2.3. Data and Statistical Analysis
Statistical analysis was done using the Statistical Package for Social
Science (SPSS) version 18. A difference between mean values is
considered significant at p < 0.05. The results were
presented as mean ± SD. All the figures were made by GraphPad Prism
version 8.
Results
CSCs were effectively enriched by 50 µg/mL CDDP
The percentage of CD44+ CD24- cells
was increased using different concentrations of DOX, CDDP, and PTX,
however, the most significant enrichment was observed with 50 µg/mL CDDP
(50 µg/mL CDDP vs . EAC; p = 0.000000363) (Fig.
1 ). This concentration was therefore selected for subsequent in
vivo experiments.
Tumorigenicity of CDDP-Resistant EAC Cells vs. Parent
EAC Cells
Based on the proportion of CD44+CD24- cells, CDDP-resistant cells had a higher
proportion of CSCs than the parent EAC cells (3.4% vs . 0.1%).
CDDP-resistant cancer cells showed a heightened ability to form tumors
compared with the sensitive cells where the CDDP-resistant cells-derived
tumors were initiated faster and significantly larger than their parent
counterparts (Fig. 2A ).
On day 17 when the tumors were harvested, all the resistant
cells-derived tumors were significantly larger than their corresponding
sensitive tumors except for indomethacin where the difference between
indomethacin-receiving resistant and sensitive tumors was not
statistically significant (p = 0.44) (Fig. 2B ).
Indomethacin effectively and selectively reduced the
tumorigenicity of CDDP-resistant cells in in vivo mouse
experiments
Indomethacin in combination with CDDP decreased the tumor volumes in
mice bearing parent SEC more significantly than CDDP (SEC vs .
SEC-combination; p = 0.00015212, SEC vs. SEC-CDDP;p = 0.00627464), whereas indomethacin alone was not able to
significantly reduce the tumor volumes (SEC vs. SEC-indomethacin;p = 0.143). In contrast, for CDDP-resistant tumors, indomethacin
dramatically reduced the tumor volumes compared to CDDP alone or
combination of both (SEC/CDDP vs. SEC/CDDP-indomethacin; p= 0.00000037, SEC/CDDP vs. SEC/CDDP-CDDP; p = 0.01159254,
SEC/CDDP vs. SEC/CDDP-combination; p = 0.00002975)
(Fig. 2 ).
The growth rates of the sensitive SEC tumors exceeded those of
the resistant tumors
The growth rates of the tumors were computed after the start of the
treatment, unexpectedly, the growth rates of all the resistant tumors
were lower than their analogous sensitive ones (Fig. 3A ).
Noteworthily, the growth rates of the CDDP-resistant tumors were
initially faster than the SEC tumors before the treatment has been
commenced (growth rate of SEC/CDDP = 0.093952 vs. SEC = 0.058813)
(Fig. 3B ).
In a similar pattern to the tumor volumes, the combination of
indomethacin and CDDP more effectively impeded the growth rates of the
parent SEC tumors than either drug alone (SEC vs.SEC-combination; p = 0.00005500, SEC vs. SEC-CDDP;p = 0.00233442, SEC vs. SEC-indomethacin; p =
0.12394883), while the growth rates of the resistant tumors were slowed
down the most by indomethacin (SEC/CDDP vs.SEC/CDDP-indomethacin; p = 0.00013892, SEC/CDDP vs.SEC/CDDP-CDDP; p = 0.04591864, SEC/CDDP vs.SEC/CDDP-combination; p = 0.00503803) (Fig. 3A ).
CDDP-resistant tumors exhibited extensively deregulated miRNAs
expressions
We determined the relative expressions of 4 miRNAs that are deregulated
in murine breast cancer and involved in drug resistance. Compared with
the untreated sensitive tumors, the untreated resistant tumors showed
lower expressions of miR-7 and miR-145 besides overexpression of miR-21
and miR-22 (p = 0.037, 0.00024, 0.001, 0.0000136, respectively)
(Fig. 4 ).
MiRNAs Expressions are Altered in Response to Indomethacin
miR-7 expression was significantly increased by indomethacin (SECvs . SEC-indomethacin; p = 2.17*10-21,
SEC/CDDP vs . SEC/CDDP-indomethacin; p =
8.33*10-7). In contrast, the conventional chemotherapy
further downregulated miR-7 in both sensitive and resistant
tumors, however, it did not reach statistical significance for the
latter (SEC vs . SEC-CDDP; p = 0.037309067230362626,
SEC/CDDP vs . SEC/CDDP-CDDP; p = 0.9029500148694073).
