Cytotoxicity Of Topotecan Research Articles

Carvacrol effect on topotecan cytotoxicity in various human cancer cells in vitro

Purpose: To investigate the modulatory effect of the natural phytochemical, carvacrol, on Topotecan (TOPO) cytotoxicity and cellular uptake in different cancer cell lines. Methods: The cytotoxicity of the carvacrol/TOPO combination therapy was determined in vitro using crystal violet assay. Coomassie blue and DAPI fluorescent stains were used for cellular morphology and molecular cell death assessments, respectively. Additionally, TOPO cellular uptake after carvacrol/TOPO combination therapy was determined. Results: Treatment of HeLa and HCT116 with carvacrol/TOPO resulted in 7.70- and 5.71-fold reduction in TOPO half maximal inhibitory concentration (IC50), respectively, relative to TOPO single treatment. On the other hand, treatment of MCF-7, HepG2, SKOV3, and A549 cancer cells with carvacrol/TOPO resulted in increasing the IC50 of TOPO by 1.49-, 1.33-, 1.50- and 1.26-fold, respectively, relative to TOPO single treatment. Conclusion: Carvacrol had enhanced TOPO cytotoxicity and cellular uptake in HeLa and HCT116 cancer cells but might cause TOPO resistance in MCF-7, HepG2, SKOV3 and A549 cells.

Abstract 2944: Identification of key transcriptomic and epigenomic factors influencing conventional and metronomic dosing in aggressive and non-aggressive prostate cancer

Abstract Repetitive, low-dose drug administration (metronomic; METRO) shows ability to overcome drug resistance and increased drug efficacy in many cancers, but the mechanisms are not understood fully. Previously we showed topotecan (TOPO) METRO dosing was more effective than conventional (CONV) dosing in aggressive prostate cancer cell lines and xenograft mouse models. To explore possible mechanisms by which METRO dosing alters tumor growth and metastases, we performed targeted mRNA and miRNA expression studies and identified potential candidate cancer pathway genes and miRNA-mRNA pairs as unique treatment-related biomarkers for alternative TOPO-METRO therapy. To determine drug response, Androgen-independent human prostate cancer cells (PC3), androgen-dependent (LNCaP) cell lines were treated with TOPO following CONV and METRO dosing schedules. Cancer pathway gene and micro-RNA expression profiles were assessed at the calculated IC50 of TOPO after each treatment. Expression signatures were identified using differential expression (DE) analysis software and a Spearman’s rank-based correlation was used to assess the association of drug response, mRNA and miRNA expression. Gene signatures associated with TOPO-METRO therapy were validated against patient profiles in The Cancer Genome Atlas (TCGA) and Genomic Data Commons (GDC) and protein expression of most significant genes by immunoblotting. We identified disparate treatment-related mRNA and miRNA expression signatures for TOPO-METRO vs CONV treatment. We also identified a gene signature (top five gene: SERPINB5, CDKN1A, TNF, FOS, & ANGPT1) for both PC-3 and LNCaP following TOPO-METRO dosing. Ingenuity Pathway Analysis identified that upregulation of tumor suppressor, anti-proliferative genes (eg, SERPINB5), genes involved in activation of the immune system (RPL13A) and down regulation of genes involved in apoptosis, invasion, metastasis, and inflammation (TNF, FOS, and MMP1) are likely vital to the observed treatment response. Epigenetic analysis showed distinct miRNA expression changes may influence differential gene expression (miRNA-mRNA pairs) for TOPO-METRO dosing. 20 miRNAs genes (including miR-30c, miR-19a, mir-20a, mir-17, let7i & let7b) were associated with TOPO cytotoxicity (p<0.05); seven of these bind to 28 mRNAs (p<0.05) as mRNA-miRNA pairs. Furthermore, we confirmed the top 5 significant genes (e.g. FOS, SERPINEB5, MMP9) for TOPO-Metro therapy by in-silico validation (TCGA). Overall, these studies address a fundamental gap in knowledge related to the effect of METRO dosing on genomic and epigenetic influences and overall treatment efficacy. Using genomics, transcriptomics and epigenomics approaches we determined gene signatures that may be used in identifying patients and personalizing their treatment with the goal of improving overall survival. Citation Format: Taraswi Mitra Ghosh, Joshua Davis, Grafton S. Barnett, Elena Skarupa, Jeff D. Warner, Brian S. Cummings, Robert D. Arnold. Identification of key transcriptomic and epigenomic factors influencing conventional and metronomic dosing in aggressive and non-aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2944.

