Lidocaine inhibits the metastatic potential of ovarian cancer by blocking NaV 1.5-mediated EMT and FAK/Paxillin signaling pathway

Abstract

Lidocaine, one of the most commonly used local anesthetics during surgery, has been reported to suppress cancer cell growth via blocking voltage-gated sodium channels (VGSCs). VGSC 1.5 (Na_V 1.5) is highly expressed in invasive cancers including ovarian cancer. This study aims to investigate whether lidocaine inhibits the malignancy of ovarian cancer through Na_V 1.5 blockage. Human ovarian cancer, its metastatic cancer and normal ovarian tissues were probed with anti-Na_V 1.5 antibody in situ. Human ovarian cancer A2780 and SKOV3 cells were cultured and their growth, epithelial-mesenchymal transition (EMT), migration, and invasion in the presence or absence of lidocaine together with underlying molecular mechanisms were assessed. Murine syngeneic ovarian cancer (ID8) model was also used to determine the chemotherapeutic efficiency of cisplatin in combination with lidocaine. The high level of Na_V 1.5 expression was found in human ovarian cancer and even higher in its metastatic cancer but not in normal ovarian tissues. Lidocaine decreased the growth, EMT, migration, and invasion of human ovarian cancer A2780 and SKOV3 cells. Lidocaine enhanced the chemotherapeutic efficiency of cisplatin in both ovarian cancer cell cultures and a murine ovarian metastatic model. Furthermore, a downregulation of Na_V 1.5 by siRNA transfection, or FAK inhibitor application, inhibited the malignant properties of SKOV3 cells through inactivating FAK/Paxillin signaling pathway. Our data may indicate that lidocaine suppresses the metastasis of ovarian cancer and sensitizes cisplatin through blocking Na_V 1.5-mediated EMT and FAK/paxillin signaling pathway. The translational value of lidocaine local application as an ovarian cancer adjuvant treatment warrants further study.

Keywords: NaV1.5; cisplatin; lidocaine; metastasis; ovarian cancer.

Figure 1

Lidocaine inhibits the proliferation of ovarian cancer cells. A and B, A2780 and SKOV3 cells were treated with lidocaine (0, 1, 2.5, 5, 7.5, and 10 mM) for 24 and 48 hrs, respectively. CCK‐8 assay was used for cell viability evaluation. C and D, A2780 and SKOV3 cells were exposed to lidocaine (0, 2.5, 5, and 7.5 mM) for 48 hrs. Representative images of FITC‐labeled EdU (green) incorporation assay were presented. Hoechst 33342 (blue) was used for nuclei staining. Bar represents 50 μm. E and F, qRT‐PCR and (G) Western blot showed the mRNA and protein expression levels of PCNA, Cyclin D1, and Cyclin E1 in control and lidocaine‐ (5 mM) treated cells. GAPDH was used as an internal control. The data were presented as mean ±SEM (n = 9); *p < 0.05, **p < 0.01, ***p < 0.001

Figure 2

Lidocaine inhibits EMT, migration, and invasion of ovarian cancer cells. A and B, qRT‐PCR and (C) Western blot analysis of EMT markers (E‐cadherin, N‐cadherin, and Vimentin) in control and lidocaine‐treated A2780 and SKOV3 cells. GAPDH was used as an internal control. D and E, Representative fluorescent images of E‐cadherin and N‐cadherin in lidocaine‐treated A2780 and SKOV3 cells. DAPI (blue) was used for nuclei staining. F and G, Scratch assay, (H and I) Transwell migration and matrigel invasion assays were performed to detect the migration and invasion potential. J and K, qRT‐PCR and (L) gelatin zymography analysis of mRNA expression levels and enzymatic activity of MMP‐2 and MMP‐9 after lidocaine treatment in A2780 and SKOV3 cells. Bars represent 20 μm (D and E) and 100 μm (H and I). The data were presented as mean ±SEM (n = 3); *p < 0.05

Figure 3

Lidocaine sensitizes ovarian cancer cells to cisplatin in vitro. A and B, A2780 and SKOV3 cells were untreated, treated with cisplatin (10 μM), or cisplatin combined with lidocaine (5 mM) for 24 and 48 hrs, respectively. Cell viability was evaluated by CCK‐8 assay. C and D, Representative images of TUNEL (green)‐labeled apoptotic A2780 and SKOV3 cells. DAPI (blue) was used for nuclei staining. E and F, Western blot analysis of apoptotic marker proteins in control, lidocaine, cisplatin, cisplatin, and lidocaine combination groups. GAPDH was used as an internal control. Bar represents 20 μm. The data were presented as mean ±SEM (n = 9, n = 3); **p < 0.01, ***p < 0.001