Strikingly, indomethacin showed an ability to overcome the
downregulatory effect of CDDP and even upregulate miR-7 when both
used together (SEC vs . SEC-combination; p = 0.00000823,
SEC/CDDP vs . SEC/CDDP-combination; p = 0.00129)
(Fig. 4A ).
Unlike miR-7 , indomethacin reduced the expression ofmiR-145 (SEC vs . SEC-indomethacin; p =
0.000000057341, SEC/CDDP vs . SEC/CDDP-indomethacin; p =
0.0098) whereas the effect of CDDP was as the same as its effect onmiR-7 (SEC vs . SEC-CDDP; p =
1.88*10-11, SEC/CDDP vs . SEC/CDDP-CDDP;p = 0.000065). Unexpectedly, the combination of both indomethacin
and CDDP downregulated miR-145 to a less extent (SEC vs .
SEC-combination; p = 0.0000000677, SEC/CDDP vs .
SEC/CDDP-combination; p = 0.046) (Fig. 4B ).
For miR-21 , indomethacin significantly downregulated its
expression (SEC vs . SEC-indomethacin; p =0.029, SEC/CDDPvs . SEC/CDDP-indomethacin; p =0.032) while it has been
overexpressed by CDDP (SEC vs . SEC-CDDP; p = 0.00000083,
SEC/CDDP vs . SEC/CDDP-CDDP; p =0.000091). The combination
of both drugs has not significantly decreased the expression of miR-21
(SEC vs . SEC-combination; p = 0.058, SEC/CDDP vs .
SEC/CDDP-combination; p = 0.059) (Fig. 4C ).
The levels of miR-22 in tumor tissues were significantly reduced
by CDDP, indomethacin, and the combination (SEC vs . SEC-CDDP;p = 0.024, SEC/CDDP vs . SEC/CDDP-CDDP; p =0.000125)
(SEC vs . SEC-indomethacin; p =0.021, SEC/CDDP vs .
SEC/CDDP-indomethacin; p =0.000027) (SEC vs .
SEC-combination; p = 0.008, SEC/CDDP vs .
SEC/CDDP-combination; p = 1.28*10-10)
(Fig. 4D ).
Indomethacin negatively affected the stem cell markers and
suppressed the CDDP-induced increase of CD44+CD24- cells
Indomethacin decreased the percentage of CD44+ cells
(p = 7.9*10-10) and increased that of
CD24+ cells (p = 1.1*10-7),
whereas CDDP showed an opposite effect (p =
3.7*10-11 and 0.003 for CD44+ and
CD24+, respectively), so as a result, the fraction of
CD44+ CD24- cells is considered to
be gone up by CDDP unlike indomethacin. Strikingly, when indomethacin
was used along with CDDP it has not only abolished the CDDP-associated
elevation of CD44+ CD24- cells, but
the proportions were further dropped below control levels (p =
1.7*10-10 and 0.019 for CD44+ and
CD24+, respectively) (Fig. 5 ).
Both CDDP and indomethacin exerted a similar effect on the percentage of
SCa-1+ cells where both drugs decreased the
SCa-1+ population (p =
4.7*10-5, 1.099*10-12, and
1.088*10-12 for CDDP, indomethacin, and the
combination, respectively) (Fig. 5 ).
Indomethacin showed favorable responses for
CD62L+, CD4+, and
CD117+ populations
Indomethacin raised the percentages of CD62L+,
CD4+, and CD117+ cells (p =
1.08*10-12, 1.08*10-12, and
1.59*10-4, respectively) while a reverse effect was
posed by CDDP, however, the decline was not statistically significant
for CD62L+ cells (p =
3.7*10-6 and 0.009 for CD4+ and
CD117+ cells, respectively). As expected, the
combination treatment showed levels that are in between those of
indomethacin and CDDP (p = 5.79*10-6, 0.022,
and 0.57 for CD62L+, CD4+, and
CD117+ cells, respectively) (Fig. 5 ).
Histological Examination
Upon examination of the H & E stained tumor sections, CDDP-resistant
tumors were of a higher grade than the parent SEC tumors featuring
extensive mitosis with pleomorphism and perineural invasion indicating
the aggressiveness of such tumors. In the case of the parent SEC tumors,
the three treatment modalities showed different degrees of necrosis with
the highest extent of necrotic cells appeared with the co-treatment
(Fig. 6 ). Remarkably, despite CDDP showed extensive areas of
necrosis, there was a detectable tumor vascular embolus suggesting that
CDDP did not target the metastasis-driving cells; CSCs (Fig.
