Yi-Xin-Shu capsule ameliorates cardiac hypertrophy by regulating RB/HDAC1/GATA4 signaling pathway based on proteomic and mass spectrometry image analysis.

BACKGROUND: Cardiac hypertrophy (CH) forms the main pathological basis of chronic heart failure (CHF). Mitigating and preventing CH is the key strategy for the treatment of ventricular remodeling in CHF. Yi-Xin-Shu capsule (YXS) has been commonly applied in the clinical treatment of CHF in Asian countries for several decades. However, the underlying mechanism of YXS has not been revealed yet. PURPOSE: To assess the efficiency of YXS in CH and identify its potential therapeutic targets for the managing of CH. METHOD: Ultrasonic cardiogram was used to evaluate the cardiac function of CH rats. Hematein Eosin (HE)-staining, Masson-staining and transmission electron microscope were used to measure the morphological changes, cardiac fibrosis degree and ultrastructure characteristics of cardiomyocytes, respectively. ELISA was used to detect the myocardial injury biomarkers. Then, the potential targets regulated by YXS were screened out via proteomic analysis and mass spectrometry image analysis. Finally, the targets were validated by real-time quantitative (RT-q) PCR, immunofluorescence, immunohistochemistry, and western-blotting methods. RESULTS: YXS improved the cardiac function of CH rats and attenuated the injuries in morphology and subcellular structure of cardiomyocytes. A core protein-protein interaction network was established on differentially expressed proteins (DEP) using proteomics analysis. GATA binding protein 4 (GATA4) was identified as the key target regulated by YXS. The results of mass spectrometry image analysis indicated that the expressions of histone deacetylase 1 (HDAC1) and retinoblastoma (RB) could also be regulated by YXS. Further valuative experiments showed that YXS may attenuate CH by regulating the RB/HDAC1/GATA4 signaling pathway. CONCLUSIONS: For the first time, this study discloses the precise mechanism investigation of the efficacy of YXS against CH. These data demonstrate that YXS may protect against CH by regulating the RB/HDAC1/GATA4 signaling pathway.

Emodin-8-O-ß-D-glucopyranoside, a natural hydroxyanthraquinone glycoside from plant, suppresses cancer cell proliferation via p21-CDKs-Rb axis.

Emodin-8-O-ß-D-glucopyranoside (EG), a natural hydroxyanthraquinone glycoside from some traditional medicinal plants, has been demonstrated to have potential antitumor effects in our previous studies. Herein, we confirm that EG remains stable in the cell culture medium. It suppresses cell viability and proliferation and induces G1 cell cycle arrest in human colorectal cancer and neuroblastoma cells in vitro. EG inhibits tumor growth in human colorectal cancer cell HCT 116-bearing xenograft mice with low toxicity in the liver and kidney. The transcriptome analysis shows that the p53 signaling pathway is the most enriched cellular pathway and EG affects the proliferation of HCT 116 cells through modulating cell cycle related genes, such as CDKN1A and Cyclin-dependent kinases (CDKs). We demonstrate that the protein expression level of p21 was up-regulated, and CDK1/CDK2 were reduced significantly in both HCT 116 and SH-SY5Y cells after EG treatment. The switch from hypo- to hyper-phosphorylated Retinoblastoma (Rb), which is believed as a result of activated CDKs, was inhibited when cells were treated with EG. These findings indicate that EG suppresses cancer cell proliferation via p21-CDKs-Rb axis.

The effect of exogenous ciliary neurotrophic factor on cell cycle and neural differentiation markers of in vitro model cells: New insights for future therapeutic approaches.

