Korean J Obstet Gynecol.  2010 Jan;53(1):43-52. 10.5468/kjog.2010.53.1.43.

Detection of chemosensitivity using K18-Asp(396) (M30) antibody in HeLa and OVCAR-3 cell lines treated with anticancer agents

• Song MK
• Park SH
• Kwack HS
• Ryu KS
• Han KT

• KMID: 2077959
• DOI: http://doi.org/10.5468/kjog.2010.53.1.43

Abstract

OBJECTIVE
The aim of this study was to detect the levels of M30-antigens as a biomarker of apoptosis in cells and their culture media after treatments with anticancer drugs as a preclinical study.
METHODS
After HeLa and OVCAR-3 cells were treated respectively with paclitaxel, cisplatin, and camptothecin, the harvested cells were stained sequentially with M30 monoclonal antibodies and propidium iodide (PI). Afterwards, they were analyzed using a FACScan flow cytometer and observed under an immunofluorescence microscope for M30-FITC immunofluorescences. Levels of M30 antigens were also detected in their culture media using M30-Apoptosense ELISA kit.
RESULTS
The levels of M30-FITC immunofluorescences were elevated in both cell lines after each drug treatments compared with those of control cells. The levels of M30 antigens detected by ELISA in media culturing each cell line treated with each of drugs were elevated compared with those of control cells.
CONCLUSION
This study suggests that M30-antigens representing chemotherapy induced apoptosis may be a useful biomarker for predicting and monitoring the response of neoadjuvant chemotherapy in patients with gynecologic cancers.

Keyword

Apoptosis; Cytokeratin 18-Asp(396) (M30); Flow cytometry; ELISA; HeLa; OVCAR-3

MeSH Terms

Antibodies, Monoclonal
Antineoplastic Agents
Apoptosis
Camptothecin
Cell Line
Cisplatin
Culture Media
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Fluorescent Antibody Technique
Humans
Paclitaxel
Propidium
Antibodies, Monoclonal
Antineoplastic Agents
Camptothecin
Cisplatin
Culture Media
Paclitaxel
Propidium

Figure

• 

  Fig. 1 DNA histograms (left) and linear diagrams (right) showing the changes of cell cycle phases and apoptotic populations (sub-G1 peaks) stained with propodium iodide (P1) and measured by FACScan flow cytometer in HeLa and OVCAR-3 cells. The blue peaks represent apoptotic populations (sub-G0G1 peaks). The first red peaks). The first red peaks represent the G0G1 phases of the cell cycles. The second red peaks represent G2M phases of cell cycles. The mid portions between two red peaks with diagonal lines represents S phases of cell cycles. (A) Untreated control cells, (B) cells treated with 1,000 nM of paclitaxel, (C) cells treated with 250 ng/mL of cisplatin, and (D) cells treated with 30 nM camptothecin. (E) and (F) The linear diagrams showing distributions of cell cycle phases and apoptotic sub-G0G1 peaks in HeLa and OVCAR-3 cells teated with 1,000 nM of paclitaxel, 250 ng/mL of cisplatin, and 30 nM of camptothecin.

• 

  Fig. 2 The histograms are showing the levels of M30-FITC immunofluorescences (Number vs M30-FITC immunofluorescence) detected by flow cytometer in HeLa and OVCAR-3 cells treated with anti-cancer agents and the comparisons between sub-G0G1 phase fractions and positive M30-FITC immunofluorescences. The popilations in gate R2 indicate negative M30-FITC immunofluorescences and those in gate R3 indicate the apoptotic populations with positive M30-FITC immunofluorescences. (A) Untreated control cells, (B) cells treated with 1,000 nM of paclitaxel, (C) cells treated with 250 ng/mL of cisplatin, and (D) cells treated with 30 nM of camptothecin. (E) and (F) The correlation between the detected levels of sub-G0G1 fractions and M30-FITC immunofluorescences were evaluated by Spearman's Methods (correlation coefficient=0.59848, P=0.0003). There were no significant differences between them when compared by Wilcoxon Rank Sum Test.

• 

  Fig. 3 The M30-FITC immunofluorescences observed under the immunofluorescence microscopy. (A) Untreated control HeLa and OVCAR-3 cells. There can be seen some positive cells with scanty intracytoplasmic M30-FITC immunofluorescences compared to the treated cells. (B~D) Cells treated with 1,000 nM of paclitaxel, 250 ug/mL of cisplatin, and 30 uM of camptothecin in order. There can be seen many cells with strong intracytoplasmic immunofluorescences in all of the treated cells, PAC: paclitaxel, CIS: cisplatin, CAM: camptothecin.

• 

  Fig. 4 The levels of M30 antigen in each cell line groups. This figure showing the comparison between the levels of CK18-Asp396 (M30) antigen detected by ELISA in the culture media of untreated controls and HeLa and OVCAR-3 cell lines treated with 1,000 nM of paclitaxel, 250 ng/mL of cisplatin, and 30 uM of camptothecin. PAC: paclitaxel, CIS: cisplatin, CAM: camptothecin.

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