Cytotoxicity of Trimetrexate against Antifolate-resistant Human T-Cell Leukemia Cell Lines Developed in Oxidized or Reduced Folate

Cytotoxicity of trimetrexate (TMQ), a lipophilic dihydrofolate reductase inhibitor, was examined in antifolate-resistant human T-cell leukemia cell lines developed in oxidized or reduced folate. An approximately 60-fold methotrexate (MTX)-resistant subline was developed in oxidized folate (pter-oylglutamic acid: PGA) (CCRF-CEM/MTX₆₀-PGA) from human T-cell leukemia cell line CCRF-CEM; this line exhibited impaired membrane transport of the drug. Further enhancement of MTX resistance resulted in selection of an approximately 5000-fold MTX-resistant subline (CCRF-CEM/ MTX₅₀₀₀-PGA), which showed increased dihydrofolate reductase activity due to gene amplification in addition to further impairment of MTX transport. An approximately 140-fold MTX-resistant subline, and then a 1500-fold MTX-resistant subline were developed in reduced folate (10 nM leucovorin) (CCRF-CEM/MTXm-LV and CCRF-CEM/MTX₁₄₀-LV); they exhibited increased dihydrofolate reductase due to gene amplification accompanied by increased intracellular drug accumulation of MTX. While CCRF-CEM/MTX₁₄₀-LV and CCRF-CEM/MTX₁₅₀₀-LV cells showed cross-resistance to TMQ, CCRF-CEM/MTX₆₀-PGA and CCRF-CEM/MTX₅₀₀₀-PGA cells were at least as sensitive to TMQ as the parent cells. TMQ was more potent against approximately 200-fold N¹⁰-propargyl-5,8-dideazafolic-acid (CB3717)-resistant human T-cell leukemia MOLT-3 sublines developed in PGA (MOLT-3/CB3717₂₀₀-PGA) or leucovorin (MOLT-3/CB3717₂₀₀-LV), as compared to the parent cells; MOLT-3/CB3717₂₀₀-PGA and MOLT-3/CB3717₂₀₀-LV cells were resistant to CB3717 by virtue of impaired transport, only the former possessing gene amplification of thymidylate synthase. The Cytotoxicity of TMQ in both MOLT-3/CB3717₂₀₀-PGA and MOLT-3/CB3717₂₀₀-LV cells was reduced by addition of leucovorin in a dose-dependent manner, suggesting intracellular folate deficiency as a cause of TMQ sensitivity. These results demonstrate that TMQ overcomes transport-impaired antifolate resistance, irrespective of gene amplification of dihydrofolate reductase or thymidylate synthase. Types of folate used during the development of antifolate resistance seem to be important in relation to the mechanism of TMQ responsiveness as well as that of antifolate resistance.     

A new analogue of 10-deazaaminopterin with markedly enhanced curative effects against human tumor xenografts in mice

Purpose: These studies sought to evaluate the biochemical and cellular pharmacokinetic properties, cytotoxicity and antitumor efficacy of a new analogue of 10-deaza-aminopterin (PDX) against human tumors. Methods: Studies were conducted with a group of human tumor cell lines in culture examining PDX and other folate analogues as permeants for mediated membrane transport, as inhibitors of dihdrofolate reductase and as substrates for folylpolyglutamate synthetase. These same analogues were examined for their cytotoxicity following a 3-h pulse exposure, in experiments providing a value for IC₅₀. Other studies with these analogues were conducted in nude mice bearing subcutaneously implanted human tumors. Treatment of the mice was initiated 4 days after implantation of the tumor using a schedule of administration of one dose per day for 5 days. The tumors were measured 6 days after cessation of therapy and compared to controls for assessment of response. Results: In the CCRF-CEM cell system, PDX was 2- to 3-fold less effective as an inhibitor of dihydrofolate reductase than aminopterin (AMT), methotrexate (MTX) or edatrexate (EDX) but much more effective as a permeant for one-carbon, reduced folate transport inward (PDX >AMT ≃ EDX >MTX) and substrate for folylpolyglutamate synthetase (PDX >AMT >EDX >MTX). As predicted by these results, PDX was 15- to 40-fold more cytotoxic than MTX and 3- to 4-fold more cytotoxic than the highly potent EDX following a 3-h pulse exposure in culture of CCRF-CEM cells and cells from a panel of three human breast and two human nonsmall-cell (NSC) lung cancers. The same relative differences were shown for the therapeutic efficacy of these three analogues at equitoxic doses in studies with the human MX-1 and LX-1 tumors and the human A549 NSC lung tumor xenografted in nude mice. On a schedule of qd × 5 given 3–4 days posttransplant, MTX was minimally active (modest tumor growth delay) against all three tumors. EDX was highly active (25–35% complete regressions and 5–10% cures) against the MX-1 and LX-1 tumors but very modestly active (no regressions) against the A549 tumor. In contrast, PDX was even more active (75–85% complete regressions and 25–30% cures) than EDX against the MX-1 and LX-1 tumors and highly active (30% complete regressions and 20% cures) against the A549 tumor. Conclusions: These studies showed significantly enhanced antitumor properties of PDX compared with MTX and EDX. Based upon these results, clinical trials of PDX in patients with metastatic breast and NSC lung cancer appear to be warranted. Received: 24 September 1997 / Accepted: 16 December 1997     

