Proteomic investigation of anti-tumor activities exerted by sinularin against A2058 melanoma cells

The extracts from soft corals have been increasingly investigated for biomedical and therapeutic purposes. The aim of this study is to examine and analyze the anti-tumor effects of the genus Sinularia extract sinularin on A2058 melanoma cells using MTT assay, cell migration assay, wound healing assay, flow cytometric analysis, and proteomic analysis. Sinularin dose-dependently (1-5 μg/mL) inhibited melanoma cell proliferation while the treatment at identical concentrations suppressed cell migration. Sinularin dose-dependently enhanced apoptotic melanoma cells and caused tumor cell accumulation at G2/M phase, indicating that sinularin exerts apoptosis-induced and cell cycle-delayed activities in A2058 melanoma cells. Comparative proteomic analysis was conducted to investigate the effects of sinularin at the molecular level by comparison between the protein profiling of melanoma cells treated with sinularin and without the treatment. Thirty-five differential proteins (13 upregulated and 22 downregulated) concerning the treatment were identified by liquid chromatography-tandem mass spectrometry. Proteomic data and Western blot displayed the levels of several tumor inhibitory or apoptosis-associated proteins including annexin A1, voltage-dependent anion-selective channel protein 1 and prohibitin (upregulated), heat shock protein 60, heat shock protein beta-1, and peroxiredoxin-2 (downregulated) in A2058 melanoma cells exposed to sinularin. Increased expression of p53, cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, p21, and Bax and decreased expression of Bcl-2 in sinularin-treated melanoma cells suggest that the anti-tumor activities of sinularin against melanoma cells are particularly correlated with these pro-apoptotic factors. These data provide important information for the mechanisms of anti-tumor effects of sinularin on melanoma cells and may be helpful for drug development and progression monitoring of human melanoma.     

Activation of heat shock protein 70 promoter with meso-tetrahydroxyphenyl chlorin photodynamic therapy reported by green fluorescent protein in vitro and in vivo

Cellular responses to photodynamic therapy (PDT) include induction of heat shock proteins (HSP). We examined meso-tetrahydroxyphenyl chlorin (mTHPC) PDT-mediated HSP activation in EMT6 cells stably transfected with a plasmid containing the gene for green fluorescent protein (GFP) driven by an hsp70 promoter. mTHPC incubation induced concentration-dependent GFP expression. Irradiation of cells exposed to a sensitizer concentration that induced a slight increase in GFP and no loss of cell viability resulted in fluence-dependent GFP accumulation. In response to drug only and to PDT, GFP levels increased to a maximum of four- to five-fold above control levels with increasing drug or fluence and then decreased at higher doses. A trypan blue-exclusion assay confirmed that decreased GFP levels in both cases were due to a loss of cell viability. For initial evaluation in vivo, HSP70/ GFP-transfected EMT6 tumors were grown in BALB/c mice and subjected to mTHPC-PDT with a fluence of 1 J/cm2. Six hours after PDT, GFP fluorescence was imaged in these tumors through the intact skin in vivo. These results indicate that sublethal doses of mTHPC-PDT stimulate GFP expression under the control of an hsp70 promoter and illustrate the potential of noninvasively monitoring reporter protein fluorescence as a measure of molecular response to PDT.     

alpha-Mangostin enhances betulinic acid cytotoxicity and inhibits cisplatin cytotoxicity on HCT 116 colorectal carcinoma cells

