Combination Of VP-16 Research Articles

Optimal chemo-mobilization for the collection of peripheral blood stem cells in patients with multiple myeloma

BackgroundFor successful autologous stem cell transplantation, the collection of a sufficient number of hematopoietic stem cells after induction therapy is essential for transplant candidates with multiple myeloma (MM).MethodsIn this study, we compared the efficacy and safety of stem cell mobilization using cyclophosphamide (CY; 3.0 g/m2 on day 1) or etoposide (VP-16; 375 mg/m2 on days 1 and 2) in patients with MM. Granulocyte-colony stimulating factor (G-CSF, 10 μg/kg/day, subcutaneously) was administered from the onset of neutropenia to the final day of collection.ResultsSixty-five patients were mobilized with a combination of CY and G-CSF, and 63 were mobilized with a combination of VP-16 and G-CSF. All patients were mobilized within 7 months of beginning frontline treatment. The median number of CD34+ cells collected was significantly higher in the VP-16 mobilization group than in the CY mobilization group (27.6 × 106 CD34+/kg vs. 9.6 × 106 CD34+/kg, P < 0.001). The rate of mobilization failure, defined as < 2.0 × 106 CD34+/kg collected in three apheresis procedures, was lower in the VP-16 group than in the CY group (1.6% vs. 10.8%, P = 0.062). Severe infections during the mobilization period were more frequent in the CY group than in the VP-16 group (18.5% vs. 7.9%, P = 0.117).ConclusionIn conclusion, an intermediate dose of VP-16 with G-CSF appears to be an effective and tolerable chemo-mobilization method compared to CY and G-CSF, particularly in cases where use plerixafor in MM is difficult.

Schedule-Dependent Cytotoxicity of Etoposide (VP-16) and Cyclophosphamide in Leukemia Cell Line K-562

In allogeneic bone marrow transplantation (allo-BMT) in patients with leukemia, the combination of VP-16 and cyclophosphamide (CY) is commonly used for the conditioning regimen. In the present study, we demonstrated schedule-dependent cytotoxicity of VP-16 and CY in K-562 cells. K-562 cells were pretreated with low concentrations (2.5 and 5 µg/mL) of 4-hydroperoxycyclophosphamide (40487S), which is a preactivated analog of CY. It was confirmed that these concentrations did not influence cell viability. Cells subsequently exposed to 0.5-100 µg/mL of VP-16 showed reduced the viability compared to that of control cells not treated with 40487S. In contrast, there was no change in the viability of K-562 cells pretreated with low concentrations (0.5 and 1 µg/mL) of VP-16. It was confirmed that these concentrations did not influence cell viability. Viability of subsequently exposed to 1-20 µg/mL was not different from that of control cells not treated with VP-16. VP-16 caused cell cycle arrest at G₂/M phase. On the other hand, 40487S arrested the cell cycle at S phase. Thymidine-synchronized cells, VP-16 showed cell cycle specificity for cell killing from early-S to mid-S phase. On the other hand, 40487S showed cell cycle-independent cytotoxicity. Exposure of cells to VP-16 after 40487S induced a greater cytotoxic effect on K-562 cells. The findings may lead to improvements in clinical combination chemotherapy.

Etoposide, mitomycin, and methotrexate combination in heavily treated breast cancer: a retrospective study

Since 2004, metastatic breast cancer patients pretreated with anthracyclines, taxanes, and capecitabine have been treated in our institution with a combination of mitomycin C, methotrexate, and VP-16 (VMM). We report in this study a retrospective analysis of the activity and safety of the VMM combination. Patients were treated with a combination of VP-16 (100 mg/m2 on day 1), mitomycin C (MMC, 10 mg/m2 on day 1), and methotrexate (MTX, 12.5 mg/m2 twice a day on day 2 and 3) in a 21-day cycle. Seventy-five patients were treated. Median age was 48 years. A total of 256 cycles were administered. Median relative dose intensities were 0.87, 0.87, and 0.95 for VP-16, MMC, and MTX, respectively. Objective response rate was 31%, with a clinical benefit rate of 47%. Median response duration was 5.8 months. Median disease stabilization duration was 9.1 months. Median progression-free survival (PFS) was 4.2 months with a 14% 1-year PFS rate. Median overall survival (OS) was 6.2 months, with a 25% 1-year OS rate. Myelosuppression was the most common toxicity. The most commonly reported extra-hematological adverse event (AE) was fatigue. Emesis and alopecia were rarely reported. This combination appears to be effective and well tolerated in this heavily pretreated metastatic breast cancer population.

