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Entamoeba invadens: Protein Kinase C Inhibitors Block the Growth and Encystation
Experimental Parasitology 95, 288–290 (2000) doi:10.1006/expr.2000.4538, available online at http://www.idealibrary.com on
RESEARCH BRIEF Entamoeba invadens: Protein Kinase C Inhibitors Block the Growth and Encystation
Asao Makioka,* Masahiro Kumagai,* Hiroshi Ohtomo,* Seiki Kobayashi,† and Tsutomu Takeuchi† *Department of Tropical Medicine, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan; and †Department of Tropical Medicine and Parasitology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Makioka, A., Kumagai, M., Ohtomo, H., Kobayashi, S., and Takeuchi, T. 2000. Entamoeba invadens: Protein kinase C inhibitors block the growth and encystation. Experimental Parasitology 95, 288– 290. q 2000 Academic Press Index Descriptors and Abbreviations: Entamoeba invadens; protozoa; encystation; signal transduction; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DMSO, dimethyl sulfoxide.
growth medium BI-S-33 (Diamond et al. 1978) at 268C. For experiments on the effect of PKC inhibitors on cell growth, duplicate cultures inoculated with 104 trophozoites/ml included various concentrations (1 nM–50 mM) of the drugs and were incubated for 7 days. The cells were counted in a hemocytometer and the viability was determined by trypan blue dye exclusion. Four PKC inhibitors, staurosporine (Tamaoki et al. 1986), chelerythrine chloride (Herbert et al. 1990; hereafter termed chelerythrine), calphostin C (Kobayashi et al. 1989), and Derythro-sphingosine (Hannun et al. 1986; hereafter termed sphingosine), were used. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, and 4a-phorbol, an inactive analog of PMA, were also used at concentrations of 1–10 mM. All these chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and were initially dissolved into dimethyl sulfoxide (DMSO). Control cultures contained the same volume of DMSO. For experiments on the effect of PKC inhibitors on the encystation, trophozoites (5 3 105 cells/ml) were transferred to encystation medium called 47% LG (LG is BI without glucose; Sanchez et al. 1994). Duplicate cultures that included various concentrations (1 nM–50 mM) of PKC inhibitors were incubated for 3 days. The cysts and trophozoites were counted for determination of the percentage of cysts and the viability was determined by trypan blue dye exclusion. All experiments in the present study were performed at least three times and similar results were obtained. Therefore, representative data from duplicate cultures are shown. The effects of four PKC inhibitors on the growth and encystation are shown in Fig. 1. Among these inhibitors, staurosporine was most potent for inhibition of both growth and encystation, the former being more sensitive to the drug than the latter (Fig. 1A). Chelerythrine also inhibited both growth and encystation, the latter being more sensitive to the drug than the former (Fig. 1B). Calphostin C showed little effect on both growth and encystation (Fig. 1C). Sphingosine was also less
The axenic encystation in vitro of Entamoeba invadens, a reptilian parasite, is a useful model for the encystation of E. histolytica because there is no available encystation medium for the human parasite (reviewed by Lo´pez-Romero and Villago´mez-Castro 1993). Transfer of trophozoites of E. invadens in growth medium to encystation medium triggers the encystation so that signal of the medium change should be transduced from the membrane to the nucleus to commence the encystation. No studies, however, on signaling in the encystation of E. invadens have so far been reported. Protein kinase C (PKC), a phospholipid-dependent serine/threonine kinase, has a crucial role in signal transduction for a variety of cellular responses, including cell proliferation and differentiation (Nishizuka 1986). Involvement of PKC has been inferred by use of specific activators such as phorbol esters or specific inhibitors of the enzyme. For E. histolytica, it was demonstrated using these agents that PKC plays an important role in the adhesion and killing of target cells by the parasite (Weikel et al. 1988; Santiago et al. 1994). We therefore considered it of interest to examine the effect of PKC inhibitors on the growth and encystation of E. invadens. Trophozoites of E. invadens strain IP-1 were cultured in axenic
288
0014-4894/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
Entamoeba invadens: PKC INHIBITORS BLOCK GROWTH AND ENCYSTATION
289
FIG. 1. Effects of protein kinase C (PKC) inhibitors on the growth and encystation of Entamoeba invadens. For growth, trophozoites were cultured for 7 days in the presence of various concentrations of four PKC inhibitors, staurosporine (A), chelerythrine (B), calphostin C (C), and sphingosine (D). Mean values 6 SE for duplicate cultures are plotted at each concentration (black circles). The control growth was measured in the absence of drug and was expressed as 100%. For encystation, trophozoites were transferred to encystation medium containing various concentrations of the PKC inhibitors. The cysts and trophozoites were counted for determination of the percentage of cysts 3 days later. The control encystation without the drug was expressed as 100%. Mean values 6 SE for duplicate cultures are plotted (white circles).
