Skip to main content

Advertisement

SpringerLink
Log in

Novel vitamin E analogue and 9-nitro-camptothecin administered as liposome aerosols decrease syngeneic mouse mammary tumor burden and inhibit metastasis

  • Original Article
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript

Abstract

Purpose

To test the anticancer properties of a nonhydrolyzable ether-linked acetic acid analogue of vitamin E, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid (α-TEA), and a derivative of camptothecin, 9-nitrocamptothecin (9-NC) singly and in combination against mouse mammary tumor cells (line 66 clone 4 stably transfected with green fluorescent protein; 66c1-4-GFP) cultured in vitro or transplanted subcutaneously into the inguinal region of female BALB/c mice to form established tumors.

Methods

Following in vitro treatment of 66cl-4-GFP cells with α-TEA and suboptimal concentrations of 9-NC, singly or in combination, apoptosis was measured by morphological evaluation of nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI), and DNA synthesis arrest was measured by tritiated thymidine uptake. For in vivo analyses α-TEA and 9-NC, both water-insoluble compounds, were formulated into liposomes using dilauroylphosphatidylcholine and administered by aerosol to deliver doses calculated to be 36 and 0.4 μg/mouse per day, respectively, (singly or each separately for combined treatments) 7 days per week.

Results

Treatment of 66cl-4-GFP cells in culture for 3 days with a combination of α-TEA (10 μg/ml; singly produces 38% apoptosis), and suboptimal concentrations of 9-NC (15.6, 31.3, 62.5, or 125 ng/ml; singly produce 2–7% apoptosis), produced 47%, 58%, 64%, and 69% apoptosis. Likewise, combinations of α-TEA + 9-NC inhibited DNA synthesis more than either agent administered singly. A significant reduction (P<0.001) in growth of subcutaneous transplanted tumors was observed with liposome-formulated and aerosolized delivery of α-TEA + 9-NC to BALB/c mice. The incidence of macroscopic lung metastasis was 83% in control vs 8% in α-TEA-, 9-NC-, or combination-treated mice. Fluorescence microscopic examination of lungs and axillary and brachial lymph nodes showed a statistically significant decrease in metastasis observed in α-TEA-, 9-NC-, and combination- vs control-treated animals. Analyses of primary tumor tissue for proliferation and apoptosis showed treatment groups to have lower Ki-67 and higher terminal deoxynucleotidyl transferase-mediated nick end labeling, respectively. Treatments showed no measurable effects on two angiogenesis parameters, namely intratumoral blood volume as assessed by hemoglobin content and intratumoral blood vessel density as assessed with CD31 staining.

Conclusions

Combination treatments enhanced antiproliferative and proapoptotic activities in cell culture, and when formulated in liposomes and delivered via aerosolization to treat an aggressive and metastatic syngeneic murine mammary tumor, the combination treatment showed a significant reduction in tumor volume in comparison to either treatment alone. Mechanistically, it appears that neither enhanced apoptosis, reduced cell proliferation, nor reduced blood vessel density can fully account for the enhanced effects of the combination treatment.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2a, b
Fig. 3
Fig. 4a, b
Fig. 5a, b
Fig. 6a, b

Similar content being viewed by others

Abbreviations

α-TEA:

2,5,7,8-Tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid

9-NC:

9-Nitro-camptothecin

66cl-4-GFP:

BALB/c mouse mammary tumor line 66 clone 4 stably transfected with GFP

DAPI:

4′,6-Diamidino-2-phenylindole

DMSO:

Dimethylsulfoxide

GFP:

Green fluorescent protein

PECAM-1:

Platelet-endothelial cell adhesion molecule

THF:

Tetrahydrofuran

TUNEL:

Terminal deoxynucleotidyl transferase-mediated nick end labeling

References

  1. Bandyopadhyay A, Lopez-Castillas F, Malik SN, Montiel JL, Mendoza V, Yang J, Sun L-Z (2002) Antitumor activity of a recombinant soluble betaglycan in human breast cancer xenograft. Cancer Res 62:4690

    CAS  PubMed  Google Scholar 

  2. Bernacki RJ, Pera P, Gambacorta P, Brun Y, Greco WR (2000) In vitro antitumor activity of 9-nitro-camptothecin as a single agent and in combination with other antitumor drugs. Ann N Y Acad Sci 922:293

