Combined Drug Exposure Research Articles

Optimization of a tri-drug treatment against lung cancer using orthogonal design in preclinical studies.

A growing body of evidence suggests that anesthetics impact the outcome of patients with cancer after surgical intervention. However, the optimal dose and underlying mechanisms of co-administered anesthetics in lung tumor therapy have been poorly studied. Here, we aimed to investigate the role of combined anesthetics propofol, sufentanil, and rocuronium in treating lung cancer using an orthogonal experimental design and to explore the optimal combination of anesthetics. First, we evaluated the effects of the three anesthetics on the proliferation and invasion of A-549 cells using Cell Counting Kit 8 and Transwell migration and invasion assays. Subsequently, we applied the orthogonal experimental design (OED) method to screen the appropriate concentrations of the combined anesthetics with the most effective antitumor activity. We found that all three agents inhibited the proliferation of A-549 cells in a dose- and time-dependent manner when applied individually or in combination, with the highest differences in the magnitude of inhibition occurring 24 h after combined drug exposure. The optimal combination of the three anesthetics that achieved the strongest reduction in cell viability was 1.4 µmol/L propofol, 2 nmol/L sufentanil, and 7.83 µmol/L rocuronium. This optimal 3-drug combination produced a more beneficial result at 24 h than either single drug. Our results provide a theoretical basis for improving the efficacy of lung tumor treatment and optimizing anesthetic strategies.

Combined Treatment with JQ1 and Curcumin Synergically Modulates Apoptosis, Cell Cycle Features, Micrornas and Gene Expression in High-Risk Pediatric Acute Myeloid Leukemia (AML)

BackgroundDespite recent progresses in the treatment of childhood AML, the outcome of high-risk patients (carrying selected molecular aberrations and/or having high levels of minimal residual disease at the end of therapy) remains dismal. In particular, patients harboring FLT3-ITD or some translocations involving MLL (such as MLL-AF10 or MLL AF4) still have a poor prognosis, mainly due to leukemia recurrence.A key feature of FLT3-ITD and MLL-rearranged leukemia is cMYC overexpression, a well-known oncogene often involved in cancer. JQ1 is a BET inhibitor, preventing cMYC expression through the binding to the BET bromodomain protein, BRD4. Moreover, Curcumin is a compound able to induce epigenetic changes, including inhibition of histone-acetyltransferases (HATs), resulting in a decrease of acetylated lysine residues, a target for BRD4 bound on DNA.AimWe evaluated whether the combination of JQ1 and Curcumin has a potential synergic effect on AML cell lines or primary blasts harboring FLT3-ITD and MLL-rearrangement. These experimental data could be instrumental for developing novel treatment options alternative or complementary to standard chemotherapy.MethodsCell lines with FLT3-ITD and/or MLL fusion proteins used in this study were: MV-4-11, THP1, MOLM-13 and RS-4-11 [ALL cell line harboring t(4;11)]. Cells were all treated with JQ1 [250nM], while the dose of Curcumin varied according to the MLL-rearrangement (5μM for MV-4-11 and RS-4-11 carrying MLL-AF4 rearrangement and 10μM for THP1 and MOLM-13 harboring MLL-AF9 rearrangement). After 48 hours of either single-drug or combined treatment (J+C), western blot, XTT assay, RT-qPCR and Annexin V FACS analysis were carried out to assess proliferation rates, apoptosis and expression of their related genes, as well as the expression of cMYC and AcH3K14. Capacity to form colonies after either single or combined drug exposure was tested on bone marrow derived CD34+ cells from 4 patients with either MLL-rearranged acute leukemia (AML MLL-AF9; ALL MLL-AF4; AML MLL-AF4) or FLT3-ITD using Methocult media.miRNA expression profile of 20 BM samples of either FLT3-ITD or MLL-rearranged AML collected at both diagnosis (Dx) and at relapse (Rel) was assessed with Nanostring platform. Results were compared with those obtained analyzing BM samples of 8 healthy controls (HCs) and of 12 patients with the same molecular lesions who did not relapse (NRel). Expression levels of the modulated miRNAs were also evaluated on the cell lines reported above. A supplemental miRNA profiling analysis was conducted comparing cells harboring FLT3-ITD with those carrying MLL-rearrangements with respect to HCs.ResultsIn comparison with treatment with either of the 2 agents, the J+C combination resulted into to a synergic effect on apoptosis rate of MOLM-13 cells and of RS-4-11 cells. In particular, the apoptosis rates of the former cell line were 16% with JQ1, 10% with Curcumin and 56% with J+C, while those of the latter cell line were 20% with JQ1, 4% with Curcumin and 52% with J+C, respectively. The XTT metabolic assay showed significant reduction in 24 and 72-hour proliferation rates after both single-agent and combined treatments in all cell lines compared to DMSO (2WayANOVA; P<0.05)RT-qPCR and western blot experiments showed an increased expression of BCL2, cleaved CASPASE-3, cleaved PARP1 and p21 levels, as well as a decrease of cMYC and AcH3K14. Single-agent and combined treatment also resulted into a significant decrease of number of colonies in all the 4 primary cultured patient blasts (Fisher's exact t-test; P<0.05). 118 miRNAs were found to be significantly modulated in samples of Rel and NRel groups compared with those of HCs, while 18 miRNAs were found to be modulated in either MLL-rearranged or FLT3-ITD AML as described in the Figure1, 2, and Table 1, 2. We also noticed an opposite expression trend for miR-99 and miR-34a in MLL-AF4 AML and MLL-AF9 AML, and significant modulations of expression after single-agent and J+C treatment for miR-99, miR-34a, miR-451 and miR-196a in our cell lines.ConclusionsOur data demonstrate that J+C exert an intense synergic effect on AML cell lines and primary blasts, leading to increased apoptosis, reduced proliferation rate, modulation in expression of some key genes, such as cMYC, BCL2 and AcH3K14. We also identified distinct miRNA signatures in patients collected at relapse as compared to those obtained at diagnosis or HCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proinflammatory cytokine profile is critical in autocrine GH-triggered curcumin resistance engulf by atiprimod cotreatment in MCF-7 and MDA-MB-231 breast cancer cells.