Moxifloxacin enhances etoposide-induced cytotoxic, apoptotic and anti-topoisomerase II effects in a human colon carcinoma cell line

Etoposide (VP-16) is a topoisomerase-II (topo II) inhibitor chemotherapeutic agent. Studies have shown that a combination of VP-16 with other drugs demonstrates better clinical responses. The aim of this study was to investigate the effects of moxifloxacin (MXF) and VP-16 on cellular topo II activity in drug-treated cells and evaluate the influence of MXF on the mode of action of VP-16, on proliferation and apoptosis of HT-29 cells. Decatenation assay, band depletion and Western blot analysis, cytotoxic assay (MTT), flow cytometric studies (cell cycle and survivin expression), apoptosis (DAPI-sulforhodamine staining and caspase 3 activity) and IL-8 and VEGF secretion were determined. MXF or VP-16 slightly affected cellular topo II activity in nuclear extracts derived from drug-treated cells while the combination enhanced inhibitory activity and the reduction in band depletion of topo II. VP-16 induced cell cycle arrest at G2/M and the appearance of the subG1 peak which was increased by the addition of MXF. Apoptosis studies (DAPI staining and caspase 3 activity) showed a marked increase in the presence of MXF and VP-16 compared to VP-16 alone. VP-16 induced the release of IL-8, and addition of MXF reduced enhanced release and the spontaneous release of VEGF from the cells. In conclusion, the results suggest that the enhancement in the reduction of topo II activity by the combined MXF/VP-16 treatments was probably due to the increase in the level of the DNA-enzyme cleavable complexes formed by both drugs. The unique combination of MXF/VP-16 may have clinical benefits and a cytotoxic drug 'sparing effect' and should be further studied in vivo.

Abstract 5431: Histone deacetylase inhibitor (HDAC) panobinostat (LBH589) enhances the antiproliferative effect of a topoisomerase II (topo II) inhibitor in doxorubicin-resistant HL-60 cells, despite high MDR expression

Abstract Acute myeloid leukemia (AML) is difficult to treat, particularly in the relapsed or refractory setting due to drug resistance. Topo II inhibitors are effectively used to treat AML, and histone deacetylase (HDAC) inhibitors have been used pre-clinically and clinically with some success. We previously reported that vorinostat (SAHA) was effective in combination with topo II inhibitors in AML cells (Proc. AACR, 2009, Abstract 4567). In the present study we evaluate LBH589, a novel class I/II HDAC inhibitor. Since resistance to HDAC inhibitors has not been well described, we hypothesized that LBH589 would be effective in combination with a topo II inhibitor in a doxorubicin-resistant AML cell line that expresses MDR and resistant by MDR-independent mechanism. A sensitive HL60 parent line (S) and the doxorubicin-resistant sub-line (R) were used. Early studies revealed that vorinostat (SAHA) had comparable anti-proliferative effects in S &amp; R cells but the R cells were relatively more resistant to LBH589. This resistance was likely MDR-mediated. We then tested whether sub-lethal doses of SAHA or LBH589, used in combination with sub-lethal doses of etoposide (VP-16), would be effective. S and R cells were treated with VP-16 for 1 h, washed, and re-incubated with SAHA or LBH, or drug-free media, for a total of 144 hours. Cell viability and apoptosis were measured at 72 h and 144 h following treatment. At 72 h and at 144 h, the combination of VP-16 and LBH was significantly more effective (p&amp;lt;0.01) in reducing proliferation in HL60/S and HL60/R cells, compared to either agent alone. At both 72 h and 144 h the combination of VP-16 and LBH also led to significantly (p&amp;lt;0.05) greater apoptosis in both S &amp; R cells. Western blotting for phospho-histone H2AX, a marker of DNA damage, showed increased DNA damage in the R cells treated with VP-16 and LBH589 compared to either drug alone. Overall, the effects of LBH589 in combination with VP-16 were significantly (p&amp;lt;0.05) more effective than SAHA in the S &amp; R cells. In summary, our results demonstrate that MDR may mediate resistance to LBH589. Further, in spite of MDR expression, LBH589 is a useful adjunct to VP-16 treatment and enhances the anti-proliferative effect of the topo II inhibitor via increased apoptosis, even in the doxorubicin-resistant cells. Thus, post-treatment with LBH589 in combination with a topo II inhibitor may be useful treatment strategy in relapsed / refractory AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5431.

