EFFECT OF ANTITUMOR DRUGS ON THE ACTIVITY OF DNA TOPOISOMERASE I

The activity of DNA topoisomerase Ⅱ prepared from either normal or tumor tissues were compared. It was found that the unknotting activity of the enzyme in malignant tumor cells was higher than that in normal cells. We selected some antitumor drugs including Chinese traditional medicine, and observed their effects on the unknotting activity of topoisomerase Ⅱ. The results showed that inhibition of the unknotting activity of the enzyme required very low concentrations of drugs, but much higher concentrations were required for other tested. Some antitumor drugs had no effect on the enzyme were also proved. It is interesting that carrageenan, an antiviral drug, strongly blocked the unknotting activity although its antitumor activity has not been reported.

Determination of Residues and Safety Analysis of Metal Impurities in Platinum-based Antitumor Drugs

The residual metal impurities in cisplatin, carboplatin and oxaliplatin were determined by ICP-AES. The samples were ignited and dissolved with HCl:HNO 3 (3:1). The method is simple and accurate. By the determination of the metal residues in the samples, the calculated actual daily exposure and concentration of the metal Pd, Ir, Rh, Ru, Mo, Ni, Cr, V, Cu, Mn, Fe and Zn that were less than the permitted daily exposures (PDE) and the limited concentration permitted in the EMEA guideline on the specification limits for residues of metal catalysts or metal reagents [1] . The metal residues can de adequately removed from the active pharmaceutical ingredients and the corresponding drugs. The trace metal residues will not affect human health and lead to the safety hazard by the intravenous injection.

A NEW EXPERIMENTAL AND CLINICAL APPROACH OF COMBINING USAGE OF HIGHLY ACTIVE TUMOR-INFILTRATING LYMPHOCYTES AND HIGHLY SENSITIVE ANTITUMOR DRUGS FOR THE ADVANCED MALIGNANT TUMOR

In recent years, tumor-nfiltrating lymphocytes (TILs) have been reported to be effective for tumors in experimental and clinical research. In order to increase the therapeutical effect, we modified some steps of Rosenberg's approach a. cold digestion with collagenase at 4C for 24 hours; b. sedimentation instead of centrifugation; c. elimination of tumor cells before the cultivation procedure. Compared with the original approach, the proliferation, activity and cytotoxicity of TILs obtained by the modified procedure were much improved. TILs' expansion-old was greater than that with the original approach. Cytotoxicity against rumor cells was more potent. Increased TILs' subsets were CD3 and CD8 cells. Meanwhile, we took tumor cells from tumor tissues to test their in vitro chemosensitivities to different drugs in order to select highly sensitive antitumor drugs for treatment of cases with advanced tumors. According to the design of using highly active TILs and highly sensitive drugs (H & H therapy), preliminary clinical results of 50 cases showed higher response rates than those in treatment with TIL / IL2, LAK / 1L2 and TIL+IL2+CTX. Less toxic side effects were observed in 14 patients.

Effects of antibiotic antitumor drugs on nucleotide levels in cultured tumor cells:an exploratory method to distinguish the mechanisms of antitumor drug action based on targeted metabolomics

Nucleotide pools in mammalian cells change due to the influence of antitumor drugs,which may help in evaluating the drug effect and understanding the mechanism of drug action.In this study,an ion-pair RP-HPLC method was used for a simple,sensitive and simultaneous determination of the levels of 12 nucleotides in mammalian cells treated with antibiotic antitumor drugs(daunorubicin,epirubicin and dactinomycin D).Through the use of this targeted metabolomics approach to find potential biomarkers,UTP and ATP were verified to be the most appropriate biomarkers.Moreover,a holistic statistical approach was put forward to develop a model which could distinguish 4 categories of drugs with different mechanisms of action.This model can be further validated by evaluating drugs with different mechanismsof action.This targeted metabolomics study may provide a novel approach to predict the mechanism of action of antitumor drugs.

Conformation and Antitumor Activity of Novel Metal Complexes of Piperazinedithioformates

The in vitro antitumour efficacy of some novel metal complexes of piperazinedi thioformates （SR-M） was examined in six cancer cell lines. The activity was compared with that of piperazinedithioformates. Biological evaluations of a series of SR-M compounds suggest that the compounds 1 c （SR-Cu） and If （SR-Sn） show a potent antitumor activity. The stable conformation structure of SR-Cu as a representative of SR-M was investigated and confirmed by computer workshop.

