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. Author manuscript; available in PMC: 2022 Jun 5.
Published in final edited form as: Eur J Pharmacol. 2021 Mar 26;900:174065. doi: 10.1016/j.ejphar.2021.174065

Central and peripheral emetic loci contribute to vomiting evoked by the Akt inhibitor MK-2206 in the least shrew model of emesis

Weixia Zhong 1, Seetha Chebolu 1, Nissar A Darmani 1
PMCID: PMC8085164  NIHMSID: NIHMS1687744  PMID: 33775646

Abstract

Akt (protein kinase B) signaling is frequently activated in diverse cancers. Akt inhibitors such as perifosine and MK-2206 have been evaluated as potential cancer chemotherapeutics. Although both drugs are generally well tolerated, among their most common side-effects vomiting is a major concern. Here we investigated whether these Akt inhibitors evoke emesis in the least shrew model of vomiting. Indeed, both perifosine and MK-2206 induced vomiting with maximal efficacies of 90% at 50 mg/kg (i.p.) and 100% at 10 mg/kg (i.p.), respectively. MK-2206 (10 mg/kg, i.p.) increased c-Fos immunoreactivity both centrally in the shrew brainstem dorsal vagal complex (DVC) emetic nuclei, and peripherally in the jejunum. MK-2206 also evoked phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in both the DVC emetic nuclei and the enteric nervous system in the jejunum. The ERK1/2 inhibitor U0126 suppressed MK-2206-induced emesis dose-dependently. We then evaluated the suppressive efficacy of diverse antiemetics against MK-2206-evoked vomiting including antagonists/inhibitors of the: L-type Ca2+ channel (nifedipine at 2.5 mg/kg., subcutaneously (s.c.)); glycogen synthase kinase 3 (GSK-3) (AR-A014418 at 10 mg/kg and SB216763 at 0.25 mg/kg., i.p.); 5-hydroxytryptamine 5-HT3 receptor (palonosetron at 0.5 mg/kg, s.c.); substance P neurokinin NK1 receptor (netupitant at 10 mg/kg., i.p.) and dopamine D2/3 receptor (sulpride at 8 mg/kg, s.c.). All tested antagonists/blockers attenuated emetic parameters to varying degrees. In sum, this is the first study to demonstrate how pharmacological inhibition of Akt evokes vomiting via both central and peripheral mechanisms, a process which involves multiple emetic receptors.

Keywords: PI3K, Akt, MK-2206, perifosine, emesis, least shrew

Graphical Abstract

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1. Introduction

Akt (protein kinase B) signaling pathway hyperactivation is frequently observed in diverse cancers (Manning and Toker, 2017). A variety of inhibitors have been developed to target this pathway to improve cancer treatment (Al-Saffar et al., 2018; Brown and Banerji, 2017; Nitulescu et al., 2016; Yap et al., 2011) and to reduce impending common side-effects such as nausea and vomiting (Nunnery and Mayer, 2019). MK-2206 is a potent, orally-effective, allosteric pan-Akt inhibitor with potential antineoplastic activity. It is currently evaluated in numerous clinical trials (Chung et al., 2017; Kalinsky et al., 2018; Ma et al., 2017; Yap et al., 2011) with grade 1 to 2 nausea and vomiting as side-effects (Yap et al., 2011). The alkylphospholipid Akt inhibitor perifosine, has also been reported to cause grade 3 nausea and vomiting during a single-agent trial (Becher et al., 2017).

We have previously demonstrated that the Akt signaling pathway is involved in emesis (Zhong et al., 2019). In fact, a time-dependent upregulation of phosphorylation of Akt at Ser473 site in the least shrew brainstem occurs following intraperitoneal (i.p.) administration of the selective substance P neurokinin type 1 receptor (NK1R) agonist GR73632 (Zhong et al., 2019). Activation of Akt signaling can be followed by phosphorylation of its downstream target protein glycogen synthase kinase (GSK-3α/β) at Ser21/9 and its subsequent inactivation (Matsuda et al., 2019). We recently found that phospho-GSK-3α/β Ser21/9 levels in the least shrew brainstem were upregulated by diverse emetogens including the nonselective and/or selective agonists of 5-hydroxytryptamine (5-HT, serotonin) type 3 (5-HT3) (e.g. 5-HT or 2-Methyl-5-HT)-, neurokinin NK1 (e.g. GR73632)-, dopamine D2/3 (e.g. apomorphine or quinpirole)-, and muscarinic M1 (e.g. pilocarpine or McN-A-343)-receptors; as well as the L-type Ca2+ channel (LTCC) agonist (FPL64176) and the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (Zhong et al., 2020). Moreover, pretreatment with GSK-3-specific inhibitors, AR-A014418 or SB216763, suppressed the emetic efficacy of the above discussed emetogens.

