The expression of CB2 receptors and its alteration by DOX and/or BCP is presented in Figure 4.9. Using western blot, we investigated whether BCP treatment is capable of selectively enhancing the expression of CB2 receptors in heart tissue.
DOX injections significantly downregulated the expression of CB2 receptors as compared to normal control rats. BCP treatment upregulated the expression of CB2
receptors in comparison to DOX-injected rats. However, AM630, a selective CB2
receptor antagonist, administered prior to BCP treatment in DOX-challenged rats, caused a noticeable decline in the expression of CB2 receptors as compared to DOX+BCP rats.
Figure 4.9: Effect of BCP on (A) CB2 receptor expression in heart tissue.
Representative immunoblot shown in (B). *p<0.05 vs Control, #p<0.05 vs DOX,
**p<0.05 vs DOX+BCP.
*
#
**
0 0.5 1 1.5 2 2.5
Control DOX BCP DOX + BCP DOX + BCP +
AM630 AM630
CB2/β-actin
CB2 (A)
CB2
Β-Actin
Control
DOX
BCP
DOX+BCP
DOX+AM 630+BCP
AM 630
(B)
Discussion
Doxorubicin (DOX) is a potent, effective and widely used chemotherapeutic agent. It is effective against both hematological and solid tumors. However, the major limitation to its clinical use is the induction of cardiotoxicity (Simůnek et al., 2009). The occurrence of fatal cardiotoxicity in all age groups is characterized by an irreversible cardiomyopathy which compromises the clinical utility of DOX and accounts for the major cause of the DOX-related morbidity and mortality (Simůnek et al., 2009). Whilst the advent of newer classes of monoclonal antibodies has revolutionized cancer chemotherapy, this approach is very expensive and is associated with a myriad of adverse effects (Jerjian, Glode, Thompson, & O’Bryant, 2016). Thus, the use of DOX continues to be a preferred mode for the treatment of cancer. Until now, several drugs, such as beta blockers, angiotensin receptor blockers, amifostine, dexrazoxane, mesna, leucovorin and erythropoietin have been evaluated as cardioprotective adjuvants to limit DOX-induced cardiotoxicity. The imperfections in currently used drugs to curb DOX-induced cardiotoxicity has emphasized the need to develop novel therapeutic agents possessing both antioxidant and anti-inflammatory properties. Therefore, finding an agent which could be useful in conferring cardioprotection against DOX-induced cardiotoxicity is vital. The sustained and overt occurrence of oxidative stress, lipid peroxidation, calcium dysregulation, endoplasmic reticulum stress, impairment of progenitor cells, activation of immune responses with subsequent inflammation, dysregulation of autophagy and the ubiquitin which leads cell death appear to play an important role in both the initiation and progression of cardiotoxicity (Renu, V G, P B, &
Arunachalam, 2018). Despite the availability of synthetic antioxidants, the adverse
effects and pro-oxidant action of these agents bring concerns in therapeutics. This shifted the focus of drug discovery to the exploration of natural compounds which target novel therapeutic targets and manipulate the immune-inflammatory changes in DOX-induced cardiotoxicity. Rats given DOX develop cardiotoxicity characterized by hemodynamic alteration, cardiomyocyte injury markers rise in serum, biochemical and histopathological changes and are widely used models for evaluation of drugs to be used as adjuvants for cardioprotection.
Among the many new therapeutic targets, the endogenous cannabinoid system, which comprises cannabinoid ligands and cannabinoid type 1 (CB1) and type 2 (CB2) receptors represents one of the newest and most promising drug targets.
