Baicalein activates 5' adenosine monophosphate-activated protein kinase, inhibits the mammalian target of rapamycin, and exhibits antiproliferative effects in pancreatic neuroendocrine tumors in vitro and in vivo.

BACKGROUND: The mammalian target of rapamycin inhibition has been shown to prolong progression-free survival in patients with pancreatic neuroendocrine tumors. The natural compound baicalein indirectly inhibits the mammalian target of rapamycin, but it is unknown if baicalein exhibits such effects at physiologically achievable concentrations or exhibits synergy. METHODS: Pancreatic neuroendocrine tumor cell lines were cultured with baicalein, everolimus, and/or a synthetic 5' adenosine monophosphate-activated protein kinase activating agent alone and in combination. Cell viability assays and immunoblotting were performed. Female severe combined immunodeficient-beige mice were injected with BON-1 cells and treated with baicalein and COH-SR4 solutions via oral gavage. Tumor volumes were compared at 30 days. RESULTS: Immunoblotting revealed that treatment of baicalein induced 5' adenosine monophosphate-activated protein kinase activation and the mammalian target of rapamycin inhibition. Treatment with baicalein alone led to a significant decrease in the ratio of viable cells compared with controls at 72 hours at concentrations ≥5 µM (P = .021). The addition of COH-SR4 led to significantly greater effect on cell viability than with baicalein alone (P < .001, P < .001). The combination of baicalein with everolimus resulted in significantly lower cell viability than with everolimus alone (P = .005, P < .001). Tumor volume in vivo was significantly decreased with the combination of baicalein and COH-SR4 compared with controls (P = .003). CONCLUSION: Baicalein exhibits antiproliferative effects against pancreatic neuroendocrine tumor cell lines at doses ≥5 µM and demonstrates synergy.

Eribulin Synergistically Increases Anti-Tumor Activity of an mTOR Inhibitor by Inhibiting pAKT/pS6K/pS6 in Triple Negative Breast Cancer.

Unlike other breast cancer subtypes, patients with triple negative breast cancer (TNBC) have poor outcomes and no effective targeted therapies, leaving an unmet need for therapeutic targets. Efforts to profile these tumors have revealed the PI3K/AKT/mTOR pathway as a potential target. Activation of this pathway also contributes to resistance to anti-cancer agents, including microtubule-targeting agents. Eribulin is one such microtubule-targeting agent that is beneficial in treating taxane and anthracycline refractory breast cancer. In this study, we compared the effect of eribulin on the PI3K/AKT/mTOR pathway with other microtubule-targeting agents in TNBC. We found that the phosphorylation of AKT was suppressed by eribulin, a microtubule depolymerizing agent, but activated by paclitaxel, a microtubule stabilizing agent. The combination of eribulin and everolimus, an mTOR inhibitor, resulted in an increased reduction of p-S6K1 and p-S6, a synergistic inhibition of cell survival in vitro, and an enhanced suppression of tumor growth in two orthotopic mouse models. These findings provide a preclinical foundation for targeting both the microtubule cytoskeleton and the PI3K/AKT/mTOR pathway in the treatment of refractory TNBC.

Combination therapy with BYL719 and LEE011 is synergistic and causes a greater suppression of p-S6 in triple negative breast cancer.

A third of patients with triple negative breast cancer (TNBC) have relapsed disease within 2-5 years from initial diagnosis, leaving an unmet need for therapeutic targets. TNBC frequently harbors alterations of the PI3K/AKT/mTOR pathway, but single agent PI3K/AKT/mTOR inhibitors have not shown marked efficacy. In this study, we investigated a strategy to improve efficacy of PI3K-α inhibitor BYL719 (alpelisib) in TNBC. While BYL719 is effective at inhibiting cell proliferation in T47D, a triple positive cell line, it had limited activity in TNBC. This may be partially due to persistent phosphorylation of RB, and incomplete inhibition of p-S6 in TNBC, since the inhibitory effect of BYL719 on p-RB and p-S6 was significantly reduced in TNBC compared to T47D cells. Addition of the CDK4/6 inhibitor LEE011 to BYL719 caused a simultaneous reduction of p-RB and p-S6, and a more complete inhibition of p-S6, leading to decreased expression of the pro-survival protein MCL-1, an induction of apoptosis, and an enhanced reduction of tumor growth in a PDX model of TNBC. These findings suggest that inhibition of p-RB and p-S6 is important for an effective response to the treatment of TNBC, and provides a strong rationale for clinical development of combination therapy with BYL719 and LEE011 for treatment of metastatic TNBC with intact RB.Presentation: This study was presented in part as an abstract at the 2016 San Antonio Breast Cancer Symposium (P3-03-15) and the 2018 Cancer Research and Targeted Therapy in London.

Baicalein upregulates DDIT4 expression which mediates mTOR inhibition and growth inhibition in cancer cells.