In brief the emetic loci involved in vomiting may include: i) the central brainstem dorsal vagal complex (DVC) [area postrema (AP), nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMNX)], and ii) the peripheral loci involving the enteric nervous system (ENS), enterochromaffin cells (EC cells) of the gastrointestinal tract (GIT), and vagal afferents which carry emetic input from the GIT to the brainstem DVC (Babic and Browning, 2014; Darmani and Ray, 2009). The involvement of jejunal ENS in the vomiting induced by various specific and nonspecific emetogens has previously been confirmed in least shrews (Chebolu et al., 2010; Darmani et al., 2009; Ray et al., 2009). In the present study, we sought to determine the emetic effects of Akt inhibitors and their potential mechanisms. Initially, we investigated whether systemic administration of varying doses of the Akt inhibitors perifosine and MK-2206, can induce vomiting in least shrews. Secondly, we investigated the involvement of central and peripheral emetic loci underlying a fully-effective emetic-dose of MK-2206 by means of c-Fos and phospho-ERK1/2 immunostaining to indicate whether MK-2206 activates the brainstem DVC (the AP, NTS and DMNX), and/or the ENS of the jejunum. Afterwards we examined the antiemetic potential of the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor (U0126) (Hotokezaka et al., 2002) and GSK-3 inhibitors (AR-A014418 and SB216763) (Coghlan et al., 2000; Mazzardo-Martins et al., 2012), as well as antagonists targeting the emetic LTCC (nifedipine) (Triggle, 2007), serotonin 5-HT3 (palonosetron)- (Navari, 2013; Rojas et al., 2014), neurokinin NK1 (netupitant) (Navari, 2013; Rojas et al., 2014)- and dopamine D2/3 (sulpride) (Jaworski et al., 2001; Naef et al., 2017)-receptors against MK-2206-evoked vomiting. Systemic administration of the above drugs exhibited antiemetic effects against various emetogens in our previous studies (Darmani et al., 1999; 2014; 2015; Zhong et al., 2019; 2020). The findings of the present study indicate that MK-2206 causes emesis involving both central and peripheral emetic loci and systemic administration of the above antiemetics can suppress the evoked vomiting in least shrews to varying degrees.

2. Materials and methods

2.1. Animals

A colony of adult least shrews from the Western University of Health Sciences Animal Facilities were housed in groups of 5–10 on a 14:10 light:dark cycle, and were fed and watered ad libitum. The experimental shrews were 45–60 days old and weighed 4–6 g. Animal experiments were conducted in accordance with the principles and procedures of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by the Institutional Animal Care and Use Committee of Western University of Health Sciences (Protocol Approval # R20IACUC018). All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiments.

2.2. Chemicals

The following drugs were used in the present studies: AR-A014418, SB216763 and U0126 were purchased from Tocris (Minneapolis, MN). Perifosine, nifedipine and sulpride were obtained from Sigma Sigma/RBI (St. Louis, MO). MK-2206 was acquired from Santa Cruz Biotechnology (Dallas, TX). The serotonin 5-HT3R antagonist palonosetron and the NK1R antagonist netupitant were kindly provided by Helsinn Health Care (Lugano, Switzerland).

Palonosetron was dissolved in water. MK-2206 and AR-A014418 and were dissolved in 25% DMSO in water. SB216763 was dissolved in 2.5% DMSO in water. Sulpride was dissolved in distilled water with a 10 μl volume of 1/3 concentrated HCl which was then back titrated to pH 5 by the addition of NaOH. Nifedipine, netupitant and U0126 were dissolved in a mixture of ethanol/Tween 80/saline at a volume ratio of 1:1:18 so that administration of large amounts of DMSO to the shrews could be avoided. All drugs were administered at a volume of 0.1 ml/10 g of body weight.

2.3. Behavioral emesis studies

On the day of the experimentation shrews were brought from the animal facility, separated into individual cages and allowed to adapt for at least two hours (h). Daily food was withheld 2 h prior to the start of the experiment but shrews were given 4 mealworms each prior to emetogen injection, to aid in identifying wet vomits as described previously (Darmani, 1998). For dose-response emesis studies with Akt inhibitors, different groups of shrews were injected with varying doses of either perifosine (0, 1, 10, 20 and 50 mg/kg, i.p., n = 6–10) or MK-2206 (0, 5, 7.5 and 10 mg/kg, i.p., n = 10). Based on the results obtained, i.p. administration of a 10 mg/kg dose of MK-2206 caused maximal frequency of emesis in all tested shrews, and thus was chosen for subsequent antagonist interaction and immunostaining studies. To evaluate drug interaction studies, different groups of shrews were pretreated with an injection of either corresponding vehicle or varying doses, or a designated dose based on our previously published reports of the following antiemetics: ERK1/2 inhibitor U0126 (2.5 and 10 mg/kg, i.p., n = 6) (Zhong et al., 2019); GSK-3 inhibitor AR-A014418 (10 mg/kg, i.p., n = 6) (Zhong et al., 2020); GSK-3 inhibitor SB216763 (0.25 mg/kg, i.p., n = 6) (Zhong et al., 2020); LTCC inhibitor nifedipine (2.5 mg/kg, s.c., n = 6) (Darmani et al., 2014); serotonin 5-HT3 receptor antagonist palonosetron (0.5 mg/kg, s.c, n = 6) (Darmani et al., 2014); neurokinin NK1 receptor antagonist netupitant (5 and 10 mg/kg, i.p., n = 6) (Zhong et al., 2019); dopamine D2/3 receptor antagonist sulpride (8 mg/kg., s.c., n = 7) (Darmani et a., 1999). Following 30 min, each pretreated shrew was challenged with a single fully-effective emetic-dose of MK-2206 (10 mg/kg., i.p.). GSK-3 inhibitors and nifedipine were tested against both Akt inhibitors perifosine (50 mg/kg., i.p.) and MK-2206 (10 mg/kg., i.p.). Immediately following injection of emetogens, each shrew was placed in the observation cage and the frequency of emesis was recorded for the next 30 min. In the emesis studies the observer was blinded to animals’ treatment conditions. Each shrew was used once and then euthanized with isoflurane following the termination of each experiment.