Retinoblastoma is known as childhood rare malignancy of the retina. Ciliary neurotrophic factor (CNTF) was previously found to reduce degeneration and promote retina survival. This work investigated the effects of CNTF supplementation on in-vitro model cells including retinoblastoma (Y79) and adipose-derived mesenchymal stem cells (AMSCs) viability, proliferation, gene expression and cell cycle. A drop of viability was detected in Y79 treated with CNTF in a dose-dependent manner (P < .05). However, the proliferation of AMSCs was increased at lower concentrations of CNTF (5 ng/mL), but declined in higher doses (50 and 100 ng/mL). The BrdU assay confirmed the MTT assay results. Cell cycle was arrested in both Y79 and AMSCs in the G0/G1 phase by CNTF treatment. A considerable down-regulation of Bcl2, CycD1 and N-Myc genes expression (P < .05) inversely, P15 and P21 genes up-regulation in treated Y79 cells was observed. Besides, stemness genes' transcription was reduced in AMSCs (P < .05), and levels of neuronal-specific markers such as neuron-specific enolase (NSE) and neuronal nuclei (NeuN) were increased (P < .05). The findings of this study suggest a promising potential of CNTF in terms of arresting Y79 retinoblastoma cells, and differentiation-inducing to AMSCs, which could be valuable for managing future innovative treatments targeting retinoblastoma. SIGNIFICANCE OF THE STUDY: We demonstrate that CNTF has the potential to reduce proliferation of Y79 cells and induce the cell cycle arrest of them. Also, down-regulation of oncogenes (such as N-Myc) while up-regulation of tumour suppressor genes (such as P21) was detected by exposure of Y79 cells to CNTF. Furthermore, we observed the cell cycle arrest, reduction of stemness gene and up-regulation of neural differentiation markers in AMSCs treated with CNTF. These results support the probable promising effects of CNTF for controlling retinoblastoma.

Ascorbate-induced oxidative stress mediates TRP channel activation and cytotoxicity in human etoposide-sensitive and -resistant retinoblastoma cells.

There are indications that pharmacological doses of ascorbate (Asc) used as an adjuvant improve the chemotherapeutic management of cancer. This favorable outcome stems from its cytotoxic effects due to prooxidative mechanisms. Since regulation of intracellular Ca2+ levels contributes to the maintenance of cell viability, we hypothesized that one of the effects of Asc includes disrupting regulation of intracellular Ca2+ homeostasis. Accordingly, we determined if Asc induced intracellular Ca2+ influx through activation of pertussis sensitive Gi/o-coupled GPCR which in turn activated transient receptor potential (TRP) channels in both etoposide-resistant and -sensitive retinoblastoma (WERI-Rb1) tumor cells. Ca2+ imaging, whole-cell patch-clamping, and quantitative real-time PCR (qRT-PCR) were performed in parallel with measurements of RB cell survival using Trypan Blue cell dye exclusion. TRPM7 gene expression levels were similar in both cell lines whereas TRPV1, TRPM2, TRPA1, TRPC5, TRPV4, and TRPM8 gene expression levels were downregulated in the etoposide-resistant WERI-Rb1 cells. In the presence of extracellular Ca2+, 1 mM Asc induced larger intracellular Ca2+ transients in the etoposide-resistant WERI-Rb1 than in their etoposide-sensitive counterpart. With either 100 µM CPZ, 500 µM La3+, 10 mM NAC, or 100 µM 2-APB, these Ca2+ transients were markedly diminished. These inhibitors also had corresponding inhibitory effects on Asc-induced rises in whole-cell currents. Pertussis toxin (PTX) preincubation blocked rises in Ca2+ influx. Microscopic analyses showed that after 4 days of exposure to 1 mM Asc cell viability fell by nearly 100% in both RB cell lines. Taken together, one of the effects underlying oxidative mediated Asc-induced WERI-Rb1 cytotoxicity stems from its promotion of Gi/o coupled GPCR mediated increases in intracellular Ca2+ influx through TRP channels. Therefore, designing drugs targeting TRP channel modulation may be a viable approach to increase the efficacy of chemotherapeutic treatment of RB. Furthermore, Asc may be indicated as a possible supportive agent in anti-cancer therapies.

Eag1 Gene and Protein Expression in Human Retinoblastoma Tumors and its Regulation by pRb in HeLa Cells.

Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. Ether à-go-go-1 (Eag1) is a voltage-gated potassium channel involved in cancer. Eag1 expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate Eag1. Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with RB1. Eag1 mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RB1 transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments.

Selective nano-thermal therapy of human retinoblastoma in retinal laser surgery.

In this research, an experimental validated predictive finite element model of a cancerous human eye is developed to investigate how the tumor cells in retinoblastoma can be selectively damaged in the course of laser irradiation. In the computational modeling, the tumor is assumed to be in the initial growth stages and located in the macular zone. The statistical calculations testify that an 8.5% improvement in our estimation of the experimental temperature inside the normal human eye compared to those provided by the previous model has been achieved. Under the surgical conditions, the at-risk regions are determined, and the thermal responses of the tissue to various intrinsic and operating factors are obtained and discussed. Our findings indicate that, in the same amount of exposure time, introducing biodegradable nanoparticles in a concentration of 0.2 into the tumor tissue can increase the lethal zone area by 51 percent, and could plays an effective role in surviving of corneal injury.

Neuronal Cell Cycle Events Link Caloric Intake to Obesity.

Obesity is a neurological disorder that operates by favoring energy storage within adipose depots and increased caloric intake. Most cases of human obesity are acquired without any underlying genetic basis. Here, we suggest that obesity can impair the function of some hypothalamic neurons critical to body weight regulation. Genetic ablation of the retinoblastoma (Rb) gene within pro-opiomelanocortin (POMC) neurons leads to death of the neurons and subsequent obesity. The Rb protein (pRb), a key inhibitor of the cell cycle, can also be inactivated by cyclin dependent kinase (CDK)-mediated phosphorylation. Extensive development led to the production of FDA-approved CDK4/6 inhibitors. Based on our own results, we propose that maintaining or re-instating pRb function using CDK4/6 inhibitors are potentially effective treatments of diet-induced obesity (DIO).

The antitumor function of arctigenin in human retinoblastoma cells is mediated by jagged­1.

Retinoblastoma is an intraocular malignant tumor that may severely affect vision and represents a life­threatening disease in children. Arctigenin (ATG) is an active compound that exhibits numerous pharmacological activities, which is isolated from the seeds of greater burdock (Arctium lappa Linnaeus), a plant used in traditional Chinese herbal medicine. The present study aimed to investigate the effects of ATG on cancer progression by analyzing the retinoblastoma cell line Y79. ATG exhibited a significant inhibitory effect on the viability of Y79 cells in a dose­dependent manner. Furthermore, treatment with ATG promoted apoptosis, and increased the protein expression levels of B­cell lymphoma 2 (BCL­2)­associated X protein and decreased the protein expression levels of BCL­2. Cell migration was suppressed following treatment with ATG, as assessed by Transwell migration assay. Furthermore, the protein expression levels of jagged­1 (JAG1) were decreased, and various factors involved in the Notch signaling pathway, including the Notch intracellular domain (NICD), transcription factor HES (HES)5 and HES1 were downregulated following treatment with ATG. The decreased expression levels of JAG1 were restored in response to JAG1 overexpression, alongside increases in the protein expression levels of NICD, HES5 and HES1. Furthermore, overexpression of JAG1 partly restored the cell viability and migration suppressed following treatment with ATG. In addition, ATG­induced apoptosis was reduced by JAG1 overexpression. Collectively, the present results suggested that ATG may serve as an antitumor compound by suppressing the proliferation and migration of retinoblastoma cells, inducing apoptosis, downregulating the protein expression levels of JAG1, and decreasing the activity of the Notch signaling pathway.

Methyl eugenol induces potent anticancer effects in RB355 human retinoblastoma cells by inducing autophagy, cell cycle arrest and inhibition of PI3K/mTOR/Akt signalling pathway.

