Paris Saponin I Sensitizes Gastric Cancer Cell Lines to Cisplatin via Cell Cycle Arrest and Apoptosis.

BACKGROUND Dose-related toxicity is the major restriction of cisplatin and cisplatin-combination chemotherapy, and is a challenge for advanced gastric cancer treatment. We explored the possibility of using Paris saponin I as an agent to sensitize gastric cancer cells to cisplatin, and examined the underlying mechanism. MATERIAL AND METHODS Growth inhibition was detected by MTT assay. The cell cycle and apoptosis were detected using flow cytometry and Annexin V/PI staining. The P21waf1/cip1, Bcl-2, Bax, and caspase-3 protein expression were detected using Western blot analysis. RESULTS The results revealed that PSI sensitized gastric cancer cells to cisplatin, with low toxicity. The IC50 value of cisplatin in SGC-7901 cell lines was decreased when combined with PSI. PSI promoted cisplatin-induced G2/M phase arrest and apoptosis in a cisplatin concentration-dependent manner. Bcl-2 protein expression decreased, but Bax, caspase-3, and P21waf1/cip1 protein expression increased with PSI treatment. CONCLUSIONS The underlying mechanism of Paris saponin I may be related to targeting the apoptosis pathway and cell cycle blocking, which suggests that PSI is a potential therapeutic sensitizer for cisplatin in treating gastric cancer.

Antitumor activity of Lobaplatin against esophageal squamous cell carcinoma through caspase-dependent apoptosis and increasing the Bax/Bcl-2 ratio.

Esophageal cancer is one of the most aggressive malignancies with poor prognosis. The administration of the first- and second-generation platinum drugs is frequently accompanied by drug resistance and severe toxicity. The aim of present study is to investigate the anti-tumor activity of the third-generation platinum drug Lobaplatin against esophageal squamous cell carcinoma in vitro and in vivo, and clarify the underlying molecular mechanism. The cytotoxicity of Lobaplatin against esophageal squamous cell carcinoma cell lines was determined by the MTT and clonogenic assay. Cell apoptosis was assessed by Annexin V-FITC/PI apoptosis assay using flow cytometry. The expression of proteins was determined by western blot analysis. The in vivo anti-tumor activity was evaluated in nude mice xenograft. Lobaplatin significantly inhibited the growth of KYSE-410 and EC-109 cells in a dose- and time-dependent manner and induced cell apoptosis by increasing expressions of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax, decreasing expression of Bcl-2. In vivo study showed that Lobaplatin suppressed tumor growth of EC-109 xenograft. Lobaplatin significantly inhibited the growth of esophageal squamous cell carcinoma by inducing apoptosis through the caspase-dependent pathway. Lobaplatin is an effective anti-cancer agent against esophageal cancer.

Paris Saponins enhance radiosensitivity in a gefitinib-resistant lung adenocarcinoma cell line by inducing apoptosis and G2/M cell cycle phase arrest.

Acquired resistance to epidermal growth factor inhibitors has been reported to be associated with cross­resistance to radiation. Paris Saponins (PSs) exert a wide range of pharmacological activities, including cell apoptosis induction, multidrug resistance inhibition, angiogenesis inhibition and tumor cell migration by modulating various signaling pathways. The present study aimed to investigate the radiosensitization effects of PSII, PSVI and PSVII in a gefitinib­resistant PC­9­ZD lung adenocarcinoma cell line, and the possible mechanism underlying their function. A clonogenic assay was performed to determine the effects of PS radiosensitization on the PC­9­ZD cell line. The cell cycle was analyzed by flow cytometry, and cell apoptosis was analyzed with Annexin V/propidium iodide and Hoechst staining. Protein expression levels were detected by western blotting. The results of the present study revealed a significant increase in PC­9­ZD cell line radiosensitivity following treatment with PSs. PSs induced G2/M cell cycle phase arrest and apoptosis of the irradiated PC­9­ZD cells. Notably, the expression levels of B cell lymphoma 2 (Bcl­2) were downregulated, and those of caspase­3, Bcl­2­associated X protein (Bax) and p21/Waf1/Cip1 were upregulated following treatment with PSs. The present results demonstrated that PSs induced radiosensitivity in gefitinib­resistant cells by inducing G2/M phase arrest and by enhancing the apoptotic response via the modulation of caspase­3, Bax, Bcl­2 and p21/Waf1/Cip1 expression.

