5-Methoxytryptophan enhances the sensitivity of sorafenib on the inhibition of proliferation and metastasis for lung cancer cells

Abstract

Background: Lung cancer is a leading cause of cancer-related mortality worldwide, and effective therapies are limited. Lung cancer is a leading cause of cancer-related mortality worldwide with limited effective therapy. Sorafenib is a multi-tyrosine kinase inhibitor frequently used to treat numerous types of malignant tumors. However, it has been demonstrated that sorafenib showed moderate antitumor activity and is associated with several side effects in lung cancer, which restricted its clinical application. This study aimed to examine the antitumor effect of the combination treatment of sorafenib and 5-methoxytryptophan (5-MTP) on cell growth and metastasis of Lewis lung carcinoma (LLC) cells.

Method: The anticancer effect of the combination treatment of sorafenib and 5-MTP was determined through cytotoxicity assay and colony forming assays. The mechanism was elucidated using flow cytometry and western blotting. Wound healing and Transwell assays were conducted to evaluate the impact of the combination treatment on migration and invasion abilities. An in vivo model was employed to analyze the effect of the combination treatment on the tumorigenic ability of LLC cells.

Result: Our results demonstrated that the sorafenib and 5-MTP combination synergistically reduced viability and proliferation compared to sorafenib or 5-MTP treatment alone. Reduction of cyclin D1 expression was observed in the sorafenib alone or combination treatments, leading to cell cycle arrest. Furthermore, the sorafenib-5-MTP combination significantly increased the inhibitory effect on migration and invasion of LLC cells compared to the single treatments. The combination also significantly downregulated vimentin and MMP9 levels, contributing to the inhibition of metastasis. The reduction of phosphorylated Akt and STAT3 expression may further contribute to the inhibitory effect on proliferation and metastasis. In vivo, the sorafenib-5-MTP combination further reduced tumor growth and metastasis compared to the treatment of sorafenib alone.

Conclusions: In conclusion, our data indicate that 5-MTP sensitizes the antitumor activity of sorafenib in LLC cells in vitro and in vivo, suggesting that sorafenib-5-MTP has the potential to serve as a therapeutic option for patients with lung cancer.

Keywords: 5-methoxytryptophan; Lung cancer; Lung metastasis; Oncogenesis; Sorafenib.

Fig. 1

Reduction effect of sorafenib and 5-methoxytryptophan (5-MTP) on the cell viability and proliferation of Lewis lung cancer (LLC) cells. The viability of LLC cells was reduced by sorafenib (A) or 5-MTP (B) treatments in cytotoxicity assay. The proliferation of LLC cells was inhibited by sorafenib (C) or 5-MTP (D) treatments in colony forming assay. Asterisks indicate a significant difference in drug-treated cells at indicated concentrations singly compared to DMSO-treated cells. *P < 0.05

Fig. 2

Synergistic inhibitory effect of the combination treatment of sorafenib and 5-MTP on the cell viability and proliferation of LLC cells. (A) Combination treatment showed a synergistic inhibitory effect on the growth of LLC cells compared to the single treatments in cytotoxicity assay. (B) The combination treatment induced the inhibition effect on cell proliferation. Asterisks indicate a significant difference in drug-treated cells at indicated concentrations singly compared to DMSO-treated cells. *P < 0.05

Fig. 3

Induction of cell cycle arrest by sorafenib or combination treatment of sorafenib and 5-MTP in LLC cells. (A) Sorafenib and combination treatment induced cell cycle arrest, as observed by DNA staining with Propidium iodide and flow cytometry in LLC cells. (B) Cell cycle arrest was confirmed by western blot analysis 24 h after Sorafenib, 5-MTP, or combination treatment in LLC cells. Membranes were incubated with the indicated antibodies against cell cycle regulator proteins. GAPDH was used as a control for equal loading. *P < 0.05

Fig. 4

Enhanced inhibitory effect of sorafenib by 5-MTP on cell migration and invasion measured by wound-healing and transwell assay. (A) Representative bright-field images show that 5-MTP enhanced the reduction effect of sorafenib on migration compared to single compound treatment in wound-healing assay. (B) 5-MTP enhanced the reduction effect of sorafenib on the number of migration cells. (C and E) Representative bright-field images showed the effect of 5-MTP and sorafenib on cell migration and invasion in a transwell assay. (D and F) Statistic results showed that 5-MTP enhanced the inhibitory effect on cell migration and invasion. Asterisks indicate a significant difference in drug-treated cells at indicated concentrations singly compared to DMSO-treated cells. *P < 0.05

Fig. 5

The effect of combination treatment of 5-TMP and sorafenib on the expression of EMT markers by mediating the expression of phosphorylated STAT3 and Akt. (A) Combination treatment of 5-MTP and sorafenib reduced the expression of vimentin and MMP9 and increased the E-cadherin level. (B) The level of phosphorylated STAT3 and Akt showed in western blotting. Asterisks indicate significant difference in drug-treated cells at indicated concentrations singly in comparison with DMSO-treated cells. *P < 0.05

Fig. 6

The inhibitory effect of sorafenib and combination treatment of sorafenib and 5-MTP on lung colony formation in vivo. (A) Morphological observation and digital outlines of representative tongues of the DMSO- or drug-treated group in the LLC mouse model. (B) hematoxylin–eosin staining of lung cancer with DMSO or drug treatment. *P < 0.05