PURPOSE: Retinoblastoma is one of the lethal malignancies in children. Approximately half of the children that are diagnosed with retinoblastoma die of this disease. Enucleation is commonly used for the treatment of retinoblastoma but this leads to loss of vision. Therefore there is an urgent need to look for viable chemotherapeutic agents for the treatment of retinoblastoma. Consistent with this, natural products can produce efficient anticancer agents and in the present study a plant-derived phenylpropene methyl eugenol (ME) was evaluated against retinoblastoma RB355 cells. METHODS: The cytotoxic activity of this molecule was evaluated by MTT cell viability assay, while autophagic effects were evaluated by flow cytometry using acridine orange (AO)/monodansylcadaverine (AO/MDC) staining dyes. The effects on the cell cycle progression were analyzed by flow cytometry while the effects on mTOR/PI3K/Akt signalling pathway were assessed by western blot method. RESULTS: The results indicated that ME exhibited an IC50 value of 50 µM and exerted its cytotoxic effects in a dosedependent manner in RB355 cells. Moreover, it was observed that the ME lessens the cell viability and triggers G2/M cell cycle arrest. It was also observed that ME induced autophagy dose-dependently in retinoblastoma RB355 cells. Western blot analysis revealed that ME could modulate the mTOR/ PI3K/Akt signalling pathway in RB355 cells at the IC50 concentration. CONCLUSIONS: The above results clearly indicate that ME is a potent anticancer agent and may be developed further as a possible anticancer lead molecule against retinoblastoma provided further in depth in vivo studies are carried out.

Antitumor effect of Quercetin on Y79 retinoblastoma cells via activation of JNK and p38 MAPK pathways.

BACKGROUND: Quercetin (QCT) is a flavonol present in many vegetables, it is proved to show chemo preventive effect against lung, cervical, prostate, breast and colon cancer due to its anti-inflammatory, anti-tumor and anti-oxidant property. Looking into the reported chemo-preventive effect we speculated antitumor activity in retinoblastoma (RB) Y79 cells, we also studied the molecular mechanism for antitumor activity. METHODS: The effect of QCT on Y79 cell viability count was done by cell counting kit, cell cycle distribution, apoptosis studies and mitochondrial membrane potential was evaluated by flow cytometry. Protein expression was done by western blot analysis. RESULTS: The outcomes of study showed that QCT reduced Y79 cell viability and caused arrest of G1 phase in cell cycle via decreasing the expression levels of cyclin-dependent kinase (CDK)2/6 and cyclin D3 and by increasing the levels of both CDK inhibitor proteins p21 and p27. Apoptosis of Y79 cells mediated by QCT occurred via activation of both caspases-3/-9. Flow cytometry studies showed that QCT caused collapse in mitochondrial membrane potential (ΔΨm) in Y79 cells. Western blot studies confirmed that QCT brought about phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). We also established that inhibitors of JNK and p38 MAPK suppressed QCT mediated activation of both caspases-3/-9 and subdued the apoptosis of cancerous Y79 cells. CONCLUSION: All the results of the study suggest that QCT induced the apoptosis of Y79 cells via activation of JNK and p38 MAPK pathways, providing a novel treatment approach for human RB.

Tetramethylpyrazine-mediated regulation of CXCR4 in retinoblastoma is sensitive to cell density.

