Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Dec 15;117(24):5519-28.
doi: 10.1002/cncr.26215. Epub 2011 Jun 20.

Eradication of chemotherapy-resistant CD44+ human ovarian cancer stem cells in mice by intraperitoneal administration of Clostridium perfringens enterotoxin

Francesca Casagrande  1 , Emiliano Cocco, Stefania Bellone, Christine E Richter, Marta Bellone, Paola Todeschini, Eric Siegel, Joyce Varughese, Dan Arin-Silasi, Masoud Azodi, Thomas J Rutherford, Sergio Pecorelli, Peter E Schwartz, Alessandro D Santin
Affiliations

Eradication of chemotherapy-resistant CD44+ human ovarian cancer stem cells in mice by intraperitoneal administration of Clostridium perfringens enterotoxin

Francesca Casagrande et al. Cancer. .

Abstract

Background: Emerging evidence has suggested that the capability to sustain tumor formation, growth, and chemotherapy resistance in ovarian as well as other human malignancies exclusively resides in a small proportion of tumor cells termed cancer stem cells. During the characterization of CD44(+) ovarian cancer stem cells, we found a high expression of the genes encoding for claudin-4. Because this tight junction protein is the natural high-affinity receptor for Clostridium perfringens enterotoxin (CPE), we have extensively investigated the sensitivity of ovarian cancer stem cells to CPE treatment in vitro and in vivo.

Methods: Real-time polymerase chain reaction and flow cytometry were used to evaluate claudin-3/-4 expression in ovarian cancer stem cells. Small interfering RNA knockdown experiments and MTS assays were used to evaluate CPE-induced cytotoxicity against ovarian cancer stem cell lines in vitro. C.B-17/SCID mice harboring ovarian cancer stem cell xenografts were used to evaluate CPE therapeutic activity in vivo.

Results: CD44(+) ovarian cancer stem cells expressed claudin-4 gene at significantly higher levels than matched autologous CD44(-) ovarian cancer cells, and regardless of their higher resistance to chemotherapeutic agents died within 1 hour after exposure to 1.0 μg/mL of CPE in vitro. Conversely, small-interfering RNA-mediated knockdown of claudin-3/-4 expression in CD44(+) cancer stem cells significantly protected cancer stem cells from CPE-induced cytotoxicity. Importantly, multiple intraperitoneal administrations of sublethal doses of CPE in mice harboring xenografts of chemotherapy-resistant CD44(+) ovarian cancer stem cells had a significant inhibitory effect on tumor progression leading to the cure and/or long-term survival of all treated animals (ie, 100% reduction in tumor burden in 50% of treated mice; P < .0001).

Conclusions: CPE may represent an unconventional, potentially highly effective strategy to eradicate chemotherapy-resistant cancer stem cells.

PubMed Disclaimer

Conflict of interest statement

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

Figures

Figure 1
Figure 1
(Top) Quantitative real-time polymerase chain reaction analysis of claudin-3 and claudin-4 expression is shown. The Y axis represents the fold induction relative to normal ovary expression. The X axis represents CD44+ cancer stem cells and autologous matched CD44− control sample tested for claudin-3 and claudin-4. Claudin-4 expression was significantly higher in CD44+ cancer stem cell lines and cancer stem cell clones when compared with matched autologous CD44− control samples (P < .004). (Middle) Representative flow cytometry histograms are shown of claudin-3 and claudin-4 expression in a representative CD44+ cancer stem cell population (OVA-1R-CSC, left panel) and 1 CD44+ cancer stem cell clone (CLONE-1-CSC, right panel) after labeling with fluorescein isothiocyanate (FITC)-conjugated-C-CPE290-319 peptide. Dashed lines indicate isotype; solid black peaks indicate FITC-conjugated-C-CPE290-319 peptide. (Bottom) Representative dose-response curves after exposure to carboplatin and paclitaxel of OVA-1R-CSC line and matched autologous CD44− control tumor cells (right panel) and of 1 representative cancer stem cell clone (CLONE-1-CSC) versus matched autologous CD44− control tumor cells (left panel) are shown. Survival was assessed by MTS assay as described in the Materials and Methods section. Each point on the cell line graph represents the mean of 3 estimations ± standard error. A significant difference was observed in the IC50 of the CD44+ cancer stem cell populations versus autologous CD44− matched tumor cells when treated with carboplatin or paclitaxel (P < .01).
Figure 2
Figure 2
Representative dose-dependent Clostridium perfringens enterotoxin (CPE)-mediated cytotoxicity of multiple CD44+ cancer stem cell (CSC) populations and CSC clones, positive control cells (ie, VERO cells), negative controls including normal ovarian epithelium (NOVA), normal cervical keratinocytes (NORM CX), normal human fibroblasts (FIBRO), and lymphoblastoid B cells (LCL) after 24 hours incubation with CPE is shown. With no exception, all 8 CD44+ CSC populations tested were found highly sensitive to CPE exposure in vitro (P = .001). Viability was determined by counting the number of trypan blue-positive cells and the total cell number and comparing such numbers to those of the untreated control cells (ie, 100% viable cells).
Figure 3
Figure 3
(Top) Representative knockdown of the Clostridium perfringens enterotoxin (CPE) receptors by anticlaudin-3 and anticlaudin-4 small interfering RNA (siRNA) in ovarian cancer stem cells (CSCs) is shown. The levels of claudin-3 and claudin-4 mRNA in OVA-1R-CSC CD44+ cells transfected with claudin-3 and claudin-4 siRNA duplexes (gray bars) were analyzed using quantitative real-time polymerase chain reaction and expressed as percentage of control cells (black bars). (Bottom) Representative dose-dependent CPE-mediated cytotoxicity of OVA-1R-CSCs transfected with claudin-3/-4 siRNA duplexes after 1 hour exposure to 0.5 μg/mL (black bars) and 1 μg/mL (gray bars) of CPE (P = .004) is shown. Mock-transfected cells and untransfected cancer stem cells were used as controls. Results are presented as percentage of viable cells when compared with number of untreated control cells (ie, 100% viable cells).
Figure 4
Figure 4
Survival of C.B-17/SCID mice after intraperitoneal (i.p.) injection of 5 × 106 viable ovarian CD44+ cancer stem cells is shown. Animals harboring ovarian CD44+ cancer stem cell tumors established for 1 week were injected i.p. with 7.5 μg of Clostridium perfringens enterotoxin (CPE) as described in the Material and Methods section for a total of 8× 72 to 96 hours apart. Mice were evaluated on a daily basis and sacrificed when showing significant sign of disease and/or disease-induced distress as per Yale Institutional Animal Care and Use Committee regulations. The log-rank test yielded P < .0001 for the differences in survival.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009;59:225–249. - PubMed
    1. Copeland L. Epithelial ovarian cancer. In: DiSaia PJ, Creasman WT, editors. Clinical Gynecologic Oncology. St Louis: Mosby Year Book; 1997. pp. 313–367.
    1. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414:105–111. - PubMed
    1. Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer. 2005;5:275–284. - PubMed
    1. Hermann PC, Huber SL, Heeschen C. Metastatic cancer stem cells: a new target for anti-cancer therapy? Cell Cycle. 2008;7:188–193. - PubMed

Publication types

MeSH terms