Anti-Human EGFR Recombinant Antibody (Cetuximab) (CAT#: TAB-003)

Figure 1 Phosphoarray analysis of signaling responses to sequential treatments.

(A) HCA7 cells were subjected to different regimens containing 10 μg/ml cetuximab and/or 50 μM oxaliplatin: (1) control medium for 24 hours (white), (2) control medium for 24 hours followed by oxaliplatin for 1 hour (black), (3) cetuximab for 24 hours followed by oxaliplatin for 1 hour (red), or (4) oxaliplatin for 24 hours followed by cetuximab for 1 hour (green). Phosphorylation profiles were analyzed using Proteome Profiler Human Phospho-Kinase Array Kit and quantified by densitometry. Mean +/− range is shown for four selected proteins. All results are shown in Suppl. Fig. 3. (B) HCA7 cells were subjected to different regimens containing or not 10 μg/ml cetuximab and/or 50 μM oxaliplatin as indicated in the figure. The expression or phosphorylation of selected proteins was analyzed by Western blotting. Full-length blots are presented in Supplementary Fig. 8. (C) Apoptosis of HCA7 cells was analyzed by annexin V staining after treating the cells first with or without 200 μM of the STAT3 inhibitor S31-201 for 8 hours followed by 18 hour treatment with or without 10 μg/ml cetuximab.

Narvi, E., Vaparanta, K., Karrila, A., Chakroborty, D., Knuutila, S., Pulliainen, A.,... & Elenius, K. (2018). Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin. Scientific reports, 8(1), 16579.

Figure 2 Block result.

(A) Schematic representation of hypothetical cellular responses to the opposite sequences of cetuximab and oxaliplatin. Left side: When cetuximab is administered first, it promotes arrest in the G1 phase of the cell cycle and stimulates apoptosis by blocking EGFR-activated survival pathways. The subsequent administration of oxaliplatin (ox) is largely ineffective due to the G1-arrest. Right side: When oxaliplatin is administered first, it actively induces DNA damage provoking apoptosis, as well as DNA repair that partially compensates for the damage. In the absence of cetuximab, the cell cycle is not arrested at G1 allowing for full activity of oxaliplatin. However, oxaliplatin-induced DNA damage itself does eventually cause cell cycle arrest by activating checkpoints in the S and G2 phases (not indicated in the figure). When cetuximab is then added after oxaliplatin, apoptosis is enhanced compared to all other regimens, as the apoptotic effect of both drugs are active, and also, as cetuximab may be suppressing the DNA repair mechanisms caused by oxaliplatin-induced DNA damage. (B) HCA7, DLD-1 and RKO cells were arrested in G1 by 8 hour treatment with or without 3 µM of the CDK4/6 inhibitor abemaciclib followed by 18 hour treatment with or without 50 μM oxaliplatin.(C) Flow cytometry analysis of cell surface EGFR after 24 hour treatment with or without 10 μg/ml cetuximab or 50 μM oxaliplatin.

Narvi, E., Vaparanta, K., Karrila, A., Chakroborty, D., Knuutila, S., Pulliainen, A.,... & Elenius, K. (2018). Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin. Scientific reports, 8(1), 16579.

Figure 3 Sequence of administration determines the effect of the regimen including cetuximab and oxaliplatin.

(A) Representative MTT experiments of HCA7 (left panel) and DLD-1 (right panel) cells. Mean +/− SD is shown. (B) Oxaliplatin ED50 values of sequential treatments from 3–4 independent experiments with HCA7 and DLD-1 cells. (C) Oxaliplatin ED50 values of sequential treatments from single independent experiments with the seven indicated cell lines. (D) HCA7 (left panel) and DLD-1 (right panel) cells suspended in soft agar were (1) treated for 48 hours (for days 4 to 5 of each treatment cycle) with oxaliplatin alone (black curves), or (2) in simultaneous combination with cetuximab (grey curves), (3) treated first for 72 hours (days 1 to 3) with cetuximab followed by 48 hour (days 4 to 5) treatment with oxaliplatin (red curves), or (4) treated first for 48 hours (days 4 to 5) with oxaliplatin followed by 72 hour (days 6 to 8) treatment with cetuximab (green curves). The cycle was repeated three times every 21 days as indicated by black dots.(E) Nude mice carrying HT-29 cell xenografts were treated with i.p. injections of (1) vehicle alone on day 1 of each treatment cycle (white dots), (2) oxaliplatin alone on day 1 (black dots), (3) cetuximab on day 1 followed by oxaliplatin on day 2 (red dots), or (4) oxaliplatin on day 1 followed by cetuximab on day 2 (green dots). The cycle was repeated three times every seven days as indicated by black dots. The concentrations used for oxaliplatin and cetuximab were 10 mg/kg and 40 mg/kg, respectively.

