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Previous studies have demonstrated that diallyl disulfide (DADS) exhibits potent anti-tumor activity. However, the pharmacological actions of DADS in inhibiting the growth of colorectal cancer (CRC) cells have not been clarified. Herein, we show that DADS treatment impairs the activation of the pentose phosphate pathway (PPP) to decrease PRPP (5-phosphate ribose-1-pyrophosphate) production, enhancing DNA damage and cell apoptosis, and inhibiting the growth of CRC cells. Mechanistically, DADS treatment promoted POU2F1 K48-linked ubiquitination and degradation by attenuating the PI3K/AKT signaling to up-regulate TRIM21 expression in CRC cells. Evidently, TRIM21 interacted with POU2F1, and induced the K272 ubiquitination of POU2F1. The effects of DADS on the enhanced K272 ubiquitination of POU2F1, the PPP flux, PRPP production, DNA damage and cell apoptosis as well as the growth of CRC tumors in vivo were significantly mitigated by TRIM21 silencing or activating the PI3K signaling in CRC cells. Conversely, the effects of DADS were enhanced by TRIM21 over-expression or inhibiting the PI3K/AKT signaling in CRC cells. Collectively, our findings reveal a novel mechanism by which DADS suppresses the growth of CRC by promoting POU2F1 ubiquitination, and may aid in design of novel therapeutic intervention of CRC.

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ETHNOPHARMACOLOGICAL RELEVANCE: Rhubarb is a natural herbal medicine widely used clinically with numerous pharmacological activities including anti-cancer. Specifically, several studies reported that free anthraquinones from Rhubarb suppressed the proliferation of hepatoma cells. Nonetheless, recent studies revealed that Rhubarb caused hepatotoxicity in vivo, confirming its "two-way" effect on the liver. Therefore, the efficacy and safety of Rhubarb in the in vivo treatment of liver cancer should be further elucidated. AIM OF THE STUDY: This study investigated the presence of hepatoprotection or hepatotoxicity of Rhubarb in diethylnitrosamine (DEN)-induced hepatocarcinogenesis. MATERIAL AND METHODS: A total of 112 male Sprague-Dawley rats weighing 190-250 g were enrolled. The rats were induced hepatocarcinogenesis using diethylnitrosamine (0.002 g/rat) until 17 weeks. Starting at week 11, Rhubarb granules (4 g/kg and 8 g/kg) were intragastrically administered daily for 7 weeks. All rats were euthanized at week 20 and the livers were analyzed via non-targeted metabolomics analysis. We established hepatic glucose 6 phosphate (6PG) levels and glucose 6 phosphate dehydrogenase (G6PD) activities to assess the pentose phosphate pathway (PPP). And the liver injuries of rats were analyzed via histological changes, hepatic function, as well as hepatic protein levels of alpha-fetoprotein (AFP), pyruvate kinase isozyme type M2 (PKM2), and proliferating cell nuclear antigen (PCNA). Furthermore, polydatin (0.1 g/kg/d) as a specific inhibitor of G6PD was used to treat rats. Notably, their histological changes, hepatic function, hepatic 6PG levels, hepatic G6PD activities, PCNA levels, and PKM2 levels were recorded. RESULTS: Non-targeted metabolomics revealed that Rhubarb regulated the PPP in the liver of Rhubarb-DEN-treated rats. Besides, Rhubarb activated the oxidative branch of the PPP by activating G6PD (a rate-limiting enzyme in the oxidative PPP) in the liver of Rhubarb-DEN-treated rats. Meanwhile, Rhubarb promoted DEN-induced hepatocarcinogenesis. Moreover, polydatin attenuated the promoting effect of Rhubarb on DEN-induced hepatocarcinogenesis. CONCLUSIONS: Rhubarb promoted DEN-induced hepatocarcinogenesis by activating the PPP, indicating that the efficacy and safety of Rhubarb in the treatment of liver cancer deserve to be deliberated.

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The pentose phosphate pathway (PPP) branches from glucose 6-phosphate (G6P), produces NADPH and ribose 5-phosphate (R5P), and shunts carbons back to the glycolytic or gluconeogenic pathway. The PPP has been demonstrated to be a major regulator for cellular reduction-oxidation (redox) homeostasis and biosynthesis. Enzymes in the PPP are reported to play important roles in many human diseases. In this review, we will discuss the role of the PPP in type 2 diabetes and cancer.

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Manipulation of metabolic pathways (e.g., ketogenic diet (KD), glycolytic inhibition) alters neural excitability and represents a novel strategy for treatment of drug-refractory seizures. We have previously shown that inhibition of glycolysis suppresses epileptiform activity in hippocampal slices. In the present study, we aimed to examine the role of a "branching" metabolic pathway stemming off glycolysis (i.e., the pentose-phosphate pathway, PPP) in regulating seizure activity, by using a potent PPP stimulator and glycolytic intermediate, fructose-1,6-bisphosphate (F1,6BP). Employing electrophysiological approaches, we investigated the action of F1,6BP on epileptiform population bursts, intrinsic neuronal firing, glutamatergic and GABAergic synaptic transmission and voltage-activated calcium currents (ICa) in the CA3 area of hippocampal slices. Bath application of F1,6BP (2.5-5 mM) blocked epileptiform population bursts induced in Mg2+-free medium containing 4-aminopyridine, in ~2/3 of the slices. The blockade occurred relatively rapidly (~4 min), suggesting an extracellular mechanism. However, F1,6BP did not block spontaneous intrinsic firing of the CA3 neurons (when synaptic transmission was eliminated with DNQX, AP-5 and SR95531), nor did it significantly reduce AMPA or NMDA receptor-mediated excitatory postsynaptic currents (EPSCAMPA and EPSCNMDA). In contrast, F1,6BP caused moderate reduction (~50%) in GABAA receptor-mediated current, suggesting it affects excitatory and inhibitory synapses differently. Finally and unexpectedly, F1,6BP consistently attenuated ICa by ~40% without altering channel activation or inactivation kinetics, which may explain its anticonvulsant action, at least in this in vitro seizure model. Consistent with these results, epileptiform population bursts in CA3 were readily blocked by the nonspecific Ca2+ channel blocker, CdCl2 (20 µM), suggesting that these bursts are calcium dependent. Altogether, these data demonstrate that the glycolytic metabolite, F1,6BP, blocks epileptiform activity via a previously unrecognized extracellular effect on ICa, which provides new insight into the metabolic control of neural excitability.