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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.

Orellanine is a nephrotoxin found in various Cortinaceae mushroom species. Unintentional consumption after these species were confused with edible mushrooms such as Cantharellus tubaeformis has caused several casualties. In this work, a quantitative HPLC-ESI-MS/MS method for total orellanine in Cortinarius rubellus, spiked blood plasma, and a mushroom stew prepared from C. tubaeformis with the addition of a single specimen of C. rubellus is presented. The existence of mono- and diglucosylated orellanine in C. rubellus was also proven, although quantitative analysis could not be obtained for the glucosides due to rapid hydrolyzation to orellanine in the extract. Extraction with 3 M HCl or water mainly yielded orellanine, while MeOH or acidified MeOH mainly extracted mono- and diglucosylated orellanine. The highest recovery of total orellanine was obtained with 3 M HCl, which was subsequently used for quantitative analysis. A C₁₈ HPLC column and low pH in the eluents retained all these toxins. Orellanine could be detected at a 4.9 ng/mL level in all extracts, which is well below the threshold for acute toxic effects. Additionally, the fragmentation pattern of orellanine upon electrospray MS/MS was probed. The method described is useful for two important applications. First, it allows quantitative analysis of processed food products that may be contaminated by orellanine from Cortinaceae mushrooms. Second, orellanine is currently being evaluated as a potential cure of metastatic renal cancer, and this work provides a method for monitoring orellanine at low concentrations within the therapeutic interval in blood serum.

Renal cancer causes over 100,000 annual deaths worldwide and constitutes about 3% of all solid cancers. The majority of renal cell carcinomas (85%), referred to as clear cell renal cell carcinoma (CCRCC), are notorious for their metastatic frequency and high resistance to conventional cancer therapies. Several new targeted therapies have been approved for treatment of metastatic renal cell carcinoma (mRCC), and although they induce a longer progression free survival, the effect on overall survival has not been statistically significant. One third of the patients with CCRCC present with advanced disease at diagnosis, and another third will develop metastases eventually, even if the original tumor is successfully removed. A metastatic form of the disease correlates with a bad poor prognosis reflected in the 5-year survival, which is less than 11% for this patient group. A fungal toxin from the Cortinarius-family, NC-001, have been shown to be specifically toxic to the proximal tubular cells in the kidney. We have shown that this sensitivity also is extended to renal cancer cells which have evolved from these. Preliminary data demonstrate that NC-001 reduces viability in five renal cancer cell lines, originating both from primary tumors and metastatic lesions. HUVEC cells seem unaffected, however at the highest dose (800µM) a reduction in viability is observed. Presently, we are evaluating the molecular mechanism behind the effect of NC-001. In vitro results show an induction of apoptosis via the intrinsic pathway, by activation of caspase-8 and further downstream by caspase-3. Caspase-9 seems to bewas unaffected. FACS analysis with Alexa Flour488 conjugated Annexin V and phosphatidylserine (PI) of SKRC-52 cells treated with 400µM for 24 hours, shows increased apoptosis and necrosis. A xenograft model of CCRCC, in radiated athymic rats, was developed to further investigate NC-001's effects in vivo. Since NC-001 affects the proximal tubular cells in the kidney and induces severe renal failure, an automated peritoneal dialysis system for rats was developed as renal replacement therapy. The rats were treated via the dialysis solution containing 40µM NC-001 for two days. Six days later, extensive necrosis was evident in the tumors of treated rats (n=5) compared to controls (n=5). In summary, our results indicate that NC-001 induces apoptosis, mainly via the intrinsic pathway, and necrosis in SKRC-52 cells and also in solid CCRCC xenograft tumors. NC-001 has therefore potential as a curative treatment of metastatic CCRCC. The sole expected side effect of NC-001, based on hundreds of case reports of Cortinarius intoxications in the literature, is collateral loss of kidney function. While this may appear dramatic, it is surely preferable to death from metastatic cancer, especially if long term survival can be achieved. Besides, it can be well managed with dialysis, and later on, kidney transplantation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-118. doi:1538-7445.AM2012-LB-118