Orellanine specifically targets renal clear cell carcinoma

Abstract

Renal cell carcinoma (RCC), arising from the proximal tubule in the kidney, accounts for approximately 85% of kidney cancers and causes over 140,000 annual deaths worldwide. In the last decade, several new therapies have been identified for treatment of metastatic RCC. Although these therapies increase survival time compared to standard care, none of them has curative properties. The nephrotoxin orellanine specifically targets proximal tubular epithelial cells, leaving other organs unaffected. We therefore hypothesized that the selective toxicity of orellanine extends to clear cell RCC (ccRCC) cells since they emanate from proximal tubular cells. Orellanine would thus target both primary and metastatic ccRCC in vitro and in vivo. We found that orellanine induces dose-dependent cell death in proximal tubular cells and in all ccRCC cells tested, both primary and cell lines, with no toxicity detected in control cells. The toxic action of orellanine involve decreased protein synthesis, disrupted cell metabolism and induction of apoptosis. In nude rats carrying human ccRCC xenografts, brief orellanine treatment eliminated more than 90% of viable tumor mass compared to control rats. This identifies orellanine as a potential treatment concept for ccRCC patients on dialysis, due to its unique selective toxicity towards ccRCC.

Keywords: anti-carcinogenic treatment; apoptosis; clear cell renal cell carcinoma; necrosis; nephrotoxin.

Conflict of interest statement

CONFLICTS OF INTEREST The following authors are shareholders in a small company, Oncorena AB, formed to commercially explore the potential of orellanine and finance a Phase I/II study: BH, JN, LB, UN, HH, JT and ER. JT is the CEO of Oncorena. Apart from that, there is nothing to disclose.

Figures

Figure 1. Orellanine is selectively toxic to human tubular epithelial cells and clear cell renal carcinoma cells

(A) Viability of HTEC treated for 24 hours with orellanine, normalized to vehicle treated control (n = 6, mean ± SEM). (B) HTEC were exposed to different concentrations of orellanine for 24 hours and their viability was estimated using Alamar Blue technique at 72 hours, n = 6 for each data point. ED50 equals 4.1 ± 1.2 μg/ml. (C) Viability of HTEC, liver cells (HEPG2), breast cancer cells (MDA-MB-231) and HUVEC at 144 hours post 24 hour orellanine treatment (n = 6, mean ± SEM). (D) Viability of orellanine-treated ccRCC cell lines at 144 h, normalized to vehicle-treated controls (n = 6). One of the the two cell lines showing lowest sensitivity in vitro (SKRC-17 ) was chosen for the in vivo experiments. (E) The SKRC-17 cells were exposed to different concentrations of orellanine for 24 hours. The graphs represent repeated incubation at the doses (♦ 4 and ○ 20 μg/ml), single treatment (□ 4 and ▲ 20 μg/ml) and doubling of the incubation time from 24 to 48 hours (■ 20 μg/ml), respectively. (F) Orellanine treatment of primary renal cancer cells obtained from 7 patients with clear cell RCC. Data are presented as mean ± SEM and p-values are determined by one way ANOVA with Tukey’s post hoc test, where p < 0.05 was considered significant, **p < 0.01 ***, p < 0.001.

Figure 2. Orellanine induces oxidative stress and down-regulates cell metabolism

(A) Oxidative stress in HTEC and SKRC-17 cells after 24 hours of vehicle or orellanine treatment, using carboxy-H2DCFDA for detection of ROS; scale bar 20 µm. Seahorse experiment showing: (B) Diagram of Basal Oxygen consumption rate (OCR) (basal), ATP production (ATP), maximum OCR (Max), spare respiratory capacity (SRC) and proton leak calculated from the OCR curve. (C) Glycolysis, glycolysis capacity and glycolysis reserve calculated from the Extracellular acidification rate (ECAR) curve. (D) Diagram showing the cell energy phenotype shift during mitochondrial stress conditions.

