Camptothecin/*pharmacology ; Liver Neoplasms, Experimental/*immunology ; Neoplasm Transplantation ; Sarcoma, Experimental/*immunology ; Transplantation, Isogeneic

Animals ; Camptothecin ; pharmacology ; Liver Neoplasms, Experimental ; immunology ; Mice ; Neoplasm Transplantation ; Sarcoma, Experimental ; immunology ; Transplantation, Isogeneic

Camptothecin is a strong anti-tumor compound isolated from Camptotheca acuminata. One of the most important way for the production of Camptothecin is by cell cultures of Camptotheca acuminata. The effect of Cu2+ on camptothecin accumulation in Camptotheca acuminata cell line was described in this paper. The results showed that the optimum CuCl2 concentration in B5 medium was 0.008 mg/mL, which increased camptothecin production for 30 times compare to the control while has no inhibitive effects on cell growth, at the same time, the peroxidase activity was increased and the anthocyanidin accumulation was inhibited. The promotive effects of Cu2+ on camptothecin accumulation in light was higher than that in dark.
Anthocyanins ; biosynthesis ; Antineoplastic Agents, Phytogenic ; biosynthesis ; Camptotheca ; growth & development ; metabolism ; Camptothecin ; biosynthesis ; Copper ; pharmacology ; Light

OBJECTIVETo investigate the effects of nitrogen concentration on the camptothecin (CPT) content in Camptotheca acuminata seedlings:

METHODThe seedlings of C. acuminata with 6 pair of leaves were subjected to five nitrogen concentrations treatments by sand culture in a greenhouse. The CPT content in the seedlings was determined by HPLC on the 20th, 35th, 50th, 65th and 80th day respectively.

RESULTThe CPT content in the young leaves of C. acuminata seedlings supplied with different nitrogen concentration was significantly higher than that in other organs (P < 0.01), and it showed a single peak curve with the time course, the highest CPT content was observed on the 50th day after treatment. The CPT content in the young leaves obviously declined with increasing nitrogen concentration, and it reached the highest (6.72%) when nitrogen concentration was 4 mmol x L(-1), equal to 1.1 times that of 16 mmol x L(-1) nitrogen.

CONCLUSIONThe results demonstrate that proper deficient nitrogen stress can significantly enhance CPT accumulation in young leaves of C. acuminata seedlings.

Camptotheca ; drug effects ; metabolism ; Camptothecin ; metabolism ; Chromatography, High Pressure Liquid ; Nitrogen ; pharmacology ; Seedlings ; drug effects ; metabolism

OBJECTIVE: To investigate the effect of Bmi-1 expression on the chemosensitivity of THP-1 cells and its relative mechanism. METHODS: The pGenesil-2-Bmi-1 1 siRNA, p-MSCV-Bmi-1 plasmid was transfected into THP-1 cells to reduce or increase the expression of Bmi-1. The expression of Bmi-1 mRNA and protein was verified by PCR and Western blot. The effect of camptothecin (CPT) on the proliferation and chemosensitivity of THP-1 cells affected by Bmi-1 gene were detected by MTT assay. The expression of DNA double-strand breaks marker-γ-H2AX was detected by immunofluorescence assay. Mitochondrial membrane potential and apoptosis were observed by flow cytometry. The expression of Cytochrome C, Caspase 3, Bax and BCL-2 was detected by Western blot. RESULTS: Silencing Bmi-1 could inhibit proliferation and enhance the sensitivity of THP-1 cells to CPT, while overexpressed Bmi-1 could promote the cell proliferation and attenucate sensitivity of THP-1 cells to CPT. Silencing Bmi-1 could enhance CPT-induced DNA double-strand breaks, decrease mitochondrial membrane potential and promote CPT-induced apoptosis. While increasing Bmi-1 gene expression could attenuate CPT-induced DNA double-strand breaks, enhamce mitochondrial membrane potential and significantly reduce CPT-induced apoptosis of cells. CONCLUSION Bmi-1 expression could influence the sensitivity of THP-1 cells to CPT, and its relative mechanism may relate to DNA double-strand breaks and endogenous apoptotic pathways.
Apoptosis ; Camptothecin/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; THP-1 Cells

OBJECTIVETo investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by comparative proteomic analysis.

METHODSApoptosis of SMMC-7721 cells were induced by using HCPT and their mitochondria were isolated with a mitochondria isolation kit for cultured cells. Three different solubility protein fractions were extracted with ReadyPrep Sequential Extraction Kit and were separated by two-dimensional gel electrophoresis (2-DE). PDQuest software was used to differentiate mitochondrial proteins between control cells and HCPT-treated cells. Matrix assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) was used to identify some of the different proteins.

RESULTSHighly purified mitochondria and high resolution 2-DE patterns of the proteins were obtained. Forty-four mitochondrial protein spots from the HCPT-treated cells showed different expressions compared to those of the control cells. Twenty of the different protein spots were analyzed by MALDI-TOF-MS.

CONCLUSIONDifferently expressed mitochondrial proteins in HCPT-treated cells and control cells were obtained in this study. This will be of help to understand the mechanism by which HCPT induces cell apoptosis.