Antitumor activity and cytotoxicity of a new ankinomycin derivative, 3′-, 11-dibutyryl ankinomycin

Ankinomycin is a new antitumor antibiotic found in the culture broth ofStreptomyces sp. SF2587. Ankinomycin showed marked cytotoxicity and antitumor activity against some murine leukemias, but the activity against murine solid tumors was rather weak because of its strong acute toxicity. We synthesized ankinomycin acyl derivatives and examined their antitumor activity. Among the derivatives, 3, 11-dibutyryl ankinomycin (AN1006) exhibited the highest antitumor activity. The antitumor activity of AN1006 was dependent on the administration schedule, and on the most effective schedule, AN1006 showed activity comparable with that of Adriamycin (ADM) against murine solid tumors and leukemias. AN1006 showed a cytotoxic spectrum different from that of ADM, exhibiting cytotoxicity stronger than that of ADM against colon carcinoma, stomach carcinoma, and some leukemia cell lines. According to these in vitro effects, AN1006 showed antitumor activity superior to and equal to that of ADM against human colon xenografts and stomach carcinoma xenografts in athymic nude mice, respectively. AN1006 was effective against multidrugresistant tumors in vitro and in vivo. AN1006 is an interesting candidate for further evaluation.Abbreviations ANK ankinomycin - AN1006 3-, 11-dibutyryl ankinomycin - ADM Adriamycin - ILS increase in life span - ILS_max the maximal value of increase in life span - IC₅₀ concentration required for 50% inhibition of cell growth - P388/ADM Adriamycin-resistant P388 cells - LLC Lewis lung carcinoma - CEM/VLB₁₀₀ vinblastine-resistant CCRF-CEM cells - 2780AD Adriamycin-resistant A2780 cells - KBC-4 colchicine-resistant KB3-1 cells - K562/ADM Adriamycin-resistant K562 cells - MCF-7/ADM Adriamycin-resistant MCF-7 cells This research was supported by grants from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare, Japan     

The protein kinase C inhibitor CGP 41251, a staurosporine derivative with antitumor activity,reverses multidrug resistance

Multidrug resistance (MDR) is frequently associated with overexpression of a 170-kDa P-glycoprotein (Pgp). Data suggest altered protein kinase C (PKC) activity in cells expressing the multidrug-resistant phenotype. The staurosporine derivative CCP A1251, an experimental anticancer drug, has been shown to exert selectivity for inhibition of protein kinase C activity and to exhibit antitumor activity in Wtro and in vivo. Here we show that CGP A1251 is also able to reverse MDR. After treatment of the mult id rug-resistant human lymphoblastoid cell line CCRF-VCR1000 with 500 nM Adriamydn, cell proliferation was reduced to 81% of untreated controls. A combination of 500 nM Adriamydn with a non-toxic concentration of 150 nM CGP 41251 (IC₅0 for inhibition of cell proliferation 420 nM CGP 41251) inhibits cell proliferation of CCRF-VCR1000 cells to 29% of untreated controls. In sensitive CCRF-CEM cells no enhancement of Adriamycin-induced cytotoxicity was observed upon addition of 150 nM CGP 41251. Strong synergism of the inhibition of cell proliferation was also observed after concomitant treatment of KB-851 I cells with CGP 41251 and Vinblas-tine or Adriamydn. Drug-sensitive KB-31 cells could not be further sensitized to Adriamycin or Vinblastine with CGP 41251 doses above 100 nM. Pretreatment with 50-1000 nM CGP 41251 for 30 min led to a dose-dependent increase in the intracellular accumulation of rhodamine 123, a substrate of P-glycoprotein. Treatment of multidrug-resistant CCRF-VCR1000 cells with CGP 41251 for 10 min was sufficient to inhibit the efflux of rhodamine 123. Preincubation with CGP 41251 for 12 or 24 hr did not alter multidrug resistance gene (mdrl)-mRNA levels. CGP 41251, a drug with antitumor efficacy in experimental systems, might offer an attractive combination partner for the treatment of tumors expressing the MDR phenotype. © Wiley-Liss, Inc.     