Active growth hormone (GH) signaling triggers cellular growth and invasion-metastasis in colon, breast, and prostate cancer. Curcumin, an inhibitor of NF-ҡB pathway, is assumed to be a promising anti-carcinogenic agent. Atiprimod is also an anti-inflammatory, anti-carcinogenic agent that induces apoptotic cell death in hepatocellular carcinoma, multiple myeloma, and pituitary adenoma. We aimed to demonstrate the potential additional effect of atiprimod on curcumin-induced apoptotic cell death via cytokine expression profiles in MCF-7 and MDA-MB-231 cells with active GH signaling. The effect of curcumin and/or atiprimod on IL-2, IL-4, and IL-17A levels were measured by ELISA assay. MTT cell viability, trypan blue exclusion, and colony formation assays were performed to determine the effect of combined drug exposure on cell viability, growth, and colony formation, respectively. Alteration of the NF-ҡB signaling pathway protein expression profile was determined following curcumin and/or atiprimod exposure by RT-PCR and immunoblotting. Finally, the effect of curcumin with/without atiprimod treatment on Reactive Oxygen Species (ROS) generation and apoptotic cell death was examined by DCFH-DA and Annexin V/PI FACS flow analysis, respectively. Autocrine GH-mediated IL-6, IL-8, IL-10 expressions were downregulated by curcumin treatment. Atiprimod co-treatment increased the inhibitory effect of curcumin on cell viability, proliferation and also increased the curcumin-triggered ROS generation in each GH+ breast cancer cells. Combined drug exposure increased apoptotic cell death through acting on IL-2, IL-4, and IL-17A secretion. Forced GH-triggered curcumin resistance might be overwhelmed by atiprimod and curcumin co-treatment via modulating NF-ҡB-mediated inflammatory cytokine expression in MCF-7 and MDA-MB-231 cells.

