Corrigendum: ß-Sitosterol and Gemcitabine Exhibit Synergistic Anti-Pancreatic Cancer Activity by Modulating Apoptosis and Inhibiting Epithelial-Mesenchymal Transition by Deactivating Akt/GSK-3ß Signaling.

[This corrects the article DOI: 10.3389/fphar.2018.01525.].

Proteomics analysis demonstrating rosmarinic acid suppresses cell growth by blocking the glycolytic pathway in human HepG2 cells.

Rosmarinic acid (RA), isolated from herbal balm mint plants, has demonstrated potent anti-tumor properties against liver cancer. However, the precise underlying mechanisms remain unclear. This study aimed to investigate the molecular mechanisms of RA in HepG2 cells. RA anti-tumor activity was assessed using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and Hoechst 33258 staining. Apoptosis and the cell cycle distribution were evaluated by flow cytometry. A proteomics approach was used to identify differentially expressed proteins following RA treatment in HepG2 cells, and quantitative reverse transcription-quantitative polymerase chain reaction was used to validate the results. Bioinformatics analysis was also implemented to further understand the identified proteins, and western blotting was used to analyze the associated proteins. Our results suggested that RA treatment significantly inhibits the viability of HepG2 cells. The MTT and LDH assays indicated dose-dependent decreases in cell proliferation following RA treatment. Hoechst 33258 staining and flow cytometry analysis showed that RA exhibits an apoptosis-inducing effect and induces cell cycle arrest in G1. The proteomics analysis successfully identified 16 differentially expressed proteins. Bioinformatics analysis indicated that the identified proteins participated in several biological processes and exhibited various molecular functions, mainly related to inactivation of the glycolytic pathway. Further western blotting analysis showed that RA could downregulate the expression of glucose transporter-1 and hexokinase-2, leading to the suppression of glucose consumption and generation of lactate and ATP. Taken together, our study found that RA exhibits significant cytotoxic effects by inhibiting cell proliferation and inducing apoptosis and cell cycle arrest, possibly by blocking the glycolytic pathway in human HepG2 cells.

Lariciresinol induces apoptosis in HepG2 cells via mitochondrial-mediated apoptosis pathway.

Lariciresinol (LA) is one of the main active ingredients in many traditional medicinal plants such as Patrinia, and has the role of anti-liver cancer. However, the precise mechanisms are unclear. This study investigated the molecular mechanisms of LA against HepG2 cells. LA anti-tumor activity was assessed with the CCK-8, Ki-67, and immunofluorescence staining. Cells apoptotic ratio was evaluated by Annexin V/PI double-staining assay. A proteomic approach was used to identify differentially expressed proteins after LA treatment. JC-1 staining was carried out to detect the mitochondrial membrane potential (ΔΨm), and the Western blot analysis was used to analyse the apoptosis-associated proteins. Our results suggested that LA significantly suppressed the viability of HepG2 cells. The CCK-8 and Ki-67 expression indicated dose-dependent decreases in cell proliferation. Flow cytometry analysis showed that LA exhibited a apoptosis-inducing effect. The proteomic study observed the presence of apoptosis-associated proteins and mitochondrial dysfunction in HepG2 cells after LA-treatment. Further analysis showed that LA could trigger the mitochondrial-mediated apoptosis pathway, based on a decrease in ΔΨm; deliver of cytochrome c; activation of caspase-9/-3 and poly(ADP-ribose) polymerase; and decrease of the proportion of Bcl-2/Bax. Collectively, our studies found that LA exhibits significant cytotoxic effects by inhibiting cell proliferation, inducing apoptosis, possibly via activation of the mitochondrial-mediated apoptosis pathway.

ß-Sitosterol and Gemcitabine Exhibit Synergistic Anti-pancreatic Cancer Activity by Modulating Apoptosis and Inhibiting Epithelial-Mesenchymal Transition by Deactivating Akt/GSK-3ß Signaling.

ß-sitosterol (BS), a major bioactive constituent present in plants, has shown potent anti-cancer activity against many human cancer cells, but its activity in pancreatic cancer (PC) cells has rarely been reported. Gemcitabine (GEM) is one of the first-line drugs for PC therapy, however, the treatment effect is not sustained due to prolonged drug resistance. In this study, we firstly studied the anti-PC activity and the mechanism of BS alone and in combination with GEM in vitro and in vivo. BS effectively inhibited the growth of PC cell lines by inhibiting proliferation, inducing G0/G1 phase arrest and apoptosis, suppressed the NF- kB activity, and increased expression of the protein Bax but decreased expression of the protein Bcl-2. Moreover, BS inhibited migration and invasion and downregulated epithelial-mesenchymal transition (EMT) markers and AKT/GSK-3ß signaling pathways. Furthermore, the combination of BS and GEM exhibited a significant synergistic effect in MIAPaCa-2 and BXPC-3 cells. More importantly, the combined treatment with BS and GEM lead to significant growth inhibition of PC xenografts. Overall, our data revealed a promising treatment option for PC by the combination therapy of BS and GEM.