Abstract IS04: PARP INHIBITOR COMBINATIONS FOR THE TREATMENT OF OVARIAN CANCER

Abstract Poly(ADP-ribose) polymerase (PARP) inhibitors have shown promising single-agent activity in ovarian cancers with BRCA1 and BRCA2 mutations (C. Scott, JCO 33:1397, 2015). Ongoing studies are testing the efficacy of PARP inhibitors in ovarian cancers with other genetic or epigenetic alterations that impair the homologous recombination (HR) pathway of DNA repair. It is less clear whether PARP inhibitor clinical activity can be enhanced in HR deficient cancers or even extended to HR proficient ovarian cancers through judicious combinations. Our preclinical studies have demonstrated that PARP inhibitors enhance the cytotoxic effects of selected therapeutic agents through a variety of mechanisms: By trapping PARP at sites of DNA damage, PARP inhibitors enhance the cytotoxicity of topotecan (A. Patel, J. Biol. Chem. 287: 4198, 2012).By inhibiting base excision repair, PARP inhibitors enhance the cytotoxicity of floxuridine (A. Huehls, Cancer Res. 71: 4944, 2011), an agent previously shown to have clinical activity in ovarian cancer. Building on the first of these observations, we conducted a phase 1 clinical trial of topotecan weekly x 3 in combination with veliparib (NCT01012817). Using a standard 3+3 design, patients were treated with veliparib PO twice daily on days 1-3, 8-10 and 15-17 and topotecan IV on days 2, 9 and 16 every 28 days. The dose-limiting toxicity was neutropenia. The MTD was veliparib 300 mg PO twice daily and topotecan 3 mg/m2/dose. Among 52 evaluable patients, objective responses (1 CR, 3 PRs) and stable disease (22 patients, 6 lasting ≥ 8 cycles) were observed. While there was no clear correlation between germline BRCA1 or BRCA2 mutations and clinical benefit, a patient with a germline RAD51D mutation had stable disease for 14 cycles. A phase 2 cohort in platinum-resistant ovarian cancer has just opened. Building on the second set of preclinical observations, we are also conducting a phase 1 trial of veliparib and IP floxuridine in patients with recurrent ovarian cancer confined to the peritoneal cavity (NCT01749397). Using a rolling 6 design, patients are treated with veliparib PO twice daily on days 1-10 and floxuridine IP on days 3-5 every 21 days. The trial is currently enrolling at dose level 5 (veliparib 300 mg PO twice daily on days 1-10 and floxuridine 3 gm/dose IP on days 3-5). Grade 3 toxicities in any cycle have included nausea (1), fatigue (1) and neutropenia (3). Among 20 evaluable patients, treatment duration has ranged from 2-33 cycles. These studies, which build on two different aspects of PARP1 function, illustrate two different approaches to potentially enhancing the activity of PARP inhibitors in relapsed ovarian cancer. These studies were funded in part by NIH grants P50 CA136393, R01 CA190423, and UM1 CA186686 as well as SU2C-AACR DT16-15. Citation Format: Scott H. Kaufmann, Andrea E. Wahner Hendrickson, Maria I Harrell, Michael E. Menefee, Edward J. Tanner, David G, Mutch, Elizabeth M. Swisher, and Larry M. Karnitz. PARP INHIBITOR COMBINATIONS FOR THE TREATMENT OF OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr IS04.

Abstract 2194: Tyrosyl DNA phosphodiesterase I is a prognostic factor and its inhibition synergizes response to topoisomerase poisons in malignant glioma.

