ETME, a novel β-elemene derivative, synergizes with arsenic trioxide in inducing apoptosis and cell cycle arrest in hepatocarcinoma cells via a p53-dependent pathway

Arsenic trioxide (ATO) has been identified as an effective treatment for acute promyelocytic leukemia (APL) but is much less effective against solid tumors such as hepatocellular carcinoma (HCC). In the search for ways to enhance its therapeutic efficacy against solid tumors, we have examined its use in combination with a novel derivative of β-elemene, N-(β-elemene-13-yl)tryptophan methyl ester (ETME). Here we report the effects of the combination on cell viability, apoptosis, the cell cycle and mitochondria membrane potential (MMP) in HCC SMMC-7721 cells. We found that the two compounds acted synergistically to enhance antiproliferative activity and apoptosis. The combination also decreased the MMP, down-regulated Bcl-2 and pro-proteins of the caspase family, and up-regulated Bax and BID, all of which were reversed by the p53 inhibitor, pifithrin-α. In addition, the combination induced cell cycle arrest at the G2/M phase and reduced tumor volume and weight in an xenograft model of nude mice. Overall, the results suggest that ETME in combination with ATO may be useful in the treatment of HCC patients particularly those unresponsive to ATO alone.

Molecular mechanism of ophiopogonin B induced cellular autophagy of human cervical cancer HeLa cells / 药学学报

This study is to investigate the antitumor activity of ophiopogonin B (OP-B). MTT assay, flow cytometric analysis, acridine orange staining, Lyso-Tracker Red staining and HeLa-GFP-LC3 transfect cells assay were used to detect the proliferation activity, apoptosis and autophagy of HeLa cells. The results showed that OP-B exerted potent antiproliferative activity on HeLa cells, the cell growth inhibition effect of OP-B was not due to apoptosis and OP-B could induce autophagy of HeLa cells. OP-B also induced the protein expression up-regulation of Beclin-1 and promoted LC3 I transformation LC3 II, which were representative proteins of autophagy. Furthermore, 3-MA, an inhibitor of autophagy, not only inhibited OP-B-mediated autophagy but also almost completely reversed the antiproliferative effect of OP-B, suggesting that the growth inhibition effect of OP-B was autophagy dependent. Western blotting demonstrated that OP-B inhibited the phosphorylation of Akt and its' downstream vital protein, such as mTOR and p70S6K. In addition, OP-B also induced the protein expression up-regulation of PTEN, which is a negative regulation protein for Akt/mTOR signaling pathway. However, OP-B did not affect the protein expression of total Akt. Collectively, the antitumor effects of OP-B were autophagy-dependent via repression Akt/mTOR signaling pathway. Therefore, OP-B is a prospective inhibitor of Akt/mTOR and may be used as an alternative compound to treat cervical carcinoma.

Synergistic antitumor effects of tanshinone II A in combination with cisplatin via apoptosis in the prostate cancer cells / 药学学报

Treatment with the combination of Chinese herbs and cytotoxic chemotherapies showed a higher survival rate in clinical trials. In this report, the results demonstrated that the tanshinone II A, a key component of Salvia miltiorrhiza bunge, when it is combined with the cytotoxic drug cisplatin showed synergistic antitumor effects on human prostate cancer PC3 cells and LNCaP cells in vitro. Antiproliferative effects were detected with MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometer. Protein expression was detected by Western blotting. The intracellular concentration of cisplatin was detected by high performance liquid chromatography. The results demonstrated that tanshinone II A significantly enhanced the antiproliferative effects of cisplatin on human prostate cancer PC3 cells and LNCaP cells with the increase of the intracellular concentration of cisplatin. These effects were correlated with cell cycle arrested at S phase and cell apoptosis. The apoptosis might be achieved through death receptor pathway and mitochondrial pathway. Furthermore, the Bcl-2 family members were also involved in this apoptotic process. Collectively, these results indicated that the combination of tanshinone II A and cisplatin had a better treatment effect in vitro not only on androgen-dependent LNCaP cells but also on androgen-independent PC3 cells.