Figure 3. Orellanine promotes cell death in clear cell renal carcinoma cells

(A) FACS scatter plots of vehicle or orellanine-treated SKRC-52 cells, treated for 24 hours with 100 μg orellanine/ml and analyzed 24, 48 and 72 hours post treatment initiation, for the presence of Annexin V and/or PI (n = 12, 9, 8 and 8 for controls, 24 h, 48 h and 72 h respectively). (B) The FACS plot presented graphically. PI indicates the cells in Q1 of Figure 3A (Necrosis). Annexin indicates PI+Annexin (late apoptosis) and Annexin (Early apoptosis), i.e. panel Q2 and Q4 in Figure 3A. Data are presented as mean +/- SEM and p values are determined by ANOVA with Tukey's post hoc test where p < 0.05 was considered significant. **p < 0.01, ***p < 0.001. Caspase 3, 8 and 9 activity in (C) SKRC-17 cells and (D) SKRC-52 cells treated with vehicle or 100 μg orellanine/ml for 24 hours, (n = 3, mean ± SEM, students t-test, *p < 0.05, **p < 0.01) (E) Western blots showing duplicate samples of total protein and phosphorylated p44/p42 MAPK (ERK1/2) (Thr202/Tyr204), AKT (Ser473), p53 (Ser15) and cleaved caspase-3 following 100 μg/ml orellanine exposure for 0, 2, 6 or 24 h. Beta actin served as a loading control.

Figure 4. PD Dialysis and the effect of orellanine on kidney function in rat

(A) The measured concentration of urea (full curve) and creatinine (dotted curve) in dialysate over plasma (D/P) concentrations in orellanine-treated anuric RNU-rats (n = 5) (B) Steady state serum orellanine concentration in rats weighing 150 grams, treated repeatedly with 15 ml dialysis solution containing 40 µM (i.e. 10 mg/L) of orellanine for 45 minutes at each cycle. The Figure is based on a kinetic modeling using an estimated D/D0 of 0.4 for orellanine after 45 minutes of dialysis. (C) Serum urea and (D) creatinine in subcutaneously orellanine treated Sprague Dawley rats, 72 hours post treatment at the doses indicated (n = 3 for each dose). (E) Orellanine concentration in serum over time after a bolus injection of 10 mg/kg orellanine intravenously in anesthetized RNU rats with ccRCC metastases during control and in rats with ligated renal arteries after a 10 mg/kg intravenous bolus dose of orellanine. Data are presented as mean as mean ± SEM and p-values are determined by t test, where p < 0.05 was considered significant. *p < 0.05.

Figure 5. The human ccRCC xenograft model

(A) Tumor growth of a xenograft model of human renal cancer (SKRC-17) in whole body irradiated RNU-rats at different radiation doses (n = 3 for each dose) (B) Leukocyte blood count post whole body irradiation of RNU-rats (n = 3). (C) Tumor volume in RNU-rats radiated with 5 gray 4 days pre inoculation of SKRC-17 cells subcutaneously (n = 3). (D) Necrotic area (%) over time in hematoxylin and eosin stained tumor sections from RNU-rats. (n = 6 at day 11, 7 at day 18 and 12 at day 26).

Figure 6. Orellanine significantly reduces tumor growth and induces necrosis

Tumor volume in control rats and in rats receiving orellanine treatment, 10 mg/L for 48 hours via the dialysis solution at Day 8–9 (controls n = 5, treated n = 6). (B–C) Representative photos of hematoxylin and eosin stained tumor sections of control rats (B) and orellanine-treated rats (C), analyzed at day 16 post inoculation. (D) Necrotic area (%) of total tumor area and (E) viable tumor mass after subtraction of necrotic areas (controls n = 5, treated n = 6). (F) Representative photo of (F) control tumor and (G) orellanine-treated tumor. Data in a, d–e are presented as mean ± SEM and p-values are determined by t test, where p < 0.05 was considered significant. ***p < 0.001.

Figure 7. Orellanine-treated tumors are considerably smaller and display an evident increase in apoptosis

(A) Tumor volume in RNU-rats, with or without one 15ml cycle of dialysis per day, irradiated with 4 gray 4 days pre implantation of SKRC-17 cells subcutaneously (n = 6). (B) Tumor weight of vechicle treated control tumors (n = 5) and tumors exposed to orellanine for 5 days (10 mg/L, n = 5) or 3 days (20 mg/L, n = 5). (C) TUNEL staining in tumor sections. From left; control, 10 mg/L orellanine-treated tumors and 20 mg/L orellanine-treated tumors. (D) Diagram showing TUNEL-positive cells in tumor sections (n = 25 for each concentration and n = 20 for control sections) from control tumors and tumors treated with 10 mg/L or 20 mg/L of orellanine. Data are presented as mean ± SEM and p-values are determined by one way ANOVA with Tukey’s post hoc test, where *p < 0.05 was considered significant. **p < 0.01, ***p < 0.001.