ABSTRACT
Phenylalanine ammonia-lyase (PAL) is the key entry enzyme of plant phenylpropanoid pathway. It plays an important role in the biosynthesis of podophyllotoxin, an anti-tumor lignan that is currently produced from its main natural source Sinopodophyllum hexandrum (Royle) Ying. In this study, we cloned the gene ShPAL encoding phenylalanine ammonia-lyase by RT-PCR from the root of S. hexandrum ecotype inhabited in the Aba' district, Sichuan, based on its public SRA transcriptome data-package. Bioinformatics analyses showed that the ShPAL-encoded protein is composed of 711 amino acids, contains the conserved domains of PAL, and has the signature motif within the active center of aromatic ammonia-lyases. Moreover, ShPAL protein was predicted to have a secondary structure mainly composed of α-helix and random coil, a typical 'seahorse' shape monomer tertiary structure, and a homologous tetramer three-dimensional structure by Swiss-Modelling. The phylogenetic lineage analysis indicated ShPAL was of the highest sequence identity and the shortest evolutionary distance with the PAL of Epimedium sagittatum from the same Berberidaceae family. Subcellular localization experiments showed that ShPAL protein was mainly distributed in the cytoplasm, despite of a minority on the endoplasmic reticulum membrane. Furthermore, ShPAL protein was recombinantly expressed in Escherichia coli and purified by histidine-tag affinity chromatography. Its enzymatic activity was determined up to 20.91 U/mg, with the optimum temperature of 41 ℃ and pH of 9.0. In contrast, the enzyme activity of its F130H mutant decreased by about 23.6%, yet with the same trends of change with temperature and pH, confirming that phenylalanine at this position does affect the substrate specificity of PAL. Both the wild type and the mutant have relatively poor thermostability, but good pH-stability. These results may help to further investigate the regulatory role of PAL in the process of podophyllotoxin biosynthesis and advance the heterologous synthesis of podophyllotoxin to protect the germplasm resource of S. hexandrum. They also demonstrate that ShPAL has a potential application in biochemical industry and biomedicine.
Subject(s)
Phenylalanine Ammonia-Lyase/metabolism , Podophyllotoxin , Phylogeny , Cloning, MolecularABSTRACT
Podophyllotoxin (PTOX) is an aryl-tetralin lignan of plant origin found in some species of Podophyllum such as Dysosma versipellis, Diphylleia sinensis, and Sinopodophyllum hexandrum. Etoposide and teniposide are produced semisynthetically from PTOX and used clinically to treat several forms of cancer. As a typical representative of new drug discovery from natural products, the production of PTOX solely depends on extraction from plants, resulting in severe contradiction between supply and demand. With the advantages of unconstrained resources and eco-friendly reaction conditions, biosynthesis method has become a trend in the production of PTOX and its derivatives. In this review, we summarize the research progress of PTOX biosynthesis in plants and expound the functions of the key enzymes as well as their subcellular location. The synthetic biology for production of PTOX intermediates in a tobacco chassis is also introduced. Finally, the heterologous expression and biotransformation of PTOX in microorganisms is summarized, which sets the foundation for the efficient microbial production of PTOX using cell factories.
Subject(s)
Genes, Plant , Podophyllotoxin/biosynthesis , Podophyllum/geneticsABSTRACT
According to drug design flattening principle,a series of novel indole podophyllotoxin derivatives which were introduced different indole substituents in C-4 position on the basis of podophyllotoxin nucleus were synthesized with the starting material podophyllotoxin and 1 H-indole-5-carboxylic acid. Its anti-tumor activity in vitro was tested in order to screen for high-efficiency and low-toxic compounds. Six target compounds were synthesized,and were confirmed by~1 H-NMR,~(13)C-NMR,HR-ESI-MS and melting point determination analysis. All these target compounds were not reported by previous literature. Using etoposide as positive control drug,all the target compounds were screened for cytotoxicity against He La cells,K562 cells and K562/A02 cell in vitro by MTT method. The antitumor activity screening results showed that compounds 4 b,4 e,4 f exhibited higher inhibitory rate against He La cells and K562 cells than those of control drug VP-16. This route has the advantages on simple operation and reasonable design,provides some practical reference value for the further development on the structure modification of podophyllotoxin and study on anti-tumor activity.