Despite the progress in colon cancer treatment, relapse is still a major obstacle. Hence, new drugs or drug combinations are required in the battle against colon cancer. α-Mangostin and betulinic acid (BA) are cytotoxic compounds that work by inducing the mitochondrial apoptosis pathway, and cisplatin is one of the most potent broad spectrum anti-tumor agents. This study aims to investigate the enhancement of BA cytotoxicity by α-mangostin, and the cytoprotection effect of α-mangostin and BA on cisplatin-induced cytotoxicity on HCT 116 human colorectal carcinoma cells. Cytotoxicity was investigated by the XTT cell proliferation test, and the apoptotic effects were investigated on early and late markers including caspases-3/7, mitochondrial membrane potential, cytoplasmic shrinkage, and chromatin condensation. The effect of α-mangostin on four signalling pathways was also investigated by the luciferase assay. α-Mangostin and BA were more cytotoxic to the colon cancer cells than to the normal colonic cells, and both compounds showed a cytoprotective effect against cisplatin-induced cytotoxicity. On the other hand, α-mangostin enhanced the cytotoxic and apoptotic effects of BA. Combination therapy hits multiple targets, which may improve the overall response to the treatment, and may reduce the likelihood of developing drug resistance by the tumor cells. Therefore, α-mangostin and BA may provide a novel combination for the treatment of colorectal carcinoma. The cytoprotective effect of the compounds against cisplatin-induced cytotoxicity may find applications as chemopreventive agents against carcinogens, irradiation and oxidative stress, or to neutralize cisplatin side effects.     

Involvement of Heat-Shock Protein 70 and P53 Proteins in Attenuation of UVC-lnduced Apoptosis by Thermal Stress in Hepatocellular Carcinoma Cells

Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. In this study, we found that a prior exposure to mild stress (42 degrees C) can protect HepG2 (p53+/+) cells from a subsequent UVC-induced apoptosis determined by DNA fragmentation and ratio of sub-G1 peak, but no heat-enhanced protection was found in Hep3B (p53-/-) cells. Although a similar inductive pattern of HSP70 protein and mRNA was detected in the two cell lines under thermal stress, the effect of thermal stress on UVC-induced apoptosis in HepG2 and Hep3B cells was obviously different. Overexpression of HSP70 by transient transfection of HSP70 expression vector in HepG2 cells significantly inhibited UVC-induced cell death; however, this inhibitory effect did not occur in transfected-Hep3B cells. Treatment of HepG2 cells with p53-specific antisense oligonucleotide could effectively block the antiapoptotic effect of thermal stress on UVC-induced apoptosis and increase of intracellular wild-type p53 protein by transfecting wtp53 expression plasmid into Hep3B cells yielded more resistance to UVC irradiation after prior thermal stress exposure. The results reveal an involvement of p53 in the antiapoptotic effect of thermal stress on UVC irradiation. Finally, a p53 protein increase was detected in UVC-treated HepG2 cells and could be coimmunoprecipitated with HSP70 after a thermal stress treatment. Prolonged p53 binding activity and enhanced expression of p53-controlled genes such as G1 arrest and DNA damage 45 and wild-type p53 activation factor 1/Cdk-interacting protein 1 by thermal stress are also observed in UVC-irradiated HepG2 cells. Based on these results, we propose that the antiapoptotic effect of thermal stress is mediated by increasing HSP70 and modulating intracellular p53 function.     

Betulinic Acid Restricts Human Bladder Cancer Cell Proliferation In Vitro by Inducing Caspase-Dependent Cell Death and Cell Cycle Arrest,and Decreasing Metastatic Potential

Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid and generally found in the bark of birch trees (Betula sp.). Although several studies have been reported that BA has diverse biological activities, including anti-tumor effects, the underlying anti-cancer mechanism in bladder cancer cells is still lacking. Therefore, this study aims to investigate the anti-proliferative effect of BA in human bladder cancer cell lines T-24, UMUC-3, and 5637, and identify the underlying mechanism. Our results showed that BA induced cell death in bladder cancer cells and that are accompanied by apoptosis, necrosis, and cell cycle arrest. Furthermore, BA decreased the expression of cell cycle regulators, such as cyclin B1, cyclin A, cyclin-dependent kinase (Cdk) 2, cell division cycle (Cdc) 2, and Cdc25c. In addition, BA-induced apoptosis was associated with mitochondrial dysfunction that is caused by loss of mitochondrial membrane potential, which led to the activation of mitochondrial-mediated intrinsic pathway. BA up-regulated the expression of Bcl-2-accociated X protein (Bax) and cleaved poly-ADP ribose polymerase (PARP), and subsequently activated caspase-3, -8, and -9. However, pre-treatment of pan-caspase inhibitor markedly suppressed BA-induced apoptosis. Meanwhile, BA did not affect the levels of intracellular reactive oxygen species (ROS), indicating BA-mediated apoptosis was ROS-independent. Furthermore, we found that BA suppressed the wound healing and invasion ability, and decreased the expression of Snail and Slug in T24 and 5637 cells, and matrix metalloproteinase (MMP)-9 in UMUC-3 cells. Taken together, this is the first study showing that BA suppresses the proliferation of human bladder cancer cells, which is due to induction of apoptosis, necrosis, and cell cycle arrest, and decrease of migration and invasion. Furthermore, BA-induced apoptosis is regulated by caspase-dependent and ROS-independent pathways, and these results provide the underlying anti-proliferative molecular mechanism of BA in human bladder cancer cells.     

Annexin II, a novel HSP27-interacted protein, is involved in resistance to UVC-induced cell death in human APr-1 cells

Heat shock protein 27 (HSP27) is implicated in diverse biologic functions as a molecular chaperone. We found that HSP27 is involved in the protection of human cells against UVC lethality. To elucidate the molecular mechanisms underlying UVC resistance, we searched for HSP27-interacted proteins related to resistance in UVC-resistant human cells, APr-1. Three candidates for HSP27-interacted proteins were found from cell lysates using an affinity column coupled with GST-fused HSP27 protein. Interaction between HSP27 and two candidates, annexin II and HSP70, was confirmed by immunoprecipitation analysis. After UVC irradiation, the amount of the complex of HSP27 and annexin II decreased in the postnuclear fraction, while it increased in the nuclear fraction. Cells transfected with annexin II-siRNA were more susceptible to UVC lethality. These results suggest that annexin II is a novel HSP27-interacted protein which is involved in UVC resistance in human cells, at least those tested here.     

HnRNP K and PDI marked response to chemotherapy to human colorectal cancer cells

5‐Fluorouracil has been the chemotherapy agent of first‐choice for colorectal cancer for many years, but since there are no proven predictors of a patient's response to therapy, all patients receive similar treatment. Consequently, identification of biomarkers for therapeutic effect is crucial for the development of novel therapeutic strategies. Two human colorectal cancer cell lines of different metastatic potential (LoVo and SW480) were studied. IC50 of 5‐FU for both cell lines were measured by 3‐(4,5‐dimethy‐lthiazol‐2‐yl)‐2,5‐diphenyltetrazolium assay and validated by cell cycle analysis. Then the cell lines were treated with 5‐FU at IC50 concentration and protein was extracted for 2‐DE. Differential protein spots were examined by MALDI‐TOF/TOF MS. The expression levels of the different proteins were further confirmed by Western blot and immunofluorescence analyses. Eleven proteins were identified. Expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in LoVo cells was higher than in SW480 cells, while protein disulfide isomerase (PDI) displayed the opposite trend. After treatment with 5‐FU, the expression of hnRNP K in LoVo decreased more significantly than in SW480, while PDI in SW480 increased more significantly than in LoVo cells. Conclusion: hnRNP K and PDI in the two cell lines have different expression characteristics. The sensitivity to 5‐FU is not consistent in tumor progression. It may assist in development of novel treatment strategies for colorectal cancer metastasis.     

