c-MYC-mediated TRIB3/P62+ aggresomes accumulation triggers paraptosis upon the combination of everolimus and ginsenoside Rh2

The mammalian target of rapamycin (mTOR) pathway is abnormally activated in lung cancer. However, the anti-lung cancer effect of mTOR inhibitors as monotherapy is modest. Here, we identified that ginsenoside Rh2, an active component of Panax ginseng C. A. Mey., enhanced the anti-cancer effect of the mTOR inhibitor everolimus both in vitro and in vivo. Moreover, ginsenoside Rh2 alleviated the hepatic fat accumulation caused by everolimus in xenograft nude mice models. The combination of everolimus and ginsenoside Rh2 (labeled Eve-Rh2) induced caspase-independent cell death and cytoplasmic vacuolation in lung cancer cells, indicating that Eve-Rh2 prevented tumor progression by triggering paraptosis. Eve-Rh2 up-regulated the expression of c-MYC in cancer cells as well as tumor tissues. The increased c-MYC mediated the accumulation of tribbles homolog 3 (TRIB3)/P62+ aggresomes and consequently triggered paraptosis, bypassing the classical c-MYC/MAX pathway. Our study offers a potential effective and safe strategy for the treatment of lung cancer. Moreover, we have identified a new mechanism of TRIB3/P62+ aggresomes-triggered paraptosis and revealed a unique function of c-MYC.

Myricetin inhibits interferon-γ-induced programmed death ligand-1 and indoleamine 2, 3-dioxygenase 1 expression in lung cancer cells / 中国药理学与毒理学杂志

OBJECTIVE Programmed death ligand-1 (PD-L1) and indoleamine 2, 3-dioxygenase 1 (IDO1) are immune checkpoints which can be induced by interferon-γ(IFN-γ) in the tumor microenvironment, leading to immune escape of tumors. Myricetin (MY) is a flavonoid distributed in many edible and medicinal plants. The aim of this study is to clarify the effect and the mechanism of MY on inhibiting IFN-γ-induced PD-L1 and IDO1 in lung cancer cells. METHODS Expressions of PD-L1 and major histocompatibility complex-I (MHC-I) were evaluated by flow cytometry and Western blotting, and the expression of IDO1 was measured by Western blotting. qRT-PCR was used to detect their mRNA levels. The function of T cells was evaluated using a co-culture system consist of lung cancer cells and the Jurkat-PD-1 T cell line that overexpressing PD-1. Molecular docking analysis, Western blotting and immunofluorescence were used for mechanism study. RESULTS MY potently inhibited IFN-γ-induced PD-L1 and IDO1 expression in human lung cancer cells, while didn't show obvious effect on the expression of MHC-I. In addition, MY restored the survival, proliferation, CD69 expression and interleukin-2 (IL-2) secretion of Jurkat-PD-1 T cells suppressed by IFN-γ-treated lung cancer cells in the co-culture system. Mechanistically, IFN-γ up-regulated PD-L1 and IDO1 at the transcriptional level through the JAK-STAT-IRF1 axis, which was targeted and inhibited by MY. CONCLUSION Our research revealed a new insight into the anti-tumor effects of MY which inhibited IFN-γ-induced PD-L1 and IDO1 expression, supporting the potential of MY in anti-tumor immunotherapy.

Nagilactone E increases PD-L1 expression through activation of c-Jun in lung cancer cells / 中国天然药物

Nagilactone E (NLE), a natural product with anticancer activities, is isolated from Podocarpus nagi. In this study, we reported that NLE increased programmed death ligand 1 (PD-L1) expressions at both protein and mRNA levels in human lung cancer cells, and enhanced its localization on the cell membrane. Mechanistically, NLE increased the phosphorylation and expression of c-Jun, and promoted the localization of c-Jun in the nucleus, while silencing of c-Jun by small interfering RNA (siRNA) reduced NLE-induced PD-L1. Further study showed that NLE activated the c-Jun N-terminal kinases (JNK), the upstream of c-Jun, and its inhibitor SP600125 reversed the NLE-increased PD-L1. Moreover, NLE-induced PD-L1 increased the binding intensity of PD-1 on the cell surface. In summary, NLE upregulates the expression of PD-L1 in lung cancer cells through the activation of JNK-c-Jun axis, which has the potential to combine with the PD-1/PD-L1 antibody therapies in lung cancer.