Synergistic cytotoxicity of ampelopsin sodium and carboplatin in human non-small cell lung cancer cell line SPC-A1 by G1 cell cycle arrested.

OBJECTIVE: To evaluate the cytotoxic effects of ampelopsin sodium (Amp-Na) and carboplatin (CBP) used alone or in combination on human non-small cell lung cancer (NSCLC) cells SPC-A1 in vitro and its related mechanism. METHODS: Cytotoxic effects were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The synergistic effects of the drugs were calculated with coefficient of drug interaction (CDI). Cell cycle was determined by flow cytometry (FCM). The levels of p53, p21, cyclinE, cyclinD1, and phosphorylated cyclin-dependent kinase-2 (p-CDK2) were evaluated by Western blot. RESULTS: Amp-Na (6.25-200 µg/mL) and CBP (3.13-100 µg/mL) alone exhibited prominent cytotoxic activity in a concentration-dependent manner on SPC-A1 cells with 50% inhibitive concentration values of 57.07±14.46 and 34.97±6.30 µg/mL, respectively. Drug combinations were associated with significantly higher cytotoxic effects than each drug alone (P<0.05 or 0.01). The CDI analysis confirmed the synergy of Amp-Na and CBP on inhibiting cancer cell viability across a wide concentration range (CDI <1). FCM and Western blot showed that synergistic cytotoxic effects of Amp-Na and CBP were related to G1 arrested which mainlym ediated by p 21 through the inhibition of CDK2 activity independent of the p53 tumor suppressor pathway. CONCLUSIONS: Amp-Na exhibits anticancer activities and enhances the antitumor activities of CBP through up-regulation of p21 and inhibition of CDK2 activity in human NSCLC cells SPC-A1. These results suggest that Amp-Na may be applied to enhance the anticancer action of CBP.

Proteomic analysis of apoptosis induction by lariciresinol in human HepG2 cells.

Lariciresinol (LA) is a traditional Chinese medicine possessing anticancer activity, but its mechanism of action remains unclear. The present study explored the effects of LA on human HepG2 cells and the underlying mechanism. Our data indicated that LA inhibited cell proliferation and induced cell cycle arrest in S phase, subsequently resulting in apoptosis in HepG2 cells. Using a proteomics approach, eight differentially expressed proteins were identified. Among them, three proteins, glyceraldehyde-3-phosphate, UDP-glucose 4-epimerase, and annexin A1, were upregulated, while the other five proteins, heat shock protein 27, haptoglobin, tropomodulin-2, tubulin alpha-1A chain, and brain acid soluble protein 1, were downregulated; all of these proteins are involved in cell proliferation, metabolism, cytoskeletal organization, and movement. Network analysis of these proteins suggested that the ubiquitin-conjugating enzyme (UBC) plays an important role in the mechanism of LA. Western blotting confirmed downregulation of heat shock protein 27 and upregulation of ubiquitin and UBC expression levels in LA-treated cells, consistent with the results of two-dimensional electrophoresis and a STRING software-based analysis. Overall, LA is a multi-target compound with anti-cancer effects potentially related to the ubiquitin-proteasome pathway. This study will increase our understanding of the anticancer mechanisms of LA.

Comparative pharmacokinetics of a tumour-targeting therapy candidate rh-IFNα2a-NGR with rh-IFNα2a administered intravenously in mice and rats.

