J Korean Surg Soc.  2012 Jul;83(1):21-29. 10.4174/jkss.2012.83.1.21.

Characterization of biological responses of colorectal cancer cells to anticancer regimens

• Roh SA
• Choi EY
• Cho DH
• Yoon YS
• Kim TW
• Kim YS
• Kim JC

• KMID: 2212275
• DOI: http://doi.org/10.4174/jkss.2012.83.1.21

Abstract

PURPOSE
Identification of subgroups of patients who differ in their response to treatment could help to establish which of the best available chemotherapeutic options are best, based on biological activity. In metastatic colorectal cancer (CRC), novel molecular-targeted agents that act on pathways that regulate cell growth, the cell cycle, apoptosis, angiogenesis, and invasion are being developed. Here, we employed an in vitro chemosensitivity assay to evaluate the biological efficacy of conventional monotherapies and combination chemotherapy with targeted drugs.
METHODS
The chemosensitivities of 12 CRC cell lines to the established regimens FOLFOX (5-fluorouracil [5-FU] + leucovorin + oxaliplatin) and FOLFIRI (5-FU + leucovorin + irinotecan) and to therapy with these regimens in combination with the biologically targeted drugs bevacizumab or cetuximab were comparatively evaluated for their effects on apoptotic and autophagic cell death processes, angiogenesis, and invasion.
RESULTS
Each of the chemotherapeutic regimens promoted apoptotic cell death and invasion. All drug regimens caused significantly greater apoptotic cell death with activation of caspase-3 in SW480 cells compared to other cells, effects that were associated with a remarkable reduction in matrix metalloproteinase-9 activity. The FOLFOX regimen more effectively promoted apoptotic cell death, angiogenesis, and invasion than the FOLFIRI regimen. Combination therapy with FOLFOX/FOLFIRI regimen and bevacizumab produced a moderate angiogenesis-blocking effect in most cell lines.
CONCLUSION
The results validate our in vitro chemosensitivity assay, and suggest that it may be applied to help determine adequate regimens in individual CRC patients based on the biological characteristics of their tumors.

Keyword

Colorectal neoplasms; Chemotherapy; Pharmacological biomarkers

MeSH Terms

Antibodies, Monoclonal, Humanized
Antineoplastic Combined Chemotherapy Protocols
Apoptosis
Autophagy
Bevacizumab
Biomarkers, Pharmacological
Caspase 3
Cell Cycle
Cell Death
Cell Line
Cetuximab
Colorectal Neoplasms
Drug Therapy, Combination
Fluorouracil
Humans
Leucovorin
Matrix Metalloproteinase 9
Organoplatinum Compounds
Population Characteristics
Antibodies, Monoclonal, Humanized
Antineoplastic Combined Chemotherapy Protocols
Biomarkers, Pharmacological
Caspase 3
Fluorouracil
Leucovorin
Matrix Metalloproteinase 9
Organoplatinum Compounds

Figure

• 

  Fig. 1 Flowcytometric detection of apoptosis in the SW480 cell line. Cells treated with the respective drug regimens and untreated controls were dual-labeled with propidium iodide (PI) and Annexin V fluorescence and analyzed by flow cytometry. (A) Untreated control; (B) FOLFOX (FX), 5-fluorouracil (5-FU) + leucovorin + oxaliplatin; (C) FOLFOX + bevacizumab (FXB); (D) FOLFOX + cetuximab (FXC); (E) FOLFIRI (FR), 5-FU + leucovorin + irinotecan; (F) FOLFIRI + bevacizumab (FRB); (G) FOLFIRI + cetuximab (FRC). Dual-parameter dot-plot of fluorescein isothiocyanate-fluorescence (x-axis) versus PI-fluorescence (y-axis) showing fluorescence intensity (log scale). Lower left quadrants, live cells; lower right quadrants, apoptotic cells; upper left quadrants, necrotic cells; upper right quadrants, apoptotic and necrotic cells. The percentage of apoptotic cells is indicated on the plots. (H) Bar diagram showing the percentage of apoptotic cells after different treatments. The percentage of apoptotic cells increased after treatment with bevacizumab-containing regimens, but decreased after treatment with combinations containing cetuximab.

• 

  Fig. 2 Caspase-3 activities were analyzed by Western blot analysis. SW480 cells (A) and DLD-1 cells (B) were treated with FOLFOX (FX), FOLFOX + bevacizumab (FXB), FOLFOX + cetuximab (FXC), FOLFIRI (FR), FOLFIRI + bevacizumab (FRB), or FOLFIRI + cetuximab (FRC) for 48 hours. Harvested-cell lysates were analyzed by Western blotting with an anti-caspase-3 antibody. C, control; FOLFOX (FX), 5-fluorouracil (5-FU) + leucovorin + oxaliplatin; FOLFIRI (FR), 5-FU + leucovorin + irinotecan.

• 

  Fig. 3 Gelatinolytic matrix metalloproteinase (MMP) activity in the SW480 cell line detected by quantitative zymography. (A) Molecular markers indicate MMP-9 and MMP-2 as 92 and 62 kDa proteins, respectively. (B) Zymographic band densities were quantified by densitometry. For all panels, data are presented ± standard errors (error bars) of three independent experiments. C, control; FX, FOLFOX (5-fluorouracil [5-FU] + leucovorin + oxaliplatin); FXB, FOLFOX + bevacizumab; FXC, FOLFOX + cetuximab; FR, FOLFIRI (5-FU + leucovorin + irinotecan); FRB, FOLFIRI + bevacizumab; FRC, FOLFIRI + cetuximab.

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