3D bioprinting of in vitro porous hepatoma models:establishment,evaluation,and anticancer drug testing

Traditional tumor models do not tend to accurately simulate tumor growth in vitro or enable personalized treatment and are particularly unable to discover more beneficial targeted drugs.To address this,this study describes the use of threedimensional(3D)bioprinting technology to construct a 3D model with human hepatocarcinoma SMMC-7721 cells(3DP-7721)by combining gelatin methacrylate(GelMA)and poly(ethylene oxide)(PEO)as two immiscible aqueous phases to form a bioink and innovatively applying fluorescent carbon quantum dots for long-term tracking of cells.The GelMA(10%,mass fraction)and PEO(1.6%,mass fraction)hydrogel with 3:1 volume ratio offered distinct pore-forming characteristics,satisfactorymechanical properties,and biocompatibility for the creation of the 3DP-7721 model.Immunofluorescence analysis and quantitative real-time fluorescence polymerase chain reaction(PCR)were used to evaluate the biological properties of the model.Compared with the two-dimensional culture cell model(2D-7721)and the 3D mixed culture cell model(3DM-7721),3DP-7721 significantly improved the proliferation of cells and expression of tumor-related proteins and genes.Moreover,we evaluated the differences between the three culture models and the effectiveness of antitumor drugs in the three models and discovered that the efficacy of antitumor drugs varied because of significant differences in resistance proteins and genes between the three models.In addition,the comparison of tumor formation in the three models found that the cells cultured by the 3DP-7721 model had strong tumorigenicity in nude mice.Immunohistochemical evaluation of the levels of biochemical indicators related to the formation of solid tumors showed that the 3DP-7721 model group exhibited pathological characteristics of malignant tumors,the generated solid tumors were similar to actual tumors,and the deterioration was higher.This research therefore acts as a foundation for the application of 3DP-7721 models in drug development research.

High-density lipoprotein as a potential carrier for delivery of a lipophilic antitumoral drug into hepatoma cells

AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells. METHODS: Recombinant complex of HDL and aclacinomycin (rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine. Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles, morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method. RESULTS: The density range of rHDL-ACM was 1.063-1.210 g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%. Encapsulated efficiencies of rHDL-ACM were more than 90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26±5.62 nm by measure of 110 rHDL-ACM particles in the range of diameter of lipoproteins. rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 μg/mL (P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 ug/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM (1.68 nmol/L vs3 nmol/L). Compared to L02 hepatocytes, a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 μg/mL.Cytotoxicity of the rHDL-ACM to SMMC- 7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5μg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells. CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721 hepatoma to normal L02 hepatocytes, HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.

THE ANTITUMOR ACTIVITIES OF GNIDIMACRIN ISOLATED FROM STELLERA CHAMAEJASMEL．

Gnidimacrin, a diterpene compound, isolated from the methanol extract of Stellera, chamaejasme L., showed significant antitumor activities against mouse leukemia P-388 and L-1210 in vivo, such as Lewis lung carcinoma, B-16 melanoma and Colon cancer 26. It showed ILSs of 40%.49% and 41% at the dosages of 0.01-0.02 mg/kg ip,respectively. Gnidimacrin strongly inhibited cell Proliferation of human cancer cell lines such as leukemia K562, stomach cancers Kato-III, MKN-28, MKN-45, and mouse leukemia L-1210 by the MTT assay and colony forming assay in vitro. The IC50 of gnidimacrin was 0.007-0.00012 μg/ml. It is concluded that gnidimacrin is the principal antitumor element in Stellera chemaejasme L. with strong antitumor activities.

Antitumor drug-induced acute pancreatitis:report of a special case

Drug-induced acute pancreatitis （DAP） is defined as pancreatitis with corresponding clinical manifestations resulted from drug-induced pancreatic secretion dysfunction and pancreatic tissue damage. One case of DAP resulted from chemotherapeutics （Nimustine） was diagnosed and treated our in hospital in 2009. This patient belonged to pancreatitis induced by anticancer drugs, and the toxicity of anticancer drugs acted directly on pancreatic cells, leading to the occurrence of pancreatitis. After treatment, the pancreatitis was effectively treated in this patient, but the final the patient and his family eventually gave up the treatment due to aggravated primary diseases

