Targeting IL8 as a sequential therapy strategy to overcome chemotherapy resistance in advanced gastric cancer

Abstract

Systemic chemotherapy with multiple drug regimens is the main therapy option for advanced gastric cancer (GC) patients. However, many patients develop relapse soon. Here, we evaluated the therapeutic potential of targeting interleukin-8 (IL8) to overcome resistance to chemotherapy in advanced GC. RNA sequencing revealed crucial molecular changes after chemotherapy resistance, in which the expression of IL8 was significantly activated with the increase in drug resistance. Subsequently, the clinical significance of IL8 expression was determined in GC population specimens. IL8-targeted by RNA interference or reparixin reversed chemotherapy resistance with limited toxicity in vivo and vitro experiments. Sequential treatment with first-line, second-line chemotherapy and reparixin inhibited GC growth, reduced toxicity and prolonged survival. Collectively, our study provides a therapeutic strategy that targeting IL8 as a sequential therapy after chemotherapy resistance in advanced GC.

Fig. 1. First-line chemotherapy effectively inhibits GC growth but develops drug resistance.

A Schematic representation of the BGC823-bearing model of first-line chemotherapy resistance. B The growth curves of tumor (n = 8 per group). C Hierarchical clustering gene transcription altered at different periods of chemotherapy. D, E The half-maximal inhibitory concentration (IC50) was measured by CCK8 assay. Primary cells from dissected tumor tissues were treated with different concentrations of 5-Fu (0, 5, 10, 20, 40, and 80 μM) and oxaliplatin (0, 2, 4, 8, 16 and 32 μM). F, G Flow cytometric assay of primary cells from dissected tumor tissues. Data are mean ± standard deviation. *P < 0.05. H Protein expression of apoptosis-related proteins (PARP, cleaved PARP, Bcl2, and Bax) were detected by western blot analysis in dissected tumor tissues. I The mRNA expression of Bax, Bcl2, and ki-67 in dissected tumor tissues were detected by qRT-PCR assay. Data are mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. Second-line chemotherapy reverses resistance to first-line chemotherapy of GC.

A Schematic representation of the BGC823-bearing model of sequential chemotherapy resistance. B The growth curves of tumor (n = 12 per group). C Kaplan–Meier survival curve after 5-Fu plus oxaliplatin or paclitaxel treatments. *P < 0.05. D–H Blood routine index (WBC, NEU%, RBC, HGB, and PLT) were measured. Data are mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. I–M Blood biochemical index (ALT, AST, DBIL, UA, and LDH) were measured. Data are mean ± standard deviation. *P < 0.05. N, O The representative images of tumor, liver, kidney, spleen, heart and lung by H&E staining. N Scale bars, 2000 μm, O Scale bars, 50 μm.

Fig. 3. Screening IL8 as the therapeutic target after chemotherapy resistance.

A The volcano plot of RNA transcription sequencing of the first-line chemotherapy group and the sequential chemotherapy group. A total of 256 differentially expressed genes (DEGs) with fold-change greater than 2. B KEGG pathways enriched by DEGs in the transcriptome affected by sequential chemotherapy compared with first-line chemotherapy. C qRT-PCR assays were performed to detect the changes of the DEGs in the three enriched signaling pathways most associated with cancer in tumor tissues treated with sequential and first-line chemotherapy. D, E IL8 expression at different periods of chemotherapy was detected by qRT-PCR assays and western blot analysis. Data are mean ± standard deviation. *P < 0.05, **P < 0.01. F Upregulation of IL8 in GC samples was obtained in the GEPIA database. G Kaplan–Meier OS curves in GC patients according to the expression of IL8. H Kaplan–Meier OS curves in GC patients after 5-Fu therapy according to the expression of IL8. I Western blot analysis of IL8 expression in BGC823 cells transfected with siRNAs against IL8. J CCK8 assays was carried out to detect the viability of BGC823 cells transfected with si-IL8#2 and si-IL8#3. **P < 0.01, ***P < 0.001. K, L Representative images (K) and quantification (L) of BGC823 cells transfected with si-IL8#2 and si-IL8#3 by EdU staining assays. Data are mean ± standard deviation. **P < 0.01. M–O Representative images (M) and quantification (N, O) of BGC823 cells transfected with si-IL8#2 and si-IL8#3 by colony-forming experiments and transwell assays. Data are mean ± standard deviation. **P < 0.01, ***P < 0.001.

Fig. 4. Sequential treatment with first-line, second-line chemotherapy, and reparixin preserves efficacy to GC cells and ameliorates toxicity to normal cells.

A Schematic representation of sequential treatment with first-line, second-line chemotherapy, and reparixin in BGC823 or GES-1 cells. B, C Colony formation assays were performed on BGC823 and GES-1 cells treated with different drugs as in Fig. 4A. Data are mean ± standard deviation. *P < 0.05. D–F BGC823 and GES-1 cells were treated as in Fig. 4A and subjected to flow cytometric analysis. G–J Representative images (G, H) and quantification (I, J) of BGC823 and GES-1 cells treated as in Fig. 4A and then stained for γH2AX and DAPI. Data are mean ± standard deviation. ***P < 0.001. K–M BGC823 and GES-1 cells were treated as in Fig. 4A and subjected to Comet analysis. DNA damage is quantified as percent DNA in tails. Data are mean ± standard deviation. *P < 0.05, **P < 0.01.

Fig. 5. Sequential treatment with first-line, second-line chemotherapy, and reparixin inhibits GC growth in vivo.

A Schematic representation of the BGC823-LPC cells from tumor tissues subjected to first-line and second-line chemotherapy xenograft model for reparixin treatment. B The growth curves of tumor (n = 6 per group). C The tumor weights were measured and recorded after the tumors were harvested. Data are mean ± standard deviation. ***P < 0.001. D, E Blood routine index (WBC and RBC) were measured. Data are mean ± standard deviation. *P < 0.05. F, G Blood biochemical index (DBIL and UA) were measured. Data are mean ± standard deviation. *P < 0.05. H The representative images of liver, kidney, and spleen by H&E staining. Scale bars, 50 μm.

Fig. 6. Sequential treatment with first-line, second-line chemotherapy and reparixin prolong the survival time of the GC-bearing mice.

A Schematic representation of the survival model that subjected to sequential treatment with first-line, second-line chemotherapy and reparixin (n = 15 per group). B The body weights at the indicated time points after indicated treatments. C Kaplan–Meier survival curve after indicated treatments. D The graphic illustration of sequential treatment with first-line, second-line chemotherapy and reparixin in suppressing GC growth.