Potent Inhibition of the Growth and Induction of Apoptosis in Lymphoma By the Anthelminthic Drug Niclosamide: In Vitro Data

Background and Purpose: Niclosamide is an anthelminthic drug which has been used for the treatment of human parasitic infections for many years. Niclosamide interacts with lysosomes and induces autophagy. In recent years, it has demonstrated anti-cancer potential in leukemia, breast cancer, colon cancer, myeloma, ovarian, prostate and lung cancer models. Multiple pathways like Wnt/β-catenin, mTORC1, STAT3, NF-κB and notch signaling were reported to be involved. Only limited studies were done in lymphoma models. We hypothesized that niclosamide may also have in vitro and in vivo activities in lymphomas. Non-Hodgkin lymphomas generally respond well to chemotherapy and/ or immunotherapy, however many patients relapse and ultimately become refractory. Relapses are often caused by tumor stem cells not eliminated by cytostatic drugs. Therefore new treatment approaches and new targets are desirable.

Materials and Methods: Established B lymphoma cell lines were exposed to different concentrations of niclosamide (0.1-4µM) and IC₅₀ was calculated at 24, 48 and 72 hours. The cell concentration, viability and proliferation were assessed by CellTiter-Blue viability and trypan blue exclusion assays. Apoptosis was assessed by a combined annexin-V/ propidium iodide stain. Gene expression changes were studied using GeneChip Human Transcriptome Array 2.0 (Affymetrix) with 44 699 annotated genes. Colony forming assays were performed in methylcellulose. Ultrastructural changes were studied using a Hitachi electron microscope. As normal controls, peripheral blood mononuclear cells from individuals without active cancer were incubated with niclosamide for up to 72 hours. Samples from patients with chronic lymphocytic leukemia were also treated under the same conditions.

Results: Treatment with niclosamide at doses as low as 0.1 μM resulted in time-and dose- dependent apoptosis, cytotoxicity and inhibition of proliferation in aggressive lymphoma cell lines. The 50% inhibitory concentration in a proliferation assay (mean of data at 24, 48 and 72 hours) is shown in the Table below. Niclosamide also inhibited clonal growth in semi-solid media. Electron microscopy showed that filopodia increased and lipid vacuoles developed whereas mitochondria were less numerous and had fewer cristae (when KOPN-8 was treated with 0.5 μM for 48 hours). The viability of mononuclear cells from 8 individuals without lymphoma was unchanged (or minimally decreased) when incubated with niclosamide. As far as cells from two patients with untreated chronic lymphocytic leukemia are concerned, no cytotoxicity was observed at doses between 0.5 and 5 μM. Gene expression changes were studied the cell lines Daudi and KOPN-8 treated with 2.5 μM for 3 and 6 h. 96 genes were consistently overexpressed , 59 down-regulated. Ten out of the 96 overexpressed genes involved the TNF pathway and immunoregulation including CD95. Thirteen out of the 59 down-regulated genes are involved in mitochondrial function.

Conclusion: Niclosamide effectively inhibits the proliferation of B lymphoma cell lines and induces apoptosis. Preliminary data show that Niclosamide targets genes involved in the TNF pathway and interferes with mitochondrial function. Normal lymphocytes are not sensitive to niclosamide. The in-vitro activity of niclosamide is at least comparable or superior to the activity seen in other malignancies. Niclosamide may target drug-resistant lymphoma stem cells and has clinical potential. We plan to study combination treatments and perform in vivo studies.

Acknowledgments: The authors thank Drs. Borje Andersson, Shile Huang, Nakle Saba, Ben Valdez and Ellen Vitetta for their kind gift of cell lines.