous and ethanol extract induced cell cycle arrest in sub-Gl phase, which represents apoptotic cells. The percentage of sub-Gl phase was increased in a time and concentration dependent manner after the treatment of ASMq aqueous or ethanol extract, indicating that the anticancer effect of ASMq extract on HepG2 cells was mediated by the induction of cell apoptosis.Many genes such as p53, p21, and genes in Bcl-2 family have been demonstrated to play important roles in deciding the initiation and execution of apoptosis in cancer cells exposed to anticancer drugs. To further investigate the molecular mechanism responsible for the ASMq aqueous or ethanol extract-induced apoptosis in HepG2 cells, the gene expression of some apoptosis-related genes such as p53, p21, Bax and Bcl-2 was analyzed by RT-PCR after 48h treatment. The results showed that ASMq aqueous extract (0.5-7.5mg/ml) caused the up-regulation of p53 and p21 gene expression in a concentration manner, while other genes were not affected. Expression of p53 and p21 increased and the expression of Bcl-2 decreased concentration-dependently in HepG2 cells after ASMq ethanol treatment, but no significant regulation on the expression of Bax.Caspase-3 activation is known to play a critical role in executing the apoptosis process induced by many anticancer agents. The enzymatic activity of Caspase-3, after cells were treated with ASMq aqueous and ethanol extract, increased concentration-dependently after 48h incubation. The relative intensity of Caspase-3 was 3.7, 4.5, 9.6 and 3.9, 3.7, 9.1 for cells treated with 2.5, 5.0 and 7.5mg/ml ASMq aqueous extract and ASMq ethanol extract, respectively.Otherwise, analysis of 5-methyldeoxycytosine (nrdC) and 8-hydroxy-2'- deoxyguanosine (8-OH-dG) were detected by HPLC-UV detection to estimate the epigenetic effects of ASMq aqueous and ethanol extract in HepG2 cells after 48h treatment. Compared to untreated control, ASMq aqueous and ethanol extract concentration-dependently inhibited the percentage of methylation ([m5dC/(m5dC + dC)]xlOO%) and oxidation ([8-OH-dG/l05dG]xl00%) (p<0.05). The percentage of m5dC was 27.0, 24.2,18.6% for ASMq aqueous extract, and 23.5, 20.2, 16.8% for ASMq ethanol extract, while the production of 8-OH-dG was 0.58, 0.45, 0.32% for ASMq aqueous extract, and 0.46, 0.36, 0.23% for ASMq ethanol extract at the concentration of 2.5, 5.0 and 7.5mg/ml, respectively.Taken together, the findings of the present study suggest that the anticancer activity of ASMq aqueous and ethanol extract relates to a cytotoxic effect as shown by the effect on cell viability and proliferation, LDH leakage and inhibition of protein, DNA and RNA synthesis in HepG2 cells. The apoptosis induced by ASMq aqueous and ethanol extract in HepG2 cells was associated with the induction of DNA fragmentation, cell cycle arrest in sub-Gl phase, regulation of Bcl-2, p53, p21 and Bax expression, stimulation of Caspase-3 activity and inhibition of m5dC and 8-OH-dG formation. The activity of ASMq ethanol extract on the proliferation, apoptosis and correlative gene expression of HepG2 cells was stronger than ASMq aqueous in these aspects.Of course, as a herbal preparation, ASMq aqueous or ethanol extract contains a variety of compounds such as alkaloids, flavonoids, polysaccharides, terpenes as well as coumarins, which may act individually or collectively on different pathways of cancer cell growth and survival. The molecular mechanisms responsible for the putative anticancer effect of ASMq and the isolation of its active components still need to be further investigated.Part 3The effect of fractions of Abnormal Savda Munziq Ethanolextract on the proliferation, apoptosis and correlative geneexpression in human hepatoma (HepG2) cellsIn the present study, MTT assay, DNA fragmentation assay, flow cytometry and RT-PCR methods were used to observe the effects of fractions and water soluble remains of Abnormal Savda Munziq Ethanol extract on the proliferation, apoptosis and correlative gene expression of HepG2 cells.The results of MTT assay demonstrated that ethyl acetate, n-bothanol extract and water soluble remains of ASMq ethanol extract showed different effects on HepG2 cell grows at 72h incubation, but it was clear that ethyl acetate extract was more efficient fraction than that of n-bolthanol extract and water soluble remains which was compared by IC50, they were 0.49, 1.56 and 2.87mg/ml for ethyl acetate, n-bothanol extract and water soluble remains of ASMq ethanol