Therapeutic Effect of Y-27632 on Tumorigenesis and Cisplatin-Induced Peripheral Sensory Loss through RhoA-NF-κB

Abstract

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect of cancer therapy that frequently requires a reduction or cessation of treatments and negatively impacts the patient's quality of life. There is currently no effective means to prevent or treat CIPN. In this study, we developed and applied CIPN in an immunocompetent, syngeneic murine Lewis Lung Carcinoma (LLCab) model that enabled the elucidation of both tumor and host responses to cisplatin and treatments of Y-27632, a selective inhibitor of Rho kinase/p160^ROCK. Y-27632 not only preserved cisplatin's efficacy toward tumor suppression but also the combination treatment inhibited tumor cell proliferation and increased cellular apoptosis. By alleviating the cisplatin-induced loss of epidermal nerve fibers (ENFs), Y-27632 protected tumor-bearing mice from cisplatin-induced reduction of touch sensation. Furthermore, quantitative proteomic analysis revealed the striking cisplatin-induced dysregulation in cellular stress (inflammation, mitochondrial deficiency, DNA repair, etc.)-associated proteins. Y-27632 was able to reverse the changes of these proteins that are associated with Rho GTPase and NF-κB signaling network, and also decreased cisplatin-induced NF-κB hyperactivation in both footpad tissues and tumor. Therefore, Y-27632 is an effective adjuvant in tumor suppression and peripheral neuroprotection. These studies highlight the potential of targeting the RhoA-NF-κB axis as a combination therapy to treat CIPN. IMPLICATIONS: This study, for the first time, demonstrated the dual antineoplastic and neuroprotective effects of Rho kinase/p160^ROCK inhibition in a syngeneic immunocompetent tumor-bearing mouse model, opening the door for further clinical adjuvant development of RhoA-NF-κB axis to improve chemotherapeutic outcomes.

Figure 1.. Y-27632 enhanced the antineoplastic effect of cisplatin in lung cancer cells in culture.

(A). LLCab Cells were treated with DMSO (Control), cisplatin 8μg/ml, Y-27632 1μM, cisplatin 8μg/ml + Y-27632 1μM, cisplatin 8μg/ml + Y-27632 10 μM, or cisplatin 8μg/ml + Y-27632 100 μM for 24 hours, and the cell lysates collected from each treatment group were subjected to Western blot analysis of Cleaved-PARP and Total PARP. (B). Analysis of the ratio of Cleaved-PARP/Total PARP. Values represent mean±SEM. **p<0.01, ****p<0.0001, relative to Control; ΔΔΔΔp<0.0001, relative to Cisplatin 8μg/ml; •p<0.05, ••••p<0.0001, relative to Y-27632 1μM. ■■p<0.01, ■■■p<0.0005. (C). LLCab cells were treated with DMSO (Control), cisplatin 8μg/ml, Y-27632 1μM, cisplatin 8μg/ml + Y-27632 1μM, cisplatin 8μg/ml + Y-27632 10 μM, or cisplatin 8μg/ml + Y-27632 100 μM for 24 hours. Then, MTT assay analysis was applied to evaluate percentage of viable cells. Values represent mean±SEM. **p<0.001, ***p<0.0001, relative to Control; •p<0.05, •••p<0.0001, relative to Y-27632 1μM; Δp<0.05, relative to Cisplatin 8μg/ml.

Figure 2.. Y-27632 suppressed tumor growth in vivo and promoted cisplatin-induced apoptosis in the tumor tissues.

(A). Drug treatment began on day 5 post LLCab injection. Day 6 to Day 20, n=10 animals/all groups. Day 20 to Day 30: n=10 animals/group Saline, Y-27632 and Cis+Y-27632, n=8 animals/group Cisplatin. Values represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 relative to Saline; Δp<0.05, ΔΔΔΔp<0.0001, relative to Y-27632. (B and C). Images of anti-Ki67 (B) and TUNEL (C) immunofluorescence staining of tumor tissues from all treatment groups. Scale bar: 30μm. (D). Density analysis of anti-Ki67 positive cells in the tumor tissues. Values represent mean±SEM. **p<0.01, ***p<0.0001, relative to Saline; •••p<0.005, relative to Y-27632; Δp<0.05, relative to Cisplatin. (E). Density analysis of TUNEL positive cells in the tumor tissues. Values represent mean±SEM. *p<0.05, ***p<0.001 relative to Saline; ••p<0.01, relative to Y-27632; Δp<0.05, relative to Cisplatin.