potent for both growth and encystation, although higher concentration (50 mM) of the drug inhibited the growth (Fig. 1D). PMA (1–10 mM) and 4a-phorbol had no or little effect on the growth and encystation under conditions used (data not shown). Thus, the results indicate participation of PKC in both growth and encystation of E. invadens, although there is a difference in potency among these PKC inhibitors used. The reason for this difference is not clear, but the site of interaction of these inhibitors with PKC appears be related to it because both staurosporine and chelerythrine interact with the catalytic domain of the
FIG. 2. Reversibility of the chelerythrine effect on the encystation of E. invadens. After exposure to 10 mM chelerythrine in encystation medium for 1 day, the drug was removed by replacement of the medium with drug-free encystation medium and the cultures were further incubated for 2 days. The cysts and trophozoites were counted.
enzyme (Tamaoki et al. 1986; Herbert et al. 1990), whereas calphostin C and sphingosine do so with the regulatory domain (Kobayashi et al. 1989; Hannun et al. 1986). Although both domains of PKC include conserved regions, sequence homology in the conserved region of the catalytic domain between E. invadens and mammals would be higher than that of the regulatory domain. Studies on signal transduction in Entamoeba are still limited and those have focused mainly on the adherence and cytolytic activity of trophozoites, where PMA has been used to demonstrate involvement of PKC in it (Weikel et al. 1988; Santiago et al. 1994). However, neither enhanced growth nor enhanced encystation by PMA was observed in the present study. The reason for this remains unclear but exposure time of PMA would be related to it because longer preincubation of the amoebae with PMA did not enhance subsequent killing of target cells and such condition may lead to down-regulation of PKC, preventing a subsequent biologic effect (Weikei et al. 1988). As staurosporine is potent but not a selective inhibitor of PKC, which blocks other protein kinases (Tamaoki et al. 1986; Ru¨egg and Burgess 1989), it would not be suitable for studies on the role of PKC. Therefore, chelerythrine was used for further experiments. Reversibility of the chelerythrine effect on encystation was studied in duplicate cultures containing 5 3 105 trophozoites/ml and 10 mM chelerythrine. Except for the control tubes, after 1 day incubation, cells were washed twice with fresh encystation medium and incubated for another 2 days. As shown in Fig. 2, no recovery of the encystation was observed after removal of chelerythrine, suggesting that, once trophozoites are exposed to chelerythrine in encystation medium, they lose their ability to encyst. The reason for this remains to be clarified. In summary, the present study indicates that PKC participates in the growth and encystation of E. invadens, contributing to further understanding of those of Entamoeba.
290 ACKNOWLEDGMENTS
We thank Dr. L. S. Diamond for supplying the E. invadens and T. Okita for technical assistance. This work was supported by a Grantin-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by a Health Sciences Research Grant for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health and Welfare of Japan.
MAKIOKA ET AL.