NNIspm, a polyamine derivative, induces cellular senescence of human hepatoma HepG2 cells and its molecular mechanism / 药学学报

This study is to examine the effects of NNIspm-mediated cellular senescence of HepG2 cells and elucidate its potential molecular mechanism. Cellular senescence was detected with senescence-associated beta-galactosidase staining. Cell cycle distribution, intracellular fluorescence intensity and accumulation of intracellular reactive oxygen species (ROS) were detected by high content screening (HCS). Protein expression was detected by Western blotting. Polyamines content was analyzed by high performance liquid chromatography (HPLC). The results demonstrated that NNIspm significantly induced HepG2 cells senescence. This effect was due to the decrease of intracellular polyamines, the arrest at G0/G1 phase and an increase of ROS level. The molecular senescence marker p21 increased significantly after NNIspm treatment. In contrast, the protein expressions of Cyclin E and CDK2 were obvious down-regulation. The results indicated that cellular senescence induced by NNIspm was one of its antitumor mechanisms.

Part IV. Synthesis and antitumor evaluation of s-triazolothiadiazines and pyrazolo s-triazoles derived from ciproxacin / 药学学报

An efficient modified route based on the targeting mechanism of antibacterial fluoroquinolones for the shift from the antibacterial activity to the antitumor one was further developed. Using a fused heterocyclic ring, s-triazolothiadiazine as a carboxyl bioisostere of ciprofloxacin, the title compounds, 1-cyclopropyl-6-fluoro-7-piperazin-1-yl-3-(6-substituted-phenyl-7H-[1, 2, 4]triazolo[3, 4-b][1, 3, 4]thiadiazin-3-yl)-quinolin-4(1H)-ones (5a-5e) and their corresponding N-acetyl products (6a-6e), were designed and synthesized, separately. Meaningfully, a ring-contraction of fused six-membered thiadiazine occurred by a sulfur extrusion reaction gave new tri-acetylated fused heterocycles related to pyrazolo[5, 1-c][1, 2, 4] triazoles (7a-7e). The in vitro antitumor activity against L1210, CHO and HL60 cell lines was also evaluated for the synthesized fifteen heterocycles compared to parent ciprofloxacin by methylthiazole trazolium (MTT) assay. Interestingly, the results displayed that fifteen fused heterocyclic compounds showed more significant growth inhibitory activity (IC50 < 25.0 micromo x L(-1)) than that of parent ciprofloxacin (IC50 > 150.0 micromol x L(-1)), and the active order decreased from 7a-7e to 5a-5e to 6a-6e, respective.

Part IV: Design, synthesis and antitumor activity of fluoroquinolone C-3 heterocycles: bis-oxadiazole methylsulfide derivatives derived from ciprofloxacin / 药学学报

To explore an efficient strategy for further development of anticancer fluoroquinolone candidates derived from ciprofloxacin, a heterocyclic ring as the bioisosteric replacement of C3 carboxyl group led to a key intermediate, oxadiazole thiol (5), which was further modified to the bis-oxadiazole methylsulfides (7a-7h) and the corresponding dimethylpiperazinium iodides (8a-8h), respectively. Structures were characterized by elemental analysis and spectra data, and their anticancer activities in vitro against CHO, HL60 and L1210 cancer cells were also evaluated by MTT assay. The preliminary results show that piperazinium compounds (8) possess more potent activity than that of corresponding free bases (7).