Figure 3.. Y-27632 ameliorated cisplatin-induced reduction of touch sensation and protected ENFs and Meissner corpuscles from cisplatin-induced impairment in the mouse hind paw.

(A).Von Frey analysis was conducted weekly on each treatment group of mice. Values represent mean±SEM. *p<0.05, ****p<0.0001, relative to Saline. •p<0.05, ••••p<0.0001, relative to Y-27632; ■p<0.05, ■■■■p<0.0001, relative to Cis+Y-27632. Baseline to Day 20: n=10 animals/all groups. Day 20 to Day 30: n=10 animals/group Saline, Y-27632 and Cis+Y-27632, n=8 animals/group Cisplatin. Day 30 to Day 34: n=10 animals/group Saline, n=9 animals/group Y-27632 and Cis+Y27632, n=7 animals/group Cisplatin. (B and C). Footpad tissues were excised and immunofluorescence analysis was performed with anti-PGP 9.5 (pan ENF marker) and anti-S-100 (schwann cells marker). ENFs and MCs were indicated with white arrowheads, F-actin were stained by FITC-Phalloidin and nuclei were indicated by Hoechst staining. Scale bar: 50μm. (D). Density analysis of anti-PGP9.5 positive ENFs in the footpad tissues from each treatment group. Values represent mean±SEM. ****p<0.0001, relative to Saline. n=5 animals per each treatment group. (E). Density analysis of MCs in the footpad tissues from each treatment group. Values represent mean±SEM. ****p<0.0001, relative to Saline. n=5 animals per each treatment group.

Figure 4.. Y-27632 attenuated the cisplatin induced inflammation/DNA repairing/oxidative stress responses in the mouse foodpad tissues.

(A). Hierarchical clustering of the z-score normalized log2 label-free quantification (LFQ) values averaged across four biological replicates for ANOVA significant proteins (p-value <0.05). Protein levels correspond to the color scale. A color gradient from red to blue represented a high level to low level of proteomic responses (grey = missing data). (B). Ratio of protein expression level change of each treatment group compared to Saline group. Listed proteins were all upregulated by cisplatin treatment while being reverted towards control level under Cis+Y-27632 combination treatment. (C). Footpad tissues were stained with anti-DHFR, anti-SMC1a, anti-HMGCS2 and anti-AKAP8 antibodies. ENFs were revealed by anti-PGP9.5 antibody and were indicated by white arrowheads, and nuclei were indicated by Hoechst staining. Scale bar: 30μm. (D). STRING analysis revealed the interaction network of cisplatin induced-upregulated gene encoded proteins with RhoA-NF-κB signaling axis (inside red circle). (E). Western blot analysis of footpad tissue lysates collected from each treatment group. Among the expression of Stat3, phospho-Stat3, NF-κB and phospho-NF-κB, phospho-NF-κB was most dramatically increased. (F). Quantification analysis of the ratio of pNF-κB/NF-κB in all treatment groups. Values represent mean±SEM. **p<0.0001, relative to Saline. N=5 animals per each treatment group.

Figure 5.. Schematic illustration of potential therapeutic mechanisms of Y-27632 on cisplatin-induced suppression of lung cancer and its neuroprotective roles in the peripheral nerves.

Cisplatin suppresses tumor growth but also induces cellular stress. At the peripheral nerve-epidermis interface in the mouse footpad, cisplatin-induced RhoA activation increases NF-κB hyper-activation, which led to peripheral neuropathy. Functional inhibition of RhoA signaling pathway by Y-27632 can promote cancer cell apoptosis, decrease NF-κB-induced chemoresistance, and prevent the loss of ENFs in the mouse footpad.