Long‐term renal and hearing toxicity of carboplatin in infants treated for localized and unresectable neuroblastoma: Results of the SFOP NBL90 study

A secondary end point of the NBL90 protocol (Rubie H et al. Pediatr Oncol 2001;36:247-250) was the concern in this infant population for possible carboplatin-(CBDCA) induced late side effects including impaired renal and hearing functions. Glomerular filtration rate (GFR), tubular function (TF), pure tone audiometry (PTA), high-frequency, and transient evoked-otoacoustic emission were prospectively assessed in 30 children alive and disease-free 6 years after the end of the treatment. Median age at diagnosis and at assessment was 4.7 months and 7 years, respectively. Blood pressure was < or =97.5 centile in all children. The mean estimated GFR was 114 +/- 13 ml/min/1.73 m(2) by Schwartz formula [range 87-145]. TF assessment failed to demonstrate any impairment. 29/30 children had grade 0 ototoxicity and all transient evoked otoacoustic emission were normal. With a 6-year follow-up the combination of VP16 and carboplatin given at conventional doses is safe on renal and hearing functions in infants with unresectable neuroblastomas treated according to SFOP NB90.

Direct interaction of the Krüppel-like family (KLF) member, BTEB1, and PR mediates progesterone-responsive gene expression in endometrial epithelial cells.

The present study was undertaken to evaluate the underlying mechanism(s) by which PR and a Krüppel-like family member, basic transcription element binding protein (BTEB1), mediate endometrial epithelial expression of pregnancy-associated genes. Human endometrial carcinoma cell lines (Hec-1-A) expressing high and low levels of BTEB1 were transiently transfected with a human PR isoform (PR-B) expression construct and a luciferase reporter gene driven by the uteroferrin gene promoter that is responsive to both BTEB1 and the PR ligand progesterone. Unliganded PR inhibited luciferase activity in low and high BTEB backgrounds, and this effect was reversed by the synthetic progestin R5020 in both lines. Transactivation by PR of uteroferrin promoter activity (approximately 4-fold) was maximal at lower R5020 concentrations (10 nM) in endometrial cells with higher BTEB1 expression, suggesting that nuclear BTEB1content influenced target gene promoter sensitivity to progesterone. BTEB1 and PR-B were found to physically interact in a progesterone-independent manner, using a coimmunoprecipitation assay that employed antibodies specific to either protein. Moreover, the formation of the BTEB1/PR complex, independent of progesterone, occurred within the context of uterine endometrial proteins and was diminished in late-pregnancy endometrium. Mammalian two-hybrid assays using the entire open reading frame of BTEB1 and/or PR-B fused to either the GAL4 DNA-binding domain or VP16 activation domain and a reporter gene (pG5CAT) under the control of GAL4-binding sites were used to evaluate the formation of functional PR-B/BTEB1 dimer in Cos-1 cells. GAL4/PR-B and VP16/PR-B induced ( approximately 3- to 4-fold) chloramphenicol acetyltransferase (CAT) activity in a progesterone-dependent manner, suggesting PR-dimer formation. By contrast, VP16/PR-B and GAL4/BTEB1 had no effect on basal CAT activity. The combination of VP16- and GAL4-PR-B fusion proteins with the BTEB1 expression construct, pCDNA3-BTEB1 enhanced ligand-bound PR-mediated CAT activity by approximately 3-fold. In transient cotransfection assays using the CAT reporter gene driven by the mouse mammary tumor virus-long terminal repeat promoter, which is responsive to ligand-bound PR but not BTEB1, BTEB1 increased PR-B-mediated CAT activity in a progesterone-dependent manner, consistent with a BTEB1/PR-dimer complex occurring independent of BTEB1 binding to DNA. Unliganded PR-B disrupted the DNA-binding activity of BTEB1 in gel retardation assays, and this effect was enhanced by the presence of PR ligand. Together, these findings support the conclusion that BTEB1 and PR-B are coregulatory proteins that mediate progesterone responsiveness of target genes by direct interactions, leading to the formation of a functional BTEB1/PR-dimer complex.

