Definition of the “Drug-Angiogenic-Activity-Index” that Allows the Quantification of the Positive and Negative Angiogenic Active Drugs: A Study Based on the Chorioallantoic Membrane Model

Since the introduction of the angiogenic therapy by Folkman et al. in the 1970’ies many antiangiogenic drugs were identified. Only few of them are still now in clinical use. Also the Vascular Endothelial Growth Factor (VEGF), the cytokine with the highest angiogenic activity, has been identified. Its antagonist, Bevacizumab, is produced and admitted for the angiogenic therapy in first line for metastatic colorectal cancer. When we look at preclinical studies, they fail of in vivo models that define the “Drug-AngiogenicActivity-Index” of angiogenic or antiangiogenic drugs. This work proposes a possible standardized procedure to define the “Drug Angiogenic Activity Index” by counting the vascular intersections (VIS) on the Chorioallantoic Membrane after drug application. The equation was defined as follows: {ΔVIS[Drug]−ΔVIS[Control]} / Δ VIS[Control]. For VEGF a Drug-Angiogenic-Activity-Index of 0.92 was found and for Bevacizumab a −1. This means almost that double of the naturally angiogenic activity was achieved by VEGF on the Chorioallantoic membrane. A complete blocking of naturally angiogenic activity was observed after Bevacizumabs application. Establishing the “Drug-Angiogenic-Activity-Index” in the preclinical phase will give us an impact of effectivness for the new constructed antiangiogenic drugs like the impact of effectiveness in the cortisone family.     

The role of cordycepin in cancer treatment via induction or inhibition of apoptosis: implication of polyadenylation in a cell type specific manner

Purpose Most anticancer drugs show their antiproliferative and cytotoxic activity via induction of apoptosis. In the present study we assessed the implication and role of cordycepin, a polyadenylation-specific inhibitor and a well-known chemotherapeutic drug, in apoptosis, induced by the anticancer drug etoposide. Methods For this purpose, a variety of leukemia and lymphoma cell lines (U937, K562, HL-60, Daudi, Molt-4) were treated with the anticancer drugs etoposide and/or cordycepin and assessed for poly(A) polymerase (PAP) activity and isoforms by the highly sensitive PAP activity assay and western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, DAPI staining, caspase-6 activity assay and ΔΨ_m reduction, whereas cytotoxicity and cell cycle status were assessed by Trypan blue staining, MTT assay and flow cytometry. Results and conclusions The results showed that PAP changes in all cell lines, in response to apoptosis induced by etoposide, in many cases even prior to hallmarks of apoptosis (endonucleosomal cleavage of DNA, ΔΨ_m reduction). A further elucidation to this apoptosis–polyadenylation correlation was added, by cell treatment with cordycepin, resulting in either suppression (U937, K562) or induction (HL-60) of the apoptotic process, according to the cell type. However, inhibition of polyadenylation did not influence the cell lines Daudi and Molt-4 used, where alternative apoptotic pathways are induced through cleavage of DNA into high molecular weight fragments.     

Thymidylate synthase inhibition induces S-phase arrest, biphasic mitochondrial alterations and caspase-dependent apoptosis in leukaemia cells

In this study, the downstream effects of thymidylate synthase (TS) inhibition in L1210 (p53 mutant) and HL60 (p53 null) leukaemia cells were investigated. TS inhibition was induced by the specific TS inhibitor Thymitaq. Within 24 h, TS inhibition resulted in S-phase cell cycle arrest in both cell lines and subsequent apoptotic cell death as characterized by nuclear condensation, DNA fragmentation and the formation of apoptotic bodies. A biphasic hyper/hypopolarization of the mitochondrial membrane potential (ΔΨm) was also observed. The mitochondrial permeability transition inhibitor, cyclosporin A, increased the baseline level of ΔΨm in L1210 cells. However, along with bongkrekic acid, it did not influence the changes in ΔΨm induced by TS inhibition in either cell line. In both cell lines the broad spectrum caspase inhibitor, zVAD.fmk as a single agent, induced a significant downward shift in the baseline of ΔΨm. However, only in HL60 cells was this accompanied by a slight increase in cytotoxicity. In L1210 cells zVAD.fmk inhibited DNA fragmentation induced by Thymitaq but did not influence other cell cycle events (S-phase arrest) or the biphasic mitochondrial alterations, indicating caspase involvement downstream but not upstream of the mitochondria following TS inhibition. In HL60 cells, zVAD.fmk reduced the hyperpolarization of ΔΨm observed with Thymitaq alone and failed to inhibit the increase in the sub-G₁ population induced by Thymitaq. Moreover, zVAD.fmk significantly increased the cell death response of these cells following TS inhibition. In conclusion, cell death induced by TS inhibition is mediated via the apoptotic pathway which clearly involves biphasic alterations in ΔΨm. In L1210 cells, but not in HL60 cells, caspases function as the final executioner of apoptosis. Received: 5 January 2000 / Accepted: 26 May 2000     