Abstract Malignant Glioma is the most common type of primary brain tumors in adults accounting for about 80% of all gliomas. Despite the advances in treatment and diagnosis, the prognosis of malignant glioma is still poor with a median survival less than a year for glioblastoma patients. Targeting DNA-repair mechanisms is a clinically relevant option for enhancing response to DNA-damaging anticancer agents. TDP1 has been recently discovered to be responsible for the repair of the irreversible DNA-topoisomerase I covalent complexes produced by topoisomerase I poisons. Other studies have suggested its role in the repair of the DNA damage produced by ionizing radiation and temozolomide. However, TDP1 relevance has not yet been studied in glioma. Thus the aims of this project are to identify the role of TDP1 in glioma aggressiveness and resistance to therapies. We hypothesize that high-grade gliomas possess higher TDP1 levels which are associated with lower survival and resistance to therapy. Quantitative real-time PCR analysis of TDP1 in glioma biopsies revealed a negative prognostic effect of TDP1, where the level of TDP1 mRNA was inversely correlated with patient survival. Moreover, western blot showed an overexpression of TDP1 in glioma biopsies compared to normal tissues and increased expression with higher grades. TDP1 overexpression in U87 cell lines did not induce resistance to temozolomide, topotecan, etoposide or doxorubicin. This may be related to other downstream DNA repair enzymes that need to be also modulated to fully induce drug resistance. However, TDP1 inhibition using small molecules synergizes the cytotoxicity of topotecan, etoposide, and doxorubicin, confirming thus its critical role in response to several therapeutic agents. As a conclusion, TDP1 is a new and promising prognostic factor and a modulator of therapy response in malignant glioma that may help in stratification of patients. Additionally, targeting TDP1 is a novel strategy that will widen the therapeutic options for malignant glioma where topoisomerase I and II poisons can be part of the gold standard therapy when used in combination with TDP1 inhibitors. Citation Format: Maha Al-Keilani, Supreet Agarwal, Mohammad Alqudah, Zita Sibenaller, Timothy Ryken, Mahfoud Assem. Tyrosyl DNA phosphodiesterase I is a prognostic factor and its inhibition synergizes response to topoisomerase poisons in malignant glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2194. doi:10.1158/1538-7445.AM2013-2194

Enhanced antitumor activity of low-dose continuous administration schedules of topotecan in prostate cancer

Purpose: The objective of this study was to determine the antitumor effects of alternate dosing schedules of topotecan in prostate cancer.Results: A concentration-dependent increase in cytotoxicity was observed in PC-3 and LNCaP cells after topotecan treatment using conventional and metronomic protocols. A significant increase in potency (2.4-18 fold, after 72 hr) was observed following metronomic dosing compared to conventional dosing administration in both cell lines. Metronomic dosing also increased the percentage of PC-3 cells in the G2/M, compared to control, but did not alter LNCaP cell cycle distribution. Metronomic dosing increased p21 protein expression in LNCaP and PC-3 cells compared to conventional dosing. The observed in vitro activity was confirmed using an in vivo model of human prostate cancer. Metronomic dosing and continuous infusion decreased tumor volume significantly (p < 0.05) compared to control and conventional topotecan treatment, but had no effect on tumor vascular staining.Methods: The cytotoxicity of topotecan after conventional or metronomic dosing was determined by examining cellular morphology, mitochondrial enzymatic activity (MTT), total cellular protein (SRB), annexin V and propidium iodine (PI) staining, cell cycle and western blot analysis in human prostate cancer cell lines (PC-3 and LNCaP) and the effects metronomic or continuous infusion on tumor growth in an in vivo tumor xenograft model.Conclusions: These data support the hypothesis that low-dose continuous administration of topotecan increases potency compared to conventional dosing in prostate cancer. These data also suggest the novel finding that the enhanced antitumor activity of topotecan following low-dose exposure correlates to alterations in cell cycle and increased p21 expression.

Subconjunctival Topotecan in Fibrin Sealant in the Treatment of Transgenic Murine Retinoblastoma