Reversible Thiol-Dependent Activation of Ryanodine-Sensitive Ca2+Release Channel by Etoposide (VP-16) Phenoxyl Radical

Many phenolic compounds can act as antioxidants by donating a proton to peroxyl radicals and quenching lipid peroxidation. Phenoxyl radicals produced this way or from metabolism by peroxidases, tyrosinase, or mixed-function oxidases, however, may react with sulfhydryl groups of proteins and other endogenous thiols. In this regard, phenolic compounds may have cytotoxic potential instead of antioxidant effects. We employed the anticancer drug, etoposide (VP-16), as a model phenolic compound to study the sensitivity of ryanodine-sensitive Ca2+ channel (RyR) to VP-16 phenoxyl radicals. The combination of VP-16 and tyrosinase, used to generate the etoposide phenoxyl radical, produced marked Ca2+ release from Ca2+-loaded RyR-rich vesicles prepared from terminal cisternae fraction of sarcoplasmic reticulum (SR). This effect was reversed by the SH-reagent, dithiothreitol (DTT), suggesting that cysteines within the RyR-protein complex were targets for modification by VP-16 phenoxyl radicals. VP-16/tyrosinase-induced release of Ca2+ was attenuated in vesicles prepared from longitudinal SR, which contain relatively little RyR. The effects of the VP-16 phenoxyl radical on Ca2+-ATPase in SR vesicles resembled those observed with caffeine or 4,4'-dithiodipyridine, both of which activate RyR Ca2+ release and lead to activation of Ca2+-ATPase via prolonged Ca2+ cycling. The addition of ruthenium red returned Ca2+-ATPase to its original level. Thus, under these conditions Ca2+-ATPase was not directly affected by VP-16 phenoxyl radical. The hypersensitive SH-groups on RyR are shown to be targets for oxidation of VP-16 phenoxyl radical, and suggest that other phenolic compounds could similarly disrupt Ca2+ homeostasis.

Salvage chemotherapy for oligodendroglioma.

The authors present their experience with salvage chemotherapy for oligodendroglioma, an uncommon brain tumor that responds predictably to PCV (procarbazine, lomustine (CCNU), and vincristine) when given as initial therapy. The authors reviewed the records of patients with oligodendrogliomas who received a second, third, or fourth cytotoxic regimen prescribed to combat tumor recurrence documented by computerized tomography or magnetic resonance imaging following an initial chemotherapy program. Initial regimens were prescribed at various time points: as neoadjuvant therapy prior to radiotherapy, as adjuvant therapy in conjunction with radiotherapy, or at recurrence following radiotherapy. Response criteria were based on measurable changes in tumor size following published guidelines. Twenty-three patients (14 men and nine women) aged 25 to 58 years (median 36 years) received 33 salvage regimens. When non-PCV chemotherapy had been the prior regimen, seven (88%) of eight patients responded to salvage chemotherapy, all seven (100%) responding to PCV. Administration of PCV was effective after regimens of carmustine and CCNU but was ineffective after prior administration of PCV. When PCV had been given any time previously, only four (19%) of 21 patients responded to salvage chemotherapy; however, four (40%) of 10 patients who received etoposide (VP-16)/cisplatin (CDDP) responded. Despite the small number of patients, two noteworthy trends emerge from these data: first, PCV is a highly effective salvage treatment when used at tumor recurrence following non-PCV chemotherapy regimens, and second, the synergistic combination of VP-16 and CDDP may exert substantial anti-oligodendroglioma activity, and it warrants further evaluation.

