Neuroprotective and anticancer effects of 7­Methoxyheptaphylline via the TAK1 pathway.

7­Methoxyheptaphylline (7­MH) is a carbazole extracted from Clausena harmandiana, a medicinal plant that is used to treat headaches and stomachaches. The aim of the present study was to examine the neuroprotective effects and anticancer activity of 7­MH. Cell death was assessed using an MTT assay and flow cytometry. The expression of apoptosis­related proteins was determined by western blot analysis. An animal model was used to test anti­metastasis. The interactions between 7­MH and the molecular target were observed using molecular docking. The results revealed that 7­MH provided protection against hydrogen peroxide (H2O2)­induced neuronal cell death. In cancer cells, 7­MH induced SH­SY5Y, 4T1, HT29, HepG2, and LNCaP cell death. 7­MH inhibited metastasis of HT29 cells in vitro and 4T1­Luc cells in vitro and in vivo. 7­MH inhibited proteins, including P­glycogen synthase kinase (GSK)­3, and cleaved caspase­3, but it activated anti­apoptotic proteins in H2O2­induced SH­SY5Y cell death. By contrast, 7­MH activated the cleaving of caspase­3 and GSK­3, but it suppressed anti­apoptotic proteins in SH­SY5Y cells. 7­MH reduced the levels of NF­κB and STAT3 in 4T1 cells; phospho­p65, Erk, and MAPK13 in LNCaP cells; and phospho­Erk and matrix metalloproteinase­9 in HT29 cells. Molecular docking analysis showed that 7­MH targets TAK1 kinase. The present study indicated that 7­MH induced apoptosis of cancer cells and provided protection against H2O2­induced neuron cell death via TAK1 kinase.

Toxicity Profiles of Kleeb Bua Daeng Formula, a Traditional Thai Medicine, and Its Protective Effects on Memory Impairment in Animals.

Kleeb Bua Daeng (KBD) formula has long been used in Thailand as a traditional herbal medicine for promoting brain health. Our recent reports illustrated that KBD demonstrates multiple modes of action against several targets in the pathological cascade of Alzheimer's disease (AD). The main purpose of the present study was to determine the protective effect and mechanism of KBD in amyloid beta (Aß)-induced AD rats and its toxicity profiles. Pretreatment with the KBD formula for 14 days significantly improved the short- and long-term memory performance of Aß-induced AD rats as assessed by the Morris Water Maze (MWM) and object-recognition tests. KBD treatment increased the activities of the antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase; reduced the malondialdehyde content, and; decreased the acetylcholinesterase activity in the rat brain. An acute toxicity test revealed that the maximum dose of 2000 mg/kg did not cause any mortality or symptoms of toxicity. An oral, subchronic toxicity assessment of KBD at doses of 125, 250, and 500 mg/kg body weight/day for 90 days showed no adverse effects on behavior, mortality, hematology, or serum biochemistry. Our investigations indicate that KBD is a nontoxic traditional medicine with good potential for the prevention and treatment of AD.

Multitarget Activities of Kleeb Bua Daeng, a Thai Traditional Herbal Formula, Against Alzheimer's Disease.

The Kleeb Bua Daeng formula (KBD) is a Thai traditional medicine for brain health promotion. On the basis of the activities of its individual components, the KBD could have good potential for the treatment of Alzheimer's disease (AD). Herein, we investigated the KBD as an AD treatment. The ethanol extracts of KBD and its components, i.e., Nelumbo nucifera (NN), Piper nigrum fruits (BP), and the aerial part of Centella asiatica (CA) exhibited antioxidant activity, as determined by both ABTS and DPPH assays. The Ellman's assay revealed that the KBD, NN, and BP showed an ability to inhibit acetylcholinesterase. The thioflavin T assay indicated that the KBD, NN, BP, and CA inhibited beta-amyloid aggregation. The neuroprotection and Western blot analysis revealed that the KBD reduced H2O2-induced neuronal cell death by inhibiting the expression of pro-apoptotic factors, i.e., cleaved caspase-9 and -3, p-P65, p-JNK, and p-GSK-3ß, as well as by inducing expression of anti-apoptotic factors, i.e., MCl1, BClxl, and survivin. Furthermore, the KBD could improve scopolamine induced memory deficit in mice. Our results illustrate that the KBD with multimode action has the potential to be employed in AD treatment. Thus, the KBD could be used as an alternative novel choice for the prevention and treatment of patients with AD.

Combination of Nimbolide and TNF-α-Increases Human Colon Adenocarcinoma Cell Death through JNK-mediated DR5 Up- regulation.

