New abietane and tigliane diterpenoids from the roots of Euphorbia fischeriana and their cytotoxic activities.

Three new abietane and two new tigliane diterpenoids were isolated from the roots Euphorbia fischeriana. Their structures were elucidated by spectroscopic methods and quantum chemical calculation. Compounds 4 and 5 exhibited the inhibitory activities against human cancer cells HeLa and HepG2, with IC50 ranging from 3.54 to 11.45 µM.

Crotonianoids A-C, Three Unusual Tigliane Diterpenoids from the Seeds of Croton tiglium and Their Anti-Prostate Cancer Activity.

Crotonianoids A-C (1-3), three unusual tigliane diterpenoids, were isolated from the seeds of Croton tiglium. Compound 1 is a 13,14:13,15-diseco-tigliane featuring a unique spiro[bicyclo[5.3.0]decane-2,5'-2'(3'H,4'H)-furanone] core; 2 is a 13,15-seco-tigliane incorporating a rare peroxide bridge between C-13 and C-15; and 3 is the first example of a phorbol ester with a 10R-configuration. Their structures were determined by spectroscopic, computational, and X-ray diffraction methods. Compounds 1 and 2 markedly inhibited the growth and survival of prostate cancer cell C4-2B at micromolar concentrations and induced cell apoptosis. Mechanistic study revealed that 1 and 2 could suppress androgen receptor (AR) signaling pathway by promoting the degradation of AR protein.

Development of a novel gene silencer pyrrole-imidazole polyamide targeting human connective tissue growth factor.

Pyrrole-imidazole (PI) polyamide can bind to specific sequences in the minor groove of double-helical DNA and inhibit transcription of the genes. We designed and synthesized a PI polyamide to target the human connective tissue growth factor (hCTGF) promoter region adjacent to the Smads binding site. Among coupling activators that yield PI polyamides, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU) was most effective in total yields of PI polyamides. A gel shift assay showed that a PI polyamide designed specifically for hCTGF (PI polyamide to hCTGF) bound the appropriate double-stranded oligonucleotide. A fluorescein isothiocyanate (FITC)-conjugated PI polyamide to CTGF permeated cell membranes and accumulated in the nuclei of cultured human mesangial cells (HMCs) and remained there for 48 h. The PI polyamide to hCTGF significantly decreased phorbol 12-myristate acetate (PMA)- or transforming growth factor-ß1 (TGF-ß1)-stimulated luciferase activity of the hCTGF promoter in cultured HMCs. The PI polyamide to hCTGF significantly decreased PMA- or TGF-ß1-stimulated expression of hCTGF mRNA in a dose-dependent manner. The PI polyamide to hCTGF significantly decreased PMA- or TGF-ß1-stimulated levels of hCTGF protein in HMCs. These results indicate that the developed synthetic PI polyamide to hCTGF could be a novel gene silencer for fibrotic diseases.

Structural investigation of protein kinase C inhibitors.

The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

[Tissue polypeptide antigen in gynecological cancer].

Serum tissue polypeptide antigen (TPA), CEA and IAP were measured simultaneously in cervical cancer, corpus cancer and ovarian cancer. TPA levels were increased (more than 110 U/1) in 39% (32/82) of the cervical cancer and in 46% (6/12) of the corpus cancer patients, respectively. In ovarian cancer, TPA levels were elevated (146 U/1 or higher) in 64% (13/22). The positive rate of CEA was somewhat lower than that of TPA and LAP levels were as high as TPA. Moreover, TPA levels were correlated in clinical course. In immunohistochemical examination, TPA was present in cancer cells and absent in normal tissue.

Radioimmunoassay for phorbol esters using rabbit antisera against phorbol succinate.

The phorbol nucleus was succinylated and then conjugated to bovine albumin using dicyclohexylcarbodiimide. Rabbits given injections of the conjugate developed antibodies which rose in titer progressively with repeated immunization. By the ninth bleeding, the binding of one antiserum, diluted 1:15,000, was saturated with about 10 nM [3H]phorbol-12,13-dibutyrate [( 3H]-PDBU) and had an average association constant, Ka, of 2.6 X 10(8) M-1. The serological specificity of the antisera was characterized by examining the inhibition of the [3H]PDBU-anti-phorbol succinate immune system by 18 phorbol-related compounds. The specificities of antibodies from two rabbits tested in detail were qualitatively similar. The rank order of inhibitory activity for certain phorbol-related compounds was PDBU [concentration of inhibitor required to give 50% inhibition of PDBU binding (IC50) = 7.6 nM] = phorbol-13-acetate [IC50 = 8.2 nM] greater than phorbol-12,13-dibenzoate greater than 4-beta-phorbol [IC50 = 124 nM] greater than or equal to phorbol-12,13-diacetate greater than or equal to phorbol-12-myristate-13-acetate [IC50 = 184 nM] greater than phorbol-13,20-diacetate greater than phorbol-12-acetate [IC50 = 2300 nM]. The following compounds showed no detectable serological activity: mezerein, 4-0-methylphorbol-12-myristate-13-acetate, ingenol, 4-alpha-phorbol, teleocidin B, and dihydroteleocidin B. These and other results indicated that the 4-beta-phorbol nucleus was required for serological activity, that esterification of the C-13 position with benzoate, acetate, or butyrate enhanced the immunoreactivity of 4-beta-phorbol, and that among the phorbol-related compounds examined there was no direct relationship between serological activity and biological potency as tumor promoters. Using the [3H]PDBU-anti-phorbol succinate immune system, we measured the concentrations of immunoreactive phorbol-related material in crude mixtures such as croton oil and performed pharmacokinetic studies in rats given PDBU s.c.

Direct evidence of incorporation of 12-O-[20-2H1]tetradecanoylphorbol-13-acetate into artificial membranes as determined by deuterium magnetic resonance.

In order to elucidate the manner of interaction of 12-O-tetradecanoylphorbol-13-acetate (TPA), 2H-NMR spectra of [20-2H1]TPA and 31P-NMR spectra were recorded in the presence of multibilayers of dimyristoylphosphatidylcholine (DMPC). Observation of several pairs of quadrupole splittings directly proves the TPA molecules are intercalated into the multibilayers. Further, reduction of the 31P chemical shift anisotropy of DMPC multibilayers by TPA is more pronounced than that of phorbol 12,13-diacetate (PDA) whose activity of tumor promotion is very weak.