Lethal Effect of Benzene Nitrogen Mustard Glucoside Derivate on K562 Cells

A new synthesized benzene nitrogen mustard was converted into glycosyl donor-trichloroacetimidate that was glycosylated with p-nitrophenol（glycosyl donors） to form β-lactosyl p-nitrobenzene under the protection of acetyl in a stereoselective manner, was prepared and evaluated for its cytotoxicity towards cultured K562 cell line. Methylthiazoy tetrazolium（MTT） assay, transmission electron microscopy（TEM）, flow cytometry（FCM） and immunohistochemistry were utilized to explore the mechanisms of how the compound arrests the growth of HCT-T cells. This new synthesed benzene nitrogen mustard glucoside derivate（BNMGD） presented a lower toxicity to normal cells, but is significantly more toxic to K562 cells compared with nitrogen mustard, meanwhile it can induce the apoptosis of K562 cells. These results indicate that the new synthesized BNMGD can inhibit the growth of K562 cells and induce the apoptosis, and its cytotoxicity towards cultured K562 cell line is much more effective than that of nitrogen mustard.

ANALYSIS OF APOPTOSIS BY DNA END LABELING METHOD (TDT) IN LEUKEMIA CELL LINES HL-60 AND U937

Antitumor-drug-indnced apoptosis in leukemia cell lines HL-60 and U937 was quantitatively analyzed with TDT (terminal deoxynuleotidyl transferase-mediated nick-end labeling) approach, which allows to labal the ends of DNA broken strands in apoptotic cells by biotinlated dUTP which to avidin-FITC. In this way the apoptotic cells show nuorescent when the labeling cells were emitted by UV light microscope or laser-activated now cell sorting at r=480. In our study, HL-6o and U937 cell lines were cocultured respectively with cisdiaminodicaicroplatinum (CDDP), hydroxycamptothecinum (HCT) and vindesini sulfa (VCR) for 18 hours.By calculating percentages of apoptotic cells with TDT mothod, we were able to show that the two cell lines gave different sensitivity to the drugs. HL-60 showed high sensitivity to CDDP but U937 cells were more sensitive to other two drugs, HCT and VCR. Meanwhile we compared the results of obtained by DNA gel electrophoresis with that by TDT. We found that gel electrophoresis is not sensitive enough to reveal apoptosis since there was no ladder structure, a typical electrophoresis pattern for apoptosis, appeared until the apoptotic cells reached or over 13%. And we report in this paper as first time that three forms of apoptotic cells could be detected under fluorescent microscope, which we called as spot form and crescent form and assembling form in terms of distribution of light spots within cell nuclei. It seemed that the spot form was at an early stage of apoptosis and the crescent form rcprtscntcd a later stage of apoptosis.

Theoretical Investigation of Detailed Thermodynamic Character of Possible Difunctional Adducts Model

The B3LYP/6-31G^＊ level of theory was used to optimize trans-[Pt（NH3）（Am）G-L], where Am = quinoline or thiazole and L is chosen as the model for functional groups of peptide side chains, and for adenine and guanine sites of DNA as the ultimate target of platinum anticancer drugs. Bond dissociating energy and stability energy of complexes are chosen to study detailedly thermodynamic character of possible difunctional adducts model. In order to investigate the influence of a polarizable environment on the energy of the Pt-L bond formation, we adopt a new bonding energy formula brought forward by Lippard and his coworkers： △H（Sol） = △H（SCF） ＋ △G（Solv）, which is quite appropriate to compare with what is found in experimental studies. Our calculated results demonstrate that N-containing ligands are more favored in view of thermodynamics both in gas phrase and in solution. However, it is worthly to be noted that addition of solvation free energies result in moderate correction of bonding energy in relative ordering, and the largest ones both present in imidazole ligand, not in guanine ligand. Finally, the nature of bond is analyzed in terms of partial charges distribution based on NBO population.

Improved Synthesis of Miriplatin

Miriplatin, a novel lipophilic platinum complex has been developed to treat hepatocellular carcinoma. An improvd synthetic route was designed and used to prepare the target compound. The intermediate Pt(C6H14N2)(I) 2 was synthesized from K 2 PtCl 4 , KI and (1R,2R)-1,2-cyclohexanediamine, Pt(C 6 H 14 N 2 )(I) 2 was reacted with AgNO 3 to prepare Pt(C 6 H 14 N 2 )(H 2 O) 2 (NO 3 ) 2 solution then was subsequently reacted with CH 3 (CH 2 ) 12 COONa in n-butanol to give target compound with satisfied yield 81%(based on Pt(C 6 H 14 N 2 )I 2 ). The structure of the target compound was identified by elemental analysis, ESI-MS, FT-IR, 1H-NMR, thermal analysis, the structure was consistent with the target compound.