6B2 ).
Discussion and Conclusion
Breast cancer patient’s mortality is on the rise due to the lack of
medications that could curb the activity of CSCs which drive distant
metastasis and promote disease recurrence after chemotherapy, both of
which are the primary causes of cancer-related deaths [22, 23].
Current directions in cancer treatment aim to identify therapies that
target CSCs thereby eliminating the roots of the disease.
NSAIDs have proven their chemoprophylactic effects in BC, and several
studies have demonstrated that NSAIDs can even reduce the risk of BC
recurrence [11]. NSAIDs reduced metastasis in cancer patients, of
them, BC patients were included [24]. Since CSCs are the ones
responsible for relapse and metastasis [6], we propose, based on
these studies and our findings, that the therapeutic effects of NSAIDs
might be ascribed to their anti-CSC effect.
Although there is extensive research on the CSC-inhibitory effect of
NSAIDs, much of the research done investigated the modulation of
stemness genes and epithelial mesenchymal transition (EMT) markers by
NSAIDs as possible mechanisms for their CSC-suppressing ability
[25-27] but little has been done on whether NSAIDs combat CSCs by
affecting either miRNAs that are implicated in the drug resistance or
the immune system whom CSCs can circumvent.
Herein, we aimed to examine the effect of indomethacin on two oncomirs
and two tumor suppressor miRNAs in BC besides showcasing a little cue on
the likely involvement of the antitumor immunity as a mechanism
underlying the CSC-suppression mediated by indomethacin.
Much evidence indicate that CSCs subset markedly expands following
chemotherapy [9] and we used this approach to enrich CSCs originally
existing in EAC as these cells represent only a very small fraction of
cancer population (0.1%) which could have hindered our ability to
either effectively induce resistant tumors or discover a potentially
pronounced anti-CSC effect of the candidate drugs. However, the
percentage of the CDDP-enriched CSCs was 3.4% which is 34-fold higher
than the portion in the parent EAC. As such, this CSCs-enriched
population proved to be highly tumorigenic upon implantation in mice
compared to their more differentiated progeny when an equivalent number
of cells were injected and our result is in harmony with a previous
report [28]. Intriguingly, although the tumors derived from the
CDDP-resistant cells exhibited larger volumes, it grew slower than the
parent tumors. These seemingly paradoxical behaviors could be
potentially accredited to a relative shortage of oxygen and nutrients in
quite big tumors which in turn retards the growth rate of such tumors
[21].
Liu et al. studied the growth kinetics of BCSCs and proposed
mathematical models thereafter. According to his study, non-stem cancer
cells exert negative feedback on the proliferation rate and self-renewal
ability of CSCs [29]. That could elaborate on why the antitumor
effect of indomethacin on CDDP-resistant tumors was far more significant
than the co-treatment. As CDDP preferentially targets the bulk tumor
cells, it will be successful in letting the tumor sizes down, but
meanwhile, the negative feedbacks on CSCs will be eliminated, therefore,
CSCs will symmetrically proliferate at a high rate, and this is
consistent with the dramatic increase in the CSCs fraction after CDDP
treatment.
On the other hand, indomethacin selectively targets CSCs leaving the
differentiated cancer cells and their negative feedbacks in action. As a
result, the division of CSCs will be shifted towards an asymmetric one
at a slower pace and this agrees well with the sharp drop in the CSCs
population post indomethacin treatment. It is commonly believed that the
optimum therapeutic strategy to achieve complete remission of cancer is
to combine drugs that target both CSCs and non-CSCs. Based on our
findings and the proposed models by Liu, Johnson et al. , the
removal of the differentiated tumor cells will not make the outcome any
better [29].
Concerning the more differentiated SEC tumors, although indomethacin has
not significantly reduced their tumors sizes Liu, Johnson et al. ,
expected for such CSCs-targeted therapies to suppress tumor growth and
inhibit relapse on the long term. On the contrary, relapse is probable
with conventional chemotherapies despite the powerful fall in tumor size
because of the accompanied CSCs enrichment [29]. This makes us
recall the histological examination of SEC-CDDP tumors when we found a
notable tumor vascular embolus indicating the advert of metastasis and
subsequent relapse.
