Galactose‐grafted chylomicron‐mimicking emulsion: evaluation of specificity against HepG‐2 and MCF‐7 cell lines

Objectives A chylomicron‐mimicking lipid emulsion was prepared and loaded with paclitaxel (paclitaxel‐CM) and was further grafted with galactose (paclitaxel‐GCM) using palmitoyl‐galactosamine, which was synthesized by reacting galactosamine hydrochloride with N‐hydroxy succinimide ester of palmitic acid. Palmitoyl‐galactosamine was used as a ligand for asialoglycoprotein receptors. Methods The uptake characteristics of the emulsions were evaluated in HepG‐2 cells (human hepatocarcinaoma), which express asialoglycoprotein receptors, and MCF‐7 (breast cancer) cells, which are devoid of these receptors. Key findings The incorporation efficiency of paclitaxel‐CM was 68.05 ± 4.80% and that of paclitaxel‐GCM was 72.10 ± 3.93% when the emulsion was prepared with 7.5% (w/w) paclitaxel/lipid phase. The globule size of paclitaxel‐GCM and paclitaxel‐CM was 124 ± 8.67 and 96.45 ± 5.78 nm, respectively. The release of paclitaxel from both of the formulations was fairly sustained: 50 ± 3.2% of paclitaxel in 24 h. The cytotoxicity and uptake of paclitaxel‐GCM were significantly higher (P < 0.05) in HepG‐2 cells than MCF‐7 cells, while for paclitaxel‐CM cytotoxicity and uptake were similar in the two cell lines. This study clearly demonstrates that upon surface modification palmitoyl‐galactosamine remains an integral part of the formulation. Paclitaxel solubility can be improved using optimum paclitaxel/lipid phase ratios. The paclitaxel‐GCM formulation recognizes asialoglycoprotein receptors over‐expressed on HepG‐2 cells. Conclusions Under our experimental conditions, the proposed paclitaxel‐GCM formulation is an ideal delivery vehicle for specific targeting to liver cancer cells, which is anticipated to result in improved efficacy and reduced toxicity to normal cells.     

Palmitoyl Ascorbate Liposomes and Free Ascorbic Acid: Comparison of Anticancer Therapeutic Effects Upon Parenteral Administration

Purpose  

To evaluate and compare anticancer therapeutic effect of palmitoyl ascorbate liposomes (PAL) and free ascorbic acid (AA).     

Palmitoyl Ascorbate-Loaded Polymeric Micelles: Cancer Cell Targeting and Cytotoxicity

Purpose  

To evaluate the potential of palmitoyl ascorbate (PA)-loaded micelles for ascorbate-mediated cancer cell targeting and cytotoxicity.     

Cytotoxicity of Paclitaxel Incorporated in PLGA Nanoparticles on Hypoxic Human Tumor Cells

Purpose  The aim of this work was to prepare paclitaxel-loaded PLGA nanoparticles and determine cytotoxicity of released paclitaxel for two hypoxic human tumor cell lines: breast carcinoma (MCF-7) and carcinoma cervicis (HeLa). Methods  Poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel were prepared by o/w emulsification-solvent evaporation method. Physicochemical characteristics of nanoparticles were studied. Cellular uptake of nanoparticles was evaluated by transmission electronic microscopy and fluorescence microscopy. Flow cytometry quantified the number of cells held in G₂/M phase. Cell viability was determined by the ability of single cell to form colonies. Biodistribution of nanoparticles in mice was evaluated by fluorescence microscopy. Results  The nanoparticles were spherical with average diameter 318 ± 5.1 nm. The encapsulation efficiency was 88.52%. The drug release profile in vitro exhibited a biphasic pattern. Cellular uptake was observed. Co-culture of tumor cells with paclitaxel-loaded nanoparticles demonstrated that released paclitaxel retained its bioactivity to block cells in G₂/M phase. Paclitaxel-loaded nanoparticles exhibited cytotoxic effect on both hypoxic MCF-7 and HeLa cells and its cytotoxicity was more significant than that of free paclitaxel. Fluorescent nanoparticles were mainly distributed to liver and spleen of mice. Conclusions  Paclitaxel-loaded PLGA nanoparticles may be considered a promising drug delivery system to eradicate hypoxic tumor cells. Cheng Jin and Ling Bai contributed equally to this work.     