Involvement of p38-p53 signal pathway in resveratrol-induced apoptosis in MCF-7 cells / 药学学报

This paper is to report the study of resveratrol-induced apoptosis and its mechanisms in MCF-7 cells. MTT assay was performed to assess the cytotoxicity of resveratrol on MCF-7 cells. Hoechst 33258 staining was used to observe cellular morphologic changes in apoptosis. Apoptosis was measured by flow cytometric analysis and the protein expression was examined by Western blotting analysis. The results indicated that resveratrol could inhibit MCF-7 cell growth in a time- and concentration-dependent manner. Remarkable morphologic changes in the cells after 60 micromol L(-1) resveratrol treatment, including cell nuclear shrinkage, DNA condensation and apoptotic bodies, were observed by Hoechst 33258 staining. Resveratrol could induce apoptosis and activate p38 and p53 in a time dependent manner in MCF-7 cells. In addition, the cell growth inhibitory ratio and the apoptotic ratio of resveratrol-treated group decreased markedly by the p38 MAPK inhibitor SB203580 or p53 inhibitor pifithrin-alpha. Further experiments confirmed that resveratrol-induced p53 activation was reduced by SB203580 whereas the activation of p38 was not affected by pifithrin-alpha. In conclusion, resveratrol induced apoptosis in MCF-7 cells could be through activating p38-p53 signal pathway.

Acetylsalicylic acid strengthens the effects of ANISpm against hepatocellular carcinoma and its molecular mechanism / 药学学报

The objective of this study is to examine the effects of ANISpm, a novel polyamine naphthalimide conjugate, with acetylsalicylic acid against hepatocellular carcinoma in vivo and in vitro and elucidate its potential molecular mechanism. The proliferation inhibition was detected by MTT assay. Cell apoptosis, intracellular fluorescence intensity and mitochondrial membrane potential (MMP) were detected by high content screening (HCS) analysis. Polyamines content was analyzed by reverse-phase high performance liquid chromatography Protein expression levels were quantified by Western blotting assay. The combination treatment strongly inhibited cell proliferation, induced cell apoptosis in HepG2 cells and H22 hepatoma cells, which was mediated by enhanced ANISpm uptake via up-regulation of spermidine/spermine N1-acetyltransferase (SSAT) and depression of intracellular polyamine. Furthermore, this synergistic apoptosis was involved in mitochondria and death-receptor signal pathway. All these findings demonstrated that the combination treatment with acetylsalicylic acid and ANISpm resulted in synergistic antitumor effects on hepatoma cells. Thus, combination therapy with these agents may be useful as a potential template for the development of better chemotherapeutic strategy against hepatoma.

Part II: Design, synthesis and antitumor action of C3/C3 bisfluoroquinolones linked-cross 2, 5-1, 3, 4oxadiazole / 药学学报

To develop a new small molecular probe for discovering an antitumor lead compound from the replacement of carboxylic group of two molecular antibacterial fluoroquinolones with a heterocyclic ring, a series of the C3/C3 bis-fluoroquinolones tethered with an 1, 3, 4-oxadiazole ring were synthesized as their respective HCl salts, and their structures were characterized by elemental analysis and spectral data. The in vitro antitumor activity against L1210, CHO and HL60 cell lines was also evaluated via the respective IC50 values by methylthiazole trazolium (MTT) assay.

Apoptotic mechanism of WJH-6, a novel polyamine conjugate, on K562 and HL-60 cells / 药学学报

In the present study, the apoptotic mechanism and polyamine transporter recognition of WJH-6, a novel polyamine conjugate, were investigated in K562 and HL-60 cells. The cytotoxicity of WJH-6 was assessed by MTT assay; cell cycle distribution and apoptosis were measured by flow cytometry; the protein expression of Caspase-3, Caspase-8, Caspase-9, Bid and mitochondrial membrane potential (MMP) were evaluated by high content screening (HCS) analysis; the protein expression of cytochrome c was measured by Western blotting. The results showed that WJH-6 could be recognized and transported by polyamine transporter (PAT). Furthermore, WJH-6 was able to inhibit K562 and HL-60 cells proliferation and induce apoptosis. This apoptotic effect was relative to MMP loss, cytochrome c release from mitochondria to cytoplasm and the activation of Caspase-8, Caspase-9, Caspase-3 and Bid. These results suggested that WJH-6-induced K562 and HL-60 cells apoptosis was related with mitochondrial damage.