Salvage chemotherapy for oligodendroglioma

The authors present their experience with salvage chemotherapy for oligodendroglioma, an uncommon brain tumor that responds predictably to PCV (procarbazine, lomustine (CCNU), and vincristine) when given as initial therapy. The authors reviewed the records of patients with oligodendrogliomas who received a second, third, or fourth cytotoxic regimen prescribed to combat tumor recurrence documented by computerized tomography or magnetic resonance imaging following an initial chemotherapy program. Initial regimens were prescribed at various time points: as neoadjuvant therapy prior to radiotherapy, as adjuvant therapy in conjunction with radiotherapy, or at recurrence following radiotherapy. Response criteria were based on measurable changes in tumor size following published guidelines. Twenty-three patients (14 men and nine women) aged 25 to 58 years (median 36 years) received 33 salvage regimens. When non-PCV chemotherapy had been the prior regimen, seven (88%) of eight patients responded to salvage chemotherapy, all seven (100%) responding to PCV. Administration of PCV was effective after regimens of carmustine and CCNU but was ineffective after prior administration of PCV. When PCV had been given any time previously, only four (19%) of 21 patients responded to salvage chemotherapy; however, four (40%) of 10 patients who received etoposide (VP-16)/cisplatin (CDDP) responded. Despite the small number of patients, two noteworthy trends emerge from these data: first, PCV is a highly effective salvage treatment when used at tumor recurrence following non-PCV chemotherapy regimens; second, the synergistic combination of VP-16 and CDDP may exert substantial antioligodendroglioma activity, and it warrants further evaluation.

Mechanisms of resistance of human small cell lung cancer lines selected in VP-16 and cisplatin

The combination of VP-16 and cisplatin is one of the most active regimens available for the treatment of small cell lung cancer (SCLC), however, most tumors eventually become resistant to these drugs. To investigate the problem of resistance to VP-16 and cisplatin in patients with SCLC, we established two resistant sublines from the drug sensitive human SCLC line, NCI-H209, by in vitro selection in VP-16 and cisplatin. The VP-16-selected cell line, H209/VP, was more than 100-fold resistant to VP-16, and displayed cross-resistance to VM-26 and other topoisomerase II interactive drugs, but not to vinca alkaloids. There was no difference in accumulation of VP-16 in H209/VP compared with its parent cell line. The level of topoisomerase II-alpha was reduced to 8% of that in the parent cell line, and there was an altered form of this enzyme with a molecular weight of 160 kilodaltons (kDa), in addition to the normal 170 kDa protein. The cisplatin-selected cell line, H209/CP, was 11.5-fold resistant to cisplatin, with only a low level of cross-resistance to other platinum compounds including carboplatin, tetraplatin, iproplatin, and lobaplatin. This line was highly cross-resistant to vinca alkaloids, but not to anthracyclines or epipodophyllotoxins. The H209/CP cell line was not resistant to cadium chloride, suggesting that alterations in metallothionein are unlikely to be a cause of resistance. Although glutathione (GSH) levels were increased nearly 2-fold in H209/CP, there was no difference in levels of the GSH-related enzymes glutathione-S-transferase, glutathione peroxidase, and glutathione reductase, compared with the parent line. The H209/CP line had a 1.4-fold elevation of topoisomerase II-alpha. The accumulation of cisplatin was reduced in this cell line, and there were fewer DNA-interstrand cross links formed in the presence of cisplatin in H209/CP, compared with the parent line. Neither H209/VP nor H209/CP expressed MDR1, the gene for P-glycoprotein. The MRP gene was expressed at a slightly higher level in the H209/VP cell line, but there was no significant increase in expression of this gene in the H209/CP cell line. The resistance of the H209/VP cell line is associated with an alteration of topoisomerase II-alpha, whereas the resistance in the H209/CP line is associated with reduced drug accumulation.

Difference in CDDP penetration into CSF between selective intraarterial chemotherapy in patients with malignant glioma and intravenous or intracarotid administration in patients with metastatic brain tumor.

