Synthesis and cytotoxicity evaluation of novel acylated starch nanoparticles

Starch nanoparticles (StNPs) were acylated under ambient conditions to obtain various nanosized derivatives formed stable suspension in water and soluble in organic solvents. The degree of substitution (DS) was determined using ¹H NMR technique. The cytotoxicity potential of the derivatised StNPs was evaluated in mouse embryonic fibroblast (3T3L1) cells and A549 tumor cell line using MTT cell viability assay. Other parameters that determine the oxidative stress viz., reactive oxygen species (ROS) generation, intracellular reduced glutathione (GSH), superoxide generation and acridine orange/ethidium bromide staining were also investigated. The present study led to the conclusion that cytotoxic activity of acylated starch nanoparticles was dependent on their dosage, DS and type of substitution. The non-toxic nature in non-cancerous cells reveals that the nanoparticles (NPs) can be used for cancer therapy and drug delivery. The nanoparticles also offered reasonable binding propensity with CT-DNA.     

Synergistic cytotoxicity of Rana catesbeiana ribonuclease and IFN-gamma on hepatoma cells

RC-RNase purified from Rana catesbeiana (bullfrog) oocytes is a pyrimidine-guanine sequence-specific ribonuclease. RC-RNase is derived from the RNase superfamily genes exerting distinct ribonucleolytic activity and possesses cytotoxicity to tumor cells, but rarely to primary cells. In this study, we utilized RC-RNase to function with antiproliferative cytokines. The combination with TNF-alpha or TNF-beta would not aggravate cell death. However, the combination with IFN-gamma could induce synergistic cytotoxicity verified by XTT assays toward three hepatoma cell lines bearing different differentiation stages. The distinct cytotoxicity from RC-RNase or RC-RNase/IFN-gamma on different hepatoma cells was correlated with the differentiation extent but not the proliferation rate of the cells. Despite the synergistic cytotoxicity and severe mitochondrial disruptions in the RC-RNase/IFN-gamma-treated cells, we scarcely detected any significant feature of apoptosis or necrosis by FACS analysis on annexin-V/propidium iodide staining. The mechanisms of cell death triggered by RC-RNase or RC-RNase/IFN-gamma require further investigation.     

Synthesis and cytotoxicity evaluation of novel indolylpyrimidines and indolylpyrazines as potential antitumor agents

Novel indolylpyrimidines and indolylpyrazines have been synthesized as potential antitumor agents. They were screened in a panel of 60 human tumor cell lines in vitro. Compounds 7, 9, 10, 15, 21 exhibited efficiently cytotoxic activities with GI(50) values in the low micromolar range against a variety of human cancer cell lines. 2,4-Bis(3'-indolyl)pyrimidine 8 displayed selective cytotoxic activity against IGROV1 tumor cell line with the GI(50) value below 0.01 microM.     

Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells

Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaper­ones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver.     

Synthesis of novel furo,thieno, and benzazetoazepines and evaluation of their cytotoxicity

We report the regio- and stereoselective synthesis of a novel cis-C(3)N-C(4)X-C(6)N series (X=O, S, and C(2)) from cyclic ketones. The cytotoxic activity of the new compounds was studied in five cell lines; the observed activities were in accordance with the concept of bioisosteric replacement.     

Intracellular glutathione regulates Andrographolide-induced cytotoxicity on hepatoma Hep3B cells

