Willi Kreis

The effect of antimicrotubule agents on signal transduction pathways of apoptosis: a review.

Purpose: Microtubules are important cytoskeletal components involved in many cellular events. Antimicrotubule agents including polymerizing agents (paclitaxel and docetaxel) and depolymerizing drugs (vincristine, vinorelbine, and estramustine phosphate) are widely used either alone or in combination with other anticancer drugs. These antimicrotubule agents are promoters of apoptosis in cancer cells. In this review, we discuss the role of bcl-2 family genes in the regulation of apoptosis, and summarize effects of microtubule targeting agents on apoptotic signal transduction pathways. Conclusion: Disruption of microtubule structure by antimicrotubule drugs results in induction of tumor suppressor gene p53 and inhibitor of cyclin-dependent kinases, p21WAF1/CIP1 (p21), and activation/inactivation of several protein kinases including Ras/Raf, PKC/PKA I/II, MAP kinases, and p34cdc2. These protein kinases are associated directly or indirectly with phosphorylation of bcl-2. Phosphorylation of bcl-2 and the elevations of p53 and p21 lead to apoptosis. New pathways of antitumor agents could be directed at this p53, p21 and bcl-2/bax function, and may enhance the effect of existing agents.

Phase I trial of the combination of daily estramustine phosphate and intermittent docetaxel in patients with metastatic hormone refractory prostate carcinoma

BACKGROUNDnTo apply our preclinical findings of cytotoxic synergy with the combination of estramustine phosphate (EP) and docetaxel as the basis of treatment of hormone refractory metastatic prostate cancer in man. To determine the optimal dosage and the toxicities of these two agents for future trials.nnnPATIENTS AND METHODSnSeventeen patients with hormone refractory metastatic prostate cancer who were ambulatory with performance status < or = 2, normal marrow, renal and hepatic function were entered. Prior exposure to EP or a taxane were exclusion factors. EP was given orally at a dose of 14 mg/kg of body weight daily with concurrent docetaxel administered every 21 days as an intravenous infusion over 1 hour with dexamethasone 8 mg. PO BID for five days. EP dosages were kept static; docetaxel dosages were explored in a minimum of three patients per level for dosages of 40, 60, 70, and 80 mg/m2. Patients were evaluated weekly. Prostate specific antigen (PSA) was measured every three weeks.nnnRESULTSnFive patients were entered at a docetaxel dose of 40 mg/m2, three at 60 mg/m2, six at 70 mg/m2, and three at 80 mg/m2. Only one patient had received prior chemotherapy. Grades 1 or 2 hypocalcemia and hypophosphatemia were seen at all dosage levels. Other grade 2 or less toxicities not related to dosage included alopecia, anorexia, stomatitis, diarrhea, and epigastric pain. Dose limiting toxicities (DLT) as grade 4 leukopenia and grade 4 fatigue were seen at 80 mg/m2. The phase II dose was defined at 70 mg/m2 with rapidly reversible leukopenia and minor liver function abnormalities. At this dosing level, dose intensity was 88% and 86% over consecutive cycles for docetaxel and EP, respectively. Two vascular events occurred at this dose level (70 mg/m2): one arterial and the other venous. PSA decreases greater than 50% from baseline were seen in 14 of 17 patients at all dosage levels. Four of the 17 patients demonstrated a complete biochemical response (PSA < or = 4 ng/ml). One patient had a partial response with measurable lung and liver lesions.nnnCONCLUSIONnEP given continuously with every three-week docetaxel at a dose of 70 mg/m2 is tolerable with evidence of antitumor activity based upon significant declines in PSA in the majority of patients and improvement of lung metastasis in one patient. Larger phase II studies of this combination in a homogenous population are warranted.

Synergistic and antagonistic combinations of drugs in human prostate cancer cell lines in vitro.