    CAS  PubMed  Google Scholar 

  3. Carpentier A, Groves S, Simmons-Menchaca M, Turley J, Zhao B, Sanders BG, Kline K (1993) RRR-alpha-tocopheryl succinate inhibits proliferation and enhances secretion of transforming growth factor-beta (TGF-beta) by human breast cancer cells. Nutr Cancer 19:225

    PubMed  Google Scholar 

  4. Chatterjee D, Schmitz I, Krueger A, Yeung K, Kirchhoff S, Krammer PH, Peter ME, Wyche JH, Pantazis P (2001) Induction of apoptosis in 9-nitrocamptothecin-treated DU145 human prostate carcinoma cells correlates with de novo synthesis of CD95 and CD95 ligand and down-regulation of c-FLIP. Cancer Res 61:7148

    CAS  PubMed  Google Scholar 

  5. Clarke R (1997) Issues in experimental design and endpoint analysis in the study of experimental cytotoxic agents in vivo in breast cancer and other models. Breast Cancer Res Treat 46:255

    Article  CAS  PubMed  Google Scholar 

  6. Dexter DL, Kowalski HM, Blazar BA, Fligiel Z, Vogel R, Heppner GH (1978) Heterogeneity of tumor cells from a single mouse mammary tumor. Cancer Res 38:3174

    CAS  PubMed  Google Scholar 

  7. Garcia-Carbonero R, Supko JG (2002) Current perspectives on the clinical experience, pharmacology, and continued development of the camptothecins. Clin Cancer Res 8:641

    CAS  PubMed  Google Scholar 

  8. Gilbert BE, Seryshev A, Knight V, Brayton C (2002) 9-Nitrocamptothecin liposome aerosol: lack of subacute toxicity in dogs. Inhal Toxicol 14:185

    Article  CAS  PubMed  Google Scholar 

  9. Giovanella BC, Stehlin JS, Hinz HR, Kozielski AJ, Harris NJ, Vardeman DM (2002) Preclinical evaluation of the anticancer activity and toxicity of 9-nitro-20(S)-camptothecin (rubitecan). Int J Oncol 20:81

    CAS  PubMed  Google Scholar 

  10. Knight V, Koshkina NV, Waldrep JC, Giovanella BC, Gilbert BE (1999) Anticancer effect of 9-nitrocamptothecin liposome aerosol on human cancer xenografts in nude mice. Cancer Chemother Pharmacol 44:177

    Article  CAS  PubMed  Google Scholar 

  11. Konstadoulakis MM, Antonakis PT, Tsibloulis AG, Stathopoulos GP, Manouras AP, Mylonaki DB, Golematis BX (2001) A phase II study of 9-nitrocamptothecin in patients with advanced pancreatic adenocarcinoma. Cancer Chemother Pharmacol 48:417

    Article  CAS  PubMed  Google Scholar 

  12. Koshkina NV, Gilbert BE, Waldrep JC, Seryshev A, Knight V (1999) Distribution of camptothecin after delivery as a liposome aerosol or following intramuscular injection in mice. Cancer Chemother Pharmacol 44:187

    Article  CAS  PubMed  Google Scholar 

  13. Koshkina NV, Kleinerman ES, Waldrep C, Jai S-F, Worth LL, Gilbert BE, Knight V (2000) 9-Nitrocamptothecin liposome aerosol treatment of melanoma and osteosarcoma lung metastases in mice. Clin Cancer Res 6:2876

    CAS  PubMed  Google Scholar 

  14. Lawson KA, Anderson K, Menchaca M, Atkinson J, Sun L-Z, Knight V, Gilbert BE, Conti C, Sanders BG, Kline K (2003) Novel vitamin E analogue decreases syngeneic mouse mammary tumor burden and reduces lung metastasis. Mol Cancer Ther 2:437

    CAS  PubMed  Google Scholar 

  15. Miller BE, Roi LD, Howard LM, Miller FR (1983) Quantitative selectivity of contact-mediated intercellular communication in a metastatic mouse mammary tumor line. Cancer Res 43:4102

    CAS  PubMed  Google Scholar 

  16. Miller KD, Haney LG, Guiney P, Murry DJ, Hawes JW, Lenaz L, Aun S-L, Sledge GW (2000) A phase II pharmacokinetic and pharmacodynamic trial of 9-nitrocamptothecin (9-NC) in patients with metastatic breast cancer. Breast Cancer Res Treat 64:126

