Folic acid-modified celastrol nanoparticles: synthesis, characterization, anticancer activity in 2D and 3D breast cancer models.

Celastrol is used in traditional Chinese medicine for treating cancers. However, its low water solubility and poor tumour selection represent major pitfalls for clinical application. In the present study, gold nanoparticle (AuNP) firstly was conjugated with PVP-co-2-dimethylaminoethyl methacrylate (Polymer) and celastrol then modified by folic acid. The as-prepared folate receptor-targeted celastrol AuNP (FCA) was characterized using attenuated total reflection Fourier transform infrared spectroscopy, UV-Vis spectrometry, transmission electron microscope, and inductively coupled plasma mass spectrometry. The physical properties of FCA were also determined in solubility, drug encapsulation and in vitro drug release. Its anticancer activities were assessed in the 2D and 3D breast cancer models. The results showed that FCA was synthesized successfully with good solubility, high encapsulation efficiency and loading content. FCA showed the optimal cumulative release at pH 5.0 and high cellular uptake and exhibited significant inhibition on breast cancer cells. FCA also induced more significant apoptosis either in 2D and 3D breast cancer model than the celastrol AuNP and celastrol alone. These findings demonstrate that FCA improves water solubility of celastrol and enhances its anticancer activities against breast cancer. FCA might be a potential candidate of anticancer drug for breast cancer in the future if further development.

Preparation of hypocrellin B nanocages in self-assembled apoferritin for enhanced intracellular uptake and photodynamic activity.

Photodynamic therapy (PDT) is a promising modality, which uses light energy to activate a nontoxic photosensitizer to treat various diseases like cancer and infectious diseases. Hypocrellin B (HB) is a naturally occurring photosensitizer isolated from traditional Chinese medicine Hypocrella bambusea. However, the high hydrophobicity and poor selectivity severely limit its application. Apoferritin, a macromolecular protein, can serve as an attractive nanocage to carry HB while improving its water solubility and tumor selectivity. In this study, novel HB-apoferritin nanoparticles (HB-AFT NPs) were successfully developed by assembling HB within the apoferritin nanocage. The self-assembled HB-AFT NPs have a narrow size distribution and smooth surface with an average diameter of 12 nm and a HB encapsulation efficiency of 85%. The morphological observation and circular dichroism analysis showed that the encapsulation strategy of HB did not impair apoferritin structure, and thus it potentially maintained the tumor targeting ability of apoferritin. Compared with free HB solution, HB-AFT NPs exhibited more pronounced photodynamic activity on MDA-MB-231 cells due to the higher intracellular uptake, increased reactive oxygen species (ROS) production and excellent tumor-targeting. All these results suggest that the self-assembled HB-AFT NPs can be considered as a potential photosensitive drug for tumor targeting photodynamic therapy.

Palmatine hydrochloride mediated photodynamic inactivation of breast cancer MCF-7 cells: Effectiveness and mechanism of action.

Breast cancer is one of the commonest malignant tumors threatening to women. The present study aims to investigate the effect of photodynamic action of palmatine hydrochloride (PaH), a naturally occurring photosensitizer isolated from traditional Chinese medicine (TCM), on apoptosis of breast cancer cells. Firstly, cellular uptake of PaH in MCF-7 cells was measured and the cytotoxicity of PaH itself on breast cancer MCF-7 cells was estimated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Subcellular localization of PaH in MCF-7 cells was observed using confocal laser scanning microscopy (CLSM). For photodynamic treatment, MCF-7 cells were incubated with PaH and then irradiated by visible light (470nm) from a LED light source. Photocytotoxicity was investigated 24h after photodynamic treatment using MTT assay. Cell apoptosis was analyzed 18h after photodynamic treatment using flow cytometry with Annexin V/PI staining. Nuclear was stained using Hoechst 33342 and observed under a fluorescence microscope. Intracellular production of reactive oxygen species (ROS) was studied by measuring the fluorescence of 2, 7-dichlorofluorescein (DCF) using a flow cytometry. Results showed that PaH treatment alone had no or minimum cytotoxicity to MCF-7 cells after incubation for 24h in the dark. After incubation for 40min, the cellular uptake of PaH reached to the maximum, and PaH mainly located in mitochondria and endoplasmic reticulum of MCF-7 cells. Photodynamic treatment of PaH demonstrated a significant photocytotoxicity on MCF-7 cells, induced remarkable cell apoptosis and significantly increased intracellular ROS level. Our findings demonstrated that PaH as a naturally occurring photosensitizer induced cell apoptosis and significantly killed MCF-7 cells.

Photodynamic action of palmatine hydrochloride on colon adenocarcinoma HT-29 cells.