Kobayashi, E., Nakano, H., Morimoto, M., and Tamaoki, T. 1989. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochemical and Biophysical Research Communications 159, 548–553. Lo´pez-Romero, E., and Villago´mez-Castro, J. C. 1993. Encystation in Entamoeba invadens. Parasitology Today 9, 225–227. Nishizuka, Y. 1986. Studies and perspectives of protein kinase C. Science 233, 305–312. Ru¨egg, U. T., and Burgess, G. M. 1989. Staurosporine, K-252 and UCN-01: Potent but nonspecific inhibitors of protein kinases. Trends in Pharmacological Sciences 10, 218–220.
REFERENCES
Sanchez, L., Enea, V., and Eichinger, D. 1994. Identification of a developmentally regulated transcript expressed during encystation of Entamoeba invadens. Molecular and Biochemical Parasitology 67, 125–135.
Diamond, L. S., Harlow, D. R., and Cunnick, C. C. 1978. A new medium for the axenic cultivation of Entaoemeba histolytica and other Entamoeba. Transactions of the Royal Society of Tropical Medicine and Hygiene 72, 431–432.
Santiago, A., Carbajal, M. E., Benı´tez-King, G., and Meza, I. 1994. Entamoeba histolytica: PKC transduction pathway activation in the trophozoite–fibronectin interaction. Experimental Parasitology 79, 436–444.
Hannun, Y. A., Loomis, C. R., Merrill, A. H., and Bell, R. M. 1986. Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. Journal of Biological Chemistry 261, 12604–12609. Herbert, J. M., Augereau, J. M., Gleye, J., and Maffrand, J. P. 1990. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochemical and Biophysical Research Communications 172, 993–999.
Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. 1986. Staurosporine, a potent inhibitor of phospholipid/ Ca++dependent protein kinase. Biochemical and Biophysical Research Communications 135, 397–402. Weikel, C. S., Murphy, C. F., Orozco, E., and Ravdin, J. I. 1988. Phorbol esters specifically enhance the cytolytic activity of Entamoeba histolytica. Infection and Immunity 56, 1485–1491. Received 3 March 2000; accepted with revision 13 June 2000
RESEARCH BRIEF Entamoeba invadens: Protein Kinase C Inhibitors Block the Growth and Encystation
Asao Makioka,* Masahiro Kumagai,* Hiroshi Ohtomo,* Seiki Kobayashi,† and Tsutomu Takeuchi† *Department of Tropical Medicine, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan; and †Department of Tropical Medicine and Parasitology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Makioka, A., Kumagai, M., Ohtomo, H., Kobayashi, S., and Takeuchi, T. 2000. Entamoeba invadens: Protein kinase C inhibitors block the growth and encystation. Experimental Parasitology 95, 288– 290. q 2000 Academic Press Index Descriptors and Abbreviations: Entamoeba invadens; protozoa; encystation; signal transduction; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DMSO, dimethyl sulfoxide.
