Cannabinoids in pancreatic cancer: correlation with survival and pain

Abstract

Cannabinoids exert antiproliferative properties in a variety of malignant tumors, including pancreatic ductal adenocarcinoma (PDAC). In our study, we quantitatively evaluated the immunoreactivity for cannabinoid-1 (CB1) and cannabinoid-2 (CB2) receptors as well as for the endocannabinoid metabolizing enzymes fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MGLL). Furthermore, quantitative real-time RT-PCR for CB1, CB2, FAAH and MGLL in normal pancreas and pancreatic cancer tissues was performed. Levels of endocannabinoids were determined by liquid chromatography/mass spectrometry. Immunoreactivity scores and QRT-PCR expression levels were correlated with the clinico-pathological (TNM, survival, pain) status of the patients. Evaluation of endocannabinoid levels revealed that these remained unchanged in PDAC compared to the normal pancreas. Patients with high CB1 receptor levels in enlarged nerves in PDAC had a lower combined pain score (intensity, frequency, duration; p = 0.012). There was a significant relationship between low CB1 receptor immunoreactivity or mRNA expression levels (p = 0.0011 and p = 0.026, respectively), or high FAAH and MGLL cancer cell immunoreactivity (p = 0.036 and p = 0.017, respectively) and longer survival of PDAC patients. These results are underlined by a significant correlation of high pain scores and increased survival (p = 0.0343). CB2 receptor immunoreactivity, CB2 receptor, FAAH and MGLL mRNA expression levels did not correlate with survival. Therefore, changes in the levels of endocannabinoid metabolizing enzymes and cannabinoid receptors on pancreatic cancer cells may affect prognosis and pain status of PDAC patients.

Figure 1

Cannabinoid receptor immunostaining in human pancreatic cancer tissues. Low (a, g), moderate (b, h) and strong (c, i) immunostaining for CB1 or CB2 receptors in pancreatic cancer cells, respectively. (d–f and j–l) Corresponding specificity controls pre-adsorbed with the respective blocking peptide.

Figure 2

CB1 receptor levels correlate with survival. CB receptor cancer cell protein levels as well as whole tissue mRNA expression levels were evaluated. (a) Inverse correlation (p = 0.0011) of high CB1 levels of pancreatic cancer cells and worse (shorter) patient survival. (b) No such correlation was observed for CB2 receptor immunostaining intensities. (c) Confirmation of the IHC quantitation results in a second, larger (n = 53) cohort of patients using quantitative RT-PCR: high CB1 receptor mRNA expression is related to shorter survival of the patients (p = 0.026). (d) CB2 receptor mRNA expression is not related to survival (p = 0.195).

Figure 3

FAAH and MGLL immunostaining in pancreatic cancer. Low (a, g), moderate (b, h) and strong (c, i) immunostaining for FAAH or MGLL in pancreatic cancer cells, respectively. (d–f and j–l) Corresponding specificity controls pre-adsorbed with the respective blocking peptide.

Figure 4

FAAH and MGLL cancer cell levels correlate with survival. FAAH and MGLL cancer cell protein levels as well as whole tissue mRNA expression levels were evaluated. (a) Correlation (p = 0.036) of high FAAH levels of pancreatic cancer cells and longer patient survival. (b) Correlation (p = 0.017) of MGLL cancer cell immunostaining intensity and survival. (c, d) FAAH and MGLL mRNA whole pancreatic cancer tissue expression levels are not correlated with survival (p = 0.874 and p = 0.62, respectively).

Figure 5

Cannabinoid receptor immunoreactivity of nerves in pancreatic cancer. Low (a, g), moderate (b, h) and strong (c, i) immunostaining for CB1 or CB2 receptors, respectively, in nerves of pancreatic cancer tissues. (d–f and j–l) Corresponding specificity controls pre-adsorbed with the respective blocking peptide. (m) High CB1 levels in pancreatic nerves are associated with a lower pain score (p = 0.012), while no such association is seen for CB2 levels (p = 0.35). (n) Kaplan–Meier survival curves of patients with low and high pain scores. Median survival was 11 months for patients with low pain scores and 24.5 months for patients with high pain scores (p = 0.0343).

Figure 6

FAAH and MGLL immunoreactivity of nerves in pancreatic cancer and endocannabinoid levels. Low (a, g), moderate (b, h) and strong (c, i) immunostaining for FAAH or MGLL receptors, respectively, in nerves of pancreatic cancer tissues. (d–f and j–l) Corresponding negative controls. (m) Neither FAAH nor MGLL levels in pancreatic nerves are associated with pain (p = 0.06 and p = 0.58, respectively). (n) Endocannabinoid levels: 1 + 2-AG and AEA were unchanged in a comparison of normal pancreas and pancreatic cancer tissues (p = 1.0 and p = 0.213, respectively). NP: normal pancreas; PDAC: pancreatic ductal adenocarcinoma.