Norcantharidin Inhibits cell growth by suppressing the expression and phosphorylation of both EGFR and c-Met in human colon cancer cells
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Norcantharidin (NCTD) is a Chinese FDA approved, chemically synthesized drug for cancer treatment. The effect of NCTD on signaling proteins of EGFR and c-Met was systematically elucidated in current study.
Two human colon cancer cell lines, HCT116 and HT29, were used as model systems to investigate the anti-cancer molecular mechanism of NCTD. Cell cycle arrest and early/late apoptosis were analyzed by flow cytometry. The levels of EGFR, phospho-EGFR, c-Met, phospho-c-Met and other related proteins were quantified by western blot analysis.
NCTD induced cell cycle arrest at G2/M phase in both cell lines. The early and late apoptosis was also observed. Further investigation indicated that NCTD suppressed not only the expression of the total EGFR and the phosphorylated EGFR but also the expression of the total c-Met and the phosphorylated c-Met in colon cancer cells. Moreover, EGFR expression could be mostly restored by co-treatment with MG132, a proteasome inhibitor. In addition, NCTD-induced cell death was comparable to that of the anti-cancer drug gefitinib, a tyrosine kinase inhibitor for EGFR, based on the immunoblot analysis of the expressed proteins after the drug treatment.
NCTD might be a useful and inexpensive drug candidate to substitute for gefitinib to serve the treatment needs of cancer patients.
KeywordsNorcantharidin Colon cancer Cell cycle Apoptosis EGFR c-Met
Relative fluorescence unit
Numerous studies have indicated that NCTD possessed the abilities to induce cell death for multiple types of cancer cells, for example, colorectal and lung cancer cells. The anti-cancer activity of NCTD relies on its ability in activation of the caspase signaling pathway that leads to apoptosis  accompanied with a decreased ratio of Bcl-2/Bax [8, 9], the changes in the expression of cell cycle-related proteins are co-related with cell cycle arrest , the interruption of DNA synthesis , inhibition of tumor invasion and metastasis [12, 13], MAPK activation, and protein kinase C pathway activation .
Colon cancer is the fourth most common cancer in the world found in both men and women. It is well known that two cell surface receptor tyrosine kinases, c-Met and EGFR, are co-present in 78%  to 80% [16, 17] in colon cancer cells. EGFR and c-Met corroborate the downstream signaling pathways in cancer cells in that c-Met and EGFR activate many of the same downstream signaling molecules. c-Met signaling is mainly responsible for cancer cell survival when EGFR inhibitors, such as gefitinib, are used during cancer treatment . So far, few c-Met inhibitors or c-Met pathway antagonists have been developed into drugs. Even though several novel compounds are in clinical trials, their potential use might be limited by their potency, pharmacokinetic defect, and safety profile . Therefore, it is highly desirable to find dual inhibitor for both EGFR and c-Met, especially from the existing drugs, to facilitate colon cancer treatment.
In current study, we demonstrated that NCTD suppressed the expressing level and phosphorylation of both c-Met and EGFR. Moreover, NCTD-induced cell death was comparable to that of the anti-cancer drug gefitinib, a tyrosine kinase inhibitor for EGFR, based on the immunoblot analysis of the expressed proteins after the treatment of NCTD.
Cell-related experiment and NCTD treatment
HT29 and HCT116, two human colon cancer cell lines, were obtained, maintained, and passaged using the identical protocol described in the published report . NCTD was obtained from National Institutes for Food and Drug Control (Shandong, China). NCTD was dissolved in dimethylsulfoxide (DMSO) and the final concentration of DMSO used was 0.1% in culture media for all experiments.
Cell growth inhibition assay
HT29 and HCT116 cells suspended in complete media were seeded in 96-well plates (2000 cells/well). After growing for 24 h in incubator, media were aspirated and 0.2 mL complete media containing serial concentrations of NCTD were added to each well. After incubating the plate for 24, 48, or 72 h, 20 μL of resazurin (2 mg/mL dissolved in water, Sigma) was added to each well. The fluorescent signal was monitored using 544 nm excitation and 595 nm emission wavelengths by Spectramax M5 plate reader (Molecular Devices) after incubation at 37 °C for 16 h in the incubator. The number of living cells in each well was proportional to the relative fluorescence unit (RFU) measured by the assay .
Cell cycle analyses
HCT116 and HT29 cells were added in 6-well plates with 6 × 104 cells/well with 2 mL complte media per well. After incubation for 24 h, different concentrations of NCTD in 2 mL of complete media was added to treat cells. After 24 h incubation, the cell pellets were collected using the published protocol  and resuspended in 1 mL of 70% ethanol at 4 °C overnight. For analysis, the supernatant was removed by centrifuging at 1600 g for 1 min. The cell pellets were incubated with 0.5 mL PI/RNase Staining Buffer (BD Biosciences) for 15 min at room temperature. Single-cell suspension was generated by gentle pipetting. The cells was analyzed using Beckman cell analyzer FC500-mpl. Beckman CXP software and Multicycle software were used for data analysis.
