Effects of concomitant use of THC and irinotecan on tumour growth and biochemical markers in a syngeneic mouse model of colon cancer

Irinotecan (CAS No. 100286-90-6, in the form of hydrochloride trihydrate salt) was purchased from LC Laboratories (Woburn, MA, USA) and prepared as a 20 mg/mL solution in Milli-Q water by mixing at 325 g for 5 min and heating at 70 °C. Δ⁹-tetrahydrocannabinol (Dronabinol; CAS-No. 1972-08-3) was obtained from THC Pharm GmbH (Frankfurt, Germany) and dissolved in sesame oil (Bio Primo, Ulm, Germany) to a 7 mg/mL concentration. Other chemicals and reagents were bought from Sigma-Aldrich Laborchemikalien GmbH (Steinheim, Germany).

The study was carried out in compliance with national and international animal welfare standards (17–19). The experimental protocol was approved by the Ethics Committee of the Institute for Medical Research and Occupational Health, Zagreb, Croatia (approval number: 100-21/16-16 of 30 June 2016) and the Croatian Ministry of Agriculture (approval number: 525-10/0543-19-6 of 29 October 2019).

Ten-week-old male BALB/c mice were obtained from the breeding colony at the Ruđer Bošković Institute, Zagreb, Croatia. The mice were maintained under pathogen-free conditions in a steady-state microenvironment with a 12 h light/dark cycle, 22 °C, and free access to standard GLP-certified food (4RF21, Mucedola, Settimo Milanese, Italy) and tap water. After seven days of acclimatisation, the mice were weighed and injected subcutaneously with 5x10⁶ CT26.WT colon cancer cells (ATCC^® CRL-2638™) in the right flank. When the average tumour size reached 150 mm³, the mice were randomised to four groups of fifteen animals with minimal weight variation (30 g ± 10 %), as follows: Control – tumour control (injected with CT26.WT and left untreated); IRI group – receiving IRI intraperitoneally (60 mg/kg) on days 1 and 5 of treatment; THC group – receiving THC by gavage (7 mg/kg a day) for seven days; and IRI+THC group – receiving the combination of IRI and THC as described above.

The choice of the IRI dose and intraperitoneal administration are based on the studies of Hardman et al. (20, 21), who showed that 60 mg/kg of IRI every four days effectively suppresses lung, colon, and breast cancer growth in xenografts and on the fact that intraperitoneal administration is more effective and less toxic to mice (22) than intravenous. Furthermore, intraperitoneal administration leads to the absorption of the compound through the portal circulation and passage through the liver before reaching systemic circulation (23). This is important because IRI is a pro-drug that has to metabolise into its active form SN-38 (2, 3). IRI plasma concentration in mice peaks about 1 h after intraperitoneal administration, while its and its active metabolite’s SN-38 half-life is about 6.4 h (22). There are arguments that tumour xenografts may influence irinotecan and SN-38 availability (24), but these have been declined by Guichard et al. (22), who reported no changes in the pharmacokinetic profile of IRI and its metabolites in a tumour model.

The choice of 7 mg/kg of THC and oral administration was based on the available literature reports of oral doses of various illicit preparations rich in THC (25) and usual oral intake of illicit and approved forms (26–28). Most pharmacokinetic studies of THC have been conducted in humans and show that it peaks in the plasma between 1 and 6 h (10, 29, 30). Schwilke et al. (31) reported the accumulation of THC metabolites but not parent compound in the plasma after a seven-day oral administration (2–7 daily oral doses of 20 mg Marinol capsules). This is why we opted for the seven-day treatment, considering that oral THC absorption and metabolism varies and is relatively slow, which can lead to its prolonged activity (32).

The seven-day treatment was also based on the recommendation that animals should be euthanised when the tumour reaches the volume of 1500 mm³ (33) and followed the Institutional Animal Care and Use Committee (IACUC) guidelines (34).

Animal body weight and tumour size were measured on days 1, 3, 5, and 7. Tumour was measured with callipers by the same operator and tumour volume was calculated from two-dimensional measurements as follows: tumour volume (mm³) = (a × b²) × 0.5, where a and b are the tumour length and width (mm), respectively (35).

Animals were sacrificed four and 24 hours after the first IRI dose and on day 7 (Figure 1) under an anaesthetic cocktail of 80 mg/ kg ketamine and 12 mg/kg xylazine (Narketan, Vetoquinol UK Ltd, Towchester, UK) delivered intraperitoneally.

Figure 1

Brain and liver weights were recorded immediately after the sacrifice and relative organ weights calculated as described earlier (36).