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Drug Screening Assays, Antitumor , HeLa Cells , Indoles , Pharmacology , K562 Cells , Podophyllotoxin , Pharmacology , Structure-Activity RelationshipABSTRACT
Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 μg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.
Subject(s)
Ascomycota , Berberidaceae , Chemistry , Endophytes , Plant Roots , Podophyllotoxin , Tissue Culture TechniquesABSTRACT
The therapeutic application of deoxypodophyllotoxin (DPT) is limited due to its poor water solubility and stability. In the present study, the micelles assembled by the amphiphilic block copolymers (mPEG-PDLLA) were constructed to improve the solubility and safety of DPT for their in vitro and in vivo application. The central composite design was utilized to develop the optimal formulation composed of 1221.41 mg mPEG-PDLLA, the weight ratio of 1 : 4 (mPEG-PDLLA : DPT), 30 mL hydration volume and the hydration temperature at 40 °C. The results showed that the micelles exhibited uniformly spherical shape with the diameter of 20 nm. The drug-loading and entrapment efficiency of deoxypodophyllotoxin-polymeric micelles (DPT-PM) were about (20 ± 2.84)% and (98 ± 0.79)%, respectively, indicating that the mathematical models predicted well for the results. Compared to the free DPT, the cytotoxicity showed that blank micelles possessed great safety for Hela cells. In addition, the DPT loaded micelle formulation achieved stronger cytotoxicity at the concentration of 1 × 10 mol·L, which showed significant difference from free DPT (P < 0.05). In conclusion, the micelles were highly promising nano-carriers for the anti-tumor therapy with DPT.
Subject(s)
Humans , Antineoplastic Agents , Chemistry , Toxicity , Cell Survival , Drug Carriers , Chemistry , Drug Delivery Systems , Methods , Drug Design , HeLa Cells , Micelles , Particle Size , Podophyllotoxin , Chemistry , Toxicity , Polyesters , Chemistry , Polyethylene Glycols , Chemistry , Solubility , Surface PropertiesABSTRACT
The therapeutic application of deoxypodophyllotoxin (DPT) is limited due to its poor water solubility and stability. In the present study, the micelles assembled by the amphiphilic block copolymers (mPEG-PDLLA) were constructed to improve the solubility and safety of DPT for their in vitro and in vivo application. The central composite design was utilized to develop the optimal formulation composed of 1221.41 mg mPEG-PDLLA, the weight ratio of 1 : 4 (mPEG-PDLLA : DPT), 30 mL hydration volume and the hydration temperature at 40 °C. The results showed that the micelles exhibited uniformly spherical shape with the diameter of 20 nm. The drug-loading and entrapment efficiency of deoxypodophyllotoxin-polymeric micelles (DPT-PM) were about (20 ± 2.84)% and (98 ± 0.79)%, respectively, indicating that the mathematical models predicted well for the results. Compared to the free DPT, the cytotoxicity showed that blank micelles possessed great safety for Hela cells. In addition, the DPT loaded micelle formulation achieved stronger cytotoxicity at the concentration of 1 × 10 mol·L, which showed significant difference from free DPT (P < 0.05). In conclusion, the micelles were highly promising nano-carriers for the anti-tumor therapy with DPT.
Subject(s)
Humans , Antineoplastic Agents , Chemistry , Toxicity , Cell Survival , Drug Carriers , Chemistry , Drug Delivery Systems , Methods , Drug Design , HeLa Cells , Micelles , Particle Size , Podophyllotoxin , Chemistry , Toxicity , Polyesters , Chemistry , Polyethylene Glycols , Chemistry , Solubility , Surface PropertiesABSTRACT
OBJECTIVE@#To test the acute and chronic toxicity of topical application of 0.5% podophyllotoxin-loaded nanostructured lipid carriers (POD-NLC) to the vaginal mucosa.@*METHODS@#Twelve New Zealand rabbits were randomized into 3 groups and subjected to daily topical applications of normal saline (control group), 0.5% podophyllotoxin tincture (POD-T) or 0.5% POD-NLC on the vaginal mucosa for 10 consecutive days, and the pathological changes in the mucosa were graded using the Eckstein scoring system.The acute toxicity of POD-NLC was tested in 20 SD female rats, which received intravaginal administration of POD-NLC or vehicle for 3 times within 24 h; After 14 days of continuous observation, the rats were dissected for calculating the viscera coefficient.For testing the chronic toxicity of POD-NLC, 80 SD female rats were randomized into 4 groups and subjected to daily intravaginal administration of the vehicle or POD-NLC at low, moderate or high doses for 13 consecutive weeks.The rats were weighed once a week and at the end of the experiment, 2/3 of the rats from each group were sacrificed to collect blood samples, calculate the viscera coefficient, and examine the pathological changes in the liver.The remaining 1/3 rats were observed for another 2 weeks without further drug treatment and the same examinations were performed.@*RESULTS@#In the rabbits, 0.5% POD-NLC elicited only mild irritation while POD-T caused moderate irritation of the vaginal mucosa.In the acute toxicity test, the organ coefficients were comparable between the rats treated with the vehicle and POD-NLC (>0.05).Long-term intravaginal administration of POD-NLC did not produce significant changes in the behavior, activity, body weight, blood biochemical profiles or organ coefficient as compared with the vehicle control group (>0.05).@*CONCLUSIONS@#Intravaginal administration of 0.5% POD-NLC causes very mild irritation without obvious acute or chronic toxicity to the vaginal mucosa in rabbits and rats.