Among cannabinoid receptors, CB2 receptors are G-protein coupled receptors that, upon activation, induce members of the MAPK family, which trigger expression of genes including those involved in stress response, inflammation, cell survival or proliferation (Howlett, 2005). CB2 receptor activation is also associated with nuclear translocation of the transcription factor NF-κB (Derocq et al., 2000). In contrast to CB1 receptors, CB2 receptor activation does not modulate ion channel function (Bosier, Muccioli, Hermans, & Lambert, 2010). As a result, CB2 receptor mediated Ca2+ responses are less pronounced (Schuehly et al., 2011) than the potent CB1
receptor-mediated effects on Ca2+ fluxes (Bosier et al., 2010). Cardioprotective effects of endocannabinoid-mediated CB2 receptor activation were first reported in LPS-induced preconditioning (Lagneux & Lamontagne, 2001). Thereafter, several recent reports using synthetic CB2 receptor agonists in vitro, ex vivo or in vivo showed protective effects on remote preconditioning, infarct size, arrhythmias and counterbalanced chronic heart failure-induced structural changes, inhibited atherogenesis and prevented myocyte enlargement (Bėla Horváth et al., 2012;
Montecucco et al., 2009; Ramirez et al., 2012; Steffens et al., 2005; Weis et al., 2010; Zarruk et al., 2012). The anti-inflammatory effects of CB2 receptor activation in the endothelium, and its inhibitory effect on monocytes/macrophages and/or leukocyte migration were diminished by pharmacological antagonism of CB2
receptors with AM630 (Zhao et al., 2010). Pretreatment with the CB2 receptor antagonist AM630 or SR144528 abolished the cardioprotective effects and reversed cardioprotection. CB2 receptors have also been implicated in the modulation of endoplasmic reticulum stress and immune cell migration (Miller & Stella, 2008) involving the PI3K/Akt and ERK ½ pathways. Many studies with synthetic cannabinoid ligands of CB2 receptor paved the way for CB2 receptor activation as a strategy in cardioprotection. However, there is no study available evaluating the effect of CB2 receptor activation by cannabinoid ligands in DOX-induced cardiotoxicity.
The adverse effects, abuse liability and addictive property of synthetic CB2
receptor ligands in humans generated interest in targeting CB2 receptors by phytocannabinoids instead, which are naturally-found ligands. Until now, several phytocannabinoids have been explored for therapeutic purposes and this is demonstrated by the availability of Sativex, nabilone and dronabinol.
Phytocannabinoids have been widely studied as potential protective agents in chronic diseases because of their characteristics of multiple targets, time tested efficacy and low cytotoxicity. However, the adverse psychoactive effects of phytocannabinoids derived from cannabis limit their therapeutic use and bring legal concerns. Recently, several of the phytocannabinoids derived from plants other than cannabis have received interest for their favourable physicochemical, pharmacokinetic and pharmacological properties including anti-inflammatory and antioxidant effects and
seem promising for pharmaceutical development (Opdyke, 1973). In recent years, there has been much interest in exploring phytocannabinoid ligands from plants other than cannabis which may provide a safe approach against DOX- induced cardiotoxicity by regulating oxidative stress, immune-inflammatory changes, autophagy and apoptosis. Therefore, we focused on phytochemicals isolated from plants other than cannabis and intended to evaluate their effects in DOX- induced cardiotoxicity. To date, many research groups focus on the DOX-induced cardiotoxicity to find new novel drugs of both synthetic or natural origin and study their pharmacological, biochemical and molecular properties. The major actions of natural cardioprotective drugs appear to lie in the restoration of antioxidant defense, preventing the occurrence of oxidative stress and subsequent mitochondrial dysfunction and curbing inflammation thereby limiting resultant necrotic or apoptotic cell death. Over the past two decades, the safety and efficacy of several herbal extracts and plant derived compounds either as monotherapy or adjunct to conventional medicines for organ toxicities appear to be favorable, due to their ability to prevent or attenuate DOX-induced cardiotoxicity as shown in many studies.
