Studies On The Anti-tumor Activity Against Human Lung Cancer Of Terpenoids From Taxus Cuspidate And Compositae Family

Part One The Anti-tumor Activity of Terpenoids from Taxus cuspidate and Two Plants from Compositae FamilyThe Taxol was isolated by Dr. Wall from Taxus brevifolia in 1971. The clinical activity of Taxol? (paclitaxel) against breast, ovarian and other carcinomas, as well as the unique mechanism of anticancer and poor solubility in water, have spurred a worldwide search for better sources and improved analogues of this drug. Taxol? has already become the biggest sale anticancer drug in the world. But limited source, low content and slowly growth seriously restrain the development and utilization of this wonderful drug. In order to reduce the damage to wild Taxus spp. resources and relieve the situation of limited supply of Taxol?, we explored this program to investigate the activity of components from Taxus cuspidata and two palnts from family of Compositae.Objective: To study the 16 components especially the non-taxanes in needles of Taxus cuspidata, and provide the foundation of searching for new anticancer leading compounds or Taxol? analoges, we detected the anti-growth activity in vitro and explored the relationship between the structure and the activity. To investigate more new anticancer components, we detected the anticancer activities of 18 sesquiterpenes in vitro from two palnts of Compositae family for obtaining the high-efficiency and low-toxin drugs.Methods: The cells were cultured in F12K medium (Applichem, Germany) containing 10% fetal bovine serum (FBS, Cellgro), penicillin (100u/ml), streptomycin (100μg/ml) and 2 mM glutamine in a humidified atmosphere with 5% CO2 at 37℃. The viability of cells treated with various chemicals was determined by a tetrazolium (MTT) assay performed in triplicate. A549 cells (1×104) were incubated in 100μl culture medium/well in 96-well plate for 12 h. Then the culture medium was exchanged by fresh medium containing various concentration of isoalantolactone (1μM, 10μM, 20μM, 40μM, 80μM) with positive control group containing cisplatin (Sigma, USA). After treatment with isoalantolactone for 12 h, 24 h, 48 h, 72 h, 10μL of 5 mg/mL MTT was added into each well respectively for another 4h. Finally, 150μL of stop solution (10 mL of 10% SDS, 6μL of 12N HCL) was added into each well, and the plate was placed in incubator with 5% CO2 at 37℃for 12 h. Absorbance at 570 nm was measured with a microplate reader using wells without cells as blanks (reference wavelength 490 nm). Cell survival was calculated from absorbance and presented as a percentage of the surviving cells.Results: Of 34 terpenes from Taxus cuspidata and Compositae, we found diterpenes includeding taxinine, 2α,10β-diacetyl-5-cinnamoyl-phototaxicin II and taxuspine A exhibited strong anticancer activity against human lung cancer cell lines A549, PC-6, QG-90 and QG-56. The proliferation inhibition rate of taxinine on A549 at different concentration 1μmol/L, 10μmol/L and 100μmol/L were 23.81%, 60.40% and 90.18%, respectively. The proliferation inhibition rate of 2α,10β-diacetyl-5- cinnamoyl-phototaxicin II on A549 at different concentration 1μmol/L, 10μmol/L and 100μmol/L were 18.31%, 48.73% and 68.19%, respectively. The IC50, the concentration to inhibit the proliferation of A549 by 50%, of taxuspine A was 9.54μmol/L.Among 18 sesquiterpenes from two palnts of Compositae family, we found that isoalantolanctone, eupatolide, 3β,9β-diacetoxy-1β,10α-epoxy- 11α,13-dihydrocostunolide, achillinin A and dehydrocostuslactone exhibited strong anticancer activity against human lung tumor cells A549, RERF-LC-jk and QG-56. The proliferation inhibition rate of dihydrodehydro-costuslactone on A549 cells was 90.75%.Conclusion: Three diterpenes from Taxus cuspidata and five sesquiterpenes from two palnts of Compositae family presented strong anticancer activity on human lung tumor cell lines in vitro. The results of anti-growth activity for the tested human lung tumor cell lines indicated that activities were closed related with C-5 cinnamoyl side chain.Part Two In vivo and In vitro Anti-human Lung Tumor Activity Studies of Isoalantolanctone from Inula helenium and Study on the Relationship between Structure and ActivityIn the past several decades, medicinal plants were regarded as important sources that could produce potential chemotherapeutic agents. There were plentful