整合素配体RGD修饰的阳离子脂质体作为siRNA输送载体的体外研究

本研究构建了能够靶向肿瘤新生血管的RGD修饰阳离子脂质体（RGD-Lipo），作为靶向耐药相关基因MDR1的siRNA输送载体并评价其相关的药剂学性质。该脂质体与siRNA形成的复合物粒径控制在200nm以内，并且对其中所包载的siRNA具有一定的保护作用。体外实验结果表明，经RGD修饰的脂质体（RGD-Lipo）细胞粘附能力显著增强，并可增加细胞内siRNA的转染效果。与利用前插法进行靶向修饰相比，利用后插法进行RGD修饰可有效地改善siRNA的溶酶体释放效率。细胞毒试验结果表明，后插法制备的pRGD-Lipo-siRNA能够有效逆转人卵巢癌SKV03／A细胞的耐药性，并增加阿霉素药物在细胞内的蓄积。体外研究结果证实，使用pRGD-Lipo-siRNA有利于提高耐药细胞对化疗药阿霉素的敏感度，并将有可能应用于临床耐药肿瘤的治疗。     

维生素C棕榈酸酯再肿瘤治疗中的研究进展

近年来,维生素C的抗肿瘤作用已引起了人们的广泛关注,但维生素C需较高剂量才可达到抗肿瘤作用,且化学性质不稳定,使其临床应用受到了限制.为了解决上述问题,研究者一直致力于维生素C衍生物的合成.维生素C棕榈酸酯作为维生素C的脂溶性衍生物,在理化性质、抗肿瘤活性等方面具有较多的优势.本文从维生素C棕榈酸酯的理化特征、抗肿瘤活性、机制和应用等方面对其在肿瘤治疗中的研究情况进行综述,为其在临床肿瘤防治中的研究提供依据.     

Cytosolic Delivery of Liposomally Targeted Proteins Induced by Photochemical Internalization

Purpose The application of therapeutic proteins is often hampered by limited cell entrance and lysosomal degradation, as intracellular targets are not reached. By encapsulation of proteins into targeted liposomes, cellular uptake via endocytosis can be enhanced. To prevent subsequent lysosomal degradation and promote endosomal escape, photochemical internalization (PCI) was studied here as a tool to enhance endosomal escape. PCI makes use of photosensitising agents which localize in endocytic vesicles, inducing endosomal release upon light exposure. Materials and Methods The cytotoxic protein saporin was encapsulated in different types of targeted liposomes. Human ovarian carcinoma cells were incubated with the photosensitiser TPPS2a and liposomes. To achieve photochemical internalization, the cells were illuminated for various time periods. Cell viability was used as read-out. Illumination time and amount of encapsulated proteins were varied to investigate the influence of these parameters. Results The cytotoxic effect of liposomally targeted saporin was enhanced by applying PCI, likely due to enhanced endosomal escape. The cytotoxic effect was dependent on the amount of encapsulated saporin and the illumination time. Conclusion PCI is a promising technique for promoting cytosolic delivery of liposomally targeted saporin. PCI may also be applicable to other liposomally targeted therapeutic proteins with intracellular targets.     

Design and synthesis of taxane derivatives of valproic acid as potent and selective cytotoxic agents

Resistance to anticancer agents has important implications for cancer chemotherapy. Small changes in chemical structures of cytotoxic agents can alter their biological interactions that can be beneficial in overcoming the drug resistance problem. Valproic acid, a well-known antiepileptic drug is in advanced clinical studies for cancer treatment. In the present study, valproic acid was incorporated into the taxane moiety at various positions and the new analogs were evaluated for their in vitro cytotoxicity. The novel analog, Valprotaxel showed comparable cytotoxicity in head and neck, and colon cancer cell lines with remarkable improvement in selectivity for cancer cells compared to paclitaxel and docetaxel.     