Baicalein is a natural flavone that exhibits anticancer properties. Using microarrays we found that DDIT4 was the highest transcript induced by baicalein in cancer cells. We confirmed in multiple cancer cell lines large, dose-related expression of DDIT4 by quantitative RT-PCR and immunoblot, which correlates with growth inhibition. Time course experiments demonstrate that DDIT4 is rapidly inducible, with high expression maintained for several days in vitro. Induction of DDIT4 expression is p53 independent based on evaluation of p53 knockout cells. Since DDIT4 is known to inhibit mTORC1 activity we confirmed that baicalein suppresses phosphorylation of mTORC1 targets. Using RNA interference we demonstrate that mTORC1 activity and growth inhibition by baicalein is attenuated by knockdown of DDIT4. We furthermore demonstrate suppression of established tumors by baicalein in a mouse model of breast cancer with increased DDIT4 expression in the tumors. Finally, we demonstrate that baicalein upregulates DDIT4 and causes mTORC1 and growth inhibition in platinum resistant cancer cells in marked contrast to platinum chemotherapy treatment. These studies demonstrate that baicalein inhibits mTORC1 through DDIT4 expression, and may be useful in cancer chemotherapy and chemoprevention.

Interferon Regulatory Factor 1 (IRF-1) induces p21(WAF1/CIP1) dependent cell cycle arrest and p21(WAF1/CIP1) independent modulation of survivin in cancer cells.

We have shown that the ectopic expression of Interferon Regulatory Factor 1 (IRF-1) results in human cancer cell death accompanied by the down-regulation of the Inhibitor of Apoptosis Protein (IAP) survivin and the induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). In this report, we investigated the direct role of p21 in the suppression of survivin. We show that IRF-1 down-regulates cyclin B1, cdc-2, cyclin E, E2F1, Cdk2, Cdk4, and results in p21-mediated G1 cell cycle arrest. Interestingly, while p21 directly mediates G1 cell cycle arrest, IRF-1 or other IRF-1 signaling pathways may directly regulate survivin in human cancer cells.

Identification of a natural compound by cell-based screening that enhances interferon regulatory factor-1 activity and causes tumor suppression.

The transcription factor interferon regulatory factor-1 (IRF-1) is induced by many tumor-suppressive stimuli and can mediate antiproliferative and proapoptotic effects in cancer cells. Thus, identifying agents that enhance IRF-1 activity may be an effective approach to cancer therapy. A cell-based screening assay was developed to identify extracts and compounds that could enhance IRF-1 activity, using an IRF-1-dependent luciferase reporter cell line. Through this approach, we identified a natural product extract and a known active component of this extract, baicalein, which causes a marked increase in IRF-1-dependent reporter gene expression and IRF-1 protein, with modulation of known IRF-1 targets PUMA and cyclin D1. Baicalein causes suppression of growth in vitro in multiple cancer cell lines in the low micromolar range. IRF-1 plays a role in this growth suppression as shown by significant resistance to growth suppression in a breast cancer cell line stably transfected with short hairpin RNA against IRF-1. Finally, intraperitoneal administration of baicalein by repeated injection causes inhibition of growth in both xenogeneic and syngeneic mouse models of cancer without toxicity to the animals. These findings indicate that identifying enhancers of IRF-1 activity may have utility in anticancer therapies and that cell-based screening for activation of transcription factors can be a useful approach for drug discovery.

[Construction of expression vector with BG2 gene and its transformation in wheat].

beta-1,3-glucanase(BG2) is one of the pathogensis-related-proteins(PR). Study of these proteins and their related genes is one of the hot points in plant genetic engineering of disease resistance for a long time. In this research, specific primers were designed with the enzyme cleavage site of Spe I in its forward one and Not I site in the backward according to the BG2 gene sequence. Using this pair of primers, BG2 gene, which was contained in the plasmid of pRTL2, was amplified and confirmed by sequencing the amplified fragment inserted into T-easy vector. The positive clone containing BG2 gene was digested with the enzymes of Spe I/Not I and then BG2 gene was inserted into the Xba I/Not I sites of super expression binary vector pATC940. The reconstructed expression vector named as pATCBG2 was introduced into the wheat of Longfumai10 and Longfumai3 (Triticum aestivum L. em. Thell) through the particle gun transformation method. The Kanamysin resistant (Km') transformants were obtained. PCR, Dot-blotting and PCR-Southern hybridization analysis showed that the BG2 gene was integrated into the genome of wheat. Result of pathogen inoculation assay on the transgenic plants showed that the transgenic plants had a higher resistant disease score of 1-2 grade than the control.

[Genetic mapping of resistant gene to southern corn rust and the tagging analysis on different genetic background].

Southern corn rust (SCR) is a destructive disease in maize. The inbred line Qi319 is highly resistant to southern corn rust. SSR technique was employed to preliminary mapping of the resistance gene. Bulked segregant analysis revealed that two primers, phi 118 and phi 041, amplified polymorphic bands. SSR analysis on populations indicated the two primers were linked to the rust resistance gene, which was mapped on the short arm of chromosome 10. In addition, comparative analysis of the amplification bands among different populations revealed that the amplification products with the same primer in different populations were dissimilar. This result indicates that the genetic background may affect results of gene mapping and tagging. So, it is important to select suitable population to performing molecular marker analysis and gene mapping.

[Advances on the plant disease resistant beta-glucanase gene].

Study of beta-1,3-glucanase is one of the hot points in plant genetic engineering of disease resistance,big progress has been made in the past few years. This paper briefly reviewed the main biological characters and mechanism involved in the disease resistance,the classification,structure,function and the transformation of beta-1,3-glucanase genes.