Platinum (Pt) levels in plasma and cerebrospinal fluid (CSF) in patients with malignant glioma were determined after initiation of selective intraarterial chemotherapy with a combination of VP-16 (etoposide) and CDDP (cisplatin), and were compared with the CSF Pt levels in patients with metastatic brain tumors after intravenous or intracarotid administration of VP-16 and CDDP. CSF Pt levels were also compared for various administration routes, doses, CSF sampling routes and blood-CSF barriers in metastatic brain tumor. Changes in the blood-CSF barrier to CDDP during treatment in a patient with meningeal lymphoma and in a patient recovering from surgical removal of a metastatic brain tumor were also examined by periodic administration of CDDP. All CSF samples were taken through Ommaya reserviors placed in the anterior horn of the lateral ventricle or the postoperative cavity. The mean peak CSF/plasma total Pt ratio (T/T ratio) and the mean CSF total Pt/plasma ultrafiltrable Pt ratio (T/U ratio) were highest (15.0% and 24.4%, respectively) following selective intraarterial infusion of CDDP in patients with malignant glioma, followed by intravenous infusion in meningeal carcinomatosis (11.5% and 18.9%), intracarotid administration (5.4% and 8.7%) and intravenous infusion (60 mg/m2 2.5% and 100 mg/m2 2.9%; and 60 mg/m2 3.5% and 100 mg/m2 7.7%) in patients with the solid type of metastatic brain tumor. In CSF obtained from the postoperative cavity in cases of metastatic brain tumor, T/T and T/U ratios were extremely high (40.9% and 62.4%). However, the CSF Pt level even after selective intraarterial administration of CDDP in malignant glioma was 0.51-1.64 micrograms/ml total Pt and 0.43-1.08 micrograms/ml ultrafiltrable Pt. Even the CSF level obtained from the postoperative cavity was 1.0-4.7 micrograms/ml total Pt. These low levels of total and ultrafiltrable Pt are considered not to be cytotoxic to disseminated cells in the CSF space and to normal brain cells. As for changes in the blood-CSF barrier, repeated administration of CDDP showed that the rate of entry of Pt into the CSF decreased in parallel with improvements apparent on CT scans in the patient with meningeal lymphoma, and also showed that the blood-CSF barrier to Pt was gradually repaired after the metastatic brain tumor had been removed.

179 P - Second line chemotherapy in HIV-related non-hodgkin's lymphoma: Evidence of activity of a combination of VP16, mitoxantrone and prednimustine in relapsed patients

Purpose To evaluate the feasibility and activity of a second line chemotherapy regimen consisting of VP16, mitoxantrone and prednimustine (VMP) in patients with relapsed or resistant HIV related non-Hodgkin's lymphoma (HIV-NHL). Patients and methods Twenty-one patients were consecutively treated Thirteen patients were resistant to primary chemotherapy and eight patients had relapsed after first complete remission (CR). VP16 and prednimustine were both given orally at doses of 80 mg/m 2 daily for 5 days, and mitoxantrone was given i.v. at a dose of 10 mg/m 2 on day 1; cycles were repeated every three weeks. Results Nineteen out of 21 patients are evaluable for response. The median number of cycles actually administered was 2 (range 1–5). A CR occurred in 5 out of 19 patients (26%; exact 95% confidence interval: 9% to 51%). Four of these CRs were observed in the 7 evaluable relapsed patients. Out of 45 cycles evaluable for toxicity, severe neutropenia ( Conclusion Although the overall prognosis of patients with resistant and relapsed HIV-NHL is very poor, palliative therapy with VMP can be effective and relatively safe. Prolonged survivals have been observed in some patients who had relapsed after initial chemotherapy. Supported by AIRC 95 grants.

Etoposide and carboplatin in extraocular retinoblastoma: a study by the Société Française d'Oncologie Pédiatrique.

A phase II study of etoposide (VP-16) and carboplatin was performed in patients with extraocular retinoblastoma to evaluate the response rate with this drug combination. Twenty patients with extraocular retinoblastoma, age 9 to 120 months, were included in a cooperative multicenter phase II study of the Société Francçaise d'Oncologie Pédiatrique (SFOP). The schedule consisted of consecutive 5-day treatment with VP-16 100 mg/m2/d and carboplatin 160 mg/m2/d. The response rate for the 20 patients was 85%; there were nine complete responses and eight partial responses. Hematologic toxicity was the only serious observed toxicity and was always manageable. This combination of VP-16 and carboplatin is highly effective in extraocular retinoblastoma. The high response rate is encouraging for further evaluation of this drug combination in adjuvant chemotherapy when necessary after enucleation or in neoadjuvant chemotherapy for intraocular tumors.

