Skip to main content

Influences of Lovastatin on membrane ion flow and intracellular signaling in breast cancer cells

Abstract

Lovastatin, an inhibitor of cellular cholesterol synthesis, has an apparent anti-cancer property, but the detailed mechanisms of its anti-cancer effects remain poorly understood. We investigated the molecular mechanism of Lovastatin anti-tumor function through the study of its effect on membrane ion flow, gap junctional intercellular communication (GJIC), and the pathways of related signals in MCF-7 mammary cancer cells. After treatment for 24–72 h with 4, 8 or 16 μmol/L Lovastatin, cellular proliferation was examined via the MTT assay, and changes in membrane potential and cellular [Ca2+]i were monitored using confocal laser microscopy. In addition, the expression of plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA was analyzed via RT-PCR, the GJIC function was examined using the scrape-loading dye transfer (SLDT) technique, and MAPK phosphorylation levels were tested with the kinase activity assay. The results showed that Lovastatin treatment significantly inhibited the growth of MCF-7 breast cancer cells. It also increased the negative value of the membrane potential, leading to the hyperpolarization of cells. Moreover, Lovastatin treatment continuously enhanced [Ca2+]i, although the levels of PMCA1 mRNA were unchanged. GJIC was also upregulated in MCF-7 cells, with transfer of LY Fluorescence reaching 4 to 5 rows of cells from the scraped line after treatment with 16 μmol/L Lovastatin for 72 h. Finally, downregulation of ERK1 and p38MAPK phosphorylation were found in Lovastatin-treated MCF-7 cells. It could be deduced that Lovastatin can induce changes in cellular hyperpolarization and intracellular Ca2+ distributions, and increase GJIC function. These effects may result in changes in the downstream signal cascade, inhibiting the growth of MCF-7 cells.

Abbreviations

[Ca2+]i :

cytosolic free Ca2+ concentration

GI:

growth inhibition

GJIC:

gap junctional intercellular communication

HMG-CoA:

3-hydroxy-3-methylglutarylcoenzyme A

LOV:

lovastatin

MVA:

mevalonic acid

PMCA1:

plasma membrane calcium ATPase isoform 1

References

  1. Lange, Y., Duan, H. and Mazzone, T. Cholesterol homeostasis is modulated by amphiphiles at transcriptional and post-transcriptional loci. Lipid Res. 37 (1996) 534–539.

    CAS  Google Scholar 

  2. Buttke, T.M. and Van Cleave, S. Adaptation of a cholesterol deficient human T cell line to growth with lanosterol. Biochem. Biophys. Res. Commun. 200 (1994) 206–212.

    PubMed  Article  CAS  Google Scholar 

  3. Shellman, Y.G., Ribble, D., Miller, L., Gendall, J., Vanbuskirk, K., Kelly, D., Norris, D.A. and Dellavalle, R.P. Lovastatin-induced apoptosis in human melanoma cell lines. Melanoma Res. 15 (2005) 83–89.

    PubMed  Article  CAS  Google Scholar 

  4. Dimitrowlakos, J., Ye, L.Y., Benzaquen, M., Moore, M.J., Kamel-Reid, S., Freedman, M.H., Yeger, H. and Penn, L.Z. Differentiation sensitivity of various pediatric cancers and squamous cell carcinomas to Lovastatin-induced apoptosis: therapeutic implications. Clin. Cancer Res. 7 (2001) 158–167.

    Google Scholar 

  5. Ruch, R.J., Madhukar, B.V., Trosko, J.E. and Klaunig, J.E. Reversal of rasinduced inhibition of gap-junctional intercellular communication, transformation, and tumorigenesis by lovastatin. Mol. Carcinog. 7 (1993) 50–59.

    PubMed  CAS  Google Scholar 

  6. Hladky, S.B. and Rink, T.J. Potential difference and the distribution of ions across the human red blood cell membrane; a study of the mechanism by which the fluorescent cation, diS-C3-(5) reports membrane potential. J. Physiol. 263 (1976) 287–319.

    PubMed  CAS  Google Scholar 

  7. Plasek, J. and Hronda, V. Assessment of membrane potential changes using the carbocyanine dye, diS-C3-(5): synchronous excitation spectroscopy studies. Eur. Biophys. J. 19 (1991) 183–188.

    PubMed  CAS  Google Scholar 

  8. Suzuki, H., Wang, Z.Y., Yamakoshi, M., Kobayashi, M. and Nozawa, T. Probing the transmembrane potential of bacterial cells by voltage-sensitive dyes. Anal. Sci. 19 (2003) 1239–1242.

