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Pancreatic cancer is one of the most lethal of human malignancies. Nearly 100% cases of pancreatic cancer carry mutations in KRas. P-21-activated kinases (PAKs) are activated by and act downstream of KRas. Gla- ucarubinone, a natural product first isolated from the seeds of the tree Simarouba glauca, was originally developed as an antimalarial drug, and has more recently been recognised as an anticancer agent. The aims of this study were to determine whether glaucarubinone, alone or in combination with the front- line chemotherapeutic agent gemcitabine, would inhibit the growth of pancreatic cancer cells in vitro or in vivo and the mechanism involved. Growth of the human pancreatic cancer cell lines PANC-1 and MiaPaCa-2 was measured by 3 H-thymidine incorporation in vitro, and by volume as xenografts in SCID mice. The expression and activities of the two serine/threonine kinases PAK1 and PAK4, which are key regulators of cancer progression, were measured by Western blotting. Here we report that glaucarubi- none decreased proliferation and migration of pancreatic cancer cells in vitro, and reduced their growth as xenografts in vivo. Treatment with glaucarubinone and gemcitabine reduced proliferation in vitro and tumor growth in vivo more than treatment with either glaucarubinone or gemcitabine alone. Treatment with glaucarubinone reduced PAK1 and PAK4 activities, which were further decreased by the combina- tion of glaucarubinone and gemcitabine. These results indicate that glaucarubinone reduced pancreatic cancer cell growth at least in part via inhibition of pathways involving PAK1 and PAK4. The synergistic inhibition by glaucarubinone and gemcitabine observed both in vitro and in vivo suggests that glaucaru- binone may be a useful adjunct to current regimes of chemotherapy.

Four quassinoids, indaquassin C (1), samaderins C (2), B (3) and A (4), isolated from the seeds and bark of Samadera indica, were tested for insect antifeedant and growth regulatory activities against the tobacco cutworm, Spodoptera litura. Indaquassin C was the most effective antifeedant. Samaderin C increased pupal duration and induced pupal mortality.

Several O -acylated derivatives of bruceolide ( 2 ) were synthesized and their anti-malarial activities together with selective toxicities were examined. It was found that 3,15-di- O -acetyl- ( 3c ), 3,15-di- O -propionyl- ( 3d ) and 15- O -propionylbruceolide ( 3b ), as well as bruceine B ( 3a ), exhibited potent anti-malarial activities with high selective toxicities.

The C-15 senecioyl side chain of brusatol was interchanged with fluorinated acyl groups, and the C-3 hydroxy group of bruceolide was esterified with fluorinated acyl chlorides. These fluorinated quassinoids 11, 12, 13, and 17 showed significant cytotoxic activity against eight human cancer cell lines including small and non-small cell lung, colon, CNS, ovarian and renal cancers, leukemia, and melanoma with 17 being about 100 times more potent than 11, 12, and 13. The activity of 17 was similar to that of bruceantin (1) in this in vitro cell line panel.

The crude methanol extract of the stem wood of Quassia amara . L. (A-1) significantly caused a reduction in the weight of the testis, epididymis and seminal vesicle, but an increase in that of the anterior pituitary gland. Epididymal sperm counts, serum levels of testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were significantly reduced when the rats were treated with the extract. Furthermore the basal and LH-stimulated testosterone secretion of Leydig cells isolated from rats pretreated with the extract was inhibited. These changes seemed to be restored eight weeks after the withdrawal from extract treatments. Two compounds — quassin and 2-methoxycanthin-6-one were isolated after fractionation of A-1. Quassin produced similar biological actions as the crude extract while the effects of 2-methoxycanthin-6-one did not seem to differ from those of the control. Quassin appears to be the antifertility principle of Quassia amara .

Cedronin was isolated from Simaba cedron Planchon (Simaroubaceae), a species popularly believed in South America to have antimalarial properties. It was examined for in vitro and in vivo antimalarial activities and for cytotoxicity against KB cells. Experimental results showed that cedronin was active against chloroquine-sensitive and resistant strain, with an IC50 of 0.25 micrograms/ml (0.65 mumol/ml). It was also found to be active in vivo against Plasmodium vinkei with an IC50 of 1.8 mg/kg (4.7 nM/kg) in the classic 4-day test. Cedronin belongs to the small group of quassinoids with a C19 basic skeleton and shows a rather low cytotoxicity against KB cells (IC50 = 4 micrograms/ml, 10.4 microM) as compared with C20 biologically active quassinoids; however its toxic/therapeutic ratio (10/1.8) remains lower than chloroquine (10/0.5).

Girodazole (RP 49532A) or 3-amino-1-[4-(2 amino-1H-imidazolyl]-propanol, 2HCl is an experimental antitumor compound which inhibits protein synthesis in cell cultures and in cell free systems. The compound has been evaluated for its capacity to inhibit specific assays of initiation, elongation and termination of protein synthesis. Girodazole inhibited the release of nascent peptides from polyribosomes in rabbit reticulocyte lysates indicating that the major effect of the compound is on the protein synthesis termination step.