Blood was drawn directly from the heart into heparinised vacutainers (Becton Dickenson, Plymouth, UK). One portion was stored at -20 °C while the other centrifuged (at 976 g, and 4 °C for 10 min) to separate and remove plasma. The remaining erythrocytes were resuspended in cold saline (0.9 % NaCl) and centrifuged at 976 g for another 10 min. Washing and centrifugation were repeated three more times. Plasma and erythrocytes were stored at -20 °C until analysis.

Blood samples were analysed for total cholinesterase (ChE), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) activities using the Ellman’s spectrophotometric method (37). Enzyme activity was measured in a 0.1 mmol/L sodium phosphate buffer, pH 7.4, at 25 °C using acetylthiocholine (ATCh) (1.0 mmol/L) and 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB) (0.3 mmol/L). Final sample dilutions were 400 times. AChE and BChE activities were determined using selective inhibitors 4,49-(3-oxo-1,5-pentanediyl)bis(N-allyl-N,N-dimethylanilinium) (10 µmol/L) and ethopropazine (20 µmol/L), respectively, as described previously (38). Increase in absorbance was monitored at 436 nm over 4 min. All measurements were taken with a Cecil 9000 spectrophotometer (Cecil Instruments Limited, Cambridge, UK). Enzyme activity is expressed as µmol/min/mL.

Statistical calculations were run on the Dell™ Statistica™ software v 13.5.0.17 (TIBCO Software Inc., Palo Alto, CA, USA). The normality of data distribution was tested with the Shapiro-Wilk test. Normally distributed data (whole body and relative brain and liver weights and tumour volumes) were compared between the groups using the parametric one-way analysis of variance (ANOVA) followed by the post-hoc Tukey’s HSD test. For the comparison of data not normally distributed (cholinesterase activities and biochemical markers of oxidative stress) we used the non-parametric Kruskal-Wallis test followed by the median test. Variable changes over time were tested with the Friedman test. Statistical significance was set at p<0.05.

During the experiment, no animal exhibited signs of diarrhoea and none died. Figure 2 shows time-dependent changes in mouse body weight. None were significant across the time points or between the groups. However, IRI treatment led to weight loss, most notably on day 3. These findings confirm our pilot study results in healthy rats (15, 16) and point to IRI systemic toxicity. Despite the absence of diarrhoea, body weight loss following the treatment with IRI could be a consequence of its adverse effect on intestinal absorption as reported elsewhere (42). Mice receiving THC alone maintained body weights across all time points, but these were about 2 % lower than control. However, it seems that combined treatment enhanced acute IRI toxicity, as mice body weights dropped 6 % compared to THC alone. This suggests a synergistic THC boost to IRI toxicity.

Figure 2

Although body weight of treated mice dropped somewhat, we found no significant changes in relative brain and liver weights between the groups (Table 1).

Figure 3 shows mean tumour volume changes. The tumours grew significantly in all groups between days 3 and 7 (Friedman ANOVA: p=0.00832–0.0150). IRI reduced tumour growth by 35 % on day 3, 22 % on day 5, and 27 % on day 7 compared to control. These findings are consistent with earlier evidence that the chemotherapeutic effect of IRI is easily reversed if therapy is stopped and that prolonged administration is essential for inhibiting tumour growth (43, 44).

Figure 3

THC, in turn, lowered the efficacy of IRI, as the tumour in the IRI+THC group shrank by 10 %, 15 %, and 14 % on respective days 3, 5, and 7 compared to control. The highest tumour volumes were determined in the THC group, but these values do not significantly differ from control. For instance, Zhu et al. (45) reported that intraperitoneal THC administration (5 mg/kg four times/week for 4 weeks) promoted tumour growth in two murine lung cancer models. That effect depended on the production of immunosuppressive cytokines and was not detected in immunodeficient SCID mice, suggesting that THC promotes immunosuppression and thereby enhances tumour growth.

Syngeneic mouse models are used in preclinical studies whose aim is to develop cancer therapy, establish drug mechanisms of action, and/or determine biomarkers that best explain these mechanisms. Our intention was to evaluate biochemical markers that help explain the mode of action of the tested compounds by analysing them in the blood of syngeneic colorectal mice over three time points, most notably cholinesterase activities and markers of oxidative stress. Taking into account known facts about IRI and THC pharmacokinetics, we selected 4 and 24 h as the first two time points in order to detect early changes in these biochemical markers. The reason to include the third day time-point (day 7) was that oral THC absorption varies a lot, is generally relatively slow, and its active metabolites exert prolonged activity (32).