Reversal of doxorubicin,etoposide, vinblastine,and taxol resistance in multidrug resistant human sarcoma cells by a polymer of spermine

We have previously descibed the synthesis of a cytotoxic polymeric conjugate of spermine (Poly-SPM) which is able to inhibit the transport of polyamines (spermine, spermidine, and putrescine) into normal and malignant cells. Recent studies examining the toxicity of Poly-SPM in parental and multidrug resistant (MDR) cancer cells have revealed a cross- resistance in the MDR variant Dx5 to the toxic effects of the conjugate in the MDR-positive cells. There were also differences in spermine and putrescine uptake rates between parental and MDR-positive cells with the MDR-positive cells having a lower V_max and a higher K_m. The ability of this Poly-SPM to reverse MDR was examined in MDR variants (Dx5 cells) of the human sarcoma cell line MES-SA. The cells express high levels of the mdr1 gene product, P-glycoprotein, and are 25- to 60-fold resistant to doxorubicin (DOX), etoposide (VP-16), vinblastine (VBL), and taxol (TAX). Cytotoxicity was measured by the MTT [3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Poly-SPM (50?μM) lowered the drug concentration IC₅₀ values in the Dx5 cells by 37-fold with VBL, 42-fold with DOX, 29-fold with VP-16, and 25-fold with TAX when compared to the control IC₅₀ values without Poly-SPM. This reversal of resistance was concentration dependent, decreasing 17-fold with DOX, 6.1-fold with VBL, 19-fold with VP-16, and 5-fold with TAX when 25?μM Poly-SPM was used. No modulation was observed in the parental cell line MES-SA, which does not express the mdr1 gene. Poly-SPM had no influence on the IC₅₀ of non-MDR chemotherapeutic agents such as cisplatin. The modulation studies correlated with the ability of Poly-SPM to reverse the cellular accumulation defect of [³H]-VBL and [³H]-TAX in the Dx5 but not MES-SA cells. Pretreatment of the Dx5 cells with α-difluoromethylornithine (DFMO at 2 and 5?μM) for 24?h increased the function of the MDR transporter to further decrease the cellular accumulation of VBL and TAX when compared to untreated cells. DFMO pretreatment is known to up-regulate the polyamine transporter(s). These findings show that, in addition to inhibiting polyamine transport, Poly-SPM reverses MDR in Dx5 cells, suggesting a potential relationship between the polyamine influx transporter and the MDR efflux pump. This potential functional link between the polyamine influx transporter(s) and the MDR efflux transporter (P-glycoprotein) offers a novel approach to inhibiting this form of drug resistance.     

Cytotoxicity of taxol in vitro against human and rat malignant brain tumors

Taxol is a novel antitumor alkaloid that has shown clinical activity against several tumors, including ovarian and breast carcinoma and melanoma. To evaluate taxol's potential as a therapy for malignant brain tumors, we measured the sensitivity of four human (U87, U373, H80, and D324) and two rat (9L, F98) brain-tumor cell lines to taxol. The cells were exposed to taxol in vitro using a clonogenic assay. Log cell kill (LD₉₀) occurred at concentrations of 42 (9L), 25 (F98), 19 (H80), 7.2 (U373), 9.1 (U87), and 3.9 nM (D324) when cells were continuously exposed to taxol for 6–8 days. The human cell lines were uniformly more sensitive to taxol than were the rat lines. The duration of exposure had a significant effect on taxol's cytotoxicity. When cells were exposed to taxol for 1 h the LD₉₀ increased to 890 nM for the 9L rat line and 280 nM for the human U373 line. On the basis of these results, we conclude that taxol has significant potency in vitro against malignant brain tumors and that the activity occurs at concentrations of taxol that have previously been shown to be effective for several tumors against which the drug is currently being evaluated clinically.     