Abstract

Andrographolide (ANDRO), a diterpenoid lactone isolated from the traditional herbal plant Andrographis paniculata, was reported to induce apoptosis in hepatoma Hep3B cells in our previous study (Ji LL, Liu TY, Liu J, Chen Y, Wang ZT. Andrographolide inhibits human hepatoma-derived Hep3B cells growth through the activation of c-Jun N-terminal kinase. Planta Med 2007; 73: 1397–1401). The present investigation was carried out to observe whether cellular reduced glutathione (GSH) plays important roles in ANDRO-induced apoptosis. ANDRO initially increased intracellular GSH levels which then decreased later, while inhibition of cellular GSH synthesis by L-Buthionine-(S,R)-sulfoximine (BSO) augmented ANDRO-induced cytotoxicity and apoptosis in Hep3B cells. On the other hand, the thiol antioxidant dithiothreitol (DTT) rescued ANDRO-depleted cellular GSH, and abrogated ANDRO-induced cytotoxicity and apoptosis. Furthermore, BSO pretreatment augmented ANDRO-decreased expression of antioxidant protein thioredoxin 1 (Trx1), while DTT reversed this decrease. Further results showed that ANDRO increased the activity of the GSH-related antioxidant enzyme glutathione peroxidase (GPx) and the production of intracellular reactive oxygen species (ROS). Taken together, this study demonstrates that the intracellular redox system plays important roles in regulating the cytotoxicity of ANDRO on hepatoma Hep3B cells.     

Synthesis and primary evaluation of novel HIV-1 inhibitors

The overcoming of antiviral drug resistance is an important challenge in the treatment of HIV-1 infection. According to the theory of viral error catastrophe, slightly increasing the mutation rate could exceed the error threshold for viability of a viral population and kill it. Investigation of this mechanism could lead to the discovery of new antiviral agents capable of bypassing viral resistance. To this aim, we designed several modified nucleosides. We describe here the synthesis and partial evaluation of 8-amido-2'-deoxyadenosine. The supplementary amide group on the base should allow base-pairing with several natural nucleosides, thus creating supplementary mutations that would kill the virus.     

Synthesis and cytotoxicity of a novel iridoid glucoside derived from aucubin

The novel iridoid glycoside 2 was prepared in six steps (15% overall yield) from natural aucubin (1) and fully characterized. Compound 2, which comprises the same conjugated cyclopentenone pharmacophore as known antitumor oxylipins and prostaglandins, displayed significant antiproliferative in vitro activity towards leukemia L1210 cells. The Michael addition of nucleophilic thiols to compound 2 occurred on a different position compared to classical delta7-prostaglandin A1 methyl ester. The resulting adducts 7a and 7b were fully characterized, and their MS fragmentation patterns were elucidated.     

Synthesis and cytotoxicity of novel indirubin-5-carboxamides

Indirubins have been reported to act as potent inhibitors of protein kinases relevant to tumorigenesis and of tumor cell growth, but their development to antitumor drugs suffer from their poor water solubility. We synthesized a novel class of indirubin derivatives, indirubin-5-carboxamides, carrying amide substituents with basic centers. Quaternization or protonation of these alkylamino substituents provided indirubins with significantly improved solubility without loss of bioactivity.     

Synthesis and cytotoxicity of novel fatty acid-nucleoside conjugates

N(3)-Hydroxyethyltegafur (3) was synthesized in 91.0% yield under a new condition. A series of novel fatty acid esters of 3 were synthesized. These fatty acid-nucleoside conjugates have shown cytotoxicities against Ec9706 cells and A549 cells, and the structure activity relationship was discussed.     

Effect of UV irradiation on aflatoxin reduction: a cytotoxicity evaluation study using human hepatoma cell line

In this proof-of-concept study, the efficacy of a medium-pressure UV (MPUV) lamp source to reduce the concentrations of aflatoxin B₁, aflatoxin B₂, and aflatoxin G₁ (AFB_1, AFB₂, and AFG₁) in pure water is investigated. Irradiation experiments were conducted using a collimated beam system operating between 200 to 360 nm. The optical absorbance of the solution and the irradiance of the lamp are considered in calculating the average fluence rate. Based on these factors, the UV dose was quantified as a product of average fluence rate and treatment time. Known concentrations of aflatoxins were spiked in water and irradiated at UV doses ranging from 0, 1.22, 2.44, 3.66, and 4.88 J cm^?2. The concentration of aflatoxins was determined by HPLC with fluorescence detection. LC-MS/MS product ion scans were used to identify and semi-quantify degraded products of AFB₁, AFB₂, and AFG₁. It was observed that UV irradiation significantly reduced aflatoxins in pure water (p < 0.05). Irradiation doses of 4.88 J cm^?2 reduced concentrations 67.22% for AFG₁, 29.77% for AFB₂, and 98.25% for AFB₁ (p < 0.05). Using this technique, an overall reduction of total aflatoxin content of ≈95% (p < 0.05) was achieved. We hypothesize that the formation of ˙OH radicals initiated by UV light may have caused photolysis of AFB₁, AFB₂, and AFG₁ molecules. In cell culture studies, our results demonstrated that the increase of UV dosage decreased the aflatoxin-induced cytotoxicity in HepG₂ cells. Therefore, our research finding suggests that UV irradiation can be used as an effective technique for the reduction of aflatoxins.     