New troglitazone derivatives devoid of PPARγ agonist activity display an increased antiproliferative effect in both hormone-dependent and hormone-independent breast cancer cell lines

Numerous recent studies indicate that most anticancer effects of PPARγ agonists like thiazolidinediones are the result of PPARγ-independent pathways. These conclusions were obtained by several approaches including the use of thiazolidinedione derivatives like Δ2-Troglitazone (Δ2-TGZ) that does not activate PPARγ. Since biotinylation has been proposed as a mechanism able to increase the specificity of drug delivery to cancer cells which could express a high level of vitamin receptor, a biotinylated derivative of Δ2-TGZ (bΔ2-TGZ) has been synthetized. In the present article, we have studied the in vitro effects of this molecule on both hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231) breast cancer cells. In both cell lines, bΔ2-TGZ was more efficient than Δ2-TGZ to decrease cell viability. bΔ2-TGZ was also more potent than Δ2-TGZ to induce the proteasomal degradation of cyclin D1 in both cell lines and those of ERα in MCF-7 cells. However, in competition experiments, the presence of free biotin in the culture medium did not decrease the antiproliferative action of bΔ2-TGZ. Besides, other compounds that had no biotin but that were substituted at the same position of the phenolic group of the chromane moiety of Δ2-TGZ decreased cell viability similarly to bΔ2-TGZ. Hence, we concluded that the increased antiproliferative action of bΔ2-TGZ was not due to biotin itself but to the functionalization of the terminal hydroxyl group. This should be taken into account for the design of new thiazolidinedione derivatives able to affect not only hormone-dependent but also hormone-independent breast cancer cells in a PPARγ-independent pathway.     

Experimental study of combination therapy with S-1 against pancreatic cancer

Purpose  To determine the most effective combination chemotherapy with S-1 against pancreatic cancer and to clarify the mechanism of synergy between S-1 and the partner drug. Methods  We tested a combination of S-1 with the following antitumor drugs in an in vitro MTT assay against pancreatic cancer cell line MIA PaCa-2: gemcitabine (GEM), cisplatin (CDDP), irinotecan (CPT-11), mitomycin C, adriamycin, and paclitaxel. The efficacy of S-1, GEM, and a combination of S-1 and GEM was also tested in vivo by administering S-1 (10 mg/kg) orally to nude mice five times a week for 3 weeks, and GEM (100 mg/kg) intravenously every 2–3 days for a total of six times. A treated-to-control ratio (T/C) of relative mean tumor weight values less than 50% was determined to be effective. Furthermore, we investigated the mechanism of the synergistic effect of S-1 and GEM on the cell cycle by flow cytometry, because both S-1 and GEM are known as antimetabolic drugs. To verify cell death induced by a change in the distribution of the cell cycle phases, we investigated apoptosis by sub-G1 analysis and a TUNEL assay. Results  From classical isobolography analysis of the in vitro MTT assay, the combination of S-1 plus GEM was found to be the most effective of the combinations tested. In vivo, T/C (percentage) with the combination of S-1 plus GEM was 48.2%, which was lower than that of S-1 or GEM alone, and the combination enhanced antitumor activity. Cell cycle analysis showed greater cell cycle delay with the combination treatment (S-1 plus GEM) than for each single drug treatment, and apoptotic cells were detected only in treatments including GEM. Conclusion  The combination chemotherapy of S-1 and GEM appears to be useful for pancreatic cancer. Both cycle delay by S-1 plus GEM and apoptosis induced by GEM are involved in this synergistic mechanism.     

