Here, we presented that treatment with cannabidiol and high-dose doxorubicin in mouse breast cancer model has a better anti-tumor effect and can reduce doxorubicin cardiotoxicity. As mentioned before breast cancer is the most common malignancy in the world and the leading cause of cancer deaths [1]. Distant metastasis accounts for the vast majority of deaths in patients with cancer like basal-like breast cancer (BLBC), and displays a lung tropism of metastasis [4]. Therefore effective treatment strategies are necessary to eradicate drug resistance, deliver a payload to the site of action in a tumor microenvironment and prevent tumor metastasis. Doxorubicin as an extensively used chemotherapeutic drug has several problems due to lack of selectivity [40, 41] which one of them is dose-dependent cardiotoxic effects [42]. Despite the enormous amount of research conducted to reduce or prevent this toxicity [43, 44] cardiac adverse effect is an uncommon but potentially serious complication of high-dose chemotherapy [10].
Cannabidiol (CBD) obtained from Cannabis sativa is able to interfere with different stages of the tumor process, it can inhibit cancer cell migrations and adhesions, and exerts anti-proliferative, pro-apoptotic, and anti-invasive effects [45]. It is proposed that cannabidiol induces endoplasmic reticulum stress and apoptosis by inhibiting the AKT/mammalian target of rapamycin (mTOR) signaling and enhances reactive oxygen species (ROS) generation for selected breast cancer cells [46].
In this study tumor size and weight decreased in pretreatment and CBD + DOX group. Accordingly, TUNEL test demonstrated that co-administration of cannabidiol and doxorubicin at the same time or as pretreatment could induce more apoptotic cells in breast tumors compared to doxorubicin or cannabidiol alone. Previous studies also reported significant synergistic interactions between cannabidiol and other chemotherapeutic drugs in breast cancer mcf7 cell line [47]. In-vitro and in-vivo study on MDA-MB-231 cells demonstrated that cannabidiol with doxorubicin can increase the expression of proteins involved in apoptosis like caspase-3 and BAX; furthermore tumor size was significantly decreased [33] as observed in this study.
Result of lung histopathology didn’t show any sing of lung metastasis in CBD + DOX group. The evidences showed that, cannabidiol can suppress the growth and metastasis of cisplatin-resistant non-small cell lung cancer (NSCLC) [48]. Furthermore in colorectal cancer protective effect of cannabidiol against tumor invasiveness, migration and metastasis were reported [49]. Cannabidiol also inhibits the invasiveness of aggressive MDA-MB-231 and MDA-MB-436 breast cancer cell lines by downregulating inhibitor of DNA binding 1 (ID-1), a transcriptional regulator, which stimulates the metastasis in a mouse model of advanced breast cancer with lung metastases [50]. Moreover other cannabidiol-related studies reported inhibition of GPR55 expression, which is related directly or indirectly to cell changes promoting malignant growth, including uncontrolled cancer cell proliferation, angiogenesis, cancer cell adhesion, cancer cell migration, and metastasis [51]. We also demonstrated that in pretreatment CBD + DOX group there was still sing of lung micro metastasis. May be cannabidiol injection with doxorubicin at the same time has a stronger effect in metastasis pathways compere to pretreatment therapy as it is routinely done for chemotherapeutic agents in different regimens [52].
To provide information on major adverse effects of maximum tolerated dose (MTD), evaluation of parameters such as body weight loss and food consumption level besides clinical signs typically performed [53]. In other hand, weight loss is a common manifestation in patients with cancer [54]. In this study cannabidiol administration with doxorubicin at the same time or as a pretreatment model could not prevent weight loss during high-dose chemotherapy. Although evidences suggest that cannabinoids are associated with reduced chemotherapy toxicity like improvement of nausea, vomiting and cachexia and also increase in appetite [55, 56], in this investigation cannabidiol could not prevent doxorubicin-induced weight loss which may be resulted as high-dose doxorubicin [34]. In the group just received cannabidiol, non-significant weight gain compared to cancerous control group observed, that seems cannabidiol can increase appetite.