Subject(s)
Animals , Female , Rabbits , Rats , Administration, Intravaginal , Liposomes , Mucous Membrane , Nanostructures , Toxicity , Podophyllotoxin , Toxicity , Random Allocation , VaginaABSTRACT
El virus del papiloma humano (VPH) es una de las enfermedades de transmisión sexual más comunes. Puede afectar tanto el aparato genital masculino y femenino, como también el área perianal, ano, y diversas áreas de cabeza y cuello y otorrinolaringológicas, ya sea como lesiones benignas o como promotor de lesiones malignas. Las lesiones benignas por VPH en genitales masculinos se caracterizan fundamentalmente por la aparición de lesiones verrugosas, aunque también puede manifestarse mediante lesiones planas atípicas. En algunos casos hay ausencia de lesiones macroscópicamente visibles que pueden hacerse evidentes con la prueba de ácido acético. La biopsia de la lesión, su evaluación anatomopatológica y, sobre todo, la determinación de la existencia y el tipo de virus involucrado mediante PCR (reacción en cadena de la polimerasa) permiten confirmar el diagnóstico. En algunas ocasiones es necesario realizar una cistoscopia para diagnosticar lesiones intrauretrales y vesicales. Los tratamientos propuestos son muy variados y de eficacia dispar, desde las topicaciones y la electrocirugía o la criocirugía, hasta el empleo de la tecnología láser. La prevención con el uso de protección durante el acto sexual así como la educación sexual son fundamentales. En los últimos 10 años se ha implementado el uso de la vacuna para el VPH en niñas con el fin de disminuir la incidencia de lesiones de alto grado y de cáncer de cuello uterino, pero su indicación en varones es menos clara y aún no ha sido consensuada. (AU)
Human papiloma virus (HPV) is one of the most common sexual transmitted diseases. It can affect the male genitalia, as well as the perianal and anal regions and multiple areas of the head and neck and otorhinolaryngological structures, as benign lesiones or as a promoter of malignant lesions. Benign male genitalia lesions are characterized mainly by verrucous lesions, although flat atypical lesions can be found, as well as the abscence of macroscopic visible lesions that in some cases can become evident using the acetic acid test. Lesion biopsy, its histological evaluation, and the determination of the existence and type of virus using PCR (Polymerase Chain Reaction) can confirm the diagnosis. In some cases is necessary to do a cistoscopy to diagnose intraurethral and vesical lesions. Proposed treatments are varied and with a wide range of efficacy, from topications to electro or cryosurgery, and the use of laser technology. Sexual education and the use of sexual protection are essential in prevention. In the last 10 years the use of VPH vaccine in girls was widely spread, in order to decrease the incidence of high grade lesions and cervix cancer. Its indication in male patients is less clear and not yet consented among specialists. (AU)
Subject(s)
Humans , Male , Papillomavirus Infections/therapy , Reproductive Tract Infections/therapy , Podophyllin/therapeutic use , Podophyllotoxin/therapeutic use , Sex Education , Trichloroacetic Acid/therapeutic use , Condylomata Acuminata/etiology , Polymerase Chain Reaction , Condoms , Papillomavirus Infections/diagnosis , Papillomavirus Infections/pathology , Papillomavirus Infections/prevention & control , Papillomavirus Infections/drug therapy , Alphapapillomavirus/pathogenicity , Papillomavirus Vaccines/therapeutic use , Laser Therapy , Reproductive Tract Infections/diagnosis , Reproductive Tract Infections/etiology , Reproductive Tract Infections/pathology , Reproductive Tract Infections/drug therapy , Fluorouracil/administration & dosage , Fluorouracil/therapeutic use , Lidocaine/administration & dosage , Lidocaine/therapeutic useABSTRACT