2.4. c-Fos immunostaining and image analysis

Following MK-2206 (10 mg/kg, i.p.) injection, vomiting shrews were subjected to c-Fos staining (n = 4 shrews/group). Following 90 min after the first emesis occurred, shrews were anesthetized with isoflurane and perfused with ice cold 4% paraformaldehyde in pH 7.4, 0.1 M phosphate-buffered saline (PBS) for 10 min. Brainstems were removed and cryoprotected with 30% sucrose in 0.01 M PBS overnight. The OCT-embedded brainstem block was cut on a freezing microtome (Leica, Bannockburn, IL, USA) into 20-μm sections, and stored in PBS with 0.03% sodium azide. Immunolabeling using rabbit anti-c-Fos polyclonal antibody (1:5000, ab190289, Abcam) were performed. Alexa Fluor 594 donkey anti-rabbit IgG (1:500, Invitrogen) was used as secondary antibody. Nuclei of cells were stained with DAPI. Images of the brainstem sections containing the DVC (AP/NTS/DMNX) were taken by a confocal microscope (Zeiss) with Zen software using 20× objective. We have already described the circumventricular organ AP and its different cytoarchitectonic detail such as cell size and packing, which differentiates DMNX from NTS within the DVC, as part of a stereotaxic atlas of the least shrew brainstem (Ray et al., 2009). This criterion has been well recognized (Chebolu et al., 2010; Darmani et al., 2008; Ray et al., 2009; Zhong et al., 2014).

Another set of c-Fos immunostaining were also performed on sections of the jejunal segment of the least shrew small intestine which was previously found to be associated with emesis (Chebolu et al., 2010; Ray et al., 2009). The jejunum was cut at an oblique angle to the longitudinal axis into 25 μm sections with a total 100 sections in 3 continuous pools, resulting in obliquely oriented slices containing layers of the intestinal wall including the enteric nervous system (Chebolu et al., 2010; Ray et al., 2009). Co-immunolabeling using rabbit anti-c-Fos polyclonal antibody and mouse anti-NeuN (neuronal marker) antibody (1:300, MAB377, Millipore) were further performed. Alexa Fluor 594 donkey anti-rabbit IgG (1:500, Invitrogen) and Alexa Fluor 488 donkey anti-mouse IgG (1:500, Invitrogen) were used as secondary antibodies. Nuclei of cells were stained with DAPI. Images were taken by a confocal microscope (Zeiss) with Zen software using 20× and 60× objectives.

Areas demonstrating Fos-immunoreactivity (IR) were marked as regions of interest (AP/NTS/DMNX/jejunum). A Fos-IR cell nucleus was only counted as positive if it retained its ovoid shape after high-pass filtering to eliminate variations in background as well as potential false positive and was fully within the defined region of interest. The numbers of Fos-positive nuclei of each region were counted by an observer blind to the animal’s treatment condition. For each region, the same number of sections were counted per animal: 3 brainstem sections at 90-μm intervals each through AP/NTS/DMNX; and 3 jejunal sections from 3 continuous pools of jejunal sections (3 images acquired for each section). The mean value per section, from an individual animal was used in statistical analysis.

2.5. Phospho-ERK1/2 immunohistochemistry

Adult least shrews were administered MK-2206 (10 mg/kg, i.p.) or vehicle (n = 3 animals per group) and rapidly anesthetized with isoflurane and subjected to perfusion at 15 min post-treatment to evaluate phospho-ERK1/2 alteration. Brain sections (20 μm) observed under a light microscope and those containing the brainstem DVC as well as the jejunal sections (25 μm) were subjected to immunostaining as described in our previous publication (Zhong et al., 2019). Co-immunostaining using rabbit anti-phospho-ERK1/2 (Thr202/Thr204) (1:500, 4370, Cell Signaling) antibody and mouse anti-NeuN antibody was followed by Alexa Fluor 594 donkey anti-rabbit IgG (1:500, Invitrogen) and Alexa Fluor 488 donkey anti-mouse IgG (1:500, Invitrogen) secondary antibodies incubation. After washing with PBS 4 times, sections were mounted with anti-fade mounting medium containing DAPI staining nuclei (Vector Laboratories). Images were acquired under a confocal microscope (Zeiss) with Zen software using 20× and 60× objectives. Integrated density of phospho-ERK1/2 immunoreactivity in each region (AP/NTS/DMNX) of 3 sections from each animal of both groups was determined with ImageJ and the mean value per section from individual animals was used in statistical analysis.

2.6. Statistical analysis

The vomit frequency data were analyzed using the Kruskal-Wallis non-parametric one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test and expressed as the mean ± S.E.M. In the case of comparing the vomit frequency data between two groups, Unpaired parametic t-test was used. The percentage of animals vomiting across groups at different doses was compared using the Chi-square test. For c-Fos and phospho-ERK1/2 immunostaining data, statistical significance for differences between MK-2206-induced vomiting and vehicle-injected, non-vomiting groups was tested by Unpaired parametic t-test. P < 0.5 was considered statistically significant.

3. Results

3.1. Akt inhibitors induce emesis

The emesis data for Akt inhibitors perifosine and MK-2206 are depicted in Fig. 1. Intraperitoneal administration of increasing doses of perifosine enhanced the frequency of emesis in the least shrew (KW (4, 33) = 22.74, P = 0.0001). Dunnett’s multiple comparisons post hoc test showed that perifosine significantly increased the vomit frequency at its 50 mg/kg dose (P = 0.0007) (Fig. 1A). Moreover, the Chi-square test indicated that the percentage of animals exhibiting emesis in response to perifosine also increased (χ2 (4, 33) = 20.81, P = 0.0003) with 50% of shrews vomiting at its 20 mg/kg dose (P = 0.0455) and 90% vomiting at its 50 mg/kg dose (P = 0.0004) (Fig. 1B). In the case of MK-2206, its intraperitoneal administration also increased the frequency of emesis in least shrews (KW (3, 36) = 27.86, P < 0.0001). Dunnett’s multiple comparisons post hoc test showed that MK-2206 significantly increased the vomit frequency at its 7.5 mg/kg dose (P = 0.0198) and 10 mg/kg dose (P < 0.0001) (Fig. 1C). In addition, the Chi-square test indicated that the percentage of animals exhibiting emesis in response to MK-2206 also increased (χ2 (3, 36) = 27.88, P < 0.0001) with 70% of tested shrews vomiting at its 7.5 mg/kg dose (p = 0.001) and all tested shrews vomiting at its 10 mg/kg dose (P < 0.0001) (Fig. 1D).

