Morphine Suppresses Liver Cancer Cell Tumor Properties In Vitro and In Vivo

Abstract

Morphine is an analgesic widely adopted to relieve cancer pain. A number of discrepancies, however, are presented by the published literature, with reports suggesting that opioids may either promote or inhibit the spread of cancer. It is of great significance to determine whether morphine may increase the risk of metastasis while utilized in liver cancer surgical treatment. In this study, we explore the effects of morphine on liver cancer cells in vitro and in vivo. Our results showed that morphine does not promote proliferative ability to cultured liver cancer cells. While morphine could increase the apoptosis rate of Hep3B/HepG2 cells. Furthermore, morphine could significantly inhibit the migratory and invasion ability of Hep3B/HepG2 cells. Subsequent investigations disclosed that morphine could inhibit sphere formation ability of Hep3B/HepG2 cells by using sphere formation assay. Based on nude mouse models, we demonstrated that morphine significantly reduced pulmonary tumorigenicity of Hep3B/HepG2 cells. In conclusion, our results found that morphine at clinical concentrations could suppress liver cancer cell tumor properties in vitro and in vivo, indicating the safety of morphine utilization in HCC patients' pain management.

Keywords: liver cancer; metastasis; morphine; opioids; tumorigenicity.

Figure 1

Morphine does not promote proliferative ability to cultured liver cancer cells. Hep3B/HepG2 cells were pretreated with morphine at the concentration of 0, 5, 10μM for 24h, 48h and 72h. Cell viabilities were measured by CCK-8 assay. Data are expressed as mean ± SE; n=3.

Figure 2

Morphine promotes the apoptosis of Hep3B/HepG2 cells in vitro. (A) Representative image of flow cytometry analysis of Annexin V and Propidium iodide as a measure of apoptosis and (B) quantitative analysis of apoptosis in Hep3B/HepG2 cells exposed to morphine for 24h. Data are expressed as mean ± SE; n=3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Morphine suppresses the migration and invasion ability of Hep3B/HepG2 in vitro. (A) Representative image of wound healing assay and (B) quantitative analysis of wound healing assay of Hep3B/HepG2 exposed to morphine for 24h. (C) Representative image of transwell analysis of migration and (D) quantitative analysis of migration of Hep3B/HepG2 exposed to morphine for 24h. (E) Representative image of transwell analysis of invasion and (F) quantitative analysis of invasion of Hep3B/HepG2 exposed to morphine for 24h. Data are expressed as mean ± SE; n=3. ***P < 0.001.

Figure 4

Morphine inhibits the sphere formation ability of Hep3B/HepG2 cells. (A) Representative pictures of spheres formed by Hep3B/HepG2 cells after treating with morphine (0, 5, 10μM) for 24h, respectively (Scale bars, 50μm). (B, C) Bar diagrams showed the diameter and number of spheres (spheres > 50μm). Data are expressed as mean ± SE; n=3. ***P < 0.001.

Figure 5

Morphine inhibits lung tumorigenicity of Hep3B/HepG2 cells in vivo. (A) Luminescence of nude mice that were tail vein injected with Hep3B cells at the initiation time point and 5 weeks after. (B) Lung sections of nude mice that were tail vein injected with Hep3B cells for H&E staining (scaled bar length = 500μm). (C) Relative quantification of luminescence of nude mice that were tail vein injected with Hep3B cells was showed. (D) Luminescence of nude mice that were tail vein injected with HepG2 cells at the initiation time point and 5 weeks after. (E) Lung sections of nude mice that were tail vein injected with HepG2 cells for H&E staining (scaled bar length = 500μm). (F) Relative quantification of luminescence of nude mice that were tail vein injected with HepG2 cells was showed. Data were shown as mean ± SD. n=9 per group. **P < 0.01.

Figure 6

Liver cancer cells were pretreated with different concentrations of morphine (0,5,10μM) for 24h followed by Western Blot to detect the protein level of OGFR, MOR, uPA and MMP-9 in Hep3B/HepG2 cells. n=3.

Figure 7

Liver cancer cells were pretreated with different concentrations of morphine (0,5,10μM) for 24h followed by qRT-PCR to detect the mRNA level of OGFR, MOR, uPA and MMP-9 in Hep3B/HepG2 cells. Data are expressed as mean ± SE; n=3. **P < 0.01, ***P < 0.001.