OBJECTIVE: The aim of this study was to determine the efficacy of 252Californium neutron intracavitary brachytherapy using a two-channel Y applicator combined with external beam radiotherapy for the treatment of endometrial cancer. METHODS: Thirty-one patients with stage I-III endometrial cancer were recruited for this study. The stage I patients received only 252Californium neutron intracavitary brachytherapy with a two-channel applicator. The stage II and III patients received both 252Californium neutron intracavitary brachytherapy using a two-channel applicator and parallel-opposed whole pelvic radiotherapy. RESULTS: The five-year local control rate was 80.6% (25/31), the overall survival rate was 51.6% (16/31), and the disease-free survival rate was 54.8% (17/31). The incidence of serious late complications was 12.9% (4/31). CONCLUSIONS: 252Californium neutron intracavitary brachytherapy using a two-channel applicator combined with external beam radiotherapy was effective for treating endometrial cancer and the incidence of serious late complications related to this combination was within an acceptable range.
Subject(s)
Adult , Aged , Female , Humans , Middle Aged , Adenocarcinoma/radiotherapy , Brachytherapy/methods , Californium/therapeutic use , Endometrial Neoplasms/radiotherapy , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Brachytherapy/instrumentation , Combined Modality Therapy , Carmustine/therapeutic use , Cytarabine/therapeutic use , Endometrial Neoplasms/mortality , Endometrial Neoplasms/pathology , Follow-Up Studies , Melphalan/therapeutic use , Podophyllotoxin/therapeutic use , Survival Rate , Treatment OutcomeABSTRACT
Podophyllotoxone (1) was isolated from the roots of Dysosma versipellis. The structure was determined by spectroscopic analysis in combination with single-crystal X-ray analysis. The absolute configuration of compound 1 was assigned based on the Flack parameter. It showed significant inhibitory activities against human prostate cancer cells PC3 and DU145 with IC50 values being 14.7 and 20.6 μmol·L(-1), respectively. It also arrested the cells at G2/M phase. Tubulin polymerization assay showed that it inhibited the tubulin polymerization in a dose-dependent manner, and molecular docking analysis revealed a different binding mode with tubulin as compared with those known tubulin inhibitors.
Subject(s)
Humans , Male , Antineoplastic Agents , Chemistry , Pharmacology , Berberidaceae , Chemistry , Cell Line, Tumor , Dose-Response Relationship, Drug , G2 Phase , Hydrogen Bonding , Molecular Conformation , Molecular Docking Simulation , Phytotherapy , Plant Extracts , Chemistry , Pharmacology , Plant Roots , Chemistry , Podophyllotoxin , Chemistry , Pharmacology , Polymerization , Prostatic Neoplasms , Drug Therapy , Tubulin , Tubulin Modulators , PharmacologyABSTRACT
In order to find the optimal topographical factor for regionslization, the content of cimetidine in 116 Sinopodophyllum hexandrum sample collected from Sichuan, Qinghai, Gansu, Tibet, Yunnan and Shaanxi provinces, was determined. Using mathematical statistics and geographical spatial analysis of GIS analysis, the relationship between content of podophyllotoxin and influencing factors including altitude gradient and gradient position was analyzed. It is found that the optimal altitude was 2 800 m to 3 600 m, the aspect of slope north or northeast and northwest and the slope 12 degrees to 65 degrees with a high suitability degree. Considering the artificial planting, the suitable planting area for S. hexandrum is comfirmed. The topographical factor is important for S. hexandrum regionalization, but has hardly effect on podophyllotoxin content. The results of the study provide an important scientific basis for S. hexandrum production development. But there are many factors which affect suitability index and podophyllotoxin content of S. hexandrum, it is necessary to consider other factors like climate and soil while exploitation and protection of S. hexandrum.
