Alan T. McGown

Tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle.

Tubulin is the biochemical target for several clinically used anticancer drugs, including paclitaxel and the vinca alkaloids vincristine and vinblastine. This review describes both the natural and synthetic agents which are known to interact with tubulin. Syntheses of the more complex agents are referenced and the potential clinical use of the compounds is discussed. This review describes the biochemistry of tubulin, microtubules, and the mitotic spindle. The agents are discussed in relation to the type of binding site on the protein with which they interact. These are the colchicine, vinca alkaloid, rhizoxin/maytansine, and tubulin sulfhydryl binding sites. Also included are the agents which either bind at other sites or unknown sites on tubulin. The literature is reviewed up to October 1997.

In vivo and in vitro evaluation of combretastatin A-4 and its sodium phosphate prodrug

SummaryThe anti-tumour effects and mechanism of action of combretastatin A-4 and its prodrug, combretastatin A-4 disodium phosphate, were examined in subcutaneous and orthotopically transplanted experimental colon tumour models. Additionally, the ability of these compounds to directly interfere with endothelial cell behaviour was also examined in HUVEC cultures. Combretastatin A-4 (150 mg kg–1, intraperitoneally (i.p.)) and its water-soluble prodrug (100 mg kg–1, i.p.) caused almost complete vascular shutdown (at 4 h), extensive haemorrhagic necrosis which started at 1 h after treatment and significant tumour growth delay in MAC 15A subcutaneous (s.c.) colon tumours. Similar vascular effects were obtained in MAC 15 orthotopic tumours and SW620 human colon tumour xenografts treated with the prodrug. More importantly, in the orthotopic models, necrosis was seen in vascularized metastatic deposits but not in avascular secondary deposits. The possible mechanism giving rise to these effects was examined in HUVEC cells. Here cellular networks formed in type I calf-skin collagen layers and these networks were completely disrupted when incubated with a non-cytotoxic concentration of combretastatin A-4 or its prodrug. This effect started at 4 h and was complete by 24 h. The same non-cytotoxic concentrations resulted in disorganization of F-actin and β-tubulin at 1 h after treatment. In conclusion, combretastatin A-4 and its prodrug caused extensive necrosis in MAC 15A s.c. and orthotopic colon cancer and metastases, resulting in anti-tumour effects. Necrosis was not seen in avascular tumour nodules, suggesting a vascular mechanism of action.

A phase I trial of bryostatin 1 in patients with advanced malignancy using a 24 hour intravenous infusion

Bryostatin 1 is a macrocyclic lactone derived from the marine invertebrate Bugula neritina. In vitro, bryostatin 1 activates protein kinase C (PKC), induces the differentiation of a number of cancer cell lineages, exhibits anti-tumour activity and augments the response of haemopoietic cells to certain growth factors. In vivo, bryostatin 1 is also immunomodulatory, but the range of tumours which respond to bryostatin 1 in xenograft tumour models is mostly the same as the in vitro tumour types, suggesting a direct mode of action. Nineteen patients with advanced malignancy were entered into a phase I study in which bryostatin 1 was given as a 24 h intravenous infusion, weekly, for 8 weeks. Myalgia was the dose-limiting toxicity and the maximum tolerated dose was 25 micrograms m-2 per week. The myalgia was cumulative and dose related, and chiefly affected the thighs, calves and muscles of extraocular movement. The mechanism of the myalgia is unknown. CTC grade 1 phlebitis affected every patient for at least one cycle and was caused by the diluent, PET, which contains polyethylene glycol, ethanol and Tween 80. Most patients experienced a 1 g dl-1 decrease in haemoglobin within 1 h of commencing the infusion which was associated with a decrease in haematocrit. Radiolabelled red cell studies were performed in one patient to investigate the anaemia. The survival of radiolabelled red cells during the week following treatment was the same as that seen in the week before treatment. However, there was a temporary accumulation of radiolabelled red cells in the liver during the first hour of treatment, suggesting that pooling of erythrocytes in the liver might account for the decrease in haematocrit. Total or activated PKC concentrations were measured in the peripheral blood mononuclear cells (PBMCs) of three patients for the first 4 h of treatment and during the last hour of the infusion. This showed that PKC activity was significantly modulated during the infusion. Bryostatin 1 is immunomodulatory in vitro, and we have confirmed this activity in vivo. An investigation of the first three cycles of treatment in seven patients showed an increased IL-2-induced proliferative response in peripheral blood lymphocytes and enhanced lymphokine-activated killer (LAK) activity. A previously reported rise in serum levels of interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF 1) was not confirmed in our study; of nine patients in this study, including patients at all dose levels, none showed an increase in these cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)