We then examined the relative expression of different miRNAs. BothmiR-7 and miR-145 were more downregulated in the
CDDP-resistant tumors. A similar finding was observed in a
study conducted by Pogribny et al. where miR-7 levels were
2.3 fold more downregulated in CDDP-resistant MCF-7 compared to MCF-7
cells [30]. Others reported that gall bladder cancer cells were
sensitized to CDDP through miR-145 -mediated regulation of MRP1
[31].
Contrarily, both miR-21 and miR-22 were overexpressed in
SEC/CDDP tumors compared to the non-resistant ones. miR-21 was
found to be involved in CDDP resistance through targeting the tumor
suppressor phosphatase and tensin homolog (PTEN) in gastric cancer
[32] and neuroblastoma and was overexpressed in CDDP-resistant cell
lines [33]. In another study, the sensitivity of MCF-7 cells to CDDP
was enhanced by knockdown of dicer which is accompanied by lower
expression of miR-21 . Collectively, these findings could explain
the decreased sensitivity of the tumors derived from the CSCs-enriched
population to CDDP treatment unlike the parent SEC tumors which
responded better.
To examine whether indomethacin is capable of antagonizing the
immunosuppressive impact of CDDP, we determined the levels of
conventional blood and immune cells markers. CD4, CD117, and CD62L
(otherwise known as L-selectin) are basic markers for T helper cells
(Th) [34], hematopoietic stem cells (HSCs) [35], and white blood
cells (WBCs) [36], respectively. CD4 is found on the surface of
other immune cells such as dendritic cells [37] and natural killer T
cells [38].
Although CDDP treatment has drastically shrunk the SEC tumors, its
effect on the CDDP-resistant tumors was subtle. CDDP enriched
CD44+ CD24- cells and further
downregulated miR-7 and miR-145 while upregulatedmiR-21 . The mechanisms by which CDDP modulated these miRNAs are
still obscure as most of the studies examined the consequence of miRNAs
expression pattern on the sensitivity of cancer to CDDP but none, to the
best of our knowledge, have tested the effect of CDDP on miRNAs
expression.
Moreover, CDDP decreased circulating CD117+,
CD62L+, and CD4+ cells as well. A
possible explanation for such deficiency is the well-known CDDP-prompted
myelosuppression effect. A study conducted by Zhao et al. showed
that CDDP-provoked G2/M phase arrest in bone marrow cells resulting in
bone marrow suppression [39].
On the other hand, albeit the SEC tumors sizes have not been
significantly decreased by indomethacin, its effect on SEC/CPPD tumors
was robust. Cells positive for CD44 or SCa-1 and those negative for CD24
have been reduced by indomethacin, additionally, it promoted the
expression of miR-7 and declined that of miR-21 andmiR-22 . Majumder et al . have found that COX-2 induces
BCSCs through EP4-mediated NOTCH/WNT axis and this effect was reversed
with COX-2 inhibitor or EP4 antagonist [40]. This could clarify the
indomethacin-associated decrease in CSCs markers. Furthermore, an
earlier study demonstrated that pre-treatment of mammary cancer cells
with COX-2 inhibitors caused suppression of radiotherapy-induced
SCa-1+ cells [41] which supports the relevant
finding of the indomethacin-accompanied reduction of
SCa-1+ pool.
Moreover, as miR-7 blocks the growth of CSCs in BC [42], it
might explain the decrease of CD44+CD24- cells in indomethacin-treated mice due to its
upregulatory effect on miR-7 . Bourguignon et al . showed
that miR-21 is induced in BC by binding of hyalouronan with CD44
[43], so miR-21 expression might be lowered in response to
indomethacin due to the associated reduction in CD44 level.
Consistently, it has been reported that the downregulation ofmiR-21 stimulated the differentiation of chemo-resistant colon
cancer and consequently enhanced the sensitivity to therapy [44].
Our findings provide an illustration of the enhanced susceptibility of
the established tumors to CDDP when used simultaneously with
indomethacin.
Of note, despite miR-145 is thought to be a tumor suppressor, it
displayed a contradictory effect where it suppressed the differentiation
of BCSCs via the 3‘-untranslated region (3‘-UTR) of insulin
receptor substrate 1 [45].
Furthermore, the percentages of cells positive for each of CD117, CD62L,
and CD4 have been raised by indomethacin. Hoggatt et al.[46], have documented that NSAIDs can mobilize the HSCs from the
bone marrow into the bloodstream. The mechanism of the NSAIDs mobilizing
effect is based on inhibition of PGE2 synthesis which disrupts the
production of osteopontin, a protein secreted by osteoblasts, that hooks
the stem cells to the bone marrow. Since these HSCs give rise to all
blood cell types, this might elucidate the indomethacin-mediated
increase in blood cells expressing CD117, CD62L, CD4, and CD24.