Retinoblastoma is the most common ocular tumor in children, and it causes extensive damage. Current treatment options for retinoblastoma include surgery, chemotherapy, radiotherapy and cryotherapy. However, the majority of chemotherapy medicines cause complications and side effects that lead to severe impairment of patient health. Previous studies have reported that tetramethylpyrazine (TMP), which is an extract of the Chinese herbal medicine Chuanxiong, reduces the risk of multidrug resistance in chemotherapy and inhibits the proliferation and metastasis of various types of cancer cells. However, the underlying molecular mechanism of TMP in retinoblastoma remains unclear. The current study demonstrated that C-X-C chemokine receptor type 4 (CXCR4) was expressed in WERI­Rb1 cells and in retinoblastoma. Using reverse transcription­quantitative polymerase chain reaction and western blotting techniques, the current study demonstrated that TMP significantly downregulated the expression of CXCR4 in WERI­Rb1 cells cultured at high density, whereas it had a minor effect in low­density WERI­Rb1 cells; additionally, this effect occurred in a time­dependent manner. TMP inhibited the proliferation of WERI­Rb1 cells as effectively as a CXCR4 antagonist, AMD3100, consistent with a role of CXCR4 in cancer development. Notably, TMP did not affect the cell cycle of cells cultured at low density (1x105 cells/ml), whereas it induced G1­phase arrest in high­density cells (7.5x105 cells/ml; P<0.05). In addition, the expression of CXCR4 in primary rat retinal neurocytes was significantly downregulated by TMP treatment, and this treatment protected primary rat retinal neurocytes from H2O2­induced damage. Thus, the results of this study indicate that TMP is a potential candidate for use in treatment of retinoblastoma, and also provides novel insights into the mechanisms of the anti­cancer and neuroprotective effects of this extract.

Synergistic Effect of Curcumin in Combination with Anticancer Agents in Human Retinoblastoma Cancer Cell Lines.

PURPOSE: Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of the herb Curcuma longa, is known to have anti-proliferative and anti-tumor properties. In this study, we evaluated the cytotoxic effect of curcumin alone and in combination with individual drugs like carboplatin, etoposide, or vincristine in a human retinoblastoma (RB) cancer cell line. MATERIALS AND METHODS: A drug-drug interaction was analyzed using the median effect/isobologram method and combination index values were used to characterize the interaction as synergistic or additive. We also performed the apoptosis and cell-cycle kinetics study with single drugs in combination with curcumin in a human RB cell lines (Y79 and Weri-Rb1). RESULTS: Curcumin caused concentration-dependent decrease in cell proliferation, cell kinetics, and also induced apoptosis in both the RB cell lines. When combination of curcumin with individual drugs like carboplatin or etoposide or vincristine was treated on to RB cells, both cell viability and cell cycling were reduced and increased apoptosis was noted, in comparison with single drug treatment. These effects were significant in both the cell lines, indicating the ability of curcumin to increase the sensitivity of RB cells to chemotherapy drugs. CONCLUSION: Our in vitro findings showed that the combination of curcumin with single drug treatment showed marked synergistic inhibitory effect against RB cell lines. These results suggest that curcumin can be used as a modulator which may have a potential therapeutic value for the treatment of RB cancer patients.

BAG3 protects against hyperthermic stress by modulating NF-κB and ERK activities in human retinoblastoma cells.

PURPOSE: BCL2-associated athanogene 3 (BAG3), a co-chaperone of HSP70, is a cytoprotective and anti-apoptotic protein that acts against various stresses, including heat stress. Here, we examined the effect of BAG3 on the sensitivity of human retinoblastoma cells to hyperthermia (HT). METHODS: We examined the effects of BAG3 knockdown on the sensitivity of Y79 and WERI-Rb-1cells to HT (44 °C, 1 h) by evaluating apoptosis and cell proliferation using western blotting, real-time quantitative PCR (qPCR), flow cytometry, and a WST-8 assay kit. Furthermore, we examined the effects of activating nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK) using western blotting and real time qPCR. RESULTS: HT induced considerable apoptosis along with the activation of caspase-3 and chromatin condensation. The sensitivity of Y79 and WERI-Rb-1 cells to HT was significantly enhanced by BAG3 knockdown. Compared to HT alone, the combination of BAG3 knockdown and HT reduced phosphorylation of the inhibitors of kappa B α (IκBα) and p65, a subunit of NF-κB, and degraded IκB kinase γ (IKKγ) during the recovery period after HT. Furthermore, BAG3 knockdown increased the HT-induced phosphorylation of ERK after HT treatment, and the ERK inhibitor U0126 significantly improved the viability of the cells treated with a combination of BAG3 knockdown and HT. CONCLUSIONS: The silencing of BAG3 seems to enhance the effects of HT, at least in part, by maintaining HT-induced inactivity of NF-κB and the phosphorylation of ERK. These findings indicate that BAG3 may be a potential molecular target for modifying the outcomes of HT in retinoblastoma.