Triptolide downregulates Rac1 and the JAK/STAT3 pathway and inhibits colitis-related colon cancer progression

Triptolide, a diterpenoid triepoxide from the traditional Chinese medicinal herb Tripterygium wilfordii Hook. f., is a potential treatment for autoimmune diseases as well a possible anti-tumor agent. It inhibits proliferation of coloretal cancer cells in vitro and in vivo. In this study, its ability to block progress of colitis to colon cancer, and its molecular mechanism of action are investigated. A mouse model for colitis-induced colorectal cancer was used to test the effect of triptolide on cancer progression. Treatment of mice with triptolide decreased the incidence of colon cancer formation, and increased survival rate. Moreover, triptolide decreased the incidence of tumors in nude mice inoculated with cultured colon cancer cells dose-dependently. In vitro, triptolide inhibited the proliferation, migration and colony formation of colon cancer cells. Secretion of IL6 and levels of JAK1, IL6R and phosphorylated STAT3 were all reduced by triptolide treatment. Triptolide prohibited Rac1 activity and blocked cyclin D1 and CDK4 expression, leading to G1 arrest. Triptolide interrupted the IL6R-JAK/STAT pathway that is crucial for cell proliferation, survival, and inflammation. This suggests that triptolide might be a candidate for prevention of colitis induced colon cancer because it reduces inflammation and prevents tumor formation and development.     

Betulinic Acid Affects the Energy-Related Proteomic Profiling in Pancreatic Ductal Adenocarcinoma Cells

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a 5-year survival rate of <8%. Therefore, finding new treatment strategies against PDAC cells is an imperative issue. Betulinic acid (BA), a plant-derived natural compound, has shown great potential to combat cancer owing to its versatile physiological functions. In this study, we observed the impacts of BA on the cell viability and migratory ability of PDAC cell lines, and screened differentially expressed proteins (DEPs) by an LC-MS/MS-based proteomics analysis. Our results showed that BA significantly inhibited the viability and migratory ability of PDAC cells under a relatively low dosage without affecting normal pancreatic cells. Moreover, a functional analysis revealed that BA-induced downregulation of protein clusters that participate in mitochondrial complex 1 activity and oxidative phosphorylation, which was related to decreased expressions of RNA polymerase mitochondrial (POLRMT) and translational activator of cytochrome c oxidase (TACO1), suggesting that the influence on mitochondrial function explains the effect of BA on PDAC cell growth and migration. In addition, BA also dramatically increased Apolipoprotein A1 (APOA1) expression and decreased NLR family CARD domain-containing protein 4 (NLRC4) expression, which may be involved in the dampening of PDAC migration. Notably, altered expression patterns of APOA1 and NLRC4 indicated a favorable clinical prognosis of PDAC. Based on these findings, we identified potential proteins and pathways regulated by BA from a proteomics perspective, which provides a therapeutic window for PDAC.     

The Proteins from Sika deer antler as potential modulators on cisplatin-induced cytotoxicity in human embryonic kidney 293 cells

Our study aimed to investigate the protective role of SDAPR on cisplatin-induced cytotoxicity and its’ possible mechanism in HEK293 cells. Cell viability was measured by MTT assay. Oxidative stress (SOD, GSH, LDH and MDA), inflammatory factors (TNF-α and IL-6) and apoptosis-related proteins (caspase-3, Bax, Bcl-2) expression were measured. The apoptotic cells were observed by TUNEL staining. Our study results indicated that non-cytotoxic levels of SDAPR significantly increased viability rate (LD₅₀ value of cisplatin is 20 μM), which improved antioxidant defence, attenuated apoptosis by decreasing expression levels of cleaved-caspase-3 and Bax, increasing Bcl-2 expression and inhibiting apoptotic positive cells in HEK 293 cells. In addition, SDAPR treatment markedly inhibited the levels of TNF-α and IL-6. In conclusion, Sika deer antler protein, a potential modulator, could alleviate cisplatin-induced cytotoxicity in HEK 293 cells.     