    Google Scholar 

  17. Pantazis P, Early JA, Mendoza JT, DeJesus AR, Giovanella BC (1994) Cytotoxic efficacy of 9-nitrocamptothecin in the treatment of human malignant melanoma cells in vitro. Cancer Res 54:771

    CAS  PubMed  Google Scholar 

  18. Shun M-C, Yu W, Atkinson J, Gapor A, Sanders BG, Kline K (2004) Pro-apoptotic mechanisms of action of a novel vitamin E analog (α-TEA) and a naturally occurring form of vitamin E (d=tocotrienol) in MDA-MB-435 human breast cancer cells. Nutr Cancer (in press)

  19. Ulukan H, Swaan W (2002) Camptothecins: a review of their chemotherapeutic potential. Drugs 62:2039

    Google Scholar 

  20. Verschraegen CF, Gupta E, Loyer E, Kavanagh JJ, Kudelka AP, Freedman RS, Edwards CL, Harris N, Steger M, Steltz V, Giovanella BC, Stehlin JS (1999) A phase II clinical and pharmacological study of oral 9-nitrocamptothecin in patients with refractory epithelial ovarian, tubal or peritoneal cancer. Anticancer Drugs 10:375

    CAS  PubMed  Google Scholar 

  21. Verschraegen CF, Gilbert BE, Huaringa AJ, Newman R, Harris N, Leyva FJ, Keus L, Campbell K, Nelson-Taylor T, Knight V (2002) Feasibility, phase I, and pharmacological study of aerosolized liposomal 9-nitro-20(S)-camptothecin in patients with advanced malignancies in the lungs. Ann N Y Acad Sci 922:352

    Google Scholar 

  22. Wani MC, Ronman PE, Lindley JT, Wall ME (1980) Plant antitumor agents. Synthesis and biological activity of camptothecin analogues. J Med Chem 23:554

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank the Histology Core Facility for preparation of H&E and immunohistochemically stained tissues and the Director of the Biostatistics and Data Processing Core, Dr. Dennis A. Johnston and Dr. Howard Thames, for help with statistical analyses (National Institute of Environmental Health Sciences Center Grant ES 07784; University of Texas M. D. Anderson). We thank Dr. Richard Willis (Division of Nutrition, University of Texas at Austin) for statistical advice and Dr. Vernon Knight’s laboratory at Baylor College of Medicine, Houston, TX, for the method of aerosol preparation, particle size determination and output studies. This work was supported by Public Health Service Grant CA59739 (to K.K., B.G.S.), CA79683 (to L.-Z.S.), the Foundation for Research (to K.K., B.G.S.), the National Institute of Environmental Health Sciences Center Grant ES 07784 (K.K., B.G.S. are members) and Toxicology Training Grant T32 ES 07247 (predoctoral support for K.A.).

Author information

Authors and Affiliations

  1. Cancer Prevention Fellowship Program, National Cancer Institute, NIH, DHHS, 6120 Executive Boulevard, Suite T-41, Bethesda, MD 20892, USA

    Karla A. Lawson

  2. Harvard Medical School, 4 Blackfan Circle, Boston, MA 02115, USA

    Kristen Anderson

  3. School of Biological Sciences, University of Texas, Austin, TX 78712, USA

    Rachel M. Snyder, Marla Simmons-Menchaca & Bob G. Sanders

  4. Department of Chemistry, Brock University, St Catharines, ON, Canada, L2S 3A1

    Jeffrey Atkinson

  5. Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA

    Lu-Zhe Sun & Abhik Bandyopadhyay

  6. Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

    Vernon Knight

  7. Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

    Brian E. Gilbert

  8. Division of Nutrition, University of Texas, Austin, TX 78712, USA

    Kimberly Kline

Authors
  1. Karla A. Lawson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kristen Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rachel M. Snyder
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marla Simmons-Menchaca
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffrey Atkinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lu-Zhe Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Abhik Bandyopadhyay
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Vernon Knight
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Brian E. Gilbert
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Bob G. Sanders
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Kimberly Kline
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kimberly Kline.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lawson, K.A., Anderson, K., Snyder, R.M. et al. Novel vitamin E analogue and 9-nitro-camptothecin administered as liposome aerosols decrease syngeneic mouse mammary tumor burden and inhibit metastasis. Cancer Chemother Pharmacol 54, 421–431 (2004). https://doi.org/10.1007/s00280-004-0817-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00280-004-0817-y

Keywords

Search

Navigation