Fig. 1. Emetic effects of Akt inhibitors in the lease shrew.

Fig. 1.

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Varying doses of perifosine (A and B) (n = 6–10) and MK-2206 (C and D) (n = 10) were administered (i.p.) to different groups of shrews and emetic responses were recorded during the next 30 min immediately following the administration of each emetogen. Statistical analysis for the mean vomit frequency was performed using Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and data are expressed as mean ± S.E.M. Statistical analysis for percentage of shrews vomiting was performed using Chi-square test and data are expressed as mean. *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. 0 mg/kg.

3.2. The Akt inhibitor MK-2206 activates both central and peripheral emetic nuclei

c-Fos induction is an indirect but classical tool to evaluate neuronal activation following peripheral agonist stimulation (Bullitt, 1990). Thus, we conducted immunohistochemistry to determine c-Fos responsiveness following systemic administration of MK-2206 (10 mg/kg, i.p.) and its vehicle (25% DMSO). Relative to the non-vomiting vehicle controls (Fig. 2A), MK-2206 (10 mg/kg) markedly evoked c-Fos induction in the brainstem emetic DVC nuclei containing the AP, NTS and DMNX (Fig. 2C). The ENS is embedded in the wall of the gastrointestinal tract (Fung and Vanden, 2020; Galligan, 2009). As shown in Fig. 2G, the jejunal ENS, but not villus epithelial cells and crypt cells, also demonstrated strong nuclear c-Fos staining signal at 90 min after MK-2206 administration (10 mg/kg., i.p.), and the c-Fos immunoreactivity was barely observed following vehicle treatment (Fig. 2E). The numbers of Fos-IR positive cell nuclei in each region of interest are delineated in Fig. 3. In vehicle-treated control shrews, the average values for Fos-IR positive cell nuclei were 13.5 ± 1.4, 31.8 ± 4.1, 20 ± 1.8 and 1.9 ± 0.8 in the AP, NTS, DMNX and ENS, respectively. Following vomiting induced by MK-2206, the mean numbers of Fos-IR positive cell nuclei were increased to 44.8 ± 3.4 in the AP (P = 0.0001 vs. Control), 96 ± 6.3 in NTS (P = 0.0001), 46 ± 3.4 in DMNX (P = 0.0005) and 35.9 ± 5.0 (P = 0.0005) in the jejunum.

Fig. 2. Effect of the Akt inhibitor MK-2206 on central and peripheral c-Fos immunoreactivity (IR).

Fig. 2.

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Brainstem sections (20 μm) and jejunal sections (25 μm) prepared from either vehicle-treated control, or shrews treated with MK-2206 (10 mg/kg, i.p.), were immunostained with c-Fos antibody. (A-D) Representative c-Fos staining images (20×) of the brainstem dorsal vagal complex containing the emetic nuclei area postrema (AP), the nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus (DMNX). Fos-IR was induced in the DVC of MK-2206-injected shrews (C). The differentiation among these three regions is described in Method section 2.4. Scale bar, 100 μm. (E–H) Representative images (20×) show the effect of MK-2206 (10 mg/kg, i.p.) on c-Fos IR in the least shrew jejunum. Very little or no c-Fos was expressed in the jejunum of vehicle-injected controls (E). c-Fos-IR was induced in the jejunum of MK-2206-injected shrews (G). Scale bar, 100 μm. Nuclei were stained with DAPI in blue (B, D, F, H).

Fig. 3.

Fig. 3.

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Quantified data for the of MK-2206-induced c-Fos expression in the least shrew brainstem dorsal vagal complex composed of the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX), as well as the in the wall of jejunum where the enteric nervous system (ENS) is embedded. Values represent the mean number of c-Fos-IR nuclei of each region of interest per section and is presented as mean ± S.E.M. (n = 4 shrews per group). ***P < 0.001 vs. Control (treated with vehicle of MK-2206), Unpaired t-test.

NeuN is considered as a marker of intrinsic primary afferent neurons of the ENS and its immunoreactivity can be found both in the nuclei and in the cytoplasm of neurons (Rivera et al., 2009; Welch et al., 2009). Consistently, in this study, both cytoplasmic and nuclear expression of NeuN was observed (Fig. 4B). Co-staining of c-Fos with NeuN suggests that some of c-Fos positive cells in the jejunal ENS are intrinsic primary afferent neurons (Fig. 4).

Fig. 4. Immunohistochemical analysis of c-Fos expression in the jejunum.

Fig. 4.

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Co-staining of jejunal sections from MK-2206 (10 mg/kg., i.p.)-treated shrews with rabbit c-Fos and mouse anti-NeuN (neuronal marker considered to label intrinsic primary afferent neurons of the enteric nervous system) antibodies was followed by Alexa Fluor 594 donkey anti-rabbit and 488 donkey anti-mouse secondary antibodies. Nuclei were stained with DAPI in blue. (A–D) Representative images (60×) show c-Fos expression seen in NeuN-positive neurons of the enteric nervous system (ENS) which are embedded in the lining of the intestine, but also seen in NeuN-negative cells. Scale bar, 50 μm.

3.3. Emesis induced by MK-2206 involves ERK1/2 phosphorylation both centrally and peripherally

In order to determine the involvement of ERK1/2 in MK-2206-induced emesis, we performed immunohistochemistry on brainstem sections containing the emetic nuclei from both vehicle control- and MK-2206 (10 mg/kg., i.p.)-pretreated animals. Representative immunoreactive brainstem sections (Fig. 5) showed that 15 min following systemic administration of MK-2206, significant increases were observed in ERK1/2 phosphorylation throughout the DVC including the AP, NTS and DMNX. A robust ERK1/2 phosphorylation was also observed in NeuN-positive neurons of the jejunal ENS after MK-2206 treatment (Fig. 6).