Abstract 3503: Gilenya and Alimta interact to kill breast cancer cells

Abstract The present studies sought to determine whether the anti-folate pemetrexed (Alimta) and the sphingosine-1-phosphate receptor modulator FTY720 (Fingolimod, Gilenya) interacted to kill tumor cells. FTY720 and pemetrexed interacted in a greater than additive fashion to kill breast, brain and colorectal cancer cells. Loss of p53 function weakly enhanced the toxicity of FTY720 whereas deletion of activated RAS strongly or expression of catalytically inactive AKT facilitated killing. In contrast, expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the toxic interaction between FTY720 and pemetrexed. Indeed, combined drug exposure reduced the activity of AKT, p70 S6K and mTOR and activated JNK and p38 MAPK. Expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the toxic interaction between FTY720 and pemetrexed. Treatment of cells with FTY720 and pemetrexed increased the numbers of autophagosomes in cells that correlated with increased LC3 processing and increased p62 levels. Knock down of ATG5 or Beclin1 suppressed autophagosome formation and cell killing by the drug combination. Sorafenib was previously shown to potently interact with pemetrexed to kill tumor cells. Sorafenib significantly enhanced the lethality of [FTY720 + pemetrexed] treatment in vitro and in vivo. Collectively our findings argue that pemetrexed, sorafenib and FTY720 could be a novel adjunct modality for breast cancer treatment. Note: This abstract was not presented at the meeting. Citation Format: Jane L. Roberts, Seyedmehrad Tavallai, Andrew Poklepovic, Laurence Booth, Paul Dent. Gilenya and Alimta interact to kill breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3503. doi:10.1158/1538-7445.AM2015-3503

Differential regulation of autophagy and cell viability by ceramide species

The present studies sought to determine whether the anti-folate pemetrexed (Alimta) and the sphingosine-1-phosphate receptor modulator FTY720 (Fingolimod, Gilenya) interacted to kill tumor cells. FTY720 and pemetrexed interacted in a greater than additive fashion to kill breast, brain and colorectal cancer cells. Loss of p53 function weakly enhanced the toxicity of FTY720 whereas deletion of activated RAS strongly or expression of catalytically inactive AKT facilitated killing. Combined drug exposure reduced the activity of AKT, p70 S6K and mTOR and activated JNK and p38 MAPK. Expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the interaction between FTY720 and pemetrexed. Treatment of cells with FTY720 and pemetrexed increased the numbers of early autophagosomes but not autolysosomes, which correlated with increased LC3II processing and increased p62 levels, suggestive of stalled autophagic flux. Knock down of ATG5 or Beclin1 suppressed autophagosome formation and cell killing. Knock down of ceramide synthase 6 suppressed autophagosome production and cell killing whereas knock down of ceramide synthase 2 enhanced vesicle formation and facilitated death. Collectively our findings argue that pemetrexed and FTY720 could be a novel adjunct modality for breast cancer treatment.

HDAC inhibitors enhance the lethality of low dose salinomycin in parental and stem-like GBM cells

The present studies determined whether the antibiotic salinomycin interacted with HDAC inhibitors to kill primary human GBM cells. Regardless of PTEN, ERBB1, or p53 mutational status salinomycin interacted with HDAC inhibitors in a synergistic fashion to kill GBM cells. Inhibition of CD95/Caspase 8 or of CD95/RIP-1/AIF signaling suppressed killing by the drug combination. Salinomycin increased the levels of autophagosomes that correlated with increased p62 and LC3II levels; valproate co-treatment correlated with reduced LC3II and p62 expression, and increased caspase 3 cleavage. Molecular inhibition of autophagosome formation was protective against drug exposure. The drug combination enhanced eIF2α phosphorylation and decreased expression of MCL-1 and phosphorylation of mTOR and p70 S6K. Activation of p70 S6K or mTOR promoted cell survival in the face of combined drug exposure. Overexpression of BCL-XL or c-FLIP-s was protective. Collectively our data demonstrate that the lethality of low nanomolar concentrations of salinomycin are enhanced by HDAC inhibitors in GBM cells and that increased death receptor signaling together with reduced mitochondrial function are causal in the combinatorial drug necro-apoptotic killing effect.