extract, respectively.DNA fragmentation observed by gel electrophoresis showed that there were no significant DNA ladders in HepG2 after 48h treatment with n-bothanol extract and water soluble remains of ASMq ethanol extract, and seems to be no detectable difference between control cultures. But ethyl acetate extract induced the typical pattern of DNA ladders in HepG2 cells at 7.5mg/ml.To study the acting mechanism of the fractions of ASMq ethanol extract, flow cytometry was used to detect the cell cycle distribution of HepG2 cells after 48h treatment with each fraction, respectively. The percentage of HepG2 cells in phase sub-Gl was concentration-dependently increased from 0.9 to 7.8% for ethyl acetate extract, from 0.6 to 2.9% for n-bothanol extract, and from 0.5 to 3.7% for water soluble remains, at the concentration from 2.5 to 7.5mg/ml.The gene expression of some apoptosis-related genes such as p53, p21, Bax and Bcl-2 was analyzed by RT-PCR. The results revealed that ethyl acetate extract concentration-dependently (2.5-7.5mg/ml) induced p53 and p21 up-regulation in HepG2 cells after 48 h incubation, but no regulation was observed in p53, p21, Bax and Bcl-2 expression in HepG2 cells treated with n-bothanol extract and water-soluble remains.In conclusion, ethyl acetate extract was the more effective anticancer fraction of ASMq ethanol extract than that of n-bothanol extract and water-soluble remains.Part 4The effect of Abnormal Savda Munziq Total Flavonoids onthe proliferation, apoptosis and correlative gene expressionin human hepatoma (HepG2) cellsFlavonoids are a group of natural products including flavones, flavanones, and isoflavones, and several beneficial biological activities of flavonoids including antioxidant, anticancer, and anti-inflammation properties have been identified in several previous studies. Our propose of the present study to examine the effects of Abnormal Savda Munziq total flavonoids on the proliferation, apoptosis and correlative gene expression of HepG2 cells, to elucidate the detailed mechanism responsible for the anticancer properties of Abnormal Savda Munziq total flavonoids, finally lay the foundation for developing new anticancer medicines.To study the potential anticancer effect of Abnormal Savda Munziq total flavonoids, we first analyzed the cell viability of HepG2 cells after Abnormal Savda Munziq total flavonoids treatment by MTT assay. The results showed that Abnormal Savda Munziq total flavonoids significantly decreased HepG2 cell viability after 72 h incubation. IC50was about 0,29 mg/ml, inhibition rate at 0.5mg/ml was about 80.2%. Indeed, the inhibition rate was ranged at a plateau about 80.2~~71.7% after the concentration of 0.5mg/ml. Because of the more marked effects of the lower concentrations, the concentration response curve was less apparent at our experimental conditions.The results of DNA fragmentation assay showed that Abnormal Savda Munziq total flavonoids induced remarkable DNA ladders in HepG2 cells after 48h incubation, but not concentration-dependently in concentrations ranging from 2.5 to 7.5 mg/ml.Flow cytometry detection of cell cycle demonstrated that incubated with increasing concentrations of ASMq total flavonoids ranged from 2.5 to 7.5 mg/ml, the percentage of sub-Gl phase in HepG2 cells significantly increased in a concentration-dependent manner from 9.3 to 12.8% at 48 h treatment, the results significantly different from untreated controls (0.4%).To further elucidate the molecular mechanism underlying the ASMq total flavonoids -induced apoptosis in HepG2 cells, we examined the expressions of p53, p21, Bax and Bcl-2 by RT-PCR analysis. The results showed that with increasing concentrations of ASMq total flavonoids ranged from 2.5 to 7.5mg/ml, the expressions of p53, p21 and Bax increased and the expression of Bcl-2 decreased concentration-dependently after 48 h treatment.Taken together, the present study suggested that the apoptosis triggered by ASMq total flavonoids was mediated through DNA fragmentation, cell cycle arrest at sub-Gl, 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 was possibly mediated through multiple pathways, suggesting multiple ingredients, rather than single component The acting ingredients in ASMq that exerted the anticancer effect may include total flavonoids which areabundant in ASMq. However, the detailed mechanism responsible for the anticancer effect of ASMq and the actual functional components still need to be further investigated in the near future. |