Microtubulin binding agents such as docetaxel have significant preclinical and clinical activity in the treatment of hormone-refractory prostate cancer. We have previously used median-effect analysis in vitro to define both synergistic and antagonistic drug combinations which may be of value in management of human disease. These studies extend our findings in defined prostate cancer cell lines. A semi-automated microtiter culture system was used. Docetaxel was combined with 18 other agents, incubated with DU 145, LnCaP or PC 3 prostate cancer cell lines for 72u2009h and the cells then incubated with MTT to determine cytotoxic effect. Both doublet and triplet combinations were examined. Synergy and antagonism as measured by the combination index were determined for each combination. The non-mutually exclusive criterion was applied. Docetaxel demonstrated cytotoxic additive effects or synergy with cis-retinoic acid, cyclosporin A and vinorelbine in all three cell lines. Docetaxel combined with either epirubicin or doxorubicin displayed cytotoxic synergistic effects in hormone-refractory DU 145 and PC 3 cell lines. In contrast, drugs which have been combined clinically to treat hormone-refractory prostate cancer, i.e. cisplatin, carboplatin or etoposide, were antagonistic when combined with docetaxel. We conclude that combinations of docetaxel with either cis-retinoic acid or vinorelbine may offer an enhanced cytotoxic effect in the management of hormone-refractory prostate cancer and need to be evaluated for therapeutic effect. The combination of docetaxel with an anthracycline was also synergistic in the two hormone-refractory cell lines, DU 145 and PC3, thus suggesting a potential role in advanced disease after endocrine failure. Combinations of docetaxel with platinum or etoposide may lead to subadditive effects in treatment.

Inhibition of HIV replication by Hyssop officinalis extracts

Crude extracts of dried leaves of Hyssop officinalis showed strong anti-HIV activity as measured by inhibition of syncytia formation, HIV reverse transcriptase (RT), and p17 and p24 antigen expression, but were non-toxic to the uninfected Molt-3 cells. Ether extracts from direct extraction (Procedure I), after removal of tannins (Procedure II), or from the residue after dialysis of the crude extract (Procedure III), showed good antiviral activity. Methanol extracts, subsequent to ether, chloroform and chloroform ethanol extractions, derived from procedure I or II, but not III, also showed very strong anti-HIV activity. In addition, the residual material after methanol extractions still showed strong activity. Caffeic acid was identified in the ether extract of procedure I by HPLC and UV spectroscopy. Commercial caffeic acid showed good antiviral activity in the RT assay and high to moderate activity in the syncytia assay and the p17 and p24 antigen expression. Tannic acid and gallic acid, common to other teas, could not be identified in our extracts. When commercial products of these two acids were tested in our assay systems, they showed high to moderate activity against HIV-1. Hyssop officinalis extracts contain caffeic acid, unidentified tannins, and possibly a third class of unidentified higher molecular weight compounds that exhibit strong anti-HIV activity, and may be useful in the treatment of patients with AIDS.

Mutational Status of Codons 12 and 13 of the N- and K-ras Genes in Tissue and Cell Lines Derived from Primary and Metastatic Prostate Carcinomas

N- and K-ras mutations at codons 12 and 13 were investigated using oligonucleotide hybridization analysis after PCR amplification and subsequent sequence analysis of the amplified DNA from the region of interest in the following prostatic primary and metastatic (met) carcinoma-derived cell lines: 1013L (primary), PC3 (bone met), DU145 (brain met), and LNCaP (lymph node met). We also examined fresh and archival primary and metastatic prostate tumor tissue and benign prostatic hypertrophy specimens. All prostatic cells and tissues examined contain at least one wild-type N- and K-ras allele with respect to codons 12 and 13. No mutations were found at N-ras codon 13. The only mutation seen in the prostatic cell lines and tissues was a K-ras codon 12 position II G-to-T transversion. Since these are established nonclonal cell lines that have adapted to tissue culture, it is possible that this mutation does not represent the mutational state of prostatic carcinoma in vivo. However, the lack of consistent mutation in the ras genes amplified directly from tumors suggests that when ras mutations occur during the progression of prostatic carcinoma, they are late-stage events not directly involved in the initial development of disease. Immunoprecipitation studies using pan-ras antibodies revealed no evidence of altered expression of Ras proteins.

In vitro evaluation of synergism or antagonism with combinations of new cytotoxic agents.

New cytotoxics with significant activity both in preclinical and clinical situations continued to be applied in the clinic by empiric means. The use of defined cell lines allows unanticipated antagonism between agents to be identified and to suggest synergistic combinations which need to be tested. By means of a semi-automated MTT assay and median effect analysis, we have identified antagonism in two couplets being evaluated in the clinic: etoposide with paclitaxel and vinorelbine with gemcitabine. Optimal use of these agents in man may require spacing these agents in time to prevent an adverse drug interaction between the agents which may diminish the potential response rate.