Treatment of hepatocellular carcinoma in a mouse xenograft model with an immunotoxin which is engineered to eliminate vascular leak syndrome.

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin and interleukin-2 therapy. It has been evidenced that VLS-inducing molecules share a three-amino acid consensus motif, (x)D(y), which may be responsible for initiating VLS. Here we have constructed a recombinant immunotoxin (SMFv-PE38KDEL) by genetically fusing PE38KDEL to a single-chain antibody derived from SM5-1 monoclonal antibody, which has a high specificity for melanoma, hepatocellular carcinoma and breast cancer. In order to eliminate VLS induced by this PE38KDEL-based immunotoxin, a panel of mutants were generated by changing amino acid residues adjacent to its three (x)D(y) motifs in the three-dimensional structure. One of the SMFv-PE38KDEL mutants, denoted as mut1, displayed a similar protein synthesis inhibitory in a reticulocyte lysate translation assay compared to the wild-type SMFv-PE38KDEL (wt). The in vitro cytotoxicity assay indicated that mut1 specifically killed SM5-1 binding protein-positive tumor cells, although its cytotoxicity was slightly less than wt. In contrast, mut1 was shown to be much weaker in inducing VLS in mice than wt. The LD(50) values of wt and mut1 in mice were investigated with the result that the LD(50) of mut1 was about tenfold higher than that of wt. The in vivo antitumor activity of wt and mut1 were also compared in tumor-bearing nude mice. Both wt and mut1 were effective in inhibiting the tumor growth but mut1 showed improved therapeutic efficacy. These studies suggest mut1 may be a novel PE-based immunotoxin with much less toxicity for clinical use.

Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli.

Pseudomonas Exotoxin A (PE) and truncated PE have been used to prepare immunotoxin with monoclonal antibodies. Truncated Pseudomonas Exotoxin A (PE38KDEL) was expressed with the pET-32a(+) vector in Escherichia coli under control of a T7 promoter. The recombinant protein was purified by His-Ni(2+) metal affinity chromatography and gel filtration. The biological activity of PE38KDEL was evaluated by the inhibition assay of protein synthesis in rabbit reticulocyte lysate system, and the cytotoxicity was tested in Hut 102 and hepatocellular cell lines by the MTS assay. PE38KDEL can significantly inhibit luciferase synthesis in cell-free protein synthesis assay and was slightly cytotoxic in the Hut 102 and hepatocellular cell lines. The results suggest that PE38KDEL would be useful for the preparation of more potent immunotoxins.

Lymphtoxin beta receptor-Ig ameliorates TNBS-induced colitis via blocking LIGHT/HVEM signaling.

LIGHT is a member of the TNF superfamily, which is transiently expressed on the surface of activated T lymphocytes and immature dendritic cells. Its known receptors are herpesvirus entry mediator (HVEM) prominently in T lymphocytes, and lymphtoxin beta receptor (LTbetaR) in stromal cells or nonlymphoid hematopoietic cells. Previous studies have shown that overexpression of LIGHT on T cells could lead to lymphocytes activation, inflammation, and tissue destruction focused on intestinal mucosal tissues. To address the role of LIGHT/HVEM signaling in colonic inflammation, an experimental colitis model induced by rectal administration of trinitrobenzene sulfonic acid (TNBS) was given a soluble LTbetaR-Ig fusion protein as a competitive inhibitor of LIGHT/HVEM pathway. Marked elevation of LIGHT expression was detected in colonic tissue of the experimental colitis. Treatment with LTbetaR-Ig significantly attenuated the progression and histological manifestations of the colonic inflammation and reduced the production of inflammatory cytokines including TNF-alpha, IL-1beta and IL-8. Moreover, LTbetaR-Ig treatment significantly down-regulated LIGHT expression, leading to reduced lymphocytes, particularly CD4+ T cells, infiltrating into the colonic inflammation tissue as shown by histological analysis. In addition, comparison of the therapeutic effects on TNBS-induced colitis between LTbetaR-Ig and mesalazine showed that both treatments were equally efficacious. We postulated that blockade of LIGHT/HVEM signaling by LTbetaR-Ig may ameliorate TNBS-induced colitis by down-regulating LIGHT expression, and therefore we envision that LTbetaR-Ig would prove to a promising strategy for the clinical treatment of inflammatory bowel disease.