In vivo inhibition of etoposide-mediated apoptosis, toxicity, and antitumor effect by the topoisomerase II-uncoupling anthracycline aclarubicin.

A number of clinically important drugs such as the epipodophyllotoxins etoposide (VP-16) and teniposide (VM-26), the anthracycline daunorubicin and doxorubicin (Adriamycin), and the aminoacridine amsacrine exert their cytotoxic action by stabilizing the cleavable complex formed between DNA and the nuclear enzyme topoisomerase II. We have previously demonstrated in several in vitro assays that the anthracycline aclarubicin (aclacinomycin A) inhibits cleavable-complex formation and thus antagonizes the action of drugs such as VP-16 and daunorubicin. The present study was performed to validate these in vitro data in an in vivo model. At nontoxic doses of 6 and 9 mg/kg, aclarubicin yielded a marked increase in the survival of non-tumor-bearing mice given high doses of VP-16 (80-90 mg/kg) in six separate experiments. In therapy experiments on mice inoculated with Ehrlich ascites tumor cells, aclarubicin given at 6 mg/kg roughly halved the increase in median life span induced by VP-16 at doses ranging from 22 to 33 mg/kg. An attempt to determine a more favorable combination of VP-16 and aclarubicin by increasing VP-16 doses failed, as the two drugs were always less effective than VP-16 alone. The way in which VP-16-induced DNA strand breaks lead to cell death remains unknown. However, VP-16 has been reported to cause apoptosis (programmed cell death) in several cell lines. To ascertain whether the protection given by aclarubicin could have a disruptive effect on the apoptotic process, we used the small intestine as an in vivo model. Whereas VP-16-induced apoptosis in crypt stem cells was detectable at a dose as low as 1.25 mg/kg, aclarubicin given at up to 20 mg/kg did not cause apoptosis. Indeed, aclarubicin caused a statistically significant reduction in the number of cells rendered apoptotic by VP-16. The present study thus confirms the previous in vitro experiments and indicates the value of including an in vivo model in a preclinical evaluation of drug combinations.

Phase II Trial of VP-16, Bleomycin, and Cisplatin in Patients with Advanced Nonsquamous Cell Head and Neck Neoplasms

We observed a 45% response rate from the combination of VP-16, bleomycin, and cisplatin among 20 patients with nonsquamous cell head and neck cancer. The regressions were partial and typically occurred within 1 to 2 months of commencing treatment. The median response duration for responding patients was 3 months. Median survival of responders was 8 months, similar to that of all study participants. Gastrointestinal and hematologic sequelae were predictable and manageable. This regimen may provide some transient palliation for selected patients with these neoplasms, but no substantive impact on survival.

Our limb salvage experiences in the patients with osteosarcoma

Our limb salvage experiences in the with osteosarcoma is the subject of this study. Twenty-five patients with osteosarcoma who were refered to our clinic were evaluated for the possibility of the Iimb salvage procedures. Findings of the preoperative examinations were discussed at the weekly council. Five of them were found to be suitable for the Iocal wide excission. Combination of VP-16, CTX, ADR and intra-arterial CDDP was prefered for the neo-adjuvant chemotherapy. Two of the five discontinued the treatment. The effect of the neoadjuvant chemotherapy was not satisfactory in two patients and radical amputations were applied. In the 5th patient at the end of the neoadjuvant chemotherapy, 90% necrosis was seen and Iocal wide excision and the arthrodesis of the knee was performed.