Combretastatin-like chalcones as inhibitors of microtubule polymerization. Part 1: Synthesis and biological evaluation of antivascular activity

The alpha-methyl chalcone SD400 is a potent inhibitor of tubulin assembly and possesses potent anticancer activity. Various chalcone analogues were synthesized and evaluated for their cell growth inhibitory properties against the K562 human chronic myelogenous leukemia cell line (SD400, IC(50) 0.21nM; combretastatin A4 CA4, IC(50) 2.0nM). Cell cycle analysis by flow cytometry indicated that these agents are antimitotic (SD400, 83% of the cells are in G(2)/M phase; CA4 90%). They inhibit tubulin assembly at low concentration (SD400, IC(50) 0.46microM; CA4, 0.10microM) and compete with [(3)H]colchicine for binding to tubulin (8% [(3)H]colchicine remained bound to tubulin after competition with SD400 or CA4). Upon treatment with SD400, remarkable cell shape changes were elicited in HUVEC cells, consistent with vasculature damaging activity.

Distribution and Clinical Significance of Heparan Sulfate Proteoglycans in Ovarian Cancer

Purpose: Heparan sulfate proteoglycans have been implicated in cancer cell growth, invasion, metastasis, and angiogenesis. This study was designed to compare their expression in normal ovary and ovarian tumors and then to examine their prognostic significance in ovarian cancer. Experimental Design: The expression of syndecan-1, -2, -3, and -4, glypican-1, and perlecan was assessed by immunohistochemistry in 147 biopsies that included normal ovary and benign, borderline, and malignant ovarian tumors. Clinical data, including tumor stage, performance status, treatment, and survival, were collected. Univariate and multivariate analyses were performed to evaluate prognostic significance. Results: The expression patterns of syndecan-1 and perlecan were altered in ovarian tumors compared with normal ovary. Syndecan-1 was not detected in normal ovary but was present in the epithelial and stromal cells of benign and borderline tumors and in ovarian adenocarcinomas. Perlecan expression was decreased in basement membranes that were disrupted by cancer cells but maintained in the basement membranes of blood vessels. Syndecan-2, -3, and -4, and glypican-1 were expressed in normal ovary and benign and malignant ovarian tumors. Stromal expression of syndecan-1 and glypican-1 were poor prognostic factors for survival in univariate analysis. Conclusion: We report for the first time distinct patterns of expression of cell surface and extracellular matrix heparan sulfate proteoglycans in normal ovary compared with ovarian tumors. These data reinforce the role of the tumor stroma in ovarian adenocarcinoma and suggest that stromal induction of syndecan-1 contributes to the pathogenesis of this malignancy.

Differential cytotoxicity of Combretastatins A1 and A4 in two daunorubicin-resistant P388 cell lines

SummaryCombretastatin A4, a novel anti-mitotic agent was effective against two P388 cell lines with acquired resistance to daunorubicin. In contrast, Combretastatin A1, a close structural analogue of A4, showed a high degree of cross-resistance. Combretastatin A1 was also more efficient at increasing intracellular daunorubicin concentrations in both resistant cell lines. Neither agent was capable of altering anthracycline accumulation in the parental (sensitive) cell line. We propose that the cross-resistance to Combretastatin A1 occurs, at least in part, as a result of the increased affinity of the drug-efflux process operative in these resistant cells for Combretastatin A1 vs Combretastatin A4. Hence, Combretastatin A4 may play a role in the treatment of tumours with acquired resistance to the anthracycline antibiotics.