Cytogenetic Effects of Valproic Acid and Ziprasidone in Human Lymphocyte Cultures

Background/Aims: Valproic acid or valproate (VA) is an anticonvulsant and mood-stabilizing drug primarily used in the treatment of epilepsy and bipolar disorder. Ziprasidone (ZPN) is an atypical antipsychotic drug used mainly for the treatment of schizophrenia. Methods: This study is a part of our investigation on the cytogenetic effects of psychotropic drugs. Lymphocytes of peripheral blood cultures from 3 healthy donors treated with VA, ZPN and combinations of these (at concentrations equivalent to the oral doses) were used for the estimation of sister chromatid exchanges (SCEs) and the proliferation rate index (PRI). As a biomarker of genotoxicity, we used SCEs, one of the most sensitive methods reflecting DNA damage and/or subsequent DNA repair, and as a marker of cytostaticity we estimated the PRI. Results: All treated lymphocyte cultures showed a statistically significant increase in SCE frequency and a significant decrease in PRI values (p < 0.001). The combined effect of the drugs induced similar or more intense results, without reaching levels indicating synergistic action. Conclusion: This in vitro study investigated the cytogenetic activity of monotherapy vs. combined chronic drug exposure, and could form a catalyst for further investigations aiming to develop more efficacious therapy with decreased cytogenetic damage.

Abstract 5380: Synergistic effects of clofarabine and decitabine in acute myeloid leukemia

Abstract Clofarabine (CLO) is a purine nucleoside analog with promising efficacy in acute myeloid leukemia (AML). Its mechanism of action involves inhibition of the ribonucleotide reductase (RR) that converts nucleotide diphosphates to the corresponding deoxynucleotide diphosphates. Expression of the small RR subunit, p53R2 (product of RRM2B), demonstrated that a shorter protein is associated with CLO resistance. We have also shown that decitabine (DEC), another active drug in AML, induces p53R2 mRNA and protein expression and therefore asked herein whether the combination will be synergistic in a human AML cell line, HEL. To understand the role of p53R2 in this interaction, we knocked it down (KD) with shRNAmir. HEL and HEL-p53R2KD cells were exposed to CLO and DEC for 24,48,72, and 96-hr. Cells were stained with trypan blue for cell viability. The Hill model was fit to each concentration-response curve for each drug. After fitting and determination of 50% inhibition concentration (IC50), five combination ratios of the IC50 (CLO:DEC; 1:1, 1:4, 4:1, 1.5:3.5; 3.5:1.5) were characterized. In addition, western blot analysis was performed for both cell lines to determine levels of P53R2 expression for the same concentration range and exposure time to each drug, and in combination. Bands in western blot were quantitated using densitometry. HSC70 was used as a house keeping gene. At 24 hr, the IC50 values for CLO and DEC in HEL cells were 500 nM. In p53R2KD cells, the IC50 values for CLO and DEC were 700 nM and 120 nM. As exposure time increased to 48, 72, or 96 hr, the potency increased 10-15-fold for clofarabine, while only 2-4-fold for decitabine. At 48 hr, IC50 values were less than 50 nM for clofarabine in both HEL and p53R2KD cells, while it was 145 and 215 nM for decitabine. For the combination treatment, a pharmacodynamic (PD) interaction model was developed to assess the degree of synergy between CLO and DEC on cell viability. A PD parameter, labeled as ψ, characterized the extent of the interaction. When ψ &amp;gt;=1, the interaction between the two drugs may be additive, while ψ&amp;lt;1 represents synergy and ψ&amp;gt;1 indicates antagonism. Following 24 hr of drug exposure of CLO and DEC at the 50% of the IC50 values for each drug combination, ψ was estimated to be 1.00 (CI 0.850-1.22) for P53R2KD and &amp;gt;1 for HEL cells. In addition, western blot analyses performed at 24-hr following combined drug exposure revealed that the upper band did not change as a function of drug concentration, while the lower band showed a concentration-dependent increase in p53R2 protein expression, especially in p53R2KD. The PD model demonstrated a synergistic effect for CLO and DEC in the AML cell line HEL; knocking down p53R2 decreased this synergism, attesting to p53R2's role in both drugs’ mechanism of action. The increase in the lower band supports its role as part of clofarabine resistance. A clinical trial combining CLO and DEC in AML is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5380. doi:10.1158/1538-7445.AM2011-5380

Transient exposure of carcinoma cells to RAS/MEK inhibitors and UCN-01 causes cell deathin vitroandin vivo