Compositae plants in China, riching of kinds of sesquiterpene lactones which had been used as folk medicines because of their exerted many pharmacologic properties. Inula helenium, a kind of Compositae plants, has been widely used as an herbal medicine in China, Japan and Europe to treat a number of diseases. Some pure compounds have been isolated from Inula helenium to prove its anticancer activity.Objective: To study the activity of isoalantolanctone on human lung cancer cell lines in vitro, we detected the anti-growth effect of isoalantolanctone on cell lines by MTT assay. To evaluate the cytotoxicity of isoalantolanctone on normal cell, we detected the effect of isoalantolanctone on CHL comparing with Cisplatin. Xenograft mice were created as a model system to study cytotoxicity effect of isoalantolactone in vivo.Methods: MTT assay was same as before. Tumor growth inhibition experiment in vivo was launched as following. Nude mice aged 6–8 weeks, weighed 18–22 g were kept under specific pathogen-free condition. The animals were s.c. implanted with 1×107 cells into the right mediodorsal flank. When the tumor reached 80–120 mm3 in volume, animals were divided randomly into 5 groups with 6 mice per group: control (intragastric administration of 1% sodium carboxymethylcellulose); positive control (intraperitoneal injection of cisplatin 2 mg/kg); test group for intragastric administration of isoalantolactone which floating in 1% sodium carboxymethylcellulose were divided to 3 groups: low dose group(1 mg/kg), middle dose group (10 mg/kg), high dose group (100 mg/kg). Daily administration of cisplatin and isoalantolactone was carried out from day 0 to 10. At the termination of experiment all animals were sacrificed, and tumors were dissected and weighed. The tumor inhibitory rates were calculated using the following formula: tumor inhibitory rate (%) = (mean tumor weight of the control mice—mean tumor weight of the treated mice)/mean tumor weight of the control mice×100%. Spleen indexs were calculated by formula: spleen index = mean spleen weight(mg)/mean body weight(g)×100%.Results: The results suggested that isoalantolactone had a strong anti-proliferative effect on A549 cells. The IC50 of isoalantolanctone on A549 cells, QG-56 cells and U251SP were 3.01μmol/L, 9.39μmol/L and 10.55μmol/L, respectively. The proliferation inhibition rate of isoalantolanctone on CHL cells at 100μmol/L was 46.33%.At the end of experiment, the mean weight of the different groups were 27.5g(control group), 26.6 g (high dose test group),27 g (middle dose test group),27.2 g (low dose test group), 26.5 g (positive control group), respectively. The mean tumor weight of the control mice was 0.53 g. The mean tumor weights of the mice treated with various concentration of isoalantolactone, 100 mg/kg, 10 mg/kg and 1 mg/kg, were 0.38 g, 0.44 g, 0.47 g, respectively. The tumor inhibitory rates were 28.3%, 17%, 11.4% respectively. The mean tumor weight and tumor inhibitory rates of the mice treated with cisplatin were 0.42 g and 20.8%. Spleen index in isoalantolanctone (high dose group to low dose group) were 5.19, 6.51, 5.12 respectively, which were bigger than in cisplatin group (3.37).Conclusion: MTT assay revealed that isoalantolactone had a strong anti-proliferative effect on A549 cells in a concentration-depent manner and time-depent manner. Comparison of CHL cell treatment with cisplatin and isoalantolactone, we concluded that isoalantolactone inhibited CHL cell not more significant than cisplatin.Our investigations showed that there was an apparently dose-dependent inhibition of tumor growth in the treated mice and isoalantolanctone suppress immune system of mice weaker than cisplatin. No body weight decrease different in treated mice compared with control mice. The results clearly demonstrated that the anti-tumor activities have close relationship with the structures,α,β-unsaturated five-membered lactone ring is necessary for the activities, whenα,β-unsaturated five-membered lactone become unsaturated led to the looses or decrease of anti-growth activity.Part Three The Mechanism of Isoalantolanctone Anticancer Activity on the Lung CancerIt had been confirmed that many anticancer drugs could induce apoptosis of kinds of tumor cells. Apoptosis could also be induced by radiotherapy, thermotherapy and biological agent in clinic. Chinese medicinal herbs contained many anticancer compounds which were proved to induce apoptosis.Objective: To explore the mechanism of isoalantolanctone anti-lung cancer, given isoalantolanctone induced apoptosis. To detecte cell cycle arrested in A549 cells treated with isoalantolanctone and expressions of proteins related with signal pathway.Methods: 1. Acridine orange stained A549 cells treated with isoalantolanctone at concentration 10μmol/L, 20μmol/L and 40μmol/L. The photo were recorded with fluorescence microscope. 