Since the recognition of DOX-associated cardiotoxicity, preclinical models including cell lines, rats and mice have been developed and extensively employed in the screening of cardioprotective agents. DOX has been used extensively in experimental studies for nearly four decades as a model toxicant that permits the investigation of drug-induced cell death in vivo and in vitro and the evaluation of cardioprotective agents of synthetic and natural origin. Thus, plants become an important source of pharmacologically active molecules that are considered to be important for the discovery and development of new drugs. These molecules are a
“stepping stone” for future drug development and usage in modern medicine. This
has been demonstrated in the past and many bioactive pure compounds isolated from these plants have become successful drugs and remain an important source of agents for prevention and treatment of various diseases. Phytochemicals appear to be an excellent source of new drug leads due to their low molecular weight, lipophilicity and chemical scaffold diversity conferring on them multifunctional properties. These phytochemicals act on multiple targets and regulate complex pathogenesis. In the past several years, there has been an upsurge in interest in the therapeutic potential of phytochemicals as antioxidants in reducing free radical-induced injuries of different organs. The concept of using herbs as antioxidants is much older, but phytochemicals have received more attention due to their multitargeted actions, as well as due to the lack of availability and adverse effects of synthetic antioxidants. As a consequence, there has been a lot of interest in investigating the beneficial properties of phytochemicals and understanding their pharmacological and molecular mechanism.
In the past few years, a large number of experimental studies have shown that phytochemicals confer cardioprotective effects against DOX-induced cardiotoxicity due to their multiple pharmacological properties including anti-inflammatory, antioxidant and antiapoptotic effects. Dietary phytochemicals exert their beneficial effects on the liver modulating cellular stress response signaling pathways. A large variety of phytochemicals have shown a cardioprotective effect in different animal models of cardiotoxicity induced by different chemical toxicants such as isoproterenol, cocaine, azidothymidine and cyclophosphamide. However, among all these models, DOX-induced cardiotoxicity is clinically more relevant and is the subject of intense research since DOX is a major cause of cardiotoxicity in cancer patients whereas the other drugs used to induce cardiotoxicity in experimental models and employed in the screening of cardioprotective drugs are of minimal
clinical and therapeutic relevance as the majority of them are considered useful for screening purposes only.
In the present study, we studied the cardioprotective effect of BCP, a dietary phytocannabinoid that has attracted attention after its approval by European regulatory agencies and the USFDA for use as food additive and flavoring agent. Chemically, BCP is a bicyclic sesquiterpene and pharmacologically, it is a selective CB2 receptor agonist. It is found abundantly in various flowering plants and spices which makes it one of the more widely available and accessible agents devoid of psychoactive properties (Gertsch, 2008). It has been recognized as a key ingredient in several traditional Chinese, European, Eastern and Indian medicines and is reported to possess a wide range of potent biological activities, including potent anti-inflammatory, antioxidant and analgesic effects. BCP’s structure contains a cyclobutane ring which provides a potent and rigid geometry to this molecule for binding to cannabinoid receptors and largely contributes to BCP’s anti-inflammatory properties. The therapeutic benefits of BCP in inflammation has been shown to be mediated by potent CB2 receptor activation (Gertsch, 2008). Additionally, the neuroprotective effects of BCP in Parkinson’s disease, cerebral ischemia, epilepsy, Alzheimer’s disease, depression, anxiety and addiction have been reported recently (Donati et al., 2015; Sain et al., 2014).
In this study, we attempted to determine the effect, if any, of BCP on DOX- induced cardiotoxicity using two models, acute and chronic. Based on our findings, we were able to demonstrate that BCP ameliorates cardiotoxicity in rat models of DCM. To our knowledge, this is the first study to report the cardioprotective effects of BCP against DCM. Concurrent administration of BCP with DOX reduced cardiac inflammation, oxidative stress and myocardial damage.
