Stereo-specific cytotoxic effects of gossypol enantiomers and gossypolone in tumour cell lines.

The naturally occurring compound, gossypol, has been previously used as a male oral contraceptive, for the treatment of benign gynaecological conditions and cancer patients. Long-term daily dosing with gossypol is associated with minimal side effects and no myelosuppression. Since gossypol exhibits atropisomerism due to the restricted rotation about the 2,2' carbon bond, we have isolated the l- and d-isomers by Schiff's base formation using a chiral amine and regenerated the enantiomers by acid hydrolysis. The enantiomers and the proposed oxidative metabolite, gossypolone, were characterized by HPLC, 1H-NMR and optical rotation. The cytotoxicity was assessed in cell cultures derived from melanoma, lung, breast, cervix, and leukaemia using the MTT viability assay. The cytotoxicity of gossypolone was similar to racemic gossypol in five out of the six cell lines studied. The l-enantiomer of gossypol induced a dose-dependent cell kill in all cell lines with a mean IC50 of 20 microM and was significantly more potent than racemic gossypol, the d-enantiomer of gossypol and gossypolone. In addition, when the leukaemia line was exposed to l-gossypol (0.5-10 microM) over a 4-day period, a schedule-dependent decrease in cell viability was observed. l-Gossypol was also compared with respective drugs used to treat patients with melanoma, lung cancer and leukaemia. The data indicate that l-gossypol was significantly more active than cisplatin, melphalan and dacarbazine in the two melanoma lines, cisplatin and daunorubicin in the lung line and hydroxyurea and busulphan in the leukaemia line. Preliminary studies using one melanoma line showed that the l-isomer induced cell shrinkage, membrane blebbing and DNA fragmentation, characteristics suggestive of apoptotic cell death.

The pentacyclic triterpenoids in herbal medicines and their pharmacological activities in diabetes and diabetic complications.

Pentacyclic triterpenoids including the oleanane, ursane and lupane groups are widely distributed in many medicinal plants, such as Glycyrrhiza species, Gymnema species, Centella asiatica, Camellia sinensis, Crataegus species and Olea europaea, which are commonly used in traditional medicine for the treatment of diabetes and diabetic complications. A large number of bioactive pentacyclic triterpenoids, such as oleanolic acid, glycyrrhizin, glycyrrhetinic acid, ursolic acid, betulin, betulinic acid and lupeol have shown multiple biological activities with apparent effects on glucose absorption, glucose uptake, insulin secretion, diabetic vascular dysfunction, retinopathy and nephropathy. The versatility of the pentacyclic triterpenes provides a promising approach for diabetes management.

Kavalactone pharmacophores for major cellular drug targets.

A number of studies have identified differential kavalactone activity against a variety of molecular targets, including P-glycoprotein (Pgp), platelet monoamine oxidase (MAO-B), transcription factor binding domains, pregnane X (PXR) and GABA receptors, and cytochrome P450 and cyclo-oxygenase (COX) enzymes. The molecular structure of the kavalactones possesses a pharmacophore for several of these targets. In most cases, conformational stability is more significant than the substituents present. The analysis of these pharmacophores provides important insights for future medicinal chemistry-based approaches to kavalactone-type drugs.

Alterations in serum lipolytic activity of cancer patients with response to therapy.

The effect of chemotherapy on the serum lipid mobilising activity of a group of cancer patients with or without weight loss has been determined. The pre-treatment level of serum lipolytic activity in all cancer patients, with or without weight loss, was higher than normal controls (0.22 +/- 0.01 versus 0.06 +/- 0.01 mumols glycerol released ml-1 serum respectively). The pre-treatment levels of lipid mobilising activity in the patients serum was proportional to the extent of weight loss (correlation coefficient 0.81), if the extent of weight loss was small (less than 14 kg). Patients who showed a positive response to chemotherapy also showed a decrease in their plasma levels of lipolytic activity, while a patient who showed no response to therapy also showed no change in the serum lipolytic activity. There was no correlation between the serum lipolytic activity and response to megestrol acetate, a synthetic orally active progestogen, which is currently under investigation as an anticachectic agent. Serum from cancer patients showed lipolytic activity which was retained on a DEAE cellulose column and eluted by a salt gradient, in contrast with normal controls. Response to chemotherapy was associated with a decrease of the retained material, although the profile did not return to the normal state. These results need confirmation in a larger group of patients using more specific methods to determine tumour lipolytic activity, but suggest that it may be possible to monitor response to therapy by measurement of the serum lipolytic activity.

Structure-activity studies on gossypol in tumor cell lines.