Characterization of the cytotoxic activity of the indoloquinoline alkaloid cryptolepine in human tumour cell lines and primary cultures of tumour cells from patients

The plant derived indoloquinoline alkaloid cryptolepine was investigated for its cytotoxic properties in 12 human tumour cell lines and in primary cultures of tumour cells from patients. The fluorometric microculture cytotoxicity assay was used to assess cytotoxicity and DNA micro-array analysis to evaluate gene expression. Cryptolepine mean IC₅₀ in the cell line panel was 0.9 μM compared with 1.0 and 2.8 μM in haematological and solid tumour malignancies, respectively. Among patient solid tumour samples, those from breast cancer were the most sensitive and essentially as sensitive as haematological malignancies. Cryptolepine activity showed highest correlations to topoisomerase II and microtubule targeting drugs. In the cell lines cryptolepine activity was essentially unaffected by established mechanisms of drug resistance. A number of genes were identified as associated with cryptolepine activity. In conclusion, cryptolepine shows interesting in vitro cytotoxic properties and its further evaluation as an anti-cancer drug seems warranted.     

Diammine Dicarboxylic Acid Platinum Enhances Cytotoxicity in Platinum-Resistant Ovarian Cancer Cells through Induction of Apoptosis and S-Phase Cell Arrest

Purpose  Polysaccharides such as chondroitin play a potent role in tumor growth, tissue repair and angiogenesis. These properties make chondroitin a good candidate for novel drug delivery systems. Diammine dicarboxylic acid platinum (DDAP), a novel polymeric platinum compound, was developed by conjugating the platinum analogue to aspartate–chondroitin for drug delivery to tumor cells. DDAP improves platinum solubility which may reduce systemic toxicity and be more efficacious than cisplatin in killing tumor cells. Methods  We tested and compared the cytotoxic effects of DDAP and CDDP on the platinum-sensitive 2008 and A2780 ovarian cancer cell lines and their platinum-resistant sublines 2008.C13 and A2780cis; we also investigated DDAP’s mechanism of action. Results  In the platinum-sensitive cell lines, the cytotoxic effects of DDAP and CDDP were comparable. However, in the platinum-resistant sublines, significantly greater cell-growth inhibition was induced by DDAP than by CDDP, especially at lower doses. DDAP also induced more apoptosis than CDDP did in the 2008.C13 subline, which was partially mediated by the caspase 3-dependent pathway. In addition, lower (but not higher) doses of DDAP arrested 90% of S-phase 2008.C13 cells, which might be associated with up-regulation of p21 and maintenance of low cyclin A expression. Furthermore, greater cellular uptake of DDAP was seen in platinum-resistant than in platinum-sensitive ovarian cancer cells. Conclusions  Low-dose DDAP enhances drug delivery to platinum-resistant ovarian cancer cells and substantially inhibits their growth by inducting apoptosis and arresting cells in the S-phase, suggesting that DDAP may overcome platinum resistance in ovarian cancer.     

Synergistic effect of paclitaxel and 4-hydroxytamoxifen on estrogen receptor-negative colon cancer and lung cancer cell lines

Antiestrogen tamoxifen (Tam) is the most prescribed drug for the treatment of estrogen receptor (ER)-positive breast cancers. It is also used in long-term clinical trials with encouraging preliminary results as a chemopreventive agent for breast cancer. The effect of Tam on ER-negative cancers, however, is unclear. Here we reported that paclitaxel and 4-hydroxytamoxifen (4-HT) have a synergistic cytotoxic effect on the ER-negative colon cancer cell line HCT15, which is refractory to paclitaxel alone. Our results showed that 4-HT at submicromolar concentrations effectively enhanced the antiproliferative effect of paclitaxel. In addition, at 1/10 of the paclitaxel concentrations used for HCT15, 4-HT and paclitaxel also showed synergistic effect on NCI H460, an ER-negative lung cancer cell line. For both cell lines, the effective concentration for paclitaxel to inhibit cell growth was 1 log lower in the combination treatment than the concentration used in the single treatment. Cell cycle analysis showed that the combination of paclitaxel and 4-HT increased the G2/M population and resulted in the increase of apoptosis in both cell lines. Enhanced early release of cytochrome c from mitochondria may be the apoptotic pathway activated in the combination treatment in HCT15 cells.     