Palmatine hydrochloride (PaH) is a natural active compound from a traditional Chinese medicine (TCM). The present study aims to evaluate the effect of PaH as a new photosensitizer on colon adenocarcinoma HT-29 cells upon light irradiation. Firstly, the absorption and fluorescence spectra of PaH were measured using a UV-vis spectrophotometer and RF-1500PC spectrophotometer, respectively. Singlet oxygen ((1)O2) production of PaH was determined using 1, 3-diphenylisobenzofuran (DPBF). Dark toxicity of PaH was estimated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake of PaH in HT-29 cells was detected at different time intervals. Subellular localization of PaH in HT-29 cells was observed using confocal laser fluorescence microscopy. For photodynamic treatment, HT-29 cells were incubated with PaH and then irradiated by visible light (470nm) from a LED light source. Photocytotoxicity was investigated 24h after photodynamic treatment using MTT assay. Cell apoptosis was observed 18h after photodynamic treatment using a flow cytometry with Annexin V/PI staining. Results showed that PaH has an absorption peak in the visible region from 400nm to 500nm and a fluorescence emission peak at 406nm with an excitation wavelength of 365nm. PaH was activated by the 470nm visible light from a LED light source to produce (1)O2. Dark toxicity showed that PaH alone treatment had no cytotoxicity to HT-29 cancer cells and NIH-3T3 normal cells after incubation for 24h. After incubation for 40min, the cellular uptake of PaH reached to the maximum and PaH was located in mitochondria. Photodynamic treatment of PaH demonstrated a significant photocytotoxicity on HT-29 cells. The rate of cell death increased significantly in a PaH concentration-dependent and light dose-dependent manner. Further evaluation revealed that the early and late apoptotic rate of HT-29 cells increased remarkably up to 21.54% and 5.39% after photodynamic treatment of PaH at the concentration of 5µM and energy density of 10.8J/cm(2). Our findings demonstrated that PaH as a naturally occurring photosensitizer has potential in photodynamic therapy on colon adenocarcinoma.

Virucidal efficacy of treatment with photodynamically activated curcumin on murine norovirus bio-accumulated in oysters.

Norovirus (NoV) is one of the most important seafood- and water-borne viruses, and is a major cause of acute gastroenteritis outbreaks. In the present study we investigated the effect of curcumin as a sensitizer to photodynamic treatment both in buffer and in oysters against murine norovirus 1 (MNV-1), a surrogate of NoV. MNV-1 cultured in buffer and MNV-1 bio-accumulated in oysters were irradiated with a novel LED light source with a wavelength of 470nm and an energy of 3.6J/cm(2). Inactivation of MNV-1 was investigated by plaque assays. After virus was extracted from the gut of oysters treated over a range of curcumin concentrations, the ultrastructural morphology of the virus was observed using electron microscopy, and the integrity of viral nucleic acids and stability of viral capsid proteins were also determined. Results showed that the infectivity of MNV-1 was significantly inhibited by 1-3logPFU/ml, with significant damage to viral nucleic acids in a curcumin dose-dependent manner after photodynamic activation. Virus morphology was altered after the photodynamic treatment with curcumin, presumably due to the change of the viral capsid protein structures. The data suggest that treatment of oysters with photodynamic activation of curcumin is a potentially efficacious and cost-effective method to inactivate food-borne NoV. Further studies are necessary to evaluate the toxicology of this approach in detail and perform sensory evaluation of the treated product.

Antidiabetic Effect of Schisandrae Chinensis Fructus Involves Inhibition of the Sodium Glucose Cotransporter.

Preclinical Research Schisandrae Chinensis Fructus (SCF), the fruit of Schisandra chinensis (Turcz.) Baill. (family Schisandraceae) is traditionally used as a tonic and antidiabetic agent in Asia. In this study, SCF was investigated for its effects on sodium glucose cotransporters 1 and 2 (SGLT 1 and 2) expressed in a COS-7 cell line for its specificity in inhibiting SGLT2, which is a novel mechanism to screen for potential antidiabetic agents. Using a bioassay-guided fractionation, we then tried to isolate and identify the active fraction(s)/component(s). The ethanol extract of SCF at a concentration of 1 mg/mL significantly inhibited 89% of SGLT1 and 73% of SGLT2 activities in a [14 C]-α-methyl-d-glucopyranoside ([14 C]-AMG) uptake assay. Fractionation of the ethanol extract yielded nine fractions, of which F8, at a concentration of 1 mg/mL, was specific in inhibiting SGLT 2 (42% inhibition, P < 0.001), without inhibiting SGLT 1. Using LC/MS-MS, three compounds, deoxyschisandrin, schisandrin B (γ-schisandrin) and schisandrin were identified in F8 and their amounts quantified. However, subsequent evaluation in the [14 C]-AMG uptake assay showed that these three compounds failed to inhibit SGLT 2 activity indicating that the SGLT active component(s) from SCF have yet to be identified. Drug Dev Res 76 : 1-8, 2015.

Apoptosis of breast cancer cells induced by hypocrellin B under light-emitting diode irradiation.