A series of brusatol, bisbrusatol, and bruceantin esters were examined for their ability to inhibit protein synthesis in P-388 lymphocytic leukemia cells. Compounds which produced high T/C % values (170-272) resulted in ID50 of 5.4-15.5 microM for inhibition of whole cell protein synthesis, ID50 of 1.3-13 microM for inhibition of endogenous protein synthesis in cell homogenates, and ID50 of 1.9-6 microM for inhibition of polyuridine directed polyphenylalanine synthesis using "runoff" ribosomes and a "pH 5" enzyme preparation. The polyuridine directed polyphenylalanine synthesis requires neither initiation nor termination factors, suggesting that quassinoids are exclusively elongation inhibitors. Bruceantin, brusatol, and bisbrusatolyl malonate allowed a runoff of the polyribosomes to 80S free ribosomes. However, formation of the ternary complex and 80S initiation complex were not inhibited by the quassinoids. Thus, these agents do not affect the individual steps leading to the formation of a stable 80S initiation complex in P-388 cells. Brusatol, bruceantin, and bisbrusatolyl malonate inhibited the formation of the first peptide bond between puromycin and [3H]methionyl-transfer RNA bound to the initiation complex, indicating peptidyl transferase activity is inhibited by the quassinoids in P-388 cells. These studies also suggest that the free 80S ribosome is the site of binding by the quassinoid. Ribosomes actively conducting protein synthesis will continue protein synthesis and terminate before the quassinoids bind. This proves quassinoids are elongation inhibitors of tumor cells. A strong correlation was observed between potent antileukemic activity and the ability to inhibit protein synthesis in P-388 lymphocytic leukemia cells.

The quassinoids bruceantin, brucein D, brucein E, bruceoside A, and brusatol significantly inhibited P-388 lymphocytic leukemic cell RNA and protein synthesis in tissue culture. However, DNA synthesis inhibition seemed to correlate more directly with the anti-neoplastic activity of these compounds in the in vivo P-338 survival system. In vitro, brusatol and bruceoside A marginally inhibited 10-day P-388 lymphocytic leukemia DNA polymerase, RNA polymerase, thymidylate synthetase, dihydrofolate reductase, phosphoribosyl pyrophosphate aminotransferase, and cathepsin protease activities. In vivo studies demonstrated similar inhibition and elevated cyclic AMP levels, correlating positively with the antineoplastic activity of individual compounds. Purine synthesis was inhibited drastically by brusatol in vivo, and one key inhibition site in purine synthesis was at phosphoribosyl pyrophosphate aminotransferase, the regulatory enzyme. Histone phosphorylation and ribonucleotide reductase activity also were inhibited marginally by brusatol.

A microtitration plate enzyme-linked immunosorbent assay has been developed for quassin and closely related seco-triterpenes. The method is much more sensitive than conventional chromatographic techniques and enables the routine analysis of large numbers of samples with a detection limit of 5 ng quassin. Antibody production was elicited by injecting a conjugate of iso-quassinic acid linked to bovine serum albumin into rabbits. Important features of the technique are (a) the use of a double-antibody, noncompetitive enzyme-linked immunosorbent assay for a low-molecular-weight, nonantigenic compound using microtitration plates; and (b) that the initial incubation of samples with primary antiserum can be carried out in buffer containing up to 20% ( v v ) methanol with minimal loss of sensitivity. The structural requirements for recognition of the hapten by the antiserum are considered based on the levels of cross-reaction found with a wide range of other quassinoids.

The effects of eleven quassinoids on Rous sarcoma virus induced cell transformation and on growth of normal cells were examined. At concentrations of 0.15-1 μg/ml they inhibited foci formation (76–99 %) without toxic effects on normal cells. The most active compounds also affected virus production by transformed cells. In intact normal and transformed cells, protein and DNA synthesis was equally affected after 3 hours of exposure to quassinoids of both cell types. RNA synthesis was not inhibited. This study has shown that the structural requirement of a C-15 ester in the quassinoids for antileukemic activity in vitro and in vivo is not essential for their antitransforming activity.

Bisbrusatolyl malonate, which was shown previously to be active against P-388 lymphocytic leukemia cell growth, was investigated for inhibitory effects on nucleic acid and protein synthesis. DNA and RNA synthesis as well as protein synthesis were markedly inhibited at 10,25, and 50 mu mole final concentrations in vitro. The major sites of inhibition of nucleic acid synthesis appeared to be DNA polymerase, messenger and transfer RNA polymerases, orotidine-5'-monophosphate decarboxylase, phosphoribosyl pyrophosphate amino transferase, and dihydrofolate reductase. Moderate inhibition of nucleotide kinase activities and oxidative phosphorylation processes occurred after drug treatment. Cyclic adenosine monophosphate levels were reduced. Protein synthesis was inhibited during the elongation step of peptide synthesis. The data suggested that bisbrusatolyl malonate interfered with the peptide bond formation. However, the ongoing polypeptide synthesis must be completed before the drug can bind to the ribosome effectively.

A series of new bisbrusatolyl and brusatolyl esters and related compounds were synthesized and tested for in vivo antileukemia activity against a quassinoid sensitive strain of P-388 lymphocytic leukemia in BDF1 mice. The bisbrusatolyl malonate, succinate, glutarate, adipate, and sebacate were as active or more active than brusatol. The C-3 esters of brusatol and bruceantin were also found to be as active or more active than brusatol or bruceantin in general. The free hydroxyl groups at C-11 and C-12 as well as the enone double bond in ring A of both bisbrusatolyl and brusatolyl esters are required for antileukemic activity. The presence of a double bond in the ester side chain contributes to the enhanced activity of these esters.

Based on the fact that some known antineoplastic agents possess an ester moiety within their structure, the esters of helenalin, a sesquiterpene lactone, and of brusatol and bruceantin, quassinoids, were synthesized and tested for antileukemic activity in the P-388 screen. These agents gave different T/C % values dependent on the P-388 lymphocytic leukemia strain and the host strain of mice used. Later studies demonstrated that the agents caused different degrees of inhibition of nucleic acid and protein synthesis in the various P-388 strains. The higher the degree of inhibition of precursor incorporation into the nucleic acid or protein, the higher was the T/C % value obtained in a given P-388 strain. The study demonstrates the lack of consistency of P-388 lymphocytic leukemia cell lines used in various laboratories and indicates that the inbred strain of mice is a critical factor in the tolerance of drug toxicity and, thus, T/C % obtained.