Subject(s)
Altitude , Berberidaceae , Chemistry , China , Ecosystem , PodophyllotoxinABSTRACT
To explore the correlation between the ecological factors and the contents of podophyllotoxin and total lignans in root and rhizome of Sinopodophyllum hexandrum, podophyllotoxin in 87 samples (from 5 provinces) was determined by HPLC and total lignans by UV. A correlation and regression analysis was made by software SPSS 16.0 in combination with ecological factors (terrain, soil and climate). The content determination results showed a great difference between podophyllotoxin and total lignans, attaining 1.001%-6.230% and 5.350%-16.34%, respective. The correlation and regression analysis by SPSS showed a positive linear correlation between their contents, strong positive correlation between their contents, latitude and annual average rainfall within the sampling area, weak negative correlation with pH value and organic material in soil, weaker and stronger positive correlations with soil potassium, weak negative correlation with slope and annual average temperature and weaker positive correlation between the podophyllotoxin content and soil potassium.
Subject(s)
Berberidaceae , Chemistry , China , Chromatography, High Pressure Liquid , Climate , Drugs, Chinese Herbal , Chemistry , Ecosystem , Lignans , Chemistry , Podophyllotoxin , Chemistry , Soil , Chemistry , TemperatureABSTRACT
Linum album, a herbaceous and medicinal plant, has been shown to accumulate anti-tumor podophyllotoxin [PTOX] and related lignans. In this study, we have verified the time-course of cell growth, lignan production in cells treated with chitosan and chitin oligomers. To study mechanism of chitosan and chitin oligomers action, expression of phenylalanine ammonio-lyase [PAL], cinnamoyl-CoA reductase [CCR], cinnamyl alcohol dehydrogenase [CAD] and pinoresinol lariciresinol reductase [PLR] genes were investigated. The cells were treated with 100 mg l[-1] chitin and chitosan oligomers. Cells were harvested for 5 days after elicitation. lignans quantified by reverse-phase high-performance liquid chromatography [HPLC]. Cell samples frozen with liquid N2 were used to elucidate the expression level of genes by quantitative PCR [qPCR]. Cells elicited with chitosan hexamer for 5 days yielded the highest amount of PTOX [73.5 micro g/g dry weight] and lariciresinol [96 micro g/g DW] which were about 3-fold and 2-fold higher than control, respectively. The expression of genes by oligomers was increased, reaching a peak at 3 day after treatment. Chitosan and chitin oligomers up-regulate the production of PTOX, by effecting on gene expression of PTOX biosynthesis pathway
Subject(s)
Chitosan , Chitin , Gene Expression , Lignans , Cell Culture Techniques , PodophyllotoxinABSTRACT
<p><b>OBJECTIVE</b>To investigate the anti-tumor effect of ZM-66 on multidrug-resistant leukemic cell line K562/ADM.</p><p><b>METHODS</b>The K562/ADM cells were treated with varying concentrations (0, 1, 2, 4 × 10⁻³ mmol/L) of ZM-66 or etoposide for 24 hours. The proliferation was detected by Sulforhodamine B Sodium Salt (SRB) assay and apoptosis was detected by flow cytometry analysis and fluorescent staining. In addition, the expression levels of p53 and bax genes in K562/ADM cells were detected by RT-PCR analysis. The level of P-glycoprotein (P-gp), P53 and Bax protein in K562/ADM cells were detected by Western blot assay.</p><p><b>RESULTS</b>SRB assay demonstrated that etoposide had little inhibitory effect on K562/ADM cells, whereas ZM-66 (1, 2, 4 × 10⁻³ mmol/L) had significantly inhibitory effect on K562/ADM cells (all P<0.01). The acridine orange/propidium iodide dual staining showed that there were typical condensation of chromatin and nuclear fragmentation nuclei with red color in ZM-66 treated cells. Flow cytometric analysis showed that there was a significantly increase of apoptotic cells in K562/ADM cells after treated with ZM-66. RT-PCR showed that the p53 and bax mRNA expression levels in K562/ADM cells treated with ZM-66 at 1, 2, 4 × 10⁻³ mmol/L were higher than those in the cell without treatment. Western blot showed that the P53 and Bax protein expression levels in K562/ADM cells treated with ZM-66 at 2, 4 × 10⁻³ mmol/L were higher than those in the cell without treatment. But the P-gp protein expression level in K562/ADM cells treated with ZM-66 at 2, 4 × 10⁻³ mmol/L was gradually lower than those in the cell without treatment.</p><p><b>CONCLUSION</b>ZM-66 is able to induce cell death by apoptosis in vitro, as a result of the reverse of the apoptosis resistance in drug-resistant K562/ADM cells by modulating expression of key factors associated with apoptosis induction.</p>