OBJECTIVES: Breast cancer is a common disease which threatens the life of women. To explore an alternative modality for combating breast cancer, a light-emitting diode (LED) that activates hypocrellin B was used in the present study to investigate apoptosis induction in breast cancer MDA-MB-231 cells. MATERIALS AND METHODS: Photocytotoxicity was investigated 24h after photodynamic treatment of hypocrellin B using MTT reduction assay and light microscopy. Apoptosis was observed 6h after photodynamic treatment using flow cytometry with Annexin V/PI staining as well as fluorescent microscopy with Hoechst33258 staining. The ultrastructure of the treated cells was observed using transmission electron microscopy (TEM). RESULTS: Hypocrellin B-induced photocytotoxicity in MDA-MB-231 cells exhibited a dose-dependent manner. The amount of MDA-MB-231 cells attached to the bottom of well decreased significantly after photodynamic treatment of hypocrellin B. Flow cytometry showed that the early and late apoptotic rate of MDA-MB-231 cells increased remarkably up to 17.46% and 32.80%, respectively, after treatment of LED-activated hypocrellin B. In addition, nuclear condensation, fragmentation and chromatin margination, and topical apoptotic body in the treated cells were observed by nuclear staining and TEM. CONCLUSION: Photodynamic action of hypocrellin B irradiated by light-emitting diodes could significantly kill breast cancer cells and induce apoptotic cell death, which suggests LED-activated hypocrellin B is a promising strategy for combating breast cancer.

Ultrasound-induced cell death of nasopharyngeal carcinoma cells in the presence of curcumin.

OBJECTIVES: Curcumin, a natural pigment from a traditional Chinese herb, has been attracting extensive attention. The present study aims to investigate cell death of nasopharyngeal carcinoma (NPC) cells induced by ultrasound sonication in the presence of curcumin in vitro. METHODS: The NPC cell line CNE2 was chosen as a tumor model, and curcumin concentration was kept constant at 10 µM while the cells were subjected to ultrasound exposure for 8 s at an intensity of 0.46 W/cm(2). Cell death was evaluated using flow cytometry with annexinV-FITC and propidium iodine staining, and nuclear staining with Hoechst 33258. Mitochondrial membrane potential and intracellular reactive oxygen species (ROS) were analyzed using flow cytometry with rhodamine 123 and dichlorodihydrofluorecein diacetate staining. RESULTS: Flow cytometry showed that the combination of ultrasound and curcumin significantly increased the necrotic or late apoptotic rate by up to 31.37% compared with the controls. Nuclear condensation was observed in the nuclear staining, and collapse of ΔΨm and ROS increase were found in the CNE2 cells after the treatment with curcumin and ultrasound. CONCLUSIONS: The findings demonstrate that the presence of curcumin significantly enhances the ultrasound-induced cell death and ROS level, and induces the collapse of ΔΨm, suggesting that ultrasound sonication can increase the cell death of NPC cells in the presence of curcumin and that the treatment using curcumin and ultrasound together is a potential therapeutic modality in the management of malignant tumors.

Hematoporphyrin monomethyl ether enhances the killing of ultrasound on osteosarcoma cells involving intracellular reactive oxygen species and calcium ion elevation.

OBJECTIVE: The present study aims to investigate the possible mechanisms of hematoporphyrin monomethyl ether (HMME) enhancing the cytotoxicity of ultrasound in osteosarcoma cells. METHODS: Osteosarcoma cell line UMR-106 was treated by HMME and ultrasound radiation, with the HMME concentration kept at 20 µg/mL and ultrasound radiation for 10 seconds at the intensity of 0.5 W/cm². Cell proliferation was investigated at 12, 24, 36, and 48 hours using MTT assay after ultrasound and HMME treatment. Ultrastructural morphology was observed using transmission electron microscopy (TEM). Intracellular reactive oxygen species (ROS) was measured using a flow cytometry with DCFH-DA staining and intracellular free calcium ion (Ca(2+)) with Fluo-3-AM staining. RESULTS: The UMR-106 cells proliferated rapidly in the sham radiation and HMME treatment alone group, but ultrasound-treated cells and HMME-ultrasound-treated cells proliferated slowly. There was a significant difference between HMME-ultrasound treatment and the controls, including ultrasound radiation, HMME treatment alone, and sham radiation (P < .05). TEM showed endoplasmic reticulum and mitochondrial swelling in the ultrasound-treated cells, and more cells presented apoptosis and necrosis after treatment with ultrasound and HMME together. Intracellular ROS and Ca(2+) in the cells increased more significantly after both ultrasound and HMME treatment than after ultrasound treatment alone. CONCLUSIONS: HMME could effectively enhance the inhibition effect of ultrasound on osteosarcoma cells. Intracellular ROS and Ca(2+) in the UMR-106 cells increased more significantly after the treatment of HMME and ultrasound together, indicating that the enhancement of HMME on ultrasound cytotoxicity to osteosarcoma cells possibly involves both intracellular ROS and Ca(2+) elevation.