Papaverine and its derivatives radiosensitize solid tumors by inhibiting mitochondrial metabolism

Abstract

Tumor hypoxia reduces the effectiveness of radiation therapy by limiting the biologically effective dose. An acute increase in tumor oxygenation before radiation treatment should therefore significantly improve the tumor cell kill after radiation. Efforts to increase oxygen delivery to the tumor have not shown positive clinical results. Here we show that targeting mitochondrial respiration results in a significant reduction of the tumor cells' demand for oxygen, leading to increased tumor oxygenation and radiation response. We identified an activity of the FDA-approved drug papaverine as an inhibitor of mitochondrial complex I. We also provide genetic evidence that papaverine's complex I inhibition is directly responsible for increased oxygenation and enhanced radiation response. Furthermore, we describe derivatives of papaverine that have the potential to become clinical radiosensitizers with potentially fewer side effects. Importantly, this radiosensitizing strategy will not sensitize well-oxygenated normal tissue, thereby increasing the therapeutic index of radiotherapy.

Keywords: hypoxia; metabolism; mitochondria; radiosensitization.

Fig. 1.

Papaverine reduces OCR by inhibition of mitochondrial complex I. (A) Representative Seahorse data showing decreased OCR in EO771 cells. (B) Competition assay between papaverine and rotenone injected as indicated in EO771 cells. (C) Succinate rescue assay in permeabilized EO771 cells. PPV or rotenone were injected alone or in combination with complex III inhibitor antimycin A (AntA) at time A; succinate (Succ) was injected at time B. (D) Dose–response analysis in A549 cell line. (E) Drug washout experiment in A549 cells, the OCR reduction at 3 h of 10 μM PPV or 1 μM rotenone (magenta), and after removal at T-2 h (blue) or 1 h (black). Error bars represent SD.

Fig. 2.

Papaverine reduces tumor hypoxia and enhances response to radiation therapy. (A) FD-NIRS analysis of baseline partial oxygen pressure (pO₂) levels in immune-deficient mice with A549 or EO771 flank xenografts (n = 3–5). P values were calculated against thigh muscle. (B and C) Normalized tissue pO₂ in EO771 (B) or A549 (C) tumor-bearing mice (n = 5) after injection of 2 mg/kg PPV or saline (readings taken every minute, and curve is average of five traces). P value was analyzed by a linear mixed model with autoregressive correlation structure at T = 30–40 min. (D) Representative immunofluorescence image showing pimonidazole (green) and Hoechst nuclear counterstain (blue) in tumor cryosections from EO771 tumors treated with saline or 2 mg/kg PPV. (E) Quantification of hypoxic fractions in D (n = 4). Values are mean pimonidazole positive area evaluated from 20 images per animal ± SEM. P value was calculated with two-tailed two-sample t test. (F) Schematic of the PPV+XRT experimental design. (G) Quantification of tumor growth delay of orthotopic EO771 tumors grown in nude mice receiving either 2 mg/kg PPV (magenta), 5 Gy XRT (blue) or 2 mg/kg PPV 35 min before 5 Gy XRT (red) (n = 9–10). Curves represent mean tumor volumes ± SEM. P values were calculated against XRT by t test. (H) Tumor growth of heterotopic A549 flank xenografts in nude mice receiving either 8 Gy XRT (magenta), 2 mg/kg PPV 35 min after (blue) or before (red) 8 Gy XRT (n = 6). Error bars are ±SEM. P values were calculated against XRT by t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.

Papaverine does not increase radiation-induced normal tissue toxicity. (A) Representative H&E- and Ki67-stained sections of jejunum harvested 24 or 72 h after treatment with papaverine and/or radiation as indicated. (Scale bar, 100 μm.) (B) Quantification of proliferating cells/crypt by Ki67 staining. Data represent mean of at least 30 counted crypts per group (n = 3) ±SEM. (C) The number of regenerating crypts/field of view (FOV). Values represent the mean number of crypts with >10 Ki67-positive cells per FOV ±SEM. P values were calculated with t test. ***P < 0.001; n.s., not significant. (D) Immunofluorescence showing hypoxic marker pimonidazole (green) and Hoechst nuclear counterstain (blue) in jejunum cryosections from MiaPaca-2 tumor-bearing mice treated with saline or 2 mg/kg PPV (n = 4). (Scale bar, 50 μm.)

Fig. 4.

Papaverine radiosensitizes through complex I inhibition. (A) Western blot of NDI1 expression in mitochondrial fractions of parent A549 and NDUFV1 KO cells. (B) Representative trypan blue viability (n = 3) of cells grown in galactose-only media (T = 96 h). Values represent mean viable cells ± SD. (C) Seahorse analysis of the response of parent A549 and NDUFV1 KO ± NDI1 cells to 10 μM papaverine or 1 μM rotenone. Values are mean ± SD. (D) Quantification of pimonidazole staining in NDUFV1 KO NDI1 flank tumors treated with 2 mg/kg PPV or vehicle (n = 3). Value is mean pimonidazole-positive cells from 20 images per tumor ± SEM. P value was calculated with t test. (E) Quantification of tumor growth delay of heterotopic NDUFV1 KO NDI1 flank xenografts receiving either 8 Gy XRT (magenta) or 2 mg/kg PPV 35 min before 8 Gy XRT (blue) (n = 4). Curves represent mean tumor volumes ± SEM. P values were calculated against XRT with t test. n.s., not significant.

Fig. 5.

Papaverine can be reengineered to remove PDE activity. (A) Structures of papaverine and the lead derivatives SMV-23 and SMV-32. (B) Calculation of OCR IC50 in A549 cells by Seahorse, n = 5 replicates per group. (C) Calculation of PDE10A IC20 by PDE10A enzymatic assay; n = 3 replicates per group. (D) Acute toxicity in wild-type C57BL/6 mice (n = 3). (E) Quantification of hypoxic fractions in orthotopic EO771 tumors treated with 2 mg/kg PPV, SMV-23, SMV-32, or vehicle (n = 3). Bar graphs represent the mean area of pimonidazole-positive cells from 20 images per animal ± SEM. P value was calculated against control by t test. (F) Tumor growth delay of heterotopic EO771 flank tumors in nude mice receiving either 5 Gy XRT (red) or 2 mg/kg SMV-23 (blue), or SMV-32 (magenta) or PPV (gray) 35 min before 5 Gy XRT (n = 8). (G) Tumor growth delay of heterotopic A549 flank tumors in nude mice receiving either 8 Gy XRT (red) or 2 mg/kg SMV-23 (blue), or SMV-32 (magenta) or PPV (gray) 35 min before 8 Gy XRT (n = 5–6). Mean volumes ± SEM. P values were calculated against XRT with t test. *P < 0.05; **P < 0.01; ***P < 0.001.