紫杉醇诱导胃癌BGC-823和SGC-7901细胞凋亡作用的比较

目的研究胃癌BGC-823细胞和SGC-7901细胞对紫杉醇诱导凋亡的敏感性。方法用0．1～0．5μmol·L^-1紫杉醇作用于BGC-823和SGC-7901细胞,再用0．3μmol·L^-1紫杉醇对BGC-823和SGC-7901细胞分别作用0、6、12、24、36、48h,通过形态学观察及流式细胞术检测细胞凋亡率的变化。结果（1）紫杉醇对两种胃癌细胞均有明显诱导凋亡作用,且均呈时间依赖性及剂量依赖性;（2）中分化的SGC-7901细胞株对紫杉醇的敏感性明显高于低分化的BGC-823细胞株。结论不同分化程度的胃癌细胞株对紫杉醇的敏感性不同。     

Design, synthesis, and QSAR study of novel 2-(2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-phenylacetamide derivatives as cytotoxic agents

This study deals with the synthesis of novel 2-(2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-phenylacetamide derivatives (6a–j) from isatin (3) and 5,7-dibromoisatin (4). All newly synthesized compounds were characterized using IR, ¹H NMR, MS, and elemental analysis followed by evaluation of their cytotoxic activity by XTT assay on breast cancer cell line MCF-7 and non-cancer African green monkey cell line VERO. Correlation study for QSAR and in vitro assay was performed. The outcomes indicated that electron withdrawing substitutions at para position of phenyl ring and 5, 7 positions of isatin ring and increasing lipophilicity of the compound increased the cytotoxic activity. The 2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-(4-nitrophenyl)acetamide (6b) was found to be the most active compound in the series and demonstrated higher selectivity toward MCF-7 cell line. The IC₅₀ values were 1.96 and 1.90 μM for test compound (6b) and vinblastin (reference drug), respectively. This indicates compound (6b) may possess equipotent cytotoxic activity to vinblastine. The compound (6b) is particularly promising, since it could kill cancer cells 19–20 times more effectively than the non-cancer cells. This property of (6b) may enable us to effectively control tumors with low side effects. Hence, we propose that 2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H- indol-1-yl)-N-(4-nitrophenyl)acetamide may be used as lead for further development.     

Cytotoxic effect induced by retinoic acid loaded into galactosyl-sphingosine containing liposomes on human hepatoma cell lines

Two retinoids, ATRA and 13cisRA, were incorporated into liposomes of different composition and charge and added to two hepatoma cell lines with different degree of transformation to measure cytotoxicity by MTT assay. Retinoid-free cationic liposomes were more toxic than the other kinds (anionic and made only of PC) but were also the best delivery system for retinoic acid to induce specific cytotoxic effects on these tumor hepatoma cell lines. Galactosyl-sphingosine containing cationic liposomes increased the cytotoxic effect induced by ATRA on Hep3B cells when compared to glucosyl-sphingosine cationic liposomes, but did not improve the effect induced by free retinoid or ATRA loaded into liposomes without glycolipids. This suggests that in this cell line, ATRA is being incorporated by a mechanism mediated by the asialoglycoprotein receptor, but at the same time, non-specific sugar-independent capture is also taking place as well as free diffusion of ATRA directly through the membrane. Galactose-specific effect was not observed in HepG2 cells treated with ATRA or both cell lines treated with 13cisRA. In fact, treatment of HepG2 cells with retinoids entrapped into liposomes likely induces proliferation instead of cytotoxicity, a result that interferes with the measurement of cell death by MTT. Compared to the specific effect of ATRA entrapped into cationic liposomes, vesicles made only by PC, did not mediate a specific mechanism, since differences between ATRA in galactosyl- and glucosyl-shpingosine PC-liposomes were not statistically significant. The specific mechanism was not present in the myoblastic cell line C2C12, where ATRA incorporated into galactosyl- and glucosyl-sphingosine containing cationic and PC-liposomes, was able to induce cytotoxicity at the same extent. Micelles containing ATRA and galactosyl-sphingosine had a significantly more toxic effect than the retinoid administered together with glucosyl-sphingosine, in Hep3B cells. Also, micelles containing ATRA were more toxic than glycolipid-containing liposomes with ATRA, for both kinds of sphingosines. The same effect was not observed in C2C12 cells, where glycolipid-containing liposomes worked better than micelles, and a sugar-specific mechanism was not seen. This suggests that, even though galactose-containing cationic liposomes could be a promising approach, a galactose-specific emulsion system could be the best strategy to specifically deliver retinoic acid to liver tumor cells, since it shows tissue specificity (perhaps induced by ASGPR-mediated internalization) and a stronger cytotoxic effect than the retinoid incorporated into liposomes.     