Endoplasmic reticulum targeted AIE bioprobe as a highly efficient inducer of immunogenic cell death

Focused oxidative stress of the specific organelles(e.g., endoplasmic reticulum(ER) and mitochondrion) of cancer cells can boost the immunogenic cell death(ICD) effect for cancer immunotherapy. Herein, an ER-targeted bioprobe with aggregationinduced emission(AIE) characteristics(TPE-PR-FFKDEL) was rationally designed and synthesized by integrating a new AIE photosensitizer with ER targeting peptide, which has been demonstrated to be able to efficiently induce ER oxidative stress to evoke ICD. Compared with the photosensitizer hypericin that is well-known as an ER-targeted ICD inducer, TPE-PR-FFKDEL can lead to more robust emission of immunostimulatory damage-associated molecular patterns such as surface-exposed calreticulin, ATP secretion, and high-mobility group protein B1(HMGB1) and heat shock protein 70(HSP 70) expression.Furthermore, a range of immune responses are activated to protect mice from the attack of cancer cells in vivo.     

Anticancer effect of Polyphyllin Ι in colorectal cancer cells through ROS-dependent autophagy and G2/M arrest mechanisms

Polyphyllin Ι is a steroidal saponin isolated from the rhizoma of Paris polyphylla. In the present study, we aimed to investigate the anticancer effects of polyphyllin Ι in colorectal cancer and to elucidate the potential underlying molecular mechanisms. Using, CCK8 assay, flow cytometry, laser confocal microscope analysis and western blot, the anticancer effects of the polyphyllin Ι were analysed in colorectal cells. Our results indicate that polyphyllin Ι significantly decreased cell viability of HCT 116 cells and induced autophagy. Furthermore, we found that polyphyllin Ι induced autophagy in an ROS-dependent cell death and not related with PI3 K/AKT/mTOR pathway. We also provide evidence that excessive ROS triggered by polyphyllin Ι could induce G2/M phase arrest via regulating cycle proteins expression of cell cycle regulators, such as p21 and cyclinB1. In conclusion, polyphyllin Ι exhibit anticancer effect through ROS-dependent autophagy and induces G2/M arrest in colorectal cancer.     

Betulinic Acid Protects DOX-Triggered Cardiomyocyte Hypertrophy Response through the GATA-4/Calcineurin/NFAT Pathway

Cardiac hypertrophy is a major risk factor for heart failure and leads to cardiovascular morbidity and mortality. Doxorubicin (DOX) is regarded as one of the most potent anthracycline antibiotic agents; however, its clinical usage has some limitations because it has serious cardiotoxic side effects such as dilated cardiomyopathy and congestive heart failure. Betulinic acid (BA) is a pentacyclic-cyclic lupane-type triterpene that has been reported to have anti-bacterial, anti-inflammatory, anti-vascular neogenesis, and anti-fibrotic effects. However, there is no study about its direct effect on DOX induced cardiac hypertrophy and apoptosis. The present study aims to investigate the effect of BA on DOX-induced cardiomyocyte hypertrophy and apoptosis in vitro in H9c2 cells. The H9c2 cells were stimulated with DOX (1 µM) in the presence or absence of BA (0.1–1 μM) and incubated for 24 h. The results of the present study indicated that DOX induces the increase cell surface area and the upregulation of hypertrophy markers including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta-myosin heavy chain (β-MHC), and Myosin Light Chain-2 (MLC2) in H9c2 cells. However, the pathological hypertrophic responses were downregulated after BA treatment. Moreover, phosphorylation of JNK, ERK, and p38 in DOX treated H9c2 cells was blocked by BA. As a result of measuring the change in ROS generation using DCF-DA, BA significantly inhibited DOX-induced the production of intracellular reactive oxygen species (ROS) when BA was treated at a concentration of over 0.1 µM. DOX-induced activation of GATA-4 and calcineurin/NFAT-3 signaling pathway were remarkably improved by pre-treating of BA to H9c2 cells. In addition, BA treatment significantly reduced DOX-induced cell apoptosis and protein expression levels of Bax and cleaved caspase-3/-9, while the expression of Bcl-2 was increased by BA. Therefore, BA can be a potential treatment for cardiomyocyte hypertrophy and apoptosis that lead to sudden heart failure.     

