Anti-pancreatic cancer activity of cassane diterpenoids isolated from the seeds of Caesalpinia sappan mediated by autophagy activation via ROS/AMPK/mTORC1 pathway.

Three undescribed cassane diterpenoids, caesalpanins D-F (1-3), and seven known ones were isolated from the seeds of Caesalpinia sappan. Structures and absolute configurations of 1-3 were elucidated based on the extensive spectroscopic analysis, single-crystal X-ray diffraction analysis, and ECD calculations. Structurally, compound 1 was the first example of 18-norcassane diterpenoid and 2 was a rare 20-norcassane diterpenoid having an unusual five-membered oxygen bridge between C-10/C-18. The anti-proliferative activity of 1, 3, and 4-10 against PANC-1 cells (pancreatic ductal adenocarcinoma cell line) was evaluated, and phanginin H (4) was found to exhibit anti-cancer activity with IC50 value of 18.13 ± 0.63 µM. Compound 4 inhibited PANC-1 cell growth by arresting the cell cycle at G2/M phase via regulation of cyclin-dependent kinases, and the self-renewal and metastasis of PANC-1 cells by suppressing cancer cell stemness. Furthermore, compound 4 induced ROS generation and subsequently activated autophagy, which was demonstrated by the formation of autophagic vacuoles and dynamic change of autophagic flux. The induced ROS accumulation resulted in AMPK activation and subsequently regulation of mTORC1 activity and ULK phosphorylation, indicating that 4 triggered autophagy through ROS/AMPK/mTORC1 pathway. These findings suggested that 4 might potentially be an autophagy inducer for the therapy of pancreatic cancer.

Diterpenoids and sesquiterpenoids from the stem bark of Metasequoia glyptostroboides.

A phytochemical study on the stem bark of Metasequoia glyptostroboides led to the isolation of sixty-one diterpenoids and sesquiterpenoids, including seventeen previously undescribed compounds, metaglyptins A-Q. Their structures were elucidated by extensive analysis of spectroscopic data (IR, UV, HRESIMS, and 1H, 13C and 2D NMR). The absolute configurations of metaglyptins I, J, and O were determined by the ECD data and single-crystal X-ray diffraction analysis. The undescribed compounds were evaluated for their cytotoxicity against HeLa, AGS, and MDA-MB-231 cancer cell lines. The results revealed that metaglyptin A exhibited moderate cytotoxicity against MDA-MB-231 cell line with IC50 value of 20.02 µM.

Vibsane-Type Diterpenoids from Viburnum odoratissimum and Their Cytotoxic and HSP90 Inhibitory Activities.

Four new vibsane-type diterpenoids, vibsanol I (1), 15-hydroperoxyvibsanol A (2), 14-hydroperoxyvibsanol B (3), 15-O-methylvibsanin U (4), and a new natural product, 5,6-dihydrovibsanin B (5), as well as six known analogues, were isolated from the twigs and leaves of Viburnum odoratissimum. Their structures were elucidated by spectroscopic analyses and chemical derivatization method. All compounds showed different levels of cytotoxicity against five cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW480). Remarkably, 14,18-O-diacetyl-15-O-methylvibsanin U (4a) showed significant cytotoxicity against HL-60, A-549, SMMC-7721, MCF-7, and SW480, with IC50 values of 0.15 ± 0.01, 0.69 ± 0.01, 0.41 ± 0.02, 0.75 ± 0.03, and 0.48 ± 0.03 µm, respectively. In addition, vibsanin K (10) was identified as a HSP90 inhibitor with an IC50 value of 19.16 µm.

Neo-clerodane and abietane diterpenoids with neurotrophic activities from the aerial parts of Salvia leucantha Cav.

Four new neoclerodane diterpenoids, leucansalvialins FI (1-4), and one rare 18(4 → 3)-abeo-abietane diterpenoid, leucansalvialin J (5), were isolated from the aerial part of Salvia leucantha Cav., along with 14 known analogues. Leucansalvialin F (1) represents the first rearranged salvigenane type clerodane-17,12:18,6-diolide. Their structures were elucidated by 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of 1, 2, 3, and 5 were determinded by X-ray diffraction crystal analysis and the ECD technique. All of the isolated components were evaluated for their neurotrophic activities on PC12 cells and all new compounds were evaluated for their cytotoxicity against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW480). Compounds 2 and 5 showed moderate neuroprotective effects in an in vitro assay.

Two New Anti-Proliferative C18 -Norditerpenes from the Roots of Podocarpus macrophyllus.

Activity-guided fractionation strategy was used to investigate chemical constituents from the roots of Podocarpus macrophyllus. Successfully, two new norditerpenes, 2ß-hydroxymakilactone A (1) and 3ß-hydroxymakilactone A (2), along with ten known analogues (3 - 12) were isolated. The structures of 1 and 2 were elucidated by spectroscopic analysis including 1D-, 2D-NMR, and HR-ESI-MS data. The previously reported structure of 2,3-dihydro-2α-hydroxypodolide was revised as 2,3-dihydro-2ß-hydroxypodolide (3) by spectroscopic analysis, and was further confirmed by X-ray crystallographic analysis. Cytotoxic activities of all isolated compounds against five human solid tumour cell lines (AGS, HeLa, MDA-MB-231, HepG-2, and PANC-1) were evaluated. All of them exhibited anti-proliferative activities (IC50  = 0.3 - 27 µm), except for 10. Compounds 1, 4, 5, 6, and 8 exhibited potent inhibitory activities with IC50  < 1 µm against HeLa and AGS cells.

[Inhibition of vascular endothelial growth factor gene expression and proliferation of leukemia cells by RNA interference].

OBJECTIVE: To explore the feasibility of selective inhibiting VEGF expression using VEGF short hairpin RNA (shRNA) interference, and observe the effects of VEGF gene silencing on NB4 cells growth. METHODS: Three 19 bp reverse repeated motifs targeting exons 3, 4, 5 respectively of VEGF gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-VR1, pGenesil-VR2, pGenesil-VR3 and pGenesil-con (plasmid containing random DNA fragment) were transfected into NB4 cells respectively through lipofectamine reagent. The alteration of VEGF expression was examined by fluorescent real time RT-PCR and Western blot. The proliferation capacity of leukemia cells was measured by trypan blue exclusion, MTT assay, colony formation assay and cell cycles analysis. RESULTS: Recombinant plasmids containing three shRNAs and random fragment were successfully constructed and transfected into NB4 cells respectively by liposome-mediated gene transfer method. shRNA in pGenesil-VR3 cells knocked down the expression of VEGF mRNA and protein dramatically in a sequence-specific manner when compared with that of pGenesil-VR1, Genesil-VR2 and pGenesil-con. The NB4 cells transfected with pGenesil-VR3 (NB4-VR3) had a more significant decrease in proliferation ability than NB4 and that transfected with pGenesil-con (NB4-con). The colony forming efficiencies of NB4-VR3, NB4-con and NB4 cell were (13.3 +/- 3.8)%, (21.3 +/- 6.4)% and (24.5 +/- 5.2)%, respectively (P < 0.05). Higher G(1) and lower S proportion were found in cell cycle distribution in comparison with the control groups by FCM. CONCLUSIONS: The shRNA can efficiently suppress VEGF expression in NB4 cells. Selective VEGF gene silence can inhibit the malignant proliferation of leukemia cells.