In our study, DOX-treated rats weighed significantly less than their control counterparts. Body weights improved after BCP treatment. The loss of body weight in DOX-treated rats could be a result of muscle cachexia (Koh et al., 2015). It has also been suggested that reduced food intake is a reason for weight loss in DOX- treated animals (Sahu et al., 2016). However, another study which measured daily food consumption between DOX-treated and saline-treated rats found no significant difference in food consumption between the two groups, which suggests that DOX- induced gastrointestinal toxicity is a likely factor in weight loss due to malabsorption of nutrients (Jenkins et al., 2016). Another possible explanation for DOX-induced weight loss is that DOX inhibits adipogenesis by down-regulating PPARg expression (Arunachalam et al., 2012). BCP, by virtue of its binding to the CB2 receptor, activates the PPARg pathway (Bento et al., 2011; Cheng et al., 2014). This could be why it prevented excessive weight loss in the BCP-treated DOX rats. However, more research is required to fully determine the DOX and BCP mechanisms regulating weight loss and/or gain.
Our study found that inflammatory cytokines were significantly reduced after BCP treatment. DOX is a well-known promoter of inflammation (Ehrke, Tomazic, Ryoyama, Cohen, & Mihich, 1983; Gaudin et al., 1993; Pecoraro et al., 2016; Tien, Peng, Yang, Subeq, & Lee, 2016; L. Wang et al., 2016). COX-2 is an enzyme which mediates inflammation by catalyzing the rate-limiting step in prostaglandin production (Hla & Neilson, 1992). The increase in COX-2 (and prostaglandin production) in DCM serves a protective mechanism against apoptosis (Dowd, Scully, Adderley, Cunningham, & Fitzgerald, 2001; Neilan et al., 2006). One study also demonstrated that COX-2 induction limits oxidative stress-induced damage in cardiomyocytes treated with DOX (Adderley & Fitzgerald, 1999). DOX treatment
also increases iNOS which forms nitric oxide (NO) which can then react with superoxide to form peroxynitrite, an inducer of cell death (Mukhopadhyay et al., 2009). DOX is also known to cause an increase in cytokine production (Bruynzeel et al., 2007; Pecoraro et al., 2016). BCP treatment reduced COX-2, iNOS and proinflammatory cytokine levels. This reduction in proinflammatory cytokines seems to be in line with the results of other studies conducted with BCP (Cho et al., 2015;
Béla Horváth et al., 2012). CB2 receptors are expressed in immune cells and many studies have shown that the CB2 receptor plays a role in immunomodulation (via both endocannabinoids and cannabinoids) (Turcotte, Blanchet, Laviolette, &
Flamand, 2016). However the exact mechanisms and pathways are still not fully understood or known (P. Pacher & Mechoulam, 2011).
The link between DOX and oxidative stress is indeed strong (Singal & Iliskovic, 1998; Sinha, Katki, Batist, Cowan, & Myers, 1987). Our study found, predictably, that antioxidant levels/activity decreased while MDA levels increased in DOX- treated rats. DOX’s structure makes it prone to free radical formation, in the form of a semiquinone free radical, which can react with molecular oxygen and produce superoxide (Keizer, Pinedo, Schuurhuis, & Joenje, 1990). Cardiomyocytes are particularly susceptible to DOX-induced oxidative damage due to a) a relatively low amount of antioxidant defenses, b) extensive oxidative metabolism, leading to increased free-radical formation and c) the presence of cardiolipin in heart mitochondrial membranes, which DOX has a high affinity for (Quiles, Huertas, Battino, Mataix, & Ramı́rez-Tortosa, 2002; Sahu et al., 2016). DOX-generated free radicals can lead to mitochondrial dysfunction, energy imbalances, DNA damage, upregulation of p53 and cell death (Deavall, Martin, Horner, & Roberts, 2012). It has been suggested that CB2 receptor activation plays a role in attenuating
ischemia/reperfusion-related oxidative/nitrosative stress (Pacher & Haskó, 2008).