growth medium BI-S-33 (Diamond et al. 1978) at 268C. For experiments on the effect of PKC inhibitors on cell growth, duplicate cultures inoculated with 104 trophozoites/ml included various concentrations (1 nM–50 mM) of the drugs and were incubated for 7 days. The cells were counted in a hemocytometer and the viability was determined by trypan blue dye exclusion. Four PKC inhibitors, staurosporine (Tamaoki et al. 1986), chelerythrine chloride (Herbert et al. 1990; hereafter termed chelerythrine), calphostin C (Kobayashi et al. 1989), and Derythro-sphingosine (Hannun et al. 1986; hereafter termed sphingosine), were used. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, and 4a-phorbol, an inactive analog of PMA, were also used at concentrations of 1–10 mM. All these chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and were initially dissolved into dimethyl sulfoxide (DMSO). Control cultures contained the same volume of DMSO. For experiments on the effect of PKC inhibitors on the encystation, trophozoites (5 3 105 cells/ml) were transferred to encystation medium called 47% LG (LG is BI without glucose; Sanchez et al. 1994). Duplicate cultures that included various concentrations (1 nM–50 mM) of PKC inhibitors were incubated for 3 days. The cysts and trophozoites were counted for determination of the percentage of cysts and the viability was determined by trypan blue dye exclusion. All experiments in the present study were performed at least three times and similar results were obtained. Therefore, representative data from duplicate cultures are shown. The effects of four PKC inhibitors on the growth and encystation are shown in Fig. 1. Among these inhibitors, staurosporine was most potent for inhibition of both growth and encystation, the former being more sensitive to the drug than the latter (Fig. 1A). Chelerythrine also inhibited both growth and encystation, the latter being more sensitive to the drug than the former (Fig. 1B). Calphostin C showed little effect on both growth and encystation (Fig. 1C). Sphingosine was also less
The axenic encystation in vitro of Entamoeba invadens, a reptilian parasite, is a useful model for the encystation of E. histolytica because there is no available encystation medium for the human parasite (reviewed by Lo´pez-Romero and Villago´mez-Castro 1993). Transfer of trophozoites of E. invadens in growth medium to encystation medium triggers the encystation so that signal of the medium change should be transduced from the membrane to the nucleus to commence the encystation. No studies, however, on signaling in the encystation of E. invadens have so far been reported. Protein kinase C (PKC), a phospholipid-dependent serine/threonine kinase, has a crucial role in signal transduction for a variety of cellular responses, including cell proliferation and differentiation (Nishizuka 1986). Involvement of PKC has been inferred by use of specific activators such as phorbol esters or specific inhibitors of the enzyme. For E. histolytica, it was demonstrated using these agents that PKC plays an important role in the adhesion and killing of target cells by the parasite (Weikel et al. 1988; Santiago et al. 1994). We therefore considered it of interest to examine the effect of PKC inhibitors on the growth and encystation of E. invadens. Trophozoites of E. invadens strain IP-1 were cultured in axenic
288
0014-4894/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
Entamoeba invadens: PKC INHIBITORS BLOCK GROWTH AND ENCYSTATION
289
FIG. 1. Effects of protein kinase C (PKC) inhibitors on the growth and encystation of Entamoeba invadens. For growth, trophozoites were cultured for 7 days in the presence of various concentrations of four PKC inhibitors, staurosporine (A), chelerythrine (B), calphostin C (C), and sphingosine (D). Mean values 6 SE for duplicate cultures are plotted at each concentration (black circles). The control growth was measured in the absence of drug and was expressed as 100%. For encystation, trophozoites were transferred to encystation medium containing various concentrations of the PKC inhibitors. The cysts and trophozoites were counted for determination of the percentage of cysts 3 days later. The control encystation without the drug was expressed as 100%. Mean values 6 SE for duplicate cultures are plotted (white circles).
potent for both growth and encystation, although higher concentration (50 mM) of the drug inhibited the growth (Fig. 1D). PMA (1–10 mM) and 4a-phorbol had no or little effect on the growth and encystation under conditions used (data not shown). Thus, the results indicate participation of PKC in both growth and encystation of E. invadens, although there is a difference in potency among these PKC inhibitors used. The reason for this difference is not clear, but the site of interaction of these inhibitors with PKC appears be related to it because both staurosporine and chelerythrine interact with the catalytic domain of the
FIG. 2. Reversibility of the chelerythrine effect on the encystation of E. invadens. After exposure to 10 mM chelerythrine in encystation medium for 1 day, the drug was removed by replacement of the medium with drug-free encystation medium and the cultures were further incubated for 2 days. The cysts and trophozoites were counted.