Therapy of refractory/relapsed acute myeloid leukemia and blast crisis of chronic myeloid leukemia with the combination of cytosine arabinoside, tetrahydrouridine, and carboplatin

SummaryEight patients, of whom four had acute myeloid leukemia (AML) and four had chronic myeloid leukemia (CML) blast crisis, were treated with a combination of cytosine arabinoside (ARA-C: 1,600 mg/m2 in three patients, 1,200 mg/m2 in five patients), tetrahydrouridine (THU: 2,800 mg/m2 in two patients, 2,646 mg/m2 in one patient, 2,100 mg/m2 in five patients), and carboplatin (900 mg/m2 in four patients, 720 mg/m2 in one patient, 450 mg/m2 in three patients). As a result of this treatment, five of the eight patients became aplastic. Two of the four patients with CML blast crisis reverted to the chronic phase and two of the four patients with acute nonlymphocytic leukemia (ANLL) attained a remission (one partial remission and one complete remission). The major toxicities included myelosuppression, unacceptable hepatotoxicity, and diarrhea. Pharmacokinetics studies revealed that the addition of carboplatin did not significantly change the disposition of ARA-C. ARA-C levels were not significantly changed in comparison with those obtained in a prior study of ARA-C with THU (ARA-C plasma levels at 3 h, 2630±1170 ng/ml).

Pharmacokinetics of ara-C and ara-U in plasma and CSF after high-dose administration of cytosine arabinoside*

SummaryCytosine arabinoside (ara-C) and uracil arabinoside (ara-U) levels were measured in the plasma, cerebrospinal fluid (CSF), and urine of 10 patients exhibiting primary central nervous system lymphoma who received 31 infusions of high-dose ara-C (3 g/m2) as part of their treatment regimen. Peak plasma and CSF ara-C levels were 10.8 and 1.5 μg/ml, respectively. Ara-C was cleared more rapidly from plasma than from CSF. Ara-U appeared rapidly in both plasma and CSF, reaching a peak that was 10 times higher than the corresponding ara-C concentration (104 and 11.2 μg/ml, respectively). Only 4%–6% of the dose was excreted unchanged in the urine, but 63%–73% of it appeared as ara-U within the first 24 h. The presence of leptomeningeal lymphoma did not affect the CSF level of ara-C or ara-U.

The Androgen Receptor: Structure, Mutations, and Antiandrogens

Androgens play a critical role not only in the physiological development of the prostate but also in the genesis of prostate cancer. The effects of andorgen on theprostate gland and on the other tisues of the body are mediated by activation of the androgen receptor. The androgen receptor is a member of the superfamily of hormome receptors with a DNA-binding site, two zinc finger domains, and a hormone-binding site. Mutations in this receptor can be associated with loss of function or chronic endogeneous activation, depending upon the site of change. Androgens effect a conformal change in the structure of the androgen receptor associated with a change in protein phosphorylation. The androgen receptor can be activated by additional ligands affecting the hormone-binding site besides androgens. Activators and repressors of the androgen receptor modify this proteins function and are very delicately balanced such that disruptions of either function are associated with a disease state. Antiandrogens, which bind to the receptor and thus down-regulate the effects of endogeneous circulating androgens, remain the first line treatment for palliation of advanced prostate cancer. Mutations in the receptor are associated with a change in function of such compounds from antagonist to agonist in vitro. Newer evidence suggests there may be a role of intermittent androgen suppression rather than continuous suppression, perhaps by preventing overgrowth of hormone independent tumor cells. Future research focuses on the development of drugs directed at suppressing the androgen drive of the androgen sensitive clone of the tumor and making the nonsenseitive subset more susceptible to cytotoxics.

A reexamination of PSC 833 (Valspodar) as a cytotoxic agent and in combination with anticancer agents

Background: The cyclosporins have been thought as being mainly immunosuppressive agents which interfere with the function of the MDR pump and thus play a role in resistance to drug anticancer effects. We reexamined their cytotoxicity in defined cell lines both as single agents and in combination with agents which may be of value in human malignant disease. Methods: Cells were grown to confluence following inoculation at 5000–8000xa0cells/well in 96-well dishes, and growth patterns and death were determined by an MTT assay. Median effect analysis was used to look for synergy, additive effects, or antagonism between the cyclosporins and drugs with antitumor effects in humans. Results: Cyclosporin A and PSC 833 were found to have cytotoxic activity at clinically achievable concentrations in breast, leukemia, and prostate cell lines. Synergistic or additive effects were demonstrated in all three prostate cell lines when PSC 833 was combined with estramustine, etoposide, ketoconazole, suramin, or vinorelbine in the prostate cancer cell lines. Cell line-selective additive effects or synergism were also identified with bicalutamide, carboplatin, cisplatinum, cis-retinoic acid, dexamethasone, 5-fluorouracil, liarozole, and trans-retinoic acid. Conclusions: PSC 833 or cyclosporin alone or in combination with other agents may have an anticancer effect independently of their modulatory action on MDR. Several of the synergistic combinations which are not mediated by the MDR pump need to be tested in vivo for efficacy.