Transportan 10 improves the anticancer activity of cisplatin

The aim of this paper was to examine whether cell-penetrating peptides (CPPs) such as transportan 10 (TP10) or protein transduction domain (PTD4) may improve the anticancer activity of cisplatin (cPt). The complexes of TP10 or PTD4 with cPt were used in the experiments. They were carried out on two non-cancer (HEK293 (human embryonic kidney) and HEL299 (human embryo lung)) and two cancer (HeLa (human cervical cancer) and OS143B (human osteosarcoma 143B)) cell lines. Both complexes were tested (MTT assay) with respect to their anticancer or cytotoxic actions. TAMRA (fluorescent dye)-stained preparations were visualized in a fluorescence microscope. The long-term effect of TP10 + cPt and its components on non-cancer and cancer cell lines was observed in inverted phase contrast microscopy. In the MTT test (cell viability assay), the complex of TP10 + cPt produced a more potent effect on the cancer cell lines (HeLa, OS143B) in comparison to that observed after separate treatment with TP10 or cPt. At the same time, the action of the complex and its components was rather small on non-cancer cell lines. On the other hand, a complex of another CPP with cPt, i.e., PTD4 + cPt, was without a significant effect on the cancer cell line (OS143B). The images of the fluorescent microscopy showed TAMRA-TP10 or TAMRA-TP10 + cPt in the interior of the HeLa cells. In the case of TAMRA-PTD4 or TAMRA-PTD4 + cPt, only the first compound was found inside the cancer cell line. In contrast, none of the tested compounds gained access to the interior of the non-cancer cells (HEK293, HEL299). Long-term incubation with the TP10 + cPt (estimated by inverted phase contrast microscopy) lead to an enhanced action of the complex on cell viability (decrease in the number of cells and change in their morphology) as compared with that produced by each single agent. With regard to the tested CPPs, only TP10 improved the anticancer activity of cisplatin if both compounds were used in the form of a complex. Additionally, the complex was relatively safe for non-cancer cells. What is more, TP10 also produced an anticancer effect on HeLa and OS143B cell lines.     

In vitro toxicity of naringin and berberine alone,and encapsulated within PMMA nanoparticles

Phytochemical compounds, such as naringin and berberine, have been used for many years due to their antioxidant activities, and consequently, beneficial health effects. In this study, it was aimed to evaluate the antioxidant properties of naringin, berberine and poly(methylmethacrylate) (PMMA) nanoparticles (NPs) encapsulated with naringin or berberine and their possible cytotoxic, genotoxic, and apoptotic effects on mouse fibroblast (NIH/3 T3) and colon cancer (Caco-2) cells. According to the results of the study, it was found that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition antioxidant activity of naringin, berberine, and naringin or berberine encapsulated PMMA NPs, was significantly increased at higher tested concentrations due to the antioxidant effects of naringin, berberine and naringin or berberine encapsulated PMMA NPs. As a result of the cytotoxicity assay, after 24-, 48- and 72-h of exposure, all of the studied compounds caused cytotoxic effects in both cell lines. Genotoxic effects of studied compounds were not registered at lower tested concentrations. Based on these data, polymeric nanoparticles encapsulated with naringin or berberine may contribute to new treatment approaches for cancer, but further in vivo and in vitro research is required.     