To test the effects of subconjunctival topotecan (TPT) in fibrin sealant (FS) in transgenic murine retinoblastoma (RB). Growth inhibitory, apoptotic, and cell cycle effects of TPT were assayed in human RB cell lines. In a dose-escalation study, eight groups of three 10- to 14-week-old wild-type mice were treated bilaterally with a single 30-microL injection of subconjunctival TPT in FS (0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, or 3.2 mg/mL). Two groups of twenty 10-week-old LHbeta-Tag transgenic mice were then treated in the right eye only with TPT in FS (3.2 mg/mL in 30 microL; 0.1-mg total dose) or with FS only. The contralateral eye in each group was left untreated to serve as an internal control. After 3 weeks, ocular tumor burden was determined by histologic examination. At 48 hours, IC(50) values of TPT in Y79 and Weri-Rb1 RB cell lines were 35 nM and 50 nM, respectively. Growth inhibitory effects were correlated with increased apoptosis and accumulation of cells in G2. Cytotoxicity of TPT was comparable in aqueous media and in FS. In the dose-escalation study, no histopathologic evidence of ocular toxicity was observed at any dose. Clinical toxicities (mild enophthalmos and eyelid alopecia) were observed only at the highest dose tested (3.2 mg/mL). In the treatment study, both eyes of TPT-treated mice demonstrated significant reduction in tumor burden compared with both eyes of mice treated with FS only (59% reduction; P = 0.04). In mice treated with TPT, tumor burden in TPT-treated eyes and in untreated contralateral eyes did not differ significantly. Subconjunctival administration of TPT in FS to one eye allows the formation of a TPT depot sufficient for an effect to occur 3 weeks after treatment. This effect -- bilateral reduction in tumor burden without a significant difference in treated versus untreated eyes -- suggests that the major route of drug delivery in this system is hematogenous rather than transscleral.

A phase I study of lapatinib and topotecan in patients with solid tumors

3598 Background: Drug resistance to topotecan can be the result of BCRP/ABCG2 expression. BCRP is a member of the ABC transporter family that pumps anticancer drugs out of the cell. Lapatinib is a potent and selective dual inhibitor of epidermal growth factor receptor (EGFR or ErbB1) and ErbB2 (Her2/Neu). 4-aminoquinazoline tyrosine kinase inhibitors have been shown to enhance the cytotoxicity of topotecan through inhibition of BCRP-mediated drug efflux in cancer cells. Methods: Thirty-seven patients with advanced stage cancers were enrolled at escalating dose levels of lapatinib and topotecan in cohorts IA, IB and IIB (MTD). Treatment schedule included lapatinib (750 - 1500 mg/d) daily for 21 (cohort IA) or 28 days (cohort IB) and topotecan (2.4 - 4.0 mg/ m2), days 1, 8 and 15; cycles were repeated every 28 days. Three patients were treated at each dose level, 18 on cohort IA, 9 on cohort IB and 10 at MTD (cohort IIB). Assessments of toxicity were performed with each cycle and clinical response was determined per RECIST criteria every other cycle. Results: The MTD for cohorts IA and IB was reached at a dose of 1250 mg of lapatinib and 3.2 mg/m2 of IV topotecan on days 1, 8 and 15. No DLT were seen during the dose escalation stage of cohorts IA and IB. Ten patients were enrolled at the MTD. There were no grade 4+ events. Thirteen grade 3+ events, considered to be related to treatment, were seen in 6 patients. The most common grade 3+ toxicities included dehydration (2) diarrhea (2), nausea (3), vomiting (2), neutropenia (1), thrombocytopenia (1), and fatigue (1). No abnormalities in left ventricular ejection fraction were noted. Stable disease was seen in 46% of the 37 patients. Conclusions: The combination of lapatinib and topotecan is a well-tolerated regimen. The MTD for the combination is lapatinib 1,250 mg orally once daily for 21 or 28 days and topotecan 3.2 mg/m2 on days 1, 8 and 15. Pharmacokinetic analysis for drug interaction will be available for presentation at the meeting. Supported in part by GSK and Mayo Clinic No significant financial relationships to disclose.

A novel stealth liposomal topotecan with amlodipine: Apoptotic effect is associated with deletion of intracellular Ca 2+ by amlodipine thus leading to an enhanced antitumor activity in leukemia