Our results show that BCP significantly increased antioxidant levels/activity when compared to DOX-treated rats. In a model of cisplatin-induced nephrotoxicity, BCP also reduced oxidative stress (Béla Horváth et al., 2012). BCP has been shown to specifically scavenge the hydroxyl radical and superoxide anion, and inhibit lipid peroxidation (Calleja et al., 2013) . This would explain why it was able to reduce MDA in BCP-treated DOX-rats when compared to DOX-alone rats. More detailed studies will be needed to fully understand the mechanisms involved with respect to the effect of BCP on free-radical formation and oxidative/nitrosative stress.
DCM causes myocardial injury which is characterized by various morphological features which include necrosis, myocardial hypertrophy, blood capillary distortion, and interstitial edema (Afsar, Razak, Batoo, & Khan, 2017), cardiomyocyte vacuolization, myofibrillar disorganization, interstitial infiltration by fibroblasts and immune cells and eventually cardiomyocyte loss (Campos et al., 2011). Our histopathological findings show extensive muscle degradation in DOX-treated rats.
This seems to correlate well with the increase in serum CK-MB and LDH which was observed in DOX-treated rats. Indeed, other studies have reported an increase in CK and LDH in rodent models of DCM (Geetha, Sankar, Marar, & Devi, 1990; Kang et al., 2002; Yagmurca et al., 2003). Calpain-mediated dystrophin loss in the cardiomyocyte membrane and disruption of sarcomeric actin/myosin may be significant contributors to myocardial structural damage in DCM (Campos et al., 2011). A study on CB2 receptor deficient mice showed that they had increased apoptosis and cardiomyocyte loss in a model of ischemic cardiomyopathy (Duerr et al., 2014). Additionally, CB2 receptor activation causes a reduction in fibrosis remodeling thereby attenuating inflammation and injury (Zubrzycki, Liebold,
Janecka, & Zubrzycka, 2014). These may be factors in the reduction of structural damage in the DOX+BCP groups (in both the acute and chronic models) when compared to the DOX-alone group. However, BCP’s effect on DOX, as it relates to apoptosis, requires further study and investigation.
Being natural, dietary, readily bioavailable, non-psychoactive and safe with a wide presence in numerous plants, including many spices, BCP could be used for nutritional supplementation over other phytocannabinoids. Translating the outcomes in humans would be promising as these agents have not only shown the ability to activate CB2 receptors only, but they also activate PPAR-γ, the target of the thiazolidinedione class of drugs which are used clinically to treat diabetes. BCP is of more therapeutic value than some other phytocannabinoids due to it being found in plants other than cannabis which may help in the legal logistics of its use. CB2
receptor selective agents may provide a new potential class of cardioprotective drugs against DOX-induced cardiotoxicity. The pharmacophore of these agents could be used for synthesizing leads in drug discovery and development.
Conclusion and Further Investigations
To the best of our knowledge, the present study is the first study to demonstrate the effect of b-caryophyllene on doxorubicin-induced cardiotoxicity. We also showed the effect in both acute and chronic models of DOX-induced cardiotoxicity and elucidated the CB2 receptor-dependent antioxidant and anti-inflammatory activity of BCP in attenuating DOX-induced cardiotoxicity. BCP demonstrated a positive effect by attenuating body weight loss and reducing oxidative stress, inflammation and inflammatory mediators along with reducing the serum levels of the cardiomyocyte injury marker enzymes CK-MB and LDH. Additionally, the structural salvaging of the myocardium further confirmed the protective effects of BCP. The use of AM630, a CB2 receptor blocker, showed us that BCP provides cardioprotection against DOX by activating CB2 receptors.
Further investigations are warranted in animals with cancer to investigate whether BCP exerts a chemosensitizing effect. Further studies are also required to determine whether BCP affects the mechanism of apoptotic cell death since, in many in vitro studies in cancer cell lines, it has been shown to affect the apoptotic cascade.
The effect of BCP on DCM should also be studied in rodents with tumors to be able to determine whether BCP interferes with antitumor activity of DOX.
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