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.