2. Apoptosis and cell cycle of A549 were determined by flow cytometry analysis. Cells were cultured in F12K medium in 100-mm dishes and treated with isoalantolactone at concentration 20μmol/L, 40μmol/L and 80μmol/L, and 24 h later a total of 1×106 cells were harvested. The stained cell samples were analyzed using a FACScan instrument. 3. Apoptotic DNA fragments were detected in A549 cell treated with isoalantolactone. Briefly, 3×105 cells were seeded in 60-mm dishes containing various concentration of isoalantolactone (5μmol/L, 10μmol/L, 20μmol/L). 48h later, all cells were harvested by trypsogen and were lysed. DNA were extracted and applied to 1% agarose gel containing 0.5μg/ml ethidium bromide, and electrophoresed in 90 mM Tris-borate (pH 8.0) and 2 mM EDTA at 50 V for 2 h. DNA ladders were photographed under UV illumination. 4. A549 cells in 100-mm dishes (1×106/dish) were treated with isoalantolactone at various concentration (5μmol/L, 10μmol/L, 20μmol/L, 40μmol/L) for 48h. After treatment, cells were collected and resuspended in 100μL Gold lysis buffer. Equal amounts of proteins (30μg) were mixed with 2×sample buffer and separated by electrophoresis on 12% (w/v)SDS-PAGE forβ-actin, Bcl-2, Bax, caspase-3, Akt, HSP90α, p53 and p21 detection. Resolved proteins were then transferred to NC membranes which probed with appropriate dilution of primary antibodies and secondary antibodies. Chemiluminescent photographic detected the expression of proteins.5. A549 cells with isoalantolactone treatment were cultured in the presence or absence of caspase inhibitors, Z-VAD-FMK. Cell viability was determined by MTT assay as described previously. Cells (1×104/well) were seeded in 100μl F12K medium in 96-well plate treated with isoalantolanctone at concentration 1μmol/L, 10μmol/L, 50μmol/L and 100μmol/L. After isoalantolactone treatment with or without Z-VAD-FMK (20μmol/L)for 48h, cell viability was measured at 570 nm absorbance wave by a microplate reader.Results: 1. Morphology changes of apoptosis were indicated by acridine orange staining. Compared to the untreated cells, A549 cell population in test group decreased and there were morphological characteristics of apoptosis included cell shrinkage, nuclear condensation and formation of apoptotic bodies in A549 cells. 2. A distinct increase in the population of cells in the sub-G1 phase was observed in the cells treated with isoalantolactone. The percentages of apoptotic cells after treating with 0μmol/L, 10μmol/L, 20μmol/L, 40μmol/L of isoalantolactone for 48 h were 0.85%, 0.82%, 6.59%, 15.32%, respectively. Percent of A549 cells in G1 stage treated with isoalantolactone 0μmol/L, 10μmol/L, 20μmol/L and 40μmol/L were 33.4%, 37.7%, 57.3%, 60.2%, respectively. 3. DNA fragmentation analysis indicated that short fragments of DNA, integral times of 180 bp-200 bp, was observed in A549 cells treated with isoalantolactone at various concentration. 4. Both Akt and HSP90αproteins expressed stable in the cells treated with isoalantolactone at different concentration. Caspase-3, Bax, p53 and p21 increased but Bcl-2 decreased in a concentration-dependent manner. 5. A549 cells were cultured mixed with isoalantolanctone at concentration 1μmol/L, 10μmol/L, 50μmol/L and 100μmol/L. 48 h later, MTT assay suggested cell viability were 81.01%, 31.03%, 22.00%, 13.96%, respectively. But added with Z-VAD-FMK (20μmol/L), cell viability were 93.34%, 68.76%, 74.80% and 70.14%.Conclusion: 1. A549 cells were induced apoptosis by isoalantolanctone and arrested in G1 stage. 2. P53 was increasely stimulated in A549 cells with isoalantolanctone. Followed P53, P21 and Bax increased and Bcl-2 decreased leading to cells arrested in G1 stage and caspase-dependent apoptosis induced. 3. There were different principles in isoalantolanctone, which arrested cells in G1 stage, and Taxol on A549 cells arrested cells in M stage. 4. The results showed there was no relationship with PI3K/Akt in A549 cells with isoalantolanctone.