Reduced tumorigenicity and drug resistance through the downregulation of octamer-binding protein 4 and Nanog transcriptional factor expression in human breast stem cells.

Breast cancer is the most common type of malignancy among females. Previous studies examining breast cancer tissue have demonstrated the presence of stem cells, and have detected octamer­binding protein 4 (Oct4) and Nanog transcription factor expression. In the present study, breast cancer stem cells (CSCs) were isolated and enriched from MDA­MB­231 breast cancer cell lines, and were defined as MDA­MB­231 stem cells using flow cytometry. The expression of Oct4 and Nanog in breast CSCs were detected by quantitative polymerase chain reaction and western blotting. RNA interference (RNAi) was used in order to downregulate the expression of Oct4 and Nanog. Drug resistance and tumor­initiating capability following in vivo injection of MDA­MB­231 stem cells trans-duced with negative RNAi, Oct4 RNAi and Nanog RNAi were compared with that of MDA­MB­231 stem cells without siRNA transfection as a control group. In addition the capability of MDA­MB­231 breast cancer cells to initiate tumor formation in mice was compared with that of MDA­MB­231 stem cells. A paclitaxel inhibition test was also conducted in order to detect resistance of MDA­MB­231 breast cancer stem cells to this treatment. The MDA­MB­231 stem cells were revealed to exhibit elevated percentages of the cluster of differentiation (CD)44+CD24­/low subset, high tumorigenicity and resistance to chemotherapy, all of which are characteristic stem cell properties. In addition, the MDA­MB­231 stem cells were more tumorigenic in vivo. Furthermore, the breast CSCs also expressed high levels of the Oct4 and Nanog transcription factors. Therefore, downregulation of Oct4 or Nanog expression may reduce chemotherapeutic drug resistance and tumorigenicity in breast CSCs. In conclusion, Oct4 and Nanog expression may be a key factor in the development of resistance to chemotherapy and tumor growth of breast CSCs. This finding indicates that Oct4 or Nanog­targeted therapy may be a promising means of overcoming resistance to chemotherapy and inhibiting tumor growth in breast cancer treatment.

[Cytosine deaminase and thymidine kinase double suicide gene system driven by carcinoembryonic antigen promoter for the treatment of colorectal carcinoma xenograft in nude mice].

OBJECTIVE: To explore the therapeutic efficacy of double suicide gene system driven by carcinoembryonic antigen (CEA) promoter (Cp-CDglyTK) on colorectal carcinoma xenograft in nude mice. METHODS: The plasmid pcDNA3.1(-)Cp-CDglyTK was transfected into the CEA-positive SW480 and CEA-negative HeLa cells respectively. The expression of suicide gene was detected by RT-PCR. And the transfected cells were treated with 5-fluorocytosine (5-FC) and ganciclovir (GCV) at different concentrations and the cell-killing and bystander effects assayed by methyl thiazolyl tetrazolium (MTT). By a transplantation of cultivated cells, SW480 or HeLa cell lines were injected subcutaneously into right axillary of nude mice to establish 96 SW480 and 72 HeLa tumor animal models. Nude mice were completely randomized with statistical software according to tumor volume. For prodrug therapy, 48 SW480-bearing mice were divided equally into 4 groups of I-IV. At the same time, 48 HeLa-bearing mice were divided equally into 4 groups of V-VIII. Groups I & V received an intratumoral injection of PBS, groups II & VIGCV and 5-FC intratumorally, groups III & VII PBS intraperitoneally and groups IV & VIII GCV and 5-FC intraperitoneally. Forty-eight SW480-bearing mice were divided equally into 4 groups of IX∼XII and 24 Hela-bearing ones into groups of & in therapy experiment by suicide gene plus prodrug. Six groups received an intratumoral injection of liposome Lipofectamine and plasmid CP-CDglyTK and then an intraperitoneal injection of drug. The groups of IX and received an injection of PBS, group X GCV, group XI 5-FC and groups XII & GCV and 5-FC. The observation parameters included tumor bulk, tumor weight, survival time and treatment effect in each group. RESULTS: SW480 cells transfected by plasmid pcDNA3.1(-)Cp- CDglyTK expressed CDglyTK gene. The inhibition rates of GCV and 5-FC were significantly higher than those of HeLa cells (59.87% ± 0.21% vs 9.90% ± 0.09%, P < 0.01). And higher inhibition rates and stronger bystander effect existed in double versus single produg (all P < 0.05). Tumor size, final tumor weight and survival time of nude mice in groups ofII, IV, VI & VIII had no significant difference with groups ofI, III, V & VII (all P < 0.05). Final tumor size and weight of group XII was significantly smaller than those of groups of IX, X and XI ((150.0 ± 3.2) vs (522.5 ± 1.9) and (256.8 ± 10.4) and (260.7 ± 2.2) mm(3), (54.1 ± 10.4) vs (682.0 ± 12.0) and (251.8 ± 15.1) and (271.6 ± 17.7) mg, all P < 0.05). Meanwhile, the tumor inhibition rate and survival time of group XII(92.1% and (25.7 ± 0.8)d) were significant higher and longer than group X (63.1% and (21.8 ± 0.5) d) and group XI (60.2% and (18.0 ± 0.9) d) (all P < 0.05). However, no significant difference existed in tumor size, final tumor weight and survival time between groups and (all P > 0.05). The inhibition rate of group was merely 0.9%. CONCLUSION: CDglyTK double suicide gene system driven by CEA promoter may inhibit CEA positive colorectal cancer xenograft in prodrug-treated nude mice.

