Combined effects of Lenvatinib and iodine-131 on cell apoptosis in nasopharyngeal carcinoma through inducing endoplasmic reticulum stress

Abstract

Nasopharyngeal carcinoma (NPC) is a type of malignant tumor characterized by high invasiveness, metastatic potential and worldwide incidence among patients with head and neck cancer. It has previously been demonstrated that Lenvatinib (LEB) is an efficient anti-cancer agent by multi-targeting of tyrosine kinase inhibitors. Iodine-131 (I-131) therapy has been accepted for the treatment of thyroid cancer and other carcinomas. In the present study, the combined effects of LEB and I-131 were investigated on NPC and the potential signal pathway mediated by LEB and I-131 on NPC cells was explored. Inhibitory effects of LEB and I-131 for NPC cells growth were investigated via MTT assay. Migration and invasion of NPC cells was studied by aggression assays following incubation of LEB and I-131. Apoptosis of NPC cells and tissues were analyzed via flow cytometry and TUNEL assay, respectively. Apoptosis-related gene expression levels in NPC cells following treatment with LEB and I-131 were determined by western blotting. Endoplasmic reticulum (ER) stress in NPC cells were analyzed in NPC cells and tumor tissues. Immunohistochemistry was used to analyze the efficacy of LEB and I-131 in NPC-tumor bearing mice. The results demonstrated that combined treatment of LEB and I-131 significantly inhibited growth, apoptosis, migration and invasion of NPC cells compared with single agent therapy. Apoptosis-related gene expression levels of caspase-3 and caspase-9 were upregulated by LEB and I-131, whereas B call lymphoma-2, and P53 were downregulated in NPC cells and tumor tissues. In addition, signal mechanism analysis demonstrated that combined treatment of LEB and I-131 promoted expression levels of activating transcription factor 6, inositol-requiring protein 1 (IER1), protein kinase RNA-like endoplasmic reticulum kinase (RERK), and C/EBP homologous protein in NPC cells. Furthermore, combined treatment of LEB and I-131 markedly inhibited in vivo growth of NPC and further prolonged survival of experimental mice compared with single agent and control groups. Immunohistochemistry indicated that c-jun N-terminal kinase and Caspase-3 were increased in NPS tumor tissues in xenograft models treated with LEB and I-131. Apoptotic bodies were also increased in tumors treated by LEB and I-131. In conclusion, these findings indicate that combined treatment of LEB and I-131 may inhibit NPC growth and aggression through upregulation of ER stress, suggesting combined treatment of LEB and I-131 may be a potential therapeutic schedule for the treatment of NPC.

Keywords: Lenvatinib; apoptosis; endoplasmic reticulum stress; iodine-131; nasopharyngeal carcinoma.

Figure 1.

Combination of LEB and I-131 inhibits growth and aggressiveness of nasopharyngeal carcinoma cells in vitro. (A) Growth of HK-1 cells following treatment with LEB or/and I-131 determined by MTT assay. (B) Migration (magnification, ×20) and (C) invasion (magnification, ×20) of HK-1 cells following treatment with LEB or/and I-131 determined via aggression assays. (D) Expression levels of MTA-1 and FIB in HK-1 cells following treatment with LEB or/and I-131 determined by western blotting. The data are presented as the mean + standard error of the mean of three independent experiments. **P<0.01. LEB, Lenvatinib; I-131, iodine-131; MTA-1, metastasis-associated protein; FIB, fibrinogen.

Figure 2.

Combination of LEB and I-131 promotes apoptosis of nasopharyngeal carcinoma cells and inhibits tumor angiogenesis-related protein in vitro. (A) Apoptosis rate of HK-1 cells following treatment with LEB or/and I-131 determined by flow cytometry. (B) Protein expression levels of Caspase-3 and Caspase-9 in HK-1 cells determined by western blotting. (C) Protein expression levels of Bcl-2 and P53 in HK-1 cells determined by western blotting. (D) VEGF and PDGF expression levels in HK-1 cells determined by western blotting. The data are presented as the mean + standard error of the mean of three independent experiments. *P<0.05, **P<0.01. LEB, Lenvatinib; I-131, iodine-131; Bcl-2, B cell lymphoma 2; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor.

Figure 3.

Combination of LEB and I-131 regulates apoptosis of nasopharyngeal carcinoma cells through ER stress. (A) ATF6, IER1, RERK and CHOP expression levels in HK-1 cells following treatment with LEB or/and I-131 determined by western blotting. (B) Analysis of mRNA expression levels of ATF6, IER1, RERK and CHOP expression levels in HK-1 cells. (C) Effects of LEB or/and I-131 on ER in HK-1 cells (magnification, ×100). (D) GRP78 expression in HK-1 cells following combined treatment of LEB and I-131 determined by immunofluorescence (magnification, ×20). (E) Adhered morphology of HK-1 cells following treatment with LEB or/and I-131 (magnification, ×20). (F) JNK and P38 expression levels in HK-1 following treatment with LEB or/and I-131. The data are presented as the mean + standard error of the mean of three independent experiments. **P<0.01. LEB, Lenvatinib; I-131, iodine-131; ATF6, activating transcription factor 6; IER1, inositol-requiring protein 1; RERK, protein kinase RNA-like endoplasmic reticulum kinase; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein 78; JNK, c-jun N-terminal kinase.

Figure 4.

In vivo effects of combined treatment of LEB and I-131 in an NPC xenograft mice model. (A) Effects of combined treatment of LEB and I-131 on tumor growth in NPC xenograft mice model. (B) Apoptotic bodies in tumors following treatment with LEB or/and I-131 determined by terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay (magnification, ×20). (C) JNK and Caspase-3 expression levels in tumors following treatment with LEB or/and I-131 determined by immunohistochemistry (magnification, ×20). (D) Expression levels of TNF-α and HIF-1α in tumors following treatment with LEB or/and I-131 determined by immunohistochemistry (magnification, ×20). (E) Angiogenesis and VEGF expression in tumors following treatment with LEB or/and I-131 determined by hematoxylin and eosin staining (magnification, ×20). **P<0.01. LEB, Lenvatinib; I-131, iodine-131; NPC, nasopharyngeal carcinoma; JNK, c-jun N-terminal kinase; TNF-α, tumor necrosis factor-α; HIF-1α, hypoxia-inducible factor-1α; VEGF, vascular endothelial growth factor.