Antineoplastic Agents, Phytogenic ; pharmacology ; Camptothecin ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Humans ; Membrane Potentials ; drug effects ; Mitochondrial Proteins ; metabolism ; Proteomics

AIMTo find new anticancer drug based on the structure of 10-hydroxy camptothecin.

METHODSSeven camptothecin derivatives (3 -9) were synthesized and the antitumor activities of these derivatives were evaluated.

RESULTSStructures of seven new compounds were determined by 1HNMR, IR, MS. Seven compounds showed inhibitory effects on Hela, BEL-7402, 7901 cell lines in vitro. Especially, compound 4 showed high bioactivities to all of the tumor cells in vitro, its anticancer activity against human cervical carcinoma Hela was much higher than that of 10-hydroxy camptothecin.

CONCLUSIONSome compounds are worth further studying.

Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Camptothecin ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Humans ; Molecular Structure

Cisplatin (CDDP), homoharringtonine (HHT), mitoxantrone (MIT) and hydroxycamptothecin (HCPT) are highly effective anti-tumor drugs. To evaluate their effects in the therapy of leukemia and establish a valuable method to estimate anti-tumor drugs, Annexin V/PI double parameter flow cytometry was used to detect the effects of these drug inducing apoptosis and death in Jurkat cell line. The results showed that MIT and HCTP-induced apoptosis effects on Jurkat cell line were obvious at 4 hours in early phase after adding drug (P < 0.05) and at 8 hours in late phase after adding drug (P < 0.05). HHT had obvious effect on inducing apoptosis of Jurkat cells, but no significant difference from low to high doses. The effect of CDDP on inducing apoptosis of Jurkat cell line was obviously weaker than that of HHT, MIT and HCPT, its weak effect on apoptosis of Jurkat cell line was found only at high concentration of drug for long time. Death effects on Jurkat cell line can not be observed in every experimental group. It is concluded that low dose of MIT can effectively induced apoptosis of Jurkat cell line. Annexin V/PI double parameter flow cytometry can be used as a reliable method for clinical screening anti-tumor drugs.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Camptothecin ; pharmacology ; Cisplatin ; pharmacology ; Drug Screening Assays, Antitumor ; methods ; Harringtonines ; pharmacology ; Humans ; Jurkat Cells ; Mitoxantrone ; pharmacology

OBJECTIVETo investigate the effects of hydroxycamptothecin (HCPT) on proliferation and apoptosis of rat hepatic stellate cells (HSC).

METHODSRat HSC line (HSC-T6) and rat hepatocyte line (BRL-3A) were treated with different concentrations of HCPT (0, 0.008, 0.016, 0.031, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 mg/L respectively) for 24 h. Cell proliferation was assessed by MTT colorimetric assay, apoptosis was detected with PI staging followed by flow cytometry, and by DNA ladder assay. The morphological change of apoptosis was observed under transmission electron microscopy (TEM).

RESULTSMTT assay indicated that HCPT significantly inhibited the proliferation of HSC-T6 and BRL-3A in a dose-dependent manner. 24 h after the treatment with different concentrations of HCPT (0.25, 0.5, 1 mg/L), the apoptosis rate (13.46%+/-2.42%, 26.25%+/-5.65%, 47.05%+/-8.76%, respectively) in HSC-T6 was significantly higher than that in control cells (4.89%+/-1.80%, F = 34.24, P less than 0.01). 24 h after 0.5 mg/L HCPT treatment, cell shrinkage, nucleoli disappearance, chromatin condensation were found under TEM, and DNA ladder was demonstrated by agarose gel electrophoresis.

CONCLUSIONHCPT could significantly inhibit proliferation and induce apoptosis of HSC-T6 in a dose-dependent manner.

Animals ; Apoptosis ; drug effects ; Camptothecin ; analogs & derivatives ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Hepatic Stellate Cells ; drug effects ; Rats

OBJECTIVESTo investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by using quantitative proteome.

METHODSSMMC-7721 cell apoptosis was induced by HCPT and the mitochondria were isolated with a mitochondria isolation kit. Mitochondrial proteins labeled with a cleavable isotope-coded affinity tag were identified and quantified using two-dimensional liquid chromatography/tandem mass spectrometry.

RESULTSHighly purified mitochondria were obtained. Seventy-four mitochondrial proteins, which were statistically significantly altered (P less than 0.05) in HCPT-treated cells, were identified and analyzed. A total of 42 proteins were significantly down-regulated, and 32 were up-regulated in the cells that responded to apoptosis. The functions of these proteins were likely involved in cell energy metabolism, nucleic acid translation and transcription, cytoskeleton, etc.

CONCLUSIONOur results about the information of differentially expressed mitochondrial proteins in HCPT-treated cells and the control cells will help to understand the mechanism by which HCPT induces cell apoptosis. The integrated techniques we used in this study will be helpful to the investigation of subcellular quantitative proteomics.

Apoptosis ; drug effects ; Camptothecin ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Humans ; Mitochondria ; metabolism ; Mitochondrial Proteins ; metabolism ; Proteome ; metabolism