Schedule-Dependent Antiangiogenic and Cytotoxic Effects of Chemotherapy on Vascular Endothelial and Retinoblastoma Cells

Abstract

Current treatment of retinoblastoma involves using the maximum dose of chemotherapy that induces tumor control and is tolerated by patients. The impact of dose and schedule on the cytotoxicity of chemotherapy has not been studied. Our aim was to gain insight into the cytotoxic and antiangiogenic effect of the treatment scheme of chemotherapy used in retinoblastoma by means of different in vitro models and to evaluate potential effects on multi-drug resistance proteins. Two commercial and two patient-derived retinoblastoma cell types and two human vascular endothelial cell types were exposed to increasing concentrations of melphalan or topotecan in a conventional (single exposure) or metronomic (7-day continuous exposure) treatment scheme. The concentration of chemotherapy causing a 50% decrease in cell proliferation (IC50) was determined by MTT and induction of apoptosis was evaluated by flow cytometry. Expression of ABCB1, ABCG2 and ABCC1 after conventional or metronomic treatments was assessed by RT-qPCR. We also evaluated the in vivo response to conventional (0.6 mg/kg once a week for 2 weeks) and metronomic (5 days a week for 2 weeks) topotecan in a retinoblastoma xenograft model. Melphalan and topotecan were cytotoxic to both retinoblastoma and endothelial cells after conventional and metronomic treatments. A significant decrease in the IC50 (median, 13-fold; range: 3–23) was observed following metronomic chemotherapy treatment in retinoblastoma and endothelial cell types compared to conventional treatment (p<0.05). Metronomic topotecan or melphalan significantly inhibited in vitro tube formation in HUVEC and EPC compared to vehicle-treated cells (p<0.05). Both treatment schemes induced apoptosis and/or necrosis in all cell models. No significant difference was observed in the expression of ABCB1, ABCC1 or ABCG2 when comparing cells treated with melphalan or topotecan between treatment schedules at the IC50 or with control cells (p>0.05). In mice, continuous topotecan lead to significantly lower tumor volumes compared to conventional treatment after 14 days of treatment (p<0.05). Continuous exposure to melphalan or topotecan increased the chemosensitivity of retinoblastoma and endothelial cells to both chemotherapy agents with lower IC50 values compared to short-term treatment. These findings were validated in an in vivo model. None of the dosing modalities induced multidrug resistance mechanisms while apoptosis was the mechanism of cell death after both treatment schedules. Metronomic chemotherapy may be a valid option for retinoblastoma treatment allowing reductions of the daily dose.

Conservative management of retinoblastoma: Challenging orthodoxy without compromising the state of metastatic grace. “Alive, with good vision and no comorbidity”

“…Melphalan concentration versus time profile in (•, green) vitreous humor after 15 μg IVi (intravitreal) to rabbits, (■, blue) aqueous humor after 15 μg IVi to rabbits, (♦, black) retina concentrations after 15 μg IVi to rabbits, and (▲, red) vitreous humor after IAC (intra-arterial chemotherapy) of 7 mg to pigs. Dotted line represents the IC50 (concentration of chemotherapy that reduces 50% the cell viability) determined in a commercial cell line of retinoblastoma (Schaiquevich et al, 2012b;Winter et al, 2016). (Data corresponds to results published in Schaiquevich et al, 2012b; Buitrago et al, 2016).…”

“…Total topotecan concentration versus time profile in (•, green) vitreous humor after 5 μg IVi (intravitreal) to rabbits, (▼, red) aqueous humor after 5 μg IVi to rabbits, (■, blue) vitreous humor after 1 mg IAC (intra-arterial) to pigs, (♦, yellow) vitreous humor after 1 mg PO (periocular) to rabbits, (▲, violet) vitreous humor after 1 mg IV (intravenous) to rabbits, (♦, black) retina concentration after 4 mg IAC to pigs, and (•) optic nerve concentrations after 4 mg IAC to pigs. Dotted line represents the IC50 (concentration of chemotherapy that reduces 50% the cell viability) determined in a commercial cell line of retinoblastoma (Laurie et al, 2005;Schaiquevich et al, 2012a;Winter et al, 2016). (Data corresponds to results published in Carcaboso et al, 2007;Schaiquevich et al, 2012a;Buitrago et al, 2010).…”

Enhanced antitumor and anti-angiogenic effects of metronomic Vinorelbine combined with Endostar on Lewis lung carcinoma

“…Previous studies have shown that metronomic topotecan administrated for two weeks compared to the maximum tolerated dose of topotecan enhanced anti-angiogenic responses and had low toxicity used in a xenograft model of retinoblastoma treatment [ 44 ]. In our study, these results also indicated that metronomic Vinorelbine combined with Endo administrated for 14 consecutive days had similar results.…”

Resistance to metronomic chemotherapy and ways to overcome it

“…Hence, the addition of VEGF to tumor endothelial cells rendered them resistant to paclitaxel, a P-glycoprotein substrate, and the P-glycoprotein inhibitor verapamil enhanced the antiangiogenic effects of metronomic paclitaxel in the A375SM human melanoma xenograft model [39]. Of note, metronomic versus MTD chemotherapy administration may impact the expression of P-glycoprotein and other drug efflux pumps in endothelial cells in a complex manner, depending on the type of endothelial cells and chemotherapeutic agents used [17,26]. Overall, MTD chemotherapy seems to have a higher propensity to induce drug efflux pumps in both endothelial and tumor cells compared to MC [17,40].…”

“…Of note, metronomic versus MTD chemotherapy administration may impact the expression of P-glycoprotein and other drug efflux pumps in endothelial cells in a complex manner, depending on the type of endothelial cells and chemotherapeutic agents used [17,26]. Overall, MTD chemotherapy seems to have a higher propensity to induce drug efflux pumps in both endothelial and tumor cells compared to MC [17,40]. …”