Small molecule RL71 targets SERCA2 at a novel site in the treatment of human colorectal cancer

Abstract

While targeted agents are an important part of the treatment arsenal for colorectal cancer, there is still a lack of efficient small-molecule targeted agents based on the understanding of pathogenic molecular mechanisms. In this study, curcumin analog RL71 displayed potent cytotoxicity towards human colon cancer cells with an IC50 of 0.8 µM in SW480 cells and inhibited xenotransplanted tumor growth in a dose-dependent manner. Using affinity chromatography, we identified sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2 as the binding target of RL71. Most notably, RL71 demonstrated special binding to SERCA2 at a novel site with the lowest estimated free energy -8.89 kcal mol(-1), and the SERCA2 residues critical for RL71 binding were identified. RL71 suppressed the Ca(2+)-ATPase activity of SERCA2 both in vitro and in vivo, accompanied by the induction of endoplasmic reticulum stress leading to apoptosis and G2/M cycle arrest in SW480 cells. In addition, RL71 showed synergistic cytotoxicity with the pan-SERCA inhibitor thapsigargin. These results suggest that RL71 could be a selective small-molecule inhibitor of SERCA2, and that it may serve as a lead compound for the study of targeted colorectal cancer therapy.

Keywords: RL71; SERCA2; colorectal cancer; novel binding site; targeted agent.

Figure 1. RL71 inhibits cell viability in human colon cancer cells

A. Chemical structures of RL71 (1) and curcumin (2). B. Dose-dependent cytotoxicity of RL71 in human colon cancer cell lines. Cell viability was determined by MTT assay after a 48 h treatment. Curcumin (Cur) was used as a control. C. Time-dependent cytotoxicity of RL71 (1 μM) in SW480 and HCT116 cells. Data are the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group without any treatment.

Figure 2. RL71 targets the SERCA2 protein

A. Cytotoxicity of RL71 analogs (3-6) in SW480 cells. The concentrations that cause a 50% inhibition (IC₅₀) of cell viability are shown. B. Chemical structure of biotinylated RL71 (RL71-biotin, 7). C. The effects of RL71 and RL71-biotin on SW480 cell viability. Cell viability was determined by MTT assay after a 24 h treatment. Data are the mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.001 versus the control group without any treatment. D. Purification of RL71-binding proteins by affinity chromatography with 7-conjugated beads, which were incubated with the whole lysates of SW480 cells. Bound proteins were separated on an SDS gel and visualized by silver staining. E. Western blot analysis using the anti-SERCA2 antibody and the eluted proteins from D.. F. The co-localization of RL71 and SERCA2 in SW480 cells. The cells were treated with 10 μM of RL71 for 2 h and stained with an anti-SERCA2 antibody. Confocal microscopy was performed after a 2 h incubation. G. Molecular docking analysis of RL71 and SERCA2. H. The binding affinity for RL71-biotin of the double mutants within the binding site. The whole lysates of the HEK293 cells overexpressing FLAG-tagged SERCA2b (WT) or its mutants were incubated with RL71-biotin and streptavidin beads. The bound fractions were separated by SDS-PAGE and analyzed by western blotting.

Figure 3. RL71 displays SERCA2-inhibiting activity and facilitates apoptosis by inducing ER stress in SW480 cells