A pharmacodynamic analysis method to determine the relative importance of drug concentration and treatment time on effect

Purpose: The pharmacodynamics of most drugs follow the empirical relationship, Cⁿ × T=h, where C is drug concentration, T is exposure time and h is drug exposure constant. The value of n indicates the relative importance of C and T in determining the effect. An n value greater than 1.0 indicates that for two infusions that produce the same C × T, a short infusion that delivers high concentrations over a short duration will produce a greater Cⁿ × T and therefore a greater effect, compared to a long infusion that delivers lower concentrations. The reverse is true for an n value less than 1.0 and would support the use of a slow infusion. Hence, it is important to determine the n values and whether the n value significantly differs from 1.0. This report describes a three-step method for this purpose. Methods: First, we obtained experimental data on the relationship between drug concentration, treatment time and effect, and analyzed the data with a three-dimensional surface response method to obtain the pharmacodynamic model parameters and the magnitude of data variability. The experiments used mitomycin C and two human cancer cell lines, i.e. bladder RT4 and pharynx FaDu cells. The n values obtained from four experiments ranged from 1.04 to 1.16 for FaDu cells and from 1.14 to 1.46 for RT4 cells. The variability in the effect data decreased from 11.9% at 0% effect to 6.14% at 100% effect. Second, these results were used with Monte Carlo simulations to generate 100 concentration-time-effect data sets, which contained randomly and normally distributed data variability comparable to the experimentally observed variability, for each experimentally determined n value. This is analogous to performing 100 experiments under the same experimental conditions. Third, we analyzed the simulated data sets to obtain 100 estimated n values. The frequency with which these estimated n values fell above or below 1.0 indicated the probability that the experimentally determined n value used in the Monte Carlo simulations was truly different from 1.0. We defined this frequency for individual experiments as F_one, and calculated the overall probability for multiple experiments (F_multiple). A probability of greater than 97.5% (i.e. P < 0.05 for a two-tailed test) was considered statistically significant. Results: Analysis of the mitomycin C pharmacodynamic data yielded F_one and F_multiple of 99% to 100% for FaDu and RT4 cells, indicating that the n values for these cells were significantly higher than 1.0. A comparison of the statistical significance of the n value analyzed by the three-step pharmacodynamic analysis method, a conventional statistical method such as the Student's t-test and nonlinear regression analysis, indicated two advantages for the pharmacodynamic method: fewer experiments were required (theoretically only one experiment with three replicates would be sufficient) and a higher statistical significance of the n value was obtained. Conclusions: In summary, the three-step pharmacodynamic study design and analysis method can be used to define the relative importance of drug concentration and treatment time on drug effect. Received: 19 May 1999 / Accepted: 15 September 1999     

Association of T916C (Y257H) mutation in Candida albicans ERG11 with fluconazole resistance

Repeated and prolonged use of fluconazole in treating candidosis leads to drug resistance. The aim of this study was to confirm the relationship between T916C (Y257H) mutation in Candida albicans ERG11 and fluconazole resistance. We replaced one copy of ERG11 with ERG11 containing the T916C mutation in C. albicans CAI4 and expressed ERG11 with the T916C mutation in Saccharomyces cerevisiae INVSc1. The MIC values were two‐ to four‐fold greater in CAI4 transformants with than without the T916C mutation and 128 and 32 μg ml^?1 for S. cerevisiae INVSc1‐containing ERG11 with and without the T916C mutation. T916C mutation may be associated with fluconazole resistance in C. albicans.     

A novel doxorubicin-mitomycin C co-encapsulated nanoparticle formulation exhibits anti-cancer synergy in multidrug resistant human breast cancer cells

Anthracycline-containing treatment regimens are currently the most widely employed regimens for the management of breast cancer. These drug combinations are often designed based on non-cross resistance and minimal overlapping toxicity rather than drug synergism. Moreover, aggressive doses are normally used in chemotherapy to achieve a greater therapeutic benefit at the cost of more acute and long-term toxic effects. To increase chemotherapeutic efficacy while decreasing toxic effects, rational design of drug synergy-based regimens is needed. Our previous work showed a synergistic effect of doxorubicin (DOX) and mitomycin C (MMC) on murine breast cancer cells in vitro and improved efficacy and reduced systemic toxicity of DOX-loaded solid polymer–lipid hybrid nanoparticles (PLN) in animal models of breast cancer. Herein we have demonstrated true anticancer synergy of concurrently applied DOX and MMC, and have rationally designed PLN to effectively deliver this combination to multidrug resistant (MDR) MDA435/LCC6 human breast cancer cells. DOX–MMC co-loaded PLN were effective in killing MDR cells at 20–30-fold lower doses than the free drugs. This synergistic cell killing was correlated with enhanced induction of DNA double strand breaks that preceded apoptosis. Importantly, co-encapsulation of dual agents into a nanoparticle formulation was much more effective than concurrent application of single agent-containing PLN, demonstrating the requirement of simultaneous uptake of both drugs by the same cells to enhance the drug synergy. The rationally designed combination chemotherapeutic PLN can overcome multidrug resistance at a significantly lower dose than free drugs, exhibiting the potential to enhance chemotherapy and reduce the therapeutic limitations imposed by systemic toxicity.     