Fig. 5. Effect of the Akt inhibitor MK-2206 on central ERK1/2 phosphorylation.

Fig. 5.

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Shrews were killed 15 min after MK-2206 (10 mg/kg, i.p.) or vehicle administration (Control) (n = 4 per group). Brainstem sections (20 μm) were stained with rabbit anti-phospho-ERK1/2 antibody and Alexa Fluor 594 donkey anti-rabbit IgG. Nuclei were stained with DAPI in blue. (A–D) Representative images (20×) show upregulation of ERK1/2 phosphorylation (pERK) in response to MK-2206 treatment in the brainstem dorsal vagal complex (DVC) emetic nuclei containing the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX). Scale bar, 100 μm. (E) Statistic analysis of integrated density of pERK per section in AP, NTS and DMNX. *P < 0.05 vs. Control, Unpaired t-test.

Fig. 6. Effect of the Akt inhibitor MK-2206 on peripheral ERK1/2 phosphorylation.

Fig. 6.

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Shrews were killed 15 min after MK-2206 (10 mg/kg, i.p.) administration (n = 3 per group). The jejunal sections (25 μm) were co-stained with rabbit anti-phospho-ERK1/2 antibody and mouse anti-NeuN antibody followed by Alexa Fluor 594 donkey anti-rabbit and 488 donkey anti-mouse secondary antibodies incubation. Nuclei were stained with DAPI in blue. (A–D) Representative images (20×) show significant upregulation of ERK1/2 phosphorylation (pERK) in response to MK-2206 administration in the wall of jejunum where the enteric nervous system (ENS) is embedded. (E–L) Representative images (60×) show MK-2206-evoked ERK1/2 phosphorylation (pERK) occurred in NeuN-positive neurons of the jejunal ENS.

To further validate the role for ERK1/2 activation in MK-2206-mediated emesis, we examined the antiemetic potential of the ERK1/2 inhibitor U0126 behaviorally (Fig. 7). U0126 (0, 2.5 and 10 mg/kg, i.p.) was administered to different groups of shrews 30 min prior to MK-2206 (10 mg/kg, i.p.) injection. As shown in Fig. 7A and 7B, U0126 pretreatment suppressed the frequency of MK-2206-induced vomiting (KW (2, 15) = 10.61, P = 0.0016), as well as the percentage of shrews vomiting (χ2 (2, 15) = 8.55, P = 0.0139) in a dose-dependent and significant manner. A significant reduction in the vomit frequency was observed at its 10 mg/kg dose (P = 0.0026) (Fig. 7A), whereas significant reductions in the percentage of shrews vomiting occurred at both 2.5 (P = 0.0455) and 10 (P = 0.0034) mg/kg doses of U0126 (Fig. 7B).

Fig. 7. The dose-response antiemetic effects of the ERK1/2 inhibitor U0126 on Akt inhibitor (MK-2206)-induced emesis.

Fig. 7.

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Varying doses of the ERK1/2 inhibitor U0126 (0, 2.5 and 10 mg/kg, n = 6 shrews per group) were administered (i.p.) to different groups of shrews 30 min prior to MK-2206 (10 mg/kg, i.p.) injection. The emetic responses were recorded for 30 min following MK-2206 administration. (A) Frequency of vomiting was analyzed with Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± S.E.M. (B) Percentage of shrews vomiting was analyzed with Chi-square test and presented as mean. *p < 0.05, **p < 0.01 vs. vehicle of U0126 pretreated animals (0 mg/kg + MK-2206).

3.4. MK-2206-evoked emesis is blocked by the selective LTCC inhibitor

A 2.5 or 5 mg/kg dose of nifedipine (s.c.) has previously been shown to significantly or completely prevent vomiting evoked by diverse emetogens including nonspecific/specific receptor agonists as well as Ca2+ mobilizers (Darmani et al., 2014; Zhong et al., 2016). In this study, pretreatment with nifedipine (2.5 mg/kg, s.c.) 30 min prior to MK-2206 (10 mg/kg, i.p.) or perifosine (50 mg/kg., i.p.) administration, completely suppressed the emetic efficacy of MK-2206 and perifosine, as shown in Fig. 8. Indeed, relative to the vehicle-pretreated control group, nifedipine (2.5 mg/kg, s.c.) caused significant decreases both in the mean vomit frequency in response to perifosine (P = 0.0184) and MK-2206 (P = 0.0003) (Fig. 8A), as well as percentage of shrews vomiting (P = 0.0034 and P = 0.0005, respectively) (Fig. 8B).

Fig. 8. Antiemetic efficacy of the LTCC blocker nifedipine against Akt inhibitor-induced emesis.

Fig. 8.

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The L-type Ca2+ channel (LTCC) blocker nifedipine (2.5 mg/kg, s.c.) or its vehicle was injected to different groups of shrews 30 min prior to administration of Akt inhibitors MK-2206 (10 mg/kg, i.p.), or perifosien (50 mg/kg, i.p.) (n = 6 shrews per group). Emetic parameters were recorded for the next 30 min. (A) The frequency of emesis was analyzed with Unpaired t-test and presented as mean ± S.E.M. (B) The percentage of shrews vomiting was analyzed with Chi-square test and presented as mean. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle of nifedipine-pretreated animals.

3.5. MK-2206-evoked emesis is blocked by GSK-3 inhibitors

Inhibitors of downstream of Akt signaling molecule GSK-3, were found to be potential antiemetics in our previous study (Zhong et al., 2020). As shown in Fig. 9, both GSK-3 inhibitors AR-A014418 (10 mg/kg; Fig. 9A and 9B) and SB216763 (0.25 mg/kg; Fig. 9C and 9D) exerted antiemetic efficacy against vomiting evoked by perifosine (50 mg/kg) or MK-2206 (10 mg/kg). Indeed, relative to the vehicle-pretreated group, AR-A014418 significantly decreased the mean vomit frequency in response to perifosine (P = 0.03) and MK-2206 (P = 0.001), as well as percentage of shrews vomiting in response to these emetogens (p = 0.0209 and p = 0.0034, respectively). SB216763 also caused significant decreases both in the mean vomit frequency in response to perifosine (P = 0.0386) and MK-2206 (P = 0.003), as well as percentage of shrews vomiting (P = 0.0209 and P = 0.0034, respectively).