Subject(s)
Humans , Apoptosis , Blotting, Western , Cell Proliferation , Flow Cytometry , K562 Cells , Podophyllotoxin , Chemistry , Pharmacology , RNA, Messenger , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To explore the mechanism of podophyllotoxin nanostructured lipid carriers (POD-NLC)-induced apoptosis of VK2/E6E7 cells mediated by endoplasmic reticulum stress (ERS).</p><p><b>METHODS</b>VK2/E6E7 cells cultured in vitro were exposed to 0.125, 0.25, and 0.5 µg/ml POD-NLC or blank NLC for 24 h. The intracellular calcium concentration was measured by laser scanning confocal microscopy (LSCM), and the expression levels of GRP78, GRP94, and calpain2 mRNA and proteins in the cells were detected using RT-PCR and Western blotting.</p><p><b>RESULTS</b>Compared with the control cells, the cells exposed to POD-NLC showed a concentration-dependent increase of intracellular calcium concentration (P<0.01), and the differences were statistically significant between different dose groups (P<0.05). RT-PCR and Western blotting showed that POD-NLC up-regulated GRP78, GRP94 and calpain2 mRNA and proteins expressions, which showed significant differences between blank-NLC and the control groups (P>0.05).</p><p><b>CONCLUSION</b>POD-NLC induces apoptosis of VK2/E6E7 cells possibly by triggering the endoplasmic reticulum stress response.</p>
Subject(s)
Humans , Apoptosis , Calcium , Metabolism , Calpain , Metabolism , Cell Line , Endoplasmic Reticulum Stress , HSP70 Heat-Shock Proteins , Metabolism , Heat-Shock Proteins , Metabolism , Membrane Proteins , Metabolism , Nanostructures , Chemistry , Podophyllotoxin , Chemistry , RNA, MessengerABSTRACT
<p><b>OBJECTIVE</b>To investigate the therapeutic value of inhibiting the expression of insulin-like growth factor-I receptor (IGF-IR) using picropodophyllin (PPP) by studying the effects on proliferative and metastatic potentials of human hepatocellular carcinoma (HCC) using an in vitro cultured cell system.</p><p><b>METHODS</b>IGF-IR expression in human HCC cell lines (Bel-7404, Bel-7402, HepG2, and Huh-7) and human hepatocytes (L02) was assessed at baseline (pre-treatment) and after PPP treatment by western blotting. Changes in cell cycle were analyzed by flow cytometry and in cell viability by sulforhodamine B staining. Early apoptosis was detected by annexin-V/FITC and propidium iodide double-staining assay. Caspase-3/7 activity was suppressed by z-VAD-FMK and analyzed by homogeneous luminescence assay. Effects on cell motility were tested by wound-scratch test. Between-group differences were assessed by t-test or one-way analysis of variance.</p><p><b>RESULTS</b>IGF-IR was markedly up-regulated in all HCC cell lines (vs. non-hepatoma hepatocytes). HCC cells with PPP-inhibited IGF-IR showed time-dependent decreases in cell motility and viability. After treatment with PPP for 24 hours, the proportion of HCC cells in G1 phase was 2.1% +/- 0.4%, in S phase was 11.0% +/- 0.7%, and in G2/M phase was 87.1% +/- 0.6%, and no healing was observed in the wound-scratch assay. The PPP treatment induced cell apoptosis, as evidenced by enhanced caspase-3/7 activity; the proportion of annexin-V+/PI- cells was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (16.4% +/- 0.4% vs. 5.8% +/- 0.2%, t = 14.05, P less than 0.01). After z-VAD-FMK treatment, the apoptosis rate was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (11.3% +/- 0.7% vs. 5.8% +/- 0.2%, t = 11.83, P less than 0.01).</p><p><b>CONCLUSION</b>IGF-IR is associated with proliferation, cell motility, and apoptosis of HCC cells, and may be a promising molecular target for HCC.</p>
Subject(s)
Humans , Apoptosis , Carcinoma, Hepatocellular , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Liver Neoplasms , Metabolism , Pathology , Podophyllotoxin , Pharmacology , Receptor, IGF Type 1 , MetabolismABSTRACT