    PubMed  Article  CAS  Google Scholar 

  9. Kao, J.P., Harootunian, A.T. and Tsien, R.Y. Photochemically generated cytosolic calcium pulses and their detection by Fluo-3. J. Biol. Chem. 264 (1989) 8179–8184.

    PubMed  CAS  Google Scholar 

  10. Roberts-Thomson, S.J., Holman, N.A., May, F.J., Lee, W.J. and Monteith, G.R. Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line. J. Pharmacol. Toxicol. Methods 44 (2000) 513–517.

    PubMed  Article  CAS  Google Scholar 

  11. el-Fouly, M.H., Trosko, J.E. and Chang, C.C. Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp. Cell. Res. 168 (1987) 422–430.

    PubMed  Article  CAS  Google Scholar 

  12. Zhuang, L., Kim, J., Adam, R.M., Solomon, K.R. and Freeman, M.R. Cholesterol targeting alters lipid raft composition and cell survival in prostate cancer cells and xenografts. J. Clin. Invest. 115 (2005) 959–968.

    PubMed  Article  CAS  Google Scholar 

  13. Waczulikova, I., Sikurova, L., Bryszewska, M.R., Kawiecka, K., Carsky, J. and Ulicna, O. Impaired erythrocyte transmembrane potential in diabetes mellitus and its possible improvement by resorcylidene aminoguanidine. Bioelectrochemistry 52 (2000) 251–256.

    PubMed  Article  CAS  Google Scholar 

  14. Toyomizu, M., Okamoto, K., Akiba, Y., Nakatsu, T. and Konishi, T. Anacardic acid-mediated changes in membrane potential and pH gradient across liposomal membrane. Biochem. Biophys. Acta 1558 (2002) 54–62.

    PubMed  CAS  Google Scholar 

  15. de Poorter, L.M. and Keltjens, J.T. Convenient fluorescence-based methods to measure membrane potential and intracellular pH in the Archaeo Methanobacterium thermoautotrophicum. J. Microbiol. Methods 47 (2001) 233–241.

    PubMed  Article  Google Scholar 

  16. Schiffenbauer, Y.S., Trubniykov, E., Zacharia, B.T., Gerbat, S., Rehavi, Z., Berke, G. and Chaitchik, S. Tumor sensitivity to anti-cancer drugs predicted by changes in fluorescence intensity and polarization in vitro. Anticancer Res. 22 (2002) 2663–2669.

    PubMed  CAS  Google Scholar 

  17. Fouty, B.W. and Rodman. D.M. Mevastatin can cause G1 arrest and induce apoptosis in pulmonary artery smooth muscle cells through a p27Kip1-independent pathway. Circ. Res. 92 (2003) 501–509.

    PubMed  Article  CAS  Google Scholar 

  18. Germano, D., Pacilio, C., Cancemi, M., Cicatiello, L., Altucci, L., Petrizzi, V.B., Sperandio, C., Salzano, S., Michalides, R.J., Taya, Y., Bresciani, F. and Weisz, A. Inhibition of human breast cancer cell growth by blockade of the mevalonate-protein prenylation pathway is not prevented by overexpression of cyclin D1. Breast Cancer Res. Treat. 67 (2001) 23–33.

    PubMed  Article  CAS  Google Scholar 

  19. Zhang, T.C., Cao, E.H., Li, J.F., Ma, W. and Qin, J.F. Induction of apoptosis and inhibition of human gastric cancer MGC-803 cell growth by arsenic trioxide. Eur. J. Cancer 35 (1999) 1258–1263.

    PubMed  Article  CAS  Google Scholar 

  20. Florio, T., Thellung, S., Arena, S., Corsaro, A., Spaziante, R., Gussoni, G., Acuto, G., Giusti, M., Giordano, G. and Schettini, G. Somatostatin and its analog lanreotide inhibit the proliferation of dispersed human nonfunctioning pituitary adenoma cells in vitro. Eur. J. Endocrinol. 141 (1999) 396–408.

    PubMed  Article  CAS  Google Scholar 

  21. Miyake, H., Hara, I., Yamanaka, K., Arakawa, S. and Kamidono, S. Calcium ionophore, ionomycin inhibits growth of human bladder cancer cells both in vitro and in vivo with alteration of Bcl-2 and Bax expression levels. J. Urol. 162 (1999) 916–921.

    PubMed  Article  CAS  Google Scholar 

  22. Popescu, B.O., Cedazo-Minguez, A., Popescu, L.M., Winblad, B., Cowburn, R.F. and Ankarcrona, M. Caspase cleavage of exon 9 deleted presenilin-1 is an early event in apoptosis induced by calcium ionophore A 23187 in SH-SY5Y neuroblastoma cells. J. Neurosci. Res. 66 (2001) 122–134.