The objectives of the present study were to define whether amlodipine induces apoptosis and what mechanism is involved in the process in human resistant and non-resistant leukemia cells following co-administration of stealth liposomal topotecan with amlodipine, a novel antiresistant liposomes developed by our institution. In three leukemias, K562, HL-60, and multidrug resistant (MDR) HL-60, cytotoxicity of topotecan was potentiated by amolodipne, while topotecan alone was resistant to MDR HL-60 cells. In two selected K562 or MDR HL-60 cells, the apoptotic effects were increased by addition of amlodipine, showing a dose-dependent manner. The activities of caspase 3 and 7 (marked as caspase 3/7), and caspase 8 were significantly activated by topotecan with amlodipine co-treated as the stealth liposomes. The deletions of intracellular Ca 2+ stores induced by amlodipine correlated with the activated activities of caspase 3/7, or 8, respectively. In xenograft model with MDR HL-60 in nude mice, antitumor activity of stealth liposomal topotecan with amlodipine was significantly enhanced as compared to that of stealth liposomal topotecan or topotecan alone. In conclusion, apoptotic effect is associated with deletion of intracellular Ca 2+ by amlodipine through activation of caspase 8 and then 3/7 activities. The enhanced antitumor activities by stealth liposomal topotecan with amlodipine are mainly due to the potentiating apoptotic effect and reversing the resistance by amlodipine. Stealth liposomal encapsulation of anticancer agent with a modulator may provide a novel strategy for improving the chemotherapeutic effects.

The Effect of Quercetin on Topotecan Cytotoxicity in MCF-7 and MDA-MB 231 Human Breast Cancer Cells 1

Topotecan, which is a Camptothecin derivative, shows a large spectrum in anti-tumor activity. Topotecan exerts its cytotoxic effect on tumor cells mainly by inhibition of topoisomerase I activity resulting in double-strand DNA breaks. In our study, we investigated the combined cytotoxic action of Topotecan and Quercetin in MCF-7 and MDA-MB 231 human breast cancer cells. To examine the possible relation between the cytotoxic activity of Topotecan and oxidative stress, we measured ROS and nitrite levels in both human breast cell lines. MCF-7 and MDA-MB 231 cells were exposed to Topotecan, Quercetin, or a combination of both agents for 24 h at 37 degrees C. The viability of the cells was measured using the colorimetric MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. We determined reactive oxygen species and nitrite levels as indicators of oxidative stress in both cell lines with and without Topotecan and/or Quercetin incubations using fluorometric dichlorofluorescin diacetate (DCFH-DA) and diaminonaphtalene (DAN) assay. The IC(50) concentration of Topotecan was 100 ng/ml in MCF-7 cell line and 160 ng/ml in MDA-MB231 cell line. Treatment with Quercetin enhanced cytotoxicity of Topotecan as 1.4-fold in MCF-7 and 1.3-fold in MDA-MB-231 cell line. A significant increment on ROS and nitrite levels was found in MCF-7 and MDA-MB-231 cells following Topotecan incubation. Our results suggest that Topotecan has cytotoxic activity against both of the breast cancer cell lines in vitro. A combination with Quercetin increases efficacy of Topotecan in the treatment of breast cancers. Our results indicate that increased oxidative stress plays a role in the cytotoxic action of Topotecan.

Interaction of ionizing radiation with topotecan in two human tumor cell lines.

The effect of topotecan, a topoisomerase I inhibitor, on ionizing radiation-induced cytotoxicity was studied in 2 human tumor cell lines characterized by a different expression of the target enzyme. The cytotoxicity of topotecan alone or in combination with radiation was assessed in exponentially growing non-small-cell lung cancer (H460) and glioblastoma (GBM) cells using the colony-forming assay. An isobologram method was used to evaluate the treatment interaction. An apparent supra-additive effect in cell killing following drug-radiation-combined treatment was observed only in GBM cells exposed to topotecan for 24 hr. In the case of H460 cells, interaction varied from a strong infra-additive effect at low radiation doses to a slight supra-additive effect when cells were exposed to radiation doses greater than 3 Gy. Northern blot analysis indicated that topoisomerase I expression in H460 cells was 8-fold higher than that of GBM cells. Although the H460 cell line exhibited an increased sensitivity to topotecan, only in the GBM cell line (which expressed a lower level of topoisomerase I) did the drug potentiate the radiation cytotoxicity. The observation that the radiosensitization by topotecan was related to topoisomerase I level is consistent with a putative role of the enzyme in processes involved in the repair of radiation damage. It is conceivable that the modulation of enzyme function results in an effective reduction of cellular capability for repair of radiation damage only if the enzyme is not over-expressed. Although a precise role of topoisomerase I in the cellular response to ionizing radiations (in particular, in DNA repair) remains to be documented, such results suggest the potential interest of topoisomerase I inhibitors in combination with radiation therapy for tumors expressing low topoisomerase I levels.