enzyme (Tamaoki et al. 1986; Herbert et al. 1990), whereas calphostin C and sphingosine do so with the regulatory domain (Kobayashi et al. 1989; Hannun et al. 1986). Although both domains of PKC include conserved regions, sequence homology in the conserved region of the catalytic domain between E. invadens and mammals would be higher than that of the regulatory domain. Studies on signal transduction in Entamoeba are still limited and those have focused mainly on the adherence and cytolytic activity of trophozoites, where PMA has been used to demonstrate involvement of PKC in it (Weikel et al. 1988; Santiago et al. 1994). However, neither enhanced growth nor enhanced encystation by PMA was observed in the present study. The reason for this remains unclear but exposure time of PMA would be related to it because longer preincubation of the amoebae with PMA did not enhance subsequent killing of target cells and such condition may lead to down-regulation of PKC, preventing a subsequent biologic effect (Weikei et al. 1988). As staurosporine is potent but not a selective inhibitor of PKC, which blocks other protein kinases (Tamaoki et al. 1986; Ru¨egg and Burgess 1989), it would not be suitable for studies on the role of PKC. Therefore, chelerythrine was used for further experiments. Reversibility of the chelerythrine effect on encystation was studied in duplicate cultures containing 5 3 105 trophozoites/ml and 10 mM chelerythrine. Except for the control tubes, after 1 day incubation, cells were washed twice with fresh encystation medium and incubated for another 2 days. As shown in Fig. 2, no recovery of the encystation was observed after removal of chelerythrine, suggesting that, once trophozoites are exposed to chelerythrine in encystation medium, they lose their ability to encyst. The reason for this remains to be clarified. In summary, the present study indicates that PKC participates in the growth and encystation of E. invadens, contributing to further understanding of those of Entamoeba.
290 ACKNOWLEDGMENTS
We thank Dr. L. S. Diamond for supplying the E. invadens and T. Okita for technical assistance. This work was supported by a Grantin-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by a Health Sciences Research Grant for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health and Welfare of Japan.
MAKIOKA ET AL.
Kobayashi, E., Nakano, H., Morimoto, M., and Tamaoki, T. 1989. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochemical and Biophysical Research Communications 159, 548–553. Lo´pez-Romero, E., and Villago´mez-Castro, J. C. 1993. Encystation in Entamoeba invadens. Parasitology Today 9, 225–227. Nishizuka, Y. 1986. Studies and perspectives of protein kinase C. Science 233, 305–312. Ru¨egg, U. T., and Burgess, G. M. 1989. Staurosporine, K-252 and UCN-01: Potent but nonspecific inhibitors of protein kinases. Trends in Pharmacological Sciences 10, 218–220.
REFERENCES
Sanchez, L., Enea, V., and Eichinger, D. 1994. Identification of a developmentally regulated transcript expressed during encystation of Entamoeba invadens. Molecular and Biochemical Parasitology 67, 125–135.
Diamond, L. S., Harlow, D. R., and Cunnick, C. C. 1978. A new medium for the axenic cultivation of Entaoemeba histolytica and other Entamoeba. Transactions of the Royal Society of Tropical Medicine and Hygiene 72, 431–432.
Santiago, A., Carbajal, M. E., Benı´tez-King, G., and Meza, I. 1994. Entamoeba histolytica: PKC transduction pathway activation in the trophozoite–fibronectin interaction. Experimental Parasitology 79, 436–444.
Hannun, Y. A., Loomis, C. R., Merrill, A. H., and Bell, R. M. 1986. Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. Journal of Biological Chemistry 261, 12604–12609. Herbert, J. M., Augereau, J. M., Gleye, J., and Maffrand, J. P. 1990. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochemical and Biophysical Research Communications 172, 993–999.
Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. 1986. Staurosporine, a potent inhibitor of phospholipid/ Ca++dependent protein kinase. Biochemical and Biophysical Research Communications 135, 397–402. Weikel, C. S., Murphy, C. F., Orozco, E., and Ravdin, J. I. 1988. Phorbol esters specifically enhance the cytolytic activity of Entamoeba histolytica. Infection and Immunity 56, 1485–1491. Received 3 March 2000; accepted with revision 13 June 2000