SYNTHESIS AND CHARACTERIZATION OF DVE-CO-MA DERIVATIVES OF CIS- PLATINUM COMPLEXES

Copolymer of divinyl ether and maleic anhydride (DVE-co-MA) derivatives of cis-platinum complexes were synthesized and characterized by elementary analysis, IR and XPS ( X-ray photoelectron spectroscopy). The behavior of the products in biological environment was also studied. UV-visible and fluorescence spectra show that these polymer derivatives are able to exchange ligands with selected nucleophilic groups in biological environment.

Crystal and Molecular Structure of N－Isopropyl－O－（p－Chlorophenyl）－O＇－（1，2：3，4－Di－O＂－isopropylidene－α－D－galactopyranosyl） Thiophosphoramidate

One pure isomer of the title compound was obtained,and its crystal structure was determined by single crystal X-ray diffraction analysis.C21H31ClNO7PS,Mr=507.97,triclinic,space group P1,crystal data:a=10.475 （4）,b=11.165（3）,c=12.484（2）,α=76.65（2）,β=81.05（2）,γ=65.54（2）°,V=1290.1（7） 3,Z=2,Dx=1.31 g/cm3,μ=3.22 cm-1,F（000）=536,the final R=0.073 and Rω=0. 083 for 2851 observed reflections[I≥3σ（I）].The configuration of the chiral phosphorus atom P（1） is R.

Studies on Antineoplastic Effect by Adjusting Ratios of Targeted-ligand and Antitumor Drug

A series of drug delivery systems based on a sodium alginate derivative were prepared by mixing glycyrrhetinic acid （GA） and doxorubicin （DOX） conjugates at different ratios. GA （a liver-targeting ligand） and DOX （an antitumor drug） were both conjugated to oligomeric glycol monomethyl ether-modified sodium alginate （ALG-mOEG） for prolonged duration of action. These NP-based delivery systems exhibited active cell uptake and cytotoxicity in vitro and liver-targeted distribution and anti-tumor activity in vivo. In addition, nanoparticles with a 1：1 （W：W） ratio of GA-ALG-mOEG and DOX-ALG-mOEG （NPs-3） showed the highest cellular uptake and cytotoxicity in vitro and liver-targeted distribution and anti-tumor activity in vivo. Specifically, when mixed nanoparticles defined as NPs-3 were injected in mice, liver DOX concentration reached 61.9 μg/g 3 h after injection, and AUC0-∞ and t1/2 of DOX in liver reached 4744.9 μg·h/g and 49.5 h, respectively. In addition, mice receiving a single injection of NPs-3 exhibited much slower tumor growth （88.37% reduction in tumor weight） 16 days after injection compared with placebo. These results indicate that effective cancer treatment may be developed using mixed NP delivery systems with appropriate ratio of targeted ligand and drug.

Design, Synthesis and Biological Evaluation of Novel Etoposide Analogues as Cytotoxic Agents

Five novel compounds composed of etoposide and 5-fluorouracil derivatives joined by an ester linkage were prepared and evaluated for their antitumor potential. Most of these analogues have exhibited promising in vitro cytotoxic activity against cell cultures of murine leukaemia P-388 and human lung carcinoma A-549. The results presented herein challenged the long-standing structure-activity relationships, which proposed that a free 4＇-hydroxyl group is essential structural requirement for etoposide-like activity. And in addition, the 4＇-position was suggested to tolerate chemical modifications such as esterification. The preliminary testing results also indicated that the design and synthesis of these compounds were beneficial for therapeutic values of etoposide.

cDNA cloning of a novel lectin that induce cell apoptosis from Artocarpus hypargyreus

We isolated a novel lectin(AHL)from Artocarpus hypargyreus Hance and showed its immunomodulatory activities.In this study,the amino acid sequence of AHL was determined by cDNA sequencing.AHL cDNA(875bp)contains a 456-bp open reading frame(ORF),which encodes a protein with 151 amino acids.AHL is a new member of jacalin-related lectin family(JRLs),which share high sequence similarities to KM+and Morniga M,and contain the conserved carbohydrate binding domains.The antitumor activity of AHL was also explored using Jurkat T cell lines.AHL exhibits a strong binding affinity to cell membrane,which can be effectively inhibited by methyl-α-D-galactose.AHL inhibits cell proliferation in a time-and dose-dependent manner through apoptosis,evidenced by morphological changes,phosphatidylserine externalization,poly ADP-ribose polymerase(PARP)cleavage,Bad and Bax up-regulation,and caspase-3 activation.We further showed that the activation of ERK and p38 signaling pathways is involved for the pro-apoptotic effect of AHL.