In vitro toxicity of camalexin derivatives in human cancer and non-cancer cells

The aim of the study was to investigate the cytotoxic activity of camalexin and its five synthetic derivatives in cancer and non-cancer cells.In cancer cells the benzocamalexin (BC) displayed the most potent activity with an IC₅₀ value of 23.3–30.1 μmol/L. On the other hand, minimal toxicity (IC₅₀ > 100.0 μmol/L) in non-cancer cells was observed. Based on these results, BC was selected for further studies.Flow cytometric analysis revealed a BC-induced arrest of the cell cycle in the G2 phase associated with downregulation of α-tubulin, α1-tubulin, β5-tubulin expression. These findings suggest that the inhibitory effect of BC is mediated via inhibition of microtubule formation. Moreover, BC downregulated the expression of microtubule-related protein indicating the effect of this compound on microtubule assembly. After treatment with BC increase of the sub-G₁ DNA content fraction was noted which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by DNA fragmentation assay. Moreover, quantitative real-time PCR showed that BC downregulated the expression of antiapoptotic genes Bcl-2 and Bcl-xL and upregulated the expression of proapoptotic Bax.Taken together, our study suggests that the blockade of cell cycle progression and initiation of apoptosis may play an important role in the antiproliferative activity of BC in human cancer cells.     

Synthesis of piperine–amino acid ester conjugates and study of their cytotoxic activities against human cancer cell lines

Abstract  

A series of piperine–amino acid ester conjugates (4a–4r) were synthesized under mild conditions and screened for their cytotoxic activities against a panel of human cancer cell lines (IMR-32, MCF-7, PC-3, DU-145, Colo-205, and Hep-2). The parent compound piperine lacked significant activity but the analogues were effective to in all tested human cancer cell lines. The introduction of d- and l-amino acid side chain to piperine through peptide linkage significantly increased cytotoxic activity. Among the tested conjugates, 4p showed significant cytotoxic activity against DU-145 cell lines with IC₅₀ of 21 μM. The synthetic protocol is suitable for generating piperine derivatives with various structural motifs for exploring the desired activity.     

Lipopeptide-based liposomes for DNA delivery into cells expressing neuropilin-1

In this paper, liposomes containing a lipopeptide bearing a ligand specifically recognized by neuropilin-1 (NRP-1) have been used to target a human breast cancer cell line overexpressing this receptor. The synthesis of this lipopeptide, C16-A7R, formed by the sequence of 7 amino acids ATWLPPR, linked to a palmitoyl fatty chain by an amide bond was described. After the characterisation of cationic liposomes formulated with the lipopeptide, the results obtained using various techniques showed that the lipopeptide-based liposomes were well accumulated in cells of the human breast cancer line MDA-MB-231 overexpressing NRP-1. Delivery of reporter genes expressing either beta-galactosidase (beta-gal) or green fluorescent protein (GFP) was selectively enhanced in these cells when compared with NRP-1-negative cells. In MDA-MB-231 cells, an increase by 250% in beta-gal activity was observed when delivered by lipopeptide-based liposomes compared to cationic liposomes alone.     

Bufadienolides from the Bufo viridis toad venom exert cytotoxic effects on cancer cells by inducing cell apoptosis and cell cycle arrest

A series of bufadienolides were isolated from the Bufo viridis toad venom, and their cytotoxic activities against three human cancer cell lines (HeLa, HT-29, MCF7) and a non-cancer cell line (L-O2) were explored using the MTT assay in vitro. All of nine compounds exhibited cytotoxic activities against the three cancer cell lines, with compound D4 exhibiting potent cytotoxic activity against HeLa cells and was better than positive control. Herein, we further evaluated the effect of compound D4 on HeLa cells. The results revealed that compound D4 has excellent cytotoxic effect on HeLa cells by inhibiting cell colony formation and migration, promoting cell apoptosis, increasing reactive oxygen species (ROS) levels and arresting of HeLa cells in S and G2/M phases. These findings encourage further work on the chemistry and bioactivity of the Bufo viridis toad venom.