Gambogic acid reverses oxaliplatin resistance in colorectal cancer by increasing intracellular platinum levels

Abstract

Resistance to oxaliplatin (L-OHP) is a major obstacle to successful chemotherapy in colorectal cancer (CRC). In the present study, the ability of gambogic acid (GA) to reverse L-OHP resistance in CRC LoVo cells was investigated. L-OHP-resistant LoVo/L-OHP cells were established by exposing them to increasing concentrations of L-OHP. GA-reversed L-OHP-sensitive LoVo/L-OHP/GA cells were established by exposure to 0.5 µmol/l GA for 2 weeks. A Cell Counting Kit-8 assay was used to assess levels of proliferation. Flow cytometry was applied to detect apoptosis rates. Transwell assays were used to analyse invasion. Inductively coupled plasma mass spectrometry was used to determine intracellular platinum (Pt) content. Western blot analysis was used to reveal the protein levels of Human copper transporter 1 (hCTR1), Copper-transporting p-type adenosine triphosphatases 1 (ATP7A) and Copper-transporting p-type adenosine triphosphatases 2 (ATP7B). LoVo/L-OHP and LoVo/L-OHP/GA cell lines were successfully established, and it was identified that L-OHP inhibited the proliferation of LoVo, LoVo/L-OHP and LoVo/L-OHP/GA cells in a dose-dependent manner. Compared with the parent LoVo cells, the anti-apoptosis and invasion properties of LoVo/L-OHP cells were enhanced, and were reversed by GA treatment. Intracellular Pt content was highest in the LoVo cells, followed by LoVo/L-OHP/GA cells, and then lowest in the LoVo/L-OHP cells. Downregulated hCTP1 and upregulated ATP7A and ATP7B were associated with L-OHP resistance, and GA reversed the resistance by increasing levels of hCTR1 and decreasing levels of ATP7A and ATP7B. In conclusion, GA has the potential ability to reverse L-OHP resistance in CRC cells by increasing intracellular Pt content, which it achieves by increasing hCTR1 levels and decreasing ATP7A and ATP7B levels. GA may represent a promising treatment agent for L-OHP resistance.

Keywords: colorectal cancer; gambogic acid; oxaliplatin; resistance.

Figure 1.

L-OHP inhibits the proliferation of LoVo, LoVo/L-OHP and LoVo/L-OHP/GA cells. (A) Survival rates of LoVo, LoVo/L-OHP and LoVo/L-OHP/GA cells in the presence of L-OHP. **P<0.01, ^#P<0.05 and ^##P<0.01 vs. LoVo cells. (B) L-OHP IC₅₀ of LoVo, LoVo/L-OHP and LoVo/L-OHP/GA cells at different times. L-OHP, oxaliplatin; GA, gambogic acid; IC₅₀, half maximal inhibitory concentration; RI, resistance index; d, days.

Figure 2.

Morphological changes of LoVo/L-OHP and LoVo/L-OHP/GA cells captured using inverted microscopy. L-OHP, oxaliplatin; GA, gambogic acid.

Figure 3.

Apoptosis rates of LoVo, LoVo/L-OHP and LoVo/L-OHP/GA cells in the presence of L-OHP (20 µmol/l for 6 h), *P<0.05 and **P<0.01. L-OHP, oxaliplatin; GA, gambogic acid; PI, propidium iodide.

Figure 4.

Invasion by LoVo, LoVo/L-OHP and LoVo/L-OHP/GA cells in the presence of L-OHP (2 µmol/l for 24 h), visualised using haematoxylin staining by light microscopy (magnification, −400). Numbers of cells represent the invasive abilities of LoVo, LoVo/L-OHP and LoVo/L-OHP/GA cells. **P<0.01. L-OHP, oxaliplatin; GA, gambogic acid.

Figure 5.

Intracellular Pt levels of LoVo, LoVo/L-OHP and LoVo/L-OHP/GA cells in the presence of L-OHP. (A) Different concentrations (0.5, 1, 2 and 4 µmol) of L-OHP for 4 h. (B) A total of 2 µmol/l L-OHP at different time intervals (1, 4, 12 and 24 h). *P<0.05 and **P<0.01. Pt, platinum; L-OHP, oxaliplatin; GA, gambogic acid.

Figure 6.

Western blotting reveals protein levels of hCTR1, ATP7A and ATP7B in LoVo, LoVo/L-OHP and LoVo/L-OHP/GA cells. **P<0.01. GA, gambogic acid; L-OHP, oxaliplatin; hCTR1, human copper transporter 1; ATP7A, copper-transporting p-type adenosine triphosphatases 1; ATP7B, copper-transporting p-type adenosine triphosphatases 2.