Reversal activity of cyclosporin A and its metabolites M1, M17 and M21 in multidrug-resistant cells

Cyclosporin A (CSA) is an effective inhibitor of the P-glycoprotein (P-gp) activity and has been shown to modulate multidrug resistance (MDR) in in vitro experimental models. During degradation of CSA, the metabolites arising from the parental compound reach high levels in the serum of patients, and it is not clear whether these metabolites maintain the reversal activity of the parental compound, like the metabolites of verapamil. In an in vitro experimental model, we compared the reversal activity of CSA and 3 CSA metabolites (M1, M17, and M21) in the range of concentrations obtained in whole blood during a clinical trial with CSA used as a revertant agent. As experimental model we used LoVo-resistant cells. Our in vitro studies indicated that the metabolic hydroxylation and demethylation of CSA lead to molecules that greatly differ from the parent drug in their reversal activity. In the range of concentration detected in the whole blood of the patients (1–3 μM), CSA had a significant reversal activity. It decreased the IC₅₀ of anti-neoplastic drugs involved in MDR (vincristine, taxol, doxorubicin and etoposide) but not the IC₅₀ of platinum or methotrexate. CSA increased intracellular doxorubicin content and inhibited P-gp ³[H]azidopine photolabeling. Conversely, CSA metabolite concentrations superimposable to those observed in the patients (0.5–2.2 μM) had no sensitizing effects on the cytotoxicity of MDR-related anti-neoplastic drugs, nor did they affect ³[H]azidopine photolabeling or doxorubicin uptake. This study demonstrates that, during degradation of CSA, metabolite derivatives arise that have a very different reversal activity from that of the parental compound. Int. J. Cancer 71: 900-906, 1997. © 1997 Wiley-Liss Inc.     

Involvement of MDR1 P-glycoprotein in multifactorial resistance to methotrexate

Cellular resistance to methotrexate (MTX) is believed to be unaffected by expression of MDRI P-glycoprotein (Pgp), a pleiotropic efflux pump acting on different hydrophobic compounds that enter cells by passive diffusion. A series of human leukemic CCRF-CEM sublines, isolated by multi-step selection for very high resistance to MTX, exhibit multiple mechanisms of MTX resistance, including decreased carrier-mediated uptake of MTX and DHFR gene amplification. These sublines show cross-resistance to drugs of the multi-drug resistance (MDR) family, which is correlated with relative resistance to MTX. The MTX-selected sublines show increased expression and function of the MDRI gene, based on the measurement of MDRI mRNA, Pgp and rhodamine 123 accumulation. Sequence analysis of the MDRI cDNA from MTX-selected CCRF-CEM cells revealed no mutations in the protein coding region. MTX resistance in these cell lines is partially reversible by a Pgp-specific monoclonal antibody (MAb) UIC2 and a monovalent Fab fragment of UIC2. Our results indicate that Pgp can contribute to multifactorial resistance to MTX. © 1996 Wiley-Liss, Inc.     

Antitumor activity of four macrocyclic ellagitannins from Cuphea hyssopifolia

We evaluated the antitumor activities of four macrocyclic hydrolyzable tannin dimers, cuphiin D₁, cuphiin D₂, oenothein B and woodfordin C isolated from Cuphea hyssopifolia (Lythraceae). All significantly inhibited the growth of the human carcinoma cell lines KB, HeLa, DU-145, Hep 3B, and the leukemia cell line HL-60, and showed less cytotoxicity than adriamycin against a normal cell line (WISH). All four compounds inhibited the viability of S-180 tumor cells in an in vitro assay and an in vivo S-180 tumor-bearing ICR mice model. Oenothein B demonstrated the greatest cytotoxicity (IC₅₀=11.4 μg/ml) against S-180 tumor cells in culture, while cuphiin D₁ resulted in the greatest increase in survival on S-180 tumor-bearing mice (%ILS=84.1%). Our findings suggest that the antitumor effects of these compounds are not only related to their cytotoxicity on carcinoma cell lines, but also depended on a host-mediated mechanism; they may therefore have potential for antitumor applications.     