The present studies were initiated to determine in greater molecular detail how MEK1/2 inhibitors [PD184352 and AZD6244 (ARRY-142886)] interact with UCN-01 (7-hydroxystaurosporine) to kill mammary carcinoma cells in vitro and radiosensitize mammary tumors in vitro and in vivo and whether farnesyl transferase inhibitors interact with UCN-01 to kill mammary carcinoma cells in vitro and in vivo. Expression of constitutively activated MEK1 EE or molecular suppression of JNK and p38 pathway signaling blocked MEK1/2 inhibitor and UCN-01 lethality, effects dependent on the expression of BAX, BAK, and, to a lesser extent, BIM and BID. In vitro colony formation studies showed that UCN-01 interacted synergistically with the MEK1/2 inhibitors PD184352 or AZD6244 and the farnesyl transferase inhibitors FTI277 and R115,777 to kill human mammary carcinoma cells. Athymic mice carrying approximately 100 mm(3) MDA-MB-231 cell tumors were subjected to a 2-day exposure of either vehicle, R115,777 (100 mg/kg), the MEK1/2 inhibitor PD184352 (25 mg/kg), UCN-01 (0.2 mg/kg), or either of the drugs in combination with UCN-01. Transient exposure of tumors to R115,777, PD184352, or UCN-01 did not significantly alter tumor growth rate or the mean tumor volume in vivo approximately 15 to 30 days after drug administration. In contrast, combined treatment with R115,777 and UCN-01 or with PD184352 and UCN-01 significantly reduced tumor growth. Tumor cells isolated after combined drug exposure exhibited a significantly greater reduction in plating efficiency using ex vivo colony formation assays than tumor cells that were exposed to either drug individually. Irradiation of mammary tumors after drug treatment, but not before or during treatment, significantly enhanced the lethal effects of UCN-01 and MEK1/2 inhibitor treatment. These findings argue that UCN-01 and multiple inhibitors of the RAS-MEK pathway have the potential to suppress mammary tumor growth, and to interact with radiation, in vitro and in vivo.

Transient exposure of mammary tumors to PD184352 and UCN-01 causes tumor cell death in vivo and prolonged suppression of tumor re-growth

Previous studies from our group have demonstrated in vitro that UCN-01 (7-hydroxystaurosporine) and inhibitors of MEK1/2 interact to cause tumor cell death in a wide variety of malignant, but not in non-transformed, cell types. The present studies determined whether UCN-01 and MEK1/2 inhibitors interacted to cause tumor cell death in vivo. In vitro colony formation studies demonstrated that UCN-01 and the MEK1/2 inhibitor PD184352 interacted to synergistically kill human mammary carcinoma cells (MDA-MB-231, MCF7) with similar combination index values. Athymic mice were implanted in the rear flank with either MDA-MB-231 or MCF7 cells and tumors permitted to form to a volume of ~100 mm3 prior to a two day exposure of either Vehicle, PD184352 (25 mg/kg), UCN-01 (0.1-0.2 mg/kg) or the drug combination. Tumor volume was measured every other day and tumor growth determined over the following ~30 days. Transient exposure of MDA-MB-231 tumors or MCF7 tumors to either PD184352 or UCN-01 did not significantly alter tumor growth rate or the mean tumor volume in vivo ~15-30 days after drug administration. In contrast, combined treatment with PD184352 and UCN-01 significantly reduced MDA-MB-231, and largely abolished MCF7 tumor growth. Tumor control values for both cell lines were 0.36. Tumor cells isolated ~30 days after combined drug exposure exhibited a significantly greater reduction in plating efficiency using ex vivo colony formation assays than tumor cells that were exposed to either drug individually. Reduced tumor growth correlated with profound tumor cell death within 5 days of combined drug exposure, which was also evident ~30 days after exposure. In addition, tumor cell death correlated with a reduction in the phosphorylation of ERK1/2 and the immuno-reactivity of Ki67 and of CD31. Collectively, these findings argue that UCN-01 and MEK1/2 inhibitors have the potential to suppress mammary tumor growth in vivo which is independent of p53 status, estrogen dependency, caspase 3 levels or oncogenic K-RAS expression.