OBJECTIVES: rh-IFNα2a-NGR is a promising anti-tumor candidate. The aim of present study was to compare pharmacokinetics of rh-IFNα2a-NGR with rh-IFNα2a. METHODS: Pharmacokinetics and elimination were investigated after intravenous administration to mice and rats. Compared tumor and tissue distribution profiles between rh-IFNα2a-NGR and rh-IFNα2a were illustrated in the tumor transplanted mice of SP2/0 myeloma. Double antibody sandwich ELISA method was used to assess the level of both rh-IFNα2a-NGR and rh-IFNα2a in serum, tissue, bile and urine. KEY FINDINGS: After a single intravenous administration, the pharmacokinetic characters of rh-IFNα2a-NGR and rh-IFNα2a were described using a two-compartment model. No significant differences were observed between the two drugs in pharmacokinetic and elimination data. However, the concentration of rh-IFNα2a-NGR in tumor was 5.34 times and 1.52 times as high as that of rh-IFNα2a at 0.5 h (P < 0.01) and 1 h. In addition, immunohistochemical stain displayed rh-IFNα2a-NGR was predominantly located in tumor vascular tissues. CONCLUSIONS: rh-IFNα2a-NGR could be an agent for tumor vascular-targeting therapy and these findings provided references for further clinical study.

[Study on the effect of grape seed proanthocyanidins on increasing the radiosensitivity for X-ray].

OBJECTIVE: To investigate the effect of Grape Seed Proanthocyanidins (GSPs) on enhancing the radiosensitivity of human hepatic carcinoma cell line HepG2, human cervical cancer cell line Hela and human leukemia cell line K562 for X-ray in vitro. METHODS: The killing effect of GSPs combined with X-ray on cells was evaluated by SRB and clone formation assay. RESULTS: The GSPs had obvious cytotoxicity on three cell lines in a dose-dependent and time-dependent manners. However, inhibition rate of different cell line were quite different, the strongest one was human leukemia K562 cells and the others were weak. The sensitization ranges calculated by univariate analysis were 6.25-12.5 microg/mL for human leukemia K562 cells. Sensitization enhancement ratio was 1.94 using curve fitting method for K562 cells. CONCLUSION: GSPs can obviously enhance the radiosensitivity of cancer cells in vitro. The mechanism of sensitization effect may be related to the effects of GSPs on oxygen balance and cell cycle.

[Effects of extracts from Patrinia heterophylla on gene expression patterns during morphogenesis of chicken limb buds in vivo].

OBJECTIVE: To study the effects of the extracts from Patrinia heterophylla on gene expression patterns during morphogenesis of chicken limb buds in vivo. METHODS: Implanted a bead into an chicken embryo, which was soaked in the extracts from Patrinia heterophylla. Detected the extracts-induced morphogenesis changes (Myf5, Myod and PCNA). RESULTS: The extracts from Patrinia heterophylla (200 mg/mL) could affect limb bud development, reduce gene expression of MyfS, MyoD and PCNA. CONCLUSION: The extracts from Patrinia heterophylla can inhibit cell differentiation and proliferation.

[Effect of Patrinia scabra extracts on the level of serum cytokine in sarcoma 180 ascitic tumor burdened mice].

OBJECTIVE: To explore the effect of Potrinia scabro extracts (PSE) on the level of serum cytokine in Sarcoma 180 ascitic tumor burdened mice and its mechanism of anti-tumor. METHODS: The mice model of Sarcoma 180 ascitic tumor were established and divided into five groups randomly, including the model group with normal saline solution, the positive group with 10 mg/kg cytoxan and PSE treated groups at doses of 2.0 g/kg, 1.0 g/kg, 0.5 g/kg intraperitoneally for 10 days. The level of serum cytokine Th1 (IL-2, IL-12, IFN-gamma, IFN-alpha) and Th2 (IL-6, IL-10) were measured by double antibody sandwich ELISA assay. RESULTS: Compared with model group of Sarcoma 180 ascitic tumor burdened mice,the level of IL-2 and IFN-gamma increased in PSE 2.0 g/kg group, but the IL-6 and IL-10 decreased in PSE 2.0 g/kg and 1.0 mg/kg groups. CONCLUSION: PSE has anti-tumor effect in vivo that could be related to the level variation of IL-2, IFN-gamma, IL-6 and IL-10 in tumor burdened mice.

[Effects of Patrinia scabra bunge macroporous adsorptive resins extracts on erythrocyte immune function in tumor bearing mice].

OBJECTIVE: To study the erythrocyte immuno-regulatory effect of Patrinia scabra Bunge extracts extracted by macroporous adsorptive resins in tumor bearing mice. METHODS: Patrinia scabra Bunge was extracted by macroporous adsorptive resins, and the amount of polysaccharides and saponins in the extract were determined. Mice bearing S180 tumor were treated with the extract and their survival prolongation rate, erythrocyte rosette formation rates of C3b receptor (ERR-CR), immune complex (ERR-IC) and tumor cell (ERR-TC), as well as the CD35 and CD44s were observed. RESULTS: Polysaccharide content was 21.4%, saponin 41.8% in the extract. As compared with the model group, the survival rate was increased, the erythrocyte immune function was improved (showed increase of ERR-CR and ERR-TC, decrease of ERR-IC), and the amount of CD35 and CD44s in red blood cell membrane increased in mice after being treated with the extract (P < 0.05 or P < 0.01). CONCLUSION: Extract of Patrinia scabra Bunge extracted by macroporous adsorptive resins can regulate the erythrocyte immune function to a certain extent.