Annexin V/propidium iodide (PI) double staining assay (BD Biosciences, San Diego CA, USA) was used to quantify the apoptotic cells by following the manufacturer’s instruction . Briefly, HCT116 and HT29 cells were treated with different concentration of NCTD in complete cell culture media for 24 h. After removing NCTD-containing media, cells were incubated in 100 μL buffer containing Annexin V and PI at room temperature for 15 min. Beckman cell analyzer FC500-mpl was then used to analyze the apoptotic cells.
Western blot analysis
HCT116 and HT29 cells were cultured in 10-cm dishes for 24 h before treated with different concentrations of NCTD or NCTD plus MG132 (Beyotime biotechnology, China). After being incubated with another 72 h, the cells were collected with cell-scrapers into 1.5 mL tube and put on ice for 30 min in whole cell lysis buffer (Cell Signaling Technology, USA) containing protease inhibitors. Proteins were quantified by BCA protein assay kit. Equal amount of proteins from different sample (50 μg) were resolved over 8 or 12% SDS-polyacrylamide gel by electrophoresis and then transferred to nitrocellulose membrane. The membranes were put in blocking buffer at room temperature for 2 h before appropriate primary antibody was added. The membrane was incubated in the presence of primary antibody at 4 °C overnight before the corresponding secondary antibodies were added. The membranes were then visualized by using Western Lightning. Antibodies for EGFR, Phospho-EGFR (Tyr1068), Her-2, Phospho-Her2/ErbB2 (Tyr1248), c-Met, Phospho-c-Met (Tyr1003), Caspase-3, Rb, β-Actin, PARP, CDK-2, CyclinD1, and Bax were purchased from Cell Signaling Technology (USA).
NCTD inhibited HCT116 and HT29 cell proliferation
NCTD suppressed the expression and activation of c-Met and EGFR in HCT116 and HT29 cells
To evaluate the potency of NCTD in killing colon cancer cells, we used gefinitib, the EGFR inhibitor, as the positive control. We compared the effect of gefinitib on attenuating the expression of the same group proteins, i.e., EGFR, Her-2 and c-Met, at two concentrations of 25 μM and 50 μM, respectively. Based on the protein band intensities, we found that NCTD exhibited stronger inhibitory effect on phosphorylation of c-Met, EGFR, and Her-2 at the concentration tested despite the expression of phosphor-Her-2 at 50 μM. Altogether, we concluded that NCTD was comparable to gefinitib in attenuating colon cancer cell growth (Fig. 3c).
In a time-dependent study, HCT116 cells were treated by NCTD at 100 μM for a period up to 72 h. The same group of proteins including c-Met, EGFR, Her-2, were analyzed again by western blot analysis. Decrease of total and phospho-c-Met, EGFR, Her-2 occurred in the 6 to 24 h and persisted for up to 72 h (Fig. 3d) by NCTD treatment.
The mechanisim of the downregulation of EGFR induced by NCTD in HCT116 cell was further invesitigated by co-treatment in combination with MG132, a proteasome inhibitor. As showed in Fig. 3e, NCTD induced 50% decrease in EGFR expression while NCTD plus MG132 induced 20% decrease in EGFR which indicated NCTD inhibited EGFR expression mainly via proteasome-mediated degradation.
G2/M cell cycle arrest induced by NCTD
NCTD induced early/late apoptosis
NCTD affected cell cycle- and apoptosis-related proteins
Accumulating evidences indicated that both c-Met and EGFR were overexpressed by 78 to 80% of colon cancers, which were associated with poor outcome. A cross-talk of c-Met and EGFR could modulate reciprocally and eventually determine the intensity of c-Met signaling pathway . One of the major findings of our study was that the mechanism underlying the cell death induced by NCTD involved in suppressing the expression and phosphorylation of c-Met and EGFR. To our knowledge, this is the first demonstration that NCTD was a dual inhibitor for c-Met and EGFR and in human colon cancers.
Another interesting finding was that signaling network might also exist between c-Met and Her-2 in colon cancer cells where the level of the two proteins decreased with the increased concentration of NCTD. However, we could not draw a conclusion how c-Met affected the expression and activation of Her-2, such as by direct suppression or indirect regulation. Additional studies were necessary to reveal the potential mechanism how c-Met downregulated Her-2 expression in colon cancer cells line.
In conclusion, NCTD suppressed the phosphorylation and expression of both EGFR and c-Met in HCT116 and HT29 human colon cancer cells. Our data provided novle molecular mechanism for further investigation if NCTD could serve as a dual inhibitor for EGFR/c-Met in terms of colon cancer treatment.
We thank Dr. Mingzhuang Zhu for helping us with the flow cytometry analysis.
This research was supported by Shandong Science and Technology Development Planning Project (Grant No.2014GGH215001), Ministry of Education New Teacher’s Fund(Grant No. 20130132120006). Natural Science Foundation of China (Grant No. 91129706 and 81672585), and NSFC-Shandong Joint Fund (U1406402).
Availability of data and materials
All data supporting the findings in this study are included within the manuscript and the supplementary figures.
LZ, PQ designed the experiments. SW, PQ, ML, MZ, LX, YC, HM, YT, and YH performed the experiments. PQ, SW, XZ, HX analyzed data. LZ, PQ wrote the manuscript. LZ and PQ revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
There was no research involving human or animal subjects in the study.
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