Potential activity of paclitaxel, vinorelbine and gemcitabine in anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma (ATC) has a rapidly fatal course in the mostly elderly patients with a median survival after diagnosis of 4-12 months. Activity of commonly used chemotherapy (doxorubicin) is low, thus more active compounds need to be introduced into the therapeutic concept of ATC. Recently, based on preclinical data Ain et al. conducted a clinical phase II study with paclitaxel 96 h infusion in ATC achieving a promising response rate of 53%. To further improve therapeutic options in ATC, we evaluated the activity of topotecan, oxaliplatin, vinorelbine, gemcitabine and paclitaxel in comparision to cisplatin and doxorubicin (1 and 96 h drug exposure) alone or in combination in the ATC cell lines SW1736 and 8505C. IC50 values were determined by the sulforhodamine B assay, potential clinical activity was estimated by relative antitumor activity (RAA) and drug interaction was analyzed using a parametric response surface approach (Greco model) of the Loewe additivity. Duration of drug effect was estimated by regrowth kinetics. We found paclitaxel, vinorelbine and gemcitabine active in ATC with RAA (1 h drug exposure) ranging from 86 to 454, 15 to 17 and 31 to 140, respectively. The activity of doxorubicin and cisplatin was moderate with RAA ranging from 1.4 to 2.2 and 0.2 to 0.3, respectively. Combined drug exposure of gemcitabine/paclitaxel and gemcitabine/vinorelbine was synergistic with a Loewe index > 0. However, these results did not reach statistical significance with p > 0.05. At clinically relevant drug concentrations paclitaxel, gemcitabine and vinorelbine but not oxaliplatin exerted a sustained growth inhibition after cessation of drug exposure for the complete assay time of 15 days. In conclusion, paclitaxel, gemcitabine and vinorelbine but not topotecan or oxaliplatin appeared to be active in anaplastic thyroid carcinoma based on RAA or growth delay at clinically relevant drug concentrations. Combinations of vinorelbine/gemcitabine and paclitaxel/gemcitabine exerted a trend to synergy. Thus, further evaluation of paclitaxel, vinorelbine and gemcitabine alone or in combination with ATC seems warranted.

Prenatal cocaine and/or nicotine exposure produces depression and anxiety in aging rats

The adult use of cocaine and nicotine has been linked to depression and/or anxiety. Changes in emotional behavior were assessed using behavioral paradigms developed as animal analogs of psychiatric disorders in 12–14 month old Sprague–Dawley rats exposed daily on gestational days 8–20 to cocaine and nicotine, either alone or in combination. Results from the elevated plus maze (EPM), used to assess anxiety-related behaviors, indicated that offspring prenatally exposed to either high-dose cocaine (40 mg/kg/day) or high-dose nicotine (5.0 mg/kg/day) were less timid/more impulsive. Animals from these two groups spent the most time on the open arms, and had the highest percentage of entries into the open arms of the EPM. Combined in utero exposure to cocaine and nicotine nullified these effects. Cocaine challenge (20 mg/kg) did not interact with prenatal treatment, but increased activity on all arms of the EPM in all groups. Sucrose preference was used as a measure of anhedonia, a cardinal symptom of depressive illness. Reduced sucrose preference was seen only in the group of offspring prenatally exposed to high-dose cocaine (40 mg/kg) plus low-dose nicotine (2.5 mg/kg/day). Exposure to a water-deprivation stress normalized sucrose preference in this group, without altering preference or intake in the other prenatal treatment groups. Transient hyperactivity was seen in the offspring of dams treated with high-dose nicotine, an effect that was again reversed in combined drug groups. Traditional gender differences in activity levels and sucrose intake, that is, females greater than males, were still evident in this population of aging rats. These data indicate that prenatal exposure to cocaine and/or nicotine has long-term effects on emotional behavior. Combined drug exposure contributed to the development of depressive symptoms, but not anxiety-like behavior, in a dose-dependent manner. In contrast, exposure to high doses of either drug alone reduced cautionary behavior. Data from this line of research could provide insight into the pathogenesis of emotional disorders, especially during the aging process.