The purpose of this study is to investigate the intracellular transporters effect and the cytotoxicity of carboxyl nanodiamond (CND) - podophyllotoxin (PPT). Nanodiamond (ND) was treated with mixed carboxylic acid and finally got 64 nm CND by centrifugation, and then it was reacted with PPT to form CND-PPT. UV spectrophotometry was used to calculate the content of PPT in CND-PPT, the particle size distribution and zeta potential were measured by Dynamic laser scattering instrument. CND, PPT, CND-PPT and CND + PPT (physical mixture of CND and PPT) were characterized by Fourier transform infrared spectroscopy, at the same time, thermal analysis and element analysis were used to estimate the content of the PPT in CND-PPT. The affect of CND, PPT, CND-PPT on HeLa cell was measured with MTT assay. The results showed that content of PPT combined with CND accounted for about 10%. MTT assay showed that CND has low cytotoxicity and CND-PPT can increase the water soluble of PPT. As a conclusion, CND as a hydrophilic pharmaceutical carrier combined with PPT is able to increase the water solubility of PPT, at low concentration, CND-PPT can enhance the antitumor activity in comparison with PPT, so CND can be used as a potential anticancer drug carrier.
Subject(s)
Humans , Antineoplastic Agents, Phytogenic , Chemistry , Pharmacology , Carboxylic Acids , Chemistry , Drug Carriers , HeLa Cells , Nanodiamonds , Chemistry , Particle Size , Podophyllotoxin , Chemistry , Pharmacology , Solubility , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform InfraredABSTRACT
No abstract available.
Subject(s)
Amaurosis Fugax , Antineoplastic Combined Chemotherapy Protocols , Blindness , Cyclophosphamide , Doxorubicin , Neurilemmoma , Podophyllotoxin , VincristineABSTRACT
<p><b>OBJECTIVE</b>To determine the content of podophyllotoxin and total lignans in Sinopodophyllum emodi Ying from different areas and evaluate the resource utilization of this endangered medicinal plant.</p><p><b>METHOD</b>HPLC and UV spectrophotometry were used to determine the content of podophyllotoxin and total lignans in 126 samples from different habitats and the total lignans content was determined by the reference wavelength method.</p><p><b>RESULT</b>According to the results, the highest content was determined from the samples from Yongdeng Nature Reserve in Gansu province, and the lowest ones was found in the samples from Tibet. The former's podophyllotoxin and total lignans contents were 7.40% and 20.66%, respectively, which were 19 times and 4 times more than those of the latter. The content of podophyllotoxin and total lignans in S. emodi were significantly positively related, meanwhile, samples from both low altitude and high latitude showed the higher content.</p><p><b>CONCLUSION</b>The two determination methods are simple, rapid, accurate and repeatable. It is more scientific and rational to evaluate the resource utilization of S. emodi with two indicators, those are the content of podophyllotoxin and the content of total lignans. This paper is instructive to the collection of wild resources and the establishment of production bases.</p>
Subject(s)
Berberidaceae , Chemistry , Chromatography, High Pressure Liquid , Lignans , Podophyllotoxin , Spectrophotometry, UltravioletABSTRACT
This study is to investigate the antitumor activity of CIP-36 on multidrug resistant human oral squamous carcinoma cell line (KBV200 cells) in vitro and the possible anticancer mechanisms. MTT assay, Hoechst fluorescein stain, RT-PCR and immunohistochemistry were carried out on KBV200 and KB cells. The growth of many tumor cells was obviously inhibited by CIP-36, especially the multidrug resistant cells KBV200. Obvious apoptosis could be observed in the Hoechst 33342 staining experiments. The results of RT-PCR showed that the levels of p53, p21, caspase-3 and bax mRNA increased, and meanwhile the expression of mdr-1 and bcl-2 mRNA decreased in a dose-dependent manner. The data were significantly different from that of vehicle. The expression of P-gp significantly decreased with the increasing dosage of CIP-36 examined by immunohistochemistry. It can be concluded that CIP-36 could change resistance-related genes and proteins to overcome multidrug resistance in the KBV200 cell line.