Tumor necrosis factor (TNF-α), an inflammatory cytokine that plays an important role in the control of cell proliferation, differentiation, and apoptosis, has previously been used in anti-cancer therapy. However, the therapeutic applications of TNF-α are largely limited due to its general toxicity and anti-apoptotic influence. To overcome this problem, the present study focused on the effect of active constituents isolated from a medicinal plant on TNF-α-induced apoptosis in human colon adenocarcinoma (HT-29) cells. Nimbolide from Azadirachta indica was evaluated for cytotoxicity by methyl tetrazolium 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and phase contrast microscopy. Effects on apoptotic signaling proteins were investigated using Western blot analysis. Nimbolide showed cytotoxicity against HT-29 cells that was significantly different from the control group (<0.01), a concentration of 10 µM significantly inducing cell death (<0.01). In combination with TNF-α, nimbolide significantly enhanced-induced cell death. In apoptotic pathway, nimbolide activated c-Jun N-terminal kinase (JNK) phosphorylation, BH3 interacting-domain death agonist (Bid) and up-regulated the death receptor 5 (DR5) level. In the combination group, nimbolide markedly sensitized TNF-α-induced JNK, Bid, caspase-3 activation and the up-regulation of DR5. Our findings overall indicate that nimbolide may enhance TNF-α-mediated cellular proliferation inhibition through increasing cell apoptosis of HT-29 cells by up-reglation of DR5 expression via the JNK pathway.

Heptaphylline induces apoptosis in human colon adenocarcinoma cells through bid and Akt/NF-κB (p65) pathways.

Heptaphylline derivatives are carbazoles in Clausena harmandiana, a medicinal plant that is utilized for headache, stomach ache, and other treatments of illness. The present study examined the effects of heptaphylline and 7-methoxyheptaphylline on apoptosis of human colon adenocarcinoma cells (HT-29 cell line). Quantification of cell viability was performed using cell proliferation assay (MTT assay) and of protein expression through immunoblotting. The results showed that only heptaphylline, but not 7-methoxyheptaphylline, significantly significantly activated cleaved of caspase-3 and poly (ADP-ribose) polymerase (PARP-1) which resulted in HT-29 cell death. We found that heptaphylline activated BH3 interacting-domain death agonist (Bid) and Bak, proapoptotic proteins. In contrast, it suppressed X-linked inhibitor-of-apoptosis protein (XIAP), Bcl-xL and survivin, inhibitors of apoptosis. In addition, heptaphylline inhibited activation of NF-κB/p65 (rel), a regulator of apoptotic regulating proteins by suppressing the activation of Akt and IKKα, upstream regulators of p65. The findings suggested that heptaphylline induces apoptosis in human colon adenocarcinoma cells .

Gomisin A enhances tumor necrosis factor-α-induced G1 cell cycle arrest via signal transducer and activator of transcription 1-mediated phosphorylation of retinoblastoma protein.

Gomisin A, a dibenzocyclooctadiene lignan isolated from the fruit of Schisandra chinensis, has been reported as an anti-cancer substance. In this study, we investigated the effects of gomisin A on cancer cell proliferation and cell cycle arrest in HeLa cells. Gomisin A significantly inhibited cell proliferation in a dose-dependent manner after 72 h treatment, especially in the presence of tumor necrosis factor-α (TNF-α), due to cell cycle arrest in the G1 phase with the downregulation of cyclin D1 expression and Retinoblastoma (RB) phosphorylation. In addition, gomisin A in combination with TNF-α strongly suppressed the expression of signal transducer and activator of transcription 1 (STAT1). Inhibition of STAT1 pathways by a small-interfering RNA against STAT1 and AG490 Janus kinase (JAK) kinase inhibitor AG490 reduced the cyclin D1 expression and RB phosphorylation, indicating that JAK-mediated STAT1 activation is involved in gomisin A-induced G1 cell cycle arrest.

Gomisin N enhances TNF-α-induced apoptosis via inhibition of the NF-κB and EGFR survival pathways.

Tumor necrosis factor (TNF-α) is a pleiotropic cytokine that plays an important role in the control of cell proliferation, differentiation, and apoptosis. TNF-α-induced apoptosis is limited by TAK1-mediated activation of NF-κB (mainly p65-p50 hetrodimer) signaling pathway. We have recently reported that TAK1 regulates phosphorylation of EGFR at Ser-1046/7 through p38 MAPK, which cooperates with NF-κB in TNF-α-induced apoptosis. The present study investigated the effect of gomisins A and N, dibenzocyclooctadiene lignans isolated from the fruit of Schisandra chinensis, on TNF-α-induced apoptosis in HeLa cells. Gomisins A and N strongly promoted TNF-α-induced cleavage of caspase-3 and PARP-1, which are key markers of apoptosis. We found that gomisin N, but not gomisin A, inhibited the TNF-α-induced activation of NF-κB by suppressing the activation of IKKα. Gomisin N also inhibited p38-mediated phosphorylation of the EGFR at Ser-1046/7 and subsequent endocytosis of EGFR, another prosurvival pathway. The findings suggested that gomisin N enhanced TNF-α-induced apoptosis by suppressing of NF-κB and EGFR signaling pathways.