EFFECT OF RECOMBINANT HUMAN TUMOR-NECROSIS-FACTOR-ALPHA AND DEQUALINIUM CHLORIDE ON HUMAN OVARIAN-CANCER CELL-LINES IN-VITRO

Due to preferential uptake and retention, the small molecular weight lipophilic, cationic antimicrobial agent dequalinium chloride (DECA) displays potent in vitro and in vivo antitumor activity against carcinoma cells. The primary mechanism of DECA activity is directed against the mitochondria where it disrupts cellular energy production. One of the direct antitumor effects of tumor necrosis factor (TNF) is also targeted against the mitochondria. The ability of DECA to synergize this effect was examined in vitro against a panel of human ovarian cancer cell lines. The data from single agent and combined drug exposure were analyzed by the isobologram methods of Tsai et al (Cancer Res 49: 2390-2397, 1989). We demonstrate that TNF and DECA strongly synergize in vitro at clinically achievable doses for TNF and potentially clinically achievable doses for DECA. The degree of synergy varied with the cell line tested with UCI-101 being the least responsive and PA-1 cells displaying the greatest synergistic effect. DECA treatment also prolonged animal survival in mice bearing the PA-1 intraperitoneal ovarian carcinoma xenograft. Single agent DECA (5 mg/kg; qod) increased animal survival by 37% (p=0.002) whereas recombinant human TNF (0.5 mug/mouse; qod) increased survival by 12% (p=0.27) in those animals treated 3 days post tumor injection. Sequential DECA/TNF enhanced animal survival by 45% (p=0.0002) in similarly treated animals. DECA, as a mitochondrial poison is an agent capable of potentiating the effects of tumor necrosis factor against ovarian cancer cell lines.

Chemosensitivity testing in ovarian cancer.

Most patients with ovarian cancer currently are treated primarily with surgery and chemotherapy. Drug selection usually is not based on individualized in vitro sensitivity studies but on reported response rates of clinical trials. Attempts to include in vitro chemosensitivity testing into the management of ovarian cancer have been disappointing to clinicians. Tumor cells from fresh human ovarian cancer do not grow well under artificial in vitro growth conditions. The selection of cells that happen to proliferate in vitro (e.g., human tumor clonogenic assay) has resulted in low plating efficiencies (0.001-0.1% of plated cells). The vigorous mechanical and enzymatic tumor disaggregation, done to obtain a single-cell suspension, further reduces the number of cells that grow in vitro, resulting in low overall evaluability rates of 40-70% for the human tumor clonogenic assay. At the University of Miami, a new in vitro chemosensitivity assay was developed that detected the decrease in total tumor cell viability by measuring intracellular adenosine triphosphate as a function of in vitro drug response. Preliminary data on 31 tumor tissues from patients, which was evaluated with this method, showed a sensitivity of 92% and a specificity of 100%. Since these initial studies, data were gathered on more than 150 fresh gynecologic tumor specimens to evaluate single drugs and drug combinations at five concentrations (range, 10-500% of reported peak plasma concentrations). The evaluability rate for ovarian tumors was more than 90%. Some tumors showed almost complete cell kill at the lowest drug concentration; others had only a limited response at the highest level. Drug-response patterns also were variable for combined drug exposure. These findings underscore the heterogeneity of drug response in morphologically similar tumors and the importance of characterizing individual chemosensitivity profiles for patients before drug treatment.

Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing

Intracellular adenosine triphosphate (ATP) is the primary energy unit of living cells, and can be quantitated by measuring the light generated with luciferase-luciferin reagent in a luminometer. The use of an ATP-bioluminescence assay, to determine tumor cell viability after exposure to chemotherapeutic agents, has been adapted to test tumor chemosensitivity in vitro. This presentation will illustrate the method of the ATP-chemosensitivity assay (ATP-CSA) using an ovarian cancer cell line NIHL: OVCAR-3 as an example and present preliminary data on 54 56 successful in vitro ATP-CSA's from 46 patients with pelvic malignancies. Fresh human tumor specimens were generally tested for single and combined drug effects at two drug concentrations (0.2 × and 1 × peak plasma concentrations). Correlation of in vitro drug sensitivity and in vivo patient response was obtained for 23 treatment regimens in 22 patients with ovarian carcinoma. The true positive rate was 100% and the true negative rate 66.7%. Our data demonstrate (a) that the ATP-CSA, measuring total cell viability, is a feasible in vitro assay for human tumor drug testing and (b) that specific criteria of in vitro chemosensitivity for this assay need to be defined by further studies, for single and combined drug exposure at different concentrations, to permit a meaningful correlation with in vivo clinical response.