Comparative in vitro cytotoxicity of taxol and Taxotere against cisplatin-sensitive and-resistant human ovarian carcinoma cell lines

Summary Using the sulforhodamine B assay, we compared the cytotoxic properties of the novel microtubule agent taxol and the semi-synthetic related compound Taxotere in nine human ovarian-carcinoma cell lines, including three pairs of cell lines rendered resistant to cisplatin or carboplatin. In addition, the cytotoxicity of the commonly used anticancer drugs cisplatin and adriamycin and the topoisomerase II inhibitor etoposide was determined. The results of continuous drug exposure showed that taxol [mean concentration producing 50% growth inhibition (IC₅₀), 1.1×10^–9 m; range, 2.8×10^–9–5×10^–10 m and Taxotere (mean IC₅₀, 5.1×10^–10 m; range, 7.2–3.3×10^–10 m) were >1,000 times more cytotoxic than either cisplatin (mean IC₅₀, 3.1×10^–6 m;P<0.05) or etoposide (mean IC₅₀, 2.3×10^–6 m;P<0.05) and >100 times more cytotoxic than Adriamycin (mean IC₅₀, 6.9×10^–8 m;P<0.05). Taxotere was more cytotoxic than taxol; following continuous exposure, the mean difference across the cell lines was 2 orders of magnitude (range, 1.1–3.9 orders of magnitude for individual lines). Although this difference did not reach statistical significance for any individual cell line (P values ranged from 0.17 for HX/62 to 0.9 for OVCAR-3), when all IC₅₀ values for the 96-h experiments were pooled, Taxotere was found to be significantly more potent than taxol (P=0.05). Following 2 h exposure, the mean cytotoxicity of Taxotere was 3.9-fold > that of taxol across the nine lines (range, 0.75- to 10-fold;P<0.05 for the CH1 cell line; overall pooled IC₅₀ data,P=0.05). Although a 71-fold range of sensitivity to cisplatin was observed across the six parent cell lines (IC₅₀ most resistant line/IC₅₀ most sensitive line), this was largely abolished by treatment with taxol (5.6-fold range) and Taxotere (2.2-fold rante). Following continuous exposure of the three pairs of lines exhibiting acquired resistance to platinum, no cross-resistance with either Taxotere or taxol was found (resistance factors, <1.5). In the 41M and 41McisR pair of lines, in which previous studies have shown resistance to be due to reduced platinum accumulation, taxol and Taxotere exhibited some collateral sensitivity (resistance factors, 0.69 and 0.66, respectively). Taxotere and, particularly, taxol showed a pronounced concentration times exposure duration (CxT) dependence as compared with cisplatin (P<0.05). The mean loss in potency across the nine lines for 2 vs 96 h exposure was 97 for taxol, 35 for Taxotere, 30 for Adriamycin and only 9.9 for cisplatin. However, these differences in potency loss observed between taxol and Taxotere did not reach statistical significance (P=0.18). These data indicate that Taxotere is approximately 2 times more cytotoxic than taxol and shows an encouraging lack of cross-resistance in three cell lines exhibiting acquired resistance to cisplatin and carboplatin.This study was supported by grants to the Institute of Cancer Research, Royal Cancer Hospital, from the Cancer Research Campaign and, through the European Organisation for Research and Treatment of Cancer (EORTC) Clonogenic Assay Screening Group, by grant 90031 from Rhone-Poulenc Rorer.     