Synthesis and primary cytotoxicity evaluation of arylmethylenenaphthofuranones derivatives

New series of 2(or 3)-arylmethylenenaphtho[2,1-b]furan-3(or 2)-ones were synthesized, characterized and tested for anticancer properties in vitro. The target compounds were prepared by Knoevenagel coupling between the naphthofuranones 3, 28–30 and formyl derivatives. 2-(4-Oxo-1-benzopyran-3-ylmethylene)naphtho[2,1-b]furan-3-one 36 was the most active compound (IC₅₀ (L1210) = 1.6 μM). These compounds were also evaluated, in an independent manner, as inhibitors of Src protein tyrosine kinase, but only minor activity was observed.     

Synthesis and cytotoxicity evaluation of oleanolic acid derivatives

Twelve derivatives of oleanolic acid (1) have been synthesized and evaluated for their inhibitory activities against the growth of prostate PC3, breast MCF-7, lung A549, and gastric BGC-823 cancer cells by MTT assays. Within these series of derivatives, compound 17 exhibited the most potent cytotoxicity against PC3 cell line (IC₅₀ = 0.39 μM) and compound 28 displayed the best activity against A549 cell line (IC₅₀ = 0.22 μM). SAR analysis indicates that H-donor substitution at C-3 position of oleanolic acid may be advantageous for improvement of cytotoxicity against PC3, A549 and MCF-7 cell lines.     

Synthesis and primary cytotoxicity evaluation of arylmethylenenaphthofuranones derivatives

New series of 2(or 3)-arylmethylenenaphtho[2,1-b]furan-3(or 2)-ones were synthesized, characterized and tested for anticancer properties in vitro. The target compounds were prepared by Knoevenagel coupling between the naphthofuranones 3, 28-30 and formyl derivatives. 2-(4-Oxo-1-benzopyran-3-ylmethylene)naphtho[2,1-b]furan-3-one 36 was the most active compound (IC50 (L1210) = 1.6 microM). These compounds were also evaluated, in an independent manner, as inhibitors of Src protein tyrosine kinase, but only minor activity was observed.     

A novel andrographolide derivative AL‐1 exerts its cytotoxicity on K562 cells through a ROS‐dependent mechanism

Andrographolide‐lipoic acid conjugate (AL‐1) is a new in‐house synthesized chemical entity, which was derived by covalently linking andrographolide with lipoic acid. However, its anti‐cancer effect and cytotoxic mechanism remains unknown. In this study, we found that AL‐1 could significantly inhibit cell viability of human leukemia K562 cells by inducing G2/M arrest and apoptosis in a dose‐dependent manner. Thirty‐one AL‐1‐regulated protein alterations were identified by proteomics analysis. Gene ontology and ingenuity pathway analysis revealed that a cluster of proteins of oxidative redox state and apoptotic cell death‐related proteins, such as PRDX2, PRDX3, PRDX6, TXNRD1, and GLRX3, were regulated by AL‐1. Functional studies confirmed that AL‐1 induced apoptosis of K562 cells through a ROS‐dependent mechanism, and anti‐oxidant, N‐acetyl‐l ‐cysteine, could completely block AL‐1‐induced cytotoxicity, implicating that ROS generation played a vital role in AL‐1 cytotoxicity. Accumulated ROS resulted in oxidative DNA damage and subsequent G2/M arrest and mitochondrial‐mediated apoptosis. The current work reveals that a novel andrographolide derivative AL‐1 exerts its anticancer cytotoxicity through a ROS‐dependent DNA damage and mitochondrial‐mediated apoptosis mechanism.     

