Apoptosis induced by the methanol extract of Salvia miltiorrhiza Bunge in non-small cell lung cancer through PTEN-mediated inhibition of PI3K/Akt pathway.

Salvia miltiorrhiza Bunge, a well-known traditional Chinese medicinal (TCM) plant, has been used to treat cardiovascular diseases since thousands of years. Many studies reported that the active component tanshinones displayed a variety of biological activities: anti-thrombous, anti-allergic, anti-inflammatory, antioxidant and anti-tumor promoting. But the mechanism of how the active components working still need to be clarified. The anti-tumor effect of compounds of tanshinone (CTN), the methanol extract of Salvia miltiorrhiza Bunge roots, was investigated. The aim of this study was to investigate the effects of CTN on the growth inhibition, apoptosis and molecular targets of human non-small cell lung cancer (NSCLC). CTN-induced cytotoxicity was determined by MTT assay. The cell survival was evaluated using clonogenic survival assay. The morphology of Glc-82 cells after treatment with CTN was determined by fluorescence microscopy. Cell cycle distribution was revealed by flow cytometry. The apoptotic cells were quantified with annexin V-FITC/PI staining and flow cytometry, and observed using Hoechst 33258 staining and TUNEL assays. The expression levels of proteins were analyzed using western blot. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice. CTN inhibited the proliferation of NSCLC in a dose-dependent manner and induced both early and late apoptosis. Treatment of Glc-82 cells with CTN (5-80μg/ml) significantly (p<0.05) suppressed the cell proliferation in a concentration and time-dependent manner. CTN induced significant (p<0.05) and dose-dependent apoptosis of Glc-82 cells. Cell cycle assay showed that CTN induced a G2/M phase arrest, and significantly (p<0.05) increased expression of p53 and p21, actived caspase-3/9 and PARP1, which suggest the involvement of the mitochondria in the apoptotic signals. In addition, CTN decreased expression of the anti-apoptotic protein Bcl-2, Bcl-xl and increased expression of the pro-apoptotic protein Bax. Result also showed that CTN could increase expression levels of PTEN, and reduce the phosphorylated levels of Akt (protein kinase B) on Thr 308 and Ser 473 domain. In vivo assay showed that the antitumor effect of CTN was significantly augmented without increasing toxicity in nude mice bearing Glc-82 xenograft. The PTEN/Akt signaling axis is defined as a critical pathway regulated by PTEN in NSCLC. CTN, the methanol extract of Salvia miltiorrhiza Bunge, are the active compounds as shown by their ability to induce apoptosis through the mitochondrial pathway of apoptosis and PTEN-mediated inhibition of PI3K/Akt pathway. CTN could inhibit tumor growth more efficiently, which supports the ethno-medicinal use of this herb as an alternative or complementary therapy for NSCLC.

Ye YT ，Zhong W ，Sun P ，Wang D ，Wang C ，Hu LM ，Qian JQ ... -  《-》

Alpinia oxyphylla oil induces apoptosis of hepatocellular carcinoma cells via PI3K/Akt pathway in vitro and in vivo.

The anti-tumor properties of Alpinia oxyphylla Miquel (A. oxyphylla) extracts and their petroleum ether (PE) fractions have long attracted scientific attention. These extracts' anti-tumor activity and mechanisms in vivo are still unclear. This study was designed to investigate the anti-tumor activity and the underlying mechanism of PE's effect on hepatocellular carcinoma (HCC) in vitro and in vivo. The anti-tumor activity of PE was evaluated by MTT assay and xenograft study. Mechanistic studies of PE were analyzed by Hoechst 33342 staining, Annexin V-FITC/PI double-staining assay, immunohistochemical staining and western blot assay. The toxicity of the PE treatment was verified by the levels of liver and kidney function in nude mice and the H&E staining of their liver and kidney tissues. PE significantly inhibited the growth of HepG2, BEL-7402, SMMC-7721 and Hep3B cells in a concentration- and time-dependent manner. Specifically, PE inhibited the growth of Hep3B cells by inducing apoptosis. PE treatment at the doses of 0.25, 0.5 and 1 g/kg for 21 days caused a respective 35.7 percent, 49.3 percent and 58.8 percent inhibition of the tumor volume, and a 14.8 percent, 40.2 percent and 55.6 percent decrease in the tumor weight, respectively, as compared with the vehicle group in tumor-loaded mice in vivo. PE promoted the release of cytochrome c from mitochondria to cytosol in a concentration-dependent manner. The expression levels of BAX (p <  0.01), cleaved caspase-9 (p <  0.01) and cleaved caspase-3 (p <  0.05) were increased significantly in the PE-treated group at the dose of 1 g/kg; the expression level of BAX (p <  0.05) was increased significantly in the PE-treated group at the dose of 0.5 g/kg, and the expression level of Bcl-2 (p <  0.01) was decreased significantly in the PE-treated group in a concentration-dependent manner. Apoptosis was induced by PE through up-regulating the expression of PTEN, down-regulating the expression of PI3K and inhibiting the phosphorylation of Akt. The liver and kidney function of the plasma and the morphology of the liver and kidney were normal in each group. These findings suggested that PE exhibited anti-cancer efficacy on Hep3B cell in vitro and in vivo. The induction of apoptosis might be one mechanism that underlies PE's ability to combat cancer by inhibiting the PI3K/Akt pathway. PE has no obvious toxicity in vivo when it exerts anti-tumor effects and has the potential to develop into an alternative anti-cancer drug for HCC treatment.

