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.
Antineoplastic Agents ; chemical synthesis ; pharmacology ; Drug Screening Assays, Antitumor ; HeLa Cells ; Humans ; Indoles ; chemical synthesis ; pharmacology ; K562 Cells ; Podophyllotoxin ; chemical synthesis ; pharmacology ; Structure-Activity Relationship

According to drug design flattening principle and using podophyllotoxin or 4'-demethylepipodophyllotoxin and aldehydes as starting material,a series of podophyllotoxin derivatives containing an imine structure with low toxicity were highly effective synthesized. Nine target compounds were successfully synthesized,and their structures were confirmed by ~1H-NMR,HR-ESI-MS and melting point data analysis. Using etoposide as positive control drug,nine target compounds were screened for cytotoxicity against He La cells in vitro by MTT method. The antitumor activity screening results showed that compound 6 b,6 d,6 e,6 f,6 g,6 i exhibited higher inhibitory rate against He La cells than those of control drug VP-16. It provides some practical reference value for the further development on the structure modification of podophyllotoxin and study on anti-tumor activity.
Antineoplastic Agents/pharmacology* ; Drug Design ; Drug Screening Assays, Antitumor ; Podophyllotoxin/pharmacology* ; Structure-Activity Relationship

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.
Antineoplastic Agents ; chemistry ; pharmacology ; Berberidaceae ; chemistry ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; G2 Phase ; drug effects ; Humans ; Hydrogen Bonding ; drug effects ; Male ; Molecular Conformation ; Molecular Docking Simulation ; Phytotherapy ; Plant Extracts ; chemistry ; pharmacology ; Plant Roots ; chemistry ; Podophyllotoxin ; chemistry ; pharmacology ; Polymerization ; Prostatic Neoplasms ; drug therapy ; Tubulin ; drug effects ; Tubulin Modulators ; pharmacology

OBJECTIVETo investigate the anti-tumor effect of ZM-66 on multidrug-resistant leukemic cell line K562/ADM.

METHODSThe 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.

RESULTSSRB 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.

CONCLUSIONZM-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.

Apoptosis ; drug effects ; Blotting, Western ; Cell Proliferation ; drug effects ; Flow Cytometry ; Humans ; K562 Cells ; Podophyllotoxin ; analogs & derivatives ; chemistry ; pharmacology ; RNA, Messenger ; genetics

OBJECTIVETo 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.

METHODSIGF-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.

RESULTSIGF-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).

CONCLUSIONIGF-IR is associated with proliferation, cell motility, and apoptosis of HCC cells, and may be a promising molecular target for HCC.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Podophyllotoxin ; analogs & derivatives ; pharmacology ; Receptor, IGF Type 1 ; metabolism

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.
Antineoplastic Agents, Phytogenic ; administration & dosage ; chemistry ; pharmacology ; Carboxylic Acids ; chemistry ; Drug Carriers ; HeLa Cells ; drug effects ; Humans ; Nanodiamonds ; chemistry ; Particle Size ; Podophyllotoxin ; administration & dosage ; chemistry ; pharmacology ; Solubility ; Spectrophotometry, Ultraviolet ; Spectroscopy, Fourier Transform Infrared

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.
ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Caspase 3 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; KB Cells ; Mouth Neoplasms ; metabolism ; pathology ; Podophyllotoxin ; administration & dosage ; analogs & derivatives ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Proto-Oncogene Proteins p21(ras) ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism

OBJECTIVETo study the effect of podophyllotoxin nanostructured lipid carriers (POD-NLC) on immortalized human cervical epithelial cells (H8) infected with HPV in vitro.

METHODSPOD-NLC was prepared by emulsion evaporation method and characterized using transmission electron microscopy, Zetasizer analyzer and high-performance liquid chromatography (HPLC). H8 cells were treated with different concentrations (0.0001-1 µg/ml) of POD-NLC, free POD, or blank nanostructured lipid carriers (NLC), and the cell proliferation was assessed using MTT assay to evaluate the cytotoxic effects. The changes of cell morphology were observed using fluorescence microscopy, and the cell cycle changes and cell apoptosis were analyzed using flow cytometry.