Narvi, E., Vaparanta, K., Karrila, A., Chakroborty, D., Knuutila, S., Pulliainen, A.,... & Elenius, K. (2018). Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin. Scientific reports, 8(1), 16579.

Figure 4 Sequential administration of cetuximab after oxaliplatin reduces G1 arrest and enhances apoptosis.

Cell cycle (A) and apoptosis (B) analyses of HCA7 cells subjected to different regimens containing 10 μg/ml cetuximab and/or 50 μM oxaliplatin: (1) control medium for 24 hours (white), (2) cetuximab for 24 hours (blue), (3) oxaliplatin for 24 hours (black), (4) simultaneous combination of oxaliplatin and cetuximab for 24 hours (grey), or treated sequentially (5) first with cetuximab for 24 hours followed by oxaliplatin for 24 hours (red) or (6) first with oxaliplatin for 24 hours followed by cetuximab for 24 hours (green). Cell cycle was analyzed by PI staining (A) and apoptosis by annexin V staining (B). Mean +/− SD is shown.

Narvi, E., Vaparanta, K., Karrila, A., Chakroborty, D., Knuutila, S., Pulliainen, A.,... & Elenius, K. (2018). Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin. Scientific reports, 8(1), 16579.

Figure 5 Low concentrations of HT and cetuximab reduce cell growth in colorectal cancer.

HT-29 (A), and WiDr (D) cells were exposed to increasing HT concentrations in presence/absence of EGF (5 ng/ml) for 48 h. Cell viability values, reported as absorbance at 540 nm, were obtained by MTT assay. HT-29 (B), and WiDr (E) cells were exposed to increasing cetuximab concentrations in presence/absence of EGF (5 ng/ml). Values obtained as in A. Numbers represent mean ± DS of three experiments run in triplicate. HT-29 (C), and WiDr (F) cells were exposed to HT (10 μM) and/or cetuximab (1 μg/ml) in presence/absence of EGF (5 ng/ml) for 48 h. These concentrations were used throughout this work, unless otherwise noted. Values obtained as in A.

Terzuoli, E., Nannelli, G., Frosini, M., Giachetti, A., Ziche, M., & Donnini, S. (2017). Inhibition of cell cycle progression by the hydroxytyrosol–cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells. Oncotarget, 8(47), 83207.

Figure 6 Combination of low concentrations of HT and cetuximab reduces colony formation of colorectal cancer cells.

Colony formation capability of HT-29 (A), and WiDr (B) cells in response to HT (10 μM) and/or cetuximab (1μg/ml) in presence/absence of EGF (5 ng/ml). Colonies (>75 cells) with 50% efficiency were counted. Results are expressed as surviving factor (SF, see material and methods). ** P <0.01, *** P <0.001, vs. untreated cells. # P <0.05, ### P <0.001 vs. EGF-treated cells.

Terzuoli, E., Nannelli, G., Frosini, M., Giachetti, A., Ziche, M., & Donnini, S. (2017). Inhibition of cell cycle progression by the hydroxytyrosol–cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells. Oncotarget, 8(47), 83207.

Figure 7 EGFR expression in colorectal cancer cells treated with HT and cetuximab alone or combined.

HT-29 (A) and WiDr (B) cells were exposed to low concentration of HT or cetuximab alone or in combination for 8 h and EGFR proteins was analyzed by western blot. Images of immunostaining in HT-29 (C) and WiDr (D) for EGFR (green) and DAPI (blue) in tumor cells exposed to 10 % FBS (a), cetuximab 1 μg/ml (b), HT 10 μM (c), cetuximab 1 μg/ml + HT 10 μM (d), cetuximab 10 μg/ml (e), HT 100 μM (f). Confocal images were captured with Leica SP5 confocal using 63x objective, scale bars 20 μm. HT-29 (E) and WiDr (F) cells were exposed to high concentration of HT or cetuximab for 8 h and EGFR proteins were analyzed by western blot. β-actin has been used to normalized loading (in Figures 3 and 7).

Terzuoli, E., Nannelli, G., Frosini, M., Giachetti, A., Ziche, M., & Donnini, S. (2017). Inhibition of cell cycle progression by the hydroxytyrosol–cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells. Oncotarget, 8(47), 83207.

Figure 8 Cell cycle analysis in cancer cells treated with low concentration of HT and cetuximab combined.