Fig. 9. Antiemetic efficacy of GSK-3 inhibitors against Akt inhibitor-induced emesis.

Fig. 9.

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GSK-3 inhibitors AR-A014418 (10 mg/kg, i.p.) (A and B), SB216763 (0.25 mg/kg, i.p.) (C and D) or their corresponding vehicles were injected to different groups of shrews 30 min prior to administration of the maximally-effective emetic-dose of Akt inhibitors MK-2206 (10 mg/kg, i.p.) or perifosine (50 mg/kg, i.p.) (n = 6 shrews per group). Emetic parameters were recorded for the next 30 min. The mean frequency of emesis was analyzed with Unpaired t-test and presented as mean ± S.E.M. Percentage of shrews vomiting was analyzed with Chi-square test and presented as mean. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle of AR-A014418 or SB216763 pretreated animals.

3.6. MK-2206-evoked emesis is blocked by antagonists targeting serotonin 5-HT3-, neurokinin NK1- or dopamine D2/3-emetic receptors

To study a role for serotonin 5-HT3-, neurokinin NK1- or dopamine D2/3- receptors in MK-2206-induced vomiting, different groups of shrews were pretreated with either the serotonin 5-HT3 receptor antagonist palonosetron (0.5 mg/kg, s.c.), or the NK1 receptor antagonist netupitant (5 and 10 mg/kg, i.p.), or the dopamine D2/3 receptor antagonist sulpride (8 mg/kg, s.c.), for 30 min prior to MK-2206 (5 mg/kg, i.p.) injection. Palonosetron (0.5 mg/kg, s.c.) pretreatment significantly reduced the mean vomit frequency (P = 0.0002) (Fig. 10A) as well as the percentage of shrews vomiting (P = 0.0034) (Fig. 10B) in response to MK-2206 (10 mg/kg, i.p.) administration. Netupitant (0, 5 and 10 mg/kg, i.p.) caused a dose-dependent decrease in the frequency of evoked vomits [(KW (2, 15) = 13.07, P < 0.0001)] with a complete blockade at its 10 mg/kg dose (P = 0.0006) (Fig. 10C). The Chi-square test indicates that the percentage of shrews vomiting in response to MK-2206 was also reduced by netupitant in a dose-dependent manner [(χ2 (2, 15) = 12.6, P = 0.0018)], with total protection occurring at its 10 mg/kg dose (p = 0.0005) (Fig. 10D). Sulpride (8 mg/kg, s.c.) also suppressed the frequency of emesis in response to MK-2206 (10 mg/kg, i.p.) (P = 0.0002) (Fig. 10E). The Chi-square test indicates that sulpride (8 mg/kg) reduced the percentage of shrews vomiting in response to MK-2206 by 57% (P = 0.0180) (Fig. 10F).

Fig. 10. Efficacy of receptor-selective antiemetics against Akt inhibitor-induced emesis.

Fig. 10.

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The serotonin 5-HT3 receptor antagonist palonosetron (0.5 mg/kg, s.c.) (A and B), neurokinin NK1 receptor antagonist netupitant (5 and 10 mg/kg, i.p.) (C and D), dopamine D2/3 receptor antagonist sulpride (8 mg/kg, s.c.) (E and F), or the corresponding vehicles (0 mg/kg) were injected to different groups of shrews 30 min prior to the Akt inhibitor MK-2206 (10 mg/kg, i.p.) (n = 6–7shrews per group). Emetic parameters were recorded for the next 30 min. The frequency of emesis was analyzed with either Unpaired t-test (A, E) or Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test (C), presented as mean ± S.E.M. Percentage of shrews vomiting was analyzed with Chi-square test and presented as mean (B, D, F). *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 mg/kg.

4. Discussion

4.1. MK-2206 is a more effective emetogen than perifosine

Akt signaling hyperactivation occurs in several types of cancers and diverse Akt inhibitors are under development as potential armamentarium for cancer treatment (e.g. McKenna et al., 2018; Noorolyai et al., 2019). Nausea and vomiting are important impending side-effects when cancer patients are treated with Akt signaling inhibitors (Mateo et al., 2017; Nunnery and Mayer, 2019). Clinical trials demonstrate that perifosine evokes vomiting in up to 63% of cancer patients (Knowling et al., 2006). Likewise, MK-2206 is an efficacious proemetic agent in such patients (Ramanathan et al., 2015). Our current findings suggest that MK-2206 is a more effective emetogen than perifosine in least shrews. Published literature indicate that the observed differences in emetic efficacy between perifosine and MK-2206 in the least shrew model may be attributed to their relative efficacy and selectivity at the target Akt. Perifosine was first reported as an Akt inhibitor with IC50 of 4.7 μM in MM.1S cells, is an alkylphospholipid that blocks membrane translocation of Akt, hence preventing Akt phosphorylation and activation (Kondapaka et al., 2003). Perifosine also has non-Akt targets, such as c-Jun N-terminal kinase and mammalian target of rapamycin signaling components (Fu et al., 2009; Gills and Dennis, 2009; Hideshima et al., 2006; Liu et al., 2012). The next-generation Akt inhibitor, MK-2206 is an allosteric inhibitor of Akt1/2/3, with a potency approximately 1 log higher than previous inhibitors with significantly improved specificity (Tan et al., 2011). Moreover, MK-2206 has a high Akt selectivity with IC50 in the nanomolar range in cell-free assays, respectively (Yan, 2009). In addition, MK-2206 has been regarded as an optimal tool for studying the effects of Akt (Tan et al., 2011).