Figure 4

Downregulation of Na_V1.5 expression suppresses the metastatic capability of ovarian cancer cells. A, qRT‐PCR, (B) Western blot, and (C) Immunofluorescent staining were used to detect the knockdown efficiency of Na_V1.5 siRNAs (−1, −2, and −3) after transfection of SKOV3 cells. D, qRT‐PCR and (E) Western blot analysis of EMT markers (E‐cadherin, N‐cadherin, and Vimentin) in scrambled siRNA and Na_V1.5 siRNA‐1 transfected cells. GAPDH was used as an internal control. F, Scratch assay, (G) Transwell migration and matrigel invasion assays were performed to detect the cellular motility of SKOV3 cells. H, qRT‐PCR and (I) Gelatin zymography showed the mRNA expression level and enzymatic activity of MMP‐2 and MMP‐9 after scramble siRNA and Na_V1.5 siRNA‐1 transfection. Bars represent 20 μm (C) and 100 μm (G). Data were presented as mean ±SEM (n = 3); *p < 0.05, **p < 0.01

Figure 5

Lidocaine suppresses cancer cell malignancy and enhances the cisplatin sensitivity by blocking Na_V1.5‐mediated FAK/paxillin signaling pathway in SKOV3 cells. A and B, Western blot analysis of p‐FAK and FAK in lidocaine and TTX‐treated cells, as well as in scramble RNA and Na_V1.5 siRNA transfected cells, respectively. C and D, Western blot analysis of p‐FAK and FAK in scrambled siRNA, FAK siRNAs (−1, −2, and −3) transfected cells, or in DMSO and FAK inhibitor‐ (1 μM, 5 μM, and 10 μM) treated cells, respectively. E, SKOV3 cells were treated with lidocaine, or transfected with scrambled siRNA, Na_V1.5 siRNA, FAK siRNA, as well as addition of FAK inhibitor, and the levels of p‐FAK, FAK, p‐Paxillin, Paxillin, N‐cadherin, and Vimentin were detected by western blot. The enzymatic activity of MMP‐9 was detected by gelatin zymography analysis. F, SKOV3 cells were treated with cisplatin, or in combination with lidocaine, scrambled siRNA, Na_V1.5 siRNA, and FAK inhibitor. The levels of p‐FAK, FAK, p‐Paxillin, Paxillin, Bcl‐2, Cleaved‐caspase‐3, and Cleaved‐PARP were detected by western blot

Figure 6

Lidocaine inhibits tumorigenesis and metastasis of ovarian cancer in vivo. A, Schematic representation of the treatment paradigm in this study. B, Representative photographs of the abdominal implantation metastasis foci of murine ovarian cancer in control (saline), lidocaine, cisplatin, or the combination of lidocaine and cisplatin group by B‐ultrasonography, respectively. Bar represents 20 μm. C, Representative pictures of the abdominal implantation metastasis foci in differently treated groups viewed after laparotomy, respectively. Bar represents 15 mm. D, Statistical analysis of the excised tumor weight in differently treated groups. E, Western blot analysis of p‐FAK, FAK, Cleaved‐caspase‐3, PCNA, and N‐cadherin of cancer tissues collected on the day 14. GAPDH was used as an internal control. F, Representative images of immunohistochemical staining of p‐FAK and Cleaved‐caspase‐3. Bar represents 50 μm. Data were presented as mean ±SEM (n = 8); *p < 0.05, **p < 0.01

Figure 7

Schematic representation of the molecular mechanism in the suppression of ovarian cancer malignancy by lidocaine. Ovarian cancer cells expressing high Na_V1.5 level present strong capacity of intraperitoneally implanted metastasis. Lidocaine binding with Na_V1.5 blocks the activation of FAK/Paxillin signaling pathway, inhibits the proliferation and metastasis, and decreases cisplatin resistance of ovarian cancer cells. The voltage‐gated sodium channels (VGSCs) blocker, tetrodotoxin (TTX), or FAK inhibitor (FAKi) reduces ovarian cancer malignancy by inactivating FAK/Paxillin signaling pathway