HT-29 (A), and WiDr (B) cells were exposed to HT or cetuximab alone or in combination in presence or absence of EGF for 48 h. The percentage of cells at each stage of the cell cycle was analyzed by flow cytometry after DNA staining with propidium iodide. Quantification of cells residing in G0 and G1 for HT-29 (C), and WiDr (D) are reported. Percent of HT-29- (C), and WiDr-cells (D) in sub Go/G1 phase.

Terzuoli, E., Nannelli, G., Frosini, M., Giachetti, A., Ziche, M., & Donnini, S. (2017). Inhibition of cell cycle progression by the hydroxytyrosol–cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells. Oncotarget, 8(47), 83207.

Figure 9 HT and cetuximab combination modulate the cell cycle checkpoint proteins in colorectal cancer cells.

HT-29 (A, B, C), and WiDr (D, E, F) cells were exposed to HT or cetuximab, alone or in combination in presence of EGF for 48 h and the cell cycle checkpoint proteins were analyzed by western blot.

Terzuoli, E., Nannelli, G., Frosini, M., Giachetti, A., Ziche, M., & Donnini, S. (2017). Inhibition of cell cycle progression by the hydroxytyrosol–cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells. Oncotarget, 8(47), 83207.

Figure 10 HT and cetuximab combination induces caspace3-independent apoptosis in colorectal cancer cells.

Phosphatidylserine (red) and DAPI (blu) exposure, assessed by immunofluorescence, in HT-29 (A, C for quantification) and WiDr (B, D for quantification) cells treated for 48 h with 10% FBS (Ctr) (a), cetuximab 1 μg/ml (b), HT HT 10 μM (c) or cetuximab 1 μg/ml+ HT 10 μM. Confocal images were captured with Leica SP5 confocal using 40 x objective, scale bars 60 μm.*P<0.05; **P <0.01 vs. untreated cells; §P<0.05; §§P<0.01 vs. HT- or cetuximab-treated cells. HT-29 (E), and WiDr (F) cells were exposed to HT or cetuximab alone or in combination for 8 h and caspase-3 activity were analyzed by western blot.

Terzuoli, E., Nannelli, G., Frosini, M., Giachetti, A., Ziche, M., & Donnini, S. (2017). Inhibition of cell cycle progression by the hydroxytyrosol–cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells. Oncotarget, 8(47), 83207.

Figure 11 HT-cetuximab combination reduces side effects of cetuximab treatment in colorectal cancer.

CCD-18Co (A), differentiated CaCo2 (B) and HaCaT (C) cells were exposed to the indicated concentration of HT and cetuximab in presence/absence of EGF (5 ng/ml) for 48 h. Cell survival values, reported as absorbance at 540 nm, were obtained by MTT assay. *P<0.05; **P<0.01 vs. untreated cells; §P<0.05; §§P<0.01 vs. cetuximab 10 μg/ml alone; çP<0.05, ççP<0.01 vs EGF-treated cells; #P<0.05, ##P<0.01 vs cetuximab 10 μg/ml plus EGF-treated cells. CCD-18Co (D, upper panel) and differentiated CaCo2 (D, bottom panel) cells were exposed to HT or cetuximab alone or in combination for 8 h and EGFR proteins was analyzed by western blot. CCD-18Co (E) and differentiated CaCo2 (F) cells were exposed to cetuximab (10 μg/ml) alone for 8 h and EGFR proteins were analyzed by western blot. β-actin has been used to normalize loading.

Terzuoli, E., Nannelli, G., Frosini, M., Giachetti, A., Ziche, M., & Donnini, S. (2017). Inhibition of cell cycle progression by the hydroxytyrosol–cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells. Oncotarget, 8(47), 83207.

Figure 1 Anti-Human EGFR Antibody (TAB-003) in ELISA

ELISA analysis of TAB-003 was performed with EGFR Protein, Human, Recombinant (Isoform vIII, His Tag).
The secondary antibody: HRP-Anti-Human IgG (H+L)

Figure 2 Anti-Human EGFR Antibody (TAB-003) in WB

Western blot analysis of TAB-003 was performed with EGFR Protein, Human, Recombinant (Isoform vIII, His Tag).
TAB-003 incubation concentration: 2ng/μL.
The secondary antibody: HRP-Anti-Human IgG (H+L)
Lane 1: Reducing antigen (0.6μg)

Figure 3 Anti-Human EGFR Antibody (TAB-003) in DB

Dot blot analysis of TAB-003 was performed with EGFR Protein, Human, Recombinant (Isoform vIII, His Tag).
TAB-003 incubation concentration: 2ng/μL.
NC: Negative control PC: Positive control