Topoisomerase Inhibitors Have Potent Differentiation‐inducing Activity for Human and Mouse Myeloid Leukemia Cells

DNA topoisomerase inhibitors, camptothecin and 4′‐demethylepipodophyllotoxin ethylidene‐jS‐D‐glucoside (VP16) had strong differentiation‐inducing activity for all five kinds of leukemia cells examined (human HL60, U937, ML1, and K562 cells and mouse Ml cells) as judged from measurements of various differentiation markers. The characteristics that appeared as a result of differentiation induced by these inhibitors were essentially similar in every cell line. Exposure to VP16 for 2 h induced both differentiation and DNA‐strand breaks in K562 cells, whereas podophyl‐lotoxin, which lacks topoisomerase II inhibitory activity, induced neither differentiation nor DNA‐strand breaks in these cells. These results suggest a parallelism between the induction of differentiation and that of DNA‐strand breaks. The combination of VP16 and recomhinant tumor necrosis factor α (rTNFα) synergistically induced differentiation of human U937, ML1, and M1 cells and had an additive effect on HL60 cells. Simultaneous treatment with rTNFa plus camptothecin or VP16, or pretreatment with camptothecin or VP16, followed by rTNFα induced marked differentiation of Ml cells. These results indicate that inhibition of topoisomerase (either topoisomerase I or II) followed by the action of rTNFα was effective in inducing differentiation of leukemia cells.

Potentiation of tnf‐mediated cell killing by VP‐16: Relationship to DNA single‐strand break formation

Interaction between tumor necrosis factor (TNF) and the DNA topoisomerase II inhibitor, etoposide VP-16, in cell killing has been studied. To accurately investigate the nature of DNA damage during the cell killing process, experiments were assessed using the highly TNF-sensitive WEHI164.13 murine fibrosarcoma clone and DNA filter elution methodology. Concomitant treatment of cells with combination of TNF/VP-16 resulted in marked enhancement of cell lysis. Using the alkaline elution technique, we show that TNF fails to induce DNA single-strand breaks as compared to those generated by VP-16. In addition, the potentiating effect of VP-16 on TNF-mediated WEHI164.13 cell killing was not associated with an increase in its intrinsic activity with respect to DNA single-strand break formation. While the 2 phospholipase A2 inhibitors, quinacrine and dexamethasone, were efficient in inhibiting TNF-mediated cell lysis, only quinacrine was efficient in selectively abrogating the TNF/VP-16 cell killing pathway. The inhibitory effect of quinacrine on VP-16/TNF-mediated cell lysis was accompanied by a marked decrease in VP-16-mediated DNA single-strand break generation. Taken together, our findings suggest that TNF and TNF/VP-16 treatments may involve different events during cell killing and support the hypothesis that 2 signals are required for optimal induction of cell lysis by the combination of VP-16/TNF: one signal provided by VP-16 resulting in topoisomerase II inhibition and subsequent DNA single-strand break generation, and a second signal involving TNF.

Response of osteogenic sarcoma to the combination of etoposide and cyclophosphamide as neoadjuvant chemotherapy

A Phase II study of the combination of etoposide (VP-16) and cyclophosphamide (CPM) was conducted in an attempt to identify active and potentially less toxic agents for treating patients with osteogenic sarcoma (OS). VP-16 was given as a 72-hour infusion for a total dose of 600 mg/m2. CPM was given as six pulses of 300 mg/m2 every 12 hours for a total dose of 1800 mg/m2. Seventeen newly diagnosed patients, including five (29%) with metastatic disease, were evaluated before and after two courses of VP-16 and CPM for clinical, radiologic, and biochemical (serum alkaline phosphatase [SAP]) responses of the primary tumor and metastases. Fifteen (88%) patients achieved complete or partial clinical responses. Fourteen (82%) patients achieved radiologic responses. Thirteen (87%) of 15 patients with higher than normal SAP levels for their age showed partial or complete responses. Three (60%) of the five patients with metastatic disease achieved complete or partial responses. The only major toxicity was myelosuppression, which led to 21 (62%) brief admissions after 34 courses of chemotherapy for intravenous antibiotic therapy for fever and neutropenia, without associated mortality. It was concluded that the combination of VP-16 and CPM is effective chemotherapy for both primary and metastatic OS. Although myelosuppression is inevitable, it is rapidly reversible in the drug dosages used. Further studies are needed to evaluate the effect of these drugs in combination with established agents in improving the disease-free survival of patients with OS.