Hui F ，Qin X ，Zhang Q ，Li R ，Liu M ，Ren T ，Zhao M ，Zhao Q ... -  《-》

Salvia miltiorrhiza polysaccharide activates T Lymphocytes of cancer patients through activation of TLRs mediated -MAPK and -NF-κB signaling pathways.

Salvia miltiorrhiza polysaccharide (SMP) is one of the most important components in the water extract of Salvia miltiorrhiza Bunge, which has been mainly applied for the prevention or treatment of ischemic encephalopathy and cardiac diseases including myocardial infarction and coronary heart diseases in clinical practice. Our object is to investigate the immune regulation effects of SMP, specifically on the proliferation and cytotoxicity of T lymphocytes through MAPK and NF-κB pathway in peripheral blood of cancer patients. SMP was prepared through refluxing with ethanol, refluxing with water, Sevage treatment and ethanol precipitation. The lymphocytes were obtained from the peripheral blood of cancer patients. The effect of SMP on T lymphocyte proliferation was investigated by cell counting and flow cytometry. The effect of SMP on the proliferation of cancer cell lines A549, hepG2 and HCT116 was examined by MTT assay. The cytotoxic activity of T lymphocytes treated with SMP was detected by Calcein-acetoxymethyl (Calcein-AM) release. The gene expression of IL-4, IL-6, IFN-γ and toll like receptors (TLRs) was detected by semi-quantitative PCR. The protein expression of mitogen activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathway were detected by western blotting. To further verify whether SMP functions through the indicated pathways,, T lymphocytes were treated with SMP and an extracellular regulated protein kinase (ERK) inhibitor (U0126), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125) or an inhibitor of NF-κB inhibitor-α (IκBα) (BAY11-7082), respectively. After 24 h co-treatment, the expressions of p-JNK, p-ERK, IκBα, inhibitory kappa B kinase α (IKKα) and inhibitory kappa B kinase β (IKKβ) protein were detected by western blotting, meanwhile cell numbers of T lymphocytes after inhibition were calculated again by cell counter. SMP dose-dependently promoted the proliferation of T lymphocytes of the cancer patients and significantly improved the cytotoxicity of T lymphocytes against cancer cells. However, SMP showed no effect on the proliferation of the tumor cells from the same source. Furthermore, the gene expression of cytokines including IL-4, IL-6 and IFN-γ were also up-regulated. Moreover, SMP enhanced gene expression of TLR1, TLR2 and TLR4; elevated protein expression of p-JNK and p-ERK; increased protein expression of IKKα, and IKKβ and decreased IκBα levels. Meanwhile, knockdown of ERK、JNK or IκBα expression with specific inhibitor significantly depressed the proliferation of T lymphocytes treated with SMP, corroborating the specific regulation effect of SMP on T lymphocytes through MAPK and NF-κB signaling pathways. SMP specifically promotes the proliferation and enhances cytotoxicity of T lymphocytes in peripheral blood of cancer patients through activation of TLRs mediated -MAPK and -NF-κB signaling pathways.

Chen Y ，Li H ，Li M ，Niu S ，Wang J ，Shao H ，Li T ，Wang H ... -  《-》

Ethyl acetate extract from Selaginella doederleinii Hieron inhibits the growth of human lung cancer cells A549 via caspase-dependent apoptosis pathway.

Selaginella doederleinii Hieron has been used as a folk medicine for the treatment of different cancers, especially for nasopharyngeal carcinoma, lung cancer and trophoblastic tumor in China. Previously, the ethyl acetate extract from S. doederleinii (SDEA extract) showed favorable anti-cancer potentials. However, the main chemical composition and anticancer mechanism of the SDEA extract were still not very clear. Until now, there are no reports available about the oral toxicity of the extract. The present study was to further elucidate the chemical composition and anti-lung cancer mechanism of the SDEA extract, and evaluate the acute oral toxicity of the extract. The SDEA extract was separated and analysed by HPLC to disclose its main chemicals. The effects of the extract were then investigated in vitro on cell viability, apoptosis and cell cycle using fluorescence microscopy and flow cytometry, and the molecular mechanism against human lung cancer cells A549 was further studied by western blot assays. The in vivo anti-cancer effect of the extract was evaluated in A549 xenograft mice model by histochemical assay, and tumor growth, microvascular density (MVD) and Ki67 expression were also measured. In addition, acute oral toxicity test of the extract was executed in mice. SDEA extract mainly contained eight biflavonoids. The extract caused the loss of mitochondrial membrane potential and induced cell apoptosis by upregulating Bax, downregulating Bcl-2, activating caspase-9 and caspase-3 and blocked the cell cycle in S phase. The extract reduced expression of antigen Ki67, decreased MVD, and significantly inhibited the tumor growth. The extract did not show apparent oral acute toxicity in healthy mice. The SDEA extract exerted anti-tumor effect through activating mitochondrial pathways and reducing Ki67 expression and MVD. Low oral acute toxicity suggested it a promising chemotherapy agent.

Sui Y ，Li S ，Shi P ，Wu Y ，Li Y ，Chen W ，Huang L ，Yao H ，Lin X ... -  《-》

Response of non-small cell lung cancer cells to the inhibitors of phosphatidylinositol 3-kinase/Akt- and MAPK kinase 4/c-Jun NH2-terminal kinase pathways: an effective therapeutic strategy for lung cancer.

Lee HY ，Oh SH ，Suh YA ，Baek JH ，Papadimitrakopoulou V ，Huang S ，Hong WK ... -  《CLINICAL CANCER RESEARCH》