Preservation of microplate-attached human hepatoma cells and their use in cytotoxicity tests

We investigated the feasibility of hypothermic- orcryogenically-preserved human hepatoma Hep G2 cell preculturedin 96-well plates in cytotoxicity testings. First, we observedthat microplates precoated with both collagen (CN) and pronectin (PN) showed significantly improved living cell adhesion (71.0 ± 5.5%) after 48 hr of cryopreservation with 10%-DMSO containing culture medium, whereas non-coated surfaces gave very low living cell adhesion (33.5 ± 2.1%). Hypothermic preservation was most suitable for short-term storage, and cryogenic preservation at –20 °C allowed cells to be used within a week of the storage period. Only cryopreservation in a deep freezer (–85 °C) gave satisfactory results in much longer period of storage. Second, we evaluated the cytotoxicity of ten chemicals during 48 hr of exposure using hypothermically – (4 °C for 2 days) or cryogenically – (–85 °C for 7 days) preserved cells cultured inCN/PN-precoated microplates in comparison with results fromfreshly inoculated cells. Although almost the same LD₅₀values were obtained, LD₁₀ values of relatively hydrophilic chemicals obtained with cryopreserved cell were significantly lowered. These results shown that CN/PN-precoating is effective in keeping cells attached even in recultivation of preserved cells and that the toxicities of relatively hydrophilic chemicals tend to be overestimated when we use preserved cells in that manner.     

Synthesis and cytotoxicity of novel phosphorusless analogues of edelfosine

Modified series of phosphorusless edelfosine analogues bearing the polar heads of aliphatic bases, N,N-dimethylethanolamine and N,N,N1,N¹-tetramethylethylenediamine, were synthesized, with the length of the spacer varying from three to four methylene units. The cytotoxic characteristics of the compounds synthesized were studied.     

Identification of a novel insulin-sensitive glycophospholipid from H35 hepatoma cells

This study identifies and partially characterizes an insulin-sensitive glycophospholipid in H35 hepatoma cells. The incorporation of [3H]glucosamine into cell lipids was investigated. A major labeled lipid was purified by sequential thin layer chromatography using first an acid followed by a basic solvent system. After hydrochloric acid hydrolysis and sugar analysis by thin layer chromatography, 80% of the radioactivity in the purified lipid was found to comigrate with glucosamine. H35 cells were prelabeled with [3H]glucosamine for either 4 or 24 h and treated with insulin causing a dose-dependent stimulation of turnover of the glycophospholipid which was detected within 1 min. The purified glycolipid was cleaved by nitrous acid deamination indicating that the glucosamine C-1 was linked to the lipid moiety through a glycosidic bond. [14C]Ethanolamine, [3H]inositol, and [3H]sorbitol were not incorporated into the purified glycolipid. The incorporation of various fatty acids into this glycolipid was also studied. [3H]Palmitate was found to be preferentially incorporated while myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid were either not incorporated or incorporated less than 10% of palmitate. The purified glycolipid labeled with [3H]palmitate was cleaved by treatment with phospholipase A2 but was resistant to mild alkali hydrolysis suggesting the presence of a 1-hexadecyl,2-palmitoyl-glyceryl moiety in the purified lipid. Treatment of labeled glycophospholipid with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus generated a compound migrating as 1-alkyl,2-acyl-glycerol and a polar head group with a size in the range from 800 to 3500. These findings coupled with the nitrous acid deamination demonstrate that glucosamine was covalently linked through a phosphodiester bond to the glyceryl moiety of the purified glycolipid. These findings suggest that insulin acts on this glycophospholipid by stimulating an insulin-sensitive phospholipase C. This unique glycophospholipid may play an important role in insulin action by serving as precursor of insulin-generated mediators.