Targeting the vasculature of colorectal carcinoma with a fused protein of (RGD)3-tTF.

PURPOSE: Truncated tissue factor (tTF) fusion protein targeting tumor vasculature can induce tumor vascular thrombosis and necrosis. Here, we generated (RGD)3-tTF in which three arginine-glycine-aspartic (RGD) targeting integrin α(v)ß3 and tTF induce blood coagulation in tumor vessels. METHODS: The bioactivities of (RGD)3-tTF including coagulation activity, FX activation, and binding with integrin α(v)ß3 were performed. The fluorescent labeled (RGD)3-tTF was intravenously injected into tumor-bearing mice and traced in vivo. The tumor growth, volume, blood vessel thrombosis, tumor necrosis, and survival time of mice treated with (RGD)3-tTF were evaluated. RESULTS: The clotting time and FX activation of (RGD)3-tTF were similar to that of TF (P > 0.05) but different with that of RGD (P < 0.05). (RGD)3-tTF presented a higher binding with α(v)ß3 than that of RGD and TF at the concentration of 0.2 µmol/L (P < 0.05). (RGD)3-tTF could specifically assemble in tumor and be effective in reducing tumor growth by selectively inducing tumor blood vessels thrombosis and tumor necrosis which were absent in mice treated with RGD or TF. The survival time of mice treated with (RGD)3-tTF was higher than that of mice treated with TF or RGD (P < 0.05). CONCLUSION: (RGD)3-tTF may be a promising strategy for the treatment of colorectal cancer.

[Expression of OCT4 in gastric cancer cell lines and its significance].

OBJECTIVE: To detect the expression of octamer-binding protein-4 (OCT4) in gastric cancer cell lines with different differentiation (MKN-28, SGC-7901, BGC-823) and normal gastric mucosal cells line GES-1, and further assess the relationship between OCT4 expression and the differentiation grade of gastric carcinoma cells. METHODS: Expression level of OCT4 in GES-1, MKN-28, SGC-7901 and BGC-823 was detected by reverse transcription-polymerase chain reaction (RT-PCR), and OCT4 siRNA was employed to interfere OCT4 expression in BGC-823 cell lines. Detect the quantity of OCT4 mRNA and OCT4 protein by fluorescent quantitative PCR and Western blot respectively. In addition, the invasion ability was analyzed via Transwell chamber. RESULTS: The normal gastric mucosal cells did not express OCT4 and there was higher expression of OCT4 in gastric cancer cell lines with poorly differentiation (P<0.05). The expression of OCT4 in BGC-823 cells was the highest. The expression of OCT4 mRNA and OCT4 protein were decreased distinctly in BGC-823 cells after being interfered by OCT4 siRNA (P<0.05). After being interfered by OCT4, BGC-823 cells were less aggressive, and the number of penetrating cells was decreased (P<0.05). CONCLUSION: The OCT4 expression level is associated with gastric cancer differentiation. OCT4 may play an important role in the differentiation and invasion of gastric cancer cell and it may serve as a reference index in predicting the malignant grade of gastric cancer.