Modulatory effect of tamoxifen and ICI 182,780 on adriamycin resistance in MCF-7 human breast-cancer cells

In this study the ability of the new pure anti-estrogen ICI 182,780 to modulate the cytotoxic action of adriamycin (ADR) on parental and ADR-resistant MCF-7 (MCF-7 ADRr) human breast-cancer cells was investigated and compared with that of tamoxifen (TAM). TAM enhanced ADR cytotoxicity in MCF-7 ADRr cells in a dose-related manner, but this effect was slight or absent in MCF-7 WT. In contrast, ICI 182,780 was able to enhance ADR toxicity both in MCF-7 ADRr and in the parental cell line. ICI 182,780 was up to 2.5-fold more effective than TAM in reducing the IC₅₀ of ADR in MCF-7 ADRr cells. Analysis of the data by the isobole method showed that the combination ADR/TAM and ADR/ICI 182,780 produced synergistic anti-proliferative activity in MCF-7 ADRr cells. Because ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of anti-estrogens on Pgp expression and activity. Both ICI 182,780 and TAM failed to modulate Pgp expression as assessed by flow cytometry and Western-blot analysis, performed using the monoclonal antibodies MM4.17 and C219, which are specific for an external or an internal determinant respectively. Pgp activity was investigated by flow cytometry measuring the extrusion of ADR and the cationic dye Rhodamine 123 (Rh 123). ICI 182,780, but not TAM, reduced the activity of Pgp in MCF-7 ADRr cells. Flow cytometry was also used to investigate cell-cycle modifications induced by ADR in MCF-7 ADRr cells, both in the presence and in the absence of anti-estrogens. After 72 hr, higher doses induced an arrest of cells at the G₂/M phase. The same effect was visible when lower doses of ADR were combined with ICI 182,780 or TAM. In terms of cell-cycle-blocking activity ICI 182,780 was largely more effective than TAM. © 1996 Wiley-Liss, Inc.     

Sensitization by interleukin-lα of carboplatinum anti-tumor activity against human ovarian (NIH:OVCAR-3) carcinoma cells in vitro and in vivo

Cytokines are directly cytotoxic to tumor cells in vitro and in vivo, and interleukin-1α (IL-1α) potentiates the cytotoxicity of a number of clinically active drugs in several human tumor cells, including carcinomas of the breast and ovary. In this study, we found that IL-1α potentiated the cytotoxicity of carboplatin in human ovarian NIH:OVCAR-3 cancer cells during simultaneous drug exposure in vitro. Human ovarian carcinoma NIH:OVCAR-3 cells are resistant to clinically relevant concentrations of chemotherapeutic agents, including cisplatin. Both carboplatin and IL-1α as single agents inhibited the growth of NIH:OVCAR-3 cells grown as xenograft in nude mice; however, carboplatin was more effective in delaying tumor growth, and this inhibition was dose-dependent. Treatment with IL-1α or followed by carboplatin caused a significant delay in tumor growth, resulting in a significant enhancement (4-fold) of the anti-tumor effect of carboplatin. In vitro potentiation of carboplatin cytotoxicity by IL-1α did not involve formation of nitric oxide as IL-1α or combinations of IL-1α with carboplatin failed to modulate basal nitric oxide production in OVCAR-3 cells. Potentiation of the anti-tumor activity of carboplatin by IL-1α was due to a significant (3- to 4-fold) increase in the accumulation of total Pt in IL-1α-treated tumor xenograft, resulting in a 2-fold increase in DNA-Pt adduct formation in these tumors. In contrast, IL-1α had no significant effect on DNA-Pt adduct formation in the kidney. The potent synergy of IL- I1α and carboplatin in vitro and in vivo against ovarian carcinoma cells suggests that combinations of carboplatinum and interleukin-1α may be effective against this disease in the clinic. (This article is a US Government work and. as such. is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Cytotoxicity of the anticancer agents cisplatin and taxol during cell proliferation and the cell cycle

The overt effects of the anti-cancer drugs cisplatin (cis-DDP) and taxol appear to be DNA modification and microtubule stabilization respectively, yet the mechanisms by which these drugs elicit tumor cell death are not well understood. In this report cell sensitivities to cis-DDP and taxol were accurately determined as a function of cell proliferation and cell cycle stage. Quiescent fibroblasts restimulated to synchronously enter the cell cycle become maximally sensitive to cis-DDP immediately preceding DNA synthesis, and resistance increases with onset of DNA synthesis. Mid-log proliferating cells were separated into progressive stages of the cell cycle by centrifugal elutriation or by double thymidine (dThd) block. Cells staged by either method are maximally sensitive to cis-DDP in G₁, just prior to the onset of DNA synthesis and minimally sensitive in peak DNA synthesis, with entry into S phase resulting in a 2-fold decrease in sensitivity. Cells that remained blocked at the G₁/S phase boundary during cis-DDP treatment remain maximally sensitive after release. Sensitivity to taxol increases at 2 points: transiently during transition of normal cells from quiescence to proliferation and steadily as proliferating cells progress from early G₁ to late G₂. This 3-fold increase in taxol sensitivity through the cell cycle is rapidly reversed upon cell division. Synchronous cells treated with either drug at points of maximum sensitivity initiate apoptotic DNA fragmentation 12-14 hr post-exposure to drug. © 1994 Wiley-Liss, Inc.     