Chebulagic acid from Terminalia chebula causes G1 arrest, inhibits NFκB and induces apoptosis in retinoblastoma cells.

BACKGROUND: Plants are the valuable source of natural products with important medicinal properties. Most of the approved anti cancer drugs have a natural product origin or are natural products. Retinoblastoma is the most common ocular cancer of children. Although chemotherapy is the preferred mode of therapy, a successful treatment for retinoblastoma requires enucleation. Chebulagic acid (CA) from Terminalia chebula was shown to have anti-proliferative properties in the studies on cancerous cell lines. Due to anti cancer properties of CA and due to limitation in treatment options for retinoblastoma, the present study is undertaken to understand the role of CA on the proliferation of retinoblastoma cells. METHODS: Anti proliferative potential of CA was determined by MTT assay. The expression levels of various cell death mediators in retinoblastoma cells with CA treatment were assessed by Western blotting. Flowcytometer analysis was used to estimate the mitochondrial membrane potential (MMP) and to determine the percentage of cells undergoing apoptosis. RESULTS: The present study showed CA inhibited the proliferation of retinoblastoma cells in a dose dependent manner. CA modulated MMP, induced release of Cytochrome c, activated caspase 3 and shifted the ratio of BAX and Bcl2 towards cell death. G1 arrest, noticed in CA treated cells, is mediated by the increase in the expression of CDK inhibitor p27. CA treatment also decreased the levels of NFκB in the nucleus. This decrease is mediated by suppression in degradation of IκBα. CONCLUSION: CA has shown significant anti proliferative potential on retinoblastoma cells. Our findings clearly demonstrate that CA induces G1 arrest, inhibits NFκB and induces apoptosis of retinoblastoma cells.

In vitro moxifloxacin drug interaction with chemotherapeutics: implications for retinoblastoma management.

Retinoblastoma (RB) is a common malignant intraocular tumor primarily affecting children. Multidrug resistance (MDR) proteins (P-gp and MRPs) mediated chemoresistance have been considered as a major cause of treatment failure in treatment of RB. Ocular cells have shown good tolerability against moxifloxacin (MFX). Hence, the aim of present study was to investigate the effect of moxifloxacin on the functionality of MDR proteins. Furthermore, we have also examined an interaction of MFX with anticancer agents (Topotecan, etoposide and vinblastine) for RB treatment. For interaction of MFX with efflux transporter, model cell lines transfected with the efflux transporters (MDCK-MDR1 and MDCK-MRP2) were used to perform uptake and bi-directional transport experiments. Modulation of anticancer induced cell cytotoxicity, pro-inflammatory cytokines (IL-6 and IL-8) release and caspase-3 enzyme activity in presence of MFX was also evaluated. Result indicates that MFX is a substrate of both MDR1 and MRP2 efflux transporters. Furthermore elevation of anticancer uptake and bi-directional transport, reduction in IC50 cytotoxic value and modulation of antiproliferative and cytokines release in presence of MFX by anticancer agents was observed. Our results demonstrate that MFX may not only modulate the permeability of anticancer agents at efflux sites but it may also potentiate antiproliferative activity of anticancer agents in retinoblastoma cells. This study may be further extended to explore in vivo outcome of this finding.

Inhibition of polo-like kinase 1 promotes hyperthermia sensitivity via inactivation of heat shock transcription factor 1 in human retinoblastoma cells.