    PubMed  Article  CAS  Google Scholar 

  23. Cronier, L., Frendo, J.L., Defamie, N., Pidoux, G., Bertin, G., Guibourdenche, J., Pointis, G. and Malassine, A. Requirement of gap junctional intercellular communication for human villous trophoblast differentiation. Biol. Reprod. 69 (2003) 1472–1480.

    PubMed  Article  CAS  Google Scholar 

  24. Evans, W.H. and Martin, P.E. Gap junctions: Structure and Function. Mol. Membr. Biol. 19 (2002) 121–136.

    PubMed  Article  CAS  Google Scholar 

  25. Alexander, D.B. and Goldberg, G.S. Transfer of biologically important molecules between cells through gap junction channels. Curr. Med. Chem. 10 (2003) 2045–2058.

    PubMed  Article  CAS  Google Scholar 

  26. Carruba, G., Webber, M.M., Quader, S.T., Amoroso, M., Cocciadiferro, L., Saladino, F., Trosko, J.E. and Castagnetta, L.A. Regulation of cell-to-cell communication in non-tumorigenic and malignant human prostate epithelial cells. Prostate 50 (2002) 73–82.

    PubMed  Article  CAS  Google Scholar 

  27. Saito, T., Tanaka, R., Wataba, K., Kudo, R. and Yamasaki, H. Overexpression of estrogen receptor-alpha gene suppresses gap junctional intercellular communication in endometrial carcinoma cells. Oncogene 23 (2004) 1109–1116.

    PubMed  Article  CAS  Google Scholar 

  28. Trosko, J.E. and Ruch, R.J. Cell-cell communication in carcinogenesis. Front Biosci. 3 (1998) D208–236.

    PubMed  CAS  Google Scholar 

  29. Trosko, J.E. and Ruch, R.J. Gap junctions as targets for cancer chemoprevention and chemotherapy. Curr. Drug Targets 3 (2002) 465–482.

    PubMed  Article  CAS  Google Scholar 

  30. Van Golen, K.L., Bao, L.W., Pan, Q., Miller, F.R., Wu, Z.F. and Merajver, S.D. Mitogen activated protein kinase pathway is involved in RhoC GTPase induced motility, invasion and angiogenesis in inflammatory breast cancer. Clin. Exp. Metastasis 19 (2002) 301–311.

    PubMed  Article  Google Scholar 

  31. Senokuchi, T., Matsumura, T., Sakai, M., Yano, M., Taguchi, T., Matsuo, T., Sonoda, K., Kukidome, D., Imoto, K., Nishikawa, T., Kim-Mitsuyama, S., Takuwa, Y. and Araki, E. Statins suppress oxidized low density lipoprotein-induced macrophage proliferation by inactivation of the small G protein-p38 MAPK pathway. J. Biol. Chem. 280 (2005) 6627–6633.

    PubMed  Article  CAS  Google Scholar 

  32. Wu, J., Wong, W.W., Khosravi, F., Minden, M.D. and Penn, L.Z. Blocking the Raf/MEK/ERK pathway sensitizes actue myelogenous leukemia cells to lovastatin-induced apoptosis. Cancer Res. 64 (2004) 6461–6468.

    PubMed  Article  CAS  Google Scholar 

  33. Holstein, S.A. and Hohl, R.J. Interaction of cytosine arabinoside and lovastatin in human leukemia cells. Leukemia Res. 25 (2001) 651–660.

    Article  CAS  Google Scholar 

  34. Johnson, M.D., Woodard, A., Okediji, E.J., Toms, S.A. and Allen, G.S. Lovastatin is a potent inhibitor of meningioma cell proliferation: evidence for inhibition of a mitogen associated protein kinase. J. Neurooncol. 56 (2002) 133–142.

    PubMed  Article  Google Scholar 

  35. Takata, R., Fukasawa, S., Hara, T., Nakajima, H., Yamashina, A., Yanase, N. and Mizuguchi, J. Cerivastatin-induced apoptosis of human aortic smooth muscle cells through partial inhibition of basal activation of extracellular signal-regulated kinases. Cardiovasc. Pathol. 13 (2004) 41–48.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Man Tian Mi.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wei, N., Mi, M.T. & Zhou, Y. Influences of Lovastatin on membrane ion flow and intracellular signaling in breast cancer cells. Cell Mol Biol Lett 12, 1–15 (2007). https://doi.org/10.2478/s11658-006-0050-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-006-0050-2

Key words

  • Lovastatin
  • Human breast cancer cells
  • Cellular membrane ion transfer
  • Gap junctional intercellular communication (GJIC)
  • MAPK activity