4.2.1. MK-2206 activates both central and peripheral emetic nuclei

Both c-Fos and pERK1/2 have been accepted as neuronal activation markers in vivo (Ferrini et al., 2014). Enteric neurons express high levels of Akt which plays a role in regulating enteric neurotransmission and gastrointestinal motility (Guerra et al., 2019). In the present study, least shrews were treated with the Akt inhibitor MK-2206 intraperitoneally, which may directly and rapidly affect Akt activity in the enteric neurons. In addition, MK-2206 circulating in the blood can also gain rapid access to the brainstem area postrema and activate the rest of the DVC emetic nuclei. The observed c-Fos and pERK1/2 induction following MK-2206 administration in the DVC emetic nuclei containing the AP, NTS and DMNV, as well as in the jejunal ENS, supports participation of both central and peripheral emetic circuits in Akt inhibitor-induced vomiting. However, the precise molecular mechanisms underlying MK-2206-induced vomiting need further investigation. In the current study, NeuN expression was observed among c-Fos positive cells in the ENS of jejunum, suggesting the intrinsic primary afferent neurons which have shown to be selectively labeled by NeuN antibody (Galligan, 2009; Van Nassauw et al., 2005) responded to MK-2206 administration. To be noted, not all the c-Fos expressing cells were co-labeled with NeuN, suggesting other types of enteric neurons or non-neuronal cells may also respond to MK-2206. Therefore, MK-2206-evoked c-Fos induction could be further investigated in other subclasses of enteric neurons (interneurons, excitatory and inhibitory motor neurons) expressing specific markers (Furness, 2000; Nezami and Srinivasan, 2010). Whether all c-Fos-expressing cells in the jejunum evoked by MK-2206 are neurons, we performed co-immunostaining c-Fos with Hu, a pan-neuronal marker (Desmet et al., 2014; Leembruggen et al., 2000; Rivera et al., 2009). Unexpectedly, not all the c-Fos expressing cells were co-labeled with HuC/D antibody (data not shown), suggesting non-neuronal cell types, such as glia and interstitial cells of Cajal which the ENS also contains (Nezami and Srinivasan, 2010), respond to MK-2206 in this study.

Our published findings have implicated that ERK1/2 signaling in both the brainstem and jejunum as a common signaling factor in the regulation of emetic responses elicited by systemic administration (i.p.) of diverse emetogens including the: i) NK1 receptor agonist GR73632 (5 mg/kg), ii) LTCC activator FPL64176 (10 mg/kg), iii) Ca2+ signaling amplifier thapsigargin (0.5 mg/kg), iv) the serotonin 5-HT3 receptor agonist 2-Methyl-5-HT (5 mg/kg) (Zhong et al., 2014; 2016; 2018; 2019), and v) chemotherapeutic agent cisplatin (Darmani et al., 2015). Consistent with these findings, in the present study we also demonstrate that the Akt inhibitor MK-2206 evokes significant ERK1/2 phosphorylation both centrally and peripherally. The elevated ERK1/2 phosphorylation occurring in NeuN-immunoreactive neurons of the least shrew jejunal ENS further supports the activation of the intrinsic primary afferent neurons in response to MK-2206 administration. In fact, the intrinsic primary sensory afferent neurons play a key role in detecting peripheral stimuli and initiating intestinal reflexes (Furness, 2000; Galligan, 2009). Thus, in the present study both c-Fos and pERK1/2 induction observed in this type of enteric neurons after MK-2206 administration suggests the peripheral participation in the MK-2206-induced vomiting. Moreover, the ERK1/2 inhibitor U0126 (10 mg/kg, i.p.) reduced MK-2206-induced vomiting, suggesting ERK1/2 signaling contributes to emesis evoked by pharmacological inhibition of Akt. The latter findings are in line with several studies that demonstrate Akt pathway inhibition leads to the activation of RAF/MEK/ERK pathway. Indeed, the Akt inhibitor VIII significantly potentiates ERK1/2 phosphorylation at the cellular level (Olianas et al., 2017). In addition, upregulation of phosphorylated ERK1/2 has also been observed following exposure of breast cancer cells to MK-2206 (Matkar et al., 2017; Serra et al., 2011), which is consistent with our current findings.

4.2.2. Akt inhibitors evoke vomiting in a Ca2+-dependent manner

Ca2+ is considered as the final intracellular messenger for muscle excitation-contraction coupling and blockade of Ca2+ entry through LTCCs cause inhibition of contraction (Godfraind et al, 1986). The LTCC blocker nifedipine suppresses vomiting in a dose-dependent fashion when evoked by diverse emetogens including agonists of LTCC (FPL64176), serotonin 5-HT3- (e.g. 5-HT or 2-Me-5-HT), substance P neurokinin NK1- (GR73632), dopamine D2/3- (apomorphine or quinpirole), and muscarinic M1- (McN-A-343) receptors (Darmani et al., 2014). Per our recent review, the mechanism underlying the broad-spectrum antiemetic potential of nifedipine against these diverse emetogens is closely related to mobilization of Ca2+ (Zhong et al., 2017). In the current study, nifedipine pretreatment at 2.5 mg/kg (s.c.) provided 100% protection against the vomiting caused by both Akt inhibitors perifosine (50 mg/kg, i.p.) and MK-2206 (10 mg/kg, i.p.). At this dose nifedipine could only partially protect shrews from vomiting evoked by the above discussed emetogens, including that caused by LTCC activator FPL64176 (Darmani et al., 2014). Thus, our current and previous findings demonstrate that Akt inhibitor-evoked emesis is highly sensitive to the LTCC blocker nifedipine, and extracellular Ca2+ entry through LTCCs plays a major role in both perifosine- and MK-2206- induced emesis.