PURPOSE: Hyperthermia (HT) has been recognized as an effective focal treatment in retinoblastoma. However, one of the problems with HT therapy is that cells acquire acquisition. The purpose of this study was to evaluate whether the inhibition of polo-like kinase 1 (PLK1) would promote HT sensitivity in human retinoblastoma cells. METHODS: We examined the effects of PLK1 knockdown by small interfering RNA (siRNA) or by the inhibition of PLK1 activity with PLK1 inhibitor (BI-2536) on the sensitivity to HT (44°C, 1 hour) in human retinoblastoma Y79 and WERI-Rb-1 cells by evaluating apoptosis and cell proliferation using flow cytometry, Western blotting, real-time quantitative polymerase chain reaction, and WST-8 assay. Furthermore, we investigated the effects of activating heat shock transcription factor 1 (HSF1) through a combination of PLK1 knockdown and HT using Western blotting and immunocytochemistry. RESULTS: The combination of PLK1 inhibition and HT enhanced sensitivity to HT synergistically. Furthermore, PLK1 knockdown inhibited HT-induced phosphorylation of HSF1, the nuclear translocation of HSF1 from the cytoplasm, and nuclear granule formation of HSF1. Heat shock transcription factor 1, inactivated by the silencing of PLK1, reduced the expression of heat shock proteins (HSPs), such as HSP70 and HSP40, as well as the expression of Bcl-2-associated athanogene 3 (BAG3). CONCLUSIONS: Polo-like kinase 1 inhibition may attenuate the thermoresistance of HT through the inactivation of HSF1 concomitant with reductions in HSPs and BAG3. The combination of PLK1 inhibition and HT may become an option for HT therapy in patients with retinoblastoma.

Artesunate exerts specific cytotoxicity in retinoblastoma cells via CD71.

Retinoblastoma (RB) is an intraocular cancer that affects young children. There is an ongoing effort to find new agents for RB management that are effective, specific and with few side-effects. In the present study, we tested artesunate (ART), a synthetic derivative from the herbal drug artemisinin, used in the clinic for the treatment of malaria. We analyzed ART cytotoxicity in an RB cell line (RB-Y79) and in a retinal epithelial cell line (hTERT-RPE1) by flow cytometric analysis (FCM). We related the effect of ART to the expression of transferrin receptor 1 (TfR-1, also known as CD71) by knocking down CD71 with RNAi and analyzing cell cycle variables by FCM. We found that the cytotoxic action of ART is specific for RB cells in a dose-dependent manner, with low toxicity in normal retina cells. ART is more effective in RB than carboplatin with a markedly strong cytotoxic effect on carboplatin-resistant RB cells. RB had higher CD71 levels at the membrane compared to normal retinal cells. We showed that ART internalization in RB cells is dependent upon the expression of the CD71. In addition, ART blocked the cell cycle progression at the G1 phase, even at low doses, and decreased the proportion of RB cells in the S phase. In conclusion, we showed that ART is a promising drug exhibiting high selective cytotoxicity even against multidrug-resistant RB cells. Thus, we suggest that ART could be used in the treatment of RB.

Antitumor activity of celastrol nanoparticles in a xenograft retinoblastoma tumor model.

BACKGROUND: Celastrol, a Chinese herbal medicine, has shown antitumor activity against various tumor cell lines. However, the effect of celastrol on retinoblastoma has not yet been analyzed. Additionally, the poor water solubility of celastrol restricts further therapeutic applications. The goal of this study was to evaluate the effect of celastrol nanoparticles (CNPs) on retinoblastoma and to investigate the potential mechanisms involved. METHODS: Celastrol-loaded poly(ethylene glycol)-block-poly(ɛ-caprolactone) nanopolymeric micelles were developed to improve the hydrophilicity of celastrol. The 2-(2-methoxy-4- nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulf-ophenyl)-2H tetrazolium monosodium salt (WST-8) assay was used to determine the inhibitory effect of CNPs on SO-Rb 50 cell proliferation in vitro. Immunofluorescence was used to evaluate the apoptotic effect of CNPs on nuclear morphology, and flow cytometry was used to quantify cellular apoptosis. The expression of Bcl-2, Bax, NF-κB p65, and phospo-NF-κB p65 proteins was assessed by Western blotting. A human retinoblastoma xenograft model was used to evaluate the inhibitory effects of CNPs on retinoblastoma in NOD-SCID mice. Hematoxylin and eosin staining was used to assess the apoptotic effects of CNPs on retinoblastoma. RESULTS: CNPs inhibit the proliferation of SO-Rb 50 cells in a dose- and time-dependent manner with an IC(50) of 17.733 µg/mL (celastrol-loading content: 7.36%) after exposure to CNPs for 48 hours. CNPs induce apoptosis in SO-Rb 50 cells in a dose-dependent manner. The expression of Bcl-2, NF-κB p65, and phospo-NF-κB p65 proteins decreased after exposure to CNPs 54.4 µg/mL for 48 hours. Additionally, the Bax/Bcl-2 ratio increased, whereas the expression of Bax itself was not significantly altered. CNPs inhibit the growth of retinoblastoma and induce apoptosis in retinoblastoma cells in mice. CONCLUSION: CNPs inhibit the growth of retinoblastoma in mouse xenograft model by inducing apoptosis in SO-Rb 50 cells, which may be related to the increased Bax/Bcl-2 ratio and the inhibition of NF-κB. CNPs may represent a potential alternative treatment for retinoblastoma.