As regards cholinesterases, scientific evidence supports their involvement in functions such as cellular proliferation and differentiation, which implies their potential involvement in tumorigenesis (47–49). Variations in ChE activity and profile in cancerous tissues seem to depend on the kind of tumour cell. For instance, colon cancer has been characterised by lower AChE and BChE activities and their lower mRNA expression (47). Moreover, Battisti et al. (49) also reported lower whole blood AChE and plasma BChE activities in patients with advanced prostate cancer. Figure 4 shows changes in whole-blood ChE, AChE, and BChE activity. Total cholinesterases activities (0.994±0.134 IU/mL at 4 h, 1.335±0.039 IU/mL at 24 h, 1.261±0.207 IU/mL on day 7) increased with time and tumour size (Figure 3), but the changes were not significant (according to Friedman test). However, they are obvious compared to the basal ChE activity of healthy nontumour bearing mice (1.097±0.035 IU/mL, unpublished result). The same was noticed for BChE (0.436±0.052 IU/mL at 4 h, 0.570±0.060 IU/mL at 24 h, 0.593±0.175 IU/mL on day 7) vs healthy animals (0.449±0.083 IU/mL, unpublished result). AChE, in contrast, dropped with time (0.542±0.054 IU/mL at 4 h, 0.495±0.123 IU/mL at 24 h, 0.433±0.047 IU/mL on day 7) and remained close to the levels observed in healthy animals (0.552±0.057 IU/mL, unpublished result).

Figure 4

According to Kimura et al. (50), serum cholinesterase can predict several cancers (50), but its clinical predictive value is not clear yet. The main reason for us to measure cholinesterase activities in the whole blood of syngeneic colorectal mice was to determine how would particular treatment affect their activity. Irinotecan is a potent AChE inhibitor, while BChE plays an important role in its hydrolysis (51). Reports of THC effects on cholinesterases, in turn, are inconclusive. Some authors hypothesised that it could bind into the AChE active site (52), which suggests that it may act similarly with BChE, as it is a homologous enzyme with a similar active site. In our study, however, neither IRI nor THC significantly changed cholinesterase activities in mice blood. Irinotecan inhibited approximately 10 % of the AChE and BChE activities compared to control, and this rate of inhibition remained similar throughout the experiment. The 10 % drop in total ChE activity was observed only 24 h after the first dose of IRI was applied. THC, in turn, inhibited AChE activities by 12 % after 4 h and 19 % after 24 h compared to control, yet total ChE and BChE activities slightly increased (about 10 %). Similar to THC, combined IRI and THC treatment led to an increase in total ChE and BChE and a decrease in AChE activities. However, we did not determine a clinically relevant inhibition of 30 %, which is consistent with the absence of the cholinergic syndrome symptoms (salivation, cramps or diarrhoea).

Most cancer cells exhibit high oxidative stress, metabolic activity, and production of ROS (53). In fact, the mechanism of action of many chemotherapy drugs involves ROS-mediated apoptosis (53). Similarly, drugs of abuse are often associated with oxidative stress, in addition to other toxicity mechanisms (54).

Figure 5 shows no significant time-dependent changes for any parameter of oxidative stress in untreated tumour control mice. However, IRI given alone increased ROS levels on days 1 (24 h) and 7. Although this effect was not accompanied by an increase in the concentration of lipid peroxidation products, it resulted in the activation of antioxidant defence. Four hours after IRI treatment, GSH level rose significantly vs control, and then significantly dropped. SOD activity was higher following IRI treatment at all time points. CAT activity increased slightly, while GPx activity remained unchanged.

Figure 5

THC treatment resulted in higher ROS and LPO levels after 24 h, but not significantly, which dropped to below control after 7 days. Neither GSH levels nor CAT and GPx activities varied significantly. Only SOD activity dropped significantly 4 and 24 h after THC treatment.

Combined IRI+THC treatment produced the highest ROS and TBARS levels. GSH levels significantly rose after 4 h and significantly dropped after 24 h and 7 days compared to tumour control. SOD activity was significantly lower than control at all time points, while GPx activity increased significantly compared to IRI given alone. Combined treatment also produced fluctuations in CAT activity: an increase at 4 h, followed by a decrease. In some instances it seems that combined treatment produced a synergistic effect (increase in ROS and TBARS levels).

High ROS levels contribute to many adverse effects that are common for IRI, such as gastrointestinal toxicity and mutagenesis. As oxidative stress causes imbalance in all cells, it is reasonable to assume that IRI should cause damage to other tissues during and after the treatment. Moreover, Rtibi et al. (55) reported that IRI caused oxidative stress-induced disturbances in water and electrolyte transport in the intestinal mucosa in rats, which suggests that such disorders are likely in the blood as well. As for THC given alone, even the early pharmacokinetic studies have established that oral administration results in erratic absorption, so the effects are sustained longer and are not as severe (10, 32).