Considering this mobilizing effect, indomethacin therapy, unlike
chemotherapy, can exclude the need for HSCs transplantation when used by
cancer patients.
Aside from counteracting CDDP-resistance, indomethacin probably
eradicates CSCs through immunostimulatory effects. It has been
documented that PGE2 drives tumor progression by inducing
myeloid-derived suppressor cells (MDSCs) and that EP antagonists block
MDSCs differentiation [47]. MDSCs are immunosuppressive and they
have been involved in BC promotion by conferring stem cell-like
qualities as well as suppressing T-cell activation [48].
Interestingly, tumor-induced MDSCs downregulate the expression of
L-selectin on CD4+ and CD8+ T cells
impairing L-selectin dependent cell-mediated antitumor immunity [49]
and this might explain the indomethacin-induced increase in
CD62L+ cells. Furthermore, PGE2 reduces dendritic
cells (DCs) differentiation and this type of immune cells is thought to
eradicate CSCs [47, 50].
Intriguingly, the activity of the immune cells is influenced by miRNAs.
For example, MDSCs highly express miR-21 which alters its
differentiation and functional activity [51]. As indomethacin
downregulates miR-21 , this could provide another support on how
indomethacin might impede MDSCs activity. Moreover, miR-21downregulates the tumor suppressor programmed cell death-4 (PDCD4).
PDCD4 inhibits the expression of the immunosuppressives IL-10 and IL-4
[52]. Both IL-4 and IL-10 promote CSCs survival [53, 54].
Furthermore, the expression of IL-12 is reciprocal to miR-21[52] and unlike IL-10 and IL-4, IL-12 inhibits CSCs [55]. Hence,
indomethacin may kill CSCs through its downregulatory impact onmiR-21 and the subsequent reduction in IL-4 and IL-10 as well as
elevation of IL-12.
Additionally, it has been shown that PGE2 suppresses T cell
proliferation [56], thus, the apparent increase in peripheral
CD4+ population could be accounted for the PGE2
synthesis inhibition by indomethacin. Moreover, we can expect that
indomethacin might augment Th1 actions as long as PGE2 suppresses Th1
responses [56]. Notably, both IL-4 and IL-10 inhibit Th1 cell
production and function, respectively [57, 58] whereas IL-12 is
indeed involved in Th1 polarization [52]. Given these, we could
deduce that the indomethacin-associated increase in peripheral
CD4+ cells is potentially in favor of Th1 cell type.
In fact, recent studies have indicated that immunotherapeutic strategies
in BC should be directed towards type 1 immune response that is rich in
Th1 cells [59]. However, whether the indomethacin-associated
CD4+ cells are tumor-antigen specific or even have a
role in fighting CSCs needs to be investigated. Interestingly, elevated
levels of antigen specific CD4+ T cells in the
peripheral blood of BC patients who undergo anticancer therapy is not
unfamiliar. This is exemplified by trastuzumab therapy which boosted the
levels of circulating HER2 specific CD4+ Th1 cells in
BC patients who otherwise normally have few circulating antigen-specific
T cells able to eradicate their tumors [59].
Impressively, one study discovered the existence of anti-CSC specific
CD4+ T cells in the peripheral blood of ovarian cancer
patients [60]. This encourages us to complement the current work by
conducting further studies on the phenotypic and functional
characterization of the indomethacin-induced CD4+cells. Moreover, whether the high levels of CD4+ cells
in the blood are correlated with the disease outcome remains to be
explored. If this is the case, this could be harnessed as a prognostic
tool instead of invasively monitoring the level of tumor-infiltrating
lymphocytes.
There is also a link between miR-22 and the immune system, for
instance, miR-22 is upregulated in DCs retarding their anti-tumor
activity [61]. Thus, the indomethacin-induced downregulation ofmiR-22 could promote the CSC-suppressing ability of
tumor-associated DCs.
In conclusion, this study shows that indomethacin has a CSC-suppressing
effect in BC and inhibits CDDP-induced increase of CSCs presumably
through modulation of miRNAs implicated in drug resistance as well as
triggering an antitumor immune response. To this end, NSAIDs sound to
have far-reaching mechanisms behind its CSC-suppressing activity that
necessitate deeper investigations to exactly unravel the pathways of
such mechanisms.