Gossypol [(2,2'-binaphthalene)-8,8'-dicarboxaldehyde-1,1',6,6',7,7'-hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl] 1a is a naturally occurring compound extracted from the cotton plant and has been extensively studied as an oral male contraceptive. Its favorable toxicity profile, and the more recent demonstration of anti-tumor activity in animals and humans, prompted us to investigate the role of the aldehyde groups in a structure-activity study in cultured tumor cells. Four racemic compounds were evaluated: gossypol 1a, gossypolone 2, the bis Schiff's base of L-phenylalanine methyl ester with gossypol (bis Schiff's base) 1c and apogossypol 1b. The former two compounds both retain the aldehyde functional groups at positions 8 and 8' of the molecule whilst in the latter two compounds the aldehydes are blocked or absent, respectively. In addition, the l- and d-isomers of gossypol 1a, the bis Schiff's base 1c and the half Schiff's base 1d (one aldehyde blocked) were tested. The cell lines studied included melanoma (SK-mel-19), cervix (Sihas), small cell lung (H69) and myelogenous leukemia (K562). Cytotoxicity was measured using the MTT and flow cytometric viability assays. Racemic gossypol 1a and gossypolone 2 induced similar dose-dependent decreases in cell viability in all the cell lines with IC50 values of 23-46 and 28-50 microM, respectively. In contrast, the racemic bis Schiff's base derivative of gossypol 1c and apogossypol 1b showed minimal activity in any cell line up to 50 microM. The l-enantiomer of gossypol 1a was significantly more active than the d-enantiomer (IC50 of 20 versus > 50 microM, respectively). When one aldehyde of either enantiomer was blocked 1d cytoxicity was comparable to the l-enantiomer of gossypol. The data suggest that only one aldehyde group is required for the cytotoxicity of gossypol 1a, irrespective of the stereoconfiguration.

Molecular modelling and biological evaluation of a series of hydroxylated benzylideneanilines and benzylamines designed as tyrosine kinase inhibitors.

A series of hydroxylated benzylideneanilines and benzylamines were prepared and tested for inhibition of epidermal growth factor receptor-associated protein tyrosine kinase (PTK) activity in vitro. Molecular modelling and analysis of the biological results lead us to propose a minimal structural pharmacophore for two distinct binding sites within the PTK domain.

Alteration of serum and urinary lipolytic activity with weight loss in cachectic cancer patients.

The possibility that weight loss in cancer patients may be augmented by tumour produced catabolic factors, which stimulate lipid mobilisation, was investigated in a group of cancer patients with total body weight loss ranging from 0 to 50%. The serum and urine lipolytic activity has been determined using freshly isolated murine adipocytes in an in vitro assay. As a control group, we have used patients with Alzheimer's disease, in which some patients may lose a considerable amount of weight, without an obvious cause. The serum lipolytic activity for the Alzheimer's group with weight loss (0.11 +/- 0.02 mumols glycerol released 10(5) adipocytes-1 ml-1 serum) was not significantly different from the group without weight loss (0.11 +/- 0.02 mumols glycerol released 10(5) adipocytes-1 ml-1) or from a healthy control group (0.07 +/- 0.02 mumols glycerol released 10(5) adipocytes-1 ml-1), but all three groups were significantly (P less than 0.005) lower than the cancer patient group (0.20 +/- 0.03 mumols glycerol 10(5) adipocytes-1 ml-1), irrespective of weight loss. A similar difference between the cancer and the control group was observed for the urinary lipolytic activity (0.67 +/- 0.03 versus 0.28 +/- 0.03 mumols glycerol released 10(5) adipocytes-1 mg creatinine-1 respectively, P less than 0.01). Weight loss in animals bearing the MAC16 adenocarcinoma was paralleled by a corresponding rise in serum lipolytic activity which peaked when the loss of carcass weight was 16%. A similar decrease in serum lipolytic activity was also observed in cancer patients at high percentages loss in body weight. However, a linear relationship was observed between both the serum and urinary lipolytic activity and weight loss in cancer patients (correlation coefficients 0.79 and 0.70 respectively) when the total body weight loss did not exceed 20%. This suggests that weight loss in cancer patients may be attributed, at least in part, to an, as yet, unidentified lipolytic factor.

Synthesis and biological evaluation of a series of flavones designed as inhibitors of protein tyrosine kinases.

A series of flavones has been prepared, which are variously substituted in the 3,3',4',5 and 7 positions with halo-, alkoxy-, nitro-, amino-, hydroxy-, acyloxy- and azido-groups, for evaluation of their cytotoxicity to ANN-1 cells (3T3 murine fibroblasts transformed with the Abelson murine leukaemia virus) which contain a tyrosine kinase. This cytotoxicity was compared to their non-transformed 3T3 counterparts. 3'-Amino-4'-methoxyflavone was the most cytotoxic compound (IC50 = 1.6 microM) and was less inhibitory to the non-transformed parent 3T3 cell line (IC50 = 8 microM). The compound was inactive at 50 microM in assays of the inhibition of the cell-associated Abelson protein tyrosine kinase but inhibited an epidermal growth factor (EGF) protein tyrosine kinase by 42% at 50 microM. Quercetin (3,3',4',5,7-pentahydroxyflavone) was the most potent inhibitor of the Abelson protein tyrosine kinase but showed no selective inhibition of the growth of ANN-1 cells compared to the parent 3T3 cell line. Different structure-activity relationships were observed between the results of the cytotoxicity assays and inhibition of protein tyrosine kinases. Inhibitors of the Abelson protein tyrosine kinase which were competitive with respect to ATP showed different potencies for inhibition of the EGF receptor kinase.