A. The dose-dependent inhibitory effect of RL71 on Ca²⁺-ATPase activity. The cells were incubated with the indicated concentrations of RL71 or Cur for 24 h. Then, the Ca²⁺-ATPase activity was measured according to the instructions of the Ca²⁺-ATPase kit provided by Nanjing Jiancheng Bioengineering Institute. B. The effects of the indicated compounds on Ca²⁺-ATPase activity. C. The increase of intracellular Ca²⁺ concentrations after RL71 treatment. Fura-2/AM loaded cells were stimulated with or without 2 μM of RL71. The y-axes represent the percentage of intracellular Ca²⁺ concentration. The x-axes depict the time in seconds, with time 0 representing the time of RL71 addition. The data are representative of at least 3 experiments. D. Changes of [Ca²⁺]_i after pretreatment with vehicle or with TG (5 μM), followed by stimulation with 2 μM of RL71. The data are representative of at least 3 experiments. E. The protein levels of ubiquitin-linked proteins, GRP78, ATF4, CHOP and PARP. Cells were treated with the indicated concentrations of RL71 or Cur for 24 h. F. The effects of SERCA2 overexpression on GRP78, CHOP and PARP expression. Cells were transiently transfected with pcDNA3.1 or SERCA2b expression plasmids. After 24 h, the cells were incubated with 2 μM of RL71 for another 24 h. G. The effects of SERCA2 knockdown on GRP78, CHOP and PARP expression (left panel) and cell viability (right panel). Cells stably expressing control virus or sh-SERCA2 virus were incubated with 2 μM of RL71 for 24 h. The protein levels were measured by western blot. The cell survival rate was determined by MTT assay. The data are mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. All of the western blot analyses are representative of at least 3 experiments.

Figure 4. Binding of RL71 at a novel site contributes to ER stress-associated apoptosis

A. The effects of SERCA2 mutants within the binding site on GRP78 and CHOP expression. The HEK293 cells were transiently transfected with FLAG-tagged SERCA2b or its mutant expression plasmids. After 24 h, the cells were incubated with 2 μM of RL71 for another 24 h. The data are representative of at least 3 experiments. B. The effects of the SERCA2 mutants on intracellular Ca²⁺ concentrations. Transfected HEK293 cells were loaded with Fura-2/AM and then stimulated with or without 2 μM of RL71. Increasing percentages of intracellular Ca²⁺ concentrations were monitored. The data are representative of at least 3 experiments. C. Synergistic cytotoxicity of RL71 with thapsigargin in SW480 cells. RL71 and thapsigargin (TG) were evaluated in a cell viability assay individually and in combination at the indicated concentrations. Cell viability was determined by MTT assay after a 48 h treatment. The data are the mean ± SEM of three independent experiments.

Figure 5. RL71 induces G2/M cell cycle arrest via ER stress in SW480 cells

A. G2/M cell cycle arrest in RL71-treated SW480 cells. Cells were incubated with indicated concentrations of RL71 or Cur for 24 h. Propidium iodide staining and flow cytometry were used to determine the proportion of cells in various phases of the cell cycle. B. The effects of SERCA2 overexpression on cell cycle arrest. SW480 cells were transiently transfected with pcDNA3.1 or SERCA2b expression plasmids. After 24 h, the cells were incubated with 2 μM of RL71 for another 24 h. C. The protein levels of the full-length p53 (p53FL) and the p53 isoform p53/47. Cells were incubated with 2 μM of RL71 or 1 μM of TG for 24 h. The data are representative of at least 3 experiments. D. Schematic representation of the mechanism by which RL71 affects ER stress-associated signaling events by targeting SERCA2.

Figure 6. RL71 suppresses the growth of SW480 cells in nude mice via inhibition of SERCA2 activity

A. Tumor volumes. SW480 cells were injected subcutaneously into the right flank of nude mice. Two weeks later, the tumor-bearing mice were distributed into 4 groups and treated with various doses of RL71 or olive oil for 14 additional days. The tumor volumes were monitored and recorded every two days (n = 8-10 mice per group). B. Tumor weight on day 15 of treatment. C. Survival curve of mice (n = 14 mice per group). D. Body weight. E. Weight of liver and spleen on day 15. F. The Ca²⁺-ATPase activity of tumor samples from RL71-treated mice at 2 mg/kg and olive oil-treated controls. The tumor tissues were excised on day 15 and measured according to the instructions of the Ca²⁺-ATPase kit. G. Protein levels of PARP, cleaved caspase-3 and CHOP in tumor samples. The tumor tissues were excised on day 15 and analyzed by western blot. H. Tumor tissues stained with H&E (left), TUNEL reagents (middle) or an antibody specific for CD31 (right). The tumor tissues were excised on day 15. The data shown are representative of three experiments. Necrotic tumor cells (Nec) and apoptotic condensed nuclei (Apo) are indicated with arrows. Original magnification, ×100. The data are the mean ± SEM of 8-10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus the olive oil group.