Discussion
OXA is an effective drug for the treatment of solid tumours such as colorectal cancer; however, OXA-induced peripheral neurotoxicity reduces the life quality of patients and greatly limits the use of OXA in the clinic. The present study confirmed that OCT2 and OCTN1, especially OCT2, play vital roles in the uptake of OXA, and MRP2 is involved in the efflux of OXA. The further study demonstrated that L-THP could inhibit OCT2 and OCTN1 activities but not MRP2, reduce the accumulation of OXA in the DRG, and subsequently attenuate OXA-induced peripheral neurotoxicity. Finally, our results demonstrated that L-THP did not reduce the accumulation of OXA in tumours; therefore, it did not impair the antitumour efficiency of OXA.
Although several published papers have shown that OCT2, OCTN1, and CTR1 contribute to the uptake of OXA, to further compare the role of individual transporters in the uptake of OXA, we first estimated the OXA-induced cell toxicity and OXA accumulation in mock, MDCK-hOCTN1, MDCK-hOCTN2, and MDCK-hOCT2 cells. Our data demonstrated that OXA is a substrate of OCTN1 and OCT2 but not that of OCTN2. Furthermore, OCT2 played a more important role in the uptake of OXA because the LD50 of OXA in MDCK-hOCT2 cells (3.4 μM) was much lower than that in MDCK-hOCTN1 (25.8 μM), and the cellular accumulation of OXA was much higher in MDCK-hOCT2 cells than that in MDCK-hOCTN1 cells (5.4 vs 2.0 folds that of the mock cells).
Because we did not have a CTR1-overexpression cell model, we did not study the role of CTR1 in the uptake of OXA. However, as reported by Burger et al., the inhibitor of CTR1, copper histidine (Ip & Liu et al., 2013), did not exhibit a significant influence on OXA uptake in DRG cells, which indicates that the role of CTR1 in OXA uptake may not be essential in DRG. Considering that CTR1 is also expressed in human malignant tissues, such as colon carcinomas, it is not a good target for the inhibition of OXA-induced peripheral neurotoxicity. Our study result demonstrated that the concentration of platinum in the tumour of the nude mice co-treated with L-THP and OXA was not obviously altered, when compared with that in the nude mice treated with OXA alone, which implies that L-THP did not inhibit CTR1; therefore, it is reasonable that L-THP did not impair the antitumour efficiency of OXA.
Additionally, ATP7A/7B (Ip & Liu et al., 2010; Li & Yin et al., 2018; Lasorsa & Nardella et al., 2019), MRP2 (Myint & Li et al., 2015; Myint & Biswas et al., 2019), and MATE1 (Nies & Koepsell et al., 2011; Fujita & Hirota et al., 2019) are involved in OXA efflux in the DRG, and MRP2 was reported as one of the most important transporters in the efflux of OXA from DRG cells. Our result showed that the OXA accumulation in MDCK-MRP2 cells was much lower than that in mock cells, and 100 μM L-THP did not obviously influence the accumulation of OXA in MDCK-MRP2 cells and mock cells, indicating that L-THP does not influence OXA accumulation in the DRG and tumour tissues via inhibiting MRP2. Moreover, our previous study revealed that L-THP did not affect the function of MATE1 (Li & Song et al., 2016). Since we did not have an ATP7A/7B-transfected cell model, ATP7A/7B was not considered in the present study. Based on the result that L-THP significantly decreased OXA accumulation in the DRG in vitro and in vivo , we speculate that L-THP mainly inhibits OXA uptake in the DRG rather than its efflux.
Although OCT2 plays the most crucial role in OXA uptake in the DRG, it is extremely down-regulated or even lost in most tumours due to epigenetic modification (Zhu & Yu et al., 2019; Chen & Qin et al., 2019). Therefore, selective inhibition of OCT2 would not impair the antitumour effect of OXA. As expected, our present results showed that co-incubation with L-THP did not decrease the cell toxicity of OXA in colorectal cancer cell lines (the main clinical indication of OXA). Thein vivo results further revealed that co-administration of L-THP did not reduce the tumour accumulation of OXA and impair the antitumour efficacy of OXA.
L-THP has been used in China as an analgesic with sedative/hypnotic properties for more than 40 years (Liu & Wang et al., 2019; Gong & Yue et al., 2016; Du & Du et al., 2017). OXA-induced peripheral neurotoxicity increases cold-stimulation sensitivity, as indicated by an increase in the frequency of lapping hind paws. To exclude the sedative/hypnotic effect of L-THP, we included a group that was treated with L-THP alone. As we observed, twenty-four hours after the L-THP administration, the mice did not exhibit sedative or hypnotic effects, and further behaviour evaluation showed no significant change between the vehicle control group and the group administered L-THP alone. The above observation implied that the reduction of cold-stimulation sensitivity in the group that was co-administered L-THP was irrelevant to its sedative/hypnotic effect. The significant reduction of OXA in the DRG of the mice by L-THP directly proved that the inhibitory effect of L-THP on OCT2 may be the important factor in the inhibition of OXA-induced peripheral neurotoxicity.
To study whether OXA-induced peripheral neurotoxicity exhibits gender difference, 16 mice were allocated in one group (half male and half female) in the behavioural test. To meet the requirements of the detection limit of platinum, the DRGs from two mice of the same group were pooled as one sample. The behavioural test (Figure 5a and 5b) showed that OXA-induced peripheral neurotoxicity showed no gender difference; therefore, the platinum concentration in the DRGs of male and female mice from the same group was combined. Originally, we had aimed to evaluate the antitumour effect of OXA in tumour-bearing nude mice for 3 weeks; however, the tumours in the vehicle group grew so fast that we had to terminate the experiment at the end of two weeks. Fortunately, we can conclude that L-THP did not impair the antitumour efficiency of OXA, based on the results from comparing the platinum concentration in the tumours and the tumour size between the OXA-alone group and L-THP co-treatment groups.
Considering that OXA can induce mitochondrial dysfunction (Cheng & Xiang et al., 2019; Trecarichi & Flatters, 2019; Toyama & Shimoyama et al., 2018), it is interesting and meaningful to identify how OXA enters the mitochondria because of its poor passive permeability. OCTN1 has been reported to be localized in the mammalian mitochondrial membrane (Lamhonwah & Tein, 2006), and our western blotting analysis also showed that OCT2 and OCTN1 were expressed in the mitochondria of rat DRGs (data not shown). Considering that L-THP passes easily across the cell membrane due to its high passive permeability, we speculated that L-THP probably inhibited OXA transport across the mitochondrial membrane. Apart from the SLC22A family, the SLC25A family has also been determined to play an important role in the mitochondrial carrier system (Taylor, 2017), which deserves consideration.
In summary, the present study confirmed the crucial role of OCT2 and OCTN1 but not OCTN2 in the uptake of OXA in the DRG and demonstrated that L-THP could prevent OXA-induced peripheral neurotoxicity through inhibiting OCT2 and OCTN1, without impairing the antitumour effect of OXA. Considering the safety of L-THP in clinical studies, L-THP is likely a potential candidate to attenuate OXA-induced peripheral neurotoxicity.