Structural requirements for the interaction of combretastatins with tubulin: how important is the trimethoxy unit?

A series of combretastatins possessing both a trimethoxy unit and other substituents on ring A has been synthesised and tested for cytotoxicity and their ability to interact with the protein tubulin. All previous studies have indicated that the trimethoxy unit is essential for interaction with tubulin. The studies herein show that molecules possessing functionalities other than trimethoxy can also interact with tubulin. Importantly a trimethyl substituted agent 52a has shown reduced cytotoxicity, but increased potency in its ability to inhibit the assembly of tubulin.

Isoflavones inhibit intestinal epithelial cell proliferation and induce apoptosis in vitro

SummaryThere have been many reports that high soya-based diets reduce the risk of certain types of cancer. This effect may be due to the presence of high levels of isoflavones derived from the soya bean, particularly genistein which has been shown to be a protein tyrosine kinase (PTK) inhibitor and have both oestrogenic and anti-oestrogenic properties. We have examined the effect of genistein and a number of novel synthetic analogues on both normal (IEC6, IEC18) and transformed (SW620, HT29) intestinal epithelial cell lines. Responses were compared to those elicited by oestradiol, the anti-oestrogen tamoxifen, and the tyrosine kinase inhibitor tyrphostin. Genistein and tamoxifen were potent inhibitors of cell proliferation. Of seven novel isoflavones tested, none were more potent inhibitors than genistein, and all displayed similar relative activities across the different cell lines. In addition to inhibiting cell proliferation, cell death via apoptosis was observed when the cells were exposed to the isoflavones and all but one exhibited PTK inhibitory activity. These data suggest that by reducing proliferation and inducing apoptosis, possibly due in part to PTK inhibition, isoflavones may have a role in protecting normal intestinal epithelium from tumour development (reducing the risk) and may reduce colonic tumour growth.

A proposed mechanism of resistance to cyclophosphamide and phosphoramide mustard in a Yoshida cell line in vitro

SummaryA Yoshida sarcoma cell line (YR/cyclo) showing decreased sensitivity to metabolically activated cyclophosphamide in vitro has been shown to be cross-resistant to phosphoramide mustard, the ultimate alkylating agent formed from cyclophosphamide. Resistance to these alkylating agents has been shown to be associated with increased activity of the glutathione S-transferase group of enzymes, and with elevated levels of glutathione, the cosubstrate of the enzyme. The resistant cell line shows lower levels of cellular damage, as measured by alkaline elution following treatment with phosphoramide mustard, than the parental (Ys) line. The mechanism of resistance is ascribed to increased deactivation of potentially damaging metabolites of cyclophosphamide by the glutathione S-transferase enzymes, resulting in decreased cellular damage in the resistant cell line.

The effect of ifosfamide and its metabolites on intracellular glutathione levels in vitro and in vivo.

The effect of ifosfamide and its metabolites on intracellular levels of glutathione in P388 cells in vitro has been studied. It is demonstrated that glutathione depletion occurs only in the presence of 4-hydroperoxyifosfamide and chloroacetaldehyde. In contrast isophosphoramide mustard had no effect on glutathione levels in intact cells. The concentration of 4-hydroperoxyifosfamide required to reduce glutathione levels by 50% was approximately 1 mM and this represents a concentration far in excess of that achievable in patients receiving the drug. However the concentration of chloroacetaldehyde (approximately 100 microM) required to reduce intracellular levels of glutathione to a similar extent is attained in patients receiving ifosfamide. The glutathione levels in lymphocytes isolated from a patient undergoing an eight hour infusion of ifosfamide showed a marked decrease to about 30% of their original value. We conclude that ifosfamide causes glutathione depletion in vivo and the majority of this can be accounted for by the production of chloroacetaldehyde.