RESULTSPOD-NLC showed a spherical or elliptical shape with good stability in vitro. The average particle size of POD-NLC was 85.6∓10.25 nm, with a Zeta potential of 26.2∓4.1 mV and entrapment efficiency of POD of (88.56∓3.1)%. POD-NLC caused a significant inhibition of H8 cell proliferation in a concentration- and time-dependent manner. At an equivalent concentration, POD-NLC produced a stronger inhibitory effect on cell proliferation than POD. The inhibition rate of H8 cells after a 48-h exposure to POD-NLC and POD reached 95.8% and 65.6%, respectively, and at the highest concentration of 1 µg/ml, the IC(50) of POD-NLC and POD was 0.015 µg/ml and 0.13 µg/ml, respectively. Blank NLC did not obviously affect the proliferation of H8 cells. POD-NLC and POD both caused obvious increases in G(2)/M phase cell percentages and induced typical apoptotic changes of the cells, and their effects were comparable (P>0.05).

CONCLUSIONCompared with POD, POD-NLC has more potent effect in inhibiting H8 cell proliferation and inducing cell apoptosis, suggesting its potential in the treatment of cervical HPV infection.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cervix Uteri ; cytology ; Drug Carriers ; pharmacology ; Epithelial Cells ; drug effects ; Female ; HIV Infections ; pathology ; Humans ; Lipids ; Nanostructures ; Particle Size ; Podophyllotoxin ; chemical synthesis ; pharmacology

OBJECTIVEto investigate the effect of deoxypodophyllotoxin (DOP) on membrane potential of dorsal unpaired median neurons (DUM, neurons) and its correlation with sodium channel.

METHODSDUM neurons were labeled with DiBAC4(3). Laser scanning confocal microscope was used to monitor the changes of membrane potential at real time on these neurons that were treated with different concentrations of the DOP. The effect of sodium channel blocker tetrodotoxin (TTX) on the changes was also observed.

RESULTSmembrane potential depolarization induced by the DOP peaked at 5 min and became stabilized after 8min. After compared with fluorescence intensity without treatment, the normalized fluorescence intensity was 69.6 ± 3.0, 72.1 ± 2.7, 77.8 ± 3.6, 86.2 ± 3.1 in cells which were treated with 1, 5, 25, 125 micromol/L DOP, respectively. These numbers were significantly lower than those from untreated control cells (P < 0.01). When DUM neurons were co-incubated with 1 micromol/L TTX for 20 min, then treated with 25 micromol/L DOP, the intensity changed to 63.6 ± 5.4, which was similar to that of the control (P > 0.05). This indicated that the effect of DOP could be completely inhibited by TTX.

CONCLUSIONDOP induced membrane depolarization of DUM neurons in the range of 1 approximately 125 micromol/L and the sodium channel should be involved in this process.

Animals ; Cells, Cultured ; Ganglia, Invertebrate ; drug effects ; physiology ; Membrane Potentials ; drug effects ; physiology ; Neurons ; drug effects ; physiology ; Periplaneta ; drug effects ; physiology ; Podophyllotoxin ; analogs & derivatives ; pharmacology ; Sodium Channels ; metabolism

OBJECTIVETo evaluate the anti-proliferative and apoptosis-inducing effect of podophyllotoxin solid lipid nanoparticles (PDP-SLN) in human cervical carcinoma cells in vitro.

METHODSHela cells were treated with PDP and PDP-SLN at different concentrations (0.0005-5 micromol/L), and the proliferation of the cells was assessed using MTT assay and the apoptotic index was determined by flow cytometry.

RESULTSBoth PDP and PDP-SLN showed obvious inhibitory effect on the cell proliferation in a dose- and time-dependent manner. At the same concentration, PDP-SLN produced stronger inhibitory effect on the cells than PDP, with IC50 24, 48, and 72 h after the cell exposure to PDP-SLN and PDP of 4.10, 0.65, 0.20 micromol/L and 9.2, 4.0, 1.3 micromol/L, respectively. Both PDP and PDP-SLN significantly induced the apoptosis of the Hela cell, and the apoptosis rates of the cells incubated in the presence of 0.5 micromol/L PDP-SLN reached 90.8% at 24 h and 94.2% at 72 h, significantly higher than the rate of cells incubated with PDP (64.1% at 24 h and 68.4% at 72 h, P<0.01).

CONCLUSIONPDP-SLN can effectively suppress the proliferation and induce apoptosis of Hela cells in vitro.

Antineoplastic Agents, Phytogenic ; chemistry ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Delayed-Action Preparations ; Drug Carriers ; Female ; HeLa Cells ; Humans ; Liposomes ; Nanoparticles ; chemistry ; Podophyllotoxin ; chemistry ; pharmacology ; Uterine Cervical Neoplasms ; pathology