Carpachromene Ameliorates Insulin Resistance in HepG2 Cells via Modulating IR/IRS1/PI3k/Akt/GSK3/FoxO1 Pathway

Insulin resistance contributes to several disorders including type 2 diabetes and cardiovascular diseases. Carpachromene is a natural active compound that inhibits α-glucosidase enzyme. The aim of the present study is to investigate the potential activity of carpachromene on glucose consumption, metabolism and insulin signalling in a HepG2 cells insulin resistant model. A HepG2 insulin resistant cell model (HepG2/IRM) was established. Cell viability assay of HepG2/IRM cells was performed after carpachromene/metformin treatment. Glucose concentration and glycogen content were determined. Western blot analysis of insulin receptor, IRS1, IRS2, PI3k, Akt, GSK3, FoxO1 proteins after carpachromene treatment was performed. Phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase (HK) enzymes activity was also estimated. Viability of HepG2/IRM cells was over 90% after carpachromene treatment at concentrations 6.3, 10, and 20 µg/mL. Treatment of HepG2/IRM cells with carpachromene decreased glucose concentration in a concentration- and time-dependant manner. In addition, carpachromene increased glycogen content of HepG2/IRM cells. Moreover, carpachromene treatment of HepG2/IRM cells significantly increased the expression of phosphorylated/total ratios of IR, IRS1, PI3K, Akt, GSK3, and FoxO1 proteins. Furthermore, PEPCK enzyme activity was significantly decreased, and HK enzyme activity was significantly increased after carpachromene treatment. The present study examined, for the first time, the potential antidiabetic activity of carpachromene on a biochemical and molecular basis. It increased the expression ratio of insulin receptor and IRS1 which further phosphorylated/activated PI3K/Akt pathway and phosphorylated/inhibited GSK3 and FoxO1 proteins. Our findings revealed that carpachromene showed central molecular regulation of glucose metabolism and insulin signalling via IR/IRS1/ PI3K/Akt/GSK3/FoxO1 pathway.     

LncRNA HSPA7 in human atherosclerotic plaques sponges miR-223 and promotes the proinflammatory vascular smooth muscle cell transition

Although there are many genetic loci in noncoding regions associated with vascular disease, studies on long noncoding RNAs (lncRNAs) discovered from human plaques that affect atherosclerosis have been highly limited. We aimed to identify and functionally validate a lncRNA using human atherosclerotic plaques. Human aortic samples were obtained from patients who underwent aortic surgery, and tissues were classified according to atherosclerotic plaques. RNA was extracted and analyzed for differentially expressed lncRNAs in plaques. Human aortic smooth muscle cells (HASMCs) were stimulated with oxidized low-density lipoprotein (oxLDL) to evaluate the effect of the identified lncRNA on the inflammatory transition of the cells. Among 380 RNAs differentially expressed between the plaque and control tissues, lncRNA HSPA7 was selected and confirmed to show upregulated expression upon oxLDL treatment. HSPA7 knockdown inhibited the migration of HASMCs and the secretion and expression of IL-1β and IL-6; however, HSPA7 knockdown recovered the oxLDL-induced reduction in the expression of contractile markers. Although miR-223 inhibition promoted the activity of Nf-κB and the secretion of inflammatory proteins such as IL-1β and IL-6, HSPA7 knockdown diminished these effects. The effects of miR-223 inhibition and HSPA7 knockdown were also found in THP-1 cell-derived macrophages. The impact of HSPA7 on miR-223 was mediated in an AGO2-dependent manner. HSPA7 is differentially increased in human atheroma and promotes the inflammatory transition of vascular smooth muscle cells by sponging miR-223. For the first time, this study elucidated the molecular mechanism of action of HSPA7, a lncRNA of previously unknown function, in humans.Subject terms: Mechanisms of disease, Atherosclerosis, Long non-coding RNAs