Synthesis and in vitro cytotoxicity of naphthalimide polyamine conjugates as antitumor agents / 药学学报

Six naphthalimide polyamine conjugates were synthesized and their structures were confirmed by elemental analysis, 1H NMR, 13C NMR and MS. Antitumor activities were evaluated in vitro using MTT assay on Leukemia cells (K562), human breast cancer cells (MB-231) and prostate cancer cells (Ln cap cell). The results showed that most of the six compounds were superior to the control (amonafide), 6d, 6e, and 6f exhibited nice selectivity in a screen of hepatoma cells (BEL-7402) and human normal hepatocytes (QSG-7701).

Synthesis and antibacterial activity of imidazothiadiazoles and heterocyclic-amine Mannich-base hydrochloride / 药学学报

To optimize the synthetic method and antibacterial activity of fused heterocyclic thiadiazole compounds, cyclocondensation of 2-(4-methoxyphenyl)-5-amino-1,3,4-thiadiazole (2) with alpha-chloro-4-chloro acetophenone (3) resulted in a key intermediate (4), 6 -(4-chlorophenyl)-2-(4-methoxyphenyl)-imidazo-[2,1-b][1,3,4]thiadiazole, which was carried out an nucleophilic substitution with substituted piperazine to give the corresponding free bases of piperazine (5a-5c), then followed by Mannich reaction with heterocyclicamines and formaldehyde to yield the corresponding Mannich bases, (1a-11) as respective hydrochloride salts. The structures were confirmed by IR, 1H NMR, MS and elemental analysis and the antibacterial activities in vitro of fifteen newly synthesized compounds were also tested against Gram positive bacteria and Gram negative bacteria with the standard 2-fold agar dilution method. The antibacterial results showed that the introduction of a polar group resulted in the enhancement of antibacterial activity in vitro. Thus, the structures of these fused compounds could further be investigated.

Synthesis and antitumor activity of C3 heterocyclic-substituted fluoroquinolone derivatives (I): ciprofloxacin aminothiodiazole Schiff-bases / 药学学报

To discover a novel antitumor lead compound derived from fluoroquinolone, C3 carboxyl group of ciprofloxacin (1) was replaced with heterocyclic ring to form cyclopropyl fluoroquinolone aminothiadiazole scaffold (2), then reacted with aromatic aldehydes to give the Schiff bases compounds (3a-3j). The structures of new compounds were characterized by element analysis and spectral data, and their in vitro antitumor activity against SMMC-7721, HL60 and L1210 cell lines was evaluated by MTT assay via the respective IC50 values. The bioactive assay showed that eleven thiadiazole-substituted ciprofloxacin derivatives displayed potential cytotoxicity against the tested cancer cell lines, where the IC50 values of compounds 3d and 3f reached micromolar concentration. Therefore, the C3 carboxyl group of fluoroquinolone is not necessary to antitumor activity. Functionally modified heterocycle-substituted fluoroquinolone as potent antitumor lead compound is valuable for further study.

Synthesis and antitumor activity of anthracene-9-carbaldehyde amino-s-triazole Schiff-bases with side-chain of S-acetic acid / 药学学报

To find out a novel lead compound from heterocyclic amine Schiff bases for developing new antitumor agents, each of (4-amino-5-substituted-s-triazol-3-ylthio) -acetic acids 2a-j was condensed with anthracene-9-carbaldehyde to obtain Schiff-bases of [4-(anthracen-9-yl methylene) amino] -5-substituted-s-triazol-3-ylsulfanyl] -acetic acids 3a-j. The structures of new compounds synthesized were characterized by elemental analysis and spectral data, and in vitro antitumor activity was also evaluated against CHO, HL60 and L1210 cell lines by MTT assay.

