The anti-tumoral effects of cannabinoids have been described in different tumor systems, including pancreatic adenocarcinoma, but their mechanism of action remains unclear. We used cannabinoids specific for the CB1 (ACPA) and CB2 (GW) receptors and metabolomic analyses to unravel the potential pathways mediating cannabinoid-dependent inhibition of pancreatic cancer cell growth. Panc1 cells treated with cannabinoids show elevated AMPK activation induced by a ROS-dependent increase of AMP/ATP ratio. ROS promote nuclear translocation of GAPDH, which is further amplified by AMPK, thereby attenuating glycolysis. Furthermore, ROS determine the accumulation of NADH, suggestive of a blockage in the respiratory chain, which in turn inhibits the Krebs cycle. Concomitantly, inhibition of Akt/c-Myc pathway leads to decreased activity of both the pyruvate kinase isoform M2 (PKM2), further downregulating glycolysis, and glutamine uptake. Altogether, these alterations of pancreatic cancer cell metabolism mediated by cannabinoids result in a strong induction of autophagy and in the inhibition of cell growth.
Cannabinoids are a class of bioactive lipids1, 2, 3 that have a range of interesting activities, including the ability to reduce the growth of tumours such as glioma,4 breast cancer,5 prostate cancer,6 and colon cancer7 in different animal models. They impair tumour progression at different levels, with the most prevalent effects being the inhibition of cell proliferation by apoptosis,8 cell cycle arrest,9 and autophagy.10 Cannabinoids induce autophagy in various types of cancer cell lines, and pharmacological or genetic inhibition of autophagy prevents their antiproliferative action, thus demonstrating that autophagy is important for cannabinoid antineoplastic activity.11 Autophagy is an evolutionarily conserved process in eukaryotes by which cytoplasmic cargo sequestered inside double-membrane vesicles are delivered to the lysosome for degradation.12 This process has the role to rid the cell of intracellular misfolded or long-lived proteins, superfluous or damaged organelles, and invading microorganisms, and also is an adaptive response to provide nutrients and energy on exposure to various stresses.13 In hepatocellular carcinoma cells, cannabinoids can trigger an ER stress-dependent activation of AMP-activated protein kinase (AMPK) that cooperates with the TRIB3-mediated inhibition of the Akt–mTORC1 axis in the stimulation of autophagy-mediated cell death.14 AMPK is a sensor of energy status that responds to the increase of AMP or ADP cellular concentration to maintain cellular energy homeostasis.15 AMPKs appear to exist universally as heterotrimeric complexes comprising catalytic α subunits and regulatory β and γ subunits.15 The α subunits contain a typical serine/threonine kinase domain at the N terminus and is significantly active only when phosphorylated by upstream kinases.15 The γ subunits contain four regulatory adenine nucleotide-binding sites, two of which competitively bind AMP, ADP and ATP, and are the sites via which cellular energy status is sensed.15 The major upstream kinase phosphorylating Thr 172 of the α subunit, and thus activating AMPK, in most mammalian cells is the tumour suppressor kinase LKB1.16, 17, 18 Although LKB1 has to be expressed in mammalian cells for agents that increase the cellular AMP/ATP and ADP/ATP ratios to cause the activation of AMPK,16 it is worth emphasizing that these effects are due to the binding of adenine nucleotides to the γ subunit of AMPK and that the LKB1 complex itself appears to be constitutively active.19 In some cell types, Thr 172 can also be phosphorylated by the Ca2+/calmodulin-dependent protein kinase, CaMKKβ, providing a Ca2+-activated pathway to switch on AMPK.20, 21, 22Activation by this mechanism can occur in the absence of any change in the adenine nucleotide ratios, although increases in Ca2+ can act synergistically with increases in AMP or ADP.23
Recently, we have demonstrated that cannabinoids and gemcitabine, a nucleoside analogue used in cancer chemotherapy, synergistically inhibit pancreatic adenocarcinoma cell growth by a ROS-mediated autophagy induction.10
Here, to shed light on the molecular mechanisms of autophagy induction by cannabinoids in pancreatic adenocarcinoma cells, we have investigated whether the AMPK has a role in this effect and whether this mechanism is related to the alteration of the energetic metabolism. For this purpose, we performed analysis of autophagy with a mutant of the γ subunit isoform 2 of AMPK unable to bind AMP, metabolomic analyses and determination of phosphorylation, activity or localization of proteins involved in the energetic metabolism or autophagy. We show that cannabinoids induce AMPK-mediated autophagy in pancreatic adenocarcinoma cells through a ROS-dependent increase of the AMP/ATP ratio.
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Cannabinoids shown to inhibit pancreatic cancer cell growth