Reversal of vinblastine resistance by a new staurosporine derivative, NA-382, in P388/ADR cells.

Activities of a newly synthesized compound, N-ethoxycarbonyl-7-oxo-staurosporine (NA-382), on cyclic AMP-dependent protein kinase (A-kinase), Ca2+/phospholipid dependent protein kinase (C-kinase), and drug resistance were investigated and compared with those of staurosporine. Protein kinase-inhibitory activity of NA-382 was lower but more selective to C-kinase than that of staurosporine. NA-382 was less toxic to P388 cells and at a non-cytotoxic concentration completely reversed the vinblastine (VBL) resistance of Adriamycin-resistant P388 (P388/ADR) cells without influence on the effect of VBL on the parental P388/S cells. However, the cytotoxicity of staurosporine was too high to give the combination effect with VBL. NA-382 dose-dependently increased VBL-accumulation and inhibited VBL-efflux in P388/ADR with higher potency than staurosporine. Both compounds inhibited the photolabeling of [3H]azidopine on 140-kDa P-glycoprotein in the plasma membrane from the resistant cells. These results suggest that a staurosporine analog, NA-382, reverses multidrug resistance by inhibiting the drug-efflux system or P-glycoprotein.     

Multidrug-resistant human kb carcinoma cells are highly resistant to the protein phosphatase inhibitors okadaic acid and calyculin A. Analysis of potential mechanisms involved in toxin resistance

In this study we show that multidrug-resistant (MDR) human KB-VI cells are highly resistant to the cytotoxicity of okadaic acid and calyculin A, 2 toxins from marine sponges that are potent inhibitors of type-I and type-2A protein phosphatases (PP1 and PP2A). Cytotoxicity and colony-forming assays indicated that, relative to parental drug-sensitive KB-3 cells, KB-VI cells are 35-fold more resistant to okadaic acid and 70-fold more resistant to calyculin A. Cytotoxicity of the toxins was associated with mitotic arrest characterized by chromosome scattering and over-condensation, with KB-3 cells being more sensitive than KB-VI cells and calyculin A being more potent than okadaic acid. The resistance of KB-VI cells to both okadaic acid and calyculin A was completely reversed by verapamil, suggesting that the toxins may be transported by P-glycoprotein (P-gp). To further assess the possibility of an interaction with P-gp, the toxins were employed as potential modulators of the photoaffinity labeling of P-gp by [³H]azidopine. Relative to vinblastine, which effectively competed with [³H]azidopine for P-gp photolabeling, calyculin A was 100-fold less potent and okadaic acid did not inhibit photolabeling at concentrations up to 50 μM. To determine whether the resistance mechanism involved differences in toxin-sensitive phosphatase activity, the activity was assayed in extracts from both cell lines and found to be slightly higher (1.6-fold) in KB-VI than in KB-3 cells. Our results demonstrate a novel, marked resistance of MDR KB-VI cells to these phosphatase inhibitors and suggest that a major mechanism of resistance may involve toxin transport by P-gp at sites apparently different from those which bind azidopine.     

Identification of novel immunogenic human leukocyte antigen-A 2402-binding epitopes of human papillomavirus type 16 E7 for immunotherapy against human cervical cancer

BACKGROUND:

A study was undertaken to identify new immunogenic human leukocyte antigen (HLA)‐A*2402‐restricted epitopes from human papillomavirus (HPV) type 16 E7 protein for immunotherapy against cervical cancer.

METHODS:

Synthetic overlapping peptides were screened by measuring the frequency of CD8⁺ cytotoxic T lymphocytes (CTLs) producing intracellular interferon‐γ (IFN‐γ) using flow cytometry and were validated in SiHa cells with a Cr release cytotoxicity assay. In vivo antitumor effects of peptide‐sensitized peripheral blood mononuclear cells (PBMCs) and isolated CD8⁺ CTLs were evaluated using BALB/c nude mice with SiHa cell xenotransplants.