Apoptosis induced by NNAMB, a novel polyamine conjugate, in human erythroleukemia K562 cells and its mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the apoptosis-inducing effects of NNAMB, a novel polyamine conjugate, in erythroleukemia K562 cells and its molecular mechanism.</p><p><b>METHODS</b>Cell viability was assessed by MTT assay and trypan blue dye exclusion method. The cell morphology was observed by fluorescence microscopy. The cell cycle distribution, apoptosis and mitochondrial membrane potential were measured by flow cytometry. The expression of caspase-3, -8, -9, cytochrome c in the K562 cells was detected by Western blot.</p><p><b>RESULTS</b>NNAMB inhibited the proliferation of K562 cells. The cells treated with NNAMB showed a typical apoptotic morphology, Sub-G1 peak and loss of mitochondrial membrane potential. Western blot assay showed that NNAMB increased the expression of caspase-3, -9, cytochrome c but not caspase-8 in a dose-and time-dependent manner.</p><p><b>CONCLUSION</b>NNAMB induces apoptosis via mitochondrial pathway in K562 cells.</p>

Synthesis and antitumor activity of benzoic nitrogen mustard derivatives / 药学学报

To study the effect of isoprenoid and aliphatic saturated alcohols as modificator on benzoic nitrogen mustard, the intermediate 4-[N,N-bis(2-chloroethyl) amino] benzoic acid 4 was prepared in four steps utilizing p-amino benzoic acid as the starting material. Target compounds were synthesized by the catalytic esterification of DCC/DMAP and the structures of the six new esters were characterized by elemental analysis, 1H NMR, 13C NMR and MS. Antitumor activities were evaluated in vitro using MTT assay. The result showed that some derivatives were more potent than the intermediate 4, and compound 5c modified with dodecanol exhibited similar activity to the commercial drug melphalan.

Synthesis and antibacterial activity of 3-(4-piperazin-1-yl-phenyl)-s-triazolo 3,4-b 1,3,4 thiadiazole hydrochlorides / 药学学报

To study the synthetic method and antibacterial activity of water-soluble fused heterocyclic compounds containing piperazine group, the nucleophilic substitution of 3-(4-chlorophenyl)-6-substituted-s-triazolo-[3, 4-b] [1, 3, 4] thiadiazoles (2a - n) with piperazine in the presence of phase transfer catalyst TBAI afforded 3-(4-piperazin-1-yl-phenyl)-6-substituted-s-triazolo [3, 4-b] [1, 3, 4] thiadiazole and then followed by acid treatment afforded 3-(4-piperazin-1-yl-phenyl)-6-substituted-s-triazolo [ 3, 4-b] [1, 3, 4] thiadiazole hydrochlorides (3a - n). Twenty-eight new compounds were synthesized and their structures were confirmed by IR, 1H NMR, MS and element analysis. The in vitro antibacterial activities of all newly synthesized compounds were tested against Gram positive bacteria and Gram negative bacteria with the standard 2-fold agar dilution method. Fourteen title compounds exhibited potential antibacterial activities in vitro. The structures of these compounds needed to be further optimized.

Synthesis and antibacterial activity of 3-(5-substituted phenyl-1,3,4 oxadiazole-2-yl-methylenethio)-5-pyridin-3-yl-1,2,4 triazole-4-yl-amines / 药学学报

<p><b>AIM</b>To study on synthesis and antibacterial activity evaluation of polyheterocycles.</p><p><b>METHODS</b>The condensation of 4-amino-3-pyridin-3-yl-4H-[1,2,4] triazole-5-thiol with 2-chloromethyl-5-substituted phenyl-[1,3,4] oxadiazoles gave the corresponding title heterocycle amines, and the in vitro antibacterial activity was primarily evaluated by the method of cup-plate diffusion solution.</p><p><b>RESULTS</b>Twelve novel compounds were synthesized, and their structures were confirmed by IR, 1H NMR, MS and element analysis. Biological screening results demonstrated that most of the compounds prepared showed good antibacterial activity.</p><p><b>CONCLUSION</b>Oxadiazoles incorporting pyridyl triazole ring may be a pharmacophor structure in the molecule for developing antibacterial candidate drugs.</p>