Efficacy of chemotherapeutic agents under hypoxic conditions in pulmonary adenocarcinoma multidrug resistant cell line

Hypoxia is often observed in solid tumors. The aim of this study was to investigate the efficacy of seven cytotoxic drugs against the pulmonary adenocarcinoma multidrug-resistant cell line A549/MDR under hypoxia (3% O(2)), and to explore the possible mechanisms for the change of efficacy. The efficacy of cytotoxic drugs under hypoxic conditions was different from that under normoxia. Proliferation of A549/MDR cells was enhanced under hypoxia and no close correlation was found between proliferation and cytotoxic effects. Under hypoxia, the efficacy of rhodamine123 efflux was unchanged; the culture medium became more acidic and the generation of reactive oxygen species (ROS) was decreased. The intracellular fluorescence intensity of daunorubicin was much lower in this acidic microenvironment. These results indicate that susceptibility to drugs was greatly influenced by hypoxia and different intracellular drug concentrations induced by microenvironment acidification which may be the main cause of the change in drug efficacy. In addition, proliferation may change resistance to study drugs under hypoxia for A549/MDR cells. The decreased generation of ROS may be another reason for the resistance of A549/MDR cell line to daunorubicin under hypoxic conditions. Drug exclusion mediated by P-gp may not be the key reason.     

化疗药物对T淋巴细胞及其亚群影响的体外探讨

目的:探讨化疗药物对T淋巴细胞及其亚群的影响.方法:取健康成年人外周血T淋巴细胞,采用MTT法观察几种常见化疗药物对T淋巴细胞的杀伤作用,在倒置显微镜下观察化疗后T淋巴细胞的转化能力,应用流式细胞仪比较化疗前后细胞亚群比例的变化.结果:化疗药物在大剂量时对T淋巴细胞的杀伤活力极大,并显著影响淋巴细胞的转化,化疗对T淋巴细胞亚群的杀伤无选择性,不改变细胞亚群的比例(P＞0.05).结论:化疗通过对免疫活性细胞的非选择性杀伤,抑制机体的细胞免疫功能.     

The impact of patient characteristics on enzalutamide pharmacokinetics and how this relates to treatment toxicity and efficacy in metastatic prostate cancer patients

The aim of the study is to investigate the influence of patient characteristics, age and body mass index (BMI), on pharmacokinetics of enzalutamide, and to study the relationships between drug exposure and enzalutamide efficacy and toxicity, in mCRPC patients. Data were collected in a longitudinal cohort study (ANDROPS) and a prospective observational study (ILUMINATE), both in mCRPC patients treated with enzalutamide. To investigate the influence of age and BMI on exposure, enzalutamide and N-desmethylenzalutamide levels were compared by ANOVA. To investigate the relation of exposure versus time to progression (TTP), the sum plasma levels were divided into quartiles and compared by Kaplan–Meier analysis. To assess the relation of exposure with fatigue, plasma levels in patients experiencing fatigue vs. no fatigue were compared by and independent t test. Data of 68 mCRPC patients were included for analysis. Plasma levels were not different for age or BMI. No difference in TTP between both studies was observed (383 days (95% CI 287–859), and 567 days (95% CI 351–NR), p = 0.36). Kaplan–Meier analysis of quartiles of sum levels showed no difference for TTP. Fatigue was reported by 22 patients, no difference in sum plasma levels was observed between patients with and without fatigue. We observed that age and BMI did not influence systemic exposure in patients treated with enzalutamide. No relation of exposure with efficacy or fatigue was observed. Further research using enzalutamide at a lower dose is needed to understand the relation of enzalutamide exposure and fatigue.     

Ex vivo cytotoxic drug evaluation by DiSC assay to expedite identification of clinical targets: results with 8-chloro-cAMP.