4.2.3. Akt/GSK-3 pathway in emesis and anti-emesis

Consistent with our published findings that both GSK-3 inhibitors AR-A014418 and SB216763, protect shrews from vomiting evoked by diverse direct-acting emetogens such as agonists of specific key emetic receptors (serotonin 5-HT3, neurokinin NK1, dopamine D2/3, muscarinic M1) and Ca2+ channel regulators; the current study provides further evidence for the broad-spectrum antiemetic potential of these GSK-3 inhibitors against vomiting evoked by the Akt inhibitors MK-2206 and perifosine. Indeed, AR-A014418 at 10 mg/kg and SB216763 at 0.25 mg/kg, suppressed the frequency of MK-2206- and perifosine-evoked emesis by approximately 90%. The discussed findings further support our notion for the broad-spectrum antiemetic efficacy of GSK-3 inhibitors in suppression of diverse causes of vomiting. It is well recognized that Akt inhibition activates GSK-3 (Liu et al., 2019). Our unpublished findings that the GSK-3 activator pyrvinium pamoate (Basu et al., 2013), is proemetic in the least shrew emesis model, is also in line with the present findings.

4.2.4. Akt inhibitor induced-emesis involves activation of multiple emetic receptors

Significant evidence indicates that cytotoxic chemotherapeutic agents such as cisplatin cause vomiting via indirect stimulation of serotonin 5-HT3-, neurokinin NK1- and dopamine D2/3- receptors subsequent to the release of the corresponding neurotransmitters in both the brainstem and GIT emetic loci (Darmani and Ray, 2009). In the current study, we investigated the possibility of involvement of these receptors in the Akt inhibitor MK-2206-evoked vomiting. Our behavioral results demonstrate that all three receptors play a role in MK-2206-induced vomiting. In fact:

  1. The selective serotonin 5-HT3 receptor antagonist palonosetron at 0.5 mg/kg significantly abolished MK-2206-induced emesis, whereas the same dose of palonosetron failed to reach a similar antiemetic efficacy in least shrews when tested against vomiting evoked by either the LTCC activator FPL64176- or the 5-HT3 receptor agonist 2-methyl-5-HT (Darmani et al., 2014). Pharmacological inhibition of Akt reduces serotonin transporter-mediated 5-HT clearance from the synaptic cleft, and inhibition of downstream GSK-3α/β promotes 5-HT clearance by serotonin transporter, indicating that Akt activity may play a role in 5-HT tissue levels and serotonergic neurotransmission (Rajamanickam et al., 2015).

  2. Netupitant and other neurokinin NK1R antagonists possess broad-spectrum antiemetic profile against various emetic challenges including centrally and peripherally acting emetogens, such as apomorphine, morphine, ipecacuanha, copper sulfate and motion-induced emesis (Aziz, 2012; Muñoz and Coveñas, 2014; Rudd and Andrews, 2004; Rudd et al., 2016). In the current study, netupitant (10 mg/kg., i.p.) exerted total protection against MK-2206-induced emesis.

  3. The selective dopamine D2/3 receptor antagonist sulpride at 8 mg/kg partially but significantly decreased both the frequency and percentage of MK-2206-induced vomiting. Although at this dose, sulpride can protect up to 80% of shrews from vomiting caused by the dopamine D2/3 receptor preferring agonist quinpirole, and its 2 mg/kg dose has been shown to fully protect shrews against vomiting caused by the nonselective dopamine receptor agonist apomorphine (Darmani et al., 1999). Akt signaling plays a key role in maintaining homoeostasis of dopaminergic system (Garcia et al., 2005). Cellular studies indicate that pharmacological inhibition of Akt decreases expression of dopamine transporter and consequently leads to dopamine accumulation (Garcia et al., 2005; Speed et al., 2010; Vaughan and Foster, 2013).

The tested antiemetics may nonspecifically attenuate vomiting via a general decrease in motor behaviors. Thus, we investigated the effect of the tested antiemetics on locomotor activity parameters of least shrews using a computerized video tracking, motion analysis and behavior recognition system (EthoVision) as described in our published studies (Darmani and Crim, 2005; Darmani et al., 2007). The tested antiemetic doses of nifedipine, GSK-3 inhihibitors (AR-A014418 and SB216763), palonosetron, netupitant or sulpiride did not affect either the velocity or distance travelled by the shrews (data not shown). Thus, motor inhibition does not contribute to the antiemetic potential of these drugs.

5. Conclusion

Taken together, our findings demonstrate that Akt inhibitors are effective emetogens in the least shrew when administered systematically, which can be prevented by GSK-3 inhibitors. This study extends the complex relationship between Akt/GSK-3 pathway and emesis as well as antiemesis. Akt inhibitor-induced emetic behavior is accompanied by central and peripheral activation of emetic loci as indicated by the evoked expression of c-Fos and ERK1/2 phosphorylation in both the brainstem DVC area and the jejunal ENS, involving complex signaling pathways including mobilization of Ca2+ signaling as well as multiple emetic receptors such as serotonin 5-HT3, neurokinin NK1 and dopamine D2/3.

Highlights.

Akt inhibitors (perifosine and MK-2206) are proemetic in the least shrew.

MK-2206 evokes c-Fos expression and ERK1/2 phosphorylation in central and peripheral emetic loci.

Pretreatment with the ERK1/2 inhibitor U0126, LTCC antagonist nifedipine or GSK-3 inhibitors reduces MK-2206-induced vomiting.

Pretreatment with 5-hydroxytryptamine 5-HT3, neurokinin NK1, or dopamine D2/3 receptor antagonists reduces MK-2206-induced vomiting.

Acknowledgements

This work was supported by the NIH-NCI grant (CA207287) and WesternU intramural startup fund (1395) to NAD.

Footnotes

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Conflicts of interest

We have no conflict of interest to declare.

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