RESULTS:

Among 14 overlapping 15‐amino acid peptides, E7_61‐75(CDSTLRLCVQSTHVD) and E7_67‐81(LCVQSTHVDIRTLED) induced significantly higher IFN‐γ production (P < .05) and showed higher in vitro cytotoxicity against SiHa cells than did cells sensitized with the negative control. To determine the exact HLA‐A*2402‐restricted epitopes, a total of 25 overlapping 9‐ or 10‐amino acid peptides spanning E7_61‐75 and E7_67‐81 were synthesized. E7_61‐69(CDSTLRLCV) and E7_67‐76(LCVQSTHVDI) induced significantly greater IFN‐γ production as well as increased in vitro cytotoxicity against SiHa cells compared with those of other peptides and the negative control (P < .01), and the antitumor effects of these peptide‐sensitized PBMCs were induced by CD8⁺ CTLs. E7_61‐69‐sensitized and E7_67‐76‐sensitized PBMCs and isolated CD8⁺ CTLs showed a much greater suppression of tumor growth in vivo compared with that of control groups treated with PBS (P < .01). The authors also confirmed the synergistic antitumor effect of cisplatin followed by E7_67‐76‐sensitized PBMCs in vivo.

CONCLUSIONS:

E7_61‐69 and E7_67‐76 were identified as novel HPV type 16 E7 epitopes for HLA‐A*2402, which could be used for immunotherapy against cervical cancer. Cancer 2012. © 2011 American Cancer Society.     

Anti-human lung giant cell cancer (PG) effect of human LAK cellsin vitro and in nude mice

Human LAK cells were prepared by culturing normal human peripheral blood mononuclear cells (PBMC) with or without rIL-2 and assayed for T cell surface markers as well as anti-tumor activity against PGin vitro and in nude mice. Although the percentages of T₃, T₄ and T₈ positive cells in rIL-2-activated cells did not differ significantly from those of control cellsin vitro, the former showed stronger cytotoxicity than control cells to PG tumor cellsin vitro. In vivo, LAK cells completely inhibited the growth of PG tumor in nude mice, whereas PBMC control cells were to be of no effect. The anti-tumor effect of human LAK cells in nude mice may offer a useful model to study the role of human LAK cells against human tumorin vivo.     

Characterization of tetrandrine,a potent inhibitor of P-glycoprotein-mediated multidrug resistance

Multidrug resistance (MDR) is one of the main obstacles in tumor chemotherapy. A promising approach to solving this problem is to utilize a nontoxic and potent modulator able to reverse MDR, which in combination with anticancer drugs increases the anticancer effect. Experiments were carried out to examine the potential of tetrandrine (Tet) as a MDR-reversing agent. Survival of cells incubated with Tet at 2.5 mol/l for 72 h was over 90%. Tet at 2.5 mol/l almost completely reversed resistance to vincristine (VCR) in KBv200 cells. Tet at a concentration as low as 0.625 mol/l produced a 7.6-fold reversal of MDR, but showed no effect on the sensitivity of drug-sensitive KB cells in vitro. In the KBv200 cell xenograft model in nude mice, neither Tet nor VCR inhibited tumor growth. However, VCR and Tet combined inhibited tumor growth by 45.7%, 61.2% and 55.7% in three independent experimental settings. In the KB cell xenograft model in nude mice, Tet did not inhibit tumor growth, but VCR and the combination of VCR and Tet inhibited tumor growth by 40.6% and 41.6%, respectively. Mechanism studies showed that Tet inhibited [³H]azidopine photoaffinity labeling of P-gp and increased accumulation of VCR in MDR KBv200 cells in a concentration-dependent manner. The results suggest that Tet is a potent MDR-reversing agent in vitro and in vivo. Its mechanism of action is via directly binding to P-gp and increasing intracellular VCR accumulation.Abbreviations DMSO Dimethyl sulfoxide - FBS Fetal bovine serum - MDR Multidrug resistance - MTT 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide - PBS Phosphate-buffered saline - P-gp P-glycoprotein - SDS Sodium dodecyl sulfate - Tet Tetrandrine - VCR Vincristine