Expression profile of genes regulated by curcumin in Y79 retinoblastoma cells.

Curcumin, a well-known chemopreventive agent from turmeric, inhibits the expression of several oncogenes and cell proliferation genes in tumor cells. This study aims to understand the precise molecular mechanism by which curcumin exerts its effects on retinoblastoma cells, by performing whole genome microarray analysis to determine the gene expression profiles altered by curcumin treatment. Curcumin suppressed cell viability and altered the cell cycle of retinoblastoma cells. We identified 903 downregulated genes and 1,319 upregulated genes when compared with the control cells after treatment with 20 µM curcumin concentration for 48 h. These genes were grouped into respective functional categories according to their biological function. We found that curcumin regulated the expression of genes that are involved in the regulation of apoptosis, tumor suppressor, cell-cycle arrest, transcription factor, and angiogenesis. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to validate the results of genome array, and the results were consistent with the obtained data. In conclusion, treatment of curcumin affects the expression of genes involved in various cellular functions and plays an important role in tumor metastasis and apoptosis. Thus, curcumin might be an effective chemopreventive agent for retinoblastoma cancer.

Effects of matrine on proliferation and apoptosis of cultured retinoblastoma cells.

BACKGROUND: Extracted from the traditional Chinese medicine of Kushen, matrine is an alkaloid with potential anti-neoplastic and anti-inflammatory effects. Here, we examined the effect of matrine on proliferation and apoptosis of cultured retinoblastoma cells. METHODS: The retinoblastoma cell lines Y79, WERI-RB1 and SO-RB50 were treated with matrine in increasing concentrations from 0.2-1.1 mg/ml for 24 hours, and the cell proliferation rate was measured. The cells were exposed to matrine at 50% inhibition concentration (IC50) for 12, 24 and 48 hours. Cell cycle was analyzed by flow cytometry, concentration of proteins regulating cell cycle and apoptosis was determined by Western blot, apoptosis rate was measured by TUNEL staining, and cell morphology was assessed by electron transmission microscopy. RESULTS: The retinoblastoma cell lines Y79, WERI-RB1 and SO-RB50 showed an increased inhibition of cell proliferation with increasing matrine concentrations. Applying the IC50 concentration of matrine, the alteration of the cell cycle, including a reduced percentage of the S phase, was significantly (P < 0.01) associated with a longer treatment time by matrine. Correspondingly, the cell-cycle-associated proteins P21 and P27 were up-regulated and the protein cyclinD1 was down-regulated. The apoptosis-associated protein Bcl-2 was down-regulated, and Bax was up-regulated. In a similar manner, the apoptosis rate was significantly increased with longer treatment time. CONCLUSIONS: Matrine added to cultures of immortalized retinoblastoma cells led to a reduced tumor cell proliferation, decreased rate of mitosis and an increased tumor cell apoptosis, paralleled by corresponding changes in the proteins regulating the cell cycle or apoptosis.