Effects of body-resistance strengthening and tumor-suppressing granules on immune adhesion function of red blood cells and expression of metastasis protein CD44 in tumor cells of patients with esophageal carcinoma.

AIM: To investigate the effects of Fuzheng Yiliu granules (body-resistance strengthening and tumor-suppressing granules) in patients with esophageal carcinoma. METHODS: We compared the immune adherent properties of red blood cells (RBCs), the expression of metastasis protein CD44, and the metastasis inhibition factor nm23, in esophageal carcinoma tumor cells of patients before and after radiotherapy in the presence and absence of orally administered Fuzheng Yiliu granules. Sixty-three hospitalized patients with esophageal carcinoma were treated with standard radiotherapy and randomly divided into treatment group (n = 30) treated with both radiotherapy and Fuzheng Yiliu granules and control group (n = 33) given radiotherapy only. Blood samples and tumor tissue were obtained before and after 21 d of treatment. The rosette rates for complement receptor type 3b (C3bRR) and immune complex receptor (ICRR) on RBCs were measured by erythrocyte immunological methods. Expression of CD44 and nm23 in tumor tissue sections was determined by immunohistochemical staining with monoclonal antibodies CD44v6 ad nm23H-1, respectively. RESULTS: The positivity of RBC-C3bRR before and after 21 d of treatment increased from 7.78% +/- 1.59% to 10.03% +/- 2.01% in the double treatment group, while it changed only slightly from 7.18% +/- 1.29% to 7.46% +/- 1.12% in the radiotherapy group. The positive rate for RBC-ICRR decreased from 37.68% +/- 2.51% to 22.55% +/- 1.65% after the double treatment, and from 37.28% +/- 2.41% to 24.69% +/- 1.91% in radiotherapy group at the same time points. The difference in erythrocyte immune adherent function between the two groups was significant (P < 0.01, t-test). The CD44(+)-cases were reduced from 21 (70.00%) to 12 (40.00%) after treatment with Fuzheng Yiliu granules, whereas the CD44(+)-cases (69.70%) in the radiotherapy group remained unchanged. The difference between the treatment (40.00%) and control (69.70%) groups was significant (P < 0.05). Although the nm23(+)-cases were increased from 4 (13.33%) to 6 (20.00%) in the double treatment group and from 6 (18.18%) to 7 (21.21%) in the radiotherapy group, the difference was not significant (P > 0.05). CONCLUSION: Fuzheng Yiliu granules enhance the immune adhesion function of RBCs and reduce the number of CD44(+)-cells in esophageal carcinoma patients, suggesting a potential role of these Chinese herbals in suppression of invasion and metastasis of malignant cells. However, this anti-metastatic effect has yet to be validated in vivo.

[Effect of Fuzheng Yiliu Granule contained-serum from tumor bearing mice on apoptotic rate, free radicals content and mitochondrial membrane potential of hepatoma cell lines H22 in vitro].

OBJECTIVE: To observe the effect of traditional Chinese herbs Fuzheng Yiliu Granule (FYG)-contained serum from tumor bearing mice on apoptotic rate, free radicals content and mitochondrial membrane potential of hepatoma cell lines H22 in vitro. METHODS: The effect of FYG drug-serum on apoptosis of hepatoma cell line H22 was determined using flow cytometry. The changes of DNA RNA, free radicals and mitochondrial membrane potential (delta psi m) in H22 cell were detected through laser scanning confocal microscope. RESULTS: FYG-contained serum can induce the apoptosis of H22 cell, enhance the free radicals content, and reduce the content of DNA RNA and delta psi m of H22 cell in vitro. CONCLUSION: The apoptosis of hepatoma cell line H22 induced by FYG is probably correlated to the change of free radicals content and delta psi m.

[Study on depressant effects of THPS on S180 tumor-bearing-mice and its mechanisms].

