Page 106 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

Serbian Association for Cancer Research SDIRSACR

that NaV1.7 protein levels were markedly elevated in both pancreatic cancer cell lines. Following siRNA-
mediated knockdown, a substantial reduction in both NaV1.7 mRNA and protein expression was achieved in
PANC-1 and MiaPaCa-2 cells (>80% downregulation, p < 0.001), confirming the high efficiency of gene silencing.
Functionally, NaV1.7 knockdown resulted in a significant decrease in pancreatic cancer cell proliferation, as assessed by MTS
assays conducted at 24, 48, and 72 hours post-transfection (p < 0.001 at all time points compared to controls). Importantly,
NaV1.7 siRNA treatment did not elicit any cytotoxic effect in normal HaCaT cells, indicating a tumor-specific impact. In
clonogenic assays, the ability of PANC-1 and MiaPaCa-2 cells to form colonies was dramatically suppressed following
NaV1.7 silencing, with an approximately 70–75% reduction in colony numbers relative to negative controls (p < 0.001).
In PANC-1 cells, combination treatments with sub-cytotoxic doses of gemcitabine and NaV1.7 siRNA demonstrated a
clear synergistic effect, significantly enhancing the cytotoxicity of gemcitabine (combination index CI < 1). The combined
treatment led to further suppression of proliferation and clonogenic potential beyond either treatment alone (p < 0.001).
Cell invasion and migration assays revealed that NaV1.7 siRNA treatment significantly impaired the invasive
capacity of both PANC-1 and MiaPaCa-2 cells, with a >60% reduction in invasion through Matrigel-coated
Transwell inserts (p < 0.001). Similarly, migration was decreased by >50% following NaV1.7 knockdown
(p < 0.001). In wound-healing assays, NaV1.7 silencing delayed wound closure kinetics by more than
60% at 24 hours compared to untreated controls, indicating marked inhibition of cancer cell motility.
Flow cytometric analysis demonstrated that NaV1.7 silencing induced robust apoptosis in both pancreatic
cancer cell lines, with significant increases in early and late apoptotic populations (Annexin V+/PI+ cells, p
< 0.001). Moreover, NaV1.7 knockdown caused a pronounced arrest in the G1 phase of the cell cycle, with
a corresponding decrease in S-phase populations (p < 0.001), suggesting impaired cell cycle progression.
At the molecular level, Western blot analyses showed significant downregulation of key proteins associated with
proliferation (Cyclin D1, Cyclin E1), survival (P-Akt, P-mTOR), and invasion/migration (P-Src, P-FAK, Integrin β1, EF2K)
following NaV1.7 knockdown in both cancer cell lines. In addition, markers of apoptosis, including cleaved PARP
and cleaved Caspase-3/9, were markedly elevated, further supporting the pro-apoptotic effect of NaV1.7 inhibition.
Collectively, these results demonstrate that NaV1.7 channel activity critically regulates multiple hallmarks of pancreatic
cancer, including proliferation, invasion, migration, apoptosis, and drug resistance, and that its silencing offers a
promising therapeutic approach.
Conclusions: Our study provides the first comprehensive and mechanistic evidence that both adult and
neonatal isoforms of the NaV1.7 voltage-gated sodium channel play a critical role in driving pancreatic cancer
progression, metastatic potential, and therapeutic resistance. siRNA-mediated targeting of NaV1.7 not only
effectively suppressed key malignant phenotypes—including proliferation, clonogenicity, invasion, migration,
and survival—but also significantly enhanced the cytotoxic efficacy of the current first-line chemotherapeutic
agent, gemcitabine, in pancreatic cancer cells. Importantly, these effects were observed without inducing
cytotoxicity in normal keratinocyte cells, highlighting the tumor-specific therapeutic potential of NaV1.7 inhibition.
Furthermore, our findings revealed that NaV1.7 silencing modulates multiple oncogenic signaling
pathways involved in cell cycle regulation, survival, and metastasis, thereby providing mechanistic insights
into its role as a key regulator of pancreatic cancer biology. These collective results position NaV1.7
as a highly promising and novel molecular target for therapeutic intervention in pancreatic cancer.
Given the urgent unmet clinical need for more effective treatment strategies in this aggressive malignancy, further
validation of these findings in preclinical in vivo models and translational studies is warranted. Ultimately, the
development of NaV1.7-targeted therapies, either as monotherapy or in combination with standard chemotherapeutics,
may offer new avenues to improve patient outcomes in pancreatic cancer.

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