Heart weight to body weight ratio increased significantly in all treatment groups compared to cancerous control group. This augmentation in doxorubicin receiving groups (DOX, pretreatment, CBD + DOX), may be related to sever body weight loss due to high-dose doxorubicin administration [34], and also doxorubicin-induced cardiac hypertrophy and congestion [57]. Despite cannabidiol known as a cardioprotective agent [58]. In cannabidiol group HW/BW increased. It is reported that in individuals with normal cardiac function, cannabidiol could be cardiotoxic and also in another study on monkeys, cannabidiol increased heart weights due to heart failure [59, 60].
Histopathological studies in this experiment did not show any interstitial cardiac fibrosis or vascular fibrosis and myocardial cell count change in the doxorubicin group, which may be as a result of short-term duration of study, and also maybe resulted because our cumulative dose didn’t reach 20 mg/kg [61], nevertheless, signs of inflammation were observed as demonstrated in previous studies [62].
As it was indicated, high-dose doxorubicin chemotherapy can cause cardiotoxicity in many ways and its pathogenesis has traditionally been attributed to the formation of reactive oxygen species (ROS) [63]. Superoxide dismutases (SODs) are a class of enzymes that catalyze the detoxification of superoxide into oxygen and hydrogen peroxide, and then converted to oxygen and water by catalase [64]. SOD2 plays a crucial role in controlling ROS production [65]. Our results also showed that administration of cannabidiol with doxorubicin at same time, significantly increased SOD2 in accordance to previous evidences demonstrating antioxidant agents can reduce doxorubicin cardiotoxicity [66]. The previous data prove that cannabidiol has antioxidant activity [67]. and also similar to our results, in disease like diabetes mellitus cardiomyopathy cannabidiol considered as a cardioprotective agent [68] with decreasing the myocardial ROS generation and expression of p22phox, p67phox, gp91phox, restoring glutathione content, improving SOD activity and reducing 3-NT formation. However, in the pretreatment group this change was not significantly. It seems cannabidiol activate antioxidant system when ROS formation rise in body therefore pretreatment therapy couldn’t activate that system.
Inducible nitric oxide (NO) synthase (iNOS) overexpression has been considered detrimental in heart failure, based on observations of NO-mediated depression of cardiomyocyte contraction in human and mice studies [69]. Our results demonstrated that CBD + DOX reduced iNOS expression and enhanced cardiac function unlike the other groups. Doxorubicin enhanced NO formation, following the induction of iNOS, that has been implicated in the pathogenesis of inflammation and reduced cardiac function [70]. In cannabidiol group iNOS increased and it may be resulted as cannabis toxicity in healthy heart [71]. In cancerous control group, in overall, protein expression enhanced and iNOS increased significantly. This can be resulted as cancer cells metabolites related ROS production and oxidative stress induction in other cells and changes in protein expression [72]. In pretreatment condition, unlike CBD + DOX group, iNOS did not change significantly, which is assumed that maybe cannabidiol administration before doxorubicin act as like as administration of cannabidiol alone and could not reduce cardiotoxicity as mentioned in SOD2 explanation.
MMPs regulate the remodeling process by facilitating extracellular matrix turnover and inflammatory signaling, especially in cardiac remodeling [73]. In this study MMP2 and MMP9 level were significantly lower in CBD + DOX unlike doxorubicin group. Similar result also reported indicating reduced MMP9 and MMP2 mRNA by coadministration of cannabidiol and doxorubicin, besides doxorubicin-induced increase in MMP9 and MMP2 level [61, 74]. Augmentation of MMP2 and MMP9 in cannabidiol and pretreatment groups, again may be related to mentioned cannabis toxicity in healthy heart [71].
In this investigation demonstrated that simultaneous administration of cannabidiol and high-dose doxorubicin in cancerous mice had a synergistic anti-tumor effect and also reduced cardiotoxicity. However cannabidiol administration alone or as a pretreatment in cancerous mice had a cardiotoxic effects. There were two limitations to this study. First, we could not rule out the other pathways of cardio protective effect of cannabidiol against doxorubicin injuries, like endothelial activation signaling pathway (eNOS/AKT). And secondly cannabidiol had a cardiotoxic effect in cancerous mice and this toxicity should be evaluated. We only focused on the cardiotoxicity effect of high dose doxorubicin, otherwise for clinical application other side-effects should be considered [75].