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Advanced Neurology TRPM7 signaling in glioblastoma

A B of migration (p < 0.05) (Figure 4A–D), consistent with
previous findings . However, naltriben was unable to
[20]
reverse the inhibitory effect of CsA. Specifically, the rate of
wound closure in the co-treatment group was significantly
lower than in the naltriben-only group (p < 0.0001) and
not significantly different compared to the CsA-only
group. Since CsA appeared to occlude naltriben’s effect
of enhancing U251 cell migration, this suggested that
calcineurin acts downstream of TRPM7 to promote GBM
C D cell migration.
In addition to migration, CsA treatment has also
been previously shown to inhibit the rate of GBM cell
invasion . Conversely, treatment with naltriben has
[24]
been reported to increase invasion . We confirmed
[20]
these findings by using the Corning BioCoat Matrigel
Figure 3. Calcineurin inhibition increases TRPM7 protein and mRNA Invasion Chamber. Specifically, we showed that the rate of
levels. (A) CCK-8 assay on U251 cells was conducted following treatment U251 cell invasion was significantly increased following
with vehicle (DMSO; control) or CsA (10, 20, 40, or 80 µM) for 24 h treatment with naltriben for 24 h (p < 0.001), whereas a
(n = 10/group). The analysis was performed with one-way ANOVA
(**p < 0.01). (B–D) Cells were treated with vehicle (DMSO; control) or 10 decreasing trend for the invasion was observed in the CsA-
µM CsA 24 h before RNA or protein isolation. (B) Representative images treated group (p = 0.131). Consistent with migration assay
and (C) exposure intensities of Western blot bands, normalized using the results, co-treatment with CsA and naltriben resulted in
loading control GAPDH, were processed with ImageJ and statistically an invasion rate that was not significantly different from
analyzed using Student’s t-test (*p < 0.05; n = 8 or 9/group). (D) TRPM7
mRNA levels were assessed using qRT-PCR and normalized using GAPDH that of the CsA-only group, while being significantly lower
as the reference gene. The difference between the control and treatment (p < 0.0001) than the naltriben-only group (Figure 4E).
groups was analyzed using Student’s t-test (***p < 0.001; n = 6/group). Thus, calcineurin may act downstream of the TRPM7
Abbreviations: TRPM7: Transient receptor potential melastatin 7; pathway that promotes GBM cell function.
CsA: Cyclosporine A.
3.5. CsA-induced changes in PI3K/AKT and
dependent decrease in U251 cell viability, consistent with a MAPK/ERK signaling pathways are not affected by
previous report (Figure 3A). Furthermore, we observed naltriben
[23]
that treatment with 10 µM CsA had no significant effect on Previous studies have demonstrated that both
the viability of U251 cells, and thus this concentration was pharmacological inhibition and genetic knockdown of
used for subsequent experiments. TRPM7 resulted in decreased phosphorylation of AKT

Our data showed that CsA treatment significantly and ERK [16,17] . In contrast, calcineurin inhibition by CsA
[26]
increased (p < 0.05) TRPM7 protein levels has been reported to increase p-AKT . In the present
(Figure 3B and C). Consistently, CsA treatment resulted study, we showed that 24-h CsA treatment elevated p-AKT
in a significant increase (p < 0.001) in mRNA levels of levels significantly, whereas naltriben treatment had no
TRPM7 compared to control (Figure 3D). These findings significant effect (Figure 5A and B). Levels of p-AKT levels
indicated that calcineurin inhibition can increase protein were significantly increased in the naltriben and CsA
and mRNA levels of TRPM7 in U251 cells. co-treatment group compared to the naltriben-only group,
but they were not significantly different from the CsA-only
3.4. Treatment with TRPM7 activator does not group.
reverse the effects of calcineurin inhibition on U251
cell migration and invasion Interestingly, although statistical significance was not
observed, treatment of U251 cells with CsA appeared
Next, we wanted to examine whether TRPM7 acted to trend towards a decrease in ERK phosphorylation.
upstream of calcineurin in regulating GBM cell function. Similarly, the naltriben and CsA co-treatment group also
After treating U251 cells for 24 h with 10 µM CsA, 25 µM showed a decreasing trend in p-ERK levels compared to the
naltriben, or co-treatment, the rate of cell migration was control group (Figure 5D and E). These findings provide
assessed using wound healing assays. We found that 10 µM further evidence that calcineurin acts as a downstream
CsA significantly lowered the rate of U251 cell migration target in the TRPM7-mediated signaling that is involved in
whereas 25 µM naltriben significantly increased the rate regulating GBM cell function.

Volume 2 Issue 3 (2023) 5 https://doi.org/10.36922/an.334