OBJECTIVE: to study depressant effect of total hedysarum polybotys saccharids (THPS) on S180 tumor-bearing-mice and its mechanisms. METHODS: THPS was extracted from Radix Xedysari with water and precipitated with ethanol, determining its molecular weight, purity, saccharide and aldonic acid content. 90 Kunming mice were divided into 9 groups randomly. One group was the normal group, the others were divided into 8 groups randomly after inculating S180 tumors and were treated with THPS and THPS combination cyclophosphamide (CY) in low, moderate and high dose, to put them to death after 14 days. To determine every tumor weight, the rate of depressant tumor, the contents of IL-2 and TNF-alpha with ELISA, and NF-kappaB with immunochemistry. RESULTS: The moderate dose THPS conspicuously possessed a depressant effect on S180 tumor and joint action with combination CY and increased the contents of IL-2, TNF-alpha and NF-kappaB of mice. CONCLUSION: THPS of moderate dose possesses a depressant effect on S180 tumor through increasing the contents of IL-2, TNF-alpha and NF-kappaB of mice.

[Erythrocyte immunologic function effects of Patrinia scabra extracts by macroporous adsorptive resins on mice burdened transplanted tumor].

OBJECTIVE: To research the erythrocyte immunoregulation effects of Patrinia scabra extracts by macroporous adsorptive resins on mice burdened transplanted tumor. METHODS: Extracts of Patrinia scabra Bunge were separated by macroporous adsorptive resins, ingredients were analysised. Mice burdened transplanted tumor were given extracted drugs. Life prolongation rate was observed, erythrocyte immunologic function and the CD35, CD44s contents of red blood cell were evaluated. RESULTS: Polysaccharide and saponin accounted for 8.4% and 48.4%. Extracts could porolong life expectancy of mice, improve erythrocyte immunolgic function and increase the CD35 and CD44s contents of red blood cell. CONCLUSION: Extracts of Patrinia scabra Bunge by macroporous adsorptive resins have erythrocyte immunoregulation effects on mice burdened transplanted tumor.

[Effects of fuzheng yiliu granules on apoptotic rate and mitochondrial membrane potential of hepatocellular carcinoma cell line H22 from mice].

OBJECTIVE: To evaluate the effects of Fuzheng Yiliu Granules (FZYLG) on apoptotic rate and mitochondrial membrane potential (Delta psi m) of hepatocellular carcinoma cell line H22 from mice. METHODS: Forty-eight mice inoculated with H22 cells were randomly divided into four groups: untreated group, cyclophosphamide-treated group, high-dose FZYLG-treated group and low-dose FZYLG-treated group. After 14 days of corresponding treatment, H22 cells in each group were stained with propidium iodide, and the apoptotic rates were detected by flow cytometry (FCM). The rhodamine 123 was used as a fluorescence probe to label the H22 cells, and the fluorescence intensities were observed with laser scanning confocal microscope. The fluorescence intensity of H22 cells indicated the Delta psi m of H22 cells. RESULTS: FZYLG could significantly increase the apoptotic rate while reduce the Delta psi m of H22 cells from mice as compared with those in the untreated group. CONCLUSION: The antitumor effects of FZYLR on H22 cells from mice are related to decreasing the Delta psi m and then inducing the apoptosis of the H22 cells.

[A study on the genital system injury induced by nickel sulfate in male rats].

OBJECTIVE: To study the mechanism of the genital system damage by nickel sulfate in male rats in order to provide the laboratory theoretical evidence for the prevention and cure of nickel genital toxicity. METHODS: Three groups of rats were injected intraperitoneally with nickel sulfate at dose of 1.25, 2.50, 5.00 mg/kg respectively for two weeks. The content of testicle nickel and blood serum testosterone (T), follicle-stimulating hormone (FSH), luteinizing hormone (LH) were assessed with atomic absorption spectrum and radioimmuno-assay, meanwhile the activity of nitric oxide synthase (NOS) and the content of nitric oxide (NO) were measured by enzyme method. RESULTS: The contents of testicle nickel [(0.22 +/- 0.03), (0.34 +/- 0.04), (0.41 +/- 0.02) micro g/g respectively] were increased, but the content of T, TSH, LH in blood serum were reduced; the activities of NOS in testicle tissue [(33.65 +/- 2.93), (26.53 +/- 9.52), (10.20 +/- 2.74) U/g respectively] were inhibited by nickel sulfate and the contents of NO [(0.26 +/- 0.03), (0.18 +/- 0.05), (0.15 +/- 0.02) mmol/g respectively] were decreased (P < 0.01). CONCLUSION: Nickel-induced genital system injury of male rats may be related to the decrease in the contents of T, TSH, LH, and the inhibition on NOS, as well as the fall of NO content.