There is a pressing need to reduce the time and cost of developing new cytotoxic agents and to accurately identify clinically active agents at an early stage. In this study, the differential staining cytotoxicity (DiSC) assay was used to assess the efficacy of the novel antitumour cAMP analogue, 8-chloro-cAMP (8-Cl-cAMP) (and its metabolite 8-Cl-adenosine) against 107 fresh specimens of human neoplastic and normal cells. Diagnoses included chronic and acute leukaemias, myeloma, non-Hodgkin''s lymphoma (NHL) and miscellaneous solid tumours. The aim was to identify targets for subsequent phase I, II and III trials. 8-Cl-cAMP was tested at 4-985 microM, along with standard chemotherapeutic drugs. 8-Cl-cAMP and its metabolite caused no morphologically observable cell differentiation but induced dose-dependent cytotoxicity. Compared with untreated patients, previously treated chronic lymphocytic leukaemia (CLL) patients showed no increase in ex vivo resistance to 8-Cl-cAMP (P = 0.878); minimal cross-resistance with other cytotoxic drugs was detected. Compared with normal cells (mean LC90 = 1803 microM), 8-Cl-cAMP showed significant ex vivo activity against CLL (117.0 microM; P < 0.0001) and NHL (140.0 microM; P < 0.0001), of which eight were mantle cell NHL (84.7 microM), and greatest activity against cells from patients with acute myeloid leukaemia (AML; mean LC90 = 24.3 microM; in vitro therapeutic index 74-fold, P < 0.0001). Solid tumour specimens were comparatively resistant to 8-Cl-cAMP. The results highlight the clinical potential of 8-Cl-cAMP, point to several new phase I, II and III trial possibilities and provide a rationale for the inclusion of ex vivo cytotoxic drug evaluation in the drug development process.     

Expression of △DNMT3B Variants and Its Association with Estrogen/Progestogen Receptor Status in Breast Cancer

Objective  Our previous study has showed that ΔDNMT3B is the predominant form of DNMT3B in non-small cell lung cancer (NSCLC). In this study, we aimed to explore the expression patterns of the ΔDNMT3B variants in breast cancer and to identify whether the pattern was similar to that in NSCLC or not and its clinical significance. Methods  Expression of seven ΔDNMT3B variants in 59 breast cancer and the corresponding normal tissue was measured using RT-PCR. The correlations between the expressions of ΔDNMT3B variants and the clinical parameters including ER/PR status, clincopathologic feature and survivals were analyzed. Results  There were significant differences in the expression ratios of ΔDNMT3B1-7 variants between breast cancer tissues and normal tissues (P<0.001). The positive ratio of ΔDNMT3B1-7 variants were 66%, 71%, 17%, 51%, 76.2%, 50% and 61% in tumor tissue, respectively; while 16%, 8.4%, 3.38%, 3.38%, 11.8%, 13.5% and 5.08% in the corresponding normal tissue, which was different from the pattern of ΔDNMT3B1-7 expression in NSCLC (62%, 76%, 2.5%, 46%, 18%, 27% and 16% in tumor tissue, respectively; while 18%, 11%, 0%, 3.3%, 0%, 0% and 0% in normal lung tissue, respectively; P<0.0001). Expressions of ΔDNMT3B2, 3B4 and 3B7 were higher in the patients with negative estrogen receptor (ER) than those with positive estrogen receptor (P=0.035, P=0.0141 and P=0.0219, respectively). ΔDNMT3B7 expression was higher for the patients with negative progestogen receptor (PR) compared to those with positive progestogen receptor (P=0.0379). Expression ratio of ΔDNMT3B5 in stage III tumors is lower than that in stage I/II ones (P= 0.041). But we did not find any relation between the ΔDNMT3B variants and the patients’ survival. Conclusion  The pattern of ΔDNMT3B variants in breast cancer is different from that in NSCLC. Expressions of ΔDNMT3B2, 3B4 and 3B7 are associated with estrogen receptors status. While ΔDNMT3B7 is associated with progestogen receptor. No relation between the ΔDNMT3B variants and the patients’ survival were found. This work was supported by the grants from the National Natural Science Foundation of China (No.30572104, 30772472).     

The antitumor efficacy of cytotoxic drugs is potentiated by treatment with PNU 145156E, a growth-factor-complexing molecule

PNU 145156E (formerly FCE 26644) is a noncytotoxic molecule whose antitumor activity is exerted through the formation of a reversible complex with growth/angiogenic factors, thus inhibiting their induction of angiogenesis. We studied in vitro and in vivo the activity of PNU145156E in combination with the four cytotoxic drugs doxorubicin, cyclophosphamide, methoxymorpholinyldoxorubicin (MMDX, FCE 23762, PNU152243), and 9-aminocamptothecin against M5076 murine reticulosarcoma. In vitro, PNU 145156E did not modify the cytotoxicity of the four drugs or the cell-cycle block induced by doxorubicin. In vivo, at the optimal dose of each compound, the antitumor activity was significantly increased in all combinations, with no associated increase in general toxicity being observed. In healthy mice treated with cyclophosphamide or doxorubicin the association with PNU 145156E did not enhance the myelotoxic effect induced by the two cytotoxics. These results indicate that two drugs affecting solid tumor growth through two different mechanisms – growth factor blockage and cell proliferation – can be combined, resulting in increased antitumor efficacy with no additive toxicity. Received: 23 January 1998 / Accepted: 27 July 1998     

The potential of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide]to circumvent three multidrug-resistance phenotypes in vitro

 The effectiveness of N-[2-(dimethylamino) ethyl]acridine-4-carboxamide (DACA) relative to that of amsacrine, idarubicin, daunorubicin and paclitaxel against three different forms of multidrug resistance (MDR) was determined using two sublines of the CCRF-CEM human leukaemia cell line, the P-glycoprotein-expressing CEM/VLB100 subline and the MRP-expressing CEM/E1000 subline, and two extended-MDR sublines of the HL60 human leukaemia cell line, HL60/E8 and HL60/V8. DACA was effective against P-glycoprotein-mediated MDR and MRP- mediated MDR, whereas the extended-MDR pheno- type showed only low levels of resistance (<2-fold) to DACA. In comparison, idarubicin was ineffec- tive against the MRP and extended-MDR phenotypes. Repeated exposure of the K562 human leukaemia cell line to DACA (55, 546 or 1092 nM for 3 days over 10 weeks) did not result in the development of any significant drug resistance. We conclude that DACA has the potential to treat refractory leukaemia. Received: 5 May 1996/Accepted: 16 August 1996     

Lack of correlation between mitotic arrest or apoptosis and antitumor effect of docetaxel

Purpose: To determine, as we did for paclitaxel, whether mitotic arrest and apoptosis induced in murine tumors in vivo by docetaxel correlate with the drug's antitumor effect and whether the antitumor efficacy of docetaxel depends on p53 mutational status of tumors. Methods: C3Hf/Kam mice were implanted with one of the following 15 syngeneic tumors: seven adenocarcinomas (MCa-4, MCa-29, MCa-35, MCa-K, OCa-I, ACa-SG, and HCa-I), two squamous cell carcinomas (SCC-IV and SCC-VII), five sarcomas (FSa, FSa-II, Sa-NH, NFSa, and Sa-4020) and one lymphoma (Ly-TH). When the tumors had grown to 8 mm in diameter, the mice were treated with 31.3 mg/kg docetaxel i.v. Tumor growth delay was the endpoint of docetaxel's antitumor effect. In separate groups of mice, mitotic arrest and apoptosis were determined micromorphometrically 1 to 72 h after docetaxel treatment. Tumors were assayed for their p53 status by sequence analysis of RNA prepared from freshly excised tumors. Results: Docetaxel caused statistically significant growth delay in six of seven adenocarcinomas, three of five sarcomas, and the lymphoma, but not in either of the squamous cell carcinomas. The drug induced mitotic arrest in all tumor types, but to various degrees ranging from 6.4 +/− 0.4% to 25.1 +/− 0.1%. In contrast, docetaxel induced appreciable apoptosis in only 5 of 15 tumors, with 10.3 +/− 1.6% being the highest apoptotic value. Neither mitotic arrest nor apoptosis were significantly correlated with tumor growth delay. However, tumors that responded to docetaxel by significant tumor growth delay histologically displayed massive cell destruction by cell lysis, and four of these tumors also showed marked infiltration with mononuclear lymphoid cells. Of the 15 tumors only 3 had mutant p53. Conclusions: Docetaxel exhibited a strong antitumor effect in two-thirds of murine tumors, and on a milligram per kilogram basis was more effective than paclitaxel against the same tumors. The drug was a potent inducer of mitotic arrest but a weak inducer of apoptosis, neither of which correlated with its antitumor effect. Tumor cell lysis appeared to be a major mode of tumor cell destruction and can be regarded as the main mechanism underlying antitumor efficacy of docetaxel. In contrast, paclitaxel's antitumor efficacy is related to its ability to induce apoptosis. At the molecular level, there was no dependency of antitumor efficacy of docetaxel on p53 mutational status of tumors. Received: 3 March 1998 / Accepted: 14 May 1998     

Flavone acetic acid increases the cytotoxicity of mitomycin C when combined with hyperthermia

 Flavone acetic acid (FAA, NSC 347512) is known to selectively reduce tumor blood flow. Taking advantage of this pharmacodynamic effect, we have previously shown that FAA in combination with hyperthermia (HT) can produce a marked improvement in antitumor response in mice. In the present study, we investigated whether FAA could increase the cytotoxicity of mitomycin C (MMC), a bioreductive drug with selective cytotoxicity against hypoxic cells, under either normothermic or hyperthermic conditions. In vitro, the cytotoxicity of MMC against B16 melanoma cells was not enhanced with exposure to FAA at concentrations less than 100 μg/ml, even when combined with HT (43°C, 60 min). The cytotoxicity of MMC (1 μg/ml) at pH 6.5, however, was enhanced by exposure of cells to hypoxia in combination with HT. In vivo, the tumor growth time, calculated as the time required to double the initial tumor volume, was 5.2, 6.8, 8.5, and 15.0 days with FAA (150 mg/kg) alone, MMC (4 mg/kg) alone, FAA+MMC, or FAA+MMC+HT (43°C, 15 min) treatment groups, respectively. Antitumor response obtained in animals treated with FAA plus MMC with HT was clearly better than that obtained in any of the other groups. Scheduling of FAA, MMC, and HT was found to be important in producing optimal antitumor response. Administration of MMC (4 mg/kg) prior to FAA (150 mg/kg) and subsequent HT treatment was superior to administration of FAA before MMC. In an attempt to explain these findings, the influence of FAA on blood flow in skeletal muscle and in tumor was examined using a laser blood flowmeter. FAA administration to mice produced a 75% reduction in blood flow to the tumor for up to 2 h but had no detectable effect on normal skeletal blood flow. Our current explanation of the increased antitumor response achieved with the combination of MMC, FAA, and HT is as follows. The FAA-mediated decrease in blood flow to the tumor, when combined with HT, may produce sufficiently hypoxic conditions to significantly increase the antitumor efficacy of the bioreductive drug, MMC. We believe that clinical testing of this combined drug treatment with hyperthermia is warranted. Received: 1 February 1995/Accepted: 6 July 1995     

Patterns of cross-resistance in a multidrug-resistant small-cell lung carcinoma cell line

Summary H69AR is a multidrug-resistant human small-cell lung carcinoma cell line that was selected in doxorubicin and has previously been shown to be cross-resistant to a variety of natural-product-type anticancer drugs. H69AR is unlike many other multidrug-resistant cell lines in that it does not overexpress P-glycoprotein. In the present study, the drug sensitivity and cross-resistance patterns of H69AR cells were further characterized. A total of 15 drugs belonging to a number of chemical classes were screened. These compounds included anthracyclines, DNA binders (anthrapyrazoles, benzothiopyranoindazoles, and pyrazoloacridines), and lipophilic antifolates. The alkylating agent melphalan and the antimetabolite cytosine arabinofuranoside (Ara-C) were also tested. In general, the drug sensitivity and cross-resistance profiles of H69AR cells were consistent with those reported by others using other drug-resistant cell lines. However, there were several unexpected instances of cross-resistance. Thus, the H69AR cell line was more resistant than its parent cell line to the potent 3’-deamino-3’-(3-cyano-4-morpholinyl) doxorubicin, bisantrene, the pyrazoloacridine PD 114541, Ara-C, and melphalan. In addition, no cross-resistance to the four lipophilic antifolates tested, including trimetrexate, was found. The absence of a consistent pattern among the various drug-resistant cell lines indicates that assumptions about the efficacy of anticancer drugs in multidrug resistance should be made with caution. Supported by a grant from the National Cancer Institute of Canada Correspondence and proofs: S. P. C. Cole, Ph. D., Cancer Research Laboratories, Botterell Hall Rm 331. Queen’s University, Kingston, Ontario K7L 3N6 Canada, TEL 613-545-6358. FAX 613-544-9708     

Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate

Background:

We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors.

Methods:

We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)).

Results:

We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA.

Conclusions:

In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required.