Pancreatic cancer is a malignant neoplasm of the pancreas. A mutation and constitutive activation of K-ras occurs in more than 90% of pancreatic adenocarcinomas. A successful approach for the treatment of pancreatic cancers is urgent. Antroquinonol, a ubiquinone derivative isolated from a camphor tree mushroom, Antrodia camphorata, induced a concentration-dependent inhibition of cell proliferation in pancreatic cancer PANC-1 and AsPC-1 cells. Flow cytometric analysis of DNA content by propidium iodide staining showed that antroquinonol induced G1 arrest of the cell cycle and a subsequent apoptosis. Antroquinonol inhibited Akt phosphorylation at Ser473, the phosphorylation site critical for Akt kinase activity, and blocked the mammalian target of rapamycin (mTOR) phosphorylation at Ser2448, a site dependent on mTOR activity. Several signals responsible for mTOR/p70S6K/4E-BP1 signaling cascades have also been examined to validate the pathway. Moreover, antroquinonol induced the down-regulation of several cell cycle regulators and mitochondrial antiapoptotic proteins. In contrast, the expressions of K-ras and its phosphorylation were significantly increased. The coimmunoprecipitation assay showed that the association of K-ras and Bcl-xL was dramatically augmented, which was indicative of apoptotic cell death. Antroquinonol also induced the cross talk between apoptosis, autophagic cell death and accelerated senescence, which was, at least partly, explained by the up-regulation of p21Waf1/Cip1 and K-ras. In summary, the data suggest that antroquinonol induces anticancer activity in human pancreatic cancers through an inhibitory effect on PI3-kinase/Akt/mTOR pathways that in turn down-regulates cell cycle regulators. The translational inhibition causes G1 arrest of the cell cycle and an ultimate mitochondria-dependent apoptosis. Moreover, autophagic cell death and accelerated senescence also explain antroquinonol-mediated anticancer effect.
During senescence, cells express molecules called senescence-associated secretory phenotype (SASP), including growth factors, proinflammatory cytokines, chemokines, and proteases. The SASP induces a chronic low-grade inflammation adjacent to cells and tissues, leading to degenerative diseases. The anti- inflammatory activity of flavonoids was investigated on SASP expression in senescent fibroblasts. Effects of flavonoids on SASP expression such as IL-1a, IL-1b, IL-6, IL-8, GM-CSF, CXCL1, MCP-2 and MMP-3 and signaling molecules were examined in bleomycin-induced senescent BJ cells. In vivo activity of apigenin on SASP suppression was identified in the kidney of aged rats. Among the five naturally-occurring flavonoids initially tested, apigenin and kaempferol strongly inhibited the expression of SASP. These flavonoids inhibited NF-kB p65 activity via the IRAK1/IkBa signaling pathway and expression of IkBz. Blocking IkBz expression especially reduced the expression of SASP. A structure-activity relationship study using some synthetic flavones demonstrated that hydroxyl substitutions at C-20,30,40,5 and 7 were important in inhibiting SASP production. Finally, these results were verified by results showing that the oral administration of apigenin significantly reduced elevated levels of SASP and IkBz mRNA in the kidneys of aged rats. This study is the first to show that certain flavonoids are inhibitors of SASP production, partially related to NF-kB p65 and IkBz signaling pathway, and may effectively protect or alleviate chronic low-grade inflammation in degenerative diseases such as cardiovascular diseases and late-stage cancer. Inhibitory activity of apigenin on IL-6, IL-8, and IL-1b was the most potent among the five flavonoids that were tested (86.5%, 60.9%, and 94.9% at 10 mM, respectively).
Natural plant flavonoid apigenin directly disrupts Hsp90/Cdc37 complex and inhibits pancreatic cancer cell growth and migration •Apigenin can be digested, released and absorbed into blood circulation to accumulate. •Apigenin directly inhibited Hsp90/Cdc37 interaction with structural specificity. •The effect of apigenin on Hsp90/Cdc37 did not rely on CK2 activity. •Apigenin induced downstream kinase client protein degradation. •Apigenin induced ROS accumulation, inhibited cell proliferation and migration.
Apigenin is a common dietary plant flavonoid widely distributed in vegetables and fruits. It exhibits chemopreventive activity against various cancer cells. In this study, we demonstrated that apigenin directly blocked heat shock protein 90 (Hsp90)and cell division cycle protein 37 (Cdc37) interaction using split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) assay. Apigenin inhibited complemented Renilla luciferase (RL) activity of NRL-Hsp90/Cdc37-CRL, while its analogues did not. Apigenin also inhibited NRL-Hsp90 and Cdc37(Ser13Ala)-CRL complementation. In addition, casein kinase II (CK2) specific inhibitor 4, 5, 6, 7-tetrabromobenzotriazole (TBB) did not affect NRL-Hsp90/Cdc37-CRL complementation, indicating that the inhibitory effect of apigenin on Hsp90/Cdc37 did not rely on CK2 activity. Moreover, apigenin blocked Hsp90/Cdc37 complex and induced kinase clients protein kinase B (Akt), cyclin-dependent-kinase 4 (CDK4) and matrix metalloproteinase-9 (MMP-9) degradation and, as a consequence, induced intracellular reactive oxygen species (ROS) accumulation and inhibited cell proliferation and migration in pancreatic cancer cells.
Apigenin has been shown to induce apoptosis in different types of cells [46, 70, 84, 85]. In human keratinocytes and organotypic keratinocyte cultures, apigenin treatment enhanced UVB-induced apoptosis more than 2-fold. In addition, apigenin stimulated changes in Bax localization, and increased the release of cytochrome c from the mitochondria. Overexpression of the antiapoptotic protein Bcl-2 and expression of a dominant-negative form of Fas-associated death domain led to a reduction in apigenin-induced apoptosis, demonstrating that enhancement of UVB-induced apoptosis by apigenin treatment involves both the intrinsic and extrinsic apoptotic pathways . In human prostate cancer cells, apigenin treatment has been shown to alter the Bax/Bcl-2 ratio in favor of apoptosis . In human promyelocytic leukemia HL-60 cells, apigenin induced caspase-3 activity and cleavage of poly-(ADP-ribose) polymerase (PARP), reduced mitochondrial transmembrane potential, released mitochondrial cytochrome c into the cytosol, and subsequently induced procaspase-9 processing . Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that kills various tumor cells without damaging normal tissues. However, many cancers remain resistant to TRAIL. Apigenin breaks TRAIL resistance by transcriptional down-regulation of c-FLIP, a key inhibitor of death receptor signaling, and by up-regulation of TRAIL receptor 2 .
Exposure of a wide array of malignant cells, including epidermal cells and fibroblasts to apigenin induces a reversible G2/M and G0/G1 arrest by inhibiting p34 (cdc2) kinase activity, accompanied by increased p53 protein stability (51, 52). Apigenin has also been shown to induce WAF1/p21 levels resulting in cell cycle arrest and apoptosis in androgen-responsive human prostate cancer, LNCaP cells and androgen-refractory DU145 cells, regardless of the Rb status and p53-dependence or p53 independence (53, 54). In addition, apigenin has been shown to induce apoptosis in a wide range of malignant cells (55–57). Apigenin treatment has been shown to alter the Bax/Bcl-2 ratio in favor of apoptosis, associated with release of cytochrome c and induction of Apaf-1, which leads to caspase activation and PARP-cleavage (54).
Apigenin-mediated cell growth inhibition along with G2/M arrest was accompanied by significant decrease in cyclin B1 and CDK1 protein levels, resulting in a marked inhibition of CDK1 kinase activity. Furthermore, apigenin treatment reduced the protein levels of CDK4, cyclin D1 and A, inhibited Rb-phosphorylation but did not affect the protein levels of cyclin E, CDK2 or CDK6. Recently, studies have shown that apigenin induces G (2)/M phase cell cycle arrest in SK-BR-3 cells which is via regulation of CDK1 and p21 (Cip1) pathway. In addition, apigenin treatment resulted in ERK MAP kinase phosphorylation and activation in MDA-MB-468 cells (86).
Effect of Luteolin and Apigenin on the Cell Senescence and Telomerase Activity of DPCs at Various Passages. Senescenceassociated b-galactosidase is caused by upregulated lysosomal activities and altered cytosolic pH, which are upregulated with senescence and aging. To elucidate the effect of luteolin and apigenin on replicative senescence state of DPCs, the senescence-associated b-galactosidase activity (SA-b-gal) was evaluated. DPCs from passages 1, 3, 5, and 7 with/without luteolin/apigenin treatment were detected, albeit only the representative results of passages 3 and 7 were presented. The result revealed that DPCs at passage 3 with luteolin/apigenin induction and the control group did not show any obvious blue staining. DPCs at passage 7 without induction showed intense blue color, albeit DPCs at passage 7 with luteolin or apigenin induction revealed weak blue staining, not as intense as the control group at passage 7. Similarly, there is no difference of the telomerase activity of DPCs at passage 3 with/without luteolin or apigenin induction albeit DPCs at passage 7 with luteolin or apigenin induction showed significantly higher telomerase activity than the control group at passage 7 (∗ 𝑃 < 0.05), which agreed with the result of b-galactosidase assay mentioned above. This result implied that luteolin and apigenin treatment significantly inhibited cell senescence and increased telomerase activity of DPCs, especially at late passages. Thus, luteolin and apigenin might be able to maintain DPCs in an undifferentiated and presenescent state.
Apigenin, a common dietary flavonoid, has been shown to induce cell growth-inhibition and cell cycle arrest in many cancer cell lines. One important effect of apigenin is to increase the stability of the tumor suppressor p53 in normal cells. Therefore, apigenin is expected to play a large role in cancer prevention by modifying the effects of p53 protein. However, the mechanisms of apigenin’s effects on p53-mutant cancer cells have not been revealed yet. We assessed the influence of apigenin on cell growth and the cell cycle in p53-mutant cell lines. Treatment with apigenin resulted in growth-inhibition and G2/M phase arrest in two p53-mutant cancer cell lines, HT-29 and MG63. These effects were associated with a marked increase in the protein expression of p21/WAF1. We have shown that p21/WAF1 mRNA expression was also markedly increased by treatment with apigenin in a dose- and time-dependent manner. However, we could not detect p21/WAF1 promoter activity following treatment with apigenin. Similarly, promoter activity from pG13-Luc, a p53-responsive promoter plasmid, was not